Biomedical subjects
G L Smith
Publications and source records attributed to G L Smith.
Effects of pH and inorganic phosphate on rigor tension in chemically skinned rat ventricular trabeculae.
1. Ventricular trabeculae from rat heart were chemically skinned with Triton X-100, which disrupts all cellular membranes including the sarcoplasmic reticulum. In the effective absence of Ca2+ (10(-9) M), trabeculae developed a maintained rigor contracture when ATP was withdrawn from the bathing solution. 2. The final level of tension obtained following withdrawal of ATP was dependent upon the pH of the bathing solution during development of rigor. Rigor tension at pH 5.5 was 10.1 +/- 0.9% (n = 8, mean +/- S.E.M.) of that at pH 7.0. Bathing the preparation in alkaline solution increased rigor force. At pH 8.0, rigor force increased to 218 +/- 6.7% (n = 4) of control responses developed at pH 7.0. The rate of development of rigor tension increased as the pH of the bathing solution was increased. Once established, rigor tension was unaffected by subsequent changes in pH. These effects of pH were fully reversible within the range 5.5-8.0. 3. The final level of rigor tension was slightly reduced when inorganic phosphate (P(i)) was included in the bathing solution prior to withdrawal of ATP. P(i) concentrations of 10, 20 and 30 mM reduced rigor tension to 87 +/- 2, 83 +/- 3 and 82 +/- 4% respectively. There was no significant effect of P(i) on the rate of development of rigor. The effect of P(i) at pH 6.0 was not significantly different from that observed at the control pH of 7.0. 4. These results suggest that the fall of intracellular pH and, to a lesser extent, the rise in [P(i)] that occurs during ischaemia will partially inhibit the development of a rigor contracture.
The metabolic consequences of an increase in the frequency of stimulation in isolated ferret hearts.
1. The metabolic consequences of an increase in the frequency of stimulation were examined in isolated ferret hearts. Intracellular pH (pHi) and the intracellular concentrations of phosphocreatine ([PCr]i), inorganic phosphate ([Pi]i) and ATP were measured by 31P nuclear magnetic resonance (NMR) spectroscopy. 2. Increasing the stimulus rate from 0.1-0.7 to 2 Hz caused an increase in [Pi]i and a decrease recovery of both [PCr]i and [Pi]i during continued stimulation. There was no change in [ATP]i during stimulation at 2 Hz. Increasing the stimulus rate caused an intracellular acidosis of around 0.1 pH units. 3. Increasing the stimulus rate generally caused an initial increase in developed pressure, followed by a decrease over 1-2 min to a steady level slightly lower than developed pressure at the low (control) stimulus rate. The increase in stimulus rate caused a 4- to 6-fold increase in time-averaged muscle activity. 4. Both oxygen uptake and production of lactate increased on 2 Hz stimulation. Lactate production accounted for less than 5% of ATP production at low or high stimulus rates, suggesting that significant anoxia was not occurring during stimulation. The observed lactate production was, however, sufficient to explain most of the intracellular acidosis observed when the stimulus rate was raised. When glycolysis was prevented by removal of glucose and depletion of glycogen stores, 2 Hz stimulation was accompanied by an intracellular alkalosis rather than an acidosis, suggesting that lactate production by glycolysis was the cause of the intracellular acidosis. 5. Reducing the rate of glycolysis increased the size of changes in [PCr]i and [Pi]i evoked by stimulation at 2 Hz. Furthermore, there was now no partial reversal of the changes in [PCr]i and [Pi]i during 2 Hz stimulation. 6. When oxidative phosphorylation was inhibited by replacing O2 with N2, increasing the rate of stimulation from 0.1-0.7 to 1-2 Hz caused an initial increase followed by a large fall in developed pressure, which declined to a level well below that at the control stimulus rate. The increase in stimulus rate was accompanied by a large fall in [PCr]i, an increase in [Pi]i, and an intracellular acidosis of 0.1-0.3 pH units. The fall in developed pressure was consistent with the known effects of the rise in [Pi]i and the fall in pHi on the contractile apparatus.
Vaccinia virus core protein VP8 is required for virus infectivity, but not for core protein processing or for INV and EEV formation.
