Susceptibility to RPR 106,972, quinupristin/dalfopristin and erythromycin among recent clinical isolates of enterococci, staphylococci and streptococci from North American medical centres

BACKGROUND: Enterotoxigenic strains of Bacteroides fragilis (ETBF) have been implicated in diarrhoeal illness in livestock and children, but their role in adult human colonic disease is unknown. AIMS: To investigate responses by primary adult human colonic epithelial cells to purified B fragilis toxin (BFT). METHODS: BFT was purified from culture supernatant of a highly toxigenic strain of ETBF. Morphological changes to primary colonic epithelial cells, in response to purified BFT, were studied in organ culture of colonic biopsy specimens from 15 adults. RESULTS: BFT induced epithelial cell cytotoxicity in colonic biopsy specimens from 12/15 subjects. The BFT induced morphological changes were characterised by epithelial cell rounding, separation from adjacent cells, and detachment from the basement membrane. In severely affected specimens, almost all the epithelial cells were affected. There was heterogeneity between subjects in the rate at which BFT induced epithelial cell cytotoxicity occurred. Furthermore, in colonic biopsy specimens from three subjects, exposure to BFT did not induce any significant morphological changes to epithelial cells. CONCLUSION: BFT is capable of inducing cytotoxicity in primary adult human colonic epithelial cells. Such an effect of ETBF derived BFT on epithelial cells in the colon in vivo would be expected to lead to mucosal inflammation and diarrhoea. Heterogeneity in responses by primary colonocytes probably reflects the outcome of host-BFT interactions. Such interactions in vivo could determine the occurrence of colonic disease in some individuals but not others.     

Molecular characterization of the fragilysin pathogenicity islet of enterotoxigenic Bacteroides fragilis

Enterotoxigenic strains of Bacteroides fragilis produce an extracellular metalloprotease toxin (termed fragilysin) which is cytopathic to intestinal epithelial cells and induces fluid secretion and tissue damage in ligated intestinal loops. We report here that the fragilysin gene is contained within a small genetic element termed the fragilysin pathogenicity islet. The pathogenicity islet of B. fragilis VPI 13784 was defined as 6,033 bp in length and contained nearly perfect 12-bp direct repeats near its ends. Sequencing across the ends of the pathogenicity islet from two additional enterotoxigenic strains, along with PCR analysis of 20 additional enterotoxigenic strains, revealed that the islet is inserted at a specific site on the B. fragilis chromosome. The site of integration in three nontoxigenic strains contained a 17-bp GC-rich sequence which was not present in toxigenic strains and may represent a target sequence for chromosomal integration. In addition to the fragilysin gene, we identified an open reading frame encoding a predicted protein with a size and structural features similar to those of fragilysin. The deduced amino acid sequence was 28.5% identical and 56.3% similar to fragilysin and contained a nearly identical zinc-binding motif and methionine-turn region.     

A fraction unresponsive to growth inhibition by TGF-beta among the high-proliferative potential progenitor cells in bone marrow of p53-deficient mice

The interaction between the respiratory pathogen Bordetella bronchiseptica and epithelial cells was studied. After 2-3 h, B. bronchiseptica strains exerted a strong cytotoxic effect on HeLa cells which was evident by rounding of the cells and detachment from the substrate and which ultimately resulted in total disintegration of the host cell. Production of this cytotoxic activity appeared to be regulated by the BvgAS sensory transduction system, which coordinately regulates many B. bronchiseptica virulence factors, since bacteria cultured in the presence of sulfate anions, inhibitors of the BvgAS response, did not exhibit this effect. Furthermore, spontaneous phase variants of B. bronchiseptica strains adhered to HeLa cells but were not cytotoxic. The cytotoxic component is presumably not secreted because the bacterial culture supernatant was not cytotoxic for HeLa cells. Besides HeLa cells other human epithelial cell lines such as Chang cells, larynx HEp-2 cells and lung NCI-H292 cells were sensitive to the cytotoxic activity of B. bronchiseptica. These results suggest the presence of a novel BvgAS-regulated virulence factor in B. bronchiseptica.     

