Inactivation and reactivation of horseradish peroxidase immobilized by various procedures.

Horseradish peroxidase (HRP) immobilized by coupling the amino acid side chain amino groups or carbohydrate spikes to the matrix has been studied for its resistance to heat, urea-induced inactivation and ability to regain activity after denaturation in order to understand the influence of the nature of immobilization procedure on these processes. The various immobilized preparations were obtained and their properties studied: Sp-HRP was obtained by direct coupling of HRP to cyanogen bromide-activated Sepharose, Sp-NHHRP by coupling periodate oxidized and diamine-treated enzyme to the cyanogen bromide activated Sepharose, SpNH-COHRP by coupling periodate-treated enzyme to amino-Sepharose and SpCon A-HRP by binding of the enzyme on Con A-Sepharose. All the immobilized preparations exhibited higher stability against heat-induced inactivation as compared to the native HRP. Sp-NHHRP was most stable followed by Sp-HRP, SpNH-COHRP and SpCon A-HRP. Sp-NHHRP was also superior in its ability to regain enzyme activity after thermal denaturation, although Sp-HRP regained maximum activity after urea denaturation. Inclusion of Ca2+ was essential for the reactivation of all preparations subsequent to denaturation by urea.     

The glycoflavone vicenin-2 its distribution in related genera within the labiatae

In a leaf flavonoid survey of Origanum the flavone C-glycoside, vicenin-2, was found to be restricted to the section Majorana. This is the first report of a glycoflavone in the Labiatae.     

Structural determinants stabilizing the axial channel of ClpP for substrate translocation

Acyldepsipeptides (ADEPs) antibiotics bind to Escherichia coli ClpP mimicking the interactions that the IGL/F loops in ClpA or ClpX ATPases establish with the hydrophobic pockets surrounding the axial pore of the tetradecamer that the protease forms. ADEP binding induces opening of the gates blocking the axial channel of ClpP and allowing protein substrates to be translocated and hydrolysed in the degradation chamber. To identify the structural determinants stabilizing the open conformation of the axial channel for efficient substrate translocation, we constructed ClpP variants with amino acid substitutions in the N‐terminal region that forms the axial gates. We found that adoption of a β‐hairpin loop by this region and the integrity of the hydrophobic cluster at the base of this loop are necessary elements for the axial gate to efficiently translocate protein substrates. Analysis of ClpP variants from Bacillus subtilis suggested that the identified structural requirements of the axial channel for efficient translocation are conserved between Gram‐positive and Gram‐negative bacteria. These findings provide mechanistic insights into the activation of ClpP by ADEPs as well as the gating mechanism of the protease in the context of the ClpAP and ClpXP complexes.     

Partial purification and characterization of glutaryl-coenzyme A dehydrogenase, electron transfer flavoprotein, and electron transfer flavoprotein-Q oxidoreductase from Paracoccus denitrificans.

Glutaryl-coenzyme A (CoA) dehydrogenase and the electron transfer flavoprotein (ETF) of Paracoccus denitrificans were purified to homogeneity from cells grown with glutaric acid as the carbon source. Glutaryl-CoA dehydrogenase had a molecular weight of 180,000 and was made up of four identical subunits with molecular weights of about 43,000 each of which contained one flavin adenine dinucleotide molecule. The enzyme catalyzed an oxidative decarboxylation of glutaryl-CoA to crotonyl-CoA, was maximally stable at pH 5.0, and lost activity readily at pH values above 7.0. The enzyme had a pH optimum in the range of 8.0 to 8.5, a catalytic center activity of about 960 min-1, and apparent Michaelis constants for glutaryl-CoA and pig liver ETF of about 1.2 and 2.5 microM, respectively. P. denitrificans ETF had a visible spectrum identical to that of pig liver ETF and was made up of two subunits, only one of which contained a flavin adenine dinucleotide molecule. The isoelectric point of P. denitrificans ETF was 4.45 compared with 6.8 for pig liver ETF. P. denitrificans ETF accepted electrons not only from P. denitrificans glutaryl-CoA dehydrogenase, but also from the pig liver butyryl-CoA and octanoyl-CoA dehydrogenases. The apparent Vmax was of similar magnitude with either pig liver or P. denitrificans ETF as an electron acceptor for these dehydrogenases. P. denitrificans glutaryl-CoA dehydrogenase and ETF were used to assay for the reduction of ubiquinone 1 by ETF-Q oxidoreductase in cholate extracts of P. denitrificans membranes. The ETF-Q oxidoreductase from P. denitrificans could accept electrons from either the bacterial or the pig liver ETF. In either case, the apparent Km for ETF was infinitely high. P. denitrificans ETF-Q oxidoreductase was purified from contaminating paramagnets, and the resultant preparation had electron paramagnetic resonance signals at 2.081, 1.938, and 1.879 G, similar to those of the mitochondrial enzyme.     

