Biomedical subjects

A G Williams

Publications and source records attributed to A G Williams.

At least 37 records · Page 2Linked to original sources

PubMed1999

Confocal imaging of the subcellular distribution of phosphatidylinositol 3,4,5-trisphosphate in insulin- and PDGF-stimulated 3T3-L1 adipocytes.

The activation of phosphatidylinositol 3-kinase (PI 3-kinase) and production of PtdIns(3,4,5)P(3) is crucial in the actions of numerous extracellular stimuli, including insulin-stimulated glucose uptake. Platelet-derived growth factor (PDGF) also stimulates PI 3-kinase, but only weakly promotes glucose uptake when compared with insulin. Insulin and PDGF have thus been proposed to have differential effects on the subcellular targeting of PI 3-kinase. However, owing to a lack of suitable methodologies, the subcellular localization of the PtdIns(3,4,5)P(3) generated has not been examined. The pleckstrin-homology (PH) domains of the nucleotide exchange factors, ADP-ribosylation factor nucleotide-binding-site opener (ARNO) and general receptor for 3-phosphoinositides (GRP1), which have a high affinity and specificity for PtdIns(3,4,5)P(3), were fused to green fluorescent protein and used to examine the subcellular localization of PtdIns(3,4,5)P(3) generation in living 3T3-L1 adipocytes. PtdIns(3,4,5)P(3) was produced almost exclusively in the plasma membrane in response to both agonists, although the response to insulin was greater in magnitude and occurred in considerably more cells. The results suggest that the greater ability of insulin to stimulate glucose uptake may be the result of its ability to generate significantly more plasma-membrane PtdIns(3, 4,5)P(3) than PDGF. ARNO and GRP1 are nucleotide exchange factors for the small GTP-binding protein ADP-ribosylation factor 6 (ARF6). The inability of a constitutively active GTPase-deficient mutant of ARF6 (ARF6-Q67L; Gln(67)-->Leu) to cause glucose transporter GLUT4 translocation suggests that activation of this pathway is not sufficient to cause GLUT4 translocation.

3T3 Cells↗

PubMed1999

A method for producing regional hypoglycemia in blood perfused tissue.

Numerous studies have focused on the metabolic contributions of glucose and other substrates in isolated tissue preparations by examining the effects of eliminating glucose from the physiologic perfusate or bath solution. To date, however, an effective method of glucose removal from the blood supply to selected tissue in the whole animal model has not been available. We have developed a method for blood glucose removal by continuous flow dialysis. This method was used to generate isolated coronary hypoglycemia for an investigation of myocardial metabolic substrate selection during hypoperfusion in open-chest, anesthetized dogs. Arterial blood was passed through the dialysis system against an isotonic and physiologic dialysate solution prior to controlled coronary perfusion. During normal perfusion pressure (100 mmHg), with a coronary blood flow of 32+/-4 ml/min, arterial blood glucose was reduced from 3.26+/-0.31 to 0.54+/-0.14 mM. When blood flow was reduced to 12+/-3 ml/min with lower perfusion pressure (40 mmHg), dialysis reduced arterial glucose from 3.53+/-0.36 to 0.15+/-0.03 mM. We conclude that this is an effective method for producing regional hypoglycemia.

Animals↗

PubMed1999

Detection of toxigenic strains of Bacillus cereus and other Bacillus spp. with an improved cytotoxicity assay.

An improved qualitative cell cytotoxicity assay for the detection of Bacillus cereus emetic and enterotoxin is described. The presence of toxin in culture supernatant fluids was detected by measurement with the tetrazolium salt MTT, as it adversely affects the metabolic status of cultured CHO cells. Psychrotrophic B. cereus isolates (65) were assessed for toxin production using the cytotoxicity assay, and 91% of culture supernatant fluids were cytotoxic. Toxin assessment using BCET-RPLA and ELISA immunoassays indicated that 51% and 85% of the cultures, respectively, were toxigenic. There were pronounced strain differences in the amount of toxin produced by the B. cereus isolates. Some isolates of B. circulans, B. laterosporus/cereus, B. lentus, B. licheniformis, B. mycoides, B. subtilis and B. thuringiensis were also toxigenic.

Animals↗

PubMed1999

Effects of basic training on material handling ability and physical fitness of British Army recruits.

