Isolation and Identification of Steroidal Saponins and Sapogenins from Aspidistra zongbayi K. Y. Lang et Z. Y. Zhu

Two steroidal saponins were isolated from the n-BuOH extract of the rhizome of Aspidistra zongbayi K. Y. Lang et Z. Y. Zhu. Their structures were elucidated as 3-O-{β-D-glucopyr-anosyl (l→2)-[β-D-xylopyranosyl (1→3)]-β-D-glucopyranosyl (l→4)-β-D-glactopyranosyl}-diosgenin (aspidistrin) and proto-aspidistrin by physical and chemical methods. Three steroidal sapogenins were isolated from the acid-treated n-BuOH extract. They were identified as△3.5 deoxyfigogenin, diosgenin and gentrogenin, β-sitosterol was isolated from the rhizome of this plant also.     

一个拖延了半个多世纪的研究课题的总结汇报

本文是汤佩松教授为北京植物生理学会1992年年会准备的发言稿,并以庆祝中国植物生理学会成立30周年。后“北京年会”因故未开,本刊应汤老要求按原样发表于此(并附英文摘要),以飨读者。     

Certain Modes of Control of Life Process Operation by Respiratory Metabolism in Plant Cells

A new concept of respiratory metabolism is proposed, which regards this process as anabolism involved in catabolism, and as the basis of self organization in living cells. In addition to furnishing material and energy for the performance of the plant as a living organism, it regulates the other physiological functions via the operation of multiple pathway. Based on the proposed concept, a bioenergetic system in he-erotrophic plant cells is described, which comprises proticity, reducing powers and water motive force, a newly coined term by the authors, generated by respiration. The feed back effects of photorespiration, or in a broad sense, photoxidatlon on photosynthesis are discussed. An analysis of regulatory effects of phytohormones on respiratory pathway is also attempted.     

Phytochrome‐mediated regulation of plant respiration and photorespiration

The expression of genes encoding various enzymes participating in photosynthetic and respiratory metabolism is regulated by light via the phytochrome system. While many photosynthetic, photorespiratory and some respiratory enzymes, such as the rotenone‐insensitive NADH and NADPH dehydrogenases and the alternative oxidase, are stimulated by light, succinate dehydrogenase, subunits of the pyruvate dehydrogenase complex, cytochrome oxidase and fumarase are inhibited via the phytochrome mechanism. The effect of light, therefore, imposes limitations on the tricarboxylic acid cycle and on the mitochondrial electron transport coupled to ATP synthesis, while the non‐coupled pathways become activated. Phytochrome‐mediated regulation of gene expression also creates characteristic distribution patterns of photosynthetic, photorespiratory and respiratory enzymes across the leaf generating different populations of mitochondria, either enriched by glycine decarboxylase (in the upper part) or by succinate dehydrogenase (in the bottom part of the leaf).     

Flavin fluorescence dynamics and photoinduced electron transfer in Escherichia coli glutathione reductase.

Time-resolved polarized flavin fluorescence was used to study the active site dynamics of Escherichia coli glutathione reductase (GR). Special consideration was given to the role of Tyr177, which blocks the access to the NADPH binding-site in the crystal structure of the enzyme. By comparing wild-type GR with the mutant enzymes Y177F and Y177G, a fluorescence lifetime of 7 ps that accounts for approximately 90% of the fluorescence decay could be attributed to quenching by Y177. Based on the temperature invariance for this lifetime, and the very high quenching rate, electron transfer from Y177 to the light-excited isoalloxazine part of flavin adenine dinucleotide (FAD) is proposed as the mechanism of flavin fluorescence quenching. Contrary to the mutant enzymes, wild-type GR shows a rapid fluorescence depolarization. This depolarization process is likely to originate from a transient charge transfer interaction between Y177 and the light-excited FAD, and not from internal mobility of the flavin, as has previously been proposed. Based on the fluorescence lifetime distributions, the mutants Y177F and Y177G have a more flexible protein structure than wild-type GR: in the range of 223 K to 277 K in 80% glycerol, both tyrosine mutants mimic the closely related enzyme dihydrolipoyl dehydrogenase. The fluorescence intensity decays of the GR enzymes can only be explained by the existence of multiple quenching sites in the protein. Although structural fluctuations are likely to contribute to the nonexponential decay and the probability of quenching by a specific site, the concept of conformational substates need not be invoked to explain the heterogeneous fluorescence dynamics.     

