Advantage of using inosine at the 3' termini of 16S rRNA gene universal primers for the study of microbial diversity

To overcome the shortcomings of universal 16S rRNA gene primers 8F and 907R when studying the diversity of complex microbial communities, the 3' termini of both primers were replaced with inosine. A comparison of the clone libraries derived using both primer sets showed seven bacterial phyla amplified by the altered primer set (8F-I/907R-I) whereas the original set amplified sequences belonging almost exclusively to Proteobacteria (95.8%). Sequences belonging to Firmicutes (42.6%) and Thermotogae (9.3%) were more abundant in a library obtained by using 8F-I/907R-I at a PCR annealing temperature of 54 degrees C, while Proteobacteria sequences were more frequent (62.7%) in a library obtained at 50 degrees C, somewhat resembling the result obtained using the original primer set. The increased diversity revealed by using primers 8F-I/907R-I confirms the usefulness of primers with inosine at the 3' termini in studying the microbial diversity of environmental samples.     

甘蓝型油菜BnCYP78A8的克隆及序列分析

采用Genome walking方法,首次克隆到甘蓝型油菜BnCYP78A8的基因组序列,根据基因特异性引物克隆到其编码序列。基因组序列长1 679bp,有1个内含子和2个外显子。编码序列长1 605bp,编码534个氨基酸。序列比对分析表明,其氨基酸序列与拟南芥细胞色素P450单加氧酶基因(AtCYP78A8)的相似度高达88%。生物信息学分析显示,该蛋白含有1个细胞色素P450特有的亚铁血红素配合基结合位点和1个跨膜结构域。实时荧光定量PCR分析结果表明,BnCYP78A8在甘蓝型油菜的各个器官组织均有表达,根中表达量最高,表明该基因可能参与根的生长发育。     

Enzyme Immunoassay for Cholecystokinin Octapeptide Sulfate and Its Application

A sensitive and specific enzyme-linked immunosorbent assay (ELISA) for cholecystokinin octapeptide sulfate (CCK-8S) has been developed using N-terminal specific antibody for CCK-8S. In this assay CCK-8S coupled with poly-L-Glu (CCK-poly-Glu), which is adsorbed on a solid phase, competes with CCK-8S for the binding sites of rabbit anti-CCK antibody, and the complex of the immobilized antibody and CCK-poly-Glu is measured using goat anti-rabbit immunoglobulin G conjugated with horseradish peroxidase. The total time for completion of the assay is less than 24 h. Near 50% bound levels, the intraassay coefficient of variation is 5.2-6.2% and the interassay coefficient of variation is 5.9-8.5%. This assay is sensitive enough to detect 9 pg of CCK-8S, and the data from rat brain regions using this ELISA are very similar to the data from those using radioimmunoassay (RIA). Therefore, this ELISA is simpler and more rapid in comparison with conventional RIA. In the preliminary experiments, we applied this method for determination of CCK content in the brain regions of adult rats treated with 6-hydroxy-dopamine or in newborn rats subjected to anoxia, and showed that this system is applicable to detection of changes of endogenous CCK content.     

Crystallization and preliminary X-ray crystallographic analysis of alginate lyase A1-II from Sphingomonas species A1

Alginate lyase A1-II of Sphingomonas species A1 was purified and crystallized using the hanging drop vapor-diffusion method in 0.1 M Tris-HCl buffer containing 43% saturated ammonium sulfate, 8% polyethylene glycol 4000 and 0.2 M Li(2)SO(4) at pH 8.5 and 20 degrees C. The crystals are tetragonal and belong to the space group P4(3)2(1)2 or P4(1)2(1)2 with unit cell dimensions of a=b=144.07 and c=296.38 A. The diffraction data up to 2.8 A were collected by a synchrotron radiation source at SPring-8 in Japan.     

