Biomedical subjects

B L Bass

Publications and source records attributed to B L Bass.

At least 37 records · Page 2Linked to original sources

PubMed1999

Subsets of the zinc finger motifs in dsRBP-ZFa can bind double-stranded RNA.

dsRBP-ZFa is a Xenopus zinc finger protein that binds dsRNA and RNA-DNA hybrids with high affinity and in a sequence-independent manner. The protein consists of a basic N-terminal region with seven C2H2 zinc finger motifs and an acidic C-terminal region that is not required for binding. The last four zinc finger motifs, and the linkers that join them, are nearly identical repeats, while the first three motifs and their linkers are each unique. To identify which regions of the protein are involved in nucleic acid binding, we examined the ability of five protein fragments to bind dsRNA and RNA-DNA hybrids. Our studies reveal that a fragment encompassing the three N-terminal, unique zinc finger motifs and another encompassing the last three of the nearly identical motifs have binding properties similar to the full-length protein. Since these two fragments do not share zinc finger motifs of the same sequence, dsRBP-ZFa must contain more than one type of zinc finger motif capable of binding dsRNA. As with the full-length protein, ssRNA and DNA do not significantly compete for dsRNA binding by the fragments.

Amino Acid Sequence↗

PubMed1999

Femur fracture induces site-specific changes in T-cell immunity.

BACKGROUND: Trauma is associated with altered host defense and susceptibility to infection, in part due to cytokine dysregulation and altered T-cell immunity. The gut-associated lymphoid tissue (GALT) provides a defense against infection and contributes to the process of mucosal healing by T-cell activation and cytokine production. OBJECTIVE: To determine whether femur fracture induces alterations in Peyer's patch and splenic T-cell phenotype, proliferative response, and cytokine expression following traumatic injury. METHODS: Mice underwent femur fracture or sham procedure and, 48 h later, lymphocytes were isolated from spleen and Peyer's patches. Lymphocytes were cultured, and lipopolysaccharide (10 microg/ml) was added in some cultures. Cells and supernatant were harvested at 48 h. Proliferation was analyzed by [3H]thymidine, and interleukin-10 (IL-10) protein was measured by ELISA in the culture supernatant. T-cell phenotype was determined by flow cytometry. RESULTS: Femur fracture induced a significant increase in proliferative response in Peyer's patch immunocytes. In contrast, no significant differences were identified in splenocyte proliferative response 48 h after femur fracture injury. Femur fracture induced a significant decrease in IL-10 protein expression of both splenocytes and Peyer's patches. Femur fracture also induced a significant increase in the fraction of CD3(+), CD4(+), and T-cell receptor-alpha beta Peyer's patch immunocytes, whereas splenocytes demonstrated no significant phenotypic change. CONCLUSION: Femur fracture is associated with significant alterations in Peyer's patch but not splenic T-cell phenotype and proliferative response early (48 h) after injury. Changes in the GALT immune response may contribute to intestinal mucosal dysfunction and increased susceptibility to gut-derived sepsis after traumatic injury.

Animals↗

PubMed1999

Interstitial cellular and matrix restoration of cardiac valves after cryopreservation.

