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The review is devoted to the inhibitors of cysteine proteinases which are believed to be very important in many biochemical processes of living organisms. They participate in the development and progression of numerous diseases that involve abnormal protein turnover. One of the main regulators of these proteinases is their specific inhibitors: cystatins. The aim of this review was to present current knowledge about endogenous inhibitors of lysosomal cysteine proteases and their synthetic analogs. [ABSTRACT FROM AUTHOR]

Protein kinase FGFR1 plays a key role in oncogenic transformation and oncovasculogenesis. Inhibitors of this kinase can be used as anticancer drugs. Protein kinase FGFR1 inhibitors searching were performed among oxindole derivatives. 9 compounds from 920 oxindole derivatives have been chosen using virtual screening. The most active compound was 2-Hydroxybenzoic acid (2-oxo-1,2-dihydroindol-3-ylidene)-hydrazide (??50 1.25 μM). Structure-activity relationship of tested compounds was investigated and optimization strategies were proposed. [ABSTRACT FROM AUTHOR]

Bioprosthetic heart valves (BHVs) are known for their lower thrombogenicity rates and excellent hemodynamic parameters similar to native valves. However, the lifespan of these medical devices is limited to 15 years due to the structural valve degeneration. One of the mechanisms underlying functional impairment and calcification of BHVs includes proteolytic degradation of biomaterials. However, proteases found in xenogeneic BHVs tissue remain poorly studied. In this study using the dot blot assay, we have performed a screening analysis of proteolytic enzymes and their inhibitors in the leaflets of five BHVs explanted due to their dysfunction. Five aortic valves (AVs) explanted due to calcific aortic valve disease were studied as a comparison group. The results of the study have demonstrated that at least 17 proteases and 19 of their inhibitors can be found in BHVs. In the AVs 20 proteases and 21 their inhibitors were identified. Small quantitative differences were noted between proteomic profiles of the BHVs and AVs. Matrix metalloproteinases (MMPs) were expressed in BHVs and AVs at comparable levels, but the level of tissue inhibitors of metalloproteinases-1/-2 and RECK protein in implant tissues was lower than in natural valves. Probably, excessive activity of MMPs cannot be counterbalanced by their inhibitors in BHVs and therefore MMPs can degrade prosthetic biomaterial. Moreover, the detection of a wide range of proteolytic enzymes and their inhibitors in the degenerated BHVs suggests the existence of several pathophysiological pathways that can lead to structural valve degeneration.

Aim: To investigate an effect of hypoxia on the formation of nitric oxide (NO) in the brain and blood of rats and an influence of L-NNA, the NO-synthase inhibitor, and sodium nitrite on NO generation in separate and simultaneous introduction of these compounds in the animal body., Material and Methods: Experiments were conducted on 42 rats of Krushinsky-Molodkina (K-M) line without adaptation to hypoxia and short-term adaptation to hypobaric hypoxia. Animals were intraperitoneally injected with sodium nitrite (0.5 mg/100 g) and the non-specific inhibitor of NO-synthase L-NNA (2.5 mg/100 g) at different times. Changes in NO content were assessed using electron paramagnetic resonance (EPR) by the intensity of nitrosyl Hb-NO complexes in ERP blood spectra., Results and Conclusion: The production of NO in the brain, compared with that in the blood, was significantly reduced, not only under physiological conditions, but also after the administration of moderate doses of NaNO2. In the short-term hypobaric hypoxia, there was the intensification of the processes of the reduction of NO2- to NO and the formation of nitrosyl heme-NO complexes not only in the blood but also in the mammalian brain.

Synergists (piperonyl butoxide, MGK-264 (N-octyl bicycloheptene dicarboximide), DEF (S, S, S, tributyl phosphorotrithioate), and diethylmaleate) were used to investigate the metabolic resistance of body lice to permethrin. Instances of the synergistic activity ofpermethrin when combined with enzyme system inhibitors have been identified. The synergic ratios were 3.3 to 52.0 for monooxygenase inhibitors; 5.2 to 7.4 for esterases, and about 4 for glutathione-S-transferases. Since the use of the synergists did not lead to full inhibition of resistance, it was concluded that the lice had also a kdr-like type of resistance.