Protein-disulfide isomerase-mediated reduction of the A subunit of cholera toxin in a human intestinal cell line

A key step in the action of cholera toxin (CT) is the reduction of its A subunit to the A1 peptide. The latter is an ADP-ribosyltransferase, which activates the alpha-subunit of the stimulatory G protein of adenylyl cyclase. In this study, the enzymatic reduction of membrane-bound CT in CaCo-2 human intestinal epithelial cells was characterized. Whereas diphtheria toxin was found to be reduced by a cell surface population of protein-disulfide isomerase (PDI) and its cytotoxicity was inhibited by p-chloromercuribenzenesulfonic acid, bacitracin, or anti-PDI antibodies, these inhibitors had no effect on CT reduction or activity in intact cells. In contrast, the reduction of CT in vitro by either postnuclear supernatants (PNS) or microsomal membranes in the presence of Triton X-100 was significantly inhibited by p-chloromercuribenzenesulfonic acid and bacitracin. Anti-PDI monoclonal antibodies likewise inhibited the in vitro reduction of CT and also were effective in depleting reductase activity from PNS. Since inhibition and depletion were not observed in the absence of detergent, these results suggested that the reductase activity was a soluble component localized to the lumen of microsomal vesicles and correlated with the presence of protein-disulfide isomerase. This was further confirmed by showing a corresponding depletion of reductase activity and PDI in alkali-treated microsomes. This activity was restored when purified bovine PDI was added back to alkali-treated microsomes in a redox buffer that reflected conditions found in the lumen of the endoplasmic reticulum (ER). When the CT-related reductase activity was assayed in subcellular fractions of PNS-derived membranes isolated on a 9-30% Iodixanol gradient, the activity, as measured by CT-A1 peptide formation localized to those fractions containing PDI. Likewise CT-A1 peptide formed in intact cells co-localized to those membrane fractions containing the majority of cellular PDI. Furthermore, the banding density corresponded to a region of the gradient containing ER-derived membranes. These results indicated that CT was a substrate for PDI-catalyzed reduction in intact cells and supported the hypothesis that CT reduction and activation occurs in the ER.     

A role for PACE4 in the proteolytic activation of anthrax toxin protective antigen

Several bacterial protein toxins require activation by eukaryotic proteases. Previous studies have shown that anthrax toxin protective antigen (PA), Pseudomonas exotoxin A (PE), and diphtheria toxin (DT) are cleaved by furin C-terminal to the sequences RKKR, RQPR, and RVRR, respectively. Because furin-deficient cells retain some sensitivity to PA and DT, it is evident that other cellular proteases can activate these toxins. Whereas furin has been shown to require arginine residues at positions -1 and -4 for substrate recognition, another protease with an activity which could substitute for furin in toxin activation, the furin-related protease PACE4, requires basic residues in the -1, -2, and -4 positions of the substrate sequence. To examine the relative roles of furin and PACE4 in toxin activation, we used furin-deficient CHO cells (FD11 cells) transfected with either the furin (FD11/furin cells) or PACE4 (FD11/PACE4 cells) gene. Mutant PA proteins containing the cleavage sequence RAAR or KR were cytotoxic toward cells expressing only PACE4. In vitro cleavage data demonstrated that PACE4 can recognize RAAR and, to a much lesser extent, KR and RR. When extracts from PACE4-transfected cells were used as a source of proteases, PACE4 had minimal activity, indicating that it had been partially inactivated or did not remain associated with the cell membranes. Cleavage of iodinated PA containing the sequence RKKR or RAAR was detected on the surface of all cell types tested, but cleavage of a dibasic sequence was detected only intracellularly and only in cells that expressed furin or PACE4. The data provide evidence that PACE4 is present at the exterior of cells, that it plays a role in the proteolytic activation of anthrax toxin PA, and that PACE4 can activate substrates at the sequence RAAR or KR.     