OBJECTIVES: We previously characterized the porcine aortic leaflet interstitial cell phenotype as having both synthetic and contractile characteristics; that is, it is a myofibroblast. In this study we hypothesized (1) that the cryopreservation of aortic valves causes a significant reduction in cell density, (2) that it simultaneously causes alterations in representative components of extracellular matrix, and (3) that both of these processes are reversible. METHODS: Seventy-two leaflets from 24 porcine aortic valves were studied. Whole valves were subjected to variable lengths of preharvest ischemia (group 1), ischemia followed by processing analogous to clinical methods (group 2), and ischemia followed processing with an organ culture type of resuscitation (group 3). Vital dye exclusion by cells enzymatically dispersed from leaflets was used to quantify viability. Electron and light microscopy, immunohistochemical assay, and a silicone rubber substratum contractility assay were used both in dispersed cell preparations and in leaflet cross sections to examine structural, ultrastructural, and functional changes across the 3 groups through a range of preharvest ischemic times. RESULTS: Results indicated that harvest ischemic periods between 2 and 24 hours after donor death were not responsible for cell number reductions. During this interval overt dissolution of chondroitin sulfate simultaneous with a relative sparing of fibronectin was evidenced by immunohistochemical staining. Although not reduced in number, ischemic interstitial cells did show significant ultrastructural evidence of injury and suppressed monoclonal binding to vimentin and alpha-smooth muscle actin. After cryopreservation, viable cell numbers were always markedly reduced at all ischemic intervals and damage to both soluble extracellular matrix components and cell ultrastructure was increased. At all time and processing points, however, some retention of matrix secretory and cellular contractile capabilities was observed among the surviving cells. After the extended periods of preharvest ischemia (2-24 hours) followed by processing, a restitution of functioning cells was accomplished by means of whole-leaflet incubation in 15% fetal bovine serum. CONCLUSIONS: After application of the described methods, new cells within restored intact leaflets as well as in single-cell preparations demonstrated normal ultrastructure and contractile and synthetic functions (normal phenotypic expression). If functioning leaflet interstitial cells can contribute to homograft durability, bioengineering methods for pretransplantation cell repopulation could be refined with these techniques and applied to clinical valve transplantation.

Actins↗

PubMed1999

A minor fraction of basic fibroblast growth factor mRNA is deaminated in Xenopus stage VI and matured oocytes.

Adenosine deaminases that act on RNA (ADARs) convert adenosine to inosine in double-stranded regions of RNA. ADAR activity is in the nucleus in Xenopus laevis stage VI oocytes, and released into the cytoplasm at oocyte maturation. We previously demonstrated that a cytoplasmic double-stranded RNA (dsRNA) binding factor(s), cyto-dsRBP, protects microinjected dsRNA from the ADAR released at maturation. Here we describe experiments to determine whether an endogenous dsRNA, the duplex formed between sense and antisense transcripts of basic fibroblast growth factor (bFGF), is protected in a similar manner. Consistent with the presence of cyto-dsRBP, we observed that the majority of bFGF RNA was not deaminated, before or after maturation. However, a minor fraction of the bFGF RNA was deaminated whether the RNA was isolated from stage VI oocytes or matured oocytes. Since ADAR activity is in the nucleus in stage VI oocytes, our results suggest that a fraction of the bFGF RNAs are hybridized in the nucleus and are ADAR substrates. Adenosine deaminations result in A-to-G changes in cDNAs, so we quantified the fraction of modified molecules using restriction-enzyme assays of RT-PCR products. Caveats due to recombination during RT-PCR are discussed.

Adenosine Deaminase↗

PubMed1999

Inhibition of polyamine synthesis induces p53 gene expression but not apoptosis.

The nuclear phosphoprotein p53 acts as a transcription factor and is involved in growth inhibition and apoptosis. The present study was designed to examine the effect of decreasing cellular polyamines on p53 gene expression and apoptosis in small intestinal epithelial (IEC-6) cells. Cells were grown in DMEM containing 5% dialyzed fetal bovine serum in the presence or absence of alpha-difluoromethylornithine (DFMO), a specific inhibitor of polyamine biosynthesis, for 4, 6, and 12 days. The cellular polyamines putrescine, spermidine, and spermine in DFMO-treated cells decreased dramatically at 4 days and remained depleted thereafter. Polyamine depletion by DFMO was accompanied by a significant increase in expression of the p53 gene. The p53 mRNA levels increased 4 days after exposure to DFMO, and the maximum increases occurred at 6 and 12 days after exposure. Increased levels of p53 mRNA in DFMO-treated cells were paralleled by increases in p53 protein. Polyamines given together with DFMO completely prevented increased expression of the p53 gene. Increased expression of the p53 gene in DFMO-treated cells was associated with a significant increase in G1 phase growth arrest. In contrast, no features of programmmed cell death were identified after polyamine depletion: no internucleosomal DNA fragmentation was observed, and no morphological features of apoptosis were evident in cells exposed to DFMO for 4, 6, and 12 days. These results indicate that 1) decreasing cellular polyamines increases expression of the p53 gene and 2) activation of p53 gene expression after polyamine depletion does not induce apoptosis in intestinal crypt cells. These findings suggest that increased expression of the p53 gene may play an important role in growth inhibition caused by polyamine depletion.

