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P L Fletcher

Publications and source records attributed to P L Fletcher.

18 recordsLinked to original sources
PubMed2001

Synthesis and release of N-acetylaspartylglutamate (NAAG) by crayfish nerve fibers: implications for axon-glia signaling.

Early physiological and pharmacological studies of crayfish and squid giant nerve fibers suggested that glutamate released from the axon during action potential generation initiates metabolic and electrical responses of periaxonal glia. However, more recent investigations in our laboratories suggest that N-acetylaspartylglutamate (NAAG) may be the released agent active at the glial cell membrane. The investigation described in this paper focused on NAAG metabolism and release, and its contribution to the appearance of glutamate extracellularly. Axoplasm and periaxonal glial cell cytoplasm collected from medial giant nerve fibers (MGNFs) incubated with radiolabeled L-glutamate contained radiolabeled glutamate, glutamine, NAAG, aspartate, and GABA. Total radiolabel release was not altered by electrical stimulation of nerve cord loaded with [(14)C]glutamate by bath application or loaded with [(14)C]glutamate, [(3)H]-D-aspartate or [(3)H]NAAG by axonal injection. However, when radiolabeled glutamate was used for bath loading, radiolabel distribution among glutamate and its metabolic products in the superfusate was changed by stimulation. NAAG was the largest fraction, accounting for approximately 50% of the total recovered radiolabel in control conditions. The stimulated increase in radioactive NAAG in the superfusate coincided with its virtual clearance from the medial giant axon (MGA). A small, stimulation-induced increase in radiolabeled glutamate in the superfusate was detected only when a glutamate uptake inhibitor was present. The increase in [(3)H]glutamate in the superfusion solution of nerve incubated with [(3)H]NAAG was reduced when beta-NAAG, a competitive glutamate carboxypeptidase II (GCP II) inhibitor, was present.Overall, these results suggest that glutamate is metabolized to NAAG in the giant axon and its periaxonal glia and that, upon stimulation, NAAG is released from the axon and converted in part to glutamate by GCP II. A quisqualate- and beta-NAAG-sensitive GCP II activity was detected in nerve cord homogenates. These results, together with those in the accompanying paper demonstrating that NAAG can activate a glial electrophysiological response comparable to that initiated by glutamate, implicate NAAG as a probable mediator of interactions between the MGA and its periaxonal glia.

Animals↗
PubMed2001

[Synthesis and release of N-acetylaspartyl glutamate (NAAG) in medial giant axons in crayfish].

Studies of crayfish Medial Giant nerve Fiber suggested that glutamate (GLU) released from the axon during action potential generation initiates metabolic and electrical responses of periaxonal glia. This investigation sought to elucidate the mechanism of GLU appearance extracellularly following axon stimulation. Axoplasm and periaxonal glial sheath from nerve fibers incubated with radiolabelled L-GLU contained radiolabeled GLU, glutamine (GLN), GABA, aspartate (ASP), and NAAG. Total radiolabel release was not altered by electrical stimulation of nerve cord loaded with [14C]-GLU by bath application or loaded with [14C]-GLU, [3H]-D-ASP, or [3H]-NAAG by axonal injection. However, radioactivity distribution among GLU and its metabolic products in the superfusate was changed, with NAAG accounting for the largest fraction. In axons incubated with radiolabeled GLU, the stimulated increase in radioactive NAAG in the superfusate coincided with the virtual clearance of radioactive NAAG from the axon. The increase in [3H]-GLU in the superfusion solution that was seen upon stimulation of nerve bathloaded with [3H]-NAAG was reduced when beta-NAAG, a competitive NAALADase inhibitor, was present. Together, these results suggest that some GLU is metabolized to NAAG in the giant axon and its periaxonal glia and that, upon stimulation, NAAG is released and converted to GLU by NAALADase. A quisqualate-, beta-NAAG-sensitive NAALADase activity was detected in nerve cord homogenates. Stimulation or NAAG administration in the presence of NAALADase inhibitor caused a transient hyperpolarization of the periaxonal glia comparable to that produced by L-GLU. The results implicate N-acetylaspartylglutamate (NAAG) and GLU as potential mediators. of the axon-glia interactions.

Action Potentials↗
PubMed2000

NMR solution structure of butantoxin.

