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G W Forsyth

Publications and source records attributed to G W Forsyth.

15 recordsLinked to original sources
PubMed1992

Effect of covalent modification on the binding of cholera toxin B subunit to ileal brush border surfaces.

A competitive binding assay has been developed to determine how modifications to the B subunit of cholera toxin affect the binding affinity of the subunit for an ileal brush border membrane surface. The Ricinus communis120 agglutinin (RCA120) specifically binds to terminal beta-D-galactosyl residues such as those found in oligosaccharide side chains of glycoproteins and ganglioside GM1. Conditions were designed to produce binding competition between the B subunit of cholera toxin and the RCA120 agglutinin. Displacement of RCA120 from brush border surfaces was proportional to the concentration of B subunit added. This assay was used to study the effect of modification of B subunit on competitive binding affinity for the ileal brush border surface. The B subunit of cholera toxin was modified by coupling an average of five sulfhydryl groups to each B subunit molecule and by reaction of the SH-modified B subunit with liposomes containing a surface maleimide group attached to phosphatidylethanolamine. SH-modified B subunit was approximately 200-fold more effective than native B subunit in displacing lectin from brush border surfaces in the competitive binding assay. The enhanced binding activity was retained on covalent attachment of the modified B subunit to the liposome surface. We conclude that the B subunit of cholera toxin may be a useful targeting agent for directing liposomes to cell surfaces that contain a ganglioside GM1 ligand.

Animals↗
PubMed1992

Inhibition of ileal brush-border chloride conductance by specific antibody.

Antibody raised in mice was used in attempting to identify proteins responsible for the conductive chloride transport that can be measured in porcine ileal brush border membrane vesicles. Ileal brush-border membrane vesicle protein from pig was separated into five different molecular mass fractions by preparative SDS polyacrylamide disc gel electrophoresis. Separated protein fractions were used to immunize mice. Antibody was screened for reactivity with antigen by Western blotting, and for effects on conductive chloride transport in ileal brush border membrane vesicles. Immunization with brush-border protein from fraction I proteins (> 110 kDa) produced polyclonal antisera which specifically inhibited the conductive component of chloride uptake by ileal brush border vesicle preparations. Western blotting of the antigen showed the presence of several protein species of molecular mass > 100 kDa that were recognized by immune serum. Spleen cells from a mouse producing antiserum that inhibited conductive chloride transport were fused with a myeloma cell line. The resulting hybridoma colonies produced antibody that reacted with at least seven distinct protein bands by Western blot assay and inhibited chloride conductance in brush-border membrane vesicles.

Animals↗
PubMed1992

Liposomes targeted to deliver antisecretory agents to jejunal mucosa.

The B subunit of cholera toxin has been covalently attached to the surface of liposomes made from a mixture of phosphatidylethanolamine, phosphatidylcholine and cholesterol. Adenylate cyclase inhibitors and chloride conductance inhibitors were encapsulated within the liposomes. These "targeted" liposomes were used to study the combined effects of this novel delivery system, and a limited number of possible antisecretory agents, on net fluid flux into the pig jejunum. A state of net secretory fluid flux was induced in isolated jejunal loops in weanling pigs by adding theophylline or cholera toxin to the lumen of the isolated loops. There was no reduction in net fluid secretion when liposome suspensions without encapsulated secretory inhibitors were added to fluid in the lumen of loops treated with theophylline. There was also no reduction in net fluid secretion when miconazole, alpha-phenylcinnamate or 5 nitro-2-(3-phenethylamino)benzoate were encapsulated within targeted liposomes added to isolated jejunal loops. The net fluid flux induced by exposure of jejunal loops to theophylline was significantly reduced by adding targeted liposomes containing 2'-deoxy-3'-AMP. The reduction involved a reversal of net secretory fluid flux to an absorptive value. The net fluid secretory response to treatment of loops with cholera toxin was also inhibited by treating loops with targeted liposomes containing 2'-deoxy-3'-AMP. However, the reversal of secretion was less complete for secretion induced by cholera toxin than for secretion induced by theophylline. The reduced antisecretory efficacy versus cholera toxin was not improved by encapsulating higher concentrations of 2'-deoxy-3'-AMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Candidate proteins for conductive chloride transport in porcine ileal brush-border membrane.

Conductive transport of chloride ion is important in controlling ion and fluid secretion by exocrine tissues. The current study was directed at identifying proteins in the intestinal brush-border membrane that may be involved with conductive chloride transport. Reaction of total brush-border membrane protein with phenyl-isothiocyanate inhibited conductive chloride transport into brush-border membrane vesicles. The conductive transport process was protected from this inhibition by including the ligands Cl- and alpha-phenylcinnamate in the reaction mixture. Brush-border membrane protein protected by this procedure and labeled with fluorescein had an apparent molecular mass in the region of 130 and 23 kDa on separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphorylation of brush-border membrane protein with [gamma-32P] ATP and endogenous protein kinase under conditions causing activation of chloride conductance in membrane vesicles caused the transfer of 32P to several proteins, including ones in the same molecular size range (130 and 23 kDa) as those identified by the fluorescein labeling procedure. Conductive chloride transport in porcine intestinal brush-border vesicles may occur via proteins identified by this differential labeling procedure.

