1. Cell membranes, their types. membrane properties. Membrane functions.

Morphological and physiological studies have shown that the cell membrane plays an important role in the functioning of the cell.

Membrane structures: nucleus, Golgi complex, ER, etc.

Membrane is a thin structure with a thickness of 7 nm. According to its chemical composition, the membrane contains 25% proteins, 25% phospholipids, 13% cholesterol, 4% lipids, 3% carbohydrates.

Structurally The basis of the membrane is a double layer of phospholipids. A feature of phospholipid molecules is that they have hydrophilic and hydrophobic parts in their composition. The hydrophilic parts contain polar groups (phosphate groups in phospholipids and hydroxide groups in cholesterol). hydrophilic parts directed towards the surface. BUT hydrophobic (fatty tails) are directed towards the center of the membrane.

The molecule has two fatty tails, and these hydrocarbon chains can be found in two configurations. Stretched - trans configuration(cylinder 0.48 nm). The second type is the gosh-trans-gosh configuration. In this case, the two fat tails diverge and the area increases to 0.58 nm.

Lipid molecules under normal conditions have a liquid crystal form. And in this state they have mobility. Moreover, they can both move within their layer and turn over. When the temperature is lowered, the transition from the liquid state of the membrane to the jelly-like one occurs, and this reduces the mobility of the molecule.

When the lipid molecule moves, microstrips are formed, which are called kings, into which substances can be trapped. The lipid layer in the membrane is a barrier to water-soluble substances, but it allows fat-soluble substances to pass through..

In addition to lipids, the membrane also contains protein molecules. Mostly glycoproteins.

Integral proteins pass through both layers. Other proteins are partially immersed in either the outer or inner layer. They are called peripheral proteins..

This membrane model is called liquid crystal model. Functionally, protein molecules perform structural, transport, enzymatic functions. In addition, they form ion channels with a diameter of 0.35 to 0.8 nm in diameter, through which ions can pass. Channels have their own specialization. Integral proteins are involved in active transport and facilitated diffusion.

Peripheral proteins on the inside of the membrane are characterized by an enzymatic function. On the inside - antigenic (antibodies) and receptor functions.

carbon chains can attach to protein molecules, and then form glycoproteins. Or to lipids, then they are called glycolipids.

Main Functions cell membranes will be:

1. Barrier function

2. Passive and active transfer of substances.

3. Metabolic function (due to the presence of enzyme systems in them)

4. Membranes are involved in the creation of electrical potentials at rest, and upon excitation - action currents.

5. Receptor function.

6. Immunological (associated with the presence of antigens and the production of antibodies).

7. Provide intercellular interaction and contact inhibition.

Upon contact of homogeneous cells, inhibition of cell division occurs. This function is lost in cancer cells. In addition, cancer cells come into contact not only with their own, but also with other cells, infecting them.

Membrane permeability function. Transport.

The transport of substances across membranes can be passive or active.

Passive transfer substances pass through the membranes without energy expenditure in the presence of gradients (differences in the concentrations of substances, differences in the electrochemical gradient, in the presence of a pressure gradient and an osmotic gradient). In this case, passive transport is carried out using:

Diffusion.

Filtration. It is carried out in the presence of a difference in hydrostatic pressure.

Osmosis. During osmosis, the solvent moves. That is, water from a pure solution will pass into a solution with a higher concentration.

In all these cases no energy wasted. Substances go through the pores that are present in the membrane.

There are pores with slow conductivity in the membrane, but there are not many such pores in the membrane. Most of the channels in the membrane also have a gate mechanism in their structure that blocks the channel. These channels can be controlled in two ways: respond to charge changes (electrically excitable or voltage-gated channels). In another case, the gate in the channel opens when a chemical (chemo-excitable or ligand-dependent) is attached.

Active transfer substances across the membrane is associated with the transport of substances against the gradient.

For active transport, integral proteins are used that have enzymatic functions. ATP is used as energy. Integral proteins have special mechanisms (protein) that are activated either when the concentration of a substance increases outside the cell, or when it decreases inside.

Resting currents.

membrane potential. The membrane is positively charged on the outside and negatively charged on the inside. 70-80 mV.

The fault current is the charge difference between the undamaged and the damaged. Damaged is negatively charged, relative to the whole.

Metabolic current is the potential difference due to the unequal intensity of metabolic processes.

Origin membrane potential explain in terms of membrane ion theory, which takes into account the unequal permeability of the membrane for ions and the different composition of ions in the intracellular and intercellular fluid. It has been established that both intracellular and intercellular fluid have the same amount of both positive and negative ions, but the composition is different. External liquid: Na + , Cl - Internal liquid: K + , A - (organic anions)

At rest, the membrane is permeable to ions in different ways. Potassium has the highest permeability, followed by sodium and chlorine. The membranes are not permeable to organic anions.

Due to the increased permeability for potassium ions, they leave the cell. As a result, org accumulate inside. anions. As a result, a potential difference (potassium diffusion potential) is created, which lasts as long as it can exit.

The calculated potassium potential is -90 mV. And the practical potential is -70 mV. This suggests that another ion also participates in the creation of the potential.

In order to contain the potential in the membrane, the cell must work, because the movement of potassium ions out of the cell, and sodium into the cell, would lead to a sign violation. The membranes are polarized. Outside the charge will be positive, and outside - negative.

State electric charge membranes.

Reversion or overshoot - change of charge sign. Return to the original charge - repolarization.

Excited currents.

When a stimulus acts on the membrane, short-term excitation occurs. The excitation process is local and spreads along the membrane, and then depolarizes. As the excitation moves, a new section of the membrane depolarizes, and so on. The action current is a two-phase current.

In each phase of the current of action, a local response can be distinguished, which is replaced by a peak potential, and after the peak potential there is a negative and positive trace potential. Occurs under the action of a stimulus. To explain the current of action, it was proposed membrane-mon theory(Hodge, Huxley, Katz). They showed that action potential greater than resting potential. When the stimulus acts on the membrane, a charge shifts to the membrane (partial depolarization) and this causes the opening of sodium channels. Sodium penetrates into the cell, gradually reducing the charge on the membrane, but the action potential does not arise during any action, but only at a critical value (change by 20-30 mV) - critical depolarization. At the same time, almost all sodium channels open, and in this case, sodium begins to avalanche-like penetrate into the cell. Complete depolarization occurs. The process does not stop at this, but continues to flow into the cell and charges up to +40. At the top of the peak potential, h gates close. At this potential value, the potassium gate opens in the membrane. And since Ka + is larger inside, then Ka + begins to leave the cell, and the charge will begin to return to its original value. It goes fast at first and then slows down. This phenomenon is called the negative tail potential. Then the charge is restored to its original value, and after that a positive trace potential is recorded, characterized by increased permeability to potassium. A state of hyperpolarization of the membrane occurs (positive trace potential). The movement of ions is passive. For one excitation, 20,000 sodium ions enter the cell and 20,000 potassium ions leave the cell.

A pumping mechanism is needed to restore concentration. 3 positive sodium ions are brought in, and 2 potassium ions go out during active transport.

The excitability of the membrane changes, and hence the action potential. During the local response, a gradual increase in excitation occurs. During the peak response, the excitement disappears.

With a negative trace potential, excitability will increase again, because the membrane is again partially depolarized. In the phase of positive light potential, there is a decrease in excitability. Under these conditions, excitability decreases.

The speed of the excitatory process - lability. Measure of lability - the number of excitations per unit of time. Nerve fibers reproduce from 500 to 1000 impulses per second. Different tissues have different lability.

2. Receptors, their classification: by localization (membrane, nuclear), by the mechanism of development of processes (iono- and metabotropic), by the speed of signal reception (fast, slow), by the type of perceiving substances.

The receipt by the cell of a signal from the primary messengers is provided by special receptor proteins, for which the primary messengers are ligands. To ensure the receptor function, protein molecules must meet a number of requirements:

• have high ligand selectivity;
• the kinetics of ligand binding should be described by a curve with saturation corresponding to the state of full employment of all receptor molecules, the number of which on the membrane is limited;
• receptors must have tissue specificity, reflecting the presence or absence of these functions in the cells of the target organ;
• ligand binding and its cellular (physiological) effect must be reversible, affinity parameters must correspond to physiological concentrations of the ligand.

Cellular receptors are divided into the following classes:

• membrane
• receptor tyrosine kinases
• G-protein coupled receptors
• ion channels
• cytoplasmic
• nuclear

Membrane receptors recognize large (eg, insulin) or hydrophilic (eg, adrenaline) signaling molecules that cannot enter the cell on their own. Small hydrophobic signaling molecules (eg, triiodothyronine, steroid hormones, CO, NO) are able to enter the cell by diffusion. The receptors for such hormones are usually soluble cytoplasmic or nuclear proteins. After the ligand binds to the receptor, information about this event is transmitted further along the chain and leads to the formation of a primary and secondary cellular response.

The two main classes of membrane receptors are metabotropic receptors and ionotropic receptors.

Ionotropic receptors are membrane channels that open or close when bound to a ligand. The resulting ion currents cause changes in the transmembrane potential difference and, as a result, the excitability of the cell, and also change the intracellular concentrations of ions, which can secondarily lead to the activation of intracellular mediator systems. One of the most fully studied ionotropic receptors is the n-cholinergic receptor.

The structure of a G-protein consisting of three types of units (heterotrimeric) - αt / αi (blue), β (red) and γ (green)

Metabotropic receptors are associated with intracellular messenger systems. Changes in their conformation upon binding to a ligand leads to the launch of a cascade of biochemical reactions, and, ultimately, a change in the functional state of the cell. The main types of membrane receptors:

Receptors associated with heterotrimeric G proteins (for example, the vasopressin receptor).

Receptors with intrinsic tyrosine kinase activity (eg insulin receptor or epidermal growth factor receptor).

G protein-coupled receptors are transmembrane proteins having 7 transmembrane domains, an extracellular N-terminus and an intracellular C-terminus. The ligand-binding site is located on extracellular loops, and the G-protein-binding domain is near the C-terminus in the cytoplasm.

Activation of the receptor causes its α-subunit to dissociate from the βγ-subunit complex and thus become activated. After that, it either activates or vice versa inactivates the enzyme that produces second messengers.

Receptors with tyrosine kinase activity phosphorylate subsequent intracellular proteins, often also protein kinases, and thus transmit a signal into the cell. Structurally, they are transmembrane proteins with a single membrane domain. As a rule, homodimers, the subunits of which are connected by disulfide bridges.

3. Ionotropic receptors, metabotropic receptors and their varieties. Secondary mediator systems for the action of metabotropic receptors (cAMP, cGMP, inositol-3-phosphate, diacylglycerol, Ca++ ions).

Receptors for neurotransmitters are located on the membranes of neurons or target cells (muscle or glandular cells). Their localization can be both on postsynaptic and presynaptic membranes. On presynaptic membranes, so-called autoreceptors are more often located, which regulate the release of the same mediator from the presynaptic ending. But there are also heteroautoreceptors that also regulate the release of a mediator, but in these receptors, the release of one mediator is regulated by another mediator or neuromodulator.

Most receptors are membrane-bound oligomeric proteins that bind a ligand (neurotransmitter) with high affinity and high selectivity. As a result of this interaction, a cascade of intracellular changes is launched. Receptors are characterized by the affinity for the ligand, the amount, saturation, and dissociation capacity of the receptor-ligand complex. Some receptors have been found to have isoforms that differ in their affinity for certain ligands. These isoforms can be in the same tissue.

Ligands are substances that selectively interact with a given receptor. If a pharmacological substance activates this receptor, it is an agonist for it, and if it reduces its activity, then it is an antagonist.

Binding of the ligand to the receptor leads to a change in the conformation of the receptor, due to which either ion channels open or a cascade of reactions is triggered, leading to changes in metabolism.

There are ionotropic and metabotropic receptors.

ionotropic receptors. Due to the formation of a postsynaptic potential, the corresponding ion channel opens either immediately under the action of a mediator, or through the activation of a G-protein. In this case, the receptor either itself forms an ion channel, or is associated with it. After the attachment of the ligand and activation of the receptor, the channel for the corresponding ion opens. As a result, a postsynaptic potential is formed on the membrane. Ionotropic receptors are a way of rapid signal transmission and the formation of PSP without changing the metabolic processes in the cell.

metabotropic receptors. This is a more complex signal transmission path. In this case, after binding of the ligand to the receptor, the phosphorylation-dephosphorylation cascade is activated. This is done either directly or through second messengers, for example, through tyrosine kinase, or through cAMP, or cGMP, or inositol triphosphate, or diacylglycerol, or by increasing intracellular calcium, which results in the activation of protein kinases. Phosphorylation most often involves the activation of cAMP-dependent or diacylglycerol-dependent protein kinase. These effects develop more slowly and last longer.

The affinity of a receptor for the corresponding neurotransmitter can change in the same way as for hormones, for example, due to allosteric changes in the receptor or other mechanisms. Therefore, receptors are now referred to as mobile and easily changeable structures. As part of the membrane, receptor proteins can interact with other membrane proteins (the so-called receptor internalization). Neuromodulators, like neurotransmitters, can affect the number and sensitivity of receptors. Long-term presence of large amounts of a neurotransmitter or neuromodulator can reduce their sensitivity (down-regulation), and the lack of ligands increase their sensitivity (up-regulation).

4. Ion channels, their structure. Classification of ion channels. sodium and potassium channels.

Structure and functions of ion channels. Ions Na + , K + , Ca 2+ , Cl - penetrate inside the cell and exit through special channels filled with liquid. The size of the channels is rather small (diameter 0.5–0.7 nm). Calculations show that the total area of ​​the channels occupies an insignificant part of the cell membrane surface.

The function of ion channels is studied in various ways. The most common is the voltage-clamp method, or "voltage-clamp" (Fig. 2.2). The essence of the method lies in the fact that with the help of special electronic systems, during the experiment, the membrane potential is changed and fixed at a certain level. In this case, the magnitude of the ion current flowing through the membrane is measured. If the potential difference is constant, then, in accordance with Ohm's law, the magnitude of the current is proportional to the conductivity of the ion channels. In response to stepwise depolarization, certain channels open, the corresponding ions enter the cell along an electrochemical gradient, i.e., an ion current arises that depolarizes the cell. This change is recorded using a control amplifier and an electric current is passed through the membrane, equal in magnitude, but opposite in direction, to the membrane ion current. In this case, the transmembrane potential difference does not change. The combined use of the potential clamp method and specific ion channel blockers led to the discovery of various types of ion channels in the cell membrane.

Currently, many types of channels for various ions are installed (Table 2.1). Some of them are very specific, the latter, in addition to the main ion, can also let other ions through.

The study of the function of individual channels is possible by the method of local fixation of the potential "path-clamp"; rice. 2.3, A). A glass microelectrode (micropipette) is filled with a saline solution, pressed against the membrane surface and a slight vacuum is created. In this case, part of the membrane is sucked to the microelectrode. If an ion channel is in the suction zone, then the activity of a single channel is recorded. The system of stimulation and registration of channel activity differs little from the voltage fixation system.

Table 2.1. The most important ion channels and ion currents of excitable cells

Channel type	Function		Channel blocker
Potassium (at rest)	resting potential generation	I K + (leakage)
sodium	Action potential generation
calcium	Generation of slow potentials		D-600, verapamil
Potassium (delayed rectification)	Ensuring repolarization	I K + (delay)
Potassium calcium-activated	Limitation of depolarization due to Ca 2+ current

Note. TEA - tetraethylammonium; TTX - tetrodotoxin.

The outer part of the canal is relatively accessible for study, the study of the inner part presents significant difficulties. P. G. Kostyuk developed a method of intracellular dialysis, which makes it possible to study the function of the input and output structures of ion channels without the use of microelectrodes. It turned out that the part of the ion channel open to the extracellular space differs in its functional properties from the part of the channel facing the intracellular environment.

It is ion channels that provide two important properties of the membrane: selectivity and conductivity.

selectivity, or selectivity, channel is provided by its special protein structure. Most of the channels are electrically controlled, i.e. their ability to conduct ions depends on the magnitude of the membrane potential. The channel is heterogeneous in its functional characteristics, especially for protein structures located at the entrance to the channel and at its exit (the so-called gate mechanisms).

5. The concept of excitability. Excitability parameters of the neuromuscular system: irritation threshold (rheobase), useful time (chronaxy). The dependence of the strength of stimulation on the time of its action (curve Goorweg-Weiss). Refractory.

Excitability- the ability of the cell to respond to stimulation by the formation of AP and a specific reaction.

1) the phase of the local response - partial depolarization of the membrane (the entry of Na + into the cell). If you apply a small stimulus, then the response is stronger.

Local depolarization - exaltation phase.

2) the phase of absolute refractoriness - the property of excitable tissues not to form AP under any stimulus of any strength

3) phase of relative refractoriness.

4) phase of slow repolarization - irritation - again a strong response

5) phase of hyperpolarization - excitability is less (subnormal), the stimulus must be large.

Functional lability- assessment of tissue excitability through the maximum possible number of AP per unit time.

Laws of excitation:

1) the law of force - the strength of the stimulus must be threshold or suprathreshold (the minimum value of the force that causes excitation). The stronger the stimulus, the stronger the excitation - only for tissue associations (nerve trunk, muscle, exception - SMC).

2) the law of time - a long acting stimulus must be sufficient for the occurrence of excitation.

There is an inversely proportional relationship between force and time within the boundaries between minimum time and minimum force. The minimum force - rheobase - is the force that causes excitation and does not depend on duration. The minimum time is the useful time. Chronaxia is the excitability of a particular tissue, the time at which excitation occurs is equal to two rheobases.

The greater the force, the greater the response up to a certain value.

Factors creating MPP:

1) the difference between the concentrations of sodium and potassium

2) different permeability for sodium and potassium

3) the operation of the Na-K pump (3 Na + is output, 2 K + is returned).

The relationship between the strength of the stimulus and the duration of its impact, necessary for the occurrence of a minimum response of a living structure, can be very well traced on the so-called force-time curve (Goorweg-Weiss-Lapik curve).

