黑发菌核糖体DNA小亚基片段的序列测定

用Rusketal 于 1995年设计的引物SMNUR10 1和Whiteetal 1990年设计的引物NS4对黑发菌 (Co matrichanigra)的rDNA小亚基片段进行了PCR扩增 ,并将扩增产物克隆到大肠杆菌JM10 9中进行测序 ,测得的序列长度为 115 4bp。同时将其与多头绒泡菌的相应序列进行了比较 ,其序列同源性为 6 6 %。

Induced Sporophore and Sclerotium of Physarum pusillum(Myxomycetes)

[Objective]The paper was to culture the plasmodia of Physarum pusillum for obtaining the optimal condition of sporophores and sclerotium,to reduce the agricultural diseases.[Method]Liquid culture combined with oat-agar culture was used to culture plasmodia of P.pusillum.Then plasmodium was induced to obtain Sporophores in lab by hunger and adjusting light（3 000,6 000,9 000,12 000 lx）and temperature（20,22,24,26 ℃）.[Result]Sporophores and sclerotium of P.pusillum were obtained from both oat-agar media and liquid media.The optimal conditions were 26 ℃,6 000 lx and 22 ℃,3 000 lx.[Conclusion]The result provided theoretical basis for reducing the loss of crops caused by myxomycete as much as possible in agricultural production.

Diarrhoea-predominant irritable bowel syndrome distinguishable by 16S rRNA gene phylotype quantification

AIM:To study whether selected bacterial 16S ribosomal RNA(rRNA)gene phylotypes are capable of disting- uishing irritable bowel syndrome(IBS). METHODS:The faecal microbiota of twenty volunteers with IBS,subdivided into eight diarrhoea-predominant (IBS-D),eight constipation-predominant(IBS-C)and four mixed symptom-subtype(IBS-M)IBS patients,and fifteen control subjects,were analysed at three time-points with a set of fourteen quantitative real-timepolymerase chain reaction assays.All assays targeted 16S rRNA gene phylotypes putatively associated with IBS,based on 16S rRNA gene library sequence analysis. The target phylotypes were affiliated with Actinobac-teria,Bacteroidetes and Firmicutes.Eight of the target phylotypes had less than 95%similarity to cultured bacterial species according to their 16S rRNA gene sequence.The data analyses were made with repeated-measures ANCOVA-type modelling of the data and principle component analysis(PCA)with linear mixed-effects models applied to the principal component scores. RESULTS:Bacterial phylotypes Clostridium cocleatum 88%,Clostridium thermosuccinogenes 85%,Coprobacillus catenaformis 91%,Ruminococcus bromii-like, Ruminococcus torques 91%,and R.torques 93%were detected from all samples analysed.A multivariate analysis of the relative quantities of all 14 bacterial 16S rRNA gene phylotypes suggested that the intestinal microbiota of the IBS-D patients differed from other sample groups.The PCA on the first principal component(PC1),explaining 30.36%of the observed variation in the IBS-D patient group,was significantly altered from all other sample groups(IBS-D vs control, P=0.01;IBS-D vs IBS-M,P=0.00;IBS-D vs IBS-C, P=0.05).Significant differences were also observed in the levels of distinct phylotypes using relative values in proportion to the total amount of bacteria.A phy- lotype with 85%similarity to C.thermosuccinogenes was quantified in significantly different quantities among the IBS-D and control subjects(-4.08±0.90 vs -3.33±1.16,P=0.04)and IBS-D and IBS-M subjects (-4.08±0.90 vs-3.08±1.38,P=0.05).Furthermore,a phylotype with 94%similarity to R.torques was more prevalent in IBS-D patients'intestinal micro- biota than in that of control subjects(-2.43±1.49 vs -4.02±1.63,P=0.01).A phylotype with 93%simi- larity to R.torques was associated with control sam- ples when compared with IBS-M(-2.41±0.53 vs -2.92±0.56,P=0.00).Additionally,a R.bromii-like phylotype was associated with IBS-C patients in com- parison to control subjects(-1.61±1.83 vs-3.69± 2.42,P=0.01).All of the above mentioned phylotype specific alterations were independent of the effect of time. CONCLUSION:Significant phylotype level alterationsin the intestinal microbiotas of IBS patients were observed,further emphasizing the possible contribution of the gastrointestinal microbiota in IBS.

