Continuous synthesis of the dnaA gene product of Escherichia coli in the cell cycle

Summary The dnaA gene product of Escherichia coli, identified as a weakly basic protein of about 48,000 daltons (Yuasa and Sakakibara 1980), can be separated from other celluar proteins by means of two-dimensional gel electrophoresis. Synthesis of the dnaA protein took place continuously during a cell growth cycle. The newly synthesized dnaA protein persisted stably for one generation. Thermosensitive dnaA protein produced by the dnaA167 mutant was stable at 30° C, but was disintegrated at 42° C. The amount of intact dnaA protein present in the mutant exposed to the high temperature for 60 min was less than a quarter of the amount at the time of the shift. The cells having the reduced amount of intact dnaA protein were capable of initiating a new round of chromosome replication at the low temperature without de novo synthesis of the dnaA protein. The potential of the mutant for initiation of DNA replication decreased with reduction in the amount of the thermoreversible dnaA protein. The mutations dnaA167 and dnaA46 had no significant effect on the syntheses of the dnaA mRNA and the protein product at the low and high temperatures.Abbreviations used SDS sodium dodecyl sulfate - kb kilobase pairs - TCA trichloroacetic acid     

Participation and Properties of Lipoxygenase and Hydroperoxide Lyase in Volatile C6-Aldehyde Formation from C18-Unsaturated. Fatty Acids in Isolated Tea Chloroplasts

Isolated tea chloroplasts utilized linoleic acid, linolenicacid and their 13-hydroperoxides as substrates for volatileC₆-aldehyde formation. Optimal pH values for oxygen uptake,hydroperoxide lyase and the overall reaction from C₁₈-fattyacids to C₆-aldehydes were 6.3, 7.0 and 6.3, respectively. Methyllinoleate, linoleyl alcohol and -linolenic acid were poor substratesfor the overall reaction, but linoleic and linolenic acids weregood substrates. The 13-hydroperoxides of the above fatty acidsand alcohol also showed substrate specificity similar to thatof fatty acids. Oxygen uptakes (relative V_max) with methyl linoleate,linoleyl alcohol, linolenic acid, -linolenic acid and arachidonicacid were comparable to or higher than that with linoleic acid.In winter leaves, the activity for C₆-aldehyde formation fromC₁₈-fatty acids was raduced to almost zero. This was due tothe reduction in oxygenation. The findings presented here provideevidence for the involvement of lipoxygenase and hydroperoxidelyase in C₆-aldehyde formation in isolated chloroplasts. (Received July 11, 1981; Accepted November 5, 1981)     

Synthesis, Excretion, and Metabolism of Glycolate under Highly Photorespiratory Conditions in Euglena gracilis Z

Glycolate was excreted from the 5% CO₂-grown cells of Euglena gracilis Z when placed in an atmosphere of 100% O₂ under illumination at 20,000 lux. The amount of excreted glycolate reached 30% of the dry weight of the cells during incubation for 12 hours. The content of paramylon, the reserve polysaccharide of E. gracilis, was decreased during the glycolate excretion, and of the depleted paramylon carbon, two-thirds was excreted to the outside of cells and the remaining metabolized to other compounds, both as glycolate. The paramylon carbon entered Calvin cycle probably as triose phosphate or 3-phosphoglycerate, but not as CO₂ after the complete oxidation through the tricarboxylic acid cycle. The glycolate pathway was partially operative and the activity of the pathway was much less than the rate of the synthesis of glycolate in the cells under 100% O₂ and 20,000 lux; this led the cells to excrete glycolate outside the cells. Exogenous glycolate was metabolized only to CO₂ but not to glycine and serine. The physiologic role of the glycolate metabolism and excretion under such conditions is discussed.     

Fungal contamination and mycotoxin-producing potential of dried beans

A total of 604 samples of about 7 different types of beans was examined to determine their mycological profiles, and suitability for use as solid substrates for mycotoxin production.All of the samples were collected from bean jam makers in Tokyo by the official food examiners.Genera Penicillium and Aspergillus were predominant, and genus Wallemia was also found commonly in all types of beans.Mycotoxin-producing Aspergillus strains were isolated from 52 samples of beans, approximately 9% of the total. The highest incidence of toxigenic Aspergillus (14.1%) was found in kidney beans. Red beans and peas inoculated with Aspergillus ochraceus were found to produce about 7 to 8 times more toxin than was obtained in a liquid medium, and red beans inoculated with A. versicolor produced more toxin than was obtained in yeast extract sucrose broth. Green peas inoculated with Fusarium graminearum produced about 8 times more T-2 toxin than was obtained in 1% peptone containing Czapek solution under comparable culture conditions.     

Development of Leucocytozoon caulleryi in chick embryos infected by biting of Culicoides arakawae through shell membrane.

Chick embryos were infected with Leucocytozoon caulleryi by biting of the midge, Culicoides arakawae, through the shell membrane. Schizonts of L. caulleryi were detected in the chorioallantoic membrane and most of the internal organs of embryos and of chicks after hatching. The development of schizonts was slower in embryos than in chickens. Soluble antigens of L. caulleryi were demonstrated by the precipitation test in the allantoic fluid and blood from the embryos and chicks. No erythrocytic stage of L. caulleryi, however, was observed in any embryo or chick.     

