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T M Smith

Publications and source records attributed to T M Smith.

At least 37 records · Page 2Linked to original sources
PubMed1998

Stored renin isoforms in the developing rat kidney.

Fetal rat kidney contains renin in renal microvasculature, whereas adult rat kidney contains renin predominantly in juxtaglomerular cells. It is hypothesized that renin isoforms stored within these renal tissues may differ chemically and functionally. To test this hypothesis, stored renin isoforms in fetal and adult rat kidney were compared by isolating renin from adult and fetal kidney homogenate with pepstatin agarose. Pepstatin-eluted renin isoforms were separated by relative molecular size using one-dimensional polyacrylamide gel electrophoresis (SDS-PAGE), or by isoelectric point (pI) and size using two-dimensional (2D) gel electrophoresis. Isoforms were identified either by silver staining or immunoblotting. One-dimensional polyacrylamide gel electrophoresis of pepstatin-treated kidney homogenates showed a silver-stained band in the range of approximately 45 kDa, which corresponded to a silver-stained spot consistently seen on 2D gels. In fetal kidney homogenate, the approximately 45 kDa band had a pI of 5.3 +/- 0.1, whereas the corresponding band in adult samples had a basic pI of 6.0 +/- 0.05. Angiotensin I generation was measured to assess renin enzymatic activity. There was significantly more inactive renin in fetal kidney homogenate than in adult kidney homogenate (60.2 +/- 22.4 v 9.6 +/- 4.0 ng AI/mg protein/h, P < .05). There was significantly less active renin in fetal kidney homogenate than in adult kidney homogenate (5.4 +/- 0.4 v 36.5 +/- 14.2 ng AI/mg protein/h, P < .05). The average total renin activity in fetal kidney homogenate was significantly higher than in adult kidney homogenate (65.6 +/- 22.3 v 46.0 +/- 15.2, P < .05). These results demonstrate major differences in the physical and enzymatic forms of stored renin found in fetal and adult kidney. It is speculated that these variations in stored renin isoforms play a role in the developmental differential regulation of the intrarenal renin angiotensin system.

Animals↗
PubMed1998

Cross-linking induces homodimer formation and inhibits enzymatic activity of chicken stomach ecto-apyrase.

We have investigated the effect of cross-linking on the enzymatic activity and oligomer formation of the chicken stomach ecto-apyrase. Cross-linking with the hydrophobic, lysine-specific dithiobis(succinimidylpropionate) (DSP) caused equal inhibition of ATPase and ADPase activity in both the membrane-bound and detergent-solubilized ecto-apyrase. The inhibitory effect of cross-linking was reversed upon the addition of the reductant dithiothreitol. Western blots of aliquots of the cross-linked samples show decreased amounts of the monomeric 80 kDa ecto-apyrase and the appearance of a 160 kDa dimer under conditions inducing enzyme inhibition. Therefore, the chicken stomach ecto-apyrase, like the chicken gizzard ecto-ATPase, is likely a homodimer in vivo. Unlike the related gizzard ecto-ATPase, however, the native stomach ecto-apyrase is not stimulated, but rather inhibited by cross-linking, presumably due to different quaternary structural stability of the two enzymes.

Animals↗
PubMed1998

Snapshot of a large dynamic replicon in a halophilic archaeon: megaplasmid or minichromosome?

