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OBJECTIVE: To find out the incidence, spectrum, and topographical distribution of brain lesions in neonatal hypernatremic dehydration. METHODS: We prospectively enrolled 100 consecutive neonates admitted with hypernatremic dehydration. 93 neonates underwent magnetic resonance imaging brain to identify the nature and site of neurological injury. RESULTS: Neuroradiological lesions were found in 42 (45.2%) babies. Edema was the most common finding in 37 (39.8%), followed by hemorrhage in 13 (13.9%) and thrombosis in 6 (6.4%). Edema predominantly affected juxtacortical/subcortical white matter followed by periventricular white matter and centrum semiovale, posterior part of internal capsule, and basal ganglia/thalamus. Occipital horns of lateral ventricle were the main sites of hemorrhage. Thrombotic lesions predominantly involved sagittal, straight and transverse sinuses. Brain lesions were observed only in severe hypernatremia group. CONCLUSIONS: In neonatal hypernatremic dehydration, edema was the most common neurological lesion, followed by hemorrhage and thrombosis. Subcortical/juxtacortical white matter was the most commonly affected site.

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Intraductal papillary mucinious neoplasm-biliary type is the biliary counterpart of intraductal papillary mucinious neoplasm-pancreatic type. We report a rare case of intraductal papillary mucinous tumor arising from extrahepatic biliary system. The diagnosis was established on histopathological analysis following endoscopic retrograde cholangiopancreatography-guided biopsy. Isolated papillary adenoma of the bile duct is extremely rare, and in this unusual case the patient was a 22-year-old young lady who had delivered a healthy infant 6 weeks previously.

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In biopharmaceutical production, the optimization of cell culture media and supplementation is a vital element of process development. Optimization is usually achieved through the screening of multiple media, feed and feeding strategies. However, most screening is performed in shake flasks, which makes the screening process very time consuming and inefficient. The use of small scale culture systems for the screening process can aid in the ability to screen multiple formulations during process development. In order to assess the suitability of 24 deep well (24DW) plates with the Duetz sandwich-covers as a small scale culture system for process development, we have tested growth and production performance of CHO cells in 24DW plates and conventional shake flask cultures. Multiple studies were performed to assess well-to-well and plate-to-plate variability in 24DW plates. Additional studies were performed to determine the applicability of 24DW plates for cell culture medium and supplement screening in batch and fed batch processes. Cultures in 24DW plates exhibited similar kinetics in growth, viability and protein production to those cultured in shake flasks, suggesting that 24DW plates with Duetz sandwich-covers can be effectively used for high throughput cell culture screening.

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BACKGROUND: Chronic administration of ethanol increases plasma prolactin levels and enhances estradiol's mitogenic action on the lactotropes of the pituitary gland. The present study was conducted to determine the changes in the pituitary levels of G proteins during the tumor development following alcohol and ethanol treatments. METHODS: Using ovariectomized Fischer-344 female rats, we have determined ethanol and estradiol actions at 2 and 4 weeks on pituitary weight and pituitary cell contents of prolactin, Gs. Gq11, Gi1, Gi2, and Gi3 proteins. Western blots were employed to measure protein contents. RESULTS: Ethanol increased basal and estradiol-enhanced wet weight and the prolactin content in the pituitary in a time-dependent manner. Chronic exposure of estradiol increased the levels of Gs protein in the pituitary. Unlike estradiol, ethanol exposure did not show significant effect on the basal level of Gs protein, but moderately increased the estradiol-induced levels of this protein. Estradiol exposure enhanced Gq11 protein levels in the pituitary after 2 and 4 weeks, while ethanol treatment failed to alter these protein levels in the pituitary in control-treated or estradiol-treated ovariectomized rats. In the case of Gi1, estradiol but not ethanol increased the level of this protein at 4 weeks of treatment. However, estradiol and ethanol alone reduced the levels of both Gi2 and Gi3 proteins at 2 and 4 weeks of treatment. Ethanol also significantly reduced the estradiol-induced Gi2 levels at 4 weeks and Gi3 level at 2 and 4 weeks. CONCLUSIONS: These results confirm ethanol's and estradiol's growth-promoting and prolactin stimulating actions on lactotropes of the pituitary and further provide evidence that ethanol and estradiol may control lactotropic cell functions by altering expression of specific group of G proteins in the pituitary.