Pharmacotherapy of prostatic adenoma is based in present-day practice primarily on two basic groups of medicines--inhibitors of 5alpha-reductase and blockers of alphal-adrenoreceptors. Our trial included 98 patients with prostatic adenoma aged 59-79 years given combined treatment with finasteride (inhibitor of 5alpha-reductase) made in Hungary (prosteride, Gedeon Richter) and an uroselective blocker of alpha1-adrenoreceptors sonisin (Gedeon Richter, Hungary). Prosteride was given for 12 months, sonisine--for the first 6 months of combined treatment. Positive results were achieved in 97 (99%) of 98 patients. After 12-month combined treatment symptoms of the disease reduced by IPSS from 17.9 +/- 1.8 to 3.9 +/- 0.9 points, Qmax rose from 8.8 +/- 0.3 to 14.6 +/- 0.3, residual urine volume diminished from 91.7 +/- 8.5 to 31.7 +/- 4.4, the size of the prostate reduced from 72.4 +/- 2.9 to 50.6 +/- 3.7, quality of life improved 2-fold. Combined administration of finasteride (prosteride) and alpha1-adrenoblocker tamsulosine (sonisine) meet three principles of pharmacotherapy of prostatic adenoma: prevents progression of the disease and surgical treatment, relieves symptoms and improves quality of life.

The presence of strongly pronounced metabolic dependence of cooling-induced action potentials (APs) in excitable cells of pumpkin stem was shown using NaN3, DCCD, and IAA. This dependence might be connected with participation of the plasma-membrane ATP-dependent pump in the excitation process. The decrease of extracellular Ca2+ concentration had as strong depressive effect on the AP as that of the inhibition of metabolism. It has been supposed that the calcium signal is important coupling factor between the AP generation and the metabolism of excitable cells.

At the end 80s of the XX century bleaching of teeth became one of the most important issues in the stomatological industry. Currently there are two methods of bleaching: external and internal (endo bleaching) methods. It is proved that the adhesive ability of the tooth tissues reduces as a result of peroxide penetration into enamel and dentine. Based on this, the aim of our study was to use the antioxidants for excluding the peroxides from tooth tissues and for rehabilitation of reduced adhesion. For this purpose we used central and side 36 healthy incisors, where after processing of tooth tissue with antioxidants and without them we studied the formation of micro rift between the operation material and dentine, and intensity of micro flowing by means of stereo microscope. We used 30% endo-opalescence as a bleacher and used 10% sodium acrobat as antioxidant. As a result of experiment we found out that the teeth where we used antioxidant were not penetrated with metaline blue; unlikely, the teeth where we did not use the same method we got the approximate result in the group of teeth, where for peroxide exclusion we performed the crown reconstruction not immediately, but after 7 days. During the whole experiment the teeth were kept in thermostat at 37 degrees C in saliva to create the natural conditions for them. In overall we can conclude that the deteriorated adhesive ability with peroxide influence can be improved by application of antioxidants.

New data were obtained for the Solanum brevidens Fill. nucleotide sequences coding for polygalacturonase inhibitor proteins (PGIPs), which are involved in plant defense against phytopathogenic fungi. Highly degenerate primers directed to the conserved regions of the known PGIP genes of tomato, kiwi, apple, carrot, and grape were used to clone four pgip genes and one pseudogene from the genome of S. brevidens, a species that is closely related to cultivated potato, forms no tubers, is highly resistant to phytopathogens, and is often employed in potato breeding. The sequenced part of the coding region of the new genes is 924 bp and codes for a protein of 308 amino acid residues (without the leader peptide). The genes were designated as pgipSbr1(1), pgipSbr1 (2). pgipSbr2, pgipSbr3, and pgipSbr4. The amino acid sequences of the S. brevidens PGIPs have 90.9-99.4% identity to each other and 94% identity to PGIP of Lycopersicon esculentum Mill., another member of the family Solanaceae. The amino acid residues differing between S. brevidens PGIPs were assumed to determine the selectivity of interactions with particular polyglucuronases of phytopathogenic fungi.

