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J A Cohn

Publications and source records attributed to J A Cohn.

At least 55 records · Page 3Linked to original sources
PubMed1992

Pituitary adenylate cyclase-activating polypeptide stimulates secretion in T84 cells.

Pituitary adenylate-cyclase-activating peptide (PA-CAP) and PACAP-27 are novel hypothalamic peptides that can stimulate adenylate cyclase in cultured anterior pituitary cells. Because these peptides are present in the gut and are homologous with vasoactive intestinal peptide (VIP), itself known to stimulate intestinal ion transport, we examined the effects of these peptides on the T84 colonocyte cell line. Using cells grown on semipermeable supports and mounted in Ussing chambers, we showed that PACAP and PACAP-27 potently activate intestinal secretion. The half-maximal secretory response was produced with 0.5 nmol/L PA-CAP and 0.1 nmol/L PACAP-27. PACAP resembled VIP in that it stimulated a secretory response potentiated by carbachol, inhibited by bumetanide and barium chloride, and not further stimulated by the subsequent addition of VIP. Like VIP, PACAP also stimulated 5' cyclic adenosine monophosphate (cAMP) production and the phosphorylation of cellular proteins known to be substrates for cAMP-dependent protein kinase. In addition, PACAP inhibited 125I-VIP binding to T84 cells, and the secretion it stimulated was reduced by the VIP receptor antagonist, L-8-K. Thus PACAP and PACAP-27 potently stimulate colonocyte ion transport via mechanisms mediated by the VIP receptor and cAMP-dependent signaling.

Cell Line↗
PubMed1992

Human cystic fibrosis transmembrane conductance regulator directed to respiratory epithelial cells of transgenic mice.

Human cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in transgenic mice under the control of transcriptional elements derived from the human surfactant protein C (SP-C) gene. The hCFTR mRNA was expressed in lungs and testes: in the lung, we found hCFTR mRNA in bronchiolar and alveolar epithelial cells, and CFTR protein in respiratory epithelial cells. While the level of expression of hCFTR mRNA varied, hCFTR mRNA and protein were detected in pulmonary epithelial cells of several lines. Lung weight, morphology, somatic growth and reproductive capacity were not altered by expression hCFTR in lung and testes of the transgenics. Our findings suggest that hCFTR can be safely transferred to lung epithelial cells for CF therapy.

Animals↗
PubMed1992

Submucosal glands are the predominant site of CFTR expression in the human bronchus.

We have used in situ hybridization and immunocytochemistry to characterize the cellular distribution of cystic fibrosis (CF) gene expression in human bronchus. The cystic fibrosis transmembrane conductance regular (CFTR) was primarily localized to cells of submucosal glands in bronchial tissues from non-CF individuals notably in the serous component of the secretory tubules as well as a subpopulation of cells in ducts. Normal distribution of CFTR mRNA was found in CF tissues while expression of CFTR protein was genotype specific, with delta F508 homozygotes demonstrating no detectable protein and compound heterozygotes expressing decreased levels of normally distributed protein. Our data suggest mechanisms whereby defects in CFTR expression could lead to abnormal production of mucus in human lung.

Bronchi↗
PubMed1991

CFTR: development of high- affinity antibodies and localization in sweat gland.

Rabbit antisera were raised to six synthetic peptides corresponding to amino acid sequences contained in the protein product of the cystic fibrosis gene, CFTR. For two peptides, [Lys102]CFTR(102-116) and CFTR(1468-1480), antibody-peptide binding was of high affinity in that half-maximal binding occurred at peptide concentrations below 10 nM. Monospecific antibodies were prepared using these peptides, and these antibodies were used to stain human skin. Specific staining was detected in the cells lining the reabsorptive duct of the sweat gland. Within these lumenal cells, staining was most prominent at the apical domain but was also detected near the basolateral surface. This finding agrees well with predictions based on the effects of cystic fibrosis on sweat gland function, and suggests that these antibodies will be useful for studying CFTR in other human tissues.

Amino Acid Sequence↗
PubMed1991

Stimulation of secretion by the T84 colonic epithelial cell line with dietary flavonols.

