Dinucleoside tetraphosphate variations in cultured tumor cells during their cell cycle and growth

Asynchronous and synchronized cultures of A549 and HTC cells were used to detect possible, cell cycle or cell density specific variations in the intracellular pools of dinucleoside tetraphosphates (Ap4X). No important variations of the nucleotide pools were observed during cell growth. When HTC cells were released from mitotic arrest, a decrease by a factor of N3 Ap4X and ATP levels was observed when the cells entered the G1 phase. This decrease is essentially due to cell doubling. When A549 cells were released from an arrest at the G1/S boundary, the nucleotide pool size increased slightly during the G2 phase just before mitosis. This result is in agreement with both earlier data from our laboratory and the observed decrease in Ap4X pool after release from mitotic-arrested HTC cells. These results suggest that the Ap4X and ATP pools are only subjected to very small variations during the cell cycle, essentially in the G2 phase and after mitosis.     

DNA synthesis is initiated at two positions within the origin of replication of plasmid R1162.

DNA synthesis of broad host-range plasmid R1162 is initiated from two positions, flanking a large (40 bp stem, 40 bp loop) inverted repeat. Each start-point is located within a highly conserved, but oppositely oriented, 10 base-pair sequence. Synthesis from the two positions converges within the intervening inverted repeat. An analysis of deletions suggests that both start positions must be present for synthesis. A model describing possible early events in replication of plasmid R1162 is presented.     

The periplasmic nitrate reductase of Rhodobacter capsulatus; purification,characterisation and distinction from a single reductase for trimethylamine-N-oxide,dimethylsulphoxide and chlorate

The periplasmic dissimilatory nitrate reductase from Rhodobacter capsulatus N22DNAR⁺ has been purified. It comprises a single type of polypeptide chain with subunit molecular weight 90,000 and does not contain heme. Chlorate is not an alternative substrate. A molybdenum cofactor, of the pterin type found in both nitrate reductases and molybdoenzymes from various sources, is present in nitrate reductase from R. capsulatus at an approximate stoichiometry of 1 molecule per polypeptide chain. This is the first report of the occurrence of the cofactor in a periplasmic enzyme. Trimethylamine-N-oxide reductase activity was fractionated by ion exchange chromatography of periplasmic proteins. The fractionated material was active towards dimethylsulphoxide, chlorate and methionine sulphoxide, but not nitrate. A catalytic polypeptide of molecular weight 46,000 was identified by staining for trimethylamine-N-oxide reductase activity after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The same polypeptide also stained for dimethylsulphoxide reductase activity which indicates that trimethylamine-N-oxide and dimethylsulphoxide share a common reductase.Abbreviations DMSO dimethylsulphoxide - LDS lithium dodecyl sulphate - MVH reduced methylviologen - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate - TMAO trimethylamine-N-oxide     

Localization of proteins L4, L5, L20 and L25 on the ribosomal surface by immuno-electron microscopy

Summary Ribosomal proteins L4, L5, L20 and L25 have been localized on the surface of the 50S ribosomal subunit of Escherichia coli by immuno-electron microscopy. The two 5S RNA binding proteins L5 and L25 were both located at the central protuberance extending towards its base, at the interface side of the 50S particle. L5 was localized on the side of the central protuberance that faces the L1 protuberance, whereas L25 was localized on the side that faces the L7/L12 stalk. Proteins L4 and L20 were both located at the back of the 50S subunit; L4 was located in the vicinity of proteins L23 and L29, and protein L20 was localized between proteins L17 and L10 and is thus located below the origin of the L7/L12 stalk.     

Molecular characterization of the diurnal/circadian expression of the chlorophyll a/b-binding proteins in leaves of tomato and other dicotyledonous and monocotyledonous plant species

Diurnal oscillations of steady-state mRNA levels encoding the chlorophyll a/b-binding proteins were monitored inLycopersicon esculentum, Glycine max, Phaseolus vulgaris, P. aureus, P. coccineus, Pisum sativum, Sinapis alba, Hordeum vulgare, Triticum aestivum andZea mays. In these plant speciescab mRNA accumulation increases and decreases periodically indicating i) that the expression of the genes for chlorophyll a/b-binding proteins (cab genes) is controlled by a circadian rhythm, and ii) that the rhythm is widely distributed among monocotyledonous and dicotyledonous plant species. A detailed characterization of the pattern ofcab mRNA expression in tomato leaves shows that the amplitude of the oscillation is dependent on i) the developmental stage of the leaves, ii) the circadian phase and duration of light and iii) the circadian phase and duration of darkness. In addition to the chlorophyll a/b-binding proteins, genes coding for other cellular functions were examined for cyclic variations of their mRNA levels. The analysis includes genes involved in i) carbon metabolism (e.g. phosphoenolpyruvate carboxylase, pyruvate orthophosphate dikinase, alpha amylase, fructose-1,6-bisphosphate aldolase, triosephosphate isomerase), ii) photosynthesis (large and small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, Q_B-binding protein, reaction-center protein of photosystem I) and iii) other physiological or morphological reactions (e.g. ubiquitin, actin). However, no periodic fluctuation pattern was detected for the mRNA levels of these genes in tomato and maize leaves.     

Regional histochemical aspects of xanthine oxidase activity in ischemic and reperfused small intestine of the rat

The study describes regional changes of xanthine oxidase and succinate dehydrogenase activities as shown by the ischemic and reperfused small intestine of the rat. The results are obtained with enzyme histochemical methods, including densitometrical verifications, and are substantiated with biochemical enzyme determinations. The decrease of xanthine oxidase activity was best visible in the anoxic duodenum and jejunum, where the findings of histochemical enzyme determinations agreed with those achieved biochemically. The activities of succinate dehydrogenase as measured densitometrically may serve as a further control, considering also the typical intracellular distribution of the reaction products.     

Non-invasive image analysis evaluation of growth during plant micropropagation

Microcomputerized video image analysis was adapted for rapid, objective, and non-intrusive quantification of shoot growth and development for plants growing in vitro. Custom-developed staging arrangements were essential to insure accurate viewing and representation of the plants in each of three standard culture vessels. Shoot length measurements from digitized culture images were strongly correlated with length measured manually ex vitro. Image analysis weighted density measurements of proliferating microcultures (even with irregular growth habits) provided a reliable indicator of shoot culture fresh weight. Non-destructive time course evaluations of growth rate and quality were demonstrated.Abbreviations FW fresh weight - WD weighted density     

Ethanol production from xylose with a pyruvate-formate-lyase mutant of Klebsiella planticola carrying a pyruvate-decarboxylase gene from Zymomonas mobilis

Summary Grown anaerobically on d-xylose, Klebsiella planticola ATCC 33531 produced acetate, formate, lactate, CO₂ and ethanol as major end-products. A Mu-insertion mutant which lacked pyruvate-formate-lyase showed among its fermentation products more than 70% d-lactate with residual acetate, 2,3-butanediol, and traces of ethanol, formate, and CO₂. After the introduction of a plasmid carrying the gene for the enzyme pyruvate decarboxylase from Zymomonas mobilis, this Klebsiella mutant became an efficient ethanol producer. The recombinant strain produced 387 mM ethanol from 275 mM xylose in 80 h, about 83% of the theoretical maximal yield. Furthermore, this mutant consumed more than double the amount of xylose (41 g/l) compared to the wild-type, due to reduced production of inhibiting acids during growth.Dedicated to Professor Dr. Zähner on the occasion of his 60th birthday