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W J Mitchell

Publications and source records attributed to W J Mitchell.

52 records · Page 3Linked to original sources
PubMed1990

Characterization of herpes simplex virus type 2 transcription during latent infection of mouse trigeminal ganglia.

Using a cornea trigeminal ganglion model, we have investigated transcription by herpes simplex virus type 2 (HSV-2) during latency in mice. Latency was verified 2 months postinoculation by reactivation of HSV-2 after explant cocultivation of trigeminal ganglia from the majority of mice (83%). Transcription during latent HSV-2 infection was limited to the repeat regions of the viral genome as determined by in situ hybridization using restriction fragment probes representing 100% of the HSV-2 genome. Further mapping of the positively hybridizing region by using subfragments showed that transcription occurred from approximately 11.5 kb of contiguous DNA fragments. A 1.0-kb PvuI-BamHI fragment within the BamHI F fragment and a 0.3-kb BamHI-SalI fragment and a 3.4-kb SalI-BamHI fragment within the BamHI P fragment hybridized more strongly than other subfragments in in situ hybridization experiments. All positive signals were confined to the nucleus. The RNA that hybridized to the 3.4-kb SalI-BamHI DNA fragment probe by in situ hybridization corresponded to a 2.3-kb transcript on Northern (RNA) blots. Under our conditions for Northern blot hybridization, the 3.4-kb SalI-BamHI probe of HSV-2 hybridized to a limited degree with the latency-associated transcripts of HSV-1. Shorter spliced species of latency-associated transcript RNA, which are seen during HSV-1 latency, have not been detected in latent HSV-2 RNA. However, viral gene expression during HSV-2 latency appears to be very similar to that during HSV-1 latency.

Animals↗
PubMed1987

Sugar transport by the bacterial phosphotransferase system. In vivo regulation of lactose transport in Escherichia coli by IIIGlc, a protein of the phosphoenolpyruvate:glycose phosphotransferase system.

Escherichia coli and Salmonella typhimurium preferentially utilize sugar substrates of the phosphoenol-pyruvate:glycose phosphotransferase system (PTS) when the growth medium also contains other sugars. This phenomenon, diauxic growth, is regulated by the crr gene, which encodes the PTS protein IIIGlc (Saffen, D.W., Presper, K.A., Doering, T.L., and Roseman, S. (1987) J. Biol. Chem. 16241-16253). We have proposed that non-PTS permeases are regulated by their interaction with IIIGlc, and in vitro studies from other laboratories have provided support for this model, but the in vivo effects of excess IIIGlc are not known. In the present studies, transformed cells that overproduced IIIGlc 2- and 10-fold, respectively, were constructed from a pts+ strain of E. coli and plasmids containing the crr gene. In the 2-fold overproducer, fermentation of, and growth on the non-PTS carbohydrates glycerol, lactose, maltose, and melibiose was generally more sensitive to the glucose analogue methyl-alpha-D-glucopyranoside than in a control strain containing normal levels of IIIGlc. In addition, inhibition of lactose permease activity by methyl-alpha-glucoside (inducer exclusion) was more effective in the 2-fold overproducer than in the control strain, particularly when the permease activity was high. The 10-fold IIIGlc overproducing strain had a requirement for the amino acids methionine, isoleucine, leucine, and valine that may or may not be related to the increased concentration of IIIGlc. Fermentation of non-PTS carbohydrates was also poor in the latter strain. Finally, lactose permease activity was 50% of that in control cells containing the same levels of beta-galactosidase, and the lactose permease activity in the IIIGlc overproducer was reduced to an extremely low level in the presence of methyl alpha-glucoside. Thus there is an inverse relationship between the cellular concentration of IIIGlc and the ability to metabolize non-PTS substrates. The results are consistent with the model where inducer exclusion is affected by a direct interaction between IIIGlc and a non-PTS transport system.

Carbohydrate Metabolism↗
PubMed1987

Sugar transport by the bacterial phosphotransferase system. Reconstitution of inducer exclusion in Salmonella typhimurium membrane vesicles.