Extremely halophilic archaea, which flourish in hypersaline environments, are known to contain a variety of large dynamic replicons. Previously, the analysis of one such replicon, pNRC100, in Halobacterium sp. strain NRC-1, showed that it undergoes high-frequency insertion sequence (IS) element-mediated insertions and deletions, as well as inversions via recombination between 39-kb-long inverted repeats (IRs). Now, the complete sequencing of pNRC100, a 191,346-bp circle, has shown the presence of 27 IS elements representing eight families. A total of 176 ORFs or likely genes of 850-bp average size were found, 39 of which were repeated within the large IRs. More than one-half of the ORFs are likely to represent novel genes that have no known homologs in the databases. Among ORFs with previously characterized homologs, three different copies of putative plasmid replication and four copies of partitioning genes were found, suggesting that pNRC100 evolved from IS element-mediated fusions of several smaller plasmids. Consistent with this idea, putative genes typically found on plasmids, including those encoding a restriction-modification system and arsenic resistance, as well as buoyant gas-filled vesicles and a two-component regulatory system, were found on pNRC100. However, additional putative genes not expected on an extrachromosomal element, such as those encoding an electron transport chain cytochrome d oxidase, DNA nucleotide synthesis enzymes thioredoxin and thioredoxin reductase, and eukaryotic-like TATA-binding protein transcription factors and a chromosomal replication initiator protein were also found. A multi-step IS element-mediated process is proposed to account for the acquisition of these chromosomal genes. The finding of essential genes on pNRC100 and its property of resistance to curing suggest that this replicon may be evolving into a new chromosome.

Chromosome Mapping↗
PubMed1997

A soluble ecto-ATPase from Tetrahymena thermophila: purification and similarity to the membrane-bound ecto-ATPase of smooth muscle.

For the first time, a soluble, dedicated E-type ecto-ATPase has been identified and purified. This fully soluble ecto-ATPase is released into the growth media of the single-celled eukaryote, Tetrahymena, at a constant rate over time (independent of the growth phase of the cells) and it has characteristics similar to those previously described for the membrane-bound ecto-enzyme in Tetrahymena. It was purified by a combination of ion-exchange, size exclusion, and affinity chromatography and nondenaturing gel electrophoresis. Its molecular weight was determined to be approximately 66,000 Da by denaturing gel electrophoresis and approximately 69,000 Da by size exclusion chromatography of the native form. The purified soluble enzyme displays the general characteristics of a dedicated E-type ecto-ATPase such as Ca2+ or Mg2+ dependence, hydrolysis of ATP and other nucleoside triphosphates (but not nucleoside diphosphates) and insensitivity to common ATPase inhibitors (vanadate, azide, ouabain, N-ethylmaleimide and p-chloromercuriphenyl sulfonate). It was further shown to be immunologically similar (by polyclonal antibodies) to both the membrane-bound ecto-ATPase of chicken gizzard smooth muscle (66 kDa) and a 66-kDa protein in Tetrahymena plasma membranes. The ecto-ATPase enzyme activity was also shown to be present in both the body plasma membrane and ciliary plasma membrane fractions but the body membrane had slightly higher specific activities. We propose that this ecto-ATPase of Tetrahymena may play a role in inactivating purinergic signals, such as in their chemorepulsion responses to external GTP and ATP. It may also play a minor role in extracellular nucleotide scavenging.

Adenosine Triphosphatases↗
PubMed1997

Segmented filamentous bacteria in the bovine small intestine.

Segmented filamentous bacteria (SFB) were observed in a 28-day-old calf, attached to the absorptive villi. Morphologically, they were similar to SFB described in other animal species. Because these organisms cannot be cultured, further characterization was not possible. The organisms were confined to the upper third of the absorptive villi and were not seen attached to the follicle-associated epithelium of the Peyer's patch, or observed in the caecum or colon. Although they were often associated with minor lesions, their pathological significance was doubtful. With this report, segmented filamentous bacteria have now been described in virtually all the commercially important livestock and poultry species, in other domestic animals, and in man.

Animals↗
PubMed1997

Force decay and deformation of orthodontic elastomeric ligatures.

