Studies on the cytochemical localization of adenylate-cyclase activity in Dugesia lugubris s.I.

The localization of adenylate-cyclase activity in Dugesia lugubris s.1. has been investigated cytochemically using 5'-adenylyl-imidodiphosphate as substrate. The enzyme was localized in mucous gland cells, in rhabdite cells, in intercellular spaces and also in nerve endings of this planarian. The presence of adenylate-cyclase on the membrane suggests that it might mediate different stimulus-secretion coupling by increasing cyclic AMP synthesis in specialized areas of the planarian.     

偶合反应化学发光分析

偶合反应化学发光分析是扩大化学发光分析应用范围的一种有效途径。本文主要从酶联化学发光分析、无机偶合反应化学发光分析、光化学反应一化学发光分析和电化学反应一化学发光分析四个方面对偶合反应化学发光分析的研究状况进行了评述。     

Administration of D-alanine did not cause increase of D-amino acid oxidase activity in mice.

D-amino acid oxidase (DAAO) activity was not altered in the liver and kidney by oral administration of D-alanine to adult mice. The enzyme was apparently not induced by the enteric microflora either, since the enzyme activity in the liver and kidney of germ-free mice was not different from that of specific-pathogen-free mice. The times of appearance of DAAO activity and of free D-amino acids in the kidney were elucidated using suckling mice. DAAO activity started to increase 7 days after birth, and reached almost the adult level by 28 days. The content of free neutral D-amino acids also increased with age, in a similar fashion. A possible conclusion is that the enzyme activity normally increases during this period, to eliminate the free D-amino acids which have increased with age in the suckling mice. Consequently, the administration of D-alanine had no further effect in increasing enzyme activity.     

Studies on the cytochemical localization of adenylate-cyclase activity inDugesia lugubris s.l.

Summary The localization of adenylate-cyclase activity inDugesia lugubris s.l. has been investigated cytochemically using 5-adenylyl-imidodiphosphate as substrate. The enzyme was localized in mucous gland cells, in rhabdite cells, in intercellular spaces and also in nerve endings of this planarian. The presence of adenylate-cyclase on the membrane suggests that it might mediate different stimulus-secretion coupling by increasing cyclic AMP synthesis in specialized areas of the planarian.Supported in part by a grant from Consiglio Nazionale delle Ricerche, Rome.Acknowledgments. This work was performed with the technical assistence of the laboratory of Electron Microscopy of the University of Perugia.     

Administration of D-alanine did not cause increase of D-amino acid oxidase activity in mice

D-amino acid oxidase (DAAO) activity was not altered in the liver and kidney by oral administration of D-alanine to adult mice. The enzyme was apparently not induced by the enteric microflora either, since the enzyme activity in the liver and kidney of germ-free mice was not different from that of specific-pathogen-free mice. The times of appearance of DAAO activity and of free D-amino acids in the kidney were elucidated using suckling mice. DAAO activity started to increase 7 days after birth, and reached almost the adult level by 28 days. The content of free neutral D-amino acids also increased with age, in a similar fashion. A possible conclusion is that the enzyme activity normally increases during this period, to eliminate the free D-amino acids which have increased with age in the suckling mice. Consequently, the administration of D-alanine had no further effect in increasing enzyme activity.     

Activation of neutrophils by a chemically separated but optically coupled neutrophil population undergoing respiratory burst

Neutrophils from pig blood were used as a model system to investigate the optical communication between cells. It was found that neutrophils stimulated to undergo respiratory burst can activate a second, chemically separated, but optically coupled population of neutrophils. The response of the latter was visualized as a temporary rising of their low-level chemiluminescence and an enhanced generation of superoxide radicals detected by both the reduction of ferricytochrome c and spin trapping. The results provide evidence that a long-range optical coupling of biological significance between living cells exists.     

Action répressive de l'oxygène sur la biosynthèse de la fumarique-réductase d'Aerobacter aerogenes

Summary The cells obtained from anaerobic cultures ofA. aerogenes contain both a hydrogenase and a fumaric reductase, but lack one or several electron transporters necessary for the coupling of the two enzymes. It is possible to measure the fumaric reductase activity of the extracts in the presence of hydrogen, hydrogenase and benzyl viologen, using the Warburg manometric technique. In anaerobic cultures the reductase is an inducible enzyme. In aerobic cultures its formation is strongly repressed by atmospheric oxygen.     

Enzyme immunoassay for arginine vasopressin

Summary A highly sensitive enzyme immunoassay for arginine vasopressin (AVP) was described. The enzyme used was -D-galactosidase fromEscherichia coli, and it was coupled to AVP by using the N-hydroxysuccinimide ester of N-(4-carboxycyclohexylmethyl)-maleimide. The complex was then utilized for the enzyme immunoassay, which could detect 0.5 fmoles (or 0.5 pg) of AVP in the assay tube.     

