静息培养条件下赤霉菌菌丝产素能力的研究

针对赤霉菌生长与产次级代谢产物赤霉素的关系进行摇瓶静息培养研究。在不同菌龄及不同底物浓度的条件下,菌丝产素能力有较大不同。实验数据表明：在菌龄96h,葡萄糖浓度为5gfL时,菌丝静息培养72h后,培养液中赤霉素含量最高,达1100μg／mg;同时单位时间单位菌丝产率也最大,为7．1μg/mg·h;在该菌龄条件下,当葡萄糖浓度为1dL时,赤霉素转化效率最高,为0．8484。该研究有助于进一步优化发酵工艺,降低赤霉素生产成本。     

Feedback regulation of GA5 expression and metabolic engineering of gibberellin levels in Arabidopsis.

The gibberellin (GA) 20-oxidase encoded by the GA5 gene of Arabidopsis directs GA biosynthesis to active GAs, whereas that encoded by the P16 gene of pumpkin endosperm leads to biosynthesis of inactive GAs. Negative feedback regulation of GA5 expression was demonstrated in stems of Arabidopsis by bioactive GAs but not by inactive GA. In transgenic Arabidopsis plants overexpressing P16, there was a severe reduction in the amounts of C20-GA intermediates, accumulation of large amounts of inactive GA25 and GA17, a reduction in GA4 content, and a small increase in GA1. However, due to feedback regulation, expression of GA5 and GA4, the gene coding for the subsequent 3beta-hydroxylase, was greatly increased to compensate for the effects of the P16 transgene. Consequently, stem height was only slightly reduced in the transgenic plants.     

16,17-dihydro gibberellin A5 competitively inhibits a recombinant Arabidopsis GA 3beta-hydroxylase encoded by the GA4 gene

Ring D-modified gibberellin (GA) A5 and A20 derivatives are structurally similar to GA20 and GA9 (the precursors to growth-active GA1 and GA4) and, when applied to higher plants, especially grasses, can reduce shoot growth with concomitant reductions in levels of growth-active GAs and increases in levels of their immediate 3-deoxy precursors. The recombinant Arabidopsis GA 3beta-hydroxylase (AtGA3ox1) protein was used in vitro to test a number of ring D-modified GA structures as possible inhibitors of AtGA3ox1. This fusion protein was able to 3beta-hydroxylate the 3-deoxy GAs, GA9 and GA20, to GA4 and GA1, respectively, and convert the 2,3-didehydro GA, GA5, to its 2,3-epoxide, GA6. Michaelis-Menten constant (Km) values of 1.25 and 10 microM, respectively, were obtained for the GA9 and GA20 conversions. We utilized the enzyme's ability to convert GA20 to GA1 in order to test the efficacy of GA5, 16,17-dihydro GA5 (dihydro GA5), and a number of other ring D-modified GAs as inhibitors of AtGA3ox activity. For the exo-isomer of dihydro GA5, inhibition increased with the dose of dihydro GA5, with Lineweaver-Burk plots showing that dihydro GA5 changed only the Km of the enzyme reaction, not the V(max), giving a dissociation constant of the enzyme-inhibitor complex (Ki) of 70 microM. Other ring D-modified GA derivatives showed similar inhibitory effects on GA1 production, with 16,17-dihydro GA20-13-acetate being the most effective inhibitor. This behavior is consistent with dihydro GA5, at least, functioning as a competitive substrate inhibitor of AtGA3ox1. Finally, the recombinant AtGA3ox1 fusion protein may be a useful screening tool for other effective 3beta-hydroxylase inhibitors, including naturally occurring ones.     

Accuracy of predicted resting metabolic rate and relationship between resting metabolic rate and cardiorespiratory fitness in obese men

[Purpose]

The purpose of this study is to examine that not only the relationship of the resting metabolic rate (RMR) and cardiorespiratory fitness(VO2peak), but also the comparison between measured and predicted results of RMR in obese men.

[Methods]

60 obese men (body fat>32%) were recruited for this study. They did not participate in regular exercising programs at least 6 months. The RMR was measured with indirect calorimetry and predicted RMR using Herris-Benedicte equation. The cardiorespiratory fitness was determined by directly measuring the oxygen consumption (VO2peak) during the exercise on the treadmill.

[Results]

The significance for the difference between the measured results and predicted result of RMR were tested by paired t-test. Correlation of measured date was obtained by Pearson correlation coefficient. The value of predicted RMR and measured RMR were significantly different in these obese subjects. (p < 0.001). The difference between RMR cardiorespiratory fitness and cardiorespiratory fitness showed significant correlation (r=0.67, p < 0.05).

