Biochemical and biophysical research communications
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Biochem. Biophys. Res. Commun. · Feb 2000
Pyrrolidine dithiocarbamate induces apoptosis by a cytochrome c-dependent mechanism.
Pyrrolidine dithiocarbamate (PDTC) is a synthetic antioxidant molecule, which has been recently proposed as an antitumoral agent on the basis of its capability of inducing apoptosis. We investigated the effect of PDTC on the proliferation and survival of the promyelocitic cell line HL-60. Concentration as low as 10 microM of PDTC induces a significant reduction of the growth rate and the contemporaneous activation of the apoptotic process. ⋯ PDTC-dependent apoptosis was associated with an early release of cytochrome c from mitochondria, while the involvement of pathways due to cell death receptors engagement was ruled out by detailed time-course analyses of caspases 3 and 8 activation. Moreover, no up-regulation of p21(CIP1) level, a pivotal cyclin-dependent kinase inhibitor, occurred at PDTC concentration able to induce apoptosis. Finally, in vitro incubation of purified mitochondria with PDTC demonstrated that the molecule is directly able to induce cytochrome c release from the intermembrane space, thus confirming that mitochondria are a primary cellular target of the molecule.
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Biochem. Biophys. Res. Commun. · Feb 2000
Rhythmic expression of BMAL1 mRNA is altered in Clock mutant mice: differential regulation in the suprachiasmatic nucleus and peripheral tissues.
BMAL1 is a putative clock gene which encodes a basic helix-loop-helix (bHLH)-PAS transcription factor. To examine whether the CLOCK protein is required for the circadian expression of BMAL1 mRNA, in situ hybridization and Northern blot analysis were performed in the suprachiasmatic nucleus (SCN) and peripheral tissues of homozygous Clock mutant mice. In the SCN of Clock mutants, BMAL1 mRNA did not oscillate significantly but apparently expressed with low levels, while in wild-type mice the mRNA was robustly oscillated in a circadian manner. ⋯ Furthermore, expressions of BMAL1 mRNA in the peripheral of Clock mutant mice were close to the peak level in wild-type mice, whereas mPer2 mRNA levels were severely blunted at trough values. Daily expression of albumin site D-binding protein (DBP), a clock controlled output gene (CCG), was also abolished at trough values by the Clock mutation in all tissues examined. These observations suggest that the circadian expression of BMAL1 mRNA is affected by the CLOCK-induced transcriptional feedback loop in the SCN and peripheral tissues in a different way and that the regulation mechanism appeared to be different from those in mPer2 and DBP expressions in vivo.
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Biochem. Biophys. Res. Commun. · Jan 2000
Coordinate recruitment of E-cadherin and ALCAM to cell-cell contacts by alpha-catenin.
Here we report on the role of alpha-catenin in the cellular localization of activated leukocyte cell adhesion molecule, ALCAM, and cadherin-mediated cell adhesion in human prostate cancer cells. Cell lines that have a functional E-cadherin-mediated cell adhesion (DU-145 and LNCaP) show ALCAM staining at cell-cell contacts. ⋯ A role of alpha-catenin in the recruitment of E-cadherin and ALCAM to cell-cell contacts was established by transfection of an alpha-N-catenin construct into cell lines ALVA-31 and PC-3. This resulted not only in the correct assembly of E-cadherin/alpha-catenin complexes at the cell membrane but also in localization of ALCAM to cell-cell contacts, indicating that indeed alpha-catenin affects ALCAM localization.
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Biochem. Biophys. Res. Commun. · Jan 1999
Cloning and characterization of DYRK1B, a novel member of the DYRK family of protein kinases.
The DYRK1A gene on human chromosome 21 encodes a protein kinase presumed to be involved in the pathogenesis of mental retardation in Down's syndrome. Here we describe a highly similar homolog, DYRK1B, which is, in contrast to DYRK1A, predominately expressed in muscle and testis. The human DYRK1B gene was mapped to chromosome 19 (19q12-13.11) by radiation hybrid analysis. ⋯ In addition, the sequence comprises a bipartite nuclear localization motif. A green fluorescent protein (GFP) fusion protein of DYRK1B was found mainly in the nucleus of transfected COS-7 cells. These data suggest that DYRK1B is a muscle- and testis-specific isoform of DYRK1A and is involved in the regulation of nuclear functions.
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Biochem. Biophys. Res. Commun. · Jan 1999
Localisation of cis regulatory elements at the beta-globin locus: analysis of hybrid haplotype chromosomes.
Several cis elements at the beta-globin gene cluster and the upstream locus control region (LCR) have been implicated in modulation of fetal haemoglobin (Hb F) level in beta-globin disorders. To determine the role of elements at the LCR and the beta-globin gene cluster on HbF level among sickle cell anaemia (SCA) patients, hybrid haplotype betaS chromosomes exhibiting variation in the association of alleles of LCR hypersensitive site 2 (HS2) and the beta-globin gene cluster restriction fragment length polymorphosim (RFLP) haplotypes were identified in an unselected population of 100 patients. On 15 chromosomes the polymorphic HS2 short tandem repeat(TA)xN10-12(TA)y containing a Hox2 binding motif differed from that typically associated with the corresponding beta-globin gene cluster RFLP haplotype. ⋯ Previously described hybrid haplotype chromosomes with the (TA)9N10(TA)10 HS2 allele associated with raised HbF by contrast arise by recombination 1 kb downstream of the pre-Ggamma framework. This study suggests that variability in HbF level associated with polymorphisn of the HS2 enhancer depend on downstream determinant (s) in tight linkage disequilibrium with HS2. The pre-Ggamma framework is the only known polymorphic cis-active determinant in this region.