The accompanying articles (Saffen, D.W., Presper, K.A., Doering, T.L., and Roseman, S. (1987) J. Biol. Chem. 262, 16241-16253; Mitchell, W.J., Saffen, D. W., and Roseman, S. (1987) J. Biol. Chem. 262, 16254-16260) show that "inducer exclusion" in intact cells of Escherichia coli is regulated by IIIGlc, a protein encoded by the crr gene of the phosphoenolpyruvate:glycose phosphotransferase system (PTS). The present studies attempt to show a direct effect of IIIGlc on non-PTS transport systems. Inner membrane vesicles prepared from a wild type strain of Salmonella typhimurium (pts+), carrying the E. coli lactose operon on an episome, showed respiration-dependent accumulation of methyl-beta-D-thiogalactopyranoside (TMG) via the lactose permease. In the presence of methyl-alpha-D-glucopyranoside or other PTS sugars, TMG uptake was reduced by an amount which was dependent on the relative concentrations of IIIGlc and lactose permease in the vesicles. The endogenous IIIGlc concentration in these vesicles was in the range 5-10 microM, similar to that found in whole cells. Methyl-alpha-glucoside had no effect on lactose permease activity in vesicles prepared from a deletion mutant strain lacking the soluble PTS proteins Enzyme I, HPr, and IIIGlc. One or more of the pure proteins could be inserted into the mutant vesicles; when one of the two electrophoretically distinguishable forms of the phosphocarrier protein, IIIGlc Slow, was inserted, both the initial rate and steady state level of TMG accumulation were reduced by up to 40%. The second electrophoretic form, IIIGlc Fast, had much less effect. A direct relationship was observed between the intravesicular concentration of IIIGlc Slow and the extent of inhibition of the lactose permease. No inhibition was observed when IIIGlc Slow was added to the outside of the vesicles, indicating that the site of interaction with the lactose permease is accessible only from the inner face of the membrane. In addition to the lactose permease, IIIGlc Slow was found to inhibit both the galactose and the melibiose permeases. Uptake of proline, on the other hand, was unaffected. The results are therefore consistent with an hypothesis that dephosphorylated IIIGlc Slow is an inhibitor of certain non-PTS permeases.

Carbohydrate Metabolism↗
PubMed1987

Addition of sulfonylurea to insulin treatment in poorly controlled type II diabetes. A double-blind, randomized clinical trial.

This study examined the potential beneficial effects of the addition of a second-generation sulfonylurea to insulin therapy for poorly controlled type II diabetes. A randomized, double-blind, crossover experimental design was utilized in 16 type II diabetic patients for a period of eight months. Treatment with glyburide, 20 mg/d (plus insulin), compared with placebo (plus insulin) resulted in a significant reduction in mean basal glucose (232 +/- 12 vs 262 +/- 11 mg/dL [12.8 vs 14.4 mmol/L]) and hemoglobin A1C (10.2% +/- 0.5% vs 10.9% +/- 03%) concentrations. Concomitant with this change, basal C-peptide and free insulin values increased with glyburide therapy, but this pharmacological agent did not alter the ability of the patient's erythrocytes to bind insulin. We conclude that in type II diabetic subjects receiving more than 28 units of insulin per day, the addition of glyburide results in a marginal, but statistically significant improvement in basal glucose concentration, but not in glucose tolerance as assessed by integrated glucose concentration. Whether this small improvement in glycemia is worth the additional cost of sulfonylureas or the risk of drug side effects is not known.

Adult↗
PubMed1987

Identification of negative strand and positive strand RNA of canine distemper virus in animal tissues using single stranded RNA probes.

In this report, we describe a technique for identifying negative strand (genome) and positive strand (messenger) RNA of canine distemper virus (CDV) in dog tissues by using single stranded RNA probes. Plasmids (pSP64-P and pSP65-P) which contain insert DNA corresponding to the P gene of CDV were transcribed by SP6 polymerase in the presence of radioisotope to produce radiolabeled single stranded RNA probes. RNA transcribed from pSP65-P is complementary to the negative strand (genome) and RNA produced from pSP64-P is complementary to the positive strand (message) of CDV. The binding specificity of the single stranded RNA probes was determined on Northern-blots. The use of these RNA probes in hybridization assays resulted in greater sensitivity and specificity than that obtained from double stranded DNA probes (either whole plasmids or purified insert DNA) which were labeled by the nick translation reaction. We also describe the making of single stranded DNA probes by reverse transcription labeling of complementary RNA. The complementary RNA was produced by the transcription of cloned DNA (pSP64-P and pSP65P). Single stranded RNA probes and single stranded DNA probes were similar in sensitivity. The single stranded RNA and DNA probes were applied to ethanolacetic acid fixed tissue sections from dogs infected with CDV-A75/17. We used 32P-labeled probes in tissue hybridizations and 35S-labeled probes in in situ hybridizations to identify negative and positive stranded CDV RNA. In this report we demonstrate that single stranded RNA and DNA probes can be used successfully in tissue hybridization and in situ hybridization assays to study viral expression in this virus-host system.