Characterization of mechanisms involved in uptake of Streptococcus dysgalactiae by bovine mammary epithelial cells

Bovine mammary epithelial cells were pretreated with inhibitors of protein kinase activity, actin polymerization and receptor-mediated endocytosis. In addition, mammary epithelial cells and Streptococcus dysgalactiae were pretreated with inhibitors of protein synthesis. Results showed that activity of tyrosine protein kinases, intact microfilaments and de novo eukaryotic protein synthesis was required for uptake of S. dysgalactiae by bovine mammary epithelial cells; a process that appeared to occur via receptor-mediated endocytosis. In contrast, de novo bacterial protein synthesis was not required for uptake of S. dysgalactiae by MAC-T cells. This study provides insight into bacterial and cellular mechanisms involved in early host-pathogen interactions, putting into perspective the role of mammary epithelial cells in the development and establishment of intramammary infections by S. dysgalactiae.     

Alterations in classical cadherins associated with progression in ulcerative and Crohn's colitis

Human colitis is a condition associated with a spectrum of altered morphologic changes and cellular adhesion. The role of cadherins, which are powerful morphoregulatory cell adhesion molecules, in colitis is provocative and as yet unknown. Herein, we present results that suggest a strong correlation between the deregulation of two cadherin molecules, E- and P-cadherins, and the progression of human colitis. We examined the expression and structural integrity of E- and P-cadherins in inflamed, dysplastic, or neoplastic human ulcerative colitis (UC) (n=58), human Crohn's colitis (n = 30), and normal tissue (n = 20) to assess cadherin function in normal and abnormal epithelium. E-cadherin is strongly expressed in normal colorectal epithelium, whereas in left-sided UC it is either down-regulated or has a single-base pair mutation in exon 4 resulting in an amino acid alteration (6 of 58 UC cases). By contrast, P-cadherin is dramatically up-regulated in both Crohn's disease and ulcerative colitis and especially in dysplastic ulcerative tissue. In vitro transfected SW-480 colorectal cells containing E-cadherin mutations identical to those in vivo were associated with increased spontaneous disaggregation compared with cells transfected with wild-type E-cadherin. Based on this evidence, we hypothesize that a small subset of colorectal cells expressing mutant E-cadherin are associated with widespread ulceration, whereas those expressing P-cadherin are associated with a rapidly dividing immature phenotype that includes dysplasia. The differential expression of mutated and wild-type cadherins examined herein are associated with a broad spectrum of abnormal epithelial phenotypes, lymphocyte integrin binding, and resistance to denudation, as is seen in the colitis adenocarcinoma sequence.     

Nasal delivery of peptides: an in vitro cell culture model for the investigation of transport and metabolism in human nasal epithelium

We investigated the transport- and metabolism properties of three peptides in monolayers of human nasal epithelial cells. The effective permeability coefficients of thyrotropin-releasing hormone, met-enkephalin and human recombinant insulin were found to be 4.5, 4.4 and 0.4 x 10(-7) cm/s, respectively. The permeability was inversely proportional to the molecular weight and one order of magnitude lower than in excised nasal mucosa of rabbits. The metabolic cleavage of thyrotropin-releasing hormone (TRH) to the free acid by cytosolic prolyl-endopeptidase was also detected in human nasal cell monolayers, suggesting that ca. 10% of the total amount of TRH is transported via a transcellular pathway. Met-enkephalin is a substrate for aminopeptidases, located on the apical membrane of nasal epithelial cells. Metabolites and enzyme activity are comparable with literature data. Our studies demonstrate that not only morphological, but also functional properties of human nasal epithelial cells are preserved under in vitro conditions. Such a cell culture model based on human nasal cells could be beneficial for the characterization of peptide transport on a cellular level and for investigation of the absorption enhancer mechanism. Further studies are necessary, however, to establish correlations between in vitro permeabilities in cell cultures and nasal drug absorption in animals and humans.     

