The anthocyanin in blue flowers ofCentaurea cyanus

The anthocyanin in the blue cornflower (Centaurea cyanus) has been known for many years to be cyanidin 3,5-diglucoside, namely cyanin. However, in the course of this study, it became evident that the major anthocyanin in the blue cornflower is not cyanin but cyanidin 3-succinyl glucoside 5-glucoside. This anthocyanin has not been reported in the literature and is tentatively called “centaurocyanin”. Centaurocyanin is chromatographically identical with the anthocyanin contained in crystalline protocyanin, the blue pigment from the cornflower. thus, there seems no doubt that this anthocyanin, but not cyanin, forms the blue complex pigment protocyanin.     

Temperature-sensitive anthocyanin production in flowers of Plantago lanceolata

Flower color in the weedy perennial Plantago lanceolata is phenotypically plastic. Darker flowers are produced at cooler ambient temperatures, and circumstantial evidence suggests that this is adaptive. The goal of this project was to investigate the chemical basis for the color plasticity. To test the hypothesis that increased anthocyanin production at low temperatures underlies the plasticity, extracts of P. lanceolata flowers produced at warm and cool temperatures were analyzed using UV/visible spectrophotometry coupled with mass spectrometry. Mass spectrometry allowed us to compare relative abundances of individual anthocyanins. Seventeen anthocyanins, derived from both cyanidin and delphinidin branches of the anthocyanin biosynthetic pathway, were detected. Most of these significantly increased in abundance under cool conditions. Genotypes differed significantly in anthocyanin levels and in their sensitivity to temperature change. Genotypes that showed greater floral color plasticity tended to show also greater temperature sensitivity with respect to anthocyanin production. Data suggest that the temperature regulation of the anthocyanin biosynthetic pathway occurs both upstream and downstream of the divergence of the cyanidin and delphinidin branches. The degree of temperature sensitivity, i.e. phenotypic plasticity, appears to be controlled downstream, whereas the overall temperature effect appears to be controlled upstream.     

Increased accumulation and decreased catabolism of anthocyanins in red grape cell suspension culture following magnesium treatment

Anthocyanins are the largest and best studied group of plant pigments. However, not very much is known about the fate of these phenolic pigments after they have accumulated in the cell vacuoles of plant tissues. We have previously shown that magnesium treatment of ornamentals during the synthesis of anthocyanins in the flowers or foliage caused an increase in the pigment concentration. In this study, we characterized the effect of magnesium on the accumulation of anthocyanin in red cell suspension originating from Vitis vinifera cv. Gamay Red grapes. Magnesium treatment of the cells caused a 2.5- to 4.5-fold increase in anthocyanin concentration, with no substantial induction of the biosynthetic genes. This treatment inhibited the degradation of anthocyanins occurring in the cells, and changed the ratio between different anthocyanins determining cell color, with an increase in the relative concentration of the less stable pigment molecules. The process by which magnesium treatment affects anthocyanin accumulation is still not clear. However, the results presented suggest at least part of its effect on anthocyanin accumulation stems from inhibition of the pigments’ catabolism. When anthocyanin biosynthesis was inhibited, magnesium treatments prevented the constant degradation of anthocyanins in the cell suspension. Future understanding of the catabolic processes undergone by anthocyanins in plants may enable more efficient inhibition of this process and increased accumulation of these pigments, and possibly of additional phenolic compounds.     

Thermophiles: A life at elevated temperatures

Interest in the ecology, physiology and evolution of microorganisms adapted to growth at relatively high temperatures (up to 110°C) has increased enormously during the past two decades. This interest was stimulated by the discovery of marine hydrothermal vent ecosystems, and also by awareness of the potential of thermophilic microbes in biotechnological processes. Subsequent attempts to isolate new thermophilic organisms have been very successful. Moreover, these results, in combination with much-improved techniques for studying the phylogeny of microorganisms, have renewed interest in the evolution of microbes and the early development of life.     