Animals↗

PubMed1999

Differentiated intestinal epithelial cells exhibit increased migration through polyamines and myosin II.

Early mucosal restitution is a rapid process by which differentiated intestinal epithelial cells migrate to reseal superficial wounds. However, most of the in vitro studies for restitution employ undifferentiated intestinal crypt cells as a model. The transcription factor, Cdx2, plays an important role in the regulation of intestinal epithelial differentiation. Forced expression of the Cdx2 gene in undifferentiated intestinal crypt cells induces the development of a differentiated phenotype. The current study was designed to determine changes in differentiated intestinal epithelial cell migration after wounding in the stable Cdx2-transfected IEC-6 cells and then to examine involvement of polyamines and nonmuscle myosin II in the process of cell motility. Cdx2-transfected IEC-6 cells were associated with a highly differentiated phenotype and exhibited increased cell migration after wounding. Migration of Cdx2-transfected IEC-6 cells were approximately four times that of nontransfected IEC-6 cells. Migration after wounding was associated with significant increases in polyamine synthesis. Depletion of cellular polyamines by 5 mM alpha-difluoromethylornithine (DFMO), a specific inhibitor of polyamine biosynthesis, inhibited cell migration without affecting the differentiated phenotype. DFMO also decreased levels of nonmuscle myosin II mRNA and protein and resulted in reorganization of myosin II, along with a marked reduction in stress fibers. Exogenous spermidine given together with DFMO not only returned nonmuscle myosin II levels and cellular distribution toward normal but also restored cell migration to control levels. These results indicate that 1) Cdx2-transfected IEC-6 cells exhibit increased cell migration after wounding and 2) cellular polyamines are absolutely required for stimulation of cell migration in association with their ability to modulate the structural organization of nonmuscle myosin II.

Animals↗

PubMed1998

Inosine exists in mRNA at tissue-specific levels and is most abundant in brain mRNA.

The general view that mRNA does not contain inosine has been challenged by the discovery of adenosine deaminases that act on RNA (ADARs). Although inosine monophosphate (IMP) cannot be detected in crude preparations of nucleotides derived from poly(A)+ RNA, here we show it is readily detectable and quantifiable once it is purified away from the Watson-Crick nucleotides. We report that IMP is present in mRNA at tissue-specific levels that correlate with the levels of ADAR mRNA expression. The amount of IMP present in poly(A)+ RNA isolated from various mammalian tissues suggests adenosine deamination may play an important role in regulating gene expression, particularly in brain, where we estimate one IMP is present for every 17 000 ribonucleotides.

Adenosine Deaminase↗

PubMed1998

Acute effects of bile acids on the pancreatic duct epithelium in vitro.

BACKGROUND: Acute pancreatitis is associated with passage of gallstones, although the mechanism(s) linking the two processes remains undefined. Bile reflux into the pancreatic duct could play a role but the experimental conditions often employed to induce pancreatitis rarely develop clinically. Here we examined whether low concentrations of bile affect ductal electrophysiology as an indirect measure of ductal epithelial integrity and function in vitro. METHODS: The main duct was dissected out of freshly harvested bovine pancreata, cut into 1- x 2-cm sections, placed in tissue culture for 48-72 h, then placed in Ussing chambers. Changes in tissue resistance (Rt) and short-circuit current (Isc) were monitored. The responses to forskolin and bile (taurodeoxycholic acid, TDCA) were examined separately and together. RESULTS: Forskolin (10 microM) produced a decrease in the Isc without a significant change in Rt, suggesting a secretory response, followed by a return to baseline. TDCA caused a similarly reversible decrease in the Isc at low doses, but a persistent drop at higher concentrations. A concurrent drop in Rt was noted at all TDCA concentrations, the duration of which correlated with dosage and degree of histological damage. Prior exposure to low (0.5 mM) doses of TDCA significantly blunted the response to subsequent forskolin challenge. CONCLUSIONS: Acute exposure to TDCA in vitro causes epithelial damage at levels lower than those normally used to induce experimental pancreatitis. At the lower concentrations, Rt returns to baseline rapidly, suggesting recovery (restitution) from epithelial damage but with a persistent loss of the response to forskolin. Reflux of minute amounts of bile into the pancreatic duct could play a significant role in the pathogenesis of gallstone pancreatitis by uncoupling the normal stimulus-secretion apparatus of the ductal system and breaking down the epithelial barrier.

