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Biological activities of Synanceja horrida (stonefish) venom.

Some biological and neurochemical properties of the venom of stonefish (Syanceja horrida) were investigated. The venom exhibited oedema-inducing, haemolytic, hyaluronidase, thrombin-like, alkaline phosphomonoesterase, 5' nucleotidase, acetylcholinesterase, phosphodiesterase, arginine esterase, and arginine amidase activities. Recalcification clotting time, prothrombin, and kaolin-cephalin clotting times were increased 1.7-2.3- and 2.4-fold respectively. The LD50 (i.v. mouse) was 300 micrograms/Kg. Its effects on uptake and stimulation of neurotransmitter synthesis and release were observed in rat brain synaptosomes. In the presence of 100 micrograms venom, uptake of [methyl-3H] choline in rat brain synaptosomes was inhibited 70%, while that of 4-amino-n-[U-14C] butyric acid was inhibited 20%. The toxin also stimulated the release of [3H]-acetylcholine from the synaptosomes.

Animals↗

Description and evaluation of a new chromogenic substrate assay kit for the determination of prekallikrein in human plasma.

Because of an increasing interest in the determination of prekallikrein a kit was made for the determination of this plasma proenzyme. The kit consists of 1) a prekallikrein activator of the cephalin-ellagic acid type containing Factor XII and HMW-kininogen to ensure a total activation of the prekallikrein even in pathological plasmas, 2) a buffer which is optimal for both activation and substrate hydrolysis and 3) the chromogenic substrate S-2302. A control plasma is also included. This kit was evaluated by thirteen research groups as well as by ourselves. Both normal and patient plasmas were analyzed. Good correlations were obtained for prekallikrein levels in plasma samples between the kit method and two other methods (immunochemical and functional). As well as in deficiency states the prekallikrein level was low in pancreatitis (n = 20), cancer (n = 16), early pregnancy with gestosis (n = 15), cirrhosis (n = 9) and cases with thromboembolic disorders (n = 5). The prekallikrein level was high in late pregnancy (n = 4).

Colorimetry↗

The haemolytic effect of phallolysin.

Phallolysin from the toadstool, Amanita phalloides, is a basic protein that causes direct haemolysis of red cells. The dose-response curve is steep; the pH optimum is in the weakly acid range. The rate of haemolysis increases with the concentration of the lysin, the optimal temperature is 20 degrees C. The percentage haemolysis-time curves are S-shaped. Haemolysis is of the non-osmotic type. Ca2+ is not required but inhibits haemolysis in a concentration-dependent fashion, as do Mg2+ and Zn2+. The red cell sensitivity of various animal species decreases in the following sequence:mouse greater than rabbit = guniea pig greater than rat greater than man greater than dog approximately or equal to pig greater than sheep = cattle. Red cells of cattle and sheep are largely resistant. Phallolysin is virtually not consumed on haemalysis: the amount of haemoglobin released increases with the number of red cells applied; on repeated addition of fresh red cells the haemolysate retains its full activity. Phallolysin is not inhibited by serum, albumin, cholesterol, lecithin, cephalin or sphingomyelin; inhibition by red cell ghosts of phallolysin haemolysis is considerably less than that of digitonin haemolysis. At sublytic concentrations phallolysin, unlike benzalkonium chloride, liberates practically no membrane lipids from human red cells. Surface activity of phallolysin does not exceed that of bovine serum albumin.-A saponin-like interaction with cholesterol as the basic mechanism of haemolysis can be disregarded. There is also no evidence suggesting a detergent-like effect.

Amanitins↗

Coagulation abnormalities in diabetic coma before and 24 hours after treatment.

A coagulation screen consisting of measurement of the prothrombin time, thrombin time, kaolin caphalin clotting time, platelet count, plasma fibrinogen level, fibrin degradation products and ethanol gelation test was performed on 24 patients with impairment of consciousness due to acute diabetic metabolic decompensation at the start of treatment and 24 hours later. 22 out of 24 patients showed at least one coagulation abnormality on admission of which the commonest were a prolonged prothrombin time, shortened kaolin cephalin clotting.time and raised plasma fibrinogen level. After 24 hours of treatment these values were more normal but 20 out of 22 patients still displayed some abnormality. 15 patients had two or more coagulation abnormalities on admission including 3 patients with haematological abnormalities suggestive of disseminated intravascular coagulation. This group was older and had higher blood ureas than those with fewer abnormalities, but plasma glucose, sodium, potassium and bicarbonate levels were similar in both groups of patients. All 5 patients with hyperosmolar non-ketotic coma and all 3 patients who died without recovering consciousness had two or more coagulation abnormalities on admission.