高效包载酪氨酸羟化酶mRNA外泌体的制备

目的:酪氨酸羟化酶(TH)是多巴胺合成的限速酶,其对于帕金森病发生有着重要意义。外泌体是由细胞分泌的直径30~200 nm的微囊泡,被认为是可通过血脑屏障的潜在药物载体。拟利用古菌核糖体蛋白L7Ae和Kt环的结合特性,获得高效包载酪氨酸羟化酶mRNA的外泌体(TH-Kt-Exo),以实现mRNA药物穿越血脑屏障的递送。方法:通过构建带有Kt环的TH mRNA重组质粒,以及外泌体膜蛋白CD63和L7Ae融合表达的重组质粒pCMV-CD63-L7Ae-His,共转染HEK293F细胞,通过超速离心方法收取细胞分泌的外泌体,通过qPCR检测外泌体中TH mRNA的含量并转染受体细胞。结果:与单一酪氨酸羟化酶质粒转染得到的外泌体TH-Exo相比,利用该方法获得的外泌体TH-Kt-Exo在TH mRNA的装载水平上有显著提高。此外,该外泌体能够将其装载的mRNA转运至受体细胞中。结论:通过L7Ae和Kt环的特异性结合,可以有效提高目的mRNA在外泌体中的包载。

Isolation and identification of a bacteria strain Enterobacter cloacae I7 for degradation of hydrolyzed polyacrylamide

A bacteria strain for the degradation of hydrolyzed polyacrylamide （HPAM） was isolated from a curing pot in HPAM distribution station of Daqing Oilfield using Hungate anaerobic technique. The isolate was investigated from morphological, physiological, biochemical and molecular characterization. It is a Gram-negative, shortbacillus, non-spore-forming anaerobic bacteria with an optimum growth at 8.0 pH at 40℃. It can reduce sulfate to I-I2S. Alignment of 16S ribosomal DNA and 16S-23S ribosomal DNA intergenic spacer sequences suggests that this isolate is closely related to the Enterobacter cloacae. The isolate is identified as a new strain belonging to Enterobacter genus, temporarily named as Enterobacter cloacae 17. Analysis results of infrared spectroscopy （IR） show that the bacteria can use HPAM as the only carbon source, change the structure of HPAM polymer surface, and realize the hydrolysis of amide to carboxyl group by hydrolysis mechanism. It can degrade the side chain and change some functional groups, which obviously decreases the viscosity. GC-MS analysis indicates that the determined low-molecular weight degradation products of HPAM are polyacrylamide fragments with duplet bond, epoxy as well as carbonyl group, but most of them are acrylamide oligomer derivatives.

A novel salt-tolerant Micrococcus sp. DUT_AHX capable of degrading nitrobenzene

A novel strain of Micrococcus sp.DUT_AHX,which was isolated from the sludge of a nitrobenzene(NB)-manufacturing plant and could utilize NB as the sole carbon source,was identified on the basis of physiological and biochemical tests and 16S ribosomal DNA(rDNA)sequence analysis.It can grow at the temperature up to 40℃or in the presence of NaCl concentration up to 12 g/L in Luria-Bertani(LB)medium.The optimal degradation conditions are as follows:temperature 37℃,pH 7.0,and shaking speed 150 r/min.The strain involves a partial reductive pathway due to the release of ammonia and can also utilize 2-aminophenol as the sole carbon source.Furthermore,the enzyme activity tests show that crude extracts of NB-grown strain DUT_AHX mainly contain 2-aminophenol 1,6-dioxygenase activity.The exploitation of salt-tolerant bacteria will be a remarkable improvement in NB bioremediation and wastewater treatment at high salinity and high temperature.

