Biomedical subjects
Publications and source records attributed to R B Clark.
A case of fatal Plesiomonas shigelloides septicaemia is reported in a splenectomised patient. One week prior to his illness the patient was exposed to river water that was potentially contaminated with plesiomonads. Autopsy findings indicated that the plesiomonad may have invaded the blood stream through the terminal ileum.
SJL/J mice are highly susceptible to actively induced experimental allergic encephalomyelitis (EAE), whereas B10.S mice are not. Yet both strains share the H-2s major histocompatibility complex (MHC) haplotype. In order to help determine the cellular basis for the disparate susceptibility to EAE, the antigen-specific in vitro proliferative responses of lymph node (LN) T cells from SJL/J and B10.S mice primed with porcine myelin basic protein (MBP) were assessed. The results indicated that SJL/J mice were high responders and B10.S mice were low responders to both porcine and murine MBP, as demonstrated by limiting dilution analyses and cloning efficiency analysis of MBP-reactive T cells. The low response of B10.S mice to MBP was not due to elevated suppressor cell activity or to a discernible defect in antigen-presenting cell activity. Rather, it appeared to be due to a paucity (or defect in function) of high affinity MBP-reactive T cells in B10.S as compared to SJL/J mice. This difference in MBP responsiveness must, by necessity, be linked to non-MHC background genes. Therefore, assuming that the relative number of MBP-reactive T cells parallels that of EAE-effector T cells in SJL/J and B10.S mice (as separate in vivo studies indicate), the present results suggest that differences in the T cell repertoire for the encephalitogenic determinants of MBP may contribute significantly to the observed differences in antigen reactivity, and may relate to differences in susceptibility to EAE.
The 1,4-dihydropyridine (DHP) Ca2+ antagonists and agonists can inhibit a time- and voltage-dependent, but intracellular Ca(2+)-independent transient outward K+ current (It), in myocytes from rabbit atrium. In the presence of 0.3 mM CdCl2, DHPs decreased the peak It slightly and markedly accelerated its apparent rate of inactivation. When the inhibition of It was measured from integrated It records, the 50% inhibitory concentrations (IC50) of nicardipine and BAY K 8644 were 630 nM and 7 microM, respectively, and the IC50 of nicardipine for inhibition of the Ca2+ current (ICa) was only approximately fourfold lower (160 nM). The inhibition of It by nicardipine was not affected by changing holding potential from -55 to -100 mV; in contrast, the inhibitory effect on ICa was significantly reduced by this hyperpolarization. We conclude that the DHP Ca2+ antagonist nicardipine blocks It at similar doses to those that block ICa and that nicardipine blocks this K+ current by mechanism different from that for ICa inhibition. This inhibitory effect on It is shared by other DHP compounds; the rank order for potency of It inhibition is nicardipine greater than benidipine greater than nisoldipine greater than BAY K 8644 greater than nitrendipine greater than nifedipine.
We have previously demonstrated that monooleylphosphatidate (MOPA) and phosphatidate inhibit adenylyl cyclase in cultured fibroblasts. In this study, the specificity of the phospholipid effect was probed by analysis of the effect of phosphonate analogs of these phospholipids on adenylyl cyclase in C6 glioma cells. The MOPA phosphonate analog inhibited adenylyl cyclase, but the comparable phosphonate analog of phosphatidate was ineffective. The IC50 for inhibition of adenylyl cyclase by the MOPA phosphonate analog was similar to that of MOPA, the maximal inhibitions were comparable (approximately 45% inhibition of hormone-stimulated adenylyl cyclase), and the effects of both appeared to be mediated by Gi, because treatment with islet-activating protein reduced the inhibition to 5-10%.
