Rapid induction by fungal elicitor of the synthesis of cinnamyl-alcohol dehydrogenase, a specific enzyme of lignin synthesis

A fivefold increase in the extractable activity of cinnamyl-alcohol dehydrogenase, an enzyme of phenylpropanoid metabolism specific for lignin synthesis, was observed within 10 h of treatment of cell-suspension cultures of bean (Phaseolus vulgaris L.) with a high-molecular-mass elicitor preparation heat-released from mycelial cell walls of the bean pathogen Colletotrichum lindemuthianum. Elicitor caused a rapid, marked but transient increase in the synthesis of cinnamyl-alcohol dehydrogenase with maximum rates 2-3 h after elicitation, concomitant with the phase of rapid increase in enzyme activity. There is a close correspondence between increased polysomal mRNA activity encoding cinnamyl-alcohol dehydrogenase, as measured by incorporation of [35S]methionine into immunoprecipitable enzyme subunits in vitro, and the stimulation of enzyme synthesis in vivo in response to elicitor. This marked increase in polysomal mRNA activity represents an increase as a proportion of total cellular mRNA activity, indicating that elicitor does not stimulate synthesis of this enzyme by selective recruitment from the total pool of cellular mRNA. Elicitor stimulation of cinnamyl-alcohol dehydrogenase activity and enzyme synthesis is more rapid than previously observed for other proteins involved inducible defense mechanisms, such as enzymes of phytoalexin biosynthesis or the apoproteins of cell-wall hydroxyproline-rich glycoproteins.     

Inhibition of CO2 production from aminopyrine or methanol by cyanamide or crotonaldehyde and the role of mitochondrial aldehyde dehydrogenase in formaldehyde oxidation

Previous results have shown that cyanamide or crotonaldehyde are effective inhibitors of the oxidation of formaldehyde by the low-Km mitochondrial aldehyde dehydrogenase, but do not affect the activity of the glutathione-dependent formaldehyde dehydrogenase. These compounds were used to evaluate the enzyme pathways responsible for the oxidation of formaldehyde generated during the metabolism of aminopyrine or methanol by isolated hepatocytes. Both cyanamide and crotonaldehyde inhibited the production of 14CO2 from 14C-labeled aminopyrine by 30-40%. These agents caused an accumulation of formaldehyde which was identical to the loss in CO2 production, indicating that the inhibition of CO2 production reflected an inhibition of formaldehyde oxidation. The oxidation of methanol was stimulated by the addition of glyoxylic acid, which increases the rate of H2O2 generation. Crotonaldehyde inhibited CO2 production from methanol, but caused a corresponding increase in formaldehyde accumulation. The partial sensitivity of CO2 production to inhibition by cyanamide or crotonaldehyde suggests that both the mitochondrial aldehyde dehydrogenase and formaldehyde dehydrogenase contribute towards the metabolism of formaldehyde which is generated from mixed-function oxidase activity or from methanol, just as both enzyme systems contribute towards the metabolism of exogenously added formaldehyde.     

Regulation of biosynthesis of secondary metabolites

The activity of decarboxylating malate dehydrogenase (NADP⁺ spec.) (EC 1.1.1.40) was studied during fermentation with the low producing and producing strain in relation to the production of chlortetracycline. The synthesis of the decarboxylating malate dehydrogenase is repressed by acetate. With both the strains the enzyme reaches a maximum activity during the logarithmic phase of growth, during which phase maximum activity of the pentose cycle was also found. The onset of the biosynthesis of chlortetracycline is accompanied by a significant decrease of the content of decaroboxylating malate dehydrogenase. The role of the enzyme as NADPH donor in the metabolism ofStreptomyces aureofaciens is discussed.     

Short-term effects of thyroid hormones on lipogenic enzymes and 14C-acetate incorporation into various lipid classes: in vivo and in vitro studies

Short-term effect of 3,5,3'-triiodothyronine (T3) and 3,5-diiodothyronine (T2) on lipid metabolism in the liver of Anabas testudineus was examined. In vivo injections of both T3 and T2 at a concentration of 10 ng/g body weight increased malic enzyme (ME), glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) activity compared to 6-propylthiouracil (6-PTU) treated group. Treatment of 6-PTU results in the accumulation 14C-acetate into fat and thyroid hormones' treatment reduce it. In vitro experiments show that malic enzyme activity is augmented only by high concentration of T3 (10(-7) M) where as all concentrations of T2 increase its activity. In vitro studies with T3 showed a biphasic effect on cholesterol content. Conversely T2 in vitro, reduced cholesterol content with all concentrations. From these results it can be concluded that both T3 and T2 have short-term effect on lipid metabolism in Anabas.     

