Volatile organic compounds cytotoxicity and expression of HSP72, HSP90 and GRP78 stress proteins in cultured human cells

The aim of this study was to determine whether overexpression of stress proteins (SPs) could be a sensitive biomarker for cell injury due to exposure to low doses of volatile organic compounds (VOCs) such as benzene, ethylbenzene, toluene, xylene, and chlorinated derivatives (ClB). Sublethal and cytotoxic threshold concentrations of the VOCs were determined by studying the growth rate of normal (fibroblasts) or tumor-derived human cell lines (A549, HepG2) exposed for 4 days to VOCs. Changes in SP expression as a function of concentrations were investigated by Western blotting.VOC toxicity was found to be correlated with their degree of chlorination and their hydrophobicity. Cytotoxic threshold concentrations (no-observed effect concentration, NOEC) were found to be similar for the three cell lines. It was observed that using a mixture of VOCs, each of them at concentration below the NOEC, resulted in an actual toxicity to the cells. This finding reveals a synergistic effect and should be taken into account when assessing threshold risk and exposure limit values in the worker's environment when several pollutants may be present. HSP72 and HSP90 expression levels were not affected whereas GRP78 expression was increased by all the VOCs. Taking into account the specific molecular function of GRP78, it suggests that VOC exposure results in misfolded or underglycosylated protein accumulation in the endoplasmic reticulum. GRP78 overexpression was closely related to the magnitude of growth inhibition due to increasing concentrations of each VOC. The overexpression was found to be significant for concentrations 5 to 30 times higher than NOEC, indicating that, under our experimental conditions, GRP78 expression cannot be considered as a sensitive biomarker of exposure to environmental VOCs.     

Interactions between the isolated oxygenase and reductase domains of neuronal nitric-oxide synthase: assessing the role of calmodulin

Nitric-oxide synthase (NOS) is a fusion protein composed of an oxygenase domain with a heme-active site and a reductase domain with an NADPH binding site and requires Ca(2+)/calmodulin (CaM) for NO formation activity. We studied NO formation activity in reconstituted systems consisting of the isolated oxygenase and reductase domains of neuronal NOS with and without the CaM binding site. Reductase domains with 33-amino acid C-terminal truncations were also examined. These were shown to have faster cytochrome c reduction rates in the absence of CaM. N(G)-hydroxy-l-Arg, an intermediate in the physiological NO synthesis reaction, was found to be a viable substrate. Turnover rates for N(G)-hydroxy-l-Arg in the absence of Ca(2+)/CaM in most of the reconstituted systems were 2.3-3.1 min(-1). Surprisingly, the NO formation activities with CaM binding sites on either reductase or oxygenase domains were decreased dramatically on addition of Ca(2+)/CaM. However, NADPH oxidation and cytochrome c reduction rates were increased by the same procedure. Activation of the reductase domains by CaM addition or by C-terminal deletion failed to increase the rate of NO synthesis. Therefore, both mechanisms appear to be less important than the domain-domain interaction, which is controlled by CaM binding in wild-type neuronal NOS, but not in the reconstituted systems.     

Dual role for phosphoinositides in regulation of yeast and mammalian phospholipase D enzymes

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that regulates diverse biological processes by binding to a family of G protein-coupled receptors or as an intracellular second messenger. Mammalian S1P phosphatase (SPP-1), which degrades S1P to terminate its actions, was recently cloned based on homology to a lipid phosphohydrolase that regulates the levels of phosphorylated sphingoid bases in yeast. Confocal microscopy surprisingly revealed that epitope-tagged SPP-1 is intracellular and colocalized with the ER marker calnexin. Moreover, SPP-1 activity and protein appeared to be mainly enriched in the intracellular membranes with lower expression in the plasma membrane. Treatment of SPP-1 transfectants with S1P markedly increased ceramide levels, predominantly in the intracellular membranes, diminished survival, and enhanced apoptosis. Remarkably, dihydro-S1P, although a good substrate for SPP-1 in situ, did not cause significant ceramide accumulation or increase apoptosis. Ceramide accumulation induced by S1P was completely blocked by fumonisin B1, an inhibitor of ceramide synthase, but only partially reduced by myriocin, an inhibitor of serine palmitoyltransferase, the first committed step in de novo synthesis of ceramide. Furthermore, S1P, but not dihydro-S1P, stimulated incorporation of [3H]palmitate, a substrate for both serine palmitoyltransferase and ceramide synthase, into C16-ceramide. Collectively, our results suggest that SPP-1 functions in an unprecedented manner to regulate sphingolipid biosynthesis and is poised to influence cell fate.     

