缢蛏C型凝集素基因ScCTL-2的序列分析及凝菌活性

为研究缢蛏C型凝集素基因ScCTL-2的结构和功能特点,克隆了该基因的全长序列,并对其mRNA的表达模式以及重组蛋白的凝菌活性进行研究。结果显示,ScCTL-2的cDNA全长2 194 bp,共编码630个氨基酸,其氨基酸序列拥有4个C型凝集素糖识别结构域和1个N端跨膜区,这一特殊的序列结构不同于任何一种已知的C型凝集素。基因mRNA在健康缢蛏肝胰腺中的表达量显著高于其他组织,在鳃中的表达量次之,而在其余组织中的表达量没有显著性差异;金黄色葡萄球菌与鳗弧菌感染能够显著上调ScCTL-2在缢蛏血细胞中的表达。ScCTL-2的重组蛋白凝菌实验显示:在Ca~(2+)存在的情况下,rScCTL-2能够对实验所采用的金黄色葡萄球菌、枯草芽孢杆菌、大肠杆菌、藤黄微球菌和酿酒酵母均产生明显的凝集反应;当溶液中不存在Ca~(2+)时,凝集反应几乎不能发生,意味着rScCTL-2的凝菌活性是严格Ca~(2+)依赖的。研究表明,ScCTL-2可能是一种新的无脊椎动物C型凝集素类型,它具有无脊椎动物C型凝集素普遍具备的免疫活性,可能在缢蛏的免疫中具有重要作用。     

Innate lymphoid cells promote anatomical containment of lymphoid-resident commensal bacteria

The mammalian intestinal tract is colonized by trillions of beneficial commensal bacteria that are anatomically restricted to specific niches. However, the mechanisms that regulate anatomical containment remain unclear. Here, we show that interleukin-22 (IL-22)-producing innate lymphoid cells (ILCs) are present in intestinal tissues of healthy mammals. Depletion of ILCs resulted in peripheral dissemination of commensal bacteria and systemic inflammation, which was prevented by administration of IL-22. Disseminating bacteria were identified as Alcaligenes species originating from host lymphoid tissues. Alcaligenes was sufficient to promote systemic inflammation after ILC depletion in mice, and Alcaligenes-specific systemic immune responses were associated with Crohn's disease and progressive hepatitis C virus infection in patients. Collectively, these data indicate that ILCs regulate selective containment of lymphoid-resident bacteria to prevent systemic inflammation associated with chronic diseases.     

The antibacterial lectin RegIIIgamma promotes the spatial segregation of microbiota and host in the intestine

The mammalian intestine is home to ~100 trillion bacteria that perform important metabolic functions for their hosts. The proximity of vast numbers of bacteria to host intestinal tissues raises the question of how symbiotic host-bacterial relationships are maintained without eliciting potentially harmful immune responses. Here, we show that RegIIIγ, a secreted antibacterial lectin, is essential for maintaining a ~50-micrometer zone that physically separates the microbiota from the small intestinal epithelial surface. Loss of host-bacterial segregation in RegIIIγ(-/-) mice was coupled to increased bacterial colonization of the intestinal epithelial surface and enhanced activation of intestinal adaptive immune responses by the microbiota. Together, our findings reveal that RegIIIγ is a fundamental immune mechanism that promotes host-bacterial mutualism by regulating the spatial relationships between microbiota and host.     

Interactions between commensal fungi and the C-type lectin receptor Dectin-1 influence colitis

The intestinal microflora, typically equated with bacteria, influences diseases such as obesity and inflammatory bowel disease. Here, we show that the mammalian gut contains a rich fungal community that interacts with the immune system through the innate immune receptor Dectin-1. Mice lacking Dectin-1 exhibited increased susceptibility to chemically induced colitis, which was the result of altered responses to indigenous fungi. In humans, we identified a polymorphism in the gene for Dectin-1 (CLEC7A) that is strongly linked to a severe form of ulcerative colitis. Together, our findings reveal a eukaryotic fungal community in the gut (the "mycobiome") that coexists with bacteria and substantially expands the repertoire of organisms interacting with the intestinal immune system to influence health and disease.     

