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The intestinal epithelium acts as the first line of defence against pathogens present in the lumen of the gastrointestinal tract. The epithelium is composed of a single monolayer that includes a variety of cell types, each of which play roles in nutrient and water absorption, antimicrobial defence, and immunomodulation to maintain a homeostatic gut environment. Tight junction (TJ) complexes between adjacent intestinal epithelial cells are responsible for the structural integrity of the gut barrier and controlling the paracellular translocation of luminal contents. The effectiveness of TJs can be impacted by both genetic and environmental factors including microbiota dysbiosis and dietary components. The increased systemic entry of luminal contents has been associated with the development, progression, and/or relapse of autoimmune diseases such as Crohn’s and rheumatoid arthritis. In particular, the extraintestinal spread of luminal microbes possessing components with structural similarity to those of the human host are thought to be involved in the breakdown of immune tolerance towards host components. Here, the structure and function of the intestinal epithelium are discussed as well as the genetic and environmental factors that influence its permeability. There is emphasis on the role of increased intestinal permeability and how the subsequent translocation of luminal contents could be involved in the development and/or exacerbation of autoimmune diseases. This review reinforces how protecting the integrity of the intestinal epithelium and minimising immunological exposure to luminal components, either directly or indirectly, could be a useful strategy in reducing the prevalence and severity of autoimmune diseases.

A total mixture of 29 persistent organic pollutants (POPs) modelled from Scandinavian blood concentrations was used to expose human A-498 kidney cells for 24 h over a concentration range spanning below to above blood level (1/10x, 1x, 50x, 100x, 500x). Its constituent submixtures (PFAA, Br, Cl) and co-mixtures (PFAA + Br, PFAA + Cl, Br + Cl) were also tested. Valinomycin (12 µM) was used as a cytotoxic comparative compound. Cell number (CN), nuclear area (NA), nuclear intensity (NI), mitochondrial membrane potential (MMP), and mitochondrial mass (MM) were assessed using high content analysis (HCA). Only the co-mixtures (PFAA + Cl, PFAA + Br) at 50x and 50x, 500x decreased CN, respectively. NI was increased by the total mixture at 500x and Cl mixture at all concentrations tested. MMP was increased by the total mixture at 100x and 500x, PFAA at 1x, Br + Cl and PFAA + Cl at 100x and 500x, respectively. MM was decreased by the total mixture at 500x. In contrast, valinomycin decreased CN and surviving cells showed a decrease in MMP and an increase in MM. In conclusion, POP exposure altered mitochondrial metabolism and induced cell death via an alternative mechanism to valinomycin. Only specific combinations of individual chemical classes, but not the total mixture, affected cell number.

Background: Certain types of hair products are more commonly used by Black women. Studies show hair products contain several endocrine-disrupting chemicals that are associated with adverse health outcomes. As chemical mixtures of endocrine disruptors, hair products may be hormonally active, but this remains unclear. Objective: To assess the hormonal activity of commonly used Black hair products. Methods: We identified six commonly used hair products (used by >10% of the population) from the Greater New York Hair Products Study. We used reporter gene assays (RGAs) incorporating natural steroid receptors to evaluate estrogenic, androgenic, progestogenic, and glucocorticoid hormonal bioactivity employing an extraction method using bond elution prior to RGA assessment at dilutions from 50 to 500. Results: All products displayed hormonal activity, varying in the amount and effect. Three samples showed estrogen agonist properties at levels from 12.5 to 20 ng/g estradiol equivalent concentrations All but one sample showed androgen antagonist properties at levels from 20 to 25 ng/g androgen equivalent concentrations. Four samples showed antagonistic and agonistic properties to progesterone and glucocorticoid. Significance: Hair products commonly used by Black women showed hormonal activity. Given their frequent use, exposure to hormonally active products could have implications for health outcomes and contribute to reproductive and metabolic health disparities.