[Conclusion]

The current formulas of predicted RMR have limited the evaluation of measured RMR for Korean obese men. Therefore, this study suggests that new formula should be designed for Korean in order to obtain more accurate results in obese.     

Comparative investigations on the metabolic fate of a GA3 amino acid conjugate with those of glucosides and derivatives of GA3

Three different radioactively 1 abelled conjugates and four derivatives of GA₃ were compared with regard to the uptake and metabolism in isolated cells ofLycopersicon esculentum L. The generally low and heavily varying rates of uptake did not correlate with the partition coefficients of the individual compounds in the system. N-GA₃-oyl-[2-¹⁴C]-glyeine was not hydrolyzed at all in tomato cell cultures whilst GA₃ glucosyl ester and GA₃-3-O-glueoside showed partial cleavage to GA₃. The stability of the amide linkage in N-GA₃-oyl-glycine was further established in metabolic experiments with intact tomato seedlings and inin vitro systems with proteases.     

Increased product formation induced by a directed secondary substrate limitation in a batch Hansenula polymorpha culture

By the use of directed limitations of secondary substrates, the metabolic flux should be deflected from biomass production to product formation. In order to study the impact of directed limitations caused by various secondary substrates on the growth and product formation of the methylotrophic yeast Hansenula polymorpha, the cultivation systems respiration activity monitoring system (RAMOS) and BioLector were used in parallel. While the RAMOS device allows the online monitoring of the oxygen transfer rate in shake flasks, the BioLector enables in microtiter plates the monitoring of scattered light and the fluorescence intensity of the green fluorescent protein (GFP). Secondary substrate limitations of phosphate, potassium, and magnesium were analyzed in batch fermentations. The sole carbon source was either 10 g/L glucose or 10 g/L glycerol. The expression of the GFP gene is controlled by the FMD promoter (formate dehydrogenase). In batch cultures with glucose as carbon source, a directed limitation of phosphate increased the GFP production 1.87-fold, compared to phosphate unlimited conditions. Under potassium-limited conditions with glycerol as sole carbon source, the GFP production was 1.41-fold higher compared to unlimited conditions. A limitation of the substrate magnesium resulted in a 1.22-fold increase GFP formation in the case of glycerol as carbon source.     

Relation between average daily metabolic rate and resting metabolic rate of the mongolian gerbil (Meriones unguiculatus)

Summary Some early reports have claimed that the Mongolian gerbil is a diurnal rodent, but we found it to be active both night and day, being more active at night. A circadian rhythm was found both in locomotory activity under laboratory conditions and in average daily metabolic rate (ADMR).The ADMR of the Mongolian gerbil observed at 20° C in our study was 2.921 ml O₂/g·h which was near the value expected from Grodzinski's equation (ADMR=19.94 W^-0.50).Both ADMR and RMR were found to be negatively correlated with ambient temperature, with the slope of the regression line for ADMR being less than that for RMR. These two lines intersected at 20° C, lower below 20° C.According to Grodzinski, the ADMR is usually lower than RMR, but in our study the ADMR was found to be lower than the RMR only below 20° C, which suggests that the ambient temperature acts differently on ADMR and RMR. The temperature-dependent variation of both ADMR and RMR as well as their differences should be taken into account in the estimation of the productivity of animal population.     

Cross talk between gibberellin and cytokinin: the Arabidopsis GA response inhibitor SPINDLY plays a positive role in cytokinin signaling

SPINDLY (SPY) is a negative regulator of gibberellin (GA) responses; however, spy mutants exhibit various phenotypic alterations not found in GA-treated plants. Assaying for additional roles for SPY revealed that spy mutants are resistant to exogenously applied cytokinin. GA also repressed the effects of cytokinin, suggesting that there is cross talk between the two hormone-response pathways, which may involve SPY function. Two spy alleles showing severe (spy-4) and mild (spy-3) GA-associated phenotypes exhibited similar resistance to cytokinin, suggesting that SPY enhances cytokinin responses and inhibits GA signaling through distinct mechanisms. GA and spy repressed numerous cytokinin responses, from seedling development to senescence, indicating that cross talk occurs early in the cytokinin-signaling pathway. Because GA3 and spy-4 inhibited induction of the cytokinin primary-response gene, type-A Arabidopsis response regulator 5, SPY may interact with and modify elements from the phosphorelay cascade of the cytokinin signal transduction pathway. Cytokinin, on the other hand, had no effect on GA biosynthesis or responses. Our results demonstrate that SPY acts as both a repressor of GA responses and a positive regulator of cytokinin signaling. Hence, SPY may play a central role in the regulation of GA/cytokinin cross talk during plant development.     

