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C J Smith

Publications and source records attributed to C J Smith.

At least 163 records · Page 9Linked to original sources
PubMed1994

Insertional activation of cepA leads to high-level beta-lactamase expression in Bacteroides fragilis clinical isolates.

Bacteroides fragilis is an important opportunistic pathogen of humans and is resistant to many drugs commonly used to treat anaerobic infections, including beta-lactams. A strain set comprised of B. fragilis isolates producing either low or high levels of the endogenous cephalosporinase activity, CepA, has been described previously (M. B. Rogers, A. C. Parker, and C. J. Smith, Antimicrob. Agents Chemother. 37:2391-2400, 1993). Clones containing cepA genes from each of seven representative strains were isolated, and the DNA sequences were determined. Nucleotide sequence comparisons revealed that there were few differences between the cepA coding sequences of the low- and high-activity strains. The cepA coding sequences were cloned into an expression vector, pFD340, and analyzed in a B. fragilis 638 cepA mutant. The results of beta-lactamase assays and ampicillin MICs showed that there was no significant difference in the enzymatic activity of structural genes from the high- or low-activity strains. Comparison of sequences upstream of the cepA coding region revealed that 50 bp prior to the translation start codon, the sequence for high-activity strains change dramatically. This region of the high-activity strains shared extensive homology with IS21, suggesting that an insertion was responsible for the increased expression of cepA in these isolates. Northern (RNA) blot analysis of total RNA by using cepA-specific DNA probes supported the idea that differential cepA expression in low- and high-activity strains was controlled at the level of transcription. However, the insertion did not alter the cepA transcription start site, which occurred 27 bp upstream of the ATG translation start codon in both expression classes. Possible mechanisms of cepA activation are discussed.

Amino Acid Sequence↗
PubMed1994

A lesson in ethics.

In arriving at an ethical decision, concern for the patient must come first. Recompense, inconvenience or embarrassment for the dentist are of secondary importance. Finally, the DSSNY Code of Ethics is at the essence of our profession. It encompasses the very image of how we wish to be viewed by society and indeed how we view ourselves. Uphold and guard it well.

Adolescent↗
PubMed1993

Actin stimulates plasmin generation by tissue and urokinase-type plasminogen activators.

The experiments reported here were carried out to define in greater detail actin's stimulation of plasmin generation by t-PA. Actin did not alter t-PA's hydrolysis of a synthetic substrate, and thus is unlikely to have a direct effect upon t-PA's proteolytic activity. When studied in a single-stage assay, actin accelerated t-PA-mediated plasmin generation from both Glu-plasminogen and Lys-plasminogen, indicating the central role of ternary complex formation. Although actin does not appear to bind two-chain urokinase (tcu-PA), it stimulates tcu-PA's cleavage of Glu-plasminogen. This finding suggests that actin alters the conformation of Glu-plasminogen to an open form. The failure of actin to increased plasmin generation by tcu-PA acting on Lys-plasminogen, which is in an open configuration, is consistent with this interpretation. Immunoglobin G, which shares with actin the property of binding to Glu-plasminogen after nicking by plasmin, did not stimulate tcu-PA's cleavage of Glu-plasminogen, indicating the uniqueness of actin's effects and suggesting interactions between actin and plasminogen at multiple binding sites. Unlike fibrin and heparin, whose stimulation of t-PA is related to polymer length actin is able to stimulate t-PA when presented in either a monomeric or polymeric form. Denaturation of actin by exposure to urea and guanidine increased its ability to stimulate plasmin generation by t-PA. Because actin's structure is maintained by a noncovalently bound adenine nucleotide (ATP or ADP), exposure to ATP/ADPases found in plasma and on cell membranes might also result in its denaturation. Actin treated with an enzyme functionally similar to such ecto-ATP/ADPases, potato apyrase, was more potent than native actin in stimulating plasmin generation by t-PA. The effects of apyrase were blocked by the addition of the plasma actin-binding proteins, gelsolin and the vitamin D-binding protein (DBP). Thus, denaturation of actin may occur in under physiologic conditions, with potential biological consequences. Actin thus appears to be unique with regard to its interactions with the fibrinolytic system and plasma actin-binding proteins may serve to protect the host from the effects of denatured actin.

Actins↗
PubMed1993

Oxidative inactivation of plasmin and other serine proteases by copper and ascorbate.