In this paper we have explored the role of different kinase pathways of signal transduction in proliferation control of E1A + Ras transformants, using specific inhibitors of MAP-kinases ERK, JNK, p38 and PI3-kinase. According to our data, suppression of signalling cascades driven by RI3K only arrested proliferation of E1A + Ras cells, while suppression of either MAP-kinase did not lead to noticeable antiproliferative effect. We have shown that suppression of RI3K with LY294002 gave rise to accumulation of cyclin-dependent kinase inhibitor p27(KiP1) but not p21(Waf1). Accumulation of p27(KiP1) in LY294002-treated E1A + Ras cells was accompanied by a decrease in Cyclin E-Cdk2 and Cyclin A-Cdk2 activity, which caused diminution of Rb phosphorylation and strengthening of E2F-Rb binding. Binding of E2F with hypophosphorylated Rb resulted in inhibition of E2F activity and reduction of E2F-regulated gene transcription, these genes being necessary for S-phase entry and DNA synthesis. Thus, RI3K--Akt cascade plays the key role in maintenance of autonomous proliferation of cells transformed with E1A and cHa-ras oncogenes. Inhibition of PI3K leads to p27(Kip1) accumulation and cell cycle arrest, consequently.

In the rat placenta symplastotrophoblast the activity of glucose-6-phosphatase increased and that of Mg-adenosine-thriphosphatase and succinate dehydrogenase decreased under the influence of joint and separate action of acetone vapour and high air humidity. The activity of alkaline phosphatase is oppressed only at high humidity or in combination of high humidity and acetone.

The effects of ligands of active and inhibitory centers of homogeneous aldose reductase from cattle eye lens on glucose reduction were studied. Using spectrophotometric titration and equilibrium gel filtration, the interaction of the enzyme active center with substrates was investigated. It was shown that the reaction kinetics obeys a mechanism with a quasi-equilibrium non-ordered attachment of substrates and isomerization of enzyme complexes with nicotinamide dinucleotide phosphates in the course of the catalytic act. It was found that the NADPH in equilibrium NADP equilibrium in the enzyme active center is shifted to the right; however, NADP dissociation may occur only as a result of the aldehyde reduction. The mechanisms of regulation of the enzyme activity by NADP, ADP and alpha-glycerol phosphate were proposed. It was shown that the binding of catalin and morine to the enzyme results in the inhibition of the enzymatic reaction and in the isomerization blocking. It was found that the inhibitory site of the isomeric form of aldose reductase displays a lower affinity for morine.

Antitryptic activity of human blood serum was decreased after incubation with metalloproteinase from Legionella pneumophila. The enzymatic activity depends on the time of incubation as well as on the ratio between the enzyme content and blood serum total protein. Cross immunoelectrophoresis, involving monospecific rabbit antiserum towards the alpha 1-antitrypsin, demonstrated highly effective hydrolysis of alpha 1-antitrypsin by the metalloproteinase. As shown by polyacrylamide gel electrophoresis the metalloproteinase hydrolyzed acid stable inhibitor of serine proteinases into fragments with distinct loss of the inhibitor activity. Thermolysine hydrolyzed similarly the proteins studied but at a lower rate. The metalloproteinase from L. pneumophila appears to be mainly responsible for production of utilizable components from protein substrates involved in vital activity of the bacteria. It may not be excluded that the enzyme is able to impair some host protective mechanisms.