Acute exercise induces biphasic increase in respiratory mRNA in skeletal muscle

Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) promotes the expression of oxidative enzymes in skeletal muscle. We hypothesized that activation of the p38 MAPK (mitogen-activated protein kinase) in response to exercise was associated with exercise-induced PGC-1α and respiratory enzymes expression and aimed to demonstrate this under the physiological level. We subjected mice to a single bout of treadmill running and found that the exercise induced a biphasic increase in the expression of respiratory enzymes mRNA. The second phase of the increase was accompanied by an increase in PGC-1α protein, but the other was not. Administration of SB203580 (SB), an inhibitor of p38 MAPK, suppressed the increase in PGC-1α expression and respiratory enzymes mRNA in both phases. These data suggest that p38 MAPK is associated with the exercise-induced expression of PGC-1α and biphasic increase in respiratory enzyme mRNAs in mouse skeletal muscle under physiological conditions.     

Inorganic types of fermentation and anaerobic respirations in the evolution of energy-yielding metabolism

We proposed long ago the following sequence as one of the main pathways in the evolution of energy-yielding metabolism: fermentation→nitrate fermentation→nitrate respiration→oxygen respiration. In the present report our concept is presented in a more general form: (1) fermentation→ →(2) fermentation with H₂ release→(3) inorganic types of fermentation→(4) anaerobic respirations →(5) oxygen respiration, based upon recent biological and physical information. The energy-yielding efficiency increased gradually together with the evolution. (2) is characterized by the participation of ferredoxin, (3) by the establishment of electron transfer chain, and (4) by the participation of cytochrome and oxidative phosphorylation. The close relationship between the primary structure of ferredoxins of anaerobic bacteria and that of a cytochrome (cytochromec ₃) was demonstrated. It reveals that the transition from inorganic types of fermentation to anaerobic respirations was direct and accompanied by the transition from ferredoxins to cytochromes, and it further supports our concept that the cytochrome system, and consequently the oxidative phosphorylation, were induced at this evolutionary step. Our concept based upon biological observations is consistent with a physical theory recently proposed by M. Shimizu.     

Differences in the distribution of energy-metabolizing enzymes in rat brain regions

The activities of several enzymes of glucose metabolism (glycolytic and tricarboxylic acid pathways) in four different regions of rat brain (cerebellum, medulla oblongata and pons, cerebral cortex and diencephalon) have been studied. Statistical differences were found in the activities of all the enzymes analyzed in the four regions, except in the case of the soluble hexokinase and pyruvate kinase. The changes observed in citrate synthase activity may account for physiological differences in those areas related to myelin formation and energy metabolism. Cerebral cortex and diencephalon showed enzyme activities which were generally greater than those of the cerebellum and medulla oblongata and pons. The results obtained lend support to the concept of a differential energy metabolism in brain regions.     

Detoxifying function of cytochrome c against oxygen toxicity

The detoxifying function of cytochrome c to scavenge O2-* and H2O2 in mitochondria is confirmed experimentally. A model of respiratory chain operating with two electron-leak pathways mediated by cytochrome c is suggested to illustrate the controlling mechanism of ROS level in mitochondria. A concept of mitochondrial radical metabolism is suggested based on the two electron-leak pathways mediated by cytochrome c are metabolic routes of O2-*. Two portions of oxygen consumption can be found in mitochondria. The main portion of oxygen consumed in the electron transfer of respiratory chain is used in ATP synthesis, while a subordinate part of oxygen consumed by the leaked electrons contributes to ROS generation. It is found that the amount of electron leak of respiratory chain is not fixed, but varies with age and pathological states. The models of respiratory chain operating with two cytochrome c-mediated electron-leak pathways and a radical metabolism of mitochondria accompanied with energy metabolism are helpful to comprehend the pathological problems caused by oxygen toxicity.     

Regulation of enzymes and isoenzymes of carbohydrate metabolism in the yeast Saccharomyces cerevisiae

An electrophoretic method has been devised to investigate the changes in the enzymes and isoenzymes of carbohydrate metabolism, upon adding glucose to derepressed yeast cell. (i) Of the glycolytic enzymes tested, enolase II, pyruvate kinase and pyruvate decarboxylase were markedly increased. This increase was accompanied by an overall increase in glycolytic activity and was prevented by cycloheximide, an inhibitor of protein synthesis. (ii) In contrast, respiratory activity decreased after adding glucose. This decrease was clearly shown to be the result of repression of respiratory enzymes. A rapid decrease within a few minutes of adding glucose, by analogy with the so-called ‘Crabtree effect’, was not observed in yeast. (iii) The gluconeogenic enzymes, fructose-1,6-bisphosphatase and malate dehydrogenase, which are inactivated after adding glucose, showed no significant changes in electrophoretic mobilities. Hence, there was no evidence of enzyme modifications, which were postulated as initiating degradation. However, it was possible to investigate cytoplasmic and mitochondrial malate dehydrogenase isoenzymes separately. Synthesis of the mitochondrial isoenzyme was repressed, whereas only cytoplasmic malate hydrogenase was subject to glucose inactivation.     

