Bacteria of the genus Acinetobacter. Their systematics and ecological analysis

The content of A. lwoffi and A. calcoaceticus in water and sewage has been determined. A considerable prevalence of A. lwoffi in both objects has been revealed. The most definite results have been obtained with the use of selective media: ethanol-ammonium medium and Baumann's modified nitrate-acetate medium. The seasonal dynamics of both species in water has been determined, the peak being observed in June and the decrease (and with A. calcoaceticus even disappearance), in August.     

Ochratoxin A in pig blood: method of analysis and use as a tool for feed studies

A procedure is presented for screening the quality of feed in respect to ochratoxin A contamination based upon the analysis of ochratoxin A in pig blood. Representative samples from large feed lots may be obtained by using pigs as in vivo sample collectors which enrich the toxin and forms homogeneous samples in the blood. The spectrofluorometric procedure for ochratoxin A analysis (K. Hult and S. Gatenbeck, J. Assoc. Off. Anal. Chem. 59:128-129, 1976) has been adapted to pig blood and has been simplified to involve only three extraction steps. A volume of 2.5 ml of blood or plasma is needed, and the detection limit is 2 ng of ochratoxin A per ml. The disappearance of ochratoxin A from pig blood as a function of time has been studied. A feeding experiment with ochratoxin A has been performed, and the time course of the concentration of ochratoxin A in blood has been followed during the experiment.     

Ochratoxin A in pig blood: method of analysis and use as a tool for feed studies.

A procedure is presented for screening the quality of feed in respect to ochratoxin A contamination based upon the analysis of ochratoxin A in pig blood. Representative samples from large feed lots may be obtained by using pigs as in vivo sample collectors which enrich the toxin and forms homogeneous samples in the blood. The spectrofluorometric procedure for ochratoxin A analysis (K. Hult and S. Gatenbeck, J. Assoc. Off. Anal. Chem. 59:128-129, 1976) has been adapted to pig blood and has been simplified to involve only three extraction steps. A volume of 2.5 ml of blood or plasma is needed, and the detection limit is 2 ng of ochratoxin A per ml. The disappearance of ochratoxin A from pig blood as a function of time has been studied. A feeding experiment with ochratoxin A has been performed, and the time course of the concentration of ochratoxin A in blood has been followed during the experiment.     

Crystallization and preliminary X-ray crystallographic studies of benzamidine-inhibited trypsin from the North Atlantic salmon (Salmo salar)

Crystals of benzamidine-inhibited trypsin from the North Atlantic salmon (Salmo salar) have been grown from ammonium sulphate solution at pH 5.0. Two crystal forms suitable for X-ray structure analysis, obtained from a hanging-drop experiment, have been characterized. Both belong to space-group P22(1)2(1) with cell dimensions a = 39.2 A, b = 62.4 A, c = 84.6 A and a = 31.4 A, b = 74.8 A, c = 83.5 A, for forms I and II, respectively. Intensity data to 1.82 A have been collected for crystal form I on a CAD4 diffractometer, and initial phases have been obtained by molecular replacement methods. The conventional R-factor after two rounds of model building and subsequent refinement is 0.25 for data between 6.0 and 2.0 A. So far no water molecules have been included in the model.     

The SPR-based biosensor test system for analysis of small compounds interaction with human cytochrome P450 51A1 (CYP51A1)

The SPR-based test for human cytochrome P450 51A1 (CYP51A1) ligand screening has been developed. Applicability of this system has been validated with known azole inhibitors of cytochromes P450. The studied azoles selectively interacted with human cytochrome P450 51A1, which showed the highest affinity towards ketoconazole. The efficiency of the SPR based assay has been tested using 19 steroid and triterpene compounds, which have not been investigated as potential ligands of CYP51A1.     