Flavonols are dietary compounds widely distributed in plants and characterized by a 2-phenyl-benzo(alpha)pyrane nucleus possessing hydroxyl and ketone groups at positions 3 and 4, respectively. Kaempferol, quercetin, and myricetin are flavonols that are further mono-, di-, or trihydroxylated on the phenyl ring, respectively. To test whether these ingested flavonols might exert a direct secretory effect on intestinal epithelial cells, monolayers of the T84 colonocyte cell line were mounted in Ussing chambers and examined for ion transport response. Twenty minutes after addition of 100 microM quercetin to either the serosal or mucosal side, the short-circuit current change was maximal at 16.6 microA/cm2. Kaempferol was less potent than quercetin, while myricetin and glycosylated quercetin (rutin) did not induce secretion. The secretion induced by quercetin did not seem to be mediated by the reactive oxygen species generated by quercetin through auto-oxidation and/or redox cycling (superoxide, hydrogen peroxide, and the hydroxyl radical) because it was neither enhanced by iron, nor inhibited by desferroxamine B or catalase (alone or in combination with superoxide dismutase). Like vasoactive intestinal peptide, quercetin induced a secretory response that was inhibited by barium chloride and bumetanide, and which exhibited synergism with carbachol. Quercetin also stimulated a modest increase in intracellular cAMP levels and the phosphorylation of endogenous protein substrates for cAMP-dependent protein kinase. Thus, quercetin is a potent stimulus of colonocyte secretion that resembles secretagogues which act via a cAMP-mediated signaling pathway.

Carbachol↗
PubMed1991

Localization of the cystic fibrosis transmembrane conductance regulator in pancreas.

Cystic fibrosis (CF) is characterized by an abnormality in cAMP-regulated chloride transport that results from a primary defect in the protein product of the CF gene, the CF transmembrane conductance regulator (CFTR). In this report, antibodies against CFTR peptides were used to localize the CFTR protein in human pancreas. An affinity purified antibody (alpha-1468) raised against a synthetic CFTR peptide identified a 155-170-kD protein on immunoblot. Cytochemical studies with alpha-1468 localized CFTR to small branching, tubular structures. The same structures were recognized by two other antibodies raised against different regions of the CFTR molecule. To identify the cells being stained, double-label immunofluorescence studies were performed using alpha-1468 and a monoclonal antibody which stains pancreatic centroacinar and intralobular duct cells. Both antibodies localized to the same population of cells, with alpha-1468 being confined to the apical domain of these cells. No conclusive staining of acinar cells was evident. These findings suggest that proximal duct epithelial cells play a key role in the early events leading to pancreatic insufficiency in CF, and imply that apical chloride transport by these cells is essential for normal pancreatic secretory function.

Amino Acid Sequence↗
PubMed1991

cAMP analogs inhibit gamma-aminobutyric acid-gated chloride flux and activate protein kinase A in brain synaptoneurosomes.

The effects of permeant cAMP analogs were studied on the function of the gamma-aminobutyric acidA (GABAA) receptor and on the activation of protein kinase A in brain synaptoneurosomes. Incubation of cerebral cortical synaptoneurosomes with permeant cAMP analogs decreased muscimol-induced 36Cl- uptake in a concentration-dependent manner. The order of potency was chlorophenylthio-cAMP (CPT-cAMP) greater than dibutyryl-cAMP greater than 8-bromo-cAMP. This order of potency was reflected by the ability of the analogs to gain access to the intravesicular compartment. cAMP, which failed to penetrate the membrane, had no effect. The half-maximal and maximal effects of the cAMP analogs were similar in the cerebral cortex, hippocampus, striatum, and cerebellum. To determine whether the cAMP analogs were acting through the activation of protein kinase A, protein kinase A activity was measured in lysed synaptoneurosomes, using kemptide as the substrate. In the lysed preparation, where the cAMP analogs have direct access to intracellular enzymes, the order of potencies of the cAMP analogs to activate protein kinase A (8-bromo-cAMP greater than CPT-cAMP greater than dibutyryl-cAMP) differed from the order of potencies to inhibit muscimol-induced 36Cl- uptake. In regional studies, the greatest effect of CPT-cAMP was observed in the cortex, whereas the smallest effect was observed in the hippocampus and cerebellum. To determine whether cAMP inhibition of GABA-gated ion flux was due to activation of protein kinase A, the time course for each response was measured. Inhibition of muscimol-induced 36Cl- uptake by cAMP analogs was nearly complete by 5 sec. Significant activation of protein kinase A by CPT-cAMP was also observed as early as 5 sec, but protein kinase A activation continued up to 10 min. The protein kinase inhibitor peptide inhibited protein kinase A activity in lysed synaptoneurosomes but had no effect on ion flux in intact synaptoneurosomes, as expected. However, a permeant kinase inhibitor, H-8, also failed to inhibit the effect of cAMP analogs on the muscimol response, yet it inhibited protein kinase A activity. The failure of H-8 to inhibit cAMP analog effects on GABAA receptor function was most likely due to the presence of ATP inside the synaptoneurosomes, because H-8 inhibition of protein kinase A was reduced in the presence of ATP. These results indicate that cAMP and cAMP analogs must penetrate the intravesicular compartment to inhibit GABAA receptor function. Although cAMP analogs decrease GABA-gated ion flux under conditions in which they activate protein kinase A, a causal relationship remains to be established.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1990

