Anticoagulant proteases from western diamondback rattlesnake (Crotalus atrox) venom

Crotalus atrox venom contains agents that render human fibrinogen and plasma incoagulable by thrombin. To elucidate the mechanism of alteration of fibrinogen clotting function by the venom, four immunochemically different proteases, I, II, III, and IV, were purified from the venom by anion-exchange chromatography and column gel filtration. All four proteases had anticoagulant activity rendering purified fibrinogen incoagulable. Proteases I and IV do not affect fibrinogen in plasma but in purified fibrinogen cleave the A alpha chain first and then the B beta and gamma chains. Both enzymes are metalloproteases containing a single polypeptide chain with 1 mol of zinc, are inhibited by (ethylenedinitrilo)tetraacetate and human alpha 2-macroglobulin, and have an optimal temperature of 37 degrees C and an optimal pH of 7. Protease I has a molecular weight (Mr) of 20 000 and is the most cationic. Protease IV has an Mr of 46 000 and is the most anionic glycoprotein with one free sulfhydryl group. Proteases II and III degrade both purified fibrinogen and fibrinogen in plasma, cleaving only the B beta chain and leaving the A alpha and gamma chains intact. Both enzymes are alkaline serine proteases, cleave chromogenic substrates at the COOH terminal of arginine or lysine, are inhibited by diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride, and have an optimal temperature of 50-65 degrees C. Protease II is a single polypeptide chain glycoprotein with an Mr of 31 000. Protease III is a two polypeptide chain protein with an Mr of 24 000, each of the two chains having an Mr of 13 000; its activity is not affected by major protease inhibitors of human plasma. Proteases II and III are enzymes with unique and limited substrate specificity by cleaving only the B beta chain, releasing a peptide of Mr 5000 and generating a fibrinogen derivative of Mr 325 000, with intact A alpha and gamma chains and poor coagulability. Since the two enzymes are active in human plasma and serum, it is postulated that proteases II and III can mediate anticoagulant effects in vivo after envenomation.     

EXAFS analysis of the pH dependence of the blue-copper site in amicyanin from Thiobacillus versutus

The room temperature Cu K-edge EXAFS (extended X-ray absorption fine structure) spectrum of reduced and oxidized amicyanin, the blue copper protein from Thiobacillus versutus, was measured at low and high pH. The data interpretation was partly based on independent NMR evidence for the occurrence of a ligand histidine protonation at low pH (pKa = 6.9) in the reduced protein. In the oxidized protein two nitrogen-donors (from two histidines; Cu-N distances 1.95-2.01 A and 1.86-1.89 A) and a sulfur-donor (from a cysteine; Cu-S distance 2.11-2.13 A) were identified and the coordination appears independent of pH. Upon reduction at high pH the Cu-S bond and one of the Cu-N bonds lengthen slightly (from 2.11 to 2.19 A and from 2.01 to 2.18 A, respectively). Upon lowering of the pH one of the N-donors of the Cu in reduced amicyanin disappears from the Cu EXAFS and a second S-donor (from a methionine) becomes visible at 2.41 A from the Cu. The Debye-Waller factors are compatible with a Cu-N vibrational stretch frequency in the range of 150-250 cm-1 and one greater than 285 cm-1, and a Cu-S vibrational stretch frequency of about 150 cm-1 (Cu-Smet; reduced amicyanin at low pH) and one in the range of 230-800 cm-1 (Cu-Scys).     

Comparison of two colorimetric assays to determine viral infectivity in micro culture virus titration

Efficacy of two colorimetric assays, viz. MTT (3-4,5-dimethylthiazol-2-(yl-2,5-diphenyl tetrazolium bromide) and neutral red (NR) assays, performed by integrating them to micro culture virus titration (MCVT), was compared with the conventional MCVT method in terms of percentages of infectivity and 50% infectivity end points by employing Polio virus type-3 and Dengue virus type 4 as the candidate viruses. The results suggested that MTT assay has an edge over NR assay as well as conventional MCVT method. For the first time, NR assay has been successfully employed for the determination of virus infectivity titre.     

Lentivirus vectors using human and simian immunodeficiency virus elements

Lentivirus vectors based on human immunodeficiency virus (HIV) type 1 (HIV-1) constitute a recent development in the field of gene therapy. A key property of HIV-1-derived vectors is their ability to infect nondividing cells. Although high-titer HIV-1-derived vectors have been produced, concerns regarding safety still exist. Safety concerns arise mainly from the possibility of recombination between transfer and packaging vectors, which may give rise to replication-competent viruses with pathogenic potential. We describe a novel lentivirus vector which is based on HIV, simian immunodeficiency virus (SIV), and vesicular stomatitis virus (VSV) and which we refer to as HIV/SIVpack/G. In this system, an HIV-1-derived genome is encapsidated by SIVmac core particles. These core particles are pseudotyped with VSV glycoprotein G. Because the nucleotide homology between HIV-1 and SIVmac is low, the likelihood of recombination between vector elements should be reduced. In addition, the packaging construct (SIVpack) for this lentivirus system was derived from SIVmac1A11, a nonvirulent SIV strain. Thus, the potential for pathogenicity with this vector system is minimal. The transduction ability of HIV/SIVpack/G was demonstrated with immortalized human lymphocytes, human primary macrophages, human bone marrow-derived CD34(+) cells, and primary mouse neurons. To our knowledge, these experiments constitute the first demonstration that the HIV-1-derived genome can be packaged by an SIVmac capsid. We demonstrate that the lentivirus vector described here recapitulates the biological properties of HIV-1-derived vectors, although with increased potential for safety in humans.     

