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Leonard R Forte - One of the best experts on this subject based on the ideXlab platform.
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in vivo imaging of human colorectal cancer using radiolabeled analogs of the uroguanylin peptide hormone
Anticancer Research, 2009Co-Authors: Dijie Liu, Leonard R Forte, Douglas Overbey, Lisa D Watkinson, Said Daibesfigueroa, Timothy J Hoffman, Wynn A Volkert, Michael F GiblinAbstract:Background: Uroguanylin is an endogenous peptide agonist that binds to the guanylate cyclase C receptor (GC-C). GC-C is overexpressed in human colorectal cancer (CRC), and exposure of GC-C-expressing cells to GC-C agonists results in cell cycle arrest and/or apoptosis, highlighting the therapeutic potential of such compounds. This study describes the first use of radiolabeled uroguanylin analogs for in vivo detection of CRC. Materials and Methods: The peptides uroguanylin and E3-uroguanylin were N-terminally labeled with the DOTA chelating group via NHS ester activation and characterized by RP-HPLC, ESI-MS, and GC-C receptor binding assays. The purified conjugates were radiolabeled with In-111 and used for in vivo biodistribution and SPECT imaging studies. In vivo experiments were carried out using SCID mice bearing T84 human colorectal cancer tumor xenografts. Results: Alteration of the position 3 aspartate residue to glutamate resulted in increased affinity for GC-C, with IC50 values of 5.0±0.3 and 9.6±2.9 nM for E3-uroguanylin and DOTA-E3-uroguanylin, respectively. In vivo, 111In-DOTA-E3-uroguanylin demonstrated tumor uptake of 1.17±0.23 and 0.61±0.07% ID/g at 1 and 4 h post injection, respectively. The specificity of tumor localization was demonstrated by coinjection of 3 mg/kg unlabeled E3-uroguanylin, which reduced tumor uptake by 69%. Uptake in kidney, however, was dramatically higher for the uroguanylin peptides than for previously characterized radiolabeled E. coli Heat-Stable Enterotoxin (STh) analogs targeting GC-C, and was also inhibited by coinjection of unlabeled peptide in a fashion not previously observed. Conclusion: Use of uroguanylin-targeting vectors for in vivo imaging of colorectal cancers expressing GC-C resulted in tumor uptake that paralleled that of higher affinity Heat-Stable Enterotoxin peptides, but also resulted in increased kidney uptake in vivo.
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in vitro and in vivo evaluation of 177lu and 90y labeled e coli heat stable Enterotoxin for specific targeting of uroguanylin receptors on human colon cancers
Nuclear Medicine and Biology, 2006Co-Authors: Leonard R Forte, Timothy J Hoffman, Wynn A Volkert, Michael F Giblin, Gary L Sieckman, Tiffani SheltonAbstract:The human E. coli Heat-Stable Enterotoxin (ST(h), amino acid sequence N1SSNYCCELCCNPACTGCY19) binds specifically to the guanylate cyclase C (GC-C) receptor, which is present in high density on the apical surface of normal intestinal epithelial cells as well as on the surface of human colon cancer cells. Analogs of ST(h) are currently being used as vectors targeting human colon cancers. Previous studies in our laboratory have focused on development of 111Indium-labeled ST(h) analogs for in vivo imaging applications. Here, we extend the scope of this work to include targeting of the therapeutic radionuclides 90Y and 177Lu. The peptide DOTA-F19-ST(h)(1-19) was synthesized using conventional Fmoc-based solid-phase techniques and refolded in dilute aqueous solution. The peptide was purified by RP-HPLC and characterized by MALDI-TOF MS and in vitro receptor binding assay. The DOTA-conjugate was metallated with nonradioactive Lu(III)Cl3 and Y(III)Cl3, and IC50 values of 2.6+/-0.1 and 4.2+/-0.9 nM were determined for the Lu- and Y-labeled peptides, respectively. 177Lu(III)Cl3 and 90Y(III)Cl3 labeling yielded tracer preparations that were inseparable by C18 RP-HPLC, indicating that putative differences between Lu-, Y- and In coordination spheres are not observed in the context of labeled ST(h) peptides. In vivo biodistribution studies of the 177Lu-labeled peptide in severe combined immunodeficient (SCID) mice bearing T-84 human cancer tumor xenografts showed rapid clearance from the bloodstream, with >90 %ID in the urine at 1 h pi. Localization of the tracer within tumor xenografts was 1.86+/-0.91 %ID/g at 1 h pi, a value higher than for all other tissues with the exception of kidney (2.74+/-0.24 %ID/g). At 24 h pi, >98 %ID was excreted into the urine, and 0.35+/-0.23 %ID/g remained in tumor, again higher than in all other tissues except kidney (0.91+/-0.46 %ID/g). Biodistribution results at 24 h pi for the 90Y-labeled peptide mirrored those for the 177Lu analog, in agreement with the identical behavior of the labeled analogs by C18 RP-HPLC. These results demonstrate the ability of 177Lu- and 90Y-labeled ST(h) molecules to specifically target GC-C receptors expressed on T-84 human colon cancer cells.
