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PubMed2002

Prospective use of molecular typing of Mycobacterium tuberculosis by use of restriction fragment-length polymorphism in a public tuberculosis-control program.

We performed a prospective, community-based evaluation of molecular typing of Mycobacterium tuberculosis isolates as a method for tuberculosis (TB) control. We performed restriction fragment-length polymorphism (RFLP) analysis of the insertion sequences IS6110 and pTBN12 for isolates recovered from 61 of 62 patients with culture-positive TB in St. Louis during 12 months. Twenty-four (39%) of the 61 patients were infected with an isolate with an RFLP pattern that was shared with >/=1 other isolate, and 11 (46%) also had epidemiologic links with patients in their cluster of cases. One case each of laboratory cross-contamination and occupational transmission were discovered. The patients in clusters were more likely to be younger, black, United States-born, to have substance abuse problems, and to live in poorer areas. A predictive algorithm for molecular identification of clusters had a sensitivity and a specificity of 75%. This study allowed the TB-control program in St. Louis to be redirected toward the affected subpopulations.

Adult↗
PubMed2000

[Papillomavirus infection in healthy women from St.Petersburg].

The study was aimed to evaluate the features of human papillomavirus (HPV) infection in healthy female inhabitants of St. Petersburg. 309 attendants of gynecological clinics were recruited for the screening. All participants were of reproductive age and were lacking the clinical or morphological symptoms of HPV-related diseases. PCR revealed HPV-specific sequences in 90 (29%) women. HPV type distribution was similar to the one in USA and Europe. HPV-positivity did not correlate with age of females, age at sexual debut, or total life-long duration of sexual activity. However, HPV were overrepresented among women reporting excessive number of contraceptive abortions. The data imply that an establishment of large-scale HPV-testing in Russia could be a desirable component of preventive anti-cancer programs.

Adult↗
PubMed2001

Biological activities of glucagon-like peptide-1 analogues in vitro and in vivo.

Studies support a role for glucagon-like peptide 1 (GLP-1) as a potential treatment for diabetes. However, since GLP-1 is rapidly degraded in the circulation by cleavage at Ala(2), its clinical application is limited. Hence, understanding the structure-activity of GLP-1 may lead to the development of more stable and potent analogues. In this study, we investigated GLP-1 analogues including those with N-, C-, and midchain modifications and a series of secretin-class chimeric peptides. Peptides were analyzed in CHO cells expressing the hGLP-1 receptor (R7 cells), and in vivo oral glucose tolerance tests (OGTTs) were performed after injection of the peptides in normal and diabetic (db/db) mice. [D-Ala(2)]GLP-1 and [Gly(2)]GLP-1 showed normal or relatively lower receptor binding and cAMP activation but exerted markedly enhanced abilities to reduce the glycemic response to an OGTT in vivo. Improved biological effectiveness of [D-Ala(2)]GLP-1 was also observed in diabetic db/db mice. Similarly, improved biological activity of acetyl- and hexenoic-His(1)-GLP-1, glucagon((1-5)-, glucagon((1-10))-, PACAP(1-5)-, VIP(1-5)-, and secretin((1-10))-GLP-1 was observed, despite normal or lower receptor binding and activation in vitro. [Ala(8/11/12/16)] substitutions also increased biological activity in vivo over wtGLP-1, while C-terminal truncation of 4-12 amino acids abolished receptor binding and biological activity. All other modified peptides examined showed normal or decreased activity in vitro and in vivo. These results indicate that specific N- and midchain modifications to GLP-1 can increase its potency in vivo. Specifically, linkage of acyl-chains to the alpha-amino group of His(1) and replacement of Ala(2) result in significantly increased biological effects of GLP-1 in vivo, likely due to decreased degradation rather than enhanced receptor interactions. Replacement of certain residues in the midchain of GLP-1 also augment biological activity.

Amino Acid Sequence↗
PubMed1992

Association of amino acid sequences in the HLA-DQB1 first domain with antitopoisomerase I autoantibody response in scleroderma (progressive systemic sclerosis).

