Prevalence and Characterization of Non-O157 Shiga Toxin-Producing Escherichia coli on Carcasses in Commercial Beef Cattle Processing Plants

Beef carcass sponge samples collected from July to August 1999 at four large processing plants in the United States were surveyed for the presence of non-O157 Shiga toxin-producing Escherichia coli (STEC). Twenty-eight (93%) of 30 single-source lots surveyed included at least one sample containing non-O157 STEC. Of 334 carcasses sampled prior to evisceration, 180 (54%) were found to harbor non-O157 STEC. Non-O157 STEC isolates were also recovered from 27 (8%) of 326 carcasses sampled after the application of antimicrobial interventions. Altogether, 361 non-O157 STEC isolates, comprising 41 different O serogroups, were recovered. O serogroups that previously have been associated with human disease accounted for 178 (49%) of 361 isolates. Although 40 isolates (11%) carried a combination of virulence factor genes (enterohemorrhagic E. coli hlyA, eae, and at least one stx gene) frequently associated with STEC strains causing severe human disease, only 12 of these isolates also belonged to an O serogroup previously associated with human disease. Combining previously reported data on O157-positive samples (R. O. Elder, J. E. Keen, G. R. Siragusa, G. A. Barkocy-Gallagher, M. Koohmaraie, and W. W. Laegreid, Proc. Natl. Acad. Sci. USA 97:2999-3003, 2000) with these data regarding non-O157-positive samples indicated total STEC prevalences of 72 and 10% in preevisceration and postprocessing beef carcass samples, respectively, showing that the interventions used by the beef-processing industry effected a sevenfold reduction in carcass contamination by STEC.     

Several clock genes polymorphisms are meaningful risk factors in the development and severity of cannabis addiction

Circadian rhythms have been related to psychiatric diseases and regulation of dopaminergic transmission, especially in substance abusers. The relationship between them remained enigmatic and no data on the role of clock genes on cannabis dependence have been documented. We aimed at exploring the role of clock gene genotypes as potential predisposing factor to cannabis addiction, using a high throughput mass spectrometry methodology that enables the large-scale analysis of the known relevant polymorphisms of the clock genes. We have conducted a case-control study on 177 Caucasians categorizing between cannabis-addicted subjects and casual consumers based on structured interviews recorded socio-demographic data, AUDIT, Fagerström test, MINI, and medical examinations. Alcohol, opiates, and stimulants’ consumption was as exclusion criteria. We report an association between several Single Nucleotide Polymorphism (SNP)s in main circadian genes SNPs, especially the gene locus HES7/PER1 on chromosome 17 and cannabis consumption as well as the development of neuropsychiatric and social disorders. This SNP’s signature that may represent a meaningful risk factor in the development of cannabis dependence and its severity requires to be deeply explored in a prospective study.     

Activities of the Porphyromonas gingivalis PrtP Proteinase Determined by Construction of prtP-Deficient Mutants and Expression of the Gene in Bacteroides Species

