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Biomedical subjects

S M Bell

Publications and source records attributed to S M Bell.

At least 55 records · Page 3Linked to original sources
PubMed1996

Characterisation of atypical biotype 3, serotype O:3 Yersinia and development of a simple identification scheme.

Five clinical strains of Yersinia isolated in Japan and identified as atypical biotype 3 or biotype 3B, serotype O:3, phage type II (3*/O:3/II) Yersinia enterocolitica were characterised since the biochemical reactions of these strains indicate they might also belong to the species Yersinia bercovieri. Biochemical tests, characterisation of the beta-lactamases and DNA-DNA hybridization studies provided strong evidence indicating that these strains should be classified as Yersinia enterocolitica. A simple scheme combining a disc diffusion test and four biochemical tests was devised for identification of these atypical strains.

Acetaminophen↗
PubMed1996

Intraventricular leptin reduces food intake and body weight of lean rats but not obese Zucker rats.

The protein encoded by the obese (ob) gene, leptin, is secreted from adipose tissue and is proposed to act in the brain as an important regulator of food intake and body weight. To investigate the direct effects of leptin within the CNS, we injected 3.5 microg of either mouse or human leptin into the third ventricle (ICV) of lean Long-Evans rats or obese (fa/fa) Zucker rats, in which obesity results from a mutation in the leptin receptor gene. ICV administration of leptin reduced 4-h food intake in both deprived and non-deprived lean rats. In addition, repeated ICV administration produced a long-lasting reduction in body weight while peripheral administration of the same dose had no effect. ICV administration of the same dose of leptin into the third ventricle of obese Zucker rats did not reduce food intake. These results are consistent with the hypothesis that leptin has direct actions in the CNS as an afferent signal related to the state of energy stores in adipose tissue. Furthermore, insensitivity to these central effects of leptin may be an important determinant of obesity.

Animals↗
PubMed1995

Transducin-alpha C-terminal mutations prevent activation by rhodopsin: a new assay using recombinant proteins expressed in cultured cells.

We have measured the activation by recombinant rhodopsin of the alpha-subunit (alpha 1) of retinal transducin (Gt, also recombinant) using a new assay. Cultured cells are transiently transfected with DNAs encoding opsin and the three subunits of Gt (alpha t, beta 1 and gamma 1). In the microsomes of these cells, incubated with 11-cis-retinal, light causes the rapid activation of Gt, as measured by the ability of GTP gamma S to protect alpha t fragments from proteolytic degradation. The activation of Gt is also observed when all-trans-retinal is added to microsomes under constant illumination. Activation depends on both opsin and retinal. Opsin mutants with known defects in activating Gt show similar defects in this assay. alpha t mutations that mimic the corresponding mutations in the alpha-subunit of Gs also produce qualitatively similar effects in this assay. As a first step in a strategy aimed at exploring the relationships between structure and function in the interactions of receptors with G proteins, we tested mutant alpha t proteins with alanine substituted for each of the 10 amino acids at the C-terminus, a region known to be crucial for interactions with rhodopsin. Alanine substitution at four positions moderately (K341) or severely (L344, G348, L349) impairs the susceptibility of alpha 1 to activation by rhodopsin. All four mutants retain their ability to be activated by AIF-4. Two other substitutions (N343 and F350) resulted in very mild defects, while substitutions at the remaining four positions (E342, K345, D346 and C347) had no effect. In combination with previous observations, these results constrain models of the interaction of the C-terminus of alpha t with rhodopsin.

Amino Acid Sequence↗
PubMed1995

Intravenous morphine self-administration by rats with low versus high saccharin preferences.

An experiment was performed to determine the relationship between saccharin preference and the self-administration of morphine via the oral and intravenous routes. On the basis of voluntary intake of a saccharin solution by male rats, low and high preference groups were formed. Rats selected for high saccharin preference self-administered more morphine intravenously than rats selected for low preference. The two groups did not differ in oral morphine intake. The positive relationship between the intake of saccharin and intravenous morphine self-administration may be due to their mediation by a common mechanism. Measures of taste sensitivity or preference may be useful in identifying individuals at risk for drug abuse.