Giant two-dimensional gel electrophoresis: methodological update and comparison with intermediate-format gel systems.

Two-dimensional (2-D) gel electrophoresis methods for separating complex mixtures of proteins have not changed fundamentally since their original description in the late 1970's. Nevertheless, 2-D gel resolution has improved substantially as a result of a series of incremental modifications. One of these was the development of the "giant-gel" format, using gels measuring at least 30 x 30 cm to provide the highest resolution 2-D gel system available. As originally described, this procedure has several important limitations: it requires custom-built equipment; it is expensive in terms of time, reagents, film and support matrices; and it generates gels which are difficult to manupulate, particularly for silver staining. This report describes modifications in the giant gel procedure to permit use of a commercially available gel apparatus and to obtain giant gels of improved mechanical strength suitable for silver staining. The resolution of giant gels is compared with that obtained using two systems currently being marketed for use by laboratories performing large numbers of 2-D gel analyses. The smaller format gels resolved fewer proteins, by 30-40%, compared with the giant gels. This difference in resolving power suggests that giant gels will continue to be useful in selected applications.

Autoradiography↗
PubMed1990

Protein kinase C mediates cholinergically regulated protein phosphorylation in a Cl(-)-secreting epithelium.

T84 cell monolayers were used to study the cholinergic regulation of protein phosphorylation in epithelial cells. When T84 cell monolayers are labeled with 32Pi and stimulated with carbachol, six proteins exhibit altered phosphorylation. The most prominent response is a fivefold increase in labeling of p83, an acidic protein of Mr 83,000. Increasing labeling of p83 parallels stimulated secretion with respect to the onset of agonist action, agonist potency, and antagonism by atropine. However, the p83 and secretory responses differ in that the p83 response is more sustained. When T84 cell fractions are incubated with [gamma-32P]ATP, Ca2(+)-phospholipid stimulates p83 labeling. Phosphorylation of p83 also occurs when a T84 cell extract is incubated with purified protein kinase C and when intact cells are exposed to phorbol myristate acetate. p83 does not become phosphorylated in cell fractions incubated with adenosine 3',5'-cyclic monophosphate (cAMP) or in monolayers stimulated with agonists acting via cAMP. Thus carbachol stimulates the phosphorylation of an endogenous substrate for protein kinase C in T84 cells. The duration of this phosphorylation response suggests that protein kinase C may mediate a sustained response to carbachol, possibly acting to limit the duration of stimulated secretion.

Animals↗
PubMed1990

Immune-related intestinal chloride secretion. II. Effect of adenosine on T84 cell line.

