Alterations in cardiac function and subcellular membrane activities after hypervitaminosis D3

The present study was designed to induce massive accumulation of calcium in the myocardium and to evaluate the effect of calcium overload on myocardial contractile function and biochemical activity of cardiac subcellular membranes. Rats were treated with an oral administration of 500,000 units/kg of vitamin D₃ for 3 consecutive days, and their hearts were sampled on the 5th day for biochemical analysis. On the 4th and 5th days, heart rate, mean aortic pressure, left ventricular systolic pressure and left ventricular dP/dt were significantly lowered in vitamin D₃-treated rats, demonstrating the existence of appreciable myocardial contractile dysfunction. Marked increases in the myocardial calcium (67-fold increase) and mitochondrial calcium contents (24-fold increase) were observed by hypervitaminosis D3. Mitochondrial oxidative phosphorylation and ATPase activity were significantly reduced by this treatment. A decline in sarcolemmal Na⁺, K⁺-ATPase activity was also observed, while relatively minor or insignificant changes in calcium uptake and ATPase activities of sarcoplasmic reticulum were detectable. Electron microscopic examination revealed calcium deposits in the mitochondria after vitamin D₃ treatment. The results suggest that hypervitaminosis D₃ produces massive accumulation of calcium in the myocardium, particularly in the cardiac mitochondrial membrane, which may induce an impairment in the mitochondrial function and eventually may lead to a failure in the cardiac contractile function.     

Cloning and expression of cDNAs from enterically-transmitted non-A, non-B hepatitis virus.

The fragment gene of enterically-transmitted non-A, non-B hepatitis virus (ET-NANBHV) was cloned as a cDNA and inserted into an expression vector pUEX2. The recombinant protein was expressed in Escherichia coli HB101 as a fusion protein with beta-galactosidase (beta-Gal). The fusion protein reacted with the sera of infected cynomolgus monkeys and of patients from Myanmar. This reaction was highly related with ET-NANBHV infection, and obviously demonstrates in that the recombinant protein can be used for the detection of ET-NANBHV infection.     

Pyruvic-acid-containing polysaccharide in the cell wall of Bacillus polymyxa AHU 1385

Three acidic polymer fractions with molecular masses of about 16 kDa, 35 kDa and 70 kDa were isolated from lysozyme digests of N-acetylated cell walls of Bacillus polymyxa AHU 1385 by ion-exchange chromatography and gel chromatography. These fractions, containing mannosamine, glucosamine and pyruvic acid in a molar ratio of about 1:1:1 together with glycopeptide components, were characterized as polysaccharide-linked glycopeptides with one, two and more polysaccharide chains. On the other hand, treatment of the cell walls with glycine/HC1 buffer, pH 2.5, at 100 degrees C for 10 min followed by separation of water-soluble products on ion-exchange chromatography gave three polysaccharide fractions, PS-I-III, which contained different amounts of pyruvic acid (0,0.6 and 0.9 residue/mannosamine residue) along with equimolar amounts of mannosamine and glucosamine. Pyruvate-free polysaccharides similar to PS-I were also obtained from PS-II, PS-III and polysaccharide-linked glycopeptides by treatment with 10 mM HC1 at 100 degrees C for 1 h. Results of analyses of these polysaccharide preparations by 1H-NMR and 13C-NMR measurement and methylation, together with data from characterization of fragments obtained by hydrogen fluoride hydrolysis, lead to the most likely structure, ----3)[4,6-O-(1-carboxyethylidene)]ManNAc(beta 1----4)GlcNac(beta 1----, for the acidic polysaccharide of this strain.     

Immunochemical and histochemical studies on a phosphonoglycosphingolipid, SGL-II, isolated from the sea gastropod Aplysia kurodai

Antiserum was raised against 3-O-MeGal beta 1----3GalNAc alpha 1----3[6' -O-(2-aminoethylphosphonyl)Gal alpha 1----2 (2-aminoethylphosphonyl----6)Gal beta 1----4Glc beta 1----1Ceramide (SGL-II) isolated from the skin of a mollusc, Aplysia kurodai. This antiserum reacted with SGL-II and other phosphonoglycosphingolipids of Aplysia such as SGL-I', F-21, and some minor glycolipids on TLC plates, but it did not react with ganglioside or globoside. The sugars recognized were 3-O-methylgalactose at the non-reducing end and galactose at the branched chain of the glycolipids. One membrane glycoprotein (Mr 280,000) reacted strongly, and some other proteins reacted weakly with the antiserum. Immunohistochemical examination of the nervous tissues revealed distinct staining in the periganglionic tissue of the ganglia, and the perineural sheath of the proximal portion of the peripheral nerves. The neuropil and satellite cells were also stained. In the skin, subcutaneous connective tissues were moderately stained, and the cytoplasm of small mononuclear cells and foamy cells was also stained. The staining patterns were essentially the same in paraffin and cryostat sections. From the findings with sections pretreated with chloroform-methanol (2 : 1, v/v), it was suggested that the periganglionic and perineural stainings were due to glycoproteins, including an SDS-soluble glycoprotein of Mr 280,000, while those of the other regions were due to SGL-II and glycolipids immunologically related to SGL-II. The stainings in the skin sections were largely due to glycoproteins.     

