非小细胞肺癌中血管生成拟态和VEGF的表达及意义

目的探讨血管生成拟态(vasculogenic mimicry,VM)与血管内皮生长因子(vascular epithelial growth factor,VEGF)在非小细胞肺癌(non-small cell lung cancer,NSCLC)中的表达及意义。方法收集NSCLC术后标本160例和20例正常肺组织,应用免疫组化法和组织化学法检测NSCLC和正常肺组织中VM和VEGF的表达情况。结果在NSCLC组织和正常肺组织中,VM和VEGF的阳性率分别为36.9%、51.3%和0%、0%,差异有统计学意义;含有VM的NSCLC的VEGF表达高于无VM者(P<0.05),且VM与NSCLC的组织学分级、淋巴结转移及临床分期等有关(P<0.05);多因素分析:PTNM分期、VM、VEGF的表达是影响NSCLC根治术后患者预后的独立因素(P<0.05);VM阳性组与阴性组的5年生存率分别为1.7%和41.6%,差异有统计学意义;VEGF阳性组与阴性组的5年生存率分别为2.4%和52.6%,差异有统计学意义。结论具有VM的NSCLC组织分化低,患者临床预后差;VEGF的表达水平和VM与NSCLC的发展及预后有一定的关系。     

Energetics of SecA dimerization

Transport of many proteins to extracytoplasmic locations occurs via the general secretion (Sec) pathway. In Escherichia coli, this pathway is composed of the SecYEG protein-conducting channel and the SecA ATPase. SecA plays a central role in binding the signal peptide region of preproteins, directing preproteins to membrane-bound SecYEG and promoting translocation coupled with ATP hydrolysis. Although it is well established that SecA is crucial for preprotein transport and thus cell viability, its oligomeric state during different stages of transport remains ill defined. We have characterized the energetics of SecA dimerization as a function of salt concentration and temperature and defined the linkage of SecA dimerization and signal peptide binding using analytical ultracentrifugation. The use of a new fluorescence detector permitted an analysis of SecA dimerization down to concentrations as low as 50 nM. The dimer dissociation constants are strongly dependent on salt. Linkage analysis indicates that SecA dimerization is coupled to the release of about five ions, demonstrating that electrostatic interactions play an important role in stabilizing the SecA dimer interface. Binding of signal peptide reduces SecA dimerization affinity, such that K_d increases about 9-fold from 0.28 μM in the absence of peptide to 2.68 μM in the presence of peptide. The weakening of the SecA dimer that accompanies signal peptide binding may poise the SecA dimer to dissociate upon binding to SecYEG.     

金黄色葡萄球菌hmp基因缺失突变株的构建及抗一氧化氮能力分析

摘要：【目的】:构建金黄色葡萄球菌RN6390黄素血红蛋白(flavohaemoglobin, HMP)基因缺失突变株,研究其抗一氧化氮(Nitric Oxide, NO) 能力及其在细菌生物被膜形成中的作用。【方法】：根据同源重组技术的原理,利用PCR扩增RN6390的hmp基因上下游同源臂,经过抗生素和温度交替培养筛选hmp基因缺失突变株,利用基因组PCR、定量PCR对突变菌株进行鉴定。以硝普钠（SNP）为NO供体,检测了hmp基因缺失菌株的抗NO能力,并初步研究了hmp基因在生物被膜形成中的作用。【结果】：成功构建了RN6390的hmp基因缺失突变株,外源NO能够诱导菌株hmp基因的表达,hmp基因缺失菌株抗NO能力明显下降,但其生物被膜形成能力有明显提高。【结论】：获得了RN6390的hmp基因缺失突变株,该突变株的获得为进一步研究hmp基因的生物功能,以及细菌内源性NO的作用奠定了良好的技术平台。     

Morphological characteristics of natural strains of certain species of basidiomycetes and biological analysis of antimicrobial activity under submerged cultural conditions

