Binge drinking and insomnia in students from health sciences at one university in Rio de Janeiro, Brazil.

In spite of the many studies examining alcohol consumption, recent reviews have indicated that binge drinking has not been extensively studied. Furthermore, it is becoming increasingly clear that sleep is associated with many physiological functions and to drug addictions. The present study aimed to evaluate the relationship between alcohol binge drinking and insomnia in college students of health sciences. All first-year health sciences students (n=286) were evaluated in a cross-sectional study. Envelopes containing the Insomnia Severity Index (ISI), the Alcohol, Smoking, and Substance Involvement Screening Test (ASSIST), and questions capturing sociodemographic data were distributed and collected in classes. It was found that most non-drinkers were female (70.6%), although there were no sex-related differences in the number of binge drinkers (more than 5 drinks on each occasion at least once a week), allowing statistical comparison. The Mann-Whitney U test indicated that the ISI scores were significantly greater in female than male binge drinkers (P=0.014). Moderate or severe insomnia was reported by 23% of the sample, with alcohol being the most frequently associated substance. A specialized intervention was suggested by ASSIST: brief for marijuana (19.2%) and tobacco (23.3%) use, and moderate (31.5%) or intensive (1.4%) for alcohol consumers. The data highlighted the need to pay attention to the habits of college students beyond obtaining scientific information. New data suggesting the influence of genetics on insomnia may be of importance when performing additional studies on the sex differences in alcohol binge drinking.

Binge drinking and insomnia in students from health sciences at one university in Rio de Janeiro, Brazil

In spite of the many studies examining alcohol consumption, recent reviews have indicated that binge drinking has not been extensively studied. Furthermore, it is becoming increasingly clear that sleep is associated with many physiological functions and to drug addictions. The present study aimed to evaluate the relationship between alcohol binge drinking and insomnia in college students of health sciences. All first-year health sciences students (n=286) were evaluated in a cross-sectional study. Envelopes containing the Insomnia Severity Index (ISI), the Alcohol, Smoking, and Substance Involvement Screening Test (ASSIST), and questions capturing sociodemographic data were distributed and collected in classes. It was found that most non-drinkers were female (70.6%), although there were no sex-related differences in the number of binge drinkers (more than 5 drinks on each occasion at least once a week), allowing statistical comparison. The Mann-Whitney U test indicated that the ISI scores were significantly greater in female than male binge drinkers (P=0.014). Moderate or severe insomnia was reported by 23% of the sample, with alcohol being the most frequently associated substance. A specialized intervention was suggested by ASSIST: brief for marijuana (19.2%) and tobacco (23.3%) use, and moderate (31.5%) or intensive (1.4%) for alcohol consumers. The data highlighted the need to pay attention to the habits of college students beyond obtaining scientific information. New data suggesting the influence of genetics on insomnia may be of importance when performing additional studies on the sex differences in alcohol binge drinking.

Dental status, salivary flow, and sociodemographic aspects in Sheehan Syndrome patients.

BACKGROUND: Sheehan's syndrome (SS) is one of the leading causes of hypopituitarism in developing countries. It occurs after postpartum necrosis of the pituitary gland, and it is considered a significant public health problem. This paper, apparently unpublished, aimed to perform an analysis on oral aspects in patients with SS. MATERIAL AND METHODS: A cross-sectional study was performed with 23 women diagnosed with SS at the Division of Endocrinology and Diabetes (Walter Cantidio University Hospital, Fortaleza, Brazil). RESULTS: Data on sociodemographic, dental and salivary flow aspects were collected through a clinical approach and a panoramic radiograph request. The mean age was 64 ± 11.5 years old, with the sample consisting mainly of married women (56.5%), socioeconomic class C2 or D / E (78.2%) and years of education up to 8 years (69.5%). The presence of horizontal bone loss (p<0.001) and bilateral pneumatization of the maxillary sinus (p=0.015) were significant data. The mean number of absent teeth considering all subjects was 23.17±9.7, being statistically significant (p<0.001). In relation to age, the mean number of missing teeth was higher in individuals over 65 years old (p=0.048). Reduced salivary flow was observed in 78.3% of the patients. In a bivariate analysis, considering the outcome variables missing teeth and reduced salivary flow, it was observed that economic class (p<0.001), family income (0.037) and maxillary sinus pneumatization (0.032) were statistically significant. CONCLUSIONS: In brief, patients with SS showed severe teeth loss, reduced salivary flow, and low educational status. This study addressed important aspects regarding oral findings in SS and highlighted the importance of researches in oral medicine.

