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G W Aponte

Publications and source records attributed to G W Aponte.

17 recordsLinked to original sources
PubMed1999

Y receptor-mediated induction of CD63 transcripts, a tetraspanin determined to be necessary for differentiation of the intestinal epithelial cell line, hBRIE 380i cells.

Peptide YY (PYY) and neuropeptide Y (NPY) are peptides that coordinate intestinal activities in response to luminal and neuronal signals. In this study, using the rat hybrid small intestinal epithelial cell line, hBRIE 380i cells, we demonstrated that PYY- and NPY-induced rearrangement of actin filaments may be in part through a Y1alpha and/or a nonneuronal Y2 receptor, which were cloned from both the intestinal mucosa and the hBRIE 380i cells. A number of PYY/NPY-responsive genes were also identified by subtractive hybridization of the hBRIE 380i cells in the presence or absence of a 6-h treatment with PYY. Several of these genes coded for proteins associated with the cell cytoskeleton or extracellular matrix. One of these proteins was the transmembrane-4 superfamily protein CD63, previously shown to associate with beta(1)-integrin and implicated in cell adhesion. CD63 immunoreactivity, using antibody to the extracellular domain, was highest in the differentiated cell clusters of the hBRIE 380i cells. The hBRIE 380i cells transfected with antisense CD63 cDNA lost these differentiated clusters. These studies suggest a new role for NPY and PYY in modulating differentiation through cytoskeletal associated proteins.

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PubMed1999

Cell cycle and hormonal control of nuclear-cytoplasmic localization of the serum- and glucocorticoid-inducible protein kinase, Sgk, in mammary tumor cells. A novel convergence point of anti-proliferative and proliferative cell signaling pathways.

The serum- and glucocorticoid-inducible kinase (sgk) is a novel serine/threonine protein kinase that is transcriptionally regulated in rat mammary tumor cells by serum under proliferative conditions or by glucocorticoids that induce a G1 cell cycle arrest. Our results establish that the subcellular distribution of Sgk is under stringent cell cycle and hormonal control. Sgk is localized to the perinuclear or cytoplasmic compartment as a 50-kDa hypophosphorylated protein in cells arrested in G1 by treatment with the synthetic glucocorticoid dexamethasone. In serum-stimulated cells, Sgk was transiently hyperphosphorylated and resided in the nucleus. Laser scanning cytometry, which monitors Sgk localization and DNA content in individual mammary tumor cells of an asynchronously growing population, revealed that Sgk actively shuttles between the nucleus (in S and G2/M) and the cytoplasm (in G1) in synchrony with the cell cycle. In cells synchronously released from the G1/S boundary, Sgk localized to the nucleus during progression through S phase. The forced retention of exogenous Sgk in either the cytoplasmic compartment, using a wild type sgk gene, or the nucleus, using a nuclear localization signal-containing sgk gene (NLS-Sgk), suppressed the growth and DNA synthesis of serum-stimulated cells. Thus, our study implicates the nuclear-cytoplasmic shuttling of sgk as a requirement for cell cycle progression and represents a novel convergence point of anti-proliferative and proliferative signaling in mammary tumor cells.

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PubMed1998

Oleic acid distribution in small intestinal epithelial cells expressing intestinal-fatty acid binding protein.

Intestinal-fatty acid binding protein (I-FABP) has been proposed to target long chain fatty acids (LCFA) to triglyceride synthesis pathways in the intestinal epithelium. In the present studies hBRIE 380i cells, which endogenously express I-FABP only when fully differentiated, were used to investigate the role of I-FABP in LCFA incorporation and targeting by examining the relative distribution of [3H]-oleic acid in cellular lipids in these cells. [3H]-oleic acid incorporation into triglyceride was significantly higher in hBRIE 380i cells expressing I-FABP than in cells not expressing I-FABP. After 15 min, 1 and 4 h of incubation, cells expressing I-FABP incorporated 24.0%, 34.0% and 43.9% of [3H]-oleic acid into triglyceride, while newly confluent cells (no I-FABP expression) incorporated 15.6%, 18.3% and 31.9%. An I-FABP negative cell line, hBRIE 380i-neg cells, was stably transfected to investigate the effect of adding I-FABP to small intestinal epithelial cell lines. No measurable differences in [3H]-oleic acid incorporation into triglyceride was detected in these transfectants. Additionally I-FABP expression had no effect on [3H]-oleic acid incorporation or distribution within phospholipid subclasses in hBRIE 380i or transfected hBRIE 380i-neg fabpi cells. Our data from hBRIE 380i cells suggest that I-FABP can target LCFA to triglyceride synthesis pathway. However, endogenous I-FABP expression was also correlated to cellular differentiation and therefore raises the possibility that other differentiation-dependent factors may have a role in LCFA targeting. Because no effects of I-FABP were detected in the transfected hBRIE 380i-neg fabpi cells, it is concluded that factors in addition to I-FABP play a major role in determining the metabolic fate of LCFA in small intestinal epithelial cells. (c) 1998 Elsevier Science B.V.

