[DNA damage by active oxygen species in cryopreservation and the antioxydative properties of cryoprotectants].

Several disorders in the DNA structure in the cells of vertebrate and invertebrate animals, plant and algae are reviewed. Some causes of the DNA disorders, the reactive oxygen species, especially, are discussed. Some data are shown, that the common used cryoprotectants such as dimethylsulfoxide, glycerol, methanol, sucrose and albumin, are OH* scavengers. Some seldom used cryoprotectors, which scavenges several forms of active oxygen, are described. It is supposed that the antioxidant properties of the cryoprotectors are essential for their mechanism of action.

[Consequences of plant tissue cryopreservation (phenotype and genome)].

The question about the correspondence of regenerants after cryopreservation to the initial organisms has been the focus of attention of cryobiologists. The correspondence has been established for morphological and physiological characteristics and the content of DNA fragment in flowering plants and algae. But no less investigations point to some changes in the phenotype and the appearance of DNA polymorphism in specimens of both plant sections. Changes in DNA methylation at cytosine after cryopreservation of flower plant tissues have attracted considerable interest. Thus, after freezing and thawing, changes in the phenotype and genome may occur, and the latter changes proceed by two mechanisms: mutations and modifications of DNA methylation.

[The problem of the stability of organisms after cryopreservation (fungi as an example)].

The results of research on the consequences of cryoconservation of fungi indicating the stability of their morphology are reviewed, and a number of facts indicating the changes in the properties of the fungus and the mutagenicity of cryopreservation are presented. The investigations of the last ten years confirm the Rosanov's hypothesis according to which cryoconservation must lead to an increase in the reserve of the hereditary variation of organisms.

[On the similarity in the effects of different cryopreservation conditions on the growth and development of plants].

The morphological characteristics of 35 wild plant species were studied after freezing of seeds under the conditions of deep, fast, and programmed freezing (-196 degrees C) and non-deep freezing (-10 degrees C). The seeds were stored frozen for a month. The seeds of all the species were characterized by a low humidity. The field and laboratory seed germination capacity, leaf growth, the quantity and length of shoots, the quantity of generative organs, and the variability of these characteristics were studied. It was shown that the direction of changes under different cooling conditions was the same except for the laboratory germination capacity of some species. The direction was determined by the species features rather than cooling conditions.

[Dehydration in the cryopreservation of moist plant tissues and in seed maturation].

The possibility of long-term cryopreservation of plant objects depends on their water content. In orthodox seeds, it decreases at the late stage of maturation and is accompanied by the synthesis of protectors--sugars and proteins. These seeds easily withstand cryopreservation. Organs with a high water content, meristems, and recalcitrant seeds are dried in presence of sucrose before plunging in liquid nitrogen. In orthodox seeds, artificially dried moist seeds, and meristems, the cellular content forms glass structures that are estimated in frozen materials by differential scanning calorimetry and electron paramagnetic resonance methods. It is proposed that the glass cellular content is connected with the duration of cryopreservation. Methodical approaches to successive cryopreservation of moist plant tissues are described.

[Effect of freezing of seeds on the morphological characteristics of four pink species].

The effect of non-deep freezing (-10 degrees C) and deep freezing (-196 degrees C) on the morphological characteristics of four pink species was investigated. It was shown that the growth and development of plants after cryopreservation changed insignificantly. Rather stable characteristics (diameter of flower, the number of cauline nodes, and root length) did not change. The length of generative shoots decreased a little, and the character of histograms changed. On the other hand, the changeable characteristics (the number of vegetative shoots and flowers) decreased after seed cryopreservation. However, the number of flowers did not vary in the next year. The parameters essential for seed cryopreservation, seed germination and the quantity of fruits, did not changed. It was also found that seed freezing exerts a stimulating effect on seed germination and the top limit of some parameters, including the quantity of generative organs.

Interaction of therapeutic ultrasound with purified enzymes in vitro.