ABSTRACTSOCS-6 is a member of the suppressor of cytokine signaling (SOCS) family of proteins (SOCS-1 to SOCS-7 and CIS) which each contain a central SH2 domain and a carboxyl-terminal SOCS box. SOCS-1, SOCS-2, SOCS-3, and CIS act to negatively regulate cytokine-induced signaling pathways; however, the actions of SOCS-4, SOCS-5, SOCS-6, and SOCS-7 remain less clear. Here we have used both biochemical and genetic approaches to examine the action of SOCS-6. We found that SOCS-6 and SOCS-7 are expressed ubiquitously in murine tissues. Like other SOCS family members, SOCS-6 binds to elongins B and C through its SOCS box, suggesting that it might act as an E3 ubiquitin ligase that targets proteins bound to its SH2 domain for ubiquitination and proteasomal degradation. We investigated the binding specificity of the SOCS-6 and SOCS-7 SH2 domains and found that they preferentially bound to phosphopeptides containing a valine in the phosphotyrosine (pY) +1 position and a hydrophobic residue in the pY +2 and pY +3 positions. In addition, these SH2 domains interacted with a protein complex consisting of insulin receptor substrate 4 (IRS-4), IRS-2, and the p85 regulatory subunit of phosphatidylinositol 3-kinase. To investigate the physiological role of SOCS-6, we generated mice lacking the SOCS-6 gene. SOCS-6−/−mice were born in a normal Mendelian ratio, were fertile, developed normally, and did not exhibit defects in hematopoiesis or glucose homeostasis. However, both male and female SOCS-6−/−mice weighed approximately 10% less than wild-type littermates.

SOCS-6 is a member of the suppressor of cytokine signaling (SOCS) family of proteins (SOCS-1 to SOCS-7 and CIS) which each contain a central SH2 domain and a carboxyl-terminal SOCS box. SOCS-1, SOCS-2, SOCS-3, and CIS act to negatively regulate cytokine-induced signaling pathways; however, the actions of SOCS-4, SOCS-5, SOCS-6, and SOCS-7 remain less clear. Here we have used both biochemical and genetic approaches to examine the action of SOCS-6. We found that SOCS-6 and SOCS-7 are expressed ubiquitously in murine tissues. Like other SOCS family members, SOCS-6 binds to elongins B and C through its SOCS box, suggesting that it might act as an E3 ubiquitin ligase that targets proteins bound to its SH2 domain for ubiquitination and proteasomal degradation. We investigated the binding specificity of the SOCS-6 and SOCS-7 SH2 domains and found that they preferentially bound to phosphopeptides containing a valine in the phosphotyrosine (pY) +1 position and a hydrophobic residue in the pY +2 and pY +3 positions. In addition, these SH2 domains interacted with a protein complex consisting of insulin receptor substrate 4 (IRS-4), IRS-2, and the p85 regulatory subunit of phosphatidylinositol 3-kinase. To investigate the physiological role of SOCS-6, we generated mice lacking the SOCS-6 gene. SOCS-6−/−mice were born in a normal Mendelian ratio, were fertile, developed normally, and did not exhibit defects in hematopoiesis or glucose homeostasis. However, both male and female SOCS-6−/−mice weighed approximately 10% less than wild-type littermates.

Inhibitor of apoptosis (IAP) proteins inhibit caspases, a function counteracted by IAP antagonists, insect Grim, HID, and Reaper and mammalian DIABLO/Smac. We now demonstrate that HtrA2, a mammalian homologue of the Escherichia coliheat shock-inducible protein HtrA, can bind to MIHA/XIAP, MIHB, and baculoviral OpIAP but not survivin. Although produced as a 50-kDa protein, HtrA2 is processed to yield an active serine protease with an N terminus similar to that of Grim, Reaper, HID, and DIABLO/Smac that mediates its interaction with XIAP. HtrA2 is largely membrane-associated in healthy cells, with a significant proportion observed within the mitochondria, but in response to UV irradiation, HtrA2 shifts into the cytosol, where it can interact with IAPs. HtrA2 can, like DIABLO/Smac, prevent XIAP inhibition of active caspase 3in vitroand is able to counteract XIAP protection of mammalian NT2 cells against UV-induced cell death. The proapoptotic activity of HtrA2 in vivoinvolves both IAP binding and serine protease activity. Mutations of either the N-terminal alanine of mature HtrA2 essential for IAP interaction or the catalytic serine residue reduces the ability of HtrA2 to promote cell death, whereas a complete loss in proapoptotic activity is observed when both sites are mutated.

