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Biomedical subjects

S Hyslop

Publications and source records attributed to S Hyslop.

At least 37 records · Page 2Linked to original sources

Lonomia obliqua caterpillar spicules trigger human blood coagulation via activation of factor X and prothrombin.

In southern Brazil, envenomation by larvae of the moth Lonomia obliqua (Walker) may result in blood clotting factor depletion, leading to disseminated intravascular coagulation with subsequent haemorrhage and acute renal failure which may prove fatal. We have examined the effect of a crude extract of spicules from these caterpillars on in vitro hemostasis. The extract alone did not aggregate platelets and had no detectable effect on purified fibrinogen, suggesting that extract induces clot formation by triggering activation of the clotting cascade. In agreement with the presence of thrombin-mediated activity, hirudin prevented clot formation. The extract was found to activate both prothrombin and factor X, suggesting that the depletion of blood clotting factors results from the steady activation of factor X and prothrombin. Heating and diisopropylfluorophosphate abolished the procoagulant activity of the extract, indicating that the active component involved is a protein that may belong to the serine protease family of enzymes. The ability of hirudin to inhibit this coagulant activity suggests that this inhibitor could be beneficial in the treatment of patients envenomed by L. obliqua caterpillars.

Animals↗

Non-specific inhibitors of nitric oxide synthase cause myocardial necrosis in the rat.

1. To study the effect of acute nitric oxide (NO) inhibition on the rat heart both in vitro and in vivo, male Wistar rats received a single bolus injection of saline, N omega-nitro-L-arginine methyl ester (L-NAME; 0.5, 1.5, 5.0, 15.0 and 45.0 mg/kg) and D-NAME (45.0 mg/kg). 2. Animals were killed 72 h after the bolus injection of L-NAME and the hearts were removed and studied under light microscopy. In other groups of animals; saline, L-NAME and D-NAME were administered as above and the mean arterial blood pressure (MABP/carotid) was recorded. Furthermore, L-NAME was also administered in the drinking water (20 mg/kg per day) for 72 h and animals were then killed and their hearts evaluated as described above. Hearts of control animals were perfused in vitro and coronary flow was measured following saline, L-NAME (45 micrograms/heart) and D-NAME (45 micrograms/heart). 3. Areas of necrosis were observed in the left ventricle of animals that had received L-NAME at 5.0, 15.0 and 45.0 mg/kg. Also, only doses higher than 1.5 mg/kg caused an important increase in MABP. The frequency and extent of the lesions paralleled the dose of L-NAME administered and no lesions were observed in D-NAME- and saline-treated animals. 4. The oral administration of L-NAME also caused myocardial lesions similar to those described above, but the frequency and extent of these lesions were more discrete compared with those observed following 5.0 mg/kg, i.v., L-NAME. 5. Bolus injection of L-NAME into control rat hearts in vitro resulted in a small and transient fall in coronary flow (17.2 +/- 1.4 and 12.2 +/- 1.2 mL/min before and after L-NAME administration, respectively) within 30 s and this was followed 4.5 min later by a further (11.5 +/- 1.6 mL/min) decrease. The administration of D-NAME to control hearts caused no change in coronary flow. 6. In conclusion, the acute inhibition of NO biosynthesis by L-NAME causes myocardial necrosis. Both high levels of MABP and a small but significant reduction in coronary flow (associated or not) can be responsible for the lesions we found.

Animals↗

The effects of Duvernoy's gland secretion from the xenodontine colubrid Philodryas olfersii on striated muscle and the neuromuscular junction: partial characterization of a neuromuscular fraction.