Intestinal microbiota promote enteric virus replication and systemic pathogenesis

Intestinal bacteria aid host health and limit bacterial pathogen colonization. However, the influence of bacteria on enteric viruses is largely unknown. We depleted the intestinal microbiota of mice with antibiotics before inoculation with poliovirus, an enteric virus. Antibiotic-treated mice were less susceptible to poliovirus disease and supported minimal viral replication in the intestine. Exposure to bacteria or their N-acetylglucosamine-containing surface polysaccharides, including lipopolysaccharide and peptidoglycan, enhanced poliovirus infectivity. We found that poliovirus binds lipopolysaccharide, and exposure of poliovirus to bacteria enhanced host cell association and infection. The pathogenesis of reovirus, an unrelated enteric virus, also was more severe in the presence of intestinal microbes. These results suggest that antibiotic-mediated microbiota depletion diminishes enteric virus infection and that enteric viruses exploit intestinal microbes for replication and transmission.     

Mammalian ribosomal protein: analysis by electrophoresis on polyacrylamide gel

Ribosomal protein from five mammalian tissues when analyzed by discontinuous electrophoresis on polyacrylamide gel at pH 4.5 yielded 24 bands. Densitometric tracings indicated that the patterns of the basic ribosomal proteins from the several tissues were qualitatively similar. Protein from Escherichia coli ribosomes analyzed at pH 4.5 gave 29 bands, and the pattern was different from that of mammalian ribosomal protein. No distinct band was found when mammalian ribosomal protein was analyzed at pH 8.3 (acidic proteins). Ribosomal protein from Escherichia coli gave eight bands at pH 8.3. Thus, the structure of the genes responsible for synthesis of ribosomal protein in several mammalian tissues is the same, and different genes direct synthesis of ribosomal protein in bacteria.     

High-frequency C-type virus induction by inhibitors of protein synthesis

When inhibitors of protein synthesis are added to BALB/c mouse cells in culture, induction of naturally integrated C-type RNA virus occurs in a high percentage of cells. The action of protein synthesis inhibitors differs from that of halogenated pyrimidines, another class of virus inducers, in their effects on biologically distinguishable viruses. The use of such inhibitors to study integrated virus expression provides a means for studying gene regulation in mammalian cells.     

Developmentally regulated piRNA clusters implicate MILI in transposon control

Nearly half of the mammalian genome is composed of repeated sequences. In Drosophila, Piwi proteins exert control over transposons. However, mammalian Piwi proteins, MIWI and MILI, partner with Piwi-interacting RNAs (piRNAs) that are depleted of repeat sequences, which raises questions about a role for mammalian Piwi's in transposon control. A search for murine small RNAs that might program Piwi proteins for transposon suppression revealed developmentally regulated piRNA loci, some of which resemble transposon master control loci of Drosophila. We also find evidence of an adaptive amplification loop in which MILI catalyzes the formation of piRNA 5' ends. Mili mutants derepress LINE-1 (L1) and intracisternal A particle and lose DNA methylation of L1 elements, demonstrating an evolutionarily conserved role for PIWI proteins in transposon suppression.     

tlr4基因对羊肠道细菌影响的研究

【目的】通过分析tlr4基因对羊肠道主要细菌数量的影响,评价转基因羊tlr4的生物安全性。【方法】收集15只转基因羊tlr4和15只非转基因羊的粪便样本,采用传统平板培养法和实时荧光定量PCR法(quantitative Real-Time PCR,qPCR)分析转基因羊tlr4和非转基因羊肠道中主要细菌数量的变化。【结果】传统培养法的结果表明,转tlr4基因对羊肠道中大肠杆菌(E.coli)、沙门氏菌(Salmonella)、双歧杆菌(Bifidobacterium)、粪肠球菌(Enterococcus)和金黄色葡萄球菌(Staphylococcus)的生长均有一定程度的抑制作用,在数量上转基因羊tlr4和非转基因羊的差异不显著(P0.05);qPCR法的结果表明,转tlr4基因对羊肠道中大肠杆菌、双歧杆菌、拟杆菌(Bacteroides)和粪肠球菌的数量亦无显著性影响(P0.05)。【结论】显示抗病基因tlr4处理对羊肠道主要细菌基本无显著性影响。     

A binding protein for fatty acids in cytosol of intestinal mucosa, liver, myocardium, and other tissues

A protein of molecular weight approximately 12,000 which binds long-chain fatty acids and certain other lipids has been identified in cytosol of intestinal mucosa, liver, myocardium, adipose tissue, and kidney. Binding is noncovalent and is greater for unsaturated than for saturated and medium-chain fatty acids. This protein appears to be identical with the smaller of two previously described cytoplasmic anion-binding proteins. Binding of long-chain fatty acids by this protein is greater than that of other anions tested, including sulfobromophthalein, and does not depend on negative charge alone. The presence of this binding protein may explain previously observed differences in intestinal absorption among fatty acids, and the protein may participate in the utilization of long-chain fatty acids by many mammalian tissues.     

