建立液相芯片方法检测鉴别结核分枝杆菌复合群、鸟分枝杆菌与副结核分枝杆菌

运用液相芯片技术原理,以分枝杆菌菌种(群)特异基因序列IS6110、IS1081、IS1245和F57为目标基因,设计筛选4套扩增引物和杂交探针,建立同时检测鉴别结核分枝杆菌复合群、鸟分枝杆菌和副结核分枝杆菌的四重液相基因芯片检测方法。对13种共54株分枝杆菌菌株以及23种常见微生物样品的检测结果显示,四重液相芯片方法可特异检测鉴别目标菌种(群),与其它分枝杆菌菌种或微生物无非特异交叉反应;检测敏感性达2.1×101-2.5×102基因拷贝或0.06-0.74 fg DNA;组内检测变异系数和组间检测变异系数均<10%。采用四重液相芯片方法从临床结核疑似人痰样和牛组织样品中检出结核致病菌,检出率分别达75.6%(99/131)和94.9%(37/39),显著高于培养法(38.9%和53.8%)。对副结核疑似临床样品的检测试验结果显示,四重液相芯片方法与荧光PCR方法的阳性符合率为83%(24/29)。对四重混合模板的检测试验结果显示该液相芯片方法可鉴别不同菌种混合感染。四重液相芯片方法的检测周期<1 d,其中对纯化DNA模板的检测时间可在2-3 h内完成。     

Isolation and characterisation of 8-hydroxy-3Z,5Z-tetradecadienoic acid,a putative intermediate in Pichia guilliermondii gamma-decalactone biosynthesis from ricinoleic acid

During a screening procedure for the discovery of a strong gamma-decalactone producer from ricinoleic acid, we observed that the yeast Pichia guilliermondii accumulated transiently 8-hydroxy-3Z,5Z-tetradecadienoic acid 1 during gamma-decalactone biosynthesis in the stationary phase of growth. The structural elucidation of 1 was based on nuclear magnetic resonance, infrared, ultraviolet and gas chromatography-mass spectrometry experiments. The occurrence of 1 is discussed in relation with previously proposed gamma-decalactone biosynthetic pathways.     

Isolation and characterization of a naturally occurring multidrug-resistant strain of the canine hookworm,Ancylostoma caninum

Soil-transmitted nematodes infect over a billion people and place several billion more at risk of infection. Hookworm disease is the most significant of these soil-transmitted nematodes, with over 500?million people infected. Hookworm infection can result in debilitating and sometimes fatal iron-deficiency anemia, which is particularly devastating in children and pregnant women. Currently, hookworms and other soil-transmitted nematodes are controlled by administration of a single dose of a benzimidazole to targeted populations in endemic areas. While effective, people are quickly re-infected, necessitating frequent treatment. Widespread exposure to anthelmintic drugs can place significant selective pressure on parasitic nematodes to generate resistance, which has severely compromised benzimidazole anthelmintics for control of livestock nematodes in many areas of the world. Here we report, to our knowledge, the first naturally occurring multidrug-resistant strain of the canine hookworm Ancylostoma caninum. We reveal that this isolate is resistant to fenbendazole at the clinical dosage of 50?mg/kg for 3?days. Our data shows that this strain harbors a fixed, single base pair mutation at amino acid 167 of the β-tubulin isotype 1 gene, and by using CRISPR/Cas9 we demonstrate that introduction of this mutation into the corresponding amino acid in the orthologous β-tubulin gene of Caenorhabditis elegans confers a similar level of resistance to thiabendazole. We also show that the isolate is resistant to the macrocyclic lactone anthelmintic ivermectin. Understanding the mechanism of anthelmintic resistance is important for rational design of control strategies to maintain the usefulness of current drugs, and to monitor the emergence of resistance. The isolate we describe represents the first multidrug-resistant strain of A. caninum reported, and our data reveal a resistance marker that can emerge naturally in response to heavy anthelminthic treatment.     

Isolation and identification of a new,naturally occurring cytokinin (6-benzylaminopurineriboside) from an anise cell culture (Pimpinella anisum L.)

