Intestinal microbiota was assessed in cirrhotic patients with hepatitis B virus infection. Intestinal microbiota of HBV cirrhotic patients

To unravel the profile of intestinal microecological parameters in Chinese patients with asymptomatic carriage of hepatitis B virus (HBV), chronic hepatitis B, decompensated HBV cirrhosis, and health controls and to establish their correlation with liver disease progression, we performed quantitative PCR and immunological techniques to investigate fecal parameters, including population of fecal predominant bacteria and the abundance of some virulence genes derived from Escherichia coli, Bacteroides fragilis, Clostridium difficile, and Clostridium perfringens in fecal crude DNA and some immunological parameters in extracts of all fecal samples. Data analysis indicated that 16S rRNA gene copy numbers for Faecalibacterium prausnitzii, Enterococcus faecalis, Enterobacteriaceae, bifidobacteria, and lactic acid bacteria (Lactobacillus, Pediococcus, Leuconostoc, and Weissella) showed marked variation in the intestine of HBV cirrhotic patients. The Bifidobacteria/Enterobacteriaceae (B/E) ratio, which may indicate microbial colonization resistance of the bowel, was decreased significantly in turn from 1.15 ± 0.11 in healthy controls, 0.99 ± 0.09 in asymptomatic carriers, and 0.76 ± 0.08 in patients with chronic hepatitis B to 0.64 ± 0.09 in patients with decompensated HBV cirrhosis (for all, P < 0.01). This suggests that B/E ratio is useful for following the level of intestinal microecological disorder in the course of liver disease progression. The data for virulence gene abundance suggested increased diversity of virulence factors during liver disease progression. Fecal secretory IgA and tumor necrosis factor-α in decompensated HBV cirrhotic patients were present at higher levels than in other groups, which indicates that a complicated autoregulatory system tries to achieve a new intestinal microecological balance.     

水的饱和辛醇溶液水分标准物质的研制

生物燃料的国家标准规定了产品的技术指标和相应的检测方法。水的饱和辛醇溶液的水分标准物质,用于生物燃料水分测量时仪器的校准和方法的验证,能够保障测量结果的准确可靠和等效一致。该标准物质采用卡尔·费休库仑法、卡尔·费休容量法和定量核磁共振等三种不同原理的方法定值。通过方法研究和改进,实现了库仑法和容量法的一致;通过引入新的核磁共振方法,提高了结果的准确性。最终标准物质的水分量值为4.76％,扩展不确定度为0.09％。     

Redundancy in B cell developmental pathways: c-Cbl inactivation rescues early B cell development through a B cell linker protein-independent pathway

Deficiency in the adaptor protein B cell linker protein (BLNK) results in a substantial but incomplete block in B cell development, suggesting that alternative pathways exist for B lineage differentiation. Another adaptor protein, c-Cbl, plays a negative regulatory role in several BCR-signaling pathways. We therefore investigated the role of c-Cbl during B cell development and addressed the possibility that redundancies in pathways for B cell differentiation could be further revealed by eliminating negative effects mediated by c-Cbl. Strikingly, c-Cbl inactivation reversed a number of the critical defects in early B cell differentiation that are seen in BLNK-deficient mice. c-Cbl(-/-)BLNK(-/-) mice exhibited normalized down-regulation of pre-BCR and CD43, up-regulation of MHC class II, and augmented L chain rearrangement, resulting in a successful transition from pre-B cells to immature B cells. c-Cbl inactivation also reversed the potentially tumor-predisposing hyperproliferative response of BLNK(-/-) pre-B cells to IL-7. Pre-BCR cross-linking induced enhanced and prolonged tyrosine phosphorylation in c-Cbl(-/-)BLNK(-/-) pre-BCR(+) pre-B cells compared with c-Cbl(+/-)BLNK(-/-) cells, including elevated phosphorylation of Lyn, Syk, Btk, and phospholipase C-gamma2. Our studies suggest that some, but not all, pre-BCR-triggered developmental events can be mediated by BLNK-independent pathways that are negatively regulated by c-Cbl, and further suggest that different events during early B cell development require different strength or duration of pre-BCR signaling.     