Impact of application of organic waste materials on microbial and enzyme activities of mine soils in the Lusatian coal mining region

The objective of the present work was to study the short-term stimulation of microbial and enzyme activity in mine soils by application of organic waste materials in lysimeter and mesocosm studies. The mine soils derived from tertiary and quaternary deposits were ameliorated with brown coal filter ash (tertiary deposits) and lime (quaternary deposits). At the beginning of recultivation the soils were treated with varying amounts of sewage sludge, coal sludge, composted sewage sludge and compost to a depth of 30 cm. In the first 2 years after application of organic waste materials we found a very low level of microbial properties especially in the sandy materials from quaternary deposits but a significant increase in microbial respiration, substrate induced respiration and enzyme activities like invertase and alkaline phosphatase with increasing application rates of sewage sludge, compost and sewage sludge mixed with coal sludge. This can be explained by an increase in organic matter and nutrient content of the soils and an improvement of soil physical properties such as water and nutrient retention capacity. Additionally it can be assumed, that constituents of the coal admixtures of tertiary deposits can be mineralised or converted by the soil microorganisms. In the tertiary materials ameliorated with brown coal ash the highest amounts of microbial and enzyme activities were measured after application of nitrogen-rich sewage sludge or very high amounts of mature compost mainly consisting of green waste. Compared with sewage sludge the stimulating effects of composted sewage sludge were quite lower because of organic matter fragmentation and a reduced energy and nutrient supply to soil microorganisms. This revised version was published online in June 2006 with corrections to the Cover Date.     

Characterization of rat lymphocyte primary culture for the development of an in-vitro mutagenesis assay: Effect of interleukin-2 and 2-mercaptoethanol on the activities of intermediary metabolism enzymes and cell proliferation

Efficient energy utilization is essential for cell growth; in an attempt to improve the growth conditions of the rat T-lymphocyte culture model for potential use in studying the mutagenic activity of carcinogens in vitro, we have investigated the effects of phytohemagglutinin (PHA), interleukin-2 (IL-2) and 2-mercaptoethanol (2-ME) on the activities of intermediary metabolism enzymes and cell proliferation. Isolated lymphocytes were cultured in the presence and absence of PHA, IL-2, or 2-ME. The intermediary metabolism enzymes investigated were glutamate dehydrogenase, glutamate-pyruvate transaminase, malate dehydrogenase, isocitrate dehydrogenase, lactate dehydrogenase, pyruvate kinase, and fatty acid synthetase (FAS). Measurable activity of all enzymes investigated, except for FAS, was detected in PHA-stimulated cells cultured with IL-2 or 2-ME. The unstimulated lymphocytes had significantly lower enzyme activity than stimulated cells. The combination of all three agents showed increased enzyme activity. This increase in activity brought about by the combination of the three agents was not reproduced by either agent acting alone. In general, the increase in enzyme activity correlated with cell proliferation as measured by [³H]thymidine uptake in PHA-stimulated cultures containing IL-2 and/or 2-ME. The results suggest that the addition of exogenous IL-2 and 2-ME enhances metabolic function and may be beneficial in in vitro culture of rat lymphocytes.Abbreviations PHA phytohemagglutinin - IL-2 interleukin-2 - 2-ME 2-mercaptoethanol - GDH glutamate dehydrogenase - GPT glutamate-pyruvate transaminase - MDH malate dehydrogenase - ICD isocitrate dehydrogenase - LDM lactate dehydrogenase - PK pyruvate kinase - FAS fatty acid synthetase     

Ethanol exposure modulates hepatic S-adenosylmethionine and S-adenosylhomocysteine levels in the isolated perfused rat liver through changes in the redox state of the NADH/NAD(+) system

Methionine metabolism is disrupted in patients with alcoholic liver disease, resulting in altered hepatic concentrations of S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and other metabolites. The present study tested the hypothesis that reductive stress mediates the effects of ethanol on liver methionine metabolism. Isolated rat livers were perfused with ethanol or propanol to induce a reductive stress by increasing the NADH/NAD(+) ratio, and the concentrations of SAM and SAH in the liver tissue were determined by high-performance liquid chromatography. The increase in the NADH/NAD(+) ratio induced by ethanol or propanol was associated with a marked decrease in SAM and an increase in SAH liver content. 4-Methylpyrazole, an inhibitor the NAD(+)-dependent enzyme alcohol dehydrogenase, blocked the increase in the NADH/NAD(+) ratio and prevented the alterations in SAM and SAH. Similarly, co-infusion of pyruvate, which is metabolized by the NADH-dependent enzyme lactate dehydrogenase, restored the NADH/NAD(+) ratio and normalized SAM and SAH levels. The data establish an initial link between the effects of ethanol on the NADH/NAD(+) redox couple and the effects of ethanol on methionine metabolism in the liver.     