The inflammatory mediator adenosine caused sustained Cl- secretion across monolayers of T84 cells. The effect was promptly reversed by the adenosine receptor antagonist 8-phenyltheophylline and appeared to be mediated through an adenosine A2-receptor [rank order of potency: 5'-(N-ethyl)-carboxamido-adenosine (NECA) greater than adenosine greater than (-)-N6-(phenylisopropyl)adenosine (PIA) greater than or equal to (+)-PIA]. High doses of adenosine and its analogues increased cellular adenosine 3',5'-cyclic monophosphate (cAMP) but not guanosine 3',5'-cyclic monophosphate (cGMP) or free cytosolic Ca2+. However, lower concentrations of adenosine had maximal effects on Cl- secretion with little or no effect on cAMP. In other respects, Cl- secretion resembled that induced by cAMP-mediated secretagogues such as vasoactive intestinal peptide (VIP). Addition of both low and high doses of NECA activated basolateral K+ and apical Cl- channels, exhibited synergism with Ca2(+)-mediated secretagogues, did not produce additive effects with VIP or Escherichia coli heat-stable enterotoxin, and was associated with cAMP-dependent protein kinase-mediated protein phosphorylation. The results suggest that either adenosine mobilizes an intracellular pool of cAMP that is extremely efficiently coupled to the cAMP-dependent protein kinase and is thereafter rapidly destroyed or that second messenger(s) other than cAMP, cGMP, or Ca2+ are able to activate Cl- secretion in the T84 cell line. In the latter case, such messenger(s), as yet unidentified, might represent a final common pathway for cyclic nucleotide-activated Cl- secretion.

2-Chloroadenosine↗
PubMed1989

Histamine stimulates calcium-mediated protein phosphorylation in a colonic epithelial cell line.

Protein phosphorylation responses in intact enterocytes were examined by stimulating 32Pi-labeled T84 cell monolayers with histamine and resolving proteins by two-dimensional gel electrophoresis. Histamine increases 32P-incorporation into two acidic proteins of Mr 83,000 and of Mr 29,000, designated p83 and p29. Labeling of p83 and p29 is also increased in cells exposed to ionomycin, but not in cells exposed to vasoactive intestinal peptide under conditions resulting in cAMP-mediated secretion and cAMP-stimulated protein phosphorylation. When T84 cell fractions are incubated with [gamma-32P]ATP, labeling of p83 is stimulated by Ca++, but not by cAMP. Thus, histamine stimulates Ca++-mediated protein phosphorylation during the regulation of Cl- secretion.

Animals↗
PubMed1989

Evaluation of the policy of empiric treatment of suspected Toxoplasma encephalitis in patients with the acquired immunodeficiency syndrome.

PURPOSE: This study was designed to measure response rates and survival in patients with acquired immunodeficiency syndrome (AIDS) and suspected Toxoplasma encephalitis treated empirically and in AIDS patients treated for biopsy-proven toxoplasmosis. PATIENTS AND METHODS: AIDS patients identified at Bellevue Hospital between August 1985 and May 1986, who had abnormal computed tomographic scans of the brain and who received empiric treatment for toxoplasmosis, constitute the empirically treated cohort. A cohort with biopsy-proven toxoplasmosis was identified from Bellevue Hospital neuropathology records spanning 1981 through 1986. Patient records were reviewed with a standardized data form, and tomograms were evaluated by neuroradiologists unaware of the identity of the scans. Survival analysis was performed by the product limit method. RESULTS: Of 38 empirically treated patients, 26 responded clinically and radiographically within four weeks of initiation of therapy. Four of nine patients who underwent biopsy responded to treatment. There was no difference in these response rates (68% versus 44%, p = 0.24). The median survival of the empirically treated responders, from first diagnosis of AIDS to last follow-up, was 422 days. Among the 30 responders, five patients discontinued therapy and four of them had relapses. No relapses occurred in the 25 patients who continued full-dose therapy indefinitely (p = 0.0004). Sixteen of 30 patients (53%) receiving continuous therapy developed toxicity, which required a change in medication. There was no difference in the survival of patients who continued to receive sulfadiazine and pyrimethamine compared with those in whom clindamycin was substituted for sulfadiazine (median, 311 days versus 422 days, p = 0.25). CONCLUSION: A policy of empiric treatment of suspected Toxoplasma encephalitis is satisfactory, and patients who respond to such therapy and continue to take full therapeutic doses of anti-Toxoplasma drugs have relatively long survivals.

Acquired Immunodeficiency Syndrome↗
PubMed1987

Purification and properties of a multifunctional calcium/calmodulin-dependent protein kinase from rat pancreas.