This study evaluated commercially available molded gray elastomeric ligatures from seven companies for force decay, dimensional change, and the relationship between ligature dimension and force. The initial wall thickness, inside diameter, outside diameter, and force levels of each ligature were measured. Three of four test groups of ligatures were stretched over stainless steel dowels with a circumference approximating that of a large orthodontic twin bracket. Test group 1 was kept at room temperature and humidity for 28 days and test group 2 in a synthetic saliva bath at 37 degrees C, pH 6.84 for 28 days. The residual forces and dimensional changes were measured. The third test group was placed in a synthetic saliva bath at 37 degrees C, pH 6.84, and force levels recorded at initial, 24 hours, 7 days, 14 days, and 28 days. The fourth test group of unstretched samples was placed in a synthetic saliva bath at 37 degrees C, pH 6.84 for 28 days to evaluate dimensional changes due solely to moisture sorption. The results for stretched samples in a simulated oral environment revealed the following: (1) Moisture and heat had a pronounced effect on force decay and permanent deformation, (2) a positive correlation existed between the wall thickness and force, (3) a negative correlation existed between the inside diameter and force, (4) a weak correlation existed between outside diameter and force, (5) the greatest force loss occurred in the first 24 hours and the decay pattern was similar for all ligatures tested, and (6) unstretched ligatures absorbed moisture in the range of 0.060% to 3.15%. The ligatures tested appear to be suitable for use during initial aligning and leveling. However, the rapid force loss and permanent deformation of these products may preclude their use for rotational and torque corrections.

Analysis of Variance↗
PubMed1997

Food control systems in Canada.

This paper provides an overview of the responsibilities and jurisdictional boundaries of Health Canada (HC) and Agriculture and Agri-Food Canada (AAFC) with regard to food regulation in Canada. It examines their interagency coordination within the federal structure and with other levels of government, industry, and the consumer. The international developments are considered with the North American Free Trade Agreement (NAFTA) and the Canada, United States Trade Agreement (CUSTA) being regarded as likely to have a significant future impact. The federal food safety and quality system is complex and fragmented. Federal food regulation comes under the jurisdiction of four federal departments: HC, AAFC, Industry Canada (IC), and Fisheries and Oceans Canada (FOC). All four departments are involved with inspection, surveillance, and the analysis of food sold in Canada. In addition, Canada's ten provincial and two territorial governments have provincial-, regional-, municipal-, and local-level governments that also have jurisdiction over food safety and quality. Consideration is first given to the main legislative provision covering food--the Federal Food and Drugs Act. This Act is administered by several of the Federal Government departments. The role of these departments is examined individually along with additional, more specific legal provisions for which responsibility is not divided (in particular, the Canada Agricultural Products [CAP] Act administered by AAFC, and the Consumer Packaging and Labeling Act [CPLA] administered by IC). The various reviews that have taken place in the recent past and those still in progress are considered, and the final part of this paper looks at the international developments that are likely to have a major impact on the future development of the Canadian food control system.

Canada↗
PubMed1997

Hopper: software for automating data tracking and flow in DNA sequencing.

MOTIVATION: Genome-scale DNA sequencing is a multistep process in which large numbers of small template clones are propagated, purified, sequenced and analyzed on acrylamide gels. A significant challenge to these projects is the scale at which the data handling must be done. Hence, large-scale sequencing facilities will benefit from tracking template DNA information (purification methods, reaction and electrophoresis conditions) in a systematic fashion. A lack of software tools that support automated sample entry, and automatic data storage, retrieval and analysis are a major hindrance to recording and using laboratory workflow information to monitor the overall quality of data production. RESULTS: The UNIX file system has been used to prototype automation of the flow of data from the ABI sequencer to a data repository. Data are automatically processed by a central Perl program, Hopper, which runs a series of programs that analyze data quality (read length estimate, fraction of indeterminate bases, and number of contaminating and repetitive sequences), assemble shotgun sequence data, and generates simple reports describing the results.

Database Management Systems↗
PubMed1996

Testing for adverse reactions using prescription event monitoring.

The Drug Safety Research Unit's current methods of investigating adverse drug reactions using prescription event monitoring are discussed. The statistical properties of estimators of rates of occurrence of events in post-marketing surveillance using prescription event monitoring are considered, and a simple model is proposed based on an exponential distribution of time to first occurrence of the event. It is shown that current methodology closely relates to the use of maximum likelihood estimation under this assumption and the distributions of the estimators are shown to be approximately normal, which allows simple confidence intervals and tests to be developed. Two recent applications are considered and corresponding simulations are presented to verify the approximate properties of the test statistics, based on ratios of rates over time and between drugs. Sources of bias in the rates and rate ratios are considered, including under-reporting in later months. A rule-of-thumb, developed from many years experience, is shown to be generally conservative, except when these under-reporting biases are large.

