Biochemistry (Moscow) (v.74, #3)

Ganglioside GM3 and its biological functions by N. V. Prokazova; N. N. Samovilova; E. V. Gracheva; N. K. Golovanova (235-249).
Metabolism, topology, and possible mechanisms for regulation of the ganglioside GM3 content in the cell are reviewed. Under consideration are biological functions of GM3, such as involvement in cell differentiation, proliferation, oncogenesis, and apoptosis.
Keywords: ganglioside GM3; GM3 synthase; metabolism; cell differentiation; proliferation; oncogenesis; apoptosis

Posttranslational chemistry of proteins of the GFP family by A. A. Pakhomov; V. I. Martynov (250-259).
This review focuses on the current knowledge about posttranslational chemistry underlying the diverse optical properties of GFP-like proteins.
Keywords: posttranslational modifications; fluorescence; chromophore; GFP; DsRed; KFP; Kaede

Use of buckwheat seed protease inhibitor gene for improvement of tobacco and potato plant resistance to biotic stress by N. V. Khadeeva; E. Z. Kochieva; M. Yu. Tcherednitchenko; E. Yu. Yakovleva; K. V. Sydoruk; V. G. Bogush; Y. E. Dunaevsky; M. A. Belozersky (260-267).
The possibility to use agrobacterial transformation of leaf discs to produce resistance to bacterial infections in tobacco and potato plants by introduction of a single gene encoding the serine proteinase inhibitor BWI-1a (ISP) from buckwheat seeds is shown. All studied PCR-positive transgenic plants exhibited antibacterial activity in biotests. It was shown that the presence of just a single gene of serine proteinase inhibitor provides sufficient protection at least against two bacterial phytopathogens, Pseudomonas syringae pv. tomato and Clavibacter michiganensis sbsp. michiganensis. The biotest including tobacco plant infection by the white wings butterfly in the green house has also demonstrated the existence of protective effect in transgenic tobacco plants. Significant genotypic variations in the protection efficiency were found between members of different genera of the same family (potato and tobacco) as well as between different lines of the same species. Northern blot analysis of four transgenic potato lines and three tobacco lines transformed by a vector plasmid containing the ISP gene of serine proteinases BWI-1a from buckwheat seeds has shown the presence of the expected size mRNA transcript.
Keywords: agrobacterial transformation; serine proteinase inhibitor gene; tobacco; potato; protection against phytopathogens

In this work we comparatively analyzed interleukin-2 (IL-2) and interferon γ production (IFN-γ) and also CD69 and CD25 expression by activated T-cells depending on extracellular calcium concentration ([Ca2+]e), which was varied with EGTA. The expression of CD69 molecules on the surface of T-cells depended only on the presence of phorbol myristate acetate, occurred at [Ca2+]e higher than 0.2 mM, and did not require the presence of ionomycin. The increase in [Ca2+]e by itself cannot induce expression of CD25 and CD69 molecules by activated cells. The values of [Ca2+]e, at which maximal fractions of CD3+CD69+(IL-2)+, CD3+CD69+(IFN-γ)+, and CD3+CD25+ activated T-cells were reached, never coincided with mean values of [Ca2+]e for healthy donors and were different from each other. So, there is different [Ca2+]e dependence for initial stages of activated T-cells differentiation. The relation between T-cells activation parameters and their differentiation is discussed.
Keywords: human peripheral blood; ionomycin; PMA; cell activation; EGTA; IL-2; IFN-γ

In the present study, we demonstrate for the first time that the potent neurotoxin vipoxin from the venom of Vipera ammodytes meridionalis exhibits hemolytic and anticoagulant properties. By investigating the effects of phospholipids and calcium ions on hemolysis, we established that the phospholipase A2 (PLA2) enzyme activity is responsible for the hemolytic properties. This was confirmed by chemical modification of the PLA2 active-site histidine residue with p-bromophenacylbromide. Applying different clotting assays, we show that the PLA2 is a weakly anticoagulant enzyme, which affects intrinsic tenase complex by the hydrolysis of procoagulant phospholipids, rather than by nonenzymatic mechanisms (binding to specific coagulation factors). The whole complex—vipoxin—does not affect the coagulation system.
Keywords: vipoxin; phospholipase A2 ; hemolysis; anticoagulant activity

