HLA class II DRB1 resistance and susceptible markers in hydatidosis Saudi patients in association to the clinical course and gender.

Hydatidosis constitutes a major public health problem in kingdom of Saudi Arabia. The variability on susceptibility to hydatidosis has been related to the HLA system. So this study aimed to identifying the possible association between the class-II HLA-DRB1 alleles with the occurrence of hydatidosis and the clinical course in Saudi patients using Micro SSP HLA-DRB1 kits. Since HLA-disease associations might vary in relation to gender, so this study aims also to determining the HLA-DRB1 alleles- hydatidosis association in relation to gender. The results proved that HLA-DR16 and HLA-DR7 alleles were the suitable markers of susceptibility association while HLA-DR1 and HLA-DR10 alleles might confer protection against hydatidosis. All the previous susceptible and resistance associations were statistically significant. Also, the majority of the female patients (81.8%) had HLA-DR14 compared to 9.1% of female controls while 50% of the male patients had HLA-DR7 compared to 4.5% of male controls. These results were statistically associated and this is the first study that found an association between certain HLA-DRB 1 alleles and the occurrence of human hydatidosis in relation to gender. Also, significant relations were detected between HLA-DR16 and hepatic and single cyst, HLA-DR7 and lung cyst, HLA-DR14 and combined cysts. Also, no statistical significance was found between other cystic characteristics and any one of the susceptible HLA-DRBs. On conclusion beside the role HLA-DRB 1 on the susceptibility or the resistance to hydatidosis disease occurrence among Saudi population also it may have an important role in the prevalence of the disease in relation to gender.

Purification of Synechocystis sp. strain PCC6308 cyanophycin synthetase and its characterization with respect to substrate and primer specificity.

Synechocystis sp. strain PCC6308 cyanophycin synthetase was purified 72-fold in three steps by anion exchange chromatography on Q Sepharose, affinity chromatography on the triazine dye matrix Procion Blue HE-RD Sepharose, and gel filtration on Superdex 200 HR from recombinant cells of Escherichia coli. The native enzyme, which catalyzed the incorporation of arginine and aspartic acid into cyanophycin, has an apparent molecular mass of 240 +/- 30 kDa and consists of identical subunits of 85 +/- 5 kDa. The K(m) values for arginine (49 microM), aspartic acid (0.45 mM), and ATP (0.20 mM) indicated that the enzyme had a high affinity towards these substrates. During in vitro cyanophycin synthesis, 1.3 +/- 0.1 mol of ATP per mol of incorporated amino acid was converted to ADP. The optima for the enzyme-catalyzed reactions were pH 8.2 and 50 degrees C, respectively. Arginine methyl ester (99.5 and 97% inhibition), argininamide (99 and 96%), S-(2-aminoethyl) cysteine (43 and 42%), beta-hydroxy aspartic acid (35 and 37%), aspartic acid beta-methyl ester (38 and 40%), norvaline (0 and 3%), citrulline (9 and 7%), and asparagine (2 and 0%) exhibited an almost equal inhibitory effect on the incorporation of both arginine and aspartic acid, respectively, when these compounds were added to the complete reaction mixture. In contrast, the incorporation of arginine was diminished to a greater extent than that of aspartic acid, respectively, with canavanine (82 and 53%), lysine (36 and 19%), agmatine (33 and 25%), D-aspartic acid (37 and 30%), L-glutamic acid (13 and 5%), and ornithine (23 and 11%). On the other hand, canavanine (45% of maximum activity) and lysine (13%) stimulated the incorporation of aspartic acid, whereas aspartic acid beta-methyl ester (53%) and asparagine (9%) stimulated the incorporation of arginine. [(3)H]lysine (15% of maximum activity) and [(3)H]canavanine (13%) were incorporated into the polymer, when they were either used instead of arginine or added to the complete reaction mixture, whereas L-glutamic acid was not incorporated. No effect on arginine incorporation was obtained by the addition of other amino acids (i.e., alanine, histidine, leucine, proline, tryptophan, and glycine). Various samples of chemically synthesized poly-alpha,beta-D,L-aspartic acid served as primers for in vitro synthesis of cyanophycin, whereas poly-alpha-L-aspartic acid was almost inactive.

Molecular characterization of the cyanophycin synthetase from Synechocystis sp. strain PCC6308.

A 3878-bp genomic region from the cyanobacterium Synechocystis sp. strain PCC6308, amplified by inverse PCR, harbored the structural genes cphA (2625 bp) and cphB (819 bp) encoding cyanophycin synthetase and cyanophycinase, respectively. Both primary structures exhibited a high degree of similarity to the corresponding translational products from other cyanobacteria. Five regions were localized in the cyanophycin synthetase consensus sequence by their resemblance to conserved sites of ATP-dependent carboxylate-amine/thiol ligases and three substrate ligases. The functionality of cphA was proven by heterologous expression of active enzyme and synthesis of cyanophycin in Escherichia coli, which led to a maximum cyanophycin content of 26.6% (w/w) of cell dry mass. Furthermore, a modified radiometric enzyme assay for a more reliable and feasible measurement of cyanophycin synthetase activity was developed and applied to reveal the substrate specificity of the enzyme.