Development of a hybrid cell for energy production.

This study focuses on the development of a new hybrid biological material to be applied in the production of electrical energy. These organo-metallic cells are constituted by cyanobacteria (Fischerella muscicola) and silver nanoparticles (AgNPs). AgNPs were obtained by green synthesis using the extract of the fruit of theBerberis halliiplant as reducing agent with two different concentrations of silver nitrate (AgNO3), 1 and 10 mM. The morphology, physicochemical and electrical properties of the cyanobacteria with and without AgNPs were evaluated. To verify the efficacy of this new material, and the effect of the medium used, Nitrofoska or BG-11, the growth kinetics was evaluated by UV-vis up tot= 63 d with and without renewal of the culture medium and O2/CO2exchange. Through morphological characterizations ofFischerella muscicolait was possible to identify the presence of an associated bacterium identified using molecular techniques asPseudomona guguanensithat could act as a supporting organism in the growth of this cyanobacteria. The studies carried out did not shown cell toxicity for the cultures that have AgNPs and on the other hand, it was observed that the hybrid cells (Cy-AgNPs) are electron carriers recording an increase of up to 57% and 18% in their electrical potential with BG-11 and Nitrofoska culture media, respectively and an increase in the anodic current peak of 6.5% of Cy-AgNPs respect to onlyF. musicola.

Sputum induction for the diagnosis of pulmonary tuberculosis: a systematic review and meta-analysis.

Sputum induction (SI) has been proposed as the optimal sample collection method for patients with paucibacillary tuberculosis (TB). Studies reporting the culture of Mycobacterium tuberculosis from SI were reviewed. A random-effects meta-analysis of diagnostic yield (numerator M. tuberculosis SI culture-positive cases; denominator all culture-positive cases) was conducted. Diagnostic yields (95% confidence intervals, CIs) were displayed as Forest plots. Heterogeneity was evaluated using Chi-squared and I-squared tests and meta-regression analysis. Ninety publications were screened, 28 full-text papers reviewed, and 17 analyzed. Collectively, n=627 SI culture-positive cases among n=975 culture-confirmed TB cases were reported. The diagnostic yield of SI ranged from 35 to 95%. The pooled diagnostic yield was 74% (CI 65-81%), with significant heterogeneity (p<0.0001, I2=86%). There were no statistically significant differences in the yield between sub-groups defined by human immunodeficiency virus (HIV) prevalence or age. Univariate analysis demonstrated that the use of fiberoptic bronchoscopy (FOB) as the comparator method was associated with a 22% reduction (CI 2-42%) in the diagnostic yield of SI. However, after adjustment for confounding, the meta-regression analysis showed that FOB usage (p=0.21) and saline concentration (p=0.31) were not independently associated with the diagnostic yield. SI will detect approximately three-quarters of M. tuberculosis culture-positive cases under study conditions. Significant heterogeneity in the diagnostic yield was not explained by HIV prevalence, age, or the use of FOB as the comparator method. The use of a particular nebulized saline concentration for SI cannot be recommended on the basis of this meta-regression analysis.

Tyr-->Trp-substituted peptide 115-129 of a Lys49 phospholipase A(2) expresses enhanced membrane-damaging activities and reproduces its in vivo myotoxic effect.