A new, naturally occurring cytokinin was isolated from a cell culture of anise (Pimpinella anisum L.). Its structure was determined as 6-benzylaminopurine riboside by the use of gas chromatography and mass spectrometry. Quantitation of this new plant hormone was accomplished by single-ion-monitoring measurements.     

Isolation,identification and biological activity of chlamydosporol from Fusarium culmorum HM-8

An isolate of Fusarium culmorum (W.G. Smith), Sacc. HM-8 from a scabby wheat kernel sample from England produced a novel toxin when grown in culture on rice. This toxin, which was given the trivial name of HM-8, was purified, its toxic properties demonstrated and its structure determined by spectroscopic methods. Subsequent to preliminary reports of our findings, two other laboratories have independently isolated the same substance from F. chlamydosporum and F. acuminatum, and demonstrated the same structure by spectroscopic methods and X-ray crystallography. Identity of the metabolite from F. culmorum HM-8 with the published structure is based on (1) identical elemental composition derived from both elemental analysis and high resolution mass spectrometry in electron impact and fast atom bombardment modes, (2) comparable melting point and chromatographic properties, and (3) the presence of characteristic absorption bands in the infrared, ultraviolet, and nuclear magnetic resonance (proton and carbon-13) spectra. Because both laboratories which subsequently isolated this metabolite have used the name chlamydosporol, it is being adopted here. The mass spectral properties of chlamydosporol are described. The derivative 8-O-acetylchlamydosporol was prepared and characterized. Chlamydosporol caused food refusal and weight loss in rats, cytotoxic effects to cultured mouse and human fibroblast cells at concentrations of 2.5 g/ml and 7.5 g/ml, respectively, and mortality to chick embryos (10 to 70%) over a concentration range from 0.5 mg to 4 mg per egg.     

Molecular dynamics analysis of the effects of GTP,GDP and benzimidazole derivative on structural dynamics of a cell division protein FtsZ from Mycobacterium tuberculosis

Abstract

The prevailing multi-drug resistance in Mycobacterium tuberculosis continues to remain one of the main challenges to combat tuberculosis. Hence, it becomes imperative to focus on novel drug targets. Filamenting temperature-sensitive mutant Z (FtsZ) is an essential cell division protein, a eukaryotic tubulin homologue and a promising drug target. During cytokinesis, FtsZ polymerises in the presence of GTP to form Z-ring and recruits other proteins at this site that eventually lead to the formation of daughter cells. Benzimidazoles were experimentally shown to inhibit Mtb-FtsZ, with one of the benzimidazole derivatives, M1, being reported to have the minimum inhibitory concentration (MIC) value of 3.13 µg/mL. In the present study, mechanism of destabilisation of FtsZ in the presence of M1 was computationally investigated in the presence of its substrate GTP/GDP employing molecular dynamics (MD) simulation analysis, principal component analysis (PCA), molecular mechanics combined with the generalised Born and surface area continuum salvation (MM-GBSA) and density functional theory (DFT). From the analyses, it is proposed that binding of M1 in the inter-domain cleft induces structural changes in the GTP-binding region that affect GTP binding, thus switching the preference of this protein towards depolymerised state and eventually inhibiting the cell division. Hence, this study provides mechanistic insights into the design of novel benzimidazole inhibitors against Mtb-FtsZ.

Communicated by Ramaswamy H. Sarma     

Inactivation and the site of labeling of F1-ATPase from Mycobacterium phlei by dicyclohexylcarbodiimide, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and quinacrine mustard

The F₁-ATPase from Mycobacterium phlei is inactivated by dicyclohexylcarbodiimide (DCCD), 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) and quinacrine mustard. The inactivation is both time-and concentration-dependent and in the case of DCCD being more pronounced at acidic pH. The minimum inactivation half-time ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) for DCCD, NBD-Cl and quinacrine mustard was observed to be 14, 6 and 7 min, respectively. Inactivation of F₁-ATPase resulted in the incorporation of [¹⁴C]DCCD as well as [¹⁴C]NBD-Cl into α and γ subunits. The incorporation of label into α and γ subunits, utilizing [¹⁴C]NBD-Cl, was reversible by dithiothreitol. Complete inactivation, by linear extrapolation to zero activity, revealed that 4 mol [¹⁴C]DCCD and 4 mol [¹⁴C]NBD-Cl bind per mol F₁-ATPase. Kinetic and binding studies show that these probes bind to site(s) distinct from ATP-binding site in F₁-ATPase from M. phlei.     

