Power-line frequency electromagnetic fields do not induce changes in phosphorylation, localization, or expression of the 27-kilodalton heat shock protein in human keratinocytes.

The linkage of the exposure to the power-line frequency (50-60 Hz) electromagnetic fields (EMF) with human cancers remains controversial after more than 10 years of study. The in vitro studies on the adverse effects of EMF on human cells have not yielded a clear conclusion. In this study, we investigated whether power-line frequency EMF could act as an environmental insult to invoke stress responses in human keratinocytes using the 27-kDa heat shock protein (HSP27) as a stress marker. After exposure to 1 gauss (100 micro T) EMF from 20 min to 24 hr, the isoform pattern of HSP27 in keratinocytes remained unchanged, suggesting that EMF did not induce the phosphorylation of this stress protein. EMF exposure also failed to induce the translocation of HSP27 from the cytoplasm to the nucleus. Moreover, EMF exposure did not increase the abundance of HSP27 in keratinocytes. In addition, we found no evidence that EMF exposure enhanced the level of the 70-kDa heat shock protein (HSP70) in breast or leukemia cells as reported previously. Therefore, in this study we did not detect any of a number of stress responses in human keratinocytes exposed to power-line frequency EMF

HIV-1 integrase crosslinked oligomers are active in vitro

The oligomeric state of active human immunodeficiency virus type 1 (HIV-1) integrase (IN) has not been clearly elucidated. We analyzed the activity of the different purified oligomeric forms of recombinant IN obtained after stabilization by platinum crosslinking. The crosslinked tetramer isolated by gel chromatography was able to catalyze the full-site integration of the two viral LTR ends into a target DNA in vitro, whereas the isolated dimeric form of the enzyme was involved in the processing and integration of only one viral end. Accurate concerted integration by IN tetramers was confirmed by cloning and sequencing. Kinetic studies of DNA-integrase complexes led us to propose a model explaining the formation of an active complex. Our data suggest that the tetrameric IN bound to the viral DNA ends is the minimal complex involved in the concerted integration of both LTRs and should be the oligomeric form targeted by future inhibitors

DNA compaction by the higher-order assembly of PRH/Hex homeodomain protein oligomers

Protein self-organization is essential for the establishment and maintenance of nuclear architecture and for the regulation of gene expression. We have shown previously that the Proline-Rich Homeodomain protein (PRH/Hex) self-assembles to form oligomeric complexes that bind to arrays of PRH binding sites with high affinity and specificity. We have also shown that many PRH target genes contain suitably spaced arrays of PRH sites that allow this protein to bind and regulate transcription. Here, we use analytical ultracentrifugation and electron microscopy to further characterize PRH oligomers. We use the same techniques to show that PRH oligomers bound to long DNA fragments self-associate to form highly ordered assemblies. Electron microscopy and linear dichroism reveal that PRH oligomers can form protein–DNA fibres and that PRH is able to compact DNA in the absence of other proteins. Finally, we show that DNA compaction is not sufficient for the repression of PRH target genes in cells. We conclude that DNA compaction is a consequence of the binding of large PRH oligomers to arrays of binding sites and that PRH is functionally and structurally related to the Lrp/AsnC family of proteins from bacteria and archaea, a group of proteins formerly thought to be without eukaryotic equivalents

DNA compaction by the higher-order assembly of PRH/Hex homeodomain protein oligomers

Protein self-organization is essential for the establishment and maintenance of nuclear architecture and for the regulation of gene expression. We have shown previously that the Proline-Rich Homeodomain protein (PRH/Hex) self-assembles to form oligomeric complexes that bind to arrays of PRH binding sites with high affinity and specificity. We have also shown that many PRH target genes contain suitably spaced arrays of PRH sites that allow this protein to bind and regulate transcription. Here, we use analytical ultracentrifugation and electron microscopy to further characterize PRH oligomers. We use the same techniques to show that PRH oligomers bound to long DNA fragments self-associate to form highly ordered assemblies. Electron microscopy and linear dichroism reveal that PRH oligomers can form protein–DNA fibres and that PRH is able to compact DNA in the absence of other proteins. Finally, we show that DNA compaction is not sufficient for the repression of PRH target genes in cells. We conclude that DNA compaction is a consequence of the binding of large PRH oligomers to arrays of binding sites and that PRH is functionally and structurally related to the Lrp/AsnC family of proteins from bacteria and archaea, a group of proteins formerly thought to be without eukaryotic equivalents

