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Biomedical subjects

A J Williams

Publications and source records attributed to A J Williams.

At least 55 records · Page 3Linked to original sources
PubMed2001

Markovian models of low and high activity levels of cardiac ryanodine receptors.

The modal gating behavior of single sheep cardiac sarcoplasmic reticulum (SR) Ca2+-release/ryanodine receptor (RyR) channels was assessed. We find that the gating of RyR channels spontaneously shifts between high (H) and low (L) levels of activity and inactive periods where no channel openings are detected (I). Moreover, we find that there is evidence for multiple gating modes within H activity, which we term H1 and H2 mode. Our results demonstrate that the underlying mechanisms regulating gating are similar in native and purified channels. Dwell-time distributions of L activity were best fitted by three open and five closed significant exponential components whereas dwell-time distributions of H1 activity were best fitted by two to three open and four closed significant exponential components. Increases in cytosolic [Ca2+] cause an increase in open probability (Po) within L activity and an increase in the probability of occurrence of H activity. Open lifetime distributions within L activity were Ca2+ independent whereas open lifetime distributions within H activity were Ca2+ dependent. This study is the first attempt to estimate RyR single-channel kinetic parameters from sequences of idealized dwell-times and to develop kinetic models of RyR gating using the criterion of maximum likelihood. We propose distinct kinetic schemes for L, H1, and H2 activity that describe the major features of sheep cardiac RyR channel gating at these levels of activity.

Animals↗
PubMed2001

Light at the end of the Ca(2+)-release channel tunnel: structures and mechanisms involved in ion translocation in ryanodine receptor channels.

RyR and InsP3R are Ca(2+)-release channels. When induced to open by the appropriate stimulus, these channels allow Ca2+ to leave intracellular storage organelles at an astonishing rate. Investigations of the ion-handling properties of isolated RyR channels have demonstrated that, at least in comparison to voltage-gated channels of surface membranes, these channels display limited powers of discrimination between physiologically relevant cations and this relative lack of selectivity is likely to contribute to the ability of Ca(2+)-release channels to maintain high rates of cation translocation without compromising function. A range of ion-handling properties in RyR are consistent with the proposal that this channel functions as a single-ion channel and theoretical considerations indicate that the high rates of ion translocation monitored for RyR would require the pore of such a structure to be short and possess a large capture radius. Measurements of the dimensions of regions of RyR involved in ion conduction and discrimination indicate that this is likely to be the case. In each monomer of RyR/InsP3R, residues making up the last two trans-membrane spanning domains and a luminal loop linking these two helices contribute to the formation of the channel pore. The luminal loops of both RyR and InsP3R contain amino acid sequences similar to those known to form the selectivity filter of K+ channels. In addition the luminal loops of both Ca(2+)-release channels contain sequences that are likely to form helices that may be analogous to the pore helix visualised in KcsA. The correlation in structural elements of the luminal loops of RyR/InsP3R and KcsA has prompted us to speculate on the tertiary arrangement for this region of the Ca(2+)-release channels using the established structure of KcsA as a framework.

Animals↗
PubMed2001

Metabolism of the veterinary fluoroquinolone sarafloxacin by the fungus Mucor ramannianus.

To investigate the microbial biotransformation of veterinary fluoroquinolones, Mucor ramannianus was grown in sucrose/peptone broth with sarafloxacin for 18 days. Cultures were extracted with ethyl acetate and extracts were analyzed by liquid chromatography. The two metabolites (26% and 15% of the A280, respectively) were identified by mass and 1H nuclear magnetic resonance spectra as N-acetylsarafloxacin and desethylene-N-acetylsarafloxacin. The biological formation of desethylene-N-acetylsarafloxacin has not been previously observed.

Animals↗
PubMed2001

Quantitative electroencephalography spectral analysis and topographic mapping in a rat model of middle cerebral artery occlusion.

