Cholinergic systems in muscle and brain in vitamin E-deficient rats

Rats fed a vitamin E-deficient diet for 7–8 weeks postweaning showed no change in brain weight or the activity in brain of various enzymes involved in neurotransmitter synthesis and metabolism. Body and muscle weights were markedly reduced. Muscle choline acetyltransferase and acetylcholinesterase activities were significantly elevated on a protein basis, but the total amount of choline acetyltransferase/muscle was essentially normal and total acetylcholinesterase activity was slightly reduced. Total carnitine acetyltransferase and butyrylcholinesterase activities were markedly decreased. The results are quite different from those found in hereditary murine muscular dystrophy and suggest a myogenic etiology for the vitamin E-deficiency-induced condition.     

METABOLISM OF THE PHOSPHOINOSITIDES IN GUINEA-PIG BRAIN SYNAPTOSOMES

Abstract— The subcellular distribution of a number of enzymes concerned with inositol lipid metabolism has been studied in sub-fractions of disrupted guinea-pig brain synaptosomes. The enzymes were CDP-diglyceride: inositol phosphatidate transferase, phospha-tidylinositol kinase, diphosphoinositide kinase, diphosphoinositide phosphomonoesterase and diphosphoinositide diesterase. The distribution of phosphatidylinositol kinase in sub-fractions from water-treated synaptosomes was compared with that of other plasma membrane enzymes. After partial solubilization of synaptosomes by Triton X-100 the activities of phosphatidylinositol kinase and several other enzymes were examined.
Distribution of phosphatidylinositol kinase closely resembled that of acetylcholinesterase in sub-fractions of synaptosomes. Both enzymes appeared to be localised in the outer membrane of the synaptosome. CDP-diglyceride: inositol phosphatidate transferase was present in all types of synaptosomal membrane. All three enzymes concerned with diphosphoinositide metabolism were found in the cytoplasm of the synaptosome.     

Calorie restriction in the rotifer Brachionus plicatilis enhances hypoxia tolerance in association with the increased mRNA levels of glycolytic enzymes

The rotifer Brachionus plicatilis shows a typical sigmoid growth curve, where calorie restriction (CR) and hypoxia are thought to be introduced at high population density in the stationary phase. CR may induce a shift from aerobic to anaerobic metabolism in this stationary phase, possibly contributing to an increased hypoxia tolerance. This study was undertaken to investigate the effect of CR on hypoxia tolerance at the molecular level. When rotifers were cultured under CR (fed every second day) or fed ad libitum (AL), and subsequently exposed to hypoxia, those in the CR group had a higher survival rate than their AL counterparts. We then cloned cDNAs encoding three glycolytic enzymes, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), enolase (ENO), and phosphoglycerate mutase (PGM) and compared their accumulated mRNA levels between CR and AL rotifers at ages of 1–8 days by quantitative real-time PCR. The CR group showed significantly higher mRNA levels of GAPDH and ENO than their AL counterparts. Furthermore, rotifers in the stationary phase showed higher mRNA levels of these enzymes than those in the exponential growth phase. These results suggest that CR induces anaerobic metabolism, which possibly contributes to population stability under hypoxia in the stationary phase.     

Metabolic alteration in tumorigenesis

Altered metabolism in cancer was first discovered by Otto Warburg early last century.Although the Warburg Effect has been widely used in tumor detection,relatively little progress had been made in mechanistic understanding of cancer metabolism in the subsequent eight decades.Genetic studies have recently identified mutations in human cancer targeting multiple enzymes involved in intermediate metabolism.One emerging mechanism common to these mutant enzymes is the accumulation of a metabolite that alters the epigenetic control.     

Cytokinin biosynthesis and interconversion

To maintain hormone homeostasis, the rate of cytokinin biosynthesis, interconversion, and degradation is regulated by enzymes in plant cells. Cytokinins can be synthesized via direct (de novo) or indirect (tRNA) pathways. In the de novo pathway, a cytokinin nucleotide is synthesized from 5'-AMP and isopentenyl pyrophosphate; a key enzyme which catalyzes this synthesis has been isolated from plant tissues, slime mold, and some microorganisms. Studies on the in vitro synthesis of the isopentenyl side chain of cytokinin in tRNA demonstrated that the isopentenyl group was derived from mevalonate, and turnover of the cytokinin-containing tRNA may serve as a minor source of free cytokinins in plant cells. The interconversion of cytokinin bases, nucleosides and nucleotides is a major feature of cytokinin metabolism; and enzymes that regulate the interconversion have been identified. The N⁶-side chain and purine moiety of cytokinins are often modified and some of the enzymes involved in the modifications have been isolated. Most of the cytokinin metabolites have been characterized but very few enzymes regulating their metabolism have been purified to homogeneity. It remains a significant challenge to isolate plant genes involved in the regulation of cytokinin biosynthesis, interconversion and degradation.     