应用蛋白质组学筛选宫颈癌患者尿液中的肿瘤标志物

通过比较健康女性和宫颈癌患者的尿蛋白质组,发现并分析差异表达蛋白,从中筛选潜在的宫颈癌的标志物。研究对象由43名宫颈癌患者（CC）和47名健康女性（HW）组成。用超速离心法沉淀尿蛋白,再用一维凝胶电泳（SDS-PAGE）与液相色谱-质谱联用技术（LC-MS/MS）鉴定尿液中的蛋白质,蛋白质定量采用无标定量。比较患者尿蛋白质组、健康对照的尿蛋白质组和宫颈癌组织蛋白质组,有1910个蛋白质是患者和健康对照共有的尿蛋白,这其中有746个蛋白质也存在于宫颈癌组织蛋白质组。在这746个蛋白质中找到84个上调蛋白和82下调蛋白。通过生物信息学分析发现牛皮癣素（S100A7）和癌胚抗原相关细胞黏附分子8（CEACAM8）是宫颈癌尿液样本独有蛋白质。在验证组的70例样本中,双盲法测试S100A7、CEACAM8以及两者联合诊断宫颈癌的敏感性能达到73%、87%、93%。结果提示,宫颈癌患者的尿蛋白质组与健康女性的尿蛋白质组不同,并且S100A7和CEACAM8可以作为宫颈癌潜在的肿瘤标志物。     

Advantage of Using Inosine at the 3′ Termini of 16S rRNA Gene Universal Primers for the Study of Microbial Diversity

To overcome the shortcomings of universal 16S rRNA gene primers 8F and 907R when studying the diversity of complex microbial communities, the 3′ termini of both primers were replaced with inosine. A comparison of the clone libraries derived using both primer sets showed seven bacterial phyla amplified by the altered primer set (8F-I/907R-I) whereas the original set amplified sequences belonging almost exclusively to Proteobacteria (95.8%). Sequences belonging to Firmicutes (42.6%) and Thermotogae (9.3%) were more abundant in a library obtained by using 8F-I/907R-I at a PCR annealing temperature of 54°C, while Proteobacteria sequences were more frequent (62.7%) in a library obtained at 50°C, somewhat resembling the result obtained using the original primer set. The increased diversity revealed by using primers 8F-I/907R-I confirms the usefulness of primers with inosine at the 3′ termini in studying the microbial diversity of environmental samples.     

Comparative studies of phenotypic and genetic characteristics between two desulfurizing isolates of Rhodococcus erythropolis and the well-characterized R. erythropolis strain IGTS8

Two Rhodococcus erythropolis isolates, named A66 and A69, together with the well-characterized R. erythropolis strain IGTS8 were compared biochemically and genetically. Both isolates, like strain IGTS8, desulfurized DBT to 2-hydroxybiphenyl (2-HBP), following the 4S pathway of desulfurization. Strain IGTS8 showed the highest (81.5%) desulfurization activity in a medium containing DBT at 30 °C. Strain A66 showed approximately the same desulfurization activity either when incubated at 30 °C or at 37 °C, while strain A69 showed an increase of desulfurization efficiency (up to 79%) when incubated at 37 °C. Strains A66 and A69 were also able to grow using various organosulfur or organonitrogen-compounds as the sole sulfur or nitrogen sources. The biological responses of A66, A69 and IGTS8 strains to a series of mutagens and environmental agents were evaluated, trying to mimic actual circumstances involved in exposure/handling of microorganisms during petroleum biorefining. The results showed that strains A69 and IGTS8 were much more resistant to UVC treatment than A66. The three desulfurization genes (dszA, dszB and dszC) present in strains A66 and A69 were partially characterized. They seem to be located on a plasmid, not only in the strain IGTS8, but also in A66 and A69. PCR amplification was observed using specific primers for dsz genes in all the strains tested; however, no amplification product was observed using primers for carbazole (car) or quinoline (qor) metabolisms. All this information contributes to broaden our knowledge concerning both the desulfurization of DBT and the degradation of organonitrogen compounds within the R. erythropolis species.     