Drosophila Aurora A kinase is required to localize D-TACC to centrosomes and to regulate astral microtubules

Disruption of the function of the A-type Aurora kinase of Drosophila by mutation or RNAi leads to a reduction in the length of astral microtubules in syncytial embryos, larval neuroblasts, and cultured S2 cells. In neuroblasts, it can also lead to loss of an organized centrosome and its associated aster from one of the spindle poles, whereas the centrosome at the other pole has multiple centrioles. When centrosomes are present at the poles of aurA mutants or aurA RNAi spindles, they retain many antigens but are missing the Drosophila counterpart of mammalian transforming acidic coiled coil (TACC) proteins, D-TACC. We show that a subpopulation of the total Aurora A is present in a complex with D-TACC, which is a substrate for the kinase. We propose that one of the functions of Aurora A kinase is to direct centrosomal organization such that D-TACC complexed to the MSPS/XMAP215 microtubule-associated protein may be recruited, and thus modulate the behavior of astral microtubules.     

A role for the exosome in the in vivo degradation of unstable mRNAs

In mammals, the mRNAs encoding many proteins involved in inflammation bear destabilizing AU-rich elements (AREs) in the 3'-untranslated region. The exosome, a complex of 3' --> 5' exonucleases, is rate limiting in the destruction of such mRNAs in a mammalian in vitro system, but a role in vivo has not been demonstrated. The phenomenon of ARE-mediated degradation also occurs in the protist parasite Trypanosoma brucei. Messenger RNAs with 3'-untranslated region U-rich elements, which strongly resemble AREs, are extremely unstable in the trypanosome form that parasitizes mammals. The first step in degradation of these mRNAs in vivo is rapid destruction of the 3'-untranslated region; subsequently the mRNA is destroyed by exonucleases acting in both 5' --> 3' and 3' --> 5' directions. We here investigated the roles of three subunits of the trypanosome exosome complex, RRP45, RRP4, and CSL4, in this process, depleting the individual subunits in vivo by inducible RNA interference. RRP45 depletion, which probably disrupts exosome integrity, caused a delay in the onset of degradation of the very unstable RNAs, but did not affect degradation of more stable species. Depletion of RRP4 or CSL4 does not affect the stability of the residual exosome and did not change mRNA degradation kinetics. We conclude that the exosome is required for the initiation of rapid degradation of unstable mRNAs in trypanosomes.     

BAG-1--a nucleotide exchange factor of Hsc70 with multiple cellular functions

BAG-1 (Bcl-2-associated athanogene) is a multifaceted protein implicated in the modulation of a large variety of cellular processes. Elucidating the molecular mechanisms that underlie the cellular functions of BAG-1 becomes an increasingly important task, particularly in light of the growing evidence connecting aberrant BAG-1 expression to certain human cancers. A common element of the remarkable functional diversity of BAG-1 appears to be the interaction with molecular chaperones of the Hsp70 family. In fact, BAG-1 functions as a nucleotide exchange factor of mammalian cytosolic Hsc70, thereby triggering substrate unloading from the chaperone. In addition, recent findings reveal an association of BAG-1 with the proteasome, which suggests a role in coordinating chaperone and degradation pathways.     

Q-Gene: processing quantitative real-time RT-PCR data

SUMMARY: Q-Gene is an application for the processing of quantitative real-time RT-PCR data. It offers the user the possibility to freely choose between two principally different procedures to calculate normalized gene expressions as either means of Normalized Expressions or Mean Normalized Expressions. In this contribution it will be shown that the calculation of Mean Normalized Expressions has to be used for processing simplex PCR data, while multiplex PCR data should preferably be processed by calculating Normalized Expressions. The two procedures, which are currently in widespread use and regarded as more or less equivalent alternatives, should therefore specifically be applied according to the quantification procedure used. AVAILABILITY: Web access to this program is provided at http://www.biotechniques.com/softlib/qgene.html     

Digital extractor: analysis of digital differential display output

Digital Extractor is a program for the high-throughput processing of data sets derived from digital differential display-based comparisons of EST libraries. These comparisons can be utilized to identify discrete subsets of genes whose expression is restricted to distinct tissue types. The program facilitates these investigations by permitting parallel annotation of genes identified as being differentially expressed.     

SCide: identification of stabilization centers in proteins

SUMMARY: SCide is a program to identify stabilization centers from known protein structures. These are residues involved in cooperative long-range contacts, which can be formed between various regions of a single polypeptide chain, or they can belong to different peptides or polypeptides in a complex. The server takes a PDB file as an input, and the result is presented in graphical or text format. AVAILABILITY: SCide is available on the web at http://www.enzim.hu/scide. The source code can be obtained from the authors on request.