Fibrin thrombi form at sites of injury, where leukocytes release a variety of oxidants. To determine whether oxidants might affect proteins of the fibrinolytic system, we examined the effects of various oxidants on plasmin. Plasmin was not inhibited by micromolar concentrations of hypochlorous acid, chloramine T, or H2O2. Neither Fe nor Cu affected plasmin alone or in the presence of H2O2. However, incubation of plasmin with 5 mumol/L Cu(I or II) in the presence of the reducing agent ascorbic acid resulted in a loss of its hydrolytic activity towards proteins as well as towards small synthetic substrates. The addition of EDTA, but not mannitol, prevented its inactivation. Inactivation was prevented by the addition of catalase and accelerated by hydrogen peroxide. Preincubation of plasmin with the competitive inhibitor alpha-N-acetyl-L-lysine methyl ester prevented inactivation by Cu(II) and ascorbate. These results together suggest site-specific oxidation of plasmin's active site. Treatment of the plasminogen activators tissue plasminogen activator and two-chain urokinase-type plasminogen activator, as well as trypsin, neutrophil elastase, and thrombin with Cu(II) and ascorbate resulted in a loss of their amidolytic and proteolytic activity, indicating the general susceptibility of serine proteases to this type of oxidation. Oxidation of the zymogens Glu-plasminogen and single-chain urokinase-type plasminogen activator by Cu(II) and ascorbate resulted in the failure of these molecules to generate active enzymes when treated with plasminogen activators or plasmin, respectively. The active site His residue may be the target of oxidative inactivation, as evidenced by the partial protection afforded plasmin by the addition of Zn(II), histidine, or the platinum derivative, platinum(II) (2,2':6',2"-terpyridine) chloride. Because platelets contain micromolar concentrations of Cu and leukocytes are rich in ascorbate, Cu-dependent site-specific oxidation might play a role in modulating proteolytic events and the life span of thrombi formed at sites of tissue injury.

Ascorbic Acid↗
PubMed1993

Differentiation of monocytoid THP-1 cells with phorbol ester induces expression of prostaglandin endoperoxide synthase-1 (COX-1).

Prostaglandin endoperoxide synthase (PGH synthase) is responsible for converting arachidonic acid to PGH2, the common precursor of prostaglandins. It has been shown previously that phorbol ester-induced differentiation of the THP-1 human monocytic leukemia cell line is accompanied by induction of PGH synthase enzyme and enhanced capacity to produce prostaglandins. However, the identity of the isoform of PGH synthase that is induced under these conditions has not been established. Northern and Western analyses revealed a dramatic increase in levels of PGH synthase-1 mRNA and protein levels within 24 hr after treatment of THP-1 cells with phorbol ester. No significant increase in PGH synthase-2 mRNA or protein was observed. The increases in PGH synthase-1 were accompanied by enhanced capacity of the cells to produce PGE2. These findings indicate that expression of PGH synthase-1 is greatly enhanced in THP-1 cells by treatment with an agent that induces differentiation.

Cell Differentiation↗
PubMed1993

The energetics and cooperativity of protein folding: a simple experimental analysis based upon the solvation of internal residues.

The reversible unfolding of two dissimilar proteins, phosphoglycerate kinase from Bacillus stearothermophilus (PGK) and Staphylococcus aureus nuclease (SAN), was induced with two denaturants, urea and guanidinium chloride (GuHCl). For each protein, structural transitions were monitored by intrinsic fluorescence intensity changes arising from a unique tryptophan residue. In the case of SAN the single, native tryptophan residue was used, whereas for PGK two versions, one with a tryptophan at position 315 and one at 379, were constructed genetically. The resultant folding curves were analyzed by considering the change in the solvation free energy of internal amino acid residues as the denaturant concentration was varied. We derive the following simple relationship: -RT ln K = delta Gw + n delta Gs,m[D]/Kden. + [D]) where K is the equilibrium constant describing the distribution of folded and unfolded forms at a given denaturant concentration [D], delta Gw is the free energy change for the transition in the absence of denaturant, and n is the number of internal side chains becoming exposed. delta Gs,m and Kden. are constants derived empirically from the solvation energies of model compounds and represent the behavior of an average internal side chain between 0 and 6 M GuHCl and 0 and 8 M urea. For proteins of known structure these values can easily be derived, and for others, average values in guanidinium chloride (delta Gs,m = 0.775 kcal/mol and Kden. = 5.4 M) or urea (delta Gs,m = 1.198 kcal/mol and Kden. = 25.25 M) can be used in the analysis. Results show that the parameters n and delta Gw are independent of the denaturant used for all 12 transitions studied. This supports the hypothesis that the unfolding activity of urea and GuHCl can be accounted for by their effect on the solvation energy of amino acid side chains which are buried in the folded but exposed in the unfolded protein. This simple analytical treatment allows the "cooperativity" of protein folding to be interpreted in terms of the number of side chains becoming exposed to the solvent in a given step and allows accurate estimation of the free energy irrespective of the denaturant concentration needed to induce the transition.

