[Anticancer activity of oxovanadium compounds].

Cytotoxic and antitumor activity of the biligand vanadyl derivative of L-malic acid (bis(L-malato)oxovanadium(IV) (VO(mal)2) was investigated in comparison with inorganic vanadium(IV) compound--vanadyl sulfate (VOSO4) and also with oxovanadium monocomplex with L-malic acid (VO(mal)) and vanadyl biscomplex with acetylacetonate. In this purpose the effect of vanadyl compounds on growth of normal human skin fibroblasts and tumor cells of different lines: mouse fibrosarcoma (L929), rat pheochromocytome (PC12), human liver carcinoma (HepG2), virus transformated mouse fibroblast (NIN 3T3), virus transformated cells of human kidney (293) were investigated. The results showed that VO(mal)2 was not toxic for normal human skin fibroblasts but considerably inhibited growth of cancer cells in culture. Cytotoxic antitumor effect of vanadium complexes was found to be dependent on incubation time and concentration and on type of cells and nature of ligands of the central group of the complex (VO2+). These studies provide evidence that VO(mal)2 may be considered as a potential antitumor agent due to its low toxicity in non-tumor cells and significant anticancer activity.

[Synthesis and hypoglycemic activity of bis(L-malato)oxovanadium(IV)].

In order to create new oral vanadyl organic complexes-based drugs for the treatment of diabetes mellitus biligand vanadyl derivative of L-malic acid (bis(L-malato)oxovanadium(IV) was prepared and its potential as a novel hypoglycemic agent was studied in the streptozotocin-diabetic rats. We show that the oral administration of bis(L-malato)oxovanadium(IV) with drink water significantly reduced glucose concentration in blood and urine, as well as the level of glycated proteins in the streptozotocin-diabetic rats.

[Study of proteomic profile of Danio rerio embryos using one-dimensional electrophoresis and mass spectrometry].

In the present study, a proteomic technology combining one-dimensional gel electrophoresis (1DE) with subsequent mass spectrometry (MALDI-TOF-PMF) has been successfully applied for revelation of changes in the protein profile of zebrafish (Danio rerio) 52 hpf embryos. Prior to 1DE separation of zebrafish embryonic proteins, the procedure for obtaining embryos homogenate was optimized by ultrasonic treatment. A total of 84 proteins, including 15 vitellogenins, were identified. It was shown that growing ofzebrafish embryos in the medium with doxorubicin (DOX) stimulated Caspase-3 induction and promoted the disappearance of cardiac troponins, both these findings being consistent with literature data on doxorubicin-induced cardiotoxicity. The 1DE-based proteomic mapping approach proposed herein enabled not only to identify proteins but also to register those changes in embryos' proteomic profile that were caused by doxorubicin.

[Zebrafish as a model organism for biomedical studies].

Zebrafish (Danio rerio) are now firmly established as a powerful research model for many areas of biology and medicine. Here, we review some achievements of zebrafish-based assays for modeling human diseases and for drug discovery and development. For drug discovery, zebrafish are especially valuable in the earlier stages of research as they provide a model organism to demonstrate a new treatment's efficacy and toxicity before more costly mammalian models are used. This review provides examples of compounds known to be toxic to humans that have been demonstrated to functional similarly in zebrafish. Major advantages of zebrafish embryons are that they are readily permeable to small molecules added to their incubation medium and the transparent chorion enables the easy observation of development. Assay of acute toxicity (LC50 estimation) in embryos can also include the screening for developmental disorders as an indicator of teratogenic effects. We used zebrafish for toxicity testing of new drugs on the base of phospholipid nanoparticles. The organization of the genome and the pathways controlling signal transduction appear to be highly conserved between zebrafish and humans that allow using zebrafish for modeling of human diseases some examples of which are illustrated in this paper.

[Modern approaches to pharmacotherapy of diabetes mellitus type II]. / Sovremennye podkhody k farmakoterapii sakharnogo diabeta II tipa.

The pharmacological approaches to the treatment of type 2 diabetes mellitus were reviewed. Special attention was paid to the new therapeutic agents that are able to decrease plasma glucose levels. The possible mechanisms of the hypoglycemic effects are discussed. Briefly, repaginide, nateglinede and alpha-glucosidase inhibitors prevent postprandial hypoclycemia while thiazaolidinediones improve the sensitivity to insulin and vanadium compounds act as an insulin action enhancer.