Effect of spinal cord stimulation on the metabolism of developing latissimus dorsii muscles in chick embryo

Influence of chronic spinal cord stimulation upon some characteristic enzyme activities of energy metabolism was investigated in slow anterior (ALD) and fast posterior (PLD) latissimus dorsii muscles of the chick embryo. During embryonic life, oxidative metabolism (as evaluated by the activity of malate dehydrogenase (MDH] represents the main energetic pathway in both slow and fast muscles. At the end of embryonic life, an increase in anaerobic (as evaluated by the activity of lactate dehydrogenase (LDH] and creatine phosphokinase (CPK) activities occurs in PLD muscle. Chronic spinal cord stimulation at a low frequency was performed from the 10th day to the 16th day of embryonic development. In ALD, the enzyme activities were unaffected, while in PLD a concomitant decrease in LDH and CPK activities was observed.     

Early and late changes in the metabolic pattern of the working myocardial fibres and Purkinje fibres of the human heart under ischaemic and inflammatory conditions: An enzyme histochemical study

Summary In this communication, the results of an enzyme histochemical study on the working myocardial fibres and Purkinje fibres of the atrioventricular conducting system of the human heart under ischaemic and inflammatory conditions are presented. The material was selected from patients showing changes which could be classified in three major groups: (1) early changes due to acute ischaemia either in the myocardial fibres or in the conducting system or in both; (2) chronic ischaemic changes due to cardiovascular insufficiency, such as old infarction, or coronary arteriosclerosis or both; and (3) inflammatory conditions such as myocarditis.The activity and location of about 20 enzymes that play a role in the aerobic and anaerobic pathways of energy metabolism were examined. The activity and location of some hydrolytic enzymes and the glycogen and lipid content were also studied.The most important findings were an obvious depletion of the glycogen reserves under acute ischaemic changes in both types of fibre. This was associated with a transient or permanent reduction in activity of many enzymes that play a role in aerobic and anaerobic metabolism. Further, there was an instantaneous and persistent increase in the activity of the NADPH-regenerating enzymes of the pentose phosphate pathway and of glyceraldehyde-3-phosphate dehydrogenase, the rate-limiting enzyme of glycolysis under ischaemic conditions. Chronic ischaemic changes were characterized by a gradual long-term increase in the activity of many anaerobic glycolytic enzymes. Moreover, there was an absence of activity of acetylcholine esterase immediately after the onset of infarction in the fibres of the conducting system. Lastly, a slight increase in lipid content was found in the hypertrophic chronic ischaemic fibres and in old infarcted areas. Cardiac fibres in inflamed areas showed a marked increased activity of the pentose phosphate shunt enzymes and a less pronounced increased activity of most anaerobic and hydrolytic enzymes. In contrast to the cardiac fibres in infarcted areas, the fibres in inflamed areas did not reveal a decrease or absence of activity of aerobic enzymes such as succinate dehydrogenase.     

不同年限对栽参土壤生物活性的影响

通过对不同年限栽参土壤微生物功能多样性和土壤酶活性的动态变化分析,探究人参生长年限与微生物碳源利用、土壤酶活性之间的相互关系,寻找人参生长与土壤生物活性之间的变化规律,旨在从生物活性角度分析人参连作障碍成因及为科学指导栽参提供理论依据。分析表明,随着人参生长年限的增加,AWCD值、物种丰富度指数和碳源利用丰富度指数均降低,而优势度度指数升高。土壤脲酶,蔗糖酶活性随栽培年限增加活性降低,而过氧化氢酶活性升高。相关性分析表明,微生物代谢与土壤酶活性关系密切,微生物种类多,奇异度高,微生态系统稳定,土壤酶活性高。因此,保证土壤微生物的多样性,保持土壤酶活性,是提高土壤质量的重要手段,也是解决人参连作障碍的有效途径。     

Post-transcriptional analysis of rat mitochondrial D-3-hydroxybutyrate dehydrogenase control through development and physiological stages.