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comparison of effects of uroguanylin guanylin and escherichia coli heat stable Enterotoxin sta in mouse intestine and kidney evidence that uroguanylin is an intestinal natriuretic hormone
Journal of Investigative Medicine, 1997Co-Authors: Richard Greenberg, William J. Krause, Sammy L. Eber, F K Hamra, Michael S Hill, Jessica Crytzer, Leonard R ForteAbstract:BACKGROUND: Uroguanylin and guanylin are intestinal peptides that activate a receptor-guanylate cyclase, which is also a receptor for Escherichia coli Heat-Stable Enterotoxin (STa). These peptides may have a role in the body's regulation of fluid and electrolytes. METHODS: STa, bioactive guanylin, and bioactive uroguanylin were evaluated for effects in: 1) the suckling mouse intestinal fluid secretion assay; 2) an in vitro suckling mouse intestinal loop assay; 3) an intestinal receptor autoradiography assay; 4) a control or agonist-stimulated assay for cGMP response in T84 cells; and 5) an in vivo renal function assay in mice. RESULTS: In vivo, orally administered uroguanylin and STa but not guanylin, stimulated intestinal fluid secretion. All three peptides activated intestinal guanylate cyclase and had common intestinal receptors. In vitro, after pretreatment with chymotrypsin, only uroguanylin and STa retained agoinst activity. Chymostatin preserved guanylin activity. STa and uroguanylin induced diuresis, natriuresis, and kaliuresis. Guanylin was less potent than uroguanylin and STa. CONCLUSIONS: The results suggest that the endogenous intestinal peptides, uroguanylin and guanylin, regulate water and electrolyte homeostasis both through local effects on intestinal epithelia and endocrine effects on the kidney.
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distribution of escherichia coli heat stable Enterotoxin guanylin uroguanylin receptors in the avian intestinal tract
Cells Tissues Organs, 1995Co-Authors: William J. Krause, Sammy L. Eber, F K Hamra, R H Freeman, K F Fok, M G Currie, Leonard R ForteAbstract:Pathogenic strains of enteric bacteria secrete small Heat-Stable toxins (STs) that activate membrane guanylyl cyclase receptors found in the intestine. The intestinal peptide agonists, guanylin and uroguanylin, are structurally related to STs. Receptors for 125I-ST were found throughout the entire length of the intestinal tract of all the birds examined. These receptors were restricted to intestinal epithelial cells covering villi and forming intestinal glands and were not observed in other strata of the gut wall. The most intense labeling of receptors by 125I-ST occurred in the region of the microvillus border of individual enterocytes. There appeared to be a decrease in receptor density distally along the length of the small intestine, although labeling of receptors by 125I-ST was observed throughout the small intestine and colon. Cellular cGMP accumulation responses to Escherichia coli ST and rat guanylin in the domestic turkey and duck were greater in the proximal small intestine compared to the distal small intestine or colon. Brush border membranes (BBM) isolated from the mucosa of proximal small intestine of turkeys exhibited agonist-stimulated guanylyl cyclase activity. The rank order potency for enzyme activation was E. coli ST > uroguanylin > guanylin. Competitive radioligand binding assays using 125I-ST and turkey intestine BBM revealed a similar rank order affinity for the receptors that was exemplified by the Kd values of ST 2.5 nM, uroguanylin 80 nM and guanylin 2.6 microM. It may be concluded that functional receptors for the endogenous peptides, guanylin and uroguanylin, occur in the apical membranes of enterocytes throughout the avian intestine. The receptor-guanylyl cyclase(s) of proximal small intestine were preferentially activated by uroguanylin relative to guanylin, but both endogenous peptides were less potent than their molecular mimic, E. coli ST.
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distribution of escherichia coli heat stable Enterotoxin guanylin uroguanylin receptors in the avian intestinal tract
Cells Tissues Organs, 1995Co-Authors: William J. Krause, Sammy L. Eber, F K Hamra, R H Freeman, K F Fok, M G Currie, Leonard R ForteAbstract:Pathogenic strains of enteric bacteria secrete small Heat-Stable toxins (STs) that activate membrane guanylyl cyclase receptors found in the intestine. The intestinal peptide agonists, guanylin and ur
Ralph A Giannella - One of the best experts on this subject based on the ideXlab platform.
