Development of specific DNA probes and their usage in the detection of Plasmodium vivax infection in blood.

The application of nucleic acid probes, in the detection of pathogenic micro-organisms, has become an integral part of diagnostic technologies. In this study, Plasmodium vivax-specific DNA probes have been identified by carrying out genomic subtractive hybridization. In this approach, the recombinant clones from a P. vivax genomic library are screened with radiolabelled human and P. falciparum DNA. The colonies which react with labelled P. falciparum and human DNA are eliminated and those which do not produce any autoradiographic signal have been subjected to further screening procedures. Three P. vivax specific DNA probes have been obtained by these repeated screenings. Further analyses indicate that these probes are specific and sensitive enough to detect P. vivax infection in clinical blood samples when used in a non-radioactive DNA hybridization assay.

Partial restoration of activity to Lactobacillus casei thymidylate synthase following inactivation by domain deletion.

Thymidylate synthase (TS) from Lactobacillus casei has a 50 amino acid insert (residues 90-139) in the small domain that is found in only one other TS. A deletion mutant was constructed which lacked the entire insert, thereby reducing the small domain to the size found in Escherichia coli TS. This mutant did not catalyze the formation of dTMP. From the crystal structure of L. casei TS, we surmised that the loss of activity might have resulted from the exposure of residues of helices C and D, which were previously buried by the insert. To restore the local structure of helices C and D in the deletion mutants, we replaced several residues in this region by the corresponding residues found in E. coli TS. The mutant whose sequence most closely resembled that of E. coli TS carried six mutations and possessed partially restored TS activity. The mutant which had all those mutations except F87D did not catalyze any dTMP formation. The crucial role of F87D was proven in a deletion mutant which had only this change and showed greatly increased activity. All of the mutants catalyzed the debromination of BrdUMP in the absence of cofactor about as well as wild type TS. The kinetic parameters for dTMP formation of the active mutants show that the deletion has its major effect on kcat and binding of cofactor CH2H4folate, with less effect on binding of the substrate dUMP. Removal of residues 90-139 is believed to disorder helices C and D, which in turn decreases cofactor binding and catalysis.

Thermal stabilization of thymidylate synthase by engineering two disulfide bridges across the dimer interface.

Thermal inactivation of oligomeric enzymes is most often irreversible and is frequently accompanied by precipitation. We have engineered two symmetry related disulfide bridges (155-188' and 188-155') across the subunit interface of Lactobacillus casei thymidylate synthase, at sites chosen on the basis of an algorithm for the introduction of stereochemically unstrained bridges into proteins. In this communication, we demonstrate a remarkable enhancement in the thermal stability of the covalently cross-linked double disulfide containing dimeric enzyme. The mutant enzyme remains soluble and retains secondary structure even at 90 degrees C, in contrast to the wild-type enzyme which precipitates at 52 degrees C. Furthermore, the mutant enzyme has a temperature optimum of 55 degrees C and possesses appreciable enzymatic activity at 65 degrees C. Cooling restores complete activity, in the mutant protein, demonstrating reversible thermal unfolding. The results suggest that inter-subunit crosslinks can impart appreciable thermal stability in multimeric enzymes.

Evaluation of a crude soluble antigen from in-vitro culture of Plasmodium falciparum for ELISA.

The present study was to evaluate the soluble antigen prepared from the in-vitro cultured P. falciparum (FCR3) as an alternative source of antigen to those obtained from in-vivo models for enzyme linked immunosorbent assay (ELISA) in serology of malaria. Results obtained on known positive and negative reference sera revealed good correlation between the ELISA and the indirect fluorescent antibody (IFA) technique (rs = 0.797; p less than 0.001). However such close correlation was not observed on six local sera from patients whose peripheral blood smear showed ring stage of P. falciparum (rs = 0.43; p greater than 0.05), though all the six sera were positive by the IFA and ELISA tests. Test was repeated to establish its reproducibility. The results indicate that antigen prepared from in vitro culture and stored in liquid nitrogen was found sensitive in ELISA for serology of malaria.