Cross-cultural adaptation, reliability and construct validity of the Tampa scale for kinesiophobia for temporomandibular disorders (TSK/TMD-Br) into Brazilian Portuguese.

Fear of movement (kinesiophobia) seems to play an important role in the development of chronic pain. However, for temporomandibular disorders (TMD), there is a scarcity of studies about this topic. The Tampa Scale for Kinesiophobia for TMD (TSK/TMD) is the most widely used instrument to measure fear of movement and it is not available in Brazilian Portuguese. The purpose of this study was to culturally adapt the TSK/TMD to Brazilian Portuguese and to assess its psychometric properties regarding internal consistency, reliability, and construct and structural validity. A total of 100 female patients with chronic TMD participated in the validation process of the TSK/TMD-Br. The intraclass correlation coefficient (ICC) was used for statistical analysis of reliability (test-retest), Cronbach's alpha for internal consistency, Spearman's rank correlation for construct validity and confirmatory factor analysis (CFA) for structural validity. CFA endorsed the pre-specified model with two domains and 12-items (Activity Avoidance - AA/Somatic Focus - SF) and all items obtained a loading factor greater than 0·4. Acceptable levels of reliability were found (ICC > 0·75) for all questions and domains of the TSK/TMD-Br. For internal consistency, Cronbach's α of 0·78 for both domains were found. Moderate correlations (0·40 < r < 0.60) were observed for 84% of the analyses conducted between TSK/TMD-Br scores versus catastrophising, depression and jaw functional limitation. TSK/TMD-Br 12 items and two-factor demonstrated sound psychometric properties (transcultural validity, reliability, internal consistency and structural validity). In such a way, the instrument can be used in clinical settings and for research purposes.

Serum levels of nitric oxide and cytokines in smokers at the beginning and after 4months of treatment for smoking cessation.

INTRODUCTION: Nitric oxide (NO) modulates inflammatory reactions, having beneficial or toxic effects depending on the concentration. Its elevation can cause proinflammatory effects amplifying the inflammatory process with the participation of cytokines. Smoking has a negative impact on health and is considered one of the risk factors that influence disease development facilitating inflammatory processes. AIM: To compare the serum concentration of NO and cytokines in smokers at baseline and after 4months of abstinence treatment. METHODS: Blood samples which were collected to obtain the serum, at baseline and after 4months, were stored at -80°C until analysis. NO was measured by the total dose of nitrite determined by the Greiss method. CBA was the used technique to determine the concentration of cytokines in supernatants serum. The initial and final results of NO, TNF-α, IL-1, IL-6, IL-8, IL-10 and IL-12 that remained after 4months treatment were compared. Wilcoxon test was used to compare the data and Spearman test for correlations between NO and other variables. A significance level of p<0.05 was adopted. RESULTS: The analysis of NO observed a significant reduction (p=0.001) of the initial median value of 18.80 (3.55-80.01) µmol/L to 8.10 (2.85-14.97) µmol/L after 4months of treatment. There were no significant differences in cytokines from baseline to the end of treatment. CONCLUSION: The results may not mean harm to the body, but an adaptive process, decreasing the metabolism of abstinents due to the reduction of the use of nicotine.

Effects of exercise on mitochondrial function, neuroplasticity and anxio-depressive behavior of mice.

