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PubMed1997

Modulation of protein kinetics in chronic renal failure.

Many factors are involved in inducing muscle wasting and derangements of protein metabolism in chronic renal failure. Anorexia, low protein intake, hormonal abnormalities (increased cortisol and parathyroid hormone secretion, and insulin resistance), acidosis, abnormalities of the cytokine system, and other unidentified uremic toxins create a negative nitrogen balance and stimulate protein catabolism. The protein turnover rate (i.e., synthesis and degradation) is generally decreased in non-acidotic uremic patients. However, in uncorrected acidosis, protein degradation is accelerated and a rapid loss of body proteins supervenes. Nutrition can be improved in chronically uremic patients through pharmacological therapy that can control protein catabolism. Administration of growth hormone and insulin-like growth factor-1 has been shown to be effective. These hormones influence both glucose and glutamine metabolism. Another approach is the administration of pentoxifylline, which may have an anticatabolic effect by interfering with the tumor necrosis factor-alpha system.

Amino Acids↗

PubMed2006

Parallel signaling pathways of melatonin in the pancreatic beta-cell.

Previous results demonstrated that melatonin inhibits cAMP production and stimulates IP(3) liberation in rat insulinoma INS1 cells, a model for the pancreatic beta-cell. This study addresses the impact of melatonin on insulin release. Insulin, cAMP and IP(3) levels of INS1 cells in a superfusion system were measured. Initially, forskolin was used to stimulate cAMP and subsequently insulin release. Incubation of forskolin (5 micromol/L)-stimulated cells with melatonin (100 nmol/L) inhibited cAMP and insulin levels (down to 60% of insulin and cAMP release). The G(i)alpha-protein-inhibitor pertussis toxin (PTX) was used to distinguish between the G(i)alpha-dependent cAMP pathway and the G(i)alpha-independent IP(3) pathway. In our experiments we employed a specific stimulation pattern to prove proper inhibition of G(i)alpha-proteins by PTX. In INS1 cells incubated with 250 ng/mL PTX for 24 hr, melatonin was no longer able to inhibit the forskolin-induced cAMP and insulin release. In a study, carbachol was used to stimulate IP(3) and subsequently insulin release. Surprisingly, incubation of carbachol (300 micromol/L)-stimulated cells with melatonin (100 nmol/L) inhibited insulin release (down to 75% of insulin release). Finally, in PTX-incubated INS1 cells, melatonin (100 nmol/L) increased carbachol (300 micromol/L)-induced insulin release (up to 124% of insulin release). In conclusion, we found that the melatonin MT(1)-receptor on pancreatic beta-cells is coupled to parallel signaling pathways, with opposite influences on insulin secretion. The cAMP- and subsequently insulin-inhibiting signaling pathway involves PTX-sensitive G(i)alpha-proteins and is predominant in terms of insulin release.

Animals↗

PubMed1983

Yeast DNA plasmids.

The study of yeast DNA plasmids has been initiated with the discovery of the 2-micron DNA in Saccharomyces cerevisiae. This multiple copy plasmid, organized into chromatin structure in vivo, probably exists in the nucleus and provides a good system to obtain information on eukaryotic DNA replication. Yeast transformation with the 2-micron DNA or artificially constructed chimeric plasmids had contributed significantly to the study of the molecular biology of yeast and eukaryotes, allowing the isolation and characterization of various genes, ars, centromeres, and telomeres, and also serving as a tool to study the expression of various heterologous genes. Encouraged by these fruitful results, new yeast plasmids have been screened among phylogenetically distant yeasts. The linear DNA plasmids (pGKl1 and pGKl2) from Kluyveromyces lactis are the first case of yeast plasmids associated with biological function (killer phenotype). This plasmid system would be ideal as a model to study the structure and function of eukaryotic linear chromosomes. The extracellular secretion of protein toxin suggests the plasmids to be an excellent candidate for a secretion vector. The importance of yeasts as suitable materials for the study of eukaryotic cell biology would be much enhanced by the advent of new transformation systems with diverse host yeasts of genetically and phylogenetically distinct properties.

Chimera↗

PubMed2000

Bordetella pertussis infection: pathogenesis, diagnosis, management, and the role of protective immunity.