Myotoxin II is a group II Lys49 phospholipase A(2) (PLA(2)) isolated from the venom of the snake Bothrops asper. Previous studies on a synthetic peptide derived from its heparin-binding, cationic/hydrophobic sequence 115-129 demonstrated a direct functional role of this particular region in the in vitro cytolytic and bactericidal actions of the protein. Nevertheless, no significant myonecrosis has been observed after local intramuscular injection of peptide 115-129 (p115-129) in mice. Since the membrane-damaging action of p115-129 was proposed to depend on its amphiphilic character, the present study examined the effects of substituting its cluster of three tyrosine residues by tryptophan residues, on its toxic/pharmacological activities in vitro and in vivo. This substitution resulted in a drastic enhancement of the membrane-damaging activities of the peptide (p115-W3), together with the clear expression of myotoxic activity in vivo. Both the heparin-binding and antigenic characteristics of p115-129 were essentially conserved in p115-W3, suggesting that the modification did not lead to radical structural alterations. In addition to myotoxicity, cytotoxicity, and bactericidal action, p115-W3 exerted edema-forming activity in the mouse footpad assay. Thus, the synthetic 13-mer p115-W3 reproduced all the known toxic effects of myotoxin II. In spite of its potent membrane-damaging actions, p115-W3 did not acquire direct hemolytic activity upon mouse erythrocytes, an effect which is not present in myotoxin II, but that has been ascribed to the presence of tryptophan in other cationic, membrane-damaging peptides such as mellitin from bee venom. The myotoxic activity of p115-W3 herein described constitutes the first example of a short, PLA(2)-based linear synthetic peptide with the ability to reproduce this effect of a parent protein in vivo. This finding is in clear support of the proposed relevance of the C-terminal region 115-129 in all the membrane-damaging mechanisms exerted by myotoxin II, including the myotoxic mechanism.

Isolation and characterization of myotoxin II from Atropoides (Bothrops) nummifer snake venom, a new Lys49 phospholipase A2 homologue.

Myotoxic phospholipases A2 of class II are commonly found in the venoms of crotalid snakes. As an approach to understanding their structure-activity relationship, diverse natural variants have been characterized biochemically and pharmacologically. This study describes a new myotoxic phospholipase A2 homologue, isolated from the venom of Atropoides (Bothrops) nummifer from Costa Rica. A. nummifer myotoxin 1 is a basic protein, with an apparent subunit molecular mass of 16 kDa, which migrates as a dimer in sodium dodecylsulfate-polyacrylamide gel electrophoresis under nonreducing conditions. It is strongly recognized by antibodies generated against Bothrops asper myotoxin II, by enzyme-immunoassay. The toxin induces rapid myonecrosis upon intramuscular injection in mice (evidenced by an early increase in plasma creatine kinase activity), and significant edema in the footpad assay. It also displays cytolytic activity upon cultured murine endothelial cells. The toxin (up to 50 microg) has no detectable phospholipase A2 activity on egg yolk phospholipids, and does not show an anticoagulant effect on sheep platelet-poor plasma in vitro. N-terminal sequence determination (53 amino acid residues) demonstrated that A. nummifer myotoxin II is a new Lys49 variant of the family of myotoxic, class II phospholipases A2. Sequence comparison with other phospholipases A2 revealed Asn53 as a novel substitution. In addition, this myotoxin is the first Lys49 variant presenting Asn in its N-terminus. Consequently, these findings suggest that neither Ser1 or Lys53, usually found in this family of proteins, are essential amino acid residues for their myotoxic, cytolytic, or edema-inducing effects.

Myotoxic phospholipases A(2) in bothrops snake venoms: effect of chemical modifications on the enzymatic and pharmacological properties of bothropstoxins from Bothrops jararacussu.

Venoms from eight Bothrops spp. were fractionated by ion-exchange chromatography on CM-Sepharose at pH 8.0 for the purification of myotoxins. Chromatographic profiles showed differences regarding myotoxic components among these venoms. B. alternatus, B. atrox and B. jararaca venoms did not show the major basic myotoxic fractions identified in the other venoms. Polyacrylamide gel electrophoresis for basic proteins also showed distinct patterns for these toxins. In vivo, all the isolated myotoxins induced release of creatine kinase due to necrosis of muscle fibers, accompanied by polymorphonuclear cell infiltration, and edema in the mouse paw. In addition, the toxins showed cytotoxic and liposome-disrupting activities in vitro. B. jararacussu bothropstoxins-I (BthTX-I) and II (BthTX-II) were submitted to chemical modifications of: His, by 4-bromophenacyl bromide (BPB) or photooxidation by Rose Bengal (RB); Tyr, by 2-nitrobenzenesulphonyl fluoride (NBSF); and Trp, by o-nitrophenylsulphenyl chloride (NPSC). The myotoxic and cytotoxic activities of BthTX-I, a Lys49 PLA(2) homologue, after modification by BPB, RB, NBSF and NPSC, were reduced to 50%, 20%, 75%, 65% and 13%, 0.5%, 76%, 58%, respectively. However, the edema-inducing and liposome-disrupting activities were not significantly reduced by the above modifications. BPB-treated BthTX-II, an Asp49 PLA(2) homologue, lost most of its catalytic, indirect hemolytic, anticoagulant, myotoxic and cytotoxic activities. The edema-inducing and liposome-disrupting activities were reduced to 50% and 80%, respectively. Lethality caused by BthTX-I and -II was strongly reduced after treatment with BPB or RB, but only partially with NBSF or NPSC. BthTX-I and -II, both native or modified, migrated similarly in a charge-shift electrophoresis. Antibodies raised against BthTX-I or -II, B. asper Basp-II and the C-terminal 115-129 peptide from Basp-II did not show significant differences in their cross-reactivity with the modified toxins, except with RB photooxidized toxins.

Clinical and laboratory alterations in horses during immunization with snake venoms for the production of polyvalent (Crotalinae) antivenom.

Six horses were immunized with the venoms of Bothrops asper, Crotalus durissus durissus and Lachesis muta stenophrys for the production of polyvalent (Crotalinae) antivenom. During the immunization, clinical and laboratory alterations were evaluated in these animals, and the development of humoral immune response was followed. Only moderate local tissue changes (edema, abscesses, fistules and fibrosis) were observed in these animals, whereas no systemic alterations occurred. Regarding laboratory tests, there was a drop in hemoglobin concentration and hematocrit, together with an increment in total serum protein. Horses developed a moderate leukocytosis, with increments in polymorphonuclear leukocytes and lymphocytes. No significant changes were observed in prothrombin time or platelet count. There were no alterations in serum lactic dehydrogenase and gamma glutamyl transferase activities, whereas minor increments in creatine kinase and alanine aminotransferase activities were observed, together with a decrease in aspartate aminotransferase. All these changes occurred after the injection of 9 mg venom, when sodium alginate was first used as adjuvant. Creatinine levels had a small increment, although no changes were observed in urea levels or in the urea/creatinine ratio. An important individual variability was observed in the humoral immune response, as judged not only by enzyme-linked immunosorbent assay, but also by assessing the neutralization of the indirect hemolytic activity of venoms.

Identification of the myotoxic site of the Lys49 phospholipase A(2) from Agkistrodon piscivorus piscivorus snake venom: synthetic C-terminal peptides from Lys49, but not from Asp49 myotoxins, exert membrane-damaging activities.

Group II phospholipase A(2) (PLA(2)) myotoxins found in the venoms of Crotalidae snakes can be divided into 'Asp49' and 'Lys49' isoforms, the latter being considered catalytically-inactive variants. Previous studies on one Lys49 isoform, myotoxin II from Bothrops asper, indicated that its myotoxic activity is due to the presence of a short cationic/hydrophobic sequence (115-129) near its C-terminus, which displays membrane-damaging properties. Since the C-terminal region of different group II PLA(2) myotoxins presents considerable sequence variability, synthetic peptides homologous to region 115-129 of myotoxin II, but corresponding to B. asper myotoxin III (Asp49), Agkistrodon piscivorus piscivorus Asp49 PLA(2) and Lys49 PLA(2), were studied to determine the possible functional relevance of such region for the toxic activities of these proteins. Results showed that both Lys49-derived peptides (p-BaK49 and p-AppK49) were able to lyse skeletal muscle C2C12 cells in culture, and to induce edema in the mouse footpad assay. Moreover, p-AppK49, which showed a markedly stronger cytotoxic potency than p-BaK49, additionally induced skeletal muscle necrosis when injected into mice. These observations unequivocally identify the sequence 115-129 (KKYKAYFKLKCKK) of the Lys49 PLA(2) of A. p. piscivorus as containing the key structural determinants needed for myotoxicity, and represent the first report of an unmodified, PLA(2)-derived short synthetic peptide with the ability to reproduce this effect of a parent toxin in vivo. On the other hand, the two Asp49-derived peptides did not show any toxic effects in vitro or in vivo, even at high concentrations. These findings suggests that Lys49 and Asp49 group II PLA(2)s might exert their myotoxic actions through different molecular mechanisms, by implying that the latter may not utilize their C-terminal regions as main membrane-destabilizing elements.