Twenty-one strains belonging to 18 species of basidiomycetes from different ecological groups of fungi were isolated from natural sources. Light and electron microscopy was used to determine the morphological properties of the cultures, which confirmed their classification as basidiomycetes and facilitated their identification in monocultures. The capacity of the fungal strains for biosynthesis of antibiotics was determined by one- or two-stage cultivation on seven nutrient media. It was established that, under submerged cultivation, antimicrobial substances were formed by 13 strains (81.25%) of 12 fungal species (Armillaria sp., Coprinus comatus, Flammulina velutipes, Hypsizygus ulmarius, Lentinus tigrinus, Lycoperdon pyriforme, Macrolepiota procera, Panellus serotinus, Pholiota aurivella, Pholiota lenta, Rhodocollybia maculate, and Sparassis crispa). The antibiotics formed were efficacious against bacterial test strains, including the methicillin-resistant strain Staphylococcus aureus (MRSA) and the strain Leuconostoc mesenteroides VKPM B-4177 that is resistant to the glycopeptide antibiotics. No antibiotic activity was revealed against fungal test cultures (Aspergillus niger INA 00760 and Saccharomyces cerevisiae RIA 259).     

Evaluation of inert and organic carriers for <Emphasis Type="Italic">Verticillium lecanii</Emphasis> spore production in solid-state fermentation

Growth and sporulation of Verticillium lecanii on inert and organic carriers (sugar-cane bagasse, corncob, rice straw, polyurethane foam and activated carbon) in a solid-state fermentation process was studied. Sugar-cane bagasse and polyurethane foam produced 10¹⁰ spores g⁻¹ dry carrier whereas corncob, rice straw, and activated carbon yielded, respectively 8 × 10⁹, 4 × 10⁹, and 3 × 10⁸ spores g⁻¹. Chitinase activity of the conidia was in the following order: sugar-cane bagasse (3.3 U mg⁻¹) > wheat bran (3.0 U mg⁻¹) > polyurethane foam (2.7 U mg⁻¹). There was no significant difference (2.5–2.7 U mg⁻¹) in the proteinase activity among the conidia from the three cultures. Scanning electron microscopy shows that aerial mycelium freely penetrated into the internal area of polyurethane foam. Sugar-cane bagasse provided enough area for vegetative hyphae to attach. Of the carriers analyzed, polyurethane foams and sugar-cane bagasse were the best carriers for V. lecanii growth and spore production.     

Effects of 20-HETE on Na+ transport and Na+ -K+ -ATPase activity in the thick ascending loop of Henle

Previous studies have indicated that 20-hydroxyeicosatetraenoic acid (20-HETE) inhibits Na+ transport in the medullary thick ascending loop of Henle (mTALH), but the mechanisms involved remain uncertain. The present study compared the effects of 20-HETE with those of ouabain and furosemide on intracellular Na+ concentration ([Na+]i), Na+ -K+ -ATPase activity, and 86Rb+ uptake, an index of Na+ transport, in mTALH isolated from rats. Ouabain (2 mM) increased, whereas furosemide (100 microM) decreased, [Na+]i in the mTALH of rats. Ouabain and furosemide inhibited 86Rb+ uptake by 91 and 30%, respectively. 20-HETE (1 microM) had a similar effect as ouabain and increased [Na+]i from 19 +/- 1 to 30 +/- 1 mM. 20-HETE reduced Na+ -K+ -ATPase activity by 30% and 86Rb+ uptake by 37%, but it had no effect on 86Rb+ uptake or [Na+]i in the mTALH of rats pretreated with ouabain. 20-HETE inhibited 86Rb+ uptake by 12% and increased [Na+]i by 19 mM in mTALH pretreated with furosemide. These findings indicate that 20-HETE secondarily inhibits Na+ transport in the mTALH of the rat, at least, in part by inhibiting the Na+ -K+ -ATPase activity and raising [Na+]i.     