分枝杆菌TZh51菌株的分离鉴定及其生物修复污染土壤的特性

[目的]为获得降解芘的微生物菌株,并用其生物修复被多环芳烃污染的土壤.[方法]芘降解菌的分离采用平板升华法.根据表型观察、生理生化特性和16S rDNA的序列同源性分析,对菌株进行分类学鉴定.通过活菌计数、HPLC测定多环芳烃的残留量,研究菌株在固体、液体无机盐培养基以及在污染土壤中降解多环芳烃(polycyclic aromatic hydrocarbons,PAHs)的能力.[结果]分离到4株能降解芘的菌株TZh51、TZh52、TG42和TG52.实验结果表明,TZh51降解PAHs的能力强于其余3株菌.TZh51被鉴定为分枝杆菌属(Mycobacterium sp.),但与已发表的分枝杆菌菌株M11为不同的种.TZh51接种在芘膜的固体无机盐培养基上,测定获得最大芘降解量的条件是培养温度为3512和芘膜厚度为130 ng/mm2.在芘浓度为50、100 mg/L的液体无机盐培养基中培养,6天时TZh51的芘降解率分别达到91.9%、71.8%,10天时菌体数量分别达到最大值为2.0、6.0×108cfu/mL;TZh51降解芘的效果强于M11.在种植作物的处理中,到第6周时TZh51的菌体数量达到每克干土含7.2×108个菌落数,到第8周时菲、荧蒽和芘的降解率分别达到91.4%、86.9%和85.8%;[结论]TZh51具有很强降解PAHs的能力;另外,TZh51与作物联合生物修复污染土壤的效果明显.     

DNA repair and replication in human fibroblasts treated with (±)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene

DNA repair and replication were examined in diploid human fibroblasts after treatment with (±)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I). Unscheduled DNA synthesis exhibited a linear response to BPDE-I concentrations up to 1.5 μM and a saturation plateau after higher concentrations. Maximal unscheduled DNA synthesis was observed in the first hour after treatment with synthesis diminishing progressively thereafter. Half-maximal unscheduled DNA synthesis was seen within 4–6 h after treatment with 0.7 μM BPDE-I. DNA replication was inhibited by BPDE-I in a dose- and time-dependent fashion. The mechanisms of this inhibition were characterized by velocity sedimentation of pulse-labeled nascent DNA in alkaline sucrose gradients. Very low concentrations of BPDE-I (0.03 and 0.07 μM) were found to inhibit replicon initiation by up to 50% within 30–60 min after treatment. Recovery of initiation following these low concentrations was evident within 3 h after treatment. Higher concentrations of carcinogen inhibited DNA synthesis in active replicons. This effect was manifested by a reduction in incorporation of precursor into replication intermediates of greater than 1·10⁷ Da with the concurrent production of abnormally small nascent DNA. When viewed 45 min after treatment with 0.17 μM BPDE-I the combination of these two effects partially masked the inhibition of replicon initiation. However, even after treatment with 0.33 μM BPDE-I an effect on initiation was evident. These results reveal a pattern of response to BPDE-I that is quite similar to that produced by 254 nm radiation.     