Many hormones have been shown to activate phospholipase C, which results in the hydrolysis of membrane polyphosphoinositides, such as phosphatidylinositol 4,5-bisphosphate (PIP2). Two second messengers are known to be produced by PIP2 hydrolysis, 1,2-diacylglycerol, an endogenous activator of a family of enzymes called protein kinase C (PKCs), and inositol 1,4,5-trisphosphate, which raises free levels of intracellular Ca2+. Treatment of various cells with 4 beta-phorbol 12-myristate 13-acetate (PMA), a specific exogenous activator of PKCs, causes an enhancement or sensitization of adenylyl cyclase activities. This finding prompted us to examine the effects of direct hormonal activation of PIP2 hydrolysis on the sensitization of adenylyl cyclase. Liao et al. [J. Biol. Chem. 265:11273-11284 (1990)] have shown that P2 purinergic receptor agonists such as ATP and muscarinic receptor agonists such as carbachol stimulate PIP2 hydrolysis in L cells expressing the M5 muscarinic acetylcholine receptor. We investigated the effects of these hormones on adenylyl cyclase and contrasted these effects with the sensitizing effects of PMA. We found that ATP pretreatment of two different types of L cells resulted in a rapid 50-150% sensitization of prostaglandin E1-, epinephrine-, and forskolin-stimulated adenylyl cyclase activity, with an EC50 of 3 microM ATP. This effect was qualitatively similar to that caused by 10 nM PMA. The enhancement of adenylyl cyclase activity was associated with an increase in the Vmax for hormonal stimulation and with a lack of significant effects of ATP on the EC50. The effect was completely eliminated when adenylyl cyclase was assayed in the presence of high free Mg2+ levels (10 mM). Down-regulation of PKCs with long term PMA treatment did not affect the ATP-induced sensitization of adenylyl cyclase, although the PMA-induced sensitization of adenylyl cyclase was eliminated. In contrast to the effects of ATP and PMA, treatment of the cells with carbachol alone had no effect on adenylyl cyclase; however, in combination with nanomolar concentrations of PMA, synergism of the sensitization of adenylyl cyclase was observed. These data indicate that the activation of P2 purinergic receptors by ATP, and possibly activation of M5 muscarinic receptors by carbachol, may be important in the signal transduction pathways leading to the increases in the responsiveness of hormone-stimulated adenylyl cyclase.
Uncertainty regarding pathogenic mechanisms has been a major impediment to effective prevention and treatment for human neurologic diseases such as multiple sclerosis, tropical spastic paraparesis, and AIDS demyelinating disease. Here, we implicate lymphotoxin (LT) (tumor necrosis factor beta [TNF-beta]) and TNF-alpha in experimental allergic encephalomyelitis (EAE), a murine model of an autoimmune demyelinating disease. In this communication, we report that treatment of recipient mice with an antibody that neutralizes LT and TNF-alpha prevents transfer of clone-mediated EAE. LNC-8, a myelin basic protein-specific T cell line, produces high levels of LT and TNF-alpha after activation by concanavalin A, antibody to the CD-3 epsilon component of the T cell receptor, or myelin basic protein presented in the context of syngeneic spleen cells. LNC-8 cells transfer clinical signs of EAE. When LNC-8 recipient mice were also treated with TN3.19.12, a monoclonal antibody that neutralizes LT and TNF-alpha, the severity of the transferred EAE was reduced, while control antibodies did not alter the disease. The effect of anti-LT/TNF-alpha treatment was long lived and has been sustained for 5 mo. These findings suggest that LT and TNF-alpha and the T cells that produce them play an important role in EAE.
Recent studies with phorbol esters have suggested that protein kinase C (PKC) may play a role in the regulation of adenylate cyclase in mammalian cells. Since D-sphingosine has been reported to specifically inhibit PKC in many cell types, we evaluated its effects on stimulation of cyclic AMP accumulation by adrenaline in S49 lymphoma cells. We found sphingosine to have multiple non-specific effects which could not be explained by an inhibition of PKC. These effects included: (i) inhibition by sphingosine (50 microM) of adrenaline-stimulated cyclic AMP accumulation and sphingosine permeation of the cells which rendered them leaky to ATP; (iii) sphingosine (20 microMs) augmentation of adrenaline-stimulated cyclic AMP accumulation; (iii) inhibition by sphingosine of adrenaline-stimulated adenylate cyclase in isolated membranes by up to 95%; and (iv) sphingosine (20 microM) inhibition of cellular mechanisms for the elimination of cyclic AMP. These results demonstrate the importance of evaluating the non-specific effects of sphingosine before concluding that its actions are the consequences of a specific inhibition of PKC.
