Expression of heat shock protein and hemoglobin genes in Chironomus tentans (Diptera, chironomidae) larvae exposed to various environmental pollutants: a potential biomarker of freshwater monitoring.

To identify a sensitive biomarker of freshwater monitoring, we evaluated pollutant-induced expression of heat shock proteins (HSPs) and hemoglobins (Hbs) genes in the larvae of the aquatic midge Chironomus tentans (Diptera, Chironomidae). As pollutants, we examined nonylphenol, bisphenol-A, 17alpha-ethynyl estradiol, bis(2-ethylhexyl) phthalate, endosulfan, paraquat dichloride, chloropyriphos, fenitrothion, cadmium chloride, lead nitrate, potassium dichromate, benzo[a]pyrene and carbon tetrachloride. We also investigated larval growth as a physiological descriptor by measuring changes in the body fresh weight and dry weight after chemical exposure. The response of the HSPs gene expression by chemical exposure was rapid and sensitive to low chemical concentrations but it was not stressor specific. Interestingly, an increase in the expression of HSPs genes was observed not only in a stress inducible form (HSP70), but also in a constitutively (HSC70) expressed form. The expression of Hb genes showed chemical-specific responses: that is, alkyl phenolic compounds increased the expression of hemoglobin genes, whereas pesticides decreased the expression. As expected, molecular-level markers were more sensitive than physiological endpoints, suggesting that gene expression could be developed as an early warning biomarker in this animal. The overall results suggest that the expression of HSP and Hb genes in Chironomus could give useful information for diagnosing general health conditions in fresh water ecosystem. The expression of Hb genes, in particular, seems to be a promising biomarker, especially in view of the potential of Chironomus larvae as a biomonitoring species and of the physiological particularities of their respiratory pigments.

Redox regulation of OxyR requires specific disulfide bond formation involving a rapid kinetic reaction path.

The Escherichia coli OxyR transcription factor is activated by cellular hydrogen peroxide through the oxidation of reactive cysteines. Although there is substantial evidence for specific disulfide bond formation in the oxidative activation of OxyR, the presence of the disulfide bond has remained controversial. By mass spectrometry analyses and in vivo labeling assays we found that oxidation of OxyR in the formation of a specific disulfide bond between Cys199 and Cys208 in the wild-type protein. In addition, using time-resolved kinetic analyses, we determined that OxyR activation occurs at a rate of 9.7 s(-1). The disulfide bond-mediated conformation switch results in a metastable form that is locally strained by approximately 3 kcal mol(-1). On the basis of these observations we conclude that OxyR activation requires specific disulfide bond formation and that the rapid kinetic reaction path and conformation strain, respectively, drive the oxidation and reduction of OxyR.

Structure of human FIH-1 reveals a unique active site pocket and interaction sites for HIF-1 and von Hippel-Lindau.

The master switch of cellular hypoxia responses, hypoxia-inducible factor 1 (HIF-1), is hydroxylated by factor inhibiting HIF-1 (FIH-1) at a conserved asparagine residue under normoxia, which suppresses transcriptional activity of HIF-1 by abrogating its interaction with transcription coactivators. Here we report the crystal structure of human FIH-1 at 2.8-A resolution. The structural core of FIH-1 consists of a jellyroll-like beta-barrel containing the conserved ferrous-binding triad residues, confirming that FIH-1 is a member of the 2-oxoglutarate-dependent dioxygenase family. Except for the core structure and triad residues, FIH-1 has many structural deviations from other family members including N- and C-terminal insertions and various deletions in the middle of the structure. The ferrous-binding triad region is highly exposed to the solvent, which is connected to a prominent groove that may bind to a helix near the hydroxylation site of HIF-1. The structure, which is in a dimeric state, also reveals the putative von Hippel-Lindau-binding site that is distinctive to the putative HIF-1-binding site, supporting the formation of the ternary complex by FIH-1, HIF-1, and von Hippel-Lindau. The unique environment of the active site and cofactor-binding region revealed in the structure should allow design of selective drugs that can be used in ischemic diseases to promote hypoxia responses.