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G L Smith

Publications and source records attributed to G L Smith.

At least 163 records · Page 9Linked to original sources
PubMed1993

Simultaneous hybridization and subsequent colour detection of subgingival bacterial DNA on colony lifts.

This paper reports the development of a protocol allowing hybridization and detection of DNA fixed to nylon colony lifts from up to three species of bacteria simultaneously. Half ml samples of serial dilutions of pure cooked-meat broth (CMB) cultures of Capnocytophaga ochracea, Actinobacillus actinomycetemcomitans and Prevotella intermedia were grown on trypticase soy blood agar (TSBA) plates for 7 days in an anaerobic chamber. From the same CMBs a further set of dilutions was completed that contained all three species. Samples from these dilutions produced mixed-growth TSBA plates following anaerobic incubation for 7 days. After incubation, colony counts on pure-growth TSBA plates were enumerated by colony counter. Colony counts of C. ochracea, A. actinomycetemcomitans and P. intermedia on mixed-growth TSBA plates were enumerated by nylon colony lift, simultaneous hybridization with non-isotopic whole chromosomal DNA probes and alkaline phosphatase substrates generating three colours. The results indicate that the protocol correctly identified and differentiated between the three species on mixed-growth TSBA plates. The proportions of each species and mean total colony count expected by counting pure plates were in agreement with the proportions of each species and total colony counts enumerated by DNA probes on mixed growth plates. The development of simultaneous hybridization and multicolour detection will result in improved data recovery from dental plaque samples, in addition to reducing the cost and labour required in current colony-lift protocols, without affecting the specificity or sensitivity of the probes used.

Aggregatibacter actinomycetemcomitans↗
PubMed1993

Duplicated genes within the variable right end of the genome of a pathogenic isolate of African swine fever virus.

The right variable region of the genome of a pathogenic strain of African swine fever virus (ASFV), Malawi LIL20/1, has been sequenced and 15 open reading frames (ORFs) identified by computer analysis. Eight of these ORFs were found to be similar to previously described ASFV ORFs and three of these belong to two previously described multiple gene families (MGF), 360 and 110. Four of the remaining five ORFs belong to a novel MGF, designated MGF 100, and the last ORF encodes a protein that is similar to the virus structural protein, p22. Copies of MGF 110 and the gene coding for p22 have previously been characterized only at the left end of the ASFV genome. The organization of these genes suggests evolution by duplications, deletions and sequence transposition from one end of the genome to the other. Sequence comparisons of members of MGF 360 suggest that the Malawi LIL20/1 genome has undergone separate DNA rearrangements compared to the Ba71V genome. Lastly, one ORF was found to be similar to the myeloid differentiation primary response protein, MyD116 and to the herpes simplex virus neurovirulence-associated factor ICP34.5.

African Swine Fever Virus↗
PubMed1993

Characterization of vaccinia virus gene B12R.

We report the characterization of vaccinia virus gene B12R which is predicted to encode a 33K protein with 36% amino acid identity to the serine/threonine protein kinase encoded by vaccinia virus gene B1R. S1 nuclease protection experiments showed that gene B12R is transcribed early during infection from an initiation site 11 bp upstream of the open reading frame (ORF). The gene encodes a 33K polypeptide that is not required for virus replication in tissue culture nor for virus virulence in a murine intranasal model. Expression of the B12R gene in Escherichia coli produced an abundant 33K polypeptide which lacked protein kinase activity under conditions in which the protein kinases encoded by vaccinia virus gene B1R and African swine fever virus gene j9L are active.

Animals↗
PubMed1993

Three African swine fever virus genes encoding proteins with homology to putative helicases of vaccinia virus.

Sequence analysis of the SalI g, h, i and j restriction fragments of the African swine fever virus (ASFV) genome from the virulent isolate Malawi (LIL20/1) identified three open reading frames (ORFs) encoding predicted proteins of 125.0K (g10L), 80.4K (j10L) and 58.0K (j11L) which showed homology to members of the DNA and RNA helicase superfamily. ORFj10L was related to protein 4 of the Kluyveromyces lactis killer plasmid pKG12 and to two putative helicases, D6R and D11L, of vaccinia virus. ORF g10L was most closely related to ASFVj10L and to vaccinia virus D11L. ORFj11L was homologous to A18R, a third putative helicase of vaccinia virus. The possible functions of these genes in the replication of ASFV are discussed and the evolutionary implications are considered.

African Swine Fever Virus↗
PubMed1993

Effects of pH and inorganic phosphate on force production in alpha-toxin-permeabilized isolated rat uterine smooth muscle.

