Response of aromatic-pathway enzymes to changing physiological states of growth in suspension cultures of Nicotiana silvestris

The activity levels of enzymes of aromatic amino acid biosynthesis respond to changing physiological states of growth, as illustrated by results obtained from suspension-cultured cells of Nicotiana silvestris Speg. et Comes line ANS 1 (2N=24). The experimental system provides a foundation for interpretations about overall regulation of enzyme levels in relationship to growth physiology. Levels of activity for shikimate dehydrogenase (EC 1.1.1.25), prephenate aminotransferase and arogenate dehydrogenase were followed throughout a growth cycle obtained by a conventional subculture protocol. Enzyme date were also obtained from cell cultures maintained in continuous exponential growth for greater than 10 generations (EE cells). Both shikimate dehydrogenase and prephenate aminotransferase exhibited elevated stationary-phase levels of enzyme, much of which was carried over into a subsequent subculture. At least 4 generations of exponential growth were required before diminution of the latter two enzymes to the levels characteristic of truly exponential-phase growth (EE cells) occurred. This is reminiscent of the overall behavior of 3-deoxy-D- arabino -heptulosonate 7-phosphate (DAHP) synthase (EC 4.1.2.15), specifically attributed to the properties of the cytosolic isozyme species (DAHP synthase-Co). Elevation of arogenate dehydrogenase also occurred in stationary-phase cells, but diminished rapidly during lag phase to reach the level characteristic of EE cells.     

HPLC separation and indirect ultraviolet detection of phosphorylated sugars

An HPLC method for the separation and analysis of phosphorylated sugars is presented. Ion-exchange chromatography coupled to indirect ultraviolet detection has produced good resolution and sensitivity. Fructose 6-P, glucose 6-P, ribose 5-P, 3-phosphoglyceric acid, ribulose 1,5-P₂, fructose 1,6-P₂, and sedoheptulose 1,7-P₂ can be separated at a sensitivity down to 10 nanomoles. The system resolves 2-carboxy-D-arabinitol 1,5-P₂ from 2-carboxy-D-ribitol 1,5-P₂. The natural inhibitor of ribulose bisphosphate carboxylase, 2-carboxy-D-arabinitol 1-P, has been separated from its 5-P isomer and most other phosphorylated compounds. This method is applied to identification of the products obtained upon ion-exchange purification of synthetic 2-carboxyarabinitol 1-P.     

Increased working capacity with hyperoxia in humans

The purpose of the study was to examine the influence of oxygen-breathing on maximal oxygen uptake (VO2max) and submaximal endurance performance. Six young women and five men rode a cycle-ergometer while breathing compressed air (normoxia, NOX) or a 55% O2 in N2 mixture (hyperoxia, HOX). The VO2max increased significantly by 12% (P less than 0.01) with HOX in the women but not in the men (+4%; nonsignificant). Maximal heart rate was also increased with HOX in the women but not in the men. Endurance time during work to exhaustion at 80% of normoxic VO2max was 41% longer in HOX than in NOX (P less than 0.025) with no significant difference between the men and the women. The variation among individuals was large. The oxygen uptake and respiratory quotient were not different in the two endurance tests, but pulmonary ventilation (VE) and blood lactate concentration were lower in HOX than in NOX, especially during the latter part of the task. Plasma base deficit (BDpl) increased initially by 3.5 mmol.l-1 during HOX and then stabilized. In NOX, a continuous increase was seen and the change was more than twice as large. Relative to BDpl, VE was higher in HOX than in NOX indicating a more efficient ventilatory compensation of the metabolic acidosis. The reduced ventilatory demand and lower metabolic acidosis in HOX in combination with lower relative exercise intensity may have contributed to the longer time to exhaustion. However, the pattern of individual variation suggested that other mechanisms were also involved.     

Induction and modification of dinitrogenase reductase in the unicellular cyanobacterium Synechocystis BO 8402

Oxygen is an important regulatory factor of nitrogenase induced in a unicellular cyanobacterium, Synechocystis BO 8402, during nitrogen starvation. Synthesis of the enzyme is limited by the efficiency of the cells to remove oxygen by respiration, supported by hydrogenases and, in the light, by inhibition of photosynthesis. With a polyclonal antibody against dinitrogenase reductase (the Fe protein of nitrogenase) a single polypeptide is detected, indicative of an active dimeric enzyme in dense cell suspensions. Inhibition of nitrogenase by addition of oxygen is accompanied by the appearance of a second polypeptide of the Fe protein having a 1.5 kDa higher molecular weight. This disappears upon removal of oxygen from the gas phase while nitrogenase activity is restored. No protein synthesis is required indicating that a fraction of the existing polypeptides is reversibly modified in response to oxygen. After induction of nitrogenase activity in dilute culture suspensions, both forms of the Fe-protein are found in variable amounts possibly due to oxygen contamination during the experiment.Abbreviations CAM chloramphenicol - Chl chlorophyll a - CHO carbohydrates - DCMU 3,4-dichlorophenyl-1,1-dimethylurea (diuron) - kDa kilodalton - SDS sodium dodecylsulphate     

Molecular genetic analysis of 67 patients with duchenne/becker muscular dystrophy

Summary A total of 56 Duchenne muscular dystrophy (DMD) patients and 11 Becker muscular dystrophy (BMD) patients was analyzed by extended multiplex amplification of the DMD/BMD gene; deletions were found in 60% of these patients. The data obtained were used to test the frameshift hypothesis and to compare the distribution of familial versus isolated cases. A significant correlation was found between deletions and isolated cases. Additional experiments were performed in order to determine the deletion breakpoints more precisely. These data are a prerequisite for carrier analysis in the respective families by detection or exclusion of aberrant cDNA fragments derived from ectopic lymphocyte RNA. This diagnostic technique is illustrated by 5 examples.     

Organotin compounds induce aneuploidy in human peripheral lymphocytes in vitro

In vitro exposure of PHA-stimulated human lymphocytes to organotin compounds resulted in statistically significant increases in the frequencies of hyperdiploid cells. When taken together with our previous study demonstrating spindle inhibiting effects of the same organotin compounds by an indirect method (Jensen et al., 1989), the present study strongly indicates that organotin compounds are able to induce aneuploidy, probably by affecting spindle function.     

Drug Stimulated DNA Cleavage Mediated by Cauliflower Topoisomerase II

We have investigated cauliflower (Brassica oleracea) topoisomerase II with respect to its interaction with DNA and demonstrate that the enzyme shares the characteristics of topoisomerase II purified from a variety of phylogenetically remote organisms. In the presence of the 2-nitroimidazole Ro 15-0216, cauliflower topoisomerase II-mediated DNA cleavage is extensively stimulated (approximately 20-fold) only at a site recognized as a major cleavage site for the enzyme in the absence of drug. The conservation of the enzyme's DNA specificity in the presence of Ro 15-0216 is in contrast to the effect exerted by traditional topoisomerase II inhibitors, which cause enzyme-mediated cleavage to take place at a multiple number of DNA sites. Ro 15-0216 may therefore prove useful as a tool in the elucidation of the enzyme's DNA interaction sites and its involvement in nucleic acid metabolism in plant cells.