The NMR structure of a new toxin, butantoxin (BuTX), which is present in the venoms of the three Brazilian scorpions Tityus serrulatus, Tityus bahiensis, and Tityus stigmurus, has been investigated. This toxin was shown to reversibly block the Shaker B potassium channels (K(d) approximately 660 nM) and inhibit the proliferation of T-cells and the interleukin-2 production of antigen-stimulated T-helper cells. BuTX is a 40 amino acid basic protein stabilized by the four disulfide bridges: Cys2-Cys5, Cys10-Cys31, Cys16-Cys36, and Cys20-Cys38. The latter three are conserved among all members of the short-chain scorpion toxin family, while the first is unique to BuTX. The three-dimensional structure of BuTX was determined using (1)H-NMR spectroscopy. NOESY, phase sensitive COSY (PH-COSY), and amide hydrogen exchange data were used to generate constraints for molecular modeling calculations. Distance geometry and simulated annealing calculations were performed to generate a family of 49 structures free of constraint violations. The secondary structure of BuTX consists of a short 2(1/2) turn alpha-helix (Glu15-Phe23) and a beta-sheet. The beta-sheet is composed of two well-defined antiparallel strands (Gly29-Met32 and Lys35-Cys38) connected by a type-I' beta-turn (Asn33-Asn34). Residues Cys5-Ala9 form a quasi-third strand of the beta-sheet. The N-terminal C2-C5 disulfide bridge unique to this toxin does not appear to confer stability to the protein.

Amino Acid Sequence↗
PubMed1996

Toxic peptides and genes encoding toxin gamma of the Brazilian scorpions Tityus bahiensis and Tityus stigmurus.

Seven toxic peptides from the venom of Tityus bahiensis and Tityus stigmurus was isolated and sequenced, five of them to completion. The most abundant peptide from each of these two species of scorpion was 95% identical with that of toxin gamma from the venom of Tityus serrulatus. They were consequently named gamma-b and gamma-st respectively. The genes encoding these new gamma-like peptides were cloned and sequenced by utilizing oligonucleotides synthesized according to known cDNA sequences of toxin gamma, and amplified by PCR on templates of DNA purified from both T. bahiensis and T. stigmurus. They contain an intron of approx. 470 bp. Possible mechanisms of processing and expressing these peptides are discussed, in view of the fact that glycine is the first residue of the N-terminal sequence of T. stigmurus, whereas lysine is the residue at position 1 of toxin gamma from T. serrulatus and T. bahiensis. In addition, chemical characterization of the less abundant toxic peptides showed the presence of at least four distinct families of peptides in all three species of the genus Tityus studied. There is a large degree of similarity among peptides from different venoms of the same family. By using specific horse and rabbit antisera, the venoms of T. bahiensis, T. serrulatus and T. stigmurus were compared. They showed an extended degree of cross-reactivity. Thus these three species of scorpion have similar toxic components, the genes of which are similarly organized, processed and expressed.

Amino Acid Sequence↗
PubMed1994

Morphological studies by light and electron microscopy of pancreatic acinar cells under the effect of Tityus serrulatus venom.

We studied in vivo and in vitro morphological aspects of pancreatic acinar cells after treatment with Tityus serrulatus venom (TSV). After three hours in an in vitro system, positive secretagogue effects of the venom were identifiable both at the light-microscopic (LM) and the electron-microscopic (EM) levels. At 1 microgram/ml TSV, maximal secretion (as measured in a concomitant radiolabeling dose-response experiment) of exocrine proteins at 58% was manifest as a discharge of most zymogen granules (ZG) and consequent appearance of secretory material in acinar lumina. At the supramaximal dose of 10 micrograms/ml TSV, exocytotic images were often observed also with secretory contents previously discharged. The lowest dose of venom at 0.01 microgram/ml caused no stimulation of zymogen discharge above resting secretion levels; however, morphological changes were observed. At high doses of TSV, both in vivo and in vitro, large aggregates associated with the cis-Golgi develop between this region and the endoplasmic reticulum (ER). Since Tityus venoms have been associated with causation of pancreatitis, we were interested in comparisons of our experimental tissue with parameters attributed to development of the disease. Our studies have demonstrated considerable evidence that large intracellular vacuoles, discharged ZG, effaced acinar lumina with disappearance of microvilli and other manifestations of possible early events in pancreatitis are indeed frequently observed both in pancreatic lobules in vitro and in whole pancreas in vivo when exposed to TSV.

Animals↗
PubMed1994

Isolation and pharmacological characterization of four novel Na+ channel-blocking toxins from the scorpion Centruroides noxius Hoffmann.