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heat stable Enterotoxin of escherichia coli sta can stimulate duodenal hco3 secretion via a novel gc c and cftr independent pathway
The FASEB Journal, 2008Co-Authors: Zachary M Sellers, Mitchell B Cohen, Ralph A Giannella, Elizabeth A. Mann, Anders Smith, Kim E Barrett, Hui DongAbstract:The Heat-Stable Enterotoxin of Escherichia coli (STa) is a potent stimulant of intestinal chloride and bicarbonate secretion. Guanylyl cyclase C (GC-C) has been shown to be the primary receptor involved in mediating this response. However, numerous studies have suggested the existence of an alternative STa-binding receptor. The aims of this study were to determine whether a non-GC-C receptor exists for STa and what is the functional relevance of this for intestinal bicarbonate secretion in mice. (125)I-STa-binding experiments were performed with intestinal mucosae from GC-C knockout (KO) and wild type (WT) mice. Subsequently, the functional relevance of an alternative STa-binding receptor was explored by examining STa-, uroguanylin-, and guanylin-stimulated duodenal bicarbonate secretion (DBS) in GC-C KO mice in vitro and in vivo. Significant (125)I-STa-binding occurred in the proximal small intestines of GC-C KO and WT mice. Analysis of binding coefficients and pH dependence showed that (125)I-STa-binding in GC-C KO mice involved a receptor distinct from that of WT mice. Functionally, STa, uroguanylin, and guanylin all stimulated a significant increase in DBS in GC-C KO mice. Uroguanylin- and guanylin-stimulated DBS were significantly inhibited by glibenclamide, but not by 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS). However, STa-stimulated DBS was unaffected by glibenclamide but inhibited by DIDS. Taken together, our results suggest that alternative, non-GC-C, receptors likely exist for STa, uroguanylin, and guanylin in the intestines of mice. While uroguanylin- and guanylin-stimulated DBS are cystic fibrosis transmembrane conductance regulator (CFTR) dependent, STa-stimulated DBS is CFTR independent. Further understanding of this alternative receptor and its signaling pathway may provide important insights into rectification of intestinal bicarbonate secretion in cystic fibrosis.
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heat stable Enterotoxin of escherichia coli sta can stimulate duodenal hco3 secretion via a novel gc c and cftr independent pathway
The FASEB Journal, 2008Co-Authors: Zachary M Sellers, Mitchell B Cohen, Ralph A Giannella, Elizabeth A. Mann, Anders Smith, Kim E Barrett, Kwang Hyun Ko, Hui DongAbstract:The Heat-Stable Enterotoxin of Escherichia coli (STa) is a potent stimulant of intestinal chloride and bicarbonate secretion. Guanylyl cyclase C (GC-C) has been shown to be the primary receptor involved in mediating this response. However, numerous studies have suggested the existence of an alternative STa-binding receptor. The aims of this study were to determine whether a non-GC-C receptor exists for STa and what is the functional relevance of this for intestinal bicarbonate secretion in mice. 125I-STa-binding experiments were performed with intestinal mucosae from GC-C knockout (KO) and wild type (WT) mice. Subsequently, the functional relevance of an alternative STa-binding receptor was explored by examining STa-, uroguanylin-, and guanylin-stimulated duodenal bicarbonate secretion (DBS) in GC-C KO mice in vitro and in vivo. Significant 125I-STa-binding occurred in the proximal small intestines of GC-C KO and WT mice. Analysis of binding coefficients and pH dependence showed that 125I-STa-binding in G...
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lack of guanylyl cyclase c the receptor for escherichia coli heat stable Enterotoxin results in reduced polyp formation and increased apoptosis in the multiple intestinal neoplasia min mouse model
International Journal of Cancer, 2005Co-Authors: Elizabeth A. Mann, Mitchell B Cohen, Kris A Steinbrecher, Carmen Stroup, David P Witte, Ralph A GiannellaAbstract:Guanylyl cyclase C (GC-C), a transmembrane receptor for bacterial Heat-Stable Enterotoxin and the mammalian peptides guanylin and uroguanylin, mediates intestinal ion secretion and affects intestinal cell growth via cyclic GMP signaling. In intestinal tumors, GC-C expression is maintained while guanylin and uroguanylin expression is lost, suggesting a role for GC-C activation in tumor formation or growth. We show by in situ hybridization that GC-C expression is retained in adenomas from multiple intestinal neoplasia (Apc(Min/+)) mice. In order to determine the in vivo role of GC-C in intestinal tumorigenesis, we generated Apc(Min/+) mice homozygous for a targeted deletion of the gene encoding GC-C and hypothesized that these mice would have increased tumor multiplicity and size compared to wild-type Apc(Min/+) mice on the same genetic background. In contrast, the absence of GC-C resulted in a reduction of median polyp number by 55%. There was no change in the median diameter of polyps, suggesting no effect on tumor growth. Somatic loss of the wild-type Apc allele, an initiating event in intestinal tumorigenesis, also occurred in polyps from GC-C-deficient Apc(Min/+) mice. We have found increased levels of apoptosis as well as increased caspase-3 and caspase-7 gene expression in the intestines of GC-C-deficient Apc(Min/+) mice compared with Apc(Min/+) mice. We propose that these alterations are a possible compensatory mechanism by which loss of GC-C signaling also affects tumorigenesis.