Bacterial metabolism of polychlorinated biphenyls

Microbial metabolism is responsible for the removal of persistent organic pollutants including PCBs from the environment. Anaerobic dehalogenation of highly chlorinated congeners in aquatic sediments is an important process, and recent evidence has indicated that Dehalococcoides and related organisms are predominantly responsible for this process. Such anaerobic dehalogenation generates lower chlorinated congeners which are easily degraded aerobically by enzymes of the biphenyl upper pathway (bph). Initial biphenyl 2,3-dioxygenases are generally considered the key enzymes of this pathway which determine substrate range and extent of PCB degradation. These enzymes have been subject to different protein evolution strategies, and subsequent enzymes have been considered as crucial for metabolism. Significant advances have been made regarding the mechanistic understanding of these enzymes, which has also included elucidation of the function of BphK glutathione transferase. So far, the genomes of two important PCB-metabolizing organisms, namely Burkholderia xenovorans strain LB400 and Rhodococcus sp. strain RHA1, have been sequenced, with the rational to better understand their overall physiology and evolution. Genomic and proteomic analysis also allowed a better evaluation of PCB toxicity. Like all bph gene clusters which have been characterized in detail, particularly in strains LB400 and RHA1, these genes were localized on mobile genetic elements endowing single strains and microbial communities with a high flexibility and adaptability. However, studies show that our knowledge on enzymes and genes involved in PCB metabolism is still rather fragmentary and that the diversity of bacterial strategies is highly underestimated. Overall, metabolism of biphenyl and PCBs should not be regarded as a simple linear pathway, but as a complex interplay between different catabolic gene modules.     

The time course of development of acetylcholinesterase and choline acetyltransferase in Drosophila melanogaster

Twenty stages in the life cycle of Canton-S, a normal strain of Drosophila melanogaster, were investigated for protein content and the activities of choline acetyltransferase and acetylcholinesterase, enzymes associated with the metabolism of acetylcholine. The maximum protein content is reached at the prepupal stage. Specific activities of choline acetyltransferase and acetylcholinesterase were high in the larval stages and again in the mature fly. The activities of these enzymes expressed on a per fly basis were compared with the activities of other enzymes, previously published by other workers, expressed on the same basis. The developmental pattern of acetylcholinesterase and choline acetyltransferase differed from the patterns exhibited by the other enzymes described earlier. It was possible to relate the different enzyme patterns to known changes occurring in the life cycle of Drosophila melanogaster.Supported by grants from the National Multiple Sclerosis Society (347), and from the National Institutes of Health (FR 05471; NB 08864 and NB 08014).     

Bridging epigenetics and metabolism

Recent research suggests that chromatin-modifying enzymes are metabolic sensors regulating gene expression. Epigenetics is linked to metabolomics in response to the cellular microenvironment. Specific metabolites involved in this sensing mechanism include S-adenosylmethionine, acetyl-CoA, alphaketoglutarate and NAD⁺. Although the core metabolic pathways involving glucose have been emphasized as the source of these metabolites, the reprogramming of pathways involving non-essential amino acids may also play an important role, especially in cancer. Examples include metabolic pathways for glutamine, serine and glycine. The coupling of these pathways to the intermediates affecting epigenetic regulation occurs by “parametabolic” mechanisms. The metabolism of proline may play a special role in this parametabolic linkage between metabolism and epigenetics. Both proline degradation and biosynthesis are robustly affected by oncogenes or suppressor genes, and they can modulate intermediates involved in epigenetic regulation. A number of mechanisms in a variety of animal species have been described by our laboratory and by others. The challenge we now face is to identify the specific chromatin-modifying enzymes involved in coupling of proline metabolism to altered reprogramming of gene expression.     

Effects of 5-(3,3-dimethyl-1-triazeno) imidazole-4-carboxamide [NSC 45388, DTIC] on neuroblastoma cells in culture.

The effects of 5-(3,3-dimethyl-1-triazeno) imidazole-4-carboximide (DTIC) on morphological and biochemical parameters of differentiation were studied in mouse neuroblastoma cells in culture. DTIC (10 μg/ml) did not induce formation of neurites in the cells but inhibited cell division, and produced a marked increase in cell size and in activity of three enzymes (tyrosine hydroxylase, choline acetyltransferase and acetylcholinesterase) involved in neurotransmitter metabolism. These effects were apparently not related to an increase in the intracellular level of cyclic AMP.     