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Thrombospondin-1 (TSP-1), a multifunctional matrix glyco-protein, has been shown to control tumor growth by inhibiting angiogenesis in various tissues. However, the role of this glycoprotein in pituitary angiogenesis is not well studied. In this report, we determined the changes in the production and action of TSP-1 on endothelial cells in anterior pituitary following estradiol treatment, which is known to increase prolactin-secreting tumor growth and vascularization in this tissue. We showed that TSP-1 immunoreactive protein is distributed in the anterior pituitary, particularly in the endothelial cells. Estradiol treatment for 2 and 4 weeks decreased the total tissue immunoreactive level of TSP-1 as well as the endothelial cell-specific immunoreactive level of this protein in the anterior pituitary. The steroid treatment also decreased the protein levels of TSP-1 in anterior pituitary tissues and in purified pituitary endothelial cells in primary cultures. Determination of the effects of TSP-1 on proliferation and migration of pituitary-derived endothelial cells in primary cultures elucidated an inhibitory action of TSP-1 on these vascular cell functions. These results suggest that locally produced TSP-1 may regulate estrogen angiogenic action on the pituitary.

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This study was designed to test the hypothesis that inhibition of agonist-induced delta-receptor down-regulation would block the development of opioid tolerance in a cell-based model. A human embryonic kidney 293 cell line was established that expressed an epitope-tagged delta-opioid receptor (DOR). Treatment of DOR cells with Tyr-d-Ala-Gly-Phe-d-Leu-enkephalin (DADL) resulted in a time-dependent decrease in the B(max) of delta-opioid receptor binding sites and immunoreactive receptor protein. When cells were coincubated with the proteasome inhibitor N-benzyloxycarbonyl-l-leucyl-l-leucyl-l-leucinal (ZLLL) and DADL, the magnitude of the agonist-induced decrease in B(max) and immunoreactive receptor protein was reduced compared with DADL treatment alone. Acute treatment of DOR cells with DADL caused a 3-fold increase in the level of phosphorylated mitogen-activated protein (MAP) kinase. Prior exposure of DOR cells to DADL completely abrogated the agonist-induced activation of MAP kinase. When DOR cells were coincubated with DADL and ZLLL, the proteasome inhibitor prevented the loss of agonist activation of MAP kinase. Acute treatment of DOR cell membranes with DADL stimulated [(35)S]guanosine 5'-3-O-(thio-)triphosphate (GTPgammaS) binding. When DOR cells were preincubated with DADL, the agonist-induced increase in [(35)S]GTPgammaS binding was attenuated. Coincubation of ZLLL and agonist partially prevented the decreased responsiveness to agonist stimulation. The results of this study demonstrated that inhibition of agonist-induced down regulation with a proteasome inhibitor attenuated opioid tolerance in a cellular model, and suggest that coadministration of a proteasome inhibitor with chronic opioid agonist treatment may be useful for limiting opioid tolerance in vivo.

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Most previous studies that determined the effect of estradiol on angiogenesis used endothelial cells from nonpituitary sources. Because pituitary tumor tissue receives its blood supply via portal and arterial circulation, it is important to use pituitary-derived endothelial cells in studying pituitary angiogenesis. We have developed a magnetic separation technique to isolate endothelial cells from pituitary tissues and have characterized these cells in primary cultures. Endothelial cells of the pituitary showed the existence of endothelial cell marker, CD31, and of von Willebrand factor protein. These cells in cultures also showed immunoreactivity of estrogen receptors alpha and beta. The angiogenic factors, vascular endothelial growth factor and basic fibroblast growth factor, significantly increased proliferation and migration of the pituitary-derived endothelial cells in primary cultures. These results suggest that a magnetic separation technique can be used for enrichment of pituitary-derived endothelial cells for determination of cellular mechanisms governing the vascularization in the pituitary.

