Long-term primary culture of secretory cells of Bothrops jararaca venom gland for venom production in vitro

This protocol details the optimal conditions to establish a long-term primary culture of secretory cells from the venom gland of the Bothrops jararaca snake. Furthermore, these conditions allow the production and secretion of venom into the culture medium. Snake venom is a rich source of active molecules and has been used for bioprospection studies. However, obtaining enough venom from snakes is a major obstacle. Secretory cells of venom glands are capable of producing active toxins. Therefore, a culture of secretory cells is a good in vitro system to acquire the venom of snakes without capturing the animal from the wild. The protocol described here provides a rapid (approximately 4 h) and reproducible means of producing sufficient amounts of snake venom for biological investigations.     

Immunolocalization of venom metalloproteases in venom glands of adult and of newborn snakes of Bothrops jararaca

Using immunoelectronmicroscopy we analyzed qualitative and quantitatively the intracellular distribution of bothropasin, hemorrhagic factor 2 (HF2) and hemorrhagic factor 3 (HF3) in the venom secretory cells from adult snakes in the active (7 days after venom extraction) and in the resting (without venom extraction for 40 days) stages of protein synthesis. Glands from the newborn Bothrops jararaca were also studied. The results lead to the conclusion that all the secretory cells and the secretory pathway in the cells are qualitatively alike in regard to their content of the three metalloproteases. Secretory cells from the resting glands, unlike the active ones and the newborn glands, did not present immunolabeling in the narrow intracisternal spaces of the rough endoplasmic reticulum (RER). The label intensity for bothropasin was greater than that for the other proteins in the adults. HF3 and HF2 labeling densities in the newborn were higher than in the adults and HF3 labeling was not different from that of bothropasin. Co-localization of the three metalloproteases was detected in the RER cisternae of the active gland secretory cells, implying that mixing of the proteases before co-packaging into secretory vesicles occurs at the beginning of protein synthesis in the RER cisternae.     

Cytochemical analysis of acid phosphatase activity in the venom secretory cells of Bothrops jararaca

A study of the histochemical reaction for acid phosphatase (AcPase) in venom gland secretory cells from Bothrops jararaca was done to investigate the distribution of lysosomes and related structures in stages of high- and low-protein synthesis. From this analysis, it was expected to gain insight into the cellular pathway by which AcPase is secreted into the venom. Two subtypes of AcPase reactivities were detected in the venom gland secretory cells: one was found in lysosomes and related structures and in some trans-Golgi network (TGN) elements and reacts with beta-glycerophosphate (betaGP) as substrate; the other was found in secretory vesicles, apical plasmalemma, lysosomes and related structures, and in some TGN elements, and reacts with cytidine monophosphate (CMP). The results are compatible with the possibility that there is a secretory via for AcPase in the venom gland of B. jararaca and that the elements composing this pathway are noted only when CMP is used as substrate. Large autophagosomes reactive to both betaGP and to CMP were commonly observed in the basal region of the secretory cells, and they were more abundant in the glands during the stage of low activity of protein synthesis.     

Angiostatin-like molecules are generated by snake venom metalloproteinases

Angiostatin is a plasminogen-derived anti-angiogenic factor composed of its first four kringle structures. This molecule is generated by proteolytic cleavage of plasminogen by some proteolytic enzymes in vitro. Since venoms of viper snakes are a rich source of both serine- and metalloproteinase, we hypothesized that angiostatin-like polypeptides could be generated during the envenomation after snake bites and play a pathophysiological role in the local tissue damage and regeneration. Our results showed that crude venoms from several species of Bothrops snakes were able to generate angiostatin-like polypeptides and purified metalloproteinases but not serine proteinases from Bothrops jararaca and Bothrops moojeni venoms were responsible for their generation in vitro. The putative plasminogen cleavage sites by the crude venoms and purified proteinases were determined by N-terminal amino acid sequencing of the angiostatin-like molecules. Angiostatin-like peptides derived from human plasminogen digestion by jararhagin, a metalloproteinase isolated from B. jararaca venom, inhibited endothelial cell proliferation in vitro. These results indicate that angiostatin-like molecules can be generated upon snakebite envenomations and may account for the poor and incomplete regenerative response observed in the damaged tissue.     