Previous studies in Caucasians with progressive systemic sclerosis (PSS) have suggested associations of antitopoisomerase I (antitopo I) autoantibodies with either serologically defined HLA-DR2 or DR5. To better define class II HLA associations with the antitopo I response, 161 PSS patients (132 Caucasians and 29 American blacks) were studied for antitopo I autoantibodies by immunodiffusion and immunoblotting, and their HLA-DRB1, DRB3, DQA1, and DQB1 alleles were determined by restriction fragment length polymorphic analysis and DNA oligotyping. Among Caucasians with antitopo I, HLA-DR5(DRB1*1101-*1104), DRB3*0202 and DQw3 (DQw7,8,9) were significantly increased in frequency. In American blacks, however, only HLA-DQB1*0301(DQw7) was significantly increased. The presence of HLA-DQB1*0301(DQw7) and other HLA-DQB1 alleles bearing the uncharged polar amino acid residue tyrosine at position 30 of the outermost domain was found in all antitopo I-positive Caucasian PSS patients compared with 66% of antitopo I-negative PSS patients (pc = 0.007) and 70% of normal controls (pc = 0.008), as well as all antitopo I-positive black patients. The association with HLA-DQB1 was independent of HLA-DR5(DRB1*1101-*1104) or any other HLA-DRB1, DRB3, or DQA1 alleles. Alternative or additional candidate epitopes for this autoimmune response include alanine at position 38 and threonine at position 77 of these same DQB1 alleles. These data suggest that genetic predisposition to the antitopo I response in PSS is associated most closely with the HLA-DQB1 locus.

Alleles↗
PubMed2006

First insights into the evolution of Streptococcus uberis: a multilocus sequence typing scheme that enables investigation of its population biology.

Intramammary infection with Streptococcus uberis is a common cause of bovine mastitis throughout the world. Several procedures to differentiate S. uberis isolates have been proposed. However, all are prone to interlaboratory variation, and none is suitable for the description of the population structure. We describe here the development of a multilocus sequence typing (MLST) scheme for S. uberis to help address these issues. The sequences of seven housekeeping gene fragments from each of 160 United Kingdom milk isolates of S. uberis were determined. Between 5 and 17 alleles were obtained per locus, giving the potential to discriminate between 1.3 x 10(7) sequence types. In this study, 57 sequence types (STs) were identified. Statistical comparisons between the maximum-likelihood trees constructed by using the seven housekeeping gene fragments showed that the congruence was no better than that between each tree and trees of random topology, indicating there had been significant recombination within these loci. The population contained one major lineage (designated the ST-5 complex). This dominated the population, containing 24 STs and representing 112 isolates. The other 33 STs were not assigned to any clonal complex. All of the isolates in the ST-5 lineage carried hasA, a gene that is essential for capsule production. There was no clear association between ST or clonal complex and disease. The S. uberis MLST system offers researchers a valuable tool that allows further investigation of the population biology of this organism and insights into the epidemiology of this disease on a global scale.

Animals↗
PubMed1995

Pattern of muscle fiber type formation in the pig.

The aim of this study was to analyze the temporal sequence of expression of the myosin isoforms in the populations of muscle fibers in the pig and to bring more information on the origin of the strikingly different pattern of fiber composition and distribution between the deep medial red (oxido-glycolytic) and superficial white (glycolytic) portions of semitendinosus (ST) muscle. Muscle samples were taken from 49-, 55-, 75-, 90-, 103-, and 113- (birth) day-old fetuses, from 6-, 11-, 21-, 35-, 50-, and 80-day-old piglets, and from a 3-year-old pig. Our results confirm the sequential formation of primary and secondary generation fibers. The use of immunohistochemistry and heterologous monoclonal antibodies (mAb) directed against specific myosin heavy chain (MHC) isoforms revealed a different pattern of gene expression between the two portions of the ST muscle for both generations of fibers. By 75 days of gestation (dg), primary myotubes from the deep medial portion stained positively for the anti-slow MHC mAb and negatively for the adult anti-fast MHC, whereas the opposite was observed in the superficial portion. Secondary fibers never expressed slow MHC until late gestation. Instead, they expressed an adult fast MHC isoform as soon as they formed in the deep medial portion and later on in the superficial portion. From late gestation to the first 3 postnatal weeks, slow MHC began to be expressed in a subpopulation of secondary fibers. These fibers were in the direct vicinity of primary myotubes in the deep medial portion, whereas their location could not be established in the superficial portion. The remaining secondary fibers matured to type IIA in the direct vicinity of these type I fibers and to type IIB at the periphery of the islets. In both portions of the muscle, a subpopulation of secondary fibers, the first ones to express slow MHC, also transitorily expressed a MHC that was identical or closely related to the alpha-cardiac MHC during the early postnatal period. A third generation of small diameter fibers was observed shortly after birth and reacted with the anti-fetal MHC mAb; their destiny remains to be established.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1997

A yeast ATP-binding cassette-type protein mediating ATP-dependent bile acid transport.

