Scombroid poisoning revisited.
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Animals↗
Biomedical subjects
R B Clark
Publications and source records attributed to R B Clark.
Voltage-sensitive dye recordings of electrophysiological activation in a Langendorff-perfused mouse heart.
The pattern of electrophysiological activation of adult mouse ventricles was measured with the use of voltage-sensitive dye methods. Di-4-ANEPPS was used to monitor membrane potential as small changes in fluorescence, which were detected by a state of the art, cooled, charged coupled device camera/image intensifier system. The extremely rapid conduction velocity, coupled with the small size of this preparation, necessitated taking these measurements at room temperature (22 to 23 degrees C). Initial experiments demonstrate that ventricular activation can be identified and its conduction pattern can be monitored reproducibly and with high resolution for extended time periods (10 to 20 mins) during spontaneous activity.
Animals↗
Elevation of intracellular cyclic AMP induces an anergic-like state in Th1 clones.
Because elevated intracellular cAMP suppresses T cell receptor (TCR)-mediated effector activity and/or proliferation in response to antigen but does not always affect IL-2-stimulated proliferation, the effects of cAMP on a T lymphocyte response to antigen resemble antigen-induced anergy. To test the hypothesis that elevated cAMP induces anergy in T lymphocytes, we have precultured murine Th1 clones responsive to porcine myelin basic protein (PMBP) with dibutyryl cyclic AMP (dbcAMP) or forskolin and subsequently removed the dbcAMP or forskolin and measured the proliferative response of the clones to antigen and antigen-presenting cells (APC) in the presence or absence of exogenously added interleukin-2 (IL-2). Cells precultured with dbcAMP or forskolin for 3 days did not proliferate or produce IL-2 in response to antigen and APC, but did proliferate to antigen and APC in the presence of IL-2. Cells that had not been stimulated recently with antigen/APC or IL-2 were not affected by dbcAMP, while cells stimulated recently with antigen/APC and IL-2 were susceptible to the anergizing effect of dbcAMP. These observations support the hypothesis that elevation in intracellular cAMP in antigen-activated Th1 clones, prior to subsequent culture with antigen, induces a state of anergy.
Animals↗
Action potential duration modulates calcium influx, Na(+)-Ca2+ exchange, and intracellular calcium release in rat ventricular myocytes.
The experimental work summarized in this paper and described in more detail in our previous publication demonstrates a very important functional role for Na(+)-Ca2+ exchange in intracellular Ca2+ homeostasis in ventricular myocytes from rat hearts. Ca2+ homeostasis in mammalian cardiac myocytes can be considered to be the result of four interactive processes: (i) Ca2+ influx through L-type Ca2+ channels, (ii) Ca2+ release from the SR and its subsequent re-uptake, (iii) intracellular Ca/+ buffering, and (iv) Ca2+ extrusion across the sarcolemma. Our results demonstrate a number of interesting features of these processes. (1) When the action potential voltage-clamp technique is used to identify the size and time-course of Ca2+ fluxes during the action potential, both the peak current and the associated influx of Ca2+ are relatively large as was previously demonstrated by Isenberg and his colleagues. (2) Nevertheless, this source of Ca2+ is unable, by itself, to produce a significant twitch, which is consistent with previous data from rat ventricle. (3) This Ca2+ influx, however, does represent the trigger for SR Ca2+ release. (4) The Na(+)-Ca2+ exchanger on the SR is able, on average, to extrude all the Ca2+ which enters through L-type Ca2+ channels, although it provides relatively little Ca2+, i.e., during the course of the normal action potential there is no significant reverse Na(+)-Ca2+ exchange activity, at least under our experimental conditions. Our results also suggest that although the L-type Ca2+ current cannot by itself trigger and control contraction its amplitude, frequency, and time-course can alter the rate and the extent of Ca2+ release from the SR. Recently, detailed mathematical formulations and a direct demonstration of some of these phenomena have been published. Stern and Stern and Lakatta predicted more than three years ago that the concentration and the time-course of change in concentration of Ca2+ very near the release sites of the SR may be critical determinants of the overall release process. Within the past year Wier and his colleagues and also Lederer et al. have combined electrophysiological measurements with recordings of localized intracellular Ca2+ (made using a confocal microscope) and have shown that rapid, and relatively large, but very localized changes in intracellular Ca2+ due to Ca2+ influx through L-type Ca2+ channels are responsible for triggering, and to some extent, controlling the release of Ca2+ from the SR. However, it has also been shown that this release depends importantly on the loading or priming state of the SR. Perhaps not surprisingly, the massive release of Ca2+ from the SR can, itself, alter the pattern of subsequent SR release events (cf. Ref. 46) and the time-course of Ca2+ influx through the L-type Ca2+ channels. Thus, although our relatively crude measurements have clearly demonstrated the relationship between L-type Ca2+ channel activity and Na+-Ca2+ exchanger function during a normal cardiac action potential in rat ventricle, they fall far short of any delineation of the functional roles of either of these processes in overall Ca2+ homeostasis. This additional information can, in principle, be obtained from studies in which cellular microanatomy can be visualized dynamically in conjunction with localized changes in intracellular Ca2+ as well as Ca2+ of L-type Ca2+ channels, SR release, and cell shortening.
