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L L Simpson

Publications and source records attributed to L L Simpson.

At least 55 records · Page 3Linked to original sources
PubMed1992

Role of protein kinase C in short-term transmission at the mammalian neuromuscular junction.

Neuronal cells grown in culture were exposed to drugs that stimulate protein kinase C (phorbol myristate acetate), inhibit the catalytic site in protein kinase C (H7, staurosporine) or inhibit the regulatory site in protein kinase C (calphostin, sphingosine). In NG-108 and N1E-115 cells, phorbol myristate acetate produced substantial stimulation of protein kinase C activity (0.1 microM produced approximately 75% stimulation). In these same cells, H7 [100% inhibition concentration (IC100) approximately 1 mM] and staurosporine (IC100 approximately 0.2 microM) inhibited the catalytic site in the enzyme, and calphostin (IC80-IC90 approximately 2.0 microM) and sphingosine (IC80-IC90 approximately 1 microM) inhibited the regulatory site in the enzyme. Phorbol myristate acetate, as well as drugs that inhibit the catalytic and regulatory sites in protein kinase C, were tested for their effects on phrenic nerve-hemidiaphragm preparations. At concentrations that stimulated enzyme activity in neuronal cells in culture, phorbol myristate acetate did not augment normal transmission, nor did it restore transmission to preparations bathed in medium with low calcium (0.4-0.6 mM). At concentrations equivalent to the IC80 to IC100 values in neuronal cells in culture, H7, staurosporine, calphostin and sphingosine did not paralyze short-term transmission, nor did they depress transmission in tissues bathed in low calcium. Pretreatment of neuromuscular preparations with phorbol myristate acetate, H7, staurosporine, calphostin or sphingosine did not alter the amount of time necessary for botulinum neurotoxin type A, botulinum neurotoxin type B or tetanus toxin to paralyze transmission. The data indicate that protein kinase C is not required for short-term neuromuscular transmission.(ABSTRACT TRUNCATED AT 250 WORDS)

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Botulinum C2 toxin and steroid production in adrenal Y-1 cells: the role of microfilaments in the toxin-induced increase in steroid release.

Exposure of adrenal Y-1 cells to C2 toxin results in an increase in steroid release that is accompanied by a rounding of the cell. The actions of C2 toxin mimic those of adrenocorticotropin and cholera toxin except that there is no increase in intracellular cyclic AMP content. In the present study we provide evidence that C2 toxin increases steroid output from Y-1 cells through an alteration in the microfilament network of the cell. C2 toxin significantly increased steroid output after 3 hr of exposure. This effect was accompanied by a significant increase in the transport of [3H]cholesterol to the mitochondrial fraction, independent of cholesterol uptake by the cell. The toxin was unable to increase steroid output from cells prerounded in suspension culture. The protease inhibitors benzamidine and phenylmethylsulfonyl fluoride did not attenuate the ability of C2 toxin to alter the morphology of Y-1 cells. A 3-hr exposure to C2 toxin resulted in the ADP-ribosylation of 50 to 60% of the total actin pool. Fluorescein isothiocyanate-labeled phalloidin visualization of the cytoskeleton of toxin-treated cells confirmed that the toxin caused a decrease in the stress fiber network. C2 toxin treatment of a protein kinase A mutant Y-1 cell (Kin 8) resulted in morphological changes and an increase in steroid output that was not different from that observed for wild type Y-1 cells. The data suggest that C2 toxin increases steroid output from adrenal Y-1 cells by a cyclic AMP-independent mechanism that involves the microfilament network of the cell.

Actin Cytoskeleton↗
PubMed1991

Cellular and molecular actions of binary toxins possessing ADP-ribosyltransferase activity.