ATP-dependent transport of bile acids is a key determinant of bile flow in mammalian liver and is associated with cholesterol excretion, gallstone formation, and numerous inherited and acquired hepatobiliary diseases. Secretory vesicles and a vacuole enriched fraction purified from Saccharomyces cerevisiae also exhibit ATP-dependent bile acid transport. ATP-dependent transport of bile acids by the vacuolar fraction was independent of the vacuolar proton ATPase, responded to changes in the osmotically sensitive intravesicular space, and was saturable, exhibiting a Km of 63 microM for taurocholate. The BAT1 (bile acid transporter) gene was isolated from yeast DNA by polymerase chain reaction amplification using degenerate oligonucleotides hybridizing to conserved regions of ABC-type proteins. ATP-dependent bile acid transport was abolished when the BAT1 coding region was deleted from the genome and restored upon reintroduction of the gene. The deduced amino acid sequence predicts that Bat1p is an ABC-type protein 1661 amino acids in length, similar to mammalian cMOAT/cMRP1 and MRP1 transporters, yeast Ycf1p, and two yeast proteins of unknown function. Information obtained from the yeast BAT1 gene may aid identification of the gene encoding the mammalian bile acid transporter.

ATP-Binding Cassette Transporters↗
PubMed1999

Sialyltransferase isoforms are phosphorylated in the cis-medial Golgi on serine and threonine residues in their luminal sequences.

ST6Gal-I (alpha2,6-sialyltransferase) is expressed as two isoforms, STTyr and STCys, which exhibit differences in catalytic activity, trafficking through the secretory pathway, and proteolytic processing and secretion. We have found that the ST6Gal-I isoforms are phosphorylated on luminal Ser and Thr residues. Immunoprecipitation of 35S- and 32P-labeled proteins expressed in COS-1 cells suggests that the STTyr isoform is phosphorylated to a greater extent than the STCys isoform. Analysis of domain deletion mutants revealed that STTyr is phosphorylated on stem and catalytic domain amino acids, whereas STCys is phosphorylated on catalytic domain amino acids. An endoplasmic reticulum retained/retrieved chimeric Iip33-ST protein demonstrates drastically lower phosphorylation than does the wild type STTyr isoform. This suggests that the bulk of the ST6Gal-I phosphorylation is occurring in the Golgi. Treatment of cells with the ionophore monensin does not significantly block phosphorylation of the STTyr isoform, suggesting that phosphorylation is occurring in the cis-medial Golgi prior to the monensin block. This study demonstrates the presence of kinase activities in the cis-medial Golgi and the substantial phosphorylation of the luminal sequences of a glycosyltransferase.

Animals↗
PubMed1999

PHA synthase from chromatium vinosum: cysteine 149 is involved in covalent catalysis.

Polyhydroxyalkanoate synthase (PHA) from Chromatium vinosum catalyzes the conversion of 3-hydroxybutyryl-CoA (HB-CoA) to polyhydroxybutyrate (PHB) and CoA. The synthase is composed of a approximately 1:1 mixture of two subunits, PhaC and PhaE. Size-exclusion chromatography indicates that in solution PhaC and PhaE exist as large molecular weight aggregates. The holo-enzyme, PhaEC, has a specific activity of 150 units/mg. Each subunit was cloned, expressed, and purified as a (His)6-tagged construct. The PhaC-(His)6 protein catalyzed polymerization with a specific activity of 0.9 unit/mg; the PhaE-(His)6 protein was inactive (specific activity <0.001 unit/mg). Addition of PhaE-(His)6 to PhaC-(His)6 increased the activity several 100-fold. To investigate the priming step of the polymerization process, the PhaEC was incubated with a trimer of HB-CoA in which the terminal hydroxyl was replaced with tritium ([3H]-sT-CoA). After Sephadex G50 chromatography, the synthase contained approximately 0.25 equiv of the labile label per PhaC. Incubation of [3H]-sT-synthase with HB-CoA resulted in production of [3H]-polymer. Digestion of [3H]-sT-synthase with trypsin and HPLC analysis resulted in isolation of three labeled peptides. Sequencing by ion trap mass spectrometry showed that they were identical and that they each contained an altered cysteine (C149). One peptide contained the [3H]-sT while the other two contained, in addition to the [3H]-sT, one and two additional monomeric HBs, respectively. Mutation of C149 to alanine gave inactive synthase. The remaining two cysteines of PhaC, 292 and 130, were also mutated to alanine. The former had wild-type (wt) activity, while the latter had 0.004 wt % activity and was capable of making polymer. A mechanism is proposed in which PhaC contains all the elements essential for catalysis and the polymerization proceeds by covalent catalysis using C149 and potentially C130.

