Effect of dopamine, bombesin and cysteamine hydrochloride on plasma growth hormone response to synthetic growth hormone-releasing factor in rats

In urethane anesthetized rats, an intracerebroventricular (icv) injection of 2 micrograms bombesin 5 min prior to the administration of synthetic human growth hormone-releasing factor (GRF) (1 microgram/kg, iv) inhibited plasma growth hormone (GH) response, while cysteamine hydrochloride (90 mg/kg, sc) administered 150 min beforehand depleted immunoreactive somatostatin content in the pituitary-stalk median eminence and consequently potentiated the response to GRF. Under the same experimental conditions, central injection of 1.89 micrograms (10(-8)M) dopamine hydrochloride or iv administration of L-DOPA (10 mg/kg) did not influence the subsequent plasma GH response to GRF. Results suggest indirectly that bombesin and cysteamine, but not dopamine, predominantly modulate somatostatin release from the hypothalamus.     

CRF-containing neurons of the rat hypothalamus

Summary The immunoreactive CRF-neurons of the rat hypothalamus have been examined immunohistochemically employing anti-rat CRF serum. These neurons are confined to the paraventricular nucleus, dorsomedial-lateral hypothalamic area, and suprachiasmatic nucleus, and are, respectively, also immunoreactive to anti-Met-enk, -alpha-MSH, and -VIP sera. Intraventricular administration of colchicine (50 g/5 l/rat) induces a dramatic enhancement of the immunostainability of the cell somata, and also accelerates the development of immunoreactivity of other stored peptides, especially in the paraventricular nucleus.The CRF-neurons respond to adrenalectomy by showing increased immunoreactivity and an increase in the number of cell bodies; in the dorsomedial-lateral area and suprachiasmatic nucleus, there is also an enhanced immunoreactivity for alpha-MSH and VIP, respectively. CRF-cells in the paraventricular nucleus become markedly hypertrophied, but do not show any enhanced immunoreactivity for Met-enk. Since the axons of the paraventricular neurons run to the median eminence, it is probable that they are involved with the endocrine control of hypophysial ACTH release. It is concluded that the CRF-containing neurons in rat hypothalamus consist of three types which are functionally and morphologically different.     

Immunoreactive-beta-endorphin, -adrenocorticotropin and -calcitonin in extracts of anaplastic or differentiated (rat) medullary thyroid carcinoma.

Fifteen analogs of luliberin (2, LRH) were synthesized by the solid phase method and examined for their ability to block ovulation in the rat. Two analogs, [Ac-DAla¹,DPhe²,DTrp^3,6]-LRH and [Ac-DPhe¹,DPhe²,DTrp^3,6]-LRH, each blocked ovulation at a single injection dose of 250 μg administered at noon on the day of proestrus; three peptides, [Ac-DPro¹,DPhe²,DTrp^3,6]-LRH, [Ac-DThi¹,DPhe²,DTrp^3,6]-LRH and [Ac-DTrp¹,DPhe²,DTrp^3,6]-LRH, were effective at doses of 500 μg each; and four others, [Ac-DTrp¹,DPhe²,DTrp³,DTrp(Nps)⁶]-LRH, [Chlorambucil-DPhe¹, DPhe², DTrp^3,6]-LRH, [1,DThi²,DTrp^3,6]-LRH and [(2-DLys⁶]-LRH, gave partial inhibition at doses tested.     

Deoxyribonucleic acid strand breaks during drying of Escherichia coli on a hydorohobic filter membrane.

Cells of Escherichia coli mounted on a hydrophobic filter membrane were dried under various vapor pressures. A mutant defective in deoxyribonucleic acid repair (uvrA recA) was more sensitive to drying at a water activity of 0.53 or below than the parent strain but not at a water activity of 0.75 and above. Sucrose gradient studies showed that single- and double-strand breaks of deoxyribonucleic acid occurred at a water activity of 0.53 or below, but no breaks could be observed at a water activity of 0.75 or above. These results were observed in all cells rehydrated with 0.03 M tris (hydroxymethyl) aminomethane-hydrocholoride buffer solution at 0 or 37 degrees C, in the presence or absence of oxygen, with saturated water vapor or with a hypertonic solution followed by a gradual dilution. Freezable water was detected in the cells only at a water activity above 0.75 by differential scanning calorimetry. Removal of unfreezable water of cells in the drying, therfore, might induce deoxyribonucleic acid strand breaks.     