It follows from the analysis of the curve that, no matter how great the strength of the stimulus, if the duration of its action is insufficient, there will be no response (points to the left of the ascending branch of the hyperbola). A similar phenomenon is observed with prolonged action of subthreshold stimuli. The minimum current (or voltage) that can cause excitation is called by Lapik the rheobase (the segment of the ordinate OA). The smallest period of time during which a current equal in strength to twice the rheobase causes excitation in the tissue is called chronaxia (abscissa segment OF), which is an indicator of the threshold duration of stimulation. Chronaxy is measured in δ (thousandths of a second). By the magnitude of chronaxia, one can judge the rate of occurrence of excitation in the tissue: the smaller the chronaxia, the faster the excitation occurs. The chronaxy of human nerve and muscle fibers is equal to thousandths and ten thousandths of a second, and the chronaxy of the so-called slow tissues, for example, the muscle fibers of the frog's stomach, is hundredths of a second.

The definition of chronaxy of excitable tissues has become widespread not only in the experiment, but also in the physiology of sports, in the clinic. In particular, by measuring the chronaxia of the muscle, the neuropathologist can establish the presence of damage to the motor nerve. It should be noted that the stimulus can be quite strong, have a threshold duration, but a low rate of rise in time to the threshold value, excitation does not occur in this case. The adaptation of an excitable tissue to a slowly growing stimulus is called accommodation. Accommodation is due to the fact that during the increase in the strength of the stimulus, active changes have time to develop in the tissue that increase the threshold of irritation and prevent the development of excitation. Thus, the rate of increase of stimulation over time, or the gradient of stimulation, is essential for the onset of excitation.

The excitation gradient law. The reaction of a living formation to a stimulus depends on the irritation gradient, i.e., on the urgency or steepness of the growth of the stimulus in time: the higher the irritation gradient, the stronger (up to certain limits) the response of the excitable formation.

Consequently, the laws of stimulation reflect the complex relationship between the stimulus and the excitable structure during their interaction. For the occurrence of excitation, the stimulus must have a threshold strength, have a threshold duration, and have a certain rate of increase in time.

6. Ion pumps (ATPases):K+- Na+-left,Ca2+ (plasmolemma and sarcoplasmic reticulum),H+- K+-exchanger.

According to modern concepts, biological membranes contain ion pumps that operate due to the free energy of ATP hydrolysis - special systems of integral proteins (transport ATPases).

Currently, three types of electrogenic ion pumps are known that carry out active transfer of ions through the membrane (Fig. 13).

The transfer of ions by transport ATPases occurs due to the conjugation of transfer processes with chemical reactions, due to the energy of cell metabolism.

During the work of K + -Na + -ATPase due to the energy released during the hydrolysis of each ATP molecules, two potassium ions are transferred into the cell and three sodium ions are pumped out of the cell at the same time. Thus, an increased concentration of potassium ions in the cell and a reduced concentration of sodium are created compared to the intercellular medium, which is of great physiological importance.

Signs of a "bio pump":

1. Movement against the gradient of the electrochemical potential.

2. the flow of matter is associated with the hydrolysis of ATP (or other energy source).

3. asymmetry of the transport vehicle.

4. The in vitro pump is capable of hydrolyzing ATP only in the presence of those ions that it carries in vivo.

5. when embedding the pump in an artificial environment, it is able to maintain selectivity.

The molecular mechanism of the work of ionic ATPases is not fully understood. Nevertheless, the main stages of this complex enzymatic process can be traced. In the case of K + -Na + -ATPase, there are seven stages of ion transfer associated with ATP hydrolysis.

The diagram shows that the key stages of the enzyme are:

1) the formation of an enzyme complex with ATP on the inner surface of the membrane (this reaction is activated by magnesium ions);

2) binding by the complex of three sodium ions;

3) phosphorylation of the enzyme with the formation of adenosine diphosphate;

4) flip (flip-flop) of the enzyme inside the membrane;

5) the reaction of ion exchange of sodium for potassium, occurring on the outer surface of the membrane;

6) reverse turnover of the enzyme complex with the transfer of potassium ions into the cell;

7) the return of the enzyme to its original state with the release of potassium ions and inorganic phosphate (P).

Thus, for a complete cycle, three sodium ions are released from the cell, the cytoplasm is enriched with two potassium ions, and one ATP molecule is hydrolyzed.

7. Membrane potential, magnitude and origin.

Many theories have been proposed to explain the origin of biopotentials. The membrane theory proposed by the German researcher Bernstein (1902, 1912) is most fully experimentally substantiated. In the modern period, this theory has been modified and experimentally developed by Hodgkin, Huxley, Katz (1949-1952).

It has been established that the basis of bioelectrical phenomena is the uneven distribution (asymmetry) of ions in the cytoplasm of the cell and its environment. Thus, the protoplasm of nerve and muscle cells contains 30-50 times more potassium ions, 8-10 times less sodium ions and 50 times less chloride ions than the extracellular fluid. In addition, the cell cytoplasm contains organic anions (large molecular compounds that carry negative charge), which are absent in the extracellular environment.

Proponents of the membrane theory believe that the main cause of ionic asymmetry is the presence of a cell membrane with specific properties.

The cell membrane is a compacted layer of cytoplasm, the thickness of which is about 10 nm (100 A). The use of electron microscopic research methods made it possible to determine the fine structure of the membrane (Fig. 55). The cell membrane consists of a double layer of phospholipid molecules, which is covered on the inside with a layer of protein molecules, and on the outside with a layer of complex carbohydrate molecules - mucopolysaccharides. The membrane has special channels - "pores" through which water and ions penetrate into the cell. It is assumed that there are special channels for each ion. In this regard, the permeability of the membrane for certain ions will depend on the size of the pores and the diameters of the ions themselves.

In a state of relative physiological rest, the membrane has an increased permeability for potassium ions, while its permeability for sodium ions is sharply reduced.

Thus, the permeability features of the cell membrane, as well as the size of the ions themselves, are one of the reasons that ensure the asymmetry of the distribution of ions on both sides of the cell membrane. Ionic asymmetry is one of the main reasons for the emergence of the resting potential, while the leading role belongs to the uneven distribution of potassium ions.

Hodgkin performed classical experiments on a giant squid nerve fiber. The concentration of potassium ions was equalized inside the fiber and in the surrounding liquid - the resting potential disappeared. If the fiber was filled with an artificial saline solution close in composition to the intracellular fluid, a potential difference was established between the inner and outer sides of the membrane, approximately equal to the resting potential of a normal fiber (50–80 mV).

The mechanism of action potential formation is much more complicated. The main role in the occurrence of action currents belongs to sodium ions. Under the action of a threshold force stimulus, the permeability of the cell membrane for sodium ions increases by 500 times and exceeds the permeability for potassium ions by 10-20 times. In this regard, sodium rushes into the cell like an avalanche, which leads to a recharge of the cell membrane. The outer surface is charged negatively with respect to the inner one. There is a depolarization of the cell membrane, accompanied by a reversion of the membrane potential. Membrane potential reversal is the number of millivolts (mV) by which the action potential exceeds the resting potential. Restoration of the initial level of the membrane potential (repolarization) is carried out due to a sharp decrease in sodium permeability (inactivation) and active transfer of sodium ions from the cell cytoplasm to the environment.

Evidence for the sodium action potential hypothesis was also provided by Hodgkin. Indeed, if the action potential has a sodium nature, then by varying the concentration of sodium ions, it is possible to change the magnitude of the action potential. It turned out that when replacing 2/3 of sea water, which is the normal environment for the giant squid axon, with an isotonic dextrose solution, i.e., when the sodium concentration in the environment changes by 2/3, the action potential decreases by half.

Thus, the occurrence of biopotentials is a function of a biological membrane with selective permeability. The magnitude of the rest potential and action potential is determined by ionic asymmetry in the cell-environment system.

8. Electrical phenomena in the nervous and muscular tissues during excitation. Action potential, its magnitude, phases and duration. The ratio of the phases of the action potential to the phases of excitability.

We have already shown above that the conduction of excitation in nerve and muscle fibers is carried out with the help of electrical impulses propagating along the surface membrane. The transmission of excitation from the nerve to the muscle is based on a different mechanism. It is carried out as a result of the release of highly active chemical compounds by the nerve endings - mediators of the nerve impulse. In skeletal muscle synapses, such a mediator is acetylcholine (ACh).

In the neuromuscular synapse, there are three main structural elements - presynaptic membrane on the nerve postsynaptic membrane on the muscle, between them - synaptic cleft . The shape of the synapse can be varied. At rest, ACh is contained in the so-called synaptic vesicles inside the end plate of the nerve fiber. The cytoplasm of the fiber with synaptic vesicles floating in it is separated from the synaptic cleft by the presynaptic membrane. When the presynaptic membrane is depolarized, its charge and permeability change, the bubbles come close to the membrane and pour out into the synaptic cleft, the width of which reaches 200-1000 angstroms. The mediator begins to diffuse through the gap to the postsynaptic membrane.

The postsynaptic membrane is not electrogenic, but has a high sensitivity to the mediator due to the presence in it of the so-called cholinergic receptors - biochemical groups that can selectively react with ACh. The latter reaches the postsynaptic membrane in 0.2-0.5 msec. (so-called "synaptic delay") and, interacting with cholinergic receptors, causes a change in the membrane permeability for Na, which leads to depolarization of the postsynaptic membrane and the generation of a depolarization wave on it, which is called excitatory postsynaptic potential, (EPSP), the value of which exceeds the Ek of neighboring, electrogenic sections of the muscle fiber membrane. As a result, an AP (action potential) arises in them, which spreads over the entire surface of the muscle fiber, then causing its contraction, initiating the process of the so-called. electromechanical interface (Kapling). The mediator in the synaptic cleft and on the postsynaptic membrane works for a very short time, as it is destroyed by the enzyme cholinesterase, which prepares the synapse to receive a new portion of the mediator. It has also been shown that part of the unreacted ACh can return to the nerve fiber.

With very frequent stimulation rhythms, postsynaptic potentials can be summed up, since cholinesterase does not have time to completely break down the ACh released in the nerve endings. As a result of this summation, the postsynaptic membrane becomes more and more depolarized. At the same time, neighboring electrogenic sections of the muscle fiber come into a state of depression, similar to that which develops during prolonged action of the DC cathode. (Verigo's cathodic depression).

Excitation in the tissue is manifested in the appearance of a function specific to it (conduction of excitation nervous tissue, muscle contraction, gland secretion) and non-specific reactions (action potential generation, metabolic changes).

Action current (AP and PKP) - an electric current that occurs in nerve, muscle and some plant cells between their excited and adjacent resting areas. It is caused by changes in the ionic permeability of the membrane and the potential that develop in the excited area. Plays an important role in the propagation of the action potential along the cell (fiber). An action potential is a shift in the membrane potential that occurs in the tissue under the action of a threshold and suprathreshold stimulus, which is accompanied by a recharge of the cell membrane.

Under the action of a threshold or suprathreshold stimulus, the permeability of the cell membrane for ions changes to varying degrees. For Na ions, it increases by 400-500 times, and the gradient grows rapidly, for K ions - 10-15 times, and the gradient develops slowly. As a result, the movement of Na ions occurs inside the cell, K ions move out of the cell, which leads to a recharge of the cell membrane. The outer surface of the membrane is negatively charged, while the inner surface is positive. Accurate measurements have shown that the amplitude of the action potential is 30-50 mV higher than the value of the resting potential.

PD phases. PD consists of 2 phases:

1. Depolarization phase. Corresponds to a rapid change in membrane potential (membrane depolarization) of approximately 110 mV. The membrane potential changes from a resting level (about -70mV) to a value close to the equilibrium potential - the potential at which the incoming current takes on zero value (ENa+ (about 40mV)).

2. Phase of repolarization. The membrane potential again reaches the resting level (the membrane repolarizes), after which hyperpolarization occurs to a value of about 10 mV less (more negative) than the resting potential, i.e. approximately -80 mV.

The duration of the action potential in nerve and skeletal muscle fibers varies within 0.1 - 5 ms, while the repolarization phase is always longer than the depolarization phase.

The ratio of the phases of the action potential and excitability. The level of cell excitability depends on the AP phase. In the local response phase, excitability increases. This phase of excitability is called latent addition. In the AP repolarization phase, when all sodium channels open and sodium ions rush into the cell like an avalanche, no even superstrong stimulus can stimulate this process. Therefore, the phase of depolarization corresponds to the phase of absolute refractoriness. During the repolarization phase, more and more sodium channels close. However, they can reopen under the action of a suprathreshold stimulus. This corresponds to the phase of relative refractoriness. During trace depolarization, the MP is at a critical level, so even pre-threshold stimuli can cause cell excitation. Therefore, at this moment, her excitability is increased. This phase is called the supernormal excitability phase. At the time of trace hyperpolarization, the MP is higher than the initial level. She is in a phase of subnormal excitability.

9. The structure of skeletal muscles and their innervation. motor unit. Physiological properties muscles, their features in the newborn.

Morpho-functional classification of muscles:

1. Cross-striped

a) skeletal - multinucleated cells, transversely striated, nuclei closer to the sarcolemma. Weight 40%.

b) cardiac - transversely striated, mononuclear cells, the nucleus in the center. Weight 0.5%.

2. Smooth - single-nuclear cells, do not have transverse striation. They are part of other organs. total weight 5-10%.

General properties of muscles.

1) Excitability. PP = - 90mV. PD amplitude = 120 mV - +30 mV sign reversal.

2) Conductivity - the ability to conduct PD along the cell membrane (3-5 m/s). Provides delivery of PD to T-tubules and from them to L-tubules releasing calcium.

3) Contractility - the ability to shorten or develop tension when excited.

4) Elasticity - the ability to return to the original length.

Skeletal Muscle Functions:

1. Movement of the body in space

2. Moving parts of the body relative to each other

3. Maintain posture

4. Heat generation

5. Movement of blood and lymph (dynamic work)

6. Participation in lung ventilation

7. Protection of internal organs

8. Anti-stress factor

Levels of organization of skeletal muscle:

The whole muscle is surrounded by epimysium, vessels and nerves approach it. Separate muscle bundles are covered with perimysium. A bundle of cells (muscle fiber or symplast) - covered with endomysium. The cell contains myofibrils from myofilaments, the main proteins - actin, myosin, tropomyosin, troponin, calcium ATPase, creatine phosphokinase, structural proteins.

Motor (motor, neuromotor units) are isolated in the muscle - this is a functional combination of a motor neuron, its axon and muscle fibers innervated by this axon. These muscle fibers can be located in different parts (bundles) of the muscle.

The motor unit (MU) is the functional unit of skeletal muscle. ME includes a motor neuron and the group of muscle fibers innervated by it.

Types of muscle fibers:

1) slow phasic fibers of the oxidative type

2) fast phasic fibers of the oxidative type (type 2a)

3) fast phasic fibers of the glycolytic type (type 2b)

4) tonic fibers

Mechanisms of muscle contraction.

A) a single muscle fiber

B) a whole muscle

Skeletal muscle has the following essential properties:

1) excitability - the ability to respond to the action of the stimulus by changing the ionic conductivity and membrane potential. Under natural conditions, this stimulus is the neurotransmitter acetylcholine.

2) conductivity - the ability to conduct an action potential along and deep into the muscle fiber along the T-system;

3) contractility - the ability to shorten or develop tension when excited;

4) elasticity - the ability to develop stress when stretched.

10. Modes of muscle contraction: isotonic and isometric. Absolute muscle strength. Age-related changes in muscle strength.

The contractility of the skeletal muscle is characterized by the force of contraction that the muscle develops (usually estimated general strength, that a muscle can develop, and absolute, i.e., the force per 1 cm 2 of the cross section). The length of shortening, the degree of tension of the muscle fiber, the speed of shortening and development of tension, the speed of relaxation. Since these parameters are largely determined by the initial length of muscle fibers and the load on the muscle, studies of muscle contractility are carried out in various modes.

Irritation of a muscle fiber by a single threshold or suprathreshold stimulus leads to the occurrence of a single contraction, which consists of several periods (Fig. 2.23). The first, the latent period, is the sum of time delays caused by the excitation of the muscle fiber membrane, the spread of AP along the T-system into the fiber, the formation of inositol triphosphate, an increase in the concentration of intracellular calcium, and the activation of cross-bridges. For the frog sartorius muscle, the latency period is about 2 ms.

The second is the period of shortening, or the development of tension. In the case of free shortening of the muscle fiber, they speak of isotonic contraction, at which the tension practically does not change, but only the length of the muscle fiber changes. If the muscle fiber is fixed on both sides and cannot be freely shortened, then they speak of isometric mode Strictly speaking, under this mode of contraction, the length of the muscle fiber does not change, while the size of the sarcomeres changes due to the sliding of the actin and myosin filaments relative to each other. In this case, the resulting stress is transferred to the elastic elements located inside the fiber. Elastic properties are possessed by cross bridges of myosin filaments, actin filaments, Z-plates, a longitudinally located sarcoplasmic reticulum and a muscle fiber sarcolemma.

In experiments on an isolated muscle, stretching of the connective tissue elements of the muscle and tendons is revealed, to which the tension developed by the transverse bridges is transmitted.

In the human body, in an isolated form, isotonic or isometric contraction does not occur. As a rule, the development of tension is accompanied by a shortening of the muscle length - auxotonic mode contraction

The third is a period of relaxation, when the concentration of Ca 2+ ions decreases and myosin heads detach from actin filaments.

It is believed that for a single muscle fiber, the voltage developed by any sarcomere is equal to the voltage in any other sarcomere. Since sarcomeres are connected in series, the rate at which a muscle fiber contracts is proportional to the number of its sarcomeres. Thus, with a single contraction, the rate of shortening of a long muscle fiber is higher than that of a shorter one. The amount of effort developed by a muscle fiber is proportional to the number of myofibrils in the fiber. During muscle training, the number of myofibrils increases, which is the morphological substrate for increasing the strength of muscle contraction. At the same time, the number of mitochondria increases, which increase the endurance of the muscle fiber during physical activity.

In an isolated muscle, the magnitude and speed of a single contraction are determined by a number of additional factors. The magnitude of a single contraction will primarily be determined by the number of motor units involved in the contraction. Since muscles are composed of muscle fibers with different levels of excitability, there is a certain relationship between the magnitude of the stimulus and the response. An increase in the force of contraction is possible up to a certain limit, after which the amplitude of contraction remains unchanged with an increase in the amplitude of the stimulus. In this case, all muscle fibers that make up the muscle take part in the contraction.

The importance of the participation of all muscle fibers in contraction is shown when studying the dependence of the rate of shortening on the magnitude of the load.