1. Strips of longitudinal smooth muscle isolated from rat uterus were permeabilized using crude alpha-toxin from the bacterium Staphylococcus aureus. This treatment rendered the surface membrane permeable to small molecular weight substances. Simultaneous measurements of tension and calcium concentration ([Ca2+]) (using indo-1 fluorescence) were used to investigate the effects of pH and inorganic phosphate concentration ([Pi]) on Ca(2+)-activated force generated by the contractile proteins. 2. Raising the [Pi] from 1 to 11 mM at a pH of 7.2 depressed both maximal and submaximal Ca(2+)-activated force. This effect of Pi was concentration dependent having the majority of its effect by 6 mM. 3. Further experiments at a submaximal [Ca2+] showed that Ca(2+)-activated force was enhanced by raising [Pi] from 6 to 11 mM suggesting that Pi increased the Ca2+ sensitivity of tension production. Based on these results, calculations indicate that the apparent affinity constant of Ca2+ for the contractile proteins increased from 4 x 10(6) M-1 to 6 x 10(6) M-1 on raising [Pi] from 1 to 11 mM. 4. Lowering pH from 7.2 to 6.7 at a [Pi] of 1 mM potentiated Ca(2+)-activated force with a small depression in the apparent Ca2+ sensitivity of tension production. This effect of pH on maximum (100 microM Ca2+) and submaximum (0.3 microM Ca2+) Ca(2+)-activated force was observed over a range of acidic pHs (7.0-6.7). 5. Increasing pH from 7.2 to 7.7 at a [Pi] of 1 mM depressed Ca(2+)-activated force with no effect on Ca2+ sensitivity of tension production. 6. Spontaneous contractions in intact rat myometrium are abolished under hypoxic conditions. Under these same conditions intracellular [Pi] rises and pH falls. The results of this study suggest that taken individually neither the effect of a rise in [Pi] nor a fall in pH on Ca(2+)-activated force generated by the contractile proteins can account for the effect of hypoxia on spontaneous contractions.

Animals↗
PubMed1993

Relationship between intracellular pH and metabolite concentrations during metabolic inhibition in isolated ferret heart.

1. Intracellular pH (pHi) and phosphorus metabolites were measured in isolated ferret hearts with 31P nuclear magnetic resonance (NMR). 2. The application of cyanide (to mimic hypoxia) produced a fall of the concentration of phosphocreatine ([PCr]) and a rise of those of inorganic phosphate ([Pi]) and sugar phosphates. These were accompanied by an intracellular acidosis. 3. If glycolysis was partly inhibited by prior exposure to a glucose-free solution then the application of cyanide also produced a fall of [ATP]. The acidosis was similar to that observed in the presence of glucose. 4. If glycolysis was completely inhibited by iodoacetate then the acidosis produced by subsequent addition of cyanide developed more quickly. 5. The results are reproduced by a model which incorporates lactic acid production as well as the effects of protons released and absorbed by the changes in metabolite concentrations. The results suggest that the acidosis produced by cyanide (without inhibition of glycolysis) is largely due to lactic acid production. When glycolysis is partly inhibited (glucose-free solution) the acidosis produced by cyanide is partly due to protons released by ATP breakdown. Finally, when glycolysis is entirely inhibited the acidosis is completely due to ATP breakdown. There is no need to postulate a contribution on this time scale from inhibition of pH regulating mechanisms.

Acidosis↗
PubMed1993

African swine fever virus encodes a serine protein kinase which is packaged into virions.

Nucleotide sequencing of the SalI j region of the virulent Malawi (LIL20/1) strain of African swine fever virus (ASFV) identified an open reading frame (ORF), designated j9L, with extensive similarity to the family of protein kinases. This ORF encodes a 35.1-kDa protein of 299 amino acids which shares 24.6% amino acid identity with the human pim-1 proto-oncogene and 21.0% identity with the vaccinia virus B1R-encoded protein kinase. The ASFV ORF contains the motifs characteristic of serine-threonine protein kinases, with the exception of the presumed ATP-binding site, which is poorly conserved. The ORF was expressed to high levels in Escherichia coli, and the recombinant enzyme phosphorylated a calf thymus histone protein on serine residues in vitro. An antibody raised to an amino-terminal peptide of the ASFV protein kinase was reactive with the recombinant protein in Western immunoblot analyses and was used to demonstrate the presence of the protein kinase in ASF virions.

African Swine Fever Virus↗
PubMed1993

Thromboxane A2 analogue, U-46619, potentiates calcium-activated force in human umbilical artery.

