Features of myoneural junctions in the extraocular muscles of the opossum (Didelphis albiventris)

The myoneural junctions present in the opossum (Didelphis albiventris) extraocular muscles were studied through histochemical and ultrastructural techniques. Three different types of junction were shown: two single types, 'en grappe' and 'en plaque', present in the middle third of the muscle fibers; and one multiple type, more rare and observed in the distal third of the muscle.     

Ultrastructural pattern of glucagon producing-cells in the gastric mucosa of the developing opossum Didelphis albiventris (Marsupialia)

We present the case of a 59-year-old woman with an International Federation of Gynecology and Obstetrics stage IIIB serous psammocarcinoma. Only 12 cases of this rare ovarian neoplasm have been reported previously. These tumors act like borderline tumors and, therefore, do not usually require chemotherapy or radiation therapy after appropriate surgical debulking and staging.     

Inhibitory properties of the antibothropic complex from the South American opossum (Didelphis marsupialis) serum

The South American opossum Didelphis marsupialis is known to be highly resistant to snake envenomation. In this paper it is shown that the opossum serum inhibits haemorrhage induced by both Crotalinae and Viperinae venoms. Tested against Bothrops jararaca (jararaca) venom, the antibothropic complex (ABC) isolated from the opossum serum was at least six times more antihaemorrhagic than the commercial antivenom. ABC showed no proteolytic activity by itself and was not hydrolysed by the venom. It inhibited the hydrolysis of casein by B. jararaca venom, but did not inhibit its hydrolytic activities upon N alpha-benzoyl-L-arginine ethyl ester (BAEE) and N alpha-benzoyl-DL-arginine p-nitroanilide (BAPNA). The inhibitor did not interfere with trypsin and bacterial collagenase activities on BAPNA and N-(3-[2-furyl]acryloyl)-Leu-Gly-Pro-Ala (FALGPA), respectively. It reduced chymotrypsin hydrolysis of N-acetyl-L-tyrosine ethyl ester (ATEE) because ABC is also a substrate for this enzyme. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, B. jararaca venom preferentially degraded fibrinogen A alpha-chain and fibrin alpha-chain. Tested on extracellular matrix proteins, the venom hydrolysed collagen IV, gelatins I and V, laminin and fibronectin, besides depolimerizing collagen I alpha-chain dimers. Fibrillar collagen V was not digested. These hydrolyses were inhibited by ABC and by EDTA. Our results show that the antibothropic complex is a venom metalloproteinase inhibitor, which could, at least partially, account for its antihaemorrhagic activity. Electrophoretic evidence indicated non-covalent complex formation between the antihaemorrhagic factor and component(s) of B. jararaca venom.     

The detection of hemorrhagic proteins in snake venoms using monoclonal antibodies against Virginia opossum (Didelphis virginiana) serum

Most snakes and a few warm-blooded animals have a resistance to snake venoms because of naturally occurring antihemorrhagins found in their sera. The antihemorrhagins in serum of Virginia opossum (Didelphis virginiana) neutralize hemorrhagic activity by binding to hemorrhagins in snake venoms. The binding characteristic of antihemorrhagins in D. virginiana serum was used to develop a five-step western blot. The detection of hemorrhagic proteins were measured indirectly with antihemorrhagins in Virginia opossum serum and with DV-2LD#2, a monoclonal antibody specific for Virginia opossum antihemorrhagins. Snake venoms were separated by native-PAGE, transferred to a Millipore Immobilon-P membrane and then incubated with crude Virginia opossum serum. The hemorrhagins in snake venom bind to antihemorrhagins in Virginia opossum serum which react with DV-2LD#2 a monoclonal antibody that is specific for Virginia opossum antihemorrhagins. DV-2LD#2 monoclonal antibody inhibits antihemorrhagic activity in Virginia opossum serum when mixed in equal amounts. The inhibition of antihemorrhagins by DV-2LD#2 monoclonal antibody suggests specificity. DV-2LD#2 monoclonal antibody does not recognize antihemorrhagins in gray woodrat (Neotoma micropus) serum. The five-step western blot reveals two well-defined bands which represent hemorrhagins found in Western diamondback rattlesnake (Crotalus atrox) venom. Venoms from 15 different snake species were examined to determine the usefulness of the five-step western blot. Other hemorrhagic venoms (Great Basin rattlesnake (C. viridis lutosus), Prairie rattlesnake (C. viridis viridis), Tancitaran dusky rattlesnake (C. pusillus), Northern Mojave rattlesnake (C. scutulatus scutulatus type B) and Northern Pacific rattlesnake (C. v. oreganus)) had one single band in the five-step western blot. DV-2LD#2 did not bind to the non-hemorrhagic venoms and reacted with 50% of the hemorrhagic venoms used in this study. The monoclonal antibody, CAH, reacted with all the hemorrhagic venoms except for the venom of the King cobra (Ophiophagus hannah) and did not react with the non-hemorrhagic venoms. The hemorrhagic binding site of CAH monoclonal antibody and the antihemorrhagin in Virginia opossum are different binding sites. The five-step western blot will be a very useful assay for determining hemorrhagic activity without using live animals.     

Isolation in immunodeficient mice of Sarcocystis neurona from opossum (Didelphis virginiana) faeces, and its differentiation from Sarcocystis falcatula

BACKGROUND: There has been conflicting evidence of the effect of angiotensin-converting enzyme (ACE) inhibitors on exercise tolerance. Meta-analysis of published results has suggested that a beneficial effect of ACE inhibitors is demonstrated if a trial design is adequate. SETTING: Multicentre International Trial. METHODS: In a double-blind, randomized, multicentre trial, 292 patients with moderate (New York Heart Association Grades II and III) heart failure were treated with trandolapril or placebo in addition to diuretics, and followed for 16 weeks. Exercise tolerance on a treadmill was assessed at baseline and after 4, 8, 12 and 16 weeks of treatment. Both a modified Bruce and a modified Naughton protocol were used. RESULTS: Exercise tolerance improved in both treatment groups, with no significant benefit from trandolapril treatment. CONCLUSION: Trandolapril does not improve exercise tolerance as measured by treadmill testing.     

Primary structure of two-chain botrocetin, a von Willebrand factor modulator purified from the venom of Bothrops jararaca

The complete amino acid sequence and location of the disulfide bonds of two-chain botrocetin, which promotes platelet agglutination in the presence of von Willebrand factor, from venom of the snake Bothrops jararaca are presented. Sequences of the alpha and beta subunits were determined by analysis of peptides generated by digestion of the S-pyridylethylated protein with Achromobacter protease I or alpha-chymotrypsin and by chemical cleavage with cyanogen bromide or 2-(2'-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine. Two-chain botrocetin is a heterodimer composed of the alpha subunit (consisting of 133 amino acid residues) and the beta subunit (consisting of 125 amino acid residues) held together by a disulfide bond. Seven disulfide bonds link half-cystine residues 2 to 13, 30 to 128, and 103 to 120 of the alpha subunit; 2 to 13, 30 to 121, and 98 to 113 of the beta subunit; and 80 of the alpha subunit to 75 of the beta subunit. In terms of amino acid sequence and disulfide bond location, two-chain botrocetin is homologous to echinoidin (a sea urchin lectin) and other C-type (Ca(2+)-dependent) lectins.     

Effect of metoclopramide, ranitidine, and droperidol on the lower sphincter of the esophagus. Experimental study on the opossum (Didelphis albiventris)

Electromanometric measures of the gastroesophageal junction were performed in 20 adult, male and female, anesthetised opossums. The electromanometric examinations were performed according the intermitent pull through technique. The research was divided in four groups, according to the drug to be analysed: group 1 (20 animals) IM injection of physiological solution (control group); group 2 (20 animals) IM injection of metoclopramide; group 3 (20 animals) IM injection of ranitidine; group 4 (20 animals) IM injection of droperidol. Electromanometry was done 15 minutes before the drug injection, just after the injection and 15, 30, 45 and 60 minutes after the injection. In each one of the moments the pressure of the lower esophageal sphincter (LES-mmHg) was analysed. Considering LES pressure the results observed were: in group 1 it was not observed any significative alteration after IM injection of physiologic solution; in group 2 it was observed significative pressure increase, 15 minutes after metoclopramide IM injection; in group 3 it was observed pressure increase, being significative at 15 and 30 minutes after IM injection; in group 4 it was observed significative increase in LES pressure in every moment, 15 minutes after droperidol IM injection.     

