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Biomedical subjects

R B Clark

Publications and source records attributed to R B Clark.

At least 127 records · Page 7Linked to original sources
PubMed1988

Properties of the transient outward current in rabbit atrial cells.

1. Whole-cell and patch clamp techniques have been used to study the steady-state voltage dependence and the kinetics of a transient outward current, It, in single cells from rabbit atrium. 2. The steady-state voltage dependence of both activation and inactivation of It are well described by Boltzmann functions. Inactivation is fully removed at potentials negative to -70 mV and it is complete near 0 mV. The threshold for activation of It is near -30 mV and it is fully activated at +30 mV. The region of overlap between the activation and inactivation curves indicates that a steady non-inactivating current will be recorded over a membrane potential range from approximately -30 to 0 mV. 3. In general, the time course of inactivation at potentials in the range 0 to +50 mV is best described as a sum of two exponential functions. The kinetic parameters controlling these processes exhibit only very weak voltage dependence. 4. Comparison of the time course of the development of inactivation in response to long depolarizing voltage clamp steps with the development of inactivation in response to trains of brief depolarizing pulses indicates that inactivation develops very quickly and decays relatively slowly at potentials near the resting potential (e.g. -70 mV). Thus, in response to (i) a train of voltage-clamp pulses or (ii) a series of action potentials, the magnitude of It decreases due to a progressive increase in the amount of inactivation. 5. A simple model of channel gating is presented: it can account for the major aspects of the voltage dependence and kinetics of It (cf. Aldrich, 1981). 6. Cell-attached patch clamp recordings have been used to identify the single-channel or unitary events underlying the current, It. In general, only one active channel is present per patch. The single-channel conductance in normal Tyrode solution is approximately 14 pS and the current-voltage relationship is approximately linear between +50 and +150 mV with respect to rest. This information, in combination with the fully activated current-voltage characteristics from the whole-cell data, can be used to estimate the number and density of It channels per cell: these are 1600 and one per 3-4 micron 2, respectively. 7. Ensemble averages obtained from patch recordings are very similar in time course to the macroscopic or whole-cell current itself: the ensemble current rises to a peak within approximately 5 ms and decays with a biexponential time course in response to depolarizations to approximately +50 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Aminoglycoside resistance among Pseudomonas aeruginosa isolates with an unusual disk diffusion antibiogram.

In recent years, a number of clinical microbiology laboratories have isolated Pseudomonas aeruginosa with the unusual aminoglycoside disk diffusion result of resistance to both amikacin and gentamicin but susceptibility to tobramycin (ArGrTs). A total of 39 isolates of P. aeruginosa reported to have this resistance pattern were retested by the standard National Committee for Clinical Laboratory Standards disk diffusion procedure; 30 strains (77%) were confirmed to be ArGrTs. These 30 isolates were further examined for susceptibility to those aminoglycosides by agar dilution and broth micro- and macrodilution methods. Only 27, 27, and 23% of the isolates appeared to be ArGrTs by agar, broth microdilution, and broth macrodilution testing, respectively. Most of the remaining isolates were resistant to all three aminoglycosides when tested by broth dilution and resistant only to gentamicin when tested by agar dilution. The percentages of strains resistant to any particular aminoglycoside by agar dilution, broth microdilution, and broth macrodilution, respectively, were 43, 80, and 70 for amikacin, 97, 93, and 100 for gentamicin, 100, 100, and 100 for netilmicin, 30, 87, and 93 for sisomicin, and 13, 57, and 50 for tobramycin. These results indicate that strains showing the unusual aminoglycoside antibiogram are less susceptible to aminoglycosides in general and should probably be considered borderline resistant to all aminoglycosides. The efficacy of aminoglycosides in the treatment of infections produced by these strains is unknown.

Amikacin↗
PubMed1988

Beta-lactam resistance in Aeromonas spp. caused by inducible beta-lactamases active against penicillins, cephalosporins, and carbapenems.

Use of cefoperazone in a patient with Aeromonas caviae in the respiratory tract selected a mutant that constitutively produced beta-lactamase. This mutant, in contrast to its parental strain with an inducible beta-lactamase, showed enhanced resistance to newer cephalosporins and aztreonam. This observation suggested that species of Aeromonas, like those of other genera with inducible beta-lactamases, may pose therapeutic problems associated with the rapid development of multiple beta-lactam resistance. Thus, a study was designed to identify the beta-lactamases in 12 strains representing four species of Aeromonas and assess their role in drug resistance. Eleven strains possessed inducible beta-lactamases. One strain showed no detectable activity. An analysis of substrate and inhibitor profiles, isoelectric points, and beta-lactam susceptibility patterns revealed the presence of at least four distinguishable inducible beta-lactamases. These enzymes were involved in the resistance of strains within the genus to penicillins, cephalosporins, aztreonam, and imipenem but not cefoxitin. Unlike most other organisms with inducible beta-lactamases, all four strains of A. caviae, one of four strains of A. sobria, and one of three strains of A. hydrophila possessed two distinct inducible beta-lactamases. Furthermore, substrate and inhibitor profiles revealed that many of these Aeromonas beta-lactamases were distinct from inducible enzymes that have been characterized in other genera of gram-negative bacteria.