The present study was aimed at analyzing the effects of physical exercise on mitochondrial physiology, anxio-depressive-like behaviors and neuroplasticity in mice. Adult C57BL/6J male mice were isolated in home cages equipped or not with free-running wheels. After 6weeks of exercise, mice were tested in various behavioral paradigms to evaluate anxiety- and depressive-like behaviors. The hippocampi were dissected for neurochemical assays, including mitochondrial activity, monoamines content and the expression of genes involved in energy metabolism and brain-derived neurotrophic factor (BDNF) regulation. Exercise decreased anxiety-like behaviors in the open field and elevated plus maze, and exerted antidepressant-like effects in the tail suspension test. Exercise stimulated brain mitochondrial activity and increased resistance against rotenone, an inhibitor of complex I activity. Furthermore, mRNA expression of Bdnf, Gdnf, Tfam (mitochondrial transcription factor A), and Ndufa6 (mitochondrial I subunit) genes, as well as the phosphorylation of cAMP response element-binding protein were increased after exercise. In summary, exercise appears to engage mitochondrial pathways and to potentiate neuroplasticity and might be associated to mood improvement.

Exercise attenuates levodopa-induced dyskinesia in 6-hydroxydopamine-lesioned mice.

L-DOPA alleviates the motor symptoms of Parkinson's disease, but its long-term use is associated with undesirable dyskinesia. We now tested whether exercise can attenuate this L-DOPA-induced dyskinesia (LID). We tested the effects of exercise on LID in 6-hydroxydopamine hydrochloride-hemiparkinsonian mice. Animals were treated with L-DOPA/benserazide (25/12.5 mg/kg, i.p.) without and with possibility to exercise (running wheel) during 2 weeks. Exercise drastically prevented the development of LID, and its associated aberrant striatal signaling, namely the hyperphosphorylation of dopamine and cAMP-regulated phosphoprotein 32 kDa protein and c-Fos expression. Our results indicate that exercise can partially prevent the development of LID through the normalization of striatopallidal dopaminergic signaling.

RNA splicing and editing modulation of 5-HT(2C) receptor function: relevance to anxiety and aggression in VGV mice.

Changes in serotonin(2C) receptor (5-HTR2c) editing, splicing and density were found in conditions such as depression and suicide, but mechanisms explaining the changes in 5-HTR2c function are unknown. Thus, mice expressing only the fully edited VGV isoform of 5-HTR2c, in which clinically relevant behavioral changes are associated with alterations in splicing and receptor density, were studied. VGV mice displayed enhanced anxiety-like behavior in response to a preferential 5-HTR2c agonist in the social interaction test. Nearly half of interactions between pairs of VGV congeners consisted of fighting behaviors, whereas no fighting occurred in wild-type (WT) mice. VGV mice also exhibited a striking increase in freezing behaviors in reaction to an innately aversive ultrasonic stimulus. This behavioral phenotype occurred in conjunction with decreased brain 5-HT turnover during stress. These functional data were put in relation with the 5-HTR2c mRNA splicing process generating a truncated protein (5-HTR2c-Tr) in addition to the full-length receptor (5-HTR2c-Fl). 5-HTR2c-Tr mRNA was less abundant in many brain regions of VGV mice, which concomitantly had more 5-HTR2c than WT mice. Fluorescence resonance energy transfer and bioluminescence resonance energy transfer studies in transfected living HEK293T cells showed that 5-HTR2c-Tr interacts with 5-HTR2c-Fl. The 5-HTR2c-Tr was localized in the endoplasmic reticulum where it retained 5-HTR2c-Fl, preventing the latter to reach the plasma membrane. Consequently, 5-HTR2c-Tr decreased (3)H-mesulergine binding to 5-HTR2c-Fl at the plasma membrane in a concentration-dependent manner and more strongly with edited 5-HTR2c-Fl. These results suggest that 5-HTR2c pre-mRNA editing and splicing are entwined processes determining increased 5-HTR2c levels in pathological conditions through a deficit in 5-HTR2c-Tr.

