25-OHD3-26,23-lactone: a metabolite of vitamin D3 that is 5 times more potent than 25-OHD3 in the rat plasma competitive protein binding radioassay.

A new metabolite of Vitamin D₃ (25-OHD₃-26,23-lactone) has been found in the plasma of Vitamin D₃-toxic pigs and cows. This metabolite is at least 5 times more potent than 25-OHD₃ in the displacement of [³H]-25-OHD₃ from rat plasma protein binding sites under short-term incubation. This metabolite co-migrates with 24,25-(OH)₂D₃ on Sephadex LH-20 columns developed in chloroform:hexane 65:35 and with 25,26-(OH)₂D₃ on Sephadex LH-20 columns developed in hexane:chloroform:methanol 9:1:1. The presence of 25-OHD₃-26,23-lactone represents a possible contaiminant in the assay of 24,25-(OH)₂D₃ or 25,26-(OH)₂D₃ if only Sephadex LH-20 is used for pre-assay purification. 25-OHD₃-26,23-lactone is, however, resolved from 24,25-(OH)₂D₃ by high pressure liquid chromatography (HPLC) using Zorbax Sil silicic acid columns developed in either isopropanol:hexane 8:92 or isopropanol:methylene chloride 2.5:96.5. We assayed for the presence of this new metabolite of Vitamin D₃ and found it to be present in normal pig plasma and undetectable in normal cow plasma. Concentrations were elevated to 10–20 ng/ml following massive injection of Vitamin D₃ to both species.     

Metabolism and binding in vitro of 5 alpha-androstane-3 beta, 17 beta-diol and of 5 alpha-androstane-3 alpha, 17 beta-diol in cell fractions of rat ventral prostate and liver.

Rat ventral prostate and liver were investigated for the binding in vitro to particulate fractions and for the metabolism of 5 alpha-androstane-3 beta, 17 beta-diol. Comparative investigations were carried out on the metabolism of 5 alpha-androstane-3 alpha, 17 beta-diol. Preparations of the liver were investigated in order to establish the organ specificity of the method. In the prostate, the bulk of the metabolites of 5 alpha-androstane-3 beta, 17 beta-diol was present as steroids of high polarity. Of the less polar metabolites, 17 beta-hydroxy-5 alpha-androstan-3-one, 3 beta-hydroxy-5 alpha-androstan, 17-one and 5 alpha-androstane-3 alpha, 17 beta-diol were detectable. The binding of a 5 alpha-androstane-3 beta, 17 beta-diol to mitochondria and microsomes was unspecific. In the liver, among the less polar metabolites, 3 beta-hydroxy-5 alpha-androstan-17-one was the main metabolite, and the binding was unspecific. The main metabolite in the prostate homogenate of 5 alpha-androstane-3 alpha, 17 beta-diol was 17 beta-hydroxy-5 alpha-androstan-3-one. The portion of highly polar steroids was very low. The portion of unmetabolized hormone was distributed almost equally among the different cell preparations except the nuclei, in which 17 beta-hydroxy-5 alpha-androstan-3-one was higher and 5 alpha-androstane-3 alpha, 17 beta-diol was lower than in the remaining cell fractions.     

Post-transformational rearrangement of an in vitro reconstructed group-A streptococcal erythromycin resistance plasmid

Cleavage of the group-A streptococcal macrolide, lincosamide, and streptogramin B (MLS) resistance plasmid pSM19035 yields 2 fragments [13 and 4 megadaltons (MD)] with EcoRI, and 15 fragments with HindIII, 12 of which are 6 pairs of identical fragments derived from the inverted repeats that comprise about 80% of the pSM19035 genome. The large EcoRI fragment was isolated, ligated, and used to transform the Challis strain of Streptococcus sanguis to erythromycin resistance. Plasmids (pDB101, pDB102, and pDB103) isolated from three different transformants had lower molecular masses than the original large EcoRI fragment. HindIII digestion of these molecules and subsequent analysis of fragment radioactivity distributions indicated the loss of plasmid segments of various sizes. The deletions, all of which occurred in the palindrome, did not affect the level and the inducible nature of pSM19035-determined antibiotic resistance. Only pDB101 retained the unique EcoRI cleavage site. The results of this analysis allowed the construction of an EcoRI and HindIII cleavage-site map of pSM19035 and promise to simplify future studies of genetic functions specified by streptococcal MLS resistance plasmids.     