Clostridial organisms produce a number of binary toxins. Thus far, three complete toxins (botulinum, perfringens and spiroforme) and one incomplete toxin (difficile) have been identified. In the case of complete toxins, there is a heavy chain component (Mr approximately 100,000) that binds to target cells and helps create a docking site for the light chain component (Mr approximately 50,000). The latter is an enzyme that possesses mono(ADP-ribosyl)transferase activity. The toxins appear to proceed through a three step sequence to exert their effects, including a binding step, an internalization step and an intracellular poisoning step. The substrate for the toxins is G-actin. By virtue of ADP-ribosylating monomeric actin, the toxins prevent polymerization as well as promoting depolymerization. The most characteristic cellular effect of the toxins is alteration of the cytoskeleton, which leads directly to changes in cellular morphology and indirectly to changes in cell function (e.g. release of chemical mediators). Binary toxins capable of modifying actin are likely to be useful tools in the study of cell biology.

ADP Ribose Transferases↗
PubMed1991

Tetanus toxin inhibits neurotensin-induced mobilization of cytosolic protein kinase C activity in NG-108 cells.

There is considerable literature on the pathogenesis of tetanus toxin poisoning; however, the mechanism of action and intracellular substrate of this toxin have not been defined. It was demonstrated that the NG-108 neuroblastoma x glioma cell line is a suitable model in which to study the mechanism of tetanus toxin action, from binding of the toxin to inhibition of transmitter release. Further, it has been shown that tetanus toxin pretreatment attenuates the ability of phorbol myristate acetate to mobilize cytosolic protein kinase C (PKC) in this cell line. In the present study a 4-hr tetanus toxin pretreatment (10(-10)-10(-13) M) completely inhibited the mobilization of cytosolic PKC induced by a 30-min exposure to 10 microM neurotensin. Pretreatment with 10(-10) M tetanus toxin for periods as short as 1 hr was sufficient to attenuate the ability of neurotensin to mobilize cytosolic PKC; however, a 30-min pretreatment had no significant effect. At a concentration of 10(-11) M, it was necessary to pretreat the cells for greater than 1 hr to significantly attenuate neurotensin-mobilized PKC activity. The exact role that PKC plays in the secretory process is not yet known; however, these findings suggest that the effect of tetanus toxin on neurotransmitter release is accompanied by an alteration in PKC metabolism in differentiated NG-108 cells.

Animals↗
PubMed1991

Lectins from Triticum vulgaris and Limax flavus are universal antagonists of botulinum neurotoxin and tetanus toxin.

Lectins from Anguilla anguilla, Artocarpus integrifolia, Canavalia ensiformis, Datora stramonium, Glycine max, Limax flavus, Ricinus communis and Triticum vulgaris were tested for their abilities to antagonize the binding of botulinum neurotoxin and tetanus toxin to rat brain membranes and to antagonize the ability of these toxins to block neuromuscular transmission in mouse phrenic nerve-hemidiaphragm preparations. Lectins from Limax flavus and Triticum vulgaris, both of which have affinity for sialic acid, were antagonists of the various serotypes of botulinum neurotoxin and tetanus toxin. When tested against the high affinity binding site for botulinum neurotoxin type B, the lectin from Limax flavus had a Ki of 3.1 x 10(-7) M and the lectin from Triticum vulgaris had a Ki of 3.75 x 10(-7) M. When tested against the high affinity binding site for tetanus toxin, the lectins from Limax flavus and Triticum vulgaris had Ki values of 1.5 x 10(-7) and 1 x 10(-6) M, respectively. In all cases the lectins behaved as competitive antagonists. In reverse experiments, neither botulinum toxin nor tetanus toxin was a very effective antagonist of lectin binding to brain membranes. Studies on isolated neuromuscular preparations showed that the lectin from Triticum vulgaris did not affect transmission at concentrations of 10(-6) to 10(-3) M, but at a concentration of 3 x 10(-5) M the lectin produced highly statistically significant antagonism of the neuromuscular blocking properties of botulinum neurotoxin types A, B, C, D, E and F as well as tetanus toxin. The lectin did not antagonize beta-bungarotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Tetanus toxin and neuronal membranes: the relationship between binding and toxicity.