When a second stimulus is applied during the period of shortening or development of muscle tension, the summation of two consecutive contractions occurs and the resulting response in amplitude becomes significantly higher than with a single stimulus; if a muscle fiber or muscle is stimulated with such a frequency that repeated stimuli will occur during the period of shortening, or the development of tension, then a complete summation of single contractions occurs and develops smooth tetanus (Fig. 2.25, B). Tetanus is a strong and prolonged contraction of a muscle. It is believed that this phenomenon is based on an increase in the calcium concentration inside the cell, which allows the reaction of interaction between actin and myosin and the generation of muscle strength by transverse bridges for quite a long time. With a decrease in the frequency of stimulation, a variant is possible when a repeated stimulus is applied during the relaxation period. In this case, the summation of muscle contractions will also occur, however, a characteristic retraction will be observed on the curve of muscle contraction (Fig. 2.25, D) - incomplete summation, or serrated tetanus.

With tetanus, the summation of muscle contractions occurs, while the PD of muscle fibers are not summed up.

Under natural conditions, single contractions of skeletal muscles do not occur. addition occurs, or superposition, contractions of individual neuromotor units. At the same time, the contraction force can increase both due to a change in the number of motor units involved in the contraction, and due to a change in the frequency of motoneuron impulses. In the case of an increase in the frequency of impulses, a summation of contractions of individual motor units will be observed.

One of the reasons for the increase in contraction force in natural conditions is the frequency of impulses generated by motor neurons. The second reason for this is an increase in the number of excitable motor neurons and synchronization of the frequency of their excitation. An increase in the number of motor neurons corresponds to an increase in the number of motor units involved in the contraction, and an increase in the degree of synchronization of their excitation contributes to an increase in the amplitude during the superposition of the maximum contraction developed by each motor unit separately.

The force of contraction of an isolated skeletal muscle, other things being equal, depends on the initial length of the muscle. Moderate stretching of the muscle leads to the fact that the force developed by it increases compared to the force developed by the unstretched muscle. There is a summation of passive tension, due to the presence of elastic components of the muscle, and active contraction. The maximum contraction force is achieved with a sarcomere size of 2-2.2 microns (Fig. 2.26). An increase in the length of the sarcomere leads to a decrease in the force of contraction, since the area of ​​mutual overlap of actin and myosin filaments decreases. With a sarcomere length of 2.9 µm, the muscle can develop only 50% of its maximum force.

Under natural conditions, the force of contraction of skeletal muscles during their stretching, for example, during massage, increases due to the work of gamma efferents.

Absolute muscle strength is the ratio of the maximum muscle strength to its physiological diameter, i.e. the maximum load that a muscle lifts divided by the total area of ​​all muscle fibers. The strength of the contraction does not remain constant throughout life. As a result of prolonged activity, the performance of skeletal muscles decreases. This phenomenon is called fatigue. At the same time, the strength of contractions decreases, the latent period of contraction and the period of relaxation increase.

11. Single muscle contractions, its phases. Phases of changes in muscle excitability. Features of a single contraction in newborns.

Irritation of a muscle or motor nerve innervating it with a single stimulus causes a single muscle contraction. It distinguishes two main phases: the contraction phase and the relaxation phase. The contraction of the muscle fiber begins already during the ascending branch of the AP. The duration of contraction at each point of the muscle fiber is tens of times greater than the duration of AP. Therefore, there comes a moment when the AP has passed along the entire fiber and ended, while the contraction wave has covered the entire fiber and it continues to be shortened. This corresponds to the moment of maximum shortening or tension of the muscle fiber.

The contraction of each individual muscle fiber during single contractions obeys the law " all or nothing". This means that the contraction that occurs both with threshold and supra-threshold stimulation has a maximum amplitude. The magnitude of a single contraction of the entire muscle depends on the strength of the irritation. With threshold stimulation, its contraction is barely noticeable, but with an increase in the strength of irritation it increases, until it reaches a certain height, after which it remains unchanged (maximum contraction). This is due to the fact that the excitability of individual muscle fibers is not the same, and therefore only part of them is excited with weak irritation. At maximum contraction, they are all excited. The speed of the wave of muscle contraction is the same with the speed of propagation of AP.In the biceps muscle of the shoulder, it is 3.5-5.0 m/sec.

Single contraction - contraction by one stimulus. It includes a latent period, a contraction phase and a relaxation phase. At the time of the latent period, the refractory phase occurs. But already at the beginning of the shortening phase, it is restored.

12. Summation of muscle contractions. tetanic contractions.

If, in an experiment, an individual muscle fiber or the entire muscle is affected by two rapidly following each other strong single stimuli, then the resulting contraction will have a greater amplitude than the maximum single contraction. The contractile effects caused by the first and second irritation seem to add up. This phenomenon is called the summation of contractions. For summation to occur, it is necessary that the interval between stimuli has a certain duration - it must be longer than the refractory period, but shorter than the entire duration of a single contraction, so that the second stimulus acts on the muscle before it has time to relax. In this case, two cases are possible. If the second stimulation arrives when the muscle has already begun to relax, on the myographic curve the top of the second contraction will be separated from the first by a depression. If the second irritation acts when the first contraction has not yet reached its peak, then the second contraction, as it were, merges with the first, forming with it a single summed peak. Both with full and incomplete summation, PDs are not summed up. Such a summed contraction in response to rhythmic stimuli is called tetanus. Depending on the frequency of irritation, it is serrated and smooth.

The reason for the summation of contractions in tetanus lies in the accumulation of Ca ++ ions in the interfibrillar space up to a concentration of 5 * 10 6 mM / l. After reaching this value, further accumulation of Ca++ does not lead to an increase in the tetanus amplitude.

After the termination of tetanic irritation, the fibers do not relax completely at first, and their original length is restored only after some time has passed. This phenomenon is called post-tetanic, or residual contracture. She is connected to it. that it takes more time to remove from the interfibrillar space all Ca ++ that got there with rhythmic stimuli and did not have time to completely withdraw into the cisterns of the sarcoplasmic reticulum by the work of Ca-pumps.

If, after reaching a smooth tetanus, the frequency of stimulation is increased even more, then the muscle at a certain frequency suddenly begins to relax. This phenomenon is called pessimism . It occurs when each next impulse falls into refractoriness from the previous one.

13. Ultrastructure of myofibrils. Contractile proteins (actin, myosin). Regulatory proteins (troponin, tropomyosin) in thin protofibrils. The theory of muscle contraction.

Myofibrils are the contractile apparatus of the muscle fiber. In striated muscle fibers, myofibrils are divided into regularly alternating sections (discs) with different optical properties. Some of these sections are anisotropic, i.e. have double refraction. In ordinary light they look dark, but in polarized light they are transparent in the longitudinal direction and opaque in the transverse direction. Other areas are isotropic, and appear transparent in ordinary light. Anisotropic regions are denoted by the letter BUT, isotropic - I. In the middle of disk A there is a light strip H, and in the middle of disk I there is a dark stripe Z, which is a thin transverse membrane through the pores of which myofibrils pass. Due to the presence of such a support structure, parallel single-valued disks of individual myofibrils within one fiber do not move relative to each other during contraction.

It has been established that each of the myofibrils has a diameter of about 1 micron and consists of an average of 2500 protofibrils, which are elongated molecules polymerized by the protein myosin and actin. Myosin filaments (protofibrils) are twice as thick as actin filaments. Their diameter is approximately 100 angstroms. In the resting state of the muscle fiber, the filaments are located in the myofibril in such a way that the thin long actin filaments enter with their ends into the gaps between the thick and shorter myosin filaments. In such a section, each thick thread is surrounded by 6 thin ones. Due to this, disks I consist only of actin filaments, and disks A also consist of myosin filaments. The light stripe H is a zone free from actin filaments during the dormant period. Membrane Z, passing through the middle of disc I, holds the actin filaments together.

Numerous cross-bridges on myosin are also an important component of the ultramicroscopic structure of myofibrils. In turn, there are so-called active centers on actin filaments, at rest covered, like a sheath, with special proteins - troponin and tropomyosin. Contraction is based on the sliding of actin filaments relative to myosin filaments. Such sliding is caused by the work of the so-called. "chemical gear", ie. periodically occurring cycles of changes in the state of cross bridges and their interaction with active centers on actin. ATP and Ca+ ions play an important role in these processes.

When the muscle fiber contracts, the actin and myosin filaments do not shorten, but begin to slide over each other: the actin filaments move between the myosin filaments, as a result of which the length of the I disks is shortened, and the A disks retain their size, approaching each other. The H strip almost disappears, because the ends of the actin are in contact and even go behind each other.

14. Relationship of excitation and contraction (electromechanical coupling) in muscle fibers. The role of calcium ions. Function of the sarcoplasmic reticulum.

In skeletal muscle, under natural conditions, the initiator of muscle contraction is the action potential, which propagates upon excitation along the surface membrane of the muscle fiber.

If the tip of the microelectrode is applied to the surface of the muscle fiber in the area of ​​the Z membrane, then when a very weak electrical stimulus is applied that causes depolarization, the I disks on both sides of the stimulation site will begin to shorten. in this case, the excitation propagates deep into the fiber, along the Z membrane. Irritation of other sections of the membrane does not cause such an effect. From this it follows that the depolarization of the surface membrane in the region of disc I during AP propagation is the trigger of the contractile process.

Further studies showed that an important intermediate link between membrane depolarization and the onset of muscle contraction is the penetration of free CA++ ions into the interfibrillar space. At rest, most of the Ca++ in the muscle fiber is stored in the sarcoplasmic reticulum.

In the mechanism of muscle contraction, a special role is played by that part of the reticulum, which is localized in the region of the Z membrane. triad (T-system), each of which consists of a thin transverse tubule located centrally in the Z membrane region, running across the fiber, and two lateral cisterns of the sarcoplasmic reticulum, in which bound Ca ++ is enclosed. AP propagating along the surface membrane is conducted deep into the fiber along the transverse tubules of the triads. Then the excitation is transferred to the cisternae, depolarizes their membrane and it becomes permeable to CA++.

It has been experimentally established that there is a certain critical concentration of free Ca++ ions, at which the contraction of myofibrils begins. It is equal to 0.2-1.5*10 6 ions per fiber. Increasing the concentration of Ca++ to 5*10 6 already causes the maximum reduction.

The onset of muscle contraction is timed to the first third of the ascending AP knee, when its value reaches about 50 mV. It is believed that it is at this depolarization level that the concentration of Ca++ becomes the threshold for the beginning of the interaction between actin and myosin.

The Ca++ release process stops after the end of the AP peak. Nevertheless, the contraction continues to grow until the mechanism that ensures the return of Ca ++ to the reticulum cisterns comes into action. This mechanism is called the "calcium pump". To carry out its work, the energy obtained from the breakdown of ATP is used.

In the interfibrillar space, Ca++ interacts with proteins that close the active centers of actin filaments - troponin and tropomyosin, providing an opportunity for the reaction of myosin cross-bridges and actin filaments.

Thus, the sequence of events leading to contraction and then to relaxation of the muscle fiber is currently drawn as follows:

15. Fatigue during muscular work. Reasons for fatigue. The concept of active recreation.

Fatigue is a temporary decrease in the efficiency of a cell, organ or the whole organism, which occurs as a result of work and disappears after rest.

If for a long time an isolated muscle, to which a small load is suspended, is irritated with rhythmic electrical stimuli, then the amplitude of its contractions gradually decreases until it drops to zero. The fatigue curve is recorded. Along with a change in the amplitude of contractions during fatigue, the latent period of contraction increases, the period of muscle relaxation lengthens, and the stimulation threshold increases, i.e. excitability decreases. All these changes do not occur immediately after the start of work, there is a certain period during which there is an increase in the amplitude of contractions and a slight increase in muscle excitability. At the same time, it becomes easily stretchable. In such cases, they say that the muscle is "worked in", i.e. adapts to work in a given rhythm and strength of irritation. After a period of workability, a period of stable performance begins. With further prolonged irritation, fatigue of the muscle fibers occurs.

The decrease in the efficiency of a muscle isolated from the body during its prolonged irritation is due to two main reasons. The first of these is that during contractions, metabolic products accumulate in the muscle (phosphoric acid, which binds Ca ++, lactic acid, etc.), which have a depressing effect on muscle performance. Some of these products, as well as Ca ions, diffuse out of the fibers into the pericellular space and have a depressing effect on the ability of the excitable membrane to generate AP. So, if an isolated muscle placed in a small volume of Ringer's fluid is brought to complete fatigue, then it is enough just to change the solution washing it to restore muscle contractions.

Another reason for the development of fatigue in an isolated muscle is the gradual depletion of energy reserves in it. With prolonged work, the content of glycogen in the muscle decreases sharply, as a result of which the processes of regeneration are disrupted. ATP synthesis and CF needed to implement the reduction.

It should be noted that under the natural conditions of the organism's existence, fatigue of the motor apparatus during prolonged work develops in a completely different way than in an experiment with an isolated muscle. This is due not only to the fact that in the body the muscle is continuously supplied with blood, and, therefore, receives the necessary nutrients with it and is released from metabolic products. The main difference is that in the body, excitatory impulses come to the muscle from the nerve. The neuromuscular synapse gets tired much earlier than the muscle fiber, due to the rapid depletion of the accumulated mediator. This causes a blockade of the transmission of excitations from the nerve to the muscle, which prevents the muscle from exhaustion caused by prolonged work. In a whole organism, the nerve centers (nerve-nerve contacts) get tired even earlier during work.

Role nervous system in fatigue of the whole organism is proved by studies of fatigue in hypnosis (kettlebell-basket), the establishment of the influence of "active rest" on fatigue, the role of the sympathetic nervous system (the Orbeli-Ginetsinsky phenomenon), etc.

Ergography is used to study muscle fatigue in humans. The shape of the fatigue curve and the amount of work done vary enormously in different individuals and even in the same subject under different conditions.

16. Physiological features of smooth muscles. Plastic tone of smooth muscles.

An important property of smooth muscle is its large plastic , those. the ability to maintain the length given by stretching without changing the stress. Skeletal muscle, on the other hand, shortens immediately after the load is removed. A smooth muscle remains stretched until, under the influence of some kind of irritation, its active contraction occurs. The property of plasticity is of great importance for the normal activity of hollow organs - thanks to it, the pressure inside a hollow organ changes relatively little with different degrees of its filling.

There are different types of smooth muscles. In the walls of most hollow organs there are muscle fibers 50–200 microns long and 4–8 microns in diameter, which are very closely adjacent to each other, and therefore, when viewed under a microscope, it seems that they are morphologically one. Electron microscopic examination shows, however, that they are separated from each other by intercellular gaps, the width of which can be equal to 600-1500 angstroms. Despite this, smooth muscle functions as a single entity. This is expressed in the fact that AP and slow waves of depolarization propagate freely from one fiber to another.

In some smooth muscles, for example, in the ciliary muscle of the eye, or the muscles of the iris, the fibers are located separately, and each has its own innervation. In most smooth muscles, motor nerve fibers are located on only a small number of fibers.

The resting potential of smooth muscle fibers with automaticity exhibits constant small fluctuations. Its value at intracellular assignment is 30-70 mV. The resting potential of smooth muscle fibers that do not have automaticity is stable and equal to 60-70 mV. In both cases, its value is less than the resting potential of the skeletal muscle. This is due to the fact that the membrane of smooth muscle fibers at rest is characterized by a relatively high permeability to Na ions. Action potentials in smooth muscle are also somewhat lower than in skeletal muscle. The excess over the resting potential is no more than 10-20 mV.

The ionic mechanism of AP occurrence in smooth muscles is somewhat different from that in skeletal muscles. It has been established that the regenerative depolarization of the membrane, which underlies the action potential in a number of smooth muscles, is associated with an increase in the permeability of the membrane for Ca++ ions, rather than Na+.

Many smooth muscles are characterized by spontaneous, automatic activity. It is characterized by a slow decrease in the resting membrane potential, which, when a certain level is reached, is accompanied by the onset of AP.

In nerve and skeletal muscle fibers, excitation spreads through local electric currents that arise between the depolarized and neighboring resting sections of the cell membrane. The same mechanism is characteristic of smooth muscles. However, unlike in skeletal muscle, in smooth muscle an action potential originating in one fiber can propagate to adjacent fibers. This is due to the fact that in the membrane of smooth muscle cells in the area of ​​contacts with neighboring ones there are areas of relatively low resistance through which the current loops that have arisen in one fiber easily pass to the neighboring ones, causing depolarization of their membranes. In this respect, smooth muscle is similar to cardiac muscle. The only difference is that in the heart, the entire muscle is excited from one cell, while in smooth muscles, AP that arises in one area propagates from it only a certain distance, which depends on the strength of the applied stimulus.

Another essential feature of smooth muscles is that propagating AP occurs downward only if the applied stimulus simultaneously excites a certain minimum number of muscle cells. This "critical zone" has a diameter of about 100 microns, which corresponds to 20-30 parallel cells. The rate of excitation conduction in various smooth muscles ranges from 2 to 15 cm/sec. those. much less than in skeletal muscle.

As well as in skeletal muscles, in smooth action potentials they have a starting value for the start of the contractile process. The connection between excitation and contraction is also carried out here with the help of Ca ++. However, in smooth muscle fibers, the sarcoplasmic reticulum is poorly expressed; therefore, the leading role in the mechanism of contraction is assigned to those Ca ++ ions that penetrate into the muscle fiber during AP generation.

With a large force of a single irritation, smooth muscle contraction may occur. The latent period of its contraction is much longer than the skeletal period, reaching 0.25-1 sec. The duration of the contraction itself is also large - up to 1 minute. Relaxation is especially slow after contraction. The contraction wave propagates through the smooth muscles at the same speed as the excitation wave (2-15 cm/sec). But this slowness of contractile activity is combined with a great force of smooth muscle contraction. So, the muscles of the stomach of birds are capable of lifting 2 kg per 1 sq. mm. its cross section.

Due to the slowness of contraction, smooth muscle, even with rare rhythmic stimulation (10-12 per minute), easily passes into a long-term state of persistent contraction, resembling tetanus of skeletal muscles. However, the energy costs of such a reduction are very low.

The ability to automate smooth muscles is inherent in their muscle fibers and is regulated by nerve elements that are located in the walls of smooth muscle organs. The myogenic nature of automaticity has been proven by experiments on strips of muscles of the intestinal wall, freed from nerve elements. Smooth muscle responds to all external influences by changing the frequency of spontaneous rhythm, resulting in contraction or relaxation of the muscle. The effect of irritation of the smooth muscles of the intestine depends on the ratio between the frequency of stimulation and the natural frequency of spontaneous rhythm: with a low tone - rare spontaneous AP - the applied irritation increases the tone, with a high tone, relaxation occurs in response to irritation, since an excessive increase in impulses leads to the fact that each next impulse falls into the phase of refractoriness from the previous one.

