Alkylation of histidine residues of Bothrops jararacussu venom proteins and isolated phospholipases A2: a biotechnological tool to improve the production of antibodies.

Crude venom of Bothrops jararacussu and isolated phospholipases A2 (PLA2) of this toxin (BthTX-I and BthTX-II) were chemically modified (alkylation) by p-bromophenacyl bromide (BPB) in order to study antibody production capacity in function of the structure-function relationship of these substances (crude venom and PLA2 native and alkylated). BthTX-II showed enzymatic activity, while BthTX-I did not. Alkylation reduced BthTX-II activity by 50% while this process abolished the catalytic and myotoxic activities of BthTX-I, while reducing its edema-inducing activity by about 50%. Antibody production against the native and alkylated forms of BthTX-I and -II and the cross-reactivity of antibodies to native and alkylated toxins did not show any apparent differences and these observations were reinforced by surface plasmon resonance (SPR) data. Histopathological analysis of mouse gastrocnemius muscle sections after injection of PBS, BthTX-I, BthTX-II, or both myotoxins previously incubated with neutralizing antibody showed inhibition of the toxin-induced myotoxicity. These results reveal that the chemical modification of the phospholipases A2 (PLA2) diminished their toxicity but did not alter their antigenicity. This observation indicates that the modified PLA2 may provide a biotechnological tool to attenuate the toxicity of the crude venom, by improving the production of antibodies and decreasing the local toxic effects of this poisonous substance in animals used to produce antivenom.

Mycophenolate mofetil has potent anti-inflammatory actions in a mouse model of acute lung injury.

Septic shock is a systemic inflammatory response syndrome, and it is the leading cause of death in intensive care units. Mycophenolate mofetil (MMF) is an immunosuppressant that has been shown to be effective in the treatment of various inflammatory diseases. In this study, the anti-inflammatory effect of MMF in a mouse model of acute lung injury (ALI) induced by lipopolysaccharide (LPS) was evaluated. ALI was induced by intrapleural injection of LPS (250 ng/cavity). The leukocyte migration, exudation, myeloperoxidase and adenosine deaminase activities, nitric oxide products, tumor necrosis factor alpha (TNF-α), and interleukin 1 beta (IL-1ß) levels, as well as mRNA expression of TNF-α and IL-1ß, were evaluated. This study showed that MMF significantly decreased all parameters studied in a manner comparable to treatment with dexamethasone. In conclusion, MMF has important anti-inflammatory effects that may be useful as an auxiliary treatment for septic shock.

I3-naringenin-II8--4'OMe-eriodictyol: a new potential analgesic agent isolated from Rheedia gardneriana leaves.

This paper describes the isolation, identification and analgesic activity of a new biflavonoid from Rheedia gardneriana leaves, which correspond to I3-naringenin-II8-4'-OMe-eriodictyol (GB-2a-II-4'-OMe) (1), with a methoxyl group in position 4 of ring-II. Its structure was determined by spectroscopic data and confirmed by an alkaline hydrolysis. Its analgesic effect was evaluated in a writhing test and a formalin test in mice. It was found that this compound exhibits potent and dose-related analgesic action in both experimental models, with ID50's values of 4.5 micromol/kg against the writhing test and 8.2 and 6.8 micromol/kg against the first and second phase of the formalin test, respectively. It was several times more potent than some well-known analgesic drugs used as reference.

Mixed endothelin ET(A) and ET(B) antagonist bosentan inhibits oleic acid-induced lung plasma extravasation in mouse.

The possible participation of endogenous endothelins (ETs) in enhancement of plasma extravasation induced by oleic acid was assessed in mice. Oleic acid (0.5-2%/kg, i.v.) increased accumulation of Evans blue in lungs in dose-dependent fashion, with a clearcut peak at 1 h (lung Evans blue content: control 0.17 +/- 0.01; oleic acid 1%/kg 0.63 +/- 0.04 microg per 10 mg wet tissue). Pretreatment with the mixed endothelin-ET(A) and ET(B) (ET(A)/ET(B)) receptor antagonist bosentan (30 mg/kg, i.v., 30 min before oleic acid) markedly reduced lung Evans blue content (to 0.24 +/- 0.04), as did pretreatments with prazosin (1 mg/kg), meloxicam (5 mg/kg) and dexamethasone (1 mg/kg/day, for 3 days). Thus, ETs play a pivotal role in the increase in lung microvascular permeability caused by oleic acid in the mouse. The ET receptors involved in the pulmonary vascular changes associated with this experimental model of adult respiratory distress syndrome (ARDS), as well as the relationship between ETs and the sympathetic system, eicosanoids and cytokines remain to be clarified.

Isolation of biflavonoids with analgesic activity from Rheedia gardneriana leaves.

