Correlation of Staphylococcus Epidermidis Phenotype and Its Corneal Virulence.

PURPOSE: S. epidermidis is an ocular pathogen and a leading cause of keratitis. It produces hemolysins and at least 3 proteases. The purpose of the present study is to compare the secretion of hemolysins and proteases between 28 ocular isolates and one non-ocular strain and to determine their relationship to ocular virulence in selected strains using a rabbit model of infection. MATERIALS AND METHODS: Culture supernatants were compared for protease production and hemolysis. Selected strains were injected into rabbit corneas and their virulence and pathology recorded. The major protease activity in a virulent strain was identified and the gene was cloned and expressed as a recombinant protein. The corneal toxicity of this protease was determined. Antibodies to the native protease were generated and tested for neutralizing activity in vivo and in vitro. The corneal pathology of the S. epidermidis protease was compared to the pathology of S. aureus V8 protease. RESULTS: Strains that exhibited the least protease activity in vitro caused significantly less ocular pathology in vivo (p ≤ 0.003). Strains that were hemolytic and secreted a major protease had numerically higher SLE scores. This protease was identified as the serine protease Esp. The recombinant Esp protease caused extensive pathology when injected into the corneal stroma (7.62 ± 0.33). Antibody generated against native Esp did not neutralize the activity of the protease in vivo or in vitro. The antibody reacted with Esp proteases secreted by other S. epidermidis strains. S. epidermidis Esp protease and its homologue in S. aureus caused similar ocular pathology when injected in the rabbit corneal stroma. CONCLUSION: Hemolysins and proteases seem to be important in corneal pathology caused by S. epidermidis infections. The Esp protease mediates significant corneal damage. S. epidermidis Esp and S. aureus V8 protease caused similar and extensive edema in rabbit corneas.

Repeat-dose animal toxicity studies and genotoxicity study with deactivated alkaline serine protease (DASP), a protein low in phenylalanine (PHE).

Phenylketonuria (PKU) is an autosomal recessive inherited disorder affecting one in every 10,000 to 15,000 newborn children in the US each year. PKU patients' metabolism of an essential amino acid, phenylalanine (PHE), is impaired, resulting in concentrations of PHE in the circulating blood and brain that are potentially toxic. Individuals with PKU restrict dietary intakes of PHE by consuming medical foods formulated with low PHE concentrations. In this study, an alkaline serine protease (ASP) expressed in Bacillus licheniformis strain 2709, which is composed of >90% protein with a concentration of <0.25% PHE, was heat deactivated (becoming deactivated ASP (DASP)) and evaluated for safe use as an ingredient in foods, including medical foods. DASP was non-mutagenic with and without metabolic activation up to 5000 µg DASP/plate. 14-Day dietary studies supported a Maximum Tolerated Dose (MTD) of 115000 ppm DASP. In a 90-day dietary toxicity study, CRL SD CD® rats were administered 0, 28750, 57500, 115500 ppm DASP in the diet. No DASP-related adverse effects were observed at the high dose. As such, a No Observable Adverse Effect Level (NOAEL) of 115,500 ppm DASP or 6224.1 mg DASP/kg bw/day (males) and 7500.9 mg DASP/kg bw/day (females) was established.

Enterohemorrhagic E. coli (EHEC)-Secreted Serine Protease EspP Stimulates Electrogenic Ion Transport in Human Colonoid Monolayers.