Electroencephalography (EEG) has a long history in clinical evaluations of cerebrovascular disease. Distinct EEG abnormalities, such as increased slow delta activity, voltage depression and epileptiform discharge, have been identified in stroke patients. However, preclinical use of EEG analysis of cerebral ischaemia is less documented. We report a new rat model of EEG topographic mapping during permanent and transient middle cerebral artery occlusion. Ten EEG electrodes were implanted on the rat skull, symmetrically covering the cortical regions of two hemispheres. Monopolar EEG recordings were acquired from each animal at multiple time points during the initial 24 h, and again once daily for 7 days. Traditional EEG examinations, quantitative EEG (qEEG) spectral analysis and topographic EEG mapping were employed for comprehensive data analyses. Several distinct spatiotemporal EEG abnormalities were identified in the ischaemic rat brain. In the ipsilateral hemisphere, pronounced increase in delta activity was observed in each recorded area within 24 h of injury. While sharp waves and spike complexes dominated the parietal region, a nearly isoelectric EEG state was seen in the temporal region. After 48 h, spontaneous, albeit incomplete, recovery of EEG activities developed in all rats. Reperfusion appeared to promote delta and alpha recovery more efficiently. The contralateral EEG changes were also recorded in two phases: an acute moderate increase in delta activities with intermittent rhythmic activities, followed by a delayed and significant increase in beta activities across the hemisphere. The similarities of rat qEEG profiles identified in this study to that of stroke patients and the application of topographic mapping broaden our research technology for preclinical experimental studies of brain injury.

Animals↗
PubMed2001

Oxidation of phenothiazine and phenoxazine by Cunninghamella elegans.

1. To determine the ability of fungi to metabolize sulphur- and oxygen-containing azaarenes, Cunninghamella elegans ATCC 9245 was grown in 125-ml flasks containing fluid Sabouraud medium. The cultures and controls were incubated at 28 degrees C with shaking and dosed with 16.7 mM phenothiazine or phenoxazine. After incubation for 72h, the mycelia and filtrates were extracted with ethyl acetate and the combined residues analysed by high-performance liquid chromatography. Residual phenothiazine and phenoxazine were 21 and 22%, respectively, of the total UV absorbance at 254 nm. 2. The metabolites were identified by mass spectrometry and proton nuclear magnetic resonance spectroscopy. The fungus oxidized phenothiazine to phenothiazine sulphoxide, 3-hydroxyphenothiazine sulphoxide, phenothiazin-3-one, and 3-hydroxyphenothiazine and oxidized phenoxazine to phenoxazin-3-one. 3. Three of the four compounds produced by C. elegans from phenothiazine were identical to those produced by mammals, supporting the use of the fungus as a microbial model for drug metabolism.

Chromatography, High Pressure Liquid↗
PubMed2001

Ryanoid modification of the cardiac muscle ryanodine receptor channel results in relocation of the tetraethylammonium binding site.

The interaction of ryanodine and derivatives of ryanodine with the high affinity binding site on the ryanodine receptor (RyR) channel brings about a characteristic modification of channel function. In all cases, channel open probability increases dramatically and single-channel current amplitude is reduced. The amplitude of the ryanoid-modified conductance state is determined by structural features of the ligand. An investigation of ion handling in the ryanodine-modified conductance state has established that reduced conductance results from changes in both the affinity of the channel for permeant ions and the relative permeability of ions within the channel (Lindsay, A.R.G., A. Tinker, and A.J. Williams. 1994. J. Gen. Physiol. 104:425-447). It has been proposed that these alterations result from a reorganization of channel structure induced by the binding of the ryanoid. The experiments reported here provide direct evidence for ryanoid-induced restructuring of RyR. TEA+ is a concentration- and voltage-dependent blocker of RyR in the absence of ryanoids. We have investigated block of K+ current by TEA+ in the unmodified open state and modified conductance states of RyR induced by 21-amino-9alpha-hydroxyryanodine, 21-azido-9alpha-hydroxyryanodine, ryanodol, and 21-p-nitrobenzoylamino-9alpha-hydroxyryanodine. Analysis of the voltage dependence of block indicates that the interaction of ryanoids with RyR leads to an alteration in this parameter with an apparent relocation of the TEA+ blocking site within the voltage drop across the channel and an alteration in the affinity of the channel for the blocker. The degree of change of these parameters correlates broadly with the change in conductance of permeant cations induced by the ryanoids, indicating that modification of RyR channel structure by ryanoids is likely to underlie both phenomena.

Animals↗
PubMed2001

Determination of absorbed dose calibration factors for therapy level electron beam ionization chambers.