A recombinant vaccinia virus, vDW4, has been constructed in which the L4R gene encoding the VP8 core DNA-binding protein is inducibly regulated by IPTG (isopropyl beta-D-thiogalactopyranoside). This virus produced normal-sized plaques in the presence of IPTG, but tiny plaques in the absence of inducer. Production of infectious progeny virus was reduced by 97% when VP8 synthesis was repressed and immature virions which had defective interactions between the granular viroplasm and the surrounding virion membrane were evident. However, despite the formation of these abnormal immature virions, virus maturation was able to proceed with the production of mature intracellular naked virus and extracellular enveloped virus particles of normal density. These mature particles were produced to approximately 80% of wild-type levels, but were around 100-fold less infectious. Consistent with the formation of mature virions, the repression of VP8 synthesis did not inhibit proteolytic processing of the major core proteins p4a and p4b. These results suggest that VP8 is required for correct association of the viroplasm and the immature virion envelope and that VP8 must be present during virus assembly for the production of fully infectious progeny virus.
Vaccinia virus gene A36R encodes a M(r) 43-50 K protein on the surface of extracellular enveloped virus.
A characterization of vaccinia virus strain Western Reserve (WR) open reading frame (ORF) A36R is described. This ORF is predicted to encode a 221-amino-acid protein (M(r) 25.1 K) with an amino-terminal hydrophobic sequence, seven potential sites for attachment of N-linked carbohydrate, but no carboxy-terminal transmembrane anchor. It is identical in vaccinia strain Copenhagen and shares 94.6% amino acid identity with the corresponding ORF in variola virus strains Harvey, India-1967, and Bangladesh-1975. RNA analyses detected a 600-nucleotide, early transcript that initiated 10-13 nucleotides upstream of the A36R ORF, and heterogeneously sized late transcripts running across the ORF. A rabbit antiserum raised against an Escherichia coli glutathione S-transferase fusion protein identified M(r) 43-50 K proteins that accumulated late during vaccinia virus infection and fractionated as integral membrane proteins during Triton X-114 partitioning. Similar polypeptides were expressed by vaccinia virus strains Tian Tan, Tashkent, Lister, Wyeth, Copenhagen, and IHD-J and by rabbitpox virus and cowpox virus (strain Brighton Red). Immunoblot analysis of purified and protease-digested intracellular mature virus (IMV) and extracellular enveloped virus (EEV) showed that the A36R proteins were present on the surface of EEV with type II membrane topology, but were absent from IMV. A WR deletion mutant lacking the A36R ORF (delta A36R) had a small plaque phenotype on all cell lines tested. IMV formation by delta A36R was unaltered but EEV formation was reduced approximately fivefold compared to wild-type (WT) when measured either by density gradient analysis of isotopically labeled virions or by infectivity assays. Thus the loss of the A36R protein from the EEV surface did not reduce EEV specific infectivity in vitro. Despite this, delta A36R showed striking attenuation compared with WT in a murine intranasal model. Finally, a revertant virus in which the A36R ORF was restored showed WT plaque size, EEV formation, and virulence, demonstrating that all the phenotypic differences of delta A36R were attributable to loss of the A36R gene and not to other mutations acquired during its construction.
Virus strategies for evasion of the host response to infection.
The attack on viruses and virus-infected cells by the mammalian immune system has provided considerable selective pressure for viruses that have evolved vigorous countermeasures to pre-empt, neutralize or evade this host attack. These countermeasures are astonishingly diverse, and their study imparts fundamental information about immunology and the mechanisms enabling viruses to survive and cause disease.
Effects of oxidants on the sarcoplasmic reticulum of saponin treated rat ventricular trabeculae.
OBJECTIVE: The aim was to determine the effects of the superoxide anion, hydrogen peroxide, and hypochlorous acid on in situ cardiac sarcoplasmic reticulum. METHODS: Simultaneous measurement of caffeine induced calcium release from the sarcoplasmic reticulum and subsequent myofilament tension generation was made on saponin permeabilised rat ventricular trabeculae before, during, and after exposure to various reactive oxygen species. The superoxide anion (O2.-) was generated using the xanthine-xanthine oxidase system, hypochlorous acid (HOCl) was prepared from NaOCl, and hydrogen peroxide (H2O2) was added from a 30% stock solution. RESULTS: At [O2.-] sufficient to abolish tension generation by the myofilaments completely, the associated caffeine induced calcium release was unaffected. Both H2O2 and HOCl diminished caffeine induced calcium release but had differential effects on the associated tension response. Exposure to HOCl favoured the occurrence of spontaneous calcium releases from the sarcoplasmic reticulum. CONCLUSIONS: The reactive oxygen species H2O2 and HOCl reduced the amount of calcium release from the sarcoplasmic reticulum while O2.- was without effect. In some preparations it was observed that HOCl favoured spontaneous calcium release which might additionally reduce the calcium content of the sarcoplasmic reticulum.
