Hsp90 mediates activation of the heme regulated eIF-2 kinase during oxidative stress.

The heme-regulated inhibitor (HRI), a member of the eIF-2 kinase family is crucial for regulating protein synthesis during stress. In addition to heme, stress proteins Hsp90 and Hsp70 are known to regulate HRI. The present study aims to determine the physical association of these Hsps in the regulation of HRI activation during oxidative stress using human K562 cells as a model. Extracts from the stress-induced cells were used for determining HRI kinase activity by measuring eIF-2 phosphorylation, and Hsp-HRI interaction by immunoprecipitation and immunoblot analyses. The results indicate a significant increase in both Hsp70 and Hsp90 expression during AAPH (2, 2’-azobis (2-amidinopropane) dihydrochloride)-induced oxidative stress. Further, their interaction with HRI, which correlates well with its increased HRI kinase activity leads to inhibition of protein synthesis. Thus, we demonstrate that Hsps play an important role in the regulation of initiation of protein synthesis during oxidative stress.

Curcumin reduces cold storage-induced damage in human cardiac myoblasts

Curcumin is a polyphenolic compound possessing interesting anti-inflammatory and antioxidant properties and has the ability to induce the defensive protein heme oxygenase-1 (HO-1). The objective of this study was to investigate whether curcumin protects against cold storage-mediated damage of human adult atrial myoblast cells (Girardi cells) and to assess the potential involvement of HO-1 in this process. Girardi cells were exposed to either normothermic or hypothermic conditions in Celsior preservation solution in the presence or absence of curcumin. HO-1 protein expression and heme oxygenase activity as well as cellular damage were assessed after cold storage or cold storage followed by re-warming. In additional experiments, an inhibitor of heme oxygenase activity (tin protoporphyrin IX, micrometer) or siRNA for HO-1 were used to investigate the participation of HO-1 as a mediator of curcumin- induced effects. Treatment with curcumin produced a marked induction of cardiac HO-1 in normothermic condition but cells were less responsive to the polyphenolic compound at low temperature. Cold storage-induced damage was markedly reduced in the presence of curcumin and HO-1 contributed to some extent to this effect. Thus, curcumin added to Celsior preservation solution effectively prevents the damage caused by cold- storage; this effect involves the protective enzyme HO-1 but also other not yet identified mechanisms.

Dose-dependent differential effect of hemin on protein synthesis and cell proliferation in Leishmania donovani promastigotes cultured in vitro.

Leishmania donovani requires an exogenous source of heme for growth and transformation. In in vitro culture of the free-living promastigotes, exogenously added hemin enhances cell proliferation. In this investigation, the question of the function of heme with particular reference to protein synthesis and cell proliferation has been addressed. The results of in vitro cell culture experiments demonstrated that hemin (10 microM) alone is suitable for supporting optimum level of protein synthesis, and thereby cell proliferation of promastigotes to an extent that it can replace fetal bovine serum. However, in situ labelling experiments along with Western blots revealed that high concentration of hemin (50 microM) reduced the level of protein synthesis in general and of beta-tubulin in particular with a concomitant induction of hsp90, and induced consequent morphological changes that are observed during in situ transformation of promastigotes in mammalian macrophages. These results therefore suggest that sudden exposure to high concentration of heme in mammalian macrophages may be one of the key factors that trigger promastigote to amastigote transformation in L. donovani. Furthermore, hemin with its dual characteristic could be used as a tool to understand molecular mechanism of cell proliferation and transformation in these parasites.

Estudos espectroscopicos da agregacao de ferriprotoporfirina IX em meio aquoso e em membranas. Implicacoes para o mecanismo de lise celular / Spectroscopic studies of ferriprotoporphyrin IX in aqueous media in membranes. Implications for the mechanism of cell lysis

A tese descreve o estudo da agregacao de hemina (ferriprotoporfirina IX) em meio aquoso e em presenca de membranas. Foi estudada a dependencia de efeitos causados por hemina em bicamadas lipidicas (catalise de peroxidacao lipidica, alteracoes estruturais e de permeabilidade) do estado de agregacao desse composto. Foram empregadas tecnicas de espectroscopia optica e de ressonancia paramagnetica eletronica (e marcadores de spin). Foram utilizados lipossomos multilamelares, vesiculas unilamelares e membranas planas como modelos de membranas. Verificou-se que a agregacao de hemina em meio aquoso aumenta com o abaixamento do pH e com o aumento da forca ionica em presenca de membranas. A catalise de peroxidacao lipidica e efetuada por hemina monomerica ou dimerica , enquanto que agregados maiores se intercalam na bicamada aumentando a desordem e, consequentemente, a permeabilidade. Os resultados obtidos fornecem uma hipotese para o mecanismo, a nivel molecular da lise celular causada por hemina

Hemolysin production by Leptospira interrogans serovar canicola in a protein-free medium with hemin.

Leptospira interrogans serovar canicola strain moulton was grown to a high cell density in a protein-free medium. When hemin was added to this medium, hemolysin was produced. Hemolysin was not detected when other porphyrins or cytochrome C were substituted for hemin in the medium.