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Biomedical subjects

R A Harris

Publications and source records attributed to R A Harris.

At least 127 records · Page 7Linked to original sources
PubMed1998

Aortic reconstructive surgery for limb ischaemia: immediate and long-term follow-up to provide a standard for endovascular procedures.

PURPOSE: Aortobifemoral and aortobiliac bypass has been a preferred treatment of severe aortoiliac occlusive disease. Recently, endovascular procedures and unilateral bypass grafting have been introduced. We report the results of aortic reconstructive surgery over a 19-year period (1975-1994). METHODS: A total of 285 patients underwent surgery. Follow-up data from clinical notes, general practitioner questionnaire or phone interview was available in 93.3% (266/285). RESULTS: Of the total 68% were male (180/226) and the median age was 63 years (range 22-85 years). Indications for surgery were tissue loss/gangrene (n = 32), rest pain (n = 84), severe claudication (n = 40), moderate claudication (n = 110). Total of 177 bifurcated grafts, 51 iliofemoral, eight left/right aortofemoral, and 30 other procedures were performed. The median duration of follow-up was 49.8 months. Cumulative secondary graft patency for 2, 5 and 10 years was 97.7%, 95.9%, and 94.4%. The amputation rate was 4.5% (12/266). Early mortality occurred in eight of 266 patients (3%) and late mortality in 60 patients. The most common cause of late death was ischaemic heart disease (24/60 followed by cancer (14/60). Data was further analysed by type of graft, risk factors, complications and previous and subsequent surgery. In our experience, aortic reconstructive surgery is a successful option for the management of severe aortoiliac occlusive disease. The outcome of other methods of maintaining arterial patency should be measured against this standard.

Adult↗
PubMed1998

The crystal structure of H-2Dd MHC class I complexed with the HIV-1-derived peptide P18-I10 at 2.4 A resolution: implications for T cell and NK cell recognition.

The structure of H-2Dd complexed with the HIV-derived peptide P18-I10 (RGPGRAFVTI) has been determined by X-ray crystallography at 2.4 A resolution. This MHC class I molecule has an unusual binding motif with four anchor residues in the peptide (G2, P3, R/K/H5, and I/L/F9 or 10). The cleft architecture of H-2Dd includes a deep narrow passage accomodating the N-terminal part of the peptide, explaining the obligatory G2P3 anchor motif. Toward the C-terminal half of the peptide, p5R to p8V form a type I' reverse turn; residues p6A to p9T, and in particular p7F, are readily exposed. The structure is discussed in relation to functional data available for T cell and natural killer cell recognition of the H-2Dd molecule.

Binding Sites↗
PubMed1998

Full-dose and half-dose Klean Prep produce clearer images with iliac duplex examination than picolax.

OBJECTIVES: Iso-osmotic bowel preparation (Klean Prep) improves the accuracy of iliac duplex examination and reduces the time of each examination. Full-dose Klean Prep entails 4 l of fluid. We studied the effect of 2 l of Klean Prep (half-dose) and Picolax on image quality. DESIGN: Prospective study comparing clarity of duplex examination after three different bowel preparation regimes with that after 12 h starvation. MATERIALS AND METHODS: Thirty patients underwent iliac duplex examination after 12 h starvation. Scans were scored subjectively for grey scale and colour image quality, and Doppler signal-to-noise ratio. Patients were allocated blindly to: (a) full-dose Klean Prep, (b) half-dose Klean Prep, or (c) Picolax. After out-patient preparation, the scan was repeated and scored by the same observer, blinded to the preparation. RESULTS: Both full- and half-dose Klean Prep produced significant improvements in image quality for all three modalities; Picolax produced minimal change. There was minimal advantage of full-dose over half-dose Klean Prep. Patients preferred half-dose Klean Prep to full-dose. CONCLUSION: Klean Prep significantly improves the image obtained by iliac duplex examination; Picolax does not. Half-dose Klean Prep is an acceptable preparation to patients.

Aged↗
PubMed1998

Enhancement of glycine receptor function by ethanol: role of phosphorylation.

