Proadrenomedullin N-Terminal 20 Peptide (PAMP), an Endogenous Anticholinergic Peptide: Its Exocytotic Secretion and Inhibition of Catecholamine Secretion in Adrenal Medulla

Abstract: In cultured bovine adrenal medullary cells, stimulation of nicotinic receptors by carbachol evoked the Ca²⁺-dependent exocytotic cosecretion of proadrenomedullin N-terminal 20 peptide (PAMP) (EC₅₀ = 50.1 µ M ) and catecholamines (EC₅₀ = 63.0 µ M ), with the molar ratio of PAMP/catecholamines secreted being equal to the ratio in the cells. Addition of PAMP[1–20]NH₂ inhibited carbachol-induced ²²Na⁺ influx via nicotinic receptors (IC₅₀ = 2.5 µ M ) in a noncompetitive manner and thereby reduced carbachol-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels (IC₅₀ = 1.0 µ M ) and catecholamine secretion (IC₅₀ = 1.6 µ M ). It did not alter high K⁺-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels or veratridine-induced ²²Na⁺ influx via voltage-dependent Na⁺ channels. PAMP seems to be a novel antinicotinic peptide cosecreted with catecholamines by a Ca²⁺-dependent exocytosis in response to nicotinic receptor stimulation.     

Comparison of the DNA content of megakaryocytes identified immunologically with that identified morphologically

 We devised a new microfluorometric method for determining the ploidy of megakaryocytes identified immunologically in bone marrow smears. The smears were immunostained by incubation with mouse monoclonal anti-glycoproteins (GP) IIb antibodies, followed by fluorescein isothiocyanate-conjugated goat anti-mouse IgG antibodies. They were then stained with 4′,6-diamidino-2-phenylindole (DAPI). Megakaryocytes were identified by their GPIIb immunofluorescence using a microfluorometer and, after the filters were changed, their DNA content was assayed by measuring the intensity of DAPI fluorescence. This intensity was shown to be proportional to the DNA content when the aperture of the objective lens was reduced. We compared these results with those obtained when megakaryocytes were identified morphologically, using DAPI staining after Wright-Giemsa destaining. In all 12 normal controls, the ploidy peaks were shown to be 16N by both methods, and the mean ploidy detected by the immunological method was only reduced 0.961 times relative to the estimate from the morphological method. In contrast, in eight myelodysplastic syndrome (MDS) patients, the ploidy peaks were either 8N or 4N and the mean was reduced by 0.906 times (P=0.018). Thus we could immunologically identify small megakaryocytes which we could not identify morphologically. Therefore, this method is useful for measuring megakaryocytic ploidy, especially in the pathological megakaryocytes of MDS patients. Accepted: 29 April 1997     

Changes of an androgen-dependent nuclear protein during functional differentiation and by dedifferentiation of the dorsolateral prostate of rats

In contrast with previous results that indicate that Saccharomyces cerevisiae fructose-1,6-bisphosphatase is a dimer of 56,000 molecular weight subunits, we find that the subunit Mr of the enzyme purified from baker's yeast is 40,000. The same subunit Mr was observed in immunoprecipitates of crude supernatants of baker's yeast and S. cerevisiae cultures, as well as in acid-extracts of cells detected by immunoblotting, suggesting that the native subunit indeed has a Mr of 40,000 and it has not been produced from a larger polypeptide. Complete immunoprecipitation of fructose-1,6-bisphosphatase activity with saturating concentrations of specific antibody suggests that there is only one fructose-1,6-bisphosphatase isozyme in S. cerevisiae. The Mr of the purified enzyme determined by size exclusion HPLC suggests that it has a tetrameric structure characteristic of fructose-1,6-bisphosphatases from a broad phylogenetic spectrum.     

Aldosterone biosynthesis by a reconstituted cytochrome P-45011 beta system

[3H]Corticosterone was incubated with cytochrome P-45011 beta purified to electrophoretic homogeneity from bovine adrenocortical mitochondria, and the reaction products were analyzed by high performance liquid chromatography. The production of aldosterone (21.2 pmol/nmol P-450/min) and 18-hydroxycorticosterone (1.17 nmol/nmol P-450/min) was observed. When lipidic extracts from mitochondria of bovine adrenocortical zona glomerulosa were added to the reaction mixture, the rate of production of aldosterone was increased 28-fold. When [3H]18-hydroxycorticosterone was incubated with cytochrome P-45011 beta, the amount of aldosterone produced was 55.7 pmol/nmol P-450/min in the absence of the lipidic extracts and the enhancing effect of the lipidic extracts was 4-fold.     

