肠道中特异性菌群与结直肠癌的发生发展关系

结直肠癌（colorectal cancer,CRC）是常见的恶性肿瘤之一,并且发病率有逐年上升的趋势。建立有效的早期筛查方法是当今防治结直肠癌的首要任务。随着肠道菌群研究的深入,越来越多的研究结果显示肠道菌群与CRC的发生发展有一定的关系,尤其是具核梭杆菌、产肠毒素脆弱拟杆菌、大肠埃希菌等这些特异性菌群与CRC的发展密切相关。本文结合国内外最新研究进展,就这些特异性菌群在CRC发病机制中的作用作一综述,为进一步研究CRC和实现对其早期筛查提供新的思路。     

Construction of normal human IgE phage antibody library and the screening of the anti—trichosanthin IgE

Allergen specific IgE response is the major cause of immediate hypersensitivity.However the number of IgEproducing B cells and the amount of IgE,especially the specific IgE,are so low,it greatly impedes the study of the allergic-specifc antibody responses.Here we report the construction of a normal human IgE combinatorial library.The repertoire of IgE VH genes and of κ genes were separately amplified from normal human peripheral blood lymphocytes through RT-PCR,and were then constructed to form the phage surface display human Fab(IgEVH) library.A plant protein allergen,trichosanthin(TCS),was used to affinity-enrich and to screen the anti-TCS phage HuFab clones from the library.Human IgE(Fab) to TCS were detected.     

Role of neuraminidase-dependent adherence in Bacteroides fragilis attachment to human epithelial cells

Of 50 B. fragilis strains isolated from clinical samples we have demonstrated that 24 (48%) possess an adhesin that mediates a neuraminidase-dependent attachment of B. fragilis to mammalian epithelial cells, but does not mediate any association with human polymorphonuclear leucocytes. This ligand interacts with a mammalian cell receptor that contains a galactoside residue, exposed after neuraminidase pretreatment. Our results suggest a possible role for cell associated neuraminidase in mediating a two step adherence mechanism.     

IgE peptide-specific CTL inhibit IgE production: a transient IgE suppression model in wild-type and HLA-A2.1 transgenic mice

Effect of IgE peptide-specific CTL on IgE antibody production was studied in mouse models. CTL elicited in B6.A2Kb tg mice against a human IgE peptide nonamer, pWV, lysed human IgE-secreting U266 myeloma cells and inhibit IgE production by these cells. U266 transfected with mouse A2Kb transgene (U266-A2Kb) were optimally lysed by these CTL, because the α3 domain of A2Kb interacts well with the CD8 co-receptors. The CTL generated were more effective in inhibiting IgE production by U266-A2Kb cells than lysing these cells. IgE production by and progression of U266 myeloma were suppressed in B6.A2Kb tg mice rendered tolerant to these cells and vaccinated with pWV along with CpG. We also studied the CTL response elicited in wild-type mice by a mouse nonameric IgE peptide, PI-1, along with CpG. This treatment caused a transient suppression of the IgE response in mice previously sensitized to an antigen. In mice treated with this regimen repeatedly, the IgE response was fully recovered 20 days after each treatment. Notably, while IgE peptide/CpG-treated mice remained unresponsive to antigen challenge in vivo, antigen-specific IgE production can be elicited by antigen in cultured splenocytes from these mice. Moreover, IgE peptide/CpG also inhibited an on-going IgE response, including IgE production by bone marrow cells. Taken together, these observations indicate that a CTL-based IgE peptide vaccine targeting IgE-secreting B/plasma cells may be safely employed as a therapeutic approach for suppressing IgE production.     

Immunological evidence for a bacterial toxin aetiology in sudden infant death syndrome

Toxin-specific antibodies to clostridial, enterobacterial and staphylococcal toxins implicated in sudden infant death syndrome were studied in sera from sudden infant death syndrome infants and a comparison group of infants (babies with phenylketonuria). The results indicated a higher proportion of sera from sudden infant death syndrome infants contained IgA that bound to clostridial and enterobacterial toxins but a higher proportion of sera from the phenylketonuria comparison group contained IgA that bound staphylococcal toxins. The higher proportion of serum samples with IgG and IgM in the healthy comparison babies serum probably indicated immunity in this group of babies to these toxins. The effect of gender and age had a minimal effect on the incidence of these antibodies. The presence of toxin-specific antibodies in sudden infant death syndrome and the of comparison infants suggests that all infants are exposed to these toxins and most babies successfully overcome the toxic challenge. Some infants with predisposing risk factors (temperature change, smoking, infection, immune development, sleeping position, etc.) that could affect the baby's immune competency could succumb to these and possibly other toxins. This immunological evidence further strengthens the view that bacterial toxins are a significant cause of sudden infant death syndrome.     