Central infusion of angiotensin IV or its more stable analogues facilitates memory retention and retrieval in normal animals and reverses amnesia induced by scopolamine or by bilateral perforant pathway lesions. These peptides bind with high affinity and specificity to a novel binding site designated the angiotensin AT4receptor. Until now, the AT4receptor has eluded molecular characterization. Here we identify the AT4receptor, by protein purification and peptide sequencing, to be insulin-regulated aminopeptidase (IRAP). HEK 293T cells transfected with IRAP exhibit typical AT4receptor binding characteristics; the AT4receptor ligands, angiotensin IV and LVV-hemorphin 7, compete for the binding of [125I]Nle1-angiotensin IV with IC50values of 32 and 140 nm,respectively. The distribution of IRAP and its mRNA in the brain, determined by immunohistochemistry and hybridization histochemistry, parallels that of the AT4receptor determined by radioligand binding. We also show that AT4receptor ligands dose-dependently inhibit the catalytic activity of IRAP. We have therefore demonstrated that the AT4receptor is IRAP and propose that AT4receptor ligands may exert their effects by inhibiting the catalytic activity of IRAP thereby extending the half-life of its neuropeptide substrates.

gp130 is the common signal transducing receptor subunit for the interleukin-6-type family of cytokines. Its extracellular region (sgp130) is predicted to consist of five fibronectin type III-like domains and an NH2-terminal Ig-like domain. Domains 2 and 3 constitute the cytokine-binding region defined by a set of four conserved cysteines and a WSXWS motif, respectively. Here we determine the disulfide structure of human sgp130 by peptide mapping, in the absence and presence of reducing agent, in combination with Edman degradation and mass spectrometry. Of the 13 cysteines present, 10 form disulfide bonds, two are present as free cysteines (Cys279and Cys469), and one (Cys397) is modified byS-cysteinylation. Of the 11 potentialN-glycosylation sites, Asn21, Asn61, Asn109, Asn135, Asn205, Asn357, Asn361, Asn531, and Asn542are glycosylated but not Asn224and Asn368. The disulfide bonds, Cys112–Cys122and Cys150–Cys160, are consistent with known cytokine-binding region motifs. Unlike granulocyte colony-stimulating factor receptor, the connectivities of the four cysteines in the NH2-terminal domain of gp130 (Cys6–Cys32and Cys26–Cys81) are consistent with known superfamily of Ig-like domains. An eight-residue loop in domain 5 is tethered by Cys436–Cys444. We have created a model predicting that this loop maintains Cys469in a reduced form, available for ligand-induced intramolecular disulfide bond formation. Furthermore, we postulate that domain 5 may play a role in the disulfide-linked homodimerization and activation process of gp130.

Syntaxin 7 is a mammalian target solubleN-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) involved in membrane transport between late endosomes and lysosomes. The aim of the present study was to use immunoaffinity techniques to identify proteins that interact with Syntaxin 7. We reasoned that this would be facilitated by the use of cells producing high levels of Syntaxin 7. Screening of a large number of tissues and cell lines revealed that Syntaxin 7 is expressed at very high levels in B16 melanoma cells. Moreover, the expression of Syntaxin 7 increased in these cells as they underwent melanogenesis. From a large scale Syntaxin 7 immunoprecipitation, we have identified six polypeptides using a combination of electrospray mass spectrometry and immunoblotting. These polypeptides corresponded to Syntaxin 7, Syntaxin 6, mouse Vps10p tail interactor 1b (mVti1b), α-synaptosome-associated protein (SNAP), vesicle-associated membrane protein (VAMP)8, VAMP7, and the protein phosphatase 1M regulatory subunit. We also observed partial colocalization between Syntaxin 6 and Syntaxin 7, between Syntaxin 6 and mVti1b, but not between Syntaxin 6 and the early endosomal t-SNARE Syntaxin 13. Based on these and data reported previously, we propose that Syntaxin 7/mVti1b/Syntaxin 6 may form discrete SNARE complexes with either VAMP7 or VAMP8 to regulate fusion events within the late endosomal pathway and that these events may play a critical role in melanogenesis.