Interaction of Escherichia coli heat-stable enterotoxin B with cultured human intestinal epithelial cells

Binding of Escherichia coli heat-stable enterotoxin B (STb) to the human intestinal epithelial cell lines T84 and HT29 and to polarized T84 cells was studied to define the initial interaction of this peptide toxin with target cells. Equilibrium and competitive binding isotherms showed that 125I-STb bound specifically to T84 and HT29 cells; however, the toxin-epithelial cell interactions could be characterized by low-affinity binding (< or = 10(5) M(-1)) to a high number of binding sites (> or = 10(6) per cell). STb binding to T84 and HT29 cells as a function of 125I-STb concentration did not approach saturation at levels well above the effective biological concentration of STb for fluid secretion. Treatment of the 125I-STb-bound T84 and HT29 cells with an acidic saline solution to remove surface-bound toxin revealed that only approximately 55% +/- 10% of 125I-STb could be removed by this treatment at 4 degrees C, suggesting that approximately half of the bound STb was stably associated with the plasma membrane and/or internalized into the cytoplasm. Similar results were obtained when binding and internalization experiments were conducted at 22 and 37 degrees C. Immunofluorescence studies demonstrated that the strongest signal for STb appeared in the plasma membrane even after acid treatment. Toxin-treated cells also displayed diffuse cytoplasmic staining, indicating that once cell bound, STb did not appear to preferentially associate with membrane vesicles or cellular organelles. Binding and subsequent internalization of 125I-STb were not affected by treatment of the cells with trypsin, endoglycosidase F/peptide N-glycosidase F, Vibrio cholerae neuraminidase, tunicamycin, or 5 mM sodium chlorate, which blocks sulfation of surface proteoglycans. In addition, the internalization process was not altered by preincubation of the cells with the cytoskeleton inhibitors cytochalasin D and colchicine or cellular perturbants (i.e., 0.45 M sucrose and 5 mM sodium azide), indicating that cell surface proteins or carbohydrates did not function as STb receptors. The binding of 125I-STb to polarized T84 cells was also examined, and the total and nonspecific binding isotherms were found to overlap, indicating that the apical surface of polarized T84 cells did not contain a specific receptor for STb. In comparison to undifferentiated cells, twice the amount of bound STb (approximately 80% +/- 10%) was removable from polarized T84 cells after treatment with acidic solution. The percentage of surface-bound STb to polarized T84 cells did not vary significantly with the transepithelial electrical resistance of the cells or when STb was applied basolaterally. Together, our results indicate that STb binds with relatively low affinity to the plasma membrane of cultured intestinal epithelial cells and polarized T84 cells, probably to membrane lipids, and becomes stably associated with the lipid bilayer. The fact that a significant portion of the bound STb becomes free in the cytoplasm, even at a low temperature, suggests that the bound toxin may directly traverse the membrane bilayer.     

Staphylococcal enterotoxin B primes cytokine secretion and lytic activity in response to native bacterial antigens

Superantigens stimulate T-lymphocyte proliferation and cytokine production, but the effects of superantigen exposure on cell function within a complex, highly regulated immune response remain to be determined. In this study, we demonstrate that superantigen exposure significantly alters the murine host response to bacterial antigens in an in vitro coculture system. Two days after exposure to the superantigen staphylococcal enterotoxin B, splenocytes cultured with Streptococcus mutans produced significantly greater amounts of gamma interferon (IFN-gamma) and interleukin-12 than did sham-injected controls. The majority of IFN-gamma production appeared to be CD8(+) T-cell derived since depletion of this cell type dramatically reduced the levels of IFN-gamma. To study host cell damage that may occur following superantigen exposure, we analyzed cytotoxicity to "bystander" fibroblast cells cultured with splenocytes in the presence of bacterial antigens. Prior host exposure to staphylococcal enterotoxin B significantly enhanced fibroblast cytotoxicity in the presence of bacteria. Neutralization of IFN-gamma decreased the amount of cytotoxicity observed. However, a greater reduction was evident when splenocyte-bacterium cocultures were separated from the bystander cell monolayer via a permeable membrane support. Increased cytotoxicity appears to be primarily dependent upon cell-cell contact. Collectively, these data indicate that overproduction of inflammatory cytokines may alter the activity of cytotoxic immune cells. Superantigen exposure exacerbates cytokine production and lytic cell activity when immune cells encounter bacteria in vitro and comparable activities could possibly occur in vivo.     