The regulation of cambial division and secondary xylem differentiation in xanthium by auxins and gibberellin

Cambial division continued in decapitated Xanthium plants without concomitant xylem fiber differentiation. The application of indoleacetic acid to these plants did not affect the production of cambial derivatives or induce xylem fiber differentiation. When naphthaleneacetic acid was applied either to the second internode or to the stump of a lateral shoot, xylem fiber differentiation was induced in the newly formed cambial derivatives on the xylem side of the cambium in the stem. When naphthaleneacetic acid was applied unilaterally, xylem fiber differentiation was restricted to that side of the stem in the first internode and hypocotyl. Naphthaleneacetic acid also enhanced the production of cambial derivatives. Gibberellic acid enhanced cambial derivative production but did not affect the differentiation of xylem fibers. Similar numbers of cambial derivatives were produced in some naphthaleneacetic acid-treated plants in which xylem fiber differentiation was induced and in gibberellic acid-treated plants which did not differentiate xylem. When naphthaleneacetic acid was applied 72 hours after decapitation, the oldest of the cambial derivatives on the xylem side failed to develop into fibers although younger cells did. These results suggest that auxin has its direct effect on the induction of xylem differentiation rather than the induction of divisions prerequisite to differentiation.     

Effects of kinetin and gibberellin a3 on callus growth and organ formation inLimnophila chinensis tissue culture

The action of exogenously applied hormones in the induction of morphogenesis inLimnophila chinensis (Osb.) Merr. tissue culture has been demonstrated. Stem expiants were grown on Murashige and Skoog’s medium containing various levels of kinetin, gibberellic acid and indole-3-acetic acid. Formation of roots, shoots (normal or abnormal), plantlets and friable, hard or nodulated calluses depended largely on the hormone levels used. The formation of normal shoots and roots were stimulated by treatment with kinetin. GA₃ treatment stimulated the bud differentiation but inhibited the root initiation. A combination of kinetin and GA₃ gave variable results.     

Interaction between transcobalamin II and immobilized Cibacron Blue F3GA

Human serum transcobalamin II (TC II), a vitamin B₁₂ (Cbl) transport protein, complexes with Cibacron Blue F3GA, a reactive blue dye which can bind to proteins that require nucleotides as cofactors. Apo-TC II and holo-TC II both bind, but intrinsic factor (IF) and R-type binders of Cbl do not. Other mammalian species TC II also complex with the dye. Greater than 87% of the applied TC II-CN-[⁵⁷Co]Cbl remains bound to the dye even at pH 4.0. At pH values below this, the CN-[⁵⁷Co]Cbl dissociates off TC II which remains bound to the dye. High salt concentrations will break the TC II-dye complex. Ionic forces were considered not to be involved since complexing also occurred at pH 9.0, 2.5 pH units above the isoelectric point of TC II. Failure to dissociate the TC II-dye complex with 50% glycerol makes hydrophobic interactions unlikely. In addition to the potential uses of TC II-Cibacron Blue F3GA complexes in a total scheme for protein purification, the possibility that TC II is a nucleotide-requiring protein should be explored.     

Engineering of gibberellin levels in citrus by sense and antisense overexpression of a GA 20-oxidase gene modifies plant architecture

Carrizo citrange (Citrus sinensisxPoncirus trifoliata) is a citrus hybrid widely used as a rootstock, whose genetic manipulation to improve different growth characteristics is of high agronomic interest. In this work, transgenic Carrizo citrange plants have been produced overexpressing sense and antisense CcGA20ox1 (a key enzyme of GA biosynthesis) under control of the 35S promoter to modify plant architecture. As expected, taller (sense) and shorter (antisense) phenotypes correlated with higher and lower levels, respectively, of active GA1 in growing shoots. In contrast, other phenotypic characteristics seemed to be specific to citrus, or different from those described for similar transgenics in other species. For instance, thorns, typical organs of citrus at juvenile stages, were much longer in sense and shorter in antisense plants, and xylem tissue was reduced in leaf and internode of sense plants. Antisense plants presented a bushy phenotype, suggesting a possible effect of GAs on auxin biosynthesis and/or transport. The main foliole of sense plants was longer, although total leaf area was reduced. Leaf thickness was smaller in sense and larger in antisense plants due to changes in the spongy parenchyma. Internode cell length was not altered in transgenic plants, indicating that, in citrus, GAs regulate cell division rather than cell elongation. Interestingly, the phenotypes described were not apparent when transgenic plants were grafted on non-transgenic rootstock. This suggests that roots contribute to the GA economy of aerial parts in citrus and opens the possibility of using the antisense plants as dwarfing rootstocks.     