Whooping cough is presently one of the ten most common causes of death from infectious disease worldwide. Despite a high vaccine uptake, resurgences of this disease have been observed in several countries. Virulence factors of Bordetella pertussis include agglutinogens, fimbriae, P.69/pertactin, pertussis toxin, filamentous haemagglutinin, adenylate cyclase, tracheal cytotoxin, dermonecrotic toxin, lipopolysaccharide, tracheal colonisation factor, serum resistance factor, and type III secretion. Virulence factor expression is regulated by the bvgAS locus, a two-component signal transduction system. The pathophysiologic sequence consists of attachment (fimbriae, P.69/pertactin, tracheal colonisation factor, pertussis toxin, filamentous haemagglutinin), evasion of host defence (adenylate cyclase, pertussis toxin, serum resistance factor), local effects (tracheal cytotoxin), and systemic effects (pertussis toxin). Bordetella pertussis is transmitted by respiratory droplets and causes disease only in humans. Various diagnostic methods are available, including culture, serological methods, and the polymerase chain reaction. Serotyping of isolates to detect agglutinogens 2 and 3 is useful because serotype 1,2 may be associated with higher mortality, and antibodies to these antigens (agglutinins) may be protective in both animals and humans. Immunisation using whole-cell vaccine is effective but is reactogenic. Acellular vaccines containing one to five components are being used increasingly in various countries. Protective immunity to pertussis correlates with high levels of antibody to each of pertactin, fimbriae, and pertussis toxin; however, doubt remains as to the relationship between agglutinogen 3 and fimbria 3, making results of trials investigating these virulence factors difficult to interpret.

Animals↗

PubMed2002

Enteric nerves and diarrhoea.

This review article discusses the importance of the enteric nervous system for the fluid and electrolyte secretion evoked by luminal secretagogues in the small intestine. The first part of the review summarizes observations on augmented secretion caused by cholera toxin, which has been the subject of extensive studies in the past. The latter part reviews studies of the participation of the enteric nervous system in other secretory states of the gut. The involvement of the enteric nervous system in the pathophysiology of intestinal secretory states opens up potential new sites of actions for drugs in the treatment of diarrhoea. This is discussed in the final part of this review.

Antidiarrheals↗

PubMed1981

Is ethanol a testicular toxin?

A method is described by which the testis of the rat can be isolated and selectively perfused in vitro to examine the effect of potential gonadal toxins. Using this system, the effect of ethanol at levels commonly found in the plasma of nonalcoholic men who drink alcohol was evaluated. Ethanol was found to significantly reduce (p less than 0.01) testosterone production and secretion by the isolated perfused testis. The advantage of such a system is that it permits examination of the effect of specific testicular toxins, such as alcohol, independent of hepatic metabolism and/or hypothalamic-pituitary suppression by such substances. These results suggest that ethanol, independent of hepatic metabolism and/or hypothalamic-pituitary suppression, is a direct testicular toxin.

Animals↗

PubMed2002

Purification of secreted leukotoxin (LtxA) from Actinobacillus actinomycetemcomitans.

The RTX (repeats in toxin) family of toxins is important in the pathogenesis of many Gram-negative bacteria. The oral and systemic human pathogen Actinobacillus actinomycetemcomitans produces a member of this family known as leukotoxin (LtxA). Previously, we found that LtxA is secreted into culture supernatants of A. actinomycetemcomitans and that this protein is abundant and relatively pure. Here, we report a large-scale method for the isolation and purification of LtxA from culture supernatants of A. actinomycetemcomitans strain JP2. The purification scheme involves ammonium sulfate precipitation of culture supernatants, dialysis, and ultrafiltration to concentrate LtxA to approximately 10mg/ml. We found that LtxA remained soluble in buffer that contained at least 250mM NaCl. Purified LtxA was >98% pure and the final preparations were active against HL-60 cells. The entire purification protocol can be completed within 2 days. The ability to readily obtain a large amount of purified leukotoxin should accelerate investigations into the structure and biology of this important virulence factor.

Actinobacillus↗

PubMedDate not supplied

Rho GTPases as targets of bacterial protein toxins.

Several bacterial toxins target Rho GTPases, which constitute molecular switches in several signaling processes and master regulators of the actin cytoskeleton. The biological activities of Rho GTPases are blocked by C3-like transferases, which ADP-ribosylate Rho at Asn41, but not Rac or Cdc42. Large clostridial cytotoxins (e. g., Clostridium difficile toxin A and B) glucosylate Rho GTPases at Thr37 (Rho) or Thr35 (Rac/Cdc42), thereby inhibiting Rho functions by preventing effector coupling. The 'injected' toxins ExoS, YopE and SptP from Pseudomonas aeruginosa, Yersinia and Salmonella ssp., respectively, which are transferred into the eukaryotic target cells by the type-III secretion system, inhibit Rho functions by acting as Rho GAP proteins. Rho GTPases are activated by the cytotoxic necrotizing factors CNF1 and CNF2 from Escherichia coli and by the dermonecrotizing toxin DNT from B. bronchiseptica. These toxins deamidate/transglutaminate Gln63 of Rho to block the intrinsic and GAP-stimulated GTP hydrolysis, thereby constitutively activating the GTPases. Rho GTPases are also activated by SopE, a type-III system injected protein from Salmonella ssp., that acts as a GEF protein.

