Isolation and identification of 25-hydroxy-24-oxocholecalciferol: A metabolite of 25-hydroxycholecalciferol

A metabolite of 25-hydroxycholecalciferol has been isolated in pure form from chicken kidney homogenates. It has been identified as 25-hydroxy-24-oxocholecalciferol by means of ultraviolet absorption spectrophotometry, mass spectrometry, infrared spectrometry, nuclear magnetic resonance spectrometry, and specific chemical reactions.     

Heterotrophic bacteria accompanying Stichococcus bacillaris in laboratory cultures

The number and composition of bacterial microflora accompanying Stichococcus bacillaris in various media with urea was determined. The number of heterotrophic bacteria during 5 days of incubation increased 10-fold. Only some of the isolated bacterial strains were able to grow in medium for algae supplemented with dead S. bacillaris cells. It is suggested that bacteria utilize organic matter released to the medium by S. bacillaris. The most numerous among the isolated bacteria were Enterobacteriaceae. The order of domination by bacterial populations distinguishes nonaxenic cultures of S. bacillaris from cultures of other algae.     

The MAS-encoded processing protease of yeast mitochondria. Interaction of the purified enzyme with signal peptides and a purified precursor protein

The matrix of yeast mitochondria contains a chelator-sensitive protease that removes matrix-targeting signals from most precursor proteins transported into this compartment. The enzyme consists of two nonidentical subunits that are encoded by the nuclear genes MAS1 and MAS2. With the aid of these cloned genes, we have now overexpressed the active holoenzyme in yeast, purified it in milligram amounts, and studied its biochemical and physical properties. Atomic absorption analysis shows that the purified enzyme lacks significant amounts of zinc, manganese, or cobalt; if none of these metal ions is added during the assay, the enzyme is catalytically inactive but can still cleave substoichiometric amounts of substrate. The amino-terminal sequences of the two mature subunits were determined; comparison with the deduced amino acid sequences of the corresponding precursors revealed that the MAS1 and MAS2 subunits are synthesized with prepeptides composed of 19 and 13 residues, respectively, which have similar sequences. The enzyme is inhibited competitively by chemically synthesized matrix-targeting peptides; the degree of inhibition correlates with the peptides' targeting efficacy. Matrix-targeting peptides containing the cleavage site of the corresponding authentic precursor protein are cleaved correctly by the purified enzyme. A purified artificial precursor protein bound to the holoenzyme can be photocross-linked to the MAS2 subunit.     

Mechanism of action of amylin in bone.

Amylin is a 37 amino acid peptide produced mainly by beta-cells of the endocrine pancreas. Human amylin has 43% homology with human calcitonin gene-related peptide (CGRP) and 13% homology with human calcitonin (CT). Amylin and CGRP have been reported to have CT-like hypocalcemic activity in vivo. To investigate the role of amylin in bone, we examined the mechanisms of action of human amylin, CGRP, and CT in osteoclasts and osteoblasts. Both human amylin and CGRP inhibited 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3]- induced bone resorption in an organ culture system, and the potencies of the two peptides were similarly approximately 60-fold lower than that of human CT. Using a recently developed procedure for preparing large numbers of osteoclast-like multinucleated cells (MNCs) formed in co-cultures of mouse osteoblasts and bone marrow cells in the presence of 1 alpha,25(OH)2D3, we found that both human amylin and CGRP stimulated cAMP production in osteoclast-like MNCs, but only at 60-fold higher concentrations than human CT. Specific binding of [125I]-human CT to osteoclast-like MNCs was detected (dissociation constant, 3 x 10(-8) M; binding sites, 3 x 10(7) per cell). To displace the bound [125I]-human CT from osteoclast-like MNCs, about 170-fold higher concentrations of human amylin and CGRP were required. No specific bindings of [125I]-amylin and [125I]-CGRP to osteoclast-like MNCs could be detected. Human CGRP stimulated cAMP production both in established mouse osteoblast-like cells (KS-4) and in mouse primary osteoblast-like cells. Amylin was a weak agonist for cAMP production in KS-4 cells. The increment in cAMP production induced by CGRP and amylin was abolished by the addition of human CGRP(8-37), a selective antagonist for CGRP receptors. CT did not stimulate cAMP production in KS-4 cells. Amylin, but not CT, displaced the bound [125I]-human CGRP from rat brain membranes. These results indicate that amylin binds not only to CT receptors in osteoclast-like MNCs but also to CGRP receptors in osteoblasts. The relative potencies of these compounds to induce cAMP production was CT greater than amylin not equal to CGRP in osteoclast-like MNCs and CGRP greater amylin much greater than CT in osteoblast-like cells.     

Clinical significance of adrenal computed tomography in Addison's disease.

Adrenal computed tomographic (CT) scanning was conducted in twelve patients with Addison's disease during the clinical course. In tuberculous Addison's disease (n = 8), three of four patients examined during the first two years after disease onset had bilaterally enlarged adrenals, while one of four had a unilaterally enlarged one. At least one adrenal gland was enlarged after onset in all six patients examined during the first four years. Thereafter, the adrenal glands may atrophy bilaterally, in contrast to adrenal glands in idiopathic Addison's disease, which atrophy bilaterally from disease onset (n = 2). Adrenal calcification was a less sensitive clue in tracing pathogenesis, i.e., adrenal calcification was observed in five of eight patients with tuberculous Addison's disease, but not in idiopathic patients. Thus, adrenal CT scanning could show the etiology of Addison's disease (infection or autoimmunity) and the phase of Addison's disease secondary to tuberculosis, which may be clinically important for initiating antituberculous treatment.     

