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Results for “toxin secretion system”

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At least 397 records · Page 22Linked to original sources

PubMed1997

A recombinant adenovirus that directs secretion of biologically active kappa-bungarotoxin from mammalian cells.

A novel Cre-lox system was used to construct an adenovirus encoding kappa-bungarotoxin (kappa-Bgt), modified to be secreted by attachment of a bovine prolactin signal sequence at the N-terminus of the toxin. Western blot of medium from HEK-293 cells infected with the virus demonstrated that recombinant kappa-Bgt (R-kappa-Bgt) was secreted. The biological activity of the secreted R-kappa-Bgt was investigated in Xenopus oocytes that expressed neuronal nicotinic acetylcholine receptor (nAChR) subtypes alpha3beta2 and alpha2beta2. The recombinant toxin inhibited the response of alpha3beta2 type AChRs to ACh, but did not inhibit the response of alpha2beta2 type AChRs. These data demonstrated that the recombinant adenovirus directs the secretion of biologically active kappa-Bgt from a mammalian cell line. Because adenovirus can be used to infect post-mitotic cells, recombinant adenoviruses encoding biologically active peptides may be of use as delivery vehicles for in vivo experiments where repeated application of the purified peptide is unfeasible.

Adenoviridae↗

PubMed1975

Lymphocyte depletion induced by cholera toxin; relationship to adrenal cortical function.

Intravenous injection of cholera toxin (choleragen) into mice caused a profound lymphocytopenia associated with marked cellular depletion of the lymph nodes, spleen, and thymus. After administration of 1 mu g of choleragen, lymphocytopenia was mot marked at 24 hr; recovery occurred 6 to 10 days later. Similarly depletion of lymph nodes and spleen was maximal at 24 hr with recovery by 14 days. Choleragen also caused a marked elevation of serum corticosterone and lymphocyte depletion was not observed in adrenalectomized mice. These results suggested that the lymphocytopenic effect of choleragen was mediated by increased production and secretion of adrenal cortical hormones secondary to a rise in intracellular cAMP induced by cholera toxin. The site of action of choleragen may be the adrenal cortex itself and/or the hypothalamic-pituitary system.

Adrenal Cortex↗

PubMed1994

Stimulation of reactive oxidant production in neutrophils by soluble and insoluble immune complexes occurs via different receptors/signal transduction systems.

Cell-free synovial fluid from patients with rheumatoid arthritis contains soluble and insoluble IgG-containing immune complexes which activate reactive oxidant production in human neutrophils. In this report we have measured the effects of inhibitors of signal transduction pathways on neutrophil activation by these complexes and also following activation by synthetic soluble and insoluble immune complexes made from human serum albumin (HSA) and anti-(HSA) antibodies. In all aspects studied, the soluble rheumatoid complexes and the soluble synthetic complexes were indistinguishable in the ways in which they activated neutrophils. Activation of reactive oxidant production in response to these soluble complexes was completely inhibited by pertussis toxin (indicating G-protein coupling of receptor occupancy), completely insensitive to staurosporine (indicating that oxidant production did not require protein kinase C activity), only marginally (< 30%) inhibited by butanol (indicating that dependence upon activity of phospholipase D was minimal), and completely inhibited by chloracysine, an inhibitor of phospholipase A2. In contrast, activation of reactive oxidant production in response to the insoluble rheumatoid or insoluble synthetic immune complexes was largely pertussis toxin insensitive, inhibited by > 50% by staurosporine, inhibited by > 50% by butanol, and completely inhibited by chloracysine. These results show that the receptor-mediated signal transduction systems activated by the soluble and insoluble immune complexes are different. Because the soluble complexes activate a transient burst of reactive oxidant secretion from primed neutrophils, the mechanisms regulating either the release or the intracellular production of oxidants within rheumatoid joints are distinct and hence may be pharmacologically modified independently of each other.

Alkaloids↗

PubMed1995

Effects of domoic acid on serum levels of TSH and thyroid hormones.

