The seemingly static architecture of interphase and mitotic chromatin betrays an otherwise elegantly dynamic entity capable of remodelling itself to facilitate DNA replication, transcription, repair and recombination. Remodelling of local chromatin domains in response to physiological cues proceeds, at least in part, through transient cycles of relaxation and condensation that require use of histone variants and post-translational modifications of histones. Studies have connected poly(ADP-ribosyl)ation of histones with virtually every aspect of DNA metabolism and function over the years, most notably with the response to DNA damage, where convincing evidence supports its essential role granting repair machinery access to damaged DNA. Recent reports extend this notion to transcription and the maintenance of genomic stability, thereby supporting a general role for nuclear poly(ADPribosyl)ation in many aspects of genomic activity. The phenomenon might contribute to the 'histone code' by dictating levels of local chromatin compaction.

Periodic elevation of ambient particulate matter and ozone levels is linked to acute cardiac morbidity and mortality. Increased plasma levels of the potent vasoconstrictor endothelin (ET)-1, a prognostic indicator of cardiac mortality, have been detected in both animal models and humans after exposure to air pollutants. The lungs are the primary source of circulating ET-1, but the direct effects of individual air pollutants and their interaction in modulating the pulmonary endothelin system are unknown. Fischer-344 rats were exposed to particles (0, 5, 50 mg/m3 EHC-93), ozone (0, 0.4, 0.8 ppm), or combinations of particles and ozone for 4 h. Changes in gene expression were measured using real-time reverse transcription polymerase chain reaction immediately after exposure and following 24 h recovery in clean air. Both pollutants individually increased preproET-1, endothelin converting enzyme-1, and endothelial nitric oxide synthase mRNA levels in the lungs shortly after exposure, consistent with the concomitant increase in plasma of the 21 amino acid ET-1[1-21] peptide measured by HPLC-fluorescence. PreproET-1 mRNA remained elevated 24 h after exposure to particles but not after ozone, in line with previously documented changes of the peptide in plasma. Both pollutants transiently increased endothelin-B receptor mRNA expression, while ozone decreased endothelin-A receptor mRNA levels. Coexposure to particles plus ozone increased lung preproET-1 mRNA but not plasma ET-1[1-21], suggesting alternative processing or degradation of endothelins. This coincided with an increase in the lungs of matrix metalloproteinase-2 (MMP-2), an enzyme that cleaves bigET-1 to ET-1[1-32]. Taken together, our data indicate that ozone and particulate matter independently regulate the expression of lung endothelin system genes, but show complex toxicological interaction with respect to plasma ET-1.

Thiazide diuretics have been shown to decrease bone-loss rate and to improve bone mineral density in patients using this medication. However, the exact role of thiazides on bone cells is still debated. In the present work, we studied whether thiazides could affect the normal features of osteoblasts using the human model cell line MG-63. Hydrochlorothiazide (HCTZ) did not affect cell growth nor DNA synthesis in these cells, yet slightly increased alkaline phosphatase activity in these cells at pharmacologically relevant concentrations. Under similar conditions, HCTZ dose-dependently inhibited 1,25(OH)2D3-induced osteocalcin secretion by these cells (maximal effect, -40 to 50%, P < 0.005). However, HCTZ did not inhibit the basal production of osteocalcin in MG-63 cells (without 1,25(OH)2D3 induction), which was very low to undectable. Two different thiazide derivatives, chlorothiazide and cyclothiazide, and two structurally related sulfonamides with selective inhibition of carbonic anhydrase (Acetazolamide) or hyperglycemic effects (Diazoxide) were also tested. Chlorothiazide (1000 microM) inhibited osteocalcin secretion (-42 +/- 12.7%) at doses 10-fold higher than HCTZ (100 microM) while cyclothiazide was effective at doses of 1 microM (-27 +/- 3.6%), and hence 100-fold lower than HCTZ, compatible with the relative natriuretic effect in vivo of these compounds. Acetazolamide (10 microM) poorly affected osteocalcin secretion at doses 100-fold higher than those needed in vivo to inhibit carbonic anhydrase. Likewise, Diazoxide (100 microM) poorly affected osteocalcin secretion at doses known to promote its biological effect. Higher doses of acetazolamide and diazoxide induced cell death. Neither Acetazolamide nor Diazoxide affected alkaline phosphatase, