Effect of beta-n-oxalyl-L-alpha, beta-diaminopropionic acid on glutamate uptake by synaptosomes

THE unusual amino acid beta-N-oxalyl-L-alpha, beta-diaminopropionic acid (ODAP), isolated from the seeds of Lathyrus sativus is a potent neurotoxin1−3. It produces biochemical changes in the brain typical of an excitant amino acid and is implicated in the aetiology of human neurolathyrism caused by eating the seeds of L. sativus 4−6. It may act as a glutamate antagonist: ODAP inhibits glutamate oxidation7 possibly by inhibiting glutamate uptake in bovine brain mitochondria; it also acts as a competitive inhibitor of glutamate uptake in certain strains of yeast8, and a similar process might occur at the synaptic level. Any effect of ODAP on glutamate uptake at synapses is significant in view of the neurotransmitter function of glutamate, which seems to be neuroexcitory as well as neurotoxic9−12. But Balcar and Johnston13 have shown with rat brain slices that ODAP does not inhibit the glutamate uptake by the high affinity system.

Oxidase-peroxidase enzymes of Datura innoxia. Oxidation of formylphenylacetic acid ethyl ester

An enzyme system from Datura innoxia roots oxidizing formylphenylacetic acid ethyl ester was purified 38-fold by conventional methods such as (NH4)2SO4 fractionation, negative adsorption on alumina Cy gel and chromatography on DEAE-cellulose. The purified enzyme was shown to catalyse the stoicheiometric oxidation of formylphenylacetic acid ethyl ester to benzoylformic acid ethyl ester and formic acid, utilizing molecular O2. Substrate analogues such as phenylacetaldehyde and phenylpyruvate were oxidized at a very low rate, and formylphenylacetonitrile was an inhilating agents, cyanide, thiol compounds and ascorbic acid. This enzyme was identical with an oxidase-peroxidase isoenzyme. Another oxidase-peroxidase isoenzyme which separated on DEAE-chromatography also showed formylphenylacetic acid ethyl ester oxidase activity, albeit to a lesser extent. The properties of the two isoenzymes of the oxidase were compared and shown to differ in their oxidation and peroxidation properties. The oxidation of formylphenylacetic acid ethyl ester was also catalysed by horseradish peroxidase. The Datura isoenzymes exhibited typical haemoprotein spectra. The oxidation of formylphenylacetic acid ethyl ester was different from other peroxidase-catalysed reactions in not being activated by either Mn2+ or monophenols. The oxidation was inhibited by several mono- and poly-phenols and by catalase. A reaction mechanism for the oxidation is proposed.

Oxidase-peroxidase enzymes of Datura innoxia. Oxidation of reduced nicotinamide-adenine dinucleotide in the presence of formylphenylacetic acid ethyl ester

The oxidase-peroxidase from Datura innoxia which catalyses the oxidation of formylphenylacetic acid ethyl ester to benzoylformic acid ethyl ester and formic acid was also found to catalyse the oxidation of NADH in the presence of Mn2+ and formylphenylacetic acid ethyl ester. NADH was not oxidized in the absence of formylphenylacetic acid ethyl ester, although formylphenylacetonitrile or phenylacetaldehyde could replace it in the reaction. The reaction appeared to be complex and for every mol of NADH oxidized 3-4 g-atoms of oxygen were utilized, with a concomitant formation of approx. 0.8 mol of H2O2, the latter being identified by the starch-iodide test and decomposition by catalase. Benzoylformic acid ethyl ester was also formed in the reaction, but in a nonlinear fashion, indicating a lag phase. In the absence of Mn2+, NADH oxidation was not only very low, but itself inhibited the formation of benzoylformic acid ethyl ester from formylphenylacetic acid ethyl ester. A reaction mechanism for the oxidation of NADH in the presence of formylphenylacetic acid ethyl ester is proposed.

A new mode of ring cleavage of 2,3-dihydroxybenzoic acid in Tecoma stans (L.). Partial purification and properties of 2,3-dihydroxybenzoate 2,3-oxygenase.

2,3-Dihydroxybenzoic acid has been shown to be oxidized via the 3-oxoadipate pathway in the leaves of Tecoma stans. The formation of 2-carboxy-cis,cis-muconic acid, a muconolactone, 3-oxoadipic acid and carbon dioxide during its metabolism has been demonstrated using an extract of Tecoma leaves. The first reaction of the pathway, viz., the conversion of 2,3-dihydroxybenzoate to 2-carboxy-cis,cis-muconic acid has been shown to be catalysed by an enzyme designated as 2,3-dihydroxybenzoate 2,3-oxygenase. The enzyme has been partially purified and a few of its properties studied. The enzyme is very labile with a half-life of 3--4 h. It is maximally active with 2,3-dihydroxybenzoate as the substrate and does not exhibit any activity with catechol, 4-methyl catechol, 3,4-dihydroxybenzoic acid, etc. However, 2,3-dihydroxy-p-toluate and 2,3-dihydroxy-p-cumate are also oxidized by the enzyme by about 38% and 28% respectively, compared to 2,3-dihydroxybenzoate. Sulfhydryl reagents inhibit the enzyme reaction and the inhibition can be prevented by preincubation of the enzyme with the substrate. Substrate also affords protection to the enzyme against thermal inactivation. Sulfhydryl compounds strongly inhibit the reaction and the inhibition cannot be prevented by preincubation of the enzyme with its substrates. Data on the effect of metal ions as well as metal chelating agents suggest that copper is the metal cofactor of the enzyme. Evidence is presented which suggests that iron may not be participating in the overall catalytic mechanism.