Ultrastructural and histochemical study of collagen fibres types I and III in the venom gland of Bothrops jararaca during secretory cycle

Fragments of snake (Bothrops jararaca) venom gland were analysed by light and transmission electron microscopy in order to characterize the changes in collagen fibres types I and III in the intertubular gland septa during the secretory cycle. The snakes were sacrificed at 45 days (unmilked group), 6 h, 4 and 8 days after manual extraction of the venom. The fragments were fixed, processed according to standard histologic technique for embedding in paraffin, and stained with haematoxylin-eosin and Gomori's trichrome and submitted to Gomori's silver impregnation technique and picrosirius-polarization method. For transmission electron microscopy the fragments were fixed and processed for embedding in Spurr's medium. At the 45th day (the gland at rest), when the secretory activity was at a minimum, the septa were narrow and filled with densely packed collagen fibrils. At 6 h, the septa were enlarged and exhibited wide spaces filled with finely granular Alcian Blue-positive material. Until the 8th day, the septa were narrower and the histologic aspect resembled that of the gland at rest. The results demonstrated structural modifications in the glandular septa according to the different periods of the secretory cycle. These modifications can be associated with the transformation in the secretory epithelium during the venom synthesis cycle.     

The mechanism of venom secretion from Duvernoy's gland of the snake Thamnophis sirtalis

The venom glands of snakes of the families Elapidae and Viperidae are thought to have evolved from Duvernoy's gland of colubrid ancestors. In highly venomous snakes elements of the external adductor musculature of the jaw insert fibers directly onto the capsule of the venom gland. These muscles, upon contraction, cause release of contents by increasing intraglandular pressure. In Thamnophis sirtalis, a colubrid, there is no direct connection between Duvernoy's gland and the adductor musculature. The anatomical arrangement of the gland, skull, adductor muscles, and the integument is such that contraction of the muscles may facilitate emptying of the gland. This hypothesis was tested by electrical stimulation of the muscles, which resulted in significantly greater release of secretion than elicited by controls. The results suggest a possible early step in the evolution of a more intimate association between venom glands and adductor musculature in highly venomous snakes.     

Effect of beta-antagonists on isoprenaline-induced secretion of fluid, amylase and protein by the parotid gland of the red kangaroo, Macropus rufus

Selective and non-selective beta-adrenoceptor antagonists were used to block the increases in fluid, protein and amylase secretion caused by sympathomimetic stimulation of the parotid gland of red kangaroos during intracarotid infusion of isoprenaline. ICI118551 at antagonist/agonist ratios up to 300:1 caused increasing but incomplete blockade of fluid secretion, and protein/amylase release. Atenolol at antagonist/agonist ratios up to 300:1 was only marginally more potent than ICI118551 at blocking the fluid, protein and amylase responses. Propranolol at antagonist/agonist ratios of 30:1 was as effective at blocking fluid and protein secretion as the highest ratios of either atenolol or ICI118551. Simultaneous administration of atenolol (30:1) with ICI118551 (30:1) was not as potent as propranolol (30:1). Thus, the beta-adrenoceptor/s in the acini of the kangaroo parotid gland appear to have antagonist-binding affinities atypical of those found for eutherian tissues. The data are consistent with the gland possessing either a single anomalous beta-adrenoceptor or functional beta(2)-receptors in addition to the beta(1)-receptors which are characteristic of eutherian salivary glands.     

