Common characteristics shared by different differential phase contrast imaging methods.

There are many variations of differential phase contrast imaging methods. Although these imaging methods are different in configuration, they are alike in imaging by extracting differential phase information through the evaluation of the refraction angles. In this paper, we investigate common characteristics shared by various different differential phase contrast imaging methods.

Intrauterine growth retardation leads to the functional change of insulin secretion in the newborn rats.

To investigate the observed variation in glucose tolerance and insulin secretion in intrauterine growth retarded newborn rats and to explore the mechanism of the variations, Sprague-Dawley pregnant rats were allocated into two groups: a control group and an intrauterine energy restricted group. The intrauterine growth retardation (IUGR) in the rats was induced by 50% calorie restriction in pregnant rats from gestational day 15 until term as compared to the control group. The pancreata of control and IUGR newborn rats were dissected respectively. RT-PCR was used to study the mRNA level related to insulin synthesis and exocytosis. Intraperitoneal glucose tolerance tests were done to study the function of the pancreatic islet. We found that birth weight and pancreas mass of IUGR newborn rats were significantly lower than those of controls. Although no significant differences were observed in mRNA level of insulin and PDX-1, the expression of genes related to insulin exocytosis such as munc13-1, vamp-2, syntaxin1a, rab3a were reduced significantly in IUGR newborn rats. IUGR animals were glucose-intolerant. The observed blood insulin level and insulin secretion response to glucose challenge were both found to be at reduced level in IUGR newborn rats as compared with the normal control group rats. With these findings, we hypothesize that IUGR can induce changes in glucose homeostasis due to, at least in part, a reduced function of insulin exocytosis in newborn rats.

Characterization of Munc13-1 and insulin secretion during pancreatic development in rats.

Munc13-1 may be a key factor in regulating insulin exocytosis, but its exact expression and role have not been clarified yet, especially during pancreatic development. We attempted to investigate the expression and function of Munc13-1 during embryonic pancreatic development in rats and determine the effects on insulin secretion. In the present study, pancreata of rats at embryonic day 12.5 (E12.5), E15.5, E18.5, new-born, 21 after birth (P21), and adult stage were dissected under microscope. The rat model of intrauterine growth retardation (IUGR) was made by 50% calorie restriction in pregnant rats from gestational day 15 until term. The expression of Munc13-1 and insulin secretion was studied by the techniques of RTPCR, real-time PCR, Western blot, and enzyme-linked immunosorbent assay. Immunohistochemistry and immunofluorescence were used to define the location of Munc13- 1. We found that Munc13-1 was located at islet along with insulin. Insulin- and Munc13-1-specific mRNA were not detected until E12.5 and E15.5, respectively, and increased with the development of the fetus. Western blot showed that Munc13-1 was low at E15.5 and E18.5 and increased later. The blood insulin level and Munc13-1 were reduced simultaneously in IUGR newborn rats compared with normal ones. These results suggest that Munc13-1 exists in pancreas islets during fetus development and its deficiency in the pancreas, as occurs in IUGR, was in accordance with decreased blood insulin level. Munc13-1 may play an essential role in insulin exocytosis.

A new iterative algorithm to reconstruct the refractive index.

The latest developments in x-ray imaging are associated with techniques based on the phase contrast. However, the image reconstruction procedures demand significant improvements of the traditional methods, and/or new algorithms have to be introduced to take advantage of the high contrast and sensitivity of the new experimental techniques. In this letter, an improved iterative reconstruction algorithm based on the maximum likelihood expectation maximization technique is presented and discussed in order to reconstruct the distribution of the refractive index from data collected by an analyzer-based imaging setup. The technique considered probes the partial derivative of the refractive index with respect to an axis lying in the meridional plane and perpendicular to the propagation direction. Computer simulations confirm the reliability of the proposed algorithm. In addition, the comparison between an analytical reconstruction algorithm and the iterative method has been also discussed together with the convergent characteristic of this latter algorithm. Finally, we will show how the proposed algorithm may be applied to reconstruct the distribution of the refractive index of an epoxy cylinder containing small air bubbles of about 300 micro of diameter.