[Vanadium compounds--a new class of therapeutic agents for the treatment of diabetes mellitus]. / Vanadiisoderzhashchie soedineniia--novyi klass terapevticheskikh sredstv dlia lecheniia sakharnogo diabeta.

Vanadium compounds as insulin mimics with promising therapeutic properties are reviewed. The biological effects of both inorganic forms of vanadium and vanadyl organic complexes are decried for various animal models. These effects include hypoglycemic and insulin reserve actions, insulin sensitivity enhance, cholesterol lowering and other manifestations. The effectiveness of vanadium compounds in diabetes treatment is confirmed with clinical trials. The possible mechanisms of insulin-like effects of vanadium are discussed. The various nutritional supplements for patients with diabetes mellitus including vanadium-contained used in Russia and abroad are also considered.

[Comparative characteristics of vanadium-containing compounds, possessing insulin-like effects]. / Sravnitel'naia kharakteristika vanadii-soderzhashchikh soedinenii, obladaiushchikh insulino-podobnym deistviem.

The serum glucose concentration, HbAc1, fructoseamine, the activity of AST, ALT, amylase, glycogen content and glucokinase activity in rat hepatocytes were examined in streptozotocin-diabetic rats during long-term orthovanadate and vanadyl sulphate treatment. The improvement of this parameters were demonstrated after oral orthovanadate and vanadyl sulphate administration. However, insulin-mimetic properties of vanadyl sulphate were more marked in comparison with orthovanadate. LD-50 and cytotoxicity analysis in isolated hepatocytes showed that vanadyl sulphate was less toxic than orthovanadate.

[Changes in the level of fructose-2,6-biphosphate in peripheral blood lymphocytes in patients with diabetes mellitus]. / Izmenenie soderzhaniia fruktozo-2,6-bifosfata v limfotsitakh perifericheskoi krovi bol'nykh sakharnym diabetom.

Variation of fructose 2,6-bisphosphate (F-2,6-P2) content in the peripheral blood lymphocytes of patients with diabetes mellitus (type II) was investigated. The 2.5-fold increase of F-2,6-P2 level compared with to the control group was observed in untreated patients with early stage of the disease. In patients using of peroral antidiabetic drugs that corrected diabetes F-2,6-P2 level was approached to the normal one. At the same time F-2,6-P2 content decreases in patients with severe (noncorrected) diabetes mellitus. The possible mechanisms of regulation of lymphocyte F-2,6-P2 concentration in normal conditions and diabetes mellitus are discussed.

[Functional characteristics of fructose-2,6-bisphosphate system in the liver during experimental streptozotocin diabetes]. / Nekotorye osobennosti funktsionirovaniia sistemy fruktozo-2,6-bisfosfata v tkani pecheni pri éksperimental'nom streptozototsinovom diabete.

The activity of phosphofructokinase-2, fructose, 1,6-bisphosphatase, glucokinase, and also the level of fructose 2,6-bisphosphate and glycogen were examined in the liver of normal, and streptozotocin-diabetic rats. It was shown that the activity of phosphofructokinase-2 was decreased in the liver of diabetic rats. Besides that the activity determined at pH 6.6 (the "active" or unphosphorylated enzyme form) was 3-fold reduced whereas the "total" enzyme activity as measured at pH 8.5 was lowered 1,7-fold. The phosphofructokinase-2 activity assay at two pH values allows to estimate a degree of phosphorylation of bifunctional enzyme which is markedly enhanced in diabetes. The fall of the bifunctional enzyme k in case activity is accompanied by the lowered fructose 2.6-bisphosphate level, increased fructose 1,6-bisphosphatase activity that in turn favours the liver tissue glycolysis inhibition and gluconeogenesis enhanced in diabetes.

[Fructose-2,6-bisphosphate system and experimental states]. / Sistema fruktozo-2,6-bisfosfata i éksperimental'nye sostoianiia.