Characterization of the antibiotic resistance plasmid ERL1 from Streptococcus pyogenes

Summary The streptococcal plasmid ERL1 determining inducible resistance to erythromycin, lincomycin, and staphylomycin S was isolated by dye-buoyant density centrifugation and shown to have a molecular weight of about 17.5 Mdal, as revealed by sedimentation through neutral sucrose gradients. In SM60 cells entering the stationary phase its covalently closed circular form was present to the extent of 5 copies per chromosomal genome equivalent. ERL1 was subject to the DNA restriction and modification mechanism discovered in strain 56188. It did not apear to exercise restriction of phage DNA but mediated a partial release of the restricted growth of A25.     

A quench-flow kinetic investigation of calcium ion accumulation by isolated cardiac sarcoplasmic reticulum. Dependence of initial velocity on free calcium ion concentration and influence of preincubation with a protein kinase,MgATP, and cyclic AMP

Ca²⁺ accumulation at pH 6.8 by isolated rabbit heart microsomes derived chiefly from sarcoplasmic reticulum was investigated by a quench-flow technique. The reaction was terminated at preset times by addition to the reaction mixture of an equal volume of 10 to 50 mM ethyleneglycol-bis-(β-aminoethyl ether)-N,N′-tetraacetic acid buffered at pH 6.0. The initial velocity of Ca²⁺ accumulation by microsomal preparations exhibiting a steady state Ca²⁺ accumulation of 25.6 nmol Ca²⁺/mg increased from 3.67 to 33.4 nmol Ca²⁺/mg · s as the free Ca²⁺ concentration was raised from 0.2 to 18.9 μM. Preincubation of the cardiac microsomes with a partly purified soluble cardiac cyclic AMP-dependent protein kinase, MgATP, and cyclic AMP lead to a significant increase in the initial Ca²⁺ accumulation rate. The amounts of Ca²⁺ that were found to accumulate in the first 200 ms of the reaction are comparable to the quantities of the ion that according to literature data need to be removed from the myofilaments and the myoplasm for induction of relaxation of the myocardial fibers.     

Incorporation of isolated chromosomes and induction of hypoxanthine phosphoribosyltransferase in Chinese hamster cells.

Evidence is presented for the uptake of radioactive-labeled isolated Chinese hamster chromosomes following incubation with Chinese hamster cells. Metaphases were found which contained radioactive labeled chromosomes in a very low frequency, and in some of the labeled chromosomes only one chromatid was labeled. Incubation of hypoxanthine phosphoribosyltransferas (HPRT)-deficient Chinese hamster cells with chromosomes isolated from HPRT+ Chinese hamster or human cells resulted in the appearance of HPRT+ cells. Clones derived from these cells were isolated in HAT medium. Cells in mitosis during incubation with the chromosomes yielded thr-e times more HPRT+ clones than did cells in interphase. The intraspecies combination involving recipient cells and chromosomes from Chinese hamster origin yielded significantly higher numbers of HPRT+ clones than did the interspecies system using human chromsomes and Chinese hamster recipient cells (5 X 10(-5) and 6 X 10(-6) respectively). Electrophoresis of HPRT from Chinese hamster cells treated with human chromosomes revealed the pattern of the human enzyme.     

Protoplasmatische Anatomie des fixierten Helodea-Blattes

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Photoreactivity of buckminsterfullerene-bis(triphenylphosphite)platinum(0) induced by metal-to-ligand charge transfer excitation

The complex C₆₀Pt[P(OPh)₃]₂ displays C₆₀ ππ^* intraligand bands in the UV-Vis region and a long-wavelength absorption at λ_max = 770 nm which is assigned to a metal-to-ligand charge transfer (MLCT) transition from platinum to fullerene. The irradiation of the complex leads to the population of the reactive MLCT state and subsequently to the dissociation (C₆₀Pt[P(OPh)₃]₂ → C₆₀ + Pt[P(OPh)₃]₂) in the primary photochemical step. Product formation takes place by the interception of Pt[P(OPh)₃]₂ with suitable scavengers such as CHCl₃ or O₂.