The results of the comparative study on the contents and activity of the enzymes of the system of oxidases of mixed function in the lungs and liver in rabbits, the specific features of phospholipid spectrum in the supernatant and microsomal fractions of the lungs and liver tissues are presented. The same parameters were investigated in experimental chronic inflammatory diseases of the lungs. The degree of the damage of the monoxygenase capacity of the lungs and the degree of the liver involvement in the process were established. The peculiarities of the inhibitory action of cimetidine on the detoxication capacity of the lungs and liver and on the disorder of the reproduction of phospholipids in these organs were revealed. The substrate specificity of cimetidine in inhibition of microsomal NADPN-cytochrome-C-reductase both in the liver and in the lungs, the probability of blocking the synthesis of phospholipids at the level of phosphatide acid and citidinephosphatecholine were noted. The results obtained should be taken into consideration during drug therapy.

An unusual ("zig-zag") temperature dependence of the rate of irreversible thermoinactivation of enzymes was observed for native and covalently modified alpha-chymotrypsin and trypsin. This dependence was characterized by alternation of plots with positive and negative apparent values of activation energy for the thermoinactivation process. A kinetic scheme which reflects the observed regularities in thermoinactivation for which the temperature-dependent conformational transition is an essential feature, is proposed. Fluorescence spectroscopy data suggest that the conformational transition predicted by the scheme is of the unfolding type. Substantial differences in thermostabilities of "high temperature" and "low temperature" conformations of enzymes may be due to different mechanisms of their irreversible thermoinactivation.

In experimental canine myocardial infarction induced by an hour coronary artery occlusion, followed by 5-hour reperfusion, quercetin solution, 10 mg/kg, was demonstrated to improve left ventricular myocardial contractility, to reduce the incidence of rhythm and conduction disturbances, to promote the limitation of an ischemic lesion area, to produce a protective effect on the ultrastructure of coronary artery, contributing to the maintenance of vessel integrity, improvement of coronary blood flow, and prevention of intravascular thrombus formation.

The fungi of the genus Candida isolated from patients with oral mucosa candidiasis and from candidiasis carriers have been studied for their antilysozyme activity. These fungi (Candida albicans, C. tropicalis, C. krusei, C. quilliermondii) are antilysozyme-active. A high antilysozyme activity of the fungi isolated from patients with oral mucosa candidiasis permits supposing that the presence of this trait may be one of the factors of microorganism pathogenicity. The effective antimycotic therapy (clotrimasole, sanguirhitrin) decreases the antilysozyme activity of fungi of the genus Candida.

The experiments on rats were undertaken to study the action of pepsidil to make up for the biosynthesis deficiency of digestive enzymes in the stomach and pancreas caused by antituberculous drugs (methazide + PAS, isoniazid + PAS, isoniazid + rifampicin + ethambutol). It was proved that pepsidil reduced an inhibiting action of antituberculous drugs on the activity of gastroenteric tract proteinases. The given condition should be taken into account when antituberculous treatment is conducted.

Preparations of human erythrocyte acetyl cholinesterase (HEACE) that differed in their specific activities (0.7 to 4.1 U/mg) and the final purification method were examined for protein and isoenzymic spectrum (by polyacrylamide gel disc electrophoresis), catalytic properties in the reaction of acetytriocholine hydrolysis, sensitivity to the specific organophosphorus inhibitor Gd-42, concentrations of active centres of HEACE, and activity of one catalytic centre. The preparations showed a stable spectrum of 11-12 proteins: HEACE had molecular heterogeneity and was electrophoretically separated into 3 fractions that differed in their aggregation level. Preparations with varying specific activity contained different HEACE quantities. Regardless of the purification degree, HEACE displayed similar activity of the catalytic centre and enzymic properties in reactions with the substrate and inhibitor.