The molecular mechanisms of the inhibitory action of fructose- 2,6-bisphosphate (F-2,6-P2) on fructose-1,6-biphosphatase (FB-Pase-1), the key enzyme of gluconeogenesis, and the those of the activating action of F-2,6-P2 on phosphofructo-1-kinase (PFK-1), the key enzyme of glycolysis, NMR spectroscopy first provided direct evidence for the fact that F-2,6-P2 was involved in the regulation of the sedoheptulose cycle of a nonoxidative stage of the pentosephosphate pathway. Procedures were developed in measuring the levels of F-2,6-P2 in the cell of experimental animal tissues and human blood lymphocytes. Naturally different emergencies substantially affected the F-2,6-P2 system by triggering these or those mechanisms controlling the activity of enzymes of this system. Vanadium-containing compounds were demonstrated to have a positive action on carbohydrate metabolism in diabetic (streptozotocin-induced) rat hepatocytes.

[The allosteric nature of substrate inhibition of rabbit skeletal muscle fructose-1,6-diphosphatase]. / Allostericheskaia priroda substratnogo ingibirovaniia fruktozo-1,6-bisfosfatazy iz skeletnykh myshts krolika.

The mechanism of substrate inhibition of rabbit skeletal muscle fructose-1.6-bisphosphatase was examined. Analysis of substrate saturation curves obtained at different concentrations of Mg2+ revealed that the inhibiting effect of the substrate is manifested only within the complex with Mg2+, whereas the free form of the substrate causes no inhibition. Evidence for the allosteric nature of substrate inhibition was obtained by partial desensitization of the enzyme in the presence of salicylates. It was shown that fructose-1.6-bisphosphatase inhibition by the substrate obeys positively cooperative kinetics and is noncompetitive with respect to the substrate involved in the catalytic process. Studies on enzyme modification in the presence of DTNB and pyridoxal-5'-phosphate demonstrated that the inhibiting concentrations of the substrate are bound to the center which differs from the allosteric site for AMP. It is suggested that the antagonism of simultaneous action of AMP and high substrate concentrations may be due to the competition of the phosphate groups of these ligands for binding to the common lysine residue located in the overlapping region of two allosteric sites.

[Determination of the concentration of fructose-2,6-bisphosphate using phosphofructokinase]. / Opredelenie kontsentratsii fruktozo-2,6-bisfosfata s ispol'zovaniem fosfofruktokinazy

Measurement of fructose-2,6-bisphosphate (F-2,6-P2) level in biologic material is based on its ability to eliminate allosteric inhibition of phosphofructokinase (PFK) with high ATP concentrations. Effects of the buffer types and length of storage of commercial PFK from rabbit muscle, manufactured by Boehringer, on the enzyme ability to inhibit ATP and activate F-2,6-P2 were examined. The enzyme showed the highest sensitivity to ATP inhibition in tris buffer and the least one in morpholine propane sulfonic acid. If PFK is stored for a year its sensitivity to ATP inhibition and F-2,6-P2 activation essentially reduces and it becomes unfit for measurement of F-2,6-P2 concentration in biologic material.

[The mechanism of rat liver fructose-1,6-bisphosphate inhibition by fructose-2,6-bisphosphate]. / O mekhanizme ingibirovaniia fruktozo-1,6-bisfosfatazy iz pecheni krysy fruktozo-2,6-bisfosfatom.

It was found that a decrease in the activating cation (Mg2+) concentration below [A]0.5 causes the disappearance of cooperativity of the fructose 1.6-bisphosphatase substrate binding sites induced by high fructose 2.6-bisphosphate concentrations without any significant alteration in the extent of the enzyme inhibition. Under these conditions, a competitive type of inhibition (with respect to the substrate) is transformed into a non-competitive type with an increase in the fructose 2.6-bisphosphate concentration. The data obtained confirm the viewpoint that fructose 2.6-bisphosphate binds to the enzyme at two distinct sites, a catalytic and an allosteric ones, differing in their affinity for the inhibitor. It is supposed that the interaction between the allosteric fructose 2.6-bisphosphate binding site and the activator site occupied by Mg2+ is necessary for the cooperative response of the enzyme to the substrate.

