Identification and characterization of venom proteins of two solitary wasps, Eumenes pomiformis and Orancistrocerus drewseni

Secretory proteins were identified in the venoms of two solitary hunting wasps, Eumenes pomiformis and Orancistrocerus drewseni, by SDS-PAGE in conjunction with mass analysis. More than 30 protein bands (2-300 kDa) were detected from the crude venom of each wasp. With the aid of the previously constructed venom gland/sac-specific EST libraries, a total of 31 and 20 proteins were identified from 18 to 20 distinctive protein bands of E. pomiformis and O. drewseni venoms, respectively. Arginine kinase was the most predominant protein in both wasp venoms. Along with the full-length arginine kinase, a truncated form, which was known to have paralytic activity on a spider, was a common predominant protein in the two wasp venoms. Insulin/insulin-like peptide-binding protein was abundantly found only in E. pomiformis venom, which might be due to its unique behaviors of oviposition and provision. The presence of various immune response-related proteins and antioxidants suggested that wasps might use their venom to maintain prey fresh while feeding wasp larvae by protecting the prey from microbial invasion and physiological stresses. It seemed that some venom proteins are secreted into venom fluid from venom gland cells via exosomes, not by signal sequence-mediated transport processes.     

Venom apparatus of braconid wasps: comparative ultrastructure of reservoirs and gland filaments

Two types of venom apparatus present in female braconid wasp were examined in nine species and compared ultrastructurally. The reservoir of type 1 venom apparatus has a relatively thick muscular sheath which is innervated, while the longitudinal and circular muscles of the type 2 reservoir consist of scattered fibers which are not innervated. The intima of the reservoir of type 1 venom apparatus is unevenly thickened. In contrast, the reservoir of type 2 venom apparatus has a relatively thinner and more uniform intima. The gland filaments of both types of venom apparatus are ultrastructurally similar. Distinct particles found in the venom apparatus of two of the nine species are described.     

Functional structure of Agelena labyrinthica's (Araneae:Agelenidae) venom gland and electrophoresis of venom.

The funnel-web spider, Agelena labyrinthica, is widely distributed throughout Turkey. The objective of the present study was to describe the histological and functional fine structure of A. labyrinthica's venom gland by using light microscope, scanning (SEM) and transmission electron microscope (TEM). We have also preliminarily analyzed venom components by SDS-PAGE. Each venom gland has surrounded by a thin adventitia and gross striated muscular bundles. Basal lamina underlies between muscular bundles and the inner glandular epithelium, and ties up them each other. The striated muscular bundles spirally covered venom gland has been observed by SEM. Intricate relations formed between motor neuron axons and the muscle fibers have been revealed by TEM. The secretory epithelium, which made up of simple columnar cells, formed the secretory region of the venom gland. The secretory surface of the gland was increased by a sort of fringes extended from basal membrane into the gland lumen. The epithelial cells have many rough endoplasmic reticulum, mitochondria and different size and shape of secretory granules. These granules have been accumulated in apical portion of the secretory cells. After the gland is emptied, the apical portions of secretory cells deteriorate and the basal epithelial cells regenerate the columnar cells. The analysis of A. labyrinthica venom, which was achieved by SDS-PAGE showed that there have been at least seven components ranging from 10 to 40 kDa molecular weight.     

大连地区涎腺肿瘤337例临床病理分析

目的: 本研究的主要目的是检测不同类型的涎腺肿瘤的不同年龄组男女发生率的不同以及评价良恶性肿瘤的症状和表征的区别.方法: 我们从肿瘤发生的年龄、性别、部位、诊断类型、病史和临床表现几方面对337例涎腺肿瘤的蜡块标本进行研究.结果: 良性肿瘤约占全部被研究肿瘤的79.5%,恶性肿瘤为20.5%.良恶性涎腺肿瘤在发病率上几乎无性别差异.腮腺发生的肿瘤占大涎腺的大多数,为57%(192/337).本次研究中,多形性腺瘤,腺淋巴瘤和腺样囊性癌位居涎腺肿瘤的前三位.多数肿瘤无论男女都在20～59岁的年龄段.结论: 本次研究回顾分析的涎腺肿瘤的发生率相似于其他地区,主要发生在大涎腺,其中主要是腮腺,小涎腺中主要是发生在腭部.肿瘤的快速增长并不是恶性肿瘤特有的,疼痛和活动度差却有较高的恶性肿瘤倾向.儿童没有发现涎腺肿瘤.     