Tetanus toxin labeled by the Bolton-Hunter technique possesses high specific activity and retains substantial biological activity. This material can be used to characterize tetanus toxin binding to receptors in brain membrane preparations. In experiments aimed at measuring the absorption of labeled toxin, the displacement of labeled toxin by unlabeled toxin and the on-rate and off-rate constants, the data revealed two binding sites. The high affinity site had a Kd of 0.033 to 0.070 nM and a Bmax of 0.26 to 0.4 pmol/mg of protein; the low affinity site had a Kd of 0.89 to 6.9 nM and a Bmax of 1.55 to 3.0 pmol/mg of protein. The binding of tetanus toxin to brain membranes was enhanced greatly by low pH and ionic strength. Similarly to tetanus toxin, botulinum neurotoxin could be labeled by the Bolton-Hunter technique, and its binding to brain membranes was also enhanced by low pH and ionic strength. In studies with a neutralizing monoclonal antibody against tetanus toxin, the antigen-antibody interaction was not significantly altered by media with low ionic strength and pH. On the other hand, the ability of the antibody to block toxin binding to brain membranes was reduced substantially in nonphysiologic media. In a bioassay aimed at determining the effect of pH and tonicity on tissue association by toxin, low pH and ionic strength did not enhance toxicity. The biological activity of tetanus toxin was unaffected and that of botulinum neurotoxin was greatly diminished. The present findings confirm the widely reported observation that low pH and ionic strength promote tissue association by tetanus toxin, but they challenge the premise that this binding is relevant to the normal process of cell poisoning.

Animals↗
PubMed1990

Cardiorespiratory function is altered by picomole injections of 5'-N-ethylcarboxamidoadenosine into the nucleus tractus solitarius of rats.

A limited occipital craniotomy was conducted on urethane-anesthetized, spontaneously breathing rats to expose the caudal medulla in the region of the obex. Microinjections of 5'-N-ethylcarboxamidoadenosine (NECA), an adenosine analog, were made into the medial region of the caudal nucleus tractus solitarius (NTS) at the level of the caudal tip of the area postrema, an area of the NTS in which there is known to be a functional co-existence of cardiovascular and respiratory-related neuronal elements. Cardiorespiratory responses were subsequently recorded for a 60 min test period. Microinjections of NECA, in the dose range of 0.35-350 pmol per rat, produced significant dose-related reductions in respiratory rate which were accompanied by dose-dependent increases in tidal volume and these pronounced effects on respiration persisted throughout the test period. In contrast, the effects of NECA microinjections on cardiovascular parameters in this region of the NTS were bidirectional and elicited considerably more complex responses during the test period. During the initial period (2-5 min) following injection, NECA elicited significant hypotension (at lower doses) and pressor responses (at higher doses) in addition to significant bradycardia (at lower doses) whereas by the end of the 60 min test period, almost all doses of NECA had resulted in hypertension and tachycardia. Multivariate analysis of variance (MANOVA) and correlation statistics indicated that the effects of NECA on blood pressure during the initial 2-5 min were dose-dependent and unlikely related to depression of respiratory frequency. A further examination of the data by MANOVA indicated that the pharmacological effects of NECA during the 60 min test period exhibited a highly significant and specific dose-dependent and time-related response pattern for the respiratory, but not the cardiovascular, parameters. Taken together, these manifold response patterns suggest that the respiratory effects of NECA may be mediated by different intrinsic mechanisms in the NTS than are the cardiovascular effects of NECA. At the end of the 60 min test period following the administration of NECA, the respiratory rate remained profoundly depressed. In view of previous studies showing that microinjections of cyclic AMP analogs, forskolin, isoproterenol and adenosine into the same NTS sites elicit a similar depression of respiration, the results with NECA in the present study further support the notion that cyclic AMP may serve as a second messenger in NTS respiratory control regions and these respiratory depressant effects may be mediated by a single adenosine receptor subtype.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine↗
PubMed1990

Tetanus toxin attenuates the ability of phorbol myristate acetate to mobilize cytosolic protein kinase C in NG-108 cells.

