The preparation of an enzyme associated with aflatoxin biosynthesis by affinity chromatography

An affinity matrix for the purification of norsolorinic acid dehydrogenase, an enzyme involved in aflatoxin biosynthesis, was prepared by coupling norsolorinic acid to an agarose gel. This matrix was found to be ineffective in isolating active enzyme, and was therefore modified by methylation, using diazomethane. The methylated matrix produced a one-step purification of the enzyme from a crude homogenate, resulting in a 138-fold purification. The active isolate was found to contain one major and two minor bands upon nondenaturing electrophoresis, and all the norsolorinic acid dehydrogenase activity was associated with the major band. It was concluded that the matrix exhibited true affinity for the enzyme, and that affinity chromatography was a valuable approach to isolating other secondary metabolic enzymes involved in the biosynthesis of the aflatoxins.     

Interaction of the Bacillus sphaericus mosquito larvicidal proteins

Genes for 51.4- and 41.9-kDa insecticidal proteins of Bacillus sphaericus were separately cloned and expressed in Escherichia coli. Both proteins were required for toxicity. Approximately equal numbers of cells containing the 51.4- and 41.9-kDa proteins produced the greatest toxicity; excess 41.9-kDa protein did not affect toxicity, whereas excess 51.4-kDa protein reduced activity. Larvae were killed when 41.9-kDa protein was fed up to 24 h after the 51.4-kDa protein, but not when the order of feeding was reversed. Radiolabelled toxins bound in approximately equal amounts to the gastric caecum and posterior midgut of Culex quinquefasciatus larvae. Radiolabelled 51.4-kDa protein was rapidly degraded by ca. 12-13 kDa in the larval gut, while 41.9-kDa protein was degraded by 1-2 kDa. Nonreduced toxin extracted from B. sphaericus produced a band on SDS-PAGE of ca. 68-74 kDa that contained both 51.4- and 41.9-kDa proteins based on sequence analysis, and a band of ca. 51 kDa that contained primarily 41.9-kDa protein. Escherichia coli containing 51.4-kDa protein enhanced toxicity of the latter eluted SDS-PAGE band. These proteins may associate very strongly, and trace amounts of 51.4-kDa protein in preparations of 41.9-kDa protein from B. sphaericus may be responsible for the previously reported toxicity of the latter.     

Enzymatic studies during spontaneous cell rupture of Saccharomyces cerevisiae

Summary Some of the extract and intracellular enzyme activities in K2nB strain of Saccharomyces cerevisiae that growing in the condition which induce spontaneous cell rupture, were measured. B-1-3-glucanase, invertase, acid phosphatase and active chitin synthetase zymogen showed a reduced activity in ruptured cell while alkaline phosphatase shows no differences in its activity.     

Stepwise isolation and properties of unstable Chinese hamster cell variants that overproduce adenylate deaminase.

Addition of coformycin (0.5 microgram/ml) to a culture medium containing adenine causes in Chinese hamster fibroblasts a lethal depletion of IMP. Resistant variants have been recovered, some of which exhibit increased adenylate deaminase activity. (Debatisse et al., J. Cell. Physiol., 106:1-11, 1981). The selective medium was made more specific for the isolation of this class of variants by supplementation with azaserine. The hyperactive variants remained sensitive to coformycin concentrations above that used for their selection and were unstable. Their frequency was not increased by ethyl methane sulfonate mutagenesis. The resistant phenotype and the increased activity of adenylate deaminase behaved as semidominant traits in hybrids. No change was detected in the Km for AMP, the cofactor requirement, or the chromatographic properties of adenylate deaminase in the variants. Through stepwise selection in media supplemented with increasing coformycin concentrations, unstable clones with adenylate deaminase activity up to 150-fold the wild-type level were isolated; from an unstable clone, a stable subclone with reduced resistance and enzyme activity was recovered. Evidence that increased adenylate deaminase activity is the manifestation of overaccumulation of the enzyme protein was supplied by the correlation of enzyme activity with the intensity of a protein band comigrating with purified adenylate deaminase during sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell extracts. Several unidentified additional bands showed comparable quantitative changes. The striking similarity between the adenylate deaminase-overproducing lines and unstable dihydrofolate reductase-overproducing lines generated by gene amplification strongly suggests that the coformycin-resistant variants also resulted from amplification of an adenylate deaminase gene.     

Growth Temperature-Induced Alterations in the Thermotropic Properties of Nerium oleander Membrane Lipids

The temperature boundary for phase separation of membrane lipids extracted from Nerium oleander leaves was determined by analysis of spin label motion using electron spin resonance spectroscopy and by analysis of polarization of fluorescence from the probe, trans-parinaric acid. A discontinuity of the temperature coefficient for spin label motion, and for trans-parinaric acid fluorescence was detected at 7°C and −3°C with membrane lipids from plants grown at 45°C/32°C (day/night) and 20°C/15°C, respectively. This change was associated with a sharp increase in the polarization of fluorescence from trans-parinaric acid indicating that significant domains of solid lipid form below 7°C or −3°C in these preparations but not above these temperatures. In addition, spin label motion indicated that the lipids of plants grown at low temperatures are more fluid than those of plants grown at higher temperatures.

