A glutamate dehydrogenase-based method for the assay of -glutamic acid: Formation of pyridine nucleotide fluorescent derivatives

A method for the quantitation of -glutamic acid in the picomole range was developed by finding conditions which allowed the production of NADH by the action of the -glutamate dehydrogenase (EC 1.4.1.3) and its subsequent transformation to a highly fluorescent derivative. The method measures linearly glutamate from 250 pmol to 5 nmol. For its simplicity and low cost it is ideally suited to the assay of a large number of samples within a single working day. Its application to the determination of regional glutamate levels in the rat brain, as well as to the measurement of ornithine aminotransferase (EC 2.6.1.13) activity from several tissues is described. The results are similar to those obtained by different methodologies in several laboratories, but the present method offers additional advantages.     

Plant glutathione peroxidases

Oxidative stress in plants causes the induction of several enzymes, including superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11) and glutathione reductase (EC 1.6.4.2). The first two are responsible for converting superoxide to H₂O₂ and its subsequent reduction to H₂O, and the third is involved in recycling of ascorbate. Glutathione peroxidases (GPXs, EC 1.11.1.9) are a family of key enzymes involved in scavenging oxyradicals in animals. Only recently, indications for the existence of this enzyme in plants were reported. Genes with significant sequence homology to one member of the animal GPX family, namely phospholipid hydroperoxide glutathione peroxidase (PHGPX), were isolated from several plants. Cit-SAP, the protein product encoded by the citrus csa gene, which is induced by salt-stress, is so far the only plant PHGPX that has been isolated and characterized. This protein differs from the animal PHGPX in its rate of enzymatic activity and in containing a Cys instead of selenocysteine (Sec) as its presumed catalytic residue. The physiological role of Cit-SAP and its homologs in other plants is not yet known.     

Assay of L-3-hydroxyacyl-coenzyme A dehydrogenase with substrates of different chain lengths

A method for assaying L-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) which permits rate measurements with L-3-hydroxyacyl-CoA substrates of various chain lengths at physiological pH is described. The method is based on a coupled assay system in which 3-ketoacyl-CoA compounds formed by the dehydrogenase are cleaved by 3-ketoacyl-CoA thiolase (EC 2.3.1.16) in the presence of CoASH. The advantages of this assay method are its irreversibility and elimination of product inhibition. The assay procedure was used to determine the kinetic parameters (Km, Vmax) of pig heart L-3-hydroxyacyl-CoA dehydrogenase with several substrates of various chain lengths. The data obtained show the enzyme to be most active with medium-chain substrates whereas Km values for medium-chain and long-chain substrates are almost equal but much lower than those previously reported.     

Pathogenicity ofBacillus cereus for insects

The conditions of phospholipase C (EC 3.1.4.3) production by a strain ofBacillus cereus and a method of its preparation are described. The molecular weight of the enzyme as determined by gel filtration is 24,000. Comparison of several characteristics of the enzyme (pH optimum, immunospecificity, mobility in discelectrophoresis) with that fromClostridium perfringens shows them to be different molecules.     

Properties of two phosphatases and a cyclic phosphodiesterase of Salmonella typhimurium.

The properties of three phosphatases from Salmonella typhimurium have been examined. A cyclic 2',3'-nucleotide phosphodiesterase (EC 3.1.4.d) hydrolyzes cyclic 2',3'-purine and -pyrimidine nucleotides, as well as 3'-mononucleotides, and has a pH optimum of about 7.5. It requires divalent cations for activity and has a molecular weight of 67,000. Acid hexose phosphatase (EC 3.1.2.2) possesses activity towards hexose phosphates as well as other sugar phosphates. The enzyme is apparently a dimer of 37,000-dalton subunits. Nonspecific acid phosphatase (EC 3.1.3.2) hydrolyzes a variety of phosphate esters, including nucleotides and sugar phosphates. The enzyme also hydrolyzes the phosphoric anhydride bonds of pyrophosphate and nucleotides. Michaelis constants of the nonspecific acid phosphatase for several of its substrates are in the 1 to 2 mM range. Nonspecific acid phosphatase is a dimer of 27,000-dalton subunits.     