A secreted caspase-3-substrate-cleaving activity at low pH belongs to cathepsin B: a study on primary brain cell cultures by M. V. Onufriev; A. A. Yakovlev; A. A. Lyzhin; M. Yu. Stepanichev; L. G. Khaspekov; N. V. Gulyaeva (281-287).
The cysteine proteases caspase-3 and cathepsins are involved in both neuronal plasticity and neuropathology. Using primary neuroglial and glial cerebellar cultures, the pH dependence of cleavage of a synthetic caspase-3 substrate, Ac-DEVD-AMC, was studied. At acidic pH, cathepsin B cleaved Ac-DEVD, this activity being significantly higher than that of caspase-3 at pH 7.4. This activity is blocked by peptide inhibitors of both caspase-3 and cathepsin B. Substitution of culture medium for balanced salt solution stimulated cathepsin B secretion in both types of cultures. Ischemia (oxygen-glucose deprivation) significantly decreased secretion of cathepsin B activities into the culture medium.
Keywords: cathepsin B; caspase-3; ischemia/reoxygenation; neurons; glia

Metabolic control analysis of L-cysteine producing strain TS1138 of Pseudomonas sp. by Lihua Huai; Ning Chen; Wenbo Yang; Gang Bai (288-292).
A kinetic model describing the biosynthesis of L-cysteine by Pseudomonas sp. TS1138 has been developed. The two enzymes catalyzing this pathway, L-cysteine synthetase (CS) and L-cysteine desulfhydrase (CD), follow Michaelis-Menten kinetics with noncompetitive inhibition of CS by L-cysteine. From measurements of intermediates and end products that were made during L-cysteine enzymatic synthesis, metabolic control analysis of the pathway was carried out using the kinetic model. The elasticity coefficients and the flux control coefficients were calculated, and the analysis revealed a shift in the flux control from CS to CD during the reaction. The findings further implicate potential targets and strategies for increasing L-cysteine production; for example, the strain TS1138 could be manipulated by site-directed mutagenesis to reduce CD activity.
Keywords: Pseudomonas sp. TS1138; L-cysteine; kinetic model; metabolic control analysis; enzymatic synthesis

New function of the amino group of thiamine diphosphate in thiamine catalysis by L. E. Meshalkina; G. A. Kochetov; G. Hubner; K. Tittmann; R. Golbik (293-300).
In this work, we investigated the rate of formation of the central intermediate of the transketolase reaction with thiamine diphosphate (ThDP) or 4′-methylamino-ThDP as cofactors and its stability using stopped-flow spectroscopy and circular dichroism (CD) spectroscopy. The intermediates of the transketolase reaction were analyzed by NMR spectroscopy. The kinetic stability of the intermediate was shown to be dependent on the state of the amino group of the coenzyme. The rates of the intermediate formation were the same in the case of the native and methylated ThDP, but the rates of the protonation or oxidation of the complex in the ferricyanide reaction were significantly higher in the complex with methylated ThDP. A new negative band was detected in the CD spectrum of the complex transketolase—4′-methylamino-ThDP corresponding to the protonated dihydroxyethyl-4′-methylamino-ThDP released from the active sites of the enzyme. These data suggest that transketolase in the complex with the NH2-methylated ThDP exhibits dihydroxyethyl-4′-methylamino-ThDP-synthase activity. Thus, the 4′-amino group of the coenzyme provides kinetic stability of the central intermediate of the transketolase reaction, dihydroxyethyl-ThDP.
Keywords: transketolase; thiamine diphosphate; dihydroxyethyl-thiamine diphosphate; 4′-methylamino-thiamine diphosphate; stopped-flow spectroscopy; CD; NMR

Dihydroquercetin (taxifolin) and other flavonoids as inhibitors of free radical formation at key stages of apoptosis by Yu. A. Vladimirov; E. V. Proskurnina; E. M. Demin; N. S. Matveeva; O. B. Lubitskiy; A. A. Novikov; D. Yu. Izmailov; A. N. Osipov; V. P. Tikhonov; V. E. Kagan (301-307).
Formation of free radicals in mitochondria plays a key role in the development of apoptosis, which includes formation of superoxide by the respiratory chain, formation of radicals by cytochrome c-cardiolipin complex in the presence of hydrogen peroxide or lipids, and chain lipid peroxidation resulting in cytochrome c release from mitochondria and initiation of the apoptotic cascade. In this work the effect of taxifolin (dihydroquercetin) and some other antioxidants on these three radical-producing reactions was studied. Peroxidase activity of the complex of cytochrome c with dioleyl cardiolipin estimated by chemiluminescence with luminol decreased by 50% with quercetin, taxifolin, rutin, Trolox, and ionol at concentrations 0.7, 0.7, 0.8, 3, and 10 μM, respectively. The lipid radical production detected by coumarin C-525-activated chemiluminescence decreased under the action of rutin and taxifolin in a dose-dependent manner, so that a 50% inhibition of chemiluminescence was observed at the antioxidant concentrations of 3.7 and 10 μM, respectively. Thus, these two radical-producing reactions responsible for apoptosis onset are inhibited by antioxidants at rather low concentrations. Experiments performed on liver slices and mash showed that taxifolin, quercetin, naringenin, and Trolox have low inhibitory effect on the lucigenin-dependent chemiluminescence in the tissue only at concentrations higher than 100 μM.
Keywords: dihydroquercetin; antioxidants; apoptosis; free-radical reactions; cytochrome c ; cardiolipin