Acute Disease↗

PubMed1998

Hepatitis delta virus RNA editing is highly specific for the amber/W site and is suppressed by hepatitis delta antigen.

RNA editing at adenosine 1012 (amber/W site) in the antigenomic RNA of hepatitis delta virus (HDV) allows two essential forms of the viral protein, hepatitis delta antigen (HDAg), to be synthesized from a single open reading frame. Editing at the amber/W site is thought to be catalyzed by one of the cellular enzymes known as adenosine deaminases that act on RNA (ADARs). In vitro, the enzymes ADAR1 and ADAR2 deaminate adenosines within many different sequences of base-paired RNA. Since promiscuous deamination could compromise the viability of HDV, we wondered if additional deamination events occurred within the highly base paired HDV RNA. By sequencing cDNAs derived from HDV RNA from transfected Huh-7 cells, we determined that the RNA was not extensively modified at other adenosines. Approximately 0.16 to 0.32 adenosines were modified per antigenome during 6 to 13 days posttransfection. Interestingly, all observed non-amber/W adenosine modifications, which occurred mostly at positions that are highly conserved among naturally occurring HDV isolates, were found in RNAs that were also modified at the amber/W site. Such coordinate modification likely limits potential deleterious effects of promiscuous editing. Neither viral replication nor HDAg was required for the highly specific editing observed in cells. However, HDAg was found to suppress editing at the amber/W site when expressed at levels similar to those found during HDV replication. These data suggest HDAg may regulate amber/W site editing during virus replication.

Base Sequence↗

PubMed1998

Synergistic induction of ornithine decarboxylase by asparagine and gut peptides in intestinal crypt cells.

The objective of this study was to determine whether the amino acid asparagine stimulated the activity of ornithine decarboxylase (ODC) synergistically with epidermal growth factor (EGF) or gastrin in IEC-6 cells, a line of normal rat small intestinal crypt cells. Cells were grown in DMEM containing 5% dialyzed fetal bovine serum, and serum was deprived for 24 h before experiments. Exposure to EGF or gastrin alone increased ODC activity 4.5- to 6-fold. Asparagine alone increased the enzyme activity 10- to 13-fold in IEC-6 cells. Simultaneous addition of asparagine and EGF or gastrin, however, increased ODC activity more than 40-fold. In contrast, there was no synergistic induction of ODC activity when gastrin and EGF were added together. Increased ODC activity in cells treated with asparagine and EGF or gastrin was associated with an increase in ODC mRNA and protein levels. The rate of transcription of the ODC gene was significantly increased by exposure to EGF or gastrin. Asparagine alone had little or no effect on the rate of transcription of the ODC gene. When given together with EGF or gastrin, asparagine also had no additional effect on the transcription rate of the ODC gene. The half-life of mRNA for ODC in unstimulated IEC-6 cells was approximately 30 min and increased to more than 2 h in cells exposed to asparagine, although neither gastrin nor EGF prolonged the stability of ODC mRNA. The half-life of mRNA for ODC after combined addition of asparagine and EGF or gastrin was extended to approximately 2 h, similar to asparagine alone. Combined addition of asparagine and EGF or gastrin also significantly increased DNA synthesis compared with cells exposed to each of the three agents alone. In conclusion, 1) simultaneous addition of asparagine and EGF or gastrin increases ODC activity in a synergistic manner and 2) asparagine increases ODC mRNA levels through completely distinct mechanisms from EGF or gastrin. EGF or gastrin specifically stimulates transcription of the ODC gene, whereas asparagine affects a posttranscriptional process.

