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Conserved Sites (conserved + site)
Selected AbstractsDetection of human sapovirus by real-time reverse transcription-polymerase chain reactionJOURNAL OF MEDICAL VIROLOGY, Issue 10 2006Tomoichiro Oka Abstract Sapovirus (SaV) is an agent of gastroenteritis for humans and swine, and is divided into five distinct genogroups (GI,GV) based on its capsid gene sequences. Typical methods of SaV detection include electron microscopy (EM), enzyme-linked immunosorbent assay (ELISA), and reverse transcription-polymerase chain reaction (RT-PCR). A novel TaqMan-based real-time RT-PCR assay was developed that is sensitive and has the ability to detect the broad range of genetically diverse human SaV strains. A nucleotide alignment of 10 full-length SaV genome sequences was subjected to similarity plot analysis, which indicated that the most conserved site was the polymerase-capsid junction in open reading frame 1 (ORF1). Based on multiple alignments of the 27 available sequences encoding this junction, we designed sets of primers and TaqMan MGB probes that detect human SaV GI, GII, GIV, and GV sequences in a single tube. The reactivity was confirmed with SaV GI, GII, GIV, and GV control plasmids, and the efficiency ranged from 2.5,×,107 to 2.5,×,101 copies per tube. Analysis using clinical stool specimens revealed that the present system was capable of detecting SaV GI, GII, GIV, and GV sequences, and no cross-reactivity was observed against other enteric viruses, including norovirus (NoV), rotavirus, astrovirus, and adenovirus. This is the first real-time RT-PCR system that could detect all genogroups of human sapoviruses. J. Med. Virol. 78:1347,1353, 2006. © 2006 Wiley-Liss, Inc. [source] A non-synonymous mutation in a conserved site of the MTTP gene is strongly associated with protein activity and fatty acid profile in pigsANIMAL GENETICS, Issue 6 2009J. Estellé Summary Despite the economic interest of the fatty acid profile in pigs, no gene has been convincingly associated with this trait so far. Here, the porcine microsomal triglyceride transfer protein (MTTP) gene, which plays a crucial role in the assembly of nascent lipoproteins, has been analysed as a positional candidate gene for a QTL affecting the fatty acid composition that was previously identified on chromosome 8 in an Iberian by Landrace F2 cross. By resequencing a panel of different breeds, a non-synonymous polymorphism in a conserved residue of the lipid transfer domain of MTTP was identified. Association analyses with this polymorphism showed a strong association with the fatty acid composition of porcine fat, much stronger than the QTL effect, in the F2 cross and in a synthetic Sino-European line. In addition, in vitro activity assays in liver protein extracts have shown that this mutation is also associated with the lipid transfer activity of the MTTP protein (P < 0.1). These results suggest that the detected polymorphism is a potential causal factor of the fatty acid composition QTL. There appears to be an interaction between the porcine MTTP genotype and the type of fat source in the pig diet, which would agree with the previous results on the biology of MTTP biology. [source] Commercial manufacturing scale formulation and analytical characterization of therapeutic recombinant antibodiesDRUG DEVELOPMENT RESEARCH, Issue 3 2004Reed J. Harris Abstract Stable therapeutic antibody dosage forms present production technology challenges, particularly when high-concentration formulations are needed to meet the elevated dose requirements that are generally required for successful antibody therapy. Solid dosage forms, such as lyophilized powders, are generally more stable than liquid formulations. High-concentration drug products can be achieved by reconstitution of the lyophilisate in a smaller volume than its initial (pre-lyophilization) volume, but requires a significant vial overfill. High-concentration liquid formulations are becoming feasible as new techniques and technologies become available. Analytical methods to detect subtle molecular variations have been developed to demonstrate manufacturing consistency. Some molecular heterogeneity is contributed by conserved sites, such as Asn297 glycosylation and the loss of heavy chain C-terminal Lys residues. Characteristics that affect potency, stability, or immunogenicity must be elucidated for each therapeutic antibody. Drug Dev. Res. 61:137,154, 2004. © 2004 Wiley-Liss, Inc. [source] HTLV-1 infection in blood donors from the Western Brazilian Amazon region: Seroprevalence and molecular study of viral isolatesJOURNAL OF MEDICAL VIROLOGY, Issue 11 2008Aline Cristina Mota-Miranda Abstract To determine the seroprevalence of HTLV-1 in Brazil, and to review the virus molecular epidemiology in this Amazon population (Rio Branco-Acre), 219 blood donors were screened for HTLV-1. Only one case of infection (0.46% seroprevalence) was detected during July 2004 screening at the Acre Hospital Foundation (FUNDACRE). Neighbor-joining and Maximum Likelihood phylogenetic analyses of two (n,=,2) complete LTR region sequences were performed with the PAUP* software. Since the HTLV-1 envelope surface (gp46) and transmembrane (gp21) glycoproteins are important for virus fitness, three envelope glycoproteins sequences (n,=,3) were analyzed using the Prosite tool to determinate potential protein sites. Phylogenetic analysis demonstrated that the new isolate described in this study, and the unpublished LTR strain described in a previous report belong to the Transcontinental subgroup of the Cosmopolitan subtype, inside the Latin American cluster. A similar result was obtained when submitting, to the Automated Genotyping System, three LTR partial sequences from a previous study of the seroprevalence of HTLV-1 in the same Amazon population. In all analyzed env sequences, the potential protein site was found: two PKC phosphorylation sites at amino acid (aa) positions 310,312 and 342,344, one CK2 phosphorylation site at 194,197aa, three N -glycosylation sites at 222,225aa, 244,247aa and 272,275aa, and a single N -myristylation site at 327,338aa. In conclusion, potential protein sites described in HTLV-1 gp46 and gp21 confirm the presence of conserved sites in the HTLV-1 envelope proteins, likewise phylogenetic analysis suggests a possible recent introduction of the virus into North Brazil. J. Med. Virol. 