Necrotic Activity Research Articles

Anti Necrotic and Anticlastogenic Effect of Aloe Vera Extract in Swiss Albino Mice

Aloe vera L (A. barbadensis) is commonly used medicinal and cosmetic plant. In the present investigations the anti necrotic and anticlastogenic activity of Aloe vera extract was studied using Skin Papilloma, Melanoma Bone marrow micronucleus and chromosomal assays in Swiss albino and C57 Bl mice. It has been observed that topical application of Aloe vera extract on the skin tumour induced by DMBA (7.12 Dimethyl Benz (a) nthracene) and croton oil have significantly reduced the tumour size, weight and histological changes as compared to control group. Antinecrotic activity was also observed in skin melanoma tumour induced by subcutaneous administration of B6 F10 cell lines in C57 Bl mice. It reduced the tumour volume and increases the significant survival of animals as compared to control group. In anticlastogenic activity, Aloe vera extract have significantly prevented chromosomal aberrations and micronucleus formations in dose dependent manner. Results suggest the antinecrotic and anticlastogenic potential of Aloe vera extract in the above test systems.

Anti Necrotic and Anticlastogenic Effect of Aloe Vera Extract in Swiss Albino Mice

Aloe vera L (A. barbadensis) is commonly used medicinal and cosmetic plant. In the present investigations the anti necrotic and anticlastogenic activity of Aloe vera extract was studied using Skin Papilloma, Melanoma Bone marrow micronucleus and chromosomal assays in Swiss albino and C57 Bl mice. It has been observed that topical application of Aloe vera extract on the skin tumour induced by DMBA (7.12 Dimethyl Benz (a) nthracene) and croton oil have significantly reduced the tumour size, weight and histological changes as compared to control group. Antinecrotic activity was also observed in skin melanoma tumour induced by subcutaneous administration of B6 F10 cell lines in C57 Bl mice. It reduced the tumour volume and increases the significant survival of animals as compared to control group. In anticlastogenic activity, Aloe vera extract have significantly prevented chromosomal aberrations and micronucleus formations in dose dependent manner. Results suggest the antinecrotic and anticlastogenic potential of Aloe vera extract in the above test systems.

Isolation, identification, characterization, and screening of rhizospheric bacteria for herbicidal activity

The consistent application of agrochemical herbicides has been reported to impact negatively on human health, environment, and food safety, and facilitated the emergence of weed resistances. Rhizosphere bacteria (RB) of different crops were screened for antagonism against Amaranthus hybridus L. (pigweed) and Echinochloa crus-galli (L.) Beauv. (barnyard grass) using necrosis assay technique. A total of eight rhizosphere bacterial isolates (B1–B8) produced different degrees of leaf necrosis on target weeds with isolate B2 manifesting the most significant necrotic activity. The rhizospheric bacterium (B2) with the highest necrotic activity was identified using 16S rRNA sequencing technique and further investigated. Molecular, morphological, and biochemical characterizations confirmed B2 isolate to be Pseudomonas aeruginosa. On isolation with ethyl acetate, separation, defatting, purification, and flash chromatography, seven different fractions (fraction 1–fraction 7) were obtained out of which fraction 4 showed the highest necrotic activity in necrosis assay experiment. Preparative HPLC of fraction 4 resulted in a pure compound that completely inhibited seed germination and seedling development of pigweed and barnyard grass but remained non-antagonistic to other tested soil fungi used in this study. The result obtained from this present study consequently confirmed the antagonistic behavior of rhizosphere-inhabiting P. aeruginosa to the target weeds and qualified the suitability of bacterium as good alternative source of bioherbicide. Potential herbicidal formulation from P. aeruginosa will help reduce crop loss due to weed challenges while offering a partial solution to the use of agrochemicals and food security.