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guanylin and e coli heat stable Enterotoxin induce chloride secretion through direct interaction with basolateral compartment of rat and human colonic cells
Pediatric Research, 2005Co-Authors: Fabio Albano, Ralph A Giannella, Giulio De Marco, Roberto Berni Canani, Pia Cirillo, Vittoria Buccigrossi, Alfredo GuarinoAbstract:We previously detected specific binding activity of Escherichia coli Heat-Stable Enterotoxin (ST), the guanylin exogenous ligand, in rat colonic basolateral membranes. Because guanylin circulates in the bloodstream, we tested the hypothesis that it modulates intestinal ion transport by acting on the serosal side of intestinal cells. The effects of the mucosal and serosal addition of ST and guanylin on ion transport were investigated in the rat proximal colon and in Caco-2 cells in Ussing chambers, by monitoring short-circuit current (Isc). cGMP concentration was measured in Caco-2 cells by RIA. Mucosal ST addition induced an increase in Isc in rat proximal colon consistent with anion secretion. Serosal addition induced the same effects but to a lesser extent. The electrical effects observed in Caco-2 cells paralleled those observed in rat proximal colon. A pattern similar to the electrical response was observed with cGMP concentration. Guanylin addition to either side of Caco-2 cells induced the same effects as ST, although to a lesser extent. In all conditions, the electrical effect disappeared in the absence of chloride. ST directly interacts with basolateral receptors in the large intestine inducing chloride secretion through an increase of cGMP. However, the serosal effects are less pronounced compared with those observed with mucosal addition. Guanylin shows the same pattern, suggesting that it plays a role in the regulation of ion transport in the colon, but the relative importance of serosally mediated secretion remains to be determined.
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A gradient in expression of the Escherichia coli Heat-Stable Enterotoxin receptor exists along the villus-to-crypt axis of rat small intestine.
Biochemical and biophysical research communications, 1992Co-Authors: Elizabeth A. Mann, Chantal Lau, Susan J. Henning, Ralph A GiannellaAbstract:Binding of Escherichia coli Heat-Stable Enterotoxin to its receptor is critical to the initiation of toxin-induced secretion and diarrheal disease; it is also likely, however, that this receptor binds an endogenous ligand. In order to characterize the expression of the Heat-Stable Enterotoxin receptor in the small intestine, we isolated epithelial cells from villus tip to crypt in rat jejunum and ileum. Binding of radiolabeled toxin was maximal in the villus preparations and gradually decreased along the villus-to-crypt axis, paralleling the decline of sucrase activity. Northern blots of total RNA identified a single heat stable Enterotoxin receptor transcript (3.8 kb), predominantly in the villus cell fractions. In situ hybridization demonstrated clear signal in the villus cells with no apparent signal in the crypt cells, lamina propria or muscularis. Expression of this receptor was greatest after enterocytes leave the proliferative cycle and enter villi. This pattern of gene and protein expression may reflect a role of this receptor in binding endogenous ligands which in turn may regulate intestinal ion flux along the villus-to-crypt axis.
Mitchell B Cohen - One of the best experts on this subject based on the ideXlab platform.