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PubMed1997

Evidence for a role of the gut hormone PYY in the regulation of intestinal fatty acid-binding protein transcripts in differentiated subpopulations of intestinal epithelial cell hybrids.

Peptide tyrosine tyrosine (PYY) is a gut hormone present in endocrine cells in the lower intestine that can be released by the presence of luminal free fatty acids (FFAs). The biological action of this peptide includes inhibition of gut motility and gastrointestinal and pancreatic secretions. Intestinal fatty acid-binding protein (I-FABP) binds FFA and may be involved in their cytosolic trafficking. Quantitative in situ hybridization on heterogeneous populations of small intestinal somatic cell hybrids selected for endogenous I-FABP expression (hBRIE 380i cells) demonstrated a 5-fold increase in I-FABP transcripts in response to PYY (within 6 h) that was confined to clusters of differentiated cells, whereas ribonuclease protection assays performed on heterogeneous populations of these cells showed no significant differences. High affinity PYY receptors, with an IC50 of 5-50 pM, were identified in both differentiated and nondifferentiated cell populations, as determined by competitive binding assays and autoradiography. In situ hybridization of rat ileal tissue also revealed differing patterns of mRNA expression for liver fatty acid-binding protein (L-FABP) and I-FABP. Only I-FABP mRNA was detected in the villus tips. This localization correlated with the expression pattern of I-FABP mRNA in the hBRIE 380i cells where changes in transcripts were observed only in differentiated cells that did not incorporate bromodeoxyuridine. The sustained expression of I-FABP transcripts in the villar tips suggests (unlike L-FABP) that older terminally differentiated cell populations of the mucosa can still be PYY responsive. These studies demonstrate that physiological concentrations of PYY can regulate I-FABP and place this peptide in a key position as part of a feedback system that determines the processing of cytosolic FFA in the enterocyte. In addition, these studies suggest a mechanism whereby luminal agents can modulate expression of proteins in terminally differentiated cells in the gastrointestinal mucosa.

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PubMed1995

Intra-islet regulation of hormone secretion by glucagon-like peptide-1-(7--36) amide.

Glucagon-like peptide (GLP)-1-(7--36) amide, a peptide product of the posttranslational processing of pancreatic and intestinal proglucagon, has been shown to regulate insulin secretion. Monoclonal antibodies to glucagon and GLP-1-(7--36) amide were generated to localize GLP-1-(7--36) amide in the pancreatic islets by immunocytochemistry and radioimmunoassay. GLP-1-(7--36) amide immunoreactivity was found in some, but not all, glucagon-containing alpha-cells. Displaceable receptor binding for GLP-1-(7--36) amide and nonamidated GLP-1-(7--37) on hormone secretion were investigated using isolated pancreatic islet preparations. GLP-1-(7--37) and -(7--36) amide significantly increased insulin and somatostatin release in the concentration range of 0.01-100 nM in 11.0 mM glucose. GLP-1-(7--37) and -(7--36) amide had no effect on glucagon secretion in the presence of 11.0 mM glucose. GLP-1-(7--36) amide was released from isolated islets in response to 2.25, 5.5, and 11.0 mM glucose. These results suggest that pancreatic GLP-1 may be important in the regulation of intra-islet hormone secretion.

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PubMed1994

Expression of intestinal fatty acid binding protein in intestinal epithelial cell lines, hBRIE 380 cells.