Comparative HPLC-DAD and UHPLC-ESI(-)-HRMS & MS/MS profiling of Hypericum species and correlation with necrotic cell-death activity in human leukemic cells

In the search for potential new anti-cancer agents, the possible anti-proliferative effect of various plant extracts from an in-house extract library was investigated in vitro. The cytotoxic, cytostatic and the pro-apoptotic capacity of the extracts was estimated on the HL-60 human promyelocytic leukemia cell line by the trypan-blue exclusion method, the MTT assay and flow-cytometric analysis. Interestingly, out of all extracts screened, three methanol extracts, namely Hypericum perforatum, Hypericum triquetrifolium and Hypericum rumeliacum, exhibited the most significant activity by promoting necrotic cell-death, accompanied by alterations in the mRNA expression levels of the BCL2 and BAX genes. All the three bioactive extracts were subjected to HPLC-DAD and UHPLC-ESI(−)-HRMS & MS/MS dereplication procedures for correlation purposes. Interesting comparative results related to known constituents such as naphodianthrones, phloroglucinol derivatives and flavonoids were elucidated. To our knowledge, this is the first time that H. triquetrifolium and H. rumeliacum are investigated for both their anti-proliferative properties and their detailed composition.

Functional genomics-guided discovery of a light-activated phytotoxin in the wheat pathogen Parastagonospora nodorum via pathway activation.

Parastagonospora nodorum is an important pathogen of wheat. The contribution of secondary metabolites to this pathosystem is poorly understood. A biosynthetic gene cluster (SNOG_08608-08616) has been shown to be upregulated during the late stage of P. nodorum wheat leaf infection. The gene cluster shares several homologues with the Cercospora nicotianae CTB gene cluster encoding the biosynthesis of cercosporin. Activation of the gene cluster by overexpression (OE) of the transcription factor gene (SNOG_08609) in P. nodorum resulted in the production of elsinochrome C, a perelyenequinone phytotoxin structurally similar to cercosporin. Heterologous expression of the polyketide synthase gene elcA from the gene cluster in Aspergillus nidulans resulted in the production of the polyketide precursor nortoralactone common to the cercosporin pathway. Elsinochrome C could be detected on wheat leaves infected with P. nodorum, but not in the elcA disruption mutant. The compound was shown to exhibit necrotic activity on wheat leaves in a light-dependent manner. Wheat seedling infection assays showed that ΔelcA exhibited reduced virulence compared with wild type, while infection by an OE strain overproducing elsinochrome C resulted in larger lesions on leaves. These data provided evidence that elsinochrome C contributes to the virulence of P. nodorum against wheat.

Lethal, Hemorrhagic, and Necrotic Effects ofMontivipera bornmuelleriVenom

Montivipera bornmuelleri is an endangered viper that has been described as endemic to the Lebanese mountains. The lethal dose 50% (LD50) of M. bornmuelleri venom has been determined by performing intravenous, intraperitoneal (IP), and subcutaneous injections. However, to date, intramuscular injection has not been performed to determine LD50, nor have the minimum hemorrhagic (MHD) or the minimum necrotic doses (MND) been evaluated. Thus, this study was conducted to complete the characterization of the M. bornmuelleri venom. Our results showed that intramuscularly injected LD50 is around 5.39 mg/kg, and IP LD50 is around 1.93 mg/kg. After intradermal injection, our findings showed significant necrotic and medium hemorrhagic activities, and MHD was around 250 µg/kg. By comparing with other local venomous snakes, these results indicate that the lethality of M. bornmuelleri venom is similar to that of Daboia palaestinae venom (IP LD50=1.9 mg/kg) and is higher than that of Macrovipera lebetina venom (IP LD50=7.58 mg/kg) and that the hemorrhagic activity of M. bornmuelleri venom is lower than that of the Macrovipera lebetina (MHD=200 µg/kg). On the other hand, necrotic activity was detected at low doses of injected venom: MND was estimated at 20 µg/kg. Altogether, our findings could extend the scope of characterization of M. bornmuelleri venom and provide useful data for further in vivo studies.