The bactericidal activity of aminoglycosides alone and in combination with various beta-lactams was studied by the time-kill technique against ten Pseudomonas aeruginosa isolates with an unusual antibiogram (amikacin-resistant, gentamicin-resistant, tobramycin-susceptible [ArGrTs]). Previous studies have indicated that ArGrTs isolates are moderately resistant to all aminoglycosides and many are multiply resistant to beta-lactams. Aminoglycoside-beta-lactam combinations showed infrequent synergistic (16%) or enhanced killing (12%) against the ArGrTs isolates. Synergistic activity, when present, was more likely to occur with tobramycin and amikacin than with gentamicin, even though these differences were not statistically significant. Antibiotic resistance patterns were not predictive of synergy or enhanced killing. Systemic infections produced by ArGrTs isolates that are multiply resistant to the beta-lactams may not respond to combination therapy with an aminoglycoside and beta-lactam. Alternative treatment with polymyxin B or a quinolone may be required for these infections.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Cytoplasmic extracts prepared from T cell lines undergoing antigen-specific, interleukin-2 (IL-2)-dependent proliferation were tested for their ability to induce DNA synthesis in isolated, quiescent nuclei. A tetanus toxoid (TET)-specific T cell line, established from peripheral blood of a normal human volunteer, was stimulated in the presence of relevant antigen and 1 unit/ml IL-2. Cytoplasmic extracts prepared from these cells were capable of inducing DNA synthesis in isolated, quiescent nuclei. The ability of cytoplasmic extracts to induce DNA synthesis in isolated, quiescent nuclei. The ability of cytoplasmic extracts to induce DNA synthesis in isolated nuclei correlated positively with the degree of proliferation induced in these cells. In contrast, incubation of this T cell line in the absence of antigen failed to induce proliferation and cytoplasmic extracts prepared from these cells induced little to no DNA synthesis in isolated, quiescent nuclei. The factor present in the cytoplasm of T cells stimulated with relevant antigen in the presence of IL-2 is similar, if not identical, to a factor which we have previously demonstrated in cytoplasmic extracts prepared from transformed lymphoblastoid cell lines and from mitogenically stimulated normal human peripheral blood mononuclear cells. This factor, which we have called activator of DNA replication (ADR) is a heat-labile protein, and is inactivated by treatment with protease inhibitors, including aprotinin. The ability of cytoplasmic extracts from T cells undergoing antigen-specific, IL-2-dependent proliferation to induce DNA synthesis in isolated, quiescent nuclei was markedly inhibited in the presence of aprotinin, providing strong evidence that a cytoplasmic activator of DNA replication, ADR, is involved in the signal transduction process for antigen-specific, IL-2-dependent T cell proliferation. ADR may represent a common intracellular mediator of DNA synthesis in activated and transformed lymphocytes.
1. Single atrial myocytes were enzymatically isolated from the bull-frog as previously described (Hume & Giles, 1981), and patch-clamp techniques were used in an attempt to identify and separate two inwardly rectifying K+ channels in this tissue. 2. Single-channel measurements consistently demonstrated the existence of two different resting K+ channels, which both exhibited strong inward rectification. The unitary conductances of these K+ channels were 34 +/- 4 and 22 +/- 3 pS (mean +/- S.D., at 22-24 degrees C) when measured with 110 mM-K+ in the pipette solution, and their mean open times were 0.87 +/- 0.33 and 129.9 +/- 49.4 ms, respectively. 3. In the absence of acetylcholine (ACh) in the pipette, openings of the larger channels with the shorter open times occurred at a very low frequency. When ACh was present in the patch pipette, the activity of this channel increased significantly, although the single-channel conductance and gating behaviour were very similar either with or without ACh in the pipette. 4. The zero-current voltage (extrapolated from the inward currents through these types of channels) depended on the extracellular K+ concentration. [K+]o, in the fashion expected for a predominantly K(+)-selective channel: it shifted by 58 mV for a tenfold change in [K+]o. Very similar results were obtained from whole-cell voltage-clamp measurements (53 mV for a tenfold change in [K+]o). 5. The conductance of both types of K+ channels depended on [K+]o. The single-channel conductances were 25 +/- 3 and 13 +/- 2 pS with 50 mM [K+]o, and 19 +/- 4 and 9 +/- 2 pS with 20 mM [K+]o, respectively. 6. These results demonstrate that two types of resting inwardly rectifying K+ channels can be identified in single atrial myocytes. One of these is an inwardly rectifying K+ channel (IK1) previously identified in whole-cell voltage-clamp experiments (Hume & Giles, 1983). The second channel is the muscarinic receptor-regulated K+ channel (IK(ACh) which was first described in mammalian nodal and atrial cells. 7. N-Ethylmaleimide (NEM), a reagent which alkylates sulphydryl groups, affects these two types of K+ channels differentially. In the cell-attached patch configuration, bath application of NEM (50 microM) completely abolished the activity of IK(ACh), without affecting the IK1 channel activity. 8. To obtain further evidence that these two currents, IK1 and IK(ACh), were different, the inside-out patch-clamp technique was used.(ABSTRACT TRUNCATED AT 400 WORDS)