Mechanism of metabolic regulation in photoassimilation of propionate in Euglena gracilis z

Euglena gracilis showed a typical photoassimilation of propionate when cultured on propionate as a sole carbon source. While the acid is metabolized by the methylmalonyl-coenzyme A (CoA) pathway under illumination, supporting growth of Euglena (K. Hosotani, A. Yokota, Y. Nakano, and S. Kitaoka, 1980, Agr. Biol. Chem.44, 1097–1103), it does not allow the protozoon to grow in the dark although it was actively taken up and metabolized. Kinetics of incorporation of radioactivity of labeled propionate, trapping effect of exogenous lactate in the incorporation of labeled propionate and radiorespirometric pattern revealed that propionate was metabolized by the lactate pathway in Euglena in the dark. Enzymes involved in the lactate pathway were located in mitochondria. The reason why Euglena can not grow on propionate in the dark is explained by the failure of producing C₄ dicarboxylic acids essential for biosynthesis of amino acids and sugars, like the mitochondrial oxidation of fatty acids in higher animals. The Euglena cells cultured in the dark contained enzymes of both methylmalonyl-CoA and lactate pathways, but lack of photosynthetically generated ATP has been suggested to force Euglena to select the less-ATP-requiring but futile pathway.     

Protoclonal variation of plant regeneration in rice

Protoplasts were isolated from callus derived from a single homozygous seed of Oryza sativa L. var. Norin 8. Thirty protoclones were randomly selected and these showed variation in regeneration frequency ranging from 0–87% with an average of 52%. The potential for regeneration of each protoclone as reflected in the regeneration frequency was analyzed five times over a period of 250 days and showed that the protoclones can be classified into three types, namely: protoclones with high regeneration frequency; protoclones with low regeneration frequency, both of which maintained their respective levels of regeneration potential; and protoclones with gradually decreasing regeneration frequency. Secondary protoclones established from protoplasts isolated from some of these protoclones and regenerated 2–3 times for a period of 120 days also showed further reduction in regeneration frequency. The polypeptide composition analyzed by two dimensional gel electrophoresis suggests the presence of specific polypeptides related to regeneration potential. Analysis of ploidy level based on plant morphology and pollen size suggests the predominance of tetraploids among the regenerated plants.     

Pyruvate:NADP+ oxidoreductase from Euglena gracilis: mechanism of O2-inactivation of the enzyme and its stability in the aerobe

O2-inactivation of pyruvate:NADP+ oxidoreductase from mitochondria of Euglena gracilis was studied in vitro, and a mechanism which consists of two sequential stages was proposed. Initially, the enzyme is inactivated by the direct action of O2 in a process obeying second-order kinetics. Although the catalytic activity for pyruvate oxidation is lost by this initial inactivation, NADPH oxidation with artificial electron acceptors still occurs. Subsequently, a secondary, O2-independent inactivation occurs, rendering the enzyme completely inactive. Pyruvate stimulates the O2-inactivation while CoA and NADP+ protect the enzyme from O2. The O2-inactivation is accelerated by reduction of the enzyme with pyruvate and CoA. Reactivation of the O2-inactivated enzyme was studied in Ar by incubation with Fe2+ in the presence of some other reducing reagent such as dithiothreitol. The evidence obtained indicates that the partially inactivated enzyme, which retains catalytic activity for NADPH oxidation, can be reactivated, but the completely inactivated enzyme is not. When Euglena cells were exposed to 100% O2 the enzyme in the cells was inactivated by O2, but the rate was quite slow compared with that observed in vitro. The enzyme inactivated by O2 in the cells was almost completely reactivated in vitro by incubation with Fe2+ and other reducing reagents in Ar, suggesting that the secondary, O2-independent inactivation does not occur in situ. When the cells were returned to air, reactivation of the O2-inactivated enzyme in the cells began immediately. The enzyme, kept in isolated, intact mitochondria, was stable in air; however, the enzyme was inactivated by O2 when the mitochondria were incubated with a high concentration of pyruvate.     

Negative ion fast atom bombardment-tandem mass spectrometry for structural analysis of isoprenoid diphosphates

Applicability of negative ion fast atom bombardment (FAB)-tandem mass spectrometry (MS/MS) was examined in trace mixture analyses and structural assignments of some isoprenoid diphosphates. Negative ion FAB-MS spectra using a glycerol matrix of these isoprenoid diphosphates showed predominantly molecular ions (M-H)- together with fragment ions at m/z 177 (H3P2O7)-, 176 (H2P2O7)-, 159 (HP2O6)-, and 79 (PO3)- which were characteristic of the diphosphate ester moiety. The molecular ions did not overlap with peaks arising from any impurities even when crude sample such as butanol extracts from enzymatic reaction mixtures were directly analyzed without any purification. Moreover, collisionally activated dissociation spectra of the molecular ion showed many structurally significant fragment ions which enabled us to elucidate the structures of such irregular alkyl chain moieties as those having a homoisoprenoid skeleton or substituted structures. These studies indicate that negative ion FAB-MS/MS is a simple and useful technique for trace mixture analysis and structure elucidation of isoprenoid diphosphates.     

Temperature dependence of optical properties of chlorophyll a incorporated into phosphatidylcholine liposomes

Temperature-dependent spectral changes of chlorophyll a (Chl a) incorporated into liposomes of two types of phosphatidylcholine are studied. When Chl a incorporated into the liposomes is cooled down to 5°C from the temperature of the gel-to-liquid crystalline phase transition of the lipid, the red shift as well as the increase in half-bandwidth of the red peak of Chl a are only slight. By measuring the difference spectra produced by substracting the absorption spectrum at the phase transition temperature of the lipid from that at lower temperature, it is shown that the component absorbing at longer wavelength (675–685 nm) than the peak of the red maximum (about 670 nm) significantly increases at the expense of the component absorbing at shorter wavelength (657–668 nm). The positions of positive and negative peaks depend on the temperature and the molar ratio of the lipid to Chl a. The absorbance change is most pronounced on cooling below the phase transition temperature of the lipid. The temperature-induced absorbance change is almost completely reversible. The results indicate that the aggregated forms of Chl a in liposomes can be spectrophotometrically detected in the gel phase of the lipid.