Numerous examples of protein inactivation are considered. Several principal inactivation mechanisms are distinguished: aggregation, thiol-disulfide exchange, alteration in the protein primary structure, dissociation of cofactor molecules from the enzyme active site, dissociation of proteins with quaternary structure into subunits, and conformational changes. All the mechanisms interweave during protein inactivation. In many cases, however, conformational transformations in protein play an important role; they accompany and often trigger other mechanisms of inactivation which would be impossible without preliminary alterations in protein structure. The published data on reactivation of inactivated proteins are considered. Reactivation was a success when inactivation was caused by aggregation, chemical modification of SH-groups of S-S bonds, or resulted from "irreversible" conformational changes.

The binding of antibodies to enzymes-antigens markedly affects the activity and structure of the latters. The mechanisms of activation and inhibition of enzymes by antibodies and the use of immunochemical methods for the study of the active sites structure, conformational changes during interaction with substrates and other ligands and the quaternary structure of enzymes are discussed.

It was demonstrated that the relations between substrate and product concentrations for a reaction catalyzed by michaelian enzyme incorporated in a multienzyme system can be graphically represented by a diverging set of straight lines intersecting in one point, the flux velocity being treated as a parameter. A competitive inhibitor shifts the intersection point along the line of equilibrium state. The relations between the concentrations of more than two reagents are represented by a set of equivelocity surfaces. The relations between substrate and product concentrations for a kinetically cooperative reaction conforming to the graphical representation by the second--order curves were analyzed. The stability criterion was obtained for a multienzyme system with the first enzyme allosterically regulated by products of subsequent reactions.

The analysis of the interaction of ethanolamine-O-sulphate with 4-aminobutyrate transaminase revealed that the inhibitory effect is exerted upon the substrate subsite of the active site of the enzyme in aldimine form. The inhibition in irreversible. The inactivation rate versus pH-curve was shown to have a sigmoid character with inclination point at neutral pH. The study of inhibition kinetics by the Kitz and Wilson method revealed a complex inhibitory pattern compatible with a minimal two-step mechanism. Rate constant of inactivation was found to be equal to 0.22 min-1 and the value of the inhibitory constant--to 1.1-10(-2) M.

Determination of inhibitory constants and antienzymic activity of reversible retardants of different type of action by the generalized inhibitory constant K sigma is estimated, results being presented. To determine more precisely the values of inhibitory constants of the competitive (Ki), noncompetitive (K'i) inhibition and of the generalized K sigma it is suggested to conduct linearization of the experimental data in coordinates: 1/K sigma, s; 1/[S], where K sigma, s is a total inhibitory constant whose value depends on the substrate [S] concentration and to make calculations by the least-square method with the allowance for principal intervals of the Km values and the maximal rate of the enzymic reaction V. Comparative calculations are made by a computer using the BASIC/RT-60 language programme through the example of the acetyl choline acetyl hydrolase (EC 3.1.1.7) interaction with the quaternary phosphonium salts--reversible retardants of this enzyme.

The kinetics of inhibition of penicillinase of Bac. licheniformis 749/c by various alkyl-sulphates (with a long hydrocarbon chain from C8 to C16), n-toluolsulphoacid and alkylbenzol-sulphonate (R-C12--C16) was studied. The inhibition rate increased with elongation of the alkyl radical and rising of the inhibitor concentraiton. This means that the determining factor in inhibition process was hydrophobic interaction of the alkyl chains and not the electrostatic interaction with the enzyme. In-vitro experiments with penicillinase-producing strains of staphylococcus showed that non-bactericidal concentrations of the alkylsulphates and alkylben zolsulphonates increased the effect of benzylpenicillin, ampicillin and methicillin. The highest increase in the antibiotic activity, as well as in the enzyme inhibition was observed with respect to the compound with the hydrocarbon chain fron C12 to C16. The increase in the activity of methicillin against the staphylococcal strains resistant simultaneously to benzylpenicillin, ampicillin and methicillin was indicative of possible using of the above surface active substances as inhibitors of the realization process of various mechanisms of penicillin resistance.