Effect of Clostridium difficile toxin A on human colonic lamina propria cells: early loss of macrophages followed by T-cell apoptosis

We have previously shown that Clostridium difficile toxin A induces detachment of human colonic epithelial cells from the basement membrane and subsequent cell death by apoptosis. Because these cells require adhesion-dependent signalling from the extracellular matrix for survival, their detachment from the basement membrane by other means also induces apoptosis. The role of toxin A in the induction of apoptosis therefore remains to be determined. In addition, sensitivities to C. difficile toxin A of lamina propria lymphocytes, macrophages, and eosinophils, which lie below the surface epithelium, are not known. In contrast to epithelial cells, these lamina propria cells do not require adhesion-dependent signalling from the extracellular matrix for survival, and this may allow the mechanisms of toxin A-induced cell death to be further investigated. The aim of this study was to investigate the effect of purified C. difficile toxin A on human colonic lamina propria T cells, macrophages, and eosinophils. We show that C. difficile toxin A induces loss of viability in isolated colonic lamina propria cell preparations containing the three different cell types in a dose- and time-dependent fashion. Exposure to high concentrations of the toxin led to loss of macrophages within 72 h. T-lymphocyte and eosinophil cell death was prominent at later time points and occurred by apoptosis. Exposure to toxin A also induced the production of tumor necrosis factor alpha by the isolated colonic lamina propria cells. However, the presence of neutralizing antibodies to this cytokine did not influence C. difficile toxin A-induced T-cell apoptosis. Moreover, purified T cells also underwent apoptosis following exposure to toxin A, implying that apoptosis occurred as a consequence of a direct interaction between T cells and the toxin. Our studies suggest that C. difficile toxin A is capable of suppressing human colonic mucosal immune responses by inducing early loss of macrophages followed by T-cell apoptosis.     

Implications of bacterial protein toxins in infectious and food-borne diseases

Among the 315 protein toxins elicited by gram positive and gram negative bacteria so far characterized, about 50 toxins are currently considered as totally or partially, responsible of the pathological manifestations and/or lethality resulting from host infection or intoxication (contaminated food) by relevant toxinogenic bacteria. A certain number of criteria are required for the assessment of indisputable involvement of a toxin or an array of toxins (from the same bacteria) in infectious diseases: 1) The bacterial microorganism clearly identified as the pathogenic agent of the disease produces component(s) considered as toxin(s); 2) The administration to appropriate animal(s) of the toxin(s) separated from the relevant bacteria or produced by genetic engineering from a heterologous tox+ recombinant bacterial strain produces symptoms and pathophysiological disorders that mimic those observed in the natural disease or at least those elicited in experimental animals by the cognate toxin-producing bacteria; 3) The in vitro incubation of the isolated toxin(s) with appropriate animal organs, tissues or cells elicits certain pathophysiological, biochemical or metabolic manifestions observed in the host infected with the relevant toxinogenic bacteria; 4) Toxin concentration in the organism of the host infected by the toxinogenic bacteria should be compatible with the characteristics of the relevant disease. The toxins of pathogenic interest exhibit a variety of effects in bacterial diseases. Bacteria that colonize a wound or mucosal surface but do not invade target cells can produce toxins that act locally or enter the bloodstream and attack internal organs (e.g. Corynebacterium diphtheriae, Vibrio cholerae, ...). Bacteria growing in a wound can produce toxins that destroy host tissue and kill phagocytes in the immediate vicinity of the bacteria, thus facilitating bacterial growth and spread. On the basis of the above mentioned criteria, the following bacterial diseases among many others are toxin-associated (toxinoses): diphtheria, tetanus, botulism, whooping cough, diarrhea, bloody diarrhea, hemolytic uremic syndrome, cholera, scarlet fever, toxic shock syndrome, gas gangrene, B. fragilis diarrhea, anthrax, pseudomembranous colitis.     