Action Potentials↗
Positive chronotropic responses of rabbit sino-atrial node cells to flash photolysis of caged isoproterenol and cyclic AMP.
The kinetics of onset and the intracellular biochemical signalling mechanisms which are responsible for the positive chronotropic effect of sympathetic stimulation in rabbit cardiac pacemaker cells were examined by using flash photolysis of caged isoproterenol (ISO) and cyclic AMP (cAMP). When caged ISO (10 microM) was present in the superfusate, a single ultraviolet flash caused gradual increases in the spontaneous beating frequency and action potential height of S-A node cells. Both these effects developed after an initial latency of approximately 5 s. Photorelease of ISO also increased the L-type Ca2+ current (ICa-L) with a time-course similar to that of the changes in action potential waveform and heart rate. All of these ISO-induced effects were blocked completely by 1 microM propranolol, demonstrating that they were beta-adrenergic responses. Flash photolysis of caged cAMP (50 microM) also resulted in increased firing frequency and ICa-L. However, these responses to cAMP developed with little or no latency. Intracellular dialysis with a selective inhibitor of the cAMP-dependent protein kinase, Rp-cAMPS, completely abolished the increase in ICa-L demonstrating that it is mediated exclusively via cAMP-dependent activation of protein kinase A, as opposed to a direct G-protein mediated mechanism.
Action Potentials↗
Comparison of five different susceptibility test methods for detecting antimicrobial agent resistance among Haemophilus influenzae isolates.
The detection of antimicrobial agent resistance among ninety-eight Haemophilus influenzae isolates was assessed by six different antibiotic test methods: agar dilution on Mueller-Hinton agar supplemented with 5% lysed horse blood (MH-LHB), E-test using both Haemophilus test medium (HTM) agar and chocolate Mueller-Hinton (CMH) agar plates, Vitek Haemophilus susceptibility cards, and three overnight microdilution systems that included two commercial systems, Micro-Media and MicroScan, and the reference broth microdilution method using HTM broth. Agents tested in the study included ampicillin, amoxicillin/clavulanic acid (A/C), cefaclor, cefuroxime, cefotaxime, ceftriaxone, chloramphenicol, and trimethoprim/sulfamethoxazole. Both the reference HTM microbroth dilution method and agar dilution correctly classified all nine of the beta-lactamase negative ampicillin resistant (BLNAR) isolates. Each of the other test methods failed to detect one of the BLNAR strains, either because of growth failure (Micro-Media and MicroScan) or miscategorization of an isolate as susceptible (E-Test HTM, E-Test CMH, and Vitek). None of the test methods detected all six isolates identified as A/C resistant by HTM microbroth dilution. Of the remaining antimicrobials tested, ampicillin and cefuroxime yielded data that could be compared by all test methods. The very major, major, and minor errors for these two antimicrobials in comparison to the reference HTM microdilution method were as follows: Micro-Media (1.7%, 0%, and 4.8%); MicroScan (11.9%, 0%, and 8.1%); E-Test HTM (1.6%, 0%, and 2.0%); E-Test CMH (1.6%, 1.6%, and 4.6%); Vitek (8.1%, 0%, and 3.1%); and agar dilution on MH-LHB (0%, 0%, and 4.6%). Micro-Media and MicroScan panels failed to support the growth of 4.1% and 5.1% of the isolates, respectively.