Reevaluation of immunoreactive gonadotropin-releasing hormone (GnRH) levels in general circulation in women: changes in levels and episodic patterns before, during and after gonadotropin surges

In order to reevaluate the earlier varying data regarding circulatory gonadotropin-releasing hormone (GnRH), we assayed extracted GnRH from the plasma frequently collected at mid-cycle in 11 women. For the analysis of episodic GnRH patterns and basal levels, blood samples were obtained at 6 h intervals for 72 h and at 15 min intervals for 2 h every 12 h throughout the experimental period. All blood samples were assayed for GnRH and selected samples for LH, FSH, estradiol and progesterone. For GnRH assay, 5 or 6 ml of blood was mixed with 60 mg of ethylenediaminetetraacetic acid, disodium salt, and 3 mg of phenylmethylsulfonyl floride immediately after blood collection. These enzyme inhibitors prevented the destruction of GnRH in the blood at room temperature for at least 4 h. Plasma GnRH was extracted through several steps including florisil absorption, acidic extraction and washing with organic solvent. Nonspecific immunoreactivity in the plasma was markedly decreased through this extraction process. Our assay values (approximate range, 0.1-2.0 pg/ml) of plasma GnRH in normal women corresponded to the low range of those obtained by others who used the alcohol extraction method. The basal levels of GnRH did not change significantly throughout 3 different periods, i.e., before, during and after the LH surges, and fluctuated between a small range of 0.11 and 1.44 pg/ml. Although the peak levels of GnRH observed in its episodic patterns did not change between the periods before and during the LH surges, they decreased significantly after the LH surge compared with those seen during the LH surges (0.93 +/- 0.07 vs 1.17 +/- 0.09 pg/ml, p less than 0.05). The present data demonstrate that immunoreactive GnRH in the extracted peripheral plasma does not change significantly in its mean, basal and peak levels during the periovulatory period except for a minor but significant decrease in the peak levels shortly after an LH surge.     

The bialaphos biosynthetic genes ofStreptomyces hygroscopicus: Molecular cloning and characterization of the gene cluster

Summary We have isolated and studied the organization ofStreptomyces hygroscopicus genes responsible for the biosynthesis of the antibiotic herbicide bialaphos. Bialaphos production genes were cloned from genomic DNA using a plasmid vector (pIJ702). Three plasmids were isolated which restored productivity toS. hygroscopicus mutants blocked at different steps of the biosynthetic pathway. Subcloning experiments using other nonproducing mutants showed that four additional bialaphos production genes were also contained on these plasmids. A gene conferring resistance to bialaphos, which was independently cloned using the plasmid vector pIJ61, and an antibiotic-sensitive host (S. lividans), was also linked to the production genes. Cosmids were isolated which defined the location of these genes in a 16 kb cluster.     

Characterization of a diphosphonopentaosylceramide containing 3-O-methylgalactose from the skin of Aplysia kurodai (sea hare)

The complete structure is proposed for a ceramide (Cer), bis(2-aminoethylphosphono)-pentaoside, isolated from the skin of Aplysia kurodai. This new phosphonoglycosphingolipid was purified using two systems of column chromatography on silicic acid. The purity of the glycolipid was confirmed by thin-layer chromatography, analysis of its composition, and proton magnetic resonance spectrometry. The component carbohydrates were glucose, galactose, N-acetylgalactosamine, and 3-O-methylgalactose. Most (90%) of the fatty acid was palmitic acid and the major sphingosine bases were octadeca-4-sphingenine (51%) and anteisononadeca-4-sphingenine (38%). 2-Aminoethylphosphonyl-6-galactose was identified after its partial hydrolysis. From studies by methanolysis, permethylation, mild acid hydrolysis, hydrogen fluoride treatment, chromium trioxide oxidation combined with thin-layer chromatography, gas liquid chromatography, gas chromatography-mass spectrometry, and proton magnetic resonance spectrometry, the structure of the glycolipid was concluded to be 3-OMeGal beta 1----3GalNAc alpha 1----3[6'-O-(2-aminoethylphosphonyl)-Gal alpha 1----2](2-aminoethylphosphonyl----6)Gal beta 1----4Glc beta 1----1Cer.