PubMed Health⌕ Search

Biomedical subjects

S R Murray

Publications and source records attributed to S R Murray.

14 recordsLinked to original sources
PubMed2003

Comprehensive aligned sequence construction for automated design of effective probes (CASCADE-P) using 16S rDNA.

MOTIVATION: Prokaryotic organisms have been identified utilizing the sequence variation of the 16S rRNA gene. Variations steer the design of DNA probes for the detection of taxonomic groups or specific organisms. The long-term goal of our project is to create probe arrays capable of identifying 16S rDNA sequences in unknown samples. This necessitated the authentication, categorization and alignment of the >75 000 publicly available '16S' sequences. Preferably, the entire process should be computationally administrated so the aligned collection could periodically absorb 16S rDNA sequences from the public records. A complete multiple sequence alignment would provide a foundation for computational probe selection and facilitates microbial taxonomy and phylogeny. RESULTS: Here we report the alignment and similarity clustering of 62 662 16S rDNA sequences and an approach for designing effective probes for each cluster. A novel alignment compression algorithm, NAST (Nearest Alignment Space Termination), was designed to produce the uniform multiple sequence alignment referred to as the prokMSA. From the prokMSA, 9020 Operational Taxonomic Units (OTUs) were found based on transitive sequence similarities. An automated approach to probe design was straightforward using the prokMSA clustered into OTUs. As a test case, multiple probes were computationally picked for each of the 27 OTUs that were identified within the Staphylococcus Group. The probes were incorporated into a customized microarray and were able to correctly categorize Staphylococcus aureus and Bacillus anthracis into their correct OTUs. Although a successful probe picking strategy is outlined, the main focus of creating the prokMSA was to provide a comprehensive, categorized, updateable 16S rDNA collection useful as a foundation for any probe selection algorithm.

Algorithms↗
PubMed2001

Application of polymerase chain reaction (PCR) and TaqMan PCR techniques to the detection and identification of Rhodococcus coprophilus in faecal samples.

Rhodococcus coprophilus, a natural inhabitant of herbivore faeces, has been suggested as a good indicator of animal (as opposed to human) faecal contamination of aquatic environments. However, conventional detection methods limit its use for this as they require up to 21 days to obtain a result. In this paper an optimised method for extracting R. coprophilus DNA from faecal samples is described. PCR and 5'-nuclease (TaqMan) PCR methods were developed to allow the detection and enumeration of R. coprophilus in faecal samples within 2-3 days. Both PCR methods targeted the 16S rRNA gene, producing an amplicon of 443 bp which was specific for R. coprophilus. Sixty cells were required to produce an amplification product by conventional PCR, while as little as one cell was required for the TaqMan PCR method. The latter approach gave a linear quantitative response over at least four log units with both bacterial cells and DNA. Successful amplification by PCR was achieved using DNA extracted from cow, sheep, horse and deer faeces but was negative for samples from humans, pig, possum, duck and rabbit. These PCR methods enhance the feasibility of using R. coprophilus to distinguish faecal pollution of farmed herbivores from human pollution.

Animals↗
PubMed2001

Extragenic suppressors of growth defects in msbB Salmonella.

Lipid A, a potent endotoxin which can cause septic shock, anchors lipopolysaccharide (LPS) into the outer leaflet of the outer membrane of gram-negative bacteria. MsbB acylates (KDO)(2)-(lauroyl)-lipid IV-A with myristate during lipid A biosynthesis. Reports of knockouts of the msbB gene describe effects on virulence but describe no evidence of growth defects in Escherichia coli K-12 or Salmonella. Our data confirm the general lack of growth defects in msbB E. coli K-12. In contrast, msbB Salmonella enterica serovar Typhimurium exhibits marked sensitivity to galactose-MacConkey and 6 mM EGTA media. At 37 degrees C in Luria-Bertani (LB) broth, msbB Salmonella cells elongate, form bulges, and grow slowly. msbB Salmonella grow well on LB-no salt (LB-0) agar; however, under specific shaking conditions in LB-0 broth, many msbB Salmonella cells lyse during exponential growth and a fraction of the cells form filaments. msbB Salmonella grow with a near-wild-type growth rate in MSB (LB-0 containing Mg(2+) and Ca(2+)) broth (23 to 42 degrees C). Extragenic compensatory mutations, which partially suppress the growth defects, spontaneously occur at high frequency, and mutants can be isolated on media selective for faster growing derivatives. One of the suppressor mutations maps at 19.8 centisomes and is a recessive IS10 insertional mutation in somA, a gene of unknown function which corresponds to ybjX in E. coli. In addition, random Tn10 mutagenesis carried out in an unsuppressed msbB strain produced a set of Tn10 inserts, not in msbB or somA, that correlate with different suppressor phenotypes. Thus, insertional mutations, in somA and other genes, can suppress the msbB phenotype.