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heat stable Enterotoxin of escherichia coli sta can stimulate duodenal hco3 secretion via a novel gc c and cftr independent pathway
The FASEB Journal, 2008Co-Authors: Zachary M Sellers, Mitchell B Cohen, Ralph A Giannella, Elizabeth A. Mann, Anders Smith, Kim E Barrett, Hui DongAbstract:The Heat-Stable Enterotoxin of Escherichia coli (STa) is a potent stimulant of intestinal chloride and bicarbonate secretion. Guanylyl cyclase C (GC-C) has been shown to be the primary receptor involved in mediating this response. However, numerous studies have suggested the existence of an alternative STa-binding receptor. The aims of this study were to determine whether a non-GC-C receptor exists for STa and what is the functional relevance of this for intestinal bicarbonate secretion in mice. (125)I-STa-binding experiments were performed with intestinal mucosae from GC-C knockout (KO) and wild type (WT) mice. Subsequently, the functional relevance of an alternative STa-binding receptor was explored by examining STa-, uroguanylin-, and guanylin-stimulated duodenal bicarbonate secretion (DBS) in GC-C KO mice in vitro and in vivo. Significant (125)I-STa-binding occurred in the proximal small intestines of GC-C KO and WT mice. Analysis of binding coefficients and pH dependence showed that (125)I-STa-binding in GC-C KO mice involved a receptor distinct from that of WT mice. Functionally, STa, uroguanylin, and guanylin all stimulated a significant increase in DBS in GC-C KO mice. Uroguanylin- and guanylin-stimulated DBS were significantly inhibited by glibenclamide, but not by 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS). However, STa-stimulated DBS was unaffected by glibenclamide but inhibited by DIDS. Taken together, our results suggest that alternative, non-GC-C, receptors likely exist for STa, uroguanylin, and guanylin in the intestines of mice. While uroguanylin- and guanylin-stimulated DBS are cystic fibrosis transmembrane conductance regulator (CFTR) dependent, STa-stimulated DBS is CFTR independent. Further understanding of this alternative receptor and its signaling pathway may provide important insights into rectification of intestinal bicarbonate secretion in cystic fibrosis.
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heat stable Enterotoxin of escherichia coli sta can stimulate duodenal hco3 secretion via a novel gc c and cftr independent pathway
The FASEB Journal, 2008Co-Authors: Zachary M Sellers, Mitchell B Cohen, Ralph A Giannella, Elizabeth A. Mann, Anders Smith, Kim E Barrett, Kwang Hyun Ko, Hui DongAbstract:The Heat-Stable Enterotoxin of Escherichia coli (STa) is a potent stimulant of intestinal chloride and bicarbonate secretion. Guanylyl cyclase C (GC-C) has been shown to be the primary receptor involved in mediating this response. However, numerous studies have suggested the existence of an alternative STa-binding receptor. The aims of this study were to determine whether a non-GC-C receptor exists for STa and what is the functional relevance of this for intestinal bicarbonate secretion in mice. 125I-STa-binding experiments were performed with intestinal mucosae from GC-C knockout (KO) and wild type (WT) mice. Subsequently, the functional relevance of an alternative STa-binding receptor was explored by examining STa-, uroguanylin-, and guanylin-stimulated duodenal bicarbonate secretion (DBS) in GC-C KO mice in vitro and in vivo. Significant 125I-STa-binding occurred in the proximal small intestines of GC-C KO and WT mice. Analysis of binding coefficients and pH dependence showed that 125I-STa-binding in G...
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lack of guanylyl cyclase c the receptor for escherichia coli heat stable Enterotoxin results in reduced polyp formation and increased apoptosis in the multiple intestinal neoplasia min mouse model
International Journal of Cancer, 2005Co-Authors: Elizabeth A. Mann, Mitchell B Cohen, Kris A Steinbrecher, Carmen Stroup, David P Witte, Ralph A GiannellaAbstract:Guanylyl cyclase C (GC-C), a transmembrane receptor for bacterial Heat-Stable Enterotoxin and the mammalian peptides guanylin and uroguanylin, mediates intestinal ion secretion and affects intestinal cell growth via cyclic GMP signaling. In intestinal tumors, GC-C expression is maintained while guanylin and uroguanylin expression is lost, suggesting a role for GC-C activation in tumor formation or growth. We show by in situ hybridization that GC-C expression is retained in adenomas from multiple intestinal neoplasia (Apc(Min/+)) mice. In order to determine the in vivo role of GC-C in intestinal tumorigenesis, we generated Apc(Min/+) mice homozygous for a targeted deletion of the gene encoding GC-C and hypothesized that these mice would have increased tumor multiplicity and size compared to wild-type Apc(Min/+) mice on the same genetic background. In contrast, the absence of GC-C resulted in a reduction of median polyp number by 55%. There was no change in the median diameter of polyps, suggesting no effect on tumor growth. Somatic loss of the wild-type Apc allele, an initiating event in intestinal tumorigenesis, also occurred in polyps from GC-C-deficient Apc(Min/+) mice. We have found increased levels of apoptosis as well as increased caspase-3 and caspase-7 gene expression in the intestines of GC-C-deficient Apc(Min/+) mice compared with Apc(Min/+) mice. We propose that these alterations are a possible compensatory mechanism by which loss of GC-C signaling also affects tumorigenesis.
