Construction and characterization of a novel staphylokinase variant with thrombin-inhibitory activity

Staphylokinase (SAK) is an effective thrombolysis agent for therapy of myocardial infarction. We have constructed a fusion SAK variant (SAK-HV) with a thrombin-binding domain composed of 12 amino acids from hirudin and expressed it in Escherichia coli and purified the resultant protein. SAK-HV maintained fibrinolytic activity similar to SAK and had anticoagulant activity attributable to its hirudin segment. Measurement of thrombin-binding activity in vitro demonstrated that SAK-HV possessed binding activity with thrombin while SAK did not. SAK-HV might thus be a more potent thrombolytic agent with anticoagulation property than SAK.     

<Emphasis Type="Italic">Saprolegnia brachydanis</Emphasis>, A New Oomycete Isolated from Zebra Fish

Saprolegnia brachydanis is described from zebra fish (Brachydanio rerio) in Wuhan, Hubei Province, China. The species is illustrated and compared with other species of the genus. The distinctive characteristics of S. brachydanis are the production of glomerulate oogonia wrapped around by predominantly monoclinous antheridia which can be up to eight in one oogonium. The oogonial stalks are short, straight, or curved and the antheridia, twisted, can enwind one or more oogonia. The oospores cannot mature or easily abort. Morphological features of the oomycete and the ITS sequence of its rDNA as well as the comparison with related species are discussed in this article.     

Carnosine uptake in rat choroid plexus primary cell cultures and choroid plexus whole tissue from PEPT2 null mice

PEPT2 is functionally active and localized to the apical membrane of rat choroid plexus epithelial cells. However, little is known about the transport mechanisms of endogenous neuropeptides in choroid plexus, and the role of PEPT2 in this process. In the present study, we examined the uptake kinetics of carnosine in rat choroid plexus primary cell cultures and choroid plexus whole tissue from wild-type (PEPT2(+/+)) and null (PEPT2(-/-)) mice. Our results indicate that carnosine is preferentially taken up from the apical as opposed to basolateral membrane of cell monolayers, and that basolateral efflux in limited. Transepithelial flux of carnosine was not distinguishable from that of paracellular diffusion. The apical uptake of carnosine was characterized by a high affinity (K(m) = 34 microM), low capacity (V(max) = 73 pmol/mg protein/min) process, consistent with that of PEPT2. The non-saturable component was small (K(d) = 0.063 microL/mg protein/min) and, under linear conditions, was only 3% of the total uptake. Studies in transgenic mice clearly demonstrated that PEPT2 was responsible for over 90% of carnosine's uptake in choroid plexus whole tissue. These findings elucidate the unique role of PEPT2 in regulating neuropeptide homeostasis at the blood-cerebrospinal fluid interface.     

Telomere shortening exposes functions for the mouse Werner and Bloom syndrome genes

The Werner and Bloom syndromes are caused by loss-of-function mutations in WRN and BLM, respectively, which encode the RecQ family DNA helicases WRN and BLM, respectively. Persons with Werner syndrome displays premature aging of the skin, vasculature, reproductive system, and bone, and those with Bloom syndrome display more limited features of aging, including premature menopause; both syndromes involve genome instability and increased cancer. The proteins participate in recombinational repair of stalled replication forks or DNA breaks, but the precise functions of the proteins that prevent rapid aging are unknown. Accumulating evidence points to telomeres as targets of WRN and BLM, but the importance in vivo of the proteins in telomere biology has not been tested. We show that Wrn and Blm mutations each accentuate pathology in later-generation mice lacking the telomerase RNA template Terc, including acceleration of phenotypes characteristic of latest-generation Terc mutants. Furthermore, pathology not observed in Terc mutants but similar to that observed in Werner syndrome and Bloom syndrome, such as bone loss, was observed. The pathology was accompanied by enhanced telomere dysfunction, including end-to-end chromosome fusions and greater loss of telomere repeat DNA compared with Terc mutants. These findings indicate that telomere dysfunction may contribute to the pathogenesis of Werner syndrome and Bloom syndrome.     