Synthesis, in vitro Antiproliferative and Antiinflammatory Activities, and Kinase Inhibitory effects of New 1,3,4-triarylpyrazole Derivatives

Pyrazole derivatives have been reported as both anticancer and antiinflammatory agents. This study was conducted to develop new pyrazole derivatives as potential anticancer and/or antiinflammatory agents. Their molecular mechanisms of action have been investigated. a series of new triarylpyrazole derivatives were synthesized. Their in vitro anticancer activity was tested against NCI-58 cancer cell line panel of nine cancer types. The most active compound 1a was tested against sixteen kinases, many of them are known to be over-expressed in leukemia and breast cancer. The most sensitive kinases were V600E-B-RAF, C-RAF, FLT3, and P38α/MAPK14. Compound 1a was further tested for caspase-3/7 activity and LDH release assay as measures of its apoptotic and necrotic activities against RPMI-8226. Moreover, the ability of compounds 1a, 1b, and 1g to inhibit nitric oxide and prostaglandin Eproduction in LPS-induced RAW 264.7 macrophages was also examined. Compounds 1a, 1c, and 1g showed the highest activities against the cancer cell line panel, with more inhibitory effects against leukemia and breast cancer subpanels. The highest activity was exerted by compound 1a. Its IC50 values against RPMI-8226, K-562 leukemia cell lines, and MDA-MB-468 breast cancer cell line were 1.71 μM, 3.42 μM, and 6.70 μM, respectively. The IC50 of compound 1a against P38α/MAPK14 kinase was 0.515 μM. The caspase activity was increased by 72% and 170% at 1.23 μM and 3.70 μM concentrations of compound 1a, respectively. Furthermore, compound 1b inhibited 80.26% and 95.31% of NO and PGE2 productions, respectively, at 50 μM concentration in the LPS-induced RAW 264.7 macrophages. Compound 1a could kill the cells through induction of apoptosis rather than necrosis. Compound 1a was more selective against cancer cells than non-cancerous cells. In addition, the hydroxyl analogue 1b was the most active as antiinflammatory agent.

The Results of Re-certification of Reference Standard Sample Used for the Examination of Quality Smallpox Vaccines

The article presents the results of research quality of drugs for the prevention of smallpox during the period from 2007 to 2015 years, where the reference standard uses industry standard of activity, specificity (identification) and necrotic activity of smallpox vaccine, on indicators: «Specific activity», «The antigenic activity» and «Specific safety». Described research methods, presented results and statistical analysis of the results. The article also provided the results of the regular re-certification of reference standard sample activity, specificity (identification) and necrotic activity smallpox vaccine OOO 42-28-113-12 (ОСО), held in 2015. Branch reference preparation for the determination of activity, specificity and necrotic activity of the smallpox vaccine was re-certified with the « specific activity» assigned value of 4,9 ± 2,7 х 10 8 pfu/ml. Other certified characteristics were also confirmed. The criteria «Specific activity» and «Specificity (identification)» were determined using chorioallantoic membrane of 12-days-old chicken embryos and compared against the First International reference preparation of the smallpox vaccine. The re-certification of the branch reference preparation for the determination of activity, specificity (identification) and necrotic activity of the smallpox vaccine (42-28-113-12) confirmed the possibility of its further use. The shelf-life before the next re-certification is 3 years.

Identification of enterototxin harboring gene among Clostridium perfringens isolates with different toxin types in Iran

Background: Clostridium perfringens is known as the most widely distributed pathogenic microorganism in nature. It is an extremely important pathogen of human and domestic animals. In a commonly used classification scheme, C. perfringens is divided into five toxinotypes (A to E) based on the production of four major toxins (alpha, beta, epsilon, and iota). Enterotoxin is not usually used for C. perfringens typing but it is a fatal toxin with necrotic activity. Based on our knowledge there is no published scientific report regarding identification of enterotoxin positive C. perfringens isolates from animals in Iran. OBJECTIVES: To study the presence and frequency of enterotoxin gene among C. perfringence isolates with different types. METHODS: A specific single PCR assay was developed and used for detection of cpe gene to identify the entrerotoxin harboring isolates among different types of C. perfringens isolated from animal enteric diseases in Iran. RESULTS: It was found that cpe gene presents among C.perfringens isolates types A, B, C and D with 63.6% (7/11), 25% ( 5/20), 21.4% (3/14), 53.3% (8/15), respectively. Totally 23 of 60 ( 38.3%) isolates screened by PCR were cpe-positive. CONCLUSIONS: This is the first report of cpe- positive isolates of C.perfringens causing enterototoxemia in animals in Iran. Further studies to investigate the synergistic effect of CPE toxin in pathogenesis of enteric diseases in animals is suggested.

