Biochemical and cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. VI. The correlation between UV-induced DNA lesions and chromosomal aberrations, and their modulations with inhibitors of DNA repair synthesis

The role of UV-induced DNA lesions and their repair in the formation of chromosomal aberrations in the xrs mutant cell lines xrs 5 and xrs 6 and their wild-type counterpart, CHO-K1 cells, were studied. The extent of induction of DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs) due to UV irradiation in the presence or absence of 1-beta-D-arabinofuranosylcytosine (ara-C) and hydroxyurea (HU) was determined using the alkaline and neutral elution methods. Results of these experiments were compared with the frequencies of induced chromosomal aberrations in UV-irradiated G1 cells treated under similar conditions. Xrs 6 cells showed a defect in their ability to perform the incision step of nucleotide repair after UV irradiation. Accumulation of breaks 2 h after UV irradiation in xrs 6 cells in the presence of HU and ara-C remained at the level of incision breaks estimated after 20 min, which was about 35% of that found in wild-type CHO-K1 cells. In UV-irradiated CHO-K1 and xrs 5 cells, more incision breaks were present after 2 h compared with 20 min post-treatment with ara-C, a further increase was evident when HU was added to the combined treatment. The level of incision breaks induced under these conditions in xrs 5 was about 80% of that observed in CHO-K1 cells. UV irradiation itself did not induce any detectable DNA strand breaks. Accumulation of SSBs in UV-irradiated cells post-treated with ara-C and HU coincides with the increase in the frequency of chromosomal aberrations. These data suggest that accumulated SSBs when converted to DSBs in G1 give rise to chromosome-type aberrations, whereas strand breaks persisting until S-phase result in chromatid-type aberrations. Xrs 6 appeared to be the first ionizing-radiation-sensitive mutant with a partial defect in the incision step of DNA repair of UV-induced damage.     

Hyperoxia-induced clonogenic killing of HeLa cells associated with respiratory failure and selective inactivation of Krebs cycle enzymes

Cellular intoxication by elevated concentrations of O2 may be considered as a model for accelerated cellular aging processes resulting from excessive free radical production by normal metabolic pathways. We describe here that exposure of HeLa cell cultures to 80% O2 for 2 days causes progressive growth inhibition and loss of reproductive capacity. This intoxication was correlated with inhibition of cellular O2 consumption and inactivation of 3 mitochondrial flavoproteins, i.e., partial inactivation of NADH and succinate dehydrogenases and total inactivation of alpha-ketoglutarate dehydrogenase. As alpha-ketoglutarate dehydrogenase controls the influx of glutamine/glutamate into the Krebs cycle, which is the major pathway for oxidative ATP generation in HeLa cells, the inactivation of alpha-ketoglutarate dehydrogenase was expectedly correlated with a net fall in glutamine/glutamate utilization. Furthermore, a simultaneous increase in glucose consumption and lactate production was observed, indicating that the cellular response to respiratory failure is to generate more ATP from glycolysis. In spite of this response, extensive depletion of ATP was observed. Thus, hyperoxia-induced growth inhibition and loss of clonogenicity seem to be due primarily to an impairment of mitochondrial energy metabolism resulting from inactivation of SH-group-containing flavoprotein enzymes localized at or near the inner mitochondrial membrane. These observations may be relevant for theories implicating loss of mitochondrial function as a prime factor in the aging process.     

Studies on the biosynthesis and metabolism of the phytoalexin lubimin and related compounds in Datura stramonium L.