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the heat stable Enterotoxin guanylin receptor is expressed in rat hepatocytes and in a rat hepatoma h 35 cell line
Journal of Receptors and Signal Transduction, 1997Co-Authors: J P Balint, J L Kosiba, Mitchell B CohenAbstract:AbstractBackground/Aims: Guanylyl cyclase C (GC-C) is an intestinal transmembrane receptor which binds both guanylin, an endogenous ligand, and Escherichia coli Heat-Stable Enterotoxin (STa) resulting in 5′-cyclic guanosine monophosphate (cGMP) accumulation and chloride secretion. In the adult rat, there is a high basal level of GC-C expression in the intestine, but not in the liver. Increased expression of GC-C in the rat liver has been demonstrated during the perinatal period as well as with liver regeneration and during an acute phase response. The aim of this study was to identify and utilize cell culture models to further characterize the expression of GC-C in the liver. Methods: STa binding, STa-stimulated cGMP accumulation, and GC-C RNA expression by Northern analysis were determined in primary cultures of rat hepatocytes and H-35 cells, a rat hepatoma cell line, following treatment with dexamethasone and/or interleukin-6 (IL-6). Results: In rat hepatocytes treated with the combination of dexametha...
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escherichia coli Enterotoxin sta binds to receptors stimulates guanyl cyclase and impairs absorption in rat colon
Gastroenterology, 1992Co-Authors: Mitchell B Cohen, Ralph A Giannella, Adam Mezoff, Michael N EadeAbstract:Abstract To determine the contribution of the colon in Escherichia coli Heat-Stable Enterotoxin-mediated diarrheal disease, toxin binding, guanyl cyclase activation, and toxin-induced water flux in the rat colon and ileum were compared. Scatchard analysis suggested a single class of Heat-Stable Enterotoxin receptors with an affinity constant of binding of 10 9 L/mol in both colonocytes and ileocytes; however, the number of toxin receptors per cell was 3.5-fold greater in colonocytes than ileocytes (8.32 ± 1.33 × 10 5 vs. 2.33 ± 0.28 × 10 5 receptors per cell; P = 0.02). Heat-Stable Enterotoxin stimulated guanyl cyclase activation in an identical dose-dependent manner in proximal colonic and ileal membranes, with similar sensitivity and maximum response. Heat-Stable Enterotoxin also inhibited net water flux to a similar degree in both colon and ileum (−47.8 vs. −48.4 μL · cm −1 · h −1 , respectively) at a dose of 8 nmol/L. At this dose in the colon, because of a higher baseline of absorption, absorption continued, but at a diminished level. At this dose in the ileum, Heat-Stable Enterotoxin induced net secretion. These data are consistent with the concept that Heat-Stable Enterotoxin-induced diarrheal disease results from a decreased absorptive capacity in the colon in the face of increased small intestinal fluid secretion.
William J. Krause - One of the best experts on this subject based on the ideXlab platform.
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comparison of effects of uroguanylin guanylin and escherichia coli heat stable Enterotoxin sta in mouse intestine and kidney evidence that uroguanylin is an intestinal natriuretic hormone
Journal of Investigative Medicine, 1997Co-Authors: Richard Greenberg, William J. Krause, Sammy L. Eber, F K Hamra, Michael S Hill, Jessica Crytzer, Leonard R ForteAbstract:BACKGROUND: Uroguanylin and guanylin are intestinal peptides that activate a receptor-guanylate cyclase, which is also a receptor for Escherichia coli Heat-Stable Enterotoxin (STa). These peptides may have a role in the body's regulation of fluid and electrolytes. METHODS: STa, bioactive guanylin, and bioactive uroguanylin were evaluated for effects in: 1) the suckling mouse intestinal fluid secretion assay; 2) an in vitro suckling mouse intestinal loop assay; 3) an intestinal receptor autoradiography assay; 4) a control or agonist-stimulated assay for cGMP response in T84 cells; and 5) an in vivo renal function assay in mice. RESULTS: In vivo, orally administered uroguanylin and STa but not guanylin, stimulated intestinal fluid secretion. All three peptides activated intestinal guanylate cyclase and had common intestinal receptors. In vitro, after pretreatment with chymotrypsin, only uroguanylin and STa retained agoinst activity. Chymostatin preserved guanylin activity. STa and uroguanylin induced diuresis, natriuresis, and kaliuresis. Guanylin was less potent than uroguanylin and STa. CONCLUSIONS: The results suggest that the endogenous intestinal peptides, uroguanylin and guanylin, regulate water and electrolyte homeostasis both through local effects on intestinal epithelia and endocrine effects on the kidney.