Adverse Drug Reaction Reporting Systems↗
PubMed1996

Studies on the biosynthesis of thiostrepton: 4-(1-hydroxyethyl)quinoline-2-carboxylate as a free intermediate on the pathway to the quinaldic acid moiety.

Specifically 13C-labeled quinoline-2-carboxylate derivatives were synthesized from quinoline and used to study the biosynthesis of thiostrepton in a strain of Streptomyces laurentii. 13C NMR analysis of thiostrepton recovered after feeding methyl (RS)-[11-13C]-4-(1-hydroxyethyl)quinoline-2-carboxylate or methyl [11-13C]-4-acetylquinoline-2-carboxylate showed conclusively that these compounds are specifically and efficiently incorporated into thiostrepton. Both compounds were also detected in cultures of the producing organism by isotope dilution analysis. The significance of the relative endogenous concentrations of the two compounds and of the relative extent of the incorporation of exogenously added labeled material into thiostrepton are discussed in terms of the biosynthetic pathway linking tryptophan and 4-(1-hydroxyethyl)quinoline-2-carboxylate in S. laurentii. A highly specific enzyme activity was detected in cell-free extracts of S. laurentii that was capable of adenylating (12S)-4-(1-hydroxyethyl)quinoline-2-carboxylic acid. Partial purification of the enzyme was achieved. The enzyme was found to be specific for the enantiomer of the substrate which has the same absolute configuration as found in the natural antibiotic structure. The presence of one specific enzyme catalysing the adenylation process in S. laurentii was shown by photoaffinity labeling with [alpha-32P]-8-azido-ATP and subsequent SDS PAGE analysis of the labeled products. The native molecular weight of the active enzyme, determined by gel permeation chromatography, was found to be approximately 47 kDa, compared with a denatured weight of 50 kDa estimated for the photoaffinity-labeled protein. The enzyme is thus probably monomeric.

Affinity Labels↗
PubMed1996

Complete genomic sequence and analysis of 117 kb of human DNA containing the gene BRCA1.

Over 100 distinct disease-associated mutations have been identified in the breast-ovarian cancer susceptibility gene BRCA1. Loss of the wild-type allele in > 90% of tumors from patients with inherited BRCA1 mutations indicates tumor suppressive function. The low incidence of somatic mutations suggests that BRCA1 inactivation in sporadic tumors occurs by alternative mechanisms, such as interstitial chromosomal deletion or reduced transcription. To identify possible features of the BRCA1 genomic region that may contribute to chromosomal instability as well as potential transcriptional regulatory elements, a 117,143-bp DNA sequence encompassing BRCA1 was obtained by random sequencing of four cosmids identified from a human chromosome 17 specific library. The 24 exons of BRCA1 span an 81-kb region that has an unusually high density of Alu repetitive DNA (41.5%), but relatively low density (4.8%) of other repetitive sequences. BRCA1 intron lengths range in size from 403 bp to 9.2 kb and contain the intragenic microsatellite markers D17S1323, D17S1322, and D17S855, which localize to introns 12, 19, and 20, respectively. In addition to BRCA1, the contig contains two complete genes: Rho7, a member of the rho family of GTP binding proteins, and VAT1, an abundant membrane protein of cholinergic synaptic vesicles. Partial sequences of the 1A1-3B B-box protein pseudogene and IFP 35, an interferon induced leucine zipper protein, reside within the contig. An L21 ribosomal protein pseudogene is embedded in BRCA1 intron 13. The order of genes on the chromosome is: centromere-1FP 35-VAT1-Rho7-BRCA1-1A1-3B-telomere.