Animals↗
PubMed1979

Isolation and partial characterization of two different heat-stable enterotoxins produced by bovine and porcine strains of enterotoxigenic Escherichia coli.

Heat-stable enterotoxins (ST-124 and ST-1261) have been isolated from two different enterotoxigenic Escherichia coli of bovine (124) and porcine (1261) origin. The enterotoxin preparations were isolated by ultrafiltration and ion-exchange chromatography and were both active in the suckling mouse test and pig ligated loop test in the nanogram range. The bovine (ST-124) enterotoxin was not stable to heating in its isolated form, and significant differences in amino acid composition were observed between the two enterotoxins. Although both toxins were active at similar levels in the suckling mouse and pig ligated loop tests, ST-124 lacked the ability to cause the profound secretory responses seen with ST-1261 in the weanling pig ligated loop.

Amino Acids↗
PubMed1979

Failure to reverse cholera toxin induced intestinal secretion by agents which decrease mucosal cAMP.

The feasibility of reducing intestinal secretion by the use of agents which decrease intestinal mucosal cAMP concentration has been investigated in the weanling pig and the rabbit. Three different agents for decreasing mucosal cAMP concentration were studied. The cyclic nucleotide phosphodiesterase activator, imidazole, significantly reduced mucosal cAMP concentrations only in the weanling pig. Intraluminal 2'-deoxyadenosine-3'AMP inhibited adenylate cyclase and caused a decrease in mucosal cAMP concentration in both the pig and the rabbit. The introduction of the heat-stable enterotoxin of Escherichia coli into pig jejunal segments also gave lowered mucosal cAMP concentrations. While these three agents effectively reduced cAMP concentrations in intestinal mucosa, they were ineffective in reducing the net fluid secretory effects of cholera toxin. Secretion caused by cholera toxin apparently persists independent of the temporary changes in cAMP concentration which can be induced by pharmacological agents.

Adenylyl Cyclases↗
PubMed1978

Cholera toxin effects on fluid secretion, adenylate cyclase, and cyclic AMP in porcine small intestine.

The effects of cholera toxin on mucosal cyclic nucleotide concentrations and on net fluid secretion in the porcine small intestine are reported. Cholera toxin causes net secretion of fluid into the small intestine of weanling pigs, and secretory rates are dependent on the dose of the toxin placed in intestinal loops. Intestinal secretion due to cholera toxin exposure was not consistently accompanied by elevated concentrations of mucosal cyclic AMP or cyclic GMP. Net fluid fluxes in individual loops did not correlate with mucosal cyclic AMP concentration in the same loop. Jejunal adenylate cyclase was activated to a lesser extent in pigs, compared with rabbits, after in vivo treatment with cholera toxin. In vitro activation in cell-free homogenates was similar for both species. Papaverine was similar to cholera toxin in causing fluid secretion without cyclic AMP accumulations, but 3-isobutyl-1-methyl xanthine significantly increased cyclic AMP concentration and induced fluid secretion in pigs. Weanling pigs appeared to differ from rabbits in having a secretory response to cholera toxin which was independent of elevations in total mucosal cyclic AMP concentration.

1-Methyl-3-isobutylxanthine↗
PubMed1978

Some comparative properties and localization of porcine jejunal adenylate cyclase.

Cholera toxin is thought to cause intestinal secretion by activating adenylate cyclase and increasing intracellular 3',5'-cyclic AMP concentrations in intestinal mucosa. Cholera toxin causes profuse secretion of fluid into ligated intestinal loops of both pigs and rabbits, but cholera toxin-induced increases in 3',5'-cyclic AMP concentration are much lower in the pig than in the rabbit. Porcine jejunal adenylate cyclase was examined for unusual properties which might account for a lack of 3'-5'-cyclic AMP accumulation after treatment with cholera toxin. The divalent cation requirements, the pH optimum, and the stimulation by fluoride ion were unremarkable. The Km for ATP was 0.11 mM with negative cooperativity indicated by a Hill coefficient of 0.83. Triton X-100 was inhibitory and guanosine diphosphate methylenephosphate stimulated enzyme activity. Adenylate cyclase activity was highest in the basal and lateral membrane fractions of jejunal mucosa and relatively low in brush-border preparations. Pretreatment of pig jejunum with cholera toxin caused a 30-40% activation of the crude and of the partly purified enzyme. A relatively low activation of adenylase cyclase in pig jejunal mucosa, compared with rabbit, may account for the absence of 3',5'-cyclic AMP accumulation after cholera-toxin treatment in the pig.

Adenylyl Cyclases↗
PubMed1978

Effects of plant and animal lipids rich in docosenoic acids on the myocardium of Cynomolgus monkeys.