Entrapment of concanavalin A-glycoenzyme complexes in calcium alginate gels

Glucose oxidase, invertase, and amyloglucosidase were entrapped in calcium alginate gels as concanavalin A complexes in order to prevent the leaching out of the enzymes from the porous matrix. The free as well as the gel-entrapped concanavalin A-glycoenzyme complexes exhibited a relatively high effectiveness factor, eta, indicating good accessibility to the substrates. Concanavalin A-invertase complex exhibited marked broadening of pH-activity and temperature-activity profiles and was highly resistant to temperature inactivation even after entrapment in the alginate beads. It was possible to entrap considerable quantities of invertase as concanavalin A complex in the beads without a marked decrease in eta. A column containing crosslinked concanavalin A-invertase complex entrapped in alginate beads retained the ability to completely hydrolyze 1M sucrose even after continuous operation for over four months.     

Presence of cyclopiazonic acid in kodo millet (Paspalum scrobiculatum) causing ‘kodua poisoning’ in man and its production by associated fungi

Cyclopiazonic acid was isolated and identified from a sample of kodo millet seed that caused symptoms of kodua poisoning in man. The extract of the toxic grain when injected intraperitoneally into mice produced symptoms of depression and complete loss of mobility. The seed was infected by Aspergillus flavus and A. tamarii and both fungi produced cyclopiazonic acid. This is the first report of the association of a mycotoxin with kodua poisoning and of A. tamarii with mycotoxicoses.     

Rosuvastatin alleviates high-salt and cholesterol diet-induced cognitive impairment in rats via Nrf2–ARE pathway

Objective: The objectives of our study were to investigate the possible effect of rosuvastatin in ameliorating high salt and cholesterol diet (HSCD)-induced cognitive impairment and to also investigate its possible action via the Nrf2-ARE pathway.

Methods: In silico studies were performed to check the theoretical binding of rosuvastatin to the Nrf2 target. HSCD was used to induce cognitive impairment in rats and neurobehavioral studies were performed to evaluate the efficacy of rosuvastatin in enhancing cognition. Biochemical analyses were used to estimate changes in oxidative markers. Western blot and immunohistochemical analyses were done to check Nrf2 translocation. TUNEL and caspase 3 tests were performed to evaluate reversal of apoptosis by rosuvastatin.

Results: Rosuvastatin showed good theoretical affinity to Nrf2, significantly reversed changes in oxidative biomarkers which were induced by HSCD, and also improved the performance of rats in the neurobehavioral test. A rise in nuclear translocation of Nrf2 was revealed through immunohistochemical analysis and western blot. TUNEL staining and caspase 3 activity showed attenuation of apoptosis.

Discussion: We have investigated a novel mechanism of action for rosuvastatin (via the Nrf2–ARE pathway) and demonstrated that it has the potential to be used in the treatment of cognitive impairment.     

Dopamine Modulates Option Generation for Behavior

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Stabilization of yeast invertase by insoluble immunocomplex formation using unfractionated glycosyl-specific and peptide specific polyclonal antiinvertase antibodies

Summary A strategy for the production of non-inhibitory polyclonal antisera for the immobilization of glycoenzymes is described. Insoluble complexes of invertase prepared using antiinvertase glycosyl antisera were more resistant to heat and urea induced denaturation than those prepared using antiinvertase antisera from which the glycosyl specific antibodies were removed. Unfractionated antiinvertase antibodies however gave most stable immunocomplexes with invertase.     

Interleukin-4 receptor expression on AIDS-associated Kaposi's sarcoma cells and their targeting by a chimeric protein comprised of circularly permuted interleukin-4 and Pseudomonas exotoxin.

BACKGROUND: AIDS-associated Kaposi's sarcoma (AIDS-KS) represents one of the most common malignancies associated with human immunodeficiency virus infection. To target effective therapeutic agents to AIDS-KS, we have identified a new target in the form of interleukin-4 receptors (IL-4R). MATERIALS AND METHODS: The expression of IL-4R on AIDS-KS cells and their subunit structure was determined by radioligand receptor binding, cross-linking and Northern and RT-PCR analyses. The in vitro effect of IL-4 and recombinant fusion protein made up of circularly permuted IL-4 and a mutated form of Pseudomonas exotoxin, IL-4(38-37)-PE38KDEL, was examined by clonogenic and protein synthesis inhibition assays. RESULTS: Five AIDS-KS cell lines expressed high-affinity IL-4R with a Kd of 23.5-219 pM. IL-4 appeared to cross-link to one major protein corresponding to 140 kDa and a broad band corresponding to 60-70 kDa. Both cross-linked proteins were immunoprecipitated with an antibody to human IL-4R beta chain. AIDS-KS cells exhibited IL-4R beta-specific mRNA. IL-4 caused a modest inhibition (31-34%) of colony formation in two AIDS-KS cell lines tested. IL-4(38-37)-PE38KDEL was found to be highly effective in inhibiting the protein synthesis in all five AIDS-KS examined. The IC50 ranged from 32 to 1225 pM. The cytotoxic action of IL-4 toxin was blocked by an excess of IL-4, exhibiting the specificity of IL-4(38-37)-PE38KDEL. The cytotoxicity of IL-4 toxin observed by a clonogenic assay corroborated well with the IC50 obtained by protein synthesis inhibition assay. Normal human endothelial cells expressed a negligible number of IL-4R (< 50 sites/cell) and were less sensitive or not sensitive to IL-4(38-37)-PE38KDEL. CONCLUSION: The presence of a new plasma membrane protein in the form of IL-4R on AIDS-KS cells may be targeted by IL-4(38-37)-PE38KDEL for its potential implication in the treatment of AIDS-KS.