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.

Structural characteristics of a giant tropical liana and its mode of canopy spread in an alien environment

To circumvent the practical difficulties in research on tropical rainforest lianas in their natural habitat due to prevailing weather conditions, dense camouflaging vegetation and problems in transporting equipment for experimental investigations, Entada pursaetha DC (syn. Entada scandens Benth., Leguminosae) was grown inside a research campus in a dry subtropical environment. A solitary genet has attained a gigantic size in 17 years, infesting crowns of semi-evergreen trees growing in an area roughly equivalent to 1.6 ha. It has used aerially formed, cable-like stolons for navigating and spreading its canopy across tree gaps. Some of its parts which had remained unseen in its natural habitat due to dense vegetation are described. The attained size of this liana in a climatically different environment raises the question as to why it is restricted to evergreen rainforests. Some research problems for which this liana will be useful are pointed out.

Mode of Coordination and Chemical Reactivity of Alpha-Imine Hydrasone Ligands

Interaction of nickel(I1) and copper(I1) complexes of 4,9-dimethy1-5,8-diazadodeca-4,8diene-2,1 ldione, Ni(baen) and 4,6,9-trimethyl-5, 8diazadodeca-4,8-diene-2,ll-dione, Ni(bapn), with arene diazonium chlorides in buffered solutions of methanol yielded metal derivatives of glyoxaliminearylhydrazones. This typical electrophilic addition at the 3-carbon of the complex occurs owing to the pseudo aromatic behaviour of the chelate ring. A mechanism which predicts the attack of the diazonium cation through the coordination shell of the metal is well documented from the available experimental evidences. The chemical reactivity of a few complexes with a single residual non-substituted y-carbon is reasonably manifested by their reaction with phenyl isocyanate.

Effect of configuration of the inhibitors on the mode of binding to the enzyme, thermolysin

Preferred conformations of the competitive inhibitors glycyl-L-phenylalanine and glycyl-D-phenylalanine and their mode of binding to thermolysin have been studied. The difference in configuration is shown to affect significantly the mode of binding to thermolysin. Gly-D-Phe prefers to enter the active site in the global minimum conformation whereas Gly-L-Phe may enter in a higher energy conformation. Moreover, D-enantiomer is shown to have a better fit than the L-counterpart in the active site.

Characterization of a toxin from Parthenium hysterophorus and its mode of excretion in animals

Fractionation of methanolic extracts of air dried aerial parts ofParthenium resulted in the isolation of a toxic constituent which was identified as parthenin, the major sesquiterpene lactone from the weed. The LD50 (minimal lethal dose required to cause 50% mortality) for parthenin in rats was 42 mg/kg body weight. When [3H]-parthenin was given orally or by intravenous administration, radioactivity appeared in the milk of lactating laboratory and dairy animals. Tissue distribution of radioactivity revealed that maximum label was detectable in kidneys.

Negative resistance mode of forced oscillations of a string

A new mode of driven nonlinear vibrations of a stretched string is investigated with reference to conditions of existence, properties, and regions of stability. It is shown that this mode exhibits negative resistance properties at all frequencies and driving force amplitudes. Discovery of this mode helps to fill certain gaps in the theory of forced nonlinear vibrations of strings.

Mode of inhibition of mitochondrial energy transduction by chlorophenoxyisobutyrate

1. a-p-Chlorophenoxyisobutyric acid, the ethyl ester of which is widely used as an antihypercholesterolaemic drug, is an inhibitor of energy-transfer reactions in isolated rat liver mitochondria. 2. The compound at lower concentrations (<4.0mmol/mg of mitochondrial protein) inhibits state 3 oxidation, stimulates state 4 oxidation, abolishes respiratory control and stimulates the latent adenosine triphosphatase activity of mitochondria. The inhibition imposed on state 3 oxidation is relieved by dinitrophenol. 3. At higher concentrations it inhibits coupled phosphorylation as well as dinitrophenol-stimulated adenosine triphosphatase activity. The inhibition of state 3 oxidation under these conditions is not reversed by uncouplers. 4. The three coupling sites of phosphorylation exhibit differential susceptibility to inactivation by this compound. Coupled phosphorylation at the first site is abolished at a drug concentration of 3.0mmol/mg of protein. The third site is inactivated when the concentration of the drug reaches 5.0mmol/mg of protein. The second site is the most refractory and drug concentrations of the order of 10.0mmol/mg of protein are required effectively to inhibit phosphorylation at this site. 5. The compound also inhibits ATP-dependent reversal of electron transport as well as the adenosine triphosphatase activity in submitochondrial particles. 6. The oxidation of NADH and succinate in these particles is not inhibited. 7. These properties indicate that the compound acts as an `inhibitory uncoupler' of energy-transfer reactions in isolated mitochondria.

Characterization of Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease, reveals a unique mode of DNA-binding, helical distortion and cleavage, compared to a canonical LAGLIDADG homing endonuclease

Mycobacterium leprae, which has undergone reductive evolution leaving behind a minimal set of essential genes, has retained intervening sequences in four of its genes implicating a vital role for them in the survival of the leprosy bacillus. A single in-frame intervening sequence has been found embedded within its recA gene. Comparison of M. leprae recA intervening sequence with the known intervening sequences indicated that it has the consensus amino acid sequence necessary for being a LAGLIDADG-type homing endonuclease. In light of massive gene decay and function loss in the leprosy bacillus, we sought to investigate whether its recA intervening sequence encodes a catalytically active homing endonuclease. Here we show that the purified M. leprae RecA intein (PI-MleI) binds to cognate DNA and displays endonuclease activity in the presence of alternative divalent cations, Mg2+ or Mn2+. A combination of approaches including four complementary footprinting assays such as DNase I, Cu/phenanthroline, methylation protection and KMnO4, enhancement of 2-aminopurine fluorescence and mapping of the cleavage site revealed that PI-MleI binds to cognate DNA flanking its insertion site, induces helical distortion at the cleavage site and generates two staggered double-strand breaks. Taken together, these results implicate that PI-MleI possess a modular structure with separate domains for DNA target recognition and cleavage, each with distinct sequence preferences. From a biological standpoint, it is tempting to speculate that our findings have implications for understanding the evolution of LAGLIDADG family of homing endonucleases

Mode of action of antitumour antibiotics: Spectrophotometric studies on the interaction of chromomycin A3 with DNA and chromatin of normal and neoplastic tissue

The binding of chromomycin A3, an antitumour antibiotic, to various DNA and chromatin isolated from mouse and rat liver, mouse fibrosarcoma and Yoshida ascites sarcoma cells was studied spectrophotometrically at 29°C in 10−2 M Tris-HCl buffer, pH 8.0, containing small amounts of MgCl2 (4.5 · 10−5−25 · 10−5 M). An isobestic point at 415 nm was observed when chromomycin A3 was gradually titrated with Image and its spectrum shifted towards higher wavelength. The rates and extent of these spectral changes were found to be dependent on the concentration of Mg2+. The change in absorbance at 440 nm was used to calculate apparent binding constant (Ka p M−1) and sites per nucleotide (n) from Scatchard plots for various DNA and chromatins. As expected, values of n for chromatin (0.06–0.10) were found to be lower than that found for corresponding DNA (0.10–0.15). Apparently no such correlation exists between binding constants (Ka p M−1 · 10−4) of DNA (6.4–11.2) and of chromatin (3.1–8.3), but Ka p M−1 of chromatin isolated from mouse fibrosarcoma and Yoshida ascites sarcoma are 1.5–3 times higher than that found for mouse and rat liver chromatin. These differences may be taken to indicate structural difference in nucleoprotein complexes caused by neoplasia. The relevance of this finding to tumour suppressive action of chromomycin A3 is discussed.

