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Biomedical subjects

J A Barnard

Publications and source records attributed to J A Barnard.

At least 19 recordsLinked to original sources
PubMed2004

Differential trafficking of transforming growth factor-beta receptors and ligand in polarized epithelial cells.

Epithelial cells in vivo form tight cell-cell associations that spatially separate distinct apical and basolateral domains. These domains provide discrete cellular processes essential for proper tissue and organ development. Using confocal imaging and selective plasma membrane domain activation, the type I and type II transforming growth factor-beta (TGFbeta) receptors were found to be localized specifically at the basolateral surfaces of polarized Madin-Darby canine kidney (MDCK) cells. Receptors concentrated predominantly at the lateral sites of cell-cell contact, adjacent to the gap junctional complex. Cytoplasmic domain truncations for each receptor resulted in the loss of specific lateral domain targeting and dispersion to both the apical and basal domains. Whereas receptors concentrate basolaterally in regions of direct cell-cell contact in nonpolarized MDCK cell monolayers, receptor staining was absent from areas of noncell contact. In contrast to the defined basolateral polarity observed for the TGFbeta receptor complex, TGFbeta ligand secretion was found to be from the apical surfaces. Confocal imaging of MDCK cells with an antibody to TGFbeta1 confirmed a predominant apical localization, with a stark absence at the basal membrane. These findings indicate that cell adhesion regulates the localization of TGFbeta receptors in polarized epithelial cultures and that the response to TGFbeta is dependent upon the spatial distribution and secretion of TGFbeta receptors and ligand, respectively.

Adherens Junctions↗
PubMed2002

Relationship of high energy expenditure and variation in dietary intake with reporting accuracy on 7 day food records and diet histories in a group of healthy adult volunteers.

OBJECTIVES: To assess the accuracy of reporting from both a diet history and food record and identify some of the characteristics of more accurate reporters in a group of healthy adult volunteers for an energy balance study. DESIGN: Prospective measurements in free-living people. SETTING: Wollongong, Australia. SUBJECTS: Fifteen healthy volunteers (seven male, eight female; aged 22-59 y; body mass index (BMI) 19-33 kg/m(2)) from the local community in the city of Wollongong, Australia. INTERVENTIONS: Measurement of energy intake via diet history interview and 7 day food records, total energy expenditure by the doubly labelled water technique over 14 days, physical activity by questionnaire, and body fat by dual-energy X-ray absorptiometry. RESULTS: Increased misreporting of energy intake was associated with increased energy expenditure (r=0.90, P<0.0001, diet history; r(S)=0.79, P=0.0005, food records) but was not associated with age, sex, BMI or body fat. Range in number of recorded dinner foods correlated positively with energy expenditure (r(S)=0.63, P=0.01) and degree of misreporting (r(S)=0.71, P=0.003, diet history; r(S)=0.63, P=0.01, food records). Variation in energy intake at dinner and over the whole day identified by the food records correlated positively with energy expenditure (r=0.58, P=0.02) and misreporting on the diet history (r=0.62, P=0.01). CONCLUSIONS: Subjects who are highly active or who have variable dietary and exercise behaviour may be less accurate in reporting dietary intake. Our findings indicate that it may be necessary to screen for these characteristics in studies where accuracy of reporting at an individual level is critical.

Adult↗
PubMed1999

Heparin binding epidermal growth factor-like growth factor is a transforming growth factor beta-regulated gene in intestinal epithelial cells.