Hydrolysis of substance P and bradykinin by black widow spider venom gland extract

Black widow spider venom gland extract was found to contain significant peptidase activity. Aliquots of the venom gland extract incubated at 37 degrees inactivated substance P (SP) and bradykinin but not angiotensin II or the enkephalins. The peptide inactivation was proportional to the duration of the incubation and the amount of extract used. Analysis of the peptides on high pressure liquid chromatography demonstrated that the loss in biological activity of SP and bradykinin in the longitudinal muscle of the guinea pig ileum was correlated with cleavage of the peptides into several fragments. Kinetic studies revealed that SP was initially split into two fragments but that these products underwent further degradation into smaller peptides. The optimal pH for the peptidase activity was 6.5. At 0 degree the enzymatic activity was undetectable, and it was irreversibly destroyed by incubation at 100 degrees for 5 min or by pretreatment of the extract with 100 microM diisopropyl fluorophosphate. In addition, the gland extract preparation hydrolyzed artificial substrates designed to detect trypsin or chymotrypsin-like activity.     

Clinical concerns of immunogenicity produced at cellular levels by biopharmaceuticals following their parenteral administration into human body

Similar to the low molecular weight traditional drugs, biopharmaceuticals are capable of producing not only therapeutic effects but also side effects provided if the dose of these compounds exceeds certain concentration and/or if the exposure duration of these compounds at subtoxic doses is being lengthened. In addition, a major drawback of biopharmaceuticals is the risk of antibody formation. Following the administration of biopharmaceuticals into human body, the formation of antidrug–antibody (ADA) or neutralizing antibody and other general immune system effects (including allergy, anaphylaxis, or serum sickness) are of clinical concern regarding therapeutic efficacy and patient safety. For example, drug-induced neutralizing antibodies to erythropoietin (EPO) result in pure red cell aplasia, whereas drug-induced acquired anti-factor VIII antibodies worsen the pathology associated with hemophilia. Since most of the already developed or under development biopharmaceuticals are to some extent immunogenic, the regulatory agencies insist to conduct potential ADA formation during the drug development process itself. This review encompasses a short overview on the clinical concerns of immunogenicity produced at cellular levels by growth hormone, interferon-α, EPO, factor VIII, and factor IX following their parenteral administration into human body. Clinical concerns related to immunogenicity produced by the biosimilar versions of these drugs are also presented wherever possible.     

Lysis of malarial parasites and erythrocytes by ferriprotoporphyrin IX-chloroquine and the inhibition of this effect by proteins

Ferriprotoporphyrin IX(FP) lysed both erythrocytes and isolated Plasmodium falciparum as judged by decrease in turbidity of erythrocyte and parasite suspensions. The lytic effect of FP on erythrocytes was enhanced by chloroquine (CQ). In the presence of 2.5-20 microM CQ, 5 microM FP led to complete hemolysis within 45 min. However, the lytic effect of FP or FP-CQ on both erythrocytes and parasites was inhibited completely by proteins. The protein inhibition was non-specific. This finding, the failure of FP and FP--CQ to cause hemolysis and lysis of malarial parasites in a protein-containing medium, does not support the "FP--CQ complex hypothesis" for the antimalarial action of chloroquine.     

Specific activation of human neutrophils by scorpion venom: A flow cytometry assessment

Acute lung injury following envenomation by Tityus scorpion species is due in part to activation of the inflammatory response leading to release of cytotoxic leukocyte-derived products, including cytokines and possibly reactive oxygen species (ROS). Tityus zulianus envenomation in Venezuela produces cardiorespiratory complications and death by lung injury whereas stings by Tityus discrepans produce mainly gastrointestinal and pancreatic alterations. To ascertain the role played by granulocytes in the envenomation by T. zulianus (TzV) and T. discrepans (TdV), human peripheral blood neutrophils, eosinophils, and monocytes were exposed to scorpion venoms (0.001-5 μg/mL) and the kinetics (5-15 min) of peroxide production determined by flow cytometry, using 2′,7′-dichlorodihydrofluorescein diacetate (succinimidyl ester) as a fluorescent substrate. TzV induced a significantly (p < 0.01) more potent increase in peroxide production in neutrophils (for 5 and 10 min of incubation), and to a lesser extent in monocytes (5-15 min), compared to TdV. TzV induced necrosis in neutrophils at doses higher than 5 μg/mL. No effect was observed on eosinophils, suggesting that TzV specifically targets neutrophil intracellular ROS production. The TzV-stimulated pathway is protein kinase C-dependent because it was almost completely (>90%) abolished by staurosporine. The stimulatory effect is associated with the lowest molecular mass venom peptides as gel filtration fractions TzII and TzIII significantly enhanced peroxide production. The combined used of the intracellular ROS agonist, phorbol myristate acetate (PMA), and TzV produced a modest but significant increase in peroxide production suggesting the possibility of overlapping signaling cascades amongst PMA and TzV. Up-regulation of intracellular neutrophil ROS production may be an important in vivo target for TzV which could have a role to play in the cardiorespiratory complications elicited after envenomation by this species.     