Pathogenesis of dermonecrosis induced by venom of the spitting cobra, Naja nigricollis: An experimental study in mice

The pathogenesis of dermonecrosis induced by the venom of the African spitting cobra Naja nigricollis was investigated in a mouse model. Intradermal injection of venom induced a macroscopic necrotic lesion. Histological examination revealed early edema of the dermis, followed by blistering, loss of skin appendages and reduction in cellularity. By 24 h, necrosis of the dermis was evident, sections of epidermis were lost, and a fibrinoid hyaline material filled the damaged areas. Abundant inflammatory infiltrate was present in the hypodermis and basal dermis, and there was an increment in the expression of matrix metalloproteinases (MMPs). Thrombi were observed in blood vessels. Abundant cells were present in the dermis by 7 days. By 14 and 28 days, re-epithelization had occurred, collagen was widespread in the dermis, and few skin appendages were present. The RP-HPLC fractions that reproduced the necrotic activity were composed of low molecular mass cytotoxins of the three-finger toxin family and, to a lesser extent, of phospholipases A2 (PLA2). Inhibition of PLA2 of venom by p-bromophenacyl bromide did not reduce the area of necrosis, but modified the appearance of necrotic regions. Depletion of neutrophils and inhibition of venom metalloproteinases and tissue MMPs did not affect dermonecrosis. IgG and F(ab′)2 antivenoms were effective in the neutralization of dermonecrosis when incubated with venom prior to injection. However, when antivenoms were administered immediately after venom injection, dermonecrosis was reduced only to a partial extent, underscoring the difficulties in neutralizing this effect with antivenoms.

Synthesis, Characterization and in Vitro Evaluation of Manganese Ferrite (MnFe2O4) Nanoparticles for Their Biocompatibility with Murine Breast Cancer Cells (4T1).

Manganese ferrite (MnFe2O4) magnetic nanoparticles were successfully prepared by a sol-gel self-combustion technique using iron nitrate and manganese nitrate, followed by calcination at 150 °C for 24 h. Calcined sample was systematically characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), and vibrational sample magnetometry (VSM) in order to identify the crystalline phase, functional group, morphology, particle size, shape and magnetic behavior. It was observed that the resultant spinal ferrites obtained at low temperature exhibit single phase, nanoparticle size and good magnetic behavior. The study results have revealed the existence of a potent dose dependent cytotoxic effect of MnFe2O4 nanoparticles against 4T1 cell lines at varying concentrations with IC50 values of 210, 198 and 171 μg/mL after 24 h, 48 h and 72 h of incubation, respectively. Cells exposed to higher concentrations of nanoparticles showed a progressive increase of apoptotic and necrotic activity. Below 125 μg/mL concentration the nanoparticles were biocompatible with 4T1 cells.

The effect of the fungicide captan onSaccharomyces cerevisiaeand wine fermentation

Fungicides, particularly those used during grape maturation, as captan, can affect the natural yeast population of grapes, and can reach grape must affecting wine fermentation. The objective of the present work was to study the effect of captan on the viability and fermentative behavior of S. cerevisiae . S. cerevisiae (BY4741) on exponential phase was treated with captan (0 to 40 μM) for different periods, and their cell viability analyzed. Cell membrane integrity, thiols concentration, and reactive oxygen species (ROS) accumulation was determined. The fermentation experiments were conducted in synthetic must using wine yeast strain Y904. The results showed that under aerobic conditions, 20 μM of captan reduce 90% of yeast viability in 6 hours. Captan treated cells exhibited alteration of membrane integrity, reduction of thiol compounds and increase in intracellular ROS concentration, suggesting a necrotic and pro-oxidant activity of the fungicide. Fermentative experiments showed that concentrations above 2.5 μM captan completely inhibited fermentation, while a dose dependent fermentation delay associated with the reduction of yeast viability was detected in sub-inhibitory concentrations. Petit mutants increase was also observed. In conclusion, the captan induces yeast necrotic cell death on both aerobic and anaerobic conditions causing fermentation delay and/or sucking fermentations.

