[Determination of oligonucleotide molecular masses by MS-MALDI].

MALDI mass spectrometry (MS) of 14- to 42-mer homogeneous oligonucleotides and their mixtures was carried out using a Vision 2000 instrument (Thermo BioAnalysis, Finnigan, United States). Conditions for the determination of oligonucleotide molecular masses were optimized by applying various matrices and operation modes. The most reproducible results with minimal uncontrolled decomposition of the oligonucleotides including their apurinization during the MALDI MS registration were obtained using 2,4,6-trihydroxyacetophenone as a matrix instead of 3-hydroxypicolinic acid, usually employed in the mass spectrometry of oligonucleotides. Our approach allows the determination of molecular masses of oligonucleotides obtained by chemical synthesis and the evaluation of their component composition and purity. It was applied to the mass spectrometric analysis of oligonucleotides containing a 3'-(methyl-C-phosphonate) group or a modified 1,N6-ethenodeoxyadenosine unit.

[Fluorescent-labeled lipophilic analogues of serotonin, dopamine, and acetylcholine: synthesis, mass spectrometry, and biological activity]. / Fluorestsentnomechenye lipofil'nye analogi serotonina, dofamina i atsetilkholina: sintez, mass-spektrometriia i biologicheskaia aktivnost'.

4,4-Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl derivatives of serotonin, dopamine, choline, and N,N-dimethylaminoethanol, with the fluorescence maximum at 512 nm (lambda(exc) 470 nm), and 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl derivatives of choline and N,N-dimethylaminoethanol, with the fluorescence maximum at 554 nm (lambda(exc) 470 nm), were synthesized. These compounds yield protonated molecular ions of 100% intensity upon mass spectrometry with electrospray ionization at atmospheric pressure. The fragmentation of molecular ions under the conditions of secondary mass spectrometry mainly proceeds through the elimination of hydrogen fluoride from the fluorescent core of the molecules. Experiments on sea urchin Lytechinus variegatus embryos and larvae showed that these compounds easily penetrate into the cells and are accumulated in the cytoplasm. They do not differ in their biological activity from similar derivatives of arachidonic acid described previously and are agonists of serotonin or acetylcholine or antagonists of nicotinic acetylcholine receptors. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 5; see also http: // www.maik.ru.

[Mitochondrial H+-ATPase: identification of subunits of the F0 subcomplex contacting with membrane lipids]. / H+-ATP-asa mitokhondrii: identifikatsiia sub''edinits subkompleksa F0, kontaktiruiushchikh s lipidami membrany.

The subunits of the F0 membrane sector of bovine heart mitochondrial H(+)-ATPase that contact the lipids of the mitochondrial inner membrane were identified with the use of specially synthesized proteoliposomes that contained active mitochondrial H(+)-ATPase and a photoreactive lipid, which was 1-acyl-2-[12-(diazocyclopentadiene-2-carbonylamino)-[12-14C]dodecanoyl]-sn- glycero-3-phosphocholine, 1-acyl-2-[11-([125I]diazoiodocyclopentadiene-2-carbonyloxy)undecanoyl]-sn- glycero-3-phosphocholine, or 1-acyl-2-[12-(diazocyclopentadiene-2-carbonylamino)dodecanoyl]-sn-glycero- 3-phosphocholine, where acyl is a mixture of the residues of palmitic (70%) and stearic (30%) acids. An analysis of the cross-linked products obtained upon the UV-irradiation of these proteoliposomes indicated that subunits c and a of the F0 membrane sector contact the lipids. The cross-linked products were identified by SDS-PAGE and MALDI mass spectrometry.

Unusual fatty acid composition of cerebrosides from the filamentous soil fungus Mortierella alpina.