1. Whole-cell voltage-clamp techniques were used to study the voltage-dependent membrane conductances in parasympathetic neurones enzymatically isolated from the interatrial septum of bull-frog heart and maintained in short-term (1-10 day) tissue culture. 2. The resting potential of the isolated neurones averaged -55.4 +/- 1.1 mV (+/- S.E.M., n = 11). Action potentials evoked in the isolated cells by brief (1-2 ms) current injections were similar to those recorded from neurones in the 'intact' septum. The amplitude of action potentials of isolated neurones averaged about 113 mV, with a peak depolarization of +32.8 +/- 2.8 mV and after-hyperpolarization of -80.0 +/- 2.8 mV. 3. The pattern of membrane currents recorded using voltage clamp with 'normal' external (containing 110 mM-Na+) and internal (110 mM-K+) solutions consisted of a rapidly activating and inactivating inward current followed by a slower, sustained outward current. 4. The inward components of current were isolated by using an internal solution in which Cs+ and TEA+ (tetrathylammonium) ions replaced K+. Depolarizations from holding potentials of -50 to -70 mV produced inward currents which had an initial transient phase followed by a maintained, or very slowly inactivating, component. The current-voltage relation for the initial transient phase reached a peak at membrane potentials near 0 mV, while the maintained phase, measured, for example, at the end of 50 ms voltage-clamp steps, had its peak near +10 mV. 5. The transient component of inward current was carried primarily by Na+ ions, as replacement of Na+ by TEA+ in the external solution abolished the transient. This current was thus identified as a voltage-dependent Na+ current, INa. The maintained component was greatly attenuated by removing 80-90% of the external Ca2+ ions, and it was abolished by divalent cations such as Cd2+ (0.2-0.4 mM), Ni2+ (0.5 mM) and La3+ (10-100 microM). This maintained component was thus a voltage-dependent calcium current, ICa. 6. About 80% of INa recorded in the presence of low (0.2-0.5 mM) external Ca2+ and 2 microM-LaCl3 was blocked by tetrodotoxin (TTX) with an apparent Kd of about 8 nM. The remaining 20% of INa was resistant to block by 2-10 microM-TTX. However, the 'TTX-resistant' component of INa was blocked by Cd2+ (0.2-0.4 mM). 7. The voltage-dependent calcium current, ICa, measured in saline in which Na+ was replaced by N-methyl-D-glucamine, activated near -40 mV and reached a peak near +10 to +15 mV.(ABSTRACT TRUNCATED AT 400 WORDS)
1. The effects of activation of muscarinic receptors on the voltage-dependent calcium current, ICa, in parasympathetic neurones were examined. 2. Neurones were enzymatically isolated from the interatrial septum of bull-frog (Rana catesbeiana) heart, and were maintained in short-term (1-6 day) tissue culture. ICa was recorded from the cells using whole-cell patch-clamp methods (Clark, Tse & Giles, 1990). 3. External application of 2 nM to 10 microM acetylcholine (ACh) reduced the amplitude and slowed the time course of activation of ICa. These effects were dependent on membrane potential; they were most pronounced at potentials near the peak of the current-voltage relation for ICa (i.e. +10 to +15 mV), whereas at more-negative potentials (i.e. -15 to -25 mV) the effects on both amplitude and time course were relatively small. 4. Atropine (1 microM) completely blocked the action of 1 microM-ACh, indicating that the effects of ACh on ICa were mediated by activation of muscarinic receptors. 5. Other muscarinic agonists, such as carbamylcholine (0.1-10 microM), DL-muscarine (0.1-2.5 microM) and oxotremorine (5 microM), had similar effects on ICa to ACh. 6. A guanine nucleotide-binding protein (G-protein) is involved in this muscarinic inhibition of ICa. Inclusion of the non-hydrolysable guanosine triphosphate analogue guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S; 200 microM) in the intracellular solutions mimicked the effects of ACh, and application of external ACh in the presence of internal GTP-gamma-S produced smaller changes in ICa than in control conditions. Inclusion of another non-hydrolysable analogue, guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S; 0.5-5 mM), blocked the inhibitory effect of ACh on ICa. 7. The G-protein involved in the inhibition of ICa was sensitive to pertussis toxin (islet-activating protein; IAP). The inhibition of ICa by carbamylcholine (5 microM) was reduced by about 90% after incubating cells for 12-15 h in culture medium containing 200 ng/ml IAP. 8. The possible roles of cyclic AMP or cyclic GMP-dependent protein kinases, or protein kinase C, in the muscarinic inhibition of ICa were tested, but these enzymes appear not to be directly involved.
