Autoradiographic analysis of the distribution of vasoactive intestinal peptide binding sites in the vertebrate central nervous system: a phylogenetic study

The distribution of vasoactive intestinal peptide (VIP) binding sites in the brain of several vertebrate species was examined by in vitro autoradiography on slide-mounted sections. This study included fish, frog, snake, pigeon, rat, mouse, guinea pig, cat and monkey brain. A fully characterized, monoiodinated form of vasoactive intestinal peptide (M-¹²⁵I-VIP), which maintains the biological activity of the native peptide in the central nervous system (CNS), was used throughout the study. Among the lower vertebrate species, no significant specific binding was found in the fish brain, whereas in the frog and snake brain, specific VIP binding sites were observed, mainly in the telencephalon. In the pigeon brain, high densities of VIP binding sites were localized in the hyperstriatum, neostriatum, archistriatum, hippocampal area, dorsolateral cortical area and in the optic tectum. Ectostriatum and paleostriatum augmentatum displayed lower densities of specific binding. In mammals, the highest concentrations of VIP binding sites were observed in the rodent brain. In the rat, mouse and guinea pig brain, high densities were detected in the olfactory bulb, external layers of the cerebral cortex, dentate gyrus, midline thalamic nuclei, geniculate nuclei, some hypothalamic nuclei, superior colliculus coeruleus. Intermediate densities were found in amygdala, caudate-putamen, septum and nucleus accumbens, CA1–CA3 fields of the hippocampus and central gray. The cerebellum of these species presented high densities of VIP binding sites, with species to species differences in their localization. The non-specific binding was, however, increased in the rodent cerebellum. Lower densities of VIP binding sites were observed in the cat and monkey CNS. In these two species, the non-specific binding was considerably higher than in the lower mammals brain. In the cat and monkey brain, as in the lower mammals, the highest densities were revealed in the neocortex, dentate gyrus, thalamic nuclei and some midbrain structures including substantia nigra and locus coeruleus. In all the species studied, the white matter was never labeled with M-¹²⁵I-VIP. This study suggests that VIP binding sites appear relatively early in the evolution of the vertebrate CNS. The most important densities of specific VIP binding sites are observed in the pigeon and rodent brain, whereas the cat and monkey present a marked increase in non-specific binding. It is interesting to note that the distribution of VIP binding sites as revealed by autoradiography is quite conservative in terms of evolution and indicates an association, although non-exclusive, of VIP receptors with brain regions involved in the processing of specific sensory inputs.     

Effect of the metalloproteinase inhibitor batimastat in the systemic toxicity induced by Bothrops asper snake venom: understanding the role of metalloproteinases in envenomation.

The peptidomimetic hydroxamate metalloproteinase inhibitor batimastat (BB-94) was assessed for its ability to neutralize the systemic effects (lethality, hemorrhage and coagulopathy) induced by the venom of Bothrops asper, the most important snake from a medical standpoint in Central America. Batimastat inhibited lethality when a venom challenge dose of two LD(50)s was used by intraperitoneal and intravenous routes, with ED(50)s of 250 and 22 microM, respectively. With a challenge dose of three LD(50)s, lethality was not abrogated, but a conspicuous and dose-dependent delay in the time of death was observed in mice injected with mixtures of venom plus batimastat. Upon incubation with 500 microM batimastat, venom LD(50) increased 2.86-fold (intraperitoneal route) and 2.37-fold (intravenous route), when compared with LD(50) of venom alone. Batimastat also inhibited the hemorrhagic effect induced by venom in the lungs after intravenous injection. Moreover, batimastat exerted a significant inhibition of in vitro coagulant and in vivo defibrinogenating effects of venom, evidencing that metalloproteinases play a key role in the coagulopathy characteristic of B. asper envenomation. The remaining uninhibited coagulant effect is due to serine proteinases, i.e. thrombin-like enzymes, since this effect was completely abrogated by the combination of batimastat and PMSF. Our results stress the view that metalloproteinases play a relevant role in the systemic pathophysiology of B. asper envenomation and that metalloproteinase inhibitors may become a therapeutic alternative in this pathology.     