Genetic correlation between resting metabolic rate and exploratory behaviour in deer mice (Peromyscus maniculatus)

According to the 'pace-of-life' syndrome hypothesis, differences in resting metabolic rate (RMR) should be genetically associated with exploratory behaviour. A large number of studies reported significant heritability for both RMR and exploratory behaviour, but the genetic correlation between the two has yet to be documented. We used a quantitative genetic approach to decompose the phenotypic (co)variance of several metabolic and behavioural measures into components of additive genetic, common environment and permanent environment variance in captive deer mice. We found significant additive genetic variance for two mass-independent metabolic measures (RMR and the average metabolic rate throughout the respirometry run) and two behavioural measures (time spent in centre and distance moved in a novel environment). We also detected positive additive genetic correlation between mass-independent RMR and distance moved (r(A) = 0.78 ± 0.23). Our results suggest that RMR and exploratory behaviour are functionally integrated traits in deer mice, providing empirical support for one of the connections within the pace-of-life syndrome hypothesis.     

The gene encoding tobacco gibberellin 3beta-hydroxylase is expressed at the site of GA action during stem elongation and flower organ development

Gibberellin 3beta-hydroxylase catalyzes the final step in the biosynthetic pathway leading to the plant hormone gibberellin (GA) and, therefore, the in vivo localization of this enzyme should give a direct indication of the site of synthesis of bioactive GAs in plants. We have isolated a cDNA clone, Nty (Nicotiana tabacum GA 3beta-hydroxylase), which encodes a putative GA 3beta-hydroxylase, by RT-PCR using RNA from tobacco shoot apices. Functional analysis, using an NTY protein expressed in Escherichia coli, revealed that Nty encoded an active GA 3beta-hydroxylase. A high expression level of Nty was observed in shoot apices, flowers, roots, young internodes but not in leaves or seeds. We performed more detailed expression analyses using in situ hybridization and histochemical analyses of the GUS activity in transgenic tobacco plants carrying an Nty promoter:GUS fusion gene. These studies revealed that expression of Nty was restricted to specific regions, including actively dividing and elongating cells in the various organs; rib meristem and elongation zones of shoot apices, tapetum and pollen grains in developing anthers and root tips, which are consistent with the sites of GA action. It is proposed that GA actions depend on the modulation of endogenous bioactive GA levels through the regulation of GA 3beta-hydroxylase expression in situ.     

Bacterial secondary production in freshwater measured by3H-thymidine incorporation method

Bacterial secondary production in lake water was measured by³H-thymidine incorporation into DNA. The application of the method to freshwater systems studied required (1) thymidine concentration > 10 nM (10–25 nM) evaluated from isotope dilution by varying the specific activity of labeled thymidine, (2) short incubation periods less than 1 hour, and (3) partial purification of the DNA fraction for measuring³H-thymidine incorporation. During 2 diel studies, bacterial productivity was compared to phytoplankton primary production and extracellular release of organic carbon. Diel changes in bacterial growth suggested substantial activity during the morning and evening. Possible mechanisms of control of bacterial growth, such as extracellular release of organic carbon, are discussed.     

Chemical differences between wildtype and gibberellin mutants of tomato determined by pyrolysis-mass spectrometry

Leaf, stem and root material of wild-type and gibberellin (GA)-deficient mutants of tomato (Lycopersicum esculentum L.) were analysed by pyrolysis-mass spectrometry for possible differentiation in chemical allocation pattern among cell wall and cytoplasm. GA-deficiency is accompanied by changes in the relative growth rate (RGR). RGR-correlated changes were found in leaves in the comparative amounts of cellulose- and protein-derived fragments. The low-RGR genotypes contained more protein and nucleic acid, the high-RGR ones more cellulose. In root material, a higher contribution of cellulose, hemicellulose and G- and S-lignin was found for the lower-RGR plants and comparatively high protein in the high-RGR genotypes. For stems, less clear results were obtained, possibly because of variation in the ratio of syringyl- and guaiacyl-lignin. Part of the results might be explained by a GA-dependent change in cell size.     

Effect of culture conditions on producing and uptake hydrogen flux of biohydrogen fermentation by metabolic flux analysis method

In this work, metabolic flux analysis (MFA) method was used to estimate the effects of the culture conditions on both the producing and uptake hydrogen flux inside the cell of Klebsiella pneumoniae ECU-15. The results indicated that higher temperature could reduce the amount of the uptake hydrogen and enhance the hydrogen production from the NADH pathway. Moreover, both the producing hydrogen flux from formate and the uptake hydrogen flux were attained to the maximum at pH 7.0-7.5. The producing hydrogen flux was higher at 5 g/L initial glucose than that of the other concentrations, and the uptake hydrogen flux showed the minimum value under the same condition. The apparent hydrogen generation was caused by the combined action of producing hydrogenase, uptake hydrogenase and bidirectional hydrogenase. These results were helpful to deeply understand the mechanism of the biohydrogen evolving process and establish the suitable molecular strategies for improving hydrogen production.