Heparin binding epidermal growth factor-like growth factor (HB-EGF) is an EGF-related peptide with prominent effects on cell growth and migration. We explored potentially unique characteristics of HB-EGF in the intestinal epithelial cell line RIE-1. HB-EGF stimulated [(3)H]thymidine incorporation to a level equivalent to transforming growth factor alpha (TGFalpha). HB-EGF also rapidly activated MAPK and induced cyclin D1 in mid-G1 with kinetics similar to TGFalpha. Unlike TGFalpha, HB-EGF mRNA was induced within 1 h by a variety of stimuli, including TGFbeta1. Maximal induction by TGFbeta (7-fold) occurred within 2 h of treatment. Actinomycin D decay curves showed that TGFbeta1 had no effect on HB-EGF mRNA half-life (T(1/2) 20 min). Induction of HB-EGF by TGFbeta1 was not affected by pretreatment with the MEK inhibitor PD-98059 while inhibition of protein kinase C either partially (calphostin C) or completely (staurosporin) blocked induction. Our results suggest that major differences exist in the regulation of the closely related EGF family members TGFalpha and HB-EGF. TGFbeta and HB-EGF, structurally unrelated peptides with potent effects on wound healing, may function coordinately to mediate responses to wounding or cell injury in the intestinal epithelium.

Animals↗
PubMed1999

Pharmacological inhibition of Ras-transformed epithelial cell growth is linked to down-regulation of epidermal growth factor-related peptides.

BACKGROUND & AIMS: Posttranslational farnesylation is required for Ras activation. Farnesyl transferase inhibitors (FTIs) selectively block protein farnesylation and reduce the growth of many Ras-transformed cells in vitro and in vivo. Activated Ras transforms rat intestinal epithelial (RIE-1) cells by a mechanism distinct from NIH 3T3 fibroblasts in that an epidermal growth factor receptor (EGFR) autocrine loop contributes significantly to the Ras-transformed RIE-1 phenotype. METHODS: The ability of FTIs to block growth of Ras-transformed RIE-1 cells was evaluated, and these results were correlated with decreased EGFR ligand production. RESULTS: FTI L744,832 caused a selective, dose-dependent, reversible blockade in proliferation of H-Ras-transformed RIE-1 cells, whereas control cell lines, K-Ras-transformed cells, and activated raf-transfected RIE cells were unaffected. The growth-inhibitory effects of L744,832 correlated with loss of farnesylated H-Ras protein and a marked reduction in transforming growth factor (TGF)-alpha and amphiregulin expression. Inhibition of proliferation of H-Ras RIE-1 cells by L744,832 was overcome by exogenous TGF-alpha, and enhanced growth inhibition was achieved by EGFR blockade in combination with L744,832. + CONCLUSIONS: These data suggest that one mechanism by which FTIs inhibit growth of H-Ras-transformed epithelial cells is by reducing Ras-induced EGFR ligand production.

Animals↗
PubMed1998

Increased intestinal epithelial proliferation in metallothioneine-transforming growth factor alpha transgenic mice.

BACKGROUND AND AIM: The epidermal growth factor (EGF) peptide family includes six closely-related proteins, all of which bind to the EGF receptor. In the intestinal epithelium, transforming growth factor alpha (TGFalpha) appears to be the most physiological ligand for the EGF receptor. The present studies were designed to examine the effect of TGFalpha overexpression on duodenal epithelial proliferation using a metallothioneine-inducible promoter/enhancer transgenic mouse (MT-TGFalpha). The MT-TGFalpha mouse model was further studied to determine the in vivo effect of unregulated TGFalpha production on the physiological proliferative responses to fasting and refeeding. METHODS: MT-TGFalpha mice were given 25 mM oral ZnSO4 to induce transgene expression and were studied 1 to 2 months later. Duodenal histology was analyzed morphometrically in well-oriented transverse sections. The vincristine metaphase-arrest technique was used to assess proliferation in duodenal crypts. Immunohistochemical staining and in situ hybridization were used to assess transgenic TGFalpha protein and mRNA expression, respectively. RESULTS: Normally fed MT-TGFalpha mice had deeper crypts (0.12 vs. 0.08 mm), longer villi (0.66 vs. 0.54 mm), and greater luminal diameters (2.65 vs. 2.19 mm) compared to controls (P<0.05 for all three dimensions). The crypt cell mitotic index in normally fed transgenic mice was 1.5 fold greater than the index in normally fed controls (20+/-2 vs. 35+/-4 mitoses per crypt; P <0.05). Fasting and refeeding MT-TGFalpha mice resulted in no significant change in their high baseline rate of crypt proliferation, while proliferation in control mice rose from a lower baseline during fasting to a level with refeeding that approximated rates in MT-TGFalpha mice. Transgenic TGFalpha protein and mRNA were localized to the villus epithelial compartment with little or no evidence of mRNA or protein expression in the crypt epithelium. CONCLUSION: Overproduction of TGFalpha in the mouse duodenal epithelium results in a pronounced increase in crypt epithelial cell proliferation and a resulting increase in the dimension of the crypt/villus unit. This appears to be mediated through a paracrine and/or juxtacrine effect on crypt cells by TGFalpha produced in the villus epithelium. Fasting and refeeding experiments suggest that TGFalpha may also play a role in the proliferative response to refeeding or that the full potential for proliferation is realized by TGFalpha overexpression alone. Collectively, these studies suggest that TGFalpha is a physiological autocrine and paracrine regulator of small intestinal epithelial proliferation.

