Easy route to functionalize iron oxide nanoparticles via long-term stable thiol groups.

The functionalization of superparamagnetic iron oxide nanoparticles (SPIOs) by meso-2,3-dimercaptosuccinic acid (DMSA) was investigated. Under ambient conditions, the thiol groups from DMSA are not stable and do not allow a direct functionalization without storage in stringent conditions or a chemical regeneration of free thiols. In this study, we have developed a protocol based on poly(ethylene glycol) (PEG) grafting of SPIO prior to DMSA anchoring. We have observed that PEG helps to increase the stability of thiol groups under ambient conditions. The thiol functionalized SPIOs were stable under physiological pH and ionic strength as determined by Ellman's essay and allowed us to graft a thiol reactive fluorescent dye: tetramethylrhodamine-5-maleimide (TMRM).

Anemia prophylaxis in adolescent school girls by weekly or daily iron-folate supplementation.

OBJECTIVE: To examine the benefits of anemia prophylaxis in adolescent school.girls by weekly or daily iron-folate supplementation. DESIGN: Prospective study. SETTING: Government girl schools of northeast Delhi. SUBJECTS: 2088 subjects (with hemoglobin greater than 7.9 g/dL), including 702 on daily and 695 on weekly iron-folate administration; 691 girls served as controls. RESULTS: About 85% girls were iron deficient out of which 49.3% were anemic. Weekly administration took longer time to raise hemoglobin but was effective as well as practical. Plasma ferritin estimation in girls showed rise in level in both the treated groups. CONCLUSION: Weekly administration of iron-folate was a practical and effective strategy for anemia prophylaxis in adolescent school girls.

Expression and purification of Dengue virus type 2 envelope protein as a fusion with hepatitis B surface antigen in Pichia pastoris.

The methylotrophic yeast, Pichia pastoris, has been used as a host to express the envelope protein (Den2E) of dengue type 2 virus (NGC strain) as a chimera with hepatitis B surface antigen (HBsAg): a protein known to self assemble into virus-like particles (VLPs) and to be efficiently expressed in P. pastoris. The Den2E gene used in this study is a truncated version encoding the first 395 amino acid (aa) residues of the mature Den2E protein; the HBsAg gene encodes the full length 226 aa HBsAg protein. Two in-frame gene fusions were constructed for intracellular expression in P. pastoris. The first one contains the HBsAg gene as the 5' partner and the Den2E gene as the 3'partner (HBsAg-Den2E). In the second one, the relative positions of the two partners of the gene fusion were reversed to create the hybrid Den2E-HBsAg gene. These fusion genes were integrated into the genome of P. pastoris under the control of the methanol-inducible alcohol oxidase (AOX1) promoter. Of the two fusions, the Den2E-HBsAg gene was expressed at higher levels in P. pastoris based on Northern analysis. The hybrid protein ( approximately 68 kDa) expressed by this clone was purified to near homogeneity using a combination of acid precipitation, hydrophobic interaction, and immunoaffinity chromatographic steps. Final purification achieved was approximately 1400-fold with a yield of approximately 26%. The chimeric protein was found to possess the ability to assemble into high molecular weight aggregates (akin to HBsAg particles). The recombinant fusion protein eluted close to the void volume of a Sepharose CL-4B column indicating its macromolecular nature. On a CsCl density gradient the recombinant fusion protein sedimented to a position very similar to that of HBsAg VLPs. The hybrid protein is recognized by the two neutralizing monoclonals against the two components of the chimeric protein.

Mechanism of malarial haem detoxification inhibition by chloroquine.

The haem detoxification pathway of the malaria parasite Plasmodium falciparum is a potential biochemical target for drug development. Free haem, released after haemoglobin degradation, is polymerized by the parasite to form haemozoin pigment. Plasmodium falciparum histidine-rich protein-2 (Pfhrp-2) has been implicated as the catalytic scaffold for detoxification of haem in the malaria parasite. Previously we have shown that a hexapeptide repeat sequence (Ala-His-His-Ala-Ala-Asp), which appears 33 times in Pfhrp-2, may be the major haem binding site in this protein. The haem binding studies carried out by ourselves indicate that up to 18 equivalents of haem could be bound by this protein with an observed K(d) of 0.94 microM. Absorbance spectroscopy provides evidence that chloroquine is capable of extracting haem bound to Pfhrp-2. This was supported by the K(d) value, of 37 nM, observed for the haem-chloroquine complex. The native PAGE studies reveal that the formation of the haem-Pfhrp-2 complex is disrupted by chloroquine. These results indicate that chloroquine may be acting by inhibiting haem detoxification/binding to Pfhrp-2. Moreover, the higher affinity of chloroquine for haem than Pfhrp-2 suggests a possible mechanism of action for chloroquine; it may remove the haem bound to Pfhrp-2 and form a complex that is toxic to the parasite.