One of the characteristic manifestations of Shiga-toxin-producing Escherichia coli (E. coli) infection in humans, including EHEC and Enteroaggregative E. coli O104:H4, is watery diarrhea. However, neither Shiga toxin nor numerous components of the type-3 secretion system have been found to independently elicit fluid secretion. We used the adult stem-cell-derived human colonoid monolayers (HCM) to test whether EHEC-secreted extracellular serine protease P (EspP), a member of the serine protease family broadly expressed by diarrheagenic E. coli can act as an enterotoxin. We applied the Ussing chamber/voltage clamp technique to determine whether EspP stimulates electrogenic ion transport indicated by a change in short-circuit current (Isc). EspP stimulates Isc in HCM. The EspP-stimulated Isc does not require protease activity, is not cystic fibrosis transmembrane conductance regulator (CFTR)-mediated, but is partially Ca2+-dependent. EspP neutralization with a specific antibody reduces its potency in stimulating Isc. Serine Protease A, secreted by Enteroaggregative E. coli, also stimulates Isc in HCM, but this current is CFTR-dependent. In conclusion, EspP stimulates colonic CFTR-independent active ion transport and may be involved in the pathophysiology of EHEC diarrhea. Serine protease toxins from E. coli pathogens appear to serve as enterotoxins, potentially significantly contributing to watery diarrhea.

Toxicological profile and safety pharmacology of a single dose of fibroblast activation protein-α-based doxorubicin prodrug: in-vitro and in-vivo evaluation.

Fibroblast activation protein-α (FAPα) is a promising tumor-associated target expressed by reactive stromal fibroblasts in tumor tissue. FAPα has a postprolyl peptidase activity and can specifically cleave N-terminal benzyloxycarbonyl (Z)-blocked peptides, such as the substrate Z-Gly-Pro-AMC. Doxorubicin (DOX) is an effective antitumor drug, but its application is greatly limited by toxic adverse effects owing to poor tumor selectivity. Based on these facts, we previously designed a FAPα-targeting prodrug of doxorubicin (FTPD) which can be selectively hydrolyzed by FAPα. FTPD can retain potent antitumor efficacy and has favorable tumor targeting. The present study aimed to further evaluate the toxicological profile and the safety pharmacological property of FTPD in vitro and in vivo. The cytotoxicity assay showed that FTPD displayed markedly lower cytotoxicity to 3T3 cells and HEK-293 cells compared with DOX. In the short-term toxicity study, mice treated with 25 mg/kg of FTPD showed no obvious change in the appearance and general behavior, and no case of mortality was observed within 14 days. Unlike DOX, FTPD exhibited reduced toxicity to heart, liver, kidney, spleen as well as peripheral white blood cells in mice. Moreover, open file test and general pharmacology study were also conducted correspondingly in mice and beagle dogs. It was found that FTPD may not produce significant pharmacological effects on spontaneous locomotor activity and cardiovascular-respiratory system except for a transient decreasing in systolic blood pressure. Taken together, the results of this work suggest that FTPD has more favorable toxicological profile and better drug safety compared with its parent drug DOX.

Pet serine protease from enteroaggregative Escherichia coli stimulates the inflammatory response activating human macrophages.

BACKGROUND: Pet is a toxin from the family of Serine Protease Autotransporters of Enterobacteriaceae which was initially identified in Enteroaggregative Escherichia coli strains. This protease exhibits enterotoxin properties, damages the cell cytoskeleton and induces intestinal epithelium alterations, which are associated with a severe inflammatory process. An in-vitro study was conducted to evaluate the effect of Pet on the migration of human peripheral blood monocytes-derived macrophages and its participation in the activation of the early inflammatory response and cytokine expression. RESULTS: In the macrophage migration activation assay, Pet produced a similar effect to that induced by opsonized zymosan (ZAS). Regarding the cytokine expression, an increase of IL-8, TNF-α (pro-inflammatory) and IL-10 (anti-inflammatory) was identified. In addition to the above results, the nuclear translocation of NF-kB pp65 was also identified. These events are probably related to the inflammatory response identified in the histological examination of intestine rat samples inoculated with Pet during a ligated loop assay. CONCLUSION: The results showed that Pet participates as an immunostimulant molecule for macrophages, which activates both their mobility and cytokine expression. These observations suggest that the toxin participates in the inflammatory process that is observed during the host infection by EAEC Pet producing.

Label-Free (XIC) Quantification of Venom Procoagulant and Neurotoxin Expression in Related Australian Elapid Snakes Gives Insight into Venom Toxicity Evolution.

