Implication of a critical residue (Glu175) in structure and function of bacterial luciferase

Structural properties of a bacterial luciferase mutant, evolved by random mutagenesis, have been investigated. Bacterial luciferases (LuxAB) can be readily classed as slow or fast decay luciferases based on their rates of luminescence decay in a single turnover assay. By random mutagenesis, one of the mutants generated by a single mutation on LuxA at position 175 (E175G) resulted in the "slow decay" Xenorhabdus luminescens luciferase was converted into a luciferase with a significantly more rapid decay rate [Hosseinkhani, S., Szittner, R. and Meighen, E.A. (2005) Biochemical Journal 385, 575-580]. A single mutation (E175G), in a loop that connects alpha helix 5 and beta sheet 5 brought about changes in the kinetic and structural properties of the enzyme. Enhancement of tryptophan fluorescence was observed with a lower degree of fluorescence quenching by acrylamide upon mutation. Near- and far-UV circular dichroism spectra of the native and mutant forms suggested formation of an intermediate structure, further supported by 8-anilino-1-naphthalene-sulphonic acid (ANS) fluorescence which indicated lower exposure of hydrophobic residues as a result of mutation. Fluorescence quenching studies utilizing acrylamide indicated a more accessible fluor for the native form. Thus, the E175G point mutation appears to change the enzymatic decay rate by inducing a substantial tertiary structural change, without a large effect on secondary structural elements, as revealed by Fourier transform IR spectroscopy. Overall, the mutation caused structural changes that go beyond the simple change in orientation of Glu175.     

Three species of Ulva (Ulvophyceae) from the Persian Gulf as potential sources of protein,essential amino acids and fatty acids

Three species of the cosmopolitan genus Ulva (U. paschima , U. chaugulii and U. ohnoi ) from the Persian Gulf were identified using morphological and molecular markers, and were analyzed for total lipids, fatty acids, proteins and amino acid profiles. Our results show that the studied Ulva species have high protein content (9?25% dry weight), contain essential fatty acids and nearly all essential amino acids. This suggests Ulva could have potential as a form of human nutrition and animal feed with a low carbon footprint.     

Antagonistic Activity of <Emphasis Type="Italic">Nocardia brasiliensis</Emphasis> PTCC 1422 Against Isolated Enterobacteriaceae from Urinary Tract Infections

The main drawback of current antibiotic therapies is the emergence and rapid increase in antibiotic resistance. Nocardiae are aerobic, Gram-positive, catalase-positive, non-motile actinomycetes. Nocardia brasiliensis was reported as antibiotic producer. The purpose of the study was to determine antibacterial activity of N. brasiliensis PTCC 1422 against isolated Enterobacteriaceae from urinary tract infections (UTIs). The common bacteria from UTIs were isolated from hospital samples. Antimicrobial susceptibility test was performed for the isolated pathogens using Kirby–Bauer disk diffusion method according to clinical and Laboratory Standards Institute guideline. Antagonistic activity of N. brasiliensis PTCC 1422 was examined with well diffusion methods. Supernatant of N. brasiliensis PTCC 1422 by submerged culture was analyzed with gas chromatography–mass spectrometry. Isolated strains included Escherichia coli, Klebsiella pneumoniae, Serratia marcescens and Proteus mirabilis. The most common pathogen isolated was E. coli (72.5 %). Bacterial isolates revealed the presence of high levels of antimicrobial resistances to ceftriaxone and low levels of resistance to cephalexin. Supernatant of N. brasiliensis PTCC 1422 showed antibacterial activity against all of the isolated microorganisms in well diffusion method. The antibiotic resistance among the uropathogens is an evolving process, so a routine surveillance to monitor the etiologic agents of UTI and the resistance pattern should be carried out timely to choose the most effective empirical treatment by the physicians. Our present investigation indicates that the substances present in the N. brasiliensis PTCC 1422 could be used to inhibit the growth of human pathogen. Antibacterial resistance among bacterial uropathogen is an evolving process. Therefore, in the field on the need of re-evaluation of empirical treatment of UTIs, our present. The study has demonstrated that N. brasiliensis PTCC 1422 has a high potential for the treatment of UTIs.     

Deglycosylation of glucoamylase from Aspergillus niger: Effects on structure,activity and stability

A comparative structure–function study was performed to establish possible roles of carbohydrates in stabilization of glycoproteins, using glucoamylase (GA) as a model system. In addition to kinetic properties, stability toward elevated temperatures, extremes of pH, high salt concentrations together with circular dichroism, intrinsic/extrinsic fluorescence studies, proteolysis and affinity for interaction with hydrophobic ligands were investigated. Related to all the main properties examined, with one exception, glycosylation provided improvement in functional characteristics of the enzyme, especially in relation to its thermostability. Results are explained in terms of provision of stabilizing intermolecular interactions by the sugar molecules. The improvement in protein rigidity together with reduction of surface hydrophobicity appear to be especially important in relation to prevention of aggregation, an important mechanism of irreversible thermoinactivation, occurring at elevated temperatures.     

Plant regeneration through callus cultures derived from immature-cotyledon explants of oleaster (Elaeagnus angustifolia L.)