Chloramphenicol↗
Comparative epitope mapping of sera from United States (US) and Japanese patients with bullous pemphigoid (BP) to fusion proteins encoded by BPAG1.
Sera from patients with bullous pemphigoid (BP) from the United States (US), Japan, and Britain demonstrate similar reactivity to the major target antigens BPAG1 and BPAG2. The purpose of the present study was to determine if the epitope specificity of circulating autoantibodies in patients with BP from the US and Japan is similar as mapped by binding to fusion proteins encoded by BPAG1. Sera from patients and controls with BP from the US and Japan were assayed for reactivity to intact BPAG1 and BPAG2 by immunoblot, and to fusion proteins encoded by BPAG1 by immunoblot and enzyme-linked immunosorbant assay (ELISA). Significant reactivity to fusion proteins encoded by the carboxyl region (FP 16-8) and coiled-coil region (FP3) was seen in sera from the US and Japanese patients, but not from normal controls from the US or Japan. Sera from US and Japanese patients differed in their response to FP7; namely, the reactivity of sera from US patients but not from Japanese patients to FP7 was significantly different from the reactivity of their respective control sera. The reasons for this difference in reactivity are unknown but may reflect genetic or environmental factors relevant in the generation of an autoantibody response to these target antigens.
Aged↗
Imipenem resistance among Acinetobacter baumannii: association with reduced expression of a 33-36 kDa outer membrane protein.
The mechanism of imipenem resistance of two Acinetobacter baumannii isolates (A-1 and A-24) was characterized in this study. A spontaneous revertant (A-1 (rev)) derived from isolate A-1 showed susceptibility to imipenem. beta-Lactamase hydrolysis studies showed no evidence of an imipenem hydrolyzing enzyme among A-1, A-24, A-1 (rev), or two other imipenem susceptible A. baumannii isolates. Outer membrane protein (OMP) analysis indicated decreased expression of a 33-36 kDa protein by isolates A-1 and A-24 when compared with A-1 (rev) and the other A. baumannii isolates. In conclusion, decreased expression of a 33-36 kDa OMP is associated with imipenem resistance among A. baumannii.
Acinetobacter↗
Neostigmine, atropine, and glycopyrrolate: does neostigmine cross the placenta?
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Adjuvants, Anesthesia↗
Stable activation and desensitization of beta 2-adrenergic receptor stimulation of adenylyl cyclase by salmeterol: evidence for quasi-irreversible binding to an exosite.
The relaxation of tracheal smooth muscle by the beta 2-adrenergic receptor (beta AR) agonist salmeterol displays several unusual properties: (i) slow onset of action (t1/2 = 5-15 min), (ii) prolonged activation (t1/2 = 8-14 hr), and (iii) the ability to recover from beta AR blockade. These properties led to the hypothesis that salmeterol binds with very high affinity to an exosite in addition to the beta AR activating site. Despite extensive characterization of salmeterol-induced bronchodilation, little is known about the molecular actions of salmeterol. We report the unique properties of salmeterol binding to the beta AR, activation of adenylyl cyclase, and desensitization of the hamster beta AR expressed in L cells. First, we found that salmeterol activation of adenylyl cyclase, although rapid and potent (low EC50 relative to epinephrine), was nevertheless remarkably inefficient relative to the full agonist epinephrine. Reduced coupling efficiency of salmeterol was demonstrated using formulations recently introduced by our group. Second, we found that pretreatment of L cells with salmeterol led to a stable activation of adenylyl cyclase that survives extensive wash procedures and sucrose step gradient purification of plasma membrane fractions. This activation of basal adenylyl cyclase did not require salmeterol binding to the classic active site during pretreatment, as it occurred in the presence of an excess of a beta AR antagonist. Third, we found that the rapid phase of salmeterol-induced desensitization was much reduced relative to epinephrine, consistent with its poor coupling efficiency and with its prolonged activation of adenylyl cyclase. These unique properties of salmeterol support the proposal that it binds reversibly to the activating or active site and as well to an extremely high affinity exosite from which it has access to the active site.