Although the pathology of tetanus toxin poisoning has been linked to an inhibition of neurotransmitter release, the mechanism of this inhibition is unknown. The neuroblastoma x glioma hybrid cell NG-108 is an emerging model in which to study the biochemical effect of tetanus toxin on acetylcholine secretion. In differentiated as well as undifferentiated NG-108 cells, a 4 hr tetanus toxin (10(-8) M) pretreatment had no effect on basal levels of cyclic AMP or cyclic GMP. In addition, toxin pretreatment did not affect agonist induced increases in either cyclic nucleotide. Treatment of NG-108 cells for 4 hr with 10(-10) M tetanus toxin had no effect on the subsequently measured activity of cytosolic protein kinase C. However, a 4 hr pretreatment of undifferentiated or differentiated cells with tetanus toxin (10(-8) or 10(-10) M respectively) significantly attenuated the ability of phorbol myristate acetate to mobilize cytosolic protein kinase C. Direct addition of tetanus toxin (10(-7)-10(-10) M) to isolated protein kinase C did not alter the ability of the enzyme to phosphorylate histone protein. These results suggest that one manifestation of tetanus toxin poisoning may be a disruption in protein kinase C metabolism.

Acetylcholine↗
PubMed1990

Isolation and characterization of a novel human monoclonal antibody that neutralizes tetanus toxin.

A human monoclonal antibody, designated 53-2-4, has been isolated and characterized in terms of its ability to interact with clostridial neurotoxins. In enzyme-linked immunosorbent assay assays the antibody reacted with native tetanus toxin, tetanus toxoid and the C fragment obtained from the carboxyterminus of the toxin (AA 864-1314). The antibody did not react with the B fragment of tetanus toxin (AA 1-863) or with six serotypes of botulinum neurotoxin (A to F). Both culture supernatant from the clonal line producing the antibody as well as homogeneous protein obtained by affinity purification of the antibody neutralized tetanus toxin. When tested in vivo, the antibody provided complete production against a supralethal injection of toxin; when tested in vitro, the antibody produced at least 99% inactivation of a 1 x 10(-9) M solution of toxin. The exceptional neutralizing activity of the antibody was attributed to its high affinity for the toxin (4.2 x 10(-10) mol/liter). Animal experiments revealed a novel phenomenon that has been labeled delayed intoxication. At the appropriate ratio of antibody to antigen, the toxin was retained in the host in a latent form. After several days the biological activity of the toxin became apparent and there was onset of nervous system poisoning. Isolated tissue experiments showed that each antibody molecule is capable of associating with two antigen molecules. The antibody has greater neutralizing activity when mixed with free toxin than when mixed with toxin already bound to plasma membrane receptors.

Animals↗
PubMed1990

Use of monoclonal antibodies as probes for the structure and biological activity of botulinum neurotoxin.

Experiments were done to help clarify the structure-function relationships that govern the interaction between botulinum neurotoxin and the cholinergic neuromuscular junction. Work was done with type E toxin in three different states: 1) unactivated (post-translational product before proteolytic processing), 2) activated (proteolytically modified product) and 3) denatured. Four different monoclonal antibodies were studied (E3, E14, E17 and E32), three of which were capable of diminishing the potency of the toxin. All four antibodies had approximately equivalent affinity for the unactivated and the activated forms of the toxin. Monoclonals E17 and E32 had little ability to interact with denatured toxin, suggesting they recognized conformational epitopes; monoclonals E3 and E14 retained partial ability to bind to denatured toxin, suggesting they recognized both conformational and linear determinants. When phrenic nerve-hemidiaphragm preparations were exposed to toxin under conditions that allowed binding but retarded internalization, the toxin remained accessible to antibodies. However, when tissues were stimulated in an effort to promote endocytosis, the toxin disappeared from accessibility to antibodies. The data indicate that various antigenic domains remain exposed after binding and suggest that certain parts of the toxin molecule undergo little or no conformational change during binding. The data further indicate that the molecular domains recognized by E14, E17 and E32 are internalized simultaneously.