80:1966,1971, 2008. © 2008 Wiley-Liss, Inc. [source] Functional characterization of transcription factor binding sites for HNF1-alpha, HNF3-beta (FOXA2), HNF4-alpha, Sp1 and Sp3 in the human prothrombin gene enhancerJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 8 2003H. Ceelie Summary.,Background:,Prothrombin is a key component in blood coagulation. Overexpression of prothrombin leads to an increased risk of venous thrombosis. Therefore, the study of the transcriptional regulation of the prothrombin gene may help to identify mechanisms of overexpression. Objectives:,The aim of our study was to localize the regions within the prothrombin enhancer responsible for its activity, to identify the proteins binding to these regions, and to establish their functional importance. Methods:,We constructed a set of prothrombin promoter 5, deletion constructs containing the firefly luciferase reporter gene, which were transiently transfected in HepG2, HuH7 and HeLa cells. Putative transcription factor (TF) binding sites were evaluated by electrophoretic mobility shift assays. The functional importance of each TF binding site was evaluated by site directed mutagenesis and transient transfection of the mutant constructs. Results:,We confirmed the major contribution of the enhancer region to the transcriptional activity of the prothrombin promoter. Analysis of this region revealed putative binding sites for hepatocyte nuclear factor HNF4, HNF3-beta and specificity protein(Sp)1. We identified six different TFs binding to three evolutionary conserved sites in the enhancer: HNF4-alpha (site 1), HNF1-alpha, HNF3-beta and an as yet unidentified TF (site 2) and the ubiquitously expressed TFs Sp1 and Sp3 (site 3). Mutagenesis studies showed that loss of binding of HNF3-beta resulted in a considerable decrease of enhancer activity, whereas loss of HNF4-alpha or Sp1/Sp3 resulted in milder reductions. Conclusions:,The prothrombin enhancer plays a major role in regulation of prothrombin expression. Six different TFs are able to bind to this region. At least three of these TFs, HNF4-alpha, HNF3-beta and Sp1/Sp3, are important in regulation of prothrombin expression. [source] Fate of epiphytes on phorophytes with different architectural characteristics along the perturbation gradient of Sabal mexicana forests in Veracruz, MexicoJOURNAL OF VEGETATION SCIENCE, Issue 1 2010A. Aguirre Abstract Question: Vascular epiphytes and hemiepiphytes (E/HE) in neotropical forests account for a large fraction of plant richness, but little is known of how the interplay between phorophyte architectural characteristics and habitat perturbation affect communities of E/HE. Location: Sabal mexicana forests in a coastal area of Veracruz, Mexico. Methods: We compared communities of E/HE on phorophytes with different architectural characteristics , the palm S. mexicana and non-palm phorophytes , in three environments: conserved sites, perturbed sites and small regenerated forest fragments. We combined traditional (abundance, species richness, similarity and complementarity indices) and more recent (phylogenetic diversity) metrics to describe the communities of E/HE. Results: Overall, we recorded 924 E/HE individuals (nine families, 16 genera and 21 species). The abundance and species richness of E/HE was higher on palms than on non-palm phorophytes. Abundance-based complementarities between phorophytes and sites were high. We detected clear changes in community structure of E/HE with habitat perturbation, but there were no effects on the phylogenetic diversity of the E/HE community. Palm phorophytes hosted a more phylogenetically diverse community of E/HE than did non-palm phorophytes. Conclusions: Palm phorophytes are key elements supporting the conservation of resilient communities of E/HE in S. mexicana forest. Habitat fragmentation has a strong effect on the structure of the E/HE community in S. mexicana forests. Ferns are the group of epiphytes most severely affected by habitat perturbation, but we detected no significant effect on the phylogenetic diversity of the community. [source] Sex-linked barring in chickens is controlled by the CDKN2A,/B tumour suppressor locusPIGMENT CELL & MELANOMA RESEARCH, Issue 4 2010Anders R. Hellström Summary Sex-linked barring, a common plumage colour found in chickens, is characterized by black and white barred feathers. Previous studies have indicated that the white bands are caused by an absence of melanocytes in the feather follicle during the growth of this region. Here, we show that Sex-linked barring is controlled by the CDKN2A/B locus, which encodes the INK4b and ARF transcripts. We identified two non-coding mutations in CDKN2A that showed near complete association with the phenotype. In addition, two missense mutations were identified at highly conserved sites, V9D and R10C, and every bird tested with a confirmed Sex-linked barring phenotype carried one of these missense mutations. Further work is required to determine if one of these or a combined effect of two or more CDKN2A mutations is causing Sex-linked barring. This novel finding provides the first evidence that the tumour suppressor locus CDKN2A/B can affect pigmentation phenotypes and sheds new light on the functional significance of this gene. [source] | |||||||||||||