Antimicrobial Residues in Milk. Comparison of Different Agar Diffusion Methods

Different agar diffusion methods were compared in order to find a sensitive method for the detection of various antimicrobial residues in milk. A total of 588 producer milk samples were analyzed using subsets of the most sensitive methods. With the IDF method, 2 positive cases (0.34 %) appeared among the producer milk samples, with the Thermocult method 13 positive cases (2.21 %) and with the Test agar pH 8 method with trimethoprim and glucose 4 positive cases (0.68 %). A combination of the IDF method and the Test agar pH 8 method resulted in 6 positive cases (1.02 %) and a combination of the Thermocult method and the Test agar pH 8 method in 17 positive cases (2.89 %). With penicillinase 41 % of the positive cases were identified as β-lactam antibiotics and with p-aminobenzoic acid 18 % of the positive cases were identified as sulphonamides. 41 % of the positive cases remained unexplained. The best combination for the detection of antimicrobial agents in milk seems to be that of the Thermocult method and the Test agar pH 8 method with trimethoprim and glucose.     

Hypermethylation of the Ras association domain family 1A (RASSF1A) gene in gallbladder cancer

The tumor suppressor gene Ras association domain family 1A (RASSF1A) is highly methylated in a wide range of human sporadic tumors. The current study investigated the hypermethylation of RASSF1A, the expression of RASSF1A protein, and the correlation between these and the clinicopathological features of gallbladder (GB) cancer in Korean patients. Formalin-fixed, paraffin-embedded tumors and non-neoplastic GB tissues (22 carcinomas, 8 adenomas, 26 normal epithelia) were collected from patients who had undergone surgical resection. The methylation status of two regions of the RASSF1A CpG island was determined by methylation-specific PCR (MSP), and the expression of RASSF1A protein was examined by immunohistochemistry using tissue microarrays. The K-RAS mutation was analyzed by direct sequencing. Methylation of the RASSF1A promoter (region 1) was detected in 22.7% (5/22) of carcinomas, 12.5% (1/8) of adenomas, and 0% (0/26) of normal gallbladder epithelia (P = 0.025). Methylation of the first exon (region 2) was found in 36.4% (8/22) of carcinomas, 25.0% (2/8) of adenomas, and 8.0% (2/26) of normal gallbladder epithelia (P = 0.038). K-RAS mutations were present in 4.5% (1/22) of carcinomas and 25% (2/8) of adenomas. RASSF1A methylaton was not associated with clinicopathological factors or K-ras mutation. Reduction or loss of RASSF1A expression was observed in most methylated adenocarcinomas. Three RASSF1A-expressing human biliary tract cancer cell lines examined contained unmethylated promoters and exons 1. These results suggest that downregulation of RASSF1A expression by DNA hypermethylation may be involved in GB carcinogenesis.     

Nature and biosynthesis of sialic acids in the starfish Asterias rubens. Identification of sialo-oligomers and detection of S-adenosyl-L-methionine: N-acylneuraminate 8-O-methyltransferase and CMP-N-acetylneuraminate monooxygenase activities.

Mass spectrometric and NMR spectroscopic analyses of bound sialic acids from the starfish Asterias rubens revealed the presence of N-acetylneuraminic acid (4%), N-acetyl-8-O-methylneuraminic acid (12%), N-acetyl-9-O-acetyl-8-O-methylneuraminic acid (less than 1%), N-glycoloylneuraminic acid (19%), N-glycoloyl-8-O-methylneuraminic acid (47%), and N-glycoloyl-9-O-acetyl-8-O-methylneuraminic acid (18%). Analysis of sialo-oligomeric material, obtained after mild acid hydrolysis, demonstrated that N-glycoloyl-8-O-methylneuraminic acid can occur as di- and tri-oligomers, linked through the anomeric center and the N-glycoloyl moiety, Neu5Gc8Me-alpha(2----O5)-Neu5Gc8Me and Neu5Gc8Me-alpha(2----O5)-Neu5Gc8Me-alpha (2----O5)-Neu5Gc8Me. Studies on the biosynthesis of N-acyl-8-O-methylneuraminic acid in A rubens, using the tracer S-adenosyl-L-[methyl-14C]methionine, showed that N-acylneuraminate 8-O-methyltransferase activity was present predominantly in the membrane fraction. CMP-N-acetylneuraminic acid monooxygenase activity was found in the soluble protein fraction, in agreement with investigations on the corresponding vertebrate enzyme.     