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The mechanism by which ethanol induces beta-endorphin (beta-EP) neuronal death during the developmental period was determined using fetal rat hypothalamic cells in primary cultures. The addition of ethanol to hypothalamic cell cultures stimulated apoptotic cell death of beta-EP neurons by increasing caspase-3 activity. Ethanol lowered the levels of adenylyl cyclase (AC)7 mRNA, AC8 mRNA, and/or cAMP in hypothalamic cells, whereas a cAMP analog blocked the apoptotic action of ethanol on beta-EP neurons. The AC inhibitor dideoxyadenosine (DDA) increased cell apoptosis and reduced the number of beta-EP neurons, and it potentiated the apoptotic action of ethanol on these neurons. beta-EP neurons in hypothalamic cultures showed immunoreactivity to transforming growth factor-beta1 (TGF-beta1) protein. Ethanol and DDA increased TGF-beta1 production and/or release from hypothalamic cells. A cAMP analog blocked the activation by ethanol of TGF-beta1 in these cells. TGF-beta1 increased apoptosis of beta-EP neurons, but it did not potentiate the action of ethanol or DDA actions on these neurons. TGF-beta1 neutralizing antibody blocked the apoptotic action of ethanol on beta-EP neurons. Determination of TGF-beta1-controlled cell apoptosis regulatory gene levels in hypothalamic cell cultures and in isolated beta-EP neurons indicated that ethanol, TGF-beta1, and DDA similarly alter the expression of these genes in these cells. These data suggest that ethanol increases beta-EP neuronal death during the developmental period by cellular mechanisms involving, at least partly, the suppression of cAMP production and activation of TGF-beta1-linked apoptotic signaling.

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In the present study, we determined the interactive effects of ethanol and transforming growth factor beta-3 (TGF-beta3) on basic fibroblast growth factor (bFGF) release from folliculostellate (FS) cells and the role of the mitogen-activated protein kinase (MAPK) pathway in this interaction. We found that TGF-beta3 and ethanol alone increased release of bFGF from FS cells, but together they showed markedly increased levels of bFGF compared with the individual effect. Ethanol and TGF-beta3 alone moderately increased activation of MAPK p44/42, but together they produced marked activation of MAPK p44/42. TGF-beta3 alone increased the activation of smad2. Ethanol did not activate smad2 or alter TGF-beta3 activation of smad2. Pretreatment of FS cells with a mitogen-activated protein kinase kinase 1/2 inhibitor or with a protein kinase C (PKC) inhibitor suppressed the TGF-beta3 and ethanol actions on MAPK p44/42 activation and bFGF release. Ethanol and TGF-beta3, either alone or in combination, increased the levels of active Ras. Furthermore, the MAPK p44/42 activation by TGF-beta3 and ethanol was blocked by overexpression of Ras N17, a dominant negative mutant of Ras p21. These data suggest that the PKC-activated Ras-dependent MAPK p44/42 pathway is involved in the cross talk between TGF-beta3 and ethanol to increase bFGF release from FS cells.