Properties of fibrinogen degradation products produced by alpha- and beta- fibrinogenases of Trimeresurus mucrosquamatus snake venom.

C. Ouyang, C.-M. Teng and Y.-C. Chen. Properties of fibrinogen degradation products produced by α- and β-fibrinogenases of T. mucrosquamatus snake venom. Toxicon17, 121–126, 1979.—Human fibrinogen was digested by α- and β-fibrinogenases of T. mucrosquamatus snake venom. Fifty per cent of the unclottable fibrinogen was precipitable by protamine sulfate, while only 5% of these degradation products were soluble in trichloroacetic acid. α- and β-fibrinogenases were weak anticoagulants as measured by recalcification time and plasma prothrombin time. The fibrinogen degradation products produced by β-fibrinogenase could polymerize with normal fibrin monomer, but prolonged the reaction time of thrombin with fibrinogen. α-Fibrinogenase inhibited platelet aggregation induced by ADP, while β-fibrinogenase did not.     

Neutralization of Bothrops mattogrossensis snake venom from Bolivia: Experimental evaluation of llama and donkey antivenoms produced by caprylic acid precipitation

Polyspecific bothropic/crotalic and bothropic/lachesic antivenoms were produced in Bolivia by immunizing two donkeys with the venoms of Bothrops mattogrossensis and Crotalus durissus terrificus and one llama with the venoms of B. mattogrossensis and Lachesis muta. These antivenoms are currently being used for snakebite envenomation in Bolivia. The rationale for using these animals is that donkeys and llamas are better adapted than horses to the high altitudes in South America and constitute good alternatives for antivenom production in these regions. Plasma was fractionated by caprylic acid precipitation of non-immunoglobulin plasma proteins, to obtain whole IgG preparations. Donkey-derived antivenom showed one band of 150 kDa when analyzed by SDS–PAGE, whereas llama antivenom presented two immunoglobulin bands, of 170 kDa and 120 kDa, the latter corresponding to the heavy-chain antibodies present in camelid sera. The effectiveness of these antivenoms to neutralize lethal, hemorrhagic, myotoxic, edema-forming, and defibrinogenating activities of the venom of B. mattogrossensis from Bolivia, a species formerly known as Bothrops neuwiedii, was assessed at the experimental level. Although llama antivenom has a total protein concentration four times lower than donkey antivenom, both preparations have similar neutralizing capacity against all toxic activities assessed. Llama and donkey IgG-based antivenoms are effective in the neutralization of B. mattogrossensis venom and represent valuable alternatives for antivenom manufacture in highland regions of South America.     

Alterations in sodium channel gating produced by the venom of the marine mollusc Conus striatus.

The action of the venom from the marine mollusc Conus striatus was studied using the voltage-clamp technique on myelinated nerve. Conus venom applied to an isolated node of Ranvier at 1.1 micrograms protein/ml produced repetitive firing of action potentials when the node was depolarized under current-clamp conditions. Venom application produced a leftword (depolarizing) shift in both the peak sodium current-voltage and the permeability-voltage relationships. A concomitant decrease in maximum peak current and permeability also occurred. The time course of sodium current decline (inactivation) was slowed at all voltages by the presence of venom. Venom treatment caused only a slight depolarizing shift (5 mV) in the voltage-dependence of steady-state Na inactivation. The closing to the resting state of previously activated Na channels, "deactivation", was judged from Na "tail" currents following membrane repolarization, and was slowed more than four-fold by venom treatment. The changes in Na channel gating produced by Conus striatus venom can best be described as a stabilization of the open state of the Na channel and a shift in the voltage dependence of the opening of Na channels. The slowing of both inactivation and deactivation of Na channels can be simulated by alterations in the rate constants of a five state Markov model.     