Adenylyl Cyclases↗
Repetitive endocytosis and recycling of the beta 2-adrenergic receptor during agonist-induced steady state redistribution.
The human beta 2-adrenergic receptor (beta 2AR) rapidly internalizes after binding agonist, resulting in a dramatic redistribution of receptors from the plasma membrane and into endocytic vesicles. We sought to determine whether intracellular receptors constitute a static pool or represent a fraction of dynamically internalizing and recycling receptors. Using cells expressing a beta 2AR with an epitope tag at its amino-terminal ectodomain, changes in surface receptor levels were measured by flow cytometry and radioligand binding assays. The addition of a saturating level of a strong agonist (isoproterenol) caused the endocytosis of receptors with first-order kinetics (ke for naive cells, 0.222 min-1). After 10 min, the level of surface receptors remained stable at approximately 20% that of untreated cells, even though endocytosis continued with similar kinetics (ke for pretreated cells, 0.258 min-1), suggesting that internalized receptors were cycling in steady state with surface receptors. This prediction was confirmed directly by showing that internalized beta 2ARs recycled to the cell surface in the continued presence of agonist. The calculated transit times (1/k) in the presence of isoproterenol were 3.9 min for endocytosis and 11.2 min for recycling. The endocytic rate constant and the steady state redistribution to the internal pool were much lower after treatment with the partial agonist albuterol, suggesting a correlation between the efficiency of endocytosis and that of receptor coupling to the downstream signal transduction pathway. These findings indicate that in the presence of agonist, beta 2ARs are in a dynamic steady state between the plasma membrane and endosomes that is regulated principally by agonist efficacy.
Adrenergic beta-Agonists↗
Presence of T cells with activated and memory phenotypes in inflammatory spinal cord lesions.
The phenotypic and functional characteristics of activated T cells and recruited unactivated T cells at an inflammatory site were examined using a V beta 4+ myelin basic protein-specific T cell clone in a passively transferred model of experimental allergic encephalomyelitis. A high percentage of the T cells isolated from the central nervous system (CNS) were V beta 4+. This population exhibited the characteristics of activated T cells based on the proportion of cells in the blast state, their ability to proliferate in response to IL-2 or CNS Ag, and their expression of activation/memory cell markers. Activated V beta 4+ T cells were also observed in the periphery. Large numbers of V beta 4- T cells, which are entirely host-recruited, were also found in the CNS, where they demonstrated the properties of memory cells. There were differences in adhesion molecule expression between CNS V beta 4+ T cells and peripheral V beta 4+ T cells, although both populations were in activated state. V beta 4+ T cells at the site of Ag expression (the spinal cord) demonstrated higher levels of LFA-1 and CD44, but lower levels of VLA-4 and intercellular adhesion molecule-1, than did V beta 4+ T cells in the spleen. In contrast, the levels of all of these adhesion molecules on recruited V beta 4- T cells were higher in the CNS than in the periphery. This experimental model allows the detailed characterization of different T cell populations isolated from the same inflammatory site.
Animals↗
Effects of intracellular calcium on sodium current density in cultured neonatal rat cardiac myocytes.
