Seasonal carbon isotope discrimination in a grassland community

Summary Grassland communities of arid western North America are often characterized by a seasonal increase in ambient temperature and evaporative demand and a corresponding decline in soil moisture availability. As the environment changes, particular species could respond differently, which should be reflected in a number of physiological processes. Carbon isotope discrimination varies during photosynthetic activity as a function of both stomatal aperture and the biochemistry of the fixation process, and provides an integrated measure of plant response to seasonal changes in the environment. We measured the seasonal course of carbon isotope discrimination in 42 grassland species to evaluate changes in gas exchange processes in response to these varying environmental factors. The seasonal courses were then used to identify community-wide patterns associated with life form, with phenology and with differences between grasses and forbs. Significant differences were detected in the following comparisons: (1) Carbon isotope discrimination decreased throughout the growing season; (2) perennial species discriminated less than annual species; (3) grasses discriminated less than forbs; and (4) early flowering species discriminated more than the later flowering ones. These comparisons suggested that (1) species active only during the initial, less stressful months of the growing season used water less efficiently, and (2) that physiological responses increasing the ratio of carbon fixed to water lost were common in these grassland species, and were correlated with the increase in evaporative demand and the decrease in soil moisture.     

Sensitization of low-dose-rate irradiation by nonlethal hyperthermia

To assess whether hyperthermia could radiosensitize cells irradiated at a low dose rate, Chinese hamster V79 cells were simultaneously heated and irradiated at 0.86 Gy/h. The data showed that heat treatments at 39 and 40 degrees C, which did not induce heat killing alone or high-dose-rate radiosensitization, resulted in enhanced cell killing with low-dose-rate irradiation. The dose-modification factor (ratio of the slopes of the curves for low dose rate and high dose rate) was reduced to 1.8 at 39 degrees C and 1.4 at 40 degrees C, compared to a value of 2.1 at 37 degrees C. These data indicate that nonlethal heat treatments can cause enhanced radiosensitization under low-dose-rate conditions. The implications of these results for interstitial thermoradiotherapy are discussed.     

Suppression of hepatic prostaglandin F2 alpha in rats by dietary alpha-tocopherol acetate is independent of total hepatic alpha-tocopherol.

Groups of eight weanling female F344/N rats were fed semipurified diets that supplied 0, 50, 500, 5000, or 15,000 mg alpha-tocopherol acetate/kg diet, with and without 0.05% phenobarbital (PB) for 9 weeks. Both plasma and hepatic alpha-tocopherol levels, measured by HPLC, strongly correlated with alpha-tocopherol intake (r greater than 0.73, p less than 0.0001). Phenobarbital both depleted hepatic alpha-tocopherol and increased plasma alpha-tocopherol significantly. Although treatment with PB for 9 weeks significantly increased GST activity, PB did not affect hepatic prostaglandin (PG)F2 alpha status, as determined by radioimmunoassay. PGF2 alpha was significantly greater (by 52%) in rats fed no alpha-tocopherol than in rats fed 15,000 mg alpha-tocopherol acetate/kg diet. Hepatic PGF2 alpha status was correlated inversely but weakly with dietary alpha-tocopherol (r = -0.24, p less than 0.05). Hepatic PGF2 alpha status was not correlated with hepatic or plasma alpha-tocopherol status. This finding suggests either that there is a small depletion-resistant subcellular alpha-tocopherol pool which regulates PGF2 alpha production or that alpha-tocopherol alters PGF2 alpha production in vivo by an indirect mechanism.     

Fungal growth during rice tapé fermentation

Fungal growth was quantified during Indonesian rice tapé fermentation using an agar-film technique following sample homogenization for 1 min at 25000 rev/min. After 72 h fermentation, mould hyphal length was 0·68 km/g, yeast hyphal length 2·1 km/g and the numbers of mould chlamydospores and single yeast cells were 14 times 10⁵/g and 5·1 times 10⁷/g respectively. The estimated fungal biomass in rice tapé after 72 h was 25 mg/g dry weight with 62% of this being mould hyphae, 24% mould chlamydospores, 13% yeast hyphae and 1% yeast cells.     

Tumor suppressor p53: analysis of wild-type and mutant p53 complexes.

It has been suggested that the dominant effect of mutant p53 on tumor progression may reflect the mutant protein binding to wild-type p53, with inactivation of suppressor function. To date, evidence for wild-type/mutant p53 complexes involves p53 from different species. To investigate wild-type/mutant p53 complexes in relation to natural tumor progression, we sought to identify intraspecific complexes, using murine p53. The mutant phenotype p53-246(0) was used because this phenotype is immunologically distinct from wild-type p53-246+ and thus permits immunological analysis for wild-type/mutant p53 complexes. The p53 proteins were derived from genetically defined p53 cDNAs expressed in vitro and also from phenotypic variants of p53 expressed in vivo. We found that the mutant p53 phenotype was able to form a complex with the wild type when the two p53 variants were cotranslated. When mixed in their native states (after translation), the wild-type and mutant p53 proteins did not exhibit any binding affinity for each other in vitro. Under identical conditions, complexes of wild-type human and murine p53 proteins were formed. For murine p53, both the wild-type and mutant p53 proteins formed high-molecular-weight complexes when translated in vitro. This oligomerization appeared to involve the carboxyl terminus, since truncated p53 (amino acids 1 to 343) did not form complexes. We suggest that the ability of the mutant p53 phenotype to complex with wild type during cotranslation may contribute to the transforming function of activated mutants of p53 in vivo.     