Animals↗
PubMed1985

The phosphoenolpyruvate-dependent phosphotransferase system: a central feature of carbohydrate accumulation by enteric bacteria.

Despite the fact that bacterial solute transport has been intensively studied for the last two decades, it has not yet been possible to describe the molecular mechanism of any transport process, largely due to the difficulties involved in handling membrane proteins and the lack of convenient in vitro assay procedures. The phosphoenolpyruvate: glycose phosphotransferase, a multiprotein system consisting of both soluble and membrane-bound components and with an associated chemical reaction which can be measured in cell-free extracts, has considerable potential for attaining this goal. Although the PTS is extremely complex, our understanding of the mechanisms by which it mediates and regulates carbohydrate transport in enteric bacteria is at an advanced stage.

Carbohydrate Metabolism↗
PubMed1983

Ganglioradiculitis in the dog. A clinical, light- and electron-microscopic study.

Facial hypalgesia, sensory ataxia, depression of tendon reflexes, and masticatory muscle wasting developed acutely to subacutely in three adult dogs. These deficits were correlated with craniospinal sensory ganglioradiculitis. CNS degeneration in the dorsal funiculus, spinal tract of the trigeminal nerve, and solitary tract was secondary to loss of primary sensory neurons. Megaesophagus, which occurred in two dogs appeared to be associated with loss of vagal primary afferent neurons; however, esophageal and gastric myenteric ganglionitis was also observed in one of these animals. In the three dogs changes in the sensory ganglia and roots included non-suppurative inflammation and degeneration and loss of neurons. Perivenular and perineuronal mononuclear infiltrates were prominent. EM study revealed that the satellite cells around degenerating and necrotic cell bodies were commonly invaded and displaced by lymphocytes and macrophages. It was not clear whether these mononuclear cells effected neuronal degeneration or merely responded to it. Although the pathogenesis remained undefined, the similarity of the clinical and pathologic findings in these dogs indicated a nosologic entity that had not been reported previously. This canine ganglioradiculitis was compared with inflammatory disorders of the cranial and spinal ganglia in man.

Animals↗
PubMed1982

Sugar transport by the bacterial phosphotransferase system. Regulation of other transport systems (lactose and melibiose).

The role of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) in the phenomenon of inducer exclusion was examined in whole cells of Salmonella typhimurium which carried the genes of the Escherichia coli lactose operon on an episome. In the presence of the PTS substrate methyl alpha-D-glucopyranoside, the extent of accumulation of the lactose analog methyl beta-D-thiogalactopyranoside was reduced. A strain carrying a mutation in the gene for Enzyme I was hypersensitive to the PTS effect, while a crr mutant strain was completely resistant. Influx, efflux, and exchange of galactosides via the lactose "permease" were inhibited by methyl alpha-glucoside. This inhibition occurred in the presence of metabolic energy poisons, and therefore does not involve either the generation of metabolic energy or energy-coupling to the lactose transport system. When the cellular content of the lactose permease was increased by induction with isopropyl beta-D-thiogalactopyranoside, cells gradually became less sensitive to inducer exclusion. The extent of inhibition of methyl beta-thiogalactoside accumulation by methyl alpha-glucoside was shown to be dependent on the relative cellular content of the PTS and lactose system. The data were consistent with an hypothesis involving partial inactivation of galactoside transport due to interaction between a component of the PTS and the lactose permease. By examination of the effects of the PTS and lactose uptake and melibiose permease-mediated uptake of methyl beta-thiogalactoside, it was further shown that the manner in which inducer exclusion is expressed is independent on the routes available to the non-PTS sugar for exit from the cell.