Acyltransferases↗
PubMed2001

Human glucocorticoid feedback inhibition is reduced in older individuals: evening study.

We have previously shown that when tested in the morning, older men and women, pretreated with metyrapone to block endogenous cortisol synthesis, exhibit delayed suppression of plasma ACTH in response to cortisol infusion. To confirm this finding and to determine whether aging-related changes in feedback responsiveness are exaggerated near the time of the circadian nadir in adrenocortical secretion, we performed a similar study in the evening. Healthy young (20-35 yr, n = 22) and old (>65 yr, n = 21) men and women were administered metyrapone orally (750 mg) at 1600 and 1900 h, followed by a cortisol infusion of 0.06 mg/kg/h for 150 min. Blood samples were taken at 15-min intervals for 4 h following infusion onset for measurement of plasma ACTH, cortisol, 11-deoxycortisol, and corticosteroid binding globulin. When corrections were made for differences in circulating cortisol concentrations achieved among age and gender subgroups, feedback inhibition of ACTH was found to be significantly greater in young than in old subjects of both genders. Our studies support the hypothesis that glucocorticoid responses to stress in aging individuals are likely to be prolonged due to blunted and delayed inhibition of ACTH secretion, thus increasing the total exposure to glucocorticoids.

Adrenocorticotropic Hormone↗
PubMed1999

FACE: fear of loss of face and the five hazardous attitudes concept.

An aspect of human personality, fear of loss of face, has attracted only minimal experimental investigation, despite the widespread recognition of the condition and its potentially adverse effects on behavior. A survey of the available literature shows fear of loss of face to be an important aspect of pilot decision making. This article considers the phenomenon in more detail and recommends an amendment to the 5 hazardous attitudes concept developed at the Embry-Riddle Aeronautical University (Diehl, 1990). At the same time, recognition of the contribution of Telfer (1987, 1989) is also made, with a recommendation to incorporate Telfer's proposal to improve the concept.

Adaptation, Psychological↗
PubMed1998

Phosphorylation is not required for dynamin-dependent endocytosis of a truncated mutant opioid receptor.

Opioid receptors are regulated within minutes after activation by G protein-coupled receptor kinase-mediated phosphorylation and dynamin-dependent endocytosis. We addressed the question of whether phosphorylation is required for opioid receptor endocytosis by examining a functional, truncated mutant delta opioid receptor (DOR344T), which is missing phosphorylation sites located in the carboxyl-terminal cytoplasmic domain. DOR344T receptors expressed in Chinese hamster ovary cells remained predominantly in the plasma membrane, even in the presence of saturating concentrations of agonist, consistent with previous studies demonstrating strongly inhibited endocytosis of truncated receptors in this cell type. In marked contrast, DOR344T receptors expressed at similar levels in human embryonal kidney (HEK) 293 cells exhibited rapid, ligand-induced internalization either in the presence of peptide (DADLE) or alkaloid (etorphine) agonist. Quantitative assays using ELISA and flow cytometric techniques indicated that DOR344T receptors were endocytosed in HEK293 cells with similarly rapid kinetics as full-length DOR (t1/2 < 10 min), and both full-length DOR and DOR344T mutant receptors were endocytosed by a dynamin-dependent mechanism involving clathrin-coated pits. Nevertheless, DOR344T receptors failed to undergo any detectable constitutive or agonist-induced phosphorylation in the same cells in which dynamin-dependent endocytosis was observed. These findings establish the first example of a G protein-coupled receptor that does not require phosphorylation to undergo dynamin-dependent endocytosis, and they suggest that significant cell type-specific differences exist in the biochemical requirements for ligand-induced concentration of opioid receptors in clathrin-coated pits.