Biochemical properties of Bacillus intermedius subtilisin-like proteinase secreted by a Bacillus subtilis recombinant strain in its stationary phase of growth by E. O. Mikhailova; A. M. Mardanova; N. P. Balaban; G. N. Rudenskaya; O. N. Ilyinskaya; M. R. Sharipova (308-315).
Biochemical properties of Bacillus intermedius subtilisin-like proteinase (AprBi) secreted by a B. subtilis recombinant strain in the early and late stationary phases of growth have been determined. Protein structure was analyzed and its stability estimated. It was noted that the enzyme corresponding to different phases of bacterial growth retains activity in the presence of reducing and oxidizing agents (C2H5OH and H2O2). Different effects of bivalent metal ions on activity of two proteinase fractions were found. Calcium ions more efficiently activate proteinase secreted in the late stationary phase. Unlike the first enzyme fraction, the second forms catalytically active dimers.
Keywords: subtilisin-like proteinase; AprBi; dimers

We have studied the extent of coincidence of the pathway of unfolding of protein globules upon experimental modeling of protein unfolding caused by external actions and denaturants. To this end, we compared experimental Φ-values reported in the literature and Φ-values obtained by us upon modeling of unfolding of immunoglobulin-binding domains of proteins L and G caused by external actions at a constant rate. A comparison of the results of calculation with the experimental data shows that the folding pathways for protein L coincide, while those for protein G do not coincide despite structural similarity of these proteins.
Keywords: protein structure; molecular dynamics; folding pathway; mechanical unfolding; atom—atomic contacts; folding nucleus; transition state

A lectin was isolated from the roots of Sesbania aculeata. This is a glucose specific lectin having 39 kDa subunit molecular weight. The expression of this lectin was found to be developmentally regulated and observed to be the highest in the second week. The lectin was purified by affinity chromatography using Sephadex G-50 and found to have 28% homology with Arabidopsis thaliana lectin-like protein (accession No. CAA62665). The lectin binds with lipopolysaccharide isolated from different rhizobial strains indicating the plants interaction with multiple rhizobial species.
Keywords: Sesbania ; root lectin; lipopolysaccharides

Polyclonal antibodies to LIM proteins CRP2 and CRIP2 reveal their subcellular localizations in olfactory precursor cells by Xue Gao; Jing-Yu Sun; Zeng-Yu Cao; Ying Lin; Ding-Jun Zha; Feng Wang; Tao Xue; Li Qiao; Lian-Jun Lu; Jian-Hua Qiu (336-341).
In this study, we describe the presence of CRP2 (cysteine- and glycine-rich protein 2) and CRIP2 (cysteine-rich intestinal protein 2), which are members of group 2 LIM proteins, in rat olfactory precursor cells by reverse transcription polymerase chain reaction. We have developed polyclonal antibodies against CRP2 and CRIP2 individually. Specificity of the antibodies was demonstrated by Western blot analysis, using CRP2 and CRIP2 transfected cells. No cross-reactivity was observed between the antibodies. Furthermore, we used the antibodies to determine the expression and localization of CRP2 and CRIP2 in olfactory precursor cells by Western blot analysis and immunofluorescence staining. Our results demonstrated that in undifferentiated olfactory precursor cells CRP2 was distributed both in the nucleus and the cytoplasm, whereas CRIP2 was predominantly localized in the cytoplasm. While the olfactory precursor cells differentiated into end cells, only the expression of CRIP2 would be detected. The function of these LIM proteins in olfactory precursor cells warrants further study.
Keywords: CRP2; CRIP2; LIM protein; polyclonal antibody; olfactory precursor cells

Regulation of catalytic activity of acid phosphatase by lipids in a reverse micellar system by E. V. Kudryashova; V. L. Bronza; A. V. Levashov (342-349).
The influence of biomembrane lipids on the catalytic activity of a peripheral membrane enzyme, acid phosphatase (AP), was studied in a reverse micellar system. It was found that the interaction of AP with lipids led to a number of kinetic effects depending on lipid nature on enzyme function. The observed effects might be caused by the formation of lipoprotein complexes as well as by the influence of lipids on structure and properties of the micellar matrix. The results are important for clear understanding of molecular mechanisms of regulation of the catalytic activity of the membrane-associated enzyme in vivo. These data can also be used as a physicochemical basis for application of AP in medical fields as a diagnostic tool for diseases caused by changes in lipid metabolism, e.g. urinary, orthopedic, and allergic diseases.
Keywords: acid phosphatase; reverse micelles; lipids; catalytic activity; secondary structure