Bacterial Toxins↗

PubMedDate not supplied

Inhibition of exocytosis in bovine adrenal medullary cells by botulinum toxin type D.

Botulinum toxins are known to block transmitter release at peripheral cholinergic synapses, producing muscular weakness and paralysis. The toxins may also block adrenergic transmission, although this effect is less well understood. The mechanisms by which toxins act are unclear. They are proteins of relative molecular mass approximately 150,000 and are structurally similar to tetanus toxin. It is generally accepted that a rise in intracellular calcium concentration is sufficient to trigger secretion by exocytosis, but it is not known whether the toxins block secretion by preventing this Ca transient or whether they act downstream from Ca entry by interfering with the process of exocytosis itself. We have attempted to resolve these questions in the case of the adrenergic system by studying the effects of botulinum toxins (types A, B, D and E) on the secretory response of isolated bovine adrenal medullary cells maintained in culture. The cells were either challenged with various secretagogues or rendered leaky and challenged directly with Ca buffers. We report here that botulinum toxin type D inhibits secretion in a time- and dose-dependent manner, the results being entirely consistent with the idea that the toxin acts at or near the site of exocytosis rather than at the sites controlling the rise in free Ca.

Acetylcholine↗

PubMed1999

Mutational analysis of CvaA in the highly conserved domain of the membrane fusion protein family.

The antibacterial peptide toxin colicin V (ColV) uses a dedicated signal sequence-independent export system for its secretion in Escherichia coli that involves the products of three genes, cvaA, cvaB, and tolC in this process. As a member of the membrane fusion protein (MFP) family, the CvaA protein has been proposed to interact with an outer membrane protein TolC via its C-terminal hydrophobic domain. The importance of this domain, which is highly conserved throughout the members of MFP family, was analyzed by use of site-directed mutagenesis of missense or nonsense mutations with suppressors. All the nonsense mutations tested resulted in the loss of ColV secretion, indicating the importance of the C-terminus of CvaA, including the last 100 residue-hydrophilic domain. The missense mutations of several conserved amino acids have no drastic effects. On the other hand, when Glu-248, Ala-262, Thr-274, Leu-285, Gly-313, Ala-322, or Val-335 of CvaA protein was mutated, the secretion of ColV was greatly reduced in certain mutants. While some mutations resulted in structural instability, Glu-248 to Lys and Ala-322 to Gly proteins were relatively stable, but were not functional in ColV secretion. The results indicate that these conserved amino acids are important for the structure and functions of CvaA in the secretion of ColV.

ATP-Binding Cassette Transporters↗

PubMed2001

[Discovery of phytopathogenic bacteria 100 years ago: transatlantic controversies and polemics].

The demonstration of a bacterial cause of some plant diseases has been claimed few years after it was commonly recognized that bacteria were able to cause diseases of human and animal. Nevertheless, some sharp controversies took place, between German and American specialists (1897-1901), before the existence of bacterial diseases of plants was accepted by all phytopathologists. Nowadays, about 350 bacteria are described, which infect plants: they are pathovars, or subspecies, belonging to 21 genera. Bacterial diseases of plants can be classified into three major categories according to the type of symptoms shown by the infected plant: necrosis and wilt, soft-rot, tumour. The interaction between bacteria and plant cells is usually established from the apoplast, although some bacteria are xylem or phloem limited. This interaction involves an original protein secretion system (which is also described in bacteria pathogenic for animals), hydrolytic enzymes (pectinases, cellulases), toxins and/or phytohormones. Bacteria of one group (Agrobacterium) modify the plant metabolism after gene transfer from a plasmid. On the economic and social point of view, these diseases may be limiting factors of some key-productions (rice, cassava). In addition, they play a role in reducing the quality of agricultural products (reduced growth, spots on leaves and fruits). Control of bacterial diseases is limited. It relies usually on a combination of prophylaxy, chemical applications, and use of resistant genotypes.

Agriculture↗

PubMed1995

Processing and secretion of a virally encoded antifungal toxin in transgenic tobacco plants: evidence for a Kex2p pathway in plants.