1. Na+ channel mRNA levels in the heart can be modulated by changes in intracellular Ca2+ ([Ca2+]i). We have investigated whether this regulation of Na+ channel biosynthesis by cytosolic Ca2+ translates into functional Na+ channels that can be detected electrophysiologically. 2. Whole-cell Na+ currents (INa) were recorded using patch-clamp techniques from single ventricular myocytes isolated from neonatal rats and maintained in tissue culture for 24 h. Na+ current density, measured at a membrane potential of -10 mV, was significantly decreased in the cells which were exposed for 24 h to culture medium containing 10 mM of both external Ca2+ and K+ in order to raise [Ca2+]i compared with control cells which were maintained in culture medium containing 2 and 5 mM of Ca2+ and K+, respectively. In contrast, Na+ current density (at -10 mV) was significantly increased in cells exposed for 24 h to 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetraacetoxymethyl ester (BAPTA AM; a cell membrane-permeable Ca2+ chelator) which lowered the average [Ca2+]i compared with control. 3. Changes in current density were not associated with changes in the voltage dependence of activation and inactivation of INa. There were no changes in single-channel conductances. 4. It is concluded that Na+ current density in neonatal rat cardiac myocytes is modulated by [Ca2+]i. The findings suggest that the differences in current density are attributable to a change in Na+ channel numbers rather than to changes in single-channel conductance or gating. These changes are consistent with the previously documented modulation of Na+ channel biosynthesis by cytosolic Ca2+.
Animals↗
Quinidine-induced open channel block of K+ current in rat ventricle.
1. The effects of quinidine on calcium-independent outward K+ currents in rat ventricular myocytes were studied using whole-cell patch clamp techniques. 2. Quinidine sulphate (6 microM) significantly prolonged repolarization of the ventricular action potential. This effect was larger during early repolarization (25% level) than at later times (90% level). 3. Quinidine reduced the amplitude of a transient outward current, and accelerated its rate of decay by approximately 4 fold at membrane potentials between 0 to +50 mV. Quinidine also reduced the amplitude of a slowly inactivating, tetraethylammonium-sensitive 'pedestal' component of the outward current. 4. The quinidine-induced block of the transient outward current was dependent on time and membrane potential. Maximal block occurred with depolarizations of about 100 ms duration, and longer depolarizations (up to 1.5 s) produced little additional block. The membrane potential dependence of quinidine-induced block was very similar to the membrane potential dependence of activation of the transient outward current. The membrane potential dependence of steady-state inactivation of the transient outward current was not significantly affected by quinidine. 5. These results show that quinidine blocks outward K+ currents in rat ventricular cells. The time and potential dependence of this block suggests that quinidine blocks the transient outward K+ current by acting primarily on the open state of these channels.
Action Potentials↗
Effects of action potential duration on excitation-contraction coupling in rat ventricular myocytes. Action potential voltage-clamp measurements.
Although each of the fundamental processes involved in excitation-contraction coupling in mammalian heart has been identified, many quantitative details remain unclear. The initial goal of our experiments was to measure both the transmembrane Ca2+ current, which triggers contraction, and the Ca2+ extrusion due to Na(+)-Ca2+ exchange in a single ventricular myocyte. An action potential waveform was used as the command for the voltage-clamp circuit, and the membrane potential, membrane current, [Ca2+]i, and contraction (unloaded cell shortening) were monitored simultaneously. Ca(2+)-dependent membrane current during an action potential consists of two components: (1) Ca2+ influx through L-type Ca2+ channels (ICa-L) during the plateau of the action potential and (2) a slow inward tail current that develops during repolarization negative to approximately -25 mV and continues during diastole. This slow inward tail current can be abolished completely by replacement of extracellular Na+ with Li+, suggesting that it is due to electrogenic Na(+)-Ca2+ exchange. In agreement with this, the net charge movement corresponding to the inward component of the Ca(2+)-dependent current (ICa-L) was approximately twice that during the slow inward tail current, a finding that is predicted by a scheme in which the Ca2+ that enters during ICa is extruded during diastole by a 3 Na(+)-1 Ca2+ electrogenic exchanger. Action potential duration is known to be a significant inotropic variable, but the quantitative relation between changes in Ca2+ current, action potential duration, and developed tension has not been described in a single myocyte. We used the action potential voltage-clamp technique on ventricular myocytes loaded with indo 1 or rhod 2, both Ca2+ indicators, to study the relation between action potential duration, ICa-L, and cell shortening (inotropic effect). A rapid change from a "short" to a "long" action potential command waveform resulted in an immediate decrease in peak ICa-L and a marked slowing of its decline (inactivation). Prolongation of the action potential also resulted in slowly developing increases in the magnitude of Ca2+ transients (145 +/- 2%) and unloaded cell shortening (4.0 +/- 0.4 to 7.6 +/- 0.4 microns). The time-dependent nature of these effects suggests that a change in Ca2+ content (loading) of the sarcoplasmic reticulum is responsible. Measurement of [Ca2+]i by use of rhod 2 showed that changes in the rate of rise of the [Ca2+]i transient (which in rat ventricle is due to the rate of Ca2+ release from the sarcoplasmic reticulum) were closely correlated with changes in the magnitude and the time course of ICa-L.(ABSTRACT TRUNCATED AT 400 WORDS)
Action Potentials↗
Histochemical localization of nitric oxide synthase in the bullfrog intracardiac ganglion.