Arachidonic acid, cellulase, CuSO₄, a sonicate of Phytophthora infestans mycelium and a spore suspension of Penicillium chrysogenum all elicited the formation of the sesquiterpenoid phytoalexins lubimin, 3-hydroxylubimin and rishitin in fruit cavities of Datura stramonium. 3-Hydroxylubimin was the predominant phytoalexin formed after treatment of the fruits with arachidonic acid, cellulase and the P. infestans preparation. Copper sulphate was a potent elicitor of lubimin but not 3-hydroxylubimin. The fungus P. chrysogenum metabolized lubimin and 3-hydroxylubimin to 15-dihydrolubimin and 3-hydroxy-15-dihydrolubimin respectively, both in fruit cavities inoculated with spores of this fungus and in pure culture. The 15-dihydrolubimin formed in the fruits by the fungus was further metabolized (by the fruits) to both isolubimin and 3-hydroxy-15-dihydrolubimin. The precursor-product relationships between all of the subject compounds was investigated by feeding experiments with ³H-labelled compounds. 2-Dehydro-[15-³H₁]lubimin was rapidly and efficiently incorporated into lubimin and may be the direct precursor of lubimin in planta. 3-Hydroxy[2-³H₁]lubimin was incorporated into the nor-eudesmane rishitin but 10-epi-3-hydroxy[2-³H₁]lubimin was not. An updated scheme for the biosynthesis and metabolism of lubimin and related compounds in infected tissues of solanaceous plants is presented.We thank Mr Vic Swetez for the provision of plant material, Mrs Margaret Huffee for technical assistance, Dr David Ewing for help with obtaining NMR spectra, and the Agricultural and Food Research Council for financial support.     

Studies on the maintenance and expression of cloned DNA fragments in the nuclear genome of the green alga Chlamydomonas Reinhardtii

Using a biolistic device built here and based on the principle of the device described by Klein et al. (1987). we have reproducibly obtained transformants of Chlamydomonas reinhardtii . The reproducibility of the method has allowed us to examine the maintenance and expression of cloned DNA fragments introduced into C. Reinhardtii .     

Short-term changes in carbon-isotope discrimination identify transitions between C3 and C4 carboxylation during Crassulacean acid metabolism

Short-term measurements of instantaneous carbon-isotope discrimination have been determined from mass-spectrometric analyses of CO₂ collected online during gas exchange for the epiphytic bromeliad Tillandsia utriculata L. Using this technique, the isotopic signature of CO₂ exchange for each phase of Crassulacean acid metabolism (CAM) has been characterised. During night-time fixation of CO₂ (Phase I), discrimination () ranged from 4.4 to 6.6, equivalent to an effective carbon-isotope ratio (¹³C) of –12.3 to –14.5 versus Pee Dee Belemnite (PDB). These values reflected the gross photosynthetic balance between net CO₂ uptake and refixation of respiratory CO₂, characteristic of CAM in the Bromeliaceae. When for the relative proportion of external (p _a ) and internal (p _i) CO₂ is taken into account, calculated p _i/p _a decreased during the later part of the dark period from 0.68 to 0.48. Measurements of during Phase II, early in the light period, showed the transition between C₄ and C₃ pathways, with carboxylation being increasingly dominated by ribulose bisphosphate carboxylase (Rubisco) as increased from 10.5 to 21.2 During decarboxylation in the light period (Phase III), CO₂ leaked out of the leaf and the inherent discrimination of Rubisco was expressed. The value of calculated from on-line measurements (64.4) showed that the CO₂ lost was considerably enriched in ¹³C, and this was confirmed by direct analysis of the CO₂ diffusing out into a CO₂-free atmosphere ( ¹³C = + 51.6 versus PDB). Instantaneous discrimination was characteristic of the C₃ pathway during Phase IV (late in the light period), but a reduction in showed an increasing contribution from phosphoenolpyruvate carboxylase. The results from this non-invasive technique confirm the observations that double carboxylation involving both phosphoenolpyruvate carboxylase and Rubisco occurs during the transient phases of CAM (II and IV) in the light period.Abbreviations and Symbols CAM Crassulacean acid metabolism - H⁺ (dawn-dusk) variation in titratable acidity - ¹³C carbonisotope ratio of plant organic material, relative to Pee Dee Belemnite (vs. PDB) - discrimination against ¹³CO₂, - p _i, p _a internal, external partial pressures of CO₂ - Rubisco ribulose1,5-bisphosphate carboxylase - PAR photosynthetically active radiation - PEPCase phosphoenolpyruvate carboxylase We are grateful for financial support in respect of research grants (GR3/5360, GR3/6419) and a studentship awarded by the Natural Environment Research Council, UK.     

A modified mallory-cason staining procedure for large cryosections.

The classic Mallory-Cason staining procedure has been modified for application to sections "on tape" obtained from large deep frozen tissue specimens. These 20 microns cryosections are collected on tape from a large heavy duty cryomicrotome. The stained sections provide anatomical details that are not revealed by other techniques. The merit of this procedure is found in the support of modern medical modalities, both for research and educational purposes.     