Participation of the endothelium in the development of the atherosclerotic plaque

In the past decade, initiated by the response-to-injury hypothesis of Ross and Glomset, the endothelium has been implicated in atherogenesis but as a passive participant--more involved through its absence than its presence. The hypothesis stated that endothelial desquamation due to an undefined injury led to platelet adhesion to the exposed basement membrane, and infiltration of serum lipoproteins. The subsequent release from the platelet alpha-granule of a potent smooth muscle cell mitogen and chemoattractant--the platelet-derived growth factor (PDGF)--was postulated to cause the intimal proliferative response that is known to be important in atherosclerotic plaque development. Recent evidence from several laboratories indicates that the endothelium has the potential to play a more active role in plaque development than simply contributing to pathological sequelae resulting from the loss of the nonthrombogenic surface provided by the endothelium. First, the endothelial cell (EC) is the site of attachment, and possibly activation, of blood-borne monocytes which enter the vessel wall as an early event in experimental atherogenesis. We have obtained in vitro evidence that the expression of monocyte binding sites on the surface of EC is a regulatable process and that increased EC turnover and certain exogenous agents acting on EC cause increased monocyte adhesion. Similar events may be responsible for focal adhesion of monocytes to the endothelium in vivo following hypercholesterolemia. Secondly, EC in culture are capable of chemically modifying low density lipoprotein (LDL) by a free radical oxidation process that renders the LDL toxic to proliferating cells and recognizable to the scavenger receptor of monocyte-derived macrophages. Thus, by oxidation of LDL, the EC have the potential to play an active role both in the formation of lipid-laden foam cells and in the accumulation of necrotic tissue which are hallmarks of the atherosclerotic lesion. Thirdly, cultured EC have been recently shown to secrete multiple mitogens for cultured smooth muscle cells. One of these mitogens appears to be closely related, if not identical, to PDGF using the criteria of receptor binding and biochemical and immunological similarity. Production of growth factors by EC is a regulatable process that is stimulated by exogenous agents such as endotoxin and phorbol esters which cause severe injury to cultured EC. Such a regulatory mechanism may participate in the in vivo proliferation of vascular SMC during the atherosclerotic process.(ABSTRACT TRUNCATED AT 400 WORDS)     

Preparation of chick brain synaptosomes and synaptosomal membranes.

A method is described for the preparation of synaptosomes and synaptosomal membranes from chicken brain. Procedures for isolating rat synaptosomal membranes could not be used directly; several modifications of existing procedures are reported. Purity of the subcellular and subsynaptosomal fractions was monitored by electron microscopy and measurements of ferrocytochrome c: oxygen oxidoreductase (EC 1.9.3.)), monoamine: oxygen oxidoreductase (deaminating) EC 1.4.3.4), rotenone-insensitive NADH: cytochrome c oxidoreductase (EC 1.6.99.3), NADPH: cytochrome c oxidoreductase (EC 1.6.99.1), orthophosphoric monoester phosphohydrolase (EC 3.1.3.2), ATP phosphohydrolase (EC 3.6.1.4), and levels of RNA. Microsomes are the main contaminant of the synaptosomal membrane fraction. Mitochondrial and lysosomal enzymes occur in lesser amounts. No myelin contamination was observed. Marker enzymes for contaminants suggest that these synaptosomal membranes are as pure as membranes described by others, and the specific activity of a neuronal membrane marker, (Na+ -K+)-activated ATPase, is as high as other preparations. Levels of this enzyme in the membrane fraction are enriched 13-fold over homogenate ATPase levels.     