PrtP is a major cysteine proteinase of Porphyromonas gingivalis. The gene encoding this proteinase, prtP, was cloned into the Escherichia coli-Bacteroides shuttle vectors pFD288 and pFD340 and was expressed in Bacteroides cells, apparently under the control of its own promoter, when in pFD288, or a Bacteroides promoter present on pFD340. Proteolytically active PrtP was detected by fibrinogen zymography in cells or spent growth medium of several Bacteroides species harboring the recombinant plasmids. The proteinase was recovered from Bacteroides fragilis ATCC 25285(pFD340-prtP) cells by 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS) extraction and characterized with regard to exopeptidase specificity and sensitivity to proteinase inhibitors. Lys-amidolytic activity, but not Arg-amidolytic activity, was detected. PrtP was activated by cysteine and, to a lesser extent, dithiothreitol, and it was stimulated by glycine-containing compounds. It also was inhibited by Nα-p-tosyl-l-lysine chloromethyl ketone (TLCK) and, to a lesser extent, H-d-Tyr-l-Pro-l-arginyl chloromethyl ketone (YPRCK) and was relatively insensitive to EDTA and leupeptin. Neither B. fragilis ATCC 25285(pFD340-prtP) cells nor the CHAPS extract effected hemagglutination of sheep red blood cells or collagen cleavage, but the cells did cleave gelatin. Furthermore, P. gingivalis W12, ATCC 33277, KDP110, and HG66 with knockout mutations in prtP were constructed by allelic replacement. Unlike the parent strains, the mutant strains produced beige colonies on plates containing sheep blood. These strains also were affected in their ability to effect hemagglutination, cleave collagen, and cleave a Lys-specific peptide substrate. This report presents the results of the first characterization of the PrtP proteinase clearly in the absence of any influence by other P. gingivalis proteins and describes the properties of P. gingivalis cells defective in the production of PrtP.Periodontitis is a major cause of tooth loss in the adult population, and several recent studies suggest that it may be a significant risk factor for both cardiovascular disease and preterm labor in humans (for a review, see reference 32). The potential medical importance of these oral infections justifies an intensified effort to develop effective strategies to prevent periodontitis as well as to interfere with disease progression. Accomplishing this will require identification of the major causative agents of periodontitis, as well as an understanding of the mechanisms by which these bacteria contribute to the destruction of connective tissue and bone that characterizes periodontitis lesions. Those virulence factors which render periodontal pathogens uniquely capable of contributing to the destruction of the supporting structures of the teeth, including periodontal connective tissues, periodontal ligaments, and alveolar bone, would be logical targets for the development of novel, highly specific antimicrobial agents (20).Porphyromonas gingivalis is one of a small number of bacteria implicated in the etiology and pathogenesis of human periodontitis, and its proteinases appear to be among its most important virulence factors (16, 26, 27). These enzymes, which can be recovered from P. gingivalis cells and vesicles as well as from spent growth medium (2), are capable of degrading a variety of substrates, including gelatin, fibrinogen, fibronectin, C3, C5, and C5a receptor (14, 17–19, 51). Moreover, they clearly have the potential to contribute directly, as well as indirectly (via dysregulation of host proteinase cascade systems and the inflammatory response [16]), to destruction of periodontal tissues. Results of early studies suggested that P. gingivalis elaborates a bewildering number of distinct proteolytic enzymes, including at least one which is a collagenase (4). They also suggested that some of these proteinases can function as hemagglutinins (31) as well as adhesins for several host connective tissue matrix and plasma proteins (17, 19). The proteinase that we called porphypain was isolated from P. gingivalis W12 cells as sodium dodecyl sulfate (SDS)-stable conformers (150 and 120 kDa) of a 180-kDa proteinase (6). A proteinase called Lys-gingipain (also gingipain-K) was purified from spent culture medium, following growth of P. gingivalis HG66, as a 105-kDa complex containing a 60-kDa catalytic moiety (35, 37). While these proteinases have properties in common, significant differences in some of their properties were reported. Porphypain appears to contain two types of active sites, one with Lys-X activity and one with Arg-X activity. The amidolytic activity of both types of sites is greatly stimulated by derivatives of glycine and is inhibited by EDTA (6). Lys-gingipain, on the other hand, has been reported to have amidolytic activity exclusively for Lys-X but not Arg-X; it is inhibited by derivatives of glycine and is unaffected by EDTA (37). Since these proteinases were thought to be products of the same gene (2, 35), the difference between the reported activities of the purified enzymes was difficult to explain. Moreover, in spite of the fact that results of inhibitor studies have suggested that the collagenase of P. gingivalis has Lys-X and Arg-X activity (4), it is still unclear what, if any, relationship exists between porphypain, Lys-gingipain, and the collagenase of P. gingivalis.Results of more-recent biochemical and genetic studies (2, 11, 29, 39, 42) have suggested that 85 to 95% of the total proteolytic activity of P. gingivalis is attributable to three proteinases, PrtP (also called Kgp), Rgp-1, and Rgp-2. These enzymes are the products of three related genes (prtP [kgp], rgp-1, and rgp-2, respectively), and they appear to represent a unique