Immunocytochemical localization of heparin in secretory granules of rat peritoneal mast cells using a monoclonal anti-heparin antibody (ST-1)

We performed immunogold labeling with an ST-1 monoclonal antibody (IgM), specific for intact heparin, to define the subcellular localization of heparin in mast cells. Rat peritoneal mast cells were fixed by a modified Karnovsky method and embedded in Araldite. Ultrathin sections were first treated with sodium periodate and then sequentially incubated with MAb ST-1, rabbit anti-mouse IgM, and protein A-gold. By transmission electron microscopy, gold particles were localized inside cytoplasmic granules of peritoneal mast cells. In contrast, with the same procedure, no labeling was observed in mast cells from rat intestinal mucosa. Control sections of rat peritoneal or intestinal mucosa mast Mast cells cells treated with an irrelevant MAb (IgM) did not show any labeling. Treatment with nitrous Heparin acid abolished the reactivity of MAb ST-1 with peritoneal mast cells. These results Granules show that different mast cells can be identified regarding their heparin content by immunochemical procedures using MAb ST-1.

Phospholipase A(2) - A structural review

Phospholipases A(2) (PLA(2)) are widely distributed in nature and are well characterized proteins with respect to their catalytic and pharmacological activities, A wealth of structural information has recently become available both from X-ray diffraction and NMR studies, and although a detailed model of the catalytic mechanism of PLA(2) has been proposed, the structural bases of other aspects of PLA(2) function, such as interfacial activation and venom PLA(2) pharmacological activities, are still under debate. An appreciation of the PLA(2) protein structure will yield new insights with regard to these activities, the salient structural features of the class I, II and III PLA(2) are discussed with respect to their functional roles. Copyright (C) 1996 Published by Elsevier B.V. Ltd

A molecular mechanism for Lys(49)-phospholipase A(2) activity based on ligand-induced conformational change

Agkistrodon contortrix laticinctus myotoxin is a Lys(49)- phospholipase A(2) (EC 3.1.1.4) isolated from the venom of the serpent A contortrix laticinctus (broad-banded copperhead). We present here three monomeric crystal structures of the myotoxin, obtained under different crystallization conditions. The three forms present notable structural differences and reveal that the presence of a ligand in the active site (naturally presumed to be a fatty acid) induces the exposure of a hydrophobic surface (the hydrophobic knuckle) toward the C terminus. The knuckle in A contortrix laticinctus myotoxin involves the side chains of Phe(121) and Phe(124) and is a consequence of the formation of a canonical structure for the main chain within the region of residues 118-125. Comparison with other Lys(49)-phospholipase A(2) myotoxins shows that although the knuckle is a generic structural motif common to all members of the family, it is not readily recognizable by simple sequence analyses. An activation mechanism is proposed that relates fatty acid retention at the active site to conformational changes within the C-terminal region, a part of the molecule that has long been associated with Ca2+-independent membrane damaging activity and myotoxicity. This provides, for the first time, a direct structural connection between the phospholipase active site and the C-terminal myotoxic site, justifying the otherwise enigmatic conservation of the residues of the former in supposedly catalytically inactive molecules.

Testosterone stimulates growth and secretory activity of the female prostate in the adult gerbil (Meriones unguiculatus)

The prostate of the female gerbil (Meriones unguiculatus) is similar to the human female prostate (Skene gland) and, despite its reduced size, it is functional and shows secretory activity. However, virtually nothing is known about its physiological regulation. This study was thus undertaken to evaluate the behavior of the gerbil female prostate in a hyperandrogenic condition. Adult females received subcutaneous injections of testosterone cypionate (1 mg/kg body weight every 48 h) up to 21 days. Circulating levels of testosterone and estradiol were monitored, and the prostate and ovaries subjected to structural and immunocytochemical analyses. The treatment resulted in sustained high levels of circulating testosterone, and caused a transient increase in estradiol. There was an increase in epithelial cell proliferation accompanied by significant reorganization of the epithelium and an apparent reduction in secretory activity, followed by a progressive increase in luminal volume density and accumulation of secretory products. Immunocytochemistry identified the expression of androgen receptor and a prostate-specific antigen (PSA)-related antigen in prostatic epithelial cells. A circulating PSA-related antigen was also found, and its concentration showed strong negative correlation with circulating estrogen. Epithelial dysplasia was detected in the prostate of treated females. Analysis of the ovaries showed the occurrence of a polycystic condition and stromal cell hyperplasia. The results indicate that testosterone has a stimulatory effect on the female prostate, inducing epithelial cell proliferation, differentiation, secretory activity, and dysplasia. The results also suggest that prostatic growth and activity, polycystic ovaries, and ovarian stromal cell hyperplasia are related to a hyperandrogenic condition in females.