No study has yet evaluated the efficacy of British Army basic training in improving material handling performance. Therefore, the purpose of this study was to evaluate the efficacy of the current British Army basic training in improving material handling performance and physical fitness. Forty-seven males (19.4 (3.2) years of age, 1753 (59) mm in height, 71.0 (9.6) kg in weight) and 10 females (21.5 (3.5) years, 1623 (45) mm, 62.5 (5.2) kg) served as subjects. Testing was carried out in the week prior to, and in the final week of, an 11-week basic training course. Maximal box lifts to two different heights, and repetitive lifting and carrying of a 10 kg load did not improve with training. Static (38 cm upright pull) and dynamic (incremental dynamic lift to 145 cm) lifting strength data concurred with the maximal box lift data in that no improvement was observed. Repetitive lifting and carrying of a 22 kg load improved (29.5%, p < 0.001), as did 3.2 km loaded march performance with 25 kg (15.7%, p < 0.001), but march performance with a 15 kg load did not. Predicted VO2max improved from 48.4 to 51.4 ml.kg-1.min-1, a change of 6.1% (p < 0.05). Fat-free mass increased by 0.9 kg (1.5%, p < 0.01), and body fat reduced by 2.7% of body mass (20.1%, p < 0.001), resulting in a loss of 1.2 kg of body mass (1.7%, p < 0.01). It is concluded that basic training in the British Army produces some favourable adaptations in recruits, especially in terms of aerobic fitness. However, the poor development of strength and material handling ability during training fails to improve the ability of soldiers to perform simulated military tasks, and it does little to reduce future injury risk while performing these tasks.

Adult↗

PubMed1999

A method for collecting right coronary venous blood samples from conscious dogs.

This report describes for the first time a technique to collect right coronary venous blood samples from conscious dogs. Catheters, prepared from Micro-Renathane tubing, were surgically implanted in right ventricular superficial veins of three anesthetized dogs. Also implanted were an arterial catheter, a right coronary flow transducer, and a right coronary artery constrictor. The coronary catheter was introduced at a venous bifurcation so that its side holes were positioned above the bifurcation; both ends of the catheter were exteriorized. Heparinized saline was continuously infused through the venous catheter by a battery-powered pump. The dogs were maintained for 10-13 days after surgery, and all catheters remained patent. Multiple right coronary venous samples were collected from each dog. These samples were analyzed for venous oxygen tension (PvO2) under baseline conditions, with right coronary pressure reduced to 50 mmHg, and during the reactive hyperemia after release of the right coronary artery constriction. PvO2 was 27.7 +/- 1.0 mmHg at baseline, 23.4 +/- 1.0 mmHg during coronary artery constriction, and 34.3 +/- 1.5 mmHg during reactive hyperemia. These data and the position of the catheter at autopsy demonstrated that coronary venous blood had been sampled.

Animals↗

PubMed1998

Stimulation of neuropeptide Y gene expression by brain-derived neurotrophic factor requires both the phospholipase Cgamma and Shc binding sites on its receptor, TrkB.

In PC12 cells, it has been previously reported that nerve growth factor stimulates neuropeptide Y (NPY) gene expression. In the current study we examined the signalling pathways involved in this effect by transiently expressing in PC12 cells the receptor (TrkB) for the related neurotrophin, brain-derived neurotrophic factor (BDNF). BDNF caused a 3-fold induction of luciferase expression from a transiently co-transfected plasmid possessing the firefly luciferase gene under the control of the NPY promoter. This effect of BDNF was completely blocked by either a Y484F mutation in TrkB (which blocks high-affinity Shc binding to TrkB) or by a Y785F substitution [which blocks the binding, phosphorylation and activation of phospholipase Cgamma (PLCgamma)]. Activation of the NPY promoter by neurotrophin-3 in PC12 cells overexpressing TrkC was also completely blocked by a naturally occurring kinase insert which prevents the high-affinity binding of Shc and PLCgamma. NPY promoter activation by BDNF was blocked by PD98059, suggesting a role for mitogen-activated protein kinase (MAP kinase). Stimulation of NPY gene expression by PMA, but not by BDNF, was blocked by Ro-31-8220, a protein kinase C inhibitor, excluding a role for this serine/threonine protein kinase in the effect of BDNF. In addition, BDNF did not cause an elevation in cytosolic Ca2+ concentration. Taken together, our results suggest that stimulation of the NPY promoter by BDNF requires the simultaneous activation of two distinct pathways; one involves Shc and MAP kinase, and the other appears to be PLCgamma-independent but requires an intact tyrosine-785 on TrkB and so may involve an effector of TrkB signalling that remains to be identified.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Discrimination among Bacillus cereus, B. mycoides and B. thuringiensis and some other species of the genus Bacillus by Fourier transform infrared spectroscopy.

