[Photobiochemistry of folates: a photochemical reduction of folic acid].

Exposure of deaerated folic acid solutions containing an electron donor to UV radiation (310-390 nm, I = 0.4 W m(-2)) induced formation of dihydrofolic acid (DHFA), a photoexcitation which gave tetrahydrofolic acid (THFA). Only DHFA was formed in the presence of EDTA (E'o = +0.40 V), while the presence of stronger reductants-NADH (E'o = -0.32 V) and boron hydride (E'o = -0.48 V)-induced photoreduction to THFA. It was demonstrated that UV radiation had no effect on the THFA formylation, giving the coenzyme 5,10-methenyltetrahydrofolic acid and its transformation into another coenzyme, 5-formyltetrahydrofolic acid.

[Study of the photochemical properties of biopterin and its reduced forms].

UV-irradiation (270-390 nm, 20 min, I = 3.2 W m(-2)) of deaerated biopterin solution containing electron donor (Na2-EDTA) led to the formation of 7,8-dihydrobiopterin (H2BPT), which, when excited, underwent reduction to form 5,6,7,8-tetrahydrobiopterin (H4BPT). Protonated molecules of H4BPT were resistant to oxidation by O2 both in the "dark" incubated and UV-irradiated solutions at pH below 3.0. The rate of H4BPT oxidation dramatically increased at pH above 3.0, and, then, up to pH 10.0, it did not change, showing no dependence on UV-irradiation. At the initial stage (5 min) of H4BPT oxidation in neutral solution, UV-irradiation stimulated the accumulation of quinonoid 6,7-dihydrobiopterin (q-H2BPT) in addition to H2BPT. UV-irradiation of H2BPT induced its oxidation to biopterin and unidentified products.

[A study of the nitrate and nitrite discharge from the mutant cells of Neurospora crassa lacking nitrate and nitrite reductase activities].

The Neurospora crassa mutants nit-2 (lacking both nitrite and nitrate reductases) and nit-6 (lacking nitrite reductase) grown in the medium with ammonium chloride as a sole source of nitrogen discharged nitrate and nitrite ions into culture medium. For nit-2, the content of nitrate exceeded that of nitrite in both the homogenate of fungal cells and growth medium; moreover, this difference was more pronounced in the culture medium. Unlike nit-2, the content of nitrite in the cultivation medium of the nit-6 mutant irradiated with visible light for 30 min during the lag phase of carotenogenesis photoinduction displayed a trend of increase as compared with the dark control. Further (to 240 min) irradiation of cells, i.e., irradiation during biosynthesis of carotenoid pigments, leveled this difference.

[Photoreceptor apparatus of a fungus Neurospora crassa].

Light governs living functions of ascomycete fungus Neurospora crassa by controlling expression of the genes responsible for differentiation of reproductive structures, synthesis of secondary metabolites and the circadian oscillator activity. Illumination also influences electrogenic processes in cell membrane and the activity and molecular organization of some enzymes. The major but, probably, not the sole photoreceptor pigment in Neurospora cells is WCC, a heterodimeric complex formed by PAS-domain-containing polypeptides WC-1 and WC-2, which are the products of genes white collar-1 and white collar-2. Mutation of any of these genes arrests most of the organism's responses to light. The photoreceptor belongs to a recently discovered vast group of non-homologous light-sensitive proteins, whose molecules bind flavin coenzymes as the photosensor chromophores. The photosignal transduction is started by excitation and photochemical activity of excited FAD molecule non-covalently bound by LOV-domain (a specialized version of PAS-domain) in WC-1 protein. The presence in both WC-1- and WC-2-proteins of "zinc fingers" (the GATA recognizing sequences) suggested that these motives might act as transcription factors. The critical analysis of photoinduction mechanism has shown, however, that promoters of light-sensitive genes do not contain a common cis-acting element, what makes to look for alternative mechanisms underlying photoregulated gene activity.

[Tolerance of 5,10-methenyltetrahydrofolate to ultraviolet radiation].

Aeration of aqueous solutions of 5,10-methenyltetrahydrofolic acid (MTHF) during exposure to ultraviolet irradiation (lambda 300-390 nm, 240 W/m2, 30 min) slowed down photolysis in comparison with deaerated solutions. The rate of photolysis in the presence of oxygen depended on buffer composition. It did not exceed 6% of the starting amount of MTHF. Photolysis of MTHF included opening of the imidazoline ring, dehydrogenation of the tetrahydropterin portion, and elimination of the p-aminobenzoylglutamate moiety. 6,7-Dimethyltetrahydropterin was used as a model compound to show that protonation of the reduced pterin heterocycle increased its tolerance to oxidation, and UV irradiation did not accelerate this process. The stabilizing effect of protonation of the pterin portion and the presence of the positively charged imidazoline moiety are assumed to hamper MTHF oxidation and photolysis. It is assumed that these factors favored the choice of MTHF molecules as photosensors in light-sensitive proteins in the course of evolution.