Increased cell-surface urokinase in advanced ovarian cancer

Urokinase-type plasminogen activator (uPA), uPA receptors, and cathepsin B were quantitated by using an immunological method, enzyme-linked immunosorbent assay, and amidolytic activity assays in 15 malignant and 10 benign epithelial ovarian tumors. The levels of uPA and uPA receptors, as well as cathepsin B, were found to be higher in membrane preparations obtained from malignant tumors than in those obtained from benign tumors. Acid-treated membranes acquired the ability to bind uPA, indicating that uPA is bound to a specific surface receptor that is not completely saturated. Levels of single-chain uPA (pro-uPA) and high-molecular-weight uPA in membrane preparations were measured by immunoadsorbent-amidolytic assay. The finding of a significant increase in amidolytic activity following activation of uPAs by plasmin suggested that less than half (30-40%) of all membrane immunoreactive uPAs is present in the enzymatically inactive pro-uPA form. In the membranes of malignant tumors, levels of uPA receptor and cathepsin B did not vary with stage of disease. On the other hand, we found that the level of receptor-bound uPA antigen/activity was significantly increased in advanced malignant tumors. Receptor-bound uPA may play an important role in determining invasive potential of tumor cells. Since ovarian cancer cells produce both pro-uPA and cathepsin B, the possibility of activation of tumor cell-derived pro-uPA by cellular protease cathepsin B must be considered.     

Rho-dependent cell spreading activated by E.coli cytotoxic necrotizing factor 1 hinders apoptosis in epithelial cells

Cell-cell and cell-matrix interactions play a pivotal role in numerous cell functions including cell survival and death. In this work, we report evidence that the Rho-dependent cell spreading activated by a protein toxin from E. coli, the cytotoxic necrotizing factor 1 (CNF1), is capable of hindering apoptosis in HEp-2 cells. In addition to the promotion of cell spreading, CNF1 protects cells from the experimentally-induced rounding up and detachment and improves the ability of cells to adhere to each other and to the extracellular matrix by modulating the expression of proteins related to cell adhesion. In particular, the expression of integrins such as alpha 5, alpha 6 and alpha v, as well as of some heterotypic and homotypic adhesion-related proteins such as the Focal Adhesion Kinase, E-cadherin, alpha and beta catenins were significantly increased in cells exposed to CNF1. Our results suggest, however, that the promotion of Rho-dependent cell spreading is the key mechanism in protecting cells against apoptosis rather than cell adhesion per se. A toxin inducing cell spreading without activating Rho, such as Cytochalasin B, was in fact ineffective in favouring cell survival. These data are of relevance (i) for the understanding of the role of the actin-dependent and especially Rho-dependent cellular activities involved in apoptosis regulation and (ii) in providing some clues to understanding the mechanisms by which bacteria, by controlling cell fate, might exert their pathogenic activity.     

A bacterial protease perturbs the paracellular barrier function of transporting epithelial monolayers in culture

Tight junctions between cells and adhesion to the substratum maintain the barrier function of epithelia throughout the body. Damage to the epithelial barrier by microbial products allows penetration of bacteria and promotion of infection. We studied the effects of Pseudomonas elastase (PE) on the barrier function of epithelia by using Madin-Darby canine kidney (MDCK) epithelial cells; these cells form tight junctions (zonula occludens [ZO]) in vitro. PE decreased electrical resistance across the monolayers in a concentration- and time-dependent manner. Immunostaining of selected proteins of the ZO and zonula adherens was used to explore the effects of PE on junctional proteins. PE-treated monolayers of MDCK cells had markedly decreased immunostaining of ZO-1, a protein of the ZO, but light microscopy of PE-treated cells revealed no obvious morphologic changes. A chromium release assay indicated that, even with marked changes in transmonolayer electrical resistance, the permeability defect was not due to membrane disruption. Fluorescence staining of F-actin indicated diminution of cellular microfilaments in PE-treated cells, but E cadherin (uvomorulin), a protein of the zonula adherens, was unaffected by the enzyme. Elastases from porcine pancreas and human leukocytes with similar enzymatic activity (6 U/ml) did not decrease transmonolayer electrical resistance or degrade ZO-1. These results suggest that PE disturbs the barrier function of epithelial monolayers, in part, by changing the cell architecture and altering at least one protein of the ZO.     

