Improved synthesis of 16alpha-hydroxylated androgens: intermediates of estriol formation in pregnancy.

16alpha-Hydroxyandrostenedione (16alpha-hydroxyandrost-4-ene-3,17-dione), 16alpha-hydroxytestosterone (16alpha,17beta-dihydroxyandrost-4-en-3-one) and 16alpha-hydroxydehydroepiandrosterone 3-sulfate (3beta, 16alpha-dihydroxyandrost-5-en-17-one 3-monosulfate) were synthesized by a new chemical approach with much improved yield. 16alpha-Bromoandrostendione was converted to the hydrazone of 16alpha-hydroxyandrostenedione which gave 16alpha-hydroxyandrostenedione on acid hydrolysis in total 63% yield. Oxidation of 16alpha-hydroxydehydroepiandrosterone with Jones' reagent also selectively afforded 16alpha-hydroxyandrostenedione. 16alpha-Hydroxytestosterone was observed by selective reduction of 16alpha-hydroxyandrostenedione with sodium borohydride. Reaction of 16alpha-hydroxydehydroepiandrosterone with chlorosulfonic acid in pyridine selectively gave the 3-monosulfate. The structure of the sulfate was deduced from its solvolysis to the starting material, and its acetylation and subsequent solvolysis to 16alpha-hydroxydehydroepiandrosterone 16-acetate. All procedures are suitable for large scale synthesis without the use of microorganisms.     

Radioimmunoassay of 2-hydroxyestrone

Under the protection of ascorbic acid a 2-hydroxyestrone bovine serum albumin conjugate was prepared containing intact 2-hydroxyestrone as determined by gas chromatographymass spectrometry. Using this antigen highly specific antibodies were raised in rabbits. Cross-reactivity for 2-hydroxyestradiol and 2-hydroxyestriol was 26 and 4.5%, respectively. An assay procedure of 2-hydroxyestrone in human plasma is described. Using special precautions the assay allows the determination of 2-hydroxyestrone in plasma samples of women (50–95 pg/ml), pregnant women (105–220 pg/ml), men (45–65 pg/ml) and children (20–40 pg/ml).     

Catecholamine binding to CNS adrenergic receptors

The properties of ³H-catecholamine binding to α- and β-adrenergic receptors in CNS are reviewed. ³H-epinephrine and ³H-norepinephrine label one class of α-receptors throughout the brain, with high affinities for agonists and some antagonists. Agonist affinities at this site are increased in low temperature conditions but are reduced by guanine nucleotides and monovalent cations. Divalent cations reverse both effects. This α-receptor may be coupled to adenylate cyclase by GTP and/or sodium, and uncoupled by divalent cations. ³H-epinephrine labels β₂, but not β₁, receptors in CNS, especially in bovine cerebellum. The same β-receptor does not show agonist-specific GTP-sensitivity, but does exhibit Na⁺-sensitivity. This receptor appears to be linked to adenylate cyclase, and sodium rather than GTP may be the coupling agent.     

Mitogens for murine embryo cell lines

The growth-promoting activities of fetal bovine serum, cortisol, phorbol myristate acetate, prostaglandin F2α, insulin, epidermal growth factor, and fibroblast growth factor were evaluated on four murine embryo cell lines (Swiss 3T3, Balb 3T3, M2, and C3H10T1/2). Each cell had an unique response spectrum to this collection of reported mitogens. Phorbol myristate acetate and prostaglandin F2α were active only on selected cell lines; cortisol was inactive on all four lines. Serum, epidermal growth factor, and fibroblast growth factor were able to stimulate cell division in all four lines, albeit to varying degrees for the different target cells.     

Formation of ATP by the adenosine triphosphatase complex from spinach chloroplasts reconstituted together with bacteriorhodopsin

The energy-linked ATPase complex has been isolated from spinach chloroplasts. This protein complex contained all the subunits of the chloroplast coupling factor (CF₁) as well as several hydrophobic components. When the activated complex was reconstituted with added soybean phospholipids, it catalyzed the exchange of radioactive inorganic phosphate with ATP. Sonication of the complex into proteoliposomes together with bacteriorhodopsin yielded vesicles that catalyzed light-dependent ATP formation. Both the ³²P_i-ATP exchange reactions and ATP formation were sensitive to uncouplers such as 3-tert-butyl-5,2′-dichloro-4′-nitrosalicylanilide, bis-(hexafluoroacetonyl)acetone and carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone, that act to dissipate a proton gradient. The energy transfer inhibitors dicyclohexylcarbodiimide, triphenyltin chloride and 2-β-d-glucopyranosyl-4,6′-dihydroxydihydrochalcone were also effective inhibitors of both reactions.     

