Protein-protein complexation in bioluminescence

In this review we summarize the progress made towards understanding the role of protein-protein interactions in the function of various bioluminescence systems of marine organisms, including bacteria, jellyfish and soft corals, with particular focus on methodology used to detect and characterize these interactions. In some bioluminescence systems, protein-protein interactions involve an "accessory protein" whereby a stored substrate is efficiently delivered to the bioluminescent enzyme luciferase. Other types of complexation mediate energy transfer to an "antenna protein" altering the color and quantum yield of a bioluminescence reaction. Spatial structures of the complexes reveal an important role of electrostatic forces in governing the corresponding weak interactions and define the nature of the interaction surfaces. The most reliable structural model is available for the protein-protein complex of the Ca(2+)-regulated photoprotein clytin and green-fluorescent protein (GFP) from the jellyfish Clytia gregaria, solved by means of Xray crystallography, NMR mapping and molecular docking. This provides an example of the potential strategies in studying the transient complexes involved in bioluminescence. It is emphasized that structural studies such as these can provide valuable insight into the detailed mechanism of bioluminescence.

Molecular cloning, expression and enzymatic assay of pteridine reductase 1 from Iranian lizard leishmania

Currently, there are no effective vaccines against leishmaniasis, and treatment using pentavalent antimonial drugs is occasionally effective and often toxic for patients. The PTR1 enzyme, which causes antifolate drug resistance in Leishmania parasites encoded by gene pteridine reductase 1 [ptr 1]. Since Leishmania lacks pteridine and folate metabolism, it cannot synthesize the pteridine moiety from guanine triphosphate. Therefore, it must produce pteridine using PTR1, an essential part of the salvage pathway that reduces oxidized pteridines. Thus, PTR1 is a good drug-target candidate for anti-Leishmania chemotherapy. The aim of this study was the cloning, expression, and enzymatic assay of the ptrl gene from Iranian lizard Leishmania as a model for further studies on Leishmania. Promastigote DNA was extracted from the Iranian lizard Leishmania, and the ptrl gene was amplified using specific primers. The PCR product was cloned, transformed into Escherichia coli strain JM109, and expressed. The recombinant protein [PTR1 enzyme] was then purified and assayed. Ptr1 gene was successfully amplified and cloned into expression vector. Recombinant protein [PTR1 enzyme] was purified using affinity chromatography and confirmed by Western-blot and dot blot using anti-Leishmania major PTR1 antibody and anti-T7 tag monoclonal antibody, respectively. The enzymatic assay was confirmed as PTR1 witch performed using 6-biopterin as a substrate and nicotinamide adenine dinucleotide phosphate as a coenzyme. Iranian lizard Leishmania ptr1 was expressed and enzymatic assay was performed successfully

potential anti-inflammatory effect of tetrahydrobiopetrin administration in renal mass reduction -induced chronic renal failure

Elevated C-reactive protein [CRP], as a marker of persistent inflammation, predicts cardiovascular and over all mortality in chronic kidney disease patients. The present study aimed to investigate the impact of tetrahydrobiopetrin [BH4] supplementation on the markers of inflammation and on the histological picture of the kidney in chronic renal failure [CRF] induced in rats by subtotal nephrectomy [SNx]. This study was performed at the Faculty of Medicine, King Saud University. CRF was induced by 5/6 subtotal nephrectomy [SNx] in 20 male Wister rats and other 10 rats were sham operated by flank incision and served as controls. Ten SNx rats received l0mg/kg BH4 i.p daily for 4 weeks. Plasma CRP, IL-6, malondialdehyde [MDA], and kidney functions were measured in all rats. Histopathological examination of the kidney tissues was also performed. Untreated CRF rats showed significant elevation of plasma CRP, IL-6 and MAD levels and significant decrease in plasma albumin and total protein levels, tubuloglomerularfibrosis and, interstitial tubular infiltration with inflammatory cells in comparison to the sham operated rats. BH4 treatment significantly decreased CRP, IL-6 and MAD levels and also decreased the tubuloglomerular fibrosis and interstitial inflammation in treated CRF rats. Supplementation with exogenous BH4 decreases markers of inflammation and protects the kidney against post- renal mass reduction histologic damage. Restoration of intracellular BH4 balance could normalize NO production. Therefore, BH4 might be a promising strategy in attenuating inflammation in CRF. This may decrease endothelial dysfunction and limit the associated cardiovascular morbidity and mortality in this disease

Synthesis and antitumour activities of some pteridine derivatives / 药学学报

<p><b>AIM</b>To study the synthesis and antitumour activities of some aryl-substituted pteridines.</p><p><b>METHODS</b>A series of aryl-substituted pteridines were synthesized from 4, 6-diamino-5-nitrosopyrimidines by cyclization with 4-aminophenylacetonitriles. The antitumour activities were tested by MTT method.</p><p><b>RESULTS</b>Nine new compounds (I-III) were synthesized and their structures were characterized by EA, IR, 1HNMR and MS spectra. Compounds I-III showed antitumour activities in vitro.</p><p><b>CONCLUSION</b>Compounds I-III showed remarkable antitumour activities in vitro. No interaction was determined between the title compounds and calf thymus DNA. It indicated that these compounds possibly inhibit dihydrofolate reductase (DHFR) or other enzymes on which folic acid depends.</p>

Potentiality of a new compound for in vitro differentiation between halophilic and non-halophilic vibrios.