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distribution of escherichia coli heat stable Enterotoxin guanylin uroguanylin receptors in the avian intestinal tract
Cells Tissues Organs, 1995Co-Authors: William J. Krause, Sammy L. Eber, F K Hamra, R H Freeman, K F Fok, M G Currie, Leonard R ForteAbstract:Pathogenic strains of enteric bacteria secrete small Heat-Stable toxins (STs) that activate membrane guanylyl cyclase receptors found in the intestine. The intestinal peptide agonists, guanylin and uroguanylin, are structurally related to STs. Receptors for 125I-ST were found throughout the entire length of the intestinal tract of all the birds examined. These receptors were restricted to intestinal epithelial cells covering villi and forming intestinal glands and were not observed in other strata of the gut wall. The most intense labeling of receptors by 125I-ST occurred in the region of the microvillus border of individual enterocytes. There appeared to be a decrease in receptor density distally along the length of the small intestine, although labeling of receptors by 125I-ST was observed throughout the small intestine and colon. Cellular cGMP accumulation responses to Escherichia coli ST and rat guanylin in the domestic turkey and duck were greater in the proximal small intestine compared to the distal small intestine or colon. Brush border membranes (BBM) isolated from the mucosa of proximal small intestine of turkeys exhibited agonist-stimulated guanylyl cyclase activity. The rank order potency for enzyme activation was E. coli ST > uroguanylin > guanylin. Competitive radioligand binding assays using 125I-ST and turkey intestine BBM revealed a similar rank order affinity for the receptors that was exemplified by the Kd values of ST 2.5 nM, uroguanylin 80 nM and guanylin 2.6 microM. It may be concluded that functional receptors for the endogenous peptides, guanylin and uroguanylin, occur in the apical membranes of enterocytes throughout the avian intestine. The receptor-guanylyl cyclase(s) of proximal small intestine were preferentially activated by uroguanylin relative to guanylin, but both endogenous peptides were less potent than their molecular mimic, E. coli ST.
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distribution of escherichia coli heat stable Enterotoxin guanylin uroguanylin receptors in the avian intestinal tract
Cells Tissues Organs, 1995Co-Authors: William J. Krause, Sammy L. Eber, F K Hamra, R H Freeman, K F Fok, M G Currie, Leonard R ForteAbstract:Pathogenic strains of enteric bacteria secrete small Heat-Stable toxins (STs) that activate membrane guanylyl cyclase receptors found in the intestine. The intestinal peptide agonists, guanylin and ur
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distribution of heat stable Enterotoxin guanylin receptors in the intestinal tract of man and other mammals
Journal of Anatomy, 1994Co-Authors: William J. Krause, Sammy L. Eber, G L Cullingford, R H Freeman, K C Richardson, K F Fok, M G Currie, Leonard R ForteAbstract:The human intestinal tract, as well as that of several eutherian and metatherian mammals, was examined for the distribution of Heat-Stable Enterotoxin (ST)/guanylin receptors. These receptors were confined to the intestinal epithelium lining the lumen and forming the intestinal glands throughout the length of both the small intestine and colon of all species examined. In man and most other mammalian species, there appeared to be a decrease in receptor density distally along the longitudinal axis of the small intestine. ST/guanylin receptors were not observed in other strata forming the gut wall. Along the vertical axis of the human small intestine (villus/crypt unit), as well as that of most other mammals, receptor density was greatest in enterocytes located near the base of villi and in those forming the proximal portion of the intestinal glands. ST/guanylin receptors were for the most part confined to the region of the plasmalemma forming the microvillus border. In the colon of man and the other species examined, receptor density was greatest in enterocytes forming the proximal region of the intestinal glands. Receptors were present in the intestinal epithelium lining the lumen of the colon, but generally were fewer in number. The distribution of cellular cGMP accumulation responses to E. coli ST and guanylin in the opossum (Didelphis virginiana) and raccoon (Procyon lotor) revealed that proximal small intestine had greater magnitudes of cGMP responses than did the distal small intestine. Proximal colon had greater cGMP responses than distal colon, which had no significant cGMP responses to either ST or guanylin.
Stephen J. Savarino - One of the best experts on this subject based on the ideXlab platform.