17. Structure and functions of nerve fibers. The mechanism of excitation

myelinated and unmyelinated nerve fibers. Significance of interceptions of Ranvier.

The main function of axons is to conduct impulses arising in the neuron. Axons can be covered with a myelin sheath (myelinated fibers) or devoid of it (unmyelinated fibers). Myelinated fibers are more common in motor nerves, unmyelinated fibers predominate in the autonomic (vegetative) nervous system.

An individual myelinated nerve fiber consists of an axial cylinder covered by a myelin sheath formed by Schwann cells. The axial cylinder has a membrane and an axoplasm. The myelin sheath is a product of the Schwann cell and consists of 80% lipids with high ohmic resistance and 20% protein.

The myelin sheath does not cover the axial cylinder with a continuous cover, but is interrupted, leaving open areas of the axial cylinder, called nodal intercepts (Ranvier intercepts). The length of the sections between these intercepts is different and depends on the thickness of the nerve fiber: the thicker it is, the longer the distance between the intercepts (Fig. 2.17).

Unmyelinated nerve fibers are covered only by the Schwann sheath.

The conduction of excitation in unmyelinated fibers differs from that in myelinated fibers due to the different structure of the membranes. In unmyelinated fibers, excitation gradually covers neighboring sections of the membrane of the axial cylinder and thus spreads to the end of the axon. The speed of propagation of excitation along the fiber is determined by its diameter.

In non-myelinated nerve fibers, where metabolic processes do not provide a quick compensation for the energy expended on excitation, the spread of this excitation proceeds with a gradual weakening - with a decrement. Decrementary conduction of excitation is characteristic of a low-organized nervous system.

In higher animals, due primarily to the presence of the myelin sheath and the perfection of metabolism in the nerve fiber, excitation passes without fading, without decrement. This is facilitated by the presence of an equal charge fiber throughout the membrane and its rapid recovery after the passage of excitation.

In myelin fibers, excitation covers only areas of nodal intercepts, that is, it bypasses the zones covered with myelin. This conduction of excitation along the fiber is called saltatory (jumping). In nodal intercepts, the number of sodium channels reaches 12,000 per 1 µm, which is much more than in any other fiber section. As a result, nodal intercepts are the most excitable and provide a high speed of excitation. The time of conduction of excitation along the myelin fiber is inversely proportional to the length between intercepts.

The conduction of excitation along the nerve fiber is not disturbed for a long (many hours) time. This indicates a low fatigue of the nerve fiber. It is believed that the nerve fiber is relatively indefatigable due to the fact that the processes of energy resynthesis in it proceed at a sufficiently high speed and have time to restore the energy expenditure that occurs during the passage of excitation.

At the moment of excitation, the energy of the nerve fiber is spent on the work of the sodium-potassium pump. Particularly large energy expenditures occur in the nodes of Ranvier due to the high density of sodium-potassium channels here.

J. Erlanger and X. Gasser (1937) were the first to classify nerve fibers according to the speed of excitation conduction. A different rate of conduction of excitation along the fibers of the mixed nerve appears when using an extracellular electrode. The potentials of fibers conducting excitation at different speeds are recorded separately (Fig. 2.18).

Depending on the speed of excitation, nerve fibers are divided into three types: A, B, C. In turn, type A fibers are divided into four groups: A α , A β , A γ , A δ . Group A fibers have the highest conduction speed (up to 120 m/s). α , which is made up of fibers with a diameter of 12-22 microns. Other fibers have a smaller diameter and, accordingly, excitation through them occurs at a lower speed (Table 2.4).

The nerve trunk is formed a large number fibers, however, the excitation going through each of them is not transmitted to the neighboring ones. This feature of the conduction of excitation along the nerve is called law of isolated conduction of excitation along a single nerve fiber. The possibility of such conduction is of great physiological significance, since it ensures, for example, the isolation of the contraction of each neuromotor unit.

The ability of a nerve fiber to conduct excitation in isolation is due to the presence of sheaths, and also to the fact that the resistance of the fluid filling the interfiber spaces is much lower than the resistance of the fiber membrane. Therefore, the current, leaving the excited fiber, is shunted in the liquid and turns out to be weak for excitation of neighboring fibers. Necessary condition conducting excitation in the nerve is not just its anatomical continuity, but also its physiological integrity. In any metallic conductor, electric current will flow as long as the conductor maintains physical continuity. For the nerve "conductor" this condition is not enough: the nerve fiber must also maintain physiological integrity. If the properties of the fiber membrane are violated (ligation, blockade with novocaine, ammonia, etc.), the conduction of excitation along the fiber stops. Another property characteristic of the conduction of excitation along the nerve fiber is the ability for bilateral conduction. Applying stimulation between two lead-off electrodes on the surface of the fiber will cause electrical potentials under each of them.

Table - Speed ​​of conduction of excitation along nerve fibers

Group of fibers	Fiber diameter, µm	Conduction speed, m/s

18. Laws of conducting excitation along the nerves. Classification of nerve fibers. The speed of the conduction of excitation along the nerve fibers, its age-related features.

19. Structure of the neuromuscular synapse. The mechanism of transmission of excitation from the nerve to the muscle.End plate potential, its properties.

Synapses are the contacts that neurons establish as independent formations. The synapse is a complex structure and consists of the presynaptic part (the end of the axon that transmits the signal), the synaptic cleft and the postsynaptic part (the structure of the perceiving cell).

Neuromuscular synapses ensure the conduction of excitation from the nerve fiber to the muscle due to the mediator acetylcholine, which, when the nerve ending is excited, passes into the synaptic cleft and acts on the end plate of the muscle fiber.

In the presynaptic terminal, acetylcholine is formed and accumulates in the form of vesicles. When excited by an electrical impulse going along the axon, the presynaptic part of the synapse, its membrane becomes permeable to acetylcholine.

This permeability is possible due to the fact that as a result of depolarization of the presynaptic membrane, its calcium channels open. The Ca2+ ion enters the presynaptic part of the synapse from the synaptic cleft. Acetylcholine is released and enters the synaptic cleft. Here it interacts with its receptors on the postsynaptic membrane belonging to the muscle fiber. Receptors, being excited, open a protein channel built into the lipid layer of the membrane. Through the open channel, Na + ions penetrate into the muscle cell, which leads to depolarization of the muscle cell membrane, as a result, the so-called end plate potential (EPP) develops. Since this potential is normally always above the threshold, it causes an action potential that propagates along the muscle fiber and causes contraction. The potential of the end plate is short, since acetylcholine, firstly, quickly detaches from the receptors, and secondly, it is hydrolyzed by AChE.

The neuromuscular synapse transmits excitation in one direction: from the nerve ending to the postsynaptic membrane of the muscle fiber, which is due to the presence of a chemical link in the mechanism of neuromuscular transmission.

The speed of excitation through the synapse is much less than along the nerve fiber, since it takes time for the activation of the presynaptic membrane, the passage of calcium through it, the release of acetylcholine into the synaptic cleft, the depolarization of the postsynaptic membrane, and the development of PKP.

Objective: show that the cell membrane has selective permeability. Demonstrate the role of the membrane in the process of phagocytosis and pinocytosis.

Equipment: microscopes, coverslips and slides, scalpels, dissecting needles, cups for water and solutions, filter paper, pipettes, ink. Culture of ciliates, amoebas, elodea leaf. NaCl or KCl solutions, CaCl or MgCl solutions, 2% albumin solution, 10% NaCl solution, distilled water.

Working process:

Place ciliates in a weak solution of NaCl or KCl. Prepare a microscope slide. Wrinkling of the cells can be seen, indicating permeability of the cell wall. In this case, the water from the cell is released into the environment. Transfer the cells to a drop of distilled water or draw the solution from under the coverslip with filter paper and replace it with distilled water. Watch the cells swell as water enters them.

Place the infusoria in a low concentration CaCl or MgCl solution (same as the previous solution). The ciliates continue to live, no deformations are observed. Ca and Mg ions reduce the permeability of the cell membrane, in contrast to Na and K ions. There is no movement of water through the shell.

Place the amoeba in a drop of 2% albumin solution (chicken egg white). Prepare a microscope slide. After some time, bubbles, protrusions, tubules begin to form on the surface of the amoeba. It seems that the surface of the amoeba is "boiling". This is accompanied by intense fluid movement near the membrane surface. Fluid bubbles are surrounded by protrusions of the cytoplasm. which are then closed. Pinocytic vesicles sometimes appear suddenly, which indicates the rapid capture of a drop of liquid along with a substance soluble in it.

Place the amoeba in the sugar solution. Pinocytosis is absent. Pinocytosis is caused only by substances that lower the surface tension of the cell membrane, such as amino acids, some salts. In a drop of liquid in which amoebas are located, enter a little finely ground carcass. Prepare the preparation for the microscope. After some time, the amoebas begin to slowly move towards the grains of the carcass, releasing pseudopodia. Carcass grains are attached to the surface of pseudopodia, then slowly surrounded by them and after a while are immersed in the cytoplasm. Under a microscope, observe the phenomenon of phagocytosis in an amoeba.

In the cytoplasm of Elodea cells, many round-oval green bodies are visible - these are chloroplasts. Examine the cells near the central vein of the leaf. They can detect the movement of the cytoplasm and plastids along the walls. If the movement is hardly noticeable, heat the preparation under an electric lamp.

Sketch everything you saw on the slides. Discuss in groups the processes you have seen, try to explain them.

Laboratory work identification of aromorphosis and idioadaptation in plants and animals

Objective: show on specific examples the origin of large systematic groups by aromorphosis, get acquainted with examples of possible idioadaptation of organisms (degenerations), reveal the influence of human activity on the main directions of organic evolution

Equipment: herbariums of plants (moss, plantain, conifers, angiosperms), plants with thorns, pile (camel thorn, wild rose), drawings of the beak and legs of birds, animals with a protective (masking) color, stingray fish.

Working process:

Analyzing the main features of spore, gymnosperms and angiosperms, understand the aromorphoses of plants

Determine idioadaptation by plant thorn and glandular fibers

Analyze examples of idioadaptation: the structure of the beak and legs of birds living in different environmental conditions

To identify the causes of idioadaptation in the structure of the stingray fish

1. Students have the skills to establish cause and effect relationships.

2. Biological objects are considered as biological systems.

3. Have the skills to solve problems of part C of the KIMs of the Unified State Examination.

Laboratory work. Topic. Detection of proteins in biological objects.

Target. Prove the presence of proteins in biological objects.

Equipment.

Stand with test tubes, pipette, water bath, dropper.

Egg white solution, 10% NaOH solution, 1% copper sulfate, ninhydrin (0.5% aqueous solution), nitric acid (concentrated).

Biuret reaction to determination peptide bond. The method is based on the ability of a peptide bond in an alkaline medium to form colored complex compounds with copper sulfate.

Working process.

1. Add 5 drops of 1% egg white to a test tube (the protein is filtered through gauze, then diluted with distilled water 1:10), three drops of 10% sodium hydroxide solution and 1 drop of 1% copper sulfate solution and mix.

The contents of the tube acquire a blue-violet color.

ninhydrin reaction. Proteins, polypeptides and free amino acids give a blue or violet color with ninhydrin.

Working process.

1. Take 5 drops of a 10% solution of egg white, add 5 drops of a 0.5% aqueous solution of ninhydrin and heat.

After 2-3 minutes, a pink or blue-violet color develops.

Xantoprotein reaction (Greek xantos - yellow). With the help of this reaction, cyclic amino acids are found in the protein, which contain benzene rings (tryptophan, tyrosine and others).

Working process.

1.5 drops of 1% egg white solution, add 3 drops of concentrated nitric acid (carefully) and heat. After cooling, add drops of 10% sodium hydroxide solution to the test tube until an orange color appears (it is associated with the formation of the sodium salt of these nitro compounds).

Laboratory work. Topic. Detection of carbohydrates in biological objects.

Target. Prove the presence of carbohydrates in biological objects.

Equipment. Rack with test tubes. Pipettes, water bath.

1% starch solution, 1% sucrose solution, 1% fructose solution, 1% solution of iodine dissolved in potassium iodide, naphthol dissolved in 50 mm of alcohol (diluted 5 times with water before use), 1% alcohol solution, thymol.

Concentrated sulfuric acid, Selivanov's reagent: 0.5 g of resorcinol dissolved in 100 ml of 20% hydrochloric acid

Starch detection.

Working process.

1. Add 10 drops of a 1% starch solution and one drop of a 1% solution of iodine in potassium iodide to a test tube.

A blue-purple color is observed.

detection of carbohydrates.

Using the reaction with naphthol or thymol, small amounts of carbohydrates or carbohydrate components in complex compounds are detected.

Working process.

1. Add 10 drops of 1% sucrose solution to two test tubes.

In one add 3 drops of 1% alcohol solution of naphthol. In another test tube - 3 drops of 1% alcohol solution of thymol. Pour 0.5 ml of concentrated sulfuric acid into both (carefully) and observe a violet color in the test tube with naphthol and red in the test tube with thymol at the border of the two liquids.

Detection of fructose (Selivanov's reaction).

Fructose, when heated with hydrochloric acid and resorcinol, gives a cherry red color.

Working process.

1. Pour 10 drops of Selivanov's reagent 2 drops of a 1% fructose solution into a test tube and heat gently (a red color will appear).

Laboratory work. Topic. Detection of lipids in biological objects.

Target. Prove the presence of lipids in biological objects.

Equipment.

1. Stand with test tubes, water bath, pipette, glass cups, sticks, gauze.

2.Leticin, alcohol solution (chicken egg yolk), cholesterol, 1% chloroform solution, concentrated sulfuric acid, acetone.

detection of lecithin.

Lecithin belongs to the group of phospholipids, is part of the cell membranes. It makes up the bulk of brain tissue.

Working process.

1. Pour 10 drops of acetone into a dry test tube; put in a glass? chicken egg yolk.

While stirring with a stick, add 40 ml of hot alcohol drop by drop.

When the solution has cooled, filter it into a dry test tube. The filtrate should be clear. The reagent must be prepared before use. A white precipitate falls out.

detection of cholesterol.

Cholesterol is a fat-like substance that is of great importance for the body. Included in the membranes of many organs and tissues, is a precursor of bile acids, vitamin D, sex hormones, hormones of the adrenal cortex. The reaction is based on its ability to release water and condense into colored compounds.

Working process.

1. Pour 10 drops of 1% chloroform solution of cholesterol into a dry test tube and (carefully) pour 0.5 ml of concentrated sulfuric acid along the vessel wall. Shake (carefully). A red-orange color of the upper chloroform layer appears.

Laboratory work. Topic. Evidence for the functioning of proteins as biocatalysts (enzymes).

Target. To prove the catalytic action of protein-enzymes, to show their high specificity, the highest activity in a physiological environment.

Equipment. Stand with test tubes, 1 ml pipettes, water bath, thermostat.

1% starch solution, sucrose solution, 1% iodine solution in potassium iodide, 5% copper sulfate solution, 10% sodium hydroxide solution, 2% sucrose solution, 0.2% hydrochloric acid solution.

Working process.

1. Enzymatic hydrolysis of starch.

Salivary amylase acts as an enzyme that hydrolyzes starch into its constituent parts (maltose, glucose). Evaluation of the results of the experiment is carried out using color reactions with iodine of the Trommer reaction.

Non-hydrolyzed starch gives a blue color with iodine and a negative Trommer reaction. Starch hydrolysis products do not react with iodine, but react positively to Trommer's reagent.

1. Pour 10 drops of 1% starch solution into two test tubes.

2. Add 4 drops of water (control) to one of them (test tube No. 1).

In the second (test tube No. 2) add 4 drops of saliva solution, dilute saliva 5 times.

3. Mix and put in a water bath or thermostat for 15 minutes. at 37 deg. FROM.

4. Take 4 drops of the test substance from the test tube and add it to 2 different test tubes.

5. In one add one drop of a 1% solution of iodine in potassium iodide.

In another add one drop of 5% copper sulfate solution and 4 drops of 10% sodium hydroxide solution and heat gently to boiling (Trommer reaction).

6. We do the same with the contents of test tube No. 2. The result should show that starch hydrolysis does not occur in the presence of water and the reaction with iodine should be positive. The Trommer reaction is negative (copper oxide hydroxide is blue). In the presence of salivary amylase, the results should be the opposite, since starch hydrolysis has occurred.

There is no reaction with iodine and a brick-red color (copper oxide I) occurs in the Trommer reaction.

II. The specificity of the action of enzymes.

Each enzyme acts on only one substance or group of similar substrates. This is due to the correspondence between the structure of the enzyme, its active center and the structure of the substrate. For example, amylase only acts on starch.

Preparation of sucrose.

Grind 1.100 g of yeast and pour water (400 ml). After 2 hours, filter and store in the refrigerator.

2. In two test tubes (No. 1 and No. 2), add 10 drops of 1% starch solution.

Add 10 drops of 2% sucrose solution to test tubes No. 3 and No. 4.

3. In test tubes No. 1 and No. 3, add 4 drops of a saliva solution diluted 5 times.

Add 4 drops of sucrose to test tubes No. 2 and No. 4.

4. Mix and leave in a thermostat for 15 minutes at a temperature of 37 degrees. FROM.

5. Then, with the contents of all four test tubes, perform reactions with iodine and Trommer

Determination of the specificity of the action of enzymes

In the conclusions, it should be noted in which test tube and under what conditions the action of enzymes was found and why.

III. Influence of medium pH on enzyme activity.

For each enzyme, there is a certain value of the reaction of the environment at which it exhibits the highest activity. A change in the pH of the medium causes a decrease or complete inhibition of the activity of the enzyme.

1. Pour 1 ml of distilled water into 8 test tubes.

2. Add 1 ml of 0.2% hydrochloric acid solution to test tube No. 1. Mix.

3. Take one ml of the mixture from test tube No. 1 and transfer it to test tube No. 2. Mix, pour 1 ml and transfer to test tube No. 3, etc.

4. Take 1 ml from test tube No. 8 and pour it out. We get different pH environments.

4. In each tube add 2 ml of 1% starch solution and 1 ml of saliva solution diluted 1: 10.

5. Shake the test tubes and put in a thermostat for 15 minutes at 37 degrees. FROM.

6. Cool and add one drop of 1% solution of iodine in potassium iodide to all test tubes.

Complete hydrolysis will occur in test tubes No. 5 and No. 6, where the pH of the solution medium is in the range of 6.8-7.2, i.e., optimal for the action of amylase.