Comparative analysis of 8-oxoG:C, 8-oxoG:A,A:C and C:C DNA repair in extracts from wild type or 8-oxoG DNA glycosylase deficient mammalian and bacterial cells

We have investigated repair of DNA containing 8-oxoguanine and certain mismatches in cell-free extracts from mouse embryonic fibroblasts (MEFs) using a plasmid substrate with a single lesion at a defined position. Repair synthesis was monitored in a small restriction fragment with different size single strands in order to follow the fate of repair reactions in both strands at the same time. An important part of the study was to assess the role of OGG1 in various repair reactions and the experiments were carried out with extracts from mouse embryonic fibroblasts diploid for a mogg1 deletion (Ogg1(-/-)) as well as wild type. In wild type, DNA containing 8-oxoG:C was repaired in the expected fashion predominantly through short-patch repair. Overall repair was reduced to 20% in the Ogg1(-/-) extracts and to 40% if only long-patch repair was considered. The 8-oxoG:A pair was processed similarly in wild type and Ogg1(-/-) extracts and repair synthesis at A as well as at 8-oxoG could be demonstrated, however, to the same extent in Ogg1(-/-) and wild type for both strands. Extracts from Ogg1(-/-) behaved normally in the correction of A:C and C:C mismatches, with a strong bias for correction of A for A:C and no significant strand discrimination for C:C. Similar experiments with extracts from Escherichia coli showed a 50% reduction in the repair of 8-oxoG:C in fpg extracts and an increase to 50% above wild type in mutY. These results show that the mouse OGG1 is the major enzyme for 8-oxoG repair in the MEF cells and does not participate in mismatch repair of A:C or C:C. Furthermore, 8-oxoG opposite A appears to be repaired by a two-step repair pathway with sequential removal of A and 8-oxoG mediated by enzymes different from OGG1.     

A Yeast-Based Chemical Screen Identifies a PDE Inhibitor That Elevates Steroidogenesis in Mouse Leydig Cells via PDE8 and PDE4 Inhibition

A cell-based high-throughput screen (HTS) was developed to detect phosphodiesterase 8 (PDE8) and PDE4/8 combination inhibitors. By replacing the Schizosaccharomyces pombe PDE gene with the murine PDE8A1 gene in strains lacking adenylyl cyclase, we generated strains whose protein kinase A (PKA)-stimulated growth in 5-fluoro orotic acid (5FOA) medium reflects PDE8 activity. From our previously-identified PDE4 and PDE7 inhibitors, we identified a PDE4/8 inhibitor that allowed us to optimize screening conditions. Of 222,711 compounds screened, ∼0.2% displayed composite Z scores of >20. Additional yeast-based assays using the most effective 367 compounds identified 30 candidates for further characterization. Among these, compound BC8-15 displayed the lowest IC₅₀ value for both PDE4 and PDE8 inhibition in in vitro enzyme assays. This compound also displays significant activity against PDE10A and PDE11A. BC8-15 elevates steroidogenesis in mouse Leydig cells as a single pharmacological agent. Assays using BC8-15 and two structural derivatives support a model in which PDE8 is a primary regulator of testosterone production by Leydig cells, with an additional role for PDE4 in this process. BC8-15, BC8-15A, and BC8-15C, which are commercially available compounds, display distinct patterns of activity against PDE4, PDE8, PDE10A, and PDE11A, representing a chemical toolkit that could be used to examine the biological roles of these enzymes in cell culture systems.     

Qualitative and quantitative determination of sesquiterpenoids in Achillea species by reversed-phase high-performance liquid chromatography, mass-spectrometry and thin-layer chromatography

A reversed-phase high-performance liquid chromatographic method was developed as a universal analysis system in order to determine and quantify antiphlogistic sesquiterpenoids in different Achillea species. Identification was performed by HPLC and diode array detection as well as by monitoring the HPLC fractions by TLC and MS. Using santonin as internal standard, HPLC separations were achieved with a methanol–water gradient system using RP 8 LiChrospher 100 (5 μm) as stationary phase. For validation, sample analyses were performed, using the two tetraploid species A. collina and A. pratensis. The method allows the identification and quantification of the main compounds achillicin, 8α-tigloxy-artabsin, 8α-angeloxy-artabsin, arglanin and santamarin with variation coefficients between 3.4 and 4.7% (total content) using santonin as internal standard. For the different compounds recovery was found between 81 and 107% performing multiple analyses of A. collina and A. pratensis.     