Anti-oxidant and anti-cancer activities of Angelica dahurica extract via induction of apoptosis in colon cancer cells

IntroductionAngelica dahurica Radix is the common herbal medicine with anti-cancer activities. However, details of its anti-cancer activities are lacking. Materials and methodsWe investigated the anti-cancer effects of Angelica dahurica extract in HT-29 colon cancer cell line. Cell viability, apoptotic and necrotic activities and the mechanism of actions of the active fraction were measured. Results and discussionThe organic extract of Angelica dahurica Radi decreased significantly the gene expression of p53, Bcl, Bax and induced apoptosis via caspase cascade and cell cycle arrest. The ethanol-ethyl acetate fraction showed anti-cancer activities in HT-29 cancer cells. A HPLC-DAD analysis of the fraction indicated the presence of Imperatorin and isoimperatorin, which are the major coumarins in the active fraction that contribute to the anti-cancer activities. ConclusionsThis study has evaluated the ant-cancer activity of the organic extract of Angelica dahurica Radix against colon cancer cells and provided a basis of further development of the herbal extract for treatment of colon cancer.

Pathophysiological effects of Cerastes cerastes and Vipera lebetina venoms: Immunoneutralization using anti-native and anti-60Co irradiated venoms

Cerastes cerastes and Vipera lebetina are the most medically important vipers in Algeria. Their bite induces several pathological effects on victims of accidental envenomation. In this study we analyzed the pathogenesis induced after an experimental envenomation. Indeed, we determined, in vitro, venom enzymatic activities and we analyzed, in vivo, pathological effects induced on liver, heart, lung and skin. In addition we investigated the neutralizing potency of four experimental antivenoms elicited against native and irradiated venoms. Results revealed that V. lebetina and Cerastes cerastes venoms presented strong hemorrhagic, oedematic and necrotic activities. Histopathological study showed that both venoms induced deep damage in tissue structures leading to organ dysfunction. They also increased cellular peroxidases activities, indicating an inflammatory process that is known to amplify tissue damage. Western-blot analysis evidenced that anti-irradiated venoms recognized most components of native venoms. Antivenoms were effective in neutralizing all tested activities, with an increased protective effect obtained with anti-irradiated venoms. Anti-irradiated venoms reduced cellular peroxidases activities indicating a reduction of the inflammatory response. These results may improve our understanding of Algerian Viperidae bite pathogenesis and would encourage further studies planning to provide more proofs on the effectiveness of anti-irradiated venoms administration in the treatment of envenomation.

Hépatite auto-immune induite par la nitrofurantoïne : une complication rare à connaître

IntroductionNitrofurantoin is a commonly used drug which can have liver and pulmonary adverse effects. Among hepatic nitrofurantoin-induced adverse effects, autoimmune hepatitis is a rare complication which must not be mistaken as a toxic hepatitis. Case reportWe report an 86-year-old woman who presented with acute hepatitis after a 3-month course of nitrofurantoin administration for urinary tract infections. She reported a previous hepatitis after treatment by nitrofurantoin twenty years before. Biological analysis showed polyclonal hypergammaglobulinemia, positive test for antinuclear antibodies and smooth muscle antibodies. Finally, liver histology showed lymphocytic infiltration, marked necrotic and inflammatory activity consistent with the diagnosis of autoimmune hepatitis. Nitrofurantoin was discontinued. Outcome of autoimmune hepatitis was good with corticosteroids and azathioprine but two months later, the patient died from a refractory global heart failure. ConclusionNitrofurantoin-induced autoimmune hepatitis is a severe condition which must be systematically discussed in patients taking nitrofurantoin who present with acute hepatitis. Hypergammaglobulinemia is an easily obtained blood marker, which can suggest this diagnosis. Treatment relies on nitrofurantoin eviction, corticosteroids and sometimes azathioprine. Outcome is usually favorable.