Algorithms↗
PubMed1993

Cytosolic and sarcoplasmic reticulum-associated low Km, cGMP-inhibited cAMP phosphodiesterase in mammalian myocardium.

The distribution and phosphoprotein band patterns of low Km, cGMP-inhibited cAMP phosphodiesterase (cGI PDE) activity were examined in cytosolic and microsomal fractions of human, canine, rabbit and guinea pig left ventricular myocardium following phosphorylation by cAMP-dependent protein kinase, immunoprecipitation with anti-cGI PDE antibodies and SDS-PAGE. The recovery of cGI PDE activity in cytosolic and microsomal fractions was comparable in all four species. Microsomal cGI PDE was comprised chiefly of a approximately 135 kDa phosphoprotein. Cytosolic cGI PDE was comprised solely of approximately 116 kDa and lower molecular weight phosphoproteins. The approximately 135 kDa phosphoprotein probably corresponds to the holoenzyme encoded by the recently cloned cDNA for human myocardial cGI PDE, whose predicted molecular weight is 126 kDa. The approximately 116 kDa phosphoprotein may result from deletion or removal of putative membrane-association domains from the N-terminal region of the holoenzyme. These results suggest that the cytosolic and sarcoplasmic reticulum-associated forms of mammalian myocardial cGI PDE are separate molecular species.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1993

Autoradiographic comparison of cholinergic and other transmitter receptors in the normal human hippocampus.

The vulnerability of the human hippocampal complex to disease, trauma, and aging indicates the necessity to target this area therapeutically. The distribution and density of transmitter receptors provide a rational basis for this approach, and in this study the topography of 11 different pharmacological sites is compared with the cholinergic innervation, which is particularly vulnerable in dementia. The regional distribution of cholinergic innervation to the normal adult human hippocampus and adjacent cortex, marked by acetylcholinesterase (AChE) fiber and terminal reactivity, is notable for its concentration in CA2/3 of Ammon's horn and the dentate fascia. Neither nicotinic (high-affinity nicotine binding) nor muscarinic ("M1" or "M2") cholinergic receptor binding paralleled this distribution. In Ammon's horn, 5-HT2 and kainate receptor binding more closely resembled the pattern of AChE, being concentrated in CA2-4 compared with CA1. By contrast, muscarinic M1 and M2, 5-HT1A, benzodiazepine (including zolpidem-insensitive binding), NMDA (MK801), and AMPA/QUIS receptors were higher in CA1 and/or subiculum. Kainate binding, like AChE, was high in CA4. 5-HT2 and nicotinic binding partially mimicked the pattern of AChE around the granule layer. In the subicular complex and parahippocampal gyrus, where cholinergic activity is relatively lower, muscarinic, 5-HT1A, and benzodiazepine binding were relatively high and the nicotinic receptor was remarkable for its highest density compared to other areas examined. In stratum lacunosum-moleculare of CA1, which was relatively low in AChE activity, there was a dense band of nicotinic, M2, and benzodiazepine receptor binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholinesterase↗
PubMed1993

The atherogenic potential of carbon monoxide.

This paper reviews the available evidence concerning the atherogenic potential of carbon monoxide. The evidence comes from two different types of studies--epidemiology studies on populations of humans chronically exposed to carbon monoxide, and animal studies conducted under conditions of controlled exposure to carbon monoxide. Data from both epidemiology and animal studies suggest that carbon monoxide is not atherogenic. Therefore, the increased levels of atherosclerosis associated with smoking as reported in epidemiology studies of human smokers probably cannot be attributed to CO exposure.

Air Pollutants↗
PubMed1993

NGF deprivation and neuronal degeneration trigger altered beta-amyloid precursor protein gene expression in the rat superior cervical ganglia in vivo and in vitro.

In order to study the expression of beta-amyloid precursor protein (APP) isoforms during neuronal degeneration we have used the rat superior cervical ganglia (SCG) as an experimental model. In the neonate these sympathetic ganglia are nerve growth factor (NGF) dependent and in vivo administration of anti-NGF antiserum results in exaggerated neuronal degeneration. Analysis of APP mRNA transcripts in the SCG, following NGF deprivation, revealed a coincident decrease in APP695 and augmentation of APP751/770. These changes were specific to the SCG and were not seen in sensory ganglia. Subsequent in vitro studies, using primary dissociated cultures of sympathetic or cortical neurones, confirmed these changes in APP gene expression during neuronal degeneration. These observations may have important implications for the generation of beta-amyloid in Alzheimer's disease.