A calcium/calmodulin-dependent protein kinase (Ca/calmodulin protein kinase) was purified from rat pancreas using hydrophobic chromatography followed by gel filtration and affinity chromatography. Ca/calmodulin protein kinase from pancreas resembled previously described multifunctional Ca/calmodulin protein kinases from other tissues with respect to substrate specificity, autophosphorylation on serine and threonine residues, and catalytic and hydrodynamic properties. While Ca/calmodulin protein kinase from other tissues contains subunits of 53-60 kDa with variable proportions of a smaller 50-52 kDa subunit, pancreatic Ca/calmodulin protein kinase was found to contain a single component of 51 kDa. Experiments mixing brain Ca/calmodulin protein kinase with pancreatic homogenate suggest that the absence of a larger subunit in the pancreatic Ca/calmodulin protein kinase is not due to proteolytic degradation during enzyme preparation. Ca/calmodulin protein kinase binding to 125I-labeled calmodulin in solution was demonstrated using the photoaffinity cross-linker, N-hydroxysuccinimidyl-4-azidobenzoate. 125I-labeled calmodulin binding to Ca/calmodulin protein kinase was also demonstrated using filters containing Ca/calmodulin protein kinase transferred from polyacrylamide gels after two-dimensional gel electrophoresis. Finally, the ribosomal substrate for Ca/calmodulin protein kinase was identified as the ribosomal protein, S6. The purification procedure presented in this study promises to be useful in characterizing Ca/calmodulin protein kinase in other tissues and in clarifying the role of these enzymes in cellular function.

Animals↗
PubMed1987

Vasoactive intestinal peptide stimulates protein phosphorylation in a colonic epithelial cell line.

The T84 colonic epithelial cell line was used to examine protein phosphorylation during neurohumoral stimulation of ion transport. T84 cell monolayers grown on collagen-coated filters were mounted in Ussing chambers to measure ion transport stimulated by vasoactive intestinal peptide. Maximal stimulation of active secretion occurred after 8-10 min of stimulation. Protein phosphorylation events accompanying stimulated secretion were detected using two-dimensional gel electrophoresis to resolve phosphoproteins from monolayers previously labeled using 32Pi. Within 8 min of exposure to vasoactive intestinal peptide, several phosphorylation events were detected, including a two- to fivefold increase in 32P incorporation into four soluble proteins with apparent molecular weights of 17,000, 18,000, 23,000, and 37,000. The same phosphorylation response occurs in monolayers stimulated by dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP), suggesting that cAMP mediates these intracellular events. This study indicates that changes in protein phosphorylation accompany the secretory action of vasoactive intestinal peptide and suggests that T84 cells offer a useful model for studying the possibility that such phosphorylation events regulate enterocyte ion transport.

Bucladesine↗
PubMed1983

Calmodulin-stimulated protein kinase activity from rat pancreas.

Previous work from our laboratory has demonstrated that neurohumoral stimulation of the exocrine pancreas is associated with the phosphorylation of the Mr 29,000 ribosomal protein S6. In a cell-free system using pancreatic postmicrosomal supernatant as the kinase donor, we found that the following co-factors stimulate the phosphorylation of the Mr 29,000 ribosomal protein: calcium with calmodulin, calcium with phosphatidyl serine, and cAMP. These findings suggest that the pancreas contains a calcium-calmodulin-dependent protein kinase (CaM-PK) that can phosphorylate the Mr 29,000 ribosomal protein. A CaM-PK activity was partially purified sequentially by ion exchange, gel filtration, and calmodulin-affinity chromatography. Phosphorylation of the Mr 29,000 ribosomal protein by the partially purified CaM-PK was dependent on the presence of both calcium and calmodulin and not on the other co-factors. The CaM-PK fraction contained a phosphoprotein of Mr 51,000 whose phosphorylation was also dependent on calcium and calmodulin. When 125I-calmodulin-binding proteins from the CaM-PK fraction were identified using electrophoretic transfers of SDS-polyacrylamide gels, a single Mr 51,000 protein was labeled. The preparation enriched in CaM-PK activity contained an Mr 51,000 protein that underwent phosphorylation in a calcium-calmodulin-dependent manner and an Mr 51,000 calmodulin-binding protein. It is therefore possible that the CaM-PK may comprise a calmodulin-binding phosphoprotein component of Mr 51,000.

Animals↗