Biomedical subjects

P O'Hanley

Publications and source records attributed to P O'Hanley.

At least 19 recordsLinked to original sources

PubMed1997

Topical tetracycline: potential for allergic reaction or bacterial resistance.

The high incidence of allergic reactions to some common dental antibiotics, primarily topical penicillins, has led to general concerns about all topical antibiotics. The development of resistant bacterial strains and efforts to reserve key antibiotics for life-threatening infections have also limited topical use of antibiotics. Delivery technologies providing for site-specific drug delivery have renewed interest in the use of topical antimicrobials to treat adult periodontitis. Topical tetracycline has an extremely low sensitizing potential and is not one of the antibiotics reserved by the medical community for use in life-threatening situations. Despite tetracycline's widespread dermatologic use and increasing use in adjunctive treatment of adult periodontitis, the incidence of allergic response to topical tetracycline is very low. Also, it is unlikely to cause resistance when used locally for short durations--particularly at the high per-site concentrations achieved with tetracycline periodontal fiber. Studies with tetracycline fiber showed no significant change in the tetracycline susceptibility of gram-negative periodontal microorganisms.

Administration, Topical↗

PubMed1996

Enterotoxigenic Escherichia coli diarrhea in children less than five years of age in central Java.

A community-based prospective study was performed from December 1993 through March 31, 1994 in Indonesia in children less than five years of age. Enterotoxigenic Escherichia coli (ETEC) was identified in diarrheic stool by colony hybridization assay, using toxin probes, and this bacterium was isolated from 19% of 340 episodes of diarrhea. Sixty-one percent of ETEC produced heat-labile toxin (LT) only, 325 LT and heat-stable toxin (ST), and 75 ST only. The age-specific incidence rates of diarrhea among children 0-1 and 2-3 years of age were 77% and 61%, respectively, during the study period; ETEC was isolated from 26% of children 0-1 years of age versus 53% for children 2-3 years of age. As many as seven episodes of diarrhea were repeatedly experienced by a single child during the four-month study period; however, only two children had more than one episode of known ETEC-associated diarrheal disease during the period of observation.

Age Factors↗

PubMed1995

Molecular characterization of Vibrio cholerae O139 isolates from Asia.

In 1992, a serologically novel clone of Vibrio cholerae, designated O139, caused large epidemics of diarrhea in India and Bangladesh. To determine the extent of the spread of V. cholerae O139 worldwide, 484 V. cholerae non-O1 strains isolated from different patients with diarrhea in Thailand, Indonesia, the Philippines, and Peru in 1993 were tested for agglutination in O139 antisera. One hundred fifty-one of these 484 isolates were examined for genes encoding cholera toxin, zonula occlulans toxin, the repetitive sequence 1, and the toxin coregulated pilin A (the V. cholerae virulence gene complex). Thirty-three percent (122 of 364) of V. cholerae non-O1 strains isolated from different patients with diarrhea in Thailand agglutinated in O139 antisera. Ninety-eight percent (120 of 122) of V. cholerae O139 contained the V. cholerae virulence gene complex. None of the 104 V. cholerae non-O1 strains isolated from patients with diarrhea in Indonesia or the 14 strains from patients with diarrhea in the Philippines were serotype O139. Four different ribotypes were found in V. cholerae O139 isolated in Asia. Twenty-three (47%) of 49 Thai O139 strains examined were of different ribotypes than isolates from India and Bangladesh; V. cholerae strains that were not O1 or O139 that were isolated from flies and water in Thailand 11 years previously in 1981 contained the same V. cholerae virulence gene complex found in V. cholerae O1 and O139. This suggests that other unidentified virulence determinants are involved in V. cholerae O139 pathogenesis.

Cholera↗

PubMed1994

Antimicrobial susceptibilities of Neisseria gonorrhoeae strains isolated in Surabaya, Indonesia.