Some phytoconstituents present in the R. Gardneriana Pl. Tr. leaves, a Brazilian medicinal plant, were isolated by column chromatography. Their preliminary analgesic effects were evaluated against formalin-induced pain in mice. The results demonstrated that four biflavonoids, identified as volkensiflavone, GB-2 a, fukugetin and fukugeside are the main active components of the ethyl acetate fraction. Such compounds exhibited significative analgesic activity in relation to the second phase (inflammatory pain) of the formalin test, suggesting that they are the compounds responsible for the pharmacological effects previously observed for the hydroalcoholic extract of this plant.

Dual effects of endothelins -1, -2 and -3 on guinea pig field-stimulated ileum: possible mediation by two receptors coupled to pertussis toxin-insensitive mechanisms.

This study compared the effects of endothelin-1 (ET-1), ET-2 and ET-3 on the guinea pig field-stimulated ileum. All ETs (0.3-30 nM) caused graded inhibitions of nerve-mediated responses followed by sustained contractions. The rank order of potencies for the twitch depressor effect (IC50S) was ET-3 = ET-1 greater than ET-2, with ET-3 causing greater maximal inhibition than ET-1 or ET-2. The rank order of potencies for contraction (EC50S) was ET-1 = ET-2 greater than ET-3, with ET-1 causing greater maximal contraction than ET-2 or ET-3. Twitch inhibition by ET-1 (3 nM) was unaffected by indomethacin (5.6 microM), cromakalim (10 microM), glibenclamide (3 microM) or nicardipine (0.1 microM). ET-1-induced contraction was unaltered by tetrodotoxin (0.3 microM), atropine (0.3 microM) or glibenclamide, but was reduced by indomethacin. Cromakalim and nicardipine virtually abolished ET-1-induced contraction. ET-1 (up to 30 nM) did not potentiate submaximal contractions induced by acetylcholine, histamine, bradykinin or substance P. ET-3 relaxed ileal segments precontracted with either acetylcholine (0.3 microM) or histamine (1 microM). Pretreatment of guinea pigs with pertussis toxin (50 micrograms/kg i.p., 6 days beforehand) did not influence either effects of ET-1 on the field-stimulated ileum. Our data suggest that the dual effects of ETs on the guinea pig isolated ileum are mediated by distinct receptors and possibly involve different mechanisms of action. The transient inhibition of responses to field stimulation seems unrelated to activation of ATP-sensitive potassium channels and is rather insensitive to L-type Ca++ channel blockade.

Potent constrictor actions of endothelin-1, endothelin-2, and endothelin-3 in rat isolated portal vein.

In rings of rat portal vein, endothelin-1, endothelin-2, and endothelin-3 caused graded slow contractions and potentiated spontaneous contractions. The apparent EC50 values and maximal responses to 30 nM endothelin were 1.4 nM and 0.96 g for endothelin-1, 5.2 nM and 0.65 g for endothelin-2, and 1.7 nM and 0.62 g for endothelin-3 (n = 4-12). At concentrations producing half the contraction triggered by 80 mM KCl, the order of potencies was endothelin-1 greater than U46619 = angiotensin II greater than bradykinin greater than substance P greater than phenylephrine. Longitudinal portal-mesenteric vein preparations developed very modest contractions to endothelin-1 (0.13 g at 30 nM; n = 5), but their responses to 80 mM KCl and phenylephrine were greater than those of rings. Responses of rings to endothelin-1 were profoundly reduced in Ca(2+)-free medium, but less inhibition was obtained after incubation with nicardipine (up to 1 microM) and/or nickel (up to 0.5 mM), phorbol (up to 0.3 microM), staurosporine (up to 10 nM), or cromakalim (3 microM). Indomethacin (5.6 microM) did not affect responses to endothelin-1. Cromakalim (0.1-3 microM) also relaxed rings constricted with 0.3 nM endothelin-1, and this effect was partially reversed by glibenclamide (3 microM). Thus, endothelins, especially endothelin-1, are potent constrictors of portal vein rings but not of portal-mesenteric vein strips. Their action appears to rely largely on Ca2+ influx from the external medium (only in part via L- and T-type Ca2+ channels) and activation of protein kinase C but not on eicosanoid generation.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholera in Portugal, 1974.I. Modes of transmission.

In April-November 1974, Portugal had a cholera epidemic caused by Vibrio cholerae El Tor Inaba with 2467 bacteriologically confirmed hospitalized cases and 48 deaths. Most of the country was affected, with 17 of the 18 districts reporting cases. V. cholerae was isolated from 42 per cent of shellfish tested during the epidemic, and an epidemiologic study found that a history of consumption of raw or poorly cooked cockles was significantly more common among cholera patients than among paired controls. Water from a spring and a brand of commercially bottled water were also found to be vehicles of transmission of cholera. Although night soil was sometimes used on gardens, consumption of raw fruits and vegetables was not associated with illness.