Energy metabolism and transduction in smooth muscle

Early investigations into the nature of the coupling between energy transduction and metabolism in smooth muscle, particularly from the laboratories of Bülbring and Lundholm, suggested that specific metabolic pathways could independently supply energy for ion transport and actin-myosin interactions. Subsequent work has solidified the concept that oxidative phosphorylation is specifically coupled to tension generation and maintenance, whereas, aerobic glycolysis is not only a vital characteristic of smooth muscle metabolism, but also is likely to be independently coupled to Na-K transport at the plasmalemma. The independence of oxidative and glycolytic metabolism is reflected as a compartmentation of carbohydrate metabolism in the porcine carotid artery. The coupling of these independent metabolic pathways with specific energy utilizing processes, indicates a means by which energy production and transduction can be closely and efficiently regulated. The coupling of glycogenolysis to mitochondrial respiration may have evolved as a direct response to the energetic needs of VSM. That is, the large glycogenolytic response in the initial minutes of stimulation may be necessary to maximize the cellular production of ATP during the presteady state. Likewise, the coupling between aerobic glycolysis and Na-K transport indicates a sensitive and efficient means of coordinating energy metabolism with ion transport at the membrane level. Additionally, the regulation of substrate supply, i.e. glucose transport, also may be closely coordinated with changes in ion transport. One may speculate that alterations in the microenvironment of each compartment can independently regulate intermediary metabolism and therefore allow the cell to quickly and efficiently respond to localized stimuli. Thus, stimulation of Na-K transport could effectively regulate energy production at the membrane level without mobilizing or competing with the energy transduction of other cellular processes. This compartmentation of energy utilization may be highly advantageous, since oxidative metabolism is closely coordinated with mechanical activity and therefore regulation of blood flow. Future investigations will attempt to elucidate which intracellular signals which are responsible for the regulation of these functionally independent compartments of energy metabolism and transduction in VSM. In more general terms, our findings provide a basis from which future questions concerning the regulation of cellular metabolism must be directed. The cellular cytoplasm can no longer be envisioned as a homogeneous compartment, but rather a complex array of functional subcompartments which may be individual     

Quantification of digoxin by enzyme immunoassay: synthesis of a maleimide derivative of digoxigenin succinate for enzyme coupling

Summary We synthesized the m-maleimidobenzoyl derivative of digoxigenin-3-0-succinate (through a p-phenylenediamine bridge) as a hapten derivative directed towards coupling to sulfhydryl groups of -galactosidase. Prepared enzyme conjugate had about 97% of the enzyme labeled with the hapten derivative while retaining full enzyme activity. The enzyme immunoassay for digoxin we prepared showed a maximum sensitivity of 30 pg per assay (c.v.=3%) with minimal cross-reaction with digotoxin (3.8%). Our method for hapten conjugation to -galactosidase is highly efficient and is simple and easily replicated.     

Influence of ethanol on calcium, inositol phospholipids and intracellular signalling mechanisms

Studies have implicated Ca++ in the actions of ethanol at many biochemical levels. Calcium as a major intracellular messenger in the central nervous system is involved in many processes, including protein phosphorylation enzyme activation and secretion of hormones and neurotransmitters. The control of intracellular calcium, therefore, represents a major step by which neuronal cells regulate their activities. The present review focuses on three primary areas which influence intracellular calcium levels; voltage-dependent Ca++ channels, receptor-mediated inositol phospholipid hydrolysis, and Ca++/Mg++-ATPase, the high affinity membrane Ca++ pump. Current research suggests that a subtype of the voltage-dependent Ca++ channel, the dihydropyridine-sensitive Ca++ channel, is uniquely sensitive to acute and chronic ethanol treatment. Acute exposure inhibits, while chronic ethanol exposure increases 45Ca++-influx and [3H]dihydropyridine receptor binding sites. In addition, acute and chronic exposure to ethanol inhibits, then increases Ca++/Mg++-ATPase activity in neuronal membranes. Changes in Ca++ channel and Ca++/Mg++-ATPase activity following chronic ethanol may occur as an adaptation process to increase Ca++ availability for intracellular processes. Since receptor-dependent inositol phospholipid hydrolysis is enhanced after chronic ethanol treatment, subsequent activation of protein kinase-C may also be involved in the adaptation process and may indicate increased coupling for receptor-dependent changes in Ca++/Mg++-ATPase activity. The increased sensitivity of three Ca++-dependent processes suggest that adaptation to chronic ethanol exposure may involve coupling of one or more of these processes to receptor-mediated events.     