Presynaptic snake β-neurotoxins produce tetanic fade and endplate potential run-down during neuromuscular blockade in mouse diaphragm

The present study investigated the ability of a number of presynaptic snake neurotoxins (snake β-neurotoxins) to produce nerve-evoked train-of-four fade, tetanic fade and endplate potential run-down during the development of neuromuscular blockade in the isolated mouse phrenic-hemidiaphragm nerve-muscle preparation. All the snake β-neurotoxins tested, with the exception of notexin, produced train-of-four and tetanic fade of nerve-evoked isometric muscle contractions. Train-of-four fade was not present during the initial depressant or facilitatory phases of muscle tension produced by the snake β-neurotoxins but developed progressively during the final depressant phase that precedes complete neuromuscular blockade. The ‘non-neurotoxic’ bovine pancreatic phospholipase A₂ and the ‘low-toxicity’ phospholipase A₂ from Naja naja atra venom failed to elicit train-of-four fade, indicating that the phospholipase activity of the snake β-neurotoxins is not responsible for the development of fade. Intracellular recording of endplate potentials (EPPs) elicited by nerve-evoked trains of stimuli showed a progressive run-down in EPP amplitude during the train following incubation with all snake β-neurotoxins except notexin. Again this run-down in EPP amplitude was confined to the final depressant phase of snake β-neurotoxin action. However when EPP amplitude fell to near uniquantal levels (<3mV) the extent of toxin induced-fade was reduced. Unlike postjunctional snake α-neurotoxins, prejunctional snake β-neurotoxins interfere with acetylcholine release at the neuromuscular junction during the development of neuromuscular blockade. This study provides further support for the hypothesis that fade in twitch and tetanic muscle tension is due to an underlying rundown in EPP amplitude resulting from a prejunctional alteration of transmitter release rather than a use-dependent block of postjunctional nicotinic receptors. Received: 5 May / Accepted: 16 July 1997     

Epidemiologic surveys of dog and cat bites in the Lyon area,France

The urban pet population has increased considerably in France during the last twenty years. Two main questions need to be answered regarding rabies and other bite transmitted zoonoses: What is the actual incidence rate of dog and cat bites in an urban area; and how sensitive is the animal bite reporting system? To answer these questions, four surveys were conducted in the Lyon area, France, in 1989: 1) an analysis of the consultation reports to the Pasteur Institute and of the bite reports sent by veterinarians to the local veterinary services for 1987 and 1988; 2) a survey of 10 veterinary clinics located in the Lyon area and an analysis of their bite reports for the period May 1987 — April 1989; 3) a questionnaire survey to 175 clients of these veterinary clinics; 4) a street survey of a random sample of the Lyon adult population (310 questionnaires). Bite incidence rates ranged from 10/100,000 persons/year for rabies post-exposure treatments to 37.5/100,000 persons/year for reported bites. However, less than half of the bite reports from the ten veterinary clinics were submitted to the veterinary services. The surveys conducted among pet owners and the general population indicated that, overall, bites were common events (3.4%) and occurred more often in pet owners (8.6%). In 74% of the cases, victims belonged to the pet owner's family and one fourth of the accidents occurred when playing with the pet. However, 12% of the accidents resulted from apparently unprovoked aggressions. According to these data, estimates of the incidence rate of bites for the Lyon area were at least one hundred times higher than the official reported rate of 37.5 bites/100,000 persons/year.     

普罗托品对家兔血小板超微结构的影响

以ＡＤＰ，胶原，ＡＡ和ＴＭＶＡ诱导血小板聚集，利用电子显微镜观察普罗托品对兔血小板超微结构的影响，结果表明：普罗托品明显抑制ＡＤＰ，胶原，ＡＡ和ＴＭＶＡ诱导的兔血小板聚集、形态的改变和颗粒内含物的释放，说明：普罗托品对血小板结构具有保护作用。     

Specific identification of Lachesis muta muta snake venom using antibodies against the plasminogen activator enzyme, LV-PA.

Sandwich-type enzyme linked immunosorbent assays (ELISA) were developed to detect Lachesis muta muta (bushmaster) snake venom using antibodies against the plasminogen activator enzyme (LV-PA). Antibodies to LV-PA were obtained by immunization of one rabbit with the purified enzyme. The IgG fraction was purified from rabbit blood in a single step on a column of Sepharose-L. m. muta venom and used to coat the microtiter plates. The specificity of the assay was demonstrated by its capacity to correctly discriminate between the circulating antigens in mice that were experimentally inoculated with L. m. muta venom from those in mice inoculated with venoms from Bothrops atrox, B. brazili, B. castelnaudi, Bothriopsis taeniata, B. bilineata, Crotalus durissus ruruima and the antigenic Bothrops (AgB) and Crotalus (AgC) pools venoms used to produce Bothropic and Crotalic antivenoms at Fundacao Ezequiel Dias (FUNED). Measurable absorbance signals were obtained with 1.5 ng of venom per assay. The ELISA was used to follow the kinetic distribution of antigens in experimentally envenomed mice.     