The effect of cryptogein with changed abilities to transfer sterols and altered charge distribution on extracellular alkalinization, ROS and NO generation, lipid peroxidation and LOX gene transcription in Nicotiana tabacum

Cryptogein, a protein from oomycete Phytophthora cryptogea, induces a hypersensitive cell death in Nicotiana tabacum. We prepared a new series of cryptogein mutant proteins with altered abilities to bind sterols and with altered charge distribution in the proteins. The effect of the mutations on the cryptogein ability to induce plant defence mechanisms associated with hypersensitive cell death were examined. Our results with new mutants support the previous findings that the sterol binding does not influence synthesis of ROS, cytosol acidification and development of leaf necrosis as these events seem to be more likely affected by the charge distribution and the overall protein structure. This hypothesis was also applicable on other mechanisms involved in the execution of plant cell death such as the NO generation, the stimulation of lipid peroxidation (determination of malondialdehyde and hydroxy fatty acids levels) and LOX gene transcription. In addition, the ability to bind sterols was found to serve not only for pathogen utilisation in its own metabolism but also to have an important function for the destabilization of plant membrane facilitating the pathogen spread inside the plant tissue as well as intensively contributing to the development of plant cell death. Considering the insertion of charged amino acid residues in the protein structure, the change localized in the protein surface affected its biological activity more effectively than that change inside the protein cavity. Moreover, the insertion of negative charged amino acids influenced mainly the events involved in the early phase of defence reaction, while the positive residues affected especially the necrotic activity of cryptogein.

HISTOPATHOLOGICAL EFFECT OF SPIRULINA IN RENAL ISCHEMIA REPERFUSION IN RATS

Ischemia/reperfusion (I/R) injury occurs in renal transplantation, and it could be a risk factor for late renal allograft failure. Several exogenous antioxidants used in prevention of renal I/R injury. Multiple experimental studies demonstrated the antioxidant effect of Spirulina. This study focused on the effects of Spirulina on histopathology of kidney subjected to I/R injury. Methods: Renal I/R was induced in male Sprague-Dwaley rats by occluding the left renal artery then right nephrectomty. Group I was sham operated rats; group II, control (non treated rats); and group III, Spirulina-treated rats (1000 mg/kg BW, orally. 7 days before I/R). Rats were sacrificed 1 & 3 days post reperfusion. The effects of Spirulina on both necrosis and regeneration of kidney tubules caused by renal I/R injury were assayed and scored. Results: Spirulina pretreatment decrease the necrotic injury and improve tubular regeneration after 1 and 3days of renal I/R injury. Conclusion: Spirulina decreased the necrotic activity index in rat kidney after I/R injury and help in tubular regeneration. Keywords: Spirulina; ischemia reperfusion (I/R) injury; acute renal failure; outer strip outer medulla (OSOM); necrosis, tubular regeneration.

A rapidly evolving carcinoma of the thymus discovered in a patient with multiple endocrine neoplasia type 1 and operated on for primary hyperparathyroidism

Introduction Thymic neuroendocrine (NE) tumors associated with multiple endocrine neoplasia type 1 (MEN-1) are rare, variably documented in 1–8% cases. They are malignant and aggressive tumors and constitute a major cause of mortality in MEN-1. We report here a case of neuroendocrine carcinoma of the thymus rapidly developing after parathyroidectomy in a patient with MEN-1. Case A 45-year-old man was admitted for headaches. He had hypercalcemia, a hypophosphatemia and high PTH. Imaging procedures favor parathyroid tumor and pituitary adenoma with slightly high prolactinemia = 1768 mU/mL. Abdominal MRI, FDG PetScan, and scintigraphy with octreotide completed the evaluation. A tumor of the thymus with a necrotic and atypical aspect and high metabolic activity without evidence of metastases was observed, together with 5 pancreatic and small bilateral adrenal nodules. The patient was operated on: in the same time resection of a retrotracheal parathyroid adenoma and of a malignant thymoma type P3 with invasion of pericardium and mediastinal fat, finally pituitay adenomectomy. Surgery was completed by mediastinal radiotherapy. Genetic evaluation confirmed MEN 1. Comment and conclusion NE tumors of thymus in MEN-1 are more common in males and smokers, quite always hormonally inactive, and incidentally diagnosed. They are malignant, aggressive tumors and are widely invasive and metastatic at presentation (affecting usually bone). They traditionally never are the initial feature of MEN-1, and almost always occur after hyperparathyroidism, thus providing an opportunity for prophylaxis at the time of parathyroid surgery. Usually tumors of the thymus occur 15–20 years after discovering g of MEN1. In contrast in our patient, the malignant tumor of the thymus was rapidly developed, that does not exclude precession of an unrecognized primary hyperparathyroidism.