Breast Neoplasms↗
PubMed1995

The thiostrepton-resistance-encoding gene in Streptomyces laurentii is located within a cluster of ribosomal protein operons.

A common approach to identify and clone biosynthetic gene from an antibiotic-producing streptomycete is to clone the resistance gene for the antibiotic of interest and then use that gene to clone DNA that is linked to it. As a first step toward cloning the genes responsible for the biosynthesis of thiostrepton (Th) in Streptomyces laurentii (Sl), the Th resistance-encoding gene (tsnR) was cloned as a 1.5-kb BamHI-PvuII fragment in Escherichia coli (Ec), and shown to confer Th resistance when introduced into S. lividans TK24. The tsnR-containing DNA fragment was used as a probe to isolate clones from cosmid libraries of DNA in the Ec cosmid vector SuperCos, and pOJ446 (an Ec/streptomycete) cosmid vector. Sequence and genetic analysis of the DNA flanking the tsnR indicates that the Sl tsnR is not closely linked to biosynthetic genes. Instead it is located within a cluster of ribosomal protein operons.

Amino Acid Sequence↗
PubMed1995

Identification of Streptomyces violaceoruber Tü22 genes involved in the biosynthesis of granaticin.

A 50 kb region of DNA from Streptomyces violaceoruber Tü22, containing genes encoding proteins involved in the biosynthesis of granaticin, was isolated. The DNA sequence of a 7.3 kb fragment from this region, located approximately 10 kb from the genes that encode the polyketide synthetase responsible for formation of the benzoisochromane quinone skeleton, revealed five open reading frames (ORF1-ORF5). The deduced amino acid sequence of GraE, encoded by ORF2, shows 60.8% identity (75.2% similarity) to a dTDP-glucose dehydratase (StrE) from Streptomyces griseus. Cultures of Escherichia coli containing plasmids with ORF2, on a 2.1 kb BamHI fragment, were able to catalyze the formation of dTDP-4-keto-6-deoxy-D-glucose from dTDP-glucose at 5 times the rate of control cultures, confirming that ORF2 encodes a dTDP-glucose dehydratase. The amino acid sequence encoded by ORF3 (GraD) is 51.4% identical (69.9% similar) to that of StrD, a dTDP-glucose synthase from Streptomyces griseus. The amino acid sequence encoded by ORF4 shares similarities with proteins that confer resistance to tetracycline and methylenomycin, and is suggested to be involved in transporting granaticin out of the cells by an active efflux mechanism.

Amino Acid Sequence↗
PubMed1995

The structure of trp RNA-binding attenuation protein.

The crystal structure of the trp RNA-binding attenuation protein of Bacclius subtilis solved at 1.8 A resolution reveals a novel structural arrangement in which the eleven subunits are stabilized through eleven intersubunit beta-sheets to form a beta-wheel with a large central hole. The nature of the binding of L-tryptophan in clefts between adjacent beta-sheets in the beta-wheel suggests that this binding induces conformational changes in the flexible residues 25-33 and 49-52. It is argued that upon binding, the messenger RNA target forms a matching circle in which eleven U/GAG repeats are bound to the surface of the protein ondecamer modified by the binding of L-tryptophan.

Amino Acid Sequence↗
PubMed1995

Complications resulting from the use of Chinese herbal medications containing undeclared prescription drugs.

OBJECTIVE: Many patients with chronic disease use alternative therapies. Our objective was to investigate complications resulting from the use of Chinese herbal medications containing undeclared prescription drugs, and to analyze these pills. METHODS: Medical records of 5 patients with complications were reviewed. Pills from symptomatic and asymptomatic individuals were analyzed for possible content of undeclared prescription drugs. RESULTS: All pills analyzed contained mefenamic acid and diazepam. Complications related to the presence of these substances included, among others, massive gastrointestinal bleeding. CONCLUSION: Chinese herbal medications may contain undeclared prescription drugs including nonsteroidal antiinflammatory drugs and benzodiazepines.

Adult↗