The effect of Philodryas olfersii Duvernoy's secretion was studied in vivo in mice and chicks as well as in the mouse phrenic nerve-diaphragm and the chick biventer cervicis preparations. The whole secretion (20-40 micrograms/ml) increased the creatine kinase (CK) levels in mice but had no effect on the mouse phrenic nerve-diaphragm preparation. In the chick, the secretion caused head drop and paresia as well as irreversible blockade of the twitch-tension evoked by indirect stimulation in the chick biventer cervicis preparation (50% paralysis in 34.5 +/- 2.7 min, n = 4). The secretion also caused muscle contracture (30% of the maximal twitch-tension generated) after a latency of nearly 9 min. Following fractionation on a Superose 12 FPLC column, the neuromuscular activity was recovered in the high mol. wt fraction (Peak I). At a concentration of 10 micrograms/ml in the chick biventer cervicis preparation, Peak I caused 50% paralysis within 18.5 +/- 3.0 min (n = 4), and evoked a strong contracture (70% of the maximal twitch-tension generated). The contractile responses of the chick preparation to ACh and KCL were partially blocked (90%) by the whole secretion and totally blocked by Peak I. CK release was increased by the whole secretion but not by Peak I. The whole secretion also produced various degrees of muscle cell lysis and extensive widening of the intercellular spaces. The latter showed a loosely arranged membranous network. In general, Peak I caused only minor morphological alterations compared with the whole secretion, although these were still significantly different from those observed in the control preparations. The changes principally involved hypercontraction of the muscle fibers. Based on the above results, we conclude that Peak I contains the factor(s) responsible for the in vitro effects on neuromuscular transmission, whereas the direct myotoxic effect is apparently caused by at least one other component of the Duvernoy's secretion.

Animals↗

Inhibition of carrageenin-induced rat paw oedema by crotapotin, a polypeptide complexed with phospholipase A2.

1. The effect of purified crotapotin, a non-toxic non-enzymatic chaperon protein normally complexed to a phospholipase A2 (PLA2) in South America rattlesnake venom, was studied in the acute inflammatory response induced by carrageenin (1 mg/paw), compound 48/80 (3 micrograms/paw) and 5-hydroxytryptamine (5-HT) (3 micrograms/paw) in the rat hind-paw. The effects of crotapotin on platelet aggregation, mast cell degranulation and eicosanoid release from guinea-pig isolated lung were also investigated. 2. Subplantar co-injection of crotapotin (1 and 10 micrograms/paw) with carrageenin or injection of crotapotin (10 micrograms/paw) into the contralateral paw significantly inhibited the carrageenin-induced oedema. This inhibition was also observed when crotapotin (10-30 micrograms/paw) was administered either intraperitoneally or orally. Subplantar injection of heated crotapotin (15 min at 60 degrees C) failed to inhibit carrageenin-induced oedema. Subplantar injection of crotapotin (10 micrograms/paw) also significantly inhibited the rat paw oedema induced by compound 48/80, but it did not affect 5-HT-induced oedema. 3. In adrenalectomized animals, subplantar injection of crotapotin markedly inhibited the oedema induced by carrageenin. The inhibitory effect of crotapotin was also observed in rats depleted of histamine and 5-HT stores. 4. Crotapotin (30 micrograms/paw) had no effect on either the histamine release induced by compound 48/80 in vitro or on the platelet aggregation induced by both arachidonic acid (1 nM) and platelet activating factor (1 microM) in human platelet-rich plasma. The platelet aggregation and thromboxane B2 (TXB2) release induced by thrombin (100 mu ml-1) in washed human platelets were also not affected by crotapotin. In addition, crotapotin (10 microg/paw) did not affect the release of 6-oxo-prostaglandin Fla, and TXB2 induced by ovalbumin in sensitized guinea-pig isolated lungs.5. Our results indicate that the anti-inflammatory activity of crotapotin is not due to endogenous corticosteroid release or inhibition of cyclo-oxygenase activity. It is possible that crotapotin may interact with extracellular PLA2 generated during the inflammatory process thereby reducing its hydrolytic activity.

6-Ketoprostaglandin F1 alpha↗

Effect of arginine analogues on rat hind paw oedema and mast cell activation in vitro.

Nitro-L-arginine methyl ester (0.15 mumol/paw) significantly reduced both bradykinin- and 5-hydroxytryptamine-induced rat paw oedema. At this dose, L-arginine (L-Arg), D-Arg and nitro-D-arginine methyl ester had no effect on the oedematogenic responses induced by these agents. Nitro-L-arginine methyl ester, nitro-D-arginine methyl ester, L-Arg, D-Arg, L-arginine methyl ester and L-arginine ethyl ester, at the dose of 15 mumol/paw, significantly potentiated both bradykinin- and 5-hydroxytryptamine-induced oedema. This potentiation was not observed in animals treated with both mepyramine and methysergide or in animals chronically treated with compound 48/80. Nitro-L-arginine methyl ester (0.3-3 mM) and L-Arg (0.3-3 mM) released small amounts (< 10%) of histamine from rat peritoneal mast cells when compared to compound 48/80-induced degranulation (> 40%). Histamine release was quantified by radioimmunoassay since nitro-L-arginine methyl ester and L-Arg interfere with the fluorometric assay. The potentiation of paw oedema observed with higher doses of all arginine analogues is caused by in vivo mast cell degranulation and is probably due to the cationic charge of these substances.