The work deals with the effect of carbon sources, presence of protein synthesis inhibitors (cycloheximide and chloramphenicol) and dehydration regime on the enzyme activity of the dried yeast Saccharomyces cerevisiae. The yeast grown on molasses and dried by aeration demonstrated a notable increase of the NADH-dehydrogenase and succinate dehydrogenase activities as compared with the analogously treated yeast grown on ethanol. The latter showed a notable rise only in the activity of NADH-dehydrogenase during slow drying. Addition of protein synthesis inhibitors into the cultivation medium caused no decrease of activities of the above mentioned enzymes of the dried yeasts in any variant under study.

The effect of the number of active groups of new affinity supports--odigose and odifil (ethylsulfo-activated agarose) on the retention of the specific activity of muscle aldolase was investigated. The active center of the enzyme includes lysine able to react with activated supports. The aldolase completely retained the specific activity after immobilization on the abovementioned relatively high-substituted supports, on which other enzymes, e.g. phosphorylase B, NAD-kinase from pigeon heart, were partially or completely inactivated. The aldolase was inactivated when being immobilized on more substituted supports. The enzyme specific activity completely retained if the high substituted supports were preliminary incubated at 37 degrees to destroy some diazo-groups.

The effect of cationic polymers and surface active substances (SAS) on sensitivity of Klebsiella aerogenes 600, Escherichia coli 154 and Proteus vulgaris 7470 to kanamycin was studied. A decrease in the resistance of the above organisms to kanamycin on its use in combination with cationic polymers and SAS was observed. It was shown that such substances inhibited the activity of the enzymes inactivating kanamycin. Their effect was suggested to be due to changes in the lipid surrounding of the enzymes.

Principles of detecting organisms producing beta-lactamase inhibitors among soil actinomycetes were developed. For detecting such cultures it was recommended to use the Gauze agarized medium No. 1 supplemented with beta-lactam antibiotics. Benzylpenicillin proved to be the most efficient. Various liquid fermentation media for detecting the inhibitory activity of soil actinomycetes were compared. Two media were the most favourable i.e. the glucose-yeast medium No. 18/3 and the soybean-glucose medium with Na2SO4 and CoCl2 No. 20/3. The use of test cultures with relatively low resistance to benzylpenicillin was shown expedient in screening cultures producing beta-lactamase inhibitors. Test cultures with high resistance should be used in more detailed characterization of the selected cultures.

A generalized scheme of ordered multisubstrate enzymatic reactions under steady-state conditions is described. The equations for the reaction rate and product yield as well as the observed rate constant for the enzyme inactivation as functions of concentrations of substrates, reaction products, reversible and irreversible inhibitors were derived and analyzed. It was demonstrated that the kinetic behaviour of such systems can adequately be described in terms of interrelationship of intermediate forms of the enzyme.

Bivalent cations (Mn and Co) are found to activate ATPase activity of sarcolemma in the same degree, as Mg and Ca. The increase of the activity of Ca2+-dependent ATPase under the certain conditions of treatment of sarcolemma with KCl-Triton X-100 solution is not accompanied by the increase of ATPase activity in the presence of Mn and Co ions. ATPase activities in the presence of these ions correlate with the activity of Mg2+-dependent ATPase. Bivalent cations Mg, Mn and Co are competitive inhibitors for Ca2+-dependent ATPase. The apparent inhibition constants are determined to be 3,5-10(-5) M for Mg, 0,7-10(-3) M for Co and 1,5-10(-3) M for Mn. It is supposed that Ca2+-dependent ATPase has similar selectivity to bivalent cations as calcium influx that follows depolartization of plasma membrane.

Data available in literature on biological activity of the phosphorus analogues of amino acids are summarized. The most typical kinds of bioactivity of aminophosphonic, aminophosphinic and aminophosphonous acids are characterized. These compounds, both natural and synthetic, are shown to possess a wide spectrum of bioactivity and may be estimated as potential bioregulators.