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Biomedical subjects

J A Barnard

Publications and source records attributed to J A Barnard.

At least 37 records · Page 2Linked to original sources
PubMed1993

Butyrate rapidly induces growth inhibition and differentiation in HT-29 cells.

Selected G1 events associated with butyrate-induced differentiation were examined in HT-29 colon adenocarcinoma cells. [3H]Thymidine incorporation by HT-29 cells was decreased to 40% of control levels by treatment with 5 mM butyrate for 24 h, and cell numbers decreased to 21% of control levels after 48 h of treatment. Cells released from butyrate arrest entered S phase approximately 24 h after release, and serum-deprived HT-29 cells escaped growth inhibition if butyrate was added 8 h or more after serum restimulation. Northern analysis of RNA isolated from rapidly growing HT-29 cells showed a marked induction of alkaline phosphatase mRNA expression within 12 h of treatment with 5 mM butyrate. The appearance of alkaline phosphatase mRNA was temporally associated with a 5-fold increase in expression of transforming growth factor beta 1 (TGF-beta 1) mRNA. Expression of the nuclear protooncogene c-myc began to decrease 30 min after treatment with butyrate and was decreased 4.5-fold 4 h after treatment; however, expression of other immediate-early genes (nup/475 and zif/268) was not significantly affected. Histochemical staining of HT-29 monolayers showed that no cells were positive for alkaline phosphatase protein prior to treatment, and 90% were positive 48 h after treatment. TGF-beta 1 and TGF-beta 2 had no effect on HT-29 cell growth. TGF-beta 1 did not induce alkaline phosphatase mRNA or histochemical positivity. These data indicate that butyrate arrests HT-29 cell growth early in G1.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenocarcinoma↗
PubMed1992

Increased production of transforming growth factor alpha following acute gastric injury.

Transforming growth factor alpha (TGF-alpha) production recently has been found in normal mammalian gastric mucosa. Inasmuch as TGF-alpha and epidermal growth factor (EGF) both stimulate epithelial cell migration and proliferation and suppress gastric acid secretion, the authors of the current study proposed that these growth factors may participate in tissue repair after acute gastric mucosal injury. Consequently, TGF-alpha and EGF production were examined after orogastric administration of either acidified taurocholate or 0.6 mol/L HCl to rats. TGF-alpha messenger RNA (mRNA) expression increased in a dose- and time-dependent manner after administration of taurocholate, whereas EGF mRNA expression was not detected. Radioimmunoassay of gastric mucosal scrapings obtained 6 hours after gastric injury induced by 0.6 mol/L HCl showed a 2.1-fold increase in immunoreactive TGF-alpha but no increase in immunoreactive EGF. In addition, there was a 68-fold increase in immunoreactive TGF-alpha in gastric juice within 30 minutes of gastric instillation of HCl and, again, no increase in immunoreactive EGF. There is a rapid appearance of TGF-alpha in the gastric juice within 30 minutes of injury, which is followed by increased expression of TGF-alpha mRNA and protein in the gastric mucosa. These studies suggest that locally produced TGF-alpha may participate in gastric mucosal repair following acute gastric injury to rats.

Animals↗
PubMed1991

Idiopathic neonatal iron-storage disease.

A 21-day-old infant presented with anemia, conjugated hyperbilirubinemia, hypoproteinemia, and a severe coagulopathy. The hospital course was marked by progressive hepatic failure, encephalopathy, and renal insufficiency. The infant died on day 15 of hospitalization. Postmortem examination showed diffuse hepatic fibrosis and marked siderosis of the liver, pancreas, kidney, adrenal glands, and the duodenal epithelium, with sparing of the reticuloendothelial system. These findings were characteristic of idiopathic neonatal iron-storage disease. Previously reported cases are summarized and discussed. An increased awareness and understanding of this rapidly fatal disorder will be important for genetic counseling and possibly in defining an aberrant mechanism in the handling of iron.

Blood Coagulation Disorders↗
PubMed1991

Production of transforming growth factor-alpha by normal rat small intestine.

