Replication of X chromosomes in complete moles

Summary DNA replication patterns of X chromosomes in complete hydatidiform moles were studied using cultured fibroblasts from three 46,XX moles resulting from duplication of a haploid sperm, and from a 46,XY mole originating from dispermy. Control cultures included skin fibroblasts from an adult woman and a female fetus as well as PB lymphocytes from an adult woman. Cultures were treated with 5-bromodeoxyuridine for the last 2–4h of the S phase, and the chromosome slides prepared were stained by the Hoechst 33258-Giemsa procedure. Each of the three XX moles studied revealed one early-replicating and one late-replicating X chromosomes, while the XY mole revealed one early-replicating X chromosome. DNA replication patterns of molar X chromosomes were similar to those of adult and fetal fibroblasts, but different from those in adult lymphocytes. These findings indicate that DNA replication kinetics of molar fibroblasts are tissue-specific rather than origin- or developmental-stage specific.     

New PCR-RFLP analysis for the mouse Tnfsf6gld gene caused by a point mutation in the Tnfsf6 [tumor necrosis factor (ligand) superfamily, member 6] locus.

A new polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was developed for genetic typing of the mouse Tnfsf6gld mutation. An artificial restriction site was introduced to the mouse Tnfsf6gld mutation by PCR amplification using a modified primer. The three genotypes of the Tnfsf6 locus (Tnfsf6gld/Tnfsf6gld, Tnfsf6gld/+, and +Tnfsf6-gld/+Tnfsf6-gld) could be distinguished clearly and easily. This PCR-RFLP analysis was found to be useful for the identification of the mouse Tnfsf6gld mutation.     

Identification of 4-Chloroindole-3-acetic Acid and Its Methyl Ester in Immature Seeds of Vicia amurensis (the Tribe Vicieae), and Their Absence from Three Species of Phaseoleae

The natural chlorinated auxins 4-chloroindole-3-acetic acid(4-Cl-IAA) and its methyl ester (4-Cl-IAA Me ester) were found,in addition to IAA and its Me ester, by gas chromatography-massspectrometry in immature seeds of Vicia amurensis, a Vicieaespecies. In contrast, only non-chlorinated, IAA and IAA Me esterwere present in immature seeds of three Phaseoleae species.These results are further evidence of the wide distributionof 4-Cl-IAA and its Me ester in various Vicieae. (Received October 3, 1986; Accepted December 22, 1986)     

Metabolism of 32-hydroxy-24,25-dihydrolanosterol by purified cytochrome P-45014DM from yeast. Evidence for contribution of the cytochrome to whole process of lanosterol 14 alpha-demethylation

Metabolism of 32-hydroxy-24,25-dihydrolanosterol (lanost-8-ene-3 beta,32-diol), a posturated intermediate of the 14 alpha-demethylation (removal of C-32) of 24,25-dihydrolanosterol (lanost-8-en-3 beta-ol), by a reconstituted system consisting of yeast cytochrome P-450 which catalyzes lanosterol 14 alpha-demethylation (cytochrome P-45014DM) (Yoshida, Y., and Aoyama, Y. (1984) J. Biol. Chem. 259, 1655-1660 and Aoyama, Y., Yoshida, Y., and Sato, R. (1984) J. Biol. Chem. 259, 1661-1666) and NADPH-cytochrome P-450 reductase was studied. The reconstituted system converted both 32-hydroxy-24,25-dihydrolanosterol and 24,25-dihydrolanosterol to 4,4-dimethyl-5 alpha-cholesta-8,14-dien-3 beta-ol, the 14 alpha-demethylated product of the latter. The metabolism of these compounds was inhibited by a low concentration of ketoconazole which is a potent cytochrome P-45014DM inhibitor. Affinity of cytochrome P-45014DM for 32-hydroxy-24,25-dihydrolanosterol was about 20 times higher than for 24,25-dihydrolanosterol and the cytochrome metabolized the former about 4 times faster than the latter under the experimental conditions. Spectral analysis suggested that the 32-hydroxyl group of 32-hydroxy-24,25-dihydrolanosterol interacted with the heme iron of the oxidized cytochrome and this interaction might support the high affinity of this compound for the cytochrome. These lines of evidence indicate that 32-hydroxy-24,25-dihydrolanosterol is the intermediate of the 14 alpha-demethylation of 24,25-dihydrolanosterol by cytochrome P-45014DM. It is also clear that the cytochrome catalyzes further metabolism of the 32-hydroxylated intermediate to the 14 alpha-demethylated product with higher efficiency than the 32-hydroxylation of the substrate. Cytochrome P-45014DM is thus classified as lanosterol C14-C32 lyase.     