The effects of ultrasound on the rates of the catalytic reactions of four purified enzymes in vitro have been extensively investigated under a wide range of biochemical and physical exposure conditions. In general, it can be concluded that therapeutic intensities of continuous wave 0.88 MHz ultrasound had no detectable direct effects on the rates of the reactions catalysed by creatine kinase, lactate dehydrogenase, hexokinase and pyruvate kinase. Some minor effects were noted. These were: an indirect effect resulting from mixing within the sample chamber caused by quartz wind streaming; an effect on partially-hydrated cross-linked enzyme systems which appears to be the result of increased fluid penetration of the solid matrix in the presence of ultrasound; and an increase in the rate of spontaneous dissociation of a multimeric enzyme system. It is, therefore, concluded that a direct interaction between ultrasound and the catalytic functioning of individual enzyme molecules is unlikely to be the primary step in any acousto-biological interaction, and that this primary interaction appears to be occurring at a higher level of organizational complexity.

[X-ray diffraction study of the rabbit psoas muscle with extracted H-zone]. / Rentgenograficheskoe issledovanie spinnoi myshtsy krolika s ékstragirovannoi H-zonoi.

Fibre bundles of glycerinated rabbit psoas muscle about 0.5-1.0 mm thick were incubated in 5 mM tris-(hydroxymethyl)-aminomethane (Tris), pH 8.0 for 5-20 hours at 4 degrees C. This treatment leads to selective removal of some proteins in the M-bands and H-zones of sarcomeres. Effects of extraction were analyzed on the basis of electron micrographs of longitudinal sections of muscle specimens, gel electrophoresis patterns of myofibrils and of the extracts, and measurements of the creatine kinase activity of myofibrils. In the X-ray diffraction patterns of the fibre bundles subjected to prolonged extraction a drastic decrease in the intensity of "442 A" and "223 A" meridional reflections and a considerably smaller decrease in the intensity of "212 A" meridional reflections were observed. The "147 A" meridional reflection remains practically unchanged. It was concluded that: (1) The reflections "442 A" and "223 A" were contributed mainly by diffraction on the minor proteins located in the central part of the thick filaments in between the C-zones. This is contrary to the widely accepted viewpoint according to which the appearance of "442 A" reflection is caused only by the C-protein component of the thick filaments. (2) The "147 A" meridional reflection is contributed mainly by C-protein and light meromyosin of the thick filaments.

[Dynamics of quaternary structure of creatine kinase purified from rabbit skeletal muscles]. / Dinamika chetvertichnoi struktury kreatinkinazy iz skeletnykh myshts krolika.

Using gel filtration and ultracentrifugation, the quaternary structure of muscle creatine kinase (isoenzyme MM) has been shown to depend on pH, protein concentration and ionic strength of solution. In solution the enzyme can exist not only as a dimer, but also as a monomer and tetramer with molecular weights of 40 000 and 160 000, respectively. High dilution of the protein solution and pH 6.0--5.0 provide for the monomer formation; the dimer can arise under various conditions, while the tetramer requires pH 9.0 and high protein concentration. The monomer differs from the dimer by a higher enzymatic activity, lability and needs thiol to maintain its catalytic activity. Dissociation of creatine kinase into more active subunits accounts for the increase in specific activity induced by protein dilution. It is assumed that dissociation can have physiological significance as one of the mechanisms of creatine kinase activation in muscles.

[Inhibition of creatine kinase activity by glycolysis metabolites]. / Ingibirovanie aktivnosti kreatinkinazy metabolitami glikoliza.

It is shown that lactate is a noncompetitive inhibitor of muscle creative kinase Ki 16 mmol with respect to MgATP. When inhibitors are applied in combination (lactate with glucose-6-phosphate or inorganic phosphate, glucose-6-phosphate with Pi or all three inhibitors together) a competition arises. When applied together the combined action is weaker than the sum of inhibition of three effectors in the case of separate application. The competition is revealed in model experiments with metabolite concentrations peculiar to resting and acting muscle. The supposition is made that formation of lactate, glucose-6-phosphate and inorganic phosphate in the muscle tissue can induce partial, but not complete inhibition of the creatine kinase activity.