Saponin Adjuvants. IV. Evaluation of The Adjuvant Quil a in the Vaccination of Cattle Against Foot-And-Mouth Disease

The saponin adjuvant Quil A has been investigated in the vaccination of cattle against foot-and-mouth disease. Using a Frenkel type vaccine a dose-response relationship has been established between Quil A and neutralizing antibody titres. Ten ml of vaccine was combined with 0, 50, 200, 800, and 3200 µg of Quil A. The combinations were each injected into 4 animals. The local reaction on the site of injection produced by injection of the vaccine alone and in combination with different doses of Quil A has been estimated. On this basis a therapeutical dose at 1 mg of Quil A has been estimated to combine maximum adjuvant effect with a minimum of adverse reactions. This dose has been tested in the vaccination of cattle with FMD vaccines derived from BHK suspension cell virus of type O and A respectively. The vaccines were tested in 10 ml and 5 ml doses with or without Quil A, and each in 4 animals. It is concluded that Quil A is a valuable adjuvant for use in the induction of neutralizing antibodies against foot-and-mouth disease in cattle.     

Crystallization and preliminary X-ray analysis of flavocytochrome c(3), the fumarate reductase from Shewanella frigidimarina.

The fumarate reductase (flavocytochrome c(3)) from Shewanella frigidimarina (formerly S. putrefaciens) NCIMB400 has been crystallized in the space group P2(1), with cell dimensions of a = 45.447 A, b = 92.107 A, c = 78.311 A, and beta = 91.038 degrees and one molecule per asymmetric unit. A native data set has been collected to 1.8 A. The gene encoding Fcc(3) from the S. frigidimarina type strain ACAM591 has been cloned and sequenced and the protein crystallized in space group P2(1) with cell dimensions of a = 45.359 A, b = 88.051 A, c = 77.473 A, and beta = 104.499 degrees. Anomalous data have also been collected from the NCIMB400 crystal allowing the heme iron positions to be identified.     

Carboxylic ionophore (lasalocid A and A23187) mediated lanthanide ion transport across phospholipid vesicles

The transport kinetics of three lanthanide ions (viz., Pr3+, Nd3+, and Eu3+) across dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine unilamellar vesicles mediated by the two carboxylic ionophores lasalocid A and A23187 have been studied by proton nuclear magnetic resonance spectroscopy. Time-dependent changes in the chemical shifts of head group choline signals have been measured to calculate apparent rate constants of transport. These experiments have been done at different ionophore concentrations to determine the stoichiometry of the transporting species. The rates of transport have been found to be faster in the absence of intravesicular La3+ compared to those observed in its presence. The stoichiometry of the transporting species has been found to be 2:1 (ionophore:cation) for both lasalocid A and A23187 in dimyristoylphosphatidylcholine vesicles. However, stoichiometries of greater than 2 have been obtained for lasalocid A mediated lanthanide ion transport across dipalmitoylphosphatidylcholine vesicles. Possible reasons for the observations of such noninteger stoichiometries are discussed. Our results also indicated that A23187 is a more efficient carrier ionophore than lasalocid A.     

Proton nuclear magnetic resonance characterization of phospholipase A2 from Laticauda semifasciata

The molecular properties of phospholipases (PLases) A2 I and A2 III from a sea snake, Laticauda semifasciata, have been characterized by gel-filtration, as well as proton NMR, CD, UV absorption, and fluorescence spectroscopic methods. PLase A2 I exists as a monomer in aqueous solution in the presence or in the absence of Ca2+. The dissociation constants of the Ca2+-enzyme complexes have been determined for the two enzymes. The 270-mHz proton NMR spectra of PLases A2 I and A2 III have been measured, and the aromatic proton resonances of His-21 and His-48 in the active site have been assigned. By analyzing the pH dependence of the chemical shifts of the histidine proton resonances, pKa values have been determined for His-21 and His-48 with and without Ca2+. The conformational transitions have been found to take place at low pH or at high temperature (at approximately 65 degrees C). Fluorescence change of PLase A2 I upon addition of substrate analogs suggests that Trp-70 in PLase A2 I is involved in the binding to micellar substrates. The lack of Trp-70 in PLase A2 III is probably related to the low enzymatic activity as compared with that of PLase A2 I.     