Effect of Exogenous Gangliosides on Amino Acid Uptake and Na+,K+-ATPase Activity in Superior Cervical and Nodose Ganglia of Rats

The effects of some gangliosides on active uptake of nonmetabolizable alpha-aminoisobutyric acid (AIB) and Na+, K+-ATPase and Ca2+, Mg2+-ATPase activities in superior cervical ganglia (SCG) and nodose ganglia (NG) excised from adult rats were examined during aerobic incubation at 37 degrees C for 2 h. In NG, amino acid uptake was greatly accelerated with the addition of galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylgluc osyl ceramide (GM1) (85%) and also with N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide (GM2) or [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetyl- neuraminyl]-galactosylglucosyl ceramide (GD1a) (43% each) compared with a nonaddition control at a 5 nM concentration. Under identical conditions, Na+, K+-ATPase activity was strongly stimulated with GM1 (180%) and GD1a (93%), whereas Ca2+, Mg2+-ATPase activity showed no change. In SCG, on the other hand, AIB uptake was apparently inhibited (-27%) by addition of GM1, with a slight decrease in Na+, K+-ATPase but no change in Ca2+, Mg2+-ATPase activity in the tissue. Both asialo-GM1, in which N-acetylneuraminic acid is deficient, and Forssman glycolipid, which is not present in nervous tissue, failed to produce any significant increase in both SCG and NG not only in amino acid uptake, but also in Na+, K+-ATPase activity. A kinetic study of active AIB uptake showed that GM1 ganglioside produced an increase in Km with no change in Vmax in SCG, whereas it caused a decrease in Km with a slight increase in Vmax in NG. Treatment of NG and SCG with neuraminidase from Vibrio cholerae, an enzyme that split off sialic acid from polysialoganglioside, leaving GM1 intact, caused little inhibition of the amino acid uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stereoselective total synthesis of ceramide Di-, Tri- and tetrahexosides of wheat flour

The first total synthesis of glycosphingolipids isolated from wheat flour has been achieved in a regio- and stereo-controlled manner.Abbreviations THF tetrahydrofuran - DMF dimethylformamide Part 53 in the series Synthetic Studies on Cell Surface Glycans     

Liver glutathione S-transferase polymorphism in Japanese and its pharmacogenetic importance

Summary A total of 168 autopsy liver extracts from Japanese individuals were examined for the glutathione S-transferase (GST) isozymes by means of starch gel electrophoresis. The gene frequencies of GST1^*1, GST1^*2, and GST1^*0 in Japanese were 0.252, 0.057, and 0.691, respectively. GST1^*3 was detected as a rare variant allele. The incidence of GST1 0 in 41 liver biopsy samples from patients suffering from various liver diseases was investigated using polyacrylamide gel isoelectric focusing. The GST1 0 phenotype was found more frequently in livers with hepatitis and carcinoma than in control livers. The isozymes coded by different GST loci were partially purified and characterized to study their biochemical properties. The apparent Km values with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate for the isozymes at the GST1, GST2, GST3, and GST4 loci were 604, 1345, 776 and 591 M, respectively.     

Complete nucleotide sequence and characterization of a cryptic plasmid from Lactobacillus helveticus subsp. jugurti

A small cryptic plasmid, pLJ1, was isolated from Lactobacillus helveticus subsp. jugurti and was cloned into Escherichia coli HB101 by using pBR329 as a vector. Plasmid pLJ1 was 3,292 base pairs long and had single restriction endonuclease sites for PvuII, KpnI, AvaII, Acci, HindIII, and EcoRI. In a maxicell system, pLJ1 produced a protein of about 41 kilodaltons.     

cDNA-directed expression of rat testosterone 7 alpha-hydroxylase using the modified vaccinia virus, T7-RNA-polymerase system and evidence for 6 alpha-hydroxylation and delta 6-testosterone formation

The modified vaccinia virus, T7-RNA-polymerase cDNA-expression system was used to express rat cytochrome P-450a. Various parameters such as host-cell type and density, and duration of infection were tested to optimize the level of expression of cytochrome P-450a enzyme activity. Cytochrome P-450a expressed from the cDNA sequence was exclusively incorporated into the membrane-containing portions of the cell lysates, as expected from its normal association in the liver endoplasmic reticulum. The enzyme displayed a carbon-monoxide-reduced-cytochrome-P-450a difference spectrum with a Soret maximum of 450 nm. Activity measurements revealed that cytochrome P-450a produced three metabolites of testosterone; 7 alpha-hydroxytestosterone and 6 alpha-hydroxytestosterone and delta 6-testosterone at a ratio of about 38:1:1. Under the appropriate conditions, the vaccinia-virus, T7-RNA-polymerase system produces high levels of a single form of cytochrome P-450 in cells that are virtually devoid of endogenous cytochrome P-450. Analysis of the cytochrome P-450 in its natural membrane-bound state, as opposed to artificial-lipid reconstitution studies of purified enzymes, allows accurate and confident measurements of substrate specificities.     

The primary structure of rat ribosomal protein L18a

The amino acid sequence of rat ribosomal protein L18a was deduced from the sequence of nucleotides in a recombinant cDNA. Ribosomal protein L18a contains 175 amino acids and has a molecular mass of 20,047 Da. Hybridization of the cDNA to digests of rat nuclear DNA and to a preparation of poly(A)+ mRNA suggests that there are 8-11 copies of the L18a gene and that the mRNA for the protein is about 700 nucleotides in length. Rat L18a is related to Schizosaccharomyces pombe L17 and perhaps to Halobacterium marismortui L19.