Amyloid beta-Protein Precursor↗
PubMed1993

Housing stressors, social support and psychological distress.

This paper explores the links between housing stressors, social supports and psychological distress. It was hypothesised that exposure to housing stressors would be significantly related to psychological distress, but that the adverse effects of housing stressors would be moderated by perceptions of social support resources. In other words, the stress/symptom relationship would be attenuated for individuals who believe that they have active and supportive social relationships. The results of a study conducted in the two New Zealand cities of Auckland and Christchurch partially confirmed this hypothesis, indicating that social support plays a role in mitigating the adverse effects of housing stressors. However, this relationship depends on the severity of the housing stressors. Among our respondents, the presence of social support was indeed associated with reduced symptom levels for those exposed to moderate housing stressors. However, among respondents subjected to high levels of housing stressors, social support was not associated with reduced psychological distress, indicating the need for a more specific policy response to the issue of seriously deficient housing.

Adaptation, Psychological↗
PubMed1993

(Over)eating success: the health consequences of the restoration of capitalism in rural China.

This paper reviews and evaluates some of the changes that have occurred in the Chinese health care system during the reform era associated with Deng Xiaoping (1978-1993). The reforms have helped to enrich the long suffering peasants in the Chinese countryside, and in many areas the peasants have experienced a significant improvement in the quality of their lives, including greater access to health care facilities, and better diets. The paper also considers some of the potentially negative side effects of the reform era, including the increasing income inequality between urban and rural areas; the commodification of Chinese medicine; declining access to health care for peasants in the poorest regions; and a concern about the changing patterns of diet and nutrition in the newly enriched parts of the Chinese countryside.

Cause of Death↗
PubMed1993

Evidence that generation of inositol 1,4,5-trisphosphate and hydrolysis of phosphatidylinositol 4,5-bisphosphate are rapid responses following addition of fungal elicitor which induces phytoalexin synthesis in lucerne (Medicago sativa) suspension culture cells.

Treatment of lucerne suspension culture cells with glycoprotein elicitor from the phytopathogenic fungus Verticillium albo-atrum R & B triggers Ca(2+)-mediated induction of antimicrobial secondary metabolites termed phytoalexins. The present study investigated the possible role of polyphosphoinositide signal transduction in phytoalexin elicitation. Within 1 min of addition of elicitor to lucerne suspension culture cells we found a 100-160% (15-25 pmol/g fresh wt) increase in the level of compound with chromatographic and electrophoretic properties expected for an inositol trisphosphate (InsP3) and which was strongly bound by an inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-specific binding protein; after 3 min the level of this compound had fallen below that observed prior to elicitor challenge. In 32P-prelabelled cells, the relative proportion of radioactivity which cochromatographed with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) was found to have decreased by 48% 1 min after elicitor addition and that rapid depletion of membrane lipid radioactivity was specific to this lipid fraction. The rapid, transient increase in level of Ins(1,4,5)P3 and concomitant fall in PtdIns(4,5)P2 suggests that Ins(1,4,5)P3 generated by hydrolysis of PtdIns(4,5)P2 may provide a Ca(2+)-mobilizing signal in phytoalexin elicitation in lucerne.

Cells, Cultured↗
PubMed1993

Detection of Epstein-Barr virus (EBV) DNA by the polymerase chain reaction (PCR) in oral smears from healthy individuals and patients with squamous cell carcinoma.

To assess the presence of Epstein-Barr virus (EBV) DNA in normal oral mucosa, as well as its relationship to age, sex and different sites in the oral cavity, oral smears from healthy adults were investigated using the polymerase chain reaction (PCR). Smears taken from oral cancer patients were also examined using the same method. Sixty healthy volunteers (30 men and 30 women) were selected and divided equally into three age groups. Four cytologic samples were taken from each subject using a cytobrush. Smears from 20 patients with oral cancer were taken from similar sites and from the lesion. The Bam W region of EBV DNA was chosen as the specific genome for PCR amplification. Fifteen out of 60 healthy individuals (25%) showed EBV positivity. Of these, seven were men and eight were women. There were no significant differences between the three age groups nor between the four sites of oral mucosa. Our results also showed that EBV DNA could be identified in 10 out of 20 oral cancer patients (50%), though in only 7 (35%) of the lesions. Taken into account with the age of the patients, these findings indicate that EBV infection in the oral cavity does not appear to be directly associated with the pathogenesis of oral squamous cell carcinoma.