This study demonstrates a direct role of venom protein expression alteration in the evolution of snake venom toxicity. Avian skeletal muscle contractile response to exogenously administered acetylcholine is completely inhibited upon exposure to South Australian and largely preserved following exposure to Queensland eastern brown snake Pseudonaja textilis venom, indicating potent postsynaptic neurotoxicity of the former and lack thereof of the latter venom. Label-free quantitative proteomics reveals extremely large differences in the expression of postsynaptic three-finger α-neurotoxins in these venoms, explaining the difference in the muscle contractile response and suggesting that the type of toxicity induced by venom can be modified by altered expression of venom proteins. Furthermore, the onset of neuromuscular paralysis in the rat phrenic nerve-diaphragm preparation occurs sooner upon exposure to the venom (10 µg/mL) with high expression of α-neurotoxins than the venoms containing predominately presynaptic ß-neurotoxins. The study also finds that the onset of rat plasma coagulation is faster following exposure to the venoms with higher expression of venom prothrombin activator subunits. This is the first quantitative proteomic study that uses extracted ion chromatogram peak areas (MS1 XIC) of distinct homologous tryptic peptides to directly show the differences in the expression of venom proteins.

Streptococcus pneumoniae ClpP protease induces apoptosis via caspase-independent pathway in human neuroblastoma cells: cytoplasmic relocalization of p53.

Streptococcus pneumoniae causes the most severe form of the bacterial meningitis which is the major cause of bacterial meningitis. Virulence factors produced by S. pneumoniae have been known to contribute significantly to the disease process. ClpP protease (ClpP) which is essential for virulence and survival under stress conditions in S. pneumoniae was examined for the ability to induce apoptosis and the mechanism of the induction of apoptosis in human neuron-like cells, SK-N-SH neuroblastoma cells. ClpP inhibited cell growth and induced apoptosis in SK-N-SH cells. Treatment with ClpP resulted in hypodiploid DNA contents, increased Bax/Bcl-2 ratio and induction of reactive oxygen species (ROS) production. The release of cytochrome c from mitochondria into the cytosol, which is an initiator of the activation of caspase cascades, was not observed in ClpP-treated cells. In addition, pretreatment with Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), a broad spectrum caspase inhibitor, could not rescue apoptotic cells from ClpP toxicity. Coincidently, caspase-3 and -8 activation and cleavage of PARP were not detected. Moreover, caspase independent apoptosis-inducing factor (AIF) was released from mitochondria and translocated to the nucleus in response to ClpP. We also found that ClpP treatment resulted in the increase of p53 activity and cytoplasmic p53 levels were increased by ClpP, suggesting that functional activation of p53 is intact despite increased cytoplasmic accumulation. Taken together, these data suggest that ClpP contributes to neuronal damage in meningitis and provide further insight into the mechanisms underlying action of pneumococcal virulence factors during bacterial pathogenesis.

Effects of the plasmid-encoded toxin of enteroaggregative Escherichia coli on focal adhesion complexes.

Enteroaggregative Escherichia coli (EAEC) is an emerging diarrheal pathogen. Many EAEC strains produce the plasmid-encoded toxin (Pet), which exerts cytotoxic effects on human intestinal tissue. Pet-intoxicated HEp-2 cells exhibit rounding and detachment from the substratum, accompanied by loss of F-actin stress fibers and condensation of the spectrin-containing membrane cytoskeleton. Although studies suggest that Pet directly cleaves spectrin, it is not known whether this is the essential mode of action of the toxin. In addition, the effects of Pet on cytoskeletal elements other than actin and spectrin have not been reported. Here, we demonstrate by immunofluorescence that upon Pet intoxication, HEp-2 and HT29 cells lose focal adhesion complexes (FAC), a process that includes the redistribution of focal adhesion kinase (FAK), α-actinin, paxillin, vinculin, F-actin, and spectrin itself. This redistribution was coupled with the depletion of phosphotyrosine labeling at FACs. Immunoblotting and immunoprecipitation experiments revealed that FAK was tyrosine dephosphorylated, before the redistribution of FAK and spectrin. Moreover, phosphatase inhibition blocked cell retraction, suggesting that tyrosine dephosphorylation is an event that precedes FAK cleavage. Finally, we show that in vitro tyrosine-dephosphorylated FAK was susceptible to Pet cleavage. These data suggest that mechanisms other than spectrin redistribution occur during Pet intoxication.