Comparative evaluation of 2,3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) and 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8) rapid colorimetric assays for antimicrobial susceptibility testing of staphylococci and ESBL-producing clinical isolates

A new generation of water soluble tetrazolium salts have recently become available and in this study we compared a colorimetric assay developed using one of these salts, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8), with a previously developed 2,3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) colorimetric assay to determine which agent is most suitable for use as a colorimetric indicator in susceptibility testing. The MICs of 6 antibiotics were determined for 33 staphylococci using both colorimetric assays and compared with those obtained using the British Society for Antimicrobial Chemotherapy reference broth microdilution method. Absolute categorical agreement between the reference and test methods ranged from 79% (cefuroxime) to 100% (vancomycin) for both assays. No minor or major errors occurred using either assay with very major errors ranging from zero (vancomycin) to seven (cefuroxime). Analysis of the distribution of differences in the log(2) dilution MIC results revealed overall agreement, within the accuracy limits of the standard test (+/-1 log(2) dilution), using the XTT and WST-8 assays of 98% and 88%, respectively. Further studies on 31 ESBL-producing isolates were performed using the XTT method with absolute categorical agreement ranging from 87% (nitrofurantoin) to 100% (ofloxacin and meropenem). No errors were noted for either ofloxacin or meropenem with overall agreement of 91%. The data suggests that XTT is more reliable and accurate than WST-8 for use in a rapid antimicrobial susceptibility test.     

Polymorphisms in the RANTES gene increase susceptibility to active tuberculosis in Tunisia

RANTES plays a pivotal role in attracting and activating various leukocyte populations that control Mycobacterium tuberculosis infection. The present study investigated the relationship between the RANTES polymorphisms (-28C/G; rs2280788, and -403G/A; rs2107538) and susceptibility to active tuberculosis (TB) in Tunisian populations. A total of 168 patients with pulmonary TB (pTB), 55 with extrapulmonary TB (epTB), and 150 control subjects were studied. Genotype analyses were carried out using polymerase chain reaction-restriction fragment length polymorphism method. We found that the -28 GG genotype was significantly associated with susceptibility to pTB (odds ratio [OR]=11.19; 95% confidence intervals [CI], 5.14-25; P corrected for the number of genotypes [Pc]=10(-8)) and epTB (OR=11.67; 95% CI, 4.74-29.33; Pc=10(-8)). However, the -28 CC genotype was found to be significantly associated with resistance to pTB (OR=0.08; 95% CI, 0.04-0.16; Pc=10(-8)) and epTB development (OR=0.11; 95% CI, 0.05-0.27; Pc=10(-8)). -403A allele was associated with increased risk development of epTB (OR=2.21; 95% CI, 1.18-4.14; p=0.007). G-G and A-C haplotypes and the AG/GC diplotype were associated with increase susceptibility to pTB (OR=7.88, 95% CI, 5.38-11.55; Pc=3.10(-8); OR=2.32, 95% CI, 1.32-4.11; Pc=3.10(-3); OR=13.26, 95% CI, 6.06-29.89; Pc=3.10(-8); respectively) and epTB (OR=6.64, 95% CI, 4-11.05; Pc=3.10(-8); OR=2.6, 95% CI, 1.26-5.35; Pc=12.10(-3); OR=11.26, 95% CI, 4.44-29.28; Pc=3.10(-8); respectively). Collectively, our findings suggested an association of the RANTES -28C/G and -403G/A functional polymorphisms with susceptibility to Mycobacterium tuberculosis infection in Tunisian populations.     