Over several years the National Physical Laboratory (NPL) has been developing an absorbed dose calibration service for electron beam radiotherapy. To test this service, a number of trial calibrations of therapy level electron beam ionization chambers have been carried out during the last 3 years. These trials involved 17 UK radiotherapy centres supplying a total of 46 chambers of the NACP, Markus, Roos and Farmer types. Calibration factors were derived from the primary standard calorimeter at seven energies in the range 4 to 19 MeV with an estimated uncertainty of +/-1.5% at the 95% confidence level. Investigations were also carried out into chamber perturbation, polarity effects, ion recombination and repeatability of the calibration process. The instruments were returned to the radiotherapy centres for measurements to be carried out comparing the NPL direct calibration with the 1996 IPEMB air kerma based Code of Practice. It was found that, in general, all chambers of a particular type showed the same energy response. However, it was found that polarity and recombination corrections were quite variable for Markus chambers-differences in the polarity correction of up to 1% were seen. Perturbation corrections were obtained and were found to agree well with the standard data used in the IPEMB Code. The results of the comparison between the NPL calibration and IPEMB Code show agreement between the two methods at the +/-1% level for the NACP and Farmer chambers, but there is a significant difference for the Markus chambers of around 2%. This difference between chamber types is most likely to be due to the design of the Markus chamber.

Calibration↗
PubMed2001

Bacterial meningitis: current controversies in approaches to treatment.

Acute bacterial meningitis continues to be a disease with unacceptably high mortality and morbidity rates in both adults and children worldwide, despite advances in antibacterial therapy. Death or permanent disability occurs frequently. The causative organism varies with age, immune function and immunisation status. Infection with Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae type b (Hib) is associated with the majority of cases, with Listeria monocytogenes and Streptococcus agalactiae being more prevalent pathogens at the extremes of age (<3 months or >50 years). Antibacterial resistance is an increasing problem, particularly in pneumococcal bacteria but increasingly in other organisms. The increasing prevalence of resistance of pneumococcus to penicillin and the cephalosporins complicates therapy and may have an important impact on treatment outcome. Increased understanding of the pathophysiology has allowed advances in diagnosis and therapy. The use of adjunctive corticosteroids remains controversial, but is probably beneficial in reducing neurological sequelae in children. In adults the evidence is less clear. Vaccination has virtually eradicated Hib meningitis in some countries. Recent introduction of a conjugate vaccine against serogroup C meningococci in the UK has caused a dramatic reduction in the incidence of invasive disease due to this organism. A 7-valent pneumococcal vaccine promises a similar reduction in the incidence of invasive pneumococcal disease. In the meantime, the emergence of widespread resistance of organisms to antibacterial agents, in particular among the common organisms causing bacterial meningitis, remains the biggest challenge in therapy.

Anti-Bacterial Agents↗
PubMed2001

The high prevalence of autoantibodies to tissue transglutaminase in first-degree relatives of patients with type 1 diabetes is not associated with islet autoimmunity.

OBJECTIVE: To determine the extent of celiac autoimmunity in type 1 diabetic patients and the overlap between islet and celiac autoimmunity in their nondiabetic relatives. RESEARCH DESIGN AND METHODS: IgA antibodies to tissue transglutaminase were determined in serum taken from 433 type 1 diabetic patients and 1,442 nondiabetic first-degree relatives. Samples with transglutaminase antibodies above the 97.5th percentile of 347 schoolchildren were also assayed for IgA anti-endomysial antibodies (EMAs). Markers of islet autoimmunity (islet cell antibodies and autoantibodies to insulin, glutamate decarboxylase. and protein tyrosine phosphatase IA-2) had previously been measured in all relatives. RESULTS: In the absence of known celiac disease, the prevalence of transglutaminase antibody levels above the 97.5th percentile of the schoolchildren was 13.4% in diabetic patients and 7.0% in nondiabetic relatives. ENMAs were found in addition to transglutaminase antibodies in 2.6% of probands and in 1.9% of first-degree relatives, but none of the schoolchildren. Transglutaminase antibodies were found to persist in 10 of 30 patients and in 30 of 59 relatives with follow-up samples taken at least 2 years after the initial sample. Of 186 nondiabetic relatives with islet autoantibodies, only 10 also had transglutaminase antibodies. CONCLUSIONS: We found a high prevalence of celiac autoimmunity in patients and first-degree relatives of children with type 1 diabetes, but we found limited overlap between islet and celiac autoimmunity in nondiabetic relatives. The high prevalence of celiac autoimmunity may be explained by shared genetic susceptibility and identifies a population within which screening for the disease may be justified.