Isolation and partial characterization of a mucin-type glycoprotein from plasma membranes of human melanoma cells

Plasma membranes were isolated from HM7 melanoma cells grown in the presence of [³H]glucosamine and Na₂³⁵SO₄ or [³H]mannose and [¹⁴C]glucosamine. The labelled glucoconjugates were solubilized with 0.6 M lithium diiodosalicylate/0.5% Triton X-100. Fractionation of glycoconjugates by repeated chromatography on columns of Sepharose CL-6B and DEAE-Sepharose and by affinity chromatography on WGA-Sepharose yielded three radiochemically homogenous glycoproteins. One of these having an apparent molecular weight of 100 000 was found to contain clusters of (AcNeu)_{1 or in2} å [Gal å GalNAc] linked $\text{O-}\text{glycosidically}$ to the protein. One other glycoprotein contained both $\text{O-}\text{glycosidically}$ and $\text{N-}\text{glycosidically-linked}$ oligosaccharides, and the third contained only $\text{N-}\text{glycosidically-linked}$ carbohydrates. Preliminary results indicate that the 100 000 molecular weight mucin-type glycoprotein is present in significantly reduced quantities in cultured human fetal uveal melanocytes. Further, the bulk of the glycoproteins from the melanocytes were of lower molecular size compared to those from the melanoma cells.     

Fucopyranosyl-(1 → 4)-N-glycolylneuraminic acid,a constituent of glycoproteins of the cuvierian tubules of the sea cucumber Holothuria forskali Della Chiaje

Analysis of the sialic acid fraction obtained by mild acid hydrolysis of the Cuvierian tubules of the sea cucumber Holothuria forskali Della Chiaje revealed the presence of F-glycolylneuraminic acid and fucopyranosyl-(1 → 4)-N-glycolylneuraminic acid. Furthermore, methylation analysis of a pronase-digest of the tubules demonstrated that all N-glycolylneuraminic acid residues are substituted at C-4, explaining the earlier reported resistance of sialic acid in tubules to cleavage by neuraminidase.     

Isolation of (2S,4R)-2-amino-4-methyl-hex-5-enoic acid,a nonprotein amino acid,as an allelochemical from the fruiting bodies of Boletus fraternus Peck.

The mushroom Boletus fraternus Peck. shows allelopathy and suppresses the growth of broad leaf plants in nature. According to a bioassay-guided fractionation of the fruiting body of the fungus, a rare nonprotein amino acid was isolated as a major allelochemical. The chemical structure of the compound was determined to be (2S,4R)-2-amino-4-methyl-hex-5-enoic acid (5-dehydrohomoleucine) by analysis of ¹H- and ¹³C-nuclear magnetic resonance spectra and comparison with data from the literature. The allelochemical caused 50% inhibition of lettuce seedling radicle growth at a concentration of 34 ppm (w/v). Further, since radicle growth was directed away from the filter paper to prevent contact with the allelochemical at concentrations higher than 300 ppm (w/v), the fungus may use the allelochemical to protect its immediate environment from contamination by other plants.     

Isolation and structure of wallichoside,a novel pteroside from Pteris wallichiana

Wallichoside has been isolated from the rhizomes of Pteris wallichiana and its structure and absolute configuration has been established as the 3-β-d-glucoside of 2S, 3S-pterosin C on the basis of UV, IR, NMR and MS data of wallichoside and its derivatives.     

长春地区鸡源奇异变形杆菌的分离鉴定及毒力分析

【背景】奇异变形杆菌(Proteus mirabilis)是一种机会致病菌,广泛存在于周围环境中,常导致动物和人类感染。【目的】探究吉林省长春地区某养鸡场病鸡死亡原因,为疾病防控提供参考。【方法】从病死青年鸡脏器分离到病原菌TSA-1,通过革兰氏染色、生化试验和16S rRNA基因序列鉴定,并进行药敏试验、毒力基因检测、细胞毒性和黏附性试验、大蜡螟攻毒试验研究。【结果】分离株TSA-1在镜下呈短杆状、球状的革兰氏阴性菌,生化特性与奇异变形杆菌相一致,16S rRNA基因序列比对显示与奇异变形杆菌相似度为100%;药敏试验结果显示分离株TSA-1对氨苄西林、四环素、卡那霉素、头孢唑林等14种药物耐药,对环丙沙星、恩诺沙星等7种药物敏感;分离株还具有较强的生物被膜形成能力,携带ireA、ucaA、pmfA、atfA、ptA、zapA、hpmA和flhC这8种毒力基因,并对巨噬细胞RAW264.7表现出细胞毒性,而且对Caco-2细胞具有很强的黏附作用;同时对大蜡螟幼虫表现出比禽致病性大肠杆菌(avian pathogenic Escherichia coliO78,APEC-O78)更强的致死作用。【结论】从病死鸡脏器分离得到的奇异变形杆菌TSA-1具有多重耐药性,并携带多种毒力基因,对细胞和大蜡螟幼虫表现出强毒性作用,说明该菌是引起病鸡死亡的主要致病菌之一,应引起重视。     