Three-dimensional finite element analysis of the maxillary central incisor in two different situations of traumatic impact.

Dental trauma is one of the most common events in dental practice. However, few studies have investigated the biomechanical characteristics of these injuries. The objective of this study was to analyse the stress distribution in the dentoalveolar structures of a maxillary central incisor subjected to two situations of impact loading. The following loading forces were applied using a 3D finite element model: a force of 2000 N acting at an angle of 90°on the buccal surface of the crown and a vertical 2000 N force acting in the cleidocranial direction on the incisal surface of the tooth. Harmful stresses were observed in both situations, causing damage to both the tooth and adjacent tissue. However, the damage found in soft tissues such as periodontal ligament and dental pulp was negligible. In conclusion, injuries resulting from the traumatic situations were more damaging to the integrity of the tooth and its associated hard-tissue structures.

Finite element analysis applied to dentoalveolar trauma: methodology description.

Dentoalveolar traumatic injuries are among the clinical conditions most frequently treated in dental practice. However, few studies so far have addressed the biomechanical aspects of these events, probably as a result of difficulties in carrying out satisfactory experimental and clinical studies as well as the unavailability of truly scientific methodologies. The aim of this paper was to describe the use of finite element analysis applied to the biomechanical evaluation of dentoalveolar trauma. For didactic purposes, the methodological process was divided into steps that go from the creation of a geometric model to the evaluation of final results, always with a focus on methodological characteristics, advantages, and disadvantages, so as to allow the reader to customize the methodology according to specific needs. Our description shows that the finite element method can faithfully reproduce dentoalveolar trauma, provided the methodology is closely followed and thoroughly evaluated.

High-intensity physical exercise disrupts implicit memory in mice: involvement of the striatal glutathione antioxidant system and intracellular signaling.

Physical exercise is a widely accepted behavioral strategy to enhance overall health, including mental function. However, there is controversial evidence showing brain mitochondrial dysfunction, oxidative damage and decreased neurotrophin levels after high-intensity exercise, which presumably worsens cognitive performance. Here we investigated learning and memory performance dependent on different brain regions, glutathione antioxidant system, and extracellular signal-regulated protein kinase 1/2 (ERK1/2), serine/threonine protein kinase (AKT), cAMP response element binding (CREB) and dopamine- and cyclic AMP-regulated phosphoprotein (DARPP)-32 signaling in adult Swiss mice submitted to 9 weeks of high-intensity exercise. The exercise did not alter the animals' performance in the reference and working memory versions of the water maze task. On the other hand, we observed a significant impairment in the procedural memory (an implicit memory that depends on basal ganglia) accompanied by a reduced antioxidant capacity and ERK1/2 and CREB signaling in this region. In addition, we found increased striatal DARPP-32-Thr-75 phosphorylation in trained mice. These findings indicate an increased vulnerability of the striatum to high-intensity exercise associated with the disruption of implicit memory in mice and accompanied by alteration of signaling proteins involved in the plasticity of this brain structure.

Early malnutrition favours heavy ethanol intake in weaning rats without long-lasting effects.