Epidermal growth factor (EGF) receptor ligands such as EGF and transforming growth factor-α (TGF-α) play an important role in controlling the proliferation, survival, morphology, and motility of colonic epithelial cells. There is also increasing evidence that growth factors and extracellular matrix (ECM) proteins cooperate to regulate these cellular processes. We have reported previously that autocrine TGF-α and an unidentified ECM protein in the serum-free conditioned medium of the human colon carcinoma cell line LIM1215 synergize to induce spreading of these cells in low-density cultures. We have now purified the ECM protein secreted by LIM1215 cells and show that it synergizes with EGF to induce spreading of LIM1215 cells and other human cell lines from the colon and other tissues. The purified ECM migrated as a single protein band with an apparent molecular mass of approximately 800 kDa on SDS–PAGE under nonreducing conditions and, under reducing conditions, as three protein bands of approximately 360, 210, and 200 kDa. Immunoblotting experiments and mass spectrometry analysis of tryptic digests on the purified protein identified the 360-, 210-, and 200-kDa protein bands as laminin α5, β1, and γ1chains, respectively, indicating that LIM1215 cells secrete laminin-10 (α5β1γ1). In serum-free medium, LIM1215 cells adhere to laminin-10 primarily via α2β1and α3β1integrin receptors. EGF-induced spreading of LIM1215 cells on laminin-10 is partially inhibited by pretreatment of the cells with blocking antibodies directed against integrin α3or β1but not α2, α6, or β4subunits. Spreading is almost completely inhibited by blocking α3+ α2, α3+ α6, or β1+ β4integrin chains and results in cell death. Increased spreading in the presence of EGF correlates with up-regulation of α6β4integrins in these cells after exposure to EGF. These results indicate that colon cancer cells attach and spread on laminin-10 via multiple integrin receptors and suggest a critical role for α3β1integrins in the spreading response. Together, our results support the concept that the adhesive properties of colon cancer cells are modulated by autocrine production of TGF-α and laminin-10 and autocrine induction of appropriate integrins.

Insulin regulates glucose metabolism in adipocytes via a phosphatidylinositide 3-kinase (PI3K)-dependent pathway that appears to involve protein phosphorylation. However, the generation of phosphoinositides is not sufficient for insulin action, and it has been suggested that insulin regulation of glucose metabolism may involve both PI3K-dependent and -independent pathways, the latter being insulin specific. To test this hypothesis, we have designed a phosphoprotein screen to study insulin-specific phosphoproteins that may be either downstream or in parallel to PI3K. Nineteen insulin-regulated phosphospots were detected in the cytosol and high speed pellet fractions, only six of which were significantly regulated by platelet-derived growth factor. Importantly, almost all (92%) of the insulin-specific phosphoproteins identified using this approach were sensitive to the PI3K inhibitor wortmannin. Thus, we obtained no evidence for an insulin-specific, PI3K-independent signaling pathway. A large proportion (62%) of the insulin-specific phosphoproteins were enriched in the same high speed pellet fraction to which PI3K was recruited in response to insulin. Thus, our data suggest that insulin specifically stimulates the phosphorylation of a novel subset of downstream targets and this may in part be because of the unique localization of PI3K in response to insulin in adipocytes.