The isolation and characterization of gibberellin-deficient mutants in tomato

Summary In tomato, nine independent EMS-induced mutants representing recessive mutations at three different loci (gib-1, gib-2, and gib-3) were isolated. Six of these have an almost absolute gibberellin requirement for seed germination and elongation growth. In addition, the leaves are darker green, smaller, and changed in structure as compared to wild type. The three other mutants, which germinate without GA, are allelic to specific, nongerminating mutants and have less severe mutant characteristics. The respective loci are situated on three different chromosomes. The genes identified by these mutants control steps in gibberellin biosynthesis, as endogenous gibberellins are strongly reduced.     

Influence of dietary fish on eicosanoid metabolism in man

Two groups of 40 volunteers were given a dietary supplement consisting of 135 g of mackerel or meat (control) paste per day for 6 weeks. Compliance was about 80% in both groups and the daily intake of 20:5(n-3) and 22:6(n-3) from the mackerel supplement was about 1.3 and 2.3 g, respectively. In collagen-activated platelet rich plasma, the potency of blood platelet to produce HHT from arachidonic acid (AA) clearly reduced in the mackerel group, whereas the formation of HHTE from timnodonic acid (TA) increased slightly. Changes in the formation of HHT and HHTE, measured by HPLC, correlated significantly with those of TxB2 and TxB3, respectively, measured by GC/MS. Changes in the formation of the lipoxygenase products HETE (ex AA) and HEPE (ex TA) were qualitatively similar to that seen for the cyclo-oxygenase products, but quantitatively the responses were smaller. Formation of ir TxB2 in clotting blood significantly reduced in the mackerel group. In collagen-activated, citrated whole blood, TxB2 formation tended to be reduced in the mackerel-supplemented volunteers. Mackerel consumption was associated with the formation of considerable amounts of PGI3, as judged from the appearance of 2,3-dinor-delta 17-6-keto-PGF1 alpha in urine. The amount of the major metabolite of PGI2, 2,3-dinor-6-keto-PGF1 alpha was not reduced, or even increased. The daily amount of tetranor prostaglandin metabolites in the urine did not change significantly, which indicates that mackerel supplementation did not alter the formation of prostaglandins E and F.     

Long-term survival of and plasmid stability inPseudomonas andKlebsiella species and appearance of nonculturable cells in agricultural drainage water

One year after introduction into agricultural drainage waterPseudomonas fluorescens R2f (RP4),Pseudomonas putida CYM318 (pRK2501), andKlebsiella aerogenes NCTC418 (pBR322) could be recovered on agar media. Survival of the introduced strains depended on competition with the indigenous microflora, the presence of nutrients, and the availability of air.In contrast toK. aerogenes NCTC418 (pBR322), bothPseudomonas species lost their plasmids, as indicated by the consistently lower colony counts on selective medium compared with the counts on nonselective medium. The plasmid loss did not depend on nutrient status and oxygen supply. P. fluorescens R2f cells could be detected with the immunofluorescence (IF) technique. Total cell counts determined by IF were consistently higher than corresponding colony counts. Even in samples where no colonies were recovered, R2f cells could be detected by IF. This indicated the occurrence of nonculturable R2f cells in drainage water. Homology with³²P-labelled plasmid RP4 DNA was found in several drainage water samples that originally receivedP. fluorescens R2f (RP4), by using the cell suspension filter hybridization technique. P. putida CYM318 andK. aerogenes NCTC418 cells could also be detected in sterile drainage water samples, after nonspecific staining with fluorescein isothiocyanate. Cell counts of both strains were consistently higher than corresponding plate counts.     

Hyphal and mycelial interactions betweenAgaricus bisporus andScytalidium thermophilum on agar media

The interaction betweenAgaricus bisporus andScytalidium thermophilum on agar media was studied by differential interference contrast and phase contrast microscopy.A. bisporus combatively replacesS. thermophilum in culture on agar media. The antagonistic effect ofA. bisporus is transmissible through a cellophane membrane and causes irreversible disintegration ofS. thermophilum protoplasm, resulting in a total loss of viability after prolonged interaction between the two fungi. On compost extract agar, but not on other media, the growth rate ofA. bisporus increased from 2.7 to 5.3 mm·d^–1 following contact withS. thermophilum mycelium.