Effect of miconazole on the structure and function of plasma membrane of Candida albicans

Abstract Fructose, a rarely occurring sugar constituent of Gram-negative bacterial lipopolysaccharides (LPS), is distributed ubiquitously in LPS of 01 Vibrio cholerae so far examined, but its location in LPS has not hitherto been elucidated. It was found that hydrazinolysis of LPS successfully affords a derivative retaining virtually all the fructose of intact LPS, but no ester-bound phosphate. Structural analysis carried out on the LPS derivative prepared by the hydrazinolysis of R-type LPS isolated from a rough mutant strain (NIH 41R) of 01 V. cholerae NIH 41 (Ogawa) revealed that the fructose is present as a non-reducing terminal residue bound to position C-6 of a glucose residue in the core region. This finding is considered to exclude the possibility that, in the LPS of 01 V. cholerae , the fructose is present in the region of the inner core in place of 2-keto-3-deoxyoctonate.     

Relationship between structure and antigenicity of O1 Vibrio cholerae lipopolysaccharides

The relationship between the release of fructose from O1 Vibrio cholerae lipopolysaccharides (LPS) by dilute acetic acid hydrolysis and the decrease in their antigenicity was examined. Decrease in the antigenicity of LPS was not parallel with the release of fructose, and occurred very much later than the latter. Periodate oxidation of LPS resulted in the total elimination of the fructose and glucose, and two-thirds of the heptose constituents, but no difference in the antigenicity of LPS was observed before and after oxidation. These findings indicate that the fructose present in O1 V. cholerae LPS is not substantially involved in their specific antigenicity. In the O1 V. cholerae LPS, the fructose is in the branch structure, most probably in the core region.     

O-antigenic lipopolysaccharides isolated from a marine Vibrio, bio-serogroup 1875, possessing an antigenic factor in common with O1 Vibrio cholerae

The chemical and serological characteristics of lipopolysaccharides (LPS) isolated from Vibrio bio-serogroup 1875 were compared with those of O1 Vibrio cholerae LPS. Vibrio bio-serogroup 1875 LPS contained all the component sugars which were found in O1 V. cholerae LPS, i.e. glucose, L-glycero-D-manno-heptose, fructose, glucosamine, perosamine and quinovosamine, though the amount of perosamine, a characteristic component of O1 V. cholerae LPS, was very low compared with that of O1 V. cholerae LPS. Their LPS additionally contained mannose and two unidentified neutral sugars which are not regular constituents of O1 V. cholerae LPS. Definite serological cross-reactivity in the passive haemolysis test between LPS from Vibrio bio-serogroup 1875 and LPS from O1 V. cholerae was demonstrated.     

Immunological properties of Vibrio cholerae lipopolysaccharides.

Immunological effects of wall lipopolysaccharide (LPS) preparations obtained from Vibrio cholerae Inaba 569B, Ogawa NIH 41 and NAG 4715 strains by the hot phenol-water procedure were examined in mice. Although these LPS lack KDO, which are basic components of the core region of most gram-negative LPS, they still have potencies as B-cell mitogens, adjuvants, immunosuppressants, polyclonal B-cell activators and phagocytic stimulants for macrophages. The activities of these V. cholerae LPS on murine immune system seemed to be weaker than those of Salmonella typhimurium LT2-LPS. Among these V. cholerae LPS, NAG 4715-LPS showed the strongest mitogenic activity and phagocytic stimulation, while the potencies of this NAG 4715-LPS for the induction of polyclonal B cell activation, adjuvant effects and immunosuppression did not seem to be greater to those of the other LPS.     

Chemical structure of the 2-keto-3-deoxyoctonate (KDO) region of the lipopolysaccharide isolated from O1 Vibrio cholerae NIH 4IR (Ogawa).

The chemical structure of the 2-keto-3-deoxyoctonate (KDO) region of the lipopolysaccharide (LPS) isolated from O1 V. cholerae NIH 41R (Ogawa) was elucidated by dephosphorylation, periodate oxidation and methylation analysis. Methylation analysis of KDO in the dephosphorylated LPS revealed the presence of 5-O-acetyl-1,2,4,6,7,8-hexa-O-methyl-3-deoxy-octitol and 2-keto-3-deoxy-heptulosonic acid was detected in the methanolysate of the periodate-oxidized and dephosphorylated LPS. These results indicated that the site of binding of KDO to the core oligosaccharide is position C5 as in enteric gram-negative bacterial LPS, while only one molecule of the KDO residue carrying phosphate on position C4 is present in the inner core region of the LPS in contrast to enteric gram-negative bacterial LPS in which one molecule of KDO carrying KDO or KDO2----4KDO disaccharide instead of the phosphate group at position C4 is present in its main chain.     