Investigation of Optimal Charcoal Concentration for Heterokaryon Formation of Rhizoctonia solani

Optimal charcoal concentrations were examined for tuff formation of SBIs （single-basidiospore isolates） obtained from Rhizoctonia solani AG-1 IC, AG-2-2 IV and AG-2-3, and SPIs （single-protoplast isolates） from AG-1 IA by using PDCA with different charcoal concentrations. The best charcoal concentration for SBIs from AG-1 IC was 0.5%, from AG-2-3 was 1%, and from AG-2-2 IV was 2%. The optimal concentration was 0% for SPIs from AG-1 IA. The optimal charcoal concentrations used for tuff formation between SBIs and between SPIs are quite varied, depending on the different AGs. The results of AFLP haplotypes suggest that tuff isolates formed between SBIs/SPIs are heterokaryon.

Diagnosis of intestinal tuberculosis using a monoclonal antibody to Mycobacterium tuberculosis

AIM:To investigate the utility of immunohistochemical(IHC) staining with an antibody to Mycobacterium tuberculosis(M.tuberculosis) for the diagnosis of intestinal tuberculosis(TB).METHODS:We retrospectively identified 10 patients(4 males and 6 females;mean age = 65.1 ± 13.6 years) with intestinal TB.Clinical characteristics,including age,gender,underlying disease,and symptoms were obtained.Chest radiograph and laboratory tests,including sputum Ziehl-Neelsen(ZN) staining,M.tuberculosis culture,and sputum polymerase chain reaction(PCR) for tubercle bacilli DNA,as well as Tuberculin skin test(TST) and QuantiFERON-TB gold test(QFT),were examined.Colonoscopic records recorded on the basis of Sato's classification were also reviewed,in addition to data from intestinal biopsies examined for histopathological findings,including hematoxylin and eosin staining,and ZN staining,as well as M.tuberculosis culture,and PCR for tubercle bacilli DNA.For the present study,archived formalin-fixed paraffin-embedded(FFPE) intestinal tissue samples were immunohistochemically stained using a commercially available species-specific monoclonal antibody to the 38-kDa antigen of the M.tuberculosis complex.These sections were also stained with the pan-macrophage marker CD68 antibody.RESULTS:From the clinical data,we found that no patients were immunocompromised,and that the main symptoms were diarrhea and weight loss.Three patients displayed active pulmonary TB,six patients(60%) had a positive TST,and 4 patients(40%) had a positive QFT.Colonoscopic findings revealed that all patients had type 1 findings(linear ulcers in a circumferential arrangement or linear ulcers arranged circumferentially with mucosa showing multiple nodules),all of which were located in the right hemicolon and/or terminal ileum.Seven patients(70%) had concomitant healed lesions in the ileocecal area.No acid-fast bacilli were detected with ZN staining of the intestinal tissue samples,and both M.tuberculosis culture and PCR for tubercle bacilli DNA were negative in all samples.The histopathological data revealed that tuberculous granulomas were present in 4 cases(40%).IHC staining in archived FFPE samples with anti-M.tuberculosis monoclonal antibody revealed positive findings in 4 patients(40%);the same patients in which granulomas were detected by hematoxylin and eosin staining.M.tuberculosis antigens were found to be mostly intracellular,granular in pattern,and primarily located in the CD68 + macrophages of the granulomas.CONCLUSION:IHC staining with a monoclonal antibody to M.tuberculosis may be an efficient and simple diagnostic tool in addition to classic examination methods for the diagnosis of intestinal TB.