Effect of altered bathing pH on calcium activated force in alpha toxin permeabilised rat portal vein and human umbilical artery.
OBJECTIVE: The aim was to investigate the effect of altered bathing pH on calcium activated force generated by the contractile proteins in an alpha toxin permeabilised phasic (rat portal vein) and tonic (human umbilical artery) smooth muscle. METHODS: Strips of the two muscles were permeabilised using crude alpha toxin from Staphylococcus aureus (2 mg.ml-1). The relationship between [Ca2+] and tension was then examined at different bathing pH values. [Ca2+] was monitored using indo-1 fluorescence. RESULTS: GTP-gamma-S (100 microM) potentiated maximum calcium activated force in rat portal vein on average to 146%, but had no significant effect on human umbilical artery, confirming the difference in contractile behaviour between the two muscles. Lowering bathing pH from 7.2 to 6.7 depressed submaximal calcium activated force and increased maximum calcium activated force in rat portal vein. Raising bathing pH from 7.2 to 7.7 depressed both submaximal and maximum calcium activated force in rat portal vein. Altered bathing pH had no significant effect on either maximum or submaximal calcium activated force in human umbilical artery. The calcium sensitivity of tension production was not significantly effected by acidic pH in either preparation. However, alkaline pH caused a similar fall in the calcium sensitivity in both preparations. CONCLUSIONS: Tension generated from the contractile proteins of a phasic smooth muscle (rat portal vein) are more sensitive to altered bathing pH than those of a tonic smooth muscle (human umbilical artery).
Nucleotide sequence of a 55 kbp region from the right end of the genome of a pathogenic African swine fever virus isolate (Malawi LIL20/1).
The nucleotide sequence of a 55098 bp region from the right end of the genome of a virulent African swine fever virus (ASFV) isolate (Malawi LIL20/1) has been determined. Translation of the sequence identified 67 major open reading frames (ORFs) which are closely spaced and read from both DNA strands. At six positions intergenic tandem repeat arrays are found. Comparison of the predicted amino acid sequences of encoded proteins with protein sequence databases identified a number of homologies. These include three subunits of RNA polymerase, a protein with homology to transcription factor SII (TFSII), a DNA ligase, two subunits of mRNA capping enzyme, a DNA topoisomerase type II, a dUTPase, a protein kinase, three helicases, a ubiquitin-conjugating enzyme, a protein with homology to the nif S and nif S-like proteins identified in some bacteria and Saccharomyces cerevisiae, a protein with homology to both a myeloid differentiation primary response antigen (MyD116) and to a herpes simplex virus-encoded neurovirulence-associated protein (ICP34.5), a protein with homology to the ASFV-encoded structural protein p22, two proteins with homology to copies of the ASFV-encoded multigene family 360 and one protein with homology to the ASFV-encoded multigene family 110. Four genes encode proteins which have homology to each other and constitute a new multigene family (MGF100). Nine ORFs encode proteins which contain predicted transmembrane domains. The possible functions of these predicted ASFV-encoded proteins are discussed and the evolutionary relationship of ASFV to other viruses are considered. Despite the similarities in genome structure and replication strategy of ASFV with poxviruses, sequence similarity between them is low and the organization of ASFV-encoded genes is not colinear with that of the orthopoxviruses.
Deletion of fowlpox virus homologues of vaccinia virus genes between the 3 beta-hydroxysteroid dehydrogenase (A44L) and DNA ligase (A50R) genes.