Animals↗

Crotoxin induces aggregation of human washed platelets.

Crotoxin, the main toxic component isolated from the venom of the South American rattlesnake Crotalus durissus terrificus, is a reversible protein complex composed of a non-toxic non-enzymatic acidic polypeptide (crotapotin) and a toxic basic phospholipase A2 (PLA2). In this study, we have evaluated the ability of crotoxin to induced aggregation in human washed platelets. Human washed platelet aggregation was monitored in a Payton aggregometer and thromboxane B2 (TXB2) release measured by direct radioimmunoassay (RIA). Crotoxin (15-50 micrograms/ml) produced dose-dependent and irreversible human washed platelet aggregation, which was inhibited by pre-incubation of the platelets with sodium nitroprusside (50-500 microM) or iloprost (8-80 nM). Crotoxin also induced TXB2 release (207 +/- 8 ng/ml, n = 6), and although indomethacin significantly reduced the release of TXB2 (to 23.5 +/- 5 ng/ml, P < 0.001, n = 6), it did not inhibit crotoxin-induced aggregation. Our results clearly demonstrate that crotoxin induces human washed platelet aggregation and that this phenomenon is independent of the formation of pro-aggregatory arachidonic acid metabolites.

Amino Acid Sequence↗

Anti-inflammatory activities of Ph CL28A in rats in vivo.

The anti-inflammatory effects of Ph CL28A, a potentiator of prostacyclin output and inhibitor of leukotriene (LT) synthesis, were assessed in two models of acute inflammation. In paw oedema induced by carrageenan in rats, Ph CL28A (10-100 mg/kg), given i.p. at the same time as the carrageenan, inhibited oedema for up to 4 h. When indomethacin or Ph CL28A was given locally into the paw with carrageenan, indomethacin inhibited oedema formation but Ph CL28A potentiated the oedema for up to 4 h. As Ph CL28A does not inhibit cyclo-oxygenase, its anti-inflammatory effects in this model may reflect its ability to increase prostacyclin output. In pleurisy induced by carrageenan in rats, there were increases in leukocytes, LTB4, thromboxane B2 (TxB2) and 6-oxo-prostaglandin F1 alpha (6-oxo-PGF1 alpha) in the pleural fluid over 3 h. In this model, Ph CL28A (30 mg/kg) given i.p. decreased leukocyte numbers and LTB4 but did not affect TxB2 or 6-oxo-PGF1 alpha. Indomethacin decreased both prostanoids but did not affect leukocyte accumulation. The beneficial effects of Ph CL28A in two different models of acute inflammation suggests that it may have potential as an anti-inflammatory agent.

6-Ketoprostaglandin F1 alpha↗

Inflammatory responses induced by poly-L-arginine in rat lungs in vivo.

The inflammatory responses induced by the synthetic polycation poly-L-arginine injected either into the pleural cavity or into the trachea in rats have been investigated. Poly-L-arginine (4-40 nmol/rat) injected intrapleurally induced exudate formation and leucocyte migration (mainly polymorphonuclear cells). The exudate formation (but not cell migration) was dependent on the molecular weight of the poly-L-arginine used (24 and 115 kD). The poly-L-arginine-induced pleurisy was mainly dependent on activation of mast cells since it was significantly reduced either in rats depleted of their stores of histamine and serotonin or in rats previously treated with the serotonin receptor antagonist methysergide. The polyanions heparin and dermatan sulphate when administered intrapleurally with the polycation markedly reduced the exudate formation. Poly-L-arginine (115 kD, 8.5 nmol/rat) injected intratracheally caused lung oedema, increased leucocyte number and protein content of bronchoalveolar lavage, respiratory insufficiency and 60% mortality in 6 h. Depletion of histamine and serotonin stores or of circulating neutrophils decreased the leucocytes in bronchoalveolar lavage but did not increase survival rate, whereas the polyanion dermatan sulphate prevented the mortality completely. These results suggest that the inflammatory changes caused by poly-L-arginine are dependent on mast cell activation but that the lethality after intratracheal administration is due to electrostatic interactions of the polycation with anionic surfaces present in the pulmonary epithelium.