Adolescent↗

On two-dimensional passive random walk in lipid bilayers and fluid pathways in biomembranes.

The lateral mobility of pyrene, pyrene decanoic acid, and 1-palmitoyl-2-pyrene decanoyl-phosphatidyl choline (pyrene lecithin) in lipid bilayers is determined by the excimer formation technique. This method is applied to vesicles of lecithins differing in chain length and in the degree of saturation of the hydrocarbon chains. These values are compared with results in cephalins of different chain length and in dipalmitoyl phosphatidic acid at variable pH. The influence of cholesterol is investigated. The results are analyzed in terms of the Montroll model of two-dimensional random walk. The jump frequency of the probe molecule within the lipid lattice is obtained. The advantage of this measure of transport in lipid layers is that it does not involve lipid lattice parameters. The main results of the present work are: (i) The lateral mobility of a given solute molecule in lamellae of saturated lecithins is independent of hydrocarbon chain length and rather a universal function of temperature. (ii) In unsaturated dioleyl lecithin the amphiphatic molecules have lateral mobilities of the same size as in saturated lipids. The jump frequency of pyrene, however, is by a factor of two larger in the unsaturated lecithin. (iii) The jump frequencies in phosphatidyl ethanolamines are about equal to those in lecithins. (iv) In phosphatidic acid layers the hopping frequencies depend on the charges of the head groups of both the lipids and the probes. (v) Cholesterol strongly reduces the jump frequency in fluid layers. (vi) The lateral mobility in biological membranes is comparable to that in artificial lipid bilayers. The experimental results are discussed in terms of the free volume model of diffusion in fluids. Good agreement with the predictions made from this model is found. A striking result is the observation of a tilt in dioleyl-lecithin bilayer membranes from the hopping frequencies of pyrene and pyrene lecithin. A tilt angle of phi = 17 degrees is estimated.

Cholesterol↗

In situ thrombolysis for late occlusion of suprafemoral prosthetic grafts.

Low-dose urokinase was used to obtain in situ thrombolysis in the treatment of 20 late occlusions of suprafemoral prosthetic grafts in 19 patients. Aortofemoral or aortobifemoral bypasses with occlusion of one limb had been performed in nine patients, direct iliofemoral bypasses in six, crossover iliofemoral bypasses in two, and axillofemoral bypasses in three. Sixteen of the grafts were polytetrafluoroethylene and four were Dacron. Vascular surgery had been performed on an average of 3 years earlier. Thrombolysis was inhibited on an average of 3 days after onset of occlusion. The protocol called for penetration of the thrombus with a 5 F catheter inserted through a brachial (12 cases) or femoral (eight cases) route. After initial injection of 2500 units of urokinase a continuous infusion of urokinase was begun at a dose of 2500 units/hr and heparin at 100 units/kg/12 hr. Clinical, biochemical (fibrinogen and activated cephalin time every 6 hours), and arteriographic surveillance was carried out every 12 hours with progressive mobilization of the catheter until complete clearance of the artery. Clearance was achieved in all cases. Anteroposterior and occasionally lateral arteriograms with the hip joint in flexion were obtained. An organic cause amenable to treatment was found in 16 cases, including distal or proximal lesions (two and 10 cases, respectively) and elongation/kinking (four cases). Endoluminal angioplasty, stenting, or endarterectomy (six cases) and conventional procedures (10 cases) were also performed.(ABSTRACT TRUNCATED AT 250 WORDS)

Aorta, Abdominal↗

Composition of cocoa shell fat as related to cocoa butter.