Assay of Δ4-3-oxosteroids and their conjugates by the isonicotinic acid hydrazide method

Enzyme-catalyzed hydrogenation of Δ⁴-3-oxosteroids and their conjugates can be assayed without hydrolytic and chromatographic procedures. The assay medium is mixed with methanol, followed by centrifugation. The resultant supernatant is evaporated to dryness and subsequently condensed with the isonicotinic acid hydrazide reagent. The hydrazone of Δ⁴-3-oxosteroid thus formed is determined at 372 nm.     

霍乱弧菌脂多糖基因在大肠杆菌中的克隆

利用柯斯质粒pHC 79为载体,构建了霍乱弧菌178(埃尔托生物型,小川血清型)染色体基因文库。经血清凝集试验及菌落固相ELISA检测,从基因文库中筛选到13株能够表达霍乱弧菌脂多糖O抗原的阳性克隆。经热酚水法从转化于中提取并纯化的脂多糖能与霍乱弧菌抗血清发生特异性结合。针对重组柯斯质粒PMM—VO 38进行了多种酶切分析,测定其分子量为46kb。     

A rapid method for extraction of zearalenone and zeraralenols in fermented corn

A rapid extraction method is described for isolation of zearalenone and α- and β-trans-zearalenols from laboratory fermented corn. Corn fermented withFusarium crookwellense at 25°C for 2 weeks was agitated for 5 minutes in acetone. The acetone extract was evaporated to dryness and the remaining residue was chromatographed on a silica gel column with hexane:ethyl acetate (8:2). The products eluted with the hexane:ethyl acetate mixture in 1–1/2 column volumes and were identified by regular phase and C-18 reverse-phase thin layer chromatography. The products were verified by gas chromatography-mass spectroscopy. Product recoveries were 62.5–70% for the zearalenols and 70% for zearalenone in the range 0.5–50 mg/Kg.     

O139霍乱弧菌LPS基因在大肠杆菌中的克隆和表达

利用粘粒载体pCOS5构建了国内分离的O139霍乱弧菌的基因组文库,并从文库中筛选获得可以表达O139霍乱弧菌脂多糖的重组克隆株E.coliJM109(pMG310)。重组粘粒pMG310经酶切分析,所克隆的外源DNA片段大小为37kb。实验证明：重组克隆株E.coliJM109(pMG310)所表达的脂多糖具有良好的免疫原性及反应原性。     

Sugar composition of the polysaccharide portion of lipopolysaccharides isolated from non-O1 Vibrio cholerae O2 to O41, O44, and O68

The sugar composition of the polysaccharide portion of lipopolysaccharides (LPS) was determined for 42 serovars of non-O1 Vibrio cholerae, i.e., from serogroups O2 to O41, O44, and O68. On the basis of their compositional sugar pattern, they were classified into 24 chemotypes. 2-Keto-3-deoxyoctonate (KDO) was totally undetectable by the conventional color test (Weissbach's reaction) under the conventional hydrolysis conditions. Instead, a kind of KDO-like substance, which was positive in the reaction but not identical to KDO, was found in serogroup O19. Fructose, a characteristic sugar constituent of O1 V. cholerae LPS, was found in 33 serogroups but was absent from nine serogroups, approximately 20% of the members of this group so far examined.     

A Simple Procedure for Detecting the Presence of Cyclopropane Fatty Acids in Bacterial Lipids

Four gram-negative bacterial species, including Escherichia coli strain B, Serratia marcescens, Pseudomonas fluorescens, and Vibrio cholerae (comma) strain NIH 41, were investigated for fatty acid content by gas-liquid chromatography involving a preparatory technique which facilitated detection of cyclopropane fatty acids. Methyl esters of fatty acids were subjected to mild catalytic hydrogenation to eliminate unsaturates. Hydrogenation was followed by bromination which removed cyclopropane acids from chromatographic profile patterns. Lactobacillic acid (cis-11,12-methyleneoctanoate) and cis-9,10-methylenehexadecanoate, previously reported in lipids of E. coli and S. marcescens, were found in small amounts in P. fluorescens but were not detected in V. cholerae.     