Evidence for heterogeneous forms of the snake venom metalloproteinase jararhagin: a factor contributing to snake venom variability

The reprolysin subfamily of metalloproteinases includes snake venom metalloproteinases (SVMP) and mammalian disintegrin/metalloproteinase. These proteins are synthesized as zymogens and undergo proteolytic processing resulting in a variety of multifunctional proteins. Jararhagin is a P-III SVMP isolated from the venom of Bothrops jararaca. In crude venom, two forms of jararhagin are typically found, full-length jararhagin and jararhagin-C, a proteolytically processed form of jararhagin that is composed of the disintegrin-like and cysteine-rich domains of jararhagin. To better understand the structural and mechanistic bases for these forms of jararhagin in the venom of B. jararaca and the source of venom complexity in general, we have examined the jararhagin forms isolated from venom and the autolysis of isolated jararhagin under the conditions of varying pH, calcium ion concentration, and reducing agents. From our results, jararhagin isolated from venom appears as two forms: a predominant form that is stable to in vitro autolysis and a minor form that is susceptible to autolysis under a variety of conditions including alkaline pH, low calcium ion concentrations, or reducing agent. The autolysis site for production of jararhagin-C from isolated jararhagin was different from that observed for jararhagin-C as isolated from crude venom. Taken together, these data lead us to the conclusion that during the biosynthesis of jararhagin in the venom gland at least three forms are present: one form which is rapidly processed to give rise to jararhagin-C, one form which is resistant to processing in the venom and autolysis in vitro, and one minor form which is susceptible to autolysis under conditions that promote destabilization of its structure. The presence of these different forms of jararhagin contributes to greater structural and functional complexity of the venom and may be a common feature among all snake venoms. The biological and biochemical features in the venom gland responsible for these jararhagin isoforms are currently under investigation.     

后毒牙类毒蛇

长期以来,人们仅把具有沟牙和管牙的蛇视为毒蛇,然而,近年来发现游蛇科中的虎斑颈槽蛇、红脖颈槽蛇、颈棱蛇、赤链蛇等既无管牙,也无沟牙,却频频发生这类蛇咬伤人后引起中毒的事例,甚至出现被咬伤致严重出血休克死亡的事件。经深入研究后发现,这些蛇虽没有沟牙和管牙,但却具有产生毒性分泌物的毒腺—杜氏腺(Duvernoy′s gland)及皮下腺,且不同的毒腺具有不同的毒性作用,可表现为出血不止、溶血、呼吸困难、肾损害等。这类蛇与毒腺的导管有联系的上颌牙明显粗大,上颌牙与上颌骨、横骨连接牢固,毒腺里的毒液可顺着粗大的上颌牙流入伤口,因此,应视为"后毒牙类毒蛇"。     

Histology,histochemistry, and emptying mechanism of the venom glands of some elapid snakes

The venom glands of several species of elapid snakes are described. The main venom gland consists of many tubules which usually contain large amounts of secretion product. The accessory gland surrounds the entire venom duct and is usually composed of uniform mucous epithelium. The epithelium lining the tubules of the accessory gland of Naja naja is composed of two distinct types of cells. Histochemical tests indicate that the main venom gland reacts with mercury bromphenol blue and PAS but not with alcian blue. The accessory gland reacts with PAS and alcian blue, and not with mercury bromphenol blue. Treatment of sections with sialidase demonstrates the presence of a sialomucin in the accessory gland. Stimulation of the muscles associated with the venom gland offers an indication of the venom expulsion mechanism of Bungarus caeruleus. A comparison of the venom apparatus of elapid and viperid snakes emphasizes marked differences in the internal anatomy of the venom glands, muscles associated with the gland, and arrangement of glandular components. The morphological differences and dissimilar venom expulsion mechanisms support the recent view of the polyphyletic origin of venomous snakes.     

Cloning and expression of calglandulin,a new EF-hand protein from the venom glands of Bothrops insularis snake in E. coli

The EF-hand protein family is comprised of many proteins with conserved Ca(2+)-binding motifs with important biological roles in intracellular communication. During the generation of Expressed Sequence Tags (ESTs) from the venom glands of the Viperidae snake Bothrops insularis, we identified a cDNA coding for a putative Ca(2+) binding protein with four EF-hand motifs, named here calglandulin. The deduced amino acid sequence displayed the greatest sequence similarity with calmodulin (59%), followed by troponin-C (52%). The encoded polypeptide was first expressed in Escherichia coli as a 6XHis-tagged fusion protein. The expressed protein was purified by Ni(2+)-charged affinity chromatography and circular dichroism (CD) spectroscopy confirmed the prevalence of alpha-helix as observed in calmodulin/calmodulin-like proteins. A polyclonal antiserum was generated in mice using this recombinant calglandulin. To investigate the tissue-specific biological occurrence of this protein, this antiserum was used in Western blot experiments, which revealed an immunoreactive band in samples of venom gland extracts from different snakes, but not in the crude venom or in brain, heart and other tissues. This exclusive occurrence suggests a specialized function of calglandulin in snake venom glands.     