Intestinal fatty acid binding protein (I-FABP) is a cytosolic protein present only in differentiated intestinal epithelial cells. Here we report on an intestinal cell culture system expressing I-FABP during cell differentiation and the modulation of expression by extracellular factors. An I-FABP-expressing cell line (hBRIE 380i) was generated from Berkeley rat intestinal epithelial cells (hBRIE 380). Time- and substratum-dependent changes in I-FABP mRNA expression were paralleled by changes in protein levels. Induction of I-FABP levels observed on collagen type I gels in the presence of limiting serum was prevented by insulin. When cells were grown on collagen gels containing fibronectin and laminin, a stimulation of ultrastructural characteristics of cell differentiation was observed with no further induction of I-FABP expression. The data show that I-FABP expression is limited to a differentiated population of hBRIE 380i cells and that the expression can be regulated by factors present in the extracellular matrix as well as involved in regulation of replication or metabolic state of the cell.

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PubMed1994

Expression of peptide YY in all four islet cell types in the developing mouse pancreas suggests a common peptide YY-producing progenitor.

The islets of Langerhans contain four distinct endocrine cell types producing the hormones glucagon, insulin, somatostatin and pancreatic polypeptide. These cell lineages are thought to arise from a common, multipotential progenitor cell whose identity has not been well established. The pancreatic and intestinal hormone, peptide YY, has been previously identified in glucagon-producing cells in islets; however, transgenic mice expressing Simian Virus 40 large T antigen under the control of the peptide YY gene expressed the oncoprotein in beta, delta and pancreatic polypeptide cells, and occasionally developed insulinomas, suggesting relationships between peptide YY-producing cells and several islet cell lineages. The four established pancreatic islet cell types were examined for coexpression of peptide YY in islets of normal and transgenic mice throughout development. Peptide YY immunoreactivity was identified in the earliest endocrine cells in the fetal pancreas and was coexpressed in each islet cell type during development. Peptide YY showed a high degree of co-localization with glucagon- and insulin-producing cells in early pancreatic development, but by adulthood, peptide YY was expressed in less than half of the alpha cells and was no longer expressed in beta cells. Peptide YY was also coexpressed with somatostatin and pancreatic polypeptide when these cell types first appeared, but most delta and pancreatic polypeptide cells continued to express peptide YY throughout development. The use of conditions that distinguish peptide YY from the related peptides, pancreatic polypeptide and neuropeptide Y, as well as the ability of the peptide YY gene to direct expression of a reporter gene in islets of transgenic mice, establishes expression of peptide YY in the earliest pancreatic endocrine cells.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1991

Polarized intestinal hybrid cell lines derived from primary culture: establishment and characterization.

A cell culture system has been developed that produces stable gastrointestinal (GI) polarized cell lines capable of maintaining hormone secretion. A spontaneously transformed rat mucosal epithelial cell was selected for hypoxanthine/guanine phosphoribosyltransferase deficiency and transfected with a plasmid conferring hygromycin resistance (BRIE 291 cells). Fusion of these cells with dispersed small intestinal epithelia cells resulted in hybrid cell lines that retained characteristic properties of the native GI cell more effectively than the transformed tumorigenic parental cell line. Hybrid hBRIE 380 cells are uniformly cuboidal with microvilli, contain villin, are contact inhibited, are anchorage dependent, require serum supplementation for growth, and are more sensitive to virus infection than the parental BRIE 291 cells. Fusion of BRIE 291 with dispersed pancreatic islet cells has resulted in a variety of pancreatic-hormone-producing cell lines. One of these, hybrid hBRIE 291-i2, forms confluent monolayers capable of synthesizing insulin-like immunoreactivity. These studies demonstrate that functionally polarized GI cells can be generated from primary cultures of nondividing committed epithelial cells by somatic cell hybridization and make feasible the selection and maintenance of specific GI epithelial cell types in confluent monolayer cultures.

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PubMed1989

The pathway of infection of Autographa californica nuclear polyhedrosis virus in an insect host.

An immunohistochemical study was conducted to detect the temporal infection sequence of Autographa californica M nuclear polyhedrosis virus in Trichoplusia ni larvae. Staining patterns indicated that the initial infection occurred in the midgut, simultaneously in columnar epithelial and regenerative cells, but that subsequently this tissue recovered. A major envelope glycoprotein stained in a polar fashion when it was expressed in columnar epithelial cells, but not when expressed in other cells types. Systemic infection was mediated by free virus for some tissues whereas infected hemocytes appeared to spread virus to other tissues by an unknown mechanism. A cell to cell spread within several tissues was detected. These results have important implications for baculoviruses engineered for improving their pesticide potential.

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PubMed1989

Molecular sorting in the secretory pathway.