Transforming growth factor-alpha (TGF-alpha) and epidermal growth factor (EGF) are similar in structure and biological activity. In the present study, the distributions of TGF-alpha mRNA, TGF-alpha immunoreactivity, and TGF-alpha-EGF receptor mRNA were examined in epithelial and nonepithelial compartments of the jejunum, and the effect of TGF-alpha on growth of a jejunal crypt cell line (IEC-6) was determined. Epithelial cells eluted from the rat jejunal cryptvillus axis expressed TGF-alpha mRNA at twofold higher levels in the villus tip than in the crypt and EGF receptor mRNA at sevenfold higher levels in the villus tip. Expression of these two mRNA transcripts in the subepithelium was low. Immunohistochemical staining showed TGF-alpha immunoreactivity predominantly in the epithelium and muscularis. Immunostaining of villus cells was uniform, whereas crypt cells did not stain. IEC-6 cells bound 125I-EGF to a single class of high-affinity (dissociation constant = 833 pM) receptors. EGF and TGF-alpha (10 ng/ml) only modestly stimulated IEC-6 cell growth in the presence of 5% serum but increased expression of the protooncogenes c-jun and c-myc threefold over control cells. These findings suggest that, among the potential physiological roles for TGF-alpha produced by the jejunal epithelium, promotion of cell migration and modulation of fluid and electrolyte transport may be as relatively important as stimulation of cell proliferation.

Animals↗
PubMed1990

The cell biology of transforming growth factor beta.

The TGF beta family of polypeptide growth factors regulates a remarkable diversity of cellular functions, many of which are not directly associated with cell growth. The present review has summarized many of the recent studies that have just begun to conceptually integrate this expanding array of TGF beta functions into the context of a three-dimensional, multicellular organ or tissue, be it normal or diseased. This fascinating research strongly implicates TGF beta as a key modulator of a wide variety of important physiologic and pathophysiologic processes.

Animals↗
PubMed1990

Modulation of EGF-receptors by phorbol ester in small intestinal crypt cells.

We have previously shown that EGF promotes growth and proliferation of enterocytes isolated from the crypts of the rat small intestine (IEC-6). In the present studies we have measured the affinity of EGF for its receptor, and estimated the number of surface EGF receptors on IEC-6 cells. Scatchard analysis indicates IEC-6 cells display 45,000 EGF receptors per cell with a dissociation constant of 41 pM. Treatment with phorbol-12-myristate-13-acetate (PMA) results in a dose-dependent inhibition of cell growth which is paralleled by reduced binding of 125I-EGF. Incubation of IEC-6 cells with 10 nM PMA results in a 70 percent decrease in the number of EGF receptors without a significant change of receptor affinity (kd 68 pM vs 41 pM). PMA treatment is also associated with a significant increase of protein kinase-C activity in IEC-6 cells. The reciprocal relationship between protein kinase-C activation and EGF receptors suggests in this cell line of crypt enterocytes, protein kinase-C may inhibit cellular proliferation by modulating EGF receptors.

Animals↗
PubMed1989

Regulation of intestinal epithelial cell growth by transforming growth factor type beta.

A nontransformed rat jejunal crypt cell line (IEC-6) expresses transforming growth factor type beta 1 (TGF-beta 1) mRNA, secretes latent 125I-labeled TGF-beta 1 competing activity into culture medium, and binds 125I-labeled TGF-beta 1 to specific, high-affinity (Kd = 3.7 pM) cell surface receptors. IEC-6 cell growth is markedly inhibited by TGF-beta 1 and TGF-beta 2 with half-maximal inhibition occurring between 0.1 and 1.0 ng of TGF-beta 1 per ml. TGF-beta 1-mediated growth inhibition is not associated with the appearance of biochemical markers of enterocyte differentiation such as alkaline phosphatase expression and sucrase activity. TGF-beta 1 (10 ng/ml) increases steady-state levels of its own mRNA expression within 8 hr of treatment of rapidly growing IEC-6 cells. In freshly isolated rat jejunal enterocytes that are sequentially eluted from the crypt villus axis, TGF-beta 1 mRNA expression is most abundant in terminally differentiated villus tip cells and least abundant in the less differentiated, mitotically active crypt cells. We conclude that TGF-beta 1 is an autoregulated growth inhibitor in IEC-6 cells that potentially functions in an autocrine manner. In the rat jejunal epithelium, TGF-beta 1 expression is most prominently localized to the villus tip--i.e., the region of the crypt villus unit that is characterized by the terminally differentiated phenotype. These data suggest that TGF-beta 1 may function in coordination of the rapid cell turnover typical for the intestinal epithelium.