1. The effects of several kinase inhibitors (staurosporine, GF 109203X, H89, KN62, genistein) and of the phosphatase inhibitor calyculin A were studied on the ethanol potentiation and on the function of homomeric alpha1 glycine receptor expressed in Xenopus oocytes using a two electrode voltage clamp recording technique. 2. The function of the homomeric alpha1 glycine receptor was not modified in Xenopus oocytes pretreated with kinase inhibitors or with the phosphatase inhibitor calyculin A. 3. The potentiation of the glycine receptor function induced by ethanol (10-200 mM) was significantly reduced in Xenopus oocytes pretreated with the PKC inhibitors staurosporine or GF 109203X. 4. No differences in propofol (2.5 microM) or halothane (250 microM) actions were found after exposure of Xenopus oocytes to staurosporine. 5. No differences in ethanol sensitivity were found after exposure of Xenopus oocytes expressing glycine alpha1 receptors to H89, KN62, genistein or to the phosphatase inhibitor calyculin A. 6. The mutant alpha1 (S391A), in which the PKC phosphorylation site at serine 391 was mutated to alanine, was less sensitive to the effects of ethanol than was the alpha1 wild type receptor. Moreover, the ethanol potentiation of the glycine receptor function was not affected by treatment with staurosporine in oocytes expressing alpha1 (S391A). 7. The splice variant of the alpha1 glycine receptor subunit, alpha1ins, containing eight additional amino acids and a potential phosphorylation site for PKA, did not differ from wild type for sensitivity to ethanol. 8. These results indicate that phosphorylation by PKC of the homomeric alpha1 glycine receptor subunit modulates ethanol potentiation, but not the function of the glycine receptor.

Animals↗
PubMed1998

Gammadelta+ and CD4+ alphabeta+ human T cell subset responses upon stimulation with various Mycobacterium tuberculosis soluble extracts.

By using a flow cytometric technique which allows direct identification of proliferating cells within mixed cell populations, we have previously described that soluble extracts obtained from Mycobacterium tuberculosis or M. avium represent strong stimuli for human gammadelta+ T cells. In the present study, we demonstrate that the protocol used for the preparation of M. tuberculosis soluble extracts may have an impact on their gammadelta+ T cell stimulatory capacity. In agreement with our previous data, soluble extracts prepared from bacteria killed at 85 degrees C and directly disrupted by prolonged sonication (TBe), elicited a strong proliferation of gammadelta+ T cells after 6-7 days of stimulation. In contrast, when soluble extracts were obtained from bacteria autoclaved (121 degrees C, 25 min) and then washed by centrifugation, a predominant proportion of CD4+ alphabeta+ T cells was achieved in the responding population. The stimulatory activity for gammadelta+ T cells was recovered in the supernatant of the autoclaved bacteria, indicating that autoclaving of M. tuberculosis bacilli releases an antigen(s) into the supernatant which stimulates human gammadelta+ T cells. While protease digestion of TBe only partially reduced its stimulatory capacity on gammadelta+ T cells, the stimulatory component(s) released into the supernatant after autoclavation of bacilli was found to be sensitive to protease digestion. Interestingly, in contrast to the preponderant proportion of gammadelta+ T cells induced in the responding population by unfractionated TBe, when the extract was fractionated by fast performance liquid chromatography (FPLC), most of the fractions exhibited a strong stimulatory capacity on CD4+ alphabeta+ T cells only. The gammadelta+ T cell stimulatory activity was confined to the low molecular weight range FPLC fractions. Such results may suggest a possible regulatory role of gammadelta+ T cells on CD4+ alphabeta+ T cells.

Antigen Presentation↗
PubMed1998

Cellular and cytokine characterization of vascular inflammation in CBA/J mice chronically infected with Trypanosoma cruzi.

Continuing our characterization of the immunopathological events occurring during experimental murine Chagas' disease, an immunohistological examination was conducted of the aortas of chronically infected CBA/J mice. Compared with non-infected mice of identical age, Trypanosoma cruzi-infected mice exhibited a marked vasculitis, with significant infiltration of inflammatory cells into the adventitial layer, including CD4+, CD8+ T cells and macrophages. Production of interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-alpha) was evident in the inflammatory infiltrate in the endothelial and smooth muscle layers. Vasculitis was most apparent in proximity to the heart, but extended along the aorta. Such an inflammation could lead to an alteration of the endothelium, altering the protective properties of this layer and further contributing to the focal pathology characteristic of this stage of infection.