The Lipid Phase of Thylakoid and Cytoplasmic Membranes from the Blue-Green Algae (Cyanobacteria), Anacystis nidulans and Anabaena variabilis

The lipid phases of the thylakoid and cytoplasmic membranesfrom the blue-green alga, Anacystis nidulans, were studied bya spin-probe method using 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl.The thylakoid and cytoplasmic membranes of this alga were bothin the liquid crystalline state at growth temperature, and inthe phase separation state at about 0?C. The thylakoid membranesentered the phase separation state at a temperature higher thanthe cytoplasmic membranes. The lipid phase of the thylakoidmembranes from Anabaena variabilis was studied in a similarway, and these membranes were found also to undergo the phasetransition. The temperature for the onset of the phase separationand the fluidity of the membrane lipids of both algae dependedon the growth temperature of the culture. (Received April 9, 1984; Accepted June 1, 1984)     

Relation of mode of administration of testosterone to evocation of male sex behavior in frogs

In Rana pipiens, mating behavior could be induced readily in intact males by several pituitary implantations, but never in castrates. Systemic testosterone injection (1 mg daily), with or without pituitary implantation, failed to restore mating behavior in castrated frogs. On the other hand, intracranial implantation of testosterone (approximately 60-μg pellets in which testosterone is mixed with cholesterol 1:1) in castrates evoked mating behavior, including mating calls and clasping. The most effective implantation site was the rostral part of the preoptic nucleus. Thus, the rostral part of the preoptic nucleus is the androgen-sensitive site which governs sexual behavior in this species. The relative ineffectiveness of systemic injection of testosterone is discussed.     

Mate calling induced by electrical stimulation in freely moving leopard frogs, Rana pipiens.

Electrodes were implanted chronically into the preoptic areas of normal or castrated male frogs, Rana pipiens, and monopolar monophasic stimulating currents (100 Hz, 0.5-msec duration, and mainly 50–200 μA in intensity) were delivered through the implanted electrodes in freely moving frogs. When the electrodes were placed in the rostral part of the preoptic nucleus, mating calls (mainly trills and sometimes chuckles) were inducible. Fifty microamperes was generally an effective stimulus intensity for induction of calls, and 20 μA was the minimum effective stimulus intensity observed. There was no difference in the threshold to induce calls between pituitary-treated intact and castrated frogs.     

Primary structure of mitochondrial glutamic oxaloacetic transaminase from rat liver: Comparison with that of the pig heart isozyme

The complete amino acid sequence of the mitochondrial glutamic oxaloacetic transaminase isozyme from rat liver is presented. The sequence contained 401 amino acid residues, 10 of which are methionine. Cyanogen bromide cleavage of mitochondrial glutamic oxaloacetic transaminase produced 12 peptides, one of which contained an internal homoserine residue resulting from incomplete cleavage by cyanogen bromide. The calculated molecular weight was 44,358. The sequence showed 94% homology with that of the corresponding isozyme from pig heart. These findings support the conclusion that the rate of evolution of the mitochondrial isozymes is lower than that of their cytosolic isozymes.     

Giant oil cells in the cotyledons ofPharbitis nil and other convolvulacean plants

Suspension of protoplasts (ca. 13–25 μm in diameter) that were isolated from the mesophyll of the cotyledons ofPharbitis nil, strain Violet, contained many large spherical or spheroidal bodies (ca. 100 μm in diameter). Microscopic observation of these bodies and some anatomic studies of the cotyledons during embryogenesis and after germination showed that these bodies are giant cells containing many oil drops stainable with Sudan dyes. Such giant cells were found in four otherPharbitis nil strains, Nepal, Tendan, Africa and Tokyo-kokei, and in six other Convolvulacean plants,Ipomoea batatas, cv. Koukei-14,Calystegia japonica, Calystegia hederacea, Calonyction aculeatum, Quamoclit pennata andCuscuta japonica.