In vivo demonstration of enhanced binding between ß-clamp and DnaE of pol III bearing consensus i-CBM.

BACKGROUND: Among several key protein-protein and protein-DNA interactions within the replisome, the interaction between ß-clamp and the DNA polymerase (Pol) III is of crucial importance. This interaction is mediated by a five or six-residue conserved sequence of the DnaE subunit of Pol III, referred to as the Clamp Binding Motif (CBM). In E. coli, DnaE contains two CBMs designated as e-CBM and i-CBM. A consensus sequence (QL[S/D]LF) for the CBMs has previously been proposed and studies involving mutagenesis of both the CBMs have evaluated their protein-binding properties. Surface Plasmon Resonance has been used to show that replacing i-CBM in DnaE with the consensus sequence enhances its binding to ß-clamp 120-fold. OBJECTIVE: The current study was aimed to evaluate in vivo interaction between DnaE bearing the consensus i-CBM and ß-clamp. METHOD: The C-terminal 405 residues of DnaE, bearing either the consensus i-CBM or the WT i-CBM, with ß-clamp were co-expressed in E. coli followed by co-purification of the protein complexes. The interaction was assessed by the ability of the co-expressed proteins to form stable complexes during both affinity and gel filtration chromatography. RESULT: The interaction of ß-clamp with DnaEΔ755M containing the consensus i-CBM was found to be more stable than with WT DnaEΔ755, consistent with the in vitro data previously reported. CONCLUSION: The presence of the pieces of sheared DNA generated during sonication promote the interaction of DnaEΔ755M with ß-clamp by binding the OB-fold of DnaEΔ755M and ß-clamp and serves as a bridge between them.

Molecular surveillance of HCV mono-infection and HCV-HBV co-infection in symptomatic population at Hyderabad, Pakistan.

BACKGROUND: Pakistan is endemic to hepatitis B and C infections. Alarming rise in hepatitis C virus (HCV) infection has been noticed in some areas of Sindh with an increasing risk for co-infection frequency in this region. OBJECTIVE: To estimate the burden of HBV/HCV infection in Hyderabad Pakistan. METHODS: ELISA and Nucleic acid Amplification test were performed to detect viruses. SPSS and online calculator were used for statistical analysis. RESULTS: From a total of 108 seropositive hepatitis patients, 36.1% (n=39) were found HCV RNA-positive. Non-significant differences were observed in the frequencies of HCV infection for both genders [OR=0.735, CI (95%) 0.307-1.761, p<0.05]. The percentage of HBV DNA detection among 108 HCV-seropositive cases was 17.9% (n=19). However, HCV-HBV co-infection in HCV-RNA positive cases was determined in 48.7% (n=19) cases with non-significant difference in both genders [OR=1.51, CI (95%) = 0.38 - 5.96, p< 0.05]. Analysis suggested weakly positive correlation between HCV mono-infection and HCV-HBV co-infection and age (r =0.184, and r =0.1231), respectively. CONCLUSION: The study demonstrates a high prevalence of HBV co-infection among active hepatitis C patients of Hyderabad.

Co-expression and purification of the RadA recombinase with the RadB paralog from Haloferax volcanii yields heteromeric ring-like structures.

The study of archaeal proteins and the processes to which they contribute poses particular challenges due to the often extreme environments in which they function. DNA recombination, replication and repair proteins of the halophilic euryarchaeon, Haloferax volcanii (Hvo) are of particular interest as they tend to resemble eukaryotic counterparts in both structure and activity, and genetic tools are available to facilitate their analysis. In the present study, we show using bioinformatics approaches that the Hvo RecA-like protein RadA is structurally similar to other recombinases although is distinguished by a unique acidic insertion loop. To facilitate expression of Hvo RadA a co-expression approach was used, providing its lone paralog, RadB, as a binding partner. At present, structural and biochemical characterization of Hvo RadA is lacking. Here, we describe for the first time co-expression of Hvo RadA with RadB and purification of these proteins as a complex under in vitro conditions. Purification procedures were performed under high salt concentration (>1 M sodium chloride) to maintain the solubility of the proteins. Quantitative densitometry analysis of the co-expressed and co-purified RadAB complex estimated the ratio of RadA to RadB to be 4 : 1, which suggests that the proteins interact with a specific stoichiometry. Based on a combination of analyses, including size exclusion chromatography, Western blot and electron microscopy observations, we suggest that RadA multimerizes into a ring-like structure in the absence of DNA and nucleoside co-factor.

dRTA and hemolytic anemia: first detailed description of SLC4A1 A858D mutation in homozygous state.

Mutations in the anion exchanger 1 (AE1) gene encoding the erythroid and kidney anion (chloride-bicarbonate) exchanger 1 may result in familial distal renal tubular acidosis (dRTA) in association with membrane defect hemolytic anemia. Seven children presenting with hyperchloremic normal anion gap metabolic acidosis, failure to thrive, and compensated hemolytic anemia were studied. Analysis of red cell AE1/Band 3 surface expression by Eosin 5'-maleimide (E5M) was performed in patients and their family members using flow cytometry. Genetic studies showed that all patients carried a common SLC4A1 mutation, c.2573C>A; p.Ala858Asp in exon 19, found as homozygous (A858D/A858D) mutation in the patients and heterozygous (A858D/N) in the parents. Analysis by flowcytometry revealed a single uniform fluorescence peak, with the mean channel fluorescence (MCF) markedly reduced in cases with homozygous mutation, along with a left shift of fluorescence signal but was only mildly reduced in the heterozygous state. Red cell morphology showed striking acanthocytosis in the homozygous state [patients] and only a mild acanthocytosis in heterozygous state [parents]. In conclusion, this is the first description of a series of homozygous cases with the A858D mutation. The E5M flowcytometry test is specific for reduction in the Band 3 membrane protein and was useful in conjunction with a careful morphological examination of peripheral blood smears in our patient cohort.

Endemic fluorosis in the Ethiopian Rift Valley.

Between 1977 and 1985, the fluoride content of drinking water and the incidence of endemic fluorosis were assessed and correlated in 16 large farms, villages and towns in the Ethiopian Rift Valley. The fluoride level of drinking-water collected from wells there ranged from 1.2 mg/litre to 36.0 mg/l (mean 10.0 mg/l). Dental fluorosis was observed in more than 80% of sampled children resident in the Rift Valley since birth, with maximum prevalence in the age-group 10-14 years; 32% of the children showed severe dental mottling. Males were affected more than females. Three areas, Wonji-Shoa, Alemtena and Samiberta, were identified as having cases of skeletal fluorosis. The highest incidence was at Wonji-Shoa sugar estates, where a linear relationship was observed between the development of crippling fluorosis, fluoride concentration of drinking-water, and period of exposure to it. The first cases of skeletal fluorosis there appeared among workers (98% males) who had been consuming water with fluoride content of more than 8ppm for over 10 years. Among 30 workers with crippling skeletal fluorosis, cervical radiculo-myelopathy was found to be the commonest incapacitating neurologic complication. As a preventive measure, low-fluoride surface water should be supplied for drinking wherever feasible; if this is not possible, the development of partial defluoridation should be considered.