Transcriptional regulation of inhibin beta B messenger ribonucleic acid levels in TM.4 or primary rat Sertoli cells by 8-bromo-cyclic adenosine monophosphate.

FSH, a major regulator of inhibin production in the testis, is believed to exert its effects via cAMP second messenger system. Inhibin alpha-subunit gene appears to be regulated by cAMP and has a palindromic cAMP response element sequence TGACGTCA. However, the regulation of the inhibin beta B-subunit gene by cAMP has been less clear. It has been assumed that beta B may not be regulated by cAMP, based mainly on observations that FSH stimulates only alpha, not beta B, mRNA levels, and that the 5'-up-stream regulatory region of the beta B gene does not contain the classical cAMP response element. However, we have observed that 8-bromo-cAMP stimulates beta B mRNA levels in both primary Sertoli (approximately 2-fold) and TM.4 cells (approximately 5-fold). We examined whether this cAMP-induced increase in beta B mRNA levels is the result of increased transcription or altered mRNA stability. Data from nuclear run-on assays demonstrate about a 2-fold increase in relative mRNA synthesis rates in primary Sertoli-cells and about a 4- to 5-fold increase in TM.4 cells. Transfection studies in TM.4 and JEG.3 cell lines with beta B:luciferase chimeric reporter gene constructs containing 1.5 kilobases of the beta B 5'-up-stream regulatory region revealed marked cAMP induction of reporter gene activity in both cell types.(ABSTRACT TRUNCATED AT 250 WORDS)

Transcriptional and posttranscriptional regulation of inhibin alpha-subunit gene expression in rat Sertoli cells by 8-bromo-3',5'-cyclic-adenosine monophosphate.

FSH is a major regulator of inhibin production in the testis. FSH effects on Sertoli cell inhibin production are believed to be mediated, at least in part, via the cAMP second messenger system. Previously, it has been shown that 8-bromo-cAMP (8-Br-cAMP) stimulates inhibin-alpha mRNA levels. This study examines whether the cAMP-induced increase in inhibin-alpha mRNA levels results from increased alpha mRNA synthesis, decreased degradation of mRNA, or both. The effects of cAMP on inhibin-alpha gene transcription were examined using nuclear run-on assays. Furthermore, the ability of 8-Br-cAMP to drive the transcription of chimeric constructs containing a 2.2-kilobase (kb) segment of the 5'-regulatory region of the alpha gene placed upstream of the coding region of the luciferase reporter gene was also examined. Data from nuclear run-on assays demonstrated rapid induction of alpha gene transcription by cAMP within 2 h and maximal 4- to 5-fold increase within 4-8 h in primary Sertoli cells. Transfection of TM.4 and JEG.3 cells with an alpha (2.2 kb):luciferase chimeric construct (containing 2.2 kb of the alpha gene 5'-flanking DNA) revealed rapid time-dependent induction of luciferase activity by 8-Br-cAMP in these cell types. To examine the effects of 8-Br-cAMP on alpha mRNA stability, cells were pretreated with medium or 50 micrograms/ml 8-Br-cAMP for 24 h before addition of 5 microM actinomycin D to arrest new RNA synthesis, and the decay of alpha mRNA transcripts was assessed over 24 h by Northern analysis and nonlinear regression.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential regulation of rat luteinizing hormone alpha- and beta-subunits during the stimulatory and down-regulatory phases of gonadotropin-releasing hormone action.

Chronic treatment with agonist analogs of GnRH or long term continuous administration of GnRH results in down-regulation of pituitary LH secretion. We investigated the changes in the LH subunits during the stimulatory and down-regulatory phases of GnRH action in rat pituitary cell monolayer culture. The rat pituitary cells in culture, pretreated with medium alone or GnRH agonist for 48 h, were incubated with graded doses of GnRH for 4 h, and LH, LH alpha, and LH beta concentrations in the media and cell pellets were measured by specific and sensitive RIAs. Cells pretreated with medium alone responded to GnRH with a dose-dependent increase in LH, LH alpha, and LH beta immunoreactivity in the medium. However, rat LH, LH alpha, and LH beta concentrations in the cell pellets showed a dose-dependent decrease with GnRH treatment. Pretreatment with GnRH agonist led to a marked decrease in the LH response of cells to graded doses of GnRH. During this down-regulatory phase, the concentrations of LH beta in the medium remained undetectable even though LH alpha immunoreactivity remained persistently and disproportionately elevated. These data suggest that the alpha- and beta-subunits of rat LH are both coordinately and differentially regulated. During the stimulatory phase of GnRH action, both subunits rise concordantly, but during the down-regulatory phase the secretion of the two subunits becomes unbalanced. The changes in the beta-subunit closely parallel the changes in the concentrations of LH dimer, pointing to the key role of beta-subunit in regulation of LH secretion.