Isolation and characterization of an asparagine-linked keratan sulfate from the skin of a marine teleost, Scomber japobicus

Keratan sulfate was isolated from the skin of Pacific mackerel (Scomber japonicus) after exhaustive digestion with pronase followed by ethanol precipitation and fractionation on a cellulose column with 0.3% recovery of dried material. The keratan sulfate preparation was separated into four major fractions by Dowex-1 column chromatrography. The chemical and infrared spectrum analyses of the four fractions showed a high degree of heterogeneity in sulfation. Since the carbohydrate-peptide linkage in the teleost skin keratan sulfate was found to be stable in alkali, and asparagine was the predominant amino acid, the asparagine residue in the peptide backbone was most likely to be involved in the N-glycosyl linkage with the carbohydrate moiety. Besides the type of carbohydrate-peptide linkage, the teleost skin keratan sulfate is very similar to corneal keratan sulfate, (keretan sulfate I) in two respects: (1) The teleost skin and bovine corneal keratan sulfates were hydrolyzed much faster by endo-β-galactosidase that the whale nasal cartilage keratan sulfate (keratan sulfate II). (2) Although the teleost skin keratan sulfate showed considerable polydispersity, the molecular weight was in the same range as the corneal keratan sulfate, and it was relatively higher than that of the cartilage keratan sulfate.     

Comparison of the crystallin mRNA populations from rat,calf and duck lens. Evidence for a longer αA2-mRNA and two distinct αB2-mRNAs in the birds

Total cytoplasmic poly(A)-containing RNA from rat, calf and duck lens was fractionated by electrophoresis in methylmercury hydroxide-containing agarose gels. RNA electrophoresed in parallel lanes was either transferred onto nitrocellulose and hybridized with total cDNA synthesized on the initial mRNA or was recovered from individual gel fractions for in vitro translation in a reticulocyte cell-free system. This allowed the identification and size-characterization of individual mRNA species encoding α-, β-, γ- and δ-crystallin polypeptides. The 14 S mRNA fraction of rat lens comprises two αA₂-mRNAs of approximately 1250 and 1350 nucleotides and the αA^Ins-mRNA with a size similar to that of the largest αA₂-mRNA. The calf lens 14 S mRNA fraction harbors a heterogeneous population of αA₂-mRNA. In the same fraction another mRNA encoding a polypeptide, designated X, has been found sharing no homology with αA sequences. The duck lens αA₂-mRNA appears to be 400–450 bases longer than the rat and calf lens αA₂-mRNAs. Furthermore, in contrast to the single αB₂-mRNA in rat and calf lens, two αB₂-mRNAs have been identified in duck lens, one, the major species, similar in size to the αB₂-mRNA in rat and calf lens (800 bases), and the other species 700 nucleotides longer. The large size differences among the αA₂- and αB₂-mRNAs most likely reside in their 3′-untranslated sequences.     

The mode of action of cytotoxic and antitumor 1-nitroacridines. II. In vivo enzyme-mediated covalent binding of a 1-nitroacridine derivative, Ledakrin or Nitracrine, with dna and other macromolecules of mammalian or bacterial cells