Characterization of a basic phospholipase A2-homologeu myotoxin isolated from the venom of the snake Bothrops neuwiedii (yarará chica) from Argentina.

A basic protein was isolated by CM-Sephadex C-25 chromatography from the venom of Bothrops neuwiedii from Argentina, and named B. neuwiedii myotoxin I. This protein exerted local myotoxic and edema-forming effects in mice, with potencies comparable to other myotoxins isolated from Bothrops spp. venoms. When injected by i.v. route at doses up to 4.7 mg/kg of body weight, the toxin was not lethal. In vitro, the toxin had no detectable phospholipase A2 activity on egg yolk phospholipids. B. neuwiedii myotoxin I appeared as a homodimer in sodium dodecylsulphate-polyacrylamide gel electrophoresis, with a subunit molecular weight of 15 kD. Gel immunodiffusion revealed a pattern of partial antigenic identity between the newly isolated myotoxin and myotoxin II from Bothrops asper venom. The sequence of B. neuwiedii myotoxin I was determined for the first 40 amino acid residues, showing high homology to several class II phospholipase A2 myotoxins of the Lys-49 family from crotalids. Altogether, results suggest that this toxin is a new member of the Lys-49 phospholipase A2-homologues with myotoxic, cytolytic, and edema-inducing activities.

Immunochemical properties of the N-terminal helix of myotoxin II, a lysine-49 phospholipase A(2) from Bothrops asper snake venom.

Myotoxic class II phospholipases A(2) from snake venoms can be divided into Asp49 and Lys49 types. The latter, including Bothrops asper myotoxin II, exert membrane damage despite lacking catalytic activity. A heparin-binding, hydrophobic/cationic region, near the C-terminus of myotoxin II (115-129) has been shown to be relevant in its membrane-damaging actions. However, some observations suggest also a potential participation of its N-terminal region. An immunochemical approach was utilized to examine the properties and possible role in toxicity of the N-terminal helix of myotoxin II. Rabbit antibodies raised to a synthetic peptide comprising residues 1-15 recognized the native protein. These antibodies were utilized to compare the antigenic characteristics of the N-terminal helix of several myotoxic phospholipases A(2), showing generally stronger binding to Lys49 myotoxins, in comparison to Asp49 counterparts. However, three Lys49 myotoxins (Cerrophidion godmani myotoxin II, Atropoides nummifer myotoxin II, and Trimeresurus flavoviridis basic protein I) were not recognized by the antibodies, revealing a significant antigenic variability of the N-terminal region within this group of toxins. In neutralization experiments, pre-incubation of myotoxin II with affinity-purified antibodies to the N-terminal helix did not inhibit its myotoxic activity in mice, nor its cytotoxic effect upon cultured muscle cells. These findings argue against a critical role of the N-terminal region of this protein in toxicity. Thus, the precise role of the N-terminal helix of myotoxin II and related Lys49 phospholipases A(2), regarding their toxic mechanisms, remains controversial, and requires further experimental study to be clarified.

Comparative study of the cytolytic activity of myotoxic phospholipases A2 on mouse endothelial (tEnd) and skeletal muscle (C2C12) cells in vitro.