Crystallization and preliminary X-ray diffraction analysis of a novel Arg49 phospholipase A(2) homologue from Zhaoermia mangshanensis venom

Zhaoermiatoxin, an Arg49 phospholipase A(2) homologue from Zhaoermia mangshanensis (formerly Trimeresurus mangshanensis, Ermia mangshanensis) venom is a novel member of the PLA(2)-homologue family that possesses an arginine residue at position 49, probably arising from a secondary Lys49 -> Arg substitution that does not alter the catalytic inactivity towards phospholipids. Like other Lys49 PLA(2) homologues, zhaoermiatoxin induces oedema and strong myonecrosis without detectable PLA(2) catalytic activity. A single crystal with maximum dimensions of 0.2 x 0.2 x 0.5 mm was used for X-ray diffraction data collection to a resolution of 2.05 angstrom using synchrotron radiation and the diffraction pattern was indexed in the hexagonal space group P6(4), with unit-cell parameters a = 72.9, b = 72.9, c = 93.9 angstrom.

Structure of a calcium-independent phospholipase-like myotoxic protein from Bothrops asper venom

Myotoxin II, a myotoxic calcium-independent phospholipase-like protein isolated from the venom of Bothrops asper, possesses no detectable phospholipase activity. The crystal structure has been determined and refined at 2.8 Angstrom to an R factor of 16.5% (F>3 sigma) with excellent stereochemistry. Amino-acid differences between catalytically active phospholipases and myotoxin LI in the Ca2+-binding region, specifically the substitutions Tyr28-->Asn, Gly32-->Leu and Asp49-->Lys, result in an altered local conformation. The key difference is that the epsilon-amino group of Lys49 fills the site normally occupied by the calcium ion in catalytically active phospholipases. In contrast to the homologous monomeric Lys49 variant from Agkistrodon piscivorus piscivorus, myotoxin II is present as a dimer both in solution and in the crystalline state. The two molecules in the asymmetric unit are related by a nearly perfect twofold axis, yet the dimer is radically different from the dimer formed by the phospholipase from Crotalus atrox. Whereas in C. atrox the dimer interface occludes the active sites, in myotoxin II they are exposed to solvent.

Morphological characterization of the venom secretory epidermal cells in the stinger of marine and freshwater stingrays

Marine and freshwater stingrays are characterized by the presence of one to three mineralized serrated stingers on the tail, which are covered by epidermal cells secreting venom. When these animals are dorsally touched, the stinger can be introduced into the aggressor by a whip reflex mechanism of the tail, causing severe mechanical injuries and inoculating the venom. Accidents in humans are frequent causing intense local pain, oedema and erythema. Bacterial secondary infection is also common. In addition, injuries involving freshwater stingrays frequently cause a persistent cutaneous necrosis. The exact localization of the venom secretory epidermal cells in the stinger is controversial, but it is known that it is preferentially located in the ventrolateral grooves. A comparative morphological analysis of the stinger epidermal tissue of different marine and freshwater Brazilian stingray species was carried out. The results indicate that in freshwater species there is a larger number of protein secretory cells, of two different types, spread over the whole stinger epidermis, while in marine species the protein secretory cells are located only around or inside the stinger ventrolateral grooves. These differences between the stingers of the two groups can justify the more severe envenomation accidents with the freshwater species when compared with the marine species. (c) 2007 Elsevier Ltd. All rights reserved.

Studies on hyaluronidase, chondroitin sulphatase, proteinase and phospholipase secreted by Candida species

We studied the ability of different Candida species to produce,at the same time, hyaluronidase, chondroitin sulphatase, proteinase, and phospholipase to assess whether they could be related to Candida pathogenicity. Only C. albicans was able to produce the four enzymes tested (73%) and was highly virulent to mice. Strains, that lack the capacity to produce one or more of the enzymes assayed, seemed less virulent or avirulent, similarly to the spontaneous hyaluronidase, chondroitin sulphatase, phospholipase and proteinase-deficient C. albicans strain FCF 14, 1 which was non-pathogenic to mice. Among the other Candida species tested, none of them produced the four enzymes simultaneously, being less virulent in intravenously inoculated mice.