Animals↗
PubMed1989

Localization of transforming growth factor alpha and its receptor in gastric mucosal cells. Implications for a regulatory role in acid secretion and mucosal renewal.

Transforming growth factor alpha (TGF alpha) shares with epidermal growth factor (EGF) structural homology (35%), a common cell-surface membrane receptor (TGF alpha/EGF receptor), and a nearly identical spectrum of biological activity, including inhibition of gastric acid secretion. Herein, we report expression of TGF alpha mRNA in normal gastric mucosa of the adult guinea pig, rat, and dog. TGF alpha mRNA was also detected in matched surgically resected gastric mucosa and adjacent gastric carcinoma from 10 patients, and in gastric mucosa adjacent to a benign ulcer from an additional patient. TGF alpha protein was quantitated by radioimmunoassay and was present in tumor and adjacent mucosa. TGF alpha/EGF receptor mRNA was also detected in gastric mucosa from all species studied. Localization of TGF alpha and TGF alpha/EGF receptor mRNA expression was examined in samples of unfractionated guinea pig gastric mucosa and from chief cell-enriched and parietal cell-enriched fractions. All samples exhibited TGF alpha and TGF alpha/EGF receptor expression. The TGF alpha signal was greatest in the parietal cell fraction (5.8-fold increase), but was also enhanced in the chief cell fraction (1.9-fold increase) relative to the unfractionated gastric mucosa. Like TGF alpha expression, TGF alpha/EGF receptor mRNA expression was most intense in the parietal cell-enriched fraction (7.8-fold increase), but was also increased in the chief cell-enriched fraction (2.7-fold increase) relative to the unfractionated guinea pig gastric mucosa. We conclude that TGF alpha and TGF alpha/EGF receptor genes are expressed in normal adult mammalian gastric mucosa. These findings, when interpreted in light of described actions of TGF alpha and EGF, provide evidence that local production of TGF alpha could play an important role in the regulation of acid secretion and mucosal renewal in the stomach.

Adult↗
PubMed1989

Effects of EGF and PMA on the growth and proliferation of IEC-6 cells.

Proliferation of an epithelial line (IEC-6) derived from the crypts of rat jejunum was induced with epidermal growth factor (EGF). EGF enhanced synthesis of protein, RNA, and DNA in a dose-dependent manner. Protein synthesis increased within 6-12 hours of exposure to EGF and remained elevated for 72 hours. Maximal 3H-thymidine incorporation occurred 48 hours after addition of EGF. The stimulatory effect of EGF on 3H-thymidine incorporation was two-fold greater in serum-free media than in media containing fetal calf serum (FCS). In contrast to EGF, phorbol-12-myristate-13-acetate (PMA) decreased 3H-thymidine uptake by IEC-6 cells and had no effect on either protein synthesis or RNA synthesis. EGF did not alter protein kinase-C activity in IEC-6 cells whereas PMA induced enzyme activity: activity was translocated from cytosol to membrane. Moreover, the EGF-associated increase in 3H-thymidine uptake was not altered by amiloride. These data suggest protein kinase-C activation may not be involved in the proliferation of IEC-6 cells.

Animals↗
PubMed1988

Transforming growth factor beta in the control of epidermal proliferation.