1. The intracellular mechanism(s) underlying the decrease of a transient outward K+ current (It) induced by alpha 1-adrenergic agonists was studied in isolated adult rabbit atrial myocytes using whole-cell voltage clamp and cell-attached patch clamp techniques. Experiments were carried out at 22-23 degrees C. 2. Application of the specific alpha 1-adrenergic agonist, methoxamine, produced a decrease in It which was irreversible after the non-hydrolysable GTP analogues, GTP gamma S and Gpp(NH)p, had been introduced into cells via the recording micropipette. 3. Pre-treatment of cells with 0.1-0.15 microgram/ml pertussis toxin (PT) for 8-9 h at 30-34 degrees C did not prevent the alpha 1-induced decrease in It. Yet, this protocol, as measured by the PT-catalysed incorporation of [32P]ADP-ribose in membrane-associated 40 and 41 kDa proteins, effectively caused the ADP-ribosylation of approximately 70% of the PT-sensitive GTP-binding proteins (i.e. Gi) in these treated cells. After taking into account the proportion of non-viable cells (20-30%), the effectiveness of this treatment probably approaches 100% in the viable myocytes from which electrophysiological recordings were made. 4. Cell-attached patch recordings showed that bath application of methoxamine altered the single-channel events underlying It by decreasing their opening probability. Averaged currents from ensemble single-channel openings recorded in the presence of 0.2 mM-methoxamine outside the patch reproduced the features of alpha 1-adrenergic modulation of the macroscopic It observed during whole-cell voltage clamp measurements. This observation provides evidence for the involvement of a diffusible intracellular second messenger in the alpha 1-adrenergic modulation of It. 5. The protein kinase C (PKC) activators, 4 beta-phorbol 12-myristate 13-acetate (PMA) and 1-oleoyl-2-acetylglycerol (OAG) increased It, when included in the bath perfusate, whereas the inactive analogues, 4 alpha-phorbol and 4 alpha-phorbol 12,13-didecanoate, had no effect on It. 6. Exposure of cells to the PKC inhibitors, staurosporine and H-7, either by bath superfusion or intracellularly, via the recording micropipette, did not block the decrease in It produced by methoxamine. 7. Prolonged stimulation of atrial myocytes for 7-9 h at 22 degrees C with 500 nM-PMA produced a 'down-regulation' of endogenous PKC activity, as well as a physical loss of the immunoreactive enzyme, as measured by an in vitro assay, and an anti-PKC monoclonal antibody, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)
The antibiotic susceptibilities of 29 isolates of Plesiomonas shigelloides were studied with 24 antibiotics and antibiotic-inhibitor combinations. Results indicated that all isolates were susceptible to the cephalosporins, penicillins combined with a beta-lactamase inhibitor, aztreonam, and ciprofloxacin. Most isolates were resistant to the penicillins, possibly via production of a penicillinase.
We report a case of osteomyelitis and synovitis produced by Mycobacterium marinum in the left index finger of a fisherman. A combination of surgical intervention and antimicrobial therapy with minocycline, rifampin, and ethambutol was efficacious.
We have developed a model of cardiac atrial electrical activity based on voltage-clamp measurements obtained from single cells isolated from the bullfrog atrium. These measurements have allowed us to simulate a number of processes thought to be important in action potential initiation, repolarization, and the excitation-contraction (EC) coupling process. In this atrial model, the cell membrane contains both channel-mediated (Na+, Ca2+, inward rectifier K+, delayed rectifier K+, linear background leak) and transporter-mediated (Na(+)-K+ pump, Na(+)-Ca2+ exchanger, Ca2+ pump) currents. The cell is surrounded extracellularly by a diffusion-limited space. The intracellular volume contains Ca2(+)-binding proteins (calmodulin, troponin). The model makes several important predictions. 1) Incomplete inactivation of the Ca2+ current provides an inward current the maintains the plateau of the action potential. 2) Activation of the delayed rectifier K+ current initiates repolarization. 3) Due to Ca2+ buffering by myoplasmic proteins the Na(+)-Ca2+ exchanger current is relatively small and has little influence on repolarization. 4) The Na(+)-K+ pump current does not play a major role in repolarization. 5) K+ accumulation and Ca2+ depletion may occur in the extracellular spaces. 6) Modulation of EC coupling is governed by interactions between the myoplasmic Ca2(+)-binding proteins; specifically, the inotropic "positive staircase effect" may be explained by interactions between Ca2+ and Mg2+ at a competitive binding site on troponin. When considered in conjunction with the results of our model of primary pacemaking in the sinus venosus [Rasmusson et al., Am. J. Physiol. 259 (Heart Circ. Physiol. 28): H352-H369, 1990], this atrial model shows how the presence or absence of certain transmembrane currents can change action potential characteristics and consequently alter the relative influence of the various transporter-mediated and channel-mediated currents.