The cerebrosides produced by the soil filamentous fungus Mortierella alpina strain KG-1/95 account for about 13% of the total polar lipids extractable from lyophilised cells with chloroform/methanol mixtures. By means of 1H NMR and (13)C NMR spectroscopy, matrix-assisted laser-desorption ionisation mass spectrometry, and chemical degradation experiment, they have been shown to be 1-O-beta-D-glucopyranosyl-2-N-(2'-D-hydroxyalkanoyl)-9-methylsphinga-4(E),8(E)-dienines, the fatty acid composition of which is unusual and consists of 2-hydroxytridecanoic (4%), 2-hydroxytetradecanoic (60%), 2-hydroxypentadecanoic (20%), and 2-hydroxyhexadecanoic (16%) acids.

[Phenotypic features of Ferroplasma acidiphilum strains Yt and Y-2]. / Fenotipicheskie osobennosti shtammov Yt i Y-2 Ferroplasma acidiphilum.

Earlier, we described a new family of mesophilic, strictly autotrophic Fe(2+)-oxidizing archaebacteria, Ferroplasmaceae, which belongs to the order Thermoplasmales and includes the genus Ferroplasma and species F. acidiphilum (strain YT) [1]. The present work is concerned with a comparative study of phenotypic characteristics of the type strain YT and a new strain, F. acidiphilum Y-2, isolated from dense pulps produced during oxidation of arsenogold concentrates from the Bakyrchikskoe (Kazakhstan) and Olimpiadinskoe (Krasnoyarsk Krai) ore deposits, respectively. The G + C content of DNA from strains YT and Y-2 comprised 35.1 and 35.2 mol%, respectively; the level of DNA-DNA homology between the strains was 84%. Restriction profiles of chromosomal DNA from both strains exhibited a similarity coefficient of 0.87. Genotypic characteristics of these strains indicate their affiliation to the same species. The cells of both strains are polymorphic and lack cell walls. Strains of F. acidiphilum oxidized ferrous oxide and pyrite as the sole source of energy and fixed carbon dioxide as the sole carbon source. Strains required yeast extract as a growth factor. Optimum pH for cell growth ranged from 1.7 to 1.8; the temperature optima for the growth of strains YT and Y-2 were 34-36 and 40-42 degrees C, respectively. Comparative analysis of total lipids revealed their close similarity in the strains; two glycophospholipids comprised 90% of total lipids: lipid I, beta-D-glucopyranosylcaldarchaetidylglycerol (about 55%), and lipid II, trihexosylcaldarchaetidylglycerol (26%), whose isopranyl chains contained no cyclopentane rings. The carbohydrate fraction of lipid I hydrolysate contained only D-glucose, whereas hydrolysate of lipid II contained both D-glucose and D-galactose in a molar ratio of 2:1. Thus, it was established that the intraspecific phylogenetic divergence within F. acidiphilum is manifested in two the strains by different temperature optima against the background of similarity in other phenotypic properties.

Two unusual glycerophospholipids from a filamentous fungus, Absidia corymbifera.

The chloroform-methanol extractable lipids of the soil filamentous fungus Absidia corymbifera VKMF-965 account for about 20% by weight of dry cells and are composed of low-polarity constituents (about 75% of the total lipids), such as triacylglycerols (mainly), diacylglycerols, sterols and free fatty acids, as well as of glycolipids (about 3%) and phospholipids. The last consist largely of components common to the fungal lipids, namely, phosphatidylethanolamine (38% of the total phospholipids), phosphatidyl-myo-inositol (16%), diphosphatidylglycerol (12%), phosphatidylcholine (7%), phosphatidic acid (6%) and phosphatidylglycerol (3%), and two unusual phospholipids, PL1 (6%) and PL2 (9%). Based on the infrared (IR), (1)H-nuclear magnetic resonance (NMR), (13)C-NMR and mass spectra along with the results of degradation experiment, these two phospholipids have been established to be 1,2-diacyl-sn-glycero-3-phospho(N-acetylethanolamine), or N-acetyl phosphatidylethanolamine, and 1,2-diacyl-sn-glycero-3-phospho(N-ethoxycarbonyl-ethanolamine), respectively. These structures have been confirmed by preparing similar phospholipids from the phosphatidylethanolamine isolated from the same fungus and correlating their chromatographic behaviour, IR and (1)H-NMR spectra with those of PL1 and PL2. So far N-acetyl phosphatidylethanolamine has been detected only in cattle and human brains and a human placenta but its structure was not rigorously proved. PL2 is a novel lipid; to our knowledge no natural phospholipid with an urethane group has yet been found. The main fatty acids of both the phospholipids are n-hexadecanoic, octadecanoic and octadecadienoic ones; PL2 contains in addition a considerable amount of octadecatrienoic acid with its greater portion located at the sn-1 position.