Aeromonas↗
PubMed1988

T cell receptor beta chain gene rearrangement shared by murine T cell lines derived from a site of autoimmune inflammation.

Advances in our understanding of the structure and molecular biology of the T lymphocyte antigen-receptor have now made it feasible to study human autoimmune diseases using new approaches. One such approach involves cloning of T cells from sites of autoimmune pathology followed by identification of putative disease-related T cell oligoclonality at the level of the T cell receptor gene rearrangements. We have now tested the feasibility of this approach in an animal model of autoimmunity, murine experimental allergic encephalomyelitis (EAE). Spinal cord-derived, self (murine) myelin basic protein (MBP)-reactive T cell lines and sublines were analyzed at the level of their receptor beta chain rearrangements using Southern blots. We now report that the MBP-reactive T cell lines and sublines derived from the spinal cords of four of five SJL/J mice with EAE share a 14.5-kb rearranged T cell receptor beta 1 band on Southern blots. A spinal cord-derived T cell line that was reactive to purified protein derivative of tuberculin (PPD), several lymph node-derived ovalbumin- and PPD-reactive T cell lines, as well as one MBP-reactive spinal cord-derived T cell line did not share this 14.5-kb rearranged beta 1 band. These results suggest that analysis of the antigen receptors used by T cells cloned from sites of inflammation may be a useful initial approach for identifying pathogenetically relevant T cells in the study of certain human autoimmune diseases.

Animals↗
PubMed1987

The protein kinase C inhibitor H-7, inhibits antigen and IL-2-induced proliferation of murine T cell lines.

Activation of protein kinase C has been shown to be involved in the activation pathway of many cell types. Recently, a number of investigations have suggested that protein kinase C plays an essential role in T lymphocyte activation. The recent synthesis of the protein kinase inhibitors, H-7 and HA1004, have now made possible a new approach for testing the relevance of protein kinase C in T cell activation and proliferation. We now report that the antigen-induced and interleukin-2-induced proliferation of murine T cell lines can be consistently inhibited by the protein kinase C inhibitor, H-7. HA1004, a somewhat more potent inhibitor of cyclic nucleotide-dependent protein kinases, but a significantly weaker inhibitor of protein kinase C than H-7, demonstrated no consistent inhibition of these T cell responses. These results represent a further demonstration that protein kinase C plays an essential role in the activation of T cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1987

Complement dependent in vitro neutralization of Chlamydia trachomatis by a subspecies-specific monoclonal antibody.

Three murine monoclonal antibodies which combined specifically with Chlamydia trachomatis strain HAR-13 (Serotype A) were characterized. All antibodies cross-reacted with subspecies-specific epitopes in previously undescribed patterns. One of the antibodies showed a complement-dependent partial neutralization of strain HAR-13 in vitro, whereas the remaining two antibodies did not neutralize under any experimental conditions tested. The significance of subspecies-specific antibodies in neutralizing chlamydial infectivity in vivo remains to be determined.

Animals↗
PubMed1987

Allogeneic fibroblasts and endothelial cells support mitogen but not antigen responses of tetanus-responsive T-cell lines.

Recent studies suggest a role for endothelial cells (EC) and fibroblasts (FB) in performing certain accessory functions of monocytes in immune responses. We examined the ability of allogeneic adherent cells (AC), umbilical vein EC, and foreskin FB to support antigen and mitogen responses of tetanus toxoid-responsive human T-cell lines (TCTet). Syngeneic AC supported antigen and mitogen (phytohemagglutinin and pokeweed mitogen, PHA and PWM) responses of TCTet. Allogeneic AC, EC, and FB supported mitogen but not antigen responses of TCTet, in a dose-dependent manner. PHA-activated mononuclear cell supernates or EC or FB supernates could not replace accessory cells in mitogen responses. We provide further evidence that EC and FB can function as fully competent accessory cells, a function that may be of significance in in vivo initiation of immune responses.

Antigen-Presenting Cells↗
PubMed1987

Activity of ciprofloxacin against Mycobacteria in vitro: comparison of BACTEC and macrobroth dilution methods.