Tissue-specific induction of intestinal glutathione S-transferases by alpha beta-unsaturated carbonyl compounds

Glutathione S-transferase activity in rat intestinal mucosa was increased by the injection of alpha beta-unsaturated carbonyl compounds such as phorone and diethylmaleate, but that in the liver and kidney was not. Since the administration of cycloheximide completely blocked the increase of the enzyme activity by phorone, the increase of the activity may be due to de novo synthesis rather than enzyme activation.     

A novel method in enzyme immunoassay: maleimide derivative of hapten for enzyme coupling.

Meta-maleimidobenzoyl derivative of L-thyroxine methyl ester (MBTM) was synthesized and coupled to beta-galactosidase at molar ratio of over 5 to 1. More than 97% of the enzyme was found to be labeled with MBTM. A thyroxine enzyme immunoassay was carried out with sensitivity in 0-10 microgram/100 ml range.     

Regional distribution of thiamin pyrophosphokinase in rat brain

The highest specific activity of thiamin pyrophosphokinase was found in the cerebellum, and lower activity in cerebral cortex and midbrain. The regional difference in the enzyme activity was similar to that in thiamin content and the influx rate in rat brain, suggesting that the enzyme is involved in the thiamin transport.     

Inhibition of sarcolemmal Na, K, Mg ATPase from the guinea pig heart is not compatible with a homogeneous population of non-interacting ouabain receptors

The inhibition of sarcolemmal Na, K, Mg ATPase from the guinea pig heart by ouabain was evaluated with a coupled enzyme assay. Models of negative cooperativity and of two independent receptors fitted the inhibition data equally well. The analysis was not compatible with a homogeneous population of non-interacting ouabain receptors.     

Regional distribution of thiamin pyrophosphokinase in rat brain

Summary The highest specific activity of thiamin pyrophosphokinase was found in the cerebellum, and lower activity in cerebral cortex and midbrain. The regional difference in the enzyme activity was similar to that in thiamin content and the influx rate in rat brain, suggesting that the enzyme is involved in the thiamin transport.     

A new continuous optical assay for isocitrate lyase

Summary A new continuous optical assay method for isocitrate lyase is reported. This is a coupled assay which requires lactate dehydrogenase as an ancillary enzyme. The method yields linear data up to 0.12 units/ml. The assay is also suitable for crude extracts.Acknowledgment. This research was supported by grants of the Italian Ministero della Pubblica Istruzione.     

Superoxide dismutase activity in the Spanish population

Summary Superoxide dismutase is an enzyme that catalyzes the dismutation of superoxide radicals to hydrogen peroxide and molecular oxygen. This superoxide radical is produced by all aerobic cells as a normal metabolic intermediate of molecular oxygen, and is dangerous for the cell because it induces the inactivation of various enzymes, lipid peroxidation and mutations. Superoxide dismutase can therefore be considered as a protective enzyme. The purpose of this work was to determine the level of superoxide dismutase activity in the Spanish population, and to study the factors that influence this activity. The superoxide dismutase activity of 2397 individuals was determined using the method described by Minami and Yoshikawa. The superoxide dismutase activity level in the adult Spanish population was found to be 4.16±0.89 Units/ml of blood. No significant variations with respect to sex were detected. But it was observed that the superoxide dismutase activity level was 9% higher in the young urban Spanish population.     

Superoxide dismutase activity in the Spanish population

Superoxide dismutase is an enzyme that catalyzes the dismutation of superoxide radicals to hydrogen peroxide and molecular oxygen. This superoxide radical is produced by all aerobic cells as a normal metabolic intermediate of molecular oxygen, and is dangerous for the cell because it induces the inactivation of various enzymes, lipid peroxidation and mutations. Superoxide dismutase can therefore be considered as a protective enzyme. The purpose of this work was to determine the level of superoxide dismutase activity in the Spanish population, and to study the factors that influence this activity. The superoxide dismutase activity of 2397 individuals was determined using the method described by Minami and Yoshikawa. The superoxide dismutase activity level in the adult Spanish population was found to be 4.16 +/- 0.89 Units/ml of blood. No significant variations with respect to sex were detected. But it was observed that the superoxide dismutase activity level was 9% higher in the young urban Spanish population.     

Galactosyltransferase of Neurospora

Summary An enzyme, galactosyltransferase, able to catalyze the formation of galactose polymers was detected in cell-free extracts of a wild type strain of Neurospora crassa. Enzyme activity was found in both the supernatant and the particle fractions after centrifugation at 100,000 xg. The enzyme assayed in the 100,000 xg supernatant showed a 4fold difference in specific activity as compared to that found in the particle fraction.Acknowledgment. This work was supported by a grant from the Research Corporation.