Ornithine decarboxylase induction in cells stimulated to proliferate differs from that in continuously dividing cells

Ornithine decarboxylase activity increases at least 4–5-fold before DNA synthesis both in synchronous cycling cells and in quiescent cells stimulated to proliferate. The purpose of our experiments was to test whether the transient peaks of ornithine decarboxylase activity in both growth situations were biochemically regulated in a similar manner. We found that the regulation of this particular enzyme activity is distinct in two ways. Firstly, the addition of 2mm-hydroxyurea will block the induction of ornithine decarboxylase in continuously dividing Chinese-hamster ovary cells, while having no effect on ornithine decarboxylase induction in stimulated quiescent cells. Hydroxyurea added after the induction occurs has no effect on the enzyme activity. The apparent half-life of the enzyme is not altered in cells treated with hydroxyurea. Hydroxyurea does not affect the enzyme directly, since incubation of cell homogenates with this drug results in no loss of measurable ornithine decarboxylase activity and hydroxyurea does not markedly alter general RNA- or protein-synthesis rates. The inactivation of ornithine decarboxylase activity by hydroxyurea does not resemble the loss of activity observed with a 90min treatment with spermidine. Thiourea, a less potent inhibitor of ribonucleoside diphosphate reductase, will also inhibit ornithine decarboxylase activity, but to a lesser extent. Secondly, the expression of ornithine decarboxylase in quiescent cells stimulated to proliferate is biphasic as these cells traverse G₁ and enter S phase, whereas only one peak of activity is apparent in synchronous cycling G₁-phase cells. The time interval between the first peak of ornithine decarboxylase activity and the onset of DNA synthesis is approx. 5h longer in non-dividing cells stimulated to proliferate than in continuously dividing cells. The results suggest that the regulation of ornithine decarboxylase activity is different in the two growth systems in that the induction of ornithine decarboxylase in continuously dividing cells occurs closer in time to DNA synthesis and is dependent on deoxyribonucleoside triphosphates.     

A method for the quantitative determination of glycerolipids containing O-alkyl and O-alk-1-enyl moieties.

We have developed a spectrophotometric procedure, based on a combination of established methods, for the quantitative determination of aklyl and alk-1-enyl (plasmalogens) ether-linked glycerolipids. It depends upon the release of alkylglycerols and alk-1-enylglycerols from phospholipids by phosphlipase C (Bacillus cereus) followed by saponification or by Vitride reduction the phospholipids; aldehydes are subsequently formed and measured colorimetrically after reacting them with a fuchsin reagent. The total alkyl and alk-1-enyl content of glycerolipids is determined oxidation of the sample withperiodate to form aldehydes and alkylglycolic aldehydes. The O-alk-1-enyl lipid content is determined on a separate sample by measuring the aldehydes produced after acid hydrolysis. The quantity of O-alkyl lipids is calculated from the difference between the values obtained for the total ether-lipid content and that of the O-alk-1enyl lipid content. Alternately, direct determination of alk-1-enylglycerols and alkylglycerols can be made if these hydrolytic products are first separated by thin-layer chromatography.     

Endolithic chlorophyll d-containing phototrophs

Cyanobacteria in the genus Acaryochloris are the only known oxyphototrophs that have exchanged chlorophyll a (Chl a) with Chl d as their primary photopigment, facilitating oxygenic photosynthesis with near infrared (NIR) light. Yet their ecology and natural habitats are largely unknown. We used hyperspectral and variable chlorophyll fluorescence imaging, scanning electron microscopy, photopigment analysis and DNA sequencing to show that Acaryochloris-like cyanobacteria thrive underneath crustose coralline algae in a widespread endolithic habitat on coral reefs. This finding suggests an important role of Chl d-containing cyanobacteria in a range of hitherto unexplored endolithic habitats, where NIR light-driven oxygenic photosynthesis may be significant.     

Studies on adenosine triphosphate transphosphorylases. Human isoenzymes of adenylate kinase: isolation and physicochemical comparison of the crystalline human ATP-AMP transphosphorylases from muscle and liver

Procedures are described for the isolation, in crystalline form, of the adenylate kinases from autopsy samples of human muscle and from human liver. Weight average molecular weights were determined by sedimentation equilibrium to be 22,000 (+/- 700) and 25,450 (+/- 160) for the human muscle and liver isoenzymes, respectively. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, their molecular weights were estimated to be 21,700 and 26,500 for the muscle and liver enzymes, respectively. Both isoenzymes are accordingly monomeric proteins in their native state. Amino acid analyses are reported here for the normal human liver, calf liver, and rabbit liver adenylate kinases and compared with the normal human muscle, calf muscle, and rabbit muscle myokinases. The liver types as a group and the muscle types as a group show a great deal of homology, but some distinct differences are evident between the liver and muscle enzyme groups, especially in the number of residues of His, Pro, half-cystine, and the presence of tryptophan in the liver enzymes. The normal human liver adenylate kinase, as isolated in this report, has proved to be similar in its properties, if not identical, to the adenylate kinase isolated directly from human liver mitochondria (Hamada, M., Sumida, M., Okuda, H., Watanabe, T., Nojima, M., and Kuby, S. A. (1982) J. Biol. Chem. 257, 13120-13128). Therefore, the liver-type adenylate kinase may be considered a mitochondrial type.     

An analysis of DNA replication in synchronized CHO cells treated with benzo[a]pyrene diol epoxide

Synchronized Chinese hamster ovary (CHO) cells treated with (+/-)7 beta,8 alpha- dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-dihydrobenzo[a]pyrene (BP diol epoxide I) were used to test the 'block-gap' model of replicative bypass repair in mammalian cells. One feature of the model is that carcinogenic or mutagenic DNA adducts act as blocks to the DNA replication fork on the leading strand. Using synchronized CHO cells, the rate of S phase progression by BrdUrd labeling of newly replicated DNA was measured. The rate of S phase progression was reduced by 22% and 42%, when the cells were treated at the G1/S boundary with 0.33 and 0.66 microM BP diol epoxide I, respectively. Using the pH step alkaline elution assay, it was found that the reduced rate of S phase progression was due to a delay in the appearance of multiple replicon size nascent DNA. This observation was consistent with the frequency of BP-DNA adducts present in the leading strand. A second feature of the 'block-gap' model is that the adduct-induced blockage on the lagging strand will produce gaps. It was determined by the use of high-resolution agarose gel electrophoresis, that the ligation of Okazaki size replication intermediates was blocked in a dose-dependent manner in BP diol epoxide I treated, synchronized CHO cells. These data are consistent with a block to the leading strand of DNA replication at DNA-carcinogen adducts. An inhibition of the ligation of Okazaki size fragments by BP diol epoxide I implies a block to replication of the DNA lagging strand leading to gap formation. The data presented here are, therefore, supportive of the 'block-gap' model of replicative bypass repair in carcinogen damaged mammalian cells.