Hormonal effects of gonadotropin-releasing hormone (GnRH) agonist in men: effects of long term treatment with GnRH agonist infusion and androgen.

Constant infusion of GnRH agonist (GnRH-A) leads to far greater suppression of spermatogenesis and gonadotropins in the monkey than its intermittent administration. We assessed if greater suppression of gonadotropins and spermatogenesis could also be achieved in man by continuous GnRH-A administration. Seven normal men were given 400 micrograms GnRH-A daily by constant sc infusion using a mechanical pump device and bimonthly injections of 200 mg testosterone (T) enanthate for 16 weeks. Basal serum LH, FSH, T, and estradiol concentrations were measured every week during a 5-week control period, daily on treatment days 0, 1-11, 14, 18, 22, 26, 28, and every week thereafter until day 56, and every 2 weeks thereafter during the remainder of the treatment phase and during the 14-week recovery phase. Detailed analysis of LH and FSH secretion during the 24-h period was performed by multiple blood sampling on days 0, 1, 10, 28, 56, 84, and 112. Semen analyses were performed every week during the control phase and every 2 weeks during the treatment and recovery phases. The mean sperm count declined by 93% to a nadir of 6 +/- 3 (+/- SE) million/mL between weeks 14-16. Four men had sperm counts less than 1 million/mL, and three subjects were azoospermic during treatment. Basal serum immunoreactive LH concentrations, after an early increase, declined to near baseline by day 14. The basal and 24-h integrated serum LH concentrations and 24-h urinary LH excretion were not significantly lowered by treatment. Bioassayable serum LH concentrations, however, after an early rise, declined significantly below baseline by day 28 and remained low thereafter. The frequency and amplitude of LH pulses were reduced by GnRH agonist infusion. Basal and 24-h integrated serum FSH concentrations, after a brief initial increase, declined to baseline by day 10, but were not significantly below baseline by day 112. Serum T concentrations did not fall into the hypogonadal range at any time during the treatment period. After discontinuation of treatment, serum LH and FSH and sperm counts returned to normal in all men. Thus, this regimen, employing constant infusion of 400 micrograms GnRH agonist daily plus T led to a greater suppression of spermatogenesis than the previous regimen employing single daily injections of 200 micrograms of the same agonist plus T. Whether the higher dose or the constant infusion was responsible for the greater inhibition of spermatogenesis is not clear. It is conceivable that a still higher dose of the agonist, given by constant infusion, might induce azoospermia in all men.

Role of CYP2E1 in the pathogenesis of alcoholic liver disease: modifications by cAMP and ubiquitin-proteasome pathway.