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is1414 an escherichia coli insertion sequence with a heat stable Enterotoxin gene embedded in a transposase like gene
Infection and Immunity, 2000Co-Authors: Annette L Mcveigh, Alessio Fasano, Donald C Robertson, Daniel A Scott, Sandra Jelacic, Steve L Moseley, Stephen J. SavarinoAbstract:ABSTRACT Enteroaggregative Escherichia coli (EAEC) Heat-Stable Enterotoxin 1 (EAST1) was originally discovered in EAEC but has also been associated with enterotoxigenic E. coli (ETEC). Multiple genomic restriction fragments from each of three ETEC strains of human origin showed homology with an EAST1 gene probe. A single hybridizing fragment was detected on the plasmid of ETEC strain 27D that also encodes Heat-Stable Enterotoxin Ib and colonization factor antigen I. We isolated and characterized this fragment, showing that it (i) carries an allele of astA nearly identical to that originally reported from EAEC 17-2 and (ii) expressed enterotoxic activity. Sequence analysis of the toxin coding region revealed thatastA is completely embedded within a 1,209-bp open reading frame (ORF1), whose coding sequence is on the same strand but in the −1 reading frame in reference to the toxin gene. In vitro expression of the predicted Mr-∼46,000 protein product of ORF1 was demonstrated. ORF1 is highly similar to transposase genes of IS285 from Yersinia pestis, IS1356 from Burkholderia cepacia, and ISRm3 from Rhizobium meliloti. It is bounded by 30-bp imperfect inverted repeat sequences and flanked by 8-bp direct repeats. Based on these structural features, pathognomonic of a regular insertion sequence, this element was designated IS1414. Preliminary experiments to show IS1414 translocation were unsuccessful. Overlapping genes of the type suggested by the IS1414 core region have heretofore not been described in bacteria. It seems to offer a most efficient mechanism for intragenomic and horizontal dissemination of EAST1.
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enteroaggregative escherichia coli heat stable Enterotoxin is not restricted to enteroaggregative e coli
The Journal of Infectious Diseases, 1996Co-Authors: Stephen J. Savarino, P Echeverria, M. K. Bhan, Myron M. Levine, Alejandro Cravioto, Annette L Mcveigh, John D G Watson, Jose Molina, Alessio FasanoAbstract:Enteroaggregative Escherichia coli (EAggEC) have been implicated as diarrheal pathogens in several settings. Some EAggEC produce a distinct Heat-Stable Enterotoxin named EAST1. The distribution and prevalence of the EAST1 gene in selected groups of bacterial enteropathogens were determined by colony hybridization. One hundred percent of 75 O157 :H7 enterohemorrhagic E. coli (EHEC), 41% of 227 EAggEC, 41% of 149 enterotoxigenic E. coli, 22% of 65 enteropathogenic E. coli(EPEC), and 38% of 47 E. coli stool isolates from asymptomatic children hybridized with an EAST1 DNA probe. None of 55 enteroinvasive E. coli, 12 Yersinia enterocolitica, or 20 Vibrio cholerae non-Ol strains were EAST1 probe-positive. Concordance between EAST1 genotype and enterotoxicity was shown in examined strains of EAggEC, EHEC, and EPEC. The gene encoding EAST1 is more broadly distributed among diarrheogenic E. coli than previously known and may represent an additional determinant in the pathogenesis of E. coli diarrhea.
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enteroaggregative escherichia coli heat stable Enterotoxin 1 represents another subfamily of e coli heat stable toxin
Proceedings of the National Academy of Sciences of the United States of America, 1993Co-Authors: Stephen J. Savarino, Myron M. Levine, John D G Watson, Alessio Fasano, Brian M Martin, Stefano Guandalini, Patricia GuerryAbstract:Enteroaggregative Escherichia coli (EAggEC) are associated with persistent diarrhea in young children. Some of these organisms produce a low-molecular-weight, Heat-Stable, plasmid-encoded Enterotoxin that has been named EAggEC Heat-Stable Enterotoxin 1 (EAST1). We have cloned a 4.4-kb DNA fragment from the virulence plasmid of prototype EAggEC strain 17-2, which expresses enterotoxic activity as measured by electrogenic response in Ussing chambers mounted with rabbit ileal tissue. DNA-sequence analysis of this fragment identified an open reading frame (ORF) encoding a cysteine-rich polypeptide of 38 amino acids (M(r), 4100). Insertional and deletional mutations in this ORF resulted in loss of enterotoxic activity. The ORF was cloned into a T7 expression vector, and postinduction culture filtrates exhibited enterotoxic activity and increased ileal tissue cGMP levels. A synthetic peptide consisting of predicted amino acid residues 8-29 also showed enterotoxic activity. These data indicate that this ORF, named astA (EAggEC Heat-Stable Enterotoxin), represents the EAST1 structural gene. EAST1 shows significant homology with the enterotoxic domain of Heat-Stable Enterotoxin a (STa) of enterotoxigenic E. coli and with guanylin, a mammalian analog of STa. Unlike STa, which requires six cysteines and three disulfide linkages for full biological activity, both EAST1 and guanylin contain four cysteine residues. Based on the cGMP data and the sequence homology to STa and guanylin, it is predicted that EAST1 stimulates the particulate form of guanylate cyclase through the same receptor-binding region as STa and guanylin.