The nuclear encoded mitochondrial D-3-hydroxybutyrate dehydrogenase (BDH) is synthesized in the cytosal as a larger precursor. This membrane enzyme which requires lecithin for activity plays an essential role in energy metabolism as a ketone bodies-converting enzyme. A cDNA clone of the rat liver enzyme encompassing an antigenic determinant peptide has been isolated after immunoscreening of a lambda gt11 expression library. The nucleotide sequence of this 279-base cDNA insert contains a single open reading frame of 93 amino-acids, which represents about a third of the mature enzyme. Amino-acid sequence analysis predicts a hydrophobic stretch of 29 amino-acids long which probably functions as membrane anchor domain, or as an important region for the enzyme activation by phospholipid. By using this cDNA probe the BDH gene has been investigated at the mRNA level. There is only one mRNA (2-kb size) for BDH whatever the studied tissue. The rat gene is differently expressed since its mRNA is already present in the foetus liver while the BDH polypeptide amount is low and its enzymatic activity is not detectable even in the late stage of foetal development. The mRNA content is higher in the liver than in extrahepatic tissues. Adrenalectomy and ovariectomy increase liver mRNA content and polypeptide level, as well as activity of BDH. These effects are totally or partially abolished by corticosterone and estradiol treatments respectively. In addition, a 15-day hyperlipidic diet stimulates BDH gene expression. Present results show that the gene expression of this mitochondrial enzyme is modulated through development and hormonal and metabolic conditions mentioned above.     

Inhibition of Class-3 aldehyde dehydrogenase and cell growth by restored lipid peroxidation in hepatoma cell lines

Hepatoma cells have a below-normal content of polyunsaturated fatty acids; this reduces lipid peroxidation and the production of cytotoxic and cytostatic aldehydes within the cells. In proportion to the degree of deviation, hepatoma cells also show an increase in the activity of Class-3 aldehyde dehydrogenase, an enzyme important in the metabolism of lipid peroxidation products and also in that of several drugs. When hepatoma cells with different degrees of deviation were enriched with arachidonic acid and stimulated to peroxidize by ascorbate/iron sulphate, their growth rate was reduced in proportion to the quantity of aldehydes produced and to the activity of aldehyde dehydrogenase. Therefore, 7777 cells, less deviated and with low Class-3 aldehyde dehydrogenase activity, were more susceptible to lipid peroxidation products than JM2 cells. It is noteworthy that repeated treatments with prooxidant also caused a decrease in mRNA and activity of Class-3 aldehyde dehydrogenase, contributing to the decreased growth and viability. Thus, Class-3 aldehyde dehydrogenase could be considered relevant for the growth of hepatoma cells, since it defends them against cell growth inhibiting aldehydes derived from lipid peroxidation.     

长期不同施肥措施对双季稻田土壤活性有机碳组分和水解酶活性的影响

施肥是改善土壤质量、提高土壤肥力和影响土壤微生物多样性的关键措施。为了探明南方双季稻区长期不同施肥处理下稻田土壤活性有机碳组分和水解酶活性的变化特征,本研究以34年大田定位试验为平台,设置化肥(MF)、秸秆还田+化肥(RF)、30%有机肥+70%化肥(OM)3个处理,并以无肥处理为对照(CK),分析了长期不同施肥处理下双季稻田土壤有机碳含量、活性有机碳组分、有机碳水解酶活性及其相关性。结果表明: MF、RF和OM处理增加了稻田土壤有机碳含量,分别比CK增加4.5%、22.4%和53.5%。与MF和CK处理相比,RF和OM处理均有利于增加土壤各活性有机碳组分[累积碳矿化(Cmin)、高锰酸钾可氧化碳(KMnO₄-C)、颗粒有机碳(POC)、溶解性有机碳(DOC)、轻组有机碳(LFOC)和微生物生物量碳(MBC)]和各活性有机碳组分占总有机碳的比例。OM处理土壤Cmin、KMnO₄-C、POC、DOC、LFOC和MBC含量分别比CK增加3.5、3.1、3.7、1.9、1.2和1.9倍;RF和OM处理土壤各活性有机碳组分占总有机碳的比例均显著高于CK。各施肥处理土壤水解酶活性(α-葡萄糖苷酶、β-葡萄糖苷酶、木糖苷酶、纤维二糖水解酶和N-乙酰-β-氨基葡萄糖苷酶)的大小顺序均为: OM>RF>MF>CK,其中OM处理的各土壤水解酶活性分别比CK增加111.8%、14.1%、127.3%、285.6%和91.4%。RF和OM处理有利于增加土壤过氧化物酶活性,MF处理有利于增加土壤多酚氧化酶活性。土壤水解酶与土壤有机碳含量及其活性有机碳组分均呈显著正相关。综上,有机肥和秸秆还田与化肥配合施用是提高南方双季稻田土壤活性有机碳组分和水解酶活性的有效措施。     

Relationship between dehydrogenase activity and properties of soil aggregates

Studies of dehydrogenase activity in aggregates of chernozemic rendzina and lessivē soil proved that the diameter of aggregates significantly influences the activity of the above enzyme, subject to soil type. There is a significant positive correlation between the above activity and the percentage of particles 100 to 50 μm in aggregates of lessivē soil but not in those of chernozemic rendzina. Dehydrogenase activity is also affected by soil particles of other sizes, some pores in soil aggregates, the ratio of volume of air to water in them, content of total N and organic C as well as the total, external and internal surface areas, but the correlations in these cases are not significant at the level of 0.05.     