The ethanol inducible isoform of cytochrome P450, CYP2E1, may play a role in ethanol-induced liver injury. Therefore, the factors which govern CYP2E1 degradation and turnover were investigated. These factors include cAMP, ubiquitin, proteasomal enzymes and CYP2E1 mRNA. Rats fed ethanol or pair-fed isocaloric dextrose were pair-fed with rats fed ethanol or dextrose treated with cAMP for 2 months. The liver pathology, regenerative activity, fatty acid composition, NFkappaB activation, ubiquitin conjugates and proteasomal enzymes were measured as were the apoprotein levels of CYP2E1, CYP3A, CYP4A and mRNA levels for CYP2E1 and ubiquitin expression. The results showed, that the cAMP treatment ameliorated the increase liver fat storage and changes in the fatty acid composition in the livers of ethanol fed rats. Other histologic features of alcoholic liver disease were not changed. Western blot quantitation showed that the amount of ubiquitin and ubiquitin conjugates were markedly reduced by ethanol treatment. Similarly, ethanol decreased the level of ubiquitin mRNA. cAMP ameliorated the inhibition of the proteasomal enzyme proteolysis caused by ethanol feeding. The ethanol-induced increase in the CYP2E1 protein was partially inhibited by cAMP treatment. cAMP treatment decreased CYP2E1 mRNA levels in both ethanol-fed and pair fed control rats. Likewise NFkappaB activation was not increased by ethanol but cAMP reduced the level of NFkappaB activation. CAMP treatment also reduced CYP4A but not CYP3A. The results support the concept that cAMP treatment partially protects the liver from ethanol-induced fatty liver by reducing CYP2E1 induction through cAMP's effects on CYP2E1 synthesis.

Calcitonin gene-related peptide (CGRP) in normotensive and spontaneously hypertensive rats.

With the techniques of specific radioimmunoassay and gel filtration it was found that CGRP was distributed in various tissues of normotensive (WKY) and spontaneously hypertensive rats (SHR) with the highest concentration in the lumbar spinal cord (1197 +/- 94.8 pg/mg tissue) and the lowest in the auricle (15.0 +/- 2.1 pg/mg tissue). In comparison with WKY, CGRP concentration in the plasma was decreased and in the abdominal aorta and hypothalamus was increased in SHR. Gel filtration revealed only one major CGRP molecular form in the tissues. In addition, CGRP reduced the mean arterial pressure (MAP) in SHR in a dose-dependent manner. These data suggest that CGRP may play an important role in the pathogenesis of hypertension and its possible therapy.

Regulation of peroxisome proliferator activated receptor alpha-mediated pathways in alcohol fed cytochrome P450 2E1 deficient mice.

Fatty acids are substrates and inducers for cytochrome P450 2E1 (CYP2E1) and peroxisome proliferator activated receptor alpha (PPARalpha). Previously, we have shown that the ethanol-induced CYP2E1 expression in rat is accompanied by the inhibition of the expression of the PPARalpha gene and the reduction in polyunsaturated fatty acid content. To further analyze the effect of CYP2E1 and ethanol in PPARalpha-mediated fatty acid homeostasis, the expression of PPARalpha and retinoid x receptor alpha (RXRalpha) and their target genes was examined in ethanol fed CYP2E1 deficient mice. Our data demonstrated that the expression of PPARalpha and RXRalpha genes was activated in the livers of CYP2E1-null mice suggesting a compensatory effect for the absence of CYP2El. In addition, the expression of PPARalpha target genes, which included the liver fatty acid-binding protein, malic enzyme, and CYP4A1 genes, was induced indicating the activation of PPARalpha-mediated pathways in CYP2E1 deficient mice. Ethanol inhibited the expression of some of the PPARalpha target genes in wild-type mouse livers, and the inhibitory effect of ethanol was particularly prominent in the CYP2E1-null mice. Morphologically, centrilobular fat accumulation was detected in the ethanol fed CYP2E1-null mouse livers suggesting that inhibition of PPARalpha-mediated pathways might be responsible for the ethanol-induced fatty liver in CYP2El-null mice. In addition, the expression of CYP2E1 was not changed in the PPARalpha-null mice. These data suggest that CYP2E1 and ethanol can regulate PPARalpha-mediated fatty acid homeostasis. CYP2E1-induced lipid peroxidation might play a major role in lipid metabolism, PPARalpha only becomes important when the CYP2E1 level is low and polyunsaturated fatty acids increase.

Calcitonin gene-related peptide in the pathogenesis and treatment of hypertension.

The present study demonstrated that the plasma calcitonin gene-related peptide (CGRP) concentration was lower but the CGRP content of abdominal aorta was higher in spontaneously hypertensive rats (SHR) than in normotensive rats (WKY), using specific CGRP radioimmunoassay (P less than 0.01). In eighteen patients with essential hypertension, the concentration of plasma CGRP was also much lower than that of normal subjects, suggesting that a decreased release of arterial CGRP might be a part of the pathogenesis of essential hypertension. In SHR, a significant decrease in blood pressure was found following intravenous injection of 2.5 micrograms/kg of CGRP; similarly, in seven patients with essential hypertension intravenous injection of CGRP (50 micrograms) could induce a significant hypotensive effect. These data suggest that CGRP is a new potential drug for the treatment of essential hypertension.