Identification of proteins similar to Bothrops jararaca coagulation inhibitor (BjI) in the plasmas of Bothrops alternatus, Bothrops jararacussu and Crotalus durissus terrificus snakes

Bothrops jararaca coagulation inhibitor (BjI), a protein isolated from B. jararaca plasma, specifically inhibits the coagulant activity of thrombin. Our group previously identified proteins similar to BjI in the plasma of other snakes [Tanaka-Azevedo, A.M., Tanaka, A.S., Sano-Martins I.S., 2003. A new blood coagulation inhibitor from the snake Bothrops jararaca plasma: isolation and characterization. Biochem Biophys Res Commun 308, 706-712.]. In the present study, we analyzed the presence of BjI-like proteins in the plasmas of three different species of viperid snakes, Bothrops alternatus, Bothrops jararacussu and Crotalus durissus terrificus. These proteins exhibited 109 and/or 138 kDa and were immunologically related to BjI. They also inhibited the coagulant activity of thrombin, evaluated by the thrombin time test. These findings demonstrate the presence of proteins similar to BjI in these three species, although such inhibitor could not be observed in all samples of the specimens tested. Moreover, the presence of these proteins in the plasma is related to prolongation of thrombin time, implying a relationship between these proteins and their inhibitory coagulant activity upon thrombin. Our results suggest that BjI-like proteins are widely distributed among Crotalinae snakes found in Brazil.     

Association of turkey erythrocyte beta-adrenoceptors with a specific lipid component.

We have recently reported that the highly potent beta-adrenergic affinity label [125I]bromoacetylamino cyanopindolol ([125I]BAM-CYP) irreversibly blocks the turkey erythrocyte beta-adrenoceptor binding site by combining with a receptor-associated non-protein component. In this communication, we report: lipid labelling is inhibited by beta 1-adrenergic ligands with the potency ratio and stereospecificity characteristic for the turkey erythrocyte beta 1-adrenoceptor; the tagged component is a glycolipid, probably a ganglioside; [125I]BAM-CYP-blocked receptor, after solubilization in deoxycholate, can be separated from the [125I]BAM-CYP-glycolipid with restoration of the binding capacity of the beta 1-adrenoceptor protein; the tightly associated [125I]BAM-CYP-labelled glycolipid can be displaced by a glycolipid mixture extracted from turkey erythrocyte membranes but not by bovine brain gangliosides, when the blocked receptor is solubilized in digitonin. This is the first direct demonstration that a receptor protein is associated with a specific membrane lipid. The possibility that glycolipids play a role in receptor-mediated signal transduction is discussed in view of these findings and in view of data from the literature.     

Tamulustoxin: a novel potassium channel blocker from the venom of the Indian red scorpion Mesobuthus tamulus

We have characterized tamulustoxin, a novel 35-amino-acid peptide found in the venom of the Indian red scorpion (Mesobuthus tamulus). Tamulustoxin was identified through a [125I]toxin I screen, designed to identify toxins that block voltage-activated potassium channels. Tamulustoxin has also been cloned by RT-PCR, using RNA extracted from scorpion venom glands. Tamulustoxin shares no homology with other scorpion venom toxins, although the positions of its six cysteine residues would suggest that it shares the same structural scaffold. Tamulustoxin rapidly inhibited both peak and steady-state currents (18.9 +/- 1.0 and 37 +/- 1.1%, respectively) produced by injecting CHO cells with mRNA encoding the hKv1.6 channel.     