Animals↗
PubMed1998

mu-Opioid receptor internalization: opiate drugs have differential effects on a conserved endocytic mechanism in vitro and in the mammalian brain.

mu-Opioid receptors are the pharmacological targets of endogenous opioid peptides and morphine-like alkaloid drugs. Previous studies of transfected cells and peripheral neurons indicate that opioid receptors are rapidly internalized after activation by the alkaloid agonist etorphine but not after activation by morphine. To determine whether opioid receptors in the central nervous system are regulated by a similar process of agonist-selective internalization, mu-opioid receptors were examined in rat brain neurons after treatment of animals with opioid drugs. Internalized mu receptors were observed within 30 min after intraperitoneal injection of the alkaloid agonist etorphine, and this process was blocked by the antagonist naloxone. Colocalization of internalized opioid receptors with transferrin receptors in confocal optical sections indicated that receptor internalization observed in vivo is mediated by a membrane trafficking pathway similar to that observed previously in vitro using transfected human embryonic kidney 293 cells. Morphine failed to induce detectable rapid internalization of receptors, even when administered to animals at doses far in excess of those required to induce analgesia. To quantify these agonist-selective differences and to analyze an array of opioid ligands for their ability to trigger internalization, we used flow cytometry on stably transfected 293 cells. These studies indicated that the different effects of individual agonists are not correlated with their potencies for receptor activation and that a variety of clinically important agonists differ significantly in their relative abilities to stimulate the rapid internalization of opioid receptors.

Animals↗
PubMed1996

Morphine activates opioid receptors without causing their rapid internalization.

We have examined the endocytic trafficking of epitope-tagged delta and mu opioid receptors expressed in human embryonic kidney (HEK) 293 cells. These receptors are activated by peptide agonists (enkephalins) as well as by the alkaloid agonist drugs etorphine and morphine. Enkephalins and etorphine cause opioid receptors to internalize rapidly (t1/2 approximately 6 min) by a mechanism similar to that utilized by a number of other classes of receptor, as indicated by localization of internalized opioid receptors in transferrin-containing endosomes and inhibition of opioid receptor internalization by hypertonic media. Remarkably, morphine does not stimulate the rapid internalization of either delta or mu opioid receptors, even at high concentrations that strongly inhibit adenylyl cyclase. These data indicate that agonist ligands, which have similar effects on receptor-mediated signaling, can have dramatically different effects on the intracellular trafficking of a G protein-coupled receptor.

Cell Line↗
PubMed1994

Molecular cloning and characterization of a novel kallikrein transcript in colon and its distribution in human tissues.

1. We have cloned and characterized a new species of kallikrein cDNA from a human colon cDNA library. The new kallikrein cDNA clone contains a part of intron 2 of the tissue kallikrein gene which is spliced to the remaining exon sequences. It does not contain exons 1 and 2. 2. An in-frame open reading frame is present in the new kallikrein cDNA allowing translation of a 216-amino acid product. The intron-containing kallikrein transcript was detected in salivary glands, pancreas, kidney, colon, prostate gland, testis, spleen, and lung by reverse-transcription/polymerase chain reaction followed by Southern blot analysis using an intron-containing kallikrein-specific oligonucleotide probe. 3. The results indicate that the new species of kallikrein may be processed by alternative splicing or arises from a different transcription initiation site.

Amino Acid Sequence↗
PubMed1992

Evolution of the kallikrein gene family.