The PRH/Hex repressor protein causes nuclear retention of Groucho/TLE co-repressors

The Proline-Rich Homeodomain protein (PRH/Hex) is a transcription factor that functions as an important regulator of vertebrate development and many other processes in the adult including haematopoiesis. The Groucho/TLE family of co-repressor proteins also regulate development and modulate the activity of many DNA-binding transcription factors during a range of diverse cellular processes including haematopoiesis. We have shown previously that PRH is a repressor of transcription in haematopoietic cells and that an Eh-1 motif present within the N-terminal transcription repression domain of PRH mediates binding to Groucho/TLE proteins and enables co-repression. Here we demonstrate that PRH regulates the nuclear retention of TLE proteins during cellular fractionation. We show that transcriptional repression and the nuclear retention of TLE proteins requires PRH to bind to both TLE and DNA. In addition, we characterise a trans-dominant negative PRH protein that inhibits wild type PRH activity by sequestering TLE proteins to specific sub-nuclear domains. These results demonstrate that transcriptional repression by PRH is dependent on TLE availability and suggest that sub-nuclear localisation of TLE plays an important role in transcriptional repression by PRH

CK2 phosphorylation of the PRH/Hex homeodomain functions as a reversible switch for DNA binding

The proline-rich homeodomain protein (PRH/Hex) regulates transcription by binding to specific DNA sequences and regulates mRNA transport by binding to translation initiation factor eIF4E. Protein kinase CK2 plays multiple roles in the regulation of gene expression and cell proliferation. Here, we show that PRH interacts with the β subunit of CK2 in vitro and in cells and that CK2 phosphorylates PRH. Phosphorylation of PRH by CK2 inhibits the DNA binding activity of this protein and dephosphorylation restores DNA binding indicating that this modification acts as a reversible switch. We show that phosphorylation of the homeodomain is sufficient to block DNA binding and we identify two amino acids within this the domain that are phosphorylated by CK2: S163 and S177. Site-directed mutagenesis demonstrates that mutation of either of these residues to glutamic acid partially mimics phosphorylation but is insufficient to completely block DNA binding whereas an S163E/S177E double mutation severely inhibits DNA binding. Significantly, the S163E and S177E mutations and the S163E/S177E double mutation all inhibit the ability of PRH to regulate transcription in cells. Since these amino acids are conserved between many homeodomain proteins, our results suggest that CK2 may regulate the activity of several homeodomain proteins in this manner

Characterization of volatile compounds of Daucus crinitus Desf. Headspace Solid Phase Microextraction as alternative technique to Hydrodistillation

<p>Abstract</p> <p>Background</p> <p>Traditionally, the essential oil of aromatic herbs is obtained using hydrodistillation (HD). Because the emitted volatile fraction plays a fundamental role in a plant's life, various novel techniques have been developed for its extraction from plants. Among these, headspace solid phase microextraction (HS-SPME) can be used to obtain a rapid fingerprint of a plant's headspace. <it>Daucus crinitus </it>Desf. is a wild plant that grows along the west coast of Algeria. Only a single study has dealt with the chemical composition of the aerial part oils of Algerian <it>D. crinitus</it>, in which isochavicol isobutyrate (39.0%), octyl acetate (12.3%), and β-caryophyllene (5.4%) were identified. Using GC-RI and GC-MS analysis, the essential oils and the volatiles extracted from separated organs of <it>D. crinitus </it>Desf. were studied using HS-SPME.</p> <p>Results</p> <p>GC-RI and GC-MS analysis identified 72 and 79 components in oils extracted using HD and in the volatile fractions extracted using SPME, respectively. Two types of essential oils were produced by the plant: the root oils had aliphatic compounds as the main component (87.0%-90.1%), and the aerial part oils had phenylpropanoids as the main component (43.1%-88.6%). HS-SPME analysis showed a more precise distribution of compounds in the organs studied: oxygenated aliphatic compounds were well represented in the roots (44.3%-84.0%), hydrocarbon aliphatic compounds were in the leaves and stems (22.2%-87.9%), and phenylpropanoids were in the flowers and umbels (47.9%-64.2%). Moreover, HS-SPME allowed the occurrence of isochavicol (29.6 - 34.7%) as main component in <it>D. crinitus </it>leaves, but it was not detected in the oils, probably because of its solubility in water.</p> <p>Conclusions</p> <p>This study demonstrates that HD and HS-SPME modes could be complimentary extraction techniques in order to obtain the complete characterization of plant volatiles.</p

La question d'Alger politique, colonisation, commerce

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