Eisenstasin,new antistasin family inhibitor from the earthworm

A couple of new antistasin family serine protease inhibitors have been isolated from the non-hematophagous earthworm, Eisenia andrei. These novel inhibitors have been designated as eisenstasin I and II. Similar to other antistasin family inhibitors, eisenstasin I and II feature 3 and 4 internal repeats, respectively, of a 24–29 amino acid sequence, both of which exhibit a conserved pattern of 6-cysteine/2-glycine at an identical position between the third and fourth cysteine residues. This suggests that the eisenstasins isolated from the earthworm are members of the antistasin family. The eisenstasins are 82% similar with regard to amino acid sequences and exhibit over 70% similarity with the antistasins from the earthworm Lumbricus rubellus, while also displaying less than 40% sequence similarity with the leech antistasins. Earthworm eisenstasins are basic proteins, primarily due to the frequent occurrence of arginine residues in their structure, especially at the C-terminal region. As arginine is a key residue for the substrate specificity of some serine proteases including FXa, it is thought that these multiple arginine residues may play a role in the inhibitory characteristics of the eisenstasins. Considering the structure and number of the internal repeats derived from a variety of animal species, the deletion as well as the duplication of all or part of an internal repeat may be implicated in the evolution of the structure and function of the antistasin family inhibitors.     

An enzymatic method to distinguish tetrahydrobiopterin from oxidized biopterins using UDP-glucose:tetrahydrobiopterin glucosyltransferase

The quantitative determination of tetrahydrobiopterin (BH4) and its oxidized forms (dihydrobiopterin and biopterin) is important in searching for possible markers of neuropsychiatric and cardiovascular disorders as well as in diagnosing BH4 deficiencies. Currently, two high-performance liquid chromatography (HPLC) methods are available, although both have some limitations. We developed an enzymatic method to distinguish BH4 from the oxidized forms by employing BH4:UDP-glucose α-glucosyltransferase (BGluT), which catalyzes glucosyl transfer from UDP-glucose to BH4. The recombinant BGluT isolated from Escherichia coli converted essentially all of the BH4 in a mixture containing oxidized biopterins to the glucoside while leaving the oxidized forms intact. Therefore, acidic iodine oxidation of the reaction mixture followed by single fluorescence HPLC permitted the determination of biopterin and biopterin-glucoside, which represent oxidized biopterins and BH4, respectively. The validity of the method was evaluated using authentic biopterins and animal samples such as human urine, rat plasma, and rat liver. The BGluT-catalyzed reaction not only would reduce the burden of chromatographic separation but also would promise non-HPLC analysis of BH4.     

A Bayesian Semiparametric Survival Model with Longitudinal Markers

Summary We consider inference for data from a clinical trial of treatments for metastatic prostate cancer. Patients joined the trial with diverse prior treatment histories. The resulting heterogeneous patient population gives rise to challenging statistical inference problems when trying to predict time to progression on different treatment arms. Inference is further complicated by the need to include a longitudinal marker as a covariate. To address these challenges, we develop a semiparametric model for joint inference of longitudinal data and an event time. The proposed approach includes the possibility of cure for some patients. The event time distribution is based on a nonparametric Pólya tree prior. For the longitudinal data we assume a mixed effects model. Incorporating a regression on covariates in a nonparametric event time model in general, and for a Pólya tree model in particular, is a challenging problem. We exploit the fact that the covariate itself is a random variable. We achieve an implementation of the desired regression by factoring the joint model for the event time and the longitudinal outcome into a marginal model for the event time and a regression of the longitudinal outcomes on the event time, i.e., we implicitly model the desired regression by modeling the reverse conditional distribution.     

Intestinal Helminthic Infections Diagnosed by Colonoscopy in a Regional Hospital during 2001-2008

The present study investigated characteristics of 24 parasite infection cases detected during colonoscopy in a regional hospital from January 2001 to December 2008. Sixteen patients were confirmed with Trichuris trichiura infection, 6 patients were with Ascaris lumbricoides infection, 1 patient with Enterobius vermicularis infection, and 1 patient with Anisakis infection. Among them, 7 patients (43.8%) were asymptomatic. Colonoscopy findings were normal in 18 patients (75.0%). Among the patients with T. trichiura infection, colonoscopy showed several erosions in 2 patients (8.3%) and non-specific inflammation of the affected segment of the colon in 3 patients (12.5%). In 1 patient with anisakiasis, colonoscopy revealed a markedly swollen colonic wall. Stool examinations were performed before treatment in 7 patients (29.2%) and were all negative for parasite eggs or worms. These results suggest that colonoscopy is a useful diagnostic approach for parasitic infections even for asymptomatic patients and for patients with negative stool examinations.     