[The distribution, characteristics and effects of ir-BNP in central and peripheral tissues in rat].

The present study first investigated the distribution, biochemical characteristics, receptor binding and biological effects of immunoreactive brain natriuretic peptide (ir-BNP), in the central nervous system and some peripheral tissues in rats, using highly specific radioimmunoassay, radioreceptor assay and immunohistochemical method. The results suggest that BNP may be a novel neurotransmitter or circulatory hormone, which is widely distributed in various tissues and involved in the regulation of water electrolyte balance and cardiovascular activity.

Regulation of alpha and luteinizing hormone beta subunit messenger ribonucleic acids during stimulatory and downregulatory phases of gonadotropin-releasing hormone action.

Decreased gonadotropin responsiveness (downregulation) to gonadotropin-releasing hormone (GnRH) following chronic in vivo and in vitro exposure to GnRH or its agonist (GnRH-A) has been previously reported. In the present studies, changes in LH subunit mRNAs in rat pituitary monolayer culture during stimulatory and down regulatory phases of GnRH action are described. Rat pituitary cells in culture, pretreated with medium alone or GnRH-A (10(-6) M) for 48 h were extensively washed and treated with graded concentrations of GnRH [10(-9) to 10(-7)] for 4 h. Medium was assayed for luteinizing hormone (LH) immunoreactivity, and total cytoplasmic RNAs were extracted by the hot phenol-guanidinium isothiocyanate method. Subunit-specific mRNAs were quantified by dot-hybridization assay using 32P-labeled subunit-specific cDNA probes. Cells pretreated with medium alone showed a dose-dependent increase in medium LH immunoreactivity, but the alpha and LH beta mRNAs showed no change over the 4-h period. Cells pretreated with GnRH-A showed no significant increase in medium LH with GnRH treatment, thus demonstrating that the cells had been desensitized by prior GnRH-A treatment. Alpha and LH beta subunit mRNAs of cells pretreated with GnRH-A did not show any significant change with further GnRH treatment. In subsequent experiments, cells were incubated with medium alone or 10(-7) M GnRH for 4, 8, or 24 h. GnRH failed to increase subunit mRNAs after 4 and 8 h incubation; after 24 h, alpha subunit mRNA showed a modest but significant increase and beta subunit mRNA showed a modest decrease compared to controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms of mallory body formation induced by okadaic acid in drug-primed mice.

Drug-primed mice form Mallory bodies in their liver after various types of liver injury such as heat shock, drug refeeding, or ethanol ingestion. However, the mechanisms involved that lead to Mallory body formation after these different treatments are unknown. There may be a common pathway of Mallory body formation that is initiated by these different types of injuries. Recently it was shown that the phosphatase 1/2A inhibitor okadaic acid induced Mallory body formation, suggesting that the mechanism of formation involves hyperphosphorylation or oxidative stress-induced NFkappaB activation. To test this hypothesis we exposed drug-primed mice to okadaic acid and measured phosphorylation of Mallory body proteins immunohistochemically and by immunoblot chemiluminescence using an antibody specific for phosphothreonine. NFkappaB activation was measured by a gel shift retardation assay of nuclear lysates. Beginning 15 min after okadaic acid injection, complex changes were progressively seen in the liver cells focally including aggregation of cytokeratins 8 and 18 in hepatocytes which otherwise failed to stain normally with cytokeratin antibody. The aggregates stained positive with ubiquitin and phosphothreonine antibodies. Immunoblots showed a progressive increase in positive staining of the Mallory body band with the antibody to phosphothreonine. NFkappaB activation was progressive up to 2 h after okadaic acid treatment but was downregulated 7 days later. In summary we show for the first time the effect of okadaic acid on the liver cytokeratins in vivo. We conclude that hyperphosphorylation and NFkappaB activation may play a role in the early phases of Mallory body formation.