Animals↗
PubMed1998

Acute effects of ethanol on pharmacologically isolated kainate receptors in cerebellar granule neurons: comparison with NMDA and AMPA receptors.

Comparisons of acute ethanol's effects on individual members of the three major families of ionotropic glutamate receptors (kainate, AMPA, and NMDA) have been performed only with recombinant receptors. However, no study has compared the acute effects of ethanol on individual members of each one of these receptor families in the same neuron. We accomplished this task by using cultured cerebellar granule neurons and LY303070 (GYKI-53784), a noncompetitive and selective AMPA receptor antagonist. Ethanol concentrations of 25, 50, 75, and 100 mM decreased the amplitude of pharmacologically isolated kainate-activated currents by 3 +/- 1, 9 +/- 2, 14 +/- 2, and 22 +/- 3% (n = 8), respectively. The magnitude of the ethanol-induced inhibition of nonselective kainate-activated currents, i.e., in the absence of LY303070, and currents activated by submaximal AMPA concentrations was not significantly different from that obtained with isolated kainate currents. However, the magnitude of the ethanol-induced inhibition of NMDA receptor-activated currents was about twofold greater than that of kainate and/or AMPA receptors.

Animals↗
PubMed1998

Branched-chain alpha-keto acid dehydrogenase kinase content in rat skeletal muscle is decreased by endurance training.

The activity state of branched-chain alpha-keto acid dehydrogenase complex in skeletal muscle was elevated by running exercise in trained and untrained rats, but level of this elevation was significantly greater in the former than in the latter. To elucidate the mechanism of the training effect on the exercise-induced activation of the complex, a protein amount of branched-chain alpha-keto acid dehydrogenase kinase, which is responsible for inactivation of the complex by phosphorylation, in the muscle was measured by the Western blot analysis. Endurance training decreased the content of the kinase protein in the muscle by approximately 30%, suggesting that this decrease is involved in the mechanisms for greater activation of the complex by exercise in trained rats.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1998

A molecular model of human branched-chain amino acid metabolism.

To establish an accurate molecular model of human branched-chain amino acid (BCAA) metabolism, the distribution, activity, and expression of the first 2 enzymes in the catabolic pathway--branched-chain-amino-acid aminotransferase (BCAT) and branched-chain alpha-keto acid dehydrogenase (BCKD) complex--were determined in human tissues. The same enzyme activities were measured in rat and African green monkey tissues. Overall, the activities of BCAT and BCKD were higher in rat than in human and monkey tissues; nevertheless, the ratio of the 2 activities was similar in most tissues in the 3 species. Total oxidative capacity was concentrated in skeletal muscle and liver (> 70%) with muscle having a higher proportion of the total in humans and monkeys. In humans, brain (10-20%) and kidney (8-13%) may contribute significantly to whole-body BCAA metabolism. Furthermore, in primates the high ratio of transaminase to oxidative capacity in the entire gastrointestinal tract serves to prevent loss of essential BCAA carbon and raises the possibility that the gastrointestinal tract contributes to the plasma branched-chain alpha-keto acid pool. Quantitative polymerase chain reaction was used to examine expression of human branched-chain alpha-keto acid dehydrogenase kinase (BCKDK), the key enzyme that regulates the activity state of the human BCKD complex and human BCAT isoenzymes. To design the primers for the polymerase chain reaction, human BCKDK was cloned. BCKDK message was found in all human tissues tested, with the highest amount in human muscle. As in rats, there was ubiquitous expression of mitochondrial BCAT, whereas mRNA for the cytosolic enzyme was at or below the limit of detection outside the brain. Finally, the role of BCAA in body nitrogen metabolism is discussed.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1998

Timing of vaccination does not affect antibody response or survival after pneumococcal challenge in splenectomized rats.