Until recently, the only common strains of antimicrobial agent-resistant Neisseria gonorrhoeae detected in Indonesia were penicillinase-producing N. gonorrhoeae (PPNG) strains. Despite the spread of resistance to other antimicrobial agents among N. gonorrhoeae in Southeast Asia, surveillance for such resistance in Indonesia has been limited. We evaluated the in vitro susceptibilities of 86 N. gonorrhoeae isolates from female sex workers in Surabaya, Indonesia, to 13 antimicrobial agents. Of the 86 isolates, 89% were resistant to penicillin (MIC, > or = 2.0 micrograms/ml), 98% were resistant to tetracycline (MIC, > or = 2.0 micrograms/ml), 18.1% were resistant to spectinomycin (MIC, > or = 128.0 micrograms/ml), and 97.7% showed decreased susceptibility to thiamphenicol (MIC, 1 to 2 micrograms/ml). Thus, thiamphenicol and spectinomycin may be approaching the end of their usefulness as the drugs of choice for the treatment of gonococcal infections in Surabaya. While the susceptibilities of N. gonorrhoeae to cephalosporins (ceftriaxone, cefixime, and cefoxitin) and fluoroquinolones (ciprofloxacin and ofloxacin) are universal, these drugs have not been used because they are more expensive in Indonesia than thiamphenicol. We conclude that Surabaya had the highest reported rate of penicillin and tetracycline resistance among the Southeast Asian countries and that cephalosporins or fluoroquinolones should be reasonable alternatives for the treatment of gonorrhea in this locale.

Anti-Bacterial Agents↗

PubMed1993

Solubilization and characterization of functionally coupled Escherichia coli heat-stable toxin receptors and particulate guanylate cyclase associated with the cytoskeleton compartment of intestinal membranes.

1. Particulate guanylate cyclase and receptors for E. coli heat-stable enterotoxin were solubilized from the rat intestinal cytoskeletal compartment using Lubrol-PX and KCl. 2. Thirty to forty percent of the ST receptor and guanylate cyclase activities were extracted from the lipid layer with Lubrol-PX alone. 2. Seventy percent of the remaining activities were solubilized from the cytoskeleton with Lubrol-PX and KCl. 3. Guanylate cyclase solubilized from either compartment exhibited similar reaction kinetics. 4. Both high- and low-affinity classes of ST receptors were solubilized from the lipid and cytoskeleton compartments. 5. In the presence of ATP gamma S, ST selectively activated the guanylate cyclase solubilized from the cytoskeleton compared to that solubilized from the lipid bilayer. 6. Crosslinking experiments demonstrated a preferential solubilization of the 130 kDa receptor subunit from the cytoskeleton and the 56 kDa subunit from the lipid bilayer. 7. Development of a procedure to solubilize ST receptors and guanylate cyclase from the intestinal membrane cytoskeleton will permit purification and further detailed studies of the coupling of these activities.

Adenosine Triphosphate↗

PubMed1993

Safety, immunogenicity, and transmissibility of single-dose live oral cholera vaccine strain CVD 103-HgR in 24- to 59-month-old Indonesian children.

Recombinant A-B+ Vibrio cholerae O1 strain CVD 103-HgR is a safe, highly immunogenic, single-dose live oral vaccine in adults in industrialized countries. Safety, excretion, immunogenicity, vaccine transmissibility, and environmental introduction of CVD 103-HgR were investigated among 24- to 59-month-old children in Jakarta. In 81 households, 1 child was randomly allocated a single dose of vaccine (5 x 10(9) cfu) and another, placebo. Additionally, 139 unpaired children were randomly allocated vaccine or placebo. During 9 days of follow-up, diarrhea or vomiting did not occur more often among vaccines than controls. Vaccine was minimally excreted and was isolated from no controls and from 1 (0.6%) of 177 unvaccinated family contacts. A 4-fold or higher rise in serum vibriocidal antibody was observed in 75% of vaccines (10-fold rise in geometric mean titer over baseline). Of 135 paired placebo recipients or household contacts, 5 had vibriocidal seroconversions. Moore swabs placed in sewers and latrines near 97 households failed to detect vaccine. These observations pave the way for a large-scale field trial of efficacy.

Antibodies, Bacterial↗

PubMed1993

Genetic conservation of hlyA determinants and serological conservation of HlyA: basis for developing a broadly cross-reactive subunit Escherichia coli alpha-hemolysin vaccine.