[MALDI-mass spectrometry for identification of new proteins in snake venoms]. / MALDI-mass-spektrometriia dlia identifikatsii novykh belkov v iade zmei.

By MALDI MS, we searched cobra venoms for new low-content polypeptides. A number of new proteins with molecular masses 7-25 kDa, characteristic of the known snake protein toxins, were identified, with the content of one of them less than 0.02%.

[Novel mathematical approach to determination of antibiotics concentration by agar diffusion method. Analysis of graphical, table and mathematical options of relative biological activity estimation]. / Novyi matematicheskii podkhod pri opredelenii kontsentratsii antibiotikov metodom diffusii v agar. Analiz graficheskikh, tablichnykhi matematicheskikh variantov rascheta otnocitel'noi biologicheskoi aktivnosti.

Several varieties of relative biological activity estimation are comparatively analyzed and illustrated by an example of tylosin. For visual demonstration the estimates with the equation of a straight line are represented graphically. It is concluded that the design equations in the State Pharmacopeia XI (USSR) should be respectively replaced. The advantages of the variety for the biological activity estimation with the one-point intercept form of the equation of a straight line are illustrated by particular examples.

[A new mathematical approach for determining antibiotic concentrations using the agar diffusion method]. / Novyi matematicheskii podkhod pri opredelenii kontsentratsii antibiotikov metodom diffuzii v agar.

A mathematical approach using the one-point intercept form of the equation of a straight line for the assay of antibiotic biological activity by the agar diffusion method is described. Insignificant alteration in the experimental conditions made it possible to markedly lower the volume of work and materials for the statistic processing. As distinct from the routine methods, the assay substance in three different concentrations is applied to three cavities on a dish and the standard substance in one control concentration is applied to the other three cavities on the same dish. The antibiotic biological activity is estimated from the equation lg cn = lg cst + (dn - dst)/bi when the relationship between the dose logarithm and the size of the diameters of the test microbe inhibition growth zones is direct. When the relationship is inverse e.g. when the dose is expressed as the dilution, the equation lg cn = lg cst + (dst - dn)/bi is used. Factor bi is determined experimentally for every sample by the mean value of the sizes of the inhibition growth zone diameter by the programs of the simple linear-regression.

Fluviols, bicyclic nitrogen-rich antibiotics produced by Pseudomonas fluorescens.

Fifteen fluorescent pseudomonads, isolated from the rhizosphere of agricultural plants, were similar in both their phenotypic properties and the chemical nature of produced pigments, to the previously described Pseudomonas fluorescens var. pseudoiodinum. DNA-DNA hybridisation data showed their genetic similarity (but not identity) to different biovars of P. fluorescens. A family of antibiotics-fluviols belonging to pyrazolo-[4,3-e]as-triazine derivatives was isolated from studied strains; isolation, properties, antimicrobial and antitumour activity of fluviols are described.

[Metabolites produced by Streptomyces kanamyceticus mutants with impaired biosynthesis of kanamycin]. / Metabolity, obrazuemye mutantami Streptomyces kanamyceticus s narushennym biosintezom kanamitsina.

Metabolites produced by Streptomyces kanamyceticus mutants with impaired kanamycin biosynthesis (kan mutants) were investigated by thin layer chromatography. With spectrophotometric scanning of the chromatograms the quantitative content of kanamycin A and 2-desoxystreptamine (2-DOS) in the culture fluid was determined. Five groups of the S.kanamyceticus mutants with impaired kanamycin biosynthesis at various stages were identified: kanA produced no D-glucosamine, kanB and kanC produced no 2-DOS, kanD was not able to transfer 2-DOS to the metabolites with the antibiotic activity, kanG synthesized no kanosamine.