BACKGROUND: Pneumococcal vaccination after splenectomy for trauma decreases the incidence of overwhelming postsplenectomy infection. The optimal timing of vaccination has not been established. This study was conducted to determine whether timing of vaccination after splenectomy affects antibody response or survival after pneumococcal challenge. METHODS: Sprague-Dawley rats were used for all experiments. Control rats (n=30) were divided into three equal groups and underwent splenectomy followed by sham vaccination 1, 7, or 42 days after splenectomy. Treated rats (n=66) were divided into three equal groups and underwent splenectomy followed by vaccination with polyvalent pneumococcal vaccine 1, 7, or 42 days after splenectomy. All rats then underwent intraperitoneal Streptococcus pneumoniae inoculation with the predetermined lethal dose for 50% of the population 10 days after vaccination. Rats were observed for a 72-hour period after inoculation, and mortality was recorded. Immunoglobulin G and immunoglobulin M antibody titers were determined before vaccination and before inoculation to determine antibody response. RESULTS: Mortality was greater in the control group than in the treatment group (21 of 30 [70%] vs. 2 of 64 [3%]; p < 0.01). There were no differences in mortality within either the control group (1 day, 6 of 10; 7 days, 7 of 10; 42 days, 8 of 10; p=0.62) or the treatment group (1 day, 0 of 21; 7 days, 0 of 21; 42 days, 2 of 22; p=0.14). Immunoglobulin G and immunoglobulin M antibody responses were greater in vaccinated than in nonvaccinated rats. There was no effect of timing of vaccination on antibody response. CONCLUSION: Pneumococcal vaccine reduces mortality from postsplenectomy infection. Timing of vaccination after splenectomy does not affect survival from a pneumococcal challenge or antibody response in rats. This study supports the practice of administering vaccine within 24 hours of splenectomy when vaccine cannot be administered before surgery.

Animals↗
PubMed1998

Effects of ethanol and anesthetics on type 1 and 5 metabotropic glutamate receptors expressed in Xenopus laevis oocytes.

Previous studies have demonstrated that ethanol and volatile anesthetics inhibit the function of some metabotropic (G protein-coupled) receptors, including the 5-hydroxytryptamine2 and muscarinic cholinergic receptors. The metabotropic glutamate receptors (mGluRs) show little sequence homology with most other metabotropic receptors and are important modulators of synaptic transmission in the mammalian central nervous system. It was of interest to determine drug actions on these receptors, and we investigated the effects of ethanol, halothane, the anesthetic compound F3 (1-chloro-1,2,2-trifluorocyclobutane), and the nonanesthetics F6 (1,2-dichlorohexafluorocyclobutane) and F8 (2,3-chlorooctafluorobutane) on the function of mGluR1 and mGluR5 expressed in Xenopus laevis oocytes. Halothane, F3, and ethanol inhibited mGluR5-induced Ca(2+)-dependent Cl- currents, yet pharmacologically relevant concentrations of these compounds had little effect on the glutamate-induced currents in the oocytes expressing mGluR1. F6 had inhibitory effects on both receptors, and F8 did not affect either mGluR1 or mGluR5 function. The protein kinase C (PKC) inhibitor GF109203X enhanced the glutamate-induced current, and the PKC activator phorbol-12-myristate-13-acetate inhibited this current in the oocytes expressing mGluR5, but these compounds had little effect on mGluR1 function. GF109203X abolished the inhibitory effects of halothane, F3, and ethanol on mGluR5s. Conversely, the phosphatase inhibitor calyculin A prolonged the action of halothane and ethanol. Furthermore, mutation of a PKC consensus site (Ser890) of mGluR5 abolished the inhibitory effects of halothane, F3, and ethanol. These results suggest that ethanol and volatile anesthetics inhibit mGluR5 because they promote PKC-mediated phosphorylation.

Anesthetics, Inhalation↗
PubMed1998

Colchicine is a competitive antagonist at human recombinant gamma-aminobutyric acidA receptors.

