Effect of catecholamine on aldosterone release in isolated rat glomerulosa cell suspensions

Effect of adrenergic activity on the adrenal steroidogenesis and the modulation by catecholamines of aldosterone release were studied in isolated rat adrenal cell suspensions. Isoproterenol, norepinephrine and epinephrine, but not dopamine, caused statistically significant increase in aldosterone release. Both prazosin (alpha 1 antagonist) and yohimbine (alpha 2 antagonist) suppressed the norepinephrine-induced aldosterone release in a dose dependent manner, respectively. Both atenolol (beta 1 antagonist) and ICI 118-551 (beta 2 antagonist) also blocked (-)-isoproterenol-induced aldosterone release in a dose dependent manner, respectively. Neither (-)-isoproterenol nor (+/-)-norepinephrine at concentrations of 10(-6) M potentiated aldosterone release stimulated by angiotensin II or ACTH. These results suggest that catecholamines stimulate aldosteroidogenesis, but it appears unlikely that aldosterone release induced by ACTH or angiotensin-II is modulated by adrenergic stimulation.     

Change in metabolic turnover is an alternate mechanism increasing cell surface epidermal growth factor receptor levels in tumor cells

The biosynthesis and metabolic turnover of the epidermal growth factor (EGF) receptor was examined in a human pancreatic carcinoma cell line, UCVA-1. This cell line has been shown to possess a much higher level of EGF receptors than is expected solely from receptor gene/mRNA dosage. Analysis of the biosynthesis using metabolic labeling, immunological quantitation, and inhibitor treatment revealed that the naked EGF receptor in UCVA-1 cells is a protein of Mr 130,000 that is matured consecutively as a Mr 160,000 and 170,000 glycoprotein through post-translational glycosylation. Analysis of the metabolic turnover using pulse-chase labeling and inhibitor treatment revealed that the rate of EGF receptor synthesis in UCVA-1 cells was similar to that in two squamous cell carcinoma cell lines, NA and Ca9-22, which also have high numbers of EGF receptors, but because of gene amplification. In contrast, the rate of receptor degradation in UCVA-1 cells was significantly slower than in the other two cell lines. These results suggest that the retarded metabolic turnover may constitute a unique mechanism for elevating cell surface EGF receptor levels in some tumor cells independent of gene amplification.     

Enzymic Synthesis of Leukotriene B4 in Guinea Pig Brain

Leukotriene B4 [5(S), 12(R)-dihydroxy-6, 14-cis-8,10-trans-eicosatetraenoic acid] was obtained from endogenous arachidonic acid when slices of the guinea pig brain cortex were incubated with the calcium ionophore A 23187. Enzymes involved in its synthesis, arachidonate 5-lipoxygenase [arachidonic acid to 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid and subsequently to leukotriene A4] and leukotriene A4 hydrolase (leukotriene A4 to B4), were present in the cytosol fraction. Arachidonate 5-lipoxygenase was Ca2+-dependent, and was stimulated by ATP and the microsomal membrane, as was noted for the enzyme from mast cells. The lipid hydroperoxides stimulated 5-lipoxygenase by four- to sixfold. The leukotriene A4 hydrolase activity was rich in brain, and the specific activity (0.4 nmol/min/mg of protein) was much the same as that of guinea pig leukocytes. High activities of these enzymes were detected in the olfactory bulb, pituitary gland, hypothalamus, and cerebral cortex. Since leukotriene B4 is enzymically synthesized in the brain, possible roles related to neuronal functions or dysfunctions deserve to be examined.     

Proteinase production and pathogenicity of Candida albicans. I. Invasion into chorioallantoic membrane by C. albicans strains of different proteinase activity

In order to investigate a role of proteinase in the pathogenesis of Candida infections, invasion of C. albicans strains of different proteinase activity into the chorioallantoic membrane (CAM) of developing chicks was studied. Eight strains were used after examining the inducible proteinase activity in the culture containing bovine serum albumin as the sole source of nitrogen. Six were proteinase-producing strains (type I) and two were proteinase-deficient ones (type II). Type I strains were subdivided into type Ia strains in which the proteinase activity persisted for a week in the in vitro culture and type Ib ones in which the enzyme activity was lost by the 7th day after inoculation. By inoculation onto CAM, the type I strains could invade the tissue in which secreted proteinase was detected on the periphery of the invading Candida cells by immunohistochemical method. At an early stage of the infection, proteinase secretion was detected on the surface of the yeast cells before their entry into the tissue. The type II strains remained on the surface of the CAM and did not invade the tissue where the secretion of the enzyme was not detected. The mortality rate of the chick embryo was not correlated with the degree of proteinase production of these strains. Two type Ib strains invaded the CAM tissue and elicited some tissue reactions by the host, yielding a low mortality rate of the chick embryos. These results suggested that the secretion of proteinase was an important factor for the invasion of CAM but other factors were also involved for the pathogenicity of C. albicans.     