Influence of hydrocortisone on the microscopic changes produced by Naja nigricollis venom in kidney, liver and spleen

The effects of N. nigricollis venom, in sublethal multiple doses, on the kidney, liver and spleen were demonstrated in mice. The kidney showed degenerative changes in the tubular system together with cellular infiltration. In the liver, there was vacuolization and fatty degeneration of the parenchymal cells, together with round cellular infiltration around the bile ducts. The microscopic changes in the spleen consisted of hyperplasia of the lymph follicles as evidenced by the large number of lymphoblasts. After hydrocortisone, the renal tubules exhibited more vacuolization and round cell infiltration. The changes induced by the venom in the liver were ameliorated by hydrocortisone. In the spleen, hydrocortisone caused a marked increase in the number of megakaryocytes.     

Structure and function of snake venom proteins affecting platelet plug formation

Many snake venom proteins have been isolated that affect platelet plug formation by interacting either with platelet integrins, membrane glycoprotein Ib (GPIb), or plasma von Willebrand factor (VWF). Among them, disintegrins purified from various snake venoms are strong inhibitors of platelet aggregation. Botrocetin and bitiscetin derived from Bothrops jararaca and Bitis arietans venom, respectively, induce VWF-dependent platelet agglutination in vitro. Several GPIb-binding proteins have also been isolated from snake venoms. In this review, we focus on the structure and function of those snake venom proteins that influence platelet plug formation. These proteins are potentially useful as reagents for the sub-diagnosis of platelet disorder or von Willebrand disease, as well as for clinical and basic research of thrombosis and hemostasis.     

Production of human antibody fragments binding to melittin and phospholipase A2 in Africanised bee venom: minimising venom toxicity

The hybrid created from the crossbreeding of European and African bees, known as the Africanised bee, has provided numerous advantages for current beekeeping. However, this new species exhibits undesirable behaviours, such as colony defence instinct and a propensity to attack en masse, which can result in serious accidents. To date, there is no effective treatment for cases of Africanised bee envenomation. One promising technique for developing an efficient antivenom is the use of phage display technology, which enables the production of human antibodies, thus avoiding the complications of serum therapy, such as anaphylaxis and serum sickness. The aim of this study was to produce human monoclonal single-chain Fv (scFv) antibody fragments capable of inhibiting the toxic effects of Africanised bee venom. We conducted four rounds of selection of antibodies against the venom and three rounds of selection of antibodies against purified melittin. Three clones were selected and tested by enzyme-linked immunosorbent assay to verify their specificity for melittin and phospholipase A2. Two clones (C5 and C12) were specific for melittin, and one (A7) was specific for phospholipase A2. In a kinetic haemolytic assay, these clones were evaluated individually and in pairs. The A7-C12 combination had the best synergistic effect and was chosen to be used in the assays of myotoxicity inhibition and lethality. The A7-C12 combination inhibited the in vivo myotoxic effect of the venom and increased the survival of treated animals.     

Stabilization of proteins by recombinant human gelatins

Porcine gelatins have been widely used as stabilizers of macromolecular based pharmaceuticals but the mechanism by which they stabilize has not been precisely established. Their variability and immunogenicity, however, make them less than ideal excipients. In this work, we take advantage of the availability of recombinant human gelatins (rhGs) to explore the mechanism by which they may stabilize proteins. Three model recombinant proteins, human serum albumin (HSA), bovine granulocyte colony stimulating factor (bGCSF), and human fibroblast growth factor-20 (FGF-20) that display a range of isoionic points have been selected for this study. The interaction of these model proteins with four different molecular weight rhGs and porcine gelatin was studied using a variety of biophysical techniques including fluorescence, CD and second derivative UV spectroscopy to monitor tertiary and secondary structure as a function of temperature. The 8.5, 25, and 100 kDa rhGs had the greatest effect on conformational and colloidal stability of HSA. The 8.5, 25, and 50 kDa rhGs also increased the T(0) of aggregation of bGCSF and FGF-20. Experiments to probe the mechanism of interaction of model proteins with rhGs suggest that the rhGs might interact with the partially unfolded states of target proteins through a combination of electrostatic and other intermolecular mechanisms to inhibit aggregation.