The physical and chemical constants of cocoa shell fat (a by-product resulted during the production of cocoa butter at chocolate factories) were almost identical with those of cocoa butter obtained from the same cocoa beans except for their high acid value. Shell fat contained more amount of phospholipid content (as cephalin) than cocoa butter. The lipid classes were almost the same in cocoa butter and shell fat, however, the latter contained an unidentified constituent which was not found in cocoa butter. The fatty acids were determined quantitatively by GLC, and the results showed that the predominant acids in cocoa butter were palmitic, and oleic. Less amounts of capric, myristic, palmitoleic and linoleic were found in cocoa butter, whereas more amounts of these acids were found in shell fat. Cocoa butter gave higher values of stearic and myristic acids than those of shell fat. Seventeen compounds were detected by GLC in the unsaponifiable matter of both cocoa butter and shell fat from which eight were identified as C30 hydrocarbon, C32 hydrocarbon, squalene, alpha-tocopherol, cholesterol, campsterol, stigmasterol and beta-sitosterol in the two samples. The sterols were determined quantitatively, and it was found that the predominant sterol in cocoa butter and shell fat was B-sitosterol. Cocoa butter contained higher values of stigmasterol than that of shell fat, which contained increasing values of campsterol, low values of cholesterol were found in both samples. Stability of cocoa butter and shell fat towards oxidative rancidity at 100 degrees C was the same (10.5 hrs).

Cacao↗

A new Italian family with severe prekallikrein deficiency. Desmopressin-induced fibrinolysis and coagulation changes in homozygous and heterozygous members.

In this paper we report the case of a new Italian family with severe cross-reacting material prekallikrein deficiency (CRM-). The proposita is a 22-year-old woman referred for evaluating an extremely prolonged activated partial thromboplastin time (APTT) detected during a routine screening. No clearcut bleeding history was reported. Prekallikrein antigen and activity were not measurable. The other contact-phase factors were within the normal range. Using an electromechanical coagulometer, six different commercial reagents yielded a markedly prolonged APTT (ratio greater than 2). By prolonging the incubation time up to 10 min, APTT was normalized only with reagents employing ellagic acid as activator. On the contrary, APTT remained markedly prolonged using particulate activators (i.e. micronized silica and celite). No differences were observed using either rabbit or bovine brain cephalin. APTT was also performed on a laser automated ACL instrument; in this case reagents using ellagic acid yielded only moderately prolonged APTT values (ratio 1.3 vs 1.4). The intrinsic fibrinolytic activity, as assessed by blood activator inventory test, was found to be moderately reduced (about 50% of normal) in the proposita, whereas normal values were measured in the heterozygous relatives. After infusion of 0.3 micrograms/kg 1-desamino-8-D-arginine vasopressin (DDAVP), kallikrein levels did not change in the proposita and her heterozygous relatives. A normal release of tissue-plasminogen activator, as assessed by fibrin-plate assay, was observed in all family members including the proposita.

Adult↗

The prevalence of autoantibodies during third-trimester pregnancy complicated by hypertension or idiopathic fetal growth retardation.

Lupus anticoagulant, anticardiolipin, antinuclear, anti-deoxyribonucleic acid, antithyroglobulin, and antithyroid microsomal antibodies were assayed during third-trimester pregnancy (100 normal, 100 with complications). In spite of a normal activated partial thromboplastin time in all instances, lupus anticoagulant was further investigated by three additional procedures: tissue thromboplastin inhibition time, platelet neutralization procedure, and cephalin neutralization test. The prevalence of autoantibodies in pregnancies with hypertension reaches 16% (four with lupus anticoagulant, two with anticardiolipin, and two with antithyroid microsomal antibodies), which is significantly greater than that for idiopathic fetal growth retardation (2%) (one with lupus anticoagulant antibodies) and normal pregnancies (3%) (two with antithyroglobulin and one with autithyroid microsomal antibodies) (p less than 0.01). Autoantibodies were equally distributed between patients with gestational hypertension and those with preeclampsia. When compared with the 42 patients with hypertension and no autoantibodies, the eight patients with autoantibody had a more frequent history of fetal growth retardation (p less than 0.05), but there was no difference in the severity of hypertension, the frequency of obstetric complications, or the outcome of pregnancy. They did not require any specific treatment.

Autoantibodies↗

Non-isothermal potential of phospholipid bilayer films.