Cynomolgus monkeys (Macaca fascicularis) were fed diets containing 25% rapeseed oil (RSO), partially-hydrogenated herring oil (PHHO) or a 3:1 mixture of lard/corn oil as control (CON) for 4 months. The RSO contained approximately 25% of the fatty acids as erucic acid (cis-docos-13-enoic, 22:1w9) while the PHHO contained a similar concentration of mainly cetoleic acid (cis-docos-11-enoic, 22:1w11). The CON contained no 22:1 acids. The monkeys developed the expected myocardial lipidosis, somewhat more pronounced in the RSO than the PHHO group, but small foci of mononuclear cell infiltration, while infrequent, occurred in all three groups. Significant intergroup differences in biochemical or hematologic measurements of serum constituents were an increase in serum cholesterol concentration in the RSO group and an increase in serum glutamicoxaloacetic transaminase activity in both RSO and PHHO groups at certain intervals. The shorter proportion of M. fascicularis life span represented by this experiment may account for the absence of clear intergroup differences such as are reported in rats used in similar studies.

Animals↗
PubMed1978

The effect of cholera toxin and heat labile and heat stable Escherichia coli enterotoxin on cyclic AMP concentrations in small intestinal mucosa of pig and rabbit.

The effect of cholera toxin, heat labile and heat stable Escherichia coli enterotoxin on mucosal cyclic AMP concentrations was determined on the proximal jejunum of weanling pigs and young rabbits. Ligated loops were injected with solutions containing no enterotoxin for control and either cholera toxin, heat labile or heat stable E. coli enterotoxin. The loops were drained after either two, four or six hours incubation at which time accumulated fluid was recorded and mucosal samples removed for determination of cyclic AMP concentration. In the rabbit, cholera toxin and heat labile, but not heat stable E. coli enterotoxin stimulated intestinal secretion while in the pig all three enterotoxins induced net fluid accumulation. Cholera toxin and heat labile, but not heat stable E. coli enterotoxin elevated rabbit mucosal cyclic AMP concentrations. In the pig these enterotoxins had no significant effect on mucosal cyclic AMP concentrations. The results are inconsistent with the hypothesis that the adenyl cyclase system is an essential step for enterotoxin induced intestinal secretion. The activation of intestinal adenyl cyclase by bacterial enterotoxins may only be an associated and not a necessary event for the stimulation of intestinal secretion.

Animals↗
PubMed1978

Effect of heat stable and heat labile Escherichia coli enterotoxins and cholera toxin in combination with theophylline on unidirectional sodium and chloride flux in the small intestine of weanling swine.

The effect of heat stable and heat labile Escherichia coli enterotoxins or cholera toxin in combination with theophylline on net water, sodium and chloride and unidirectional sodium and chloride fluxes was examined in acute isolated loops of jejunum of weanling swine. The effect of heat stable enterotoxin in combination with theophylline was determined in loops located in the proximal jejunum, while combinations of theophylline and either heat labile enterotoxin or cholera toxin were studied in the distal jejunum. In each situation the addition of theophylline resulted in an additive rather than a synergistic increment of intestinal secretory activity. This study implies that the intestinal adenyl cyclase system and enterotoxin induced intestinal secretion may not be directly related in the swine small intestine.

Animals↗
PubMed1977

Heart mitochondrial metabolism after feeding herring oil to rats and monkeys.

Heart mitochondrial oxidation of palmityl CoA and pyruvic acid was studied in rats and in the monkey Macaca fascicularis to determine the effects of feeding partially hydrogenated herring oil. Herring oil glycerides contain cetoleic acid (cis-11-docosenoic) which could have a similar effect to erucic acid (cis-13-docosenoic) in causing a rat cardiomyopathy. The initial rat heart mitochondrial response to dietary cetoleic acid (67% cis, 33% trans) was an in vitro decrease in palmityl CoA oxidation. Pronlonged feeding of cetoleic acid mixture was associated with a significant metabolic adaptation, increasing pyruvate and palmityl CoA oxidation above control levels. In vitro addition of cetoleyl CoA (pure cis isomer) stimulated pyruvate dehydrogenase activity, a possible response to decreased B-oxidation. There was no significant adaptive change in pyruvate or palmityl CoA use in monkeys after prolonged feeding of partially hydrogenated herring oil. Cetoleyl CoA was a good substrate for monkey heart carnitine acyl transferase even in the presence of palmityl CoA. These observations suggest that C22 fatty acids may be metabolized more rapidly in monkey heart than in rat heart. Metabolic differences argue against using the rat as an experimental model for studying possible cardiotoxic effects of docosenoic acids in primates.

Animals↗
PubMed1977

A comparison of parameters used to assess liver damage in sheep treated with carbon tetrachloride.

Changes in serum enzyme levels, liver histology and liver function tests have been correlated to determine the usefulness of these tests in assessing liver status. The effects of carbon tetrachloride administration on these parameters has been determined in a group of 20 sheep. Normal levels, elevated levels after injury and the effect of elapsed time after injury are reported for serum glutamic dehydrogenase, sorbitol dehydrogenase, glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase, lactate dehydrogenase, fructose-1-phosphate adlolase, alkaline phosphatase, cholesterol and proteins. Variation in the time of elevation of enzyme activities may be useful in determining the elapsed time between acute injury and serum sampling. In comparison to sheep fed an adequate diet, a diet with a restricted protein intake was associated with increased severity of histological lesions and decreased liver function.

Alanine Transaminase↗