Ethylene synthesis in petunia stigma tissues governs the growth of pollen tubes in progamic phase of fertilization

In the pollen-pistil system of petunia (Petunia hybrida L.) self-compatible and self-incompatible clones within 7 h after self-pollination, we determined the content of ACC (1-aminocyclopropane-1-carboxylic acid), the activity of two enzymes (ACC synthase and ACC oxidase), and the rate of ethylene production. Depending on the type of pollination, germination of pollen on the stigma surface and the pollen tube growth in the tissues of style were accompanied by different levels of ACC and ethylene release. The pollen-pistil system of the self-compatible clone contained twice more ACC than in the self-incompatible clone, whereas the pollen-pistil system in the self-incompatible clone produced 4–5 times more ethylene than in the self-compatible clone. For both types of pollination, ACC and ethylene were predominantly produced in the stigma tissues. The rate of ethylene production therein was 50 times greater than in the styles and ovaries, and the content of ACC was 100 times higher than in the styles and ovaries. Germination of male gametophyte after both types of pollination was accompanied by elevated ACC synthase activity (especially in the case of compatible pollination), whereas notable increase in ACC oxidase activity was manifested in growing pollen tubes after self-incompatible pollination     

Inhibitory effect of naringenin chalcone on inflammatory changes in the interaction between adipocytes and macrophages

Obese adipose tissue is characterized by an enhanced infiltration of macrophages. It is considered that the paracrine loop involving monocyte chemoattractant protein (MCP)-1 and tumor necrosis factor (TNF)-alpha between adipocytes and macrophages establishes a vicious cycle that augments the inflammatory changes and insulin resistance in obese adipose tissue. Polyphenols, which are widely distributed in fruit and vegetables, can act as antioxidants and some of them are also reported to have anti-inflammatory properties. Tomato is one of the most popular and extensively consumed vegetable crops worldwide, which also contains many flavonoids, mainly naringenin chalcone. We investigated the effect of flavonoids, including naringenin chalcone, on the production of proinflammatory mediators in lipopolysaccharide (LPS)-stimulated macrophages and in the interaction between adipocytes and macrophages. Naringenin chalcone inhibited the production of TNF-alpha, MCP-1, and nitric oxide (NO) by LPS-stimulated RAW 264 macrophages in a dose-dependent manner. Coculture of 3T3-L1 adipocytes and RAW 264 macrophages markedly enhanced the production of TNF-alpha, MCP-1, and NO compared with the control cultures; however, treatment with naringenin chalcone dose-dependently inhibited the production of these proinflammatory mediators. These results indicate that naringenin chalcone exhibits anti-inflammatory properties by inhibiting the production of proinflammatory cytokines in the interaction between adipocytes and macrophages. Naringenin chalcone may be useful for ameliorating the inflammatory changes in obese adipose tissue.     

Combinatorial Screening and Intracellular Antiviral Activity of Hairpin Ribozymes Directed against Hepatitis B Virus

A combinatorial screening method has been used to identify hairpin ribozymes that inhibit hepatitis B virus (HBV) replication in transfected human hepatocellular carcinoma (HCC) cells. A hairpin ribozyme library (5 x 10(5) variants) containing a randomized substrate-binding domain was used to identify accessible target sites within 3.3 kb of full-length in vitro-transcribed HBV pregenomic RNA. Forty potential target sites were found within the HBV pregenomic RNA, and 17 sites conserved in all four subtypes of HBV were chosen for intracellular inhibition experiments. Polymerase II and III promoter expression constructs for corresponding hairpin ribozymes were generated and cotransfected into HCC cells together with a replication-competent dimer of HBV DNA. Four ribozymes inhibited HBV replication by 80, 69, 66, and 49%, respectively, while catalytically inactive mutant forms of these ribozymes affected HBV replication by 36, 28, 0, and 0%. These findings indicate that the inhibitory effects on HBV replication were largely mediated by the catalytic activity of the ribozymes. In conclusion, we have identified catalytically active RNAs by combinatorial screening that mediate intracellular antiviral effects on HBV.     

Comparative analysis of genome sizes of <Emphasis Type="Italic">Streptococcus thermophilus</Emphasis> strains

Twenty-five Streptococcus thermophilus isolates were analyzed using pulse-field gel electrophoresis (PFGE) and gene restriction profile analysis techniques. 16S rRNA gene sequences of the isolates were almost 100% homologous. However, genomic fingerprinting analysis has shown variability in both genome size and restriction fragments length. The genomes varied from 1417 to 2075 kb resulting in the difference between marginal genome sizes in about 600 kb. The results are indicative of Streptococcus thermophilus intraspecies genetic polymorphism, the origin of which requires further investigation.