The thermal electrical potential generated in isochemical conditions by a temperature gradient at the two sides of lipid bilayer leaflets is measured. The experimental results agree rather well with the theoretical predictions. The cephaline (from sheep brain) bilayer behaves like a film with zero charge while the phosphatidylcholine (from egg yolk) film performs like a charged membrane. The results presented suggest that the measurement of electrical thermal potential is an interesting method to investigate the electrical behaviour of bilayer membranes.

Ions↗

A phospholipase A2 with anticoagulant activity. II. Inhibition of the phospholiped activity in coagulation.

An anticoagulant factor with phospholipase A2 activity has been isolated from Vipera berus venom. Phospholipase activity was studied on platelet phospholipid and on brain cephalin. The venom factor showed a potent anticoagulant activity: 1 mug impaired the clotting of 1 ml of citrated recalcified platelet-poor plasma. The anticoagulant inhibited clotting by antagonism to phospholipid. The antagonism constant (Kan = 6.8-10(-9) M) demonstrated the high affinity of the inhibitor for phospholipid. As with other phospholipases A2, the venom factor was thermoresistant but very sensitive to photo-oxidation. Both activities (anticoagulant activity and phospholipase activity) were not markedly dissociated by either denaturation or neutralization processes. Slightly different curves of photo-oxidative inactivation of both activities suggested the presence, on the molecule, of two very close sites responsible for phospholipase and anticoagulant activities. The inhibitor effect on coagulation was independent of the hydrolysis process. In fact, lysoderivatives and fatty acids, resulting from complete hydrolysis with the venom factor, were as active as the native phospholipids. Moreover phospholipase A2 from other viperidae venom, which did not have anticoagulant activity, produced similarly active lysoderivatives. This showed that the cleavage of the beta-acyl bond does not interfere with the activity of phospholipid. A possible mechanism of clotting inhibition by the venom factor was proposed. Owing to its high affinity for phospholipid, the inhibitor would complex phospholipid at its protein binding site impairing the normal arrangement of coagulation protein factors and, consequently, their activation. The positive charges of the inhibitor (pI = 9.2) could bind with phosphoryl or carboxyl groups of phospholipid, making them unavailable for protein binding. The complex formation involves a loss of dissociating capacity of the enzyme towards its substrate. This required an additional interaction of the inhibitor with a coagulation protein factor. The inhibitor could be removed from the complex by specific antibodies, permitting recovery of normal phospholipid-protein interaction. The role of calcium in the complex has not yet been elucidated. This venom factor affords a useful tool for investigating the phospholipid-clotting protein interaction.

Anticoagulants↗

Serum phospholipid fractionation after the use of long-acting progestational contraceptives.

The effect of the two long-acting progestational contraceptive injection, depot-medroxyprogesterone acetate (DMPA) and norethisterone onenathate (NET-OEN), on blood phospholipid fractions were studied in eighteen women. The post-injection results showed a significant increase in total serum phospholipids, serum lecithin and serum cephalins. Serum lysolecithin was significantly decreased while serum sphingomyelin showed no significant change. The mechanisms responsible for these phospholipid changes remain somewhat unclear. However deranged liver function may share in its explanation.

Adult↗

Effects of oral contraceptives containing 50 microgram estrogen on blood coagulation in non-Caucasian women.

In one of the first longitudinal studies of 163 non-Caucasian women, comprehensive coagulation, fibrinolysis and platelet function tests were performed before and during 24 months of oral contraceptive pill usage containing 50 microgram estrogen. Results of this ongoing study indicate a state of abnormal coagulability with increase in fibrinogen, prothrombin, Factor V and Factor X. There was also a decrease in antithrombin III level. There was a shortening of overall clotting parameters measured by prothrombin time and Kaolin cephalin time. The significance of these changes in the aetiology of thromboembolism is discussed.

Asian People↗

A comparative study of the biological properties of venoms of some old world vipers (subfamily viperinae).