Neutralizing capacity of ++antiophidic sera against the venom of Bothrops moojeni (lance-headed viper)

Human oncostatin M (OM) is a M(r) 28,000 glycoprotein that has been shown to regulate cell proliferation and differentiation. The biological activities of OM can be mediated by two different heterodimeric receptor complexes, the leukemia inhibitory factor (LIF)/OM shared receptor and the OM-specific receptor. In this study, we have examined the growth-regulatory effect of OM on 10 breast cancer cell lines derived from human tumors. The cellular proliferation of seven of these breast cancer cell lines was inhibited by OM. The three cell lines that did not respond to OM treatment lacked the expression of OM receptors. The growth-inhibitory activity of OM is examined further in the H3922 breast cancer cell line, which expresses the high-affinity OM receptor at a relatively higher level. We found that the cellular proliferation of H3922 cells was induced strongly by extrogenous epidermal growth factor (EGF), EGF-like factor, and basic fibroblast growth factor. The proliferative activities of these growth factors can be abolished totally by cotreatment of H3922 cells with OM. Treatment of H3922 cells with OM for 24 h did not block EGF binding or the induction of EGF receptor tyrosine phosphorylation. This finding suggests that OM interferes with the mitogenic signal at steps distal to the EGF receptor. Examination of proto-oncogene expression demonstrated that OM down-regulates the c-myc gene in H3922 cells. The biological effects reported herein are not shared by the OM-related cytokines interleukin 6 or LIF, as demonstrated by the inability of these proteins to inhibit cell growth or modulate c-myc gene expression in breast cancer cells. Additionally, the high-affinity binding of labeled OM cannot be displaced by LIF. Together, these data suggest that OM is a growth inhibitor for breast cancer cells. The inhibitory activity is mediated predominantly through the OM-specific receptor, and activation of this receptor abrogates growth factor stimulation and down-regulates the c-myc proto-oncogene.     

Isolation: analysis and properties of three bradykinin-potentiating peptides (BPP-II, BPP-III, and BPP-V) from Bothrops neuwiedi venom

Several methods of external and internal fixation are used in urgent situations to lessen intrapelvic bleeding associated with unstable pelvic fractures. Pelvic stabilization limits pelvic expansion and thereby restricts the space for potential blood loss. This study compared several fixation methods using cadaveric pelves to determine which method best prevents pelvic expansion. Three methods of internal fixation and three methods of external fixation were compared. Anteroposterior fixation provided the greatest control against pelvic expansion; however, it is clinically impractical for emergency use. Therefore, external fixation provided the most reliable control of pelvic expansion in the emergency setting.     

Purification and characterization of a kinin-releasing and fibrinogen-clotting serine proteinase (KN-BJ) from the venom of Bothrops jararaca, and molecular cloning and sequence analysis of its cDNA

Two forms of a proteinase, KN-BJ 1 and 2, were purified to homogeneity from the venom of Bothrops jararaca. In SDS/PAGE reduced KN-BJ 1 and 2 migrated as single bands with molecular masses of 38 kDa and 39 kDa. The two enzymes have similar N-terminal amino acid sequences and specific activities on synthetic chromogenic substrates, and both release bradykinin from bovine low-molecular-mass kininogen. KN-BJ 1 and KN-BJ 2 clot fibrinogen with specific activities of 245 NIH U/mg and 219 NIH U/mg, releasing only fibrinopeptide A. The amidolytic, kinin-releasing and coagulant activities are inhibited by phenylmethylsulfonyl fluoride, demonstrating that KN-BJ is a serine proteinase. Benzamidine derivatives, which are competitive inhibitors of trypsin-like proteinases, also inhibited the amidolytic activity of KN-BJ. A cDNA clone (HS104, 2.2 kb) has been isolated from a cDNA library of B. jararaca venom glands with an ORF of 771 bp. The deduced amino acid sequence contains segments that are identical to the sequences of the N-terminus and three tryptic peptides of KN-BJ 2. Therefore, the cDNA is believed to represent the gene of KN-BJ 2. The deduced amino acid sequence indicates that KN-BJ 2 is synthesized as a prezymogen of 257 amino acids with a putative signal peptide of 18 amino acids and an activating peptide of six amino acid residues. The sequence of 233 amino acids representing the mature enzyme exhibits high similarity to sequences of serine proteinases isolated from crotalid venoms.     