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Folliculostellate (FS) cells are known to communicate with each other and with endocrine cells via gap junctions in the anterior pituitary. We investigated whether TGFbeta3 and estradiol, known to regulate FS cell production and secretion of basic fibroblast growth factor (bFGF), increases gap junctional communication to alter bFGF secretion from FS cells. FS cells in monolayer cultures were treated with TGFbeta3 or vehicle alone for 24 h and then microinjected with Lucifer Yellow and high-molecular-weight Texas Red dextran. Ten minutes later the transfer of dye among adjacent cells was recorded with a digital microscope. TGFbeta3 increased the transfer of dye. The TGFbeta3-neutralizing antibody and the gap junction inhibitor octanol reduced the effect of TGFbeta3 on the transfer of dye. The TGFbeta3-induced transfer of dye was unaltered by simultaneous treatment with estradiol. The steroid alone also had no effect. TGFbeta3 increased total and phosphorylated levels of connexin 43. Estradiol treatment did not produce any significant changes on basal or TGFbeta3-induced increases in connexin 43 levels. The gap-junction inhibitor octanol reduced TGFbeta3-increased levels of bFGF in FS cells. Taken together, these results suggest that TGFbeta3 may act on FS cells to increase gap-junctional communication to maximize its effect on bFGF secretion.

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Basic fibroblast growth factor (bFGF), which is secreted from folliculostellate cells in the anterior pituitary, is known to be involved in the communication between folliculostellate cells and lactotropes during estradiol-induced lactotropic cell proliferation. We studied the role of MAPK p44/42 in bFGF-regulated cell proliferation using enriched lactotropes and the lactotrope-derived PR1 cell line. In cell cultures, bFGF increased cell proliferation of PR1 cells and enriched lactotropes. In both of these cell populations, bFGF also increased phosphorylation of MAPK p44/42. U0126, an inhibitor of MAPK p44/42, blocked the bFGF-induced activation of MAPK p44/42 as well as the bFGF-induced cell proliferation of enriched lactotropes and PR1 cells. Treatment of PR1 cells with bFGF increased the activity of Ras p21, whereas overexpression of a dominant negative mutant of Ras p21 abrogated the bFGF-induced activation of MAPK p44/42 in these cells. Furthermore, the Src kinase inhibitor PP1 suppressed bFGF-induced activation of MAPK p44/42 in both enriched lactotropes and PR1 cells. The Src kinase inhibitor PP1 also reduced bFGF activation of Ras p21 and cell proliferation in PR1 cells. On the other hand, the bFGF-induced activation of MAPK p44/42 in enriched lactotropes and PR1 cells was not affected by protein kinase C inhibitors. These data suggest that bFGF induction of lactotropic cell proliferation is possibly mediated by activation of Src kinase, Ras p21, and MAPK p44/42.

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Naltrexone, an opioid antagonist, has been used in clinical trials to treat alcoholism. As the opioid peptides beta-endorphin and enkephalin increase splenic NK cell function in laboratory animals, it is anticipated that naltrexone treatment will cause immunosuppression. However, we report in this study that chronic naltrexone administration in laboratory rats increases the cytolytic activity of NK cells. It also prevents alcohol's suppressive effect on these cells. We identified that, in the splenocytes, delta opioid receptor expression is tightly controlled by negative feedback regulation of micro opioid receptors. Naltrexone disrupts this feedback control by reducing micro opioid receptor function, thereby up-regulating delta opioid receptor binding, which results in an enhanced NK cell cytolytic response to delta opioid receptor ligands. We conclude that naltrexone, which has been shown to be a promising agent for the clinical management of alcoholism, may have potential use in the treatment of immune deficiency in alcoholic and nonalcoholic patients.

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Estradiol is known to increase lactotropic cell proliferation, but estradiol susceptibility varies among human populations and among various strains of rats. We had reported that folliculostellate (FS) cells regulate estradiol's mitogenic action on lactotropes; therefore, we studied their role in determining the susceptibility to estradiol in a high estradiol-responsive rat strain, Fischer 344 (F344), and in a low-responsive strain, Sprague Dawley (SD). Determination of total S-100-positive FS cells in the pituitary revealed that F344 rats have significantly more FS cells than do SD rats. Estradiol treatment did not change the number of FS cells in both F344 and SD rats. When cotransplanted with F344 pituitaries under the kidney capsule or cocultured with F344-derived lactotropes in vitro, FS cells derived from F344 rats increased estradiol's mitogenic action. They also increased estradiol's mitogenic action on SD-derived lactotropes in primary cultures. However, SD-derived FS cells failed to increase estrogen's action on F344- or SD-derived lactotropes. The levels of basic fibroblast growth factor production and secretion by TGF-beta 3 and estradiol were much higher in F344-derived FS cells than in SD-derived FS cells. However, the lactotropes' growth response to basic fibroblast growth factor was similar in both strains. These data suggest that cell-cell interaction between FS cells and lactotropes regulates estradiol's mitogenic action on lactotropes and also determines lactotrope susceptibility to the steroid.