Aspects of Vibrio cholerae lipopolysaccharide

In this review information on the chemical structure, biosynthesis, antigenic and biological properties of V. cholerae lipopolysaccharide (LPS) is presented. The specific structural feature of this LPS is a small size of the polysaccharide chain of O-antigen. In vibrios of serogroup O 139 it is oligosaccharide. The modification of the O-chain (methylation of individual sugars, shortened chain, etc.) plays an essential role in the antigenic specificity of V. cholerae LPS. All these factors affect of endotoxin function, the microbial resistance to external influences. V. cholerae LPS takes part in the formation of microcapsules and biofilms. The evolutional development of V. cholerae in this direction determines, to some extent, their increased resistance in the environment. In human body the heterogeneity of the LPS composition permits the preservation of vibrios and ensures, together with cholerogen, their pathogenetic action.     

Expression of Shigella sonnei lipopolysaccharide in Vibrio cholerae

Making use of a newly designed mobilizable suicide vector, the genetic determinants encoding Shigella sonnei lipopolysaccharide (LPS) were stably integrated into the chromosome of the live attenuated Vibrio cholerae vaccine strain CVD103-HgR. Expression studies showed that the production of complete S. sonnei O-polysaccharide (O-PS)-bearing LPS was limited in bivalent recombinant strains that were also proficient in the synthesis of the host-encoded Inaba O-PS. Conversely, high amounts of LPS carrying S. sonnei O-PS are produced in monovalent Inaba-deficient derivatives, even in those strains which do not co-express the compatible R1 LPS core. Thus, the non-enterobacterial V. cholerae LPS core efficiently acts as a receptor for covalent binding of S. sonnei O-PS provided that competition with the host O-PS is avoided. Expression of the R1 core interferes with cell division in recombinant V. cholerae without affecting other physiological properties of vaccine strain CVD103-HgR. Both monovalent and bivalent strains stimulated high serum-antibody titres specific for their respective O-serotype(s) when administered to rabbits. The potential of V. cholerae as an expression carrier for heterologous O-serotypes is discussed.     

Electrophoretic mobility and immune response of outer membrane proteins of Vibrio cholerae O139

Abstract The outer membrane (OM) protein components of a Vibrio cholerae O1 and four V. cholerae O139 strains, collected from cholera patients, were analysed by SDS-PAGE. A protein of 69 kDa molecular mass was observed only when the OMPs were prepared from strains grown in synthetic broth. As a result of passage in the rabbit ileal loop (RIL), virulence was enhanced, and a protein component around 18 kDa of the V. cholerae O139 OM became the major protein component. On immunoblot analysis with rabbit antiserum against V. cholerae O139 OM, it was shown that, apart from the major protein component of V. cholerae O1 OM of around 45 kDa and that of V. cholerae O139 OM of around 38 kDa, all other minor protein components were cross-reactive between the two serogroups. In immunoblot assays with convalescent sera obtained from V. cholerae O139-infected patients, it was observed that in addition to the lipopolysaccharide (LPS)-induced antibody, only the 38 kDa major protein component elicited considerable levels of antibody in the pateint. Minor OM components of 18 kDa were detected in the immunoblot analysis by LPS-directed antibody, however, as the OM proteins are known to be associated with LPS.     

Determination of bulleyaconitine A in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry

A sensitive and specific high-performance liquid chromatography (HPLC)-electrospray ionization tandem mass spectrometry (MS-MS) was developed for the determination of bulleyaconitine A (BLA) in human plasma. BLA and internal standard (I.S.) ketoconazole were extracted from the plasma by a liquid-liquid extraction. The supernatant was evaporated to complete dryness and reconstituted with acetonitrile containing 0.1% acetic acid before injecting into an ODS MS column. The gradient mobile phase was composed of a mixture of acetonitrile (containing 0.1% acetic acid, v/v) and 0.1% acetic acid aqueous solution eluted at 0.3 ml/min. BLA and I.S. were determined by multiple reaction monitoring using precursor-->product ion combinations at m/z 644.6-->584.3 and 531.2-->81.6, respectively. Linearity was established for the concentration range of 0.12-6 ng/ml. The recoveries of BLA ranged from 96.93 to 113.9% and the R.S.D. was within 20%. The method is rapid and applicable to the pharmacokinetic studies of BLA in human.     

Studies on heterogeneous lipopolysaccharide fractions of Vibrio cholerae 569B

By SDS-PAGE or gel filtration on Sephadex G-25, lipopolysaccharide (LPS) isolated from Vibrio cholerae 569B (Inaba) can be separated into two distinct fractions, one corresponding to smooth LPS and the other to rough LPS. Pulse-labelling of LPS with [14C]glucose showed that the rough form is synthesized first followed by the biosynthesis of the smooth form. A preferential release of the smooth LPS of V. cholerae 569B was also detected during normal growth of cells.     