Histochemical and immunohistological methods have been used to demonstrate the presence of nitric oxide synthase (NOS) in the intracardiac parasympathetic ganglion of bullfrog heart. Both NADPH-diaphorase staining and indirect immunofluorescence with polyclonal antibodies raised against the C-terminal regions of a cloned NOS from rat cerebellum show that the postganglionic, parasympathetic neurons in the intracardiac ganglion contain NOS. This suggests that nitric oxide may have a role in neuronal activity in frog heart.
Amino Acid Oxidoreductases↗
cAMP-dependent protein kinase and protein kinase C consensus site mutations of the beta-adrenergic receptor. Effect on desensitization and stimulation of adenylylcyclase.
Activation of cAMP-dependent protein kinase (cAPK) or protein kinase C (PKC) causes a rapid desensitization of beta 2-adrenergic receptor (beta AR) stimulation of adenylylcyclase in L cells, which previous studies suggest involves the cAPK/PKC consensus phosphorylation site in the third intracellular loop of the beta AR, RRSSK263. To determine the role of the individual serines in the cAPK- and PKC-mediated desensitizations, wild type (WT) and mutant beta ARs containing the substitutions, Ser261-->Ala, Ser262-->Ala, Ser262-->Asp, and Ser261/262-->Ala, were constructed and stably transfected into L cells. Results showed that serine 262 was the primary site of the cAPK-induced desensitization, whereas either serine 261 or serine 262 was sufficient to confer the 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA)/PKC-mediated desensitization. Coincident stimulation of cAPK and PKC caused an additive desensitization (6-8-fold increase in the EC50) which was significantly reduced (80%) only by the double substitution mutation. Quantitative evaluation of the coupling efficiencies and the GTP-shift of the WT and mutant receptors demonstrated that only one of the mutants, Ser262-->Ala, was partially uncoupled. The Ser262-->Asp mutation did not significantly uncouple, demonstrating that introducing a negative charge did not appear to mimic the desensitized state of the receptor. The beta AR expression level played a critical role in determining the pattern of beta AR desensitization; i.e. while the overall desensitization was unaltered within a large range of beta AR expression level (10-300 fmol/mg), the increase in EC50 and decrease in Vmax were differentially affected by the change in the receptor level.
Adenylyl Cyclases↗
Effects of different test conditions on the susceptibility of Mycobacterium fortuitum and Mycobacterium chelonae to amikacin.
Eighteen rapidly growing mycobacteria were tested for susceptibility to amikacin by six different antibiotic susceptibility procedures to assess method variability and factor variation within a single method. Using amikacin MICs determined by the microdilution method as the reference standard, results for Mycobacterium chelonae were on average eight-fold higher by the macrodilution method and two-fold higher by the BACTEC, 1% standard proportion, and agar dilution methods. For Mycobacterium fortuitum, macrodilution MICs were on average four-fold higher than microdilution results; however, for this species, agar dilution, the 1% standard proportion method, and the BACTEC method showed good correlation with microdilution testing. The use of different test media and incubation in increased CO2 tension increased amikacin MICs for Mycobacterium chelonae. An inoculum effect was observed with both species, especially when the organism concentration increased from 10(5) cfu/mL to 10(6) cfu/mL for broth testing and 10(5) to 10(6) cfu per spot for agar dilution. These results indicate that different antibiotic susceptibility methods and test conditions markedly influence MICs of amikacin for these rapidly growing mycobacteria.
Amikacin↗