Proteins can be secreted from animal cells by either a constitutive or a regulated pathway; those destined for regulated secretion are actively sorted into dense-core secretory granules. Although sorting is generally assumed to be accomplished by specific carriers, the nature of these carriers remains elusive. In this study, peptide hormones were used as affinity ligands to purify a set of 25-kilodalton proteins from canine pancreatic tissue. Their ligand specificities and patterns of expression have the characteristics of sorting carriers.

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PubMed1989

Meal-induced peptide tyrosine tyrosine inhibition of pancreatic secretion in the rat.

In the present studies we examined the distribution, release, and biological actions of peptide tyrosine tyrosine (PYY) in the rat. The concentration and distribution of PYY was highest in the ileum and colon as determined by both radioimmunoassay of rat tissue extracts and immunocytochemistry. An ultrastructural comparison of rat and dog colonic PYY cells revealed a bipolar distribution of peptide-containing secretory granules in both species. Serum PYY and pancreatic exocrine secretory responses were monitored after presentation of a meal to meal-trained rats (n = 12). A significant increase in PYY concentrations was not observed until 120 min after meal presentation, a delayed response similar to that previously observed in the dog. PYY responses were also observed in rats after perfusion of the intestine at the level of the duodenum and ileum with an 80 mOsm micellar solution of sodium oleate. Duodenal instillations of the fatty acids resulted in a maximum PYY response after 120 min, whereas rats subject to ileal perfusion of fat exhibited maximum PYY release within the first hour. In other experiments, infusion of exogenous PYY at 100 pmol.kg-1.h-1, which reproduced plasma PYY levels observed after a meal and perfusion of the gut with fat, significantly inhibited CCK-stimulated bile pancreatic volume (P less than 0.02), protein (P less than 0.01), and amylase (P less than 0.01) output. These studies demonstrate a bipolar distribution of PYY-containing secretory granules in cells of the jejunal, ileal, and colonic mucosa, and show that PYY is released in response to a meal in amounts sufficient to inhibit cholecystokinin-stimulated pancreatic secretion. Evidence is presented that PYY may mediate the delayed inhibition of pancreatic secretion that is observed in the rat after ingestion of a meal.

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PubMed1988

Glucocorticoid-regulated localization of cell surface glycoproteins in rat hepatoma cells is mediated within the Golgi complex.

Glucocorticoid hormones regulate the post-translational maturation and sorting of cell surface and extracellular mouse mammary tumor virus (MMTV) glycoproteins in M1.54 cells, a stably infected rat hepatoma cell line. Exposure to monensin significantly reduced the proteolytic maturation and externalization of viral glycoproteins resulting in a stable cellular accumulation of a single 70,000-Mr glycosylated polyprotein (designated gp70). Cell surface- and intracellular-specific immunoprecipitations of monensin-treated cells revealed that gp70 can be localized to the cell surface only in the presence of 1 microM dexamethasone, while in uninduced cells gp70 is irreversibly sequestered in an intracellular compartment. Analysis of oligosaccharide processing kinetics demonstrated that gp70 acquired resistance to endoglycosidase H with a half-time of 65 min in the presence or absence of hormone. In contrast, gp70 was inefficiently galactosylated after a 60-min lag in uninduced cells while rapidly acquiring this carbohydrate modification in the presence of dexamethasone. Furthermore, in the absence or presence of monensin, MMTV glycoproteins failed to be galactosylated in hormone-induced CR4 cells, a complement-selected sorting variant defective in the glucocorticoid-regulated compartmentalization of viral glycoproteins to the cell surface. Since dexamethasone had no apparent global effects on organelle morphology or production of total cell surface-galactosylated species, we conclude that glucocorticoids induce the localization of cell surface MMTV glycoproteins by regulating a highly selective step within the Golgi apparatus after the acquisition of endoglycosidase H-resistant oligosaccharide side chains but before or at the site of galactose attachment.

Animals↗
PubMed1988

Primary culture of PYY cells from canine colon.

