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[Pharmacologic and enzymatic effects of snake venoms from Antioquia and Choco (Colombia)].

We compared several pharmacological and enzymatic effects induced by 11 snake venoms from seven species, six of them from different geographic areas of Antioquia and Choco, north-west of Colombia, South America (Bothrops atrox, B. nasutus, B. schlegelii, B. punctatus, Lachesis muta, Micrurus mipartitus), and Crotalus durissus terrificus venom, from specimens captured in other provinces of the country (Tolima, Huila, Meta and Atlantico). Differences were observed in edema-forming, hemorrhage, defibrination, indirect hemolysis, myonecrosis, proteolysis and lethal activity between venoms from different genera or species, as well as according to the geographic area of origin in B. atrox and B. nasutus snake venoms. Bothrops venoms, in particular B. atrox and L. muta, produced major local effects. All of the venoms, including M. mipartitus, had myotoxic effects. The most defibrinating venoms were B. atrox, L. muta, B. punctatus and C. d. terrificus. All of the venoms had indirect hemolytic activity; the venom of M. mipartitus being greatest. The most lethal venoms were those of C. d. terrificus and M. mipartitus. Within Bothrops species, the venom of B. schlegelii was the least active in terms of local and systemic pathologic effects.

Animals↗

Peripheral leukocytes as indicators of the enzymatic effects of N-(phosphonacetyl)-L-aspartic acid (PALA) on human L-aspartate transcarbamoylase (ATCase) activity.

The interaction of N-(phosphonacetyl)-L-aspartic acid (PALA) with L-aspartate transcarbamoylase (ATCase), the putative target for the antineoplastic activity of this drug, has been studied in the blood of patients participating in a phase I trial of PALA. ATCase activity in human blood is most abundant in granulocytes and lymphocytes; comparatively little activity is seen in erythrocytes. Utilizing peripheral leukocytes from patients given infusions of PALA, we find that leukocyte ATCase in inhibited rapidly and strongly. After cessation of therapy the rate of restitution of enzyme activity is slow: half-maximal restoration is achieved in about 280 hours. As a correlate of this gradual recovery of enzymatic activity, nanomolar concentrations of PALA are detectable in the plasma 2 weeks after infusion. The apparent Ki of PALA for leukocyte ATCase with carbamoyl phosphate as the variable substrate is 5 nM. Uptake of PALA into leukocytes in vitro is saturable and occurs at a moderate rate comparable to that measured in murine tumor cells. Correspondingly, inhibitory concentrations of PALA (approximately 10(-7) M) are found in the leukocytes of patients throughout the course of PALA treatment. It is concluded that although leukocytes are not targets for PALA toxicity, they may serve as accessible and pertinent indicators of the enzymatic effects of this new oncolytic drug.

Aspartate Carbamoyltransferase↗

Enzymatic effects of a lysine-to-glutamine mutation in the ATP-binding consensus sequence in the RecD subunit of the RecBCD enzyme from Escherichia coli.

The RecBCD-K177Q enzyme has a lysine-to-glutamine mutation in the putative ATP-binding sequence of the RecD protein (Korangy, F., and Julin, D.A. (1992) J. Biol. Chem. 267, 1727-1732). We have compared the enzymatic properties of the RecBCD-K177Q enzyme with those of the wild-type RecBCD enzyme from Escherichia coli. The purified RecBCD-K177Q enzyme has ATP-dependent nuclease activity on double-stranded or denatured DNA which is reduced (4-14-fold less) compared with the wild type. The kcat and Km(ATP) for ATP hydrolysis stimulated by double-stranded DNA are both reduced in RecBCD-K177Q, so that kcat/Km(ATP) is relatively unaffected. The mutant enzyme is impaired in its ability to unwind DNA in an assay where single-stranded DNA is trapped by the single-stranded DNA binding protein and subsequently degraded by S1 nuclease. The mutant enzyme also produces fewer acid-soluble DNA nucleotides per ATP hydrolyzed than does the wild type, at low ATP concentrations (less than 20 microM).

Adenosine Triphosphatases↗

Proteases of Antarctic krill--a new system for effective enzymatic debridement of necrotic ulcerations.