A fragment of 4156 bp of fowlpox virus (FPV) genomic DNA contains homologues of vaccinia virus 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD; A44L) and DNA ligase (A50R) genes. The FPV locus has clearly been rearranged relative to that of vaccinia virus as homologues of genes A45R to A49R, including the thymidylate kinase and a gene with homology to superoxide dismutase, are deleted. The deleted genes are replaced by two open reading frames: for a serine proteinase inhibitor with homology to vaccinia virus gene K2L and for a protein with no significant homology to proteins in the databases. In addition, the FPV homologues of A44L and A50R are in the same polarity in FPV whereas they are in opposite polarities in vaccinia virus. Increased 3 beta-HSD activity has been demonstrated in cells infected with either of two different strains of FPV or with canarypox virus.
Diagnosis of periodontal disease activity by detection of key microbial antigens.
This paper reviews the development of various rapid microbial identification techniques and their potential rôle in the diagnosis of active periodontal tissue destruction. It also explores the concerns expressed by some, that although these techniques offer enormous promise as research tools, validation and translation of test results to clinical diagnosis, prognosis and treatment planning are still problematic. It may be premature to recommend these tests to clinicians in practice. Further longitudinal studies are required to establish the best use of these techniques in a clinical setting.
The regulation of membrane 125I- and 86Rb+ permeability in a virally transformed cell line (NCL-SG3) derived from the human sweat gland epithelium.
We have explored the factors that may regulate membrane permeability in a cell line (NCL-SG3) derived from the human sweat gland epithelium. Ionomycin increased the rate of 125I-efflux from preloaded cells and this action appeared to be due to an increase in intracellular free calcium ([Ca2+]i). The ionomycin-evoked increase in 125I- efflux was reduced in cells that were exposed either to barium or to valinomycin in the presence of a high concentration of external potassium. It thus appears that a fraction of the ionomycin-evoked increase in 125I- efflux is due to the activation of potassium channels and experiments using 86Rb+ also suggested that ionomycin increased the rate of potassium efflux, an effect which was totally abolished by barium. Blockade of Na(+)-K(+)-2Cl- cotransport and of Cl- -HCO3- exchange reduced the basal rate of 125I- efflux and the ionomycin-evoked increase in 125I-efflux from control cells and from cells depolarized by valinomycin. These transport systems thus contribute to anion efflux, although [Ca2+]i-dependent chloride channels also appear to be present. Acetylcholine increases [Ca2+]i in the secretory cells of human sweat glands, but this neurotransmitter did not increase [Ca2+]i in NCL-SG3 cells and so membrane permeability was not under cholinergic control. Adrenaline did not increase [Ca2+]i, but this hormone did evoke cyclic-3',5'-adenosine monophosphate (cyclic AMP) production. However, membrane permeability was not under adrenergic control, as the cells did not appear to express functional, cyclic AMP-dependent anion channels. This may be because they were not fully differentiated under the culture conditions. ATP consistently evoked a dose-dependent increase in anion efflux that appeared to be mediated by [Ca2+]i. The increase in [Ca2+]i was initiated by the release of calcium from a limited internal store and was subsequently sustained by calcium influx. UTP and ADP also increased [Ca2+]i, whereas adenosine, AMP and alpha,beta-methylene ATP were without effect. These data thus suggest that a subclass of type 2 purine receptor, which is functionally coupled to phosphoinositidase C, is present in these cells.
Comment on the paper by Shchelkunov et al. (1993) FEBS Letters 319, 80-83. Two genes encoding poxvirus cytokine receptors are disrupted or deleted in variola virus.
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Phosphorylation of ribosomal proteins by the vaccinia virus B1R protein kinase.
Two proteins of the 40S ribosomal subunit were shown to be phosphorylated in vitro by a vaccinia virus-encoded serine/threonine protein kinase. These were identified by two-dimensional gel electrophoresis as ribosomal proteins Sa and S2 and were shown by phosphoamino acid analysis to both be phosphorylated on serine and threonine residues. The three phosphorylated forms of S2 produced by the B1R protein kinase in vitro matched the phosphorylated forms of S2 previously observed in cells infected with vaccinia virus. These data strongly suggest that this enzyme is responsible for the phosphorylation of S2 and Sa which occurs early during vaccinia virus infection.
Current classifications of HIV-associated periodontal diseases.