Factors influencing kinetic and equilibrium behaviour of sodium ion exchange with strong acid cation resin

This study reports an investigation of the ion exchange treatment of sodium chloride solutions in relation to use of resin technology for applications such as desalination of brackish water. In particular, a strong acid cation (SAC) resin (DOW Marathon C) was studied to determine its capacity for sodium uptake and to evaluate the fundamentals of the ion exchange process involved. Key questions to answer included: impact of resin identity; best models to simulate the kinetics and equilibrium exchange behaviour of sodium ions; difference between using linear least squares (LLS) and non-linear least squares (NLLS) methods for data interpretation; and, effect of changing the type of anion in solution which accompanied the sodium species. Kinetic studies suggested that the exchange process was best described by a pseudo first order rate expression based upon non-linear least squares analysis of the test data. Application of the Langmuir Vageler isotherm model was recommended as it allowed confirmation that experimental conditions were sufficient for maximum loading of sodium ions to occur. The Freundlich expression best fitted the equilibrium data when analysing the information by a NLLS approach. In contrast, LLS methods suggested that the Langmuir model was optimal for describing the equilibrium process. The Competitive Langmuir model which considered the stoichiometric nature of ion exchange process, estimated the maximum loading of sodium ions to be 64.7 g Na/kg resin. This latter value was comparable to sodium ion capacities for SAC resin published previously. Inherent discrepancies involved when using linearized versions of kinetic and isotherm equations were illustrated, and despite their widespread use, the value of this latter approach was questionable. The equilibrium behaviour of sodium ions form sodium fluoride solution revealed that the sodium ions were now more preferred by the resin compared to the situation with sodium chloride. The solution chemistry of hydrofluoric acid was suggested as promoting the affinity of the sodium ions to the resin.

Structural studies of analgesics and their interactions. II. The crystal structure of a 1:1 complex between antipyrine and salicyclic acid (salipyrine)

The complex crystallizes in the space group P21/c with four formula units in a unit cell of dimensions a= 12.747, b= 7.416, c= 17.894 A and/3= 90.2 °. The structure has been solved by the symbolic addition procedure using three-dimensional photographic data and refined to an R value of 0.079 for 2019 observed reflexions. The pyramidal nature of the two hetero nitrogen atoms in the antipyrine molecule is inter:nediate between that observed in free antipyrine and in some of its metal complexes. The molecule is more polar than that in crystals of free antipyrine but less so compared with that in metal complexes. In the salicylic acid molecule, the hydroxyl group forms an internal hydrogen bond with one of the oxygen atoms in the carboxyl group. The association between the salicylic acid and the antipyrine molecules is achieved through an intermolecular hydrogen bond with the other carboxyl oxygen atom in the salicylic acid molecule as the proton donor and the carboxyl oxygen atom of the antipyrine molecule as the acceptor.

Temperature dependence of chlorine NQR in 2-chloro 5-nitrobenzoic acid and 4-chloro 3-nitrobenzoic acid

Temperature dependence of chlorine nuclear quadrupole resonance in 2-chloro 5-nitrobenzoic acid and 4-chloro 3-nitrobenzoic acid has been investigated in the region 77° K to room temperature. No phase transition has been observed. The results are analysed to obtain the torsional frequencies and their temperature dependence. A nonlinear temperature dependence is obtained for the torsional frequencies.

The purification and properties of peroxidase in Mycobacterium tuberculosis H37Rv and its possible role in the mechanism of action of isonicotinic acid hydrazide

Peroxidase from Mycobacterium tuberculosis H37Rv was purified to homogeneity. The homogeneous protein exhibits catalase and Y (Youatt's)-enzyme activities in addition to peroxidase activity. Further confirmation that the three activities are due to a single enzyme was accomplished by other criteria, such as differential thermal inactivation, sensitivity to different inhibitors, and co-purification. The Y enzyme (peroxidase) was separated from NADase (NAD+ glycohydrolase) inhibitor by gel filtration on Sephadex G-200. The molecular weights of peroxidase and NADase inhibitor, as determined by gel filtration, are 240000 and 98000 respectively. The Y enzyme shows two Km values for both isoniazid (isonicotinic acid hydrazide) and NAD at low and high concentrations. Analysis of the data by Hill plots revealed that the enzyme has one binding site at lower substrate concentrations and more than one at higher substrate concentration. The enzyme contains 6g-atoms of iron/mol. Highly purified preparations of peroxidases from different sources catalyse the Y-enzyme reaction, suggesting that the nature of the reaction may be a peroxidatic oxidation of isoniazid. Moreover, the Y-enzyme reaction is enhanced by O2. Isoniazid-resistant mutants do not exhibit Y-enzyme, peroxidase or catalase activities, and do not take up isoniazid. The Y-enzyme reaction is therefore implicated in the uptake of the drug.