Presence of a fatty acid-binding protein in the armadillo Harderian gland

Clinical measurements on gingival indices and morphologic observations were performed in this study to verify the defending mechanism of gingival soft tissue against foreign invasions from the perspective of epithelial adhesion/attachment to implant surfaces in the monkey mandible. The following zones were observed using scanning electron microscopy: (1) plaque zone, suggesting susceptibility of the gingival tissue to bacterial invasion; (2) nude zone, demonstrating indirect adhesion of epithelial cells to the implant surface through the mucous layer and preventing bacterial invasion; and (3) epithelial cell attached zone, having greater bond strength of epithelial cells at the cell-implant interface as compared to cell-cell bonding within the epithelial cell layer. This study suggested that epithelial cell attachment/adhesion may play a dominant role in retaining the successful condition of a dental implant.     

Effects of cholera toxin on cellular and paracellular sodium fluxes in rabbit ileum

The diarrhea observed in patients with cholera is known to be related to secretion of water and electrolytes into the intestinal lumen. However, the exact mechanisms involved in these secretory processes have remained unclear. Although it is clear that purified toxin acts on epithelial cell metabolism, its activity on Na+ transport across intestinal mucosa is equivocal: reported either to prevent net Na+ absorption or to cause net secretion of Na+ from serosa to mucosa. Since total transmural Na+ fluxes across "leaky" epithelia involve very significant movement via a paracellular shunt pathway, we studied the effects of cholera toxin on the cellular and paracellular pathways of Na+ movement. Unidirectional Na+ fluxes were examined as functions of applied potential in control tissues and in tissues from the same animal treated with purified cholera toxin. Treatment of rabbit ileum in vitro with toxin simulated the cellular component of serosa-to-mucosa Na+ flux (from 2.41 +/- 0.49 muequiv./h per cm2 under control conditions to 4.71 +/- 0.43 muequiv./h per cm2 after treatment with toxin, P less than 0.01). The effect of cholera toxin on Na+ movement through the cells from mucosa to serosa appeared to be insignificant. Finally, a marked decrease in the Na+ permeability (P less than 0.01) and no detectable significant changes in transference number for Na+ of the paracellular shunt pathway were observed following treatment with cholera toxin. These results provide direct evidence for the hypothesis that purified cholera toxin stimulates active sodium secretion but has minimal effect on sodium absorption.     

Throat disorders

In nonpolarized epithelial cells, microtubules originate from a broad perinuclear region coincident with the distribution of the Golgi complex and extend outward to the cell periphery (perinuclear [PN] organization). During development of epithelial cell polarity, microtubules reorganize to form long cortical filaments parallel to the lateral membrane, a meshwork of randomly oriented short filaments beneath the apical membrane, and short filaments at the base of the cell; the Golgi becomes localized above the nucleus in the subapical membrane cytoplasm (apiconuclear [AN] organization). The AN-type organization of microtubules is thought to be specialized in polarized epithelial cells to facilitate vesicle trafficking between the trans-Golgi Network (TGN) and the plasma membrane. We describe two clones of MDCK cells, which have different microtubule distributions: clone II/G cells, which gradually reorganize a PN-type distribution of microtubules and the Golgi complex to an AN-type during development of polarity, and clone II/J cells which maintain a PN-type organization. Both cell clones, however, exhibit identical steady-state polarity of apical and basolateral proteins. During development of cell surface polarity, both clones rapidly establish direct targeting pathways for newly synthesized gp80 and gp135/170, and E-cadherin between the TGN and apical and basolateral membrane, respectively; this occurs before development of the AN-type microtubule/Golgi organization in clone II/G cells. Exposure of both clone II/G and II/J cells to low temperature and nocodazole disrupts >99% of microtubules, resulting in: 1) 25-50% decrease in delivery of newly synthesized gp135/170 and E-cadherin to the apical and basolateral membrane, respectively, in both clone II/G and II/J cells, but with little or no missorting to the opposite membrane domain during all stages of polarity development; 2) approximately 40% decrease in delivery of newly synthesized gp80 to the apical membrane with significant missorting to the basolateral membrane in newly established cultures of clone II/G and II/J cells; and 3) variable and nonspecific delivery of newly synthesized gp80 to both membrane domains in fully polarized cultures. These results define several classes of proteins that differ in their dependence on intact microtubules for efficient and specific targeting between the Golgi and plasma membrane domains.