It has previously been shown that human umbilical artery (HUA) smooth muscle produces thromboxane A2 in response to increasing oxygen levels and that this thromboxane promotes contraction. To investigate the intracellular action of thromboxane A2, strips of HUA longitudinal smooth muscle were permeabilized using alpha-toxin from the bacterium Staphylococcus aureus. This treatment rendered the surface membrane permeable to low-molecular-weight substances but left functional thromboxane A2 receptors. Tension measurements were used to investigate the effect of the stable thromboxane A2 analogue, U-46619, on the Ca2+ sensitivity of smooth muscle contractile proteins. U-46619 (1 nM to 1 microM) potentiated submaximal Ca(2+)-activated force (generated by [Ca2+], 50 nM to 3 microM) but not maximal Ca(2+)-activated force (generated by [Ca2+], 10-100 microM). The specific thromboxane A2 receptor antagonist, GR-32191B (1 microM), inhibited the action of U-46619 (0.1 microM). The potentiation of submaximal Ca(2+)-activated force produced by the muscle in response to U-46619 (0.1 microM) was antagonized by guanosine 5'-O-(2-thiodiphosphate) (1 mM), the nonhydrolyzable analogue of GDP, and mimicked by guanosine 5'-O-(3-thiotriphosphate) (100 microM), the nonhydrolyzable analogue of GTP. These results suggest that U-46619 acts via the previously identified thromboxane A2 receptor to promote Ca2+ sensitivity of tension production in HUA smooth muscle. Furthermore, this effect appears to be mediated via a G protein.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1993

Effects of 2,3-butanedione monoxime on sarcoplasmic reticulum of saponin-treated rat cardiac muscle.

We have studied the effects of 2,3-butanedione monoxime (BDM) on the sarcoplasmic reticulum (SR) of saponin-treated rat cardiac trabeculae. Rapid application of 20 mM caffeine released Ca2+ from the SR, which was detected using the fluorescent Ca2+ indicator indo 1. The amplitude of the caffeine-induced Ca2+ transient was used as an index of the Ca2+ content of the SR before, during, and after exposure to various concentrations of BDM. BDM (1-5 mM) had little effect on caffeine-induced Ca2+ release. At these levels of BDM, force was inhibited predominantly by a direct action of BDM on the myofilaments. However, with higher concentrations (5-30 mM), BDM caused a concentration-dependent decrease in the amount of Ca2+ released from the SR in response to caffeine. This action of BDM may contribute to the negative inotropic effect of the drug in intact cardiac preparations by reducing the amount of Ca2+ available for release during systole. Rapid application of BDM induced a net release of Ca2+ from the SR. Both BDM and caffeine-induced Ca2+ releases were abolished following treatment of the muscle with 10 microM ryanodine. BDM failed to release Ca2+ in the absence of ATP or after substitution of ATP with nonhydrolyzable adenine nucleotides. In contrast, caffeine released Ca2+ in the absence of ATP. The possible involvement of the Ca(2+)-uptake pump in the action of BDM on the SR is discussed.

Adenosine Triphosphate↗
PubMed1993

Investigation of stimulus-secretion coupling in equine sweat gland epithelia using cell culture techniques.

When sweat glands isolated from samples of horse skin were explanted and cultured under favourable conditions, they could exhibit cellular outgrowth. This growth could be maintained for 2-4 weeks and these primary cultures were then disaggregated and the resultant cell suspensions used to initiate epithelial cell lines. Secretion from intact equine sweat glands is regulated by beta 2-adrenoceptors and appears to be mediated by cyclic AMP, but there is evidence that calcium may also play a role. Adrenaline could increase the cyclic AMP content of the cultured cells and this response was mediated by beta 2-adrenoceptors. Adrenaline was also able to evoke a small increase in intracellular free calcium ([Ca2+]i) but the pharmacology of this response remains obscure. Adrenaline thus activates at least two potentially important second-messenger signalling pathways which have the capacity to interact, because adrenaline-evoked cyclic AMP formation was inhibited if [Ca2+]i was raised with ionomycin. The chloride permeability of mammalian epithelial cells characteristically rises during secretion, and adrenaline could increase chloride permeability in the cultured epithelia but the cells did not contain cyclic-AMP-dependent chloride channels and so this response was mediated by [Ca2+]i.

Animals↗
PubMed1993

Clinical performance of a rule-based decision support system for mechanical ventilation of ARDS patients.

We developed a clinical decision support system--ventilation protocols--that managed tidal volume and ventilator rate settings during mechanical ventilation of patients with the Adult Respiratory Distress Syndrome (ARDS). We applied these protocols for a total of 10,903 hours in 40 ARDS patients. The clinical staff suspended the protocols for only 5% of the total application time due to medical procedures, surgeries, transient clinical problems not addressed by the protocols, or because of attending physician request. Of 3,148 instructions generated by the ventilation protocols, the clinical staff followed 2,932 (93%). The staff did not follow some instructions because of patient data errors, computer software and protocol logic errors, inability of the clinical staff to implement protocol instructions because of more pressing duties, and clinical staff objections to specific instructions. Sixty percent of the patients treated by the ventilation protocols survived. Our results demonstrate that the ventilation protocols provided a practical and safe decision support system for the mechanical ventilation of ARDS patients.