Metabolic control of the expression of mitochondrial D-beta-hydroxybutyrate dehydrogenase, a ketone body converting enzyme

The effects of various metabolic conditions inducing an overproduction of ketone bodies in the rat were studied at different levels of D-beta-hydroxybutyrate dehydrogenase expression, i.e., enzymatic activity and protein content in purified mitochondria, and translational activity of isolated free cytosolic polysomes. The strongest variations were obtained in diabetes mellitus where the D-beta-hydroxybutyrate dehydrogenase expression is largely decreased. Insulin can reverse this strong effect. Modulation of liver enzyme activity and of enzyme content was observed under the other conditions tested, i.e., a decrease and an increase in starvation and hyperlipidemic conditions, respectively. A comparative study was carried out on the enzyme of extrahepatic tissues, i.e., heart, kidney and brain. The results indicate that the D-beta-hydroxybutyrate dehydrogenase function appears to be controlled, at least at the translational, post-translational and catalytic levels.     

Adjustment of growth, starch turnover, protein content and central metabolism to a decrease of the carbon supply when Arabidopsis is grown in very short photoperiods

Arabidopsis was grown in a 12, 8, 4 or 3 h photoperiod to investigate how metabolism and growth adjust to a decreased carbon supply. There was a progressive increase in the rate of starch synthesis, decrease in the rate of starch degradation, decrease of malate and fumarate, decrease of the protein content and decrease of the relative growth rate. Carbohydrate and amino acids levels at the end of the night did not change. Activities of enzymes involved in photosynthesis, starch and sucrose synthesis and inorganic nitrogen assimilation remained high, whereas five of eight enzymes from glycolysis and organic acid metabolism showed a significant decrease of activity on a protein basis. Glutamate dehydrogenase activity increased. In a 2 h photoperiod, the total protein content and most enzyme activities decreased strongly, starch synthesis was inhibited, and sugars and amino acids levels rose at the end of the night and growth was completely inhibited. The rate of starch degradation correlated with the protein content and the relative growth rate across all the photoperiod treatments. It is discussed how a close coordination of starch turnover, the protein content and growth allows Arabidopsis to avoid carbon starvation, even in very short photoperiods.     

Glucose metabolism during embryogenesis of the hard tick Boophilus microplus

Glucose metabolism plays an essential role in the physiology and development of almost all living organisms. In the present study we investigated glucose metabolism during the embryogenesis of the hard tick Boophilus microplus. An increase in glucose and glycogen content during the embryonic development of B. microplus was detected and shown to be due to the high enzyme activity of both gluconeogenesis and glycolytic pathways. Glucose 6-phosphate (G-6P), formed by hexokinase, is driven mainly to pentose-phosphate pathway, producing fundamental substrates for cellular biosynthesis. We detected an increase in glucose 6-phosphate dehydrogenase and pyruvate kinase activities after embryo cellularization. Accumulation of key metabolites such as glycogen and glucose was monitored and revealed that glycogen content decreases from day 1 up to day 6, as the early events of embryogenesis take place, and increases after the formation of embryo cellular blastoderm on day 6. Glucose and guanine (a sub-product of amino acids degradation in arachnids) accumulate almost concomitantly. The activity of phosphoenolpyruvate carboxykinase was increased after embryo cellularization. Taken together these data indicate that glycogen and glucose, formed during B. microplus embryogenesis after blastoderm formation, are produced by intense gluconeogenesis.     

Enzymatic and immunologic study of the role of the thyroid hormone in the formation of the internal mitochondrial membrane during postnatal development of the rat

Hypothyroidism induces an increase of liver D-beta-hydroxybutyrate dehydrogenase activity. Injection of thyroid hormone reverses the phenomena. The use of monospecific antibody raised against the purified enzyme indicates that there was not an increase of apoenzyme biosynthesis. The thyroid hormone negative control is due to a metabolism alteration of the membrane phospholipids which are directly involved in the apoenzyme activity. The highest difference is observed with 20 days old rats. Opposite effects were obtained on succinate cytochrome c reductase.