INTRODUCTION: Although food restriction is well known to increase ethanol intake, the subject has not been extensively studied in developing animals which could be more vulnerable to long-lasting effects. Therefore, the aim of this study was to show some findings concerning this subject. MATERIALS AND METHODS: Food restriction was used to produce malnutrition either during lactation (lactating dams food restricted 60%) or after weaning (pups food restricted 60%). At weaning, day 25, males were assigned to one of the following groups: C, food ad libitum throughout the experiment - control group; MW, malnourished only after weaning; ML, malnourished only during lactation period; and MLW, malnourished throughout the experiment, during lactation and after weaning. All rats were kept isolated in cages in which they could choose to drink either a 10% ethanol solution or tap water (from days 25 to 45). Re-exposure to this model was performed on day 49. Between exposure and re-exposure, rats drank tap water for 4 days. RESULTS: There was a significant effect of malnutrition during lactation, up to day 35, with heavy drinking patterns (ethanol intake day 2: C, 8 g/kg; MW, 9 g/kg; ML, 19 g/kg; and MLW, 22 g/kg). This heavy drinking pattern lasted until the recovery of body weight. Food restriction after weaning had significant effects after 14 days, when a statistically significant decrease in body weight occurred (body weight day 39: C, 147.8 g; MW, 98.5 g). Only rats which were persistently malnourished (MLW and MW) drank ethanol more significantly than their ad libitum-fed counterparts during the re-exposure period (ethanol intake: malnourished, 5 g/kg; and well-nourished, 2.5 g/kg). Adulteration of the ethanol solution with quinine (0.1 g/l) precluded the effect of malnutrition. CONCLUSIONS: Malnutrition during early development had no long-lasting effects on ethanol consumption. In addition, malnutrition increased ethanol consumption as long as it kept body weight low, which was apparently more significant in young animals.

Behavioral toxicity of increasing doses of ethanol in malnourished rats.

In order to verify the toxicity of ethanol in malnourished rats, the following procedure was applied to two groups of rats (n = 12 each): group W: drinking water ad libitum and group E: drinking only an ethanol solution in a gradual dosage (0, 5, 10, 20 and 40% v/v). In the well-nourished phase, all rats received food ad libitum (AW and AE). Ethanol treatment (AE) was interrupted for two weeks. Rats from both AW and AE groups were submitted to food restriction (50% of AW consumption)--malnourished phase (M)--and liquid was offered as described before. Signs of ethanol intoxication were recorded daily. Ethanol withdrawal symptoms and the open-field test were performed 24 h after the well-nourished and malnourished phases. Rats were sacrificed for macroscopic evaluation of liver, spleen, thymus and biochemical analyses of the blood (hematocrit, hemoglobin, proteins and albumin). Malnourished rats showed more signs of ethanol intoxication and withdrawal. In the open-field test, malnourished rats ambulated more and made more rearing up. This effect of malnutrition was not observed during ethanol withdrawal. Consumption of ethanol decreased the levels of hemoglobin, hematocrit and total proteins. Data suggested that toxic profile of ethanol was dependent on nutritional status.

Can calories from ethanol contribute to body weight preservation by malnourished rats?

Our objective was to compare the use of calories from ethanol by well-nourished and malnourished rats in terms of body weight. Female Wistar rats weighing 170-180 g at the beginning of the study were used. The animals were divided into two groups (N = 12 each): group W received water ad libitum and group E an ethanol solution ad libitum as the only source of liquid throughout the experiment. The concentration of ethanol was increased weekly from 0 to 5, 10, 20 and 40% (v/v). In the well-nourished phase (A), all rats received food ad libitum (AW and AE). Ethanol treatment (AE) was then interrupted and water was offered to both groups. After 2 weeks both AW and AE rats were submitted to food restriction (50% of group AW food consumption), thus initiating the malnutrition phase (M). Liquid was offered as described before to the same W (MW) and E (ME) groups. The weight gain during the 1-week treatment of AE rats was similar to that of AW animals only when AE rats received the 5% (v/v) ethanol solution (9.16 vs 10.47 g). Weight loss was observed after exposure to 10% ethanol (P < 0.05) in spite of maintenance of caloric intake. Malnourished rats presented weight loss, which was attenuated by ethanol intake up to the 20% (v/v) solution and was related to an increased caloric offer. This effect was not observed with the 40% ethanol solution (-9.98 g). These data suggest that calories from ethanol were used to maintain body weight up to the concentration of 10% (v/v) (well-nourished) and 20% (v/v) (malnourished) and that ethanol has a toxic profile which depends on nutritional status.

Can calories from ethanol contribute to body weight preservation by malnourished rats?