Structural characterization and dynamics of globotetraosylceramide in vascular endothelial cells under TNF-α stimulation

In several vascular inflammatory reactions (i.e. immunity and thrombosis) inflammatory mediators lead to the activation of vascular endothelial cells (EC). To date, a number of functional molecules induced on the surface of activated-EC have been identified. We report here that Globotetraosylceramide (Gb4), a glycosphingolipid expressed in EC, is a novel inducible molecule on EC activated by TNF-α. The cell surface expression of Gb4 is increased in a time-dependent manner under TNF-α stimulation, which shows distinct expression kinetics of major proteins induced by TNF-α on EC. MALDI-TOF-MS analysis revealed that the enhanced Gb4 predominantly contains C24:0 fatty acid in the ceramide moiety. Isolated caveolae/lipid raft-enriched detergent insoluble membrane domains in activated-EC predominantly contain this molecular species of Gb4. Gb4 containing C16:0 fatty acid in the ceramide moiety, which is known to constitute the major species of Gb4 in plasma, is also found as a major molecular species in EC. These observations indicate that Gb4, especially with very long fatty acid, is enhanced in EC during its inflammatory reaction, and suggest the potential utility of Gb4 as a biomarker for monitoring inflammation status of EC involving its related diseases.     

Stimulation of differentiation of several murine embryonal carcinoma cell lines by retinoic acid

Retinoic acid stimulates several murine embryonal carcinoma (EC) cell lines, even those previously considered to be incapable of differentiating, to give rise to cell types distinguishable from the parental phenotype in morphology, production of plasminogen activator and surface protein properties. Retinoic acid promotes these changes over a range of low concentrations (10⁻⁹–10⁻⁵ M) which are generally non-toxic to the cells. The effects are clearly demonstrated when EC cells are aggregated prior to exposure to retinoic acid. It is concluded that the observed phenotypic alterations induced by retinoic acid reflect differentiation of the EC cells since non-EC cell characteristics are maintained by cloned cells several generations after retinoic acid is removed from the cultures. Our studies suggest that although retinoic acid stimulates the conversion of EC cells to differentiated derivatives, it does not influence the direction of differentiation. Furthermore, the effectiveness of retinoic acid in stimulating differentiation of EC cells from lines such as Nulli-SCC1 raises the question of whether true ‘nullipotent’ EC lines really exist.     

Inactivation and reactivation of phosphoprotein phosphatase of rabbit skeletal muscle. Role of ATP and divalent metal ions.

The regulatory mechanism of a phosphoprotein phosphatase (EC 3.1.3.16), which is considered to catalyze the dephosphorylation reaction of several phosphoproteins (glycogen synthetase-D (EC 2.4.1.11), phospho-form of phosphorylase b kinase (EC 2.7.1.38), phosphohistone and phosphorylase a (EC 2.4.1.1)), was studied with partially purified preparations from rabbit skeletal muscle. Time- and temperature-dependent inactivation and reactivation of phosphohistone phosphatase, as well as phosphorylase phosphatase (EC 3.1.3.17), were observed on pre0incubation of the enzyme(s) with ATP, and subsequent incubation with divalent metal ions (Mg2+, Mn2+, or Co2+) without any change of molecular size. Manganese, however, instantly restored the activity of the ATP-inactivated enzyme, and increased the maximal velocity of the enzyme while decreasing its affinity to phosphorylase a. However, the metal ion inhibited the reactivated enzyme competively with respect to phosphorylase a. It is suggested that phosphoprotein phosphatase(s) is a metalloenzyme, and that ATP results in a conformational change of the enzyme protein in such a way that a metal ion can be easily released due to the chelating effect of ATP, or incorporated (in the presence of excess metal ions) into the enzyme protein.     

Exploration of DNA methylation markers for diagnosis and prognosis of patients with endometrial cancer

The accurate diagnosis of endometrial cancer (EC) holds great promise for improving its treatment choice and prognosis prediction. This work aimed to identify diagnostic biomarkers for differentiating EC tumors from tumors in other tissues, as well as prognostic signatures for predicting survival in EC patients. We identified 48 tissue-specific markers using a cohort of genome-wide methylation data from three common gynecological tumors and their corresponding normal tissues. A diagnostic classifier was constructed based on these 48 CpG markers that could predict cancerous versus normal tissue with an overall correct rate of 98.3% in the entire repository. Fifteen CpG markers associated with the overall survival (OS) and development of EC were also identified based on the methylation patterns of the EC samples. A prognostic model that aggregated these prognostic CpG markers was established and shown to have a higher discriminative ability to distinguish EC patients with an elevated risk of mortality than the FIGO staging system and several other clinical prognostic variables. This study presents the utility of DNA methylation in identifying biomarkers for the diagnosis and prognosis of EC and will help improve our understanding of the underlying mechanisms involved in the development of EC.     