family of cysteine proteinases (35). The gene referred to as rgp-1 (36) has also been designated prpRI (1), prtR (45), agpA (34), and rgpA (29) and encodes a 180-kDa protein. A second gene, which we refer to as rgp-2 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"U85038","term_id":"1814393","term_text":"U85038"}}U85038), is also called prR2 (42), prtRII (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF007124","term_id":"2253385","term_text":"AF007124"}}AF007124), agpB (28), and rgpB (29), and it encodes an 80-kDa protein that is almost identical to the catalytically active region of the product of rgp-1 (42). Finally, prtP (2), like rgp-1 (33), encodes an approximately 180-kDa proteinase. Comparisons of the deduced amino acid sequences of PrtP, Rgp-1, Rgp-2, and a fourth, related P. gingivalis protein, the hemagglutinin HagA, are shown in Fig. ​Fig.1.1. The N-terminal two-fifths of the PrtP and Rgp-1 molecules is thought to contain the active sites of the enzymes, and the catalytic Cys residues have been mapped to these domains (35). The C-terminal three-fifths of these two proteinases is composed of regions that are highly homologous to each other and to much of HagA (2). No reports suggesting that HagA has cysteine proteinase activity have appeared. Open in a separate windowFIG. 1Comparison of PrtP with Rgp-1, Rgp-2, and HagA. Each protein is represented in a linear fashion; HagA is shown at half-scale. Putative cleavage sites in PrtP and Rgp-1 are shown below each protein. Regions in PrtP, Rgp-1, Rgp-2, and HagA with 90% or greater identity are indicated by identical boxes; regions with 50 to 60% identity are similarly underlined. (Modified from reference 2).It is unclear exactly which regions of HagA mediate hemagglutination. It appeared that a peptide (G-907 to T-919) derived from the C-terminal three-fifths of Rgp-1 inhibited hemagglutination by spent culture medium from P. gingivalis W50 (8), and a monoclonal antibody capable of blocking hemagglutination mapped to an epitope containing the same amino acid residues (7). This same peptide is located in the C-terminal three-fifths of PrtP and can also be found in HagA (2). It is unclear whether other peptides shared by these three gene products can mediate hemagglutination, and it is also unclear what contribution each of these gene products makes to the total hemagglutinating activity of P. gingivalis. Finally, regions of these proteins that mediate binding of P. gingivalis to host connective tissue and plasma proteins have not yet been identified.The PrtP and Rgp proteinases have been challenging to deal with biochemically. The full-length forms of Rgp-1 and PrtP are the same size (molecular weight, ∼180,000; PrtP is 1,732 amino acids in length, and Rgp-1 is 1,704 amino acids in length); they share regions of amino acid identity with each other and with HagA; and they autodegrade and possibly process each other during purification procedures (2, 6). Much of the confusion regarding the number, sizes, and properties of the proteinases of P. gingivalis can be attributed to these properties. Furthermore, their proteolytic degradation products, some of which are similar in size and amino acid sequence, autoaggregate and tend to stay tightly, though noncovalently, associated in solution, even in the presence of SDS (6). For these reasons, they copurify even when many differently based methods are applied for their separation (1, 6, 11, 38). In addition, the catalytic domains of Rgp-1 and Rgp-2, which are virtually indistinguishable biochemically (42), copurify from the P. gingivalis background. The existence of Rgp-2 as a gene product distinct from Rgp-1 was revealed only after identification of a second genomic locus encoding an arginine-specific proteinase (1, 29).Absolute separation of Rgp-1, Rgp-2, PrtP, HagA, and their proteolytically processed products from wild-type P. gingivalis cultures may well prove impossible to achieve, which would seriously hamper structure-function studies of these proteins. Expression of these genes in a heterologous host could provide a means for examining the functions of each of these proteins in the clear absence of the others. Coupling the results of these studies with the results of characterizations of corresponding P. gingivalis knockout mutants could provide a means of elucidating the functions of these proteins in P. gingivalis. No reports describing the expression of catalytically active proteinase from the rgp-1, rgp-2, or prtP gene cloned in Escherichia coli or any other prokaryotic heterologous host have appeared, although we (1a) and others (1) have expressed in E. coli, from cloned rgp-1 and prtP genes, proteins immunoreactive with antibodies raised against these proteinases. To date, the expression using a Baculovirus system of only a portion of kgp (prtP), containing just the purported catalytic domain, has been reported (35). The authors stated that supernatants from infected Sf9 cells contained a low level of Lys-specific amidolytic activity, but apparently they conducted no further analysis of the recombinant enzyme. Furthermore, while P. gingivalis strains with inactivated rgp genes have been previously constructed and partially characterized (15, 29, 30, 50, 52), P. gingivalis cells lacking a functional prtP gene have not been described. The purpose of this study was to develop a system for expression of prtP in a heterologous prokaryotic host and to characterize the recombinant enzyme in terms of its specificity, behavior in the presence of stimulators and inhibitors of proteolysis, ability to cleave type I collagen, and ability to function as a hemagglutinin. In addition, we sought to determine the effect of deletion of PrtP functions on P. gingivalis cells.     