Laboratory work. Topic. Isolation of deoxynucleoprotein from spleen (liver) tissue. Qualitative reaction on DNA.

Target. Prove that a large number of nucleic acids are contained in the form of a compound with proteins (deoxynucleoprotein - DNP) in tissues rich in nuclei (spleen, thymus).

Equipment. Test tube rack, mortar and pestle, glass powder, pipette, crystallizer, 50 ml and 300 ml measuring cylinders, 1 ml pipettes, notched wooden sticks, water bath, filter gauze, sodium chloride, 5% solution, containing 0.04% trisubstituted sodium nitrate, 0.4% sodium hydroxide solution, diphenylamine reagent (dissolve 1 g of diphenylamine in 100 ml of glacial acetic acid. Add 2.75 ml of concentrated acid to the solution), spleen (fresh or frozen Yeast RNA, freshly prepared 0.1% solution.

Working process.

1. Isolation of deoxynucleoprotein (DNP) from the tissue of the spleen (liver).

The method is based on the ability of DNP to dissolve in salt solutions of high ionic strength and precipitate when their concentration decreases.

2 - 3 g of spleen tissue carefully grind in a mortar with glass powder, gradually pouring a solution of sodium chloride.

The resulting viscous solution is filtered through two layers of gauze into the crystallizer. Use a cylinder to measure six times (in relation to the filtrate) the volume of distilled water and slowly pour into the filtrate.

The resulting DNP threads are carefully wound on a wooden stick, transferred to a test tube for use.

2. Qualitative reaction for DNA.

The method is based on the ability of deoxyribose, which is included in the DNA of deoxyribonucleoprotein, to form blue compounds with diphenylamine when heated in a medium containing a mixture of glacial acetic acid and concentrated sulfuric acid.

With ribose RNA, a similar reaction produces a green color.

Add 1 ml of 0.4% sodium hydroxide solution to 1/4 of the DNP precipitate (until dissolved). Add 0.5% ml of diphenylamine reagent. Mix the contents of the test tube and place in a boiling water bath.

Perform a similar reaction in another test tube with 1 ml of RNA solution.

Note the characteristic coloration.

Laboratory work. Topic. Physiological properties of the cell membrane.

Target. Show that the cell membrane has selective permeability. Visually demonstrate the role of the membrane in the process of phagocytosis and pinocytosis, as well as familiarize yourself with cell plasmolysis - the process of separating the protoplast (cell contents) from the cell walls.

Equipment.

Microscopes, coverslips and slides, scalpels, dissecting needles, filter paper, pipettes, ink.

Infusoria culture or tissue culture on a nutrient medium, amoeba culture, pieces of Elodea plant.

Solutions of potassium chloride, solutions of calcium chloride, magnesium chloride, 2% albumin solution, 10% sodium chloride solution, distilled water.

Working process.

1. In a weak solution of sodium or potassium chloride, place ciliates or pieces of cultured tissue.

2. Prepare a preparation for the microscope.

3. You can see the shrinkage of the cells, indicating the permeability of the cell membrane. In this case, the water from the cell is released into the environment.

4. Transfer the cells to a drop of distilled water or pull the solution out from under the coverslip with filter paper and replace it with distilled water. Observe how the cells swell as water enters them.

5. Place ciliates or pieces of cultured tissue in a solution of calcium chloride or magnesium chloride of low concentration.

Ciliates and cultured cells continue to live. Calcium and magnesium ions reduce the permeability of the cell membrane. There is no movement of water through the shell.

6. Place the amoeba in a drop of 2% albumin solution (chicken egg protein).

Prepare a slide for the microscope. After some time, bubbles, protrusions, and tubules form on the surface of the amoeba. It seems that the surface of amoebas is "boiling". This is accompanied by intense fluid movement near the membrane surface.

Fluid bubbles are surrounded by protrusions of the cytoplasm, which then close. Pinocytic vesicles sometimes appear suddenly. This suggests that liquid droplets, together with the substance soluble in it, are captured quickly. Pinocytosis is caused by substances that lower the surface tension of the cell wall. For example, amino acids, some salts.

7.Introduce a little ink into a drop of liquid in which amoebas are located. Prepare the drug. After some time, the amoebas begin to slowly move towards the grains of the carcass, releasing pseudopodia (pseudopodia).

Carcass grains are attached to the surface of pseudopodia, surrounded by them, and after a while are immersed in the cytoplasm.

Under the microscope, the phenomenon of phagocytosis in amoeba is observed.

Primary requirements.

Students should know:

1. microscope device and work with it;

2. position of the cell theory;

3. similarity and difference between plant and animal cells;

4.role of chemicals and compounds in the cell;

5. main components and organelles of the cell;

6. features of prokaryotes and eukaryotes;

7. pathology of protein and carbohydrate metabolism;

8. value of individual mineral elements.

Students should be able to:

1.work with a microscope;

2. name the main parts of the cell, "recognize" them on the diagram, photograph;

3. to produce the simplest preparations for microscopic examination;

5. independently work with additional literature and use modern technologies.

Solving problems in molecular biology and genetics

elective course

Explanatory note

The program of the elective course was developed for students of the 11th grade and is designed for 17 hours.

The topics "Molecular Biology" and "Genetics" are the most interesting and complex topics in the course " General biology". These topics are studied in both 9th and 11th grades, but there is clearly not enough time to develop the ability to solve problems in the program. However, the ability to solve problems in genetics and molecular biology is provided for by the Standard biological education; in addition, such tasks are part of the KIM USE (tasks No. 5 and No. 6 in part C).

The purpose of the elective course: to create conditions for the formation of students' ability to solve problems in molecular biology and genetics of varying degrees of complexity.

a brief repetition of the material studied on the topics "Molecular Biology" and "Genetics"; identification and elimination of gaps in students' knowledge on topics school curriculum, as well as in the ability to solve problems; teaching students to solve problems in molecular biology and genetics of increased complexity.

Elective course program

1. Introduction. Proteins: actualization of knowledge on the topic (proteins-polymers, structures of a protein molecule, functions of proteins in a cell), problem solving - (1 hour).

2. Nucleic acids: updating knowledge on the topic ( Comparative characteristics DNA and RNA), problem solving - (1 hour).

3. Protein biosynthesis: updating knowledge on the topic (DNA code, transcription, translation - the dynamics of protein biosynthesis), problem solving - (1 hour).

4. Energy metabolism: updating knowledge on the topic (metabolism, anabolism, catabolism, assimilation, dissimilation; stages of energy metabolism: preparatory, glycolysis, cellular respiration), problem solving - (1 hour).

5. Frontier diagnostics: control work - (1 hour).

6. Genetic symbols and terms - (1 hour).

7. Laws of G. Mendel: actualization of knowledge on the topic (patterns established by Mendel during mono- and dihybrid crossing), test control of the ability to solve problems for Mendel's laws provided for by the program, solving problems for mono- and dihybrid crossing of increased complexity - (1 hour).

8. Incomplete dominance: updating knowledge on the topic, solving problems of increased complexity on the topic - (1 hour).

9. Inheritance of blood groups: updating knowledge on the topic, problem solving - (1 hour).

10. sex genetics; sex-linked inheritance: updating knowledge on the topic (chromosomal and non-chromosomal sex determination in nature), solving problems of increased complexity for sex-linked inheritance - (1 hour).

11. Solving combined problems - (1 hour).

12. Interaction of genes: actualization of knowledge on the topic (interaction of allelic and non-allelic genes), solving problems of increased complexity for all types of interaction: complementarity, epistasis, polymerization - (1 hour).

13. Frontier diagnostics: the game "Running with barriers" - (1 hour).

14. T. Morgan's law: actualization of knowledge (why did T. Morgan, setting a goal to refute the laws of G. Mendel, could not do this, although he got completely different results?), solving problems for crossing over, compiling chromosome maps - (1 hour).

15. Hardy-Weinberg law: lecture "Following Hardy and Weinberg", solving problems in population genetics - (1 hour).

16. Human genetics: actualization of knowledge on the topic, terms and symbols, problem solving - (1 hour).

17. Final lesson. Final diagnostics: solution entertaining tasks- (1 hour).

Control

The student receives a "credit" on the basis of:

fulfillment control work in molecular biology; filling in the crossword "Genetic terms"; fulfillment of tasks of test control No. 1 and No. 2; solving problems in the game "Running with barriers"; performance of the final control work (solving problems of increased complexity).

Problems in molecular biology

Theme: "Squirrels"

Necessary explanations:

the average molecular weight of one amino acid residue is taken as 120; protein molecular weight calculation:

BELARUSIAN STATE UNIVERSITY

DEPARTMENT OF BIOLOGY

Department of Plant Physiology and Biochemistry

PHYSIOLOGY

VEGETABLE

CELLS

for laboratory workshops

"Plant Physiology"

for students of the Faculty of Biology

V. M. Yurin, A. P. Kudryashov, T. I. Ditchenko, O. V. Molchan, I. I. Smolich biological sciences, Associate Professor MA Dzhus Plant cell physiology: method. recommendations for laboratory studies of the workshop "Plant Physiology" for students of the Faculty of Biology / V. M. Yurin [et al.].

- Minsk: BGU, 2009. - 28 p.

This manual is an integral element of the educational and methodological complex in the discipline "Plant Physiology" and includes laboratory work on the section "Plant Cell Physiology".

Designed for students of the Faculty of Biology, studying in the specialties "Biology" and "Bioecology".

UDC 581. LBC 28. © BSU,

FROM THE AUTHORS

Guidelines to laboratory classes are an integral part of the course "Plant Physiology". The purpose of the publication is to intensify the independent work of students, taking into account the fact that the individual learning process must be effective. The workshop on the course "Plant Physiology" is designed to consolidate theoretical material, acquisition of skills practical work and familiarization with the main methods of studying the physiological processes of plants. Students are offered assignments detailing the factual material that they must master on their own.

This will allow you to use classroom time more efficiently.

1. PLANT CELL AS

OSMOTIC SYSTEM

Osmotic systems are systems consisting of two solutions of substances of different concentrations or a solution and a solvent separated by a semipermeable membrane. An ideal semi-permeable membrane is permeable to solvent molecules and impermeable to solute molecules. Water is the solvent in all biological systems. The difference in the composition and concentration of substances on both sides of a semipermeable membrane is the cause of osmosis - the directed diffusion of water molecules through a semipermeable membrane.

If we abstract from the detailed structure of the plant cell and consider it from the point of view of the osmotic model, then it can be argued that the plant cell is a living osmotic system.

The plasma membrane is semi-permeable, and the cytoplasm and tonoplast act as a single entity. Outside of the semipermeable membrane is the cell wall, which is highly permeable to water and substances dissolved in it and does not prevent the movement of water. The main role of the osmotic space of the cell is played by the vacuole, which is filled with an aqueous solution of various osmotically active substances - sugars, organic acids, salts, water-soluble pigments (anthocyanins, etc.). However, this is a rather simplified idea of ​​a cell as an osmotic system, since any cytoplasmic organelle surrounded by a membrane is also an osmotic cell. As a result, the osmotic movement of water also occurs between an individual organelle and the cytosol.

PLANT CELL MODELS

Introductory remarks. The unique physicochemical characteristics of biomembranes ensure the inflow of water and the creation of high hydrostatic pressure (turgor) in the plant cell, the preservation of the anisotropic distribution of substances between the cell and its environment, the selective absorption and release of substances, and a number of other functions.

The hypothesis of the existence of a plasma membrane on the cell surface was put forward in the second half of the 19th century. The scientific substantiation of this hypothesis (concept) was given by W. Pfeffer based on the explanation of the phenomena of plasmolysis and deplasmolysis. According to Pfeffer, this membrane had the property of "semi-permeability", that is, it was permeable to water and impermeable to substances dissolved in water. In subsequent years, studies were carried out that made it possible not only to prove the existence of such a structure on the cell surface, but also to study some of the properties of this structure invisible to optical microscopes. However, until the second half of the twentieth century. biomembranes remained only hypothetical structures of a living cell. Therefore, researchers to demonstrate certain properties of the plasma membrane and explain the patterns of functioning of the mechanisms associated with the plasma membrane created cell models (“artificial cells”).

In different periods of time, model systems appeared - “artificial cells” by Pfeffer, Traube, Jacobs, etc. The first two of the mentioned models demonstrated the phenomena of osmosis, the third - the patterns of transfer of weak electrolytes through the biomembrane. When performing laboratory work, it is proposed to create model systems "artificial cell" according to Traub and Jacobs (modified).

When forming the models of the "artificial cell" of Pfeffer and Traube, at the contact boundary of solutions of yellow blood salt and copper sulfate, a water-insoluble amorphous mass of iron-cyanide copper is formed, which has almost ideal osmotic properties - permeability to water and impermeability to dissolved substances. Since the copper-iron membrane separates two solutions, the direction and magnitude of the water flow through it will be determined by the difference in the chemical potentials of water molecules on opposite sides of the membrane. If such a membrane separated two solutions of the same substance, then the chemical potential of water molecules would be higher in a more dilute solution, and water would move from the side of a solution of lower concentration. When determining the direction of water movement in a system containing different substances on both sides of the membrane, the degree of dissociation of substances, valency and permeability of the membrane for ions should be taken into account. To simplify the discussion of the experiment on obtaining an "artificial cell" according to Traub, we assume that the membrane of iron-cyanide copper is absolutely impermeable to dissolved substances, the degree of dissociation of yellow blood salt and copper sulphate in solutions is the same. In this case, to compare the values ​​of the chemical potential of water molecules, one can use the normal concentrations of these salts.

The main regularities of the process of diffusion of substances of different polarity through plasma membranes were established in the first half of the 20th century. According to the studies of Collander and Barlund, the permeability coefficient of a membrane to any substance can be predicted from the molecular weight of the latter and its equilibrium distribution coefficient (kp) between water and vegetable oil:

where CM and CB are the concentrations of a substance that have been established in a system of solvents in contact with each other - oil and water - in a state of equilibrium. For most substances diffusing through the plasma membrane, there is a direct proportionality between the product Pi M i and kp (Pi is the membrane permeability coefficient with respect to substance i; Mi is the molecular weight of substance i).

The coefficient kp in this case acts as a quantitative measure of the degree of hydrophobicity: more hydrophobic substances accumulate in the oil and are characterized by a large value of kp, hydrophilic substances, on the contrary, accumulate in the aqueous phase, for them the value of kp is less. In accordance with this, non-polar compounds should penetrate into the cell as a result of the diffusion process through the layer of membrane lipids more easily than polar ones. The degree of hydrophobicity is determined by the structure of the substance molecule. However, the hydrophobicity of a substance largely depends on the degree of ionization of its molecules in solution. In turn, the degree of ionization of many organic and inorganic substances(weak electrolytes) is determined by the pH value of the solution.

Jacobs' "artificial cell" models the selective permeability of the plasma membrane plant cells with respect to electrically neutral molecules of weak electrolytes. In his original design of the “artificial cell”, Jacobs used a flap of frog skin as an analogue of the plasmalemma. In the proposed work, a film of a hydrophobic (polymeric) material is used as a model of the plasmalemma. This was done not only for humanitarian reasons - the polymer film more clearly models the physicochemical properties of the lipid bilayer of the plasmalemma.

Being a weak base, ammonium exists in aqueous solutions in the form of NH3 and NH4+, the concentration ratio of which depends on the pH of the medium and for dilute aqueous solutions is determined by the dissociation constant pKa, which at 25 ° C is 9.25:

where and are the concentrations of ammonia molecules and ammonium ions, respectively.

If only uncharged ammonia molecules can penetrate the membrane, then it is easy to show that the concentrations of ammonium ions on opposite sides of the membrane in equilibrium will depend on the pH of the solutions in contact with the membrane. To demonstrate the process of transfer of ammonia through the membrane in the "artificial cell" Jacobs uses his ability to shift the pH.

Objective. Get "artificial cells" by the Traube and Jacobs methods and observe the phenomenon of osmosis - the movement of water through a semipermeable membrane along the osmotic potential gradient.

Materials and equipment: 1.0 N solutions of yellow blood salt, copper sulphate, ammonium chloride, sodium hydroxide and hydrochloric acid, 1% water-alcohol solution of neutral red, universal indicator paper, fragments of glass tubes melted from the end, polymer film, threads, test tubes , 3 glasses with a capacity of 150–200 ml, stopwatch.

1. Obtaining an "artificial cell" Traube. By dilution prepare 1.0 N solution of yellow blood salt (K4Fe(CN)6), 0.5 N and 1.N solutions of copper sulfate (CuSO45 H2O). Take two test tubes. Pour 0.5 N into one, and into the other 1.0 N a solution of copper sulfate. Carefully pipette along the wall of the test tubes into each 1.0 N solution of yellow blood salt. On the contact surface of solutions of copper sulphate and yellow blood salt, a membrane of iron-cyanide copper is formed:

The amorphous precipitate of iron-cyanide copper has almost ideal osmotic properties, therefore, with a difference in the values ​​of the chemical potential of H2O molecules, a water flow should be observed, which leads to a change in the volume of the “artificial cell”. It should be noted that the membrane made of iron-cyanide copper has a weak elasticity. Therefore, when the volume of the “artificial cell” increases, the membrane breaks.

The task. Follow the behavior of "artificial cells" in 0.5 N and 1.0 N solutions of copper sulphate. Sketch "artificial cells"

and describe the dynamics of their shape change.

2. Obtaining an "artificial cell" Jacobs. By dilution, prepare 200 ml of 0.5 N ammonium chloride solution and 100 ml of 0.5 N sodium hydroxide. Pour the sodium hydroxide solution into a glass, and divide the ammonium chloride solution into two equal parts and pour them into glasses with a capacity of 150-200 ml. Using indicator paper and 1.0 N solutions of hydrochloric acid and sodium hydroxide, bring the acidity of the solution in the first glass to pH 9.0, and in the second to pH 7.0.

Take 3 glass tube fragments. On the melted end of each, put a piece of polymer film and carefully tie them with a thread. Add 5-10 drops of neutral red solution to 50 ml of water and slightly acidify the medium with 1-2 drops of hydrochloric acid.

Fill with the specified indicator solution on Jacobs' "artificial cells" (fragments of glass tubes with membranes). Place the "artificial cells" of Jacobs in beakers with solutions of sodium hydroxide and ammonium chloride in such a way that these media are in contact with the polymer membrane.