Animals↗
PubMed1998

Recurrent abdominal pain: a potential precursor of irritable bowel syndrome in adolescents and young adults.

OBJECTIVES: To assess symptoms of irritable bowel syndrome (IBS) in patients with recurrent abdominal pain (RAP) 5 years after their initial evaluation, to identify the relation of IBS symptoms to functional disability and health service use, and to determine the extent to which IBS symptoms are associated with life stress and poor psychosocial adjustment. METHODS: Patients with RAP (n = 76) and control subjects (n = 49) completed a telephone interview; measures included the Bowel Disease Questionnaire, the Functional Disability Inventory, the Life Events Questionnaire, the Family Inventory of Life Events, the Center for Epidemiological Studies Depression Scale, the Self-Perception Profile for Adolescents, and the Health Resources Inventory. RESULTS: Five years after the initial evaluation, patients with RAP reported significantly more episodes of abdominal pain than did control subjects, as well as significantly higher levels of functional disability, school absence, and clinic visits for abdominal distress. Female patients with RAP were more likely than female control subjects to meet the Manning criteria for IBS. Among patients with RAP, higher levels of IBS symptoms were associated with significantly greater functional disability, more clinic visits, more life stress, higher levels of depression, and lower academic and social competence. CONCLUSION: Female patients with a history of RAP may be at increased risk of IBS during adolescence and young adulthood. Among adolescents and young adults with a history of RAP, IBS symptoms are likely to be associated with high levels of disability and health service use.

Abdominal Pain↗
PubMed1996

Transforming growth factor-alpha.

Major advances in understanding growth factor biology, especially in epithelial cells, have resulted from work with TGF-alpha over the past decade. It is clear that TGF-alpha is a potent epithelial oncoprotein, but equally important biological activities in normal epithelial homeostasis have been described. A number of major challenges lie ahead. Foremost is the formidable task of dissecting out the individual contributions of each EGF-related peptide in the biological response to stimulation of the EGFR. Appreciation of the complexity of heterodimerization of receptors within the EGFR family will be equally important in the final analysis. These considerations assure the continued vitality and productivity of investigation of the EGF-related peptide/EGFR axis.

Amino Acid Sequence↗
PubMed1996

Type II TGF(beta) receptor expression in intestinal cell lines and in the intestinal tract.