Adolescent↗
PubMed2001

Repeatability of dorsolateral subluxation scores in dogs and correlation with macroscopic appearance of hip osteoarthritis.

OBJECTIVE: To determine whether dorsolateral subluxation (DLS) scores in young dogs could be used to reliably predict which dogs would develop evidence of hip osteoarthritis and whether DLS scores measured at various ages correlated with each other. ANIMALS: 129 Labrador Retrievers, Greyhounds, and Labrador Retriever-Greyhound crossbreds. PROCEDURES: DLS scores were measured on radiographs taken at 4, 8, and 12 months of age and at necropsy (8 to 36 months of age). At necropsy, the hip joints were examined macroscopically and a score assigned for degree of cartilage degeneration. RESULTS: DLS scores at 4 (n = 35, r(s) = -0.62), 8 (n = 106, r(s) = -0.54), and 12 (n = 15, r(s) = -0.87) months of age were significantly correlated with cartilage degeneration scores, and DLS scores at 8 months of age were significantly correlated with scores obtained at the time of necropsy (n = 39, r(s) = 0.87). The DLS scores at 4 months of age were significantly different from scores at 8 months of age, but scores did not differ significantly thereafter. Likelihood ratios for cartilage lesions for low (< 45%), intermediate (> or = 45 but < or = 55%), and high (> 55%) DLS scores at 8 months of age were 8.0, 2.6, and 0.2, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that DLS score at 8 months of age was a reasonable, albeit imperfect, predictor of the condition of the hip joint cartilage at necropsy. Thus, the DLS method might be useful for early identification of dogs with hip dysplasia.

Animals↗
PubMed2001

Electroencephalogram analysis and neuroprotective profile of the N-acetylated-alpha-linked acidic dipeptidase inhibitor, GPI5232, in normal and brain-injured rats.

We have evaluated the effects of the N-acetylated-alpha-linked acidic dipeptidase (NAALADase) inhibitor, GPI5232 [2-[(pentafluorophenylmethyl)hydroxyphosphinyl]methyl)-pentanedioic acid], to not only decrease brain injury but also to alter the inherent electroencephalographic (EEG) changes observed in a rat model of transient middle cerebral artery occlusion (MCAo). Continuous i.v. infusion of GPI5232 starting 1 h after injury resulted in more than a 50% reduction in brain infarct volume caused by 2 h of MCAo. This effect was dose-dependent and significant even when first treatment was delayed for 2 h post-MCAo. At 24 h post-MCAo, EEG spectral analysis of the injured hemisphere revealed functional improvement in GPI5232-treated rats. Significant recovery in high-frequency EEG power (8-30 Hz) was measured in GPI5232-treated animals in both parietal and temporal brain regions but not in vehicle-treated animals. MCAo-injured rats were also predisposed to developing cortical brain seizures, and GPI5232-treated rats had significantly fewer brain seizures than vehicle-treated animals. In separate experiments, acute high doses of GPI5232 in normal rats did not significantly alter EEG brain activity as evaluated by spectral analysis and did not produce any signs of seizure activity or behavioral abnormalities. These results show GPI5232 to be an effective neuroprotective treatment when given postinjury by reducing brain infarction and ameliorating the pathological EEG associated with focal brain ischemia.

Animals↗
PubMed2000

DNA adduct formation by Fusarium culture extracts: lack of role of fusarin C.

Fusarium fungi have been shown to infect corn and other crops worldwide, and have a significant impact on human health through loss of crops or contamination of food with mycotoxins. Isolates of Fusarium fungi from an area of South Africa with high incidence of esophageal cancer have been shown to induce esophageal and liver cancer in rats. Several isolates of Fusarium fungi were grown on corn to determine if genotoxic products were produced. We report the incubation of methanol extracts of Fusarium verticillioides cultures with DNA in the presence of rat liver fractions (S9) resulted in the formation of a unique DNA adduct that was detected by (32)P-postlabeling. Fusarin C was purified from cultures of Fusarium verticillioides RRC 415, and was not responsible for the formation of the DNA adduct. Treatment of the methanolic extracts with ultraviolet B radiation reduced the fusarin C content in the extract; however, this had no effect on the formation of the DNA adduct following incubation of the extract with DNA and S9. The unique DNA adduct was formed following the incubation of several Fusarium verticillioides isolates from the US and South Africa, while extracts of cultures of Fusarium graminearium and Fusarium sacchari isolates formed very little of the DNA adduct when incubated with DNA and S9. These data suggest that neither fusarin C nor any of its metabolites are responsible for formation of the DNA adduct, and that an unidentified compound is present in F. verticillioides cultures that forms a DNA adduct, and may be important in the etiology of human esophageal cancer.