The HlyA determinant among Escherichia coli isolates from patients with symptomatic urinary tract infection was compared in this report with a prototype HlyA encoded by pSF4000 by DNA-DNA hybridization tests with 20-base synthetic oligonucleotides and monoclonal antibody binding and neutralization assays. Hybridization results demonstrated that 349 (98%) of 357 definitive reactions among 54 hemolytic strains shared homology with seven DNA probes spanning many HlyA regions corresponding to residues (R) 41 to 47, 55 to 61, 248 to 254, 306 to 312, 336 to 343, 402 to 408, and 929 to 935. Genetic divergence was identified by lack of hybridization signals among 17 to 76% of the hemolytic strains within the distal portion of a predicted hydrophobic region corresponding to R491 to 319 and within a predicted hydrophilic region corresponding to R491 to 497 and R532 to 538. Serological studies demonstrated that 26 (81%) culture supernatants of 32 hemolytic strains were bound by all 12 monoclonal anti-HlyA antibodies. Among five of six remaining strains, the culture supernatants were bound by 3 to 11 monoclonal antibody preparations. There was only one hemolytic culture supernatant that failed to be bound by any monoclonal antibody, although the strain hybridized with nine hemolysin DNA probes. In addition, hemolytic activity of all 24 different culture supernatants tested was reduced by at least twofold by one monoclonal antibody specific for R2-161. These data extend and support previous views that the HlyA determinant is conserved among E. coli strains and suggest that a broadly cross-reactive HlyA subunit vaccine can be developed.

Amino Acid Sequence↗

PubMed1993

Pharmacokinetics of zidovudine alone and in combination with oxazepam in the HIV infected patient.

This three-phase study was designed to determine if a pharmacokinetic drug-drug interaction exists between zidovudine and oxazepam. Six individuals infected with human immunodeficiency virus (HIV) and receiving zidovudine at 500 mg daily, with normal renal and hepatic function, were enrolled. During phase I, zidovudine pharmacokinetics were studied after steady-state oral administration (100 mg every 4 h) and after a single dose (70 mg) of intravenous zidovudine. Phase II consisted of a single oral dose (30 mg) of oxazepam followed by a 48-h blood sampling period. Phase III began with 48 h of concomitant zidovudine, 100 mg orally every 4 h, and oxazepam, 15 mg orally every 8 h, followed by concomitant dosing of intravenous zidovudine and oral oxazepam. Zidovudine concentrations were determined by radioimmunoassay. Oxazepam concentrations were determined with use of a fluorescence polarization immunoassay. The calculated bioavailability was 0.61 for zidovudine alone and 0.75 when administered in combination with oxazepam (p = 0.16). Plasma half-life for oral zidovudine alone and in combination with oxazepam was 1.17 h versus 0.99 h, respectively (p = 0.25), and 1.38 h versus 1.15 h (p = 0.38) for intravenous zidovudine during single and combination therapy, respectively. Total body clearance of zidovudine was not significantly altered by oxazepam (93 L/h vs. 109 L/h, p = 0.16). The mean pharmacokinetic parameters determined for a single 30-mg dose of oxazepam for oral clearance, apparent volume of distribution, and plasma half-life were 9.8 L/h, 65.7 L, and 5.1 h, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1992

Affinity purification of functional receptors for Escherichia coli heat-stable enterotoxin from rat intestine.

Active receptors for Escherichia coli heat-stable enterotoxin (ST) were partially purified by ligand-affinity chromatography. The affinity column was prepared by coupling ST to biotin derivatized with an extended N-hydroxysuccinylated spacer arm prior to binding to monomeric avidin immobilized on agarose. Detergent extracts of rat intestinal mucosa membranes were quantitatively depleted of ST binding activity when chromatographed on this affinity matrix. Biotinylated ST-receptor complexes were eluted from affinity columns with 2 mM biotin and these complexes quantitatively dissociated with bile salts. Using this technique, functional ST receptors were purified maximally about 2000-fold, with about 3% of the total activity in crude extracts recovered in these purified preparations. Analysis of affinity-purified preparations by polyacrylamide gel electrophoresis and silver staining demonstrated a major protein subunit of 74 kDa. Affinity cross-linking of these preparations to 125I-ST demonstrated specific labeling predominantly of the 74-kDa subunit. In addition, lower amounts of labeled ST were incorporated into subunits of 164 and 45 kDa, confirming the heterogeneous nature of ST receptors. Purified receptors bound ST in a concentration-dependent fashion, with an IC50 of 10(-9) M. These studies demonstrate that ligand-affinity chromatography can be employed to purify ST receptors. The availability of purified receptors will facilitate further studies of mechanisms underlying ST-induced intestinal secretion.

Animals↗

PubMed1992

Local cytokine production in a murine model of Escherichia coli pyelonephritis.