Labeling of bovine heart cytochrome c oxidase with analogues of phospholipids. Synthesis and reactivity of a new cardiolipin benzaldehyde probe

The syntheses of two new radioactive probes derived from cardiolipin and phosphatidylcholine are reported. These probes are derivatives of natural lipids and contain an amine-specific benzaldehyde in the head-group region. This functional group allows a choice of timing of the reaction (e.g., after equilibration and detergent removal) because an irreversible covalent bond is formed only upon the addition of reducing agent. These probes, as well as a benzaldehyde analogue of phosphatidic acid, and a water-soluble benzaldehyde reagent were covalently attached to bovine heart cytochrome c oxidase. After reconstitution into vesicles, the lipid-benzaldehyde probes selectively incorporated into the smaller polypeptides of the enzyme, while the remaining subunits (I-IV) exhibited little incorporation of label. The accessibility of amine groups labeled under the conditions used here was independent of the structural and charge differences between the benzaldehyde probes. This suggests that all three lipid probes react with polypeptides of the cytochrome c oxidase complex at general contact sites for membrane phospholipids. A water-soluble benzaldehyde reagent predominantly labeled subunits IV, Va, and Vb and polypeptides of VII-VIII. A comparison of these results facilitates a more refined view of the disposition of polypeptides of cytochrome c oxidase in respect to the lipid and aqueous phases.     

Generation of H2O2 is regulated by cytoplasmic free calcium in cultured porcine thyroid cells

Effects of calcium ionophore A23187 and BAY-K-8644, a calcium channel agonist, on cytoplasmic free calcium ([Ca2+]i) and H2O2 generation were studied in cultured porcine thyroid cells. We monitored continuously the effects of A23187 and BAY-K-8644 on [Ca2+]i and H2O2 generation, using the intracellularly trapped fluorescent Ca2+ indicator, fura-2, and homovanillic acid, respectively. A23187 and BAY-K-8644 induce an immediate increase in [Ca2+]i and H2O2 generation. The A23187- and BAY-K-8644-induced [Ca2+]i responses and H2O2 generation occur immediately, reach a maximum within several seconds, and then slowly decline. The minimum doses of A23187 or BAY-K-8644 to increase [Ca2+]i stimulate H2O2 generation. H2O2 generation is regulated by [Ca2+]i.     

Immobilization of cellulolytic and hemicellulolytic enzymes on inorganic supports

Commercial cellulase preparations from Trichoderma viride and Aspergillus niger were immobilized on porous silica glass and ceramics such as alumina and titania with titanium tetrachloride (TiCl(4)) and on their silanized derivatives with glutaraldehyde (GLUT). The amounts of the immobilized enzymes were in the range 10-50 mg/g carrier (dry) depending on the kind of carrier and immobilization method. Their activities toward carboxymethyl cellulose (CMC), xylan, aryl-beta-glucoside, and aryl-beta-xyloside were 3-53% of those of the native enzymes. The optimum pH of the enzymes shifted to the acidic side in most cases, whereas the optimum temperatures were nearly the same as those of native ones. The activity of immobilized enzyme preparations towards CMC did not change significantly during continuous operation over a periods of 60 days. Finally, xylan was hydrolyzed with the immobilized enzymes, and the sugars formed were investigated.     

Inherited hydrocephalus in Csk: Wistar-Imamichi rats; Hyd strain: a new disease model for hydrocephalus

Hydrocephalic rats were found in a breeding colony of Csk: Wistar-Imamichi strain rats. In males, the hydrocephalus were serious and could be detected from 7 days after birth. Survival was 3-4 weeks. In females, the hydrocephalus was moderate, there was no abnormal external appearance, and the rats were able to mature. Ventricular dilatation was excessive in males but moderate in females. The total frequency of hydrocephalus was 34.3% in both males and females. Breeding data indicated that this disease is heritable and is single dominant and X-linked (symbol, Hyd). The female moderate hydrocephalics could be detected by progeny tests without examining brain sections. No evidence of developmental anomaly was observed in the ventricles. This hydrocephalus was classified as being of the communicating type, and this strain was named the Hyd strain as an animal model for human hydrocephalus.     

Role in translation of a triple tandemly repeated sequence in the 5''-untranslated region of human thymidylate synthase mRNA.

A triple tandem repeat (TTR) consisting of 90 nucleotides exists immediately upstream of the ATG initiator codon in human thymidylate synthase (TS) cDNA (pcHTS-1). To investigate the role of the TTR in the expression of the TS cDNA, we used pcHTS-1 to construct mutant cDNA clones in which part of the TTR was deleted or an additional element was inserted. The mutant cDNA plasmid was introduced into murine TS-negative mutant cells and the relative translation efficiencies of the mutant cDNAs were determined by measuring the transient expression of TS activity and the amount of TS mRNA transcribed. The translation efficiency in transient expression of the mutants was increased by deletions covering all the first two repeated elements, and the part of the third closest to the ATG initiator codon, but was not affected by deletions of only parts of the first two repeated elements at the 5' end. The translation efficiency was also not affected by insertion of an additional repeated element into the TTR. These results suggest that the first two repeated elements at the 5' end both have inhibitory effects on translation of the TS mRNA, probably due to the unique structural feature of this element.