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.     

A long Y chromosome in man

Summary An unusually long Y chromosome was described in the phenotypically normal father and paternal grandfather of a girl with Down's syndrome, and likewise in a male infant with multiple malformations and his father, normal in phenotype. Measurements revealed that the long Y chromosome corresponded in length to autosomes of group 16–18.Information was obtained to show that the increased length of the Y chromosome was an inheritable character, and that a long Y chromosome was not always associated with an abnormal phenotype (or phenotypes).Contribution No. 585 from the Zoological Institute, Hokkaido University.     

Inactivation of mitochondrial 2-oxoglutarate dehydrogenase complex as a result of phospholipid degradation induced by freeze-thawing

The inactivation of 2-oxoglutarate dehydrogenase complex by freeze-thawing was examined along with alterations of membrane phospholipids, in order to elucidate the mechanism of freezing injury in mitochondria.The dehydrogenase complex activity in slowly frozen and thawed mitochondria decreased to 70% as compared to intact mitochondria and further decreased during incubation. This inactivation during incubation was temperature dependent, i.e., at temperatures up to 25°C there was a slight decrease, while at higher temperatures there was a marked decrease in the dehydrogenase complex activity. Simultaneously, there was a significant accumulation of free fatty acids, generated from mitochondrial phospholipids, which inhibited 2-oxoglutarate dehydrogenase and subsequently enzyme complex activity. Oxoglutarate dehydrogenase activity in mitochondria was markedly inhibited by exogenous phospholipase A, and this inhibition was partially prevented with bovine serum albumin. Furthermore, when intrinsic phospholipase A was either inhibited or stimulated, there was a respective decrease or increase in the enzyme complex inactivation.The activity of the purified enzyme complex decreased slightly after slow freezing, but remained constant even when incubated at temperatures up to 32°C. However, the activity of this enzyme complex was markedly reduced when incubated either in the presence of venom phospholipase A or with exogenous fatty acid.The relationship between inactivation of the 2-oxoglutarate dehydrogenase complex, phospholipase A activation and production of free fatty acids in frozen and thawed mitochondria is discussed.     

The recently proposed 20,22-epoxycholesterol as the intermediate in the conversion of cholesterol to pregnenolone by adrenal cortex mitochondria must be 5α,6α-epoxycholestan-3β-ol

Recently proposed 20,22-epoxycholesterol as the obligatory intermediate in the NADPH-dependent conversion of cholesterol to pregnenolone by bovine adrenal cortex mitochondria was isolated and found to be a misassigned metabolite. It was identified as 5α,6α-epoxycholestan-3β-ol by gas-liquid chromatography-mass spectrometry as well as by the reverse isotope dilution method. The thin-layer chromatographic behavior of the proposed 20,22-epoxide which had been reported to have a higher polarity than pregnenolone was in good accordance with that of the identified 5α,6α-epoxycholestan-3β-ol. All four diastereoisomers of 20,22-epoxycholesterol had much lower polarity than pregnenolone in thin-layer chromatograms obtained in various solvent systems. Endogeneous cholesterol, contained originally in the mitochondria, was also converted in the presence of NADPH to pregnenolone and 5α,6α-epoxycholestan-3β-ol as observed with radioactive cholesterol added as the exogeneous substrate.     

Hydrogen evolution by co-immobilized Chlorella vulgaris and Clostridium butyricum cells

Whole cells of Chlorella vulgaris and Clostridium butyricum were co-immobilized in 2% agar gel. NADP was suitable as an electron carrier. The rate of hydrogen evolution increased with increasing NADP concentration. The optimum conditions for hydrogen evolution were pH 7.0 and 37°C. The immobilized C. vulgaris-NADP-immobilized Cl. butyricum system continuously evolved hydrogen at a rate of 0.29–1.34 μmol/h per mg Chl for 6 days. On the other hand, the system without NADP evolved only a trace amount of hydrogen.     

Biosynthesis of branched-chain fatty acids in Bacillus subtilis. A decarboxylase is essential for branched-chain fatty acid synthetase

Branched long-chain fatty acids of the iso and anteiso series are synthesized in many bacteria from the branched-chain alpha-keto acids of valine, leucine, and isoleucine after their decarboxylation followed by chain elongation. Two distinct branched-chain alpha-keto acid (BCKA) and pyruvate decarboxylases, which are considered to be responsible for primer synthesis, were detected in, and purified in homogenous form from Bacillus subtilis 168 strain by procedures including ammonium sulfate fractionation and chromatography on ion exchange, reversed-phase, and gel absorption columns. The chemical and catalytic properties of the two decarboxylases were studied in detail. The removal of BCKA decarboxylase, using chromatographic fractionation, from the fatty acid synthetase significantly reduced its activity. The synthetase activity was completely lost upon immunoprecipitation of the decarboxylase. The removal of pyruvate decarboxylase by the above two methods, however, did not affect any activity of the fatty acid synthetase. Thus, BCKA decarboxylase, but not pyruvate decarboxylase, is essential for the synthesis of branched-chain fatty acids. The very high affinity of BCKA decarboxylase toward branched-chain alpha-keto acids is responsible for its function in fatty acid synthesis.