Enzyme activities in the sheep placenta during the last three months of pregnancy

In order to assess the extent to which metabolism within the sheep placenta may influence the transfer of metabolites between mother and foetus at different stages of gestation the activities of enzymes concerned with some aspects of carbohydrate, amino acid and ketone body metabolism were determined in placental cotyledons resected from ewes during the last three months of pregnancy.The activities of pyruvate kinase (EC 2.7.1.40), lactate dehydrogenase (EC 1.1.1.27), malate dehydrogenase (EC 1.1.1.37), ATP citrate (pro-3S)-lyase (EC 4.1.3.8), citrate (si)-synthase (EC 4.1.3.7), acetyl-Co A synthetase (EC 6.2.1.1), acetyl-CoA acetyltransferase (EC 2.3.1.9) and 3-keto acid CoA-transferase (EC 2.8.3.5) per gram wet weight cotyledon do not change during the period studied. The activities of alanine aminotransferase (EC 2.6.1.2), aspartate aminotransferase (EC 2.6.1.1), isocitrate dehydrogenase (NADP⁺) (EC 1.1.1.42), ornithine-oxoacid aminotransferase (EC 2.6.1.13) and 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) show an increase in activity between the third and fourth months of pregnancy whilst the activities of arginase (EC 3.5.3.1) and possibly pyruvate carboxylase (EC 6.4.1.1) show an increase in activity between the fourth and final months of pregnancy. Ornithine decarboxylase (EC 4.1.1.17) activity declines to one tenth of its activity during this later period. The absence of detectable activities of phosphoenolpyruvate carboxykinase (EC 4.1.1.32) and ornithine carbamoyltransferase (EC 2.1.3.3) indicate that gluconeogenesis and urea synthesis from ammonia do no occur in the sheep placenta.It appears that the ability of the placenta to metabolise several substrates is achieved by the time the placenta reaches its maximum size at approximately 90 days.     

Isolation of intact chloroplasts and other cell organelles from spinach leaf protoplasts

Freshly prepared spinach leaf protoplasts were gently ruptured by mechanical shearing followed by sucrose density gradient centrifugation to separate constituent cell organelles. The isolation of intact Class I chloroplasts (d = 1.21) in high yield, well separated from peroxisomes and mitochondria, was evidenced by the specific localization of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39), NADP triose-P dehydrogenase (EC 1.2.1.9), and carbonic anhydrase (EC 4.2.1.1) in the fractions. A clear separation of chloroplastic ribosomes from the soluble cytoplasmic ribosomes was also demonstrated by the band patterns of constituent RNA species in the polyacrylamide gel electrophoresis. Localization of several enzyme activities specific to leaf peroxisomes, e.g. catalase (EC 1.11.1.6), glycolate oxidase (EC 1.1.3.1), glyoxylate reductase (EC 1.1.1.26), glutamate glyoxylate aminotransferase (EC 2.6.1.4), serine glyoxylate aminotransferase, and alanine glyoxylate aminotransferase (EC 2.6.1.12) in the peroxisomal fractions (d = 1.25), was demonstrated. Overall results show the feasibility of the method for the isolation of pure organelle components in leaf tissues.     