Synthesis and evaluation of (S)-[18F]-fluoroethylcarazolol for in vivo beta-adrenoceptor imaging in the brain

The beta-adrenergic receptor ligand (S)-4-(3-(2'-[18F]-fluoroethylamino)-2-hydroxypropoxy)-carbazol ((S)-[18F]-fluoroethylcarazolol) was prepared by reaction of [18F]-fluoroethylamine with the corresponding (S)-epoxide and was evaluated in rats by studying its pharmacokinetics and its binding profile both in vitro and in vivo. In vitro, (S)-fluoroethylcarazolol binds preferentially to beta-adrenoceptors (pK(i)=9.3 for beta(1) and 9.4 for beta(2)) and has less affinity to 5HT(1A) and 5HT(1D) receptors (pK(i)=6.7 and 5.2). In vivo, standard uptake values (SUVs) up to 0.63+/-0.07 in cortical regions were found after 60 min. Metabolites (90%) appeared within 10 min in plasma, whereas, in brain 70-75% parent compound was found after 60 min. Clearance from plasma occurred within 5 min. Cerebral uptake could be blocked by 'cold' fluoroethylcarazolol in every region, except medulla. Uptake was also blocked by propranolol and pindolol, but not by WAY 100635. ICI 89406 hardly lowered [18F] levels in brain. ICI 118551 reduced uptake of [18F] in cerebellum (mainly beta(2)) by 30%. Specific binding (tissue minus medulla values) in various brain regions corresponded with those observed for [18F]-fluorocarazolol (r(2)=0.95) and with in vitro beta-adrenoceptor densities (r(2)=0.76). Autoradiography using phosphor images of (S)-[18F]-fluoroethylcarazolol in rat brain showed the characteristic binding pattern of beta-antagonists, while propranolol treatment resulted in low and homogenous uptake. Regional tissue minus medulla values corresponded with in vitro beta-adrenoceptor densities (r(2)=0.77). We conclude that (S)-[18F]-fluoroethylcarazolol is a high affinity ligand that binds specifically to cerebral beta-adrenoceptors in vivo and may be of use for beta-adrenoceptor imaging in the brain with PET.     

Melanin deposits associated with the venom glands of snakes

Melanin deposits in the heads of both true vipers (Viperinae) and pit vipers (Crotalinae) are concentrated over the dorsal and dorsolateral aspects of the venom glands. This pigment may occur in any or all of six sites which include the epidermis, dermis, tissues covering the venom glands, and the interior of the glands themselves. The extreme localization of these melanin deposits suggests that they shield the venom glands from light. Calculations indicate that without such shielding the light energy penetrating the venom glands in the visible and ultraviolet portions of the solar spectrum would damage the venom-synthesizing apparatus and detoxify stored venom. Elapid and hydrophiid snakes have less dense pigment over the venom gland than vipers. Literature reports indicate that elapid venom is less sensitive to photodetoxification than is venom from vipers. Most colubrid snakes, including several with protein-secreting Duvernoy's glands, have little or no melanin associated with the glands. Venomous colubrids in the genera Ahaetulla, Dryophis, Leptophis, and Oxybelis have pigment over the glands as dense as that seen in vipers. Iridophores probably also shield venom glands from radiation. In puff adders and Gaboon vipers (Bitis) there appears to be an ontogenetic change in the shielding of the venom glands from melanocytes in young individuals to iridophores in adults.     

Alteration of the protein composition of bothrops jararaca venom and venom gland by isoproterenol treatment

• 1.1. The protein composition of Bothrops jararaca venom and venom gland was analyzed through SDS-PAGE, after isoproterenol (IPR) treatment.
• 2.2. Some proteins (47, 48, 57 and 72 kDa) were detected in the gland homogenate from the control but not from the IPR-treated samples.
• 3.3. Three proteins (26.5, 44.5 and 53 kDa) were detected in the venom gland from IPR-treated snakes but not from the venom gland from the control.
• 4.4. In the venom samples proteins of 41 and 74 kDa were detected only in the IPR treated samples, while proteins of 17 and 28 kDa were detected only in the control.
• 5.5. The biological activity of the venom did not change with IPR treatment.