Mallory body induction in drug-primed mouse liver.

The aim of this study was to determine the various factors that are involved in the induction of Mallory body (MB) formation. A model was developed where MB formation was induced by refeeding either of the drugs griseofulvin or diethyl 1,4-dihydro-1,4,6-trimethyl-3,5-pyridinedicarboxylate (DDC). Mice were fed the drugs for 5 months, followed by withdrawal of the drugs for 1 month (drug-primed livers). The drugs were refed for 1,3,5,7, or 11 days. Early MBs first appeared as small, enlarged aggregates of filaments in the perinuclear or pericanalicular location on the third day of refeeding. Mature MBs appeared on the fifth day of refeeding. MBs reached maximum concentration on day 5 of refeeding. Western blots showed a progressive increase in the cytokeratin proteins (CK49 and CK55) and actin while refeeding the drugs. Liver cell regeneration, as indicated by the percent of proliferating cell nuclear antigen (PCNA)-positive nuclei, increased on the third day of refeeding. However, there was no correlation between the frequency of MBs and the percent of PCNA-positive nuclei. It is concluded that MB formation is not related to the liver cell regeneration response to injury but rather involves a separate regulation pathway. The MBs were heavily ubiquitinated and were associated with increased ubiquitin-protein conjugates as indicated by Western blotting, suggesting that ubiquitinization of cytokeratin protein are involved in the formation of MB aggregation.

Dexamethasone enhances mallory body formation in drug-primed mouse liver.

In a clinical study in which patients with alcoholic hepatitis were treated with prednisone for 1 month, posttreatment liver biopsies showed diminished inflammation, but Mallory bodies were not diminished. This suggested that steroid treatment may reduce inflammation by inhibiting NFkappaB activation. Sparing of Mallory bodies suggests that NFkappaB activation may not be involved mechanistically in Mallory body formation. To test this idea, we induced Mallory body formation in drug-primed mice with or without dexamethasone treatment. As predicted, dexamethasone decreased NFkappaB activation; however, Mallory body formation was increased. Surprisingly, TNFalpha and iNOS, which normally increase as a result of NFkappaB activation, were upregulated by the dexamethasone treatment. It was concluded that NFkappaB activation is not involved in Mallory body formation. Despite this, induced increases in TNFalpha, iNOS, c-jun/API and c-myc expression indicate that oxidative stress is likely involved in Mallory body formation.

Newborn rabbit gastric smooth muscle cell culture: EGF and TGF-alpha are potent mitogens.

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) are two in a family of growth-promoting peptides for many gastrointestinal epithelia. This study was designed to assess their mitogenic effect on cultured gastric myocytes and to characterize specific EGF receptors on these cells. Single myocytes were isolated from newborn rabbit gastric fundus and placed into tissue culture. The composition of the culture at confluence as assessed by immunostaining with smooth muscle actin-specific monoclonal antibody (CGA7) was > 95% myocytes. To assess the effect of putative growth factors, freshly isolated myocytes were incubated in Dulbecco's modified Eagle's (DME) medium containing 1% fetal bovine serum in the presence or absence of growth factors. After 6 days, cells were incubated in serum-free medium with [3H]thymidine (1 microCi/ml) in the continued presence or absence of growth factors. After 24 h, EGF and TGF-alpha but not insulin-like growth factor I (IGF-I) induced a dose-dependent increase in [3H]thymidine incorporation. Ten nanomolar EGF or TGF-alpha increased [3H]thymidine incorporation more than sixfold over control. EGF was more potent than was TGF-alpha, with apparent median effective dose (ED50) values of 64 +/- 14 pM and 166 +/- 62 pM (p < 0.05), respectively. EGF bound to cultured myocytes with Kd = 7.6 +/- 1.8 nM and Bmax = 27 +/- 11 pmol/mg DNA or 440,000 receptors/cell. TGF-alpha competed for binding at these receptors. Although IGF-I did not stimulate thymidine incorporation, specific high-affinity receptors for IGF-I were detected on gastric myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)