[Effect of 5-azacytidine on the light-sensitive formation of sexual and asexual reproductive structures in wc-1 and wc-2 mutants of Neurospora crassa]. / Vliianie 5-azatsitidina na svetochuvstvitel'noe obrazovanie polovykh i bespolykh reproduktivnykh struktur u mutantov wc-1 i wc-2 Neurospora crassa.

Under the conditions of nitrogen starvation, illumination by blue light of wc-1 and wc-2 mutants of the ascomycete Neurospora crassa failed to stimulate the formation of protoperithecia and inhibit conidiation (contrary to what was observed in the mycelium of the wild-type fungus). The data obtained indicate that wc-1 and wc-2 genes of N. crassa are involved in light-dependent formation of protoperithecia and conidia. The effects of 5-azacytidine (an inhibitor of DNA methylation) under the same experimental conditions suggest that the balance between the formation of sexual and asexual reproductive structures, maintained in N. crassa, depends on genome methylation processes sensitive to the action of light, which is mediated by the photoreceptor complex of WC proteins.

[Features of lipoxygenase from cells of Mortierella species mushrooms]. / Osobennosti lipoksigenzay iz kletok gribov roda Mortierella.

The specific activity of lipoxygenase from several strains of the zygomycete Mortierella varied from 1.02 to 2.02 microMol diene per min per mg protein). The enzyme equally used linoleic or arachidonic acid as a substrate. An increase in lipoxygenase activity was observed after adding corn oil to the culture medium. Tests with inhibitors having different chemical structures revealed that the lipoxygenase activity from Mortierella cells was inhibited only by esculetin, ethanol and nordihydroguaiaretic acid (NDGA). NDGA inhibited the enzyme in vitro (IC50 = 142 microM), but its addition in the exponential phase of growth activated the enzyme.

[Effect of inhibitors of enzymatic DNA methylation on the formation of reproductive structures and carotenoid production in Neurospora crassa]. / Vliianie ingibitorov fermentativnogo metilirovaniia DNK na obrazovanie reproduktivnykh struktur i karotinogenez u Neurospora crassa.

The effect of inhibitors of DNA methylation on light-sensitive developmental stages of the filamentous fungus Neurospora crassa was studied. Under conditions of nitrogen starvation, when blue light induced protoperithecia development and inhibited conidia formation, 5-azacytidine (3-300 microM) inhibited protoperithecia formation and stimulated conidia formation (a 700-fold increase after light induction). After treatment of the mycelium with 5-azacytidine, the protoperithecia formation was accompanied by inversely proportional changes in the formation of conidia, both in the dark and after illumination. In the mycelium cultivated on the Vogel's medium, 5-azacytidine (up to 30 microM) and methotrexate (up to 3 microM) stimulated the light-induced carotenoid synthesis by 30%, whereas higher concentrations of these agents were toxic to carotenoid synthesis and growth.

[ATP and polyphosphate-dependent bacterial NAD+-kinases]. / ATF- i polifosfat-zavisimye bakterial'nye NAD+-kinazy.

Measurable levels of activity of NAD+ kinases of actinomycetes Micrococcus luteus and Corynebacterium ammoniagenes were observed after substituting inorganic tripolyphosphate for ATP, whereas the enzyme from the eubacterium Escherichia coli was not active with this substrate. Gradient PAGE found two molecular isoforms of NAD+ kinase in C. ammoniagenes and E. coli; four forms were found in M. luteus. All isoforms of this enzyme found in C. ammoniagenes and M. luteus displayed a NADP-synthesizing activity in the presence of either ATP or tripolyphosphate. Because of its capability of utilizing inorganic tripolyphosphate, M. luteus is the most promising NADP producer organism.

[Cyclic nucleotides and membrane electrogenesis in cell differentiation in Neurospora crassa]. / Tsiklicheskie nukleotidy i membrannyi élektrogenez v iavleniiakh kletochnoi differentsirovki u Neurospora crassa.

Significance of membrane electrogenic system and cyclic nucleotides for developmental regulation in lower eukaryotes was studied in Neurospora crassa. Despite of their morphological uniformity, hyphal cells were differentiated by their electrophysiological parameters--membrane potential and input resistance. The cells maintained regulated electric communication through septal pores. Such electric communication allowed the apical "cell" to compensate the energy requirements for membrane transport by means of the electrogenic mechanism of proximal zone. The photoinduced gene expression related to differentiation of reproductive structures has been shown to be mediated via cAMP. The changes of electric parameters observed in illuminated hyphal cells were controlled by the same photoregulation mechanism but were not involved in direct control of photoinduced gene expression.