Crystal structures of human IPP isomerase: new insights into the catalytic mechanism

Type I isopentenyl diphosphate (IPP): dimethylally diphosphate (DMAPP) isomerase is an essential enzyme in human isoprenoid biosynthetic pathway. It catalyzes isomerization of the carbon-carbon double bonds in IPP and DMAPP, which are the basic building blocks for the subsequent biosynthesis. We have determined two crystal structures of human IPP isomerase I (hIPPI) under different crystallization conditions. High similarity between structures of human and Escherichia coli IPP isomerases proves the conserved catalytic mechanism. Unexpectedly, one of the hIPPI structures contains a natural substrate analog ethanol amine pyrophosphate (EAPP). Based on this structure, a water molecule is proposed to be the direct proton donor for IPP and different conformations of IPP and DMAPP bound in the enzyme are also proposed. In addition, structures of human IPPI show a flexible N-terminal alpha-helix covering the active pocket and blocking the entrance, which is absent in E. coli IPPI. Besides, the active site conformation is not the same in the two hIPPI structures. Such difference leads to a hypothesis that substrate binding induces conformational change in the active site. The inhibition mechanism of high Mn(2+) concentrations is also discussed.     

Factors influencing cell fate in the infarct rim

In focal ischemia, the fate of penumbral cells is closely linked to the infarcted tissue. Because of the release of cytosolic material from damaged cells, the biochemical and ionic alterations within the core are dramatic. Hence, adjacent cells ( infarct rim) are generally exposed to these changes and may be deleteriously affected. To mimic such conditions in vitro, we have employed a slice culture system and used an ischemic solution (IS) that resembles the milieu in the territory of infarct rim. In contrast to normal artificial cerebral spinal fluid, IS is characterized by low O(2), glucose, pH; excitotoxic levels of glutamate; and ionic alterations. In organotypic hippocampal slice cultures, we examined cell injury/death using propidium iodide following exposure to IS. Our data show significant cell injury starting at approximately 8 h following IS exposure with cell injury spreading as a function of exposure duration. We further studied the effect of each component in the IS separately, i.e., acidosis, hypoxia, ionic shifts or glutamate exitotoxicity and were able to isolate the contribution of each of these effectors to the IS-induced cell death. Our results suggest that in IS, acidosis exacerbates the potential for injury while ionic shifts, especially those of K(+) and Na(+), alleviate the potential for cell death.     

Inhibitory effect of recombinant adenovirus carrying immunocaspase-3 on hepatocellular carcinoma

Previously, Srinivasula devised a contiguous molecule (C-cp-3 or immunocaspase-3) containing the small and large subunits similar to that in the active form of caspas-3 and found C-cp-3 had similar cleavage activity to the active form of caspase-3. To search for a new clinical application of C-cp-3 to treat hepatocellular carcinoma, recombinant adenoviruses carrying the C-cp-3 and a-fetoprotein (AFP) promoter (Ad-rAFP-C-cp-3) were constructed through a bacterial homologous recombinant system. The efficiency of adenovirus-mediated gene transfer and the inhibitory effect of Ad-rAFP-C-cp-3 on the proliferation of hepatocarcinoma cells were determined by X-gal stain and MTT assay, respectively. The tumorigenicity of hepatocarcinoma cells transfected by Ad-rAFP-C-cp-3 and the antitumor effect of Ad-rAFP-C-cp-3 on transplanted tumor in nude mice were detected in vivo. The results suggested that Ad-rAFP-C-cp-3 can inhibit specifically proliferation of AFP-producing human hepatocarcinoma cells in vitro and in vivo and adenovirus-mediated C-cp-3 transfer could be used as a new method to treat human hepatocarcinoma.     