Bt蛋白对拟环纹豹蛛肠道细菌多样性的影响

采用16S r DNA分子生物学分析方法,分别对试验组(喂食含Cry1Ab蛋白培养基上饲养的果蝇)和对照组(喂食普通培养基上饲养的果蝇)的拟环纹豹雌、雄蛛肠道细菌进行分析。发现,拟环纹豹蛛肠道细菌丰富,雌蛛肠道细菌丰富度大于雄蛛肠道细菌丰富度;优势菌属分别是短杆菌属(Brevibacterium)、短波单胞菌属(Brevundimonas)、葡萄球菌属(Staphylococcus)、乳杆菌属(Lactobacillus)、丛毛单胞菌属(Comamonas)、德沃斯氏菌属(Devosia)、希瓦氏菌属(Shewanella)、Leucobacter、杆菌属(Brachybacterium)、梭菌属(Clostridium);Bt蛋白对拟环纹豹蛛肠道细菌多样性有一定的影响,对放线菌属(Actinomycetes)的影响最小,对乳杆菌属(Lactobacillus)和梭菌属(Clostridium)的影响较大。     

Linkage of lactate dehydrogenase B and C loci in pigeons

Synthesis of lactate dehydrogenase in somatic and gametic tissues of certain avian and mammalian species is controlled by alleles at three loci, A, B, and C. We report breeding experiments with pigeons that conclusively demonstrate linkage between the B and C structural loci in this species. The most probable recombination fraction is zero, and contiguity is not excluded. The upper 95 percent probability limit is 4.5 percent. This tight linkage of two loci that produce closely similar polypeptides suggests that the loci acquired their separate identities through a duplication event. Further-more, the existence of recognizable B- and C-type polypeptides in both the bird and the mammal suggests that the event and the resulting linkage preceded the separation of these fauna. If so, then the linkage has persisted for a very long time.     

Induction of protective IgA by intestinal dendritic cells carrying commensal bacteria

The enormous number of commensal bacteria in the lower intestine of vertebrates share abundant molecular patterns used for innate immune recognition of pathogenic bacteria. We show that, even though commensals are rapidly killed by macrophages, intestinal dendritic cells (DCs) can retain small numbers of live commensals for several days. This allows DCs to selectively induce IgA, which helps protect against mucosal penetration by commensals. The commensal-loaded DCs are restricted to the mucosal immune compartment by the mesenteric lymph nodes, which ensures that immune responses to commensal bacteria are induced locally, without potentially damaging systemic immune responses.     

动物肠道放线菌的研究展望

动物肠道作为一个典型的、独特的环境,其肠道微生物的优势菌群是放线菌。过去将动物粪便为对象开展肠道放线菌资源研究的极少。本文对动物肠道放线菌的研究价值、菌种的多样性及其生物活性物质做了简要概述,其中肠道放线菌中具有抗肿瘤活性的菌株较多,所产生的生物活性物质还具有抗菌性,且酶活性比例较高。因此,动物肠道放线菌是开发药物和其他产品的重要来源,是一种新的微生物资源,具有巨大的开发潜力。     

人工养殖对虾肠道内可培养细菌数量及组成分析

为深入了解人工养殖条件下养殖对虾肠道内菌群结构和携带病毒情况,应用常规细菌分离、培养与纯化,细菌16S rD NA序列分析的方法分析了我国山东、江苏、韩国不同养殖场的凡纳滨对虾(Litopenaeus vannamei)和中国明对虾(Fenneorpenaeus chinensis)肠道内可培养细菌的总数、优势菌组成和数量,并用巢式聚合酶链式反应(Nested polymerase chain reaction,Nested PCR)方法检测对虾携带病毒情况。结果显示各批次凡纳滨对虾和中国明对虾样品肠道内的可培养细菌总数在105~109cfu/g之间,并对分离出的优势菌进行属(种)鉴定,结果表明这些优势菌分别属于乳球菌属(Lactococcus sp.)、弧菌属(Vibrio sp.)、芽孢杆菌属(Bacillus sp.)、发光杆菌属(Photobacterium sp.)、希瓦氏菌属(Shewanella sp.)、节杆菌属(Arthrobacter sp.)、微小杆菌属(Microbacterium sp.)。凡纳滨对虾和中国明对虾均有样品检测为WSSV阳性,6批次WSSV阳性对虾样品中均检测到弧菌属细菌,占可培养细菌比例为33%~93.58%。2批次WSSV阳性对虾样品中检测到希瓦氏菌属细菌,占可培养细菌比例为21.67%~34.21%。4批次WSSV阳性对虾样品中检测到发光杆菌属细菌,占可培养细菌比例为21.03%~66.83%。     