The actions of Domoic Acid (Dom), a marine toxin, on the levels of serum TSH and thyroid hormones (T4 and T3) has been studied to determine if these actions could be mediated by the serotoninergic system. In all the experiments, adult male Wistar rats were used. The Dom dissolved in saline was administered via i.p. in doses of 0.5 and 1 mg/kg. The T4 and T3 concentrations were determined by enzimoinmunoassay and TSH concentration was determined by radioinmunoassay. The results show that Dom 1 mg/kg increases the serum T4 levels one hour after treatment and decreases these levels 2 and 3 hr after treatment. Dom 0.5 mg/kg decreased the serum T4 levels 2 and 3 hr after treatment. The concentrations of T3 in serum were unchanged by both doses of Dom. The concentration of TSH was increased by Dom. In order to study the possible mediation of the serotoninergic system in the effect of Dom on the hormone levels, PCPA, a tryptophan hydroxylase inhibitor, was administered i.p. 90 min before blood sampling. In this case, with both doses of Dom a decrease in the levels of both hormones occurred with respect to the PCPA group. These results indicate that the serotoninergic system could affect the actions of Dom on TSH and thyroid hormone secretion.

Animals↗

PubMed1986

Regulation of the phosphoinositide hydrolysis pathway in thrombin-stimulated platelets by a pertussis toxin-sensitive guanine nucleotide-binding protein. Evaluation of its contribution to platelet activation and comparisons with the adenylate cyclase inhibitory protein, Gi.

In platelets activated by thrombin, the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C produces inositol 1,4,5-triphosphate (IP3) and diacylglycerol, metabolites which are known to cause Ca2+ release from the platelet dense tubular system and granule secretion. Previous studies suggest that phospholipase C activation is coupled to platelet thrombin receptors by a guanine nucleotide-binding protein or G protein. The present studies examine the contribution of this protein to thrombin-induced platelet activation and compare its properties with those of Gi, the G protein which mediates inhibition of adenylate cyclase by thrombin. In platelets permeabilized with saponin, nonhydrolyzable GTP analogs reproduced the effects of thrombin by causing diacylglycerol formation, Ca2+ release from the dense tubular system and serotonin secretion. In intact platelets, fluoride, which by-passes the thrombin receptor and directly activates G proteins, caused phosphoinositide hydrolysis and secretion. Fluoride also caused an increase in the platelet cytosolic free Ca2+ concentration that appeared to be due to a combination of Ca2+ release from the dense tubular system and increased Ca2+ influx across the platelet plasma membrane. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which inhibits G protein function, inhibited the ability of thrombin to cause IP3 and diacylglycerol formation, granule secretion, and Ca2+ release from the dense tubular system in saponin-treated platelets. Increasing the thrombin concentration overcame the effects of GDP beta S on secretion without restoring diacylglycerol formation. The effects of GDP beta S on platelet responses to thrombin which had been subjected to partial proteolysis (gamma-thrombin) were similar to those obtained with native alpha-thrombin despite the fact that gamma-thrombin is a less potent inhibitor of adenylate cyclase than is alpha-thrombin. Thrombin-induced diacylglycerol formation and 45Ca release were also inhibited when the saponin-treated platelets were preincubated with pertussis toxin, an event that was associated with the ADP-ribosylation of a protein with Mr = 41.7 kDa. At each concentration tested, the inhibition of thrombin-induced diacylglycerol formation by pertussis toxin paralleled the inhibition of thrombin's ability to suppress PGI2-stimulated cAMP formation.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenylate Cyclase Toxin↗

PubMed1995

Functional expression of an alpha anti-insect scorpion neurotoxin in insect cells and lepidopterous larvae.

The Leiurus quinquestriatus hebraeus alpha anti-insect toxin (Lqh alpha IT) cDNA was engineered into the Autographa californica Nuclear Polyhedrosis Virus (AcNPV) genome. Insect cells infected with the recombinant virus secreted a functional Lqh alpha IT polypeptide. Spodoptera littoralis and Heliothis armigera larvae injected with recombinant budded virus, showed typical intoxication symptoms. This recombinant virus showed enhanced insecticidal potency against H. armigera larvae compared with wild type AcNPV. The present expression system will facilitate: (1) the future elucidation of structural elements involved in its prominent anti-insect toxicity; and (2) the future design of genetically modified alpha toxins with improved anti-insect selectivity.