Periodic phenomena in Proteus mirabilis swarm colony development.

Proteus mirabilis colonies exhibit striking geometric regularity. Basic microbiological methods and imaging techniques were used to measure periodic macroscopic events in swarm colony morphogenesis. We distinguished three initial phases (lag phase, first swarming phase, and first consolidation phase) followed by repeating cycles of subsequent swarming plus consolidation phases. Each Proteus swarm colony terrace corresponds to one swarming-plus-consolidation cycle. The duration of the lag phase was dependent upon inoculation density in a way that indicated the operation of both cooperative and inhibitory multicellular effects. On our standard medium, the second and subsequent swarm phases displayed structure in the form of internal waves visible with reflected and dark-field illumination. These internal waves resulted from organization of the migrating bacteria into successively thicker cohorts of swarmer cells. Bacterial growth and motility were independently modified by altering the composition of the growth medium. By varying the glucose concentration in the substrate, it was possible to alter biomass production without greatly affecting the kinetics of colony surface area expansion. By varying the agar concentration in the substrate, initial bacterial biomass production was unaffected but colony expansion dynamics were significantly altered. Higher agar concentrations led to slower, shorter swarm phases and longer consolidation phases. Thus, colony growth was restricted by higher agar concentrations but the overall timing of the swarming-plus-consolidation cycles remained constant. None of a variety of factors which had significant effects on colony expansion altered terracing frequencies at 32 degrees C, but the length of the swarming-plus-consolidation cycle was affected by temperature and medium enrichment. Some clinical isolates displayed significant differences in terracing frequencies at 32 degrees C. Our results defined a number of readily quantifiable parameters in swarm colony development. The data showed no connection between nutrient (glucose) depletion and the onset of different phases in swarm colony morphogenesis. Several observations point to the operation of density-dependent thresholds in controlling the transitions between distinct phases.

[Allergenic properties of the substance produced by the Streptococcus strain sp. Thom-1606]. / Izuchenie allergennykh svoistv STP (substantsiia, produtsiruemaia shtammom Streptococcus sp. Tom-1606).

The newly isolated biologically active agent "STP" (the substance produced by Streptococcus strain sp. TOM-1606) has been found to possess no allergenic properties. This new preparation has shown hyposensitizing activity on the level with the allergen. The morphological data obtained in the study of the organs of experimental animals have revealed the immunostimulating action of preparation "STP" on the body, that is confirmed by the characteristic transformation of the internal organs of experimental animals.

[Structure of the reaction adduct of xanthothricin with acetone]. / Stroenie addukta reaktsii ksantotritsina s atsetonom.

Reaction of xanthothricin demethylation by amines in acetone as a solvent yielded a new compound with a formula of C10H13N5O3. Mass spectrometry, 1H NMR-spectroscopy and x-ray analysis showed that the compound was 4H-1,6-dimethyl-4a(2-oxopropyl)-pyrimido-[5,4-e]-1 ,2,4-triazine-5,7-dione.

[Structure and physicochemical properties of isolated molecules of reumycin]. / Struktura i fiziko-khimicheskie svoistva izolirovannykh molekul reumitsina.

IR and electron spectra of an uracil derivative (antitumor antibiotic reumycin) were studied by low temperature spectroscopy. The experimental data corresponded to the quantum chemistry calculations of electron transmission. On the basis of the spectra interpretation the molecular structure of the reumycin isolated molecules was suggested. pKa of reumycin in aqueous medium was determined by UV spectroscopy.

[Study of alkaline hydrolysis of reumycin using NMR spectroscopy]. / Izuchenie shchelochnogo gidroliza reumitsina metodom spektroskopii IaMR.