A heparin sulfate-regulated human keratinocyte autocrine factor is similar or identical to amphiregulin.

A novel human keratinocyte-derived autocrine factor (KAF) was purified from conditioned medium by using heparin affinity chromatography as the first step. Purified KAF stimulated the growth of normal human keratinocytes, mouse AKR-2B cells, and a mouse keratinocyte cell line (BALB/MK). Heparin sulfate inhibited KAF mitogenic activity on all cell types tested and inhibited the ability of KAF to compete with epidermal growth factor for cell surface binding. Interestingly, KAF stimulated the growth of BALB/MK cells at high cell density but failed to stimulate these cells at clonal density. Protein microsequencing of the first 20 NH2-terminal amino acid residues of purified KAF revealed identity to the NH2 terminus of human amphiregulin (AR). Northern (RNA) blot analysis with AR-specific cRNA demonstrated that human keratinocytes, as well as mammary epithelial cell cultures, expressed high levels of AR mRNA. In contrast, AR mRNA was not detected in normal human fibroblasts or melanocytes and was present at reduced levels in several mammary tumor cell lines. The mitogenic activity of purified AR was also shown to be inhibited by heparin sulfate, and an AR-specific enzyme-linked immunosorbent assay (ELISA) revealed that KAF and AR are antigenically related. We have previously shown that human keratinocytes can grow in an autocrine manner. Our present study demonstrates that one of the growth factors responsible for this autocrine growth (KAF) is similar or identical to AR and that KAF and AR bioactivity can be negatively regulated by heparin sulfate.     

Purification of ribulose 1,5-bisphosphate carboxylase/oxygenase and of carboxysomes from Thiobacillus thyasiris the putative symbiont of Thyasira flexuosa (Montagu)

The bacterial symbionts of many marine invertebrates contain ribulose 1,5-bisphosphate (RuBP) carboxylase but apparently no carboxysomes, polyhedral bodies containing RuBP carboxylase. In the few cases where polyhedral bodies have been observed they have not been characterised enzymatically. Polyhedral bodies, 50–90 nm in diameter, were observed in thin cell sections of Thiobacillus thyasiris the putative symbiont of Thyasira flexuosa and RuBP carboxylase activity was detected in both soluble and particulate fractions after centrifugation of cell-free extracts. RuBP carboxylase purified 90-fold from the soluble fraction was of high molecular weight and consisted of large and small subunits, with molecular weights of 53,110 and 11,100 respectively. Particulate RuBP carboxylase activity was associated with polyhedral bodies 50–100 nm in diameter, as revealed by density gradient centrifugation and electron microscopy. Therefore, the polyhedral bodies were inferred to be carboxysomes. Native electrophoresis of isolated carboxysomes demonstrated a major band which comigrated with the purified RuBP carboxylase and three minor bands of lower molecular weight. Sodium dodecyl-sulphate (SDS) gel electrophoresis of SDS-dissociated carboxysomes demonstrated nine major polypeptides two of which were the large and small subunits of RuBP carboxylase. The RuBP carboxylase subunits represented 21% of the total carboxysomal protein. The most abundant polypeptide had a molecular weight of 40,500. Knowledge of carboxysome composition is necessary to provide an understanding of carboxysome function.Abbreviations FPLC fast performance liquid chromatography - IB isolation buffer - PAGE polyacrylamide gel electrophoresis - RuBP carboxylase - ribulose 1,5-bisphosphate carboxylase/oxygenase - SDS sodium dodecyl-sulphate     

Isolation of plasma membranes from cultured glioma cells and application to evaluation of membrane sphingomyelin turnover

A rapid and reliable method for the isolation of plasma membranes and microsomes of high purity and yield from cultured glioma cells is described. The procedure involves disruption by N2 cavitation, preliminary separation by centrifugation in Tricine buffer, and final separation on a gradient formed from 40% Percoll at pH 9.3. Enzyme and chemical markers indicated greater than 60% yield with six- to eightfold enrichment for plasma membranes and greater than 25% yield with three- to fourfold enrichment for a microsomal fraction consisting mainly of endoplasmic reticulum. The final fractions were obtained with high reproducibility in less than 1 h from the time of cell harvesting. Application of this procedure to human fibroblasts in culture is assessed. The isolation procedure was applied to investigations of synthesis and turnover of sphingomyelin and phosphatidylcholine in plasma membranes of glioma cells following incubation for 4-24 h with [methyl-3H]choline. These studies indicated that radioactivity from phosphatidylcholine synthesized in microsomes from exogenous choline may serve as a precursor of the head-group of sphingomyelin accumulating in the plasma membrane.     

Partial amino acid sequence of rat topoisomerase I: comparison with the predicted sequences for the human and yeast enzymes

The partial amino acid sequence of rat topoisomerase I was determined by gas-phase microsequencing. Seven tryptic peptides closely matched the sequences deduced from human topoisomerase I cDNA (94.5% homology). Similarity to sequences deduced from baker's yeast and fission yeast genomic DNA were restricted to conserved domains which may represent important sites of interaction with DNA or with other proteins.