A cytochemical study of the effect of tuftsin on the enzyme activity in functionally different formations of the brain in rats]

Quantitative cytochemical methods in functionally different rat brain formations (sensomotor cortex, visual cortex, nucleus caudatus, hippocampus) showed the peculiarities of the effect of tuftsin on the activity of some enzymes (the oxidative, neurotransmitter and protein metabolism enzymes) 15 min and 3 days after its single administration. No changes of activity of neurotransmitter metabolism enzymes (monoamine oxidase, acetylcholinesterase) were registered cytochemically. The specificity of the neuro-tropical effect of tuftsin on protein (activity of aminopeptidase and acid phosphatase) and oxidative (activity of glutamate dehydrogenase and glucose-6-phosphate dehydrogenase) metabolism in different functional brain systems is discussed.     

Mechanisms of the protease-induced aggregation of human platelets

The role of phospholipase C (an enzyme involved in the metabolism of inositol-containing phospholipids), cyclooxygenase and lipoxygenase (the enzymes of arachidonic acid metabolism), and adenylate cyclase and phosphodiesterase (the enzymes of cyclic adenosine 3,5-monophosphate (cAMP) metabolism) in the mechanisms of the aggregation of human platelets induced by the serine protease in low concentrations (thrombin 0.5 mkg per ml, trypsin 1 mkg per ml, and alpha-chymotrypsin 10 mkg per ml) have been investigated with the use of the inhibitor analysis. The effect of neomycin sulfate (phospholipase C inhibitor), indometacin (cyclooxygenase inhibitor), and nordihydrogvayaretic acid (lipoxygenase inhibitor) on protease-induced increase in the content of calcium cations in platelet plasma has been studied. The results of the inhibitor analysis indicated that the enzymes of metabolism of inositol-containing phospholipids, arachidonic acid, and cAMP are involved in the mechanisms of the protease-induced platelet aggregation. The increase in the content of calcium ions, associated with the protease-induced activation of phospholipase C, in cytoplasm may play an important role in the mechanisms of platelet aggregation.     

Critical enzymes involved in endocannabinoid metabolism

Investigations of the pathways involved in the metabolism of endocannabinoids have grown exponentially in recent years following the discovery of cannabinoid receptors (CB) and their endogenous ligands, such as anandamide (AEA) and 2-arachidonoylglycerol (2-AG). The in vivo biosynthesis of AEA has been shown to occur through several pathways mediated by N-acylphosphatidylethanolamide-phospholipase D (NAPE-PLD), a secretory PLA(2) and PLC. 2-AG, a second endocannabinoid is generated through the action of selective enzymes such as phosphatidic acid phsophohydrolase, diacylglycerol lipase (DAGL), phosphoinositide-specific PLC (PI-PLC) and lyso-PLC. A putative membrane transporter or facilitated diffusion is involved in the cellular uptake or release of endocannabinoids. AEA is metabolized by fatty acid amidohydrolase (FAAH) and 2-AG is metabolized by both FAAH and monoacylglycerol lipase (MAGL). The author presents an integrative overview of current research on the enzymes involved in the metabolism of endocannabinoids and discusses possible therapeutic interventions for various diseases, including addiction.     

Some enzymes of general metabolism in the latex of Papaver somniferum

Enzymes of general metabolism have been determined in the latex of Papaver somniferum in an attempt to elucidate further the nature of the 1000 g130 min organelles and their role in alkaloid biogenesis. A number of enzymes involved in the glyoxylic acid and tricarboxylic acid cycles have been found, namely, aconitase, isocitrate dehydrogenase, succinate dehydrogenase, fumarase, malate dehydrogenase and isocitrate lyase. Two enzymes of glycolysis, namely, pyruvate kinase and lactate dehydrogenase, as well as enzymes associated with peroxisomes (glyoxylate reductase, catalase) and lysosomes (arylesterase, acid phosphatase) have been studied. Finally, some enzymes previously reported as occurring in poppy seedlings have been investigated, namely peroxidase, glutamate—oxaloacetate and glutamate-pyruvate transaminases, together with phenylalanine, tyrosine, DOPA and glutamic acid decarboxylases.     

Synthesis and biological evaluation of indoloquinoline alkaloid cryptolepine and its bromo-derivative as dual cholinesterase inhibitors