Our objective was to compare the use of calories from ethanol by well-nourished and malnourished rats in terms of body weight. Female Wistar rats weighing 170-180 g at the beginning of the study were used. The animals were divided into two groups (N = 12 each): group W received water ad libitum and group E an ethanol solution ad libitum as the only source of liquid throughout the experiment. The concentration of ethanol was increased weekly from 0 to 5, 10, 20 and 40 percent (v/v). In the well-nourished phase (A), all rats received food ad libitum (AW and AE). Ethanol treatment (AE) was then interrupted and water was offered to both groups. After 2 weeks both AW and AE rats were submitted to food restriction (50 percent of group AW food consumption), thus initiating the malnutrition phase (M). Liquid was offered as described before to the same W (MW) and E (ME) groups. The weight gain during the 1-week treatment of AE rats was similar to that of AW animals only when AE rats received the 5 percent (v/v) ethanol solution (9.16 vs 10.47 g). Weight loss was observed after exposure to 10 percent ethanol (P < 0.05) in spite of maintenance of caloric intake. Malnourished rats presented weight loss, which was attenuated by ethanol intake up to the 20 percent (v/v) solution and was related to an increased caloric offer. This effect was not observed with the 40 percent ethanol solution (-9.98 g). These data suggest that calories from ethanol were used to maintain body weight up to the concentration of 10 percent (v/v) (well-nourished) and 20 percent (v/v) (malnourished) and that ethanol has a toxic profile which depends on nutritional status.

Pharmacological aspects of mouse hind-paw oedema induced by Lachesis muta rhombeata venom.

Pharmacological aspects of mouse hind-paw oedema induced by subplantar injections of Lachesis muta rhombeata (LMR) venom were investigated. The oedema induced by subplantar injections of 10 to 50 ng/g of LMR venom is dose dependent, with onset, peak and duration at 30, 60 and 180 min, respectively. Subplantar injection of 30 ng/g of Bothrops jararaca (BJ) venom induced oedema that has the same intensity as 30 ng/g of LMR venom but lasts for more than 4 h suggesting different time course. Systemic effects or haemorrhage were not observed with doses less than 50 ng/g. Oedema is not due to the presence of oedematogenic amines since dialysis did not change the oedema induced by 30 ng/g of LMR venom. Part of the oedema induced by LMR venom is due to a thermolabile fraction since pre-heating the venom at 100 degrees C for 15 min induced a significant reduction (56.19 +/- 6.8%) of the oedematogenic activity. The oedema induced by LMR venom is possibly induced by release of a pharmacological active substance at the site of injection. Histamine, arachidonate metabolites, nitric oxide and serotonin may play important roles in the oedematogenic effect of LMR venom since pre-treatment of mice with pyrilamine, indomethacin, dexamethasone, L-NAME and methysergide induced a significant reduction (49.86 +/- 10%; 51.06 +/- 5.9%; 77.66 +/- 3.6%; 73.30 +/- 6.1% and 93.77 +/- 2.8%, respectively) of the oedema formation. The present results demonstrate that the oedema induced by LMR and BJ venoms may be triggered and maintained by different pharmacological mechanisms. Since methysergide and L-NAME were the most active inhibitors of the oedema we can suggest that a link between serotonin release by the venom and a NO synthase activation may be an important step in the oedema formation induced by LMR venom.

Purification, properties, and N-terminal amino acid sequence of a kallikrein-like enzyme from the venom of Lachesis muta rhombeata (Bushmaster).