A rapid in vitro cytolytic effect of some myotoxic phospholipases A2 (PLA2s) isolated from the venoms of Viperidae snakes has been previously described. This study was undertaken to investigate if cytolytic activity is a common property of the myotoxic proteins from this group. Murine endothelial cells (tEnd) and skeletal muscle myotubes (C2C12) were utilized as targets. The release of lactic dehydrogenase was quantified as a measure of cell damage, 3 h after exposure of cells to the different PLA2s, including representatives from the genera Bothrops, Agkistrodon, Trimeresurus, Crotalus (family Viperidae), and Notechis (family Elapidae). All of the group II myotoxic PLA2s tested displayed rapid cytolytic activity when tested in the micromolar range of concentrations (8-32 microM). In contrast, the group I myotoxic PLA2 notexin was devoid of this activity. Aspartate-49 and lysine-49 PLA2 group II variants showed a comparable cytolytic effect. Skeletal muscle myotubes, obtained after fusion and differentiation of C2C12 myoblasts, were significantly more susceptible to the cytolytic action of myotoxins than endothelial cells, previously reported to be more susceptible than undifferentiated myoblasts under the same assay conditions. Cytolytic activity appears to be a common characteristic of group II myotoxic PLA2s of the Viperidae. Bee venom PLA2, a group III enzyme of known myotoxicity, also displayed cytotoxic activity on C2C12 myotubes, being devoid of activity on endothelial cells. These results suggest that in vitro differentiated skeletal muscle myotubes may represent a suitable model target for the study of myotoxic PLA2s of the structural group II found in snake venoms.

Frequency in allergy to proteins of latex in health care workers.

OBJECTIVE: The objective of this study is to evaluate allergy prevalence to latex proteins in health care workers at the Laboratory and Surgery Room of Hospital CIMA Chihuahua. METHODOLOGY AND RESULTS: A thorough clinical chart was recorded for all health care workers studied: hematic biometry, total IgE by ELISA method, specific IgE to latex antigen by ELISA (pharmacy CAP system), cutaneous tests with latex antigen (Aphi de México, Hevea Brasiliensis Biopal Inc. Spoken WA), through scarification, together with histamine and Evans (positive-negative control). The number included is 99 individuals. Specific IgE to latex in 4 cases was positive (4%) and in the cutaneous tests to latex, 24 cases (24%) resulted positive. CONCLUSIONS: The study reports a prevalence of (4%) when performing the specific IgE to latex and (24%) to the cutaneous test with antigen to total latex. This data allows us to continue evaluating the personnel at risk at the hospital, with better results in the administration of Labor Medicine at this medical institution.

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

A new myotoxic phospholipase A2 was isolated from the venom of the arboreal snake Bothriechis schlegelii (formerly Bothrops schlegelii) from Costa Rica, by ion-exchange chromatography on CM-Sephadex. B. schlegelii myotoxin I is a basic protein (pI > 9.3) with a subunit molecular weight of 15 kDa, which migrates as a dimer in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. This myotoxin is recognized by antibodies generated against Bothrops asper myotoxin II (a lysine-49 phospholipase A2), by both enzyme-immunoassay and gel immunodiffusion, in the latter case with a pattern of partial identity. The toxin induces rapid myonecrosis upon intramuscular injection in mice, as evidenced by the early increase in plasma creatine kinase activity and by direct intravital microscopic observation. B. schlegelii myotoxin I also induces edema in the mouse footpad assay and exerts lethal activity (LD50 approximately 2.5 microg/g) upon intravenous injection. The toxin has a low phospholipase A2 activity (4.2 microEq.mg-1.min-1) using egg yolk phospholipids as substrate. It also shows a weak anticoagulant effect in vitro. Its N-terminal sequence, SMYELGKMILLETGKNAATSYIAYG, shows 93% homology with both Bothrops asper myotoxin II and B. jararacussu bothropstoxin I, suggesting that B. schlegelii myotoxin I may be a new lysine-49 variant of this family of myotoxic phospholipases A2.

Two phospholipase A2 inhibitors from the plasma of Cerrophidion (Bothrops) godmani which selectively inhibit two different group-II phospholipase A2 myotoxins from its own venom: isolation, molecular cloning and biological properties.