1. The hemorrhagic, procoagulant, anticoagulant, phosphodiesterase, hyaluronidase, alkaline phosphomonoesterase, 5'-nucleotidase, arginine ester hydrolase, phospholipase A, L-amino acid oxidase and protease activities of 30 samples of venoms from nine species (12 taxa) of the old world vipers (Subfamily Viperinae) including snakes from the genera Bitis, Causus, Cerastes, Echis, Eristicophis and Pseudocerastes, were determined and the Sephadex G-75 gel filtration patterns for some of the venoms were also examined. 2. Examination of the biological properties of the venoms of the Viperinae tested indicates the presence of common venom biological characteristics at the various phylogenic levels. 3. Venoms of most species of the Viperinae examined exhibited characteristic biological properties at the species level, and this allows the differentiation of the Viperinae species by differences in their biological properties. 4. Particularly useful for this purpose, are the effects of venom on kaolin-cephalin clotting time of platelet poor rabbit plasma and the Sephadex G-75 gel filtration pattern and arginine ester hydrolase activity of the venom.

Animals↗

Liver microsome phospholipids and cytochrome P.450 concentration in phenobarbital treated rats fed on different diets.

Liver microsomal concentration of cytochrome P.450 is increased in animals which are fed diets rich in polyunsaturated fatty acids. On the other hand, the effects of phenobarbital are more important when the dietary fat is more unsaturated. The unsaturation index in liver microsomal phosphatidylcholines depends on the unsaturation of the dietary fats. The treatment with phenobarbital constantly results in a decrease of the unsaturation index of fatty acids both in lecithins and cephalins. The importance of the liver microsomal cytochrome P.450 increase and the importance of the unsaturation index decrease in liver microsomal lecithins, both promoted by phenobarbital, are in good agreement.

Animals↗

Lupus anticoagulant in pre-eclampsia and intra-uterine growth retardation.

In a prospective study we proposed to identify the relationship between the level of lupus anticoagulant activity in pre-eclampsia and in growth retardation among 88 pregnant women. In 23 women suffering from severe pre-eclampsia (PET), and 22 women with fetal growth retardation, the mean anticardiolipin binding index was significantly higher than 43 controls (P less than 0.01 and P less than 0.005, respectively). The mean kaolin cephalin clotting time (KCCT) was significantly higher in patients with severe PET than in the control group (P less than 0.05). In intra-uterine growth retardation (IUGR), patients with 610-2452 g infants had significantly higher anticardiolipin binding index than those with 2500-3200 g infants (P less than 0.05). The Caesarean section rate was 46% in the patients with intra-uterine growth retardation (IUGR), 43% in the PET patients, and 2% in the control group.

Adult↗

A functional assay for protein C in human plasma.

We describe a functional assay for protein C in human plasma samples based on the ability of activated protein C to prolong the kaolin-cephalin activated partial thromboplastin time of normal plasma. Protein C is separated from its inhibitor by elution of a barium citrate precipitate, and activated by incubation with human alpha-thrombin for one hour. Thrombin is then inhibited by antithrombin III and heparin, heparin neutralized by protamine sulfate, and protein C activity measured in the partial thromboplastin time. 24 normal subjects had a mean protein C level of 94 +/- 12% (SD) of the activity in pooled normal plasma. Seven patients with severe liver disease had a mean protein C of 28%. Eleven patients with disseminated intravascular coagulation had a mean protein C of 29%. Eight patients receiving warfarin therapy had a mean protein C of 17%. The assay is relatively simple and should be suitable for general laboratory use.

Disseminated Intravascular Coagulation↗

A simplified PTT-based protein C activity assay using the thrombin-thrombomodulin complex.

A simplified assay for protein C activity in plasma is described which uses the ability of rabbit lung thrombomodulin to inhibit the procoagulant activity of thrombin while stimulating protein C activation. Barium eluates of plasma are activated for one hour at 37 degrees C by a mixture of human thrombin and rabbit lung thrombomodulin at concentrations which neutralize each other's effect on the kaolin-cephalin activated partial thromboplastin time (PTT). Protein C anticoagulant activity in the activated eluates is then measured directly in the PTT. The method is independent of protein S levels in the test samples, and is suitable for warfarinized and heparinized plasma. Protein C levels obtained with this method correlate closely with functional levels of vitamin K-dependent procoagulants as measured by the prothrombin and proconvertin time (P&P) in normal subjects and in patients receiving warfarin, indicating specificity for gamma-carboxylated protein C. The method has the potential to detect molecular variants defective in any of the interactions required for generation of anticoagulant activity in vivo.

Blood Coagulation Tests↗