Fourier transform infrared spectroscopy (FTIR) in conjunction with canonical variate analysis was found to be effective in discriminating among spectra of 9 representative strains of Bacillus spp., including B. cereus, B. mycoides and B. thuringiensis. The method was also able to discriminate according to species among spectra of 14 other non-type strains of B. cereus, 12 of B. mycoides and 12 of B. thuringiensis with a success rate of > 95%, even without using a prior classification of the groups by species. FTIR spectroscopy can be used for the rapid and accurate differentiation of species in the genus Bacillus that are of importance to the food and dairy industry.

Animals↗

PubMed1998

Right coronary autoregulation in conscious, chronically instrumented dogs.

Right coronary (RC) autoregulation and right ventricular (RV) function were assessed in conscious dogs, chronically instrumented to measure RC flow and RC pressure (RCP) as a hydraulic occluder on the RC was inflated. Dogs were then anesthetized, and RC autoregulation and RV function were again assessed. In the conscious state, moderate RC autoregulation was present with closed loop gains (Gc) of 0.59-0.27 as RCP was reduced from 100 to 40 mmHg. In the anesthetized state, Gc was not significantly less than in the conscious state at RCP >50 mmHg. The range and potency of RV autoregulation were greater in both groups than for previously reported findings in anesthetized dogs with RC perfused by an extracorporeal system. RV contractile function was well maintained in conscious and anesthetized dogs at RCP >45 mmHg. We conclude the following: 1) modest RC autoregulation is present in the conscious dog, 2) anesthesia limits the range but not the degree of RC autoregulation, 3) extracorporeal perfusion systems appear to depress RC autoregulation, and 4) RV contractile function remains constant in both conscious and anesthetized dogs until RCP falls below 50 mmHg.

Anesthesia, General↗

PubMed1997

Activation of mitogen-activated protein kinase and p70S6 kinase is not correlated with cerebellar granule cell survival.

We have investigated the role of mitogen-activated protein (MAP) kinase in the survival of cerebellar granule cells in primary culture. Brain-derived neurotrophic factor (BDNF) and insulin, but not epidermal growth factor (EGF), promoted the survival of P6 cerebellar granule neurons. BDNF promoted a sustained activation of MAP kinase, whereas that induced by EGF was only transient. Insulin promoted a small but transient activation of MAP kinase that was completely blocked by PD98059, an inhibitor of MAP kinase kinase activation. PD98059 had no effect on the insulin- or BDNF-induced survival of cerebellar granule cells. We also investigated the role of p70S6 kinase in survival. The activation of p70S6 kinase by EGF was transient, whereas BDNF and insulin promoted a sustained activation of p70S6 kinase. Rapamycin, which blocked p70S6 kinase activation, had no effect on the BDNF- or insulin-induced survival of cerebellar granule cells. We conclude that sustained activation of MAP kinase is not correlated with the survival response of cerebellar granule cells; indeed insulin-mediated survival is independent of MAP kinase. Survival of cerebellar granule cells is also independent of the activation of p70S6 kinase.

Amino Acid Sequence↗

PubMed1997

Analysis of mitogen-activated protein kinase activation by naturally occurring splice variants of TrkC, the receptor for neurotrophin-3.

TrkC is a receptor tyrosine kinase that binds neurotrophin-3 (NT-3) with high affinity. A number of naturally occurring splice variants of TrkC exist, including one (TrkC kil4) with a 14 amino acid insertion between subdomains VII and VIII of the tyrosine kinase domain. This kinase insert blocks the ability of NT-3 to stimulate neurite outgrowth in PC12 cells and proliferation in fibroblasts. The inserts also block the ability of TrkC to form a high-affinity complex with Shc and phospholipase C gamma (PLC gamma) and the activation of PtdIns 3-kinase, and attenuates the sustained activation of mitogen-activated protein kinase (MAPK). In the current study we set out to determine whether the attenuation of the activation of MAPK by the insert was the result of the inability of TrkC to activate the Shc-Ras pathway, PtdIns 3-kinase activation, PLC gamma activation, or a combination thereof. Experiments with the use of cell-permeant inhibitors argue against a major role for PLC gamma and PtdIns 3-kinase in the activation of MAPK by TrkC. The introduction of the 14 amino acid kinase insert appeared to slow the kinetics of NT-3-stimulated Shc phosphorylation and Shc-Grb2 association and reduce their magnitude; an effect which was associated with a delayed, and only transient, activation of MAPK. Taken together, our data suggest that the apparent defect in MAPK activation caused by the kinase insert may result predominantly from an inhibition of high-affinity Shc binding, although a role for PLC gamma and PtdIns 3-kinase cannot be completely excluded.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Effect of sampling procedure and strain variation in Listeria monocytogenes on the discrimination of species in the genus Listeria by Fourier transform infrared spectroscopy and canonical variates analysis.