Prediction of missing enzyme genes in a bacterial metabolic network. Reconstruction of the lysine-degradation pathway of Pseudomonas aeruginosa

The metabolic network is an important biological network which consists of enzymes and chemical compounds. However, a large number of metabolic pathways remains unknown, and most organism-specific metabolic pathways contain many missing enzymes. We present a novel method to identify the genes coding for missing enzymes using available genomic and chemical information from bacterial genomes. The proposed method consists of two steps: (a) estimation of the functional association between the genes with respect to chromosomal proximity and evolutionary association, using supervised network inference; and (b) selection of gene candidates for missing enzymes based on the original candidate score and the chemical reaction information encoded in the EC number. We applied the proposed methods to infer the metabolic network for the bacteria Pseudomonas aeruginosa from two genomic datasets: gene position and phylogenetic profiles. Next, we predicted several missing enzyme genes to reconstruct the lysine-degradation pathway in P. aeruginosa using EC number information. As a result, we identified PA0266 as a putative 5-aminovalerate aminotransferase (EC 2.6.1.48) and PA0265 as a putative glutarate semialdehyde dehydrogenase (EC 1.2.1.20). To verify our prediction, we conducted biochemical assays and examined the activity of the products of the predicted genes, PA0265 and PA0266, in a coupled reaction. We observed that the predicted gene products catalyzed the expected reactions; no activity was seen when both gene products were omitted from the reaction.     

Enzyme catalyzed hydrolysis of inorganic pyrophosphate. Current view of problems in characterization of PP1-phosphohydrolytic activity associated with alkaline phosphatases (EC 3.1.3.1).

A survey of the hydrolytic activity of alkaline phosphatase (EC 3.1.3.1) reveals that PP1, like phosphomonoesters, can serve as substrate in vitro. This pp1-phosphohydrolytic activity can be distinguished from PP1-phosphohydrolytic activities of inorganic pyrophosphatases (EC 3.6.1.1) and glucose-6-phosphatase (EC 3.1.3.9) by several criteria. Discrimination among these hydrolytic enzymes is possible by their dependence on variation of pH and of magnesium to PP1 ratios in the assay solutions. The true substrates and modifiers are not simply PP1 and magnesium, but the equilibrium species in mixtures of these two. The physiological significance of each of the three enzymes is not predictable from their differential efficiency as catalysts of PP1-hydrolysis in vitro.     

Purification and partial characterization of the exotoxin of Corynebacterium ovis.

1. The toxin from Corynebacterium ovis, a phospholipase D (sphingomyelin phosphodiesterase D) that acts on 2-lysophosphatidylcholine and sphingomyelins, was purified by about 400-fold to homogeneity as judged by several criteria. [The EC number of the toxin (EC 3.1.4.41) has been allotted by the Nomenclature Committee of IUB, but has not yet been published.] 2. A new assay method performed in vitro, based on inhibition by the toxin of erythrocyte lysis by staphylococcal beta-haemolysin, was developed to facilitate the purification. 3. The toxin was found to be a basic (pI9.1) glycoprotein of mol.wt. 14,500 +/- 1,000. 4. The amino acid composition of the toxin was highly reminiscent of that of collagen, since it contained hydroxyproline, hydroxylysine and a high proportion of glycine, but preliminary tests showed no other similarities to collagen or proteins with similar compositions.     

Endocannabinoid lipids and mediated system: implications for alcoholism and neuropsychiatric disorders

Several natural lipids have emerged as candidate modulators of central nervous system (CNS) functions. Fatty acid amides and their coupled signaling pathways are known to regulate several physiological and behavioral processes. Recent studies from our laboratory and others also have implicated endogenous marijuana-like brain constituents, endocannabinoids (ECs), and cannabinoid-1 (CB1) receptors in the neural circuitry that mediate drug addiction and neuropsychiatric disorders. Neuroadaptation to chronic ethanol (EtOH) has been shown to involve changes in the EC system. These changes include alterations in the synthesis of EC, their precursors, as well as density and coupling efficacy of CB1 receptors. The evidence for the participation of the EC system in the pathophysiology of various neuropsychiatric disorders is just beginning to evolve. It is of great interest to explore the components of EC system in different areas of the CNS for further understanding of its role in health and disease. This article presents a comprehensive overview of the currently available literature pertaining to the role of the EC system in alcoholism, schizophrenia, depression and/or suicide.