Isolation and characterization of a myotoxic phospholipase A2 from the venom of the arboreal snake Bothriechis (Bothrops) schlegelii from Costa Rica

PURPOSE: To evaluate the cytotoxicity of immunotoxin 4197X-ricin A (4197X-RA) and its ability to inhibit protein synthesis and human lens epithelial cell (LEC) proliferation on the inner surface of the lens capsule. SETTING: Houston Biotechnology, Inc., The Woodlands, Texas. METHODS: A cell culture system was established using human LECs as a model for the proliferation of remnant LECs that occurs during posterior capsule opacification (PCO) after extracapsular cataract extraction. The LEC culture system was also used in vitro for testing compounds that might inhibit this process in vivo. Human LECs were cultured on the surface of the original lens capsule fixed to collagen. Variability was reduced by dissecting each lens capsule into equivalent halves and exposing the segments to immunotoxin 4197X-RA. RESULTS: Protein synthesis and LEC proliferation were almost completely inhibited at relatively low 4197X-RA concentrations after short exposure. The inhibitory effects persisted up to 3 weeks after withdrawal of the immunotoxin and after several media exchanges. CONCLUSION: Immunotoxin 4197X-RA may help prevent PCO after primary cataract surgery.     

Purification from Bothrops lanceolatus (fer de lance) venom of a fibrino(geno)lytic enzyme with esterolytic activity

Bothrops lanceolatus venom has high caseinolytic, phospholipasic, esterolytic and hemorrhagic activities. In spite of having no coagulant effect on plasma, this venom contains a thrombin-like enzyme. Using gel filtration and ion-exchange chromatographies, we have purified an esterolytic fraction (F-II-1a) from this venom with a protein yield of 4% and a 58% recovery in enzyme activity. SDS-PAGE in the presence of beta-mercaptoethanol showed that the enzyme is a single chain polypeptide with a MW=38,100. Immunodiffusion and immunoelectrophoresis of fraction F-II-1a against serum from horses immunized with B. lanceolatus venom and against rabbit antiserum prepared using fraction F-II-1a both showed a single immunoprecipitin line. The Km and Vmax values for TAME hydrolysis were 0.85 mM and 38.6 micromol/min/mg, respectively. The esterolytic activity was completely inhibited by PMSF (10 mM) but not by EDTA (20 mM). Fraction F-II-1a hydrolyzed the alpha and beta chains of fibrinogen. Degradation of the alpha chain occurred within 10 min while that of the beta-chain was slower. The enzyme had no effect on the gamma-chain even after 4 h of hydrolysis.     

Neutralization of local tissue damage induced by Bothrops asper (terciopelo) snake venom

Local tissue damage represents a serious consequence of Bothrops asper envenomations. It encompasses a complex series of alterations, including myonecrosis, dermonecrosis, hemorrhage and edema. Due to its rapid development it is difficult to neutralize by antivenoms, especially if there is a delay in serotherapy. Experimental studies with this venom and the polyvalent (Crotalinae) antivenom produced in Costa Rica indicate that antivenom is effective in neutralizing these toxic activities when incubated with the venom prior to injection. However, if venom and antivenom are injected independently in mice, neutralization of these effects is only partial. Moreover, neutralization is not complete even if homologous or heterologous antibodies are present in the circulation before venom is injected. Despite differences in their pharmacokinetic profiles, equine whole IgG and F(ab')2 antivenoms show similar efficacy in the neutralization of edema, hemorrhage and myonecrosis induced by B. asper venom, suggesting that the use of antivenoms made of antibody fragments may not improve neutralization of these effects. This is due, at least in part, to the fact that microvessel disruption by venom components favors a similar antibody concentration in the affected tissues. Recent advances in the development of neutralizing substances of rapid diffusion, that could be injected locally in the field, may contribute to the neutralization of metalloproteinases and phospholipases A2. In addition, the rapid administration of antivenoms with high antibody titers against locally-acting toxins is very important in the treatment of these effects.     