Ammonia is able to diffuse through the hydrophobic phase of the polymer membrane. And since its concentration is negligible inside the “artificial cell”, NH3 molecules are transferred from the solution into the “cell” and cause alkalization of the contents of the glass tube, which is noted by the disappearance of the crimson-red color of the “intracellular” contents.

The task. Determine the time required for the red color of the indicator to disappear in each of the variants of the experiment.

1. Why does the salt concentration increase near the surface of the "artificial cell" in a 0.5 N solution of copper sulphate?

2. Why does an “artificial cell” swell in a 0.5 N solution of copper sulphate, while its surface is stable in a 1.0 N solution?

3. What factors determine the degree of dissociation of weak acids and bases?

4. Why does the neutral red color disappear when the “artificial cell” is placed in a solution of sodium hydroxide?

5. Why is there a shift in the pH of the "intracellular" content to slightly basic values ​​when an "artificial cell" is placed in a neutral ammonium chloride solution?

6. What is osmosis?

7. What solutions are called hypo-, iso- and hypertonic?

THE PHENOMENON OF PLASMOLYSIS AND DEPLASMOLYSIS

PLANT CELL

Introductory remarks. The process of water exit from a plant cell and its entry into the cell through a semipermeable membrane can be traced by observing the phenomena of plasmolysis and deplasmolysis. Plasmolysis occurs when a cell is placed in a solution hypertonic in relation to the cell sap - separation of the protoplast from the cell wall due to a decrease in its volume due to the release of water from the cell into the external solution. During plasmolysis, the shape of the protoplast changes. Initially, the protoplast lags behind the cell wall only in some places, most often the corners. Plasmolysis of this form is called angular. With an increase in the duration of incubation of a plant cell in a hypertonic solution, the following form of plasmolysis is observed - concave plasmolysis. It is characterized by the preservation of contacts of the protoplast with the cell wall in separate places, between which the separated surfaces of the protoplast acquire a concave shape. Gradually, the protoplast breaks away from the cell walls over the entire surface and takes on a rounded shape. Such plasmolysis is called convex.

After replacing the external solution with pure water, the latter begins to enter the cell. The volume of the protoplast increases and deplasmolysis occurs. After its completion, the protoplast again fills the entire volume of the cell.

Objective. Prove on the basis of the phenomena of plasmolysis and deplasmolysis that the plant cell is an osmotic system.

Materials and equipment: microscope, slides and coverslips, safety razor blade, dissecting needle, tweezers, 1 M sucrose solution, filter paper, onion bulb.

From the convex side of the surface of the onion scales, the cells of which are colored purple due to the presence of anthocyanins in the vacuoles, the epidermis is removed with a dissecting needle, placed in a drop of water on a glass slide, covered with a cover slip and examined under a microscope. The water is then replaced with 1 M sucrose solution. To do this, a large drop of solution is applied to the glass slide next to the coverslip and the water is sucked off with a piece of filter paper, applying it on the other side of the coverslip. Repeat this technique 2-3 times until the water is completely replaced with a solution. The preparation is examined under a microscope. A gradual lagging of the protoplast from the cell walls is detected, first in the corners, and then over the entire surface of the walls. Eventually, the protoplast separates completely from the cell wall and assumes a rounded shape.

Then, as described above, replace the 1 M sucrose solution with water. Water enters the cell, which leads to an increase in the volume of the protoplast, which gradually takes its former position. The cell returns to its original state.

The task. Draw the observed forms of plasmolysis, as well as the stages of deplasmolysis. Formulate conclusions.

1. What structural features of a plant cell give it the properties of the osmotic system?

2. What is plasmolysis? Describe the main forms of plasmolysis.

3. What is deplasmolysis? Under what conditions is it observed?

DETERMINATION OF OSMOTIC PRESSURE

CELL JUICE PLASMOLYTIC

METHOD

Introductory remarks. When two solutions containing different amounts of dissolved substances come into contact, due to the thermal motion inherent in the molecules, mutual diffusion occurs, which leads to equalization of the concentration of dissolved substances in the entire volume, which is equivalent to the situation of mixing liquids. If these solutions are separated by a semi-permeable membrane that traps the molecules of solutes, then only molecules of the solvent (water) will pass through the contact boundary of the solutions. Moreover, there is a unidirectional flow of water through the membrane (osmosis). The pressure that must be applied to one of the solutions of the system in order to prevent the solvent from entering it is called osmotic pressure. The osmotic pressure of a solution is directly proportional to its concentration and absolute temperature. Van't Hoff found that the osmotic pressure of dilute solutions obeys gas laws and can be calculated by the formula:

where R is the gas constant (0.0821); T is the absolute temperature (273 °C + t °C) of the solution; C is the concentration of the solute in moles; i - isotonic coefficient.

The value of the isotonic coefficient is determined by the characteristics of the processes of dissolution of the substance. For non-electrolytes (for example, for sucrose), i is equal to 1. For electrolyte solutions, the value of i depends on the number of ions into which the molecule decomposes and on the degree of dissociation. i values ​​for NaCl solutions are given in the table.

Values ​​of the isotonic coefficient of sodium chloride solutions Concentration of NaCl The value of i The value of the osmotic pressure of cell sap expresses the ability of a plant cell to "suck" water and indicates the possibility of a plant growing on soils of different water-retaining strength. At the same time, an increase in the osmotic pressure of cell sap during drought is a criterion for plant dehydration and the need for watering.

The plasmolytic method for determining the osmotic pressure of cell contents is based on the fact that the osmotic pressure of solutions, which determines the movement of water through the membrane, can be created by various substances (osmolytics). Therefore, to determine the osmotic pressure of cell sap, knowledge of its qualitative composition and concentration of individual substances is not required, but it is necessary to find the concentration of any substance in the external solution, at which there will be no movement of water through the plasmalemma in the absence of turgor and plasmolysis. To do this, sections of the tissue under study are immersed in a series of solutions of a known concentration, and then they are examined under a microscope. Based on the fact that only hypertonic solutions can cause plasmolysis, the weakest of them is found, in which only initial plasmolysis is found in individual cells. The next more dilute solution will not plasmolyze the cells.

Consequently, the concentration of the isotonic solution for these cells will be equal (with a known margin of error) to the arithmetic mean between the concentrations of neighboring solutions.

For convenience, work is carried out with tissues whose cells contain anthocyanins in the cell sap: the epidermis of blue onion scales, the lower epidermis of the tradescantia leaf. Sucrose or NaCl solutions are used as plasmolytics.

Materials and equipment: microscope, glass slides and coverslips, safety razor blade, dissecting needle, 1 M NaCl and 1 M sucrose solutions, tradescantia leaves or blue onion bulbs.

Using 1 M sucrose or NaCl solution, prepare by diluting 5 ml of the solutions according to the table.

After thoroughly mixing the solutions, pour them into glass bottles or crucibles, where you place 2-3 sections of the tissue under study for 30 minutes.

In this case, it is necessary to ensure that the sections do not float on the surface, but are immersed in liquids (if the section floats, it should be “drowned” with a dissecting needle). Close the bottles with lids or glass slides to prevent evaporation.

After the specified incubation time, examine the sections under a microscope in a drop of the appropriate solution (not in water!) in the same sequence in which they were immersed in the solutions. After each solution, the glass rod or pipette, which was used to apply the solution to glass slides, must be thoroughly rinsed with distilled water and wiped with a napkin or filter paper.

The task. Determine the presence of plasmolysis in the examined tissue and its degree. The degree of plasmolysis is expressed by the concepts: “strong”, “weak”, “initial”, “lack of plasmolysis”. Enter the results in a table.

Degree of plasmolysis Isotonic concentration, M Osmotic pressure of cell sap in atm and kPa Set the isotonic concentration of sodium chloride, i.e. the content of NaCl, which creates an osmotic pressure similar to cell sap in the tissue under study. Calculate the osmotic pressure using equation (1). Using a factor of 101.3, calculate the osmotic pressure in kPa.

1. What is osmotic pressure?

2. How is the osmotic pressure calculated?

3. What determines the value of the isotonic coefficient?

4. The criterion of what process is the increase in the osmotic pressure of cell sap?

2. PROPERTIES OF CELL MEMBRANES

The most important property of cell membranes is selective permeability. The outer cytoplasmic membrane separates the cell from environment, controls the transport of substances between the cell and free space. Intracellular membranes, due to their inherent selective permeability, provide the function of compartmentalization, which allows the cell and organelles to retain the necessary enzymes and metabolites in small volumes, create a heterogeneous physicochemical microenvironment, and carry out various, sometimes oppositely directed, biochemical reactions on different sides of the membrane.

The permeability of cell membranes for various substances can be a criterion for cell viability. The selective permeability of the membrane is maintained as long as the cell remains alive.

SELECTIVE PERMEABILITY STUDY

PLASMALEMMAS OF A PLANT CELL

Introductory remarks. It is possible to compare the permeability of the plasma membrane for various substances on the basis of simple observations characterizing the duration of the preservation of plasmolysis in plant cells in hypertonic solutions of the substances under study. In the case of a sufficiently low permeability of the plasmalemma for the solute or the complete absence of the ability of its molecules to freely diffuse into the plant cell, persistent plasmolysis will take place, in which plasmolyzed cells can remain unchanged for a long time. However, if the solute molecules pass through the membrane, but more slowly than water molecules, then the plasmolysis that has begun is temporary and soon disappears. As a result of the gradual penetration of the solute into the cell, water will flow from the external solution along the concentration gradient, which will eventually cause the cell to go into a deplasmolyzed state.

Objective. Compare the permeability of cell membranes for various substances based on the observation of persistent and temporary plasmolysis.

Materials and equipment: microscope, slides and coverslips, safety razor blade, dissecting needle, tweezers, 1 M sucrose solution, 1 M urea solution, 1 M glycerin solution, filter paper, onion bulb.

A drop of solution is applied to three object steles: on one - 1 M sucrose solution, on the other - 1 M urea solution, on the third - 1 M glycerol solution. A fragment of the stained onion epidermis is placed in each drop, covered with coverslips and examined under a microscope. Find areas in which plasmolyzed cells are clearly visible. The time of the beginning of plasmolysis is noted - the beginning of observation. The preparations are left for 10-30 minutes, then they are again examined under a microscope. In a solution of sucrose, stable plasmolysis is observed, and in solutions of carbamide and glycerol, it is temporary. The cause of deplasmolysis in the last two solutions is the permeability of the plasmalemma for carbamide and glycerol molecules.

The task. Conduct a study of the characteristics of plasmolysis of plant cells in solutions of various substances. Record the results of observations in the table, noting the degree of plasmolysis every 10 minutes after the start of observations. Based on the analysis of the results of the experiments, identify differences in the duration of the preservation of the plasmolyzed state caused by various osmolytics, and draw a conclusion about the relative permeability of the plasmalemma for the substances under study.

Solute Note: +++ – strong plasmolysis, ++ – medium plasmolysis, + – weak plasmolysis.

1. What is the selective permeability of cell membranes?

2. What substances penetrate cell membranes more easily?

3. How can the property of selective permeability be used to determine the viability of a plant cell?

STUDYING THE DIFFUSION OF NEUTRAL

RED THROUGH THE PLASMALEMM

PLANT CELL

Introductory remarks. The plasma membrane isolates the intracellular contents from the external environment. The exchange of substances between the intracellular contents and the environment surrounding the cell occurs by their transport through the membrane. The lipid bilayer is a barrier to the movement of substances. Most exogenous physiologically significant substances enter the cell as a result of the functioning of passive and active transport systems on the plasmalemma. However, simple passive diffusion through the lipid bilayer, which is a hydrophobic phase, is also possible.

The main regularities of the diffusion of substances through the lipid bilayer were established in the late 19th and early 20th centuries, i.e., at a time when biomembranes remained only hypothetical cell structures. It is the fact that hydrophobic substances penetrate the cell better than hydrophilic ones that was the basis for the researchers' assumption about the presence of lipids in the membrane.

The process of diffusion of substances through a membrane obeys Fick's first law, the mathematical expression of which, as applied to a membrane, is described by the formula:

where Pi is the membrane permeability coefficient for substance i; CiII and CiI are the concentrations of substance i on both sides of the membrane.

Weak acids and bases are characterized by the fact that the degree of ionization of their molecules in dilute solutions depends on pH (see Laboratory work 1, formula (2)). This means that the degree of dissociation of weak electrolyte molecules in the range of pH values ​​numerically equal to pKa is 50%. With a decrease in pH by one, more than 90% of the weak base molecules will be ionized, and with an increase in pH by the same value, less than 10%.

As far back as the first half of the 20th century, it was demonstrated that electrically neutral non-ionized molecules of weak electrolytes penetrate quite well through the plasma membrane into plant cells, while the membrane is practically impermeable for the corresponding ions. For example, the plasmalemma permeability coefficients for ammonia and ammonium ion differ by more than 100-fold. Thus, the shift in pH values ​​is only 1–2 units. leads to a more than 10-fold change in the concentration of the forms of substance molecules transported through the membrane.

Among weak electrolytes, acid-base indicators are of particular interest, since the molecules of these substances are characterized by a change in their optical properties during ionization. In addition, due to the characteristic color of solutions of these compounds, it is quite easy to determine their content colorimetrically. Neutral red (NK) is a weak base. Ionized NA molecules (at pH 6.8 and below) color the solutions in an intense crimson color. With an increase in pH from 6.8 to 8.0, a gradual change in color to pale yellow occurs due to a decrease in the degree of dissociation of NA molecules. In alkaline solutions, electrically uncontaminated NA molecules, which are well transported through the lipid bilayer of the plasma membrane, predominate, while in acidic solutions, NA ions that are poorly permeable to the membrane predominate.

NA molecules entering through the plasmalemma into the cell can also diffuse through other cell membranes, however, having penetrated into the vacuole (acidic compartment of the plant cell), NA molecules are ionized, staining the contents of the vacuole in a crimson color. In this case, the NA ions turn out to be “closed” in the space of the vacuole, i.e., they tend to accumulate.

Objective. To study the patterns of diffusion of neutral red through the plasmalemma of a plant cell. Materials and equipment: scissors, a water-alcohol solution of neutral red, decinormal solutions of sodium hydroxide and hydrochloric acid, universal indicator paper, Petri dishes, a microscope, a stopwatch, a culture of Nitella flexilis algae.

Add 5 drops of neutral red solution to 100 ml of water.

Pour this solution equally into 4 Petri dishes. By controlling the acidity of the contents of the Petri dishes with universal indicator paper using HCl and NaOH solutions, bring the acidity index in the first Petri dish to pH 9.0, in the second to pH 8.0, in the third to pH 7.0, in the fourth to pH 5.0. Label the Petri dishes.

Carefully separate 8–12 algal internodal cells from the thallus of Nitella flexilis with scissors. Examining the internodes under a microscope, make sure that the dissected cells are native: living intact cells retain continuous rows of chloroplasts located parallel to the light line, in addition, there is an intensive movement of the cytoplasm - cyclosis.

Place 2–3 algae internode cells into Petri dishes.

Turn on the stopwatch.

The task. Determine the time required to stain the algae cells in each experiment. To do this, after 5 min, compare the cells of the internodes of the algae of each of the variants according to the color intensity. Repeat the operation after 10, 20, 30 minutes. Record the results of observations in the table. Draw a conclusion about the forms of the weak base diffusing through the membrane.

pH value of the medium Note: +++ – intense color, ++ – medium color, + – weak color, – no color.

1. What factors determine the degree of dissociation of weak acids and bases?

2. Why are biomembranes more permeable to non-dissociated forms of weak electrolytes?

3. Under what conditions is the accumulation of a weak electrolyte in the cell noted?

CHANGES IN TONOPLAST PERMEABILITY

AND PLASMALEMMAS FOR BETACYANIN UNDER

THE ACTION OF PHYSICAL AND CHEMICAL

FACTORS

Introductory remarks. The selective permeability of cell membranes changes under the influence of various factors. It is possible to determine the influence of any substances or conditions on membrane permeability by measuring the release of various metabolites from the cell.

Betacyanin, a red beet pigment, is a relatively large, water-soluble molecule found in cell sap.

To enter the external environment, the betacyanin molecule must pass through the tonoplast, the main cytoplasmic matrix, and the plasmalemma. The tonoplasts of living cells are impermeable to the molecules of this pigment. Diffusion of betacyanin from the vacuole into the medium can proceed quite rapidly under the action of various factors or agents that cause an increase in membrane permeability. By measuring the optical density of the incubation medium after a certain period of time, it is possible to assess the degree of influence of one or another factor on the membrane permeability.

Objective. Determine the effect of temperature, as well as acids and alcohols, on the permeability of cell membranes for betacyanin by its release into the external solution.

Materials and equipment: distilled water, 30% acetic acid solution, 50% ethanol solution, filter paper, test tubes, test tube rack, water bath, spectrophotometer or photocolorimeter, table beet root.

After removing the integumentary tissues, the beet root is cut into cubes (the side of the cube is 5 mm) and thoroughly washed with water for 5–10 minutes to remove the pigment that has come out of the damaged cells.

Then they are placed one by one in each of 4 tubes, into which 5 ml of various media are poured in accordance with the experimental scheme: distilled water (2 tubes), solutions of acetic acid and ethanol.

The first test tube with distilled water is left in a rack, and the contents of the second one are heated in a water bath for 2–3 min. After 30 minutes, all test tubes are vigorously shaken, the beet cubes are removed, and the color intensity of the solutions is determined on a photocolorimeter with a green light filter or a spectrophotometer = 535 nm.

Optical density of the solution, Staining intensity, Experience option Task. Do your research. Enter the results of measurements of optical density in the table. Identify the differences in the permeability of the tonoplast and plasmalemma for betacyanin in beet root cells exposed to various factors, and draw a conclusion about the reasons for these differences.

1. What is the significance of the selective permeability of cell membranes?

2. What determines the selective permeability of plant cell membranes?

3. PROPERTIES OF THE CYTOPLASMA

The bulk of the cytoplasm that fills the space between cell organelles is called the cytosol. The proportion of water in the cytosol is approximately 90%. Almost all major biomolecules are present in dissolved form in the cytosol. True solutions form ions and small molecules (salts of alkali and alkaline earth metals, sugars, amino acids, fatty acids, nucleotides and dissolved gases). Large molecules, such as proteins, form colloidal solutions. A colloidal solution can be a sol (non-viscous) or a gel (viscous). The intensity of most intracellular processes depends on the viscosity of the cytosol.

The most important property of the cytoplasm is its active movement.