The ability to discriminate successfully among cultures of all species of the Listeria genus by infrared spectroscopy in combination with canonical variate analysis was confirmed. The robustness of the method was demonstrated by showing that the separation of L. monocytogenes and L. grayi was hardly affected by variations in broth medium, incubation temperature, incubation time and cell washing procedure. Discrimination among 24 strains of L. monocytogenes according to serotype allowed two groups to be recognised, one comprising serotypes 4 and 4b and the other containing serotypes 1, 1/2b and 1/2c. When strain variation was included in the species discrimination model, the classification of all the L. monocytogenes strains was virtually 100% correct.

Culture Media↗

PubMed1997

Effect of Methanobrevibacter sp MF1 inoculation on glycoside hydrolase and polysaccharide depolymerase activities, wheat straw degradation and volatile fatty acid concentrations in the rumen of gnotobiotically-reared lambs.

Four naturally born lambs were placed in sterile isolators 24 h after birth before the natural establishment of cellulolytic microorganisms and archaea methanogens. At the age of 6 weeks they were inoculated with pure cultures of the strains FD1 and 007 of Ruminococcus flavefaciens and at the age of 4 months with a pure culture of Methanobrevibacter sp. MF1. Following the establishment of MF1, the population of R. flavefaciens slightly increased in the rumen of the four lambs, there was also an increase in straw degradation, in the activity of some glycoside and polysaccharide hydrolases of the adherent microbial populations and in the concentration of acetate in ruminal contents.

Journal Article↗

PubMed1996

Stability of high-energy phosphates in right ventricle: myocardial energetics during right coronary hypotension.

This study was conducted to determine if mechanisms that reduce right coronary (RC) blood flow (RCBF) and right ventricular (RV) oxygen consumption (MVO2) during moderate RC hypotension preserve RV high-energy phosphates. RC arteries of anesthetized dogs were cannulated and perfused with arterial blood supplied by a pressurized extracorporeal circuit. RC perfusion pressure (RCPP) was either kept constant at 100 mmHg or reduced to 60 or 30 mmHg for 20 min followed by a freeze-clamp biopsy of RV. Left ventricular (LV) biopsy was also performed to compare energy metabolism between RV and LV.RCBF and MVO2 significantly decreased when RCPP was reduced to 60 mmHg, but RV segment shortening (%SS) was unchanged; ATP, creating phosphate (CrP) and phosphorylation state of CrP ([CrP]/[Cr][Pi]) did not differ from control values. RV %SS, CrP, and phosphorylation state fell markedly at 30 mmHg RCPP. At 100 mmHg RCPP, CrP phosphorylation state in RV was only 35% of that in LV. These results indicate that RV increases its energetic efficiency without significant changes in high-energy phosphates or CrP phosphorylation state during moderate RC hypotension. Furthermore, the RV myocardium maintains a much lower energy level than LV myocardium, commensurate with its lower energy requirements.

Adenosine Triphosphate↗

PubMed1995

The effects of co-cultivation with the acetogen Acetitomaculum ruminis on the fermentative metabolism of the rumen fungi Neocallimastix patriciarum and Neocallimastix sp. strain L2.

The effects of co-cultivation with the hydrogen-utilizing acetogenic bacterium Acetitomaculum ruminis on the fermentative activities of the rumen fungi Neocallimastix patriciarum or Neocallimastix sp. L2 were investigated. In both co-cultures acetate production increased, making it the predominant fermentation product, as the accumulation of lactate, formate, ethanol, H2 and (in the case of Neocallimastix sp. L2) succinate all decreased. The effects of co-cultivation with Methanobrevibacter smithii were more pronounced. Decreased activities of lactate dehydrogenase, alcohol dehydrogenase and (in the case of Neocallimastix sp. L2) fumarate reductase accompanied the shift in fermentation product formation. The rate of glucose utilization and the fungal biomass yield were also increased in the co-culture.

Acetates↗

PubMed1995

Multiple origins of anaerobic ciliates with hydrogenosomes within the radiation of aerobic ciliates.