Animals↗

Effects of Phoneutria nigriventer venom on rabbit vascular smooth muscle.

1. The effects of Phneutria nigriventer venom (PNV) on rabbit vascular smooth muscle have been investigated. De-endothelialized vascular strips were superfused in a cascade system with oxygenated (95% O2 + 5% CO2) Krebs solution at 37 degrees C. 2. Phoneutria nigriventer venom (0.3-30 micrograms) produced dose-dependent and short-lived contractions of both venous (cava, mesenteric and jugular veins) and arterial (pulmonary and mesenteric arteries) tissues. 3. Methysergide (5.0 microM) did not significantly affect PNV-induced contractions in venous tissues (cava and mesenteric veins) or pulmonary artery, indicating that serotonin is not involved in the contraction. This was confirmed when PNV was dialyzed (24-48 h) since the contracting activity was still observed on the above tissues. In addition, the spasmogenic activity induced by dialyzed PNV was greatly reduced by incubating the venom with trypsin. 4. Neither tetrodotoxin (3.0 microM) nor phenoxybenzamine (0.05 microM) significantly affected PNV-induced contractions, suggesting that voltage-dependent sodium channel activation or endogenous catecholamine release from autonomic nerve endings on the vascular walls do not play a role in the response to PNV. 5. Our results demonstrate that PNV contains non-dialyzable components, probably peptides, that are responsible for the contractile activity on rabbit veins and pulmonary artery strips.

Animals↗

Comparison of the physiological effects in rabbits of gaboon viper (Bitis gabonica) venoms from different sources.

The cardiovascular, respiratory and metabolic effects of B. gabonica venoms obtained from specimens originating from Ghana, Togo, Nigeria, Uganda and Tanzania were examined in anaesthetized rabbits. Intravenous injection of all venoms (0.125-2.0 mg/kg) induced hypotension. Nigeria venom was the least potent in this respect. Following doses of all venoms there was a brief bradycardia and a transient increase in respiratory rate and depth. At high doses (greater than or equal to 1.0 mg/kg), all venoms induced severe ST depression and T wave inversion. In addition, Togo venom, and to a lesser extent Tanzania and Ghana venoms, were potent in inducing extrasystoles. None of the venoms produced any significant changes in haematocrit, plasma proteins or arterial blood gas and pH levels. All venoms increased blood glucose and lactate levels by 1.3-2.1 fold and 2.2-4.0 fold respectively while the respiratory quotient remained unchanged. Togo venom was significantly (P less than 0.05) more lethal than the other venoms. The pattern of haemorrhage observed at post-mortem was the same for all venoms with the heart, ureters, adrenals, kidneys, lungs, stomach and intestines being the most affected. When combined on a subspecies basis, the results suggest that there are no significant differences in the physiological effects of venoms representing B. g. rhinoceros (West African gaboon viper) and B. g. gabonica (East African gaboon viper).

Africa, Eastern↗

Acidotic effect of gaboon viper (Bitis gabonica) venom in the urethane-anaesthetized rat.

1. Intravenous venom (4 mg/kg) caused a non-compensated metabolic acidosis. 2. Bicarbonate concentration, base excess, standard base excess and pH all fell dramatically. 3. A respiratory impairment occurred characterized by pulmonary oedema and a fall in arterial pO2. 4. Acidosis occurred soon after venom when pO2 was still normal, indicating that changes in tissue metabolism contributed to the acidosis independently of reduced oxygen availability.

Acid-Base Equilibrium↗

The mechanisms and significance of the coupled release of endothelium-derived relaxing factor (EDRF) and prostacyclin (PGI2) from endothelial cells.

Endothelium-derived relaxing factor (EDRF) and prostacyclin (PGI2) are co-released from endothelial cells by stimuli acting via membrane-bound receptors or via non-receptor mediated mechanisms. The receptor-mediated release of EDRF and PGI2 is calcium-dependent and seems to be under the negative feedback regulation of protein kinase C. Significant interactions between EDRF and PGI2 or between their respective second messengers within the endothelial cell have not yet been conclusively demonstrated. Furthermore, although EDRF and PGI2 synergize in the inhibition of platelet aggregation, there is little evidence for such synergism in smooth muscle relaxation. These observations indicate that EDRF and PGI2 release may be coupled primarily by their requirement for raised intracellular calcium levels and by their regulation through protein kinase C.