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We have recently shown that TGF-beta3, in the presence of estradiol, increases the release of basic fibroblast growth factor (bFGF) from folliculostellate (FS) cells in the pituitary. We determined the interactive effects of TGF-beta3 and estradiol on bFGF production and release from FS cells, and the role of the MAPK pathway in TGF-beta3 and estradiol interaction. We found that TGF-beta3 and estradiol alone moderately increased cell content and release of bFGF from FS cells; but together, they markedly increased the peptide. Estradiol and TGF-beta3 alone moderately activated MAPK p44/42; together they produced marked activation of MAPK p44/42. Pretreatment of FS cells with an MAPK kinase 1/2 inhibitor or with protein kinase C inhibitors suppressed the activation of MAPK p44/42, bFGF release, and protein level increases, all of which were induced by TGF-beta3 and estradiol. Estradiol and TGF-beta3, either alone or in combination, increased the levels of active Ras. Furthermore, bFGF induction by TGF-beta3 and estradiol was blocked by overexpression of Ras N17, a dominant negative mutant of Ras p21. Estrogen receptor blocker ICI 182,780 failed to prevent estrogen's and TGF-beta3's effects on bFGF. These data suggest that an estradiol receptor-independent protein kinase C- activated Ras-dependent MAPK pathway is involved in the cross-talk between TGF-beta3 and estradiol to increase bFGF production and/or release from FS cells.

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Biogenesis of various endogenous opioid peptides, anatomical distribution and the characteristics of multiple receptors with which they interact provides an opportunity for understanding the role of opioid systems and mechanism of opioid tolerance. Cellular and anatomical distribution of opioid receptor and their function is important for identification of neuronal systems and local network involved in initiation of drug action and subsequent development of adaptations resulting from repeated drug use. The details concerning discovery and progress in endogenous opioid peptide research and their distribution in brain have been described in this review. This review also describes opioid receptors, their distribution and mechanism of down regulation, which may be one of the causes for tolerance to opioids. Agonist induced down regulation and recent evidence for involvement of ubiquitin/proteasome system in this process has been discussed.

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We reported recently that the ubiquitin-proteasome pathway is involved in agonist-induced down regulation of mu and delta opioid receptors [J. Biol. Chem. 276 (2001) 12345]. While evaluating the effects of various protease inhibitors on agonist-induced opioid receptor down regulation, we observed that while the peptide aldehyde, leupeptin (acetyl-L-Leucyl-L-Leucyl-L-Arginal), did not affect agonist-induced down regulation, leupeptin at submillimolar concentrations directly inhibited radioligand binding to opioid receptors. In this study, the inhibitory activity of leupeptin on radioligand binding was characterized utilizing human embryonic kidney (HEK) 293 cell lines expressing transfected mu, delta, or kappa opioid receptors. The rank order of potency for leupeptin inhibition of [3H]bremazocine binding to opioid receptors was mu > delta > kappa. In contrast to the effect of leupeptin, the peptide aldehyde proteasome inhibitor, MG 132 (carbobenzoxy-L-Leucyl-L-Leucyl-L-Leucinal), had significantly less effect on bremazocine binding to mu, delta, or kappa opioid receptors. We propose that leupeptin inhibits ligand binding by reacting reversibly with essential sulfhydryl groups that are necessary for high-affinity ligand/receptor interactions.

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