Acyltransferases↗
PubMed2000

Molecular scanning of the human PPARa gene: association of the L162v mutation with hyperapobetalipoproteinemia.

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a member of the steroid hormone receptor super family involved in the control of cellular lipid utilization. This makes PPARalpha a candidate gene for type 2 diabetes and dyslipidemia. The aim of this study was to investigate whether genetic variation in the human PPARalpha gene can influence the risk of type 2 diabetes and dyslipidemia among French Canadians. We therefore first determined the genomic structure of human PPARalpha, and then designed intronic primers to sequence the coding region and the exon-intron boundaries of the gene in 12 patients with type 2 diabetes and in 2 nondiabetic subjects. Sequence analysis revealed the presence of a L162V missense mutation in exon 5 of one diabetic patient. Leucine 162 is contained within the DNA binding domain of the human PPARalpha gene, and is conserved among humans, mice, rats, and guinea pigs. We subsequently screened a sample of 121 patients newly diagnosed with type 2 diabetes and their age and sex-matched nondiabetic controls, recruited from the Saguenay-Lac-St-Jean region of Northeastern Quebec, for the presence of the L162V mutation by a PCR-RFLP based method. There was no difference in L162 homozygote or V162 carrier frequencies between diabetics and nondiabetics. However, whether diabetic or not, carriers of the V162 allele had higher plasma apolipoprotein B levels compared to noncarriers (P 5 0.05). To further this association, we screened another sample of 193 nondiabetic subjects recruited in the greater Quebec City area. Carriers of the V162 allele compared with homozygotes of the L162 allele had significantly higher concentrations of plasma total and LDL-apolipoprotein B as well as LDL cholesterol (P </= 0.02). These results suggest an association between the PPARalpha V162 allele and the atherogenic/hyperapolipoprotein B dyslipidemia.

Animals↗
PubMed1997

P300 sequence effects, probability, and interstimulus interval.

Probability and interstimulus interval effects on P3(00) event-related potential (ERP) stimulus sequences were examined in 2 experiments. ERPs were elicited with an auditory-discrimination paradigm in which subjects were instructed to detect target stimuli from a series of target (T) and standard (S) tones that were varied by randomly presenting 1 of 4 sequence patterns (SS, TS, TT, ST). Experiment 1 manipulated target stimulus probability as either 0.33 or 0.67; Experiment 2 kept target probability at 0.33 and manipulated the interstimulus interval (ISI) as either 2 or 6 s. Increases in target stimulus probability produced smaller P300 amplitudes that were additive with stimulus sequence type. ISI did not reliably affect P300 amplitude, although ISI interacted with stimulus sequence. P300 latency from the stimulus sequences was influenced weakly by the probability and ISI factors, with few consistent sequence effects obtained for the N1, P2, and N2 potentials. The results suggest that even relatively short sequences can affect P300 amplitude in the same way as longer sequences: as the number of standard stimuli preceding the target increased, P300 amplitude increased. The theoretical implications of the findings are discussed in the context of applied testing situations.

Adolescent↗
PubMed2006

Evaluation of a real-time nucleic acid sequence-based amplification assay using molecular beacons for detection of human immunodeficiency virus type 1.