Cytokines may play an important role in the regulation of host defense against local bacterial infections. We have evaluated the local production of cytokines in a BALB/c mouse model of Escherichia coli pyelonephritis. Kidneys, draining lymph nodes, and spleens, were harvested at specific time intervals after bladder inoculation with E. coli corresponding to the stages of renal infection, infiltration, and bacterial clearance seen in this model. The presence of messenger RNA for specific cytokines (interleukins 1 through 6, chemotactic factors, granulocyte and granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor (TNF alpha) and beta, IFN gamma, transforming growth factor (TGF beta), and cytokine synthesis inhibitory factor (CSIF)/IL-10) was determined by polymerase chain reaction (PCR) amplification of reverse transcribed RNA. We have demonstrated mRNA encoding IL-1, IL-6, G-CSF, GM-CSF, TNF alpha, H400 (a protein homologous to a family of chemotactic factors and identical to MIP-1 beta), and CSIF/IL-10 in the kidney at 12 h and 1, 2, and 3 d after bacterial challenge. No signal was seen in normal animals or in mice after 5 d. This pattern of cytokine expression was observed only in renal tissues suggesting a localized response. IL-6 was present in the urine at 4 h with rapid resolution to baseline levels by 24 to 48 h. In contrast, IL-6 was not usually detectable in the serum. TNF alpha was not detectable in the serum or urine during the course of the infection. By immunohistochemical staining of kidney sections we have shown that IL-6 is produced predominantly by mesangial cells rather than by the inflammatory infiltrate. This study provides additional evidence utilizing novel techniques that specific cytokines are produced locally in response to bacterial infections. The time course of production demonstrated in this model supports the important role of cytokines in natural host resistance to local infection.

Animals↗

PubMed1992

Extended stability of ganciclovir for outpatient parenteral therapy for cytomegalovirus retinitis.

Cytomegalovirus retinitis in the human immunodeficiency virus-infected patient currently requires almost daily and lifelong parenteral ganciclovir therapy. Self-preparation of ganciclovir is not an option in the majority of patients with decreased visual acuity or poor muscle coordination. Therefore, based on current stability data, patients usually receive a 5-day supply of reconstituted drug. This requirement is unfortunately associated with poor patient compliance. If the extended stability of ganciclovir was known, then more efficient regimens for outpatient therapy could be formulated. This study was designed to determine the stability of reconstituted ganciclovir between 7 and 28 days. Ganciclovir diluted with normal saline or 5% dextrose in water to final drug concentrations of 5 or 10 mg/ml was evaluated for stability by high-pressure liquid chromatography under different conditions of storage. Samples were stored at both 4 degrees C and -20 degrees C in polyvinyl chloride bags and at 4 degrees C in ADFuse syringes. Ganciclovir remained stable under these conditions for 28 days. On the basis of these data, we have recommended that patients receive a 2-week supply of reconstituted ganciclovir for parenteral outpatient therapy. We have observed high levels of patient compliance with this regimen and no unexpected progression of retinitis over a 5-month period in three patients who received the drug in this fashion.

Cytomegalovirus Infections↗

PubMed1991

Identification and characterization of a new family of high-affinity receptors for Escherichia coli heat-stable enterotoxin in rat intestinal membranes.

Novel high-affinity, low-capacity binding sites in intestinal membranes for the heat-stable toxin produced by Escherichia coli have been defined. The appearance of these sites is observed in the presence of physiological concentrations of NaCl in binding reactions. Scatchard analyses of equilibrium binding in the absence of NaCl demonstrated a single class of binding sites with KD = 1.9 x 10(-9) M and Bmax = 0.75 pmol/mg of protein. In contrast, similar experiments in the presence of NaCl demonstrated, in addition to the previously described low-affinity site, a high-affinity site with a KD of 2.1 x 10(-11) M and a Bmax of 73 fmol/mg of protein. Confirmation of the presence of high- and low-affinity sites was obtained in studies of the kinetics of ST binding. These sites exhibited similar dissociation but markedly different association kinetics. Determination of the association and dissociation constants permitted calculation of the KD's for the high- and low-affinity sites, which were 1.15 x 10(-11) M and 1.89 x 10(-9) M, respectively. These data agree closely with those obtained in studies of equilibrium binding. Furthermore, similar values for the KD's of these sites were obtained in experiments of competitive displacement of labeled ST, confirming the presence of two receptors for this toxin. Binding of ST to high-affinity sites is completely reversible and does not appear to be coupled to activation of particulate guanylate cyclase. In contrast, binding of ST to low-affinity sites appears to be partially reversible and may be coupled to activation of guanylate cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Efficacy of a Proteus mirabilis outer membrane protein vaccine in preventing experimental Proteus pyelonephritis in a BALB/c mouse model.