Pit viper venoms contain multiple proteinases which cause considerable damage in tissues and systemic effects after envenomation. A proteinase, kallikrein-like enzyme, belonging to the serine group must play a very important role on systemic effects. The corresponding enzyme from Lachesis muta rhombeata venom was purified to homogeneity by a combination of isoelectrofocusing fractionation followed by one step of gel filtration HPLC. The enzyme focused with pI 5.0-6.5, it had a molecular mass of 32 kDa by gel filtration HPLC, had edematogenic activity, and induced a hypotensic effect in anesthetized rats. It exhibited strong N-alpha-tosyl-L-Arg methyl esterase (955.38 units/mg) and N-Bz-DL-Arg-pNA amidolytic (233.02 units/mg) activities, hydrolyzed tripeptide nitroanilide derivatives weakly or not at all, and cleaved selectively the A-alpha and B-beta chains of fibrinogen, apparently leaving the Y-chain unaffected. The 30 N-terminal amino acid sequence of the L. m. rhombeata protein showed greatest identity (74% in 26 amino acids) with Crotalus viridis kallikrein-like protein, but significant similarities in sequence were observed with enzymes from other snake venoms and pig pancreatic kallikrein.

Single-step purification of crotapotin and crotactine from Crotalus durissus terrificus venom using preparative isoelectric focusing.

We describe the isolation of crotoxin, a presynaptic B-neurotoxin, as well as its subunits B (crotactine) and A (crotapotin) from lyophilized Crotalus durissus terrificus venom by a single-step preparative isoelectric focusing procedure. From 98 mg of dried venom protein 20.1 mg of crotactine and 13.1 mg of crotapotin were recovered in the first step of focalization and 4.2 mg in a second run. These values correspond to 35.7% of the total venom protein applied. Crotactine separated in the 9.3-7.0 pH range (tubes 1-6) and crotapotin in the 1.8-2.8 pH range (tubes 15-19) and both were homogeneous by SDS-PAGE and N-terminal amino acid analysis. Crotactine, a 12-kDA protein, presented hemolytic and phospholipase A2 activity. Thus, using isoelectric focusing we simultaneously purified both toxins in high yields. This method can be used as an alternative for the purification and characterization of proteins from other snake venoms under conditions in which biological activity is retained.

Single-step purification of crotapotin and crotactine from Crotalus durissus terrificus venom using preparative isoeletric focusing

We describe the isolation of crotoxin, a presynaptic B-neurotoxin, as well as its subunits B (crotactine) and A (crotapotin) from lyophilized Crotalus durissus terrificus venom by a single-step preparative isoelectric focusing procedure. From 98 mg of dried venom protein 20.1 mg of crotactine and 13.1 mg of crotapotin were recovered in the first step of focalization and 4.2 mg in a second run. These values correspond to 35.7 per cent of the total venom protein applied. Crotactine separated in the 9.3-7.0 pH range (tubes 1-6) and crotapotin in the 1.8-2.8 pH range (tubes 15-19) and both were homogeneous by SDS-PAGE and N-terminal amino acid analysls. Crotactine, a 12-kDa protein, presented hemolytic and phospholipase A2 activity. Thus, using isoelectric focusing we simultaneously purified both toxins in high yields. This method can be used as an altemative for the purification and characterization of proteins from other snake venoms under conditions in which biological activity is retained.

Purification and partial characterization of a thrombin-like/gyroxin enzyme from bushmaster (Lachesis muta rhombeata) venom.

The acidic coagulating enzyme of the L. m. rhombeata venom was purified to homogeneity using one step on preparative isoelectric focusing followed by gel permeation on a high performance liquid chromatography system. The enzyme focused with pIs 3.1-5.0 and had a molecular mass of 47,000 mol. wt as determined by high performance liquid gel-filtration chromatography and about 45,000 mol. wt as judged by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. The enzyme is a glycoprotein containing sialic acid and 12.4% of neutral carbohydrates. The 30 N-terminal amino acid sequence of the L. m. rhombeata protein shows 100% identity with L. m. muta gyroxin and considerable sequence homology with gyroxin and thrombin-related proteins. The enzyme exhibits strong N-p-tosyl-L-arginine methyl esterase activity, hydrolyses tripeptide nitroanilide derivatives weakly or not at all, and cleaves specifically the fibropeptide A (alpha-chain).