Isolation of a pentraxin-like protein from rainbow trout serum

To investigate the outcome of Graves' thyrotoxicosis after antithyroid drug management, data from 81 patients, treated in Chang Gung Memorial Hospital at Taipei and Linkou from October 1981 to March 1990, were analyzed. The gender ratio of female to male was 59:22. The mean age of onset was 33.1 +/- 10.5(15-60) year-old. All the patients were treated with antithyroid drug (Thionamide group) for a duration of 11 to 63 months (mean +/- SD = 28.1 +/- 9.8 months). Forty of 81 patients (49.4%) were remained remission after up to 2 years of follow-up. Those patients relapse usually occurred within 2 years after discontinuation of treatment (34/41), and only one exceptional case relapsed after 3 years. Three conditions affected the relapse rate. Patients with larger goiter (grade II-III) and shorter duration of treatment (< 23 months) had a higher relapse rate than those-with smaller goiter (grade O-I) [29/46 vs. 12/35; chi 2 = 6.576, p = 0.010; p = 0.015 in stepwise logistic regression (LR)] and longer duration of treatment (> or = 23 months) (15/20 vs. 26/61; chi 2 = 6.316, p = 0.012; p = 0.020 in LR). Patients with higher pre-treated serum triiodothyronine (T3) level (T3 > or = 300 ng/dl) had a higher relapse rate than those with lower T3 level (T3 < 300 ng/dl) in univariate analysis (30/50 vs. 11/31, chi 2 = 4.601, p = 0.032), but no significant difference by LR (P = 0.094). Other clinical parameters including age, sex, past history, family history, thyroxine (T4) level, T3/T4 ratio, thyroid autoantibodies, staging of ophthalmopathy, responsiveness to thyrotropin-releasing hormone stimulation test at the end of treatment, and whether combined treatment with thyroxine had no significant difference between the relapse and remission groups. These data suggest: (a) patients with larger goiter (grade II-III had higher relapse rate; (b) most of the recurrent thyrotoxicosis patients relapsed within two years after drug withdrawal; (c) continuing treatment for more than twenty-three months produces better outcome; (d) patients with Graves' thyrotoxicosis should be followed up for at least three years after withdrawal of antithyroid drug.     

North American opossum Didelphis virginiana as a fetal nephrotoxicity model: histologic and ultrastructural assessment of uranyl nitrate (UN)-induced damage

Individuals report a variety of reasons for having sex. Understanding these reasons can improve HIV and STD prevention efforts because they may constitute an important component in the aetiology of sexual risk-taking behaviours. Relationships between self-reported reasons for having sex and frequency of participation in sexual practices among 146 heterosexual men recruited from public STD clinics in Southern California were examined. Using a self-administered questionnaire, respondents reported how often they engaged in sex for each of 16 reasons and how frequently they participated in high, moderate, and low-risk sexual practices. A principal components analysis identified five factors used to construct scales: love; compliance; pleasure; altered states; and potency. Higher-risk sexual practices were positively associated with the pleasure and potency scales, whereas lower-risk practices were positively associated with the love scale. These findings suggest that some reasons men report for having sex may influence sexual risk-taking. Interventions to reduce unsafe sex should explicitly address how men can practise safer sex and still experience pleasure and potency.     

Isolation of myotoxic component from rattlesnake (Crotalus viridis viridis) venom. Electron microscopic analysis of muscle damage

The pathogenesis of myonecrosis induced by a purified component of rattlesnake (Crotalus viridis viridis) venom was studied at the light and electron microscopic levels. Crude venom was fractionated by gel filtration (Sephadex G-50) followed by cation exchange chromatography (Sephadex C-25). Electrophoretic homogeneity of the isolated myotoxin (Fraction II from C-25 column) was demonstrated in isoelectric focusing and disc gel polyacrylamide gel electrophoresis. White mice were injected intramuscularly with 1.5 mug/g of the purified protein in 0.1 ml of physiologic saline. Light microscopic examination of injected muscle revealed a series of degenerative events including partial vacuolation of muscle cells at 6, 12, and 24 hours and complete vacuolation and loss of striations at 48 and 72 hours. Hemorrhage was not observed. At the electron microscopic level the perinuclear space and sarcoplasmic reticulum were dilated in all samples. By 48 and 72 hours the myofibrils lacked striations and the sarcomeres were disorganized. Plasma membranes and T tubules remained intact in all samples. These results correlated well with the myonecrosis induced by crude Crotalus viridis viridis venom except for several important aspects. The pure component altered skeletal muscle cells specifically, with the sarcoplasmic reticulum being the primary site of action.