Ustilago maydis is a fungal pathogen of maize. Some strains of U. maydis encode secreted polypeptide toxins capable of killing other susceptible strains of U. maydis. We show here that one of these toxins, the KP6 killer toxin, is synthesized by transgenic tobacco plants containing the viral toxin cDNA under the control of a cauliflower mosaic virus promoter. The two components of the KP6 toxin, designated alpha and beta, with activity and specificity identical to those found in toxin secreted by U. maydis cells, were isolated from the intercellular fluid of the transgenic tobacco plants. The beta polypeptide from tobacco was identical in size and N-terminal sequence to the U. maydis KP6 beta polypeptide. Processing of the KP6 preprotoxin in U. maydis requires a subtilisin-like processing protease, Kex2p, which is present in both animal and fungal cells and is required for processing of (among other things) small secreted polypeptide hormones and secreted toxins. Our findings present evidence for Kex2p-like processing activity in plants. The systemic production of this viral killer toxin in crop plants may provide a new method of engineering biological control of fungal pathogens in crop plants.

Cloning, Molecular↗

PubMed1981

Loperamide: studies on its mechanism of action.

The effects of loperamide on net solute and water absorption, and prostaglandin E2 (PGE2) and cholera toxin-induced secretion were studied in the rat jejunum using an in vivo steady-state perfusion technique. Loperamide stimulated absorption of fluid, electrolytes, and glucose and reversed PGE2 and cholera toxin-induced secretion to absorption; this opiate analogue had no effect on cholera toxin stimulation of adenylate cyclase activity or the rise of tissue cyclic AMP (cAMP) concentrations. The opiate antagonist, naloxone, reduced the antisecretory effects of loperamide without affecting tissue levels of cAMP. These results indicate that loperamide inhibits PGE2 and cholera toxin-induced secretion, and that this phenomenon is independent of any direct effect that cholera toxin has on the adenylate cyclase system. The action of naloxone suggests, but does not prove, that loperamide exerts its effect via opiate receptors.

Adenylyl Cyclases↗

PubMed1991

[The role of signal transduction systems in the regulation of the production and secretion of hCG (alpha, beta) by cultured human choriocarcinoma cells (BeWo)].

The BeWo cell line, derived from choriocarcinoma, produces and releases human chorionic gonadotropin (hCG) and its alpha- and beta-subunits. The authors have already reported that cAMP and EGF stimulated the production and secretion of hCG and its subunits by cultured BeWo cells. Therefore, in order to elucidate the role of signal transduction systems (cAMP-A-kinase system, DG-C-kinase system and Ca(2+)-calmodulin system) in the regulation of hCG (alpha, beta) synthesis by human choriocarcinoma cells, effects of cholera toxin (CT), an activator of adenylate cyclase, phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, and Ca2+ ionophore A23187, an activator of Ca2+ modulation on hCG (alpha, beta) production and secretion by BeWo cells cultured in a serum-free condition were evaluated. Immunoreactive hCG alpha, hCG beta and hCG in the media and cultured cells were measured by each homologous RIA for hCG alpha, hCG beta and hCG, respectively. Addition of CT at a concentration of 100 ng/ml into the medium caused extreme increases in the cellular levels of hCG alpha, hCG beta and hCG together with remarkable increases in hCG alpha, hCG beta and hCG levels in the medium. This stimulatory effect of CT was first observed on the increase of hCG alpha levels in cultured BeWo cells and medium at 3h, then observed on the increase of hCG beta levels at 6h and was last detectable on the increase of hCG levels in the cultured cells and medium at 12h. Addition of PMA at a concentration of 100 ng/ml into the medium caused an increase in the cellular and medium levels of hCG alpha, hCG beta and hCG shortly (3h) after the exposure to PMA. Addition of A23187 at a concentration of 100 ng/ml into the medium caused a slight increase in hCG alpha levels in the medium at 6h without accompanying the increase in those cellular levels. When added together, PMA potentiated the stimulatory effect of CT on hCG alpha, hCG beta and hCG levels in the cultured BeWo cells and medium, while PMA did not potentiate the effect of A23187 in this experimental condition. These findings suggest that cAMP-A-kinase system plays a major role in the signal transduction of hCG (alpha, beta) synthesis and secretion by BeWo choriocarcinoma cells, and that DG-C-kinase system interacts synergistically with cAMP-A-kinase system in the regulation of hCG (alpha, beta) synthesis and secretion by BeWo cells. Ca(2+)-calmodulin system appears to participate in the regulation of hCG alpha secretion without affecting the synthesis of hCG (alpha, beta) in BeWo cells.