Crystallographic studies on D-amino acid oxidase.

D-amino acid oxidase, a flavoprotein from hog kidneys, has been crystalized in two different forms. Orthorhombic prisms have been obtained from the enzyme.benzoate complex at pH 8.3; the space group is C2221 and the cell dimensions are a = 325A, b = 138.8 A, c = 200 A. At lower pH values, the enzyme crystallizes in trigonal prisms with a = b = 116.0 A, c = 399 A, space group P3112 or its enantiomorph. The two crystal forms have been obtained at 28 degrees C while at 4 degrees C only weak evidence of crystallization has been detected. In both crystalline modifications, the protein is highly associated.     

Determination of protein A I staphylococcus aureus strains isolated from monkeys

Protein A content in Staphylococcus aureus isolated from 6 species of monkeys at the Sukhumi Monkey Nursery has been studied. Protein A has been detected in 73% of the studied strains. One strain isolated from a rhesus macaque has been found to release high amounts of protein A into the environment.     

Crystallization and preliminary crystallographic analysis of recombinant human P38 MAP kinase.

The recombinant human p38 MAP kinase has been expressed and purified from both Escherichia coli and SF9 cells, and has been crystallized in two forms by the hanging drop vapor diffusion method using PEG as precipitant. Both crystal forms belong to space group P2(1)2(1)2(1). The cell parameters for crystal form 1 are a = 65.2 A, b = 74.6 A and c = 78.1 A. Those for crystal form 2 are a = 58.3 A, b = 68.3 A and c = 87.9 A. Diffraction data to 2.0 A resolution have been collected on both forms.     

具有中等毒性的马氏钳蝎神经毒素的纯化和初步晶体学研究

运用柱层析技术对产自淅川和常德的马氏钳蝎毒素进行分离纯化,得到８种哺乳动物神经毒素。运用制备型等电聚焦电泳技术,对常德样品中具有中等毒性的蝎神经毒素（ＢｍＫ５）进一步纯化,获得了高纯度样品。两个产地的蝎毒素ＢｍＫ５均已经成功地获得了大晶体。空间群均为Ｐ２１２１２１,晶胞参数分别为：ａ＝３８．４６埃,ｂ＝３７．２８埃,ｃ＝３６．９７埃（淅川）;ａ＝３８．４４埃,ｂ＝３７．５５埃,ｃ＝３６．８３埃（常德）。对两个产地的晶体分别收集了２．１埃（淅川）和１．６２埃（常德）分辨率的衍射数据。     

Single-strand and double-strand cleavage at half-modified and fully modified recognition sites for the restriction nucleases Sau3a and Taqi

The influence of cytosine methylation on the cleavage of DNA by the restriction nucleases Sau3A and TaqI has been investigated. Bovine satellite DNA fragments containing a GATCGA sequence, i.e. a Sau3A site overlapping with a TaqI site have been used in this study. The methylation of these fragments has been determined by sequence analysis. It has been found that a TaqI site (TCGA) methylated at cytosine in both DNA strands is still sensitive to double-strand cleavage. A Sau3A site (GATC), however, is rendered resistant to double-strand cleavage by methylation of a single cytosine. Fragments containing the "half-modified" Sau3A site are nicked in the unmethylated DNA strand. It has been shown by sequence analysis of nicked DNA that the single-strand break occurs at the same position which is cleaved in unmodified DNA.     

Crystallization and preliminary X-ray diffraction analysis of oucleoside diphosphate kinase from Myxococcus xanthus.

Nucleoside diphosphate (NDP) kinase catalyzes the transfer of the gamma-phosphate from a nucleoside triphosphate to a nucleoside diphosphate. Human and rodent forms of this enzyme have been shown to be suppressors of metastasis. Crystals that diffract X-rays to high resolution have been obtained for the recombinant Myxococcus xanthus NDP kinase expressed in and purified from Escherichia coli. Two crystal forms have been obtained. Both forms are orthorhombic, space group I222 (or I2(1)2(1)2(1)) with a = 267.1 A, b = 74.0 A and c = 75.1 A for form I and a = 53.5 A, b = 74.0 A and c = 75.1 A for form II. Form I appears to have five molecules in the asymmetric unit approximately related to each other by a translation of 0.2 along the a axis. Diffraction data have been recorded to 1.9 A for form I and to 2.2 A for form II.     