In the present study we report methods for the isolation and culture of colonic peptide YY (PYY) cells and have tested the effects of sodium oleate and other chemotransmitters on PYY release. Enzyme-dispersed canine colonic mucosa cells were separated by a Beckman elutriation rotor, and the enriched PYY fraction was cultured on type I collagen in full growth medium. After 42-48 h of culture PYY cells had selectively adhered to the collagen. Forty to 45% of the adherent cells contained PYY (5.5 +/- 0.5 pmol/24-mm well), and 10-15% of the cells contained glucagon-like immunoactivity (GL29-LI; 0.95 pmol/24-mm well). The effect of various secretagogues on PYY release from these cultures was monitored. Sodium oleate stimulated PYY release in a time-dependent fashion over a dose range from 10 microM to 10 mM. However, sodium oleate at a dose of 100 mM produced disproportionately large PYY release. During a 2-h incubation 5.1% of the total cell content of PYY was released in response to 0.1 mM sodium oleate compared with basal release of 1.1%. At doses less than 10 mM sodium oleate did not release GL29-LI, whereas concentrations of 10 and 100 mM resulted in marked GL29-LI release. These findings suggest that lower concentrations of sodium oleate selectively release PYY, whereas higher concentrations nonselectively induce peptide release probably by effects on membrane stability. We also found that bombesin, epinephrine, and forskolin, but not carbachol, produced time- and dose-dependent release of PYY from these cultures.

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PubMed1985

Identification and characterization of glycine-extended post-translational processing intermediates of progastrin in porcine stomach.

We developed a radioimmunoassay specific for glycine-extended progastrin processing intermediates (G-Gly) using antisera generated against the synthetic peptide Tyr-Gly-Trp-Met-Asp-Phe-Gly. Distribution of immunoreactivity in the porcine gastrointestinal tract obtained with this antibody paralleled that of gastrin with the mucosa containing the highest quantity, 116 +/- 22 pmol/g, wet weight (mean +/- S.E., n = 5), or roughly 4% of gastrin concentration. This immunoreactivity was localized specifically to antral mucosal G-cells by immunohistochemistry. On Sephadex G-50 column chromatography of porcine antral mucosal extracts glycine-extended progastrin processing intermediates were separated into three principal molecular forms, each corresponding to known molecular forms of gastrin, component I, tetratriacontagastrin (G34) and heptadecagastrin (G17). Following purification by antibody-coupled affinity chromatography, one molecular form corresponding to G17 in size was shown to have an amino terminus identical to that of G17. Another molecular form corresponding to G34 in size could be converted to the molecular form corresponding to G17 by tryptic digestion. Our findings indicate that glycine-extended progastrin processing intermediates may serve as immediate precursors for each molecular form of gastrin, thus suggesting an alternative pathway for gastrin biosynthesis more complex than that previously conceived.

Animals↗
PubMed1985

Regional distribution and release of peptide YY with fatty acids of different chain length.

The present study examines peptide YY responses to regional intestinal perfusion of fatty acids of different chain length--dodecanoate and oleate. Six dogs with chronic gastric, duodenal, and jejunal fistulas were studied. Proximal perfusates were administered into the duodenum and diverted through an intestinal fistula placed 45 cm beyond the duodenal cannula. Distal perfusates were administered into the caudal stoma of this intestinal stoma. Peptide YY responses to proximal, distal, and whole-gut perfusion were compared. Proximal perfusion with oleate or dodecanoate failed to release peptide YY. In contrast, distal and whole-gut perfusion with either fatty acid produced significant increases that were of similar magnitude. Immunocytochemical studies demonstrated that peptide YY cells predominated in the canine ileocolonic mucosa and decreased progressively in an orad direction. We conclude that peptide YY release is not dependent on fatty acid chain length and that the duodenum does not play a dominant role in peptide YY release. As such, peptide YY release differs from that of its cousin pancreatic polypeptide and may result at least in part from direct stimulation of the peptide YY cell in the ileocolonic mucosa.

Animals↗
PubMed1983

Somatostatin-like immunoreactivity in the glomerulus of rat kidney.

It has been shown that somatostatin inhibits the antidiuretic action of vasopressin in toad urinary bladder in vitro and in dogs and rats in vivo. The presence of somatostatin-like immunoreactivity (SLI) in the urinary bladder and kidney of the toad has suggested the possibility that somatostatin may act as a local paracrine hormone modulating the effect of vasopressin in the toad; however, the inability to localize SLI in mammalian kidney has raised doubt about the physiological role of somatostatin in the mammalian renal function. We identified SLI in rat kidney. Chromatography of rat kidney extracts of Sephadex G50 superfine revealed a single peak of SLI that co-eluted with somatostatin-14. Using the avidin-biotin-peroxidase conjugate technique, we localized SLI exclusively to small cells in the glomerulus with an estimated number of four to eight cells per glomerulus. Functional significance of somatostatin in the mammalian kidney is to be determined.

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