The recent identification and cloning of mammalian transforming growth factor beta (TGFbeta) receptors permits further analysis of the importance of the TGFbeta family in intestinal biology. Expression of the type II TGFbeta receptor was examined in gastrointestinal cell lines and tissues. The 5.5 kb type II mRNA species was detected in poly-(A) mRNA isolated from the rat small bowel and colon. Northern blot analysis of RNA isolated from epithelial and non-epithelial small intestinal cell fractions showed the majority of receptor mRNA localized in the non-epithelial compartment. Immunohistochemical localization in the small intestine and colon supported the RNA findings; that is, expression was greatest in the lamina propria and muscularis. Staining was also detectable in the epithelium, where it was most prominent in the villus tip cells and absent in crypt cells. These findings mirror expression of TGFbeta in the epithelial compartment. The IEC-6, IPEC and RIE-1 cell lines, all of which are non-transformed, were growth inhibited by TGFbeta and expressed type II receptor mRNA and protein. By contrast, the ras-transfected RIE-1, HT-29, Caco-2 and SW-620 transformed lines were not growth inhibited by TGFbeta and all demonstrated a marked reduction in type II TGFbeta receptor mRNA expression and protein abundance by cross-linking. In conclusion, (i) colocalization of both ligand and receptor establishes the existence of potential autocrine and/or paracrine pathways for TGFbeta in the normal intestine and (ii) down-regulation of the type II TGFbeta receptor occurs in association with cellular transformation and may contribute to intestinal carcinogenesis.

Animals↗
PubMed1995

Expression of a novel sodium-hydrogen exchanger in the gastrointestinal tract and kidney.

We have recently cloned a novel Na+/H+ exchanger (NHE-2) from rat small intestinal cDNA library. The present report characterizes the expression of (NHE-2) mRNA in the rat small intestine, stomach, and kidney using Northern blot analysis and in situ hybridization techniques. NHE-2 mRNA was detected in the surface epithelium of the stomach, villus but not crypt epithelial cells of the small intestine and in the distal convoluted, but not in the proximal tubules of the kidney. This distribution suggests a functional role in the transport of sodium across the apical membrane of the gastrointestinal tract and the kidney.

Alternative Splicing↗
PubMed1995

Early proliferative events following intestinal resection in the rat.

Partial resection of the small intestine results in compensatory proliferation and adaptation in the remaining small intestinal mucosa. The molecular mechanisms governing the proliferative response are not known, nor has the timing of events associated with proliferation been adequately defined, particularly during the period just after resection. We designed experiments to characterize early (within 24 h) proliferative events associated with proximal intestinal resection and sought to determine the cell type that first responds to proliferative stimuli. Twenty-one day old male Sprague-Dawley rats underwent a 70% proximal intestinal resection or transection (control). Poly(A) RNA was isolated from the distal (ileal) remnants. Northern blots showed a marked induction of the immediate early genes zif-268, nup-475, and c-myc 1-3 h following resection, but not following transection. Immunohistochemical analysis of c-myc expression in ileal crypt epithelial cells showed a biphasic induction that was most marked 6 h after resection and less prominent 24 h after resection. Immunostaining with 5-bromodeoxyuridine (5-BrdU) was restricted to ileal crypt nuclei and was maximal 24 h after resection. All these events were observed in the absence of nutrient intake. Taken together, these data show that a potent nutrient-independent stimulus for intestinal epithelial cell proliferation occurs within minutes of partial small intestinal resection and that the first targets of this stimulus are crypt epithelial cells in the residual intestine.

Animals↗
PubMed1994

Auto- and cross-induction within the mammalian epidermal growth factor-related peptide family.