Animals↗

PubMed2006

Identification of the ETT2 locus in human diarrheagenic Escherichia coli by multiplex PCR.

An Escherichia coli type III secretion system 2 (ETT2) locus was discovered in enterohemorrhagic E. coli O157:H7. To determine presence or absence of the ETT2 locus in diarrheagenic E. coli, a multiplex polymerase chain reaction (PCR) encoding Shiga toxins (stx1 and stx2), intimin (eaeA), and ETT2 (etrA) was developed for rapid detection. The ETT2 locus was identified not only in Shiga toxin-producing E. coli (STEC) but also in various non-STEC.

Diarrhea↗

PubMed1988

The relationship of insects to the deterioration of stored grain by fungi.

The data presented tests the hypothesis that knowledge of basic biological relationships between insects and fungi in the stored grain ecosystem can open new paths for management of the system. Evidence indicates: (1) some storage insect species are disseminators of storage fungi and some are exterminators; (2) some storage fungi attract storage insects and promote their population increases and other repel and secrete toxins harmful to insects; (3) certain morphophysiologic, biochemical similarities of fungi and the early developmental stages of insects suggest that new protective chemicals, 'insecticidal fungicides', could be designed or specifically sought. A specific example of a naturally occurring compound with dual action, sorbic acid (2,4-hexadienoic acid), is given and its effect on both insects and fungi is discussed. Sorbic acid is the only compound in the U.S. presently labelled by the FDA (Food and Drug Administration) as GRAS (Generally Regarded as Safe) and registered by the EPA (Environmental Protection Agency) for use on grain (maize) in storage.

Animals↗

PubMed1983

Pertussis toxin uncouples dopamine agonist inhibition of prolactin release.

Pertussis toxin, a protein exotoxin produced by Bordetella pertussis, markedly reduced or eliminated the ability of dopamine or the dopamine agonist bromocriptine to inhibit prolactin release from anterior pituitary cells in vitro. Toxin-mediated reversal of the effect on dopamine agonist inhibition of prolactin release occurred with a lag of greater than 6 h, was maximal by 24 h, and persisted for at least 6 days after removal of the toxin from the medium. The toxin reduced dopamine agonist efficacy without altering potency or directly modifying the dopamine receptor (as measured by [3H]spiperone binding). The ability of dopamine to reduce cellular cyclic AMP content was also antagonized by pertussis toxin, supporting the hypothesis that reduction of cellular cyclic AMP content and inhibition of prolactin secretion may be causally related. These data demonstrated that pertussis toxin can prevent the typical inhibitory action of dopamine agonists on anterior pituitary prolactin release and suggest that this receptor-mediated inhibitory hormone system is analogous to other inhibitory receptors coupled to adenylate cyclase.

Adenylate Cyclase Toxin↗

PubMed1988

Topology of membrane insertion in vitro and plasma membrane assembly in vivo of the yeast arginine permease.

We have examined the topology of the yeast arginine permease, a plasma-membrane protein with multiple membrane-spanning domains. Using fusions of the permease with the glycosylatable secreted yeast protein, acid phosphatase, we identified membrane-spanning sequences that can translocate adjacent acid phosphatase across the membrane of the endoplasmic reticulum (ER), as measured by in vitro glycosylation. Examination for the presence or absence of glycosylation in a systematic series of such fusions gave an internally consistent model for the lumenal or cytoplasmic disposition of the acid phosphatase reporter, defining the topology of the permease. The phenotypes of a further set of arginine permease gene fusions with portions of the gene for the secreted protein, killer toxin, suggest that the pathways of export of membrane and secreted proteins need not be functionally distinct.

Acid Phosphatase↗

PubMed1988

Effects of tetanus toxin on catecholamine release from intact and digitonin-permeabilized chromaffin cells.