Intermediates in extrachromosomal homologous recombination in Xenopus laevis oocytes: characterization by electron microscopy.

Several molecular mechanisms have been proposed to account for nonconservative homologous recombination. This type of recombination is particularly efficient in Xenopus oocytes when appropriate DNA substrates are injected. To distinguish between possible models, we have investigated recombination intermediates from oocytes by direct observation in the electron microscope. Partially recombined DNA was crosslinked with a psoralen derivative after incubation in oocytes to limit the branch migration that might occur during recovery procedures and alter the structures that were initially present. Branched structures, which we interpret as intermediates, represented approximately 10% of the DNA recovered and were readily analyzed. We did not observe any structures with internal loops predicted by invasion mechanisms. The majority of intermediates had one or two single-stranded branches on product-sized molecules, as predicted for incomplete junctions in the resection-annealing mechanism. Detailed length measurements confirmed the expectations of that model. When recovered DNA was not crosslinked, or when annealed junctions were prepared in vitro, we saw branched structures that indicated the occurrence of extensive branch migration. Comparison with the crosslinked sample confirmed the effectiveness of the crosslinking in preserving structures created in the oocytes. Our results strongly support a resection-annealing mechanism of recombination in oocytes.     

A novel extracellular phospholipase C of Pseudomonas aeruginosa is required for phospholipid chemotaxis

Pseudomonas aeruginosa and other bacterial pathogens express one or more homologous extracellular phospholipases C (PLC) that are secreted through the inner membrane via the twin arginine translocase (TAT) pathway. Analysis of TAT mutants of P. aeruginosa uncovered a previously unidentified extracellular PLC that is secreted via the Sec pathway (PlcB). Whereas all presently known PLCs of P. aeruginosa (PlcH, PlcN and PlcB) hydrolyse phosphatidylcholine (PC), only PlcB is active on phosphatidylethanolamine (PE). plcB candidates were identified based on deductions made from bioinformatics data and extant DNA microarray data. Among these candidates, a gene (PA0026) required for the expression of an extracellular PE-PLC was identified. The protein encoded by PA0026 has limited, but significant similarity, over a short region (approximately 60aa of 328), to a class of zinc-dependent prokaryotic PLCs. A conserved His residue of PlcB (His216) that is required for coordinate binding of zinc in this class of PLCs was mutated. Analysis of this mutant established that the protein encoded by PA0026 is PlcB. Three in-dependent recently published reports indicate that homoserine lactone-mediated quorum sensing regulates the expression of PA0026 (i.e. plcB). PlcB, but not PlcH or PlcN, is required for directed twitching motility up a gradient of certain kinds of phospholipids. This response shows specificity for the fatty acid moiety of the phospholipid.     

圈养环境中不同来源成年雄性马麝的行为差异

于2004年8月--2005年1月,采用焦点取样和连续记录方法,对甘肃兴隆山自然保护区马麝(Moschus sifanicus)繁育中心的雄性马麝进行了行为取样.按照动物来源,将样本动物区分为野捕雄麝(17头)和圈养繁殖雄麝(6头),记录了静卧及站立凝视等12种行为的发生频次,并分别对其在交配季节和非交配季节的行为发生频率进行比较.结果表明,由于圈养环境和管理模式相同,甘肃兴隆山繁育中心的野捕和圈养繁殖马麝的总体行为格局类似,但由于幼年期人工哺乳等因素对其行为发育的影响,野捕雄麝在非交配季节和交配季节的冲突行为的表达频次显著多于圈养繁殖雄麝(P＜0.05),而圈养繁殖雄麝在交配季节的亲和行为极显著地多于野捕雄麝(P＜0.01).此外,雄麝在非交配季节的静卧行为发生频次极显著地多于交配季节(P＜0.01),而在交配季节的站立凝视、运动和环境探究及冲突的发生频次均极显著地比非交配季节多(P＜0.01).     

In vitro assays using primary embryonic mouse lymphatic endothelial cells uncover key roles for FGFR1 signalling in lymphangiogenesis

Despite the importance of blood vessels and lymphatic vessels during development and disease, the signalling pathways underpinning vessel construction remain poorly characterised. Primary mouse endothelial cells have traditionally proven difficult to culture and as a consequence, few assays have been developed to dissect gene function and signal transduction pathways in these cells ex vivo. Having established methodology for the purification, short-term culture and transfection of primary blood (BEC) and lymphatic (LEC) vascular endothelial cells isolated from embryonic mouse skin, we sought to optimise robust assays able to measure embryonic LEC proliferation, migration and three-dimensional tube forming ability in vitro. In the course of developing these assays using the pro-lymphangiogenic growth factors FGF2 and VEGF-C, we identified previously unrecognised roles for FGFR1 signalling in lymphangiogenesis. The small molecule FGF receptor tyrosine kinase inhibitor SU5402, but not inhibitors of VEGFR-2 (SU5416) or VEGFR-3 (MAZ51), inhibited FGF2 mediated LEC proliferation, demonstrating that FGF2 promotes proliferation directly via FGF receptors and independently of VEGF receptors in primary embryonic LEC. Further investigation revealed that FGFR1 was by far the predominant FGF receptor expressed by primary embryonic LEC and correspondingly, siRNA-mediated FGFR1 knockdown abrogated FGF2 mediated LEC proliferation. While FGF2 potently promoted LEC proliferation and migration, three dimensional tube formation assays revealed that VEGF-C primarily promoted LEC sprouting and elongation, illustrating that FGF2 and VEGF-C play distinct, cooperative roles in lymphatic vascular morphogenesis. These assays therefore provide useful tools able to dissect gene function in cellular events important for lymphangiogenesis and implicate FGFR1 as a key player in developmental lymphangiogenesis in vivo.