Four novel Na+ channel-blocking toxins (numbered 6 to 9) were purified from the venom of the scorpion Centruroides noxius Hoffmann by gel filtration and high-performance liquid chromatography and their chemical and pharmacological properties were characterized. Amino acid analysis and SDS-PAGE of the pure toxins showed them to be composed of approximately 65 amino acid residues with a molecular mass of approximately 7,500 Da. The amino acid sequences of the newly isolated toxins displayed substantial similarity to those of previously isolated and characterized C. noxius toxins. Toxin 7, the most active toxin from this venom, selectively blocked the whole-cell inward Na+ current (INa) from guinea pig ventricular myocytes without altering the K+ or Ca2+ currents. Using rat brain synaptosomes, the specific binding parameters of 125I-Toxin 7 were determined: KD = 40 pM and Bmax = 1.8 pmol/mg protein. The binding was independent of membrane potential and was displaced by all Na+ channel-toxins from C. noxius venom, with K0.5s ranging from 60 pM to 60 nM. Tityus gamma-toxin, a toxin representative of beta-scorpion toxins, totally displaced 125I-Toxin 7 binding, but AaH II and Lqq V toxins, representative of alpha-scorpion toxins, had no effect. All four C. noxius toxins inhibited [3H]GABA uptake by synaptosomes with IC50 s similar to KD s. The toxin effect was not synergistic with veratridine. From these results, it was concluded that the newly purified toxins exert their effects by binding to Site 4 of the voltage-sensitive Na+ channel and must, therefore, be classified as beta-scorpion toxins.

Amino Acid Sequence↗
PubMed1992

Characteristics of pancreatic exocrine secretion produced by venom from the Brazilian scorpion, Tityus serrulatus.

The influence of venom (TSV) from the Brazilian scorpion, Tityus serrulatus, on exocrine pancreatic secretion was studied in relation to known cholinergic and peptidergic secretagogue activity. Pulse-labeling followed by chase incubation in the presence of secretagogues and various pharmacological agents revealed unique physiological characteristics of TSV in guinea pig pancreatic lobules. Exocytotic discharge of newly synthesized 3H-labeled proteins during a 3-h chase incubation showed a marked increase over basal discharge levels using logarithmic TSV doses of 0.10 to 100 micrograms/ml. This stimulation was comparable to maximal values elicited by carbachol, cholecystokinin-octapeptide (CCK-8) or caerulein and discharge kinetics were similar. TSV-mediated secretion was ATP and calcium dependent and partially inhibited by atropine. Only tetrodotoxin completely blocked TSV stimulation of newly synthesized protein discharge. Both botulinum toxin and curare had no effect on venom stimulation, indicating that TSV interaction with exocrine pancreatic cells occurs postsynaptically. Verapamil, a calcium channel antagonist, produced a moderate inhibition of TSV stimulation. When antagonists to the cholecystokinin (CCK) receptor were incubated with TSV, no change in secretory activity occurred. Therefore, TSV does not bind to CCK receptors and probably operates through its own receptor which may be an ion channel. Additionally, morphological studies in vitro revealed a high level of pancreatic secretory activity as evidenced by dense secretory acinar luminal content, reduction in zymogen granule (ZG) population, and development of exocytotic images.

Amylases↗
PubMed1991

Discharge effect on pancreatic exocrine secretion produced by toxins purified from Tityus serrulatus scorpion venom.

Three toxic polypeptides were purified from the venom of the Brazilian scorpion Tityus serrulatus by means of gel filtration in Sephadex G-50 and ion-exchange chromatography in carboxymethylcellulose. The peptides are basic molecules with molecular weights in the range of 7000 for which the amino acid compositions and sequences were determined. The effect of the purified peptides on pancreatic exocrine secretion in the guinea pig was studied. Biochemical measurements show that the cells are stimulated by these peptides to discharge their zymogen granules. Light and electron microscopic images confirm the biochemical measurements. At the light microscope level, acinar cells show dramatically fewer zymogen granules than in control pancreas with the appearance of large vacuoles and some loss of morphological integrity. Electron micrographs display apical regions devoid of zymogen granules and condensing vacuoles whereas acinar lumina contain crystalline secretory material. The secretory effect observed in vitro is comparable to that of carbamylcholine and that of the peptidergic secretagogue cholecystokinin-pancreozymin.

Amino Acid Sequence↗
PubMed1990

Characterization of the venom from Crotalus molossus nigrescens Gloyd (black tail rattlesnake): isolation of two proteases.

The venom from Crotalus molossus nigrescens contains many activities including: hyde powder azure proteinase; N-benzoyl-arginine-ethyl-ester hydrolase; phospholipase; phosphodiesterase; desoxyribonuclease; fibrinogen coagulase; collagenase, fibrinolytic activity, and hemorrhagic factors. The venom, assayed with amounts of venom up to 50 micrograms protein per assay, does not contain acetylcholinesterase, phosphatase, amylase, ribonuclease, tyrosyl-ester hydrolase or hyaluronidase activities. The venom is lethal to mice with an i.p. LD50 of 2.35 mg/kg mouse. Fractionation of soluble venom by Sephadex G-75 separates at least five families of components. Fractions I-III contains all the enzymes, and fraction V have six small peptides. Further separation of fractions II-III on diethyl-amino-ethyl-cellulose columns at pH 8.0 and 8.3 gave pure proteinase E with a mol. wt of 21,390 and the following N-terminal amino acid sequence; Phe-Ala-Lys-Arg-Tyr-Val-Glx-Leu-Val-Ile-Val-Ala. A thrombin-like enzyme with a mol. wt of 75,000 was also purified from this venom by means of affinity and ion exchange chromatographies.