The venom gland of queens of Apis mellifera (Hymenoptera, Apidae): morphology and secretory cycle

The venom gland of queens of Apis mellifera was examined through light and transmission electron microscopy and subjected to electrophoretic analyses. Virgin queens exhibited prismatic secretory cells containing large amounts of rough endoplasmic reticulum with dilated cisternae, open secretory spaces, numerous vacuoles and granules scattered in the cytoplasm, and spherical nuclei with numerous nucleoli. The secretion produced was non-refringent under polarized light and the electrophoretic analysis of glandular extracts revealed five main protein bands. In mated queens, the venom gland exhibited a high degree of degeneration. Its secretion was refringent under polarized light and one of the main bands was absent in the electrophoretic pattern obtained. The morphological aspects observed are in agreement with the function of this gland in queens, given that virgin queens use venom in battles for the dominance of the colony, a situation that occurs as soon as they emerge, while fertilized queens rarely use venom. (c) 2006 Elsevier Ltd. All rights reserved.

Crystallization of piratoxin I, a myotoxic LYS49-phospholipase A(2) homologue isolated from the venom of Bothrops pirajai

Fernanda Canduri, Lit C. Mancuso, Andreimar M. Soares, Jose R. Giglio, Richard J. Ward and Raghuvir K. Arni. Crystallization of piratoxin I, a myotoxic Lys49-phospholipase A(2) homologue isolated from the venom of Bothrops pirajai. Toxicon 36, 547-551, 1998.-Large single crystals of piratoxin I, a Lys49-PLA(2) homologue with low enzymatic activity, have been obtained. The crystals belong to the orthorhombic system space group p2(1)2(1)2(1) and diffract X-raps to a resolution of 2.8 Angstrom. Preliminary analysis reveals the presence of two molecules in the crystallographic asymmetric unit. (C) 1998 Elsevier B.V. Ltd. All rights reserved.

A structure based model for liposome disruption and the role of catalytic activity in myotoxic phospholipase A(2)S

Venom phospholipase A(2)s (PLA(2)s) display a wide spectrum of pharmacological activities and, based on the wealth of biochemical and structural data currently available for PLA(2)S, mechanistic models can now be inferred to account for some of these activities. A structural model is presented for the role played by the distribution of surface electrostatic potential in the ability of myotoxic D49/K49 PLA(2)s to disrupt multilamellar vesicles containing negatively charged natural and non-hydrolyzable phospholipids. Structural evidence is provided for the ability of K49 PLA(2)s to bind phospholipid analogues and for the existence of catalytic activity in K49 PLA(2)s. The importance of the existence of catalytic activity of D49 and K49 PLA(2)s in myotoxicity is presented. (C) 2003 Elsevier Ltd. All rights reserved.

Signaling path of the action of AVP on distal K+ secretion

Background. Previous studies from our laboratory have shown that luminal perfusion with arginine vasopressin (AVP) stimulates distal tubule secretory potassium flux (J(K)) via V1 receptors (Am J Physiol 278: F809- F816, 2000). In the present work, we investigate the cell signaling mechanism of this process.Methods. In vivo stationary microperfusion was performed in rat cortical distal tubules and luminal K was measured using double K+ resin/reference microelectrodes.Results. In control conditions, J(K) was 0.71 +/- 0.05 nmol. cm(-2).second(-1); this process was inhibited (14%) by 10(-5) mol/L 8-bromo-cyclic adenosine monophosphate (cAMP), and increased by 35% with 10(-8) mol/L phorbol ester [phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC)]. During luminal perfusion with 10(-11) mol/L AVP, J(K) increased to 0.88 +/- 0.08 nmol. cm(-2).seconds(-1). In the presence of 10(-11) mol/L AVP, J(K) was not affected by 10(-4) mol/L H89, a blocker of protein kinase A (PKA), but was inhibited (45%) by 10(-5) mol/L staurosporine, an inhibitor of PKC, and by 41% during perfusion with 5 x 10(-5) mol/L of the cell Ca2+ chelator bis (2-aminophenoxy) ethane-tetraacetic acid (BAPTA). In order to study the role of Ca2+-dependent K channels in the luminal hormonal action, the tubules were perfused with 5 mmol/L tetraethylammonium chloride (TEA) or 10(-7) mol/L iberiotoxin, in the presence of AVP, and JK was significantly reduced by both agents. Iberiotoxin reduced AVP-stimulated J(K) by 36.4%, and AVP-independent J(K) (after blocking V1 receptors) by only 16%.Conclusion. The results suggest that the luminal V1-receptor effect of AVP on J(K) was mediated by the phospholipase C (PLC)/ Ca2+/PKC signaling path and not by adenylate cyclase/cAMP/PKA, therefore probably acting on maxi-potassium channels.

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 cm(1.0 mg/ml)) 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 6-50. 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 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 LD50 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. (C) 2000 Academic Press.