A change in the molecular ordering of lipids was also detected by analysis of the separation of the hyperfine extrema of electron spin resonance spectra. This occurred at 2°C and 33°C with lipids from the high and low temperature grown plants, respectively. According to previous interpretation of spin label data the change at 29°C (or 33°C) would have indicated the temperature for the initiation of the phase separation process, and the change at 7°C (or −3°C) its completion. Because of the present results, however, this interpretation needs to be modified.

Differences in the physical properties of membrane lipids of plants grown at the hot or cool temperatures correlate with differences in the physiological characteristics of plants and with changes in the fatty acid composition of the corresponding membrane lipids. Environmentally induced modification of membrane lipids could thus account, in part, for the apparently beneficial adjustments of physiological properties of this plant when grown in these regimes.

     

Rats chronically injected with urine from Huntington's chorea patients do not striatal damage

Rats were injected subcutaneously for 147 consecutive days with large volumes of urine from control subjects and from patients with Huntington's chorea (HC) in an effort to test for presence of a possible neurotoxic substance in HC. No evidence of illness was observed in animals treated with HC urine, and their behavior did not differ from animals treated with control urine. After rats were sacrificed, striatum was examined for the biochemical and neuropathological changes seen in human striatum in HC. No deficiency of γ-aminobutyric acid content, nor reduction in activities of glutamic acid decarboxylase and choline acetyltransferase, was found in striatum of rats chronically treated with HC urine. Also, no significant differences were found between striatum of control and experimental rats by light or electron microscopy. These results neither support for exclude the possibility of a neurotoxic mechanism for the neuronal loss characteristic of HC.     

Glycolate Excretion and the Oxygen to Carbon Dioxide Net Exchange Ratio during Photosynthesis in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii cells were grown in high (5% v/v) or low (0.03% v/v) CO₂ concentration in air. O₂ evolution, HCO₃⁻ assimilation, and glycolate excretion were measured in response to O₂ and CO₂ concentration. Both low- and high-CO₂-grown cells excrete glycolate. In low-CO₂-grown cells, however, glycolate excretion is observed only at much lower CO₂ concentrations in the medium, as compared with high-CO₂-adapted cells. It is postulated that the activity of the CO₂-concentrating mechanism in low-CO₂-grown cells is responsible for the different dependence of glycolate excretion on external CO₂ concentration in low- versus high-CO₂-adapted cells.     

Membrane phospholipid phase separations in plants adapted to or acclimated to different thermal regimes

The phase separation temperatures of total leaf phospholipids from warm and cool climate plants were determined in order to explore the relationship of lipid physical properties to a species' thermal habitat. The separation temperatures were determined by measuring the fluorescence intensity and fluorescence polarization of liposomes labeled with the polyene fatty acid probe trans-parinaric acid. To focus on a single climatic region, Mojave Desert dicots (chiefly ephemeral annuals) were examined, with plants grown under identical conditions whenever possible. Winter active species showed lower phase separation temperatures than the summer active species. A group of warm climate annual grasses showed separation temperatures distinctly higher than those of a group of cool climate grasses, all grown from seed under the same conditions. Growth at low temperature seems correlated with (and may require) a low phase separation temperature. Winter active ephemerals appear genetically programmed to synthesize a mixture of phospholipids which will not phase separate in the usual growth conditions. When the lipids of desert perennials were examined in cool and warm seasons, there was a pronounced seasonal shift in the phase separation temperature, implying environmental influences on lipid physical properties. The relationship of these results to high and low temperature tolerance is discussed.     

VARIABILITY OF SHORT-TERM CULTURES OF HeLa S-3 CELLS: CHANGES IN DNA DISTRIBUTIONS AND RATES OF DNA SYNTHESIS

DNA distributions of HeLa S-3 cells in spinner culture exhibit significant time—dependent changes. The major differences appear to occur in the S-phase region. Significant changes in the rates of DNA synthesis in several S-phase subcompartments correlated well with the changes in the DNA distributions. It is proposed that fluctuations in these rates of DNA synthesis are a reflection of the inherent instability of these abnormal, heteroploid cells.     

Somatic hybrids between unilateral cross-incompatible Petunia species

Summary Somatic hybrid plants regenerated following the fusion of leaf mesophyll protoplasts of Petunia parodii with those isolated from a cell suspension of albino P. inflata. These two species exhibit a unilateral cross-incompatability with a pre-zygotic mode of reproductive isolation preventing hybridizations with P. inflata as the maternal parent. Selection of somatic hybrids relied on the fact that unfused or homokaryon protoplasts of P. parodii did not develop beyond the cell colony stage while those of the putative somatic hybrids and albino P. inflata parent produced callus. Green somatic hybrid calluses were readily identified against the white background of P. inflata following complementation to chlorophyll synthesis proficiency and continued growth in hybrid cells. Shoots, and ultimately flowering plants, were identified as somatic hybrids based on their floral morphology and colour, chromosome number and the fact that they segregated for parental characters. The frequency of somatic hybrid production was comparable to that previously established for two sexually compatible Petunia species.