Animals↗
PubMed1990

The study of clostridial and related toxins. The search for unique mechanisms and common denominators.

Experiments have been conducted that deal with the structure and biological activity of clostridial toxins. Studies have dealt mainly with botulinum neurotoxin, but work has also been done with tetanus toxin and with the binary toxin. Structural studies indicate that proteolytic processing of botulinum neurotoxin induces two major outcomes: activation and aging. The first is associated with a marked increase in toxicity and with conversion from a single chain to a dichain structure. The second is associated with nominal changes in toxicity and with molecular rearrangements in the dichain structure. Immunological studies have resulted in isolation and characterization of a monoclonal antibody that neutralizes tetanus toxin. Monoclonal antibodies have also been raised against botulinum neurotoxin, and these antibodies have been used to demonstrate that: i) activation is not due to marked conformational changes in the relevant epitopes, ii) binding of the toxin to cholinergic nerve endings does not produce detectable conformational changes, and iii) all functional domains of the toxin appear to be internalized simultaneously. Immunological studies done in vivo and in vitro suggest that certain antibodies may enter cholinergic nerves and neutralize subsequently internalized toxin. Additional work on clostridial toxins has produced the following results: i) the ligand binding assay typically used with tetanus toxin (i.e., low pH and ionic strength) is of questionable biological significance, ii) the binary toxin, like the clostridial neurotoxins, enters cells by receptor-mediated endocytosis, and iii) tetanus toxin can alter the disposition of protein kinase C in one neuroblastoma cell line.

Amino Acid Sequence↗
PubMed1989

Cardiorespiratory effects of inositol hexakisphosphate following microinjections into the nucleus tractus solitarii.

Microinjections of inositol hexakisphosphate (IP6), a metabolite of inositol recently found to occur in high concentrations in the brainstem, were made into the caudal portion of the nucleus tractus solitarii (NTS) of spontaneously breathing rats and cardiorespiratory parameters recorded for a 30 min test period. Microinjections of IP6, in the dose range of 100-500 pmol/rat, produced significant dose-related reductions in mean arterial blood pressure and respiratory rate. The onset for hypotensive action and respiratory depression following microinjections of IP6 was very rapid and a transient apnea could be elicited at the higher doses. Moreover, the sodium and calcium salts of IP6 were relatively equipotent in depressing cardiorespiratory parameters, with the exception of heart rate wherein the sodium salt elicited a much more pronounced bradycardia. These results confirm and extend the findings of a previous study suggesting that IP6 and closely related metabolites may act on extracellular receptors. Taken together, these data provide further support to the notion that inositol lipid signalling pathways may generate both intracellular and extracellular signals in the brain.

Animals↗
PubMed1989

Temporal relationships of neuropathologic conditions caused by perinatal asphyxia.

The neuropathologic conditions in 120 perinatal deaths attributed to fetal or newborn asphyxia were examined. Central nervous system necrosis was present in 16 of these deaths. The approximate time of asphyxial insult was established by determining the duration of the process, based on the findings of neuronal necrosis, macrophage response, or an astrocyte response, in conjunction with clinical data. The time of the asphyxial insult for the 16 perinatal deaths was as follows: antepartum fetal asphyxia, two cases; antepartum-intrapartum fetal asphyxia, five cases; intrapartum fetal asphyxia, four cases; and neonatal asphyxia, five cases. These observations indicate that an asphyxial insult may occur in the antepartum period, in the prodromal period of preterm labor, in the intrapartum period, and in the neonatal period. Five to ten percent of the asphyxial insults in each reproductive time period were initially sublethal, allowing necrosis of the brain of the fetus or newborn to develop.