The MDR-3-encoded P-glycoprotein (Pgp) is highly expressed in liver and is thought to function as a hepatic transporter of phospholipids into bile. However its role, if any, in other tissues remains undefined. Although transfection experiments have indicated that it may be unable to confer drug resistance, there is evidence that it may be involved in drug resistance in certain B-cell leukaemias. To date, most work on clinical samples has been performed at the mRNA level; limited work has been performed using polyclonal antibodies raised to MDR-3 and mdr-2 (the murine equivalent of MDR-3). We have generated a new monoclonal antibody, termed 6/1G, which specifically recognises the human MDR-3 gene-encoded product. Antibody 6/1G was produced by in vitro immunisation of spleen cells from BALB/c mice with a synthetic 12-amino acid peptide. Cells from MDR-3 transgenic mice showed consistent membranous staining with antibody 6/1G. Immunoblotting with 6/1G identifed a band at 170 kDa on lysates of MDR-3 transgenic cells. Preliminary results with a range of B-cell leukaemias suggest that MDR-3 Pgp positivity may be a marker for a more malignant phenotype in B-CLL. Antibody 6/1G may be useful in defining a role for MDR-3 in malignancy and drug resistance, as well as in certain liver diseases such as progressive familial intracholeostasis. Int. J. Cancer 80:265–271, 1999. © 1999 Wiley-Liss, Inc.

AbstractThis investigation examined prevailing assumptions regarding differences between school psychologists in different settings. The professional preparation, experience, employment conditions, job satisfaction, roles, continuing education needs, and perceptions of key issues of school psychologists in rural, urban, suburban, and combination settings were compared using data from a large, nationally representative sample of practitioners (N = 502). There were relatively few differences, and those differences that did emerge were not consistently in favor of the urban/suburban setting. There was limited support for the existence of a more generalist role for rural school psychologists and somewhat different continuing education needs among rural and urban groups. Other differences were inconsistent and frequently not in the direction predicted in the prior literature. Implications for research on rural variables and possible variations in graduate programs and continuing education were discussed.

The production of an antibody to detect toltrazuril or its metabolite ponazuril is complicated due to structural constraints of conjugating these coccidiostats to a carrier protein. Therefore a search was carried out for a compound that shared a common substructure to use as an antigen mimic. The chosen compound, trifluoraminoether, was conjugated to two carrier proteins (HSA and BTG) and used in the immunisation of six rabbits. Two immunogen doses (1 mg and 0.1 mg) were also used. All six rabbits produced an immunological response to the hapten regardless of the carrier protein or immunogen dose used. The most sensitive polyclonal antibody produced, designated R609, was subsequently characterised. This antiserum exhibited an IC₅₀ of 18 ng ml−1 using a competitive ELISA format. Cross reactivity studies show that this serum is specific for toltrazuril and its metabolites (toltrazuril sulfoxide and toltrazuril sulfone) but does not cross-react with other coccidiostats such as halofuginone, nitroimidazoles or nicarbazin. This is the first reported production of an antibody capable of specifically binding toltrazuril and ponazuril.

The production of an antibody to detect toltrazuril or its metabolite ponazuril is complicated due to structural constraints of conjugating these coccidiostats to a carrier protein. Therefore a search was carried out for a compound that shared a common substructure to use as an antigen mimic. The chosen compound, trifluoraminoether, was conjugated to two carrier proteins HSA and BTG) and used in the immunisation of six rabbits. Two immunogen doses 1 mg and 0.1 mg were also used. All six rabbits produced an immunological response to the hapten regardless of the carrier protein or immunogen dose used. The most sensitive polyclonal antibody produced, designated R609, was subsequently characterised. This antiserum exhibited an IC50of 18 ng ml−1using a competitive ELISA format. Cross reactivity studies show that this serum is specific for toltrazuril and its metabolites toltrazuril sulfoxide and toltrazuril sulfone but does not cross-react with other coccidiostats such as halofuginone, nitroimidazoles or nicarbazin. This is the first reported production of an antibody capable of specifically binding toltrazuril and ponazuril.