The Antarctic krill (Euphausia superba) possesses an "over-dimensioned' digestive system, which is of vital importance for the survival of this euphaucean shrimp in the extreme marine environment. The isolated enzymes contain a well-balanced mixture of both endo- and exopeptidases, assuring fast and complete breakdown of proteinaceous material. These unique properties have now been shown to be extremely valuable for the effective removal of necrotic debris, fibrin or blood crusts in vitro. Therefore the krill enzymes should be considered as an important resource in the future management of necrotic wounds.

Animals↗

Enzymatic effect of hypodermin A, a parasite protease, on bovine lymphocyte membrane antigens.

The protease hypodermin A (HA) is produced by the parasitic warble-fly larva and is implicated in the modulation of the bovine immune system. This study examines the effect of this enzyme on the cell surface markers of bovine lymphocytes. HA interfered with the binding of all anti-lymphocyte receptor antibodies tested. Anti-BoCD2 and CD5 staining was completely abolished. But the mean fluorescence intensity (MFI) only was diminished for antibodies against BoCD4, CD8 and CD18. On the contrary, the MFI for anti-MHC Cl I molecules staining was increased. This effect of HA began as early as one h, and was reversed by removal of HA. Heating or PMSF treatment, which both inhibit protease activity, abolished the action of HA on the surface antigens. The HA concentrations (100 micrograms/ml) needed to alter antibody binding were similar to those that inhibited phytohaemagglutinin (PHA)-induced proliferation. These results show that enzymatic activity of HA on lymphocyte surface markers may be implicated in the inhibition of lymphocyte proliferation.

Animals↗

Chromatographic and enzymatic effects on transfer factor-like activity from human leukocytes and porcine spleen dialysate.

1. The effect of dialysable transfer factor (TFd), derived from human leukocytes or porcine spleen cells, was measured using Listeria resistance in mice. 2. The molecular weight range of substance(s) containing TF-like activity is in the less than 3500 MW dialysis fraction on the basis of the capacity of peritoneal macrophages to produce superoxide anion (O2-). This biological activity is removed by heating at 56 degrees C. 3. After Sephadex G-10 chromatography of dialysates the significant activities are found in fractions III and IV of human leukocyte dialysate and in fractions of II and III of porcine spleen dialysate. 4. From enzymatic studies, most of the protective activity of both human leukocyte and porcine spleen dialysate is based on the action of small-molecular weight structures containing peptides and/or polynucleotides.

Animals↗

Sodium compartmentalization of the arterial wall revisited: ouabain, temperature and enzymatic effects.

The sodium (Na) fractions of the dog carotid artery were identified by analyzing the effects of different procedures on 24Na washout curves. Although these never yielded more than three exponential terms, four Na fractions were identified. The fast-exchanging component amounted to 73 mmoles Na/kg w.wt., followed the kinetics of diffusion in a flat sheet with a diffusion coefficient of 6.9 X 10(-6) cm2 X s-1, and remained unaltered under the different experimental conditions. The intermediate component exchanged with a half time (t0.5) of 5.3 min and amounted to 18 mmoles Na/kg w.wt. About one third of this (6 mmoles) was constituted by cellular Na and the remaining by non-cellular Na, probably associated with extracellular structures. These two fractions could be discriminated by: (a) decreasing the rate of exchange of the cellular fraction with ouabain or low temperature (17 degrees C); (b) cell damage by freezing and thawing combined with metabolic poisoning; (c) enzymatic digestion of extracellular structures. When procedures (a) and (c) were combined, the intermediate component entirely disappeared. A third residual component amounted to 0.6 mmoles Na/kg w.wt and exchanged with a t0.5 of 70 min. It was unaffected by cellular damage or enzymatic digestion and was masked by the cellular phase in effluxes conducted at 17 degrees C or under ouabain. Its size decreased by microscopic dissection of the dense adventitia, which is probably its source.

Animals↗

An enzymatic effect for cancer prevention and eradication.

As previously described in a series of hypotheses, cancer development depends on an unconvertable duplication mitosis caused by some abnormal supplement of the mitotic maturation promoting system which is paralyzed in the cancerous cell's cytoplasm. This abnormal supplement, which may be responsible for the cancer's development, must be a biochemical compound that could be eliminated by exposure to the action of certain enzymes. Accordingly, in order to prevent and eradicate cancer development in general, an enzyme which would destroy and eliminate the biochemical supplement needs to be identified, and a specific method of delivering the enzyme to the supplement needs to be devised.