蛇毒抗栓酶对心血管系统影响的初步研究

本文通过颈动脉插管法,ECG技术及离体心脏灌流实验,初步证明:蛇毒抗栓酶(Svate)具有降低动脉血压,增加心输出量,抑制心肌收缩力及减少冠流量等作用。在一定剂量下,对心率无明显影响。     

Dog and cat bites to the hand: treatment and cost assessment

PURPOSE: To assess the demographic patterns, clinical morbidity, and treatment costs associated with domestic animal bites to the hand. METHODS: A retrospective review was performed on 111 patients who suffered either a dog or cat bite to the hand. Demographic data were collected for both the patient and the animal involved. RESULTS: The patient population had suffered 71 dog bites and 40 cat bites. Two scenarios were identified that increased the likelihood of a bite: (1) attempting to separate fighting animals and (2) attempting to aid an injured animal. More than half of the victims (61 of 111) were bitten by an animal with which they were familiar. Bite injuries ranged from relatively minor wounds to major injuries that included open fractures, persistent deep infection including osteomyelitis, nerve laceration, tendon laceration, or tissue loss. Approximately two thirds of patients required hospital admission at least for intravenous antibiotics. Approximately one third of animal bite victims required at least 1 surgical procedure. Thirteen patients required long-term intravenous antibiotics and/or multiple surgeries and incurred medical expenses in excess of dollar 77,000. CONCLUSIONS: Domestic animal bites to the hand are common injuries that can produce considerable morbidity. Stray animals did not account for the majority of incidents. Bite prevention strategies should focus on careful handling of animals that are fighting or injured. Animal bite wounds often require intravenous antibiotics and hospitalization and the cost of care for deep infections can be enormous. Our patient population was selected from a small geographic area over a relatively short collection period, suggesting that domestic animal bite injuries may represent a major public health issue. TYPE OF STUDY/LEVEL OF EVIDENCE: Prognostic, Level IV.     

Interacting surfaces of neurotoxins and acetylcholine receptor

Binding of neurotoxin II Naja naja oxiana derivatives containing one spin label at various positions (Leu 1, Glu 2, Lys 15, Lys 25, Lys 26, His 31, Lys 44 and Lys 46) to purified solubilized acetylcholine receptor protein (AchR) from Torpedo marmorata was studied by EPR techniques. AchR interaction with several dansylated neurotoxin II derivatives was followed by difference fluorescence spectroscopy. A series of neurotoxin II p-azidobenzoyl derivatives were prepared and in three of them modified lysine residues were identified. In combination, spectroscopic data and photolabling implicate a considerable area of the neurotoxin in association with AchR. Rigidity of the neurotoxin II conformation allowed to regard its binding surface as a mould of the AchR corresponding site and to estimate the minimal size of the latter. Conformation of the long-chain neurotoxins and their binding to AchR are briefly discussed basing on the 1H and 19F NMR studies of neurotoxin I Naja naja oxiana, toxin 3 Naja naja siamensis and its acetylated or trifluoroacetylated derivatives, as well as on AchR interaction with the derivatives spin labeled at Lys 27 and His 71     

Pharmacological studies on the bungarotoxins: separation of the fractions and their neuromuscular activity

A commercial sample of Bungarus multicinctus venom was separated into its constituent fractions by column chromatography on CM-Sephadex C-50. The fractions were tested on the responses of the chick biventer cervicis preparation to nerve stimulation, and to added acetylcholine and carbachol.13 fractions were obtained: 1 was inactive, 4 exhibited weak postjunctional neuromuscular blocking activity, 3 had potent postjunctional blocking activity, and the remaining 5 fractions exhibited predominately prejunctional blocking activity. Use of higher concentrations of the prejunctional toxins revelead that 2 of these fractions also possessed some postjunctional blocking activity.Because small differences in separation procedures may result in differences in the elution pattern of the venom, and because some fractions may possess nonspecific actions only obvious at high concentrations, it is suggested that, when using venom components as experimental tools, the separation method is detalied and the fractions thoroughly tested.