A BALB/c mouse model of nonobstructive, ascending Proteus mirabilis pyelonephritis was characterized bacteriologically, histologically, and serologically from 3 to 28 days. Intravesicular administration of 2 X 10(8) P. mirabilis K7 resulted in the septic death of 9 (16%) of 57 mice by day 15. Among the survivors, K7 colonized the kidneys in great numbers until day 21. Histological examination of the kidneys revealed acute inflammation which was characterized by neutrophil infiltration by day 3, renal necrosis by day 7, and fibroblastic infiltration by day 14 which persisted at least until day 28. The immunoglobulin G response to the outer membrane proteins (OMP) was assessed by enzyme-linked immunosorbent assay and Western blotting (immunoblotting). Anti-OMP immunoglobulin G antibodies were detected as early as day 7, and the reciprocals of their titers rose progressively up to day 28 (i.e., greater than or equal to 500). This model was also used to assess the efficacy of OMP and lipopolysaccharide (LPS) immunization in preventing renal infection. K7 OMP or LPS (100 micrograms) preparations were administered intramuscularly in Freund's complete adjuvant. After 2 weeks, mice were intravesicularly challenged with 2 X 10(8) bacteria of the homologous K7 strain or one of four heterologous strains. Compared with the saline-immunized control group and K7 LPS-immunized mice, K7 OMP recipients were protected from death when challenged by homologous or heterologous strains. In addition, K7 OMP recipients were protected (P less than 0.003) from subsequent renal infection when challenged by the K7 strain and had more rapid bacterial renal clearance when challenged by three of four heterologous strains. OMP recipients produced antibodies which bound major OMP moieties (viz., 36- to 39-kDa cell wall constituents) as assessed by Western blotting. These results support the concept that immunization with selected bacterial protein surface coat constituents can prevent uromucosal infection by interfering with colonization or renal injury.

Animals↗

PubMed1991

Alpha-hemolysin contributes to the pathogenicity of piliated digalactoside-binding Escherichia coli in the kidney: efficacy of an alpha-hemolysin vaccine in preventing renal injury in the BALB/c mouse model of pyelonephritis.

Digalactoside-binding (Gal-Gal) pili and alpha-hemolysin of Escherichia coli have been implicated as important virulence determinants in the pathogenesis of human ascending, nonobstructive pyelonephritis. The pathogenic significance of these determinants was evaluated in vitro and in the BALB/c mouse pyelonephritis model by employing wild-type, avirulent laboratory, and genetically defined cosmids, transformants, and recombinant strains. In vitro data suggest that the cytolytic activity of hemolysin is significantly (P less than 0.05) enhanced among digalactoside-binding strains which agglutinate erythrocytes. The basis of increased hemolysis is related presumably to more efficient delivery of the toxin to target lipid substrate in the host plasma membrane. Intravesicular administration of bacteria that express both digalactoside binding and hemolysin generally resulted in greater mortality and renal parenchymal injury in mice than strains that expressed none or only one of these determinants. Analyses convincingly demonstrate that digalactoside-binding pili are correlated with upper urinary tract colonization and that hemolysin is correlated with septicemia and renal parenchymal damage. These determinants collectively constitute the minimal virulence factors to produce disease in this model. Their efficacy as vaccines for the prevention of pyelonephritis was also assessed. A purified Gal-Gal pilus vaccine prevented (P less than 0.05) subsequent colonization by a challenge wild-type strain that exhibited homologous pili. The hemolysin vaccine did not abrogate subsequent bacterial renal colonization on challenge, but it did protect (P less than 0.05) mice which survived challenge from subsequent renal injury compared with those in the saline control group. The combination of these determinants was also protective. The combination of Gal-Gal pili and hemolysin in a vaccine preparation represents a potentially worthwhile strategy for human immunoprophylaxis against pyelonephritis by interdicting several steps in the pathogenesis of a bacterial mucosal infection.

Animals↗

PubMed1991

Frequency and organization of papA homologous DNA sequences among uropathogenic digalactoside-binding Escherichia coli strains.