Various clinical isolates of mycobacteria were tested for susceptibility to ciprofloxacin by a standard macrobroth dilution test and the radiometric BACTEC method. Agreement between the two test systems was species dependent: Mycobacterium tuberculosis (80%), M. kansasii with M. scrofulaceum (30%), M. avium-intracellulare (20%), and 0% for the rapidly growing mycobacteria. Most mycobacterial strains other than M. tuberculosis were susceptible to the breakpoint ciprofloxacin concentration of 2 mg/l as determined by BACTEC MICs, whereas none were susceptible by macrobroth testing. Seven of nine M. tuberculosis isolates were susceptible by either method. Ciprofloxacin merits further study as a potential antimycobacterial agent.

Ciprofloxacin↗
PubMed1987

Beta-adrenergic receptor desensitization of wild-type but not cyc lymphoma cells unmasked by submillimolar Mg2+.

Treatment with low physiological concentrations of epinephrine (5-50 nM) rapidly desensitizes beta-adrenergic stimulation of cAMP formation in S49 wild-type (WT) lymphoma cells. Previous attempts to detect this early phase of desensitization in cell-free assays of adenylate cyclase (EC 4.6.1.1) after intact cell treatment were unsuccessful. We have now found that reducing the Mg2+ concentrations in the adenylate cyclase assays to less than 1.0 mM unmasked this rapid phase of desensitization of the WT cells, and that high Mg2+ concentrations (5-10 mM) largely obscured the desensitization. Submillimolar Mg2+ conditions also revealed a two- to threefold decrease in the affinity of epinephrine binding to the beta-adrenergic receptor after desensitization with 20 nM epinephrine. Detection of 4 beta-phorbol 12-myristate 13-acetate (PMA) desensitization of the WT beta-adrenergic receptor was also dependent on low Mg2+ as measured either by the decrease in epinephrine stimulation of adenylate cyclase or by the reduction in the affinity of epinephrine binding. Unexpectedly, when cyc- cells were pretreated with 50 nM epinephrine, the beta-adrenergic stimulation of reconstituted adenylate cyclase was not desensitized. The characteristics of the Mg2+ effect on epinephrine- and PMA-induced desensitizations suggest a similar mechanism of action with the most likely events being phosphorylations of the beta-adrenergic receptors. Our data indicate that cAMP-dependent protein kinase (EC 2.7.1.37) may play a role in the desensitization caused by low epinephrine concentrations inasmuch as this phase of desensitization did not occur in the cyc-. For the PMA-induced desensitization, the phosphorylation may be mediated by protein kinase C (EC 2.7.1.37).

Adenylyl Cyclases↗
PubMed1987

Sodium current in single cells from bullfrog atrium: voltage dependence and ion transfer properties.

1. Whole-cell and patch-clamp techniques (Hamill, Marty, Neher, Sakmann & Sigworth, 1981) have been used to make quantitative measurements of the transient inward sodium current (INa) in single cells from bullfrog atrium. This preparation is particularly suitable for the study of INa: (i) the current density is relatively low, (ii) the cells lack a transverse tubule system, (iii) isolated myocytes can be maintained at reduced temperatures (approximately 8-12 degrees C); therefore kinetics can be studied quantitatively. 2. INa was pharmacologically and kinetically isolated from other transmembrane currents by blocking ICa with CdCl2 (0.2-0.5 mM) or LaCl3 (5 x 10(-6) M), and by using only relatively short voltage-clamp depolarizations which did not activate IK (the delayed rectifier). 3. The voltage dependence of INa in bullfrog atrium is similar to that in amphibian node of Ranvier or fast skeletal muscle. The threshold for activation is approximately -50 mV. The peak of the INa vs. membrane potential relation is near -5 to -10 mV. The reversal potential in 'normal' (115 mM-Na+) Ringer solution is +59.0 mV (S.D. +/- 3.4, n = 10). Reduction of external Na+ concentration to one-third of normal resulted in an approximately -27 mV shift of the reversal potential, close to that expected for a highly Na+-selective conductance. 4. Steady-state inactivation of INa (h infinity), measured with a conventional two-pulse voltage-clamp protocol, spanned the membrane potential range from -90 to -50 mV. The potential dependence of h infinity was well described by a single Boltzmann function with half-inactivation at -71 mV and maximum slope of 6.0 mV. 5. Steady-state activation of INa (m infinity) was determined from fits of INa records to a Hodgkin-Huxley model. The potential dependence of m infinity was fitted to a Boltzmann function with half-activation at -33 mV and maximum slope of 9.5 mV. Thus at temperatures around 10 degrees C there was very little overlap of the m infinity and h infinity curves, and only very small steady-state 'window' currents are predicted. 6. The activation time constant, tau m, had a 'bell-shaped' dependence on membrane potential. The peak value of tau m was about 4.2 ms, at a membrane potential of -35 mV (9 degrees C). 7. The time course of inactivation of INa was consistently better described by the sum of two exponentials than by one exponential.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