We evaluated the performance characteristics of a new, real-time nucleic acid sequence-based amplification (NASBA) assay that incorporates molecular beacon technology for detection of human immunodeficiency virus type 1 (HIV-1). The quantitative results were comparable to those obtained with three leading commercially available assays. The analytical sensitivity was 37 IU/ml. The NASBA assay detected clinically relevant recombinant viruses and all group M HIV-1 subtypes.

HIV Infections↗
PubMed2003

Characterization of the natural population of Bartonella henselae by multilocus sequence typing.

Investigations of the population genetics of Bartonella henselae have demonstrated a high level of diversity among strains, and the delineation of isolates into one of two subtypes, type I (Houston) and type II (Marseille), represented by specific 16S ribosomal DNA (rDNA) sequences, has long been considered the most significant genotypic division within the species. This belief is challenged by recent work suggesting a role for horizontal gene exchange in generating intraspecies diversity. We attempted to resolve this issue and extend exploration of the population structure of B. henselae by using multilocus sequence typing (MLST) to examine the distribution of polymorphisms within nine different genes in a sample of 37 human and feline isolates. MLST distinguished seven sequence types (STs) that resolved into three distinct lineages, suggesting a clonal population structure for the species, and support for these divisions was obtained by macrorestriction analysis using pulsed-field gel electrophoresis. The distribution of STs among isolates recovered from human infections was not random, and such isolates were significantly more often associated with one particular ST, lending further support to the suggestion that specific genotypes contribute disproportionately to the disease burden in humans. All but one isolate lay on lineages that bore the representative strain of either the Houston or Marseille subtype. However, the distribution of the two 16S rDNA alleles among the isolates was not entirely congruent with their lineage allocations, indicating that this is not a sensitive marker of the clonal divisions within the species. The inheritances of several of the genes studied could not be reconciled with one another, providing further evidence of horizontal gene transfer among B. henselae strains and suggesting that recombination has a role in shaping the genetic character of bartonellae.

Animals↗
PubMed2005

Hypoderma sinense: solving a century-old enigma.

Among the species of Hypoderma (Diptera: Oestridae) that have been described and named over the last three centuries, Hypoderma sinense Pleske has been the subject of several scientific discussions. Hypoderma sinense was described by T. Pleske in 1926 on the basis of only three females collected by the Russian explorer P. K. Kozlov nearly 25 years earlier during a scientific expedition to China (1900-1901). This species was examined by the foremost oestrid authorities and synonomized with H. lineatum. Recently a unique, unidentified species of Hypoderma was observed to infect cattle and yaks in China. Molecular and morphological observations confirmed the unique nature of the third-stage larvae. This data initiated a debate within the scientific community regarding the proper name of this species, in particular with reference to previous taxonomical discussion on the validity of H. sinense. The present work provides a historical overview of the Russian scientific expeditions that collected the specimens and of the explorers and the entomologists who contributed to the description of H. sinense. The morphological examination of the original type material of H. sinense and the comparison with females of H. lineatum indicated that the H. sinense lectotype, deposited at the Zoological Institute of the Russian Academy of Sciences, St Petersburg, was within the range of variation of H. lineatum. Comparisons of the cox1 (688 bp) sequence obtained from the leg of a paralectotype of H. sinense with those of H. bovis (Linneaus), H. lineatum (De Villers) and of a sixth valid species of Hypoderma identified as "H. sinense" available in GenBank revealed differences of 9.7%, 7.2% and 0.3%, respectively. On the basis of these results, we concluded that the nominal species H. sinense should be treated as valid.

Animals↗
PubMed1994

Purification and characterization of an endo-N-acetyl-beta-D-glucosaminidase from the culture medium of Stigmatella aurantiaca DW4.

A novel endo-N-acetyl-beta-D-glucosaminidase (ENGase), acting on the di-N-acetylchitobiosyl part of N-linked glycans, was characterized in the culture medium of Stigmatella aurantiaca DW4. Purified to homogeneity by ammonium sulfate precipitation, gel filtration, and chromatofocusing, this ENGase presents, upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a molecular mass near 27 kDa. Optimal pH and pI were 4.0 and 6.8, respectively. The enzyme, named ENGase St, exhibits high activity on oligomannoside-type glycoasparagines and glycoproteins and could also hydrolyze hybrid- and complex-type glycoasparagines but does not acts as a murein hydrolase.