[NAD+-kinase from Neurospora crassa: isolation and properties]. / NAD+-kinaza Neurospora crassa: vydelenie i svoistva.

The NAD+ kinase (EC 2.7.1.23) of the filamentous fungus N. crassa is localized in cytosol. The activity in the dialyzed cell free extract has a pH optimum 8.3; it utilizes only ATP but not inorganic polyphosphates as a phosphoryl donor. A method for 200-fold purification of NAD+ kinase with a 20% yield has been developed. The procedure includes 105000 g centrifugation, fractionation with (NH4)2SO4, isoelectrofocusing in a Ultrodex layer and preparative electrophoresis in polyacrylamide gel. The molecular heterogeneity of NAD+ kinase was demonstrated by polyacrylamide gradient electrophoresis and by gel filtration through Sephadex G-200. The molecular weights of four individual forms of the enzyme are: 330000-338000, 305000-306000, 215000-229000 and 203000 Da. The Km values for the reaction catalyzed by purified NAD+ kinase for NAD+ and ATP are 3.0 X 10(-4) M and 0.9 X 10(-3) M, respectively.

[Effect of the cyclic nucleotide level on the degree of carotenoid pigment formation in the mycelial cells of Neurospora crassa]. / Vliianie urovnia tsiklicheskikh nukleotidov na intensivnost' obrazovaniia karotinoidnykh pigmentov v kletkakh mitseliia Neurospora crassa.

The relation between the content of cyclic nucleotides and the rate of formation of carotenoid pigments in the Neurospora crassa mycelium cells was investigated. Light derepression of the carotenoid synthesis during the photoinduction lag-period induced a transient decrease of the cAMP content. The intracellular cAMP content was in negative correlation with the constitutive level of carotenoid pigments. The cGMP content remained unchanged during the photoinduction lag-period and showed no correlation with the constitutive level of carotenoids in N. crassa cells.

[Influence of light on membrane potential of Neurospora crassa cells]. / Vliianie sveta na membrannyi potentsial kletok Neurospora crassa.

In experiments with left atrial and right ventricular tissues fast sodium current (INa) was decreased by substituting sucrose for sodium in Tyrode's solution and with antiarrhythmic drugs--INa blockers (lidocaine, diphenylhydantoine and ethmozine). It was shown that INa decrease results in the growth of refractoriness (R) of the cardiac tissues. The hypothesis is advanced that the R growth caused by INa decrease is one of the mechanisms of selective sensitiveness of ischemic heart tissues to antiarrhythmic drugs--INa blockers.

[Specific activity and molecular forms of NAD-kinase in Neurospora crassa ontogeny]. / Udel'naia aktivnost' i molekuliarnye formy NAD-kinazy v ontogeneze Neurospora crassa.

The specific activity and molecular forms of NAD-kinase during ontogenesis of Neurospora crassa were investigated. The specific activity of the enzyme increased drastically at critical stages of fungal development, i.e. during conidia germination and during transition from the logarithmic to stationary growth stage, reaching 85 nmole NADP/hr/mg protein. By polyacrylamide gel electrophoresis four forms of NAD-kinase were revealed that had the following molecular masses: I-338,000, II-306,000, III-229,000, and IV-203,000. The vegetative mycelium contained predominantly form III, and conidia showed a high content of high-molecular-weight forms I and II.

[Photoinduction of carotenoid synthesis by Neurospora crassa on different structural and functional states of nitrate reductase]. / Fotoinduktsiia sinteza karotinoidov u Neurospora crassa pri raznom strukturnom i funktsinal'nom sostoianii nitratreduktazy.

In order to clarify the role of nitrate reductase as a potential photoreceptor, the ability of N. crassa mycelial cells for light-dependent accumulation of carotenoid pigments has been studied. The repression of the nitrate reductase synthesis by ammonium ions has been found not to influence the rate of the photoinduced carotenogenesis. The mutant experiments have shown that damage to the structural integrity of the nitrate reductase molecule, e, g., disintegrated synthesis of the protein fragment of the enzyme molecule (mutants nit-2 and nit-3) or the molybdenum coenzyme (mutant nit-1) does not affect the activity of the photoregulatory system of N. crassa. Thus, nitrate reductase is not a necessary component of the photoregulatory mechanism of N. crassa responsible for the synthesis of carotenoids.