Some ciliates live anaerobically and lack mitochondria, but possess hydrogenosomes: organelles that contain hydrogenase and produce hydrogen. The origin of hydrogenosomes has been explained by two competing hypotheses: (i) they are biochemically modified mitochondria; or (ii) they are derived from endosymbiotic association(s) of ciliates and anaerobic eubacteria that possessed the hydrogenosome biochemistry. Phylogenetic analyses of representative aerobic, and anaerobic hydrogenosomal ciliates using host nuclear SSU rDNA sequences indicate a minimum of three, but more likely four, separate origins of hydrogenosomes. Whereas this does not refute either hypothesis, the implausibility of multiple convergent endosymbioses gives further support to the view that hydrogenosomes in ciliates derive from an existing organelle, which ultrastructural evidence suggests is the mitochondrion. Our results indicate a considerable potential for physiological-biochemical plasticity among a group of predominantly aerobic eucaryotes, and provide a phylogenetic framework to further refine and test hypotheses of the origins of the hydrogenosomal enzymes.

Aerobiosis↗

PubMed1995

Multiple protease activities in Giardia intestinalis trophozoites.

Azocasein and a wide range of chromogenic and fluorogenic peptidyl substrates, representing 21 different peptide sequences, were degraded by the intracellular proteases present in lysates of trophozoites of Giardia intestinalis (syn lamblia) Portland 1 strain. Total protease activity differed considerably with the substrate with the p-nitroanilide derivatives Bz-pro-phe-arg-pNA and Bz-phe-val-arg-pNA being most rapidly hydrolysed (specific activities 4.2 +/- 0.4 and 1.1 +/- 0.6 U/mg protein, respectively). Activities were increased (16-72%) by addition of 1 mM dithiothreitol and were maximal, for the substrates monitored, in the range pH 5.5-7.0. These data and the inhibitor susceptibilities of the trophozoite proteases confirmed that the activity was predominantly due to cysteine proteases, although the presence of some serine protease, aspartic protease and aminopeptidase activity in the lysates was also indicated. The multiplicity of the protease activities was confirmed by gelatin-polyacrylamide electrophoretic analysis. Eighteen proteolytic activities with Mr values in the range 30,000 to > 211,000 were detected. These gelatinase activities were enhanced by dithiothreitol, were maximal at or close to pH 6, and were inhibited by cysteine proteinase and some serine proteinase inhibitors. Four of the proteases present in the gels exhibited activity towards fluorogenic amidomethylcoumarin peptides, but with different substrate preferences. The results show that G. intestinalis contains multiple proteases, many of which are of the cysteine type.

Amino Acid Sequence↗

PubMed1995

Effect of anaerobic fungi on glycoside hydrolase and polysaccharide depolymerase activities, in sacco straw degradation and volatile fatty acid concentrations in the rumen of gnotobiotically reared lambs.

Four naturally born lambs were placed in sterile isolators 24 h after birth, before the natural establishment of the cellulolytic microorganisms. At the age of 4 weeks, a cellulolytic bacterial population of approximately 10(8) cells g-1 of rumen contents was established by inoculation with a 10(-6) dilution of ruminal contents taken from an adult sheep. A pure culture of Neocallimastix frontalis MCH3 and Piromyces communis FL was inoculated into the rumen 5 months after birth and a stable population of 10(3)-10(4) zoospores g-1 developed; the cellulolytic bacteria and fungi established populations in the 4 lambs that were similar to those observed in conventional animals. The presence of fungi led to an increase in the activity of most of the glycoside and polysaccharide hydrolases of the particle-associated microbial populations. However, this effect was not accompanied by an increase in the in sacco degradation of wheat straw or an increase in the volatile fatty acid concentration in the rumen contents.

Anaerobiosis↗

PubMed1994

Some rumen ciliates have endosymbiotic methanogens.

Most of the small ciliate protozoa, including Dasytricha ruminantium and Entodinium spp. living in the rumen of sheep, were found to have intracellular bacteria. These bacteria were not present in digestive vacuoles. They showed characteristic coenzyme F420 autofluorescence and they were detected with a rhodamine-labelled Archaea-specific oligonucleotide probe. The measured volume percent of autofluorescing bacteria (1%) was close to the total volume of intracellular bacteria estimated from TEM stereology. Thus it is likely that all of the bacteria living in the cytoplasm of these ciliates were endosymbiotic methanogens, using H2 evolved by the host ciliate to form methane. Intracellular methanogens appear to be much more numerous than those attached to the external cell surface of ciliates.

Animals↗