Molecular phylogeny of mangrove oysters (Crassostrea) from Brazil

As a result of phenotypic plasticity, the cupped oysters (Crassostrea)are difficult to identify by means of their morphology. However,molecular DNA markers are a useful means of discriminating amongthese species. Cupped oysters are one of the most widely culturedmarine invertebrates and correct species identification is importantin aquaculture. Moreover, the molecular phylogeny of the genusCrassostrea and the subfamily Crassostreinae is still not clear.In order to identify the Brazilian cupped oysters and to clarifythe phylogenetic relationships of these species, we sequenceda fragment of mitochondrial DNA (16S rRNA gene) from 120 specimenscollected at nine different sites distributed along the Braziliancoast. The results identified two native species of oyster:Crassostrea gasar, from the Amazon to the Parnaíba delta;and Crassostrea rhizophorae, from the northeast (Fortim) tothe south of Brazil. An exotic Crassostrea species, closelyrelated to Indo-Pacific Crassostrea, was found in one locationin the north of Brazil. Crassostrea showed monophyly and theAtlantic oysters are clearly separated from the Indo-Pacificcluster. (Received 30 May 2006; accepted 12 April 2007)     

Comparative analysis of virulence genes, genetic diversity, and phylogeny of commensal and enterotoxigenic Escherichia coli isolates from weaned pigs

If the acquisition of virulence genes (VGs) for pathogenicity were not solely acquired through horizontal gene transfers of pathogenicity islands, transposons, and phages, then clonal clusters of enterotoxigenic Escherichia coli (ETEC) would contain few or even none of the VGs found in strains responsible for extraintestinal infections. To evaluate this possibility, 47 postweaning diarrhea (PWD) ETEC strains from different geographical origins and 158 commensal E. coli isolates from the gastrointestinal tracts of eight group-housed healthy pigs were screened for 36 extraintestinal and 18 enteric VGs using multiplex PCR assays. Of 36 extraintestinal VGs, only 8 were detected (fimH, traT, fyuA, hlyA, kpsMtII, k5, iha, and ompT) in the ETEC collection. Among these, hlyA (alpha-hemolysin) and iha (nonhemagglutinating adhesin) occurred significantly more frequently among the ETEC isolates than in the commensal isolates. Clustering analysis based on the VG profiles separated commensal and ETEC isolates and even differentiated serogroup O141 from O149. On the other hand, pulsed-field gel electrophoresis (PFGE) successfully clustered ETEC isolates according to both serotype and geographical origin. In contrast, the commensal isolates were heterogeneous with respect to both serotype and DNA fingerprint. This study has validated the use of VG profiling to examine pathogenic relationships between porcine ETEC isolates. The clonal relationships of these isolates can be further clarified by PFGE fingerprinting. The presence of extraintestinal VGs in porcine ETEC confirmed the hypothesis that individual virulence gene acquisitions can occur concurrently against a background of horizontal gene transfers of pathogenicity islands. Over time, this could enable specific clonotypes to respond to host selection pressure and to evolve into new strains with increased virulence.     

Both caffeoyl Coenzyme A 3-<Emphasis Type="Italic">O</Emphasis>-methyltransferase 1 and caffeic acid <Emphasis Type="Italic">O</Emphasis>-methyltransferase 1 are involved in redundant functions for lignin,flavonoids and sinapoyl malate biosynthesis in Arabidopsis