DNA damage production in CHO cells at elevated temperatures

Induction of DNA lesions in the nucleus of Chinese hamster ovary (CHO) cells was observed at hyperthermic temperatures using the alkaline filter elution and the alkaline sucrose gradient sedimentation methods. These lesions were observed principally at temperatures greater than 45 degrees C with an activation energy of 140 kcal/mole. On alkaline sucrose gradients the cell genome was reduced to a 140 S or 2 X 10(8) dalton subunit of DNA independent of increasing exposure time at temperatures above 45 degrees C. The large thermal activation energy and the limited DNA size reduction suggest the possible involvement of thermal denaturation of a nuclear polypeptide in the production of these nuclear lesions.     

Isobutanol production at elevated temperatures in thermophilic Geobacillus thermoglucosidasius

The potential advantages of biological production of chemicals or fuels from biomass at high temperatures include reduced enzyme loading for cellulose degradation, decreased chance of contamination, and lower product separation cost. In general, high temperature production of compounds that are not native to the thermophilic hosts is limited by enzyme stability and the lack of suitable expression systems. Further complications can arise when the pathway includes a volatile intermediate. Here we report the engineering of Geobacillus thermoglucosidasius to produce isobutanol at 50 °C. We prospected various enzymes in the isobutanol synthesis pathway and characterized their thermostabilities. We also constructed an expression system based on the lactate dehydrogenase promoter from Geobacillus thermodenitrificans. With the best enzyme combination and the expression system, 3.3 g/l of isobutanol was produced from glucose and 0.6 g/l of isobutanol from cellobiose in G. thermoglucosidasius within 48 h at 50 °C. This is the first demonstration of isobutanol production in recombinant bacteria at an elevated temperature.     

A malonylated anthocyanin and flavonols in blue Meconopsis flowers

The structures of the major anthocyanin and two flavonols from the blue flowers of Meconopsis were identified by NMR spectroscopy as being cyanidin 3-O-[(6-O-malonyl-2-O-B-D-xylopyranosyl)-beta-D-glucopyranoside]-7-O-beta-D-glucopyranoside, kaempferol 3-O-(6-O-beta-D-glucopyranosyl)-beta-D-glucopyranoside and kaempferol 3-O-(6-O-beta-D-glucopyranosyl)-beta-D-galactopyranoside respectively.     

Operational stability of copolymerized enzymes at elevated temperatures

The copolymerization method of immobilization was used to obtain preparations of enzymes covalently incorporated in polyacrylamide gel. They possess properties making them suitable for practical use. First, the preparations are hundreds of times more stable against irreversible thermoinactivation than native enzymes. Second, on immobilization, the reversible conformational changes which also lower enzyme activity at elevated temperatures are completely suppressed. As a result, the temperatures of maximum activity for trypsin and alpha-chymotrypsin covalently entrapped in polyacrylamide gel are 75 and 70 degrees C, respectively-25 and 30 degrees C higher than the corresponding values for the native enzymes. Therefore, the copolymerized enzyme preparations have a high operational stability at elevated temperatures.     

Energetics of peptide bond formation at elevated temperatures

Summary The free energies of formation of the peptide bond between carbobenzoxy-glycine and L-phenylalanine amide in aqueous solution at temperatures up to 60°C were calculated from experimentally determined equilibrium constants. The reaction was catalyzed by a thermophylic enzyme. The thermodynamic energy barrier to peptide bond formation was found to decrease with increasing temperature: the standard free energy of peptide bond formation did appear to become negative in the region of 60°C. The possible significance of these results for peptide bond formation under prebiotic conditions is discussed.     

Cytotoxicity of commonly used solvents at elevated temperatures

At 43 degrees C (but not at 41 degrees C), organic solvents used to dissolve water-insoluble chemotherapeutic agents become themselves lethal to cells. This finding is not unique to Chinese hamster cells (HA-1); mouse mammary sarcoma cells (EMT-6) behave similarly. The solvent concentrations involved are in the range of those needed to make drug solutions. Hence experiments measuring drug-cell interactions at elevated temperatures must include controls which independently measure solvent effects.     