Administration, Oral↗
PubMed1995

Susceptibility to beta-lactam agents of Yersinia enterocolitica biotype 4, serotype O3 isolated in various parts of the world.

Forty-eight human isolates of Yersinia enterocolitica of biotype 4, serotype O3 from various parts of the world were examined for susceptibility to 13 beta-lactam agents. The intracellular beta-lactamases of each of the 48 strains were examined. Isolates from Europe, Asia and Brazil (phage type VIII) or South Africa and Hungary (phage type IXa) produced both enzyme A and enzyme B, whereas isolates from New Zealand and Australia (phage type IXb) lacked the cephalosporinase enzyme B. Among the seven strains isolated in Canada belonging to phage type IXb, three strains expressed only enzyme A (group I) whereas the other four strains produced both enzymes A and B (group II). The high susceptibility to the combination of amoxycillin and clavulanate observed in one subtype was explained by the absence of the cephalosporinase enzyme B. A simple disk diffusion test with this antibiotic combination was shown to be effective in the detection of enzyme B in Y. enterocolitica 4/O3.

Anti-Bacterial Agents↗
PubMed1994

Diagnostic testing during pregnancy: a descriptive analysis of utilisation data.

To describe patterns of diagnostic testing during the antenatal period and to assess the potential benefit of using Medicare claims data in monitoring testing practice, we examined the matched claims data (with identifying details removed) on approximately 10,000 women having a confinement for which a Medicare benefit was claimed between 1 July and 30 September 1990. The results showed that almost all the women included in the study sample had an ultrasound and blood group and antibody examination. A smaller proportion had serological tests for syphilis (77 per cent), rubella (51 per cent) and hepatitis B carriage (73 per cent). Two-thirds had urine microscopy and culture, and under half (40 per cent) had serum alpha-fetoprotein estimation. Few (18 per cent) had a claim processed for microscopy and culture of a genital swab and fewer than 8 per cent claimed for any other pathology tests. There were differences in the proportions having tests, depending on whether the clinician managing the confinement was a specialist obstetrician or a general practitioner, and depending on geographic area and age group. While the data do not represent all women having a confinement in New South Wales, the selective use of antenatal diagnostic tests found in this study is of considerable public health importance and analysis of claims data can provide useful information for health professionals.

Adult↗
PubMed1994

Deletion of two regions on chromosome 4 in bladder carcinoma: definition of a critical 750kB region at 4p16.3.

Loss of heterozygosity (LOH) of chromosome 4 has been identified in several human tumours including carcinomas of colorectum, ovary, liver and head and neck. LOH at loci on chromosome 4p has previously been identified in 22% of primary bladder tumours. We have assessed LOH in 178 bladder tumours using a panel of six microsatellite markers. Thirty-four tumours (19%) showed LOH at one or more loci. Twenty-three deletions involved restricted regions of 4p and could be used to define two common regions of deletion. A very small common region between D4S43 and D4S127 (estimated to be approximately 750 kB) was involved in 14/23 (61%) of 4p deletions. A second common region centromeric to D4S174 was deleted in 7/23 (30%) of tumours with deletions. Two tumours (9%) had deletions involving both regions independently. Previous functional studies have demonstrated both a senescence function and a suppressor of tumorigenicity on human chromosome 4. Localisation of the common regions of deletion in bladder tumours provides a starting point for positional cloning of the gene(s) concerned and for more precise comparative functional studies.

Chromosome Deletion↗
PubMed1993

Identification of phosphorylation sites in the recombinant catalytic subunit of cAMP-dependent protein kinase.