Several polypeptide growth factors related to epidermal growth factor (EGF) have been identified recently, including transforming growth factor-alpha (TGF-alpha), amphiregulin (AR), heparin-binding EGF-like growth factor (HB-EGF), and betacellulin (BTC). These peptides all bind to the EGF receptor (EGFr). In an effort to understand redundancy within this peptide family and interactions among these related peptides, we compared the biological activities of EGF, TGF-alpha, AR, and HB-EGF in an EGF-responsive, nontransformed intestinal epithelial line (RIE-1) and also determined the effect of individual EGF-related peptides on the expression of related family members in these cells. TGF-alpha, AR, HB-EGF, and EGF were equipotent in stimulating [3H]thymidine incorporation by RIE-1 cells and bound the EGFr with equivalent affinity. Each EGF-related peptide induced the mRNA expression of the remaining family members, including BTC. HB-EGF and AR mRNAs were induced rapidly (within 30 min) and to a greater extent than TGF-alpha and BTC mRNAs, suggesting heterogeneity in the molecular mechanisms for induction. This same pattern was observed for all EGF-related peptides tested. A similar pattern of mRNA induction was observed in secondary cultures of human keratinocytes and in LIM1215 colon adenocarcinoma cells. Nuclear run-on analysis showed that induction of AR and HB-EGF is, at least in part, regulated at the level of gene transcription. Concurrent treatment with HB-EGF and cycloheximide resulted in superinduction of HB-EGF and AR, suggesting that these peptides are immediate early genes in RIE-1 cells. Our results demonstrate an equivalent biological response to EGF-related peptides in RIE-1 cells and further indicate that extensive auto-induction and cross-induction occur within the EGF-related peptide family in several EGF-responsive epithelial cell types.

Amphiregulin↗
PubMed1993

Localization of transforming growth factor beta isoforms in the normal murine small intestine and colon.

BACKGROUND: The transforming growth factor beta (TGF-beta) proteins are key regulators of cellular growth and differentiation. Previous studies have shown that TGF-beta 1 is a potent growth inhibitor of cultured jejunal epithelial cells. The reported distribution of TGF-beta 1 messenger RNA (mRNA) expression along the intestinal villus has been controversial. The purpose of the current study is to determine the loci of TGF-beta protein expression in the normal small intestine and colon. METHODS: Intestinal localization of TGF-beta isoform mRNA and protein was examined by Northern blot analysis and immunohistochemistry using isoform specific reagents. RESULTS: TGF-beta 1, TGF-beta 2, and TGF-beta 3 mRNA were found in homogenates from the intact mouse jejunum and colon. The three isoforms colocalized in these tissues. Expression in the small intestinal epithelium was most prominent in cells located on the villus tip, and no staining was detected in the crypt. Occasional lymphocytes in the lamina propria were immunopositive, and all layers of the muscularis were moderately stained. This pattern was seen in all regions of the small intestine. The surface epithelium of the colon was intensely immunopositive, whereas cells in the glands were only weakly stained. CONCLUSIONS: TGF-beta molecules may serve overlapping functions in the intestinal tract, and expression in the epithelium may function to arrest growth of cells emerging from the crypt and induce or maintain the terminally differentiated state.

Animals↗
PubMed1993

Intestinal Na+/H+ exchanger activity is up-regulated by bowel resection in the weanling rat.

Increased Na+/H+ exchanger activity is associated with cellular hyperplasia. Cellular hyperplasia is an adaptive response to small-intestinal resection. Therefore, we hypothesized that the small-intestinal Na+/H+ exchanger activity increases in response to small-intestinal resection. Twenty-one-d-old, male Sprague-Dawley rats were randomly divided to receive either a 70% small intestinal resection (n = 59), or a mid-small intestinal transection (n = 49). Seven d postoperatively, the animals were killed and the Na+/H+ exchanger activity of the intestinal remnants was studied by a well validated brush border membrane vesicle technique. The initial rate of Na+ uptake in the presence of an outwardly directed pH gradient and the Vmax of the amiloride-sensitive Na+ uptake were significantly increased (p < 0.01 and p < 0.001, respectively) in the resection as compared with the transection remnants and to a greater magnitude in the distal as compared with the proximal remnants. Km values were not significantly different. The amiloride-sensitive Na+ uptake in the setting of various intravesicular pH was significantly greater (p < 0.001) in the distal resection as compared with the distal transection remnants, with points of enhanced Na+/H+ exchanger activity of intravesicular pH 6.62 and 6.87, respectively. The presence and activation of the Na+/H+ exchanger's internal modifier site was confirmed by demonstrating the effect of intravesicular pH on Na+ efflux. The present study demonstrates an up-regulation of intestinal Na+/H+ exchange activity in a small-bowel resection model in the weanling rat.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