Myotoxic phospholipases A(2) (PLA(2)s; group II) account for most of the muscle-tissue damage that results from envenomation by viperid snakes. In the venom of the Godman's viper (Cerrophidion godmani, formerly Bothrops godmani), an enzymically active PLA(2) (myotoxin I) and an inactive, Lys-49 variant (myotoxin II) induce extensive muscle damage and oedema. In this study, two distinct myotoxin inhibitor proteins of C. godmani, CgMIP-I and CgMIP-II, were purified directly from blood plasma by selective binding to affinity columns containing either myotoxin I or myotoxin II, respectively. Both proteins are glycosylated, acidic (pI=4) and composed of 20-25-kDa subunits that form oligomers of 110 kDa (CgMIP-I) or 180 kDa (CgMIP-II). In inhibition studies, CgMIP-I specifically neutralized the PLA(2) and the myotoxic, oedema-forming and cytolytic activities of myotoxins I, whereas CgMIP-II selectively inhibited the toxic properties of myotoxin II. N-terminal amino acid sequence analysis and sequencing of cDNAs encoding the two inhibitors revealed that CgMIP-I is similar to gamma-type inhibitors, which share a pattern of cysteine residues present in the Ly-6 superfamily of proteins, whereas CgMIP-II shares sequence identity with alpha-type inhibitors that contain carbohydrate-recognition-like domains, also found in C-type lectins and mammalian PLA(2) receptors. N-terminal sequencing of myotoxin I revealed a different primary structure from myotoxin II [De Sousa, Morhy, Arni, Ward, Díaz and Gutiérrez (1998) Biochim. Biophys. Acta 1384, 204-208], which provides insight into the nature of such pharmacological specificity.

Skeletal muscle necrosis and regeneration after injection of Thalassophryne nattereri (niquim) fish venom in mice.

Stings by Thalassophryne nattereri are responsible for envenomation of fishermen in north-eastern Brazil. Its venom induces prominent local tissue damage, characterized by pain, oedema and necrosis. The pathogenesis of acute muscle damage induced by T. nattereri venom was studied in mice. Intramuscular injection induced myonecrosis within the first hours. Some muscle cells presented a hypercontracted morphology, but most necrotic fibres were not hypercontracted, being instead characterized by a disorganization of myofibrils, with Z line loss, mitochondrial swelling and sarcolemmal disruption. In addition, thrombosis was observed histologically in venules and veins, together with vascular congestion and stasis, evidenced by intravital microscopy. Venom induced a rapid increment in serum creatine kinase (CK) levels, concomitant with a reduction in gastrocnemius muscle CK activity, whereas no increments in muscle lactic acid were detected. A rapid cytolytic effect was induced by the venom on C2C12 murine myoblasts in culture. The inflammatory reaction in affected muscle was characterized by oedema and scarce cellular infiltrate of polymorphonuclear leucocytes and macrophages, with a consequent delay in the removal of necrotic material. Skeletal muscle regeneration was partially impaired, as evidenced by the presence of regenerating fibres of variable size and by the increase of fibrotic tissue in endomysium and perimysium. It is suggested that T. nattereri venom affects muscle fibres by a direct cytotoxic effect, and that the vascular alterations described preclude a successful regenerative process.

Venom of the crotaline snake Atropoides nummifer (jumping viper) from Guatemala and Honduras: comparative toxicological characterization, isolation of a myotoxic phospholipase A(2) homologue and neutralization by two antivenoms.

A comparative study was performed on the venoms of the crotaline snake Atropoides nummifer from Guatemala and Honduras. SDS-polyacrylamide gel electrophoresis, under reducing conditions, revealed a highly similar pattern of these venoms, and between them and the venom of the same species from Costa Rica. Similar patterns were also observed in ion-exchange chromatography on CM-Shephadex C-25, in which a highly basic myotoxic fraction was present. This fraction was devoid of phospholipase A(2) activity and strongly reacted, by enzyme-immunoassay, with an antiserum against Bothrops asper myotoxin II, a Lys-49 phospholipase A(2) homologue. A basic myotoxin of 16 kDa was isolated to homogeneity from the venom of A. nummifer from Honduras, showing amino acid composition and N-terminal sequence similar to those of Lys-49 phospholipase A(2) variants previously isolated from other crotaline snake venoms. Guatemalan and Honduran A. nummifer venoms have a qualitatively similar toxicological profile, characterized by: lethal; hemorrhagic; myotoxic; edema-forming; coagulant; and defibrinating activities, although there were significant quantitative variations in some of these activities between the two venoms. Neutralization of toxic activities by two commercially-available antivenoms in the region was studied. Polyvalent antivenom produced by Instituto Clodomiro Picado was effective in the neutralization of: lethal; hemorrhagic; myotoxic; coagulant; defibrinating; and phospholipase A(2) activities, but ineffective against edema-forming activity. On the other hand, MYN polyvalent antivenom neutralized: hemorrhagic; myotoxic; coagulant; defibrinating; and phospholipase A(2) activities, albeit with a lower potency than Instituto Clodomiro Picado antivenom. MYN antivenom failed to neutralize lethal and edema-forming activities of A. nummifer venoms.