[Effect of lactate and glycolytic intermediates on muscle creatine kinase]. / Issledovanie deistviia laktata i glikoliticheskikh intermediatov na myshechnuiu kreatinkinazu.

Inhibition of rabbit muscle creatine kinase by lactate, glucose-6-phosphate and PEP occurs immediately and depends on the concentration of the effectors and enzyme properties. Desensitization of creatine kinase to these effectors is described. Partial desensitization to G-6-P and lactate occurs after the enzyme preincubation with dithiothreitol. A complete loss of sensitivity to lactate and a significant loss of sensitivity to G-6-P and PEP occurs after the enzyme storage at -15 degrees during several weeks. The data obtained support our previously made assumption on the allosteric regulation of the MM isoenzyme of creatine kinase.

[Adenylate kinase and creatine kinase activity of rabbit blood serum following application of a tourniquet to the thigh]. / Adenilatkinaznaia i kreatinkinaznaia aktivnost' syvorotki krovi krolikov posle nalozheniia zhguta na bedro.

A colorimetric method is described for estimation of the adenilate kinase activity in blood serum; the method is based on coupling of adenilate- and creatine kinase reactions and on estimation of the amount of creatine formed. Adenilate kinase activity in blood serum, estimated by the method, was shown to increase 4-fold after removing of tourniquet with subsequent normalization within the next day. The same data were obtained using a spectrophotometric method for estimation of adenilate kinase based on NADP reduction in coupled reactions with hexokinase and glucose-6-phosphate dehydrogenase. An increase in creatine kinase activity in blood serum occurred later on after removing of the tourniquet; it was more distinct (8.5-fold) and maintained longer as compared with the increase in adenilate kinase activity.

[Effect of phosphoenolpyruvate on creatine kinase activity in rabbit muscles]. / Deistvie fosfoenolpiruvata na kreatinkinaznuiu aktivnost' v myshtsakh krolei.

Phosphoenolpyruvate (PEP) is a potent inhibitor of muscle creatine kinase. The inhibition of ATP formation is more pronounced at pH 7.0. The substrates, creatine and creatine phosphate, partially prevent the inhibition of the enzyme activity by PEP, creatine being as an effector.

[Allosteric properties of muscle creatine kinase]. / Allostericheskie svoistva myshechnoi kreatinkinazy

The dependence of the reaction rate on substrate concentrations at pH 8.0--7.5 does not submit the Michaelis-Menten kinetics. The dependence of v on Mg-ATP is described with a curve having an intermediate plateau. The dependence of v on the creatine concentration is expressed by a curve, which is not hyperbolic. In this case the index of the substrate concentration, (q), is variable, and it increases with the increase of creatine concentration from 1 to 2 (at the presence of effectors, PEP and ADP,--from 1 to 3.5). The specific creatine kinase activity increases 3--4-fold with protein dilution, but this effect is not observed in the presence of inhibitors to FDP and PEP. Creatine kinase is desensibilized with respect to FDP and PEP after a prolonged storage. The data obtained and the presence of an effector set indicate, that muscle creatine kinase is a regulated allosteric enzyme.

[The effect of sugar phosphates, phosphoenolpyruvate and adenylic acid on muscle, brain and heart creatine kinases]. / Vliianie fosfatov sakharov, fosfoenolpiruvata i adenilovoi kisloty na kreatinkinazy skeletnykh myshts, mozga i serdtsa

The effect of adenylic acid, glucose-6-phosphate, fructose-1,6-diphosphate and phosphoenolpyruvate on creatine kinase isoenzymes (brain extract, muscle and heart extracts and purified muscle enzyme) was studied. These effectors, especially phosphoenolpyruvate, are shown to inhibit in different degree the reaction of ATP formation catalysed by creatine kinase from all tissues. The effectors do not inhibit the creatine phosphate synthesis in extracts, but depress purified creatine kinase. The interrelationship of the creatine kinase system and the key glycolytic enzymes (phosphofructokinase, hexokinase, pyruvate kinase) is discussed.