Transforming growth factor beta is a polypeptide growth factor with a multiplicity of diverse biologic effects. Increasingly, data support a role for TGF beta in the autocrine regulation of normal epithelial cell growth (Figure 1). Definition of the normal pathways for growth stimulation and inhibition of epithelial cell growth by autocrine peptides like TGF beta and TGF alpha undoubtedly will increase understanding of normal growth and development, embryogenesis, wound repair, and tumorigenesis.

Cell Differentiation↗
PubMed1988

Taurine transport by rat intestine.

Rat intestinal transport of taurine was studied using jejunal brushborder membrane vesicles and everted jejunal sacs. Membrane vesicle experiments showed that the initial rate (at 10 s) of 10 microM taurine uptake was stimulated 3.5-fold by an inwardly directed Na+ gradient when compared with a K+ gradient. The Cl- salt of Na+ supported uptake to a significantly greater degree than did more (SCN-) or less (SO2-4) permeant salts. Na+-stimulated uptake at 1 min achieved a value 2.5 times greater than equilibrium ("overshoot"). When 10 s taurine uptake was determined over a range of taurine concentrations (10-1,000 microM) and plotted using the Woolf-Hofstee equation, a diffusive and carrier-mediated component was apparent. Half-maximal, carrier-mediated uptake occurred at 25 +/- 9 microM taurine (Km). An inside-negative, valinomycin-induced K+ diffusion potential stimulated 10-s taurine uptake when compared with voltage-clamped conditions (4.9 vs. 2.6 pmol/mg protein; P less than 0.05). Incubation with a structural analogue, hypotaurine, reduced 10-s taurine uptake by 89%. Uptake by everted jejunal sacs also demonstrated Na+ stimulation and inhibition by hypotaurine. These data confirm the existence of a high-affinity rat jejunal brush-border membrane-associated taurine transport mechanism that is electrogenic, stimulated by a Na+ gradient, and modified by external Cl-.

Animals↗
PubMed1988

Protein synthesis by HepG2 cells infected with influenza B virus.

Reye's syndrome is an acute hepatopathy and encephalopathy affecting children during the convalescent period of a viral infection, frequently influenza B. The role of influenza B in the pathogenesis of Reye's syndrome is unknown. To investigate this relationship, an in vitro system was designed to examine the interaction of influenza B virus with a cell line of human hepatocytes (HepG2) which retains many specific hepatic synthetic characteristics. HepG2 was capable of supporting a productive infection by influenza B, although greater virus inputs were required than in fully permissive Madin-Darby canine kidney cells. Because of the recent association of Reye's syndrome with aspirin use, the kinetics of influenza B growth were studied in the presence of acetylsalicylic acid (25 and 100 micrograms/ml) and were not found to be altered. Protein synthesis by HepG2 cells was decreased by 80% in influenza B-infected cells when compared to uninfected controls. Acetylsalicylic acid, 100 micrograms/ml, did not affect the rate of 35S-methionine incorporation by infected or uninfected HepG2 cells. The rates of synthesis of specific proteins (albumin, transferrin, and apoprotein B) by HepG2 cells were determined by immunoprecipitation of 35S-methionine labeled cell lysates. After 12 h of infection, synthesis of all three plasma proteins was decreased by 40-60%. These studies describe a useful system for delineation of mechanisms by which influenza B virus interacts with host hepatocytes and the host-cell protein synthetic machinery. The studies could be pertinent to the pathogenesis of Reye's syndrome.

Aspirin↗
PubMed1987

Taurocholate transport by human ileal brush border membrane vesicles.