The catalytic subunit of cAMP-dependent protein kinase expressed in Escherichia coli is a phosphoprotein. By in vivo labeling with [32Pi]orthophosphate, the sites of phosphorylation were identified as Ser-10, Ser-139, Thr-197, and Ser-338. Two of these sites, Thr-197 and Ser-338, are found in the mammalian enzyme (Shoji, S., Titani, K., Demaille, J. G., and Fischer, E. H. (1979) J. Biol. Chem. 254, 6211-6214). The predominant isoform is phosphorylated at Ser-10, Ser-338, and Thr-197. The isoforms cannot be readily interconverted by in vitro autophosphorylation, suggesting that the phosphates are relatively stable once the mature protein is assembled. Unlike the mammalian enzyme, the recombinant enzyme is not myristylated at its animo terminus. By coexpressing the catalytic subunit and N-myristyl transferase, the recombinant catalytic subunit is myristylated, and, under these conditions, phosphorylation at Ser-10 is reduced. The fact that recombinant catalytic subunit mutants that are enzymatically impaired are not phosphorylated in vivo indicates that the phosphorylation of the catalytic subunit observed in E. coli is due to autophosphorylation. Whether this process is intramolecular or intermolecular cannot be distinguished. Although autophosphorylation accounts for the modification of the catalytic subunit when it is expressed in E. coli, there may be heterologous protein kinases that are responsible for its in vivo phosphorylation when the enzyme is expressed in eukaryotic cells.

Amino Acid Sequence↗
PubMed1993

2.0 A refined crystal structure of the catalytic subunit of cAMP-dependent protein kinase complexed with a peptide inhibitor and detergent.

. A mutant (Serl39Ala) of the mouse recombinant catalytic (C) subunit of cAMP-dependent protein kinase was co-crystallized with a peptide inhibitor, PKI(5-24), and MEGA-8 (octanoyl-N-methylglucamide) detergent. This structure was refined using all observed data (30 248 reflections) between 30 and 1.95 A resolution to an R factor of 0.186. R.m.s. deviations of bond lengths and bond angles are 0.013 A and 2.3 degrees, respectively. The final model has 3075 atoms (207 solvent) with a mean B factor of 31.9 A(2). The placement of invariant protein-kinase residues and most C:PKI(5-24) interactions were confirmed, but register errors affecting residues 55-64 and 309-339 were corrected during refinement by shifting the affected sequences toward the C terminus along the previously determined backbone path. New details of C:PKI(5-24) interactions and the Ser338 autophosphorylation site are described, and the acyl group binding site near the catalytic subunit NH(2) terminus is identified.

Journal Article↗
PubMed1993

Prognostic value of p53 overexpression and c-Ki-ras gene mutations in colorectal cancer.

BACKGROUND: Mutations in Ki-ras codon 12 and the p53 gene are common abnormalities in colorectal cancer. The occurrence of p53 overexpression and/or Ki-ras codon 12 mutations were analyzed in 100 colorectal adenomas to determine if they were related to patient survival. METHODS: p53 overexpression was identified by immunohistochemistry, and Ki-ras codon 12 mutations were detected using the polymerase chain reaction and a restriction enzyme digestion method. RESULTS: p53 overexpression was identified in 45% of tumors, with a higher frequency identified in DNA aneuploid and left-sided tumors than in DNA diploid and right-sided tumors. Mutations in Ki-ras codon 12 were identified in 24% of carcinomas. Individually, mutations in Ki-ras codon 12 or p53 overexpression were not prognostic indicators of survival. However, a statistically significant difference in survival was identified when these two oncogenic abnormalities were analyzed together. The median survival of patients whose tumors contained both oncogenic abnormalities was less than half of that of patients with either alteration alone or without either abnormality. CONCLUSIONS: Screening for multiple genetic abnormalities in colorectal cancers excised at surgery may prove to be a useful tool in determining prognosis.

Adenocarcinoma↗
PubMed1993

Water deprivation-induced oral self-administration of cocaine in the Lewis rat: evidence for locomotor effects but not reinforcement.