Spatial organization in bacterial chemotaxis

Spatial organization of signalling is not an exclusive property of eukaryotic cells. Despite the fact that bacterial signalling pathways are generally simpler than those in eukaryotes, there are several well‐documented examples of higher‐order intracellular signalling structures in bacteria. One of the most prominent and best‐characterized structures is formed by proteins that control bacterial chemotaxis. Signals in chemotaxis are processed by ordered arrays, or clusters, of receptors and associated proteins, which amplify and integrate chemotactic stimuli in a highly cooperative manner. Receptor clusters further serve to scaffold protein interactions, enhancing the efficiency and specificity of the pathway reactions and preventing the formation of signalling gradients through the cell body. Moreover, clustering can also ensure spatial separation of multiple chemotaxis systems in one bacterium. Assembly of receptor clusters appears to be a stochastic process, but bacteria evolved mechanisms to ensure optimal cluster distribution along the cell body for partitioning to daughter cells at division.     

IL-4-dependent induction of IgE+ basophils in peripheral blood and IgE+ B cells in spleen as respective indicators of allergen sensitization and a precursor of cells secreting allergen-specific IgE antibody

It was recently reported by us that either primary i.n. or i.p. injection of cedar pollen extract into BALB/c mice, or a second s.c. injection of the allergen into i.v. or s.c. sensitized mice, causes an IL-4-dependent increase in total IgE serum antibody to produce allergen-specific IgE antibody upon further s.c. sensitization. To determine the biology of total IgE antibody, in the present study IgE⁺ cells in peripheral blood or lymphoid tissues of allergen-sensitized BALB/c mice have been characterized. In peripheral blood, mice sensitized one to three times with the allergen produced a 2.5- to 4-fold increase in the number of IgE⁺ cells, with a time-course similar to that of the concentration of total IgE antibody in serum. These IgE⁺ cells were basophils. On the other hand, the number of IgE⁺ cells in the lymphoid tissues did not change significantly after an i.n., i.p., i.v. or s.c. injection of allergen into the mice, whereas a second s.c. injection of the allergen into the i.v.-, but not into the i.n.-, i.p.- or s.c.-, sensitized mice induced a small number of IgE⁺/IgM⁺/B220⁺ B cells in the spleen. In contrast, IgE⁺ cells were not seen in the blood or spleen of IL-4 -/- mice after sensitization with the allergen.
These results suggest that IgE⁺ basophils in the peripheral blood, and IgE⁺ B cells in the spleen, might be IL-4-dependently induced as an indicator of sensitization with allergen, and a precursor of cells secreting allergen-specific IgE antibody, respectively.     

Induction of apoptosis in Caco-2 and HT-29 human intestinal epithelial cells by enterohemolysin produced by classic enteropathogenic Escherichia coli

AIMS: Detect the cytotoxic effects of the Enterohemolysin from enteropathogenic Escherichia coli C3888 (O 26: H-) on Caco 2 and HT-29-human epithelial intestinal cells. METHODS AND RESULTS: The Caco 2 and HT-29 cells, which were treated with Enterohemolysin (EHly) within 10-15 min, became round, lost attachment to substrate, showed extensive surface blebbing, nucleus shrank, and the chromatin became more compact. After 10 min of exposure to the EHly, the cells showed lactate dehydrogenase (LDH) leakage and reduction of mitochondrial activity. The cells showed disorganization of the actin fibers at 15 min. The death of these human epithelial intestinal cells by apoptosis was confirmed by annexin V. CONCLUSIONS: Enterohemolysin induced apoptosis on human epithelial intestinal cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The finding of EHly cytotoxic activity suggests the involvement of this hemolysin in the (Enteropathogenic Escherichia coli) EPEC infection mechanism and may facilitate the understanding of the diarrhea caused by EPEC.     

Cloning and expression in Escherichia coli of dextranase genes from Bacteroides thetaiotaomicron

A genomic bank was constructed in Escherichia coli HB101, consisting of DNA fragments from Bacteroides thetaiotaomicron strain 489 inserted within the vector pBR322. By screening on complex medium containing blue dextran, 10 stable dextranase-positive (Dex+) clones were isolated. Seven groups of Dex+ inserts were identified on the basis of their restriction maps and hybridization responses. Dextanase activity of the recombinant clones was weak, and was revealed on the selection medium after 15 days. Subcloning of a Sau3AI partially digested 3.2-kb insert in the expression vector pDR720 greatly enhanced dextranse activity on blue dextran plates in one clone, but the delay remained unaltered. This suggested that the enzyme was released by cell lysis. Expression of this 0.7-kb subcloned insert was dependent on the promoter region of tryptophan operon carried by pDR720.     