Evolution of key cell signaling and adhesion protein families predates animal origins

The evolution of animals from a unicellular ancestor involved many innovations. Choanoflagellates, unicellular and colonial protozoa closely related to Metazoa, provide a potential window into early animal evolution. We have found that choanoflagellates express representatives of a surprising number of cell signaling and adhesion protein families that have not previously been isolated from nonmetazoans, including cadherins, C-type lectins, several tyrosine kinases, and tyrosine kinase signaling pathway components. Choanoflagellates have a complex and dynamic tyrosine phosphoprotein profile, and cell proliferation is selectively affected by tyrosine kinase inhibitors. The expression in choanoflagellates of proteins involved in cell interactions in Metazoa demonstrates that these proteins evolved before the origin of animals and were later co-opted for development.     

Multiple effects of grape seed polyphenolics to prevent metabolic diseases

Obesity is increasing in developing countries. Population studies show a relationship between affluence and obesity. Changing food intake patterns with affluence such as preference for foods with less astringent polyphenolic compounds and dietary fibers may increase risk of metabolic dysfunctions due to caloric imbalance. Animal models of obesity consistently show that grape seed procyanidins prevent increases in body and abdo- minal adipose weight gain, plasma cholesterol, liver weight gain and inflammation in animals on high fat diets. The mechanisms are not clear because the oral intake of procyanidins results in pleiotropic interactions with proteins in the mouth, stomach, small intestine, cecum and colon that affect the rate of digestion of bioavailability of macronutrients, sterols, and dietary fiber. Procyanidins also bind bile acids and reduce intestinal permeability to inflammatory bacterial cell wall fragment. Procyanidins are not degraded or metabolized until reaching the lower gut where they can be metabolized into phenolic acids by gut bacteria. While they are metabolized by gut bacteria, they also alter total numbers and distribution of phyla and species of gut bacteria. Gut bacteria are recognized as significant contributors to obesity and obesity related metabolic diseases. The review examines the different pleiotropic effects of grape seed procyanidins that have a significant effect on metabolic disease in animal models of obesity.     

粘虫肠道细菌群落多样性的PCR DGGE指纹图谱分析

研究东方粘虫[ Myth imna se parata (Walker)]肠道细菌的多样性.采用常规分离培养与分子鉴定相结合的方法,并以16S rDNA作为分子标记的变性梯度凝胶电泳(DGGE)对肠道细菌进一步分离,DGGE分离样品是研磨提取粘虫中肠组织DNA、提取培养混合菌DNA及直接以培养混合菌为模板进行PCR扩增的产物.结果表明,共得到DGGE条带19条,其中肠组织研磨提取DNA的DGGE条带最多,为17条;直接以培养混合菌液为模板进行PCR扩增次之,为13条;培养混合菌液提取DNA的条带最少,为12条.传统方法和分子生物学方法相结合研究肠道微生物多样性时能获得更全面的微生物信息,可采用培养混合菌提取DNA再结合其他方法进行后续研究.     

Alleviating cancer drug toxicity by inhibiting a bacterial enzyme

The dose-limiting side effect of the common colon cancer chemotherapeutic CPT-11 is severe diarrhea caused by symbiotic bacterial β-glucuronidases that reactivate the drug in the gut. We sought to target these enzymes without killing the commensal bacteria essential for human health. Potent bacterial β-glucuronidase inhibitors were identified by high-throughput screening and shown to have no effect on the orthologous mammalian enzyme. Crystal structures established that selectivity was based on a loop unique to bacterial β-glucuronidases. Inhibitors were highly effective against the enzyme target in living aerobic and anaerobic bacteria, but did not kill the bacteria or harm mammalian cells. Finally, oral administration of an inhibitor protected mice from CPT-11-induced toxicity. Thus, drugs may be designed to inhibit undesirable enzyme activities in essential microbial symbiotes to enhance chemotherapeutic efficacy.