New insight into the mechanism of Lonomia obliqua envenoming: toxin involvement and molecular approach.

Despite the nearly worldwide distribution of Lepidoptera, there are few species with clear documentation of adverse reactions in humans. Most syndromes caused by Lepidoptera are consequences of direct contact with the hairs or setae of caterpillars. In most instances, the adverse effects caused by moth and caterpillars are self-limited and the treatment is based on the removal of hairs, application of topical antipruritics and, in some cases, the use of oral antihistamines. However, in the case of envenoming by South American Lonomiaobliqua caterpillars, the antilonomic serum produced at Instituto Butantan in Brazil is the only effective treatment to re-establish the physiological coagulation parameters in poisoned patients and to abolish the complications seen in severe cases (e.g. consumptive coagulopathy, intracerebral hemorrhage, and acute renal failure). Many studies have been carried out to understand the pathophysiological mechanism of envenoming by L. obliqua. Several toxic principles were found in bristle extract and the hemolymph, probably related to the envenoming. An interesting fact is that some toxins from the venom usually have more than one function. With the advent of molecular biology techniques it has become possible to analyze these processes at a molecular level, thus giving rise to hypotheses on the molecular basis of envenomation. This review contributes to enhance our understanding of the dramatic alterations that hemorrhagic syndrome causes in patients, current treatment, and the diversity of the molecules involved in this pathology.

The expression of a recombinant cry1Ac gene with subtilisin-like protease CDEP2 gene in acrystalliferous Bacillus thuringiensis by Red/ET homologous recombination.

A novel cDNA encoding the subtilisin-like serine protease gene CDEP2 was isolated from Beauveria bassiana by reverse transcription polymerase chain reaction (RT-PCR). It contained an 1137 bp ORF that predicted a protein of 379 amino acids with M = 38863 Da and pI = 8.21. In an attempt to improve insecticidal activity, the CDEP2 gene and the cry1Ac gene from Bacillus thuringiensis were co-fused into the vector pHT315 as pHAc-CDEP2 plasmid by Red/ET homologous recombination. The co-fusion gene was attempted under the control of the native cry1Ac promoter. Plasmid pHAc-CDEP2 was electro-transformed into the B. thuringiensis subsp. kurstaki Cry(-)B. Analyzed by SDS-PAGE and Western blotting, the transformant Cry(-)B-pHAc-CDEP2 strain produced a 130 kDa Cry1Ac protein and 39 kDa CDEP2 protein. The 50% lethal concentration values (LC(50)) of Cry(-)B-pHAc-CDEP2 strain (8.5 microl/ml) to Helicoverpa armigera third instars larvae was clearly higher than the Cry(-)B-pHAc strain (16.7 microl/ml) at 72 h.

Human neutrophils kill Streptococcus pneumoniae via serine proteases.

Neutrophils, or polymorphonuclear leukocytes, comprise a crucial component of innate immunity, controlling bacterial and fungal infection through a combination of both oxidative and nonoxidative mechanisms. Indeed, neutrophils are believed to play an important role in controlling infection caused by the major human pathogen Streptococcus pneumoniae. However, the method by which neutrophils kill the pneumococcus as well as other Gram-positive bacteria, is not fully understood. We investigated human neutrophil killing of the pneumococcus in a complement-dependent opsonophagocytic assay. In contrast to other Gram-positive organisms, inhibition of the NADPH oxidase did not affect killing of S. pneumoniae. Supernatant from degranulated neutrophils killed the pneumococcus, suggesting a role for granular products. When neutrophil granule proteases were inhibited with either a protease mixture, or specific serine protease inhibitors 4-(2-Aminoethyl)benzenesulfonylfluoride and diisopropylfluorophosphate, killing by neutrophils was inhibited in a manner that correlated with increased intracellular survival. All three compounds inhibited intracellular activity of the three major neutrophil serine proteases: elastase, cathepsin G, and proteinase 3. Additionally, purified elastase and cathepsin G were sufficient to kill S. pneumoniae in a serine protease dependent-manner in in vitro assays. Inhibition studies using specific inhibitors of these serine proteases suggested that while each serine protease is sufficient to kill the pneumococcus, none is essential. Our findings show that Gram-positive pathogens are killed by human neutrophils via different mechanisms involving serine proteases.