Alkaloids have always been a great source of cholinesterase inhibitors. Numerous studies have shown that inhibiting acetylcholinesterase as well as butyrylcholinetserase is advantageous, and have better chances of success in preclinical/ clinical settings. With the objective to discover dual cholinesterase inhibitors, herein we report synthesis and biological evaluation of indoloquinoline alkaloid cryptolepine (1) and its bromo-derivative 2. Our study has shown that cryptolepine (1) and its 2-bromo-derivative 2 are dual inhibitors of acetylcholinesterase and butyrylcholinesterase, the enzymes which are involved in blocking the process of neurotransmission. Cryptolepine inhibits Electrophorus electricus acetylcholinesterase, recombinant human acetylcholinesterase and equine serum butyrylcholinesterase with IC₅₀ values of 267, 485 and 699 nM, respectively. The 2-bromo-derivative of cryptolepine also showed inhibition of these enzymes, with IC₅₀ values of 415, 868 and 770 nM, respectively. The kinetic studies revealed that cryptolepine inhibits human acetylcholinesterase in a non-competitive manner, with ki value of 0.88 µM. Additionally, these alkaloids were also tested against two other important pathological events of Alzheimer’s disease viz. stopping the formation of toxic amyloid-β oligomers (via inhibition of BACE-1), and increasing the amyloid-β clearance (via P-gp induction). Cryptolepine displayed potent P-gp induction activity at 100 nM, in P-gp overexpressing adenocarcinoma LS-180 cells and excellent toxicity window in LS-180 as well as in human neuroblastoma SH-SY5Y cell line. The molecular modeling studies with AChE and BChE have shown that both alkaloids were tightly packed inside the active site gorge (site 1) via multiple π-π and cation-π interactions. Both inhibitors have shown interaction with the allosteric “peripheral anionic site” via hydrophobic interactions. The ADME properties including the BBB permeability were computed for these alkaloids, and were found within the acceptable range.     

Microbial starch-binding domain

Glucosidic bonds from different non-soluble polysaccharides such as starch, cellulose and xylan are hydrolyzed by amylases, cellulases and xylanases, respectively. These enzymes are produced by microorganisms. They have a modular structure that is composed of a catalytic domain and at least one non-catalytic domain that is involved in polysaccharide binding. Starch-binding modules are present in microbial enzymes that are involved in starch metabolism; these are classified into several different families on the basis of their amino acid sequence similarities. Such binding domains promote attachment to the substrate and increase its concentration at the active site of the enzyme, which allows microorganisms to degrade non-soluble starch. Fold similarities are better conserved than sequences; nevertheless, it is possible to notice two evolutionary clusters of microbial starch-binding domains. These domains have enormous potential as tags for protein immobilization, as well as for the tailoring of enzymes that play a part in polysaccharide metabolism.     

Tyrosine metabolic enzymes from insects and mammals：A comparative perspective

Differences in the metabolism of tyrosine between insects and mammals present an interesting example of molecular evolution. Both insects and mammals possess finetuned systems of enzymes to meet their specific demands for tyrosine metabolites; however, more homologous enzymes involved in tyrosine metabolism have emerged in many insect species. Without knowledge of modem genomics, one might suppose that mammals, which are generally more complex than insects and require tyrosine as a precur sor for important catecholamine neurotransmitters and for melanin, should possess more enzymes to control tyrosine metabolism. Therefore, the question of why insects actually possess more tyrosine metabolic enzymes is quite interesting. It has long been known that insects rely heavily on tyrosine metabolism for cuticle hardening and for innate immune responses, and these evolutionary constraints are likely the key answers to this question. In terms of melanogenesis, mammals also possess a high level of regulation; yet mam malian systems possess more mechanisms for detoxification whereas insects accelerate pathways like melanogenesis and therefore must bear increased oxidative pressure. Our research group has had the opportunity to characterize the structure and function of many key proteins involved in tyrosine metabolism from both insects and mammals. In this mini review we will give a brief overview of our research on tyrosine metabolic enzymes in the scope of an evolutionary perspective of mammals in comparison to insects.     

LOCALIZATION, SOLUBILIZATION AND PROPERTIES GANGLIOSIDE TRANSFERASES IN RAT BRAIN OF N-ACETYLGALACTOSAMINYL AND GALACTOSYL GANGLIOSIDE TRANSFERASES IN RAT BRAIN

Abstract— The subcellular distributions of UDP-N-acetylgalactosamine: G_M3 N-acetyl-galactosaminyl transferase and UDP-galactose: G_M2 galactosyl transferase, two enzymes involved in the biosynthesis of gangliosides, were determined in the 7-day-old rat brain by means of synaptosomal fractionation techniques. The enzymes were located on the synaptic membranes and appeared to be closely associated with gangliosides and acetylcholinesterase. Solubilization of the transferase enzymes from the microsomal particles was achieved and differed from the solubilization of acetylcholinesterase and of the total membrane protein. Competition studies suggest that the ^N-acetylgalactosaminyl transferase involved in the formation of G_M2 from G_M3 is different from the N-acetylgalactosaminyl transferase involved in the formation of GalNAoGal-Glc-ceramide from Gal-Glc-ceramide, whereas in contrast, both the formation of G_M1 from G_M2 and of Gal-GalNAc-Gal-Glcceramide from GalNAc-Gal-Glc-ceramide appear to be catalysed by the same galactosyl transferase.