Phosphatidylinositol-4-phosphate 5-kinase gamma is associated with cell-cell junction in A431 epithelial cells

Cell to cell contact in epithelial cells is crucial for tissue integrity and is maintained by junctional complexes, such as the adherens junction (AJ). Actin polymerization has been shown to be important for AJ formation; however, the molecular mechanisms have yet to be clarified. It has been shown that increased phosphatidylinositol-4,5-bisphosphate (PIP2) induces actin polymerization. It is thus of interest to know more about the production of PIP2 during cell-cell adhesion formation in epithelial cells. The distribution of phosphatidylinositol-4-phosphate 5-kinase gamma635 (PIP5Kgamma635), an isoform of the PIP2 synthesizing enzymes, was examined in epithelial cell line A431. It was found that, in non-contact cells, PIP5Kgamma635 was not concentrated at the plasma membrane. However, in cells that were in contact, PIP5Kgamma635 localized to the intercellular contact sites and colocalized with E-cadherin and beta-catenin, two components of AJ, and with polymerized actin, but did not colocalize with focal adhesion, integrin-mediated cell-substratum complex. Decreasing calcium ion concentration induced both disruption of intercellular adhesion and the dissociation of both PIP5Kgamma635 and actin from the contact site. These results suggest that PIP5K has an important role in actin polymerization in epithelial cell-cell adhesion.     

Use of 11alpha-deuterium labeled cortisol as a tracer for assessing reduced 11beta-HSD2 activity in vivo following glycyrrhetinic acid ingestion in a human subject

This study describes an oral administration of 5 mg of [1,2,4,19-13C4,11alpha-2H]cortisol (cortisol-13C4,2H1) to a human subject performed on two separate occasions, one with cortisol-13C4,2H1 alone and the other with cortisol-13C4,2H1 plus 130 mg per day of glycyrrhetinic acid for 6 days. The stable isotope methodology employed allowed for the evaluation of the individual in vivo activities of the two isozymes of 11beta-hydroxysteroid dehydrogenase (11beta-HSD), 11beta-HSD1 and 11beta-HSD2, and to demonstrate the sensitivity of changes in cortisol elimination half-life for detecting inhibition of 11beta-HSD2 activity induced with glycyrrhetinic acid. The kinetic analysis associated with the loss of 11alpha-2H during the conversion of cortisol-13C4,2H1 to cortisone-13C4 by 11beta-HSD2 clearly indicated reduced 11beta-HSD2 activity with glycyrrhetinic acid ingestion, as observed by an increase in the elimination half-life of cortisol-13C4,2H1. The elimination half-life of cortisol-13C4,2H1 provided sensitive in vivo measures of 11beta-HSD2 activity and was more sensitive for detecting changes in renal 11beta-HSD2 activity than the measurement of the urinary ratio of free cortisol and free cortisone (UFF/UFE). The 2H-labeling in the 11alpha-position of cortisol served as an appropriate tracer for assessing the reduced 11beta-HSD2 activity in vivo induced by glycyrrhetinic acid.     