All kallikrein-like genes that have been studied to date are composed of 5 exons and the tertiary structures of the encoded enzymes are remarkably similar. In the mouse and rat, these genes are highly conserved, tightly linked and tandemly arranged. In other species, such as the human, the family is less well defined and seems to be much smaller than that of the mouse and rat. Although extensively studied, the exact physiologic significance is not known for many kallikrein gene family members, however, they are thought to play important roles in processing biologically important peptide precursors. Given the potential importance of these mammalian enzymes as a group of highly selective peptide processing enzymes, it would be helpful to know more about the ways in which this family varies from species to species, especially with respect to the size of the family in each species. The evolutionary mechanisms which have shaped this family of genes are largely unknown, however, enough data has been generated to begin understanding the pathway by which this gene family has evolved.

Animals↗
PubMed1991

Molecular cloning and analysis of the rat kallikrein-binding protein gene.

The gene encoding rat kallikrein-binding protein (RKBP), a serine protease inhibitor, has been isolated and analyzed with the aid of the polymerase chain reaction. The gene is approximately 10 kilobases in length with four introns of approximately 2.2, 1.8, 0.9, and 0.84 kilobases. This gene is composed of five exons and encodes a polypeptide of 416 amino acid residues. The reactive center region of RKBP is encoded by the fifth exon with the putative P1-P1' residues being Lys-Ser. The organization of the RKBP gene is homologous to those of human alpha 1-antitrypsin and alpha 1-antichymotrypsin in size and arrangement of exons and introns, suggesting that they belong to the same subgroup of serpins. In the 5'-flanking region of the RKBP gene, a variant TATA box sequence, ATAAATA, is found 20 base pairs upstream from the transcription initiation site. The 5'-flanking region of the RKBP gene was able to direct transcription of the reporter gene chloramphenicol acetyltransferase when transfected into a rat hepatoma cell line. An internal promoter-like region was found in the first intron of the RKBP gene, downstream from the transcription initiation site and upstream from the translation initiation codon, however, it was unable to direct expression of the chloramphenicol acetyltransferase reporter gene in our experiments. The expression of RKBP in rat liver was induced by sex hormone treatment as indicated by dot-blot analysis. A genomic Southern blot using an RKBP cDNA probe revealed multiple bands suggesting that the RKBP gene belongs to a family of highly conserved genes.

Amino Acid Sequence↗
PubMed1990

Kallikrein multigene families and the regulation of their expression.

Tissue kallikreins are a group of closely related serine proteinases that are represented by multigene families in mice and rats. The existence of similar, large, kallikrein-like gene families in other mammalian species is currently a matter of dispute. We have surveyed a number of vertebrate species using genomic DNA Southern blotting and screened a human genomic library with a monkey kallikrein cDNA probe. The hybridization patterns of the genomic Southern blots and the characterization of 19 independent human clones using restriction analysis and Southern blotting indicate that other mammalian species may have multiple kallikrein-like genes as well. The regulatory mechanisms that govern the expression, activity, and bioavailability of tissue kallikreins are likewise complex. At the level of transcription, hormones, dietary factors, and tissue-specific factors are known to affect the expression of tissue kallikrein genes. At the posttranslational level, kallikrein activity and bioavailability are regulated by enzymatic activation, circulating autoantibodies, and binding proteins. We have demonstrated the presence of kallikrein-binding proteins in humans and rats, and, furthermore, we have shown reduced levels of this binding protein in a hypertensive rat model.

Animals↗
PubMedDate not supplied

Treatment with atypical antipsychotics: new indications and new populations.

Atypical antipsychotics have revolutionized the treatment of schizophrenia, becoming the treatment of choice for patients not only during their first episode, but also throughout their life course. Of note, as of 1999 more than 70% of prescriptions for these drugs are being prescribed for conditions other than schizophrenia, such as bipolar disorder and geriatric agitation. While there have been very few controlled trials that have established the efficacy of the atypical antipsychotics for these "off-label" uses, there have been a large number of open trials and case reports. The few controlled trials suggest that the atypical antipsychotics may be useful for affective disorders (both mania and depression), geriatric conditions such as senile dementia and aggression, as well as a variety of other disorders. Atypical agents may be particularly helpful for elderly, child, or adolescent patients who are especially susceptible to the side effects of medications and whose risk of tardive dyskinesia is high but further controlled studies are necessary.

Adolescent↗