The transformation sequence of reumycin in aqueous (D2O) solutions with various pD was studied by NMR spectroscopy and the structures of the yielding products were determined. It was shown that formation of 6-(3-methylureido)-1,2,4-triazine-5-carboxylic acid was the first stage of reumycin alkaline hydrolysis. The subsequent cyclization of this compound resulted in obtaining 5-methyl-5H-imidazo [4,5-e]-1,2,4-triazin-6 (7H) one-4a-carboxylic acid in mono-,di- or trianionic form which depended on the medium pH and was due to dissociation of the carboxylic group, N(4)H group of the triazine ring and N(7)H group of the imidazolidinone ring.

[Isolation and characteristics of an antibacterial substance produced by a strain of Streptococcus sp. Thom-1606. A study of the biological activity of the metabolic products of Streptococcus sp. Thom-1606]. / Vydelenie i kharakteristika antibakterial'noi substantsii, produtsiruemoi shtammom Streptococcus sp. TOM-1606. Izuchenie biologicheskoi aktivnosti produktov metabolizma Streptococcus sp. TOM-1066.

The data on the biological properties of the culture fluid of Streptococcus strain sp. TOM-1606 are presented. The native preparation has been shown to possess the capacity for stimulating the rate of the clearance of the peritoneal cavity of mice from Staphylococcus aureus cells, strain MT-1, rif. r., found to be insensitive to the action of the above-mentioned preparation in vitro. The crude preparation produces a transitory bacteriostatic effect on the streptococcal and staphylococcal strains under study. The preparation produces a prolonged bacteriostatic effect only on Micrococcus luteus test strain. All these data suggest that the crude preparation contains at least two active principles.

[X-ray structural study of the pyrimido-[5,4-e]-1,2,4-triazine antibiotics xanthothricin and fervenulin]. / Rentgenostrukturnoe issledovanie pirimido-[5,4-e]-1,2,4-triazinovykh antibiotikov ksantotritsina i fervenulina.

The structures of xanthothricin (I) and fervenulin (II), pyrimido-[5,4-e]-1,2,4-triazine antibiotics were determined by x-ray analysis. The crystals of I and II are monoclinic: a 9.667(6), 18.270(7), b 14.029(9), 5.723(2), c 6.914(3), 18.058(7) A, beta 116.47(3), 118.90(2) degrees, Z 4,8; space group P21/c. Analysis of bond length distribution in I and II was indicative of significant alternation of the double and ordinary bonds.

[Study of the stability of pyrimido-[5,4-e]-1,2,4-triazine antibiotics in acid-base media by NMR spectroscopy]. / Izuchenie ustoichivosti pirimido-[5,4-e]-1,2,4-triazinovykh antibiotikov v kislotno-osnovnykh sredakh metodom spektroskopii IaMR.

A comparative study of the NMR 1H and 13C spectra of reumycin, fervenulin and xanthothricin in aqueous acid-base media showed that at pH or pD ranging from 8.0 to 1.0 the antibiotics were chemically stable. By the ratio of the 1H and 13C chemical shifts of reumycin at pH 4.0-10.0 the pKa values of this antibiotic were determined: 6.7 in aqueous (D2O) solution and 8.76 in dimethylsulfoxide media. Alkalization of the solutions of reumycin (pH 12.0), fervenulin (pH 9.0) and xanthothricin (pH 8.0) resulted in irreversible chemical transformation of the antibiotics. The analysis of the chemical shifts in the PMR spectra of the transformation products revealed transformation of the uracil ring in reumycin and uracil and triazine rings in fervenulin and xanthothricin. Alkalization of the xanthothricin solutions resulted also in demethylation with formation of reumycin.

[Free radical mechanism of action of pyrimido-triazine antibiotics]. / Svobodnoradikal'nye mekhanizmy deistviia pirimidotriazinovykh antibiotikov.

EPR spectra of anion radicals were recorded as a result of chemical or enzymatic reduction at various pH of the pyrimido-triazine antibiotics. These anion radicals easily form superoxide radicals in the presence of oxygen. It is supposed that a higher selectivity of reumycin action is due to difference in the redox potentials of the neutral and ionized antibiotic forms. A possibility of enhance the reumycin potency may involve the pH lowering inside the tumor cells - for example, by glucose injections.