A family of repetitive nucleotide sequences (Sau 3A family) in the genome of 2 forms of the sockeye salmon Oncorhynchus nerka

A family of DNA sequences (Sau3A repeat sequences) has been revealed in genomes of two forms of O. nerka. A DNA fragment belonging to this "family" has been cloned in bacterial plasmid. Its copy number (in genomes of normal (570 +/- 33) and dwarf (66 +/- 12) forms) has been counted. The structural organization of sequences of Sau3A family in genomes of males and females of normal form is not the same. The organization of Sau3A sequences is the same in genomes of dwarf males and dwarf females of normal form, but the quantity of Sau3A sequences copies is smaller. It is supposed that the DNA sequences of Sau3A family bear the sex determination of dwarf form of O. nerka. The fragment of rRNA gene of nerka has been cloned and the number of rRNA gene copies in genomes of normal (200 +/- 62) and dwarf (1690 +/- 24) forms has been defined.     

Kink dynamics in inhomogeneous DNA

A mathematical model for studies of peculiar features of the dynamics of local conformational distortions (kinks) has been constructed. General formulas determining the dependence of the main dynamic characteristics of kinks: size, energy, density of energy, and velocity on the composition of inhomogeneous DNA have been obtained. Quantitative estimations of the characteristics have been made for kinks activated in inhomogeneous polynucleotide chains with sequences analogous to promoter sequences A1, A2, and A3 of bacteriophage T7 genome.     

Enkephalinases

Enkephalins can be degraded by a variety of peptidases. We have characterized several membrane-associated brain peptidases in an effort to determine which if any are concerned with the physiological inactivation of synaptically released enkephalin. We have distinguished two carboxyl-directed dipeptidylpeptidases, designated enkephalinase A1 and A2, that give rise to the Tyr-Gly-Gly fragment. Both enzymes are physically separable from angiotensin converting enzyme. Regional variations in enkephalinase A1 activity and opiate receptors are similar. A novel amino-terminal-directed dipeptidylpeptidase, enkephalinase B, which generates Tyr-Gly, has been identified. All of these enzymes as well as aminopeptidase have been solubilized from brain membranes by detergent treatment and have been mutually resolved by DEAE column chromatography. Enkephalinase A1 has been purified 1500-fold, to apparent homogeneity.     

Identification and Functional Analysis of Three Isoforms of Bovine BST-2

Human BST-2 (hBST-2) has been identified as a cellular antiviral factor that blocks the release of various enveloped viruses. Orthologues of BST-2 have been identified in several species, including human, monkeys, pig, mouse, cat and sheep. All have been reported to possess antiviral activity. Duplication of the BST-2 gene has been observed in sheep and the paralogues are referred to as ovine BST-2A and BST2-B, although only a single gene corresponding to BST-2 has been identified in most species. In this study, we identified three isoforms of bovine BST-2, named bBST-2A1, bBST-2A2 and bBST-2B, in bovine cells treated with type I interferon, but not in untreated cells. Both bBST-2A1 and bBST-2A2 are posttranslationally modified by N-linked glycosylation and a GPI-anchor as well as hBST-2, while bBST-2B has neither of these modifications. Exogenous expression of bBST-2A1 or bBST-2A2 markedly reduced the production of bovine leukemia virus and vesicular stomatitis virus from cells, while the antiviral activity of bBST-2B was much weaker than those of bBST-2A1 and bBST-2A2. Our data suggest that bBST-2A1 and bBST-2A2 function as part of IFN-induced innate immunity against virus infection. On the other hand, bBST-2B may have a different physiological function from bBST-2A1 and bBST-2A2.