[Relation between Mg2+ activation and AMP inhibition of fructose-1,6-diphosphatase from rabbit skeletal muscles]. / Vzaimosviaz' mezhdu aktivatsiei Mg2+ i ingibirovaniem AMP fruktozo-1,6-bifosfatazy iz skeletnykh myshts krolika.

It was shown that AMP, an allosteric inhibitor of fructose-1.6-bisphosphatase, decreases the apparent affinity of the enzyme for the activating cation, Mg2+, which is accompanied by a decrease of the kinetic cooperativity between the Mg2+-binding sites. In its turn, the Mg2+ increase diminishes the enzyme sensitivity to the inhibiting effect of AMP and decreases the cooperativity of the inhibitor binding. The heterotropic interactions between the allosteric inhibitor and activator binding centers are consistent with the predictions of the Monod-Wyman-Changeux model which involves two conformational states of the enzyme (of which one is catalytically inactive) differing in their affinity for the ligands. An increase in pH from 7.4 to 9.0 increases the enzyme affinity for Mg2+ and causes an equilibrium shift towards the catalytically active state of the enzyme.

[Various regulatory properties of fructose-1,6-diphosphatase of the rat liver under normal conditions and in experimental autoimmune cardiomyopathy]. / Nekotorye reguliatornye svoistva fruktozo-1,6-bifosfatazy pecheni krysy v norme i pri éksperimental'noi autoimmunnoi kardiomiopatii.

An increase in fructose-1,6-biphosphatase activity in liver tissue of rats with experimental autoimmune cardiomyopathy was observed. At certain concentrations of EDTA and Mg2+ the AMP-inhibition curves exhibited an intermediate plateau, which appear under different experimental, normal and pathological conditions. The occurrence of complex kinetic curves could be attributed to simultaneous existence of several enzymatic forms differing in the "structural" sites with tightly bound metals, which defines the differences in the kinetic cooperativity and sensitivity to AMP inhibition.

[Desensitization with acetylsalicylate and salicylate of fructose-1,6-diphosphatase from the rabbit liver and skeletal muscles to allosteric inhibition of AMP]. / Desensibilizatsiia atsetilsalitsilatom i salitsilatom fruktozo-1,6-bifosfatazy iz pecheni i skeletnykh myshts krolika k allostericheskomu ingibirovaniiu AMF.

Acetylsalicylate and salicylate partially desensitized fructose 1,6-biphosphatase from rabbit liver and skeletal muscle to allosteric AMP inhibition. The desensitization was accompanied by a decrease in cooperativity between allosteric sites especially distinct for the liver isoenzyme. The effect of salicylate on both isoenzymes was more pronounced than that of acetylsalicylate. No essential differences in the effect of the compounds studied on these isoenzymes were found.

[Complex type of kinetics of fructose-1,6-diphosphate from the rat and rabbit liver]. / Slozhnyi kharakter kinetiki fruktozo-1,6-bifosfata iz pecheni krysy i krolika.

Studies on rat and rabbit liver fructose 1.6-bisphosphatase inhibition by AMP showed that with an increase in EDTA concentration the hyperbolic AMP inhibition curve is transformed into a sigmoidal one. At intermediate EDTA concentrations, the kinetic curves have a plateau. The appearance of the intermediate plateau may be due to the superposition of kinetic curves corresponding to two enzyme forms simultaneously present in the assay mixture. One of these forms deprived of endogenous Me2+ (presumably Zn2+) is inhibited by AMP in a cooperative manner, while the other one retains Me2+ which prevents the cooperative response of the enzyme to AMP.

[Purification of commercial preparations of NADP from AMP]. / Ochistka kommercheskikh preparatov NADF ot AMF.

A method in described for elimination of AMP from commercially available preparations of NADP. The method enables to obtain desalted NADP preparations of 98-99.5% purity with a yield of 70-80% using only one chromatographic step. The procedure included anion exchange chromatography on Dowex 1, HCOO- -form. This chromatographic step completely separated AMP from NADP in a gradient of HCOOH concentrations from 0 to 2 N, and NADP was eluted by 1.5 N HCOOH containing 0.15 N HCOOK; during subsequent precipitation and washing of NADP by means of acetone HCOOK remained in a solution. Addition of HCOOK was required for a decrease in NADP elution volume which was important for the subsequent precipitation with acetone.