Commercial monovalent antivenoms in Australia are polyvalent.

Monovalent antivenoms have a lower volume of specific antibodies that may reduce reactions but require accurate snake identification to be used. Polyvalent antivenoms are larger volume and may have a higher reaction rate. However, they avoid the problem of snake identification and may be more cost-effective to manufacture. We have previously shown cross-neutralisation of two Australian elapid venoms, tiger snake (Notechis scutatus) and brown snake (Pseudonaja textilis) venoms, by their respective monovalent antivenoms. In this study enzyme immunoassays were used to quantify the amount of monovalent antivenom (quantity of monovalent antibodies to a specific snake venom) in vials of commercially produced antivenom in Australia. All antivenoms tested appeared to be polyvalent and contain varying amounts of all five terrestrial snake monovalent antibodies based on their binding to the five representative venoms. Redback spider antivenom did not have any measurable binding affinity for any of the five snake venoms, showing that the observed binding is not due to non-specific interactions with equine protein. The antivenoms had expire dates over a 15 year period, suggesting that the antivenoms have been mixtures for at least this time. This study cannot be used to rationalise hospital stocks of antivenom in Australia because there is no guarantee that the antivenoms will remain as mixtures. However, it would be possible for the manufacturer to reduce the number of types of snake antivenoms available in Australia to two polyvalent antivenoms which would simplify treatment of snakebite.

A low molecular weight serine protease: Purification and characterization from Hippasa agelenoides (funnel web) spider venom gland extract.

Despite the long history [Kaiser, E., 1956. Enzymatic activity of spider venoms. In: Buckley, E.E., Porges, N. (Eds.), Venoms. American Association for the Advancement of Science, Washington, DC, pp. 91-93] on proteolytic activity, no study so far claims the isolation of a serine protease from the spider venom/venom gland extract. Therefore, the present study describes the isolation and characterization of a low molecular weight serine protease from Hippasa agelenoides venom gland extract. The protease (Hag-protease) was purified to homogeneity using the combination of gel-permeation and ion-exchange chromatography. The molecular mass was found to be 16.350 kDa by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. Hag-protease was optimally active at pH 7.5 and temperature of 37 degrees C. PMSF abolished the enzyme activity while EDTA, EGTA, IAA, 1, 10-phenanthrolene did not. It hydrolyzed proteins such as casein, fibronectin and collagen type-I dose dependently but did not degrade gelatin and collagen type-IV. The isolated protease was non-lethal and devoid of hemorrhagic, myotoxic and edema forming activities. The light microscopy of Hag-protease treated skin tissue sections at the site of injection showed extensive damage of extracellular matrix (ECM) of hypodermis without causing any damage to blood vessels and capillaries. Similar damage of ECM of muscle tissue sections without affecting myocytes was noticed. Hag-protease was found to be procoagulant in property when studied plasma recalcification time.

Purification and N-terminal sequence of a serine proteinase-like protein (BMK-CBP) from the venom of the Chinese scorpion (Buthus martensii Karsch).