Colchicine is an alkaloid that is used clinically in the treatment of arthritic gout. This potent microtubule disrupting agent has also been used extensively as an experimental tool in studies characterizing the role of the cytoskeleton in a variety of cellular processes. Colchicine has also been used as a selective neurotoxin and in animal models of Alzheimer's disease and epilepsy. Although the mechanism(s) mediating the neurotoxic actions of colchicine have not been established, most studies have attributed these effects to its microtubule depolymerizing actions. Here we report another central nervous system action of colchicine, competitive antagonism of gamma-aminobutyric acid (GABA)A receptor function. By use of a rapid drug perfusion system, colchicine (10-1000 microM) significantly inhibited GABA currents recorded from L(tk-) cells stably transfected with human alpha 1 beta 2 gamma 2L GABAA receptor subunits. The inhibition was rapid and reversible, with 100 microM colchicine shifting the GABA EC50 from 2.5 to 5.1 microM with no effect on currents evoked by saturating concentrations of GABA. Colchicine also significantly inhibited binding of the competitive GABAA receptor antagonist [3H]SR-95531. Other microtubule disrupting agents (10 microM vinblastine, 10 micrograms/ml nocodazole, 1 microM taxol) had no acute effects on GABA currents, nor did the inactive analog gamma-lumicolchicine (100 microM). Moreover, pretreating cells with colchicine, vinblastine, nocodazole or taxol for 1 to 4 hr did not occlude the acute inhibitory action of colchicine. We conclude that, in addition to its well characterized effects on microtubule assembly, colchicine can also inhibit GABAA receptor function through a direct interaction with the receptor/ion channel complex.

Animals↗
PubMed1998

Adaptation of gamma-aminobutyric acid type A receptors to alcohol exposure: studies with stably transfected cells.

We studied the adaptation of gamma-aminobutyric acid type A (GABAA) receptor function to chronic ethanol exposure in cells stably transfected with the following GABAA receptor subunits: alpha-1 beta-2 gamma-2L, alpha-1 beta-2 gamma-2S, alpha-1 beta-3 gamma-2S, alpha-1 beta-1, alpha-5 beta-3 gamma-3 and alpha-6 beta-3 gamma-2S. Chronic exposure to ethanol resulted in a decrease in muscimol-stimulated 36Cl- flux and a decrease in modulation of that flux by ethanol, flunitrazepam, methyl-6,7-4-dimethoxy-4-ethyl-beta-carboline-3-carboxylate and pregnanolone without any change in the modulation by pentobarbital or zinc. Direct activation of the GABAA receptor by pentobarbital was enhanced by chronic ethanol treatment. Reduction of the action of muscimol, ethanol and flunitrazepam differed in the duration and amount of ethanol required to see an effect. Reduction of the action of ethanol of alpha-1 beta-2 gamma-2L cells occurred within 15 min and was near-maximal for 25 mM ethanol, whereas reduction of the actions of muscimol and flunitrazepam actions required hours of exposure and higher concentrations of ethanol. Chronic ethanol exposure produced a reduction in the Emax value for the action of muscimol for all six subunit combinations, but quantification of surface receptors by immunolabeling showed no change in GABAA receptor density. The differences in alcohol sensitivity and time courses for different effects of ethanol indicate multiple mechanisms of adaptation of GABAA receptors. Use of stably transfected cells rules out "subunit substitution" as a mechanism for these changes and points to post-translational changes (e.g., phosphorylation, receptor assembly) as the most likely mechanisms. These in vitro findings are compared with results from in vivo studies.

Animals↗
PubMed1998

Trypanosoma rangeli: identification and purification of a 48-kDa-specific antigen.

Trypanosoma rangeli infects humans nonpathologically in some areas of Central and South America. Due to morphological and antigenic similarities with T. cruzi, the clear identification of this parasite is an important task. Here, we describe the identification and purification of a specific 48-kDa antigen from T. rangeli. By western blotting analysis, this molecule was not detected in T. cruzi epimastigotes and in Leishmania sp. promastigotes. Fluorescence-activated cell sorter analysis demonstrated that the protein is expressed uniformly by the T. rangeli cell population during axenic culture. Additionally, following immunostaining, a particular subcellular localization (present in organelles) is proposed for this antigen. These results suggest that this 48-kDa antigen may be a useful marker for the identification and characterization of T. rangeli isolates.

Animals↗
PubMed1998

Effects of ethanol on recombinant glycine receptors expressed in mammalian cell lines.