Brk activates rac1 and promotes cell migration and invasion by phosphorylating paxillin

Brk (for breast tumor kinase) is a nonreceptor tyrosine kinase containing SH3, SH2, and tyrosine kinase catalytic domains. Brk was originally identified from a human metastatic breast tumor, and its overexpression is frequently observed in breast cancer and several other cancer types. However, the molecular mechanism by which this kinase participates in tumorigenesis remains poorly characterized. In the present study, we not only identified paxillin as the binding partner and substrate of Brk but also discovered a novel signaling pathway by which Brk mediates epidermal growth factor (EGF)-induced paxillin phosphorylation. We show that EGF stimulation activates the catalytic activity of Brk, which in turn phosphorylates paxillin at Y31 and Y118. These phosphorylation events promote the activation of small GTPase Rac1 via the function of CrkII. Through this pathway, Brk is capable of promoting cell motility and invasion and functions as a mediator of EGF-induced migration and invasion. In accordance with these functional roles, Brk translocates to membrane ruffles, where it colocalizes with paxillin during cell migration. Together, our findings identify novel signaling and biological roles of Brk and indicate the first potential link between Brk and metastatic malignancy.     

Genotypic diversity enhances invasive ability of Spartina alterniflora

Although genetic diversity is very important for alien species, which have to cope with new environments, little is known about the role that genetic diversity plays in their invasive success. In this study, we set up a manipulation experiment including three levels of genotypic diversity to test whether genotypic diversity can enhance the invasive ability of alien species, in our case the invasive Spartina alterniflora in China, and to infer the underlying mechanisms. There was no significant relationship between genotypic diversity and parameters of performance in the first year; however, from the summer of the second year onwards, genotypic diversity enhanced four of the six parameters of performance. After two growing seasons, there were significant positive relationships between genotypic diversity and maximum spread distance, patch size, shoot number per patch, and aboveground biomass. Moreover, abundance of the native dominant species Scirpus mariqueter was marginally significantly decreased with genotypic diversity of S. alterniflora, suggesting that enhanced invasive ability of S. alterniflora may have depressed the growth of the native species. There was no significant difference in most measures of performance among six genotypes, but we observed a transgressive over performance in four measures in multiple‐genotype patches. At the end of the experiment, there were significant nonadditive effects of genotypic diversity according to Monte Carlo permutations, in six‐genotype, but not three‐genotype plots. Our results indicated that both additive and nonadditive effects played roles in the positive relationship between genetic diversity and invasion success, and nonadditive effects were stronger as duration increased.     

The polysaccharides from Ganoderma lucidum: Are they always inhibitors on human hepatocarcinoma cells?

The antitumor activity of intracellular polysaccharides from submerged fermentation of Ganoderma lucidum was investigated focusing on the inhibition on human liver cancer cells. The polysaccharides inhibited human hepatocarcinoma cell HepG2 during earlier phase with lower dosage but obviously became less functional in later phase regardless of the dosage applied. However, apoptosis of the drugged HepG2 cells appeared in later incubation phase with high dosage, and the apoptosis could be enhanced by supplemental dose of the intracellular polysaccharides. Nevertheless, the intracellular polysaccharides inhibited other human hepatocarcinoma cells such as BEL-7402 and Huh-7 but luckily stimulated human normal liver cell L02 only in a positive dose- and time-dependent manner; so did the sulfated extracellular polysaccharides when it inhibited HepG2 and L02 cells. However, the toxicity of sulfated extracellular polysaccharides to L02 cells can be eliminated by the intracellular polysaccharides.     

Codon usage comparison of novel genes in clinical isolates of Haemophilus influenzae

A similarity statistic for codon usage was developed and used to compare novel gene sequences found in clinical isolates of Haemophilus influenzae with a reference set of 80 prokaryotic, eukaryotic and viral genomes. These analyses were performed to obtain an indication as to whether individual genes were Haemophilus-like in nature, or if they probably had more recently entered the H.influenzae gene pool via horizontal gene transfer from other species. The average and SD values were calculated for the similarity statistics from a study of the set of all genes in the H.influenzae Rd reference genome that encoded proteins of 100 amino acids or longer. Approximately 80% of Rd genes gave a statistic indicating that they were most like other Rd genes. Genes displaying codon usage statistics >1 SD above this range were either considered part of the highly expressed group of H.influenzae genes, or were considered of foreign origin. An alternative determinant for identifying genes of foreign origin was when the similarity statistics produced a value that was much closer to a non-H.influenzae reference organism than to any of the Haemophilus species contained in the reference set. Approximately 65% of the novel sequences identified in the H.influenzae clinical isolates displayed codon usages most similar to Haemophilus sp. The remaining novel sequences produced similarity statistics closer to one of the other reference genomes thereby suggesting that these sequences may have entered the H.influenzae gene pool more recently via horizontal transfer.     