Sensitivity of 21 halophilic vibrios and 16 clinical isolates of non-halophilic vibrios was determined against a new possible antivibrio agent, a pyrimidine analogue, 4, 6-dimethylpyrimidine -2-thiol (4,6-DMPT). It appeared to be a vibriocidal agent, having a mean MIC and MBC of 32 microg/ml for halophilic strains and 64 microg/ml for non-halophilic strains and an LD50 of 300 mg/Kg body weight of mice. Thus, 4,6-DMPT may help an in vitro distinction between halophilic and non-halophilic vibrios. Sensitivity of these strains was also studied with respect to pteridine, crystal violet and Tween 80 hydrolysis as further markers distinguishing between these 2 groups which could also be differentiated by their growth on TCBS or/and CLED media.

Vibrio cholerae resistant to 2,4-diamino-6,7-diisopropylpteridine (O/129) isolated from patients with enteritis in Ceará, Brazil.

This paper reports the characterization of clinical Vibrio cholerae resistant to vibriostatic agent O/129, using classical and plasmid analysis. In a study conducted during December 1991-September 1993, two of 7,058 V. cholerae strains, obtained from patients suspected to have cholera in the State of Ceará, northeast Brazil, were resistant to 150 micrograms of the vibriostatic agent O/129 (2,4-diamino-6,7-diisopropylpteridine). One strain was identified as V. cholerae O1 El Tor Inaba and the other one as serogroup O22. Only one O1 strain harboured a plasmid of 147 kb transferable to Escherichia coli K12, and five strains of V. cholerae O1 and non-O1 were sensitive to O/129 and plasmid-negative at a frequency between 8 x 10(-2) and 3.6 x 10(-5). Additionally, O/129-resistant strains of V. cholerae O1 and O22 were resistant to trimethoprim/sulphamethoxazole.

Structural, functional and immunological studies on a polymeric bacterial protein

The characterization of proteins from Brucella spp, the causative agent of brucellosis, has been the subject of intensive research. We have described an 18-kDa cytoplasmic protein of Brucella abortus and shown the potential usefulness of this protein as an antigen for the serologic diagnosis of brucellosis. The amino acid sequence of the protein showed a low but significant homology with that of lumazine synthases. Lumazine is an intermediate product in bacterial riboflavin biosynthesis. The recombinant form of the 18-kDa protein (expressed in E. coli) folds like the native Brucella protein and has lumazine-synthase enzymatic activity. Three-dimensional analysis by X-ray crystallography of the homolog Bacillus subtilis lumazine synthase has revealed that the enzyme forms an icosahedral capsid. Recombinant lumazine synthase from B. abortus was crystallized, diffracted X rays to 2.7-A resolution at room temperature, and the structure successfully solved by molecular replacement procedures. The macromolecular assembly of the enzyme differs from that of the enzyme from B. subtilis. The Brucella enzyme remains pentameric (90 kDa) in its crystallographic form. Nonetheless, the active sites of the two enzymes are virtually identical at the structural level, indicating that inhibitors of these enzymes could be viable pharmaceuticals across a broad species range. We describe the structural reasons for the differences in their quaternary arrangement and also discuss the potential use of this protein as a target for the development of acellular vaccines.

Serovar, biotype, phage type, toxigenicity & antibiotic susceptibility patterns of Vibrio cholerae isolated during two consecutive cholera seasons (1989-90) in Calcutta.

Characteristics of V. cholerae isolated from patients of acute secretory diarrhoea admitted to the Infectious Diseases Hospital, Calcutta during two consecutive cholera seasons (1989 and 1990), with special emphasis on biotyping and toxigenicity, were investigated. The isolation rates of V. cholerae during 1989 and 1990 were 78 and 85.1 per cent respectively, with Inaba serotype dominating in 1989 and Ogawa in 1990. All the V. cholerae 01 strains isolated in this study belonged to biotype Eltor with phage type 4 dominating (48.8%). Most of the strains of V. cholerae were resistant to 10 and 150 micrograms/ml of 0/129 vibriostatic agent. Similarly, majority of the V. cholerae strains were resistant to furazolidone (95.7%), cotrimoxazole (83%) and tetracycline (63.1%) and several resistance patterns were encountered. All the V. cholerae 01 strains examined produced cholera toxin (CT) in amounts ranging between greater than 70 pg/ml and greater than 2.5 ng/ml. In contrast, all but one of the non-01 strains isolated in this study did not produce CT. Further studies are required to elucidate the mechanism involved in the pathogenesis of non-01 V. cholerae mediated diarrhoea.