Animals↗

PubMed1998

Expression of the transforming growth factor-beta gene during growth inhibition following polyamine depletion.

Polyamine depletion and cytokine transforming growth factor-beta (TGF-beta) inhibit cell proliferation. The current study tests the hypothesis that polyamine depletion results in growth inhibition by altering expression of the TGF-beta gene in intestinal epithelial cells. Studies were conducted in the IEC-6 cell line derived from rat small intestinal crypt cells. Cells were grown in DMEM in the presence or absence of alpha-difluoromethylornithine (DFMO), a specific inhibitor of polyamine biosynthesis, for 6 and 12 days. Administration of DFMO not only depleted intracellular polyamines but also significantly increased the mRNA levels of TGF-beta. Increased TGF-beta mRNA in DFMO-treated cells was paralleled by an increase in TGF-beta content. Depletion of intracellular polyamines by DFMO had no effect on the rate of TGF-beta gene transcription, as measured by nuclear run-on assay. The half-life of mRNA for TGF-beta in normal cells was approximately 65 min and increased to >16 h in cells treated with DFMO for 6 or 12 days. Exogenous polyamine, when given together with DFMO, prevented the increased half-life of TGF-beta mRNA in IEC-6 cells. TGF-beta added to the culture medium significantly decreased the rate of DNA synthesis and final cell number in normal and polyamine-deficient cells. Furthermore, growth inhibition caused by polyamine depletion was partially but significantly blocked by addition of immunoneutralizing anti-TGF-beta antibody. These results indicate that 1) depletion of intracellular polyamines induces the activation of the TGF-beta gene through posttranscriptional regulation and 2) increased expression of the TGF-beta gene plays an important role in the process of growth inhibition following polyamine depletion.

Animals↗

PubMed1997

A Xenopus zinc finger protein that specifically binds dsRNA and RNA-DNA hybrids.

Proteins containing C2H2 type zinc finger motifs represent one of the largest classes of nucleic acid-binding proteins found in nature. We describe a novel zinc finger protein, dsRBP-ZFa, isolated by screening an expression library with dsRNA. The dsRBP-ZFa cDNA encodes a protein containing seven zinc finger motifs and an acidic C-terminal domain. Mobility shift experiments demonstrate that dsRBP-ZFa binds dsRNA and RNA-DNA hybrids with nanomolar dissociation constants and in a sequence independent manner. We also show that DNA and single stranded RNA fail to compete with dsRNA for binding suggesting dsRBP-ZFa prefers to bind an A-form helix. Using western analyses we have localized dsRBP-ZFa primarily to the nucleus of Xenopus laevis oocytes. The identification of dsRBP-ZFa provides the first example of a zinc finger protein that is specific for dsRNA. In addition, dsRBP-ZFa does not contain the previously described dsRNA binding motif, suggesting certain zinc fingers may provide an alternative way to recognize the A-form helix.

Amino Acid Sequence↗

PubMed1997

Detection of inosine in messenger RNA by inosine-specific cleavage.

Double-stranded RNA adenosine deaminases catalyze the conversion of adenosine to inosine within double-stranded RNA. A few candidate biological substrates for these enzymes have been discovered by noticing discrepancies between genomic and cDNA sequences. Toward the goal of finding a systematic approach to identify new deaminase substrates, we developed a method to cleave RNA specifically after inosine and an amplification strategy to identify the cleavage sites. We tested our method on a candidate substrate, the messenger RNA for glutamate receptor subunit B (GluR-B). We detected cleavage of the endogenous GluR-B message from rat brain at two known RNA editing sites, thus providing the first direct evidence for the presence of inosine at these sites. The described method will facilitate the mapping of inosines within RNA and, most importantly, will provide a way to identify new deaminase substrates.

Adenosine Deaminase↗

PubMed1997

Intestinal epithelial cell regulation of macrophage and lymphocyte interleukin 10 expression.