We examined the effects of acute ethanol exposure on recombinant human glycine receptors transiently transfected into HEK 293 cells and stably transfected into Ltk- fibroblast-like cells. In our study of the effects of ethanol, we used the whole-cell patch-clamp configuration. Relatively low concentrations of ethanol (25 mM and 50 mM) did not affect glycine-gated currents in any of the cell lines studied. Higher concentrations of ethanol (100 mM and 200 mM) significantly potentiated glycine responses only in stably transfected Ltk- cells expressing alpha1 and alpha2 subunits and in HEK 293 cells transiently expressing alpha2 subunits. Cells stably expressing alpha1 versus alpha2 glycine receptors were modulated equally by ethanol. Both glycine alpha1 and glycine alpha1beta receptors transiently expressed in HEK 293 cells were insensitive to all concentrations of ethanol tested; however, there was a trend toward potentiation at 100 and 200 mM ethanol concentrations. A population of cells (41-87%) that was sensitive to the potentiating effects of 100 and 200 mM ethanol (defined as more than 10% potentiation) was identified in both cell lines tested. In these sensitive cells, ethanol (100 and 200 mM) produced significant potentiation, independent of the cell line and the glycine receptor subunit tested. Together with published results from studies with Xenopus oocytes, these data indicate that the sensitivity of recombinant glycine receptors to ethanol depends upon the expression system.

Cell Line↗
PubMed1997

Inhibitory effects of anesthetics and ethanol on muscarinic receptors expressed in Xenopus oocytes.

Anesthetics (and ethanol) are known to produce amnesia as well as immobilization. Recent identification of a nonimmobilizing (nonanesthetic) agent (F6 or 1,2-dichlorohexafluorocyclobutane) that impairs learning and memory suggests that distinct mechanisms may be responsible for these two actions of anesthetic agents. Muscarinic receptors are believed to play a role in memory and learning, and we asked if a specific subtype of these receptors is affected by anesthetics as well as the new nonanesthetic. We investigated the effects of halothane, a novel halogenated anesthetic compound F3 (1-chloro-1,2,2-trifluorocyclobutane) and ethanol on acetylcholine-induced current mediated by a muscarinic m1 receptor expressed in Xenopus oocytes. We also studied the effects of halogenated nonanesthetic compounds, F6 and F8 (2,3-chlorooctafluorobutane) on muscarinic m1 receptors. Halothane, F3, F6 and ethanol inhibited muscarinic m1 receptor-induced Ca2+-dependent Cl- currents at pharmacologically relevant concentrations. F8 had no effect on acetylcholine-induced muscarinic m1 receptor function. The protein kinase C inhibitor, bisindolylmaleimide I (GF109203X), enhanced the acetylcholine-induced current and the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), inhibited this current. GF109203X abolished the inhibitory effects of halothane, F3 and ethanol on muscarinic m1 receptors but had no effect on actions of F6. These results demonstrate that anesthetics and a nonanesthetic inhibit the function of muscarinic m1 receptors and suggest activation of protein kinase C as the mechanism of action of anesthetics and ethanol on these receptors.

Anesthetics, Inhalation↗
PubMed1997

Gender difference in regulation of branched-chain amino acid catabolism.

Regulation of the activity state of the hepatic branched-chain 2-oxo acid dehydrogenase (BCODH) complex during the light-dark cycle differs markedly in male and female rats. Female rats exhibit a profound diurnal rhythm in the activity state of the complex that is not observed in male rats. Regardless of gender, most of the complex was dephosphorylated and active in the middle of the dark period and early in the light period, and this form of the complex predominated in male rats at the end of the light period. In contrast, most of the complex in female rats became phosphorylated and inactive by the end of the light period. Gonadectomy prevented the diurnal rhythm in females but was without effect in males, indicating that female sex hormones are required for this gender difference in regulation of the BCODH complex. Changes in levels of branched-chain 2-oxo acids, known regulators of BCODH kinase, do not seem to be involved; rather, an increase in BCODH kinase activity occurring between morning and evening is responsible for inactivation of the BCODH complex in female rats. The increase in kinase activity is due to an increase in the amount of kinase protein associated with the BCODH complex. Thus a marked diurnal variation in the amount of BCODH kinase and therefore its activity results in large swings in the activity state of the liver BCODH complex in female rats. This study provides the first evidence for a gender-specific difference in the regulation of branched-chain amino acid catabolism.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