Modeling of pharmacokinetics of cocaine in human reveals the feasibility for development of enzyme therapies for drugs of abuse

A promising strategy for drug abuse treatment is to accelerate the drug metabolism by administration of a drug-metabolizing enzyme. The question is how effectively an enzyme can actually prevent the drug from entering brain and producing physiological effects. In the present study, we have developed a pharmacokinetic model through a combined use of in vitro kinetic parameters and positron emission tomography data in human to examine the effects of a cocaine-metabolizing enzyme in plasma on the time course of cocaine in plasma and brain of human. Without an exogenous enzyme, cocaine half-lives in both brain and plasma are almost linearly dependent on the initial cocaine concentration in plasma. The threshold concentration of cocaine in brain required to produce physiological effects has been estimated to be 0.22±0.07 μM, and the threshold area under the cocaine concentration versus time curve (AUC) value in brain (denoted by AUC2(∞)) required to produce physiological effects has been estimated to be 7.9±2.7 μM·min. It has been demonstrated that administration of a cocaine hydrolase/esterase (CocH/CocE) can considerably decrease the cocaine half-lives in both brain and plasma, the peak cocaine concentration in brain, and the AUC2(∞). The estimated maximum cocaine plasma concentration which a given concentration of drug-metabolizing enzyme can effectively prevent from entering brain and producing physiological effects can be used to guide future preclinical/clinical studies on cocaine-metabolizing enzymes. Understanding of drug-metabolizing enzymes is key to the science of pharmacokinetics. The general insights into the effects of a drug-metabolizing enzyme on drug kinetics in human should be valuable also in future development of enzyme therapies for other drugs of abuse.     

Protections of SMND-309, a novel derivate of salvianolic acid B,on brain mitochondria contribute to injury amelioration in cerebral ischemia rats

SMND-309, a novel compound named (2E)-2-{6-[(E)-2-carboxylvinyl]-2,3-dihydroxyphenyl}-3-(3,4-dihydroxyphenyl) propenoic acid, is a new derivate of salvianolic acid B. The present study was conducted to investigate whether SMND-309 has a protective effect on brain injury after focal cerebral ischemia, and if it did so, to investigate its effects on brain mitochondria. Adult male SD rats were subjected to middle cerebral artery occlusion (MCAO) by bipolar electro-coagulation. Behavioral tests and brain patho-physiological tests were used to evaluate the damage to central nervous system. Origin targets including mitochondria production of reactive oxygen species, antioxidant potentia, membrane potential, energy metabolism, mitochondrial respiratory enzymes activities and mitochondria swelling degree were evaluated. The results showed that SMND-309 decreased neurological deficit scores, reduced the number of dead hippocampal neuronal cells in accordance with its depression on mitochondria swelling degree, reactive oxygen species production, improvements on mitochondria swelling, energy metabolism, membrane potential level and mitochondrial respiratory chain complex activities. All of these findings indicate that SMND-309 exerted potent neuroprotective effects in the model of permanent cerebral ischemia, contributed to its protections on brain mitochondrial structure and function.     

Membrane-mediated control of hepatic beta-hydroxy-beta-methylglutaryl-coenzyme A reductase.

Previously we [Sabine & James (1976) Life Sci. 18, 1185--1192] proposed that 'the activity of hepatic beta-hydroxy-beta-methylglutaryl-coenzyme A reductase is critically regulated by the fluidity of its supporting microsomal membrane'. In the present work we examined further this concept of membrane-mediated control, with respect to the specific hypothesis that such control might function as a common mechanism both for the co-ordinated regulation of other enzymes affected by cholesterol feeding and also for the subcellular integration of the several physiological factors known to influence this enzyme's activity. Contrary to earlier expectations, this hypothesis now appears not to hold. We report here that, under those conditions of short-term cholesterol feeding that affected the reductase, a variety of other microsomal enzymes did not display membrane-function interactions, i.e. neither enzymes involved in cholesterol metabolism and also affected by cholesterol feeding (cholesterol 7 alpha-hydroxylase), nor those involved in cholesterol metabolism and not affected by cholesterol feeding (hydroxymethylglutaryl-CoA hydrolase, acyl-CoA:cholesterol acyltransferase), nor those not directly involved in cholesterol metabolism at all (glucose 6-phosphatase). Furthermore, we observed no evidence for the operation of membrane-mediated control of the reductase in other situations known to influence its activity, i.e. starvation, diurnal rhythm, the very early stages of cholesterol feeding and various manipulations in vitro.     