BACKGROUND: The intestinal mucosa is subject to daily antigenic challenge and to injury by proinflammatory cytokines. Interleukin 10 (IL-10) is an important anti-inflammatory cytokine produced by macrophages and lymphocytes that modulates this response. OBJECTIVE: To investigate the hypothesis that intestinal epithelial cells are a significant local source of IL-10 in the gut milieu and also participate in the regulation of macrophage and lymphocyte IL-10 expression in the intestinal microenvironment. METHODS: C-205 cells, a human intestinal epithelial cell line, were cultured for 2 days; lipopolysaccharide or tumor necrosis factor was then added. Media and cells were harvested at specific time points to determine the kinetics of IL-10 expression. C-205 cells were then cocultured with macrophages or lymphocytes isolated from human peripheral blood mononuclear cells, and IL-10 expression was assessed in unstimulated and stimulated conditions. Interleukin 10 protein was measured by enzyme-linked immunosorbent assay; IL-10 gene expression was measured by reverse transcriptase-polymerase chain reaction. RESULTS: Constitutive production of IL-10 protein by C-205 cells was maximal at 3 days, paralleled by a peak in IL-10 messenger RNA (mRNA) expression at 24 hours. Lipopolysaccharide or tumor necrosis factor strikingly up-regulated IL-10 mRNA and protein expression by C-205 cells. Coculture of C-205 cells with macrophages or lymphocytes significantly increased lipopolysaccharide-stimulated IL-10 protein and mRNA release compared with C-205 cells, macrophages, or lymphocytes cultured alone. CONCLUSIONS: Enterocytes are a responsive source of IL-10 and may play a role in modulating production of this important cytokine by the local inflammatory cells of the gut. These redundant mechanisms to augment IL-10 production suggest a central role for this cytokine in regulation of the local intestinal inflammatory response.

Cells, Cultured↗

PubMed1997

Kinetics of splenocyte interleukin-4 production after injury and lethal endotoxin challenge.

Cytokine aberrations may contribute to sepsis-associated mortality after trauma. We have previously documented that IL-10 (a Th-2 cytokine) is downregulated after tissue trauma, and the administration of exogenous IL-10 improved survival and anti-IL-10 antibody increased lethality in a murine injury-lethal endotox-emia model. IL-4 activates the Th-2 subset of T cells, and functions in a paracrine manner to inhibit proinflammatory cytokine synthesis. The purpose of this study was to investigate the kinetics of IL-4 production in this murine trauma-sepsis model. Mice (n = 50) were randomized to five groups: Control, Femur Fracture (FFx), FFx-lipopolysaccharide (LPS), FFx-LPS-IL10, and FFx-LPS-Anti-IL10. LPS (400 micrograms ip) was administered 4 days after FFx to induce lethal sepsis. IL-10 (0.5 microgram ip) or anti-IL-10 (100 micrograms IP) was administered at resuscitation, 30 min after LPS. IL-4 production was measured in ex vivo splenocyte culture supernatants at 24-hr intervals. Splenocyte IL-4 production was significantly upregulated in the FFx-LPS group that received anti-IL-10; maximal IL-4 production was on Day 5, with a greater than sevenfold increase compared to all other groups. A transient early rise in IL-4 production was noted in the FFx-LPS group that received exogenous IL-10; however, a subsequent rapid decline was documented. Treatment with anti-IL-10 antibody after FFx injury and septic challenge with LPS is associated with an upregulation of splenocyte IL-4 synthesis, as well as an increase in mortality in this murine model. IL-4 and IL-10 interaction postinjury may profoundly influence monocyte activation, cell-mediated immunity, and the subsequent host immune response to infection.

Animals↗

PubMed1997

Protein kinase C inhibits epidermal growth factor receptor phosphorylation in enterocytes.