Efficient plant regeneration was achieved from callus derived from immature-cotyledon explants of oleaster (Elaeagnus angustifolia L.). Calli were obtained on MS media containing 3% sucrose and different concentrations of TDZ. The highest rate of green, compact and nodular callus was formed on MS medium supplemented with 1 mg/l of TDZ. Shoot organogenesis was achieved when the callus was transferred onto MS media containing 3% sucrose and BA alone (05–4 mg/l) or BA (0.5 and 1 mg/l) combined with NAA or IAA (0.5 and 1 mg/l). Maximum organogenesis was obtained with 1 mg/l BA in combination with 0.5 mg/l NAA. Rooting of the shoots was achieved on MS medium supplemented with 0.2 mg/l IBA. Regenerated plantlets were acclimatized and successfully transplanted to soil.     

Molecular Dynamics Study of the Human Beta-defensins 2 and 3 Chimeric Peptides with the Cell Membrane Model of Pseudomonas aeruginosa

International Journal of Peptide Research and Therapeutics - Pseudomonas aeruginosa is a unique microorganism among the antibiotic-resistant microbial pathogens. Among the various therapeutic...     

Comparative analysis of oncofetal fibronectin and tenascin-C expression in right atrial auricular and left ventricular human cardiac tissue from patients with coronary artery disease and aortic valve stenosis

Aortic valve stenosis (AVS) and coronary artery disease (CAD) are accompanied by changes in the cardiac extra cellular matrix (cECM) including the re-expression of oncofetal fibronectin (Fn) and tenascin-C (Tn-C) variants. Human antibodies against these variants are usable for targeted therapy. Aim of the study was the comparative analysis of cECM remodelling in tissue samples from right atrial auricle (RAA) and left ventricular septum (LVS). RAA and LVS specimens from 30 patients (17?×?AVS; 13?×?AVS+CAD) were analysed with respect to histological changes and ECM remodelling using PCR based ECM gene expression profiling. Re-expression of ED-A(+) Fn and A1(+) Tn-C was investigated on the mRNA and on the protein level. For immunofluorescence, human recombinant small immunoprotein (SIP) format antibodies were used. There was a positive correlation of the grade of histological changes in RAA and corresponding LVS samples (r?=?0.695). ECM gene expression levels were higher in LVS compared to RAA. For 24 genes, a corresponding relevant (>2.5-fold) up- or down-regulation in RAA and LVS occurred. Using SIP antibodies, a positive correlation of protein deposition levels in RAA and corresponding LVS (r?=?0.818) could be shown for ED-A(+) Fn. Cardiac tissue remodelling is likely a process involving the entire heart reflected by intra-individually comparable histology and cECM changes in RAA and LVS samples. ED-A(+) Fn might be an excellent target for an antibody-mediated delivery of diagnostic or therapeutic agents. The RAA is a valuable and representative tool to evaluate cardiac remodelling and to plan individualized therapy.     

Acid-Induced Formation of Molten Globule States in the Wild Type <Emphasis Type="Italic">Escherichia coli</Emphasis> 5-Enolpyruvylshikimate 3-Phosphate Synthase and its Three Mutated Forms: G96A,A183T and G96A/A183T

Recent advances in protein chemistry have led to progress in the understanding of protein folding and properties of possible intermediates during the folding of proteins. The molten globule (MG) state, a major intermediate of protein folding, has a denatured state with native-like secondary structure. In the present work, the acid-induced unfolding of wild type Escherichia coli 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) and its three different variants (G96A, A183T and G96A/A183T) were studied by far- and near-UV circular dichroism (CD), intrinsic fluorescent emission spectroscopy and 1-anilino naphthalene-8-sulfonate (ANS) binding. At pH < 3.0, these EPSPS variants acquire partially folded state, which show the characteristics of the MG state, e.g., a drastic reduction of defined tertiary structure and almost no change in the secondary structure. ANS binding experiments show that hydrophobic surface of these variants is exposed to a greater extent in comparison to the native form, at acidic pH. Wild type, G96A, A183T and G96A/A183T acquire MG states at pH 2.0, 1.5, 3.0 and 3.0, respectively, which show that pH stability of MG state of G96A has increased in comparison to wild type; and pH stability of MG states of two other mutants is lower than that of the wild type. The results suggest that there is a direct relationship between stability of protein and pH stability of its folding intermediates.     

Synthesis,characterization and inhibitory potency of two oxono and thiono analogues of phosphoramidate compounds on acetylcholinesterase

Two novel structurally related phosphoramidate compounds, 1 and 2, with likely β-diketone system were synthesized and characterized by ¹H, ¹³C, ³¹P NMR, IR spectroscopy and elemental analysis. Compound 2 exhibited a ³¹P NMR signal which was significantly shielded (8 ppm) relative to compound 1. Determination of human erythrocyte acetylcholinesterase (hAChE) inhibitory activity was carried out according to Ellman's modified kinetic method and the IC₅₀ values of compounds 1 and 2 were 1.567 and 2.986 mM, respectively. The k_i values of 1 and 2 were 1.39 to 2.65 min^{? 1} respectively. A comparison of the bimolecular rate constant (k_i) and IC₅₀ values for the irreversible inhibitors 1 and 2 revealed that the oxono analogue has greater affinity for hAChE than the thiono compound. Furthermore effects of two conventional oximes paralidoxime (A) and obidoxime (B) on reactivation of the inhibited hAChE were studied but low reactivity was shown by both the oximes.