This salient feature a living plant cell, an indicator of the activity of its vital processes. The movement of the cytoplasm provides intracellular and intercellular transport of substances, the movement of organelles inside the cell, plays an important role in irritability reactions. Its implementation involves the elements of the cytoskeleton - microfilaments and microtubules. The energy source for this movement is ATP. The movement of the cytoplasm (cyclosis) is one of the most sensitive indicators of cell viability. Many even minor impacts stop or, conversely, accelerate it.

INFLUENCE OF POTASSIUM AND CALCIUM IONS ON

VISCOSITY OF THE PLANT CELL CYTOPLASMA

Introductory remarks. Individual cations can significantly change the viscosity of the cytoplasm. It has been established that potassium ions contribute to an increase in its water content and a decrease in viscosity. The lower viscosity of the cytoplasm favors the flow of synthetic processes, intracellular transport of substances, but reduces the resistance of plant cells to adverse external conditions. Unlike potassium, calcium increases the viscosity of the cytoplasm. With a higher viscosity of the cytosol, physiological processes proceed more slowly, which increases the resistance of the cell to adverse environmental conditions.

Changes in the viscosity of the cytoplasm under the action of potassium and calcium ions can be judged by the form of plasmolysis in cells in hypertonic solutions of their salts. With prolonged incubation of plant cells in solutions containing potassium ions, cap plasmolysis is observed. In this case, potassium ions pass through the plasma membrane into the cytoplasm, but rather slowly penetrate through the tonoplast into the vacuole. As a result of swelling of the cytoplasm, the protoplast takes a convex shape, separating only from the transverse sections of the cell walls, from the side of which the formation of the so-called "caps" is observed. An increase in the viscosity of the cytoplasm caused by calcium is easy to detect by observing the change in the shape of the plasmolyzing protoplast: if the plasmolytic contains calcium, then the concave plasmolysis often turns into a convulsive form.

Objective. To study the nature of the influence of potassium and calcium ions on the viscosity of the cytoplasm of a plant cell based on observations of cap and convulsive plasmolysis.

Materials and equipment: microscope, slides and coverslips, safety razor blade, dissecting needle, tweezers, 1 M KNO3 solution, 1 M Ca(NO3)2 solution, filter paper, onion bulb.

A drop of 1 M solution of potassium nitrate is applied to one glass slide, and a drop of 1 M solution of calcium nitrate is applied to the other. In both drops, a piece of onion epidermis, taken from the concave surface of the same bulb scale, is placed, covered with cover slips. After 30 minutes, the preparations are examined under a microscope in the solutions in which they were located. The phenomenon of plasmolysis is observed. In some cells of the epidermis kept in a solution of KNO3, on the side of the transverse walls of the cell, the cytoplasm forms “caps”, the appearance of which is due to an increase in the hydration of the cytosol under the action of potassium ions. Calcium ions, on the contrary, increase the viscosity of the cytoplasm, increase its cohesive forces with the cell wall, and the protoplast takes on the wrong shape, characteristic of convulsive plasmolysis.

The task. Draw the observed forms of plasmolysis. Reveal the dependence of the form of plasmolysis on the viscosity of the cytoplasm in the presence of potassium and calcium ions.

1. How do potassium and calcium ions affect the viscosity of the cytoplasm?

2. Under what conditions is convulsive plasmolysis observed?

3. What causes the formation of "caps" as a result of cell incubation in KNO3 solution?

OBSERVATION OF CYTOPLASMA MOVEMENT

PLANT CELLS AND MEASURING IT

SPEEDS

Introductory remarks. The most convenient for monitoring the movement of the cytoplasm are large plant cells with large vacuoles (cells of the internodes of charophytes, marine siphon green algae, cells of the leaves of aquatic plants of elodea, vallisneria, etc.). There are several types of cytoplasmic movement. The most widespread oscillatory movement. It is considered the least ordered, since in this case some particles are at rest, others slide to the periphery, and others to the center of the cell. The movement has an unstable, random character. Circulatory movement is characteristic of cells that have cytoplasmic strands that cross the central vacuole. The direction and speed of movement of particles located inside or on the surface of the cytoplasmic layer, as well as in the cytoplasmic layers, are not constant. During rotational movement, the cytoplasm moves only on the periphery of the cell and moves like a drive belt. The movement of this type, in contrast to the circulation, has a more or less constant and ordered character, therefore it is convenient for quantitative study. In addition to the above, there are also movements of the cytoplasm, for example, spouting and shuttle. The types of movement differ from each other conditionally and in the same cell they can move from one to another.

The movement of the cytoplasm can be characterized by determining its speed, which depends not only on driving force, but also the viscosity of the cytoplasm. The speed of the cytoplasm can be measured under a microscope by observing the movement of its particles.

Objective. Familiarize yourself with the rotational type of cytoplasmic movement and measure its speed in various plant objects.

Materials and equipment: microscope, slides and coverslips, safety razor blade, dissecting needle, artificial pond water solution, wallisneria leaf, internodal nitella cells.

A small piece is cut off from the leaf blade of Vallisneria with a sharp razor, trying to injure the leaf as little as possible, placed in a drop of water on a glass slide and examined under a microscope, first at low, then at high magnification. It is not recommended to make cuts from the sheet, since the cells are severely injured in this case, and the movement in them stops. The movement of the cytoplasm is easily observed by the movement of all chloroplasts in the same direction along the cell wall. This movement is called rotational.

To observe cyclosis in nitella cells, pre-prepared cells are placed in special chambers, which are filled with a solution of artificial pond water. All Chara algae also have a rotational type of cytoplasmic movement, but the chloroplasts in these cells are immobile. Directly to the cellulose shell they adjoin a dense and immovable layer of cytoplasm, called ectoplasm. In this layer, chromatophores are fixed, which form one layer of regular longitudinal rows tightly adjacent to each other. Between the vacuole and the ectoplasm layer is the inner liquid movable layer of the cytoplasm, the so-called endoplasm. Its intensive movement can be observed by the movement of organelles smaller than chloroplasts - small colorless inclusions suspended in the cytoplasm.

To determine the speed of movement of the cytoplasm, a stopwatch and an eyepiece ruler placed in the microscope eyepiece are used. Using a stopwatch, the time is counted during which the chloroplast or other moving particle passes the distance between two selected divisions of the eyepiece ruler. Such measurements in the same cell are carried out 3–5 times. To calculate the speed of movement of the cytoplasm, the price of dividing the eyepiece ruler is measured. For this, an object micrometer is placed on the microscope stage, which is examined in the eyepiece micrometer. The selected lens is fixed on the divisions of the object micrometer and the number of divisions of the object micrometer is counted. The price of divisions of the eyepiece micrometer is calculated by the formula where N is the price of divisions of the eyepiece micrometer; 10 µm – division value of the object micrometer; b is the number of divisions of the eyepiece micrometer that fit into (a) divisions of the object micrometer.

Particle speed is the ratio of the distance in micrometers to the number of seconds it takes a moving particle to cover this distance (µm/s).

The task. Determine the speed of movement of the cytoplasm in the cells of aquatic plants. Enter the measurement results in the table. Make schematic drawings of the cells of the considered objects and indicate the direction of the cytoplasm movement with arrows, compare the nature and speed of cyclosis.

Object Type of moving Distance Particle travel time, s Cyclosis rate, 1. What is a cytosol?

2. How does the form of plasmolysis depend on the viscosity of the cytoplasm of plant cells?

3. What is the biological significance of the movement of the cytoplasm?

4. What are the main types of cytoplasmic movement?

5. What determines the speed of the cytoplasm?

From the Authors…………………………………………………………….

1. PLANT CELL AS OSMOTIC

SYSTEM………………………………………………………….

Laboratory work Models of plant cells……………………………………... Laboratory work Phenomenon of plasmolysis and deplasmolysis of a plant cell..……. Laboratory work Determination of the osmotic pressure of cell sap by the plasmolytic method………………………………….……………. 2. PROPERTIES OF CELL MEMBRANES………..…………..

Laboratory work Studying the selective permeability of the plant cell plasmalemma…………………….………………………………….. Laboratory work Studying the diffusion of neutral red through the plasmalemma Laboratory work Changing the permeability of the tonoplast and plasmalemma for betacyanin under physical and chemical factors... 3. PROPERTIES OF THE CYTOPLASMA………………………………... Laboratory work Effect of potassium and calcium ions on the viscosity of the cytoplasm of a plant cell………………………………… ..……………………. Laboratory work Observation of the movement of the cytoplasm of plant cells and measurement of its speed………………………………………………….

PHYSIOLOGY OF THE PLANT CELL

Workshop "Plant Physiology"

for students of the Faculty of Biology Responsible for the issue A. P. Kudryashov Signed for publication on 31. 08. 2009. Format 6084/16. Offset paper.

Headset Times. Conv. oven l. 1.63. Uch.-ed. l. 1.62. Circulation 50 copies. Zach.

Belarusian State University 220030, Minsk, Independence Avenue, 4.

Printed from the original layout of the customer on the copiers of Belorussky state university.

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Explanatory note.

Proposed elective course contains information about the cell - a unit of living nature, is intended for students of specialized classes who are interested in cytology and biochemistry. The proposed elective course supports and deepens the basic knowledge of biology. Studying an elective course will help in choosing further education and professional activities.

The course is based on the knowledge and skills acquired by students in the study of biology. In the process of classes, students are supposed to acquire the experience of searching for information on the proposed issues. Students improve the skills of preparing essays, reports, messages on a chosen topic, work out the technique of the experiment.

The elective course lasts 35 hours. The program provides for the study of theoretical issues, laboratory work, seminars.

Purpose of the course. To form the ability to identify, reveal, use the relationship between the structure and function of the cell. To consolidate the skills necessary for conducting laboratory work. Engage students in independent work with additional literature.

The objective of the course: the formation of skills and abilities of a comprehensive understanding of knowledge in biology, helping students to meet the interests of those who are fond of cytology and biochemistry.

The main concept of the course.

1. An integrated approach to the study of living organisms at different levels of organization.

2. When considering questions of the structure of the cell, the main attention is paid to the formation of evolutionary thinking.

The total number of hours is 35 hours.

Topic I. Cell: history of study. Cell theory. (3 hours)

Introduction to cell cytology. Tasks of modern cytology. The cell is an integral system. History of the study of cells. Creation of the cell theory. Methods for studying cells. Parallelism in the evolution of microscopic technique and the level of cytological studies.

Laboratory work 1. Microscope device and microscopy technique.

Theme II. Chemistry of the cell (8 hours).

Chemical elements of the cell. Features of the chemical composition of living things. Ions in the cell and body. The content of chemical compounds in the cell. The role of water in a living system.

organic compounds. Chemistry of proteins. Proteins are colloids, proteins are amphoteric electrolytes, proteins are hydrophilic compounds. Pathological phenomena in the absence of proteins in food.

In violation of the exchange of nucleoproteins, padagra develops. The essence of this disease is that a large amount of uric acid salts is deposited in the body in cartilage and other tissues. The content of uric acid in the blood is increased by 2-3 times and even 5 times against the norm. This process is accompanied by pain and deformity of the joints. The deposition of uric acid in the kidneys is characterized by a decrease in its excretion from the body, as a result, the level of uric acid is even higher. rises.

Laboratory work 2. Detection of proteins in biological objects.

Carbohydrates are the most common organic matter on the ground. Relationship between the structure of carbohydrates and biological functions. Pathologies due to a violation of the metabolism of carbohydrates in the body.

The level of sugar in the blood is normal. In the blood of the fetus is 35 - 115 mg%, in newborns - 20 - 30 mg%, in children - 80 - 120 mg%, in adults - 70 - 100 mg%, in the elderly - 85 - 110 mg%. The change in blood sugar is characterized by certain disorders of carbohydrate metabolism.

Hyperglycemia is a condition of the body characterized by an increase in blood sugar levels. The causes of hyperglycemia can be physiological (consumption of large amounts of carbohydrates, various emotional states, etc.), and pathological factors (diabetes mellitus, chronic diseases, brain tumors, mental illness). A form of carbohydrate metabolism disorder is diabetes mellitus.

Laboratory work 3. Detection of carbohydrates in biological objects.

Prove the presence of carbohydrates in biological objects - the most important biological substances.

Lipids. The role of lipids in the emergence of a certain autonomy of such a biological system as a cell.

Laboratory work 4. Detection of lipids in biological objects.

Nucleic acids. Watson and Crick model.

Laboratory work 5. Qualitative reaction for DNA.

Topic III. The general plan of the structure of the cells of living organisms. (10 o'clock)

Membrane cell organelles. non-membranous cell organelles. Prokaryotes and eukaryotes. Animal and plant eukaryotic cell.

Laboratory work 6. Features of the structure of prokaryotes and eukaryotes.

Membrane. Modern model of the structure of the cell membrane.

Cytoskeleton - its components and functions in different cell types.

Endocytosis and membrane receptor function.

Large molecules of biopolymers are practically not transported through membranes, but they can get inside the cell as a result of endocytosis. It is divided into phagocytosis and pinocytosis. These processes are associated with vigorous activity and mobility of the cytoplasm.

Prospects for the development of membranology.

Laboratory work 7. Physiological properties of the cell membrane.

Topic IV. Metabolism. (6 o'clock).

Energy sources of the cell. Heterotrophs and autotrophs. Mitochondria are powerhouses. Scheme of ATP synthesis.

Mechanism of photosynthesis and chemosynthesis.

Ribosomes. Types and structures of ribosomes in prokaryotes and eukaryotes. Biosynthesis of proteins. Broadcast. regulation of transcription and translation.

Topic V. Nuclear apparatus and cell reproduction (6 hours).

1. The concept of chromatin. The nucleus of a eukaryotic cell. Karyoplasm.

2. Life cycle cells. cell reproduction. The concept of "stem cells". "Theory of stem cells" - a breakthrough in modern biology and medicine.

3. Cell aging.

Cancer is the most dangerous disease of humans and other living beings.

Laboratory work 8. Mitosis in onion root cells.

Theme VI. Cell evolution. (2 hours).

Final conference "Primary stages of biological evolution on Earth".

Theory of evolution of prokaryotes and eukaryotes.

Thematic planning of the elective course "Mysteries of the living cell".

Topic	Number
Topic 1. Cell: history of study (3 hours) 1. History of the cell. Introduction to Cytology. 2. Creation of the cell theory. Methods for studying cells. 3. L. r. No. 1. The device of the microscope and the technique of microscopy.
Topic 2. Chemistry of the cell. (8 ocloc'k) 1. Chemical elements cells. The role of water in a living system. 2. Chemistry of proteins. L.r. No. 2. Evidence of proteins as biocatalysts (enzymes) 3. Pathological phenomena in the absence of proteins in food. 4. L.r. No. 3. Detection of proteins in biological objects. 5. Carbohydrates are the most common organic substances on Earth. 6.L.r. No. 4. Detection of carbohydrates in biological objects. 7. Lipids. L.r. No. 5. Detection of lipids in biological objects. 8. N.K. L.r. No. 6. Qualitative reaction for DNA.
Topic 3. (10 hours). 1. Membrane. Modern model of the structure of the cell membrane. 2. Cytoskeleton - its components and functions in different types cells. 3. Membrane transport. 4. Endocytosis and receptor function of membranes. 5 - 6. Membrane organelles. 7 - 8. Non-membrane cell organelles. L.r. No. 7. Features of the structure of prokaryotes and eukaryotes 9.L.r. No. 8. Physiological properties of the cell membrane. 10. Seminar.
Topic 4. Metabolism (6 hours). 1. Energy sources of the cell. Heterotrophs and autotrophs. 2. Scheme of ATP synthesis. Mitochondria are powerhouses. 3. Mechanism of photosynthesis. Chemosynthesis. 5. Biosynthesis of proteins. Seminar. 6. Seminar.
Topic 5. 1. The concept of chromatin. The nucleus of a eukaryotic cell. Karyoplasm. 2. Life cycle of a cell. cell reproduction. 3.Theory of stem cells. 4. Aging and death. 5.L.r. No. 9. Mitosis in onion root cells. 6. Seminar.
Topic 6. Cell evolution (2 hours). Seminar. 1-2. Final conference "Primary stages of biological evolution on Earth".

Laboratory work. Topic. The device of light microscopes and microscopy technique.

Target. Based on the knowledge of the device of a light microscope, master the technique of microscopy and preparation of temporary micropreparations. Familiarize yourself with the rules of registration of laboratory work.

Equipment. Microscope for each student. Slides and coverslips, pipettes, cups of water, cotton wool, tweezers, scissors, notebook, album. Scheme of the device of the microscope and its parts.

Working process.

Consider the main parts of the microscope: mechanical, optical and lighting.

The mechanical part includes a tripod, an object table, a tube, a revolver, macro- and micrometer screws.

The optical part of the microscope is represented by eyepieces and objectives. The eyepiece (Latin okulus - eye) is located in the upper part of the tube and faces the eye.

This is a system of lenses enclosed in a sleeve. By the figure on the upper surface of the eyepiece, one can judge the magnification factor (x 7, x 10, x 15). The eyepiece can be removed from the tube and replaced as needed. On the opposite side of the tube is a rotating plate, or a revolver (Latin rewolvo) - I rotate), in which there are three sockets for lenses. Lens - a system of lenses, they have different magnification. There are a low magnification lens (x 8), a high magnification lens (x 40) and an immersion lens for studying small objects (x 90).

The total magnification of a microscope is equal to the magnification of the eyepiece times the magnification of the objective.

The lighting part consists of a mirror, a condenser and a diaphragm.

The condenser is located between the mirror and the stage. It consists of two lenses. To move the condenser, there is a screw located anterior to the microscope and macrometric screw. When lowering the condenser, the illumination decreases, when raised, it increases. By changing the position of the diaphragm plates, using a special knob, you can adjust the lighting.

The task. Sketch the microscope and label its parts.

Microscope rules.

1. Install the microscope with the tripod towards you, the object stage away from you.

2. Place the low magnification lens in working position.

3. Looking into the eyepiece with your left eye, rotate the mirror in different directions until the field of view is illuminated brightly and evenly.

4. Place the prepared preparation on the stage (cover slip up) so that the objective is in the center of the opening of the stage.

5. Under visual control, slowly lower the tube with a macro screw so that the lens is at a distance of 2 mm from the preparation.

6. Look through the eyepiece and slowly raise the tube until an image of the object appears.

7. In order to proceed to the examination of the object at a high magnification of the microscope, it is necessary to center the preparation, i.e. place the object in the center of the field of view.