Coordination of photosynthetic and respiratory metabolism in Chlorella vulgaris UAM 101 in the light

In Chlorella vulgaris UAM 101, the presence of glucose altered the photosynthetic and respiratory metabolism in the light. When glucose was added to the growth medium, an increase in the cellular level of enzymes involved in glucose oxidation, namely glucose-6-P dehydrogenase (EC 1.1.1.49) and NAD⁺-glyceraldehyde-3-P dehydrogenase (EC 1.2.1.12), was observed. Glucose also enhanced respiratory O₂ consumption. In addition, CO₂ released by glucose oxidation was refixed in photosynthesis. The presence of glucose also affected photosynthesis. Phosphoribulokinase (EC 2.7.1.19) and NADP⁺-dependent glyceraldehyde-3-P dehydrogenase (EC 1.2.1.13), two regulatory enzymes of the reductive pentose phosphate cycle, were increased by glucose. However, Rubisco (EC 4.1.1.39) activity of these cells was lower than that of autotrophic cells. Despite these alterations, the photosynthetic O₂ evolution was not significantly inhibited by glucose. On the other hand, an increase in the cytosolic NADP⁺-glyceraldehyde-3-P dehydrogenase (EC 1.2.1.9) that is involved in obtaining reducing power for anabolic processes was observed. The CO₂ levels in the growth medium did not significantly affect the cellular level of enzymes measured in this work, except those involved in biosynthetic pathways. These data suggest that the effect of glucose on photosynthesis and respiration can be explained by alteration of the cellular level of photosynthetic enzymes and respiratory substrates, respectively.     

Functions,structures, and applications of cellobiose 2-epimerase and glycoside hydrolase family 130 mannoside phosphorylases

Carbohydrate isomerases/epimerases are essential in carbohydrate metabolism, and have great potential in industrial carbohydrate conversion. Cellobiose 2-epimerase (CE) reversibly epimerizes the reducing end d-glucose residue of β-(1→4)-linked disaccharides to d-mannose residue. CE shares catalytic machinery with monosaccharide isomerases and epimerases having an (α/α)₆-barrel catalytic domain. Two histidine residues act as general acid and base catalysts in the proton abstraction and addition mechanism. β-Mannoside hydrolase and 4-O-β-d-mannosyl-d-glucose phosphorylase (MGP) were found as neighboring genes of CE, meaning that CE is involved in β-mannan metabolism, where it epimerizes β-d-mannopyranosyl-(1→4)-d-mannose to β-d-mannopyranosyl-(1→4)-d-glucose for further phosphorolysis. MGPs form glycoside hydrolase family 130 (GH130) together with other β-mannoside phosphorylases and hydrolases. Structural analysis of GH130 enzymes revealed an unusual catalytic mechanism involving a proton relay and the molecular basis for substrate and reaction specificities. Epilactose, efficiently produced from lactose using CE, has superior physiological functions as a prebiotic oligosaccharide.     

Patterns of enzyme activities in the gills of the catfish Heteropneustes fossilis (Bloch) exposed to the anionactive detergent Na-alkyl-benzenesulphonate (LAS)

The effect of sodium-alkyl-benzenesulphonate (LAS) on the activity of the respiratory enzymes of the gills of Heteropneustes fossilis (Bloch) was investigated. After 48 h exposure, the main injury to gills was the progressive separation of the lamellae from their vascular components. The enzymes of the aerobic part of the metabolism showed a decrease in activity, whereas the activity of lactate dehydrogenase was strongly increased, thus indicating that LAS has a high potential to interfere with aerobic mechanisms; however, the mode of action of it has yet to be clearly defined.     

Comparative study of effect of cortical nucleus of amygdala and pyriform cortex on activity of bulbar respiratory neurons in cats

Comparative microelectrophysiological study of character and peculiarities of effects of the cortical nucleus of amygdala and of the periamygdalar area of pyriform cortex on impulse activity was performed on the same single functionally identified respiratory medullar neurons. A high reactivity of bulbar respiratory neurons to stimulation is established in both studied limbic structures. There is established the qualitatively different character of their response reactions at stimulation of the cortical amygdala nucleus and the periamygdalar cortex. The cortical amygdala nucleus has been shown to produce both facilitating and inhibitory effects (with predominance of the activating one) on activity of medullar respiratory neurons (without topographical orderliness). The effect of periamygdalar cortex at stimulation of various parts was characterized by topographic differentiation. The suppressing reactions of neurons in the majority of cases were recorded at stimulation of the rostral area of periamygdalar cortex, whereas the excitatory reactions-at stimulation of its caudal part. Functional organization of respiratory control of the studied limbic system structures is discussed.