Structural and functional characterization of myotoxin I, a Lys49 phospholipase A(2) homologue from Bothrops moojeni (Caissaca) snake venom.

Myotoxin-I (MjTX-I) was purified to homogeneity from the venom of Bothrops moojeni by ion-exchange chromatography on CM-Sepharose. Its molecular weight, estimated by SDS-PAGE, was 13,400 (reduced) or 26, 000 (unreduced). The extinction coefficient (E(1.0 mg/ml)(1.0 cm)) of MjTX-I was 1.145 at lambda = 278 nm, pH 7.0, and its isoelectric point was 8.2 at ionic strength mu = 0.1. When lyophilized and stored at 4 degrees C, dimeric, trimeric, and pentameric forms of the protein were identified by SDS-PAGE. This "heterogeneous" sample could be separated into three fractions by gel filtration on Sephadex G-50. The fractions were analyzed by isoelectric focusing, immunoelectrophoresis, and amino acid composition, which indicated that heterogeneity was the result of different levels of self-association. Protein sequencing indicated that MjTX-I is a Lys49 myotoxin and consists of 121 amino acids (M(r) = 13,669), containing a high proportion of basic and hydrophobic residues. It shares a high degree of sequence identity with other Lys49 PLA(2)-like myotoxins, but shows a significantly lower identity with catalytically active Asp49 PLA(2)s. The three-dimensional structure of MjTX-I was modeled based on the crystal structures of three highly homologous Lys49 PLA(2)-like myotoxins. This model showed that the amino acid substitutions are conservative, and mainly limited to three structural regions: the N-terminal helix, the beta-wing region, and the C-terminal extended random coil. MjTX-I displays local myotoxic and edema-inducing activities in mice, and is lethal by intraperitoneal injection, with an LD(50) value of 8.5 +/- 0.8 mg/kg. In addition, it is cytotoxic to myoblasts/myotubes in culture, and disrupts negatively charged liposomes. In comparison with the freshly prepared dimeric sample, the more aggregated forms showed significantly reduced myotoxic activity. However, the edema-inducing activity of MjTX-I was independent of molecular association. Phospholipase A(2) activity on egg yolk, as well as anticoagulant activity, were undetectable both in the native and in the more associated forms. His, Tyr, and Trp residues of the toxin were chemically modified by specific reagents. Although the myotoxic and lethal activities of the modified toxins were reduced by these treatments, neither its edema-inducing or liposome-disrupting activities were significantly altered. Rabbit antibodies to native MjTX-I cross-reacted with the chemically modified forms, and both the native and modified MjTX-I preparations were recognized by antibodies against the C-terminal region 115-129 of myotoxin II from B. asper, a highly Lys49 PLA(2)-homologue with high sequencial similarity.

High resolution cytochemical study of uterine epithelial cell surface of the rat at identified sites previous to blastocyst-endometrial contract.

A transmission electron microscopy study was carried out in prospective implantation sites in uterine horns of rats of the Long Evans strain. The purpose was to search for possible modifications in glycoproteins of outer coats of endometrial epithelium shortly before blastocyst-endometrial contact Shea's technique (Lanthanum-Alcian blue) was used to visualize glycoproteins. Prospective implantation sites disclosed a rather thin finely granular electrondense layer along the plasma membrane of the surface endometrial cells. In addition, there was a decrease in the number of microvilli as well as a lack of Lanthanum precipitate in junctional complexes. Sites remote from prospective implantation areas, disclosed a thicker electrondense layer of glycoproteins in cell plasma membranes including those of well developed microvilli. The junctional complexes were well depicted by the electrodense lanthanum salt precipitates. These observations suggest that a mechanism other than a more physical contact take place and induce the above changes.