The occurrence of unusual and severe forms of periodontal disease in HIV-infected individuals is well recognised. Several classification schemes have been proposed in an effort to associate periodontal deterioration with progressive stages of HIV infection and to determine aetiological factors in tissue destruction. No classification to date has proved entirely satisfactory. This paper reviews current classification schemes and suggests an alternative based upon the periodontal status of a cohort of HIV seropositive patients in Edinburgh. The proposed classification includes 'conventional', non-specific gingivitis and adult periodontitis such as occur in HIV seronegative individuals, but which may also be seen in seropositive subjects. However, conditions termed linear gingivitis and necrotising periodontitis may be pathognomonic for HIV infection. It is recommended that the term 'HIV-associated' be dropped in relation to periodontal disease.
The vaccinia virus 42-kDa envelope protein is required for the envelopment and egress of extracellular virus and for virus virulence.
Vaccinia virus gene B5R encodes a M(r) 42K glycoprotein that is expressed throughout infection and forms part of the envelope of extracellular virus. In this paper deletion mutants (delta B5R) lacking the B5R open reading frame (ORF) from the Western Reserve (WR) and IHD-J strains of vaccinia virus have been constructed and shown to form very small plaques compared with the wild-type viruses. This phenotype was directly attributable to loss of the B5R gene since re-insertion of this gene from WR or IHD-J into the WR mutant lacking B5R (W-delta B5R) restored a normal plaque phenotype. In the latter case the failure of the revertant to form comets indicated that the nine amino acid differences in the B5R ORF between the IHD-J and WR strains of virus are not responsible for comet formation by IHD-J virus. Furthermore, the B5R deletion mutant of IHD-J (I-delta B5R) still formed small comets. Despite the small plaque phenotype of the deletion mutants, normal yields of intracellular naked virus (INV) were produced. In contrast, deletion of B5R had a profound affect on the formation of the extracellular enveloped virus (EEV). Transmission electron microscopy indicated that INV particles were not wrapped by a double layer of Golgi-derived membrane and enveloped particles were not detected within the cell or on the cell surface without expression of the B5R protein. Biochemical measurement of EEV formation, by labeling infected cells with [3H]thymidine followed by cesium chloride density gradient centrifugation of particles released from the cells 24 hr postinfection, showed that only 10% of WT levels of EEV were produced by I-delta B5R. The loss of the B5R ORF caused severe attenuation in intranasally infected mice. At doses between 10(4) and 3 x 10(7) plaque-forming units there were no signs of disease in animals infected with W-delta B5R, whereas at comparable doses the WR parent virus caused significant mortalities. Finally, an ORF with 93.4% amino acid identity to vaccinia WR B5R is present in variola major virus strain Harvey and the B5R protein was shown by Western blotting to be expressed by all orthopoxviruses tested.
African swine fever virus genome content and variability.
A 55 kilobase pair (kb) region from the right end of the virulent African swine fever virus isolate, Malawi LIL20/1, has been sequenced. The 68 major open reading frames (ORFs) encoded are generally closely spaced and read from both DNA strands across the complete sequence. Comparison of the amino acid sequences of predicted ORFs with sequence databases identified 15 ORFs which encode proteins that are similar to proteins of known function. Two ORFs are homologous to copies of multigene family 360 (MGF360) and one ORF is homologous to copies of multigene family 110 (MGF110). Both of these multigene families have been described previously.
Ca-EGTA affects the relationship between [Ca2+] and tension in alpha-toxin permeabilized rat anococcygeus smooth muscle.
The relationship between calcium concentration ([Ca2+]) and force in smooth muscle can be studied by permeabilizing the sarcolemma and bathing the preparation in a mock intracellular solution. Normally [Ca2+] is set in these solutions using the Ca2+ chelator EGTA in the concentration range of 4-10 mM. This study shows that lowering total EGTA concentration ([EGTA]t) below 10 mM depresses Ca(2+)-activated force generated in 0.1 microM Ca2+. The observed threshold for the effect of EGTAt is 0.2 mM, and the effect is maximal at approximately 10 mM. BAPTA, another Ca2+ chelator, also produces this effect. Tension production in smooth muscle is controlled by acto-myosin interaction. This in turn is mediated by the relative activities of myosin light chain kinase (MLCK) and phosphatase (MLCP). Inhibiting MLCP with Microcystin LR (10 microM), an increase [EGTAt] from 0.2 mM to 10 mM still enhanced force. This suggests that EGTA promotes phosphorylation of myosin by the activation of MLCK and not by inhibition of MLCP.