Asphyxia Neonatorum↗
PubMed1989

Production by Clostridium spiroforme of an iotalike toxin that possesses mono(ADP-ribosyl)transferase activity: identification of a novel class of ADP-ribosyltransferases.

Clostridium spiroforme iotalike toxin produced time- and concentration-dependent incorporation of ADP-ribose into homo-poly-L-arginine. Polyasparagine, polyglutamic acid, polylysine, and agmatine were poor substrates. Enzyme activity was associated with the light-chain polypeptide of the toxin. The heavy chain did not possess ADP-ribosyltransferase activity, nor did it enhance or inhibit activity of the light chain. In broken-cell assays, the toxin acted mainly on G-actin, rather than F-actin. A single ADP-ribose group was transferred to each substrate molecule (G-actin). The enzyme was heat sensitive, had a pH optimum in the range of 7 to 8, was inhibited by high concentrations of nicotinamide, and was reversibly denatured by urea and guanidine. Physiological levels of nucleotides (AMP, ADP, ATP, and ADP-ribose) and cations (Na+, K+, Ca2+, and Mg2+) were not very active as enzyme inhibitors. The toxin was structurally and functionally similar to Clostridium botulinum type C2 toxin and Clostridium perfringens iota toxin. When combined with previous findings, the data suggest that a new class of mono(ADP-ribosyl)ating toxins has been found and that these agents belong to a related and possibly homologous series of binary toxins.

ADP Ribose Transferases↗
PubMed1989

Pharmacologic experiments on the interaction between crotoxin and the mammalian neuromuscular junction.

Crotoxin and its two subunits were tested for their neuromuscular blocking activity on the phrenic nerve-hemidiaphragm preparation. Two types of experimental paradigms were used, the first of which separated the toxin binding step from subsequent events in paralysis and the second of which did not. In both paradigms the toxin produced concentration-dependent blockade of transmission. However, the results with low concentrations were variable, and in some cases complete neuromuscular blockade did not develop. The isolated acidic and basic subunits possessed little toxicity. In experiments designed to characterize binding, the intact toxin displayed the following properties: 1) the apparent half-time for tissue association was about 22 min; 2) binding was not affected by low temperature, the presence or absence of nerve stimulation and the substitution of strontium for calcium; and 3) when binding was allowed to go to completion, reversibility was negligible. Pretreatment of tissues with the isolated subunits of crotoxin did not enhance or inhibit the binding of the parent molecule. Modification of one histidine residue in the isolated basic subunit, followed by reconstitution with unmodified acidic subunit, generated a molecule that possessed only about 10% of the neurotoxicity of the native toxin. The modified toxin could not be used to antagonize binding of the native toxin. Both polyclonal and monoclonal antibodies were generated that neutralized the biologic activity of crotoxin. In experiments that separated the binding step from later events in paralysis, the polyclonal preparation continued to locate and partially neutralize tissue-bound toxin. In experiments that initiated events that follow binding, polyclonal antibodies were progressively less effective with time in neutralizing toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Respiratory effects of cyclic AMP following injection into the nucleus tractus solitarius of rats.

Rats were anesthetized with urethane and a limited occipital craniotomy was performed to expose the caudal medulla in the region of the obex. Injections were made into sites in the brainstem of spontaneously-breathing rats through glass micropipettes. Tidal volume, respiratory frequency, minute volume, blood pressure and heart rate were recorded before and after the administration of 8-bromoadenosine 3',5'-cyclic monophosphate (Br-cAMP), an analog of cyclic AMP. Injections of Br-cAMP into the ventromedial portion of the caudal nucleus tractus solitarius (NTS) produced dose-related decreases in pulmonary ventilation due to effects on both respiratory frequency, as well as minute volume. In larger doses, Br-cAMP produced periodic apnea and irregular breathing. The respiratory depression was accompanied by transient hypotension and bradycardia. The data indicate that cyclic AMP may function as a second messenger in respiratory control regions in the brainstem.

8-Bromo Cyclic Adenosine Monophosphate↗