Earlier evidence, in Part I of this paper, has shown that cytotoxic and antitumor 1-nitroacridines did not primarily exert their potent inhibitory effects on cultured mammalian cells by physicochemical binding with DNA, although it undoubtedly occurred (Chem.-Biol. Interact., 43 (1983) 131). As a result it was investigated (i) whether 9-14C- or 1'-14C-labeled derivatives of their representative, 1-nitro-9-/3'-dimethylamino-n-propylamino/acridine (Ledakrin or Nitracrine), were capable of covalent binding with nucleic acids and other suitable macromolecules in target cells in vivo and/or (ii) whether activation of the agent in the cell was a necessary prerequisite for such binding. Using the criteria of resistance to exhaustive extractions with trichloroacetic acid and/or organic solvents, [14C]Ledakrin was found to bind covalently, with relatively little discrimination, with: (i) intracellular macromolecules, including DNA, of cultured tumor HeLa cells (370-2500 DNA base pairs/one Ledakrin molecule; (ii) experimental animal tumor Ehrlich ascites (Eat) cells in vivo (650-5880 DNA base pairs/one Ledakrin molecule); (iii) bacterial Bacillus subtilis SB 1058 cells (7000-33 000 Ledakrin links/one cell genome); (iv) NADPH-fortified rat liver homogenates in vitro (25.6 nmol/mg microsomal protein under air). These results far exceed the common levels reported for alkylating agents or chemical carcinogens. Unlike [ethyl-14C]quinacrine, compared in vitro, covalent macromolecules binding with Ledakrin in vitro, and most probably in vivo, can be equated to NADPH-dependent activation(s) by oxidoreductase systems and the presence of DNA alone was not satisfactory in itself to attain Ledakrin binding. Fractionation of the enzymatic digest of 14C-associated DNA, isolated from Eat cells exposed in vivo to [9-14C]Ledakrin, by Sephadex LH-20 column chromatography followed by mass spectrometry analyses of modified nucleosides, indicated that both mono- and dinucleosidical Ledakrin metabolites were the products of an in vivo reaction. This implied that the lethal reaction of the drug could be its cross-linking of the target macromolecules and/or its monofunctional attack on vitally important cellular components.     

Structural,biochemical and biophysical characterization of four oxygen-evolving Photosystem II preparations from spinach

Four procedures utilizing different detergent and salt conditions were used to isolate oxygen-evolving Photosystem II (PS II) preparations from spinach thylakoid membranes. These PS II preparations have been characterized by freeze-fracture electron microscopy, SDS-polyacrylamide gel electrophoresis, steady-state and pulsed oxygen evolution, 77 K fluorescence, and room-temperature electron paramagnetic resonance. All of the O₂-evolving PS II samples were found to be highly purified grana membrane fractions composed of paired, appressed membrane fragments. The lumenal surfaces of the membranes and thus the O₂-evolving enzyme complex, are directly exposed to the external environment. Biochemical and biophysical analyses indicated that all four preparations are enriched in the chlorophyll $\text{a}\text{b-}\text{light-harvesting}$ complex and Photosystem II, and depleted to varying degrees in the stroma-associated components, Photosystem I and the CF₁-ATPase. The four PS II samples also varied in their cytochrome f content. All preparations showed enhanced stability of oxygen production and oxygen-rate electrode activity compared to control thylakoids, apparently promoted by low concentrations of residual detergent in the PS II preparations. A model is presented which summarizes the effects of the salt and detergent treatments on thylakoid structure and, consequently, on the configuration and composition of the oxygen-evolving PS II samples.     

Isolation and characterization of a lectin from Grifola frondosa fruiting bodies

An N-acetylgalactosamine-specific lectin (GFL) was isolated from Grifola frondosa fruiting bodies by affinity chromatographies on acid-treated Sepharose CL-4B and then GalNAc-Toyopearl. The isolated lectin agglutinated all types of erythrocytes equally. Molecular masses estimated by gel filtration under various buffers and matrices varied from 30 to 52 kDa. On the other hand, SDS-PAGE in the presence or absence of 2-mercaptoethanol showed three major bands of 33, 66 and 100 kDa and a faint band of 65 kDa. This lectin exhibited GalNAc-specificity. The protein was a glycoprotein containing 3.3% total sugar, and the amino acid analysis revealed a high content of acidic and hydroxy amino acids and a low content of methionine and histidine. GFL was cytotoxic against HeLa cells. The toxicity did not appear after preincubating the lectin with the haptenic sugar N-acetylgalactosamine.