Specificity in proteinânucleic acid interaction: Solubility study on amino acidânucleoside interaction

The chemical basis of the specificity of proteinnucleic acid interaction, as seen in many biochemical phenomena such as the organization of nucleoprotein complexes (~hro~atin. ribosomes) and gene expression and its regulation, IS not yet understood.A knowledge of such specific interactions is also essential for tracing the chemical evolution of life based an the coupling between protein and nucleic acid and the origin of genetic code [I ,I?].

Effect of allylisopropylacetamide on Nuclear Ribonucleic Acid synthesis in rat liver

The porphyrogenic drug allylisopropylacetamide, a potent inducer of delta-aminolaevulinate synthetase, specifically increases nucleoplasmic RNA synthesis in rat liver. The drug-mediated increase in nucleoplasmic RNA synthesis is blocked by cycloheximide and haemin, which also inhibit the enzyme induction.

Separation of sulfur-containing components of transfer ribonucleic acid on Bio-Gel P-2 and Sephadex G-10 columns

S-Labeled nucleosides of E. coli tRNA and some of the derivatives of thionucleosides were separated on Bio-Gel P-2 and Sephadex G-10 columns employing buffers of low salt concentration and high pH.

Positive associations of serum Perfluoroalkyl substances with uric acid and hyperuricemia in children from Taiwan

To investigate the risk of hyperuricemia in relation to Perfluoroalkyl substances (PFASs) in children from Taiwan, 225 Taiwanese children aged 12-15 years were recruited from 2009 to 2010. Linear and logistic regression models were employed to examine the influence of PFASs on serum uric acid levels. Findings revealed that eight of ten PFASs analyses were detected in > 94% of the participants' serum samples. Multivariate linear regression models revealed that perfluorooctanic acid (PFOA) was positively associated with serum uric acid levels (β=0.1463, p<0.05). Of all the PFASs analyses, only PFOA showed a significant effect on elevated levels of hyperuricemia (aOR=2.16, 95%CI: 1.29-3.61). When stratified by gender, the association between serum PFOA and uric acid levels was only evident among boys (aOR=2.76, 95%CI: 1.37-5.56). In conclusion, PFOA was found to be associated with elevated serum levels of uric acid in Taiwanese children, especially boys. Further research is needed to elucidate these links.

Metabolism of mandelic acid by Neurospora crassa

Preliminary studies on the metabolism of mandelic acid by Neurospora crassa reveal the operation of a pathway for its degradation which involves benzoyl formic acid, benzaldehyde, benzoic acid, 4-hydroxybenzoic acid, and protocatechuic acid as the intermediates. This pathway is different from that followed by bacterial systems and is the same as that observed in Aspergillus niger.

Effect of 2-allyl-2-isopropylacetamide on poly(adenylic acid)-containing ribonucleic acid.

A single administration of 2-allyl-2-isopropylacetamide, a porphyrinogenic drug, enhanced the 32P-labelling of nucleoplasmic as well as cytoplasmic poly(A)-containing RNA in rat liver. The synthesis of total microsomal RNA is only marginally increased under these conditions. The drug enhances the labelling of a variety of cytoplasmic poly(A)-containing RNA species, and this effect is counteracted by the simultaneous administration of haemin. 2-Allyl-2-isopropylacetamide also enhanced the release of RNA from the nucleus to the cytoplasm.

Molecular mechanism for the specific inhibition of reverse transcriptase of rous sarcoma virus by the copper complexes of isonicotinic acid hydrazide

Isonicotinic acid hydrazide (isoniazid), one of the most potent antitubercular drugs, was recently shown, in our laboratory, to form two different complexes with copper, depending upon the oxidation state of the metal ion. Both the complexes have been shown to possess antiviral activity against Rous sarcoma virus, an RNA tumor virus. The antiviral activity of the complexes has been attributed to their ability to inhibit the endogenous reverse transcriptase activity of RSV. More recent studies in our laboratory indicate that both these complexes inhibit both endogenous and exogenous reactions. As low a final concentration as 50 μM of the cupric and the cuprous complexes inhibits the endogenous reaction to the extent of 93 and 75 per cent respectively. Inhibition of the exogenous reaction varies with the templates. The inhibition can be reversed by either β-mercaptoethanol or ethylene-diamine-tetra-acetic acid. The specificity of this inhibition has been ascertained by using a synthetic primer-template, −(dG)not, vert, similar15−(rCm)n, which is highly specific for reverse transcriptases. The inhibition is found to be template specific. The studies carried out, using various synthetic primer-templates, show the inhibition of both the steps of reverse transcription by the copper complexes of isoniazid.