8. Rotating the revolver, move the high magnification lens to the working position.

9. Lower the tube under the control of the eye (look not through the eyepiece, but from the side) almost until it touches the preparation.

10. Looking into the eyepiece, slowly raise the tube until an image appears.

11. Use the microscopic screw for fine focusing.

12. When sketching the preparation, look into the eyepiece with your left eye.

The task. Rewrite the rules for working with a microscope in a notebook for laboratory work.

Method for preparing a temporary preparation.

1. Take a glass slide, holding it by the side edges, put it on the table.

2. Place an object in the center of the glass, for example, pieces of cotton wool 1.5 cm long. Place one drop of water on the object with a pipette.

3. Place a cover slip on the glass slide.

4. Consider the finished product.

5. Sketch in an album how cotton fibers look at low and high magnification.

Microscopy of protozoa.

1. Take water from a long time ago aquarium. Take a drop together with a sprig of algae or a duckweed leaf and look through a microscope at low magnification. A variety of protozoa are usually seen: shoes, amoeba - free-living and attached to algae (suvoyki). In the water there may be small worms and crustaceans (cyclops, daphnia). Considering this preparation, you can practice pointing the microscope at moving objects. (That is, learn to fix the microscope).

Rules for the design of laboratory work.

A necessary element of the microscopic study of an object is its sketch in an album; have an album 30x21 cm and a pencil (plain and colored).

1. You can only draw on one side of the sheet.

2.Before starting the sketch, write down the name of the topic at the top of the page.

3. The drawing must be large, the details are clearly visible.

4. The drawing must correctly display the forms; the ratio of the volume and size of individual parts and the whole.

First you need to draw the outline of the object (large), then inside the outlines of the details, and then clearly draw them.

5. Draw, clearly repeating all the lines of the object. To do this, you must not take your eyes off the microscope, but only switch your attention from the object to the drawing (this must be learned).

6. For each drawing, you must give the designation of the parts. All inscriptions must be parallel to each other. Arrows are placed to individual parts of the object, write a name against each. To perform laboratory work, you must have an album and a notebook for recording text material and performing diagrams.

Laboratory work. Topic. Structural features of prokaryotic and eukaryotic cells. Cells of plants and animals.

Target. Based on the study of cells of bacteria (prokaryotes), plants and animals (eukaryotes), to discover the main similarities in the structure of bacteria, animals and plants as an indicator of the unity of the organization of living forms.

Equipment.

1. Microscope.

2. Slides and coverslips.

3. Pipettes, glasses of water, tweezers, scalpels, iodine infusion, aqueous ink solution.

4. Magenta, methylene blue, infusion of meat, fish or vegetables, onion film.

Table of the structure of bacterial, plant and animal cells.

Working process.

1. Prepare an infusion in advance from various products: meat, fish, egg protein.

2. Grind a small amount of material and place in a flask, add chalk to the tip of the scalpel. Fill with water to 2/3 of the volume.

3. Keep the flask with the infusion warm (dark) for 3-5 days. During this time, many different bacteria accumulate in the medium.

4. Place a drop of infusion on a glass slide. Consider the preparation using a 40x objective, but you can also try 90x. (A temporary preparation is prepared according to the rules presented in the previous work).

5. Add a drop of mascara. Against the general background, bacterial cells are unstained.

6. Draw bacterial cells.

7. Prepare temporary preparations of plant and animal cells.

Separate the fleshy scale from a piece of onion. There is a thin film on the inside. Remove the film, cut off. Put on a glass slide, pick up a solution of iodine with a pipette, drop onto a film, cover with a coverslip. View at low magnification. Large rounded nuclei in the cells are stained yellow with iodine.

Turn to high magnification and find the cell membrane. In the nucleus, 1-2 nucleoli can be seen, sometimes the granular structure of the cytoplasm is visible.

Unstained voids in the cytoplasm of cells are vacuoles.

8. Sketch several cells. Designate: 1) shell; 2) cytoplasm; 3) core; 4) vacuoles (if they are visible).

You can prepare a preparation of Elodea leaf. You can see chloroplasts - green plastids. Nuclei in unstained cells are not visible.

9. Animal cells can be examined on the finished product. Sketch. The figure should indicate: 1) shell; 2) cytoplasm; 3) core.

10. Conduct a joint discussion.

What provisions of the cell theory can be confirmed by the results of the work done?

Laboratory work. Topic. Detection of proteins in biological objects.

Target. Prove the presence of proteins in biological objects.

Equipment.

Stand with test tubes, pipette, water bath, dropper.

Egg white solution, 10% NaOH solution, 1% copper sulfate, ninhydrin (0.5% aqueous solution), nitric acid (concentrated).

Biuret reaction to determine the peptide bond. The method is based on the ability of a peptide bond in an alkaline medium to form colored complex compounds with copper sulfate.

Working process.

1. Add 5 drops of 1% egg white to a test tube (the protein is filtered through gauze, then diluted with distilled water 1:10), three drops of 10% sodium hydroxide solution and 1 drop of 1% copper sulfate solution and mix.

The contents of the tube acquire a blue-violet color.

ninhydrin reaction. Proteins, polypeptides and free amino acids give a blue or violet color with ninhydrin.

Working process.

1. Take 5 drops of a 10% solution of egg white, add 5 drops of a 0.5% aqueous solution of ninhydrin and heat.

After 2-3 minutes, a pink or blue-violet color develops.

Xantoprotein reaction (Greek xantos - yellow). With the help of this reaction, cyclic amino acids are found in the protein, which contain benzene rings (tryptophan, tyrosine, and others).

Working process.

1.5 drops of 1% egg white solution, add 3 drops of concentrated nitric acid (carefully) and heat. After cooling, add 5 - 10 drops of 10% sodium hydroxide solution to the test tube until an orange color appears (it is associated with the formation of the sodium salt of these nitro compounds).

Laboratory work. Topic. Detection of carbohydrates in biological objects.

Target. Prove the presence of carbohydrates in biological objects.

Equipment. Rack with test tubes. Pipettes, water bath.

1% starch solution, 1% sucrose solution, 1% fructose solution, 1% solution of iodine dissolved in potassium iodide, naphthol dissolved in 50 mm of alcohol (diluted 5 times with water before use), 1% alcohol solution, thymol.

Concentrated sulfuric acid, Selivanov's reagent: 0.5 g of resorcinol dissolved in 100 ml of 20% hydrochloric acid

Starch detection.

Working process.

1. Add 10 drops of a 1% starch solution and one drop of a 1% solution of iodine in potassium iodide to a test tube.

A blue-purple color is observed.

detection of carbohydrates.

Using the reaction with naphthol or thymol, small amounts of carbohydrates or carbohydrate components in complex compounds are detected.

Working process.

1. Add 10 drops of 1% sucrose solution to two test tubes.

In one add 3 drops of 1% alcohol solution of naphthol. In another test tube - 3 drops of 1% alcohol solution of thymol. Pour 0.5 ml of concentrated sulfuric acid into both (carefully) and observe a violet color in the test tube with naphthol and red in the test tube with thymol at the border of the two liquids.

Detection of fructose (Selivanov's reaction).

Fructose, when heated with hydrochloric acid and resorcinol, gives a cherry red color.

Working process.

1. Pour 10 drops of Selivanov's reagent 2 drops of a 1% fructose solution into a test tube and heat gently (a red color will appear).

Laboratory work. Topic. Detection of lipids in biological objects.

Target. Prove the presence of lipids in biological objects.

Equipment.

1. Stand with test tubes, water bath, pipette, glass cups, sticks, gauze.

2.Leticin, alcohol solution (chicken egg yolk), cholesterol, 1% chloroform solution, concentrated sulfuric acid, acetone.

detection of lecithin.

Lecithin belongs to the group of phospholipids, is part of the cell membranes. It makes up the bulk of brain tissue.

Working process.

1. Pour 10 drops of acetone into a dry test tube; put in a glass? chicken egg yolk.

While stirring with a stick, add 40 ml of hot alcohol drop by drop.

When the solution has cooled, filter it into a dry test tube. The filtrate should be clear. The reagent must be prepared before use. A white precipitate falls out.

detection of cholesterol.

Cholesterol is a fat-like substance that is of great importance for the body. Included in the membranes of many organs and tissues, is a precursor of bile acids, vitamin D, sex hormones, hormones of the adrenal cortex. The reaction is based on its ability to release water and condense into colored compounds.

Working process.

1. Pour 10 drops of 1% chloroform solution of cholesterol into a dry test tube and (carefully) pour 0.5 ml of concentrated sulfuric acid along the vessel wall. Shake (gently). A red-orange color of the upper chloroform layer appears.

Laboratory work. Topic. Evidence for the functioning of proteins as biocatalysts (enzymes).

Target. To prove the catalytic action of proteins - enzymes, to show their high specificity, the highest activity in a physiological environment.

Equipment. Rack with test tubes, 1 ml pipettes, water bath, thermostat.

1% starch solution, sucrose solution, 1% iodine solution in potassium iodide, 5% copper sulfate solution, 10% sodium hydroxide solution, 2% sucrose solution, 0.2% hydrochloric acid solution.

Working process.

1. Enzymatic hydrolysis of starch.

Salivary amylase acts as an enzyme that hydrolyzes starch into its constituent parts (maltose, glucose). Evaluation of the results of the experiment is carried out using color reactions with iodine of the Trommer reaction.

Non-hydrolyzed starch gives a blue color with iodine and a negative Trommer reaction. Starch hydrolysis products do not react with iodine, but react positively to Trommer's reagent.

1. Pour 10 drops of 1% starch solution into two test tubes.

2. Add 4 drops of water to one of them (test tube No. 1) (control).

In the second (test tube No. 2) add 4 drops of saliva solution, dilute saliva 5 times.

3. Mix and put in a water bath or thermostat for 15 minutes. at 37 deg.S.

4. Take 4 drops of the test substance from the test tube and add it to 2 different test tubes.

5. In one add one drop of a 1% solution of iodine in potassium iodide.

In another add one drop of 5% copper sulfate solution and 4 drops of 10% sodium hydroxide solution and gently heat to a boil (Trommer reaction).

6. We do the same with the contents of test tube No. 2. The result should show that starch hydrolysis does not occur in the presence of water and the reaction with iodine should be positive. The Trommer reaction is negative (copper oxide hydroxide is blue). In the presence of salivary amylase, the results should be the opposite, since starch hydrolysis has occurred.

There is no reaction with iodine and a brick-red color (copper oxide I) occurs in the Trommer reaction.

II. The specificity of the action of enzymes.

Each enzyme acts on only one substance or group of similar substrates. This is due to the correspondence between the structure of the enzyme, its active center and the structure of the substrate. For example, amylase only acts on starch.

Preparation of sucrose.

Grind 1.100 g of yeast and add water (400 ml). Filter after 2 hours and store in the refrigerator.

2. In two test tubes (No. 1 and No. 2), add 10 drops of 1% starch solution.

Add 10 drops of 2% sucrose solution to test tubes No. 3 and No. 4.

3. In test tubes No. 1 and No. 3, add 4 drops of a saliva solution diluted 5 times.

Add 4 drops of sucrose to test tubes No. 2 and No. 4.

4. Mix and leave in a thermostat for 15 minutes at a temperature of 37 degrees. FROM.

5. Then, with the contents of all four test tubes, perform reactions with iodine and Trommer

Determination of the specificity of the action of enzymes

In the conclusions, it should be noted in which test tube and under what conditions the action of enzymes was found and why.

III. Influence of medium pH on enzyme activity.

For each enzyme, there is a certain value of the reaction of the environment at which it exhibits the highest activity. A change in the pH of the medium causes a decrease or complete inhibition of the activity of the enzyme.

1. Pour 1 ml of distilled water into 8 test tubes.

2. Add 1 ml of 0.2% hydrochloric acid solution to test tube No. 1. Mix.

3. Take one ml of the mixture from test tube No. 1 and transfer it to test tube No. 2. Mix, pour 1 ml and transfer to test tube No. 3, etc.

4. Take 1 ml from test tube No. 8 and pour it out. We get different pH environments.

4. In each tube add 2 ml of 1% starch solution and 1 ml of saliva solution diluted 1: 10.

5. Shake the test tubes and put in a thermostat for 15 minutes at 37 degrees. FROM.

6. Cool and add one drop of 1% solution of iodine in potassium iodide to all test tubes.

Complete hydrolysis will occur in test tubes No. 5 and No. 6, where the pH of the solution medium is in the range of 6.8 - 7.2, i.e. optimal for amylase action.

Laboratory work. Topic. Isolation of deoxynucleoprotein from spleen (liver) tissue. Qualitative DNA test.

Target. Prove that a large number of nucleic acids are contained in the form of a compound with proteins (deoxynucleoprotein - DNP) in tissues rich in nuclei (spleen, thymus).

Equipment. Test tube rack, mortar and pestle, glass powder, pipette, crystallizer, 50 ml and 300 ml measuring cylinders, 1 ml pipettes, notched wooden sticks, water bath, filter gauze, sodium chloride, 5% solution, containing 0.04% trisubstituted sodium nitrate, 0.4% sodium hydroxide solution, diphenylamine reagent (dissolve 1 g of diphenylamine in 100 ml of glacial acetic acid. Add 2.75 ml of concentrated acid to the solution), spleen (fresh or frozen Yeast RNA, freshly prepared 0.1% solution.

Working process.

1. Isolation of deoxynucleoprotein (DNP) from the tissue of the spleen (liver).

The method is based on the ability of DNP to dissolve in salt solutions of high ionic strength and precipitate when their concentration decreases.

2 - 3 g of spleen tissue carefully grind in a mortar with glass powder, gradually adding 35 - 40 ml of sodium chloride solution.

The resulting viscous solution is filtered through two layers of gauze into the crystallizer. Use a cylinder to measure six times (in relation to the filtrate) volume of distilled water and slowly pour into the filtrate.

The resulting DNP threads are carefully wound on a wooden stick, transferred to a test tube for use.

2. Qualitative reaction for DNA.

The method is based on the ability of deoxyribose, which is included in the DNA of deoxyribonucleoprotein, to form blue compounds with diphenylamine when heated in a medium containing a mixture of glacial acetic acid and concentrated sulfuric acid.

With ribose RNA, a similar reaction produces a green color.

To 1/4 of the DNP precipitate add 1 ml of 0.4% sodium hydroxide solution (until dissolved). Add 0.5% ml diphenylamine reagent. Mix the contents of the tube and place in a boiling water bath (15 - 20 min).

Perform a similar reaction in another test tube with 1 ml of RNA solution.

Note the characteristic coloration.

Laboratory work. Topic. Physiological properties of the cell membrane.

Target. Show that the cell membrane has selective permeability. Visually demonstrate the role of the membrane in the process of phagocytosis and pinocytosis, as well as familiarize yourself with cell plasmolysis - the process of separating the protoplast (cell contents) from the cell walls.

Equipment.

Microscopes, coverslips and slides, scalpels, dissecting needles, filter paper, pipettes, ink.

Infusoria culture or tissue culture on a nutrient medium, amoeba culture, pieces of Elodea plant.

Solutions of potassium chloride, solutions of calcium chloride, magnesium chloride, 2% albumin solution, 10% sodium chloride solution, distilled water.

Working process.

1. In a weak solution of sodium or potassium chloride, place ciliates or pieces of cultured tissue.

2. Prepare a preparation for the microscope.

3. You can see the shrinkage of the cells, indicating the permeability of the cell membrane. In this case, the water from the cell is released into the environment.

4. Transfer the cells to a drop of distilled water or pull the solution out from under the coverslip with filter paper and replace it with distilled water. Observe how the cells swell as water enters them.

5. Place ciliates or pieces of cultured tissue in a solution of calcium chloride or magnesium chloride of low concentration.

Ciliates and cultured cells continue to live. Calcium and magnesium ions reduce the permeability of the cell membrane. There is no movement of water through the shell.

6. Place the amoeba in a drop of 2% albumin solution (chicken egg protein).

Prepare a slide for the microscope. After some time, bubbles, protrusions, and tubules form on the surface of the amoeba. It seems that the surface of the amoeba is "boiling". This is accompanied by intense fluid movement near the membrane surface.

Fluid bubbles are surrounded by protrusions of the cytoplasm, which then close. Pinocytic vesicles sometimes appear suddenly. This suggests that liquid droplets, together with the substance soluble in it, are captured quickly. Pinocytosis is caused by substances that lower the surface tension of the cell wall. For example, amino acids, some salts.

7. Introduce a little carcass into a drop of liquid in which amoebas are located. Prepare the drug. After some time, the amoebas begin to slowly move towards the grains of the carcass, releasing pseudopodia (pseudopodia).

Carcass grains are attached to the surface of pseudopodia, surrounded by them, and after a while are immersed in the cytoplasm.

Under the microscope, the phenomenon of phagocytosis in amoeba is observed.

Primary requirements.

Students should know:

1. microscope device and work with it;

2. position of the cell theory;

3. similarity and difference between plant and animal cells;

4.role of chemicals and compounds in the cell;

5. main components and organelles of the cell;

6. features of prokaryotes and eukaryotes;

7. pathology of protein and carbohydrate metabolism;

8. value of individual mineral elements.

Students should be able to:

1.work with a microscope;

2. name the main parts of the cell, "recognize" them on the diagram, photograph;

3. to produce the simplest preparations for microscopic examination;

4.correctly draw up laboratory work;

5. independently work with additional literature and use modern technologies.

Literature for the teacher.

1.Welsh U., Storch F. Introduction to Cytology. Translation from him. M. Mir, 1986

2. Zavarzin A.A. and others. Biology of the cell. - ed. St. Petersburg State University, 1992

3. Svenson K., Webster P. - M. Mir, 1982

4. Lamb M. Biology of aging - M. Mir, 1980

5.Markosyan A.A. Physiology - M. Medicine, 1968

6. Liberman E.A. living cell. M.Mir, 1985

7.M.V.Ermolaev Biological chemistry. Moscow "Medicine", 1984

8. General biology. A.O. Ruvinsky Moscow "Enlightenment", 1993

Literature for students.

1. Green N., Stout W., Taylor D. Biology.

2. De Duve K. Journey into the world of a living cell. M.Mir, 1982

3. Liberman E.A. Living cell. M. Mir, 1987

4.Kemp P., Arms K. Introduction to biology.

The main aspect in studying the course should be directed to active work students in the classroom in the form of dialogue teacher - student, active discussion in the form of student - student, student - teacher.