Increased ovulation rate in androstenedione-immune ewes is not due to elevated plasma concentrations of FSH

Two experiments were undertaken to determine the hormonal response of Merino ewes to immunization against androstenedione (Fecundin). In Exp. 1 peripheral concentrations of LH, FSH and progesterone were monitored in spontaneously cycling ewes (20 immunized and 21 controls). In Exp. 2 (10 immunized and 10 controls) the same hormones were measured in ewes before and after prostaglandin (PG)-induced luteolysis and, in addition, the pattern of pulsatile LH secretion was determined during the luteal (PG + 12 days), early follicular (PG + 24 h) and late follicular (PG + 40 h) phase of the oestrous cycle. Ovulation rates were measured in both experiments. The results of these experiments indicate that androstenedione-immune animals have elevated ovulation rates (0.6-0.7 greater than control animals; P less than 0.05) associated with elevated plasma concentrations of LH and progesterone. The magnitude of the increase in plasma progesterone was correlated with androstenedione antibody titre (r = 0.6, P less than 0.001). LH pulse frequency of androstenedione-immune ewes tended to be higher at all stages of the oestrous cycle, but this difference was only significant (P less than 0.05) during the luteal phase. Mean plasma concentrations of FSH did not differ significantly between immunized and control ewes at any stage of the cycle. Analysis of periodic fluctuations in FSH during the luteal phase revealed that androstenedione-immune animals had a similar number of fluctuations of a similar amplitude to those of control animals, but the nadir of these fluctuations was lower (P less than 0.05) in immunized animals. A significant (P less than 0.05) negative correlation existed between androstenedione antibody titre and the interval between FSH peaks (r = -0.49) and androstenedione antibody titre and FSH nadir concentrations (r = -0.46). It is concluded that plasma FSH concentrations are not a determinant of ovulation rate in androstenedione-immune ewes and that increased LH concentrations, or perturbation of normal intraovarian mechanisms, may be responsible for the increase in ovulation rate observed in ewes immunized against androstenedione.     

Replication of measles virus in Vero cells at elevated temperatures

In one-step growth experiment of measles virus (MV) in Vero cells at 39 C, the appearance of MV infectivity was delayed for 24 hr and the maximum titer was reduced by approximately 1,000-fold, when compared with those at 35 C. MV infectivity was thermolabile at the high temperature. Penetration was rather enhanced at 39 C. By Northern blot hybridization, viral RNAs including 50S genome-sized RNA and mRNAs were first detectable 24 hr post-infection (PI) at 35 C and 36 hr PI at 39 C, respectively. Rapid degradation of viral mRNAs was not observed in the infected cells at 39 C. The synthesis of N, F, and M proteins was relatively reduced at the high temperature and appearance of the other viral protein was delayed, in agreement with the time course of viral RNA synthesis. All these data suggest that less efficient synthesis of viral RNA, restriction of synthesis of N, F, and M proteins at translational level and thermolability of infectivity are all involved in the suppressed MV production in Vero cells at 39 C.     

Solubility of saturated fatty acids in water at elevated temperatures

The solubility in water of saturated fatty acids with even carbon numbers from 8 to 18 was measured in the temperature range of 60 to 230 degrees C and at a pressure of 5 or 15 MPa. The pressure had no significant effect on the solubility. The solubility of the fatty acids increased with increasing temperature. At temperatures higher than about 160 degrees C, the logarithm of the solubility in mole fraction was linearly related to the reciprocal of the absolute temperature for each fatty acid, indicating that the water containing solubilized fatty acid molecules formed a regular solution at the higher temperatures. The enthalpy of a solution of the fatty acids in water, which was evaluated from the linear relationship at the given temperatures, increased linearly with the carbon number of the fatty acid.