Two methylation steps are necessary for the biosynthesis of monolignols, the lignin precursors. Caffeic acid O-methyltransferase (COMT) O-methylates at the C5 position of the phenolic ring. COMT is responsible for the biosynthesis of sinapyl alcohol, the precursor of syringyl lignin units. The O-methylation at the C3 position of the phenolic ring involves the Caffeoyl CoA 3-O-methyltransferase (CCoAOMT). The CCoAOMT 1 gene (At4g34050) is believed to encode the enzyme responsible for the first O-methylation in Arabidopsis thaliana. A CCoAOMT1 promoter-GUS fusion and immunolocalization experiments revealed that this gene is strongly and exclusively expressed in the vascular tissues of stems and roots. An Arabidopsis T-DNA null mutant named ccomt 1 was identified and characterised. The mutant stems are slightly smaller than wild-type stems in short-day growth conditions and has collapsed xylem elements. The lignin content of the stem is low and the S/G ratio is high mainly due to fewer G units. These results suggest that this O-methyltransferase is involved in G-unit biosynthesis but does not act alone to perform this step in monolignol biosynthesis. To determine which O-methyltransferase assists CCoAOMT 1, a comt 1 ccomt1 double mutant was generated and studied. The development of comt 1 ccomt1 is arrested at the plantlet stage in our growth conditions. Lignins of these plantlets are mainly composed of p-hydroxyphenyl units. Moreover, the double mutant does not synthesize sinapoyl malate, a soluble phenolic. These results suggest that CCoAOMT 1 and COMT 1 act together to methylate the C3 position of the phenolic ring of monolignols in Arabidopsis. In addition, they are both involved in the formation of sinapoyl malate and isorhamnetin.     

Topological analysis of the complex formed between neurokinin A and the NK2 tachykinin receptor

Neurokinin A stimulates physiological responses in the peripheral and central nervous systems upon interacting primarily with the tachykinin NK2 receptor (NK2R). In this study, the structure of NKA bound to the NK2R is characterised by use of fluorescence resonance energy transfer. Four fluorescent NKA analogues with Texas red introduced at amino acid positions 1, 4, 7 and 10 were prepared. When bound to a NK2R carrying enhanced green fluorescent protein at the N-terminus, all peptides reduce green fluorescent protein fluorescence from 10% to 50% due to energy transfer. The derived donor-acceptor distances are 46, 55, 59 and 69 A for the fluorophore linked to positions 1-10, respectively. The monotonic increase in distance clearly indicates that the peptide adopts an extended structure when bound to its receptor. The present data are used, in combination with rhodopsin structure, fluorescence studies, photoaffinity labelling and site-directed mutagenesis data to design a computer model of the NKA-NK2R complex. We propose that the N-terminus of NKA is exposed and accessible to the extracellular medium. Subsequent amino acids of the NKA peptide become progressively more buried residues up to approximately one-third of the transmembrane-spanning domain.     

Antimicrobial activity of mupirocin, daptomycin, linezolid, quinupristin/dalfopristin and tigecycline against vancomycin-resistant enterococci (VRE) from clinical isolates in Korea (1998 and 2005)

It is a hot clinical issue whether newly approved antimicrobial agents such as daptomycin, linezolid, quinupristin/dalfopristin (synercid) and tigecycline are active enough to be used for infections caused by vancomycin resistant bacteria. We performed susceptibility tests for mupirocin, which is in widespread clinical use in Korea, and four new antimicrobials, daptomycin, linezolid, quinupristin/dalfopristin and tigecycline, against vancomycin-resistant Enterococcus faecalis and Enterococcus faecium isolated from Korean patients in 1998 and 2005 to evaluate and compare the in vitro activity of these antimicrobials. Among these agents, quinupristin/dalfopristin, which is rarely used in hospitals in Korea, showed relatively high resistance to several vancomycin-resistant enterococci (VRE) isolated in 2005. Likewise, daptomycin, linezolid and tigecycline have not yet been in clinical use in Korea. However, our results showed that most of the 2005 VRE isolates were already resistant to linezolid and daptomycin (highest minimum inhibitory concentration (MIC) value >100 microg/ml). Compared with the other four antimicrobial agents tested in this study, tigecycline generally showed the greatest activity against VRE. However, four strains of 2005 isolates exhibited resistance against tigecycline (MIC >12.5 microg/ml). Almost all VRE were resistant to mupirocin, whereas all E. faecium isolated in 1998 were inhibited at concentrations between 0.8 to approximately 1.6 microg/ml. In conclusion, resistances to these new antimicrobial agents were exhibited in most of VRE strains even though these new antibiotics have been rarely used in Korean hospitals.