Clinical Protocols↗
PubMed1992

African swine fever virus encodes a gene with extensive homology to type II DNA topoisomerases.

Nucleotide sequencing of a virulent African swine fever virus (ASFV) isolate (Malawi LIL20/1) identified an open reading frame of 1191 amino acid residues encoding a protein of 134.9 kDa. This gene mapped to the SalI i and j restriction endonuclease fragments of the ASFV genome. The predicted polypeptide was found to share 21.1% identity over a 1077 amino acid region with the human type II DNA topoisomerase. The sequence is compared to other type II DNA topoisomerases and the possible roles in ASFV replication are discussed.

African Swine Fever Virus↗
PubMed1992

A soluble receptor for interleukin-1 beta encoded by vaccinia virus: a novel mechanism of virus modulation of the host response to infection.

Vaccinia virus gene B15R is shown to encode an abundant, secretory glycoprotein that functions as a soluble interleukin-1 (IL-1) receptor. This IL-1 receptor has novel specificity since, in contrast with cellular counterparts, it binds only IL-1 beta and not IL-1 alpha or the natural competitor IL-1 receptor antagonist. The vaccinia IL-1 beta receptor is secreted when expressed in a baculovirus system and competitively inhibited binding of IL-1 beta to the natural receptor on T cells. Deletion of B15R from vaccinia virus accelerated the appearance of symptoms of illness and mortality in intranasally infected mice, suggesting that the blockade of IL-1 beta by vaccinia virus can diminish the systemic acute phase response to infection and modulate the severity of the disease. The IL-1 beta binding activity is present in other orthopoxviruses.

Animals↗
PubMed1992

An African swine fever virus gene with homology to DNA ligases.

Sequence analysis of the SalI g region of the genome of a virulent isolate of ASFV (Malawi Lil 20/1) has revealed an open reading frame with the potential to encode a 48 kilodalton (kD) polypeptide which has significant homology with eukaryotic and prokaryotic DNA ligases. This ASFV encoded gene also contains the putative active site region of DNA ligases including the lysine residue which is necessary for enzyme-adenylate adduct formation, but lacks the C-terminal basic region conserved in other eukaryotic DNA ligases. A novel [32P]-labelled potential DNA ligase-adenylate adduct of M(r) 45 kD was observed upon incubation of ASFV infected cell cytoplasmic extracts with alpha-[32P]-ATP and subsequent analysis of products by SDS/PAGE. These data together suggest that ASFV encodes its own DNA ligase.

Amino Acid Sequence↗
PubMed1992

Bombesin stimulates the rapid activation of phospholipase A2-catalyzed phosphatidylcholine hydrolysis in Swiss 3T3 cells.

In Swiss 3T3 fibroblasts bombesin stimulated the release of arachidonic acid in a time- and dose-dependent manner. Arachidonate levels were significantly elevated after only a 2-s stimulation with the agonist. Furthermore, by measuring the arachidonate content of cellular phospholipids after cell activation, it was shown that there was selective depletion from phosphatidylcholine over the same time course. The corresponding production of lysophosphatidylcholine suggested the involvement of a phosphatidylcholine-specific phospholipase A2. Initial arachidonic acid release was not dependent on the presence of extracellular calcium, not activated by treatment of the cells with thapsigargin, and was unaffected by down-regulation of protein kinase C activity, or by treatment of the cells with the protein kinase C inhibitor staurosporine. These data strongly suggest that occupation of the bombesin receptor is closely coupled to activation of phospholipase A2 which results in the rapid release of arachidonic acid from phosphatidylcholine.

3T3 Cells↗
PubMed1992

Steroid hormone synthesis by a vaccinia enzyme: a new type of virus virulence factor.

Vaccinia virus open reading frame (ORF) SalF7L has 31% amino acid identity to human 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD). Here we show that SalF7L encodes an active 3 beta-HSD, by the conversion of pregnenolone to the steroid hormone progesterone. The gene is transcribed early during infection into a 1.4 kb mRNA from an initiation site 12 bp upstream of the ORF. An antiserum raised against bacterially expressed SalF7L immunoprecipitated a 38 kDa polypeptide from infected cells, but not from mock infected cells or from cells infected with a mutant virus from which the SalF7L ORF had been removed. Deletion of the gene had no effect on virus replication in CV-1 cells in culture, yet the deletion mutant was attenuated when intranasally inoculated into mice. This steroid hormone synthesizing enzyme is a novel type of virus virulence factor.

3-Hydroxysteroid Dehydrogenases↗