Asparagine↗
PubMed1994

A thyroid hormone-regulated gene in Xenopus laevis encodes a type III iodothyronine 5-deiodinase.

The type III iodothyronine 5-deiodinase metabolizes thyroxine and 3,5,3'-triiodothyronine to inactive metabolites by catalyzing the removal of iodine from the inner ring. The enzyme is expressed in a tissue-specific pattern during particular stages of development in amphibia, birds, and mammals. Recently, a PCR-based subtractive hybridization technique has been used to isolate cDNAs prepared from Xenopus laevis tadpole tail mRNA that represent genes upregulated by thyroid hormone during metamorphosis. Sequence analysis of one of these cDNAs (XL-15) revealed regions of homology to the mRNA encoding the rat type I (outer ring) 5'-deiodinase, including a conserved UGA codon that encodes selenocysteine in the mammalian enzyme. We report here that the protein encoded by the XL-15 cDNA efficiently catalyzes the (inner ring) 5-deiodination of 3,5,3'-triiodothyronine with a Km value of 2 nM and is resistant to inhibition by propylthiouracil and aurothioglucose. Our analysis confirms that the UGA codon encodes a selenocysteine that is critical for the catalytic activity of the enzyme. In addition, the direct induction of XL-15 mRNA levels by thyroid hormone in X. laevis tadpole tail tissue and cultured cell lines correlates closely with increases in 5- (but not 5'-) deiodinase activity. These findings indicate that the XL-15 cDNA encodes a type III 5-deiodinase and underscores the importance of the trace element selenium in thyroid hormone metabolism.

Amino Acid Sequence↗
PubMed1996

Characterization of novel secreted and membrane proteins isolated by the signal sequence trap method.

We recently described a method, called the signal sequence trap (SST) method, to clone cDNAs of secreted proteins and/or type I transmembrane proteins containing N-terminal signal sequences by using an epitope-tagging expression plasmid vector. In this paper we describe the summary of a large-scale screening of approximately 5900 clones of an SST cDNA library constructed from mouse bone marrow stromal cell line ST-2 cells. Of 26 positive clones obtained and sequenced, 11 clones appeared to contain authentic signal sequences. Five of the clones corresponded to the 5' ends of the cDNA of known genes containing N-terminal signal sequences. The full-length cDNA clones of the 6 other unknown clones were isolated and sequenced. One clone, termed SDF3, encoded a mouse counterpart of human pigment epithelium-derived factor. Another clone, termed SDR1, had considerable homology with basigin, a member of the immunoglobulin superfamily. A third clone, termed SDF5, had partial homology with a Drosophila tissue polarity gene frizzled (fz) and its rat homologues, fz-1 and fz-2. The other three clones had no significant homology with sequences in the databases. These results indicate that the SST method is effective and useful for the isolation of secreted and membrane proteins without knowledge of their functions.

Amino Acid Sequence↗
PubMed2006

Genetic variations of 13 indigenous Chinese goat breeds based on cytochrome B gene sequences.

Phylogenetic relationships among and genetic variability within 13 Chinese indigenous goat breeds and Boer goat were analyzed using cytochrome b gene sequences. There were 44 variable sites found in a 642 bp sequence, and 46 Cyt b haplotypes were subsequently defined. The phylogeny analysis of haplotypes in combination with goat Cyt b sequences from GenBank shows that Chinese goats are obviously separated from wild goats and might come from Capra aegagrus. Further analysis indicated that indigenous Chinese goats might descend from at least two lineages; most of the individuals analyzed could be classified into lineage A as defined by Luikart, but five other goats were of uncertain lineage. The Tibet plateau is a possible place of origin for Chinese goats. The neighbor-joining tree based on pairwise differences among populations shows that most Tibetan goats, except the Middle Tibet type, cluster closely with North China goats, and then with South China goats. This result confirms that differences in genetic structure exist among goats in different geographic locations. Nucleotide diversity varied among populations. Tibet and North China goats had higher genetic diversity than South China goats. The fixation index (F (st)=87.72%) suggested that most of the total genetic variation was due to variation within populations. In addition, the results indicate that Cyt b gene sequence information alone might not be enough for phylogeny analysis among breeds within species, as shown by fewer polymorphic sites and lower bootstrap values on the neighbor-joining tree.

Animals↗