     

Synthesis and evaluation of radiolabeled antagonists for imaging of beta-adrenoceptors in the brain with PET.

Five potent, lipophilic beta-adrenoceptor antagonists (carvedilol, pindolol, toliprolol and fluorinated analogs of bupranolol and penbutolol) were labeled with either carbon-11 or fluorine-18 and evaluated for cerebral beta-adrenoceptor imaging in experimental animals. The standard radioligand for autoradiography of beta-adrenoceptors, [125I]-iodocyanopindolol, was also included in this survey. All compounds showed either very low uptake in rat brain or a regional distribution that was not related to beta-adrenoceptors, whereas some ligands did display specific binding in heart and lungs. Apparently, the criteria of a high affinity and a moderately high lipophilicity were insufficient to predict the suitability of beta-adrenergic antagonists for visualization of beta-adrenoceptors in the central nervous system.     

Bioweapons synthesis and storage: The venom gland of front-fanged snakes

A paradoxical task of the venom gland of snakes is the synthesis and storage of an instantly available suite of toxins to immobilize prey and the protection of the snake against its own venom components. Furthermore, autolysis of the venom constituents due to the action of venom metalloproteases is an additional problem, particularly among viperid venoms, which are typically rich in lytic enzymatic proteins. To address questions concerning these problems, the structure of the venom gland was investigated using light microscopy, SEM and TEM. The composition of the venom originating from the intact venom apparatus or from the main venom gland alone was analyzed by electrophoresis, and the pH of freshly expressed venom as well as pH optima of several representative enzymes was evaluated. Results from several species of rattlesnakes demonstrated that the venom gland is structurally complex, particularly in its small rostral portion called the accessory gland, which may be a site of activation of venom components. Secreted venom is stable in extremes of temperature and dilution, and several proximate mechanisms, including pH and endogenous inhibitors, exist which inhibit enzymatic activity of the venom during storage within the venom gland but allow for spontaneous activation upon injection into prey. Whereas acid secretion by the parietal cells activates digestive enzymes in the stomach, within the venom gland acidification inhibits venom enzymes. We propose that the mitochondria-rich cells of the main venom gland, which are morphologically and histochemically very similar to the parietal cells of the mammalian gastric pit, play a central role in the stabilization of the venom by secreting acidic compounds into the venom and maintaining the stored venom at pH 5.4. Hence, our results indicate yet another trophic link between the processes of venom production and of digestion, and demonstrate that the venom glands of snakes may represent an excellent model for the study of protein stability and maintenance of toxic proteins.     

Beta-adrenoceptors in human airway tissue: relationship between functional responsiveness and receptor number.

Functional organ bath experiments and radiolabelled ligand binding studies were used to investigate the relationship between beta-adrenoceptor-mediated relaxation and the total number of beta-adrenoceptors in human lung parenchymal tissue and bronchial tissue. Sensitivity to the beta-adrenoceptor agonist isoprenaline (pD2) varied almost 10-fold (pD2 values 6.00 to 6.85) for lung parenchymal preparations and 35-fold for bronchial preparations (pD2 values 6.16 to 7.67) between patients. The total number of [3H] DHA labelled beta-adrenoceptors (Bmax) varied almost 6-fold for lung parenchymal membrane preparations (Bmax 164 to 936 fmol/mg protein) and less than 2-fold for bronchial tissue membrane preparations (Bmax 188 to 342 fmol/mg protein) between patients. Comparison of sensitivity to isoprenaline and beta-adrenoceptor number for lung parenchymal tissue from the same patient demonstrated a negative correlation (r = -0.80 [95% confidence intervals: -0.13, -0.96], 6 d.f., P less than 0.05), suggesting that beta-adrenoceptor-mediated sensitivity of lung parenchymal tissue is inversely related to the number of beta-adrenoceptors. However, there was an absence of correlation between sensitivity to isoprenaline and beta-adrenoceptor number in bronchial tissue from the same patient. Thus, the findings of the present study do not support the possibility of a direct relationship between the beta-adrenoceptor-mediated responsiveness and the beta-adrenoceptor number of human airway preparations.