Animals↗

Acid-base, plasma lactate and glucose changes in the rabbit following administration of Gaboon viper (Bitis gabonica) venom.

The acid-base and metabolic effects of Bitis gabonica venom administered intravenously to the anaesthetised rabbit were studied. Doubling doses of venom from 0.125 mg/kg to 1.0 mg/kg were used. Venom caused progressive and significant increases in plasma glucose and plasma lactate levels although oxygen consumption only became significantly lower after the fourth dose. Standard base excess (SBE) became significantly more negative after the third dose of venom and the fall in pH became significant at the same point. The results indicate that venom induces a metabolic acidosis in the rabbit and because the acidosis occurs in the absence of any fall in arterial PO2, it cannot be considered a consequence of impaired pulmonary ventilation. The reduction in oxygen uptake is likely to occur at a cellular level with a shift from aerobic to anaerobic metabolism hence the increase in plasma lactate levels. However, the magnitude of the acidosis is unlikely to be the principal cause of death under experimental conditions.

Acid-Base Equilibrium↗

Isolation and characterization of a fibrinogen-clotting enzyme from venom of the snake, Lachesis muta muta (Peruvian bushmaster).

A fibrinogen-clotting enzyme from the venom of the Peruvian bushmaster snake was purified to homogeneity by gel filtration on Sephadex G-100 followed by DEAE-cellulose ion-exchange chromatography using a linear ionic strength gradient with NaCl. The specific activity of the enzyme was 866 NIH U/mg, representing a 55-fold purification, with a recovery of 45%. The amino acid composition was Asx30, Thr14, Ser15, Glx33, Pro23, Gly22, Ala15, Val22, Cys18, Met3, Ile18, Leu23, Tyr2, Phe13, His8, Lys11, Arg11. The total carbohydrate content was 13.4%, comprised of 3.4% hexose, 8.7% hexosamine and 1.3% sialic acid. The enzyme was active against the synthetic amide substrate alpha-N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) and against the ester substrates alpha-N-benzoyl-L-arginine ethyl ester (BAEE) and tosyl-L-arginine methyl ester (TAME). Kinetic parameters for TAME esterolysis were: Vmax, 135 mumoles/min/mg and Km, 2.5 x 10(-4) M. The pH optimum was 8.0. Vmax for BAPNA amidolysis was 0.363 mumoles/min/mg and Km, 7.5 x 10(-5) M. Enzyme activity was reduced by diethylpyrocarbonate and by photo-oxidation, suggesting that the enzyme is a serine protease with a histidine residue involved in the active site. The enzyme released fibrinopeptide A rapidly from purified human fibrinogen and fibrinopeptide B more slowly. Factor XIII was not activated and the clotting activity was not inhibited by heparin. A dose of 50 micrograms/kg brought about defibrinogenation in anaesthetized rats but rabbits were unaffected. A dose of 80 micrograms/kg defibrinogenated conscious rats after 5 hr. There were no hypotensive or haemorrhagic effects.

Amino Acids↗

A clinico-epidemiological study of bites by spiders of the genus Phoneutria.

From January, 1984 to December, 1996, 422 patients (ages 9 m-99 y, median 29 y) were admitted after being bitten by spiders which were brought and identified as Phoneutria spp. Most of the bites occurred at March and April months (29.2%), in the houses (54.5%), during the day (76.5%), and in the limbs (feet 40.9%, hands 34.3%). Upon hospital admission, most patients presented only local complaints, mainly pain (92.1%) and edema (33.1%) and were classified as presenting mild (89.8%), moderate (8.5%) and severe (0.5%) envenomation. Few patients (1.2%) did not present signs of envenomation. Severe accidents were only confirmed in two children (9 m, 3 y). Both developed acute pulmonary edema, and the older died 9 h after the accident. Patients more than 70 year-old had a significantly greater (p<0.05) frequency of moderate envenomations compared to the 10-70-year-old individuals. Proceedings to relief local pain were frequently performed (local anesthesia alone 32.0%, local anesthesia plus analgesics 20.6% and oral analgesics alone 25. 1%). Only 2.3% of the patients (two cases classified as severe and eight as moderate, eight of them in children) were treated with i.v. antiarachnid antivenom. No antivenom early reaction was observed. In conclusion, accidents involving the genus Phoneutria are common in the region of Campinas, with the highest risk groups being children under 10 years of age and adults over 70 years of age. Cases of serious envenomation are rare (0.5%).

Adolescent↗