Animals↗
PubMed2000

Evidence for Ca(2+) activation and inactivation sites on the luminal side of the cardiac ryanodine receptor complex.

We have used tryptic digestion to determine whether Ca(2+) can regulate cardiac ryanodine receptor (RyR) channel gating from within the lumen of the sarcoplasmic reticulum (SR) or whether Ca(2+) must first flow through the channel and act via cytosolically located binding sites. Cardiac RyRs were incorporated into bilayers, and trypsin was applied to the luminal side of the bilayer. We found that before exposure to luminal trypsin, the open probability of RyR was increased by raising the luminal [Ca(2+)] from 10 micromol/L to 1 mmol/L, whereas after luminal trypsin exposure, increasing the luminal [Ca(2+)] reduced the open probability. The modification in the response of RyRs to luminal Ca(2+) was not observed with heat-inactivated trypsin, indicating that digestion of luminal sites on the RyR channel complex was responsible. Our results provide strong evidence for the presence of luminally located Ca(2+) activation and inhibition sites and indicate that trypsin digestion leads to selective damage to luminal Ca(2+) activation sites without affecting luminal Ca(2+) inactivation sites. We suggest that changes in luminal [Ca(2+)] will be able to regulate RyR channel gating from within the SR lumen, therefore providing a second Ca(2+)-regulatory effect on RyR channel gating in addition to that of cytosolic Ca(2+). This luminal Ca(2+)-regulatory mechanism is likely to be an important contributing factor in the potentiation of SR Ca(2+) release that is observed in cardiac cells in response to increases in intra-SR [Ca(2+)].

Animals↗
PubMed2000

The interaction of a neutral ryanoid with the ryanodine receptor channel provides insights into the mechanisms by which ryanoid binding is modulated by voltage.

In an earlier investigation, we demonstrated that the likelihood of interaction of a positively charged ryanoid, 21-amino-9alpha-hydroxyryanodine, with the sarcoplasmic reticulum Ca(2+)-release channel (ryanodine receptor, RyR) is dependent on holding potential (Tanna, B., W. Welch, L. Ruest, J.L. Sutko, and A. J. Williams. 1998. J. Gen. Physiol. 112:55-69) and suggested that voltage dependence could result from either the translocation of the charged ligand to a site within the voltage drop across the channel or a voltage-driven alteration in receptor affinity. We now report experiments that allow us to assess the validity of these alternate mechanisms. Ryanodol is a neutral ryanoid that binds to RyR and induces modification of channel function. By determining the influence of transmembrane potential on the probability of channel modification by ryanodol and the rate constants of ryanodol association and dissociation, we demonstrate that the influence of voltage is qualitatively the same for both the neutral and positively charged ryanoids. These experiments establish that most, if not all, of the modification of ryanoid interaction with RyR by transmembrane holding potential results from a voltage-driven alteration in receptor affinity.

Animals↗
PubMed2000

Gram-negative bacteria induce proinflammatory cytokine production by monocytes in the absence of lipopolysaccharide (LPS).

Tumour necrosis factor-alpha (TNF-alpha), IL-1alpha and IL-6 production by human monocytes in response to a clinical strain of the Gram-negative encapsulated bacteria Neisseria meningitidis and an isogenic lpxA- strain deficient in LPS was investigated. Wild-type N. meningitidis at concentrations between 105 and 108 organisms/ml and purified LPS induced proinflammatory cytokine production. High levels of these cytokines were also produced in response to the lpxA- strain at 107 and 108 organisms/ml. The specific LPS antagonist bactericidal/permeability-increasing protein (rBPI21) inhibited cytokine production induced by LPS and wild-type bacteria at 105 organisms/ml but not at higher concentrations, and not by LPS-deficient bacteria at any concentration. These data show that proinflammatory cytokine production by monocytes in response to N. meningitidis does not require the presence of LPS. Therapeutic strategies designed to block LPS alone may not therefore be sufficient for interrupting the inflammatory response in severe meningococcal disease.

Acyltransferases↗