The frequency of selected papA DNA sequences among 89 digalactoside-binding, uropathogenic Escherichia coli strains was evaluated with 12 different synthetic 15-base probes corresponding to papA genes from four digalactoside-binding piliated recombinant strains (HU849, 201B, and 200A). The papA probes encode amino acids which are common at the carboxy terminus of all strains, adjacent to the proximal portion of the intramolecular disulfide loop of strain 210B, or predicted to constitute the type-specific epitope for each of the four recombinant strains or other epitopes of strain HU849. The presence among the strains of DNA sequence homology to the papA probes was determined by in situ colony hybridization. Hybridization data suggest that there is a high frequency of homologous papA DNA sequences corresponding to selected regions of the papA gene from strain HU849 among the clinical strains. The following nucleotide locations which encode portions of the mature HU849 PapA are detected in a high percentage (42 to 70%) of clinical isolates: 208 to 222, 310 to 324, 478 to 492, 517 to 531, 553 to 567, and 679 to 693. These sequences encode portions of the predicted protective, immunogenic, and/or antigenic epitopes of this PapA. The data also indicate considerable heterogeneity of papA sequences among the strains, especially in the region of nucleotide bases corresponding to positions 391 to 418. These oligonucleotides encode the predicted PapA type-specific immunogenic dominant epitope. Determination of the extent of genetic variability in the papA gene among digalactoside-binding strains will require more extensive DNA sequencing of prototypic papA genes, additional hybridization studies employing other papA gene oligonucleotide probes, and assessment of the different pap operons and their copy number in each strain.

Amino Acid Sequence↗

PubMed1991

Efficacy and cost-effectiveness of antibiotic monitoring at a Veterans Administration hospital.

A rigorous, continuously monitored antibiotic programme was initiated at our Veterans Administration hospital following evidence of inappropriate antibiotic usage. Seven hundred and thirty-one patients, mainly male, with an average age of 60 years were involved in the study; 378 were evaluable prior to policy implementation and 273 subsequently. The overall prevalence of deaths and average length of antibiotic therapy were less during the post-policy period. The number of antibiotic doses per patient was decreased by 24%. Drug costs were reduced by 32% post-policy, due largely to increased use of the longer-acting cephalosporins ceftriaxone and cefotetan. A 23% reduction in antibiotic acquisition costs was achieved over the 3-year period of the study, without detrimental effects on patient outcome.

Anti-Bacterial Agents↗

PubMed1990

Rat intestinal cell atrial natriuretic peptide receptor coupled to guanylate cyclase.

The present studies were initiated to determine if cells of intestinal origin possess the molecular components supporting a response to atrial natriuretic peptides. Specific binding in cultured rat ileal cells with 125I-labeled atrial natriuretic peptide was saturable and of high affinity. Scatchard analyses showed a single population of binding sites with a Kd of 2.1 nmol/L and a Bmax of 300 fmol/mg protein. Atrial natriuretic peptide activated particulate guanylate cyclase 5- to 10-fold in a concentration- and time-dependent fashion. The EC50 for activation of enzyme by atrial natriuretic peptide was 6 nmol/L. Accumulation of cyclic guanosine monophosphate stimulated by atrial natriuretic peptide was observed in the intracellular (25-fold) and extracellular (50-fold) compartments and was dependent on concentration and time. Half-maximum intracellular accumulation was observed with 10 nmol/L atrial natriuretic peptide. These data suggest a role for atrial natriuretic peptides in the gastrointestinal tract.

Animals↗

PubMed1990

Heterogeneity of intestinal receptors for Escherichia coli heat-stable enterotoxin.

The structure of rat intestinal cell receptors for Escherichia coli heat-stable enterotoxin (ST) was investigated by affinity cross-linking to 125I-ST and analysis by denaturing gel electrophoresis. Cross-linking of labeled toxin to intestinal membranes and analysis by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed five specifically labeled proteins with molecular masses of 160, 136, 78, 71, and 56 (kilodaltons) kDa. Exhaustive reduction of these samples resulted in a similar pattern of labeling. Affinity-labeled proteins were further analyzed by nonreducing SDS-PAGE, reduction of the resulting separated proteins, and further separation by SDS-PAGE in the presence of beta-mercaptoethanol. Thus, the 160-kDa band on nonreducing gels consisted of two different receptors: a 160-kDa polypeptide not further reducible and one composed of at least two subunits, one of which was the 78-kDa subunit. Similarly, the 136-kDa band on nonreducing gels consisted of a 136-kDa polypeptide not further reducible and one composed of at least two subunits, one of which was the 71-kDa subunit. The 78-, 71-, and 56-kDa subunits were not further reducible. These data suggest heterogeneity of the ST receptor subunit structure and organization in rat intestinal epithelia.

Affinity Labels↗