Neuroinflammatory reactions in sickness behavior induced by bacterial infection: Protective effect of minocycline

The neurological changes elicited by bacterial infection are called sickness behavior. Minocycline (MIN) is neuroprotective with a remarkable brain tissue penetration. MIN was orally administered at a dose 90 mg/kg for 3 days, whereas Escherichia coli was given as a single intraperitoneal injection (0.2 mL of 24 h growth) on the third day. After 24 h of bacterial infection, behavioral tests namely open field and forced swimming were carried out, then animals were decapitated. Rats infected with E. coli displayed reduced struggling time in forced swimming test, as well as, exploration and locomotion in open field test with reduction in neurotransmitters (norepinephrine, dopamine, and serotonin) versus elevation in the inflammatory (tumor necrosis factor‐alpha, interferon‐gamma) and oxidative stress (thiobarbituric acid reactive substance, reduced glutathione) biomarkers. Inflammatory infiltrates of nuclear cells were observed in brains of infected rats. MIN administration prevented the deleterious effects of E. coli infection, thus protects against sickness behavior possibly via defending from neuroinflammation.     

The probiotic Escherichia coli strain Nissle 1917 interferes with invasion of human intestinal epithelial cells by different enteroinvasive bacterial pathogens

The probiotic Escherichia coli strain Nissle 1917 (Mutaflor) of serotype O6:K5:H1 was reported to protect gnotobiotic piglets from infection with Salmonella enterica serovar Typhimurium. An important virulence property of Salmonella is invasion of host epithelial cells. Therefore, we tested for interference of E. coli strain Nissle 1917 with Salmonella invasion of INT407 cells. Simultaneous administration of E. coli strain Nissle 1917 and Salmonella resulted in up to 70% reduction of Salmonella invasion efficiency. Furthermore, invasion of Yersinia enterocolitica, Shigella flexneri, Legionella pneumophila and even of Listeria monocytogenes were inhibited by the probiotic E. coli strain Nissle 1917 without affecting the viability of the invasive bacteria. The observed inhibition of invasion was not due to the production of microcins by the Nissle 1917 strain because its isogenic microcin-negative mutant SK22D was as effective as the parent strain. Reduced invasion rates were also achieved if strain Nissle 1917 was separated from the invasive bacteria as well as from the INT407 monolayer by a membrane non-permeable for bacteria. We conclude E. coli Nissle 1917 to interfere with bacterial invasion of INT407 cells via a secreted component and not relying on direct physical contact with either the invasive bacteria or the epithelial cells.     

Binding of purified Escherichia coli O75X adhesin to frozen sections of human kidney

Abstract Binding characteristics of the purified Escherichia coli O75X adhesin in frozen sections of human kidney were determined, using antibodies raised against the purified antigen and rhodamine-conjugated second antibodies. To identify the adhesin-binding nephron domains, the same tissue sections were double stained with fluorescein isothiocynate-conjugated nephron site-specific lectins. The results revealed that, at the tubular pole, the O75X adhesin bound selectively to the basement membrane of proximal and distal tubules and, with a slightly lower efficiency, of collecting ducts. In the glomerulus, the O75X adhesin bound only to the parietal epithelial cells (Bowman's capsule). The purified O75X adhesin bound also to exfoliated epithelial cells in human urine. Our results suggest that the O75X adhesin may contribute to the uropathogenicity of E. coli by binding the bacteria to tissue structures in the human urinary tract.     

人甲状旁腺激素在大肠杆菌中的表达及鉴定

化学合成人甲状旁腺激素（hPTH）全长基因,克隆到大肠杆菌表达载体pBV220和pET22b中,获得了高表达。经破菌、阳离子交换层析、反相层析纯化获得了纯度大于95%的纯品。N端测序、质谱分析结果表明重组hPTH 结果完整,N端无Met或fMet。生物活性试验证明重组hPTH具有激活腺苷酸环化酶、增加骨质量和骨密度等作用。     

Enhanced periplasmic expression of human activin A in Escherichia coli using a modified signal peptide

Abstract

Activin A is a member of the transforming growth factor-beta (TGF-β) protein superfamily, which acts as a hormone in regulating cell proliferation and differentiation. Structurally, activin is a dimer of two subunits linked by a disulfide bond. Since the correct folding of this protein is essential for its function, we aimed to use a modified signal peptide to target the expressed recombinant protein to the periplasm of Escherichia coli as an effective strategy to produce correctly-folded activin A. Therefore, the coding sequence of native Iranian Bacillus licheniformis α-amylase signal peptide was modified and its efficiency was checked by SignalP bioinformatics tool. Then its final sequence was cloned upstream of the activin A mature cDNA. Protein expression was done using 1?mM of isopropyl thio-β-D-galactoside (IPTG) and a post-induction time of 8?hr. Additionally, following purification of recombinant activin A, circular dichroism spectroscopy was used to determine the accuracy of secondary structure of the protein. Importantly, differentiation of K562 cells to the red blood cell was confirmed by measuring the amount of Fe⁺² ions after treatment with recombinant activin A. The results indicated that the produced recombinant activin A had the same secondary structure as the commercial human activin A and was fully functional.     