A serine proteinase-like protein was isolated from the venom of Chinese red scorpion (Buthus martensii Karsch) by combination of gel filtration, ion-exchange and reveres-phase chromatography and named BMK-CBP. The apparent molecular weight of BMK-CBP was identified as 33 kDa by SDS-PAGE under non-reducing condition. The sequence of N-terminal 40 amino acids was obtained by Edman degradation. The sequence shows highest similarity to proteinase from insect source. When tested with commonly used substrates of proteinase, no significant hydrolytic activity was observed for BMK-CBP. The purified BMK-CBP was found to bind to the cancer cell line MCF-7 and the cell binding ability was dose-dependent.

[Long-term and acute toxicity of kallikrein from the venom of Agkistrodon hlays Pallas].

A novel serine protease with high purity was extracted from the venom of Agkistrodon hlays Pallas using monoclonal antibody affinity chromatography. This protease releases bradykinin and has arginine esterase activity without being activated. After purification, its hydrolytic activity exceeded 800 U/mg, far higher than its counterparts from mammalian sources. The purity of the kininogenase could exceed 95%. The acute toxicity and the long-term toxicity of this kallikrein was studied for its potential clinical application. The maximum tolerance dose in adult was 150,000 times greater than the maximum applied dose, and long-term administration at the dose 50 times of allowed clinical dose did not obviously after the animals' body weight, survival condition, liver function, renal function, or blood routines, suggesting the extremely low toxicity of the kallidrein.

Cloning and expression of the gene encoding an extracellular alkaline serine protease from Vibrio alginolyticus strain HY9901, the causative agent of vibriosis in Lutjanus erythopterus (Bloch).

A 750-bp internal fragment of the alkaline serine protease gene (asp) from the Vibrio alginolyticus strain HY9901 was amplified by polymerase chain reaction (PCR). The flanking sequences of the 5'- and 3'- ends of the asp gene were characterized by reverse and nested PCR. Sequence analysis showed that the asp gene contained an 1893-bp ORF encoding 630 amino acids. The deduced amino acid sequence of the ASP (alkaline serine protease) precursor showed significant homology with several bacterial alkaline serine proteases. Expression of the asp gene in Escherichia coli and activity tests of the ASP indicated that the N-signal peptide of the ASP precursor was essential to autocatalyse and fold correctly the enzyme to obtain activity. The purified ASP was lethal for Lutjanus erythopterus with an LD(50) of 0.25 microg protein g(-1) body weight.

Cutaneous and systemic effects of varying doses of brown recluse spider venom in a rabbit model.

OBJECTIVE: To ascertain whether a dose response exists between the dose of brown recluse spider venom (BRSV) and the cutaneous and coagulation effects in a rabbit model. Cutaneous necrosis is a serious complication of brown recluse spider envenomation (spider bite with venom). Disseminated intravascular coagulation (DIC) is a dreaded complication of brown recluse envenomation in humans. New Zealand white (NZW) rabbits have proved to be a model for the study of therapeutic regimens to prevent skin necrosis after spider bites. We studied the venom's effects on the skin and the coagulation mechanism in this rabbit model to determine if a clear dose-response relationship could be established. Establishment of a dose-response relationship is an important first step in determining if the NZW rabbit is a suitable model to study both cutaneous and systemic effects of the venom. DESIGN: Thirty-six NZW rabbits were divided into three groups. One group received a saline injection, and the other two groups received a 4.0 microg or a 10.0 microg dose of purified BRSV intradermally into the skin on the dorsum of the back. METHODS: Blood was collected at baseline, 24, 48, and 72 hours. Tissue specimens were obtained after seven days during the animal necropsy and gross and microscopic pathology examination was conducted to assess tissue damage. Measurements included complete blood count (CBC); platelets; PT; activated partial thromboplastin time (APTT); fibrinogen (clottable, immunological); coagulation factors II, V, VII, VIII, IX, X, XI, XII; anti-thrombin (AT); alpha-2 antiplasmin (AP); Protein C (PC); mixing studies; lupus anticoagulant screening; plasminogen; thrombin-antithrombin; fibrin degradation products (FDP); d-dimer; and thrombin time. RESULTS: Gross pathology results were consistent with previous studies that used higher doses of BRSV. The WBC and platelet counts decreased at 24 hours in the two groups receiving the BRSV (p < 0.05). BRSV produced a dose related prolongation in the APTT (p < 0.05). Levels of fibrinogen as well as factors V, VII, VIII, IX, X, AT, and AP (p < 0.05) were increased in response to the BRSV. Protein C decreased at 24 hours (p < 0.05) and remained low in other time points. Mixing studies corrected the prolonged APTTs to normal ranges. Factor IIXI and XII showed no significant alteration in response to the BRSV. CONCLUSIONS: In the model, both the size and depth of the eschar were dose-related. We also observed a dose related elevation in the APTT that corrected with mixing studies. The dose-response relationship suggests direct interference by a component of the venom, rather than an idiosyncratic response. We did not detect a deficiency of commonly measured coagulation factors or evidence of a lupus anticoagulant. Protein C demonstrated a decrease. Although DIC did not occur in this rabbit model, a dose-related elevation in APTT was noted. The finding that the elevation corrected with mixing studies suggests that a plasma factor is essential in the coagulopathy associated with brown recluse envenomation. Further studies to identify this factor could shed light on human coagulopathy following envenomation.