Human ileal brush border membrane vesicles were prepared from intestines obtained from cadaveric renal allograft donors. The energetics and kinetics of taurocholate transport were studied. Fifty-five percent of equilibrium uptake (picomoles per mg protein) resulted from binding to the vesicle surface or incorporation into an osmotically insensitive compartment. The initial rate of transport was stimulated fourfold by an inwardly directed Na+ gradient when compared with a K+ gradient, and cation gradient-dependent differences persisted throughout the initial 5 min of incubation (p less than 0.05). Taurocholate uptake was half-maximally stimulated by a Na+ concentration of 23 +/- 4 mM. A Hill transformation of this plot gave a slope (n) of 0.97, indicating a 1:1 (mol/mol) Na+-taurocholate coupling ratio. Generation of a negative inside diffusion potential by anion substitution or valinomycin-induced K+ diffusion potential failed to alter bile salt uptake, suggesting an electroneutral transport mechanism. When Na+-dependent uptake velocity (10 s) was examined over a range of taurocholate concentration (0.036-0.9 mM), the plot described a rectangular hyperbola. The mean apparent Michaelis constant was 0.037 +/- 0.007 mM and maximum velocity was 1093 +/- 329 pmol taurocholate per milligram protein per 10 s. These data confirm and extend animal studies of ileal bile salt transport. Taurocholate uptake by the human ileal brush border occurs by a Na+-dependent, carrier-mediated electroneutral mechanism. According to this model, a single Na ion is coupled with a single taurocholate anion and transported across the brush border by a carrier mechanism that is driven by a transmembrane Na+ gradient.

Biological Transport↗
PubMed1986

Noncirrhotic portal fibrosis after Wilms' tumor therapy.

A 9-yr-old girl developed massive hemorrhage from esophageal varices 2 yr after combined modality therapy for Wilms' tumor. Evaluation showed a patent extrahepatic portal venous system and an elevated splenic pulp pressure. In contrast to previous reports of hepatopathy after irradiation injury, histologic sections of the liver did not demonstrate occlusion of the central veins, but rather a diffuse obliteration of intrahepatic portal venous radicles. This pattern of noncirrhotic portal fibrosis has not been described following antitumor therapy.

Child↗
PubMed1986

Methylprednisolone accelerates the ontogeny of sodium-taurocholate cotransport in rat ileal brush border membranes.

The effect of methylprednisolone on the postnatal maturation of taurocholate transport was studied by using isolated ileal brush border membrane vesicles. Vesicles were prepared from 14-day-old control, 14-day-old methylprednisolone-treated, and untreated 21-day-old rats. Methylprednisolone treatment resulted in a significant stimulation of taurocholate uptake by an inwardly directed Na+ gradient when compared with a choline gradient incubation. These differences occurred at 20 seconds and 1, 2, and 5 minutes of incubation (P less than 0.05). In 14-day-old controls, uptake was similar for Na+ and choline gradients. A plot of active uptake velocity vs. taurocholate concentration (0.1 to 1.0 mmol/L) in 14-day-old controls was linear and approached the abscissa, indicating the absence of active transport. Plots for methylprednisolone-treated rats showed saturability. An inwardly directed Na+ gradient stimulated initial taurocholate uptake rates by twofold at 37 degrees C (P less than 0.01), but not at 4 degrees C. Glycocholate and glycodeoxycholate inhibited Na+-stimulated taurocholate uptake by 50% (P less than 0.01) and 20% (P less than 0.05), respectively. These data indicate that pharmacologic doses of methylprednisolone accelerate the postnatal acquisition of Na+-dependent taurocholate cotransport in rat ileal brush border membranes.

Animals↗
PubMed1985

Necrotizing entercolitis. Variables associated with the severity of disease.

The medical records of 51 inborn infants with necrotizing enterocolitis (NEC) were studied to determine factors that may contribute to the severity of NEC. In contrast to infants requiring only medical intervention, those with severe disease requiring a surgical procedure were less likely to have a history of a symptomatic patent ductus arteriosus (14% v 45%); they also required fewer days of antibiotic therapy (three v five) and fewer days of endotracheal intubation (0.5 v three). They were fed earlier (two days v four days). The diagnosis was made at a younger age in infants needing surgery (6.5 days v 14 days). These data indicate that infants with minimal neonatal morbidity may be at risk for severe NEC, which results in bowel necrosis.

Anti-Bacterial Agents↗