Dose-dependent effects of L-carnosine on the renal sympathetic nerve and blood pressure in urethane-anesthetized rats

The physiological function of L-carnosine (beta-alanyl-L-histidine) synthesized in mammalian muscles has been unclear. Previously, we observed that intravenous (i.v.) injection of L-carnosine suppressed renal sympathetic nerve activity (RSNA) in urethane-anesthetized rats, and L-carnosine administered via the diet inhibited the elevation of blood pressure (BP) in deoxycorticosterone acetate salt hypertensive rats. To identify the mechanism, we examined effects of i.v. or intralateral cerebral ventricular (l.c.v.) injection of various doses of L-carnosine on RSNA and BP in urethane-anesthetized rats. Lower doses (1 microg i.v.; 0.01 microg l.c.v.) of L-carnosine significantly suppressed RSNA and BP, whereas higher doses (100 microg i.v.; 10 microg l.c.v.) elevated RSNA and BP. Furthermore, we examined effects of antagonists of histaminergic (H1 and H3) receptors on L-carnosine-induced effects. When peripherally and centrally given, thioperamide, an H3 receptor antagonist, blocked RSNA and BP decreases induced by the lower doses of peripheral L-carnosine, whereas diphenhydramine, an H1 receptor antagonist, inhibited increases induced by the higher doses of peripheral L-carnosine. Moreover, bilateral lesions of the hypothalamic suprachiasmatic nucleus eliminated both effects on RSNA and BP induced by the lower (1 microg) and higher (100 microg) doses of peripheral L-carnosine. These findings suggest that low-dose L-carnosine suppresses and high-dose L-carnosine stimulates RSNA and BP, that the suprachiasmatic nucleus and histaminergic nerve are involved in the activities, and that L-carnosine acts in the brain and possibly other organs.     

Prevalence of viable Toxoplasma gondii in beef, chicken, and pork from retail meat stores in the United States: risk assessment to consumers

The prevalence of viable Toxoplasma gondii was determined in 6,282 samples (2,094 each of beef, chicken, and pork) obtained from 698 retail meat stores from 28 major geographic areas of the United States. Each sample consisted of a minimum of 1 kg of meat purchased from the retail meat case. To detect viable T. gondii, meat samples were fed to T. gondii-free cats and feces of cats were examined for oocyst shedding. Initially, 100 g of meat from 6 individual samples of a given species were pooled (total, 600 g), fed to a cat over a period of 3 days, and feces were examined for oocysts for 14 days; the remaining meat samples were stored at 4 C for 14 days (until results of the initial cat fecal examination were known). When a cat fed pooled samples had shed oocysts, 6 individual meat samples from each pool were bioassayed for T. gondii in cats and mice. Toxoplasma gondii isolates were then genetically characterized using the SAG2 locus and 5 hypervariable microsatellite loci. In all, 7 cats fed pooled pork samples shed oocysts. Toxoplasma gondii oocysts were detected microscopically in the feces of 2 of the cats; 1 isolate was Type II and the second was Type III. Analyzed individually, T. gondii was detected by bioassay in 3 of the 12 associated samples with genetic data indicating T. gondii isolates present in 2. The remaining 5 pooled pork samples had so few oocysts that they were not initially detected by microscopic examination, but rather by mouse bioassay of cat feces. Two were Type I, 1 was Type II, and 2 were Type III. None of the cats fed chicken or beef samples shed oocysts. Overall, the prevalence of viable T. gondii in retail meat was very low. Nevertheless, consumers, especially pregnant women, should be aware that they can acquire T. gondii infection from ingestion of undercooked meat, and in particular, pork. Cooking meat to an internal temperature of 66 C kills T. gondii.