Biological Transport↗
PubMed1979

Quantitative analysis of proton-linked transport systems. Glutamate transport in Staphylococcus aureus.

1. The magnitude of the protonmotive force in respiring Staphylococcus aureus was measured over the range of extracellular pH from 5.6 to 7.8. 2. The membrane potential remains constant at 150 mV, inside-negative, but the pH gradient decreases from 2.1 units, inside-alkaline, at pH 5.6 to zero at pH 7.5 and above. 3. The accumulation of glutamate in the soluble cell pool is pH-independent at a value equivalent to 100 mV. 4. The results of experiments studying co-transport of protons are consistent with a proton/glutamate stoichiometry of 2 and electrogenic transport across the pH range examined. 5. The amount of glutamate uptake is the result of a kinetic steady state between influx and efflux pathways. 6. Evidence is presented for the regulation of this kinetic steady state by the response of the initial rate of uptake to changes in the protonmotive force.

Biological Transport↗
PubMed1979

Quantitative analysis of proton-linked transport systems. The lactose permease of Escherichia coli.

Evidence is presented that lactose uptake into whole cells of Escherichia coli occurs by symport with a single proton over the range of external pH 6.5--7.7. The proton/lactose stoicheiometry has been measured directly over this pH range by comparison of the initial rates of proton and lactose uptake into anaerobic resting cell suspensions of E. coli ML308. Further, the relationship between the protonmotive force and lactose accumulation has been studied in E. coli ML308-225 over the range of external pH 5.9--8.7. At no point was the accumulation of the beta-galactoside in thermodynamic equilibrium with the protonmotive force. It is concluded that the concentration of lactose within the cell is governed by kinetic factors rather than pH-dependent changes in the proton/substrate stoicheiometry. The relevance of these findings to the model of pH-dependent proton/substrate stoicheiometries derived from studies with E. coli membrane vesicles is discussed.

Biological Transport↗
PubMedDate not supplied

Regulation of mosquitocidal toxin synthesis in Bacillus sphaericus.

Translational lacZ fusions to the promoters of the parasporal, crystal protein (binary toxin) and 100-kDa ADP-ribosylating mosquitocidal toxin genes of Bacillus sphaericus were prepared and expression of the toxin genes monitored as beta-galactosidase activity. Transcription of the crystal protein gene fusion began immediately before the end of exponential growth and continued into stationary phase in both B. sphaericus and Bacillus subtilis but accompanied exponential-phase growth in Escherichia coli. Expression of this fusion was severely delayed in a B. subtilis spo0A mutant and decreased relative to the wild type in a B. subtilis spoIIAC background. beta-Galactosidase activity from the 100-kDa toxin gene fusion was restricted to early exponential phase in B. sphaericus, but in B.subtilis it continued into late exponential phase. Expression was about eightfold lower in B. sphaericus than B. subtilis suggesting an element of negative control in the native host.

Bacillus↗
PubMedDate not supplied

Intravenous home blood glucose monitoring.

This study examines the correlation between the blood glucose concentration estimated by diabetic subjects at home using a Chemstrip bG reagent strip, and the blood glucose concentration subsequently measured in a Beckman glucose analyzer. Blood samples were obtained by eight diabetic patients throughout a period of 30 days in the home environment. Using the technique of i.v. blood withdrawal, a 250-microliters portion of each blood sample was stored for later physician verification of the blood glucose concentration. The preservative in these stored sampled inhibited the utilization of glucose in the blood samples for at least 30 days. Our results demonstrate that in the home setting, a highly significant correlation (R = 0.85, P less than 0.001) can be obtained between the plasma glucose concentration as estimated by Chemstrip bGs and the blood glucose concentration as estimated by Beckman automated glucose analysis. However, within the diabetic group, marked variation existed in correlation coefficients such that the highest correlation coefficient was 0.94, and the lowest was 0.72. Furthermore, the use of Chemstrip bGs tends to influence a patient's reading toward the preselected color chart of glucose concentration on the Chemstrip bG label. We conclude that i.v. home blood glucose monitoring provides a method whereby the physician can assess the accuracy with which his patients can monitor their blood glucose concentrations in a home environment.

Adult↗