Amino Acid Sequence↗
PubMed1980

Venom from the snake Bothrops asper Garman. Purification and characterization of three phospholipases A2.

The water-soluble venom of Bothrops asper Garman (San Juan Evangelista, Veracruz, México) showed 15 polypeptide bands on polyacrylamide-gel electrophoresis. This material exhibited phospholipase, hyaluronidase, N-benzoyl-l-arginine ethyl hydrolase, N-benzoyl-l-tyrosine ethyl hydrolase and phosphodiesterase activity, but no alkaline phosphatase or acid phosphatase activity. Fractionation on Sephadex G-75 afforded seven protein fractions, which were apparently less toxic than the whole venom (LD(50)=4.3mug/g mouse wt.). Subsequent separation of the phospholipase-positive fraction (II) on DEAE-cellulose with potassium phosphate buffers (pH7.55) gave several fractions, two being phospholipase-positive (II.6 and II.8). These fractions were further purified on DEAE-cellulose columns with potassium phosphate buffers (pH8.6). Fraction II.8.4 was rechromatographed in the same DEAE-cellulose column, giving a pure protein designated phospholipase 1. The fraction II.6.3 was further separated by gel disc electrophoresis yielding two more pure proteins designated phospholipase 2 and phospholipase 3. Analysis of phospholipids hydrolysed by these enzymes have shown that all three phospholipases belong to type A(2). Amino acid analysis has shown that phospholipase A(2) (type 1) has 97 residues with a calculated mol.wt. of 10978+/-11. Phospholipase A(2) (type 2) has 96 residues with a mol.wt. of 10959+/-11. Phospholipase A(2) (type 3) has 266 residues with 16 half-cystine residues and a calculated mol.wt of 29042+/-31. Automated Edman degradation showed the N-terminal sequence to be: Asx-Leu-Trp-Glx-Phe-Gly-Glx-Met-Met-Ser-Asx-Val- Met-Arg-Lys-Asx-Val-Val-Phe-Lys-Tyr-Leu- for phospholipase A(2) (type 2).

Amino Acids↗
PubMed1979

Purification and characterization of a phospholipase A2 from the venom of the coral snake, Micrurus fulvius microgalbineus (Brown and Smith).

A phospholipase A2 was purified from the Mexican coral snake Micrurus fulvius microgalbieus (Brown and Smith). Gel filtration of the soluble crude venom on Sephadex g-50 resolved five fractions, of which fraction II had 98% of the total phospholipase activity. This fraction was rechromatographed on a CM-cellulose column that resolved eight fractions, four of which had an important phospholipase activity. The first fraction (II-1) was homogeneous by polyacrylamide-gel electrophoresis and displayed a phospholipase specific activity of 920 units/mg of protein. The apparent molecular weight as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was approx. 14000. The amino acid analysis revealed the presence of 119 amino acid residues, with 12 half-cystines. the N-terminal sequence was shown to be Ser-Leu-Leu-Asx-Phe-Lys-Asx-Met-Ile-Glu-Ser-Thr..., which is homologous with that of phospholipases from other snake venoms.

Amino Acid Sequence↗
PubMedDate not supplied

Action of New World scorpion venom and its neurotoxins in secretion.

New World scorpion venom contains protein toxins specific for ion channels in the plasmalemma of excitable cells. The effects were examined of whole venoms from Tityus serrulatus, T. bahiensis and T. stigmurus, and some purified toxins in isolated nerve endings (synaptosomes) and pancreatic acinar cells. Both systems initiated exocytosis in a dose-dependent response to the venom or its bioactive protein toxins. Actions differed, however, such that pancreatic acinar cells required Ca2+ while cerebrocortical synaptosomes responded by a Ca(2+)-dependent mechanism, except in the case of one toxin, IV-5, that elicited a Ca(2+)-independent response. Membrane depolarization caused by scorpion venom toxins was measured via radioisotopic discharge of tetra[3H]phenylphosphonium bromide. The role of protein kinase C in second-messenger coupling in pancreatic acinar cells is favored over ion-exclusive routes characteristic of synaptosomes.

Animals↗