Actin Cytoskeleton↗

Snakebites and ethnobotany in the northwest region of Colombia: Part II: neutralization of lethal and enzymatic effects of Bothrops atrox venom.

Twelve of 74 ethanolic extracts of plants used by traditional healers for snakebites in the northwest region of Colombia, were active against lethal effect of Bothrops atrox venom when they were i.p. injected into mice (18-20 g). After preincubation of sublethal doses of every extract (0.5-4.0 mg/mouse) with 1.5 i.p. lethal dose 50% (LD50) (99.3 microg) of venom, seven of them demonstrated 100% neutralizing capacity within 48 h. These were the stem barks of Brownea rosademonte (Caesalpiniaceae) and Tabebuia rosea (Bignoniaceae); rhizomes of Renealmia alpinia (Zingiberaceae) and Heliconia curtispatha (Heliconiaceae); the whole plants of Pleopeltis percussa (Polypodiaceae) and Trichomanes elegans (Hymenophyllaceae); and the ripe fruits of Citrus limon (Rutaceae). The other five extracts showing partial neutralization (45-80%; 10-30% survival rate in the control group receiving the venom alone; P<0.05) were: leaves, branches and stem of Costus lasius (Costaceae); the whole plant of Sida acuta (Malvaceae); rhizomes of Dracontium croatii (Araceae); leaves and branches of Bixa orellana (Bixaceae) and Struthanthus orbicularis (Loranthaceae). When the extracts were independently administered per oral or i.p. route 60 min before an i.m. venom injection (204 microg=1.5 i.m. LD50), C. limon, T. elegans, B. orellana and T. rosea extracts had partial and significant neutralizing capacity against B. atrox venom lethal effect. C. limon extract was also partially effective when it was administered either i.v. 15 min before or i.p. 5 min after an i.m. venom injection. Three of the 12 extracts with anti-lethal effect (C. limon, D. croatii and S. acuta) were devoid of antiphospholipase A2 activity, when they were tested against one minimum indirect hemolytic dose of B. atrox venom (2 microg) in agarose-erythrocyte-egg yolk gels.

Animals↗

Some ultrastructural and enzymatic effects of water stress in cotton (gossypium hirsutum L.) leaves.

Water stress induced by floating discs cut from cotton leaves (Gossypium hirsutum L. cultivar Stoneville) on a polyethylene glycol solution (water potential, -10 bars) was associated with marked alteration of ultrastructural organization of both chloroplasts and mitochondria. Ultrastructural organization of chloroplasts was sometimes almost completely destroyed; peroxisomes seemed not to be affected; and chloroplast ribosomes disappeared. Also accompanying water stress was a sharp increase in activity of acid phosphatase [orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2], and acid and alkaline lipase [glycerol ester hydrolase EC 3.1.1.3] within chloroplasts. Only acid lipase activity was detected inside mitochondria of stressed discs. These alterations in cell organization and enzymology may account for at least part of the previously reported effects of water stress on the CO(2) compensation point, photochemical reactions, and photorespiration.

Acid Phosphatase↗

"In vitro" synthesis of different naturally-occurring, semisynthetic and synthetic penicillins using a new and effective enzymatic coupled system.

Forty-seven different penicillins, including some of great clinical importance, have been synthesized "in vitro" by coupling the newly described enzyme phenylacetyl-CoA ligase (PCL) from Pseudomonas putida and acyl-CoA: 6-aminopenicillanic acid (6-APA) acyltransferase (AT) from Penicillium chrysogenum. Incubations were carried out at 30 degrees C in 50 mM HCl-Tris buffer pH 8.0. The reaction mixtures contained 6-APA, CoA, ATP, dithiothreitol, Mg2+ and the corresponding penicillin side-chain precursor. This is the first description of the enzymatic synthesis of all the natural penicillins known, many of the semisynthetic until now reported, and some penicillins that could only be currently obtained by chemical synthesis. The efficiency of this prokaryotic-eukaryotic enzymatic-coupled system and its application to the synthesis of different beta-lactam antibiotics are discussed.

Acyltransferases↗