The effect of iron on ferritin turnover in rat liver

125I-labelled angiotensin II (A II) specifically binds to solubilized receptors extracted from rat isolated glomeruli using CHAPS (3-[3-( cholamidopropyl ) dimethylammonio ]-1-propanesulfonate). The yield of solubilization of the binding sites was 3.3%. Equilibrium was reached after 15-20 min and specific binding represented 75% of total binding. Dissociation of the hormone-receptor complex after addition of an excess of A II was very slow in the presence of Ca2+ and Mg2+. [Sar1 Ala8] A II and [Sar1 Ile8] A II were more potent as competitive inhibitors of 125I-labelled A II than A II itself and its heptapeptide. These basic features of 125I-labelled A II binding to the extracted material were similar to those observed previously with untreated glomeruli.     

Control of the production of individual fusicoccins at different dissolved oxygen concentrations

The regulatory effect of different concentrations of dissolved oxygen on the production of fusicoccins by the fungus Fusicoccum amygdali Del. was studied. The maximum output of total fusicoccins was obtained by using a profiled dissolved oxygen tension (DOT) regime, in which the DOT was maintained at 15–20% during the biomass growth phase and at 5–8% during the fusicoccins production phase. In comparison with the profiled regime, the maintenance of DOT at 15–20% during the whole fermentation shortened the fusicoccins production phase. The fermentation performance at a low DOT (5–8%) inhibited both the accumulation of biomass and the production of fusicoccins. At high DOT (40–50%), an accelerated accumulation of the biomass with an expressed autolysis of mycelia took place, and the production of fusicoccins was lowered. The qualitative composition of individual fusicoccins varied substantially at different DOTs. Fusicoccins, A, C, D, J, H, 16-O-demethyl-J, detretpentenylfusicoccin and some minor fusicoccin metabolites were found in the fermentation broth using the method of liquid secondary ion mass spectrometry. It was established that the profiled DOT regime (15–20% to 5–8%) provided both the maximum concentration of fusicoccins and an enhanced accumulation of the main metabolite – fusicoccin A (FC A). The performance of the fermentation at a DOT of 15–20% decreased the content of FC A by 2–6% in comparison with the profiled DOT regime, and increased the content of fusicoccin C to 14–20% of the total fusicoccins. Fermentation at DOT of 5–8% was characterized by the highest content of the precursors of FC A, the less oxidized fusicoccins H and J, the contents of which were in range 7–12% and 16–17% of total fusicoccins, respectively.     

Characterization of a Novel Serotonin Receptor Subtype (5-HTls) in Rat CNS: Interaction with a GTP Binding Protein

Three pharmacologically distinct high-affinity [3H]serotonin ([3H]5-HT) binding sites were identified in spinal cord synaptosomes. [3H]5-HT competition studies using selective 5-HT1A receptor ligands indicated that approximately 25% of high-affinity synaptosomal [3H]5-HT binding was inhibited by 5-HT1A-selective compounds, an estimate consistent with [3H](+-)-8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) saturation experiments in which 5-HT1A receptors were directly labeled. [3H]5-HT competition studies using high-affinity 5-HT1B compounds performed in the presence of 100 nM 8-OH-DPAT (to block 5-HT1A receptors) indicated that approximately 26% of all specific, high-affinity [3H]5-HT binding to spinal cord synaptosomes was to 5-HT1B receptors. [3H]5-HT competition studies performed in the presence of 100 nM 8-OH-DPAT and 10 nM RU 24969 (to block 5-HT1A and 5-HT1B receptors, respectively) indicated that the remaining 49% of [3H]5-HT binding did not possess the pharmacologic profile previous reported for 5-HT1C, 5-HT1D, 5-HT1E, 5-HT2, or 5-HT3 receptors. This residual 49% of [3H]5-HT binding to spinal cord synaptosomes observed in the presence of 100 nM 8-OH-DPAT and 10 nM RU 24969 (subsequently referred to as "5-HT1S") displayed high affinity and saturability (KD = 4.7 nM) in association/dissociation and saturation experiments. Addition of 300 microM GTP or the nonhydrolyzable form of GTP, 5'-guanylylimidodiphosphate, inhibited [3H]5-HT binding to 5-HT1S receptors in saturation experiments by 35 and 57%, respectively, whereas ATP was without effect.(ABSTRACT TRUNCATED AT 250 WORDS)