Neurotoxicological effects of a thrombin-like enzyme isolated from Crotalus durissus terrificus venom (preliminary study).

A thrombin-like enzyme, purified from the venom of Crotalus durissus terrificus by gel filtration and affinity chromatography, showed a single protein band in Sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE) with a molecular weight of about 33kDa. Clear cellular morphological changes, deep ganglioside level modifications in some brain areas and behavioral alterations in pup rats injected with this protein were detected. Ganglioside composition, one of the chemical markers of brain maturation, was altered specially in the hypothalamus, hippocampus and prefrontal cortex. The most reliable behavioral effects were a delayed, maturation of the righting reflex, posture and motor response after treatment. These effects were consistent with the histological changes revealed in the cerebellum and prefrontal cortex of treated neonate rats, areas related to motor activities.

Induction of apoptosis in Vero cells by Aeromonas veronii biovar sobria vacuolating cytotoxic factor.

Recently, a cytotoxin named vacuolating cytotoxic factor (VCF) in Aeromonas sobria and Aeromonas veronii biovar sobria was described. We have now purified this factor using ion metallic affinity chromatography. The VCF is a nonhemolytic enterotoxin that acts as a serine protease. The toxin can be partially neutralized by serum antiaerolysin and it induced not only cytoplasmatic vacuole formation, but also mitochondrial disorders that must be signaling the apoptotic pathways, leading to Vero (African green monkey kidney) cell death. These results suggest that the VCF is a virulence factor of these bacteria, participating in the processes of human disease provoked by preformed toxins in food and infection.

Local and systemic pathophysiological alterations induced by a serine proteinase from the venom of the snake Bothrops jararacussu.

The local and systemic pathophysiological alterations induced by BjussuSP-I, a thrombin-like serine proteinase from the venom of the snake Bothrops jararacussu, were assessed in mice. BjussuSP-I induced a mild edema but no local myonecrosis or hemorrhage. It did not induce any microvascular alteration in the cremaster muscle. Intramuscular injection of BjussuSP-I promoted an increase in the expression of proMMP-9, but it did not induce the activation of proMMP-2 or proMMP-9 synthesized in muscle tissue injected with a myotoxic phospholipase A(2) homolog. BjussuSP-I induced defibrin(ogen)ation upon intravenous and intramuscular injections, with reduction in plasma fibrinogen concentration and increments in the levels of fibrin degradation products and D-dimer. When compared with animals having normal coagulation, mice defibrin(ogen)ated by BjussuSP-I developed a slightly larger hemorrhagic lesion in the skin when injected with metalloproteinase BaP1. Intravenous injection of sublethal doses of BjussuSP-I promoted a series of behavioral and motor changes similar to those previously described for 'gyroxin', i.e. opisthotonus and a circular body movement along the longitudinal axis.