Tetanus exotoxin inhibited Ca2+-dependent catecholamine secretion in a dose-dependent manner in digitonin-permeabilized chromaffin cells. The inhibition was specific for tetanus exotoxin and the B fragment of tetanus toxin; the C fragment had no effect. Inhibition required the introduction of toxin into the cell, and was not seen when intact cells were preincubated with the toxin or toxin fragments. The degree of inhibition was related to the length of preincubation with toxin, as well as the concentration of toxin used. A short preincubation with toxin was sufficient to inhibit secretion, and the continued presence of toxin in the incubation medium was not required during the incubation with Ca2+. The inhibition of secretion by tetanus toxin or the B fragment was not overcome with increasing Ca2+ concentrations. Tetanus toxin also inhibited catecholamine secretion enhanced by phorbol ester-induced activation of protein kinase C. Thus, the toxin or a proteolytic fragment of the toxin can enter digitonin-permeabilized cells to interact with a component of the Ca2+-dependent exocytotic pathway to inhibit secretion.

Adrenal Medulla↗

PubMed2005

Delivery of heterologous protein antigens via hemolysin or autotransporter systems by an attenuated ler mutant of rabbit enteropathogenic Escherichia coli.

In this report, we describe the use of an attenuated regulatory mutant of a rabbit enteropathogenic Escherichia coli (rEPEC) as a live vaccine vector to deliver heterologous protein antigens using two dedicated transport systems, a Salmonella autotransporter and the E. coli hemolysin apparatus. We previously reported that an isogeneic ler (LEE encoded regulator) mutant of rEPEC O103:H2 is attenuated and immunogenic in rabbits. We first evaluated the Salmonella autotransporter MisL containing the immunodominant B-cell epitope of the circumsporozoite protein from Plasmodium falciparum, (NANP)8, fused to the C-terminal translocator domain under the control of the constitutive Tac17 promoter. The rEPEC ler mutant was able to express and to translocate the (NANP)8 passenger peptide to the bacterial surface. We next investigated the delivery of Shiga toxin B subunit (Stx1B) from human enterohemorrhagic E. coli by the rEPEC ler mutant via the MisL autotransporter or the E. coli hemolysin secretion apparatus. The autotransporter and hemolysin plasmids expressed similar levels of Stx1B (30-40 ng/ml/OD600). Only 6% of Stx1B was found in the autotransporter supernatants; the rest was cell-associated, with a small fraction of the Stx1B surface-exposed as determined by immunofluorescence. In contrast, 88% of Stx1B was secreted into culture supernatants by the hemolysin secretion system. In an in vivo study, no significant protection was observed in rabbits inoculated with the ler mutant harboring the Stx1B-autotransporter plasmid following experimental challenge with RDEC-H19A, the prototype rEPEC containing an Stx-converting phage. In contrast, rabbits inoculated with the rEPEC ler mutant containing the Stx1B-hemolysin fusion were partially protected from RDEC-H19A infection as demonstrated by decreased weight loss (p<0.008) when compared to rabbits inoculated with the parent ler mutant. Our results suggest that attenuated rEPEC are capable of serving as vaccine vectors to express heterologous protein antigens from different cellular locations and deliver these antigens to the intestinal mucosa. With this system, secreted proteins may be more effective than cell-associated antigens in generating protection.

Animals↗

PubMed2006

Shiga toxin-producing Escherichia coli: an overview.

The objective of this review is to highlight the importance of cattle in human disease due to Shiga toxin-producing Escherichia coli (STEC) and to discuss features of STEC that are important in human disease. Healthy dairy and beef cattle are a major reservoir of a diverse group of STEC that infects humans through contamination of food and water, as well as through direct contact. Infection of humans by STEC may result in combinations of watery diarrhea, bloody diarrhea, and hemolytic uremic syndrome. Systems of serotyping, subtyping, and virulence typing of STEC are used to aid in epidemiology, diagnosis, and pathogenesis studies. Severe disease and outbreaks of disease are most commonly due to serotype O157:H7, which, like most other highly pathogenic STEC, colonize the large intestine by means of a characteristic attaching and effacing lesion. This lesion is induced by a bacterial type III secretion system that injects effector proteins into the intestinal epithelial cell, resulting in profound changes in the architecture and metabolism of the host cell and intimate adherence of the bacteria. Severe disease in the form of bloody diarrhea and the hemolytic uremic syndrome is attributable to Shiga toxin (Stx), which exists as 2 major types, Stx1 and Stx2. The stx genes are encoded on temperate bacteriophages in the chromosome of the bacteria, and production and release of the toxin are highly dependent on induction of the phages. Regulation of the genes involved in induction of the attaching and effacing lesion, and production of Stx is complex. In addition to these genes that are clearly implicated in virulence, there are several putative virulence factors. A major public health goal is to prevent STEC-induced disease in humans. Studies aimed at understanding factors that affect carriage and shedding of STEC by cattle and factors that contribute to development of disease in humans are considered to be important in achieving this objective.