Effect of the polypeptide binding on the thermodynamic stability of the substrate binding domain of the DnaK chaperone

The effect of polypeptide binding on the stability of the substrate binding domain of the molecular chaperone DnaK has been studied by thermodynamic analysis. The calorimetric scan of the fragment of the substrate binding domain DnaK384-638, consisting of a beta-domain and an alpha-helical lid, showed two transitions centered at 56.2 and 76.0 degrees C. On the other hand, the thermal unfolding of the shorter fragment DnaK386-561, which lacks half of the alpha-helical lid, exhibited a single transition at 57.0 degrees C. Therefore, the transition of DnaK384-638 at 56.2 degrees C is mainly attributed to the unfolding of the beta-domain. The calorimetric scan of DnaK384-638D526N showed that the unfolding of the beta-domain was composed of two transitions. The polypeptide bound DnaK384-638 exhibited a symmetrical DSC peak at 58.6 degrees C, indicating that the substrate binding shifts the beta-domain toward a single cooperative unit. A low concentration of GdnHCl (<1.0 M) induced a conformational change in the beta-domain of DnaK384-638 without changes in the secondary structure. While the thermal unfolding of the beta-domain of DnaK384-638 was composed of two transitions in the presence of GdnHCl, the beta-domain of the substrate bound DnaK384-638 exhibited a single symmetrical DSC peak in the same condition. All together, our results indicate that complex between DnaK384-638 and substrate forms a rigid conformation in the beta-domain.     

Microanalysis for MDR1 ATPase by high-performance liquid chromatography with a titanium dioxide column

MDR1 is clinically important because it is involved in multidrug resistance of cancer cells and affects the pharmacokinetics of various drugs. Because MDR1 harnesses adenosine 5'-triphosphate (ATP) hydrolysis for transporting drugs, examining the effect on ATPase activity is imperative for understanding the interactions between drugs and MDR1. However, conventional assay systems for ATPase activity are not sensitive enough for screening drugs using purified MDR1. Here we report a novel method to measure ATPase activity of MDR1 using high-performance liquid chromatography equipped with a titanium dioxide column. The amount of adenosine 5'-diphosphate (ADP) produced by the ATPase reaction was determined within 2 min with a titanium dioxide column (4.6 mm ID x 100 mm). The relationship between ADP amount and chromatogram peak area was linear from 5 pmol to 10 nmol. This method made it possible to reduce the amount of purified MDR1 required for a reaction to 0.5 ng, about 1/20th of the conventional colorimetric inorganic phosphate detection assay. This method is sensitive enough to detect any subtle changes in ATPase activity of MDR1 induced by drugs and can be applied to measure ATPase activity of any protein.     

Interaction of ATP sensor, cAMP sensor, Ca2+ sensor, and voltage-dependent Ca2+ channel in insulin granule exocytosis

ATP, cAMP, and Ca(2+) are the major signals in the regulation of insulin granule exocytosis in pancreatic beta cells. The sensors and regulators of these signals have been characterized individually. The ATP-sensitive K(+) channel, acting as the ATP sensor, couples cell metabolism to membrane potential. cAMP-GEFII, acting as a cAMP sensor, mediates cAMP-dependent, protein kinase A-independent exocytosis, which requires interaction with both Piccolo as a Ca(2+) sensor and Rim2 as a Rab3 effector. l-type voltage-dependent Ca(2+) channels (VDCCs) regulate Ca(2+) influx. In the present study, we demonstrate interactions of these molecules. Sulfonylurea receptor 1, a subunit of ATP-sensitive K(+) channels, interacts specifically with cAMP-GEFII through nucleotide-binding fold 1, and the interaction is decreased by a high concentration of cAMP. Localization of cAMP-GEFII overlaps with that of Rim2 in plasma membrane of insulin-secreting MIN6 cells. Localization of Rab3 co-incides with that of Rim2. Rim2 mutant lacking the Rab3 binding region, when overexpressed in MIN6 cells, is localized exclusively in cytoplasm, and impairs cAMP-dependent exocytosis in MIN6 cells. In addition, Rim2 and Piccolo bind directly to the alpha(1)1.2-subunit of VDCC. These results indicate that ATP sensor, cAMP sensor, Ca(2+) sensor, and VDCC interact with each other, which further suggests that ATP, cAMP, and Ca(2+) signals in insulin granule exocytosis are integrated in a specialized domain of pancreatic beta cells to facilitate stimulus-secretion coupling.