RIP1-mediated AIP1 phosphorylation at a 14-3-3-binding site is critical for tumor necrosis factor-induced ASK1-JNK/p38 activation

Previously, we have shown that ASK1-interacting protein 1 (AIP1, also known as DAB2IP), a novel member of the Ras-GAP (Ras-GTPase-activating protein) protein family, opens its conformation in response to tumor necrosis factor (TNF), allowing it to form a complex with TRAF2-ASK1 that leads to activation of ASK1-JNK/p38 signaling in endothelial cells (EC). In the present study, we show that a TNF-inducible 14-3-3-binding site on AIP1 is critical for the opening of its conformation and for the AIP1-mediated TNF signaling. Ser-604, located in the C-terminal domain of AIP1, was identified as a 14-3-3-binding site. TNF treatment of EC induces phosphorylation of AIP1 at Ser-604 as detected by a phospho-specific antibody, with a similar kinetics to ASK1-JNK/p38 activation. 14-3-3 associates with an open, active state of AIP1 assessed by an in vitro pulldown assay. Mutation of AIP1 at Ser-604 (AIP1-S604A) blocks TNF-induced complex formation of AIP1 with 14-3-3. TNF treatment normally induces association of AIP1 with TRAF2-ASK1. The interactions with TRAF2 and ASK1 do not occur with AIP1-S604A, suggesting that phosphorylation at this site not only creates a 14-3-3-binding site but also opens up AIP1, allowing binding to TRAF2 and ASK1. Overexpression of AIP1-S604A blocks TNF-induced ASK1-JNK activation. We further show that RIP1 (the Ser/Thr protein kinase receptor-interacting protein) associates with the GAP domain of AIP1 and mediates TNF-induced AIP1 phosphorylation at Ser-604 and JNK/p38 activation as demonstrated by both overexpression and small interfering RNA knockdown of RIP1 in EC. Furthermore, RIP1 synergizes with AIP1 (but not AIP1-S604A) in inducing both JNK/p38 activation and EC apoptosis. Our results demonstrate that RIP1-mediated AIP1 phosphorylation at the 14-3-3-binding site Ser-604 is essential for TNF-induced TRAF2-RIP1-AIP1-ASK1 complex formation and for the activation of ASK1-JNK/p38 apoptotic signaling.     

The Microbial Community in the Feces of the Giant Panda (<Emphasis Type="Italic">Ailuropoda melanoleuca</Emphasis>) as Determined by PCR-TGGE Profiling and Clone Library Analysis

Despite having a typical carnivorous digestive tract, the giant panda has a diet consisting exclusively of bamboo, a low-efficiency food source. Given this paradox, we sought to investigate if the giant panda digestive tract is inhabited by organisms indicative of high cellulose diet or their gastrointestinal tract anatomy. The diversity and dynamics of the predominant bacteria in the fecal flora of two adult (male and female) and one young (male) giant panda reared in two different zoos over a 2-year period was studied using 16S rDNA-based approaches. The temperature gradient gel electrophoresis (TGGE) profiles of the 16S rDNA V3 region of the three individuals were highly similar. The structure of their fecal flora remained relatively stable over the 2-year period. Both the most predominant band in TGGE patterns shared by the three pandas and the biggest operational taxonomic unit (OTU) in the clone library were phylogenetically related to Escherichia coli. Gram-negative, facultative bacteria constituted almost 60% of the whole community in the clone library. All the OTUs were related to previously described phylotypes known to reside in the intestine or rumen. The results of our study indicate that the predominant bacterial populations in the intestine of the three pandas were markedly different from that of herbivores. The unbalanced intestinal community structure may play a role in the inefficient digestion of bamboo by the giant pandas.     

The protective effects of osmolytes on arginine kinase unfolding and aggregation

Osmolytes are a series of different kinds of small molecules that can maintain the correct conformation of protein by acting as molecular chaperons. In this study, the protective effects of four compatible osmolytes, i.e., proline, sucrose, DMSO and glycerol, were studied during arginine kinase (EC 2.7.3.3) unfolding and aggregation. The results showed that all the osmolytes applied in this study obviously prevented AK unfolding and inactivation that was due to a GdnHCl denaturant by reducing the inactivation rate constants (k_i), increasing the transition free energy changes (ΔΔG_i) and increasing the value for the midpoint of denaturation (C_m). Furthermore, the osmolytes remarkably prevented AK aggregation in a concentration-dependent manner during AK refolding. Our results strongly indicated that osmolytes were not only metabolism substrates, but they were also important compounds with significant physiological protective functions for proteins, especially in some extremely harsh environments.