Bacteriophage Morphological Characterization by Using Transmission Electron Microscopy

Bacteriophages or more commonly ＂phages＂ are bacterial viruses. They are ubiquitous and good indicators of bacterial contaminations since their prevalence is high in those environments where their hosts are abundant. Phage classification is based on morphology and for this reason, even though it is considered an old technique, TEM （Transmission Electron Microscopy） still plays a key role in their characterization. In the present work, the authors focused on TEM analysis of phage ФApr-1 isolated against Lactococcuslactis （L. lactis）, implicated in industrial fermentations and of phage ФIZSAM-1, active against Listeria monocytogenes （L. monocytogenes）, isolated from the environment. For observation with TEM （EM 900T-Zeiss）, phages were harvested in liquid media and were negative stained with fosfotungstic acid 2‰. An accurate viral ultrastructure analysis by using TEM is fundamental not only in the first approach of characterization of newly isolated phages but also for providing useful information to go further to the selection process as potential bio-decontaminants.

Anti-mycobacterium and Anti-cancer Activities of Combretin, an Isolated Steroidal Alkaloid from the Seeds of Combretum quadrangulare Kurz.

Combretin is the steroidal alkaloid isolated from the seeds of Combretum quadrangulare Kurz. by macerated powder of the seeds with 95% ethanol. Purified further by avicel column chromatography and preparative thin layer chromatography. Combretin was investigated for anti-mycobacterium and anti-cancer activities. Combretin was inactive against Mycobacterium tuberculosis but showed anti-cancer activities against human hepatocarcinoma （Hep G2） ATCC HB-8065 and human caucasian colon adenocarcinoma （Caco2） ATCC HTB-39 at concentration greater than 300 mcg/ml in DMSO （Dimethyl sulfoxide）.

Characterization of the Mycobacterium Tuberculosis Protein Rv2302

The Mycobacterium tuberculosis protein Rv2302 has been characterized using nuclear magnetic resonance （NMR） and circular dichroism （CD） spectroscopy. While the biochemical function of Rv2302 is still unknown, recent microarray analyses show that Rv2302 is upregulated in response to starvation and overexpression of heat shock proteins and, consequently, may play a role in the biochemical processes associated with these events. Rv2302 is a monomer in solution as shown by size exclusion chromatography and NMR spectroscopy. CD spectroscopy suggests that Rv2302 partially unfolds upon heating and that this unfolding is reversible. Using NMR-based methods, the solution structure of Rv2302 was determined. The protein contains a five-strand, antiparalle β-sheet core with one C-terminal α-helix （A61 to A75） nestled against its side. Hydrophobic interactions between residues in the α-helix and β-strands 3 and 4 hold the α-helix near the β-sheet core. The electrostatic potential on the solvent-accessible surface is primarily negative with the exception of a positive arginine pocket composed of residues R18, R70, and R74. Steady-state {^1H}-^15N heteronuclear nuclear Overhauser effects indicate that the protein＇s core is rigid on the picosecond timescale. The absence of amide cross-peaks for residues G 13 to H 19 in the ^1H-^15N heteronuclear single quantum correlation spectrum suggests that this region, a loop between β-strands 1 and 2, undergoes motion on the millisecond to microsecond timescale. Dali searches using the structure closest to the average structure do not identify any high similarities to any other known protein structure, suggesting that the structure of Rv2302 may represent a novel protein fold.

Four new species of the genus Hymenoscyphus (fungi) based on morphology and molecular data

Four new species of Hymenoscyphus (H. brevicellulus, H. hyaloexcipulus, H. microcaudatus, and H. subsymmetricus) and a new Chinese record (H. subpallescens) are described. These five species share common characteristics: small apothecia (<3mm in diameter); hymenium whitish, pale yellow, to yellow in color; ectal excipulum of textura prismatica; asci arising from simple septa; ascospores scutuloid and guttulate; saprophytic nutrition; and leaf habitats, except for H. subsymmetricus, which grows on herbaceous stems. Phylogenetic analyses of internal transcribed spacer nuclear ribosomal DNA sequences, the universal DNA barcode for fungi, for 16 species in the genus indicated that these taxa were closely related to H. microserotinus, in accordance with their morphological features, but represented independent species. The distinguishing features of each new species from its relatives are discussed, and their phylogenetic relationships explored.