Distinct roles of the repeat-containing regions and effector domains of the Vibrio vulnificus multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin.

ABSTRACTVibrio vulnificus is a seafood-borne pathogen that destroys the intestinal epithelium, leading to rapid bacterial dissemination and death. The most important virulence factor is the multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin comprised of effector domains in the center region flanked by long repeat-containing regions which are well conserved among MARTX toxins and predicted to translocate effector domains. Here, we examined the role of the repeat-containing regions using a modified V. vulnificus MARTX (MARTXVv) toxin generated by replacing all the internal effector domains with β-lactamase (Bla). Bla activity was detected in secretions from the bacterium and also in the cytosol of intoxicated epithelial cells. The modified MARTXVv toxin without effector domains retained its necrotic activity but lost its cell-rounding activity. Further, deletion of the carboxyl-terminal repeat-containing region blocked toxin secretion from the bacterium. Deletion of the amino-terminal repeat-containing region had no effect on secretion but completely abolished translocation and necrosis. Neither secretion nor translocation was affected by enzymatically inactivating the cysteine protease domain of the toxin. These data demonstrate that the amino-terminal and carboxyl-terminal repeat-containing regions of the MARTXVv toxin are necessary and sufficient for the delivery of effector domains and epithelial cell lysis in vitro but that effector domains are required for other cytopathic functions. Furthermore, Ca2+-dependent secretion of the modified MARTXVv toxin suggests that nonclassical RTX-like repeats found in the carboxyl-terminal repeat-containing region are functionally similar to classical RTX repeats found in other RTX proteins.

Effective equine immunization protocol for production of potent poly-specific antisera against Calloselasma rhodostoma, Cryptelytrops albolabris and Daboia siamensis.

Snake envenomation has been estimated to affect 1.8 million people annually with about 94,000 deaths mostly in poor tropical countries. Specific antivenoms are the only rational and effective therapy for these cases. Efforts are being made to produce effective, affordable and sufficient antivenoms for these victims. The immunization process, which has rarely been described in detail, is one step that needs to be rigorously studied and improved especially with regard to the production of polyspecific antisera. The polyspecific nature of therapeutic antivenom could obviate the need to identify the culprit snake species. The aim of this study was to produce potent polyspecific antisera against 3 medically important vipers of Thailand and its neighboring countries, namely Cryptelytrops albolabris "White lipped pit viper" (CA), Calleoselasma rhodostoma “Malayan pit viper” (CR), and Daboia siamensis “Russell’s viper” (DS). Four horses were immunized with a mixture of the 3 viper venoms using the ‘low dose, low volume multi-site’ immunization protocol. The antisera showed rapid rise in ELISA titers against the 3 venoms and reached plateau at about the 8th week post-immunization. The in vivo neutralization potency (P) of the antisera against CA, CR and DS venoms was 10.40, 2.42 and 0.76 mg/ml, respectively and was much higher than the minimal potency limits set by Queen Soavabha Memorial Institute (QSMI). The corresponding potency values for the QSMI monospecific antisera against CA, CR and DS venoms were 7.28, 3.12 and 1.50 mg/ml, respectively. The polyspecific antisera also effectively neutralized the procoagulant, hemorrhagic, necrotic and nephrotoxic activities of the viper venoms. This effective immunization protocol should be useful in the production of potent polyspecific antisera against snake venoms, and equine antisera against tetanus, diphtheria or rabies.