Epidermal growth factor (EGF) is an important proliferative signal in the gastrointestinal tract. The EGF receptor (EGFr), which transduces the mitogenic stimulus to the cell, may be regulated by a number of factors including extracellular matrix, cell-cell contact, and other peptides. As protein kinase C (PK-C) has been shown to phosphorylate and down-regulate the EGFr in certain tumor cell lines, we propose that PK-C, an important regulatory enzyme, modulates the phosphorylation of the EGFr in the IEC 6 rat enterocyte cell line. IEC 6 cells were cultured in dishes with Dulbecco's modified Eagle's medium, (DMEM)/5% fetal bovine serum (FBS), which was changed to DMEM/1% FBS 24 hr prior to all experiments. Cells (three dishes per group) were treated with the PK-C activating phorbol ester phorbol-12-myristate-13-acetate (PMA) (100 nM) or vehicle for 1 hr and challenged with EGF (50 ng/ml) or vehicle for 15 min. Cell lysates were then prepared. EGFr tyrosine phosphorylation was determined by immunoprecipitating the EGFr and immunoblotting with an antibody against phosphotyrosine. EGFr apparent molecular weight was assessed in the same lysates by Western blot with an anti-EGFr antibody. Blots were analyzed by computer densitometry. Data are expressed as mean +/- SEM; n = 3 with P value determined by t test. Exposure of cells to PMA resulted in a decrease in the EGF-stimulated EGFr phosphotyrosine content from 96 +/- 5 U in control to 66 +/- 6 U in PMA (P < 0.01). The amount of receptor did not change, 43 +/- 3 U in control vs 44 +/- 3 U in PMA (P = 0.44). Further, exposure to PMA in the absence of EGF caused a gel shift of the EGFr band consistent with a nontyrosine phosphorylation of the protein. We demonstrate that activation of PK-C results in a modification of the EGFr coincident with inhibition of EGF-stimulated receptor tyrosine kinase activity. These data support a role for PK-C in the regulation of EGFr function and hence modulation of mitogenic signals in enterocytes.

Animals↗

PubMed1997

The pancreatic duct epithelium in vitro: bile acid injury and the effect of epidermal growth factor.

BACKGROUND: Pancreatic duct epithelial cells form a barrier against parenchymal injury. The capacity of these cells to respond to injury has not been investigated. We hypothesized that epidermal growth factor (EGF), normally found in pancreatic juice, could protect the duct epithelium from damage. METHODS: An explant system of duct cell culture developed in our lab with the bovine main pancreatic duct was used. Explants were exposed to bile acid (taurodeoxycholic acid [TDCA] 0, 0.05, 0.5, and 1 mmol/L) in the presence or absence of EGF (0, 1, 10, and 100 nmol/L) for 48 hours. Epithelial proliferation, damage, and growth out from the explant edge were assessed histologically. Expression of ductal markers and the extent of cell proliferation were determined by immunohistochemistry using specific antibodies. RESULTS: Explant duct cells proliferated and demonstrated continued expression of key duct antigens in culture. TDCA produced dose-dependent mucosal damage and reduced epithelial density and growth from the edge. EGF increased cellular density in the native epithelium, but did not significantly alter growth from the edge. Mucosal damage created by TDCA exposure was significantly decreased with EGF and both growth from the edge and cell density were preserved. CONCLUSIONS: Explants created from the bovine main pancreatic duct serve as an excellent model for the study of duct epithelial cells in vitro. These cells proliferate in response to EGF and are damaged by TDCA at concentrations below those normally associated with detergent-like activity and below levels observed in bile and duodenal secretions. The ability of EGF to protect from this injury suggests a potential physiologic role in the maintenance of the pancreatic duct mucosal barrier.

Animals↗

PubMed1997

RNA editing and hypermutation by adenosine deamination.

Double-stranded RNA adenosine deaminase (dsRAD) was discovered ten years ago. In the intervening decade, research on dsRAD has progressed not only predictably, such as with the purification of the enzyme and identification of cDNAs, but also in some quite surprising ways. This review covers both areas of progress, but will concentrate on the surprises, which include the discovery that dsRAD is a member of a larger family of deaminases and the identification of RNAs that appear to be targets for these deaminases in vivo.

Adenosine Deaminase↗