A new method for extraction of iron-molybdenum cofactor (FeMoco) from nitrogenase adsorbed to DEAE-cellulose. 2. Solubilization of FeMoco in a wide range of organic solvents

While the iron-molybdenum cofactor (FeMoco) of nitrogenase, a constituent of the active site for nitrogen reduction, can be extracted into N-methylformamide (NMF) and pyrrollidinone, the inability to solubilize it in any other organic solvents has hampered further understanding of its structure and chemical properties. A method to solubilize FeMoco, prepared in N,N-dimethylformamide (DMF) with Bu4N+ as counterion [McLean, P. A., Wink, D. A., Chapman, S. K., Hickman, A. B., McKillop, D. M., & Orme-Johnson, W. H. (1989) Biochemistry (preceding paper in this issue)], in acetonitrile, acetone, methylene chloride, tetrahydrofuran, and benzene is reported. FeMoco evaporated to dryness in vacuo dissolves readily in good yield (55-100%) and with no significant loss in specific activity. In addition, FeMoco can be extracted directly into these solvents from MoFe protein bound to a DEAE-Sepharose column if the protein is pretreated with DMF. Methods have also been developed to extract fully active FeMoco into acetone and acetonitrile in the absence of any amide solvents (NMF or DMF). Extraction of FeMoco into acetone (30% yield) involves only pretreatment of column-bound protein with methanol, while extraction into acetonitrile (22% yield) requires pretreatment with methanol followed by THF. We conclude that the presence of a suitable soluble cation confers solubility to the cofactor in many common organic solvents and that the solubility of FeMoco in a given solvent may be independent of the ability of that solvent to extract the cofactor from column-bound protein.     

Effect of lipopolysaccharide core synthesis mutations on the production of Vibrio cholerae O-antigen in Escherixhia coli K-12

The rfb genes of Vibrio cholerae O1 (Ogawa serotype) were subcloned into a derivative of pBR322. This plasmid was transformed into several Escherichia coli K-12 mutant strains which produce an incomplete lipopolysaccharide (LPS)-core-oligosaccharide region. The data indicate that the V. cholerae O-antigen is assembled onto the E. coli LPS and that at least two glucoses are needed in the core in order to achieve a high level of production. These data are consistent with the reported presence of glucose in the V. cholerae LPS-core-oligosaccharide region.     

Further purification of bovine spleen inhibitors of lymphocyte DNA synthesis (chalones).

Partially purified lymphocytic factors were obtained from boving spleen; these factors are non-cytotoxic and biologically active in vitro and in vivo: [3H]Thymidine incorporation into DNA of mitogen-stimulated mouse spleen cells in culture is inhibited; similar results are obtained with phytohaemagglutinin-stimulated peripheral human lymphocytes, where blast cells transformation is blocked. [3H]Thymidine incorporation into DNA of mitogen stimulated lymphocytes withdrawn from in vivo treated mice, is also reduced. The two factors in vitro and in vivo seem to act preferentially on mouse spleen cells compared to their action on liver, kidney and testicle cells in cluture, as far as thymidine incorporation into DNA is concerned. The following techniques were applied for their purification: 1. Homogenization of the fresh tissue in water, centrifugation, dialysis of the supernatant, centrifugation, fractionation of the supernatant by alcoholic precipitation and finally concentration in vacuo and lyophilization of the material soluble at 75% of alcohol yielded fraction F. 2. Preparation of an acetone powder from the spleen, extraction of the dry powder with water, then high speed centrifugation, followed by lyophilization of the supernatant produced fraction F'. Both fractions F and F' were further fractionated by chromatography on a Sephadex G75 column: 7 peaks were obtained (F1--F7). Biological activity was found in fraction F1, corresponding to high molecular weight material, and in fraction F6, corresponding to low molecular weight substances. By rechromatography on Sephadex G 75, it is easy to dissociate from F1 a small molecular weight fraction which might be similar to F6 as far as elution volume and biological properties are concerned.     

Potential use of serological methods in detecting cholera toxin

In the study of 50 Vibrio cholerae museum strains, 45 of them producing cholerigenic effect in suckling rabbits, cholera toxin, determined by means of the passive immune hemolysis (PIH) test, has been detected in the supernatant of the culture fluid of only two strains: V. cholerae 569 B, a well-known producer of cholera toxin, and V. cholerae (eltor) 1310, from whose population a toxigenic variant has been obtained by selection. To study the capacity of V. cholerae for producing toxin in vitro, in six cholerigenic strains, besides the supernatant of their culture fluids, also protein fractions, cell lysates and membrane fractions have been studied in the PIH test. In all these strains cholera toxin has been detected only in membrane fractions, which should be taken into consideration in the serological evaluation of the toxigenicity of V. cholerae.