Calcimycin↗

PubMed1983

Synthesis and secretion of the plasmid-coded heat-labile enterotoxin of Escherichia coli in Vibrio cholerae.

Both cholera toxin and heat-labile enterotoxin were made and secreted into culture supernatants by Vibrio cholerae containing the enterotoxin plasmid pCG86. Several regulatory mutations in V. cholerae that increased or decreased the synthesis of cholera toxin did not affect production of heat-labile enterotoxin. In contrast, a mutation in V. cholerae that interfered with the secretion of cholera toxin also decreased the secretion of heat-labile enterotoxin, indicating that they are processed by a common secretory pathway. Vibrio cholerae should be useful as a model system for analyzing the secretion of true extracellular proteins by Gram-negative bacteria.

Bacterial Toxins↗

PubMed1987

T84 cell receptor binding and guanyl cyclase activation by Escherichia coli heat-stable toxin.

Escherichia coli heat-stable enterotoxin (STa) induces intestinal secretion by binding to enterocyte receptors and activating the guanylate cyclase-guanosine 3',5'-cyclic monophosphate (cGMP) system. The intermediate steps between binding of STa and secretion are poorly understood, due in part to the lack of a convenient system to study the effects of STa at the cellular level. To establish such a model, we investigated the binding of 125I-STa, STa activation of guanylate cyclase, and STa-induced increase in cGMP production in a well-characterized human colonic cell line, T84. Binding was specific, linear with cell number, and time, temperature and pH dependent, and reversible. ST may also be internalized by these cells. Addition of unlabeled STa competitively inhibited binding of 125I-STa. These parameters closely resemble those described in intact rat enterocytes and cell-free membrane preparations. STa stimulated guanylate cyclase and cGMP production in a dose-related manner. The similar dose-response relationships for binding, guanylate cyclase stimulation by STa, and cGMP production suggest that the guanylate cyclase-cGMP system is coupled to ST occupancy of specific receptors. These data, together with the fact that STa induces chloride secretion from T84 cells suggest that T84 cells are a suitable and convenient system to study the cellular mechanism of action of STa.

Bacterial Toxins↗

PubMed1985

Characterization of two adhesins of Bordetella pertussis for human ciliated respiratory-epithelial cells.

Bordetella pertussis, the causative agent of whooping cough, adheres specifically to human ciliated respiratory-epithelial cells. In order to identify the adhesins of Bordetella, we tested mutant strains, each deficient in one of several bacterial virulence factors, for the ability to adhere to single, human ciliated cells in vitro. Loss of adherence was associated with lack of secretion of either of two surface antigens: filamentous hemagglutinin or pertussis toxin. These strains regained adherence if both components were present in the assay system (i.e., the missing protein was supplied exogenously). In this instance, both bacterial components were found bound to the surface of all the Bordetella strains tested as well as to cilia. We hypothesize that filamentous hemagglutinin and pertussis toxin, two naturally secreted surface antigens, act in concert as adhesins of Bordetella for human cilia.

Adhesiveness↗

PubMed2005

The PscE-PscF-PscG complex controls type III secretion needle biogenesis in Pseudomonas aeruginosa.

Type III secretion (T3S) systems play key roles in pathogenicity of many Gram-negative bacteria and are employed to inject toxins directly into the cytoplasm of target cells. They are composed of over 20 different proteins that associate into a basal structure that traverses both inner and outer bacterial membranes and a hollow, needle-like structure through which toxins travel. The PscF protein is the main component of the Pseudomonas aeruginosa T3S needle. Here we demonstrate that PscF, when purified on its own, is able to form needle-like fibers of 8 nm in width and >1 microm in length. In addition, we demonstrate for the first time that the T3S needle subunit requires two cytoplasmic partners, PscE and PscG, in P. aeruginosa, which trap PscF in a ternary, 1:1:1 complex, thus blocking it in a monomeric state. Knock-out mutants deficient in PscE and PscG are non-cytotoxic, lack PscF, and are unable to export PscF encoded extrachromosomally. Temperature-scanning circular dichroism measurements show that the PscE-PscF-PscG complex is thermally stable and displays a cooperative unfolding/refolding pattern. Thus, PscE and PscG prevent PscF from polymerizing prematurely in the P. aeruginosa cytoplasm and keep it in a secretion prone conformation, strategies which may be shared by other pathogens that employ the T3S system for infection.

Amino Acid Sequence↗