Animals↗

PubMed1988

The role of cyclic nucleotides in atrial natriuretic peptide-mediated inhibition of aldosterone secretion.

Using freshly isolated bovine adrenal glomerulosa cells we examined the inhibitory effect of atrial natriuretic peptide (ANP) on aldosterone secretion stimulated by agonists that use either the Ca2+-phosphoinositide or cAMP messenger system. In a continuous perifusion system, angiotensin II (AII) induces a prompt initial rise in aldosterone secretion, followed by a sustained secretory response. Both phases of secretion are rapidly and independently inhibited by ANP. The role of two cyclic nucleotides, cGMP and cAMP, as mediators of this ANP-induced inhibition was examined. The effect of 8-bromo-cGMP (1-100 microM) or (Bu)2cGMP (1-50 microM) on the AII-stimulated rate of secretion was studied in a perifusion system. Either analog, whether added early or late, maximally inhibited by 20-30% only the late or sustained phase of aldosterone secretion. The effect of ANP on cellular cAMP content was examined in a static incubation system. Although ANP caused a reduction in the cAMP content of cells stimulated with either AII or ACTH, it had little or no effect on the cAMP levels in cells stimulated with carbachol. In AII- and ACTH-stimulated cells, the relationship between reduced cAMP content and reduced secretion was explored. In the AII-stimulated cell inhibited by ANP, simple restoration of cAMP content with forskolin did not restore the secretory rate. Pertussis toxin treatment blocked the inhibitory effect of ANP on cAMP content, but did not block its inhibition of secretion. In the ACTH-stimulated cell, reversal of the ANP-induced reduction of cAMP with forskolin, partially restored the stimulated rate of secretion, although restoration of cAMP with a 10-fold higher dose of ACTH did not restore the stimulated rate of secretion in the presence of ANP. These results imply that both the ANP-induced rise in cGMP and the ANP-induced decrease in cellular cAMP content may contribute to the inhibition of steroidogenesis. However, these inhibitory messages do not induce either the magnitude or the temporal pattern of inhibition induced by ANP. Thus, in the adrenal multiple messenger systems may underlie the action of ANP.

Adenylyl Cyclase Inhibitors↗

PubMed1987

Bacterial scleral abscesses after retinal buckling operations. Pathogenesis, management, and laboratory investigations.

Between 1971 and 1985, 28 patients were treated for a scleral abscess after a scleral buckle procedure. Twenty-six of the patients had the original surgery performed between those years during which a total of 4480 buckling procedures were performed. Therefore, the incidence of scleral abscess after buckling procedures was 0.58% (26/4480). All 28 patients had excessive pain, conjunctival chemosis and proptosis, a creamy white area of retina on the buckle, and a variable amount of vitreous inflammation. Seventeen of these patients had an especially severe scleral abscess with inflammatory vitreous opacification, which precluded visualization of the retinal vessels or allowed at most a hazy view of the optic nervehead or the scleral buckle. A review of these 17 severely involved patients showed that, in most cases (13 of 17), the vitreous inflammation was sterile. Therefore, most patients can be managed simply with immediate removal of the buckle along with topical and systemic antibiotics. A laboratory investigation showed that a cell-free millipore filtrate of Staphylococcus aureus culture in trypticase soy broth caused prominent but self-limited inflammation when injected intravitreally. Therefore, bacterial toxins or secretions could be the cause of the prominent vitreous inflammation in most patients with scleral abscesses. In another experiment, sponge exoplants, which had been soaked in a highly concentrated culture of S. aureus, were sutured episclerally in rabbits. A scleral abscess developed in 19 of 23 rabbit eyes with these infective sponges. Progressive scleral abscesses, which closely resembled those seen in humans, developed in 14 of the 23 eyes.(ABSTRACT TRUNCATED AT 250 WORDS)