Heterogeneity in gene loci associated with type 2 diabetes on human chromosome 20q13.1

Human chromosome 20q12-q13.1 has been linked to type 2 diabetes mellitus (T2DM) in multiple studies. We screened a 5.795-Mb region for diabetes-related susceptibility genes in a Caucasian cohort of 310 controls and 300 cases with T2DM and end-stage renal disease (ESRD), testing 390 SNPs for association with T2DM-ESRD. The most significant SNPs were found in the perigenic regions: HNF4A (hepatocyte nuclear factor 4alpha), SLC12A5 (potassium-chloride cotransporter member 5), CDH22 (cadherin-like 22), ELMO2 (engulfment and cell motility 2), SLC13A3 (sodium-dependent dicarboxylate transporter member 3), and PREX1 (phosphatidylinositol 3,4,5-triphosphate-dependent RAC exchanger 1). Haplotype analysis found six haplotype blocks globally associated with disease (p<0.05). We replicated the PREX1 SNP association in an independent case-control T2DM population and inferred replication of CDH22, ELMO2, SLC13A3, SLC12A5, and PREX1 using in silico perigenic analysis of two T2DM Genome-Wide Association Study data sets. We found substantial heterogeneity between study results.     

Admixture mapping of white cell count: genetic locus responsible for lower white blood cell count in the Health ABC and Jackson Heart studies

White blood cell count (WBC) is an important clinical marker that varies among different ethnic groups. African Americans are known to have a lower WBC than European Americans. We surveyed the entire genome for loci underlying this difference in WBC by using admixture mapping. We analyzed data from African American participants in the Health, Aging, and Body Composition Study and the Jackson Heart Study. Participants of both studies were genotyped across ≥ 1322 single nucleotide polymorphisms that were pre-selected to be informative for African versus European ancestry and span the entire genome. We used these markers to estimate genetic ancestry in each chromosomal region and then tested the association between WBC and genetic ancestry at each locus. We found a locus on chromosome 1q strongly associated with WBC (p < 10⁻¹²). The strongest association was with a marker known to affect the expression of the Duffy blood group antigen. Participants who had both copies of the common West African allele had a mean WBC of 4.9 (SD 1.3); participants who had both common European alleles had a mean WBC of 7.1 (SD 1.3). This variant explained ~20% of population variation in WBC. We used admixture mapping, a novel method for conducting genetic-association studies, to find a region that was significantly associated with WBC on chromosome 1q. Additional studies are needed to determine the biological mechanism for this effect and its clinical implications.     

16S rRNA-Based Analysis of Microbiota from the Cecum of Broiler Chickens

The microbiota of the intestinal tract of chickens plays an important role in inhibiting the establishment of intestinal pathogens. Earlier culturing and microscopic examinations indicated that only a fraction of the bacteria in the cecum of chickens could be grown in the laboratory. Therefore, a survey of cecal bacteria was done by retrieval of 16S rRNA gene sequences from DNA isolated from the cecal content and the cecal mucosa. The ribosomal gene sequences were amplified with universal primers and cloned or subjected to temporal temperature gradient gel electrophoresis (TTGE). Partial 16S rRNA gene sequences were determined from the clones and from the major bands in TTGE gels. A total of 1,656 partial 16S rRNA gene sequences were obtained and compared to sequences in the GenBank. The comparison indicated that 243 different sequences were present in the samples. Overall, sequences representing 50 phylogenetic groups or subgroups of bacteria were found, but approximately 89% of the sequences represented just four phylogenetic groups (Clostridium leptum, Sporomusa sp., Clostridium coccoides, and enterics). Sequences of members of the Bacteroides group, the Bifidobacterium infantis subgroup, and of Pseudomonas sp. each accounted for less than 2% of the total. Sequences related to those from the Escherichia sp. subgroup and from Lactobacillus, Pseudomonas, and Bifidobacterium spp. were generally between 98 and 100% identical to sequences already deposited in the GenBank. Sequences most closely related to those of the other bacteria were generally 97% or less identical to those in the databases and therefore might be from currently unknown species. TTGE and random cloning indicated that certain phylogenetic subgroups were common to all birds analyzed, but sequence data from random cloning also provided evidence for qualitative and quantitative differences among the cecal microbiota of individual birds reared under very similar conditions.