Mode of Action of a Purified Antitumor Protein from the Proteinaceous Crystal of Bacillus thuringiensis subsp. thuringiensis on Yoshida Ascites Sarcoma Cells

A purified antitumor protein from the proteinaceous crystal of Bacillus thuringiensis subsp. thuringiensis inhibits the growth of Yoshida ascites sarcoma both in vivo and in vitro. Exogenous respiration of the tumor cells was unaffected by the protein at a concentration as high as 500 µg/ml. The antitumor protein inhibits the uptake and incorporation of labeled precursors into macromolecules. However, the ratio of incorporation over uptake is not affected by the protein. Further, the protein brings about the leakage of 260-nm-absorbing material, proteins, and 32P-labeled cellular constituents from the Yoshida ascites sarcoma cells. The results show that the action of the antitumor protein appears to alter the cellular permeability of the tumor cells.

Indoleacetaldoxime hydro-lyase (4.2.1.29). III. Further studies on the nature and mode of action of the enzyme

Further purification of indoleacetaldoxime (IAOX) hydro-lyase from Gibberella fujikuroi by DEAE-cellulose chromatography is described. The purified enzyme was activated by dehydroascorbic acid (DHA), ascorbic acid (AA), and pyridoxal phosphate (PALP) and was inhibited by thiol compounds and thiol reagents including phenylthiocyanate. Ferrous ions but not ferric ions activated the purified enzyme. The enzyme was activated by dihydrofolic acid but inhibited by tetrahydrofolic acid. Phenylacetaldoxime, a competitive inhibitor, afforded partial protection of the enzyme from the action of N-ethylmaleimide suggesting the involvement of a thiol function at the active site or substrate-binding site. The inhibition of the enzyme by 2,3-dimercaptopropanol was reversed by DHA, PALP, or frozen storage. KCN inhibition of the enzyme was reversed by PALP. NaBH4 reduction of the purified enzyme in the presence of PALP gave an active enzyme which was further activated by PALP or DHA but not by ferrous ions. These results suggested a "structural" role for PALP in the activity of IAOX hydro-lyase. Dilute solutions of the purified enzyme, obtained during DEAE-cellulose chromatography and concentrated using sucrose, showed enhanced activity upon frozen storage and thawing. The increase in activity of the enzyme during certain culture conditions, the activation and inhibition of the enzyme by several unrelated compounds, and the effect of freezing indicate that IAOX hydro-lyase is probably a metabolically regulated enzyme with a structure composed of subunits.

Effect of mode of rolling on development of texture and microstructure in two-phase (alpha plus beta) brass

The evolution of microstructure and texture during deformation of two-phase (alpha + beta) brass was studied for different initial microstructure and texture. The deformation processing involved unidirectional and multi-step cross-rolling. The bulk textures were determined by measuring the pole figures and calculating the orientation distribution function ODF for both alpha (fcc) and beta (bcc) phases, while the microstructure and other microstructural parameters were measured through optical microscopy and scanning electron microscopy with electron back scatter diffraction (SEM-EBSD). Results indicate that textures developed after unidirectional rolling and multi-step cross-rolling are significantly different. The variation in initial texture had a pronounced effect on the development of texture in the alpha phase during subsequent deformation. (C) 2010 Elsevier B.V. All rights reserved.

Mode of action of antitumour antibiotics-III: Modulation of permeability of nuclear membrane in the presence of the antibiotics

The binding characteristics of the antibiotics to nuclei and their effect on the permeability of nuclear membrane with respect to histones and ribonucleic acids have been investigated. The binding constant for chromomycin A3 was found to be 1.4 × 104M?1 and number of binding sites was equal to 3.48 ± 1.08 × 1012 molecules/nuclei. The antibiotic chromomycin A3 enhanced the uptake of lysine-rich histone, actinomycin D decreased the uptake and ethidium bromide had no effect. Chromomycin A3 also enhanced the release of acid insoluble fraction containing RNA from the nuclei, actinomycin D and ethidium bromide inhibited the release of acid insoluble fraction containing RNA. The relevance of this finding to the role of nuclear envelope in understanding the mechanism of action of the antibiotic has been discussed.