Formation of palmityl-[13'-32P]coenzyme A from [gamma-32P]ATP in mitochondrial extracts of guinea pig liver.

Investigations of the incorporation of ³²P into acyl-coenzyme A (CoA) in incubation mixtures containing a soluble protein preparation derived from mitochondria, [γ-³²P]ATP, and palmityl-CoA have led to the discovery of an enzymatic activity which catalyzes the exchange of palmityl groups between molecules of CoA: CoA¹ + palmityl-CoA ? palmityl-CoA¹ + CoA. The preparation also contains dephospho-CoA kinase and palmityl-CoA thiolester hydrolase activities. The initial detection of the exchange reaction resulted from the formation of [3′-³²P]CoA via the dephospho-CoA kinase reaction with exogenous [γ-³²P]ATP. The described preparation of palmityl-[3′-³²P]CoA and palmityl-[³⁵S]CoA facilitated demonstration of the reversibility of the reaction and ruled out the possibility that the exchange of fragments of the CoA molecule mediated the observed incorporation. The reversible palmityl group exchange does not appear to be catalyzed by a previously described enzyme. None of the possible acyl group acceptors considered in these studies participated in the reaction as efficiently as CoA itself. The possibility is discussed that the exchange reaction may explain reports of an unknown lipid formed by an oligomycin-sensitive mitochondrial ATPase preparation.     

Action of alkyl cations and the natural ATPase inhibitor from mitochondria on soluble mitochondrial ATPase

The effect of the natural ATPase inhibitor and octylguanidine on the ATPase activity of soluble oligomycin-insensitive mitochondrial F₁ were compared. Both compounds induced a maximal inhibition of 60–80% in various preparations of F₁ studied. The inhibition was of the uncompetitive type with respect to MgATP, and the action of the compounds was partially additive. The data suggest that octylguanidine reproduces the action of the natural ATPase inhibitor. Alkylammonium salts also affect the ATPase activity in a similar form. F₁ bound to Sepharose-hexylammonium is largely inactive, whilst free hexylammonium at higher concentrations induces only a partial inhibition of the activity. This suggests that the degree of immobilization of F₁ is related to the magnitude of inhibition of ATPase activity induced by alkyl cations. The binding of F₁ to Sepharose-hexylammonium is prevented by high concentrations of Na⁺ or K⁺.     

The photochemical and fluorescence properties of whole cells,spheroplasts and spheroplast particles from the blue-green alga Phormidium luridum

The photochemical activities and fluorescence properties of cells, spheroplasts and spheroplast particles from the blue-green alga Phormidium luridum were compared. The photochemical activities were measured in a whole range of wavelengths and expressed as quantum yield spectra (quantum yield vs. wavelength). The following reactions were measured: Photosynthesis (O₂ evolution) in whole cells; Hill reaction (O₂ evolution) with Fe(CN)₆^3? and NADP as electron acceptors (Photosystem II and Photosystem II+Photosystem I reactions); electron transfer from reduced 2,6-dichlorophenolindophenol to diquat (Photosystem I reaction). The fluorescence properties were emission spectra, quantum yield spectra and the induction pattern.On the basis of comparison between the quantum yield spectra and the pigments compositions the relative contribution of each pigment to each photosystem was estimated. In normal cells and spheroplasts it was found that Photosystem I (Photosystem II) contains about 90 % (10 %) of the chlorophyll a, 90 % (10 %) of the carotenoids and 15 % (85 %) of the phycocyanin. In spheroplast particles there is a reorganization of the pigments: they loose a certain fraction (about half) of the phycocyanin but the remaining phycocyanin attaches itself exclusively to Photosystem I (!). This is reflected by the loss of Photosystem II activity, a flat quantum yield vs. wavelength dependence and a loss of the fluorescence induction.The fluorescence quantum yield spectra conform qualitatively to the above conclusion. More quantitative estimation shows that only a fraction (20–40 %) of the chlorophyll of Photosystem II is fluorescent. Total emission spectrum and the ratio of variable to constant fluorescence are in agreement with this conclusion.The fluorescence emission spectrum shows characteristic differences between the constant and variable components. The variable fluorescence comes exclusively from chlorophyll a; the constant fluorescence is contributed, in addition to chlorophyll a, by phycocyanine and an unidentified long wavelength component.The variable fluorescence does not change in the transition from whole cells to spheroplasts. However, the constant fluorescence increases considerably. This indicates the release of a small fraction of pigments from the photosynthetic photochemical apparatus which then become fluorescent.