Abscess↗

PubMed2003

Reduced glutathione is required for pertussis toxin secretion by Bordetella pertussis.

The abilities of cysteine-containing compounds to support growth of Bordetella pertussis and influence pertussis toxin transcription, assembly, and secretion were examined. Cysteine is an essential amino acid for B. pertussis and must be present for protein synthesis and bacterial growth. However, cysteine can be metabolized to sulfate, and high concentrations of sulfate can selectively inhibit transcription of the virulence factors, including pertussis toxin, via the BvgAS two-component regulatory system in a process called modulation. In addition, pertussis toxin possesses several disulfide bonds, and the cysteine-containing compound glutathione can influence oxidation-reduction reactions and perhaps disulfide bond formation. Bacterial growth was not observed in the absence of a source of cysteine. Oxidized glutathione, as a sole source of cysteine, also did not support bacterial growth. Cysteine, cystine, and reduced glutathione did support bacterial growth, and none of these compounds caused modulation at the concentrations tested. Similar amounts of periplasmic pertussis toxin were detected regardless of the source of cysteine; however, in the absence of reduced glutathione, pertussis toxin was not efficiently secreted. Addition of the reducing agent dithiothreitol was unable to compensate for the lack of reduced glutathione and did not promote secretion of pertussis toxin. These results suggest that reduced glutathione does not affect the accumulation of assembled active pertussis toxin in the periplasm but plays a role in efficient pertussis toxin secretion by the bacterium.

Animals↗

PubMed1991

Synthesis in Vibrio cholerae and secretion of hepatitis B virus antigens fused to Escherichia coli heat-labile enterotoxin subunit B.

A simple and effective electroporation method for the transformation of Vibrio cholerae with nonmobilizable plasmids is described. Expression plasmids directing the synthesis of fusion proteins with the subunit B of Escherichia coli heat-labile enterotoxin B (LT-B) were transformed into nontoxinogenic V. cholerae vaccine strains. A protein consisting of two overlapping immunodominant antibody-binding sites of the hepatitis B virus (HBV) middle surface antigen fused to the C terminus of full-length LT-B was secreted into the supernatant of V. cholerae cultures, whereas two other LT-B/HBV fusion proteins were mostly retained within the cells or rapidly degraded in the culture supernatant. While the secretion of fusion proteins with cholera toxin subunit B (CT-B) from V. cholerae has been described, this is to our knowledge the first report describing extracellular secretion of defined foreign epitopes fused to LT-B in V. cholerae. The fusion of guest epitopes to LT-B or CT-B and secretion in V. cholerae could be an interesting system to rapidly produce pure fusion proteins for immunisation, functional studies or diagnostic procedures. An LT-B/pre-S2 fusion protein purified from the supernatant of recombinant V. cholerae induced serum IgG antibodies against LT-B and against the HBV middle surface antigen in mice after parenteral and oral immunisation.

Animals↗

PubMed2003

Haemolysin A and listeriolysin--two vaccine delivery tools for the induction of cell-mediated immunity.

Haemolysin A of Escherichia coli and listeriolysin of Listeria monocytogenes represent important bacterial virulence factors. While such cytolysins are usually the reason for morbidity and even mortality, vaccine researchers have turned haemolysin A and listeriolysin into tools for vaccine delivery. Both cytolysins have found widespread application in vaccine research and are highly suitable for the elicitation of cell-mediated immunity. In this paper, we will review vaccine delivery mediated by the haemolysin A secretion system and listeriolysin and will highlight their use in vaccination approaches against protozoan parasites.

Antigens↗