Comparison of Cortical and White Matter Traumatic Brain Injury Models Reveals Differential Effects in the Subventricular Zone and Divergent Sonic Hedgehog Signaling Pathways in Neuroblasts and Oligodendrocyte Progenitors

The regenerative capacity of the central nervous system must be optimized to promote repair following traumatic brain injury (TBI) and may differ with the site and form of damage. Sonic hedgehog (Shh) maintains neural stem cells and promotes oligodendrogenesis. We examined whether Shh signaling contributes to neuroblast (doublecortin) or oligodendrocyte progenitor (neural/glial antigen 2 [NG2]) responses in two distinct TBI models. Shh-responsive cells were heritably labeled in vivo using Gli1-CreER^T2;R26-YFP bitransgenic mice with tamoxifen administration on Days 2 and 3 post-TBI. Injury to the cerebral cortex was produced with mild controlled cortical impact. Yellow fluorescent protein (YFP) cells decreased in cortical lesions. Total YFP cells increased in the subventricular zone (SVZ), indicating Shh pathway activation in SVZ cells, including doublecortin-labeled neuroblasts. The alternate TBI model produced traumatic axonal injury in the corpus callosum. YFP cells decreased within the SVZ and were rarely double labeled as NG2 progenitors. NG2 progenitors increased in the cortex, with a similar pattern in the corpus callosum. To further test the potential of NG2 progenitors to respond through Shh signaling, Smoothened agonist was microinjected into the corpus callosum to activate Shh signaling. YFP cells and NG2 progenitors increased in the SVZ but were not double labeled. This result indicates that either direct Smoothened activation in NG2 progenitors does not signal through Gli1 or that Smoothened agonist acts indirectly to increase NG2 progenitors. Therefore, in all conditions, neuroblasts exhibited differential Shh pathway utilization compared with oligodendrocyte progenitors. Notably, cortical versus white matter damage from TBI produced opposite responses of Shh-activated cells within the SVZ.     

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.     

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.     

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).     

Inflammatory microcrystals induce murine macrophage survival and DNA synthesis

The interaction of particulates with resident macrophages is a consistent feature in certain forms of crystal-induced inflammation, for example, in synovial tissues, lung, and the peritoneum. The mitogenic activity of basic calcium phosphate (BCP) crystals and calcium pyrophosphate dihydrate (CPPD) crystals on synovial fibroblasts has been considered relevant to the synovial hyperplasia observed in crystal-induced arthritis. The aim of the study was to determine whether microcrystals such as these could enhance macrophage survival and induce DNA synthesis, thus indicating that they may contribute to the tissue hyperplasia.     

Bushmeat Consumption and Preferences of Two Ethnic Groups in Bioko Island, West Africa

We studied consumption and preference of meats of wild species (bushmeat) by inhabitants of Bioko Island, Equatorial Guinea. The aim of the study was to quantify frequency of consumption and stated preferences of the two main ethnic groups (Bubi and Fang) in the island. Although members of both ethnic groups lived on the island, the Fang originated from the continent and maintained strong links with this area. Thus, preference and consumption of the Fang reflected exposure to animals found in the continent as well as on Bioko. A sample of 196 subjects (115 Bubi and 81 Fang) was interviewed using semistructured questionnaires. A total of 55 different bushmeat species was identified as preferred or consumed by interviewees. Principal component analyses of stated consumption and preference indicated differences between ethnic groups in their general responses. Further analyses of the effects of preference and other factors on consumption of the three main species mentioned (blue duiker (Cephalophus monticola), Emin's rat (Cricetomys emini), and brush-tailed porcupine (Atherurus africanus) were undertaken. Proportional odds logistic regression models for ordered categorical response data were employed. Results indicated that age and sex of the respondent did not affect consumption, but ethnic group was statistically significant for the three-study species. Consumption and preference of the different meats (N = 11 species) in relation to their availability in the market and price was studied using multiple linear regressions. Consumption is driven predominantly by availability but there is some influence of preference; price of the meat did not have a significant influence.     

The thermal dependence of fast-start performance in fish

Many fish species use fast-starts to escape predators and capture prey. There is evidence for changes in fast-start behaviour with temperature, over acute, seasonal, developmental and evolutionary time scales. Maximum velocity often increases with acute temperature changes. Thermal acclimation can improve fast-start performance, although responses appear to be reduced in more eurythermal species. Changes in performance with thermal acclimation are often reflected at the molecular, biochemical and cellular levels of organisation. There appears to be little compensation in fast-start performance in Antarctic fish compared to warmer water species.