Adult↗
PubMed1993

Canalicular adenoma: report of five cases with ultrastructural observations.

Canalicular adenomas from five patients were examined by light and electron microscopy. All patients were over 50 years of age; 4 of the lesions were from the upper lip, 4 were cystic, 2 showed capsular invasion and 1 was multifocal. Histopathologically there were epithelial duct-like or canalicular structures cut at various angles and epithelial islands surrounded by hyalinized and mucinous connective tissue that contained little collagen. Ultrastructurally the epithelial islands contained many small lumina and connective tissue islands. The characteristic epithelial cell was columnar or cuboidal, extended from the lumina to the connective tissue, had almost straight lateral cell boundaries with desmosomes that predominated near the lumina, and possessed gap junctions and a small number of tight junctions. Their main cytoplasmic constituents were ribosomes, mitochondria, rough-surfaced endoplasmic reticulum and microfilaments. These findings show that canalicular adenomas are truly monomorphic and support the concept that they are derived from the cells of excretory ducts.

Actin Cytoskeleton↗
PubMed1993

Cloning and characterization of the endogenous cephalosporinase gene, cepA, from Bacteroides fragilis reveals a new subgroup of Ambler class A beta-lactamases.

Bacteroides fragilis CS30 is a clinical isolate resistant to high concentrations of benzylpenicillin and cephaloridine but not to cephamycin or penem antibiotics. beta-Lactam resistance is mediated by a chromosomally encoded cephalosporinase produced at a high level. The gene encoding this beta-lactamase was cloned from genomic libraries constructed in Escherichia coli and then mated with B. fragilis 638 for identification of ampicillin-resistant (Apr) strains. Apr transconjugants contained a nitrocefin-reactive protein with the physical and enzymatic properties of the original CS30 isolate. The beta-lactamase gene (cepA) was localized by deletion analysis and subcloned, and its nucleotide sequence was determined. The 903-bp cepA open reading frame encoded a 300-amino-acid precursor protein (predicted molecular mass, 34,070 Da). A beta-lactamase-deficient mutant strain of B. fragilis 638 was constructed by insertional inactivation with the cepA gene of CS30, demonstrating strict functional homology between these chromosomal beta-lactamase genes. An extensive comparison of the CepA protein sequence by alignment with other beta-lactamases revealed the strict conservation of at least four elements common to Ambler class A. A further comparison of the CepA protein sequence with protein sequences of beta-lactamases from two other Bacteroides species indicated that they constitute their own distinct subgroup of class A beta-lactamases.

Amino Acid Sequence↗
PubMed1993

Genetic and biochemical analysis of a novel Ambler class A beta-lactamase responsible for cefoxitin resistance in Bacteroides species.

A clinical isolate of Bacteroides vulgatus was resistant to tetracycline, clindamycin, ampicillin, cephaloridine, cefoxitin, and other beta-lactam antibiotics except imipenem. beta-Lactam resistance was mediated by a membrane-associated, clavulanate-sensitive cephalosporinase capable of degrading cephalosporins and penicillins. Cefoxitin also was degraded but at a slow rate. The cefoxitin resistance (Fxr) determinant was cloned from B. vulgatus genomic libraries that were prepared in Escherichia coli and then mated with Bacteroides fragilis for the identification of Fxr strains. Analysis of B. fragilis strains with the cloned Fxr determinant revealed the presence of a new beta-lactamase protein with the physical and enzymatic properties of the beta-lactamase found in the original B. vulgatus isolate. The beta-lactamase gene (cfxA) was subcloned on a 2.2-kb DraI-HindIII fragment, and the nucleotide sequence was determined. These results showed that cfxA encoded a protein of 321 amino acids and 35,375 molecular weight. Mutant strains in which the cfxA structural gene was disrupted by insertional inactivation lost both Fxr and beta-lactamase activity. Comparison of CfxA with other beta-lactamases showed a relationship with the active-site serine beta-lactamases in the Ambler molecular class A, although CfxA had apparently diverged significantly. This was exemplified by the substitution in CfxA at 13 of 25 amino acid residues previously identified as being invariant in class A beta-lactamases. These results suggest that CfxA may represent a new class A homology group which diverged very early.

Amino Acid Sequence↗