An endothelin receptor (ETA) antagonist isolated from Streptomyces misakiensis.

A competitive endothelin (ET) antagonist, BE-18257B, was isolated from the fermentation products of Streptomyces misakiensis. It is a novel cyclic pentapeptide, cyclo(-D-Glu-L-Ala-allo-D-Ile-L-Leu-D-Trp-), and binds to ETA receptors (ET-1 selective) in cardiovascular tissues, but not to ETB receptors (equally sensitive to isopeptides of ET family) in kidney, adrenal gland and cerebellum tissues. BE-18257B also antagonizes ET-1-induced vasoconstriction in rabbit iliac artery and pressor action in rats. Thus it is a selective ETA antagonist and should provide a valuable tool for elucidation of the pharmacological and pathophysiological roles of ET-1.     

The MAS-encoded processing protease of yeast mitochondria. Overproduction and characterization of its two nonidentical subunits

The amino-terminal presequences of proteins imported from the cytoplasm across the mitochondrial inner membrane are cleaved off by a soluble matrix-localized protease composed of two nonidentical homologous subunits. In the yeast Saccharomyces cerevisiae, these are encoded by the nuclear MAS1 and MAS2 genes. We have now constructed yeast strains in which either one or both of the genomic MAS genes are controlled by a galactose-inducible strong promoter. In these strains, the intramitochondrial concentration of each MAS-encoded subunit as well as of the holo-protease can be varied over a wide range. When overproduced, the MAS1 protein precipitates in the matrix whereas the MAS2 protein remains soluble. The MAS2 protein was obtained at a purity of 98% in milligram amounts. The purified MAS2 subunit exists largely as a soluble 52-kDa monomer. Its cleavage activity is very low and might well reflect the 2% contamination by holoprotease. Activity is restored by adding the solubilized purified MAS1 subunit. Yeast cells depleted of one or both MAS subunits continue to import precursor proteins into mitochondria, but fail to cleave them; eventually the deficient cells stop growing. This growth arrest is partly suppressed on minimal medium or under conditions in which the cells are less dependent on mitochondrial metabolism. Depletion of the MAS1 subunit causes overproduction of the MAS2 subunit.     

Identification of suberimidate cross-linking sites of four histone sequences in H1-depleted chromatin. Histone arrangement in nucleosome core

The arrangement of 8 histones in the nucleosome core has been investigated by identifying the sites of 4 histone sequences cross-linked with a bifunctional amino-group reagent, dimethyl suberimidate, selected from among 4 diimidoesters of various linker lengths examined. H1-depleted calf thymus chromatin was allowed to react with 14C-labeled suberimidate at pH 8.5 and 0 degrees C. The cross-linked chromatin was then digested exhaustively with trypsin. Almost all the histone fragments were released from the chromatin with 0.25 M HCl and chromatographed on several columns and on paper. Cross-linked peptides were detected by analyzing the content of radioactive suberimidoylbislysine after acid hydrolysis. The chromatographic procedure developed here showed that the whole histone fragments contained 29 mol% of the total linked reagent as suberimidoylbisylsine. The 5 finally purified cross-linked peptides were identified from the total and N-terminal amino acids of each pair of peptides separated by two-dimensional cellulose thin layer chromatography after cutting the linker by ammonolysis. Thus, intramolecular cross-linking was found between Lys-5 and Lys-9 of H2A, and Lys-34 and Lys-85 of H2B, while intermolecular cross-linking was found between Lys-24 (or 27) of H2B and Lys-74 of H2A, Lys-85 of H2B and Lys-91 of H4, and Lys-120 of H2B and Lys-115 of H3 and/or Lys-77 of H4. Most of these lysine residues are located in the DNA-binding segments of the 4 histone sequences identified previously [Kato, Y. & Iwai, K, (1977) J. Biochem. 81, 621--630]. All the 5 or 6 cross-links can be located in a heterotypic tetramer consisting of one molecule each of H2A, H2B, H3, and H4, and a model of the histone arrangement in the tetramer is proposed. Two such tetramers may compose to the histone octamer in the nucleosome core.     

Salmon calcitonin-induced stimulation of 1 alpha,25-dihydroxycholecalciferol synthesis in rats involving a mechanism independent of adenosine 3'':5''-cyclic monophosphate.

The effect of natural salmon calcitonin on accumulation in plasma of 1 alpha,25-dihydroxy-[3H]cholecalciferol from 25-hydroxy[3H]cholecalciferol in vivo was investigated in vitamin D-deficient thyroparathyroidectomized rats into which graded doses of the hormone were continuously infused by use of a balance study system. A dose-dependent increase in plasma concentrations of 1 alpha,25-dihydroxy[3H]cholecalciferol was observed with calcitonin infusion for 6--30h at a rate greater than 20 M.R.C. m-units/h. Infusion of parathyrin or cyclic AMP produced a similar stimulation [Horiuchi, Suda, Takahashi, Shimazawa & Ogata (1977) Endocrinoly 101, 969--974], but the maximal effect of calcitonin was additive to that of either parathyrin or cyclic AMP. Furthermore concurrent infusion of theophylline (0.5 mumol/h) did not potentiate the effect of submaximal doses (3 and 20 M.R.C. m-units/h) of calcitonin. Plasma concentrations of calcium showed a decrease with calcitonin infusion for 30h, but those of Pi remained unchanged. These results strongly suggest that the rat kidney is endowed with a calcitonin-sensitive 1 alpha-hydroxylase system that is separate from the parathyrin/cyclic AMP system and is independent of changes in plasma Pi.