Modelling the vector pathway and infection of humans in an environmental outbreak of Escherichia coli O157

Quantifying the transfer of Escherichia coli O157 from the environment to humans is essential for understanding outbreaks, establishing the infectious dose of the organism and proposing safeguards. We modelled the pathogen loading shed onto a field by sheep immediately prior to a scout camp where 18 scouts and two adults were infected with E. coli O157. We estimated the dose ingested (4-24 organisms) which is in agreement with the low infective dose reported previously for this organism in food outbreaks. These data closely fit a surrogate Shigella dose-response model which can be used as a basis for risk assessment.     

Overexpression of soluble human thymosin alpha 1 in Escherichia coli

Synthesized gene of human thymosin alpha 1 (Tα1) was inserted into pET-28a, pET-9c,pThioHis B, pGEX-2T or pBV222 and then inductively expressed in strains of Escherichia coll. Among the five expression systems, the BL21/pET-28a system provides the highest expression level of fusion protein in a soluble form, which is up to 70% of total expressed bacterial proteins as visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The resulting fusion protein purified through nickel affinity chromatography accounts for 2.53% of the wet bacterial pellet weight and reaches 94.5% purity by SDS-PAGE. These results indicate the potential of this expression system for high-throughput production of recombinant Tα1.     

Effects of citraconylation on enzymatic modification of human proinsulin using trypsin and carboxypeptidase B

Insulin is a polypeptide hormone which is produced by the β‐cell of pancreas and controls the blood glucose level in the human body. Enzymatic modification of human proinsulin using trypsin and carboxypeptidase B generally causes high accumulation of insulin derivatives, leading to more complicated purification processes. A simple method including citraconylation and decitraconylation in the enzymatic modification process was developed for the reduction of a major derivative, des‐threonine human insulin. Addition of 3.0 g citraconic anhydride per g protein into the reaction solution led to the citraconylation of lysine residues in human proinsulin and reduction of relative des‐threonine insulin content from 13.5 to 1.0%. After the enzymatic hydrolysis of the citraconylated proinsulin, 100% of lysine residues can be decitraconylated and restored by adjusting pH to 2–3 at 25 °C. Combination of hydrogen peroxide addition and citraconylation of proinsulin expressed in recombinant Escherichia coli remarkably improved the conversion yield of insulin from 52.7 to 77.7%. Consequently, citraconylation of lysine residues blocked the unexpected cleavage of human proinsulin by trypsin, minimized the formation of des‐threonine insulin and hence increased the production yield of active insulin. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Formation of bacterial streamers during filtration in microfluidic systems

Bacterial behavior during filtration is complex and is influenced by numerous factors. The aim of this paper is to report on experiments designed to make progress in the understanding of bacterial transfer in filters and membranes. Polydimethylsiloxane (PDMS) microsystems were built to allow direct dynamic observation of bacterial transfer across different microchannel geometries mimicking filtration processes. When filtering Escherichia coli suspensions in such devices, the bacteria accumulated in the downstream zone of the filter forming long streamers undulating in the flow. Confocal microscopy and 3D reconstruction of streamers showed how the streamers are connected to the filter and how they form in the stream. Streamer development was found to be influenced by the flow configuration and the presence of connections or tortuosity between channels. Experiments showed that streamer formation was greatest in a filtration system composed of staggered arrays of squares 10 μm apart.     

Evidence of horizontal gene transfer between human and animal commensal Escherichia coli strains identified by microarray

Bacteria exchange genetic material by horizontal gene transfer (HGT). To evaluate the impact of HGT on Escherichia coli genome plasticity, 19 commensal strains collected from the intestinal floras of humans and animals were analyzed by microarrays. Strains were hybridized against an oligoarray containing 2700 E. coli K12 chromosomal genes. A core (genes shared among compared genomes) and a flexible gene pool (genes unique for each genome) have been identified. Analysis of hybridization signals evidenced 1015 divergent genes among the 19 strains and each strain showed a specific genomic variability pattern. Four hundred and fifty-eight genes were characterized by higher rates of interstrain variation and were considered hyperdivergent. These genes are not randomly distributed onto the chromosome but are clustered in precise regions. Hyperdivergent genes belong to the flexible gene pool and show a specific GC content, differing from that of the chromosome, indicating acquisition by HGT. Among these genes, those involved in defense mechanisms and cell motility as well as intracellular trafficking and secretion were far more represented than others. The observed genome plasticity contributes to the maintenance of genetic diversity and may therefore be a source of evolutionary adaptation and survival.