Oral cocaine self-administration was studied in water-deprived Lewis rats. Liquid was available to rats only during daily 90-min sessions, in chambers equipped with spouts that delivered precise volumes of liquid following completion of lever-press responses. Blocks of training and testing sessions were alternately carried out during which increasing cocaine concentrations were presented: 0.0, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.282, and 0.4 mg/ml. Although high cocaine intakes (23.3-33.0 mg/kg) were obtained, neither avoidance nor preference for cocaine developed. Subsequently, fixed-ratio size was increased, and then distinctive stimulus lights were correlated with each liquid. One rat showed a preference for water following these changes, but two rats continued to show no preference. To determine if the amounts of cocaine self-administered had behavioral effects, locomotor activity tests were run immediately following self-administration sessions. Locomotor activity was substantially higher following cocaine self-administration than following water self-administration. These results demonstrate that the cocaine intakes reached under the present conditions did produce locomotor, but not reinforcing, effects.

Animals↗
PubMed1993

Orally self-administered cocaine in rhesus monkeys: transition from negative or neutral behavioral effects to positive reinforcing effects.

The establishment of orally delivered cocaine as a reinforcer was examined with nine rhesus monkeys. A 2% ethanol solution served as a reinforcer for all nine monkeys, for it maintained substantially higher response rates than did the concurrently available water vehicle. A test was initially conducted to determine whether cocaine would function as a reinforcer when substituted for 2% ethanol. When an intermediate cocaine concentration (0.2 mg/ml) was substituted for the ethanol solution, the drug maintained behavior at rates less than (seven monkeys), equal to (one monkey), or greater than (one monkey) those maintained by water. Thus, for eight of nine monkeys simple substitution of cocaine for ethanol was not sufficient to establish orally delivered cocaine as a reinforcer. In the next phase a stimulus-fading procedure was used. Blocks of training and testing sessions alternated. Across blocks of training sessions, increasing amounts of cocaine (0.0125, 0.025, 0.05, 0.1 mg/ml) were added to the 2% ethanol solution and subsequently the ethanol concentration was gradually decreased until only the 0.1 mg/ml cocaine solution remained; water was always concurrently available. Between each block of training sessions, a block of test sessions was inserted. Test sessions compared relative rates of responding maintained by two concurrently available drug solutions: (1) a solution containing the combination of ethanol and cocaine used in the prior training block and (2) a solution containing the same concentration of ethanol but with no cocaine. Thus, differences in rates of behavior maintained by the two solutions could be attributed to the presence of cocaine and the existence and degree of any such differences could be monitored at each step in the acquisition procedure. The outcome of the training procedure was that cocaine came to function as a reinforcer for six of the eight monkeys tested (the ninth monkey was not put through the fading procedure, having shown higher cocaine than vehicle rates during the initial substitution procedure). During the phase when ethanol was faded from the drug solution, differences between the combination cocaine-ethanol solution and the ethanol-only solution emerged: for the six monkeys that developed cocaine reinforced behavior, the combination solution maintained higher rates of responding than the ethanol solution alone. The opposite results were obtained with the remaining two monkeys. That cocaine had been established as a reinforcer was confirmed by persistent and orderly responding when dose and fixed-ratio size were subsequently varied.

Administration, Oral↗
PubMed1993

Rapid detection of allele loss in colorectal tumours using microsatellites and fluorescent DNA technology.

In order to investigate allele loss in colorectal tumours we have developed a rapid technique which overcomes most of the problems associated with radioactive Restriction Fragment Length Polymorphism (RFLP) analysis of allele loss. We utilise microsatellite length polymorphisms which are highly informative and are closely linked to loci of interest. Sequences containing microsatellites can be amplified from normal and tumour DNA pairs by a polymerase chain reaction (PCR) in which one of the primers is fluorescently labelled. This enables us to detect the products on polyacrylamide gels run on an automated DNA sequencer using dedicated software, by which results are automatically quantitated in terms of peak size, height, and area. Using this technique we have analysed 26 normal tissue: cancer pairs for allele loss at two loci linked to the adenomatous polyposis coli (APC) gene on chromosome 5q. Repeated assays yielded identical results for each pair. Allele loss was found in 10 out of 25 informative samples (40%).

Adenocarcinoma↗
PubMed1993

Expression of the catalytic subunit of cAMP-dependent protein kinase in Escherichia coli: multiple isozymes reflect different phosphorylation states.

The catalytic subunit of mouse cAMP-dependent protein kinase expressed in Escherichia coli was separated into three distinct species using Mono-S ion exchange chromatography. These isoenzymes corresponded to three isoelectric variants with pIs of 6.4 (30%), 7.2 (60%) and 8.2 (10%). The Stokes' radius of each form was 27.7, 27.1 and 26.3 A respectively. Using electrospray mass spectroscopy the differences between the isozymes were shown to be due to phosphorylation, with each form differing by 80 mass units corresponding to a single phosphate. The fully phosphorylated recombinant enzyme contained four phosphates while the dominant isozyme contained only three. Since the enzyme is not phosphorylated when active site mutations are introduced into the C-subunit, these phosphates are incorporated in an autocatalytic mechanism and are not due to E. coli protein kinases. When the recombinant enzyme was compared with the mammalian porcine heart enzyme significant differences in post-translational modifications were observed. The mammalian enzyme could also be separated into two isozymes. However, in contrast to the recombinant enzyme, the mammalian isozymes displayed an identical mass of 40 840. This correlated with two different post-translational modifications: two phosphates and an N-terminal myristyl moiety. The importance of post-translational modifications, and in particular the phosphorylation state, for the expression of eukaryotic proteins in E. coli is discussed.

Adenosine Triphosphate↗
PubMed1993

Differential modulation of plasminogen activator gene expression by oncogene-encoded protein tyrosine kinases.

Urokinase-type plasminogen activator (uPA) gene transcription is increased > or = 50-fold in chicken embryo fibroblasts (CEF) following transformation by the protein tyrosine kinase pp60v-src. Protein phosphorylation appears to play a critical role in uPA gene expression in these cells; protein kinase C-activating phorbol esters cooperate with pp60v-src to synergistically increase uPA mRNA, whereas cyclic AMP (cAMP)-dependent protein kinase-activating agents (e.g., 8-bromo cAMP) repress uPA mRNA levels. To explore the relationship between transforming oncogenes and uPA gene expression, uPA mRNA levels were measured in CEF infected with selected avian retroviruses. We report that v-ras and the transforming protein tyrosine kinases v-src, v-yes, and v-ros all increase cellular uPA mRNAs. However, transformation with the protein tyrosine kinase encoded by v-erbB, or the nuclear proteins encoded by v-jun, v-ski, or v-myc, did not increase uPA mRNA detectably. Ras and all of the protein tyrosine kinases analyzed, including the v-erbB product, but none of the nuclear oncoproteins sensitized cells to phorbol ester induction of uPA gene expression. Thus, increased uPA gene expression is not simply a secondary consequence of cell transformation but, rather, is regulated or comodulated by only a subset of oncogene products. Analysis of cells expressing site-directed mutants of pp60v-src showed that the induction of the uPA gene is dependent on protein tyrosine kinase catalytic activity, myristylation, and plasma membrane localization. However, these properties together are not sufficient; an additional feature in the src homology 2 domain is also required. The major sites of serine phosphorylation, serines 12 and 17, and the autophosphorylation site, tyrosine 416, are not essential for uPA gene induction. However, the reduction of uPA mRNA in pp60v-src-transformed cells by 8-bromo cAMP is dependent on tyrosine 416.

Animals↗
PubMed1993

p53 expression and K-ras mutation in colorectal adenomas.

The frequency of p53 overexpression and K-ras codon 12 mutation was investigated in a series of colorectal adenomas. p53 was detected by immunohistochemistry in only 5% of tumours, whereas K-ras mutation was found in eight of 30 adenomas examined. In vitro, mutant p53 and ras genes cooperate to transform primary rat cells into a tumourigenic cell line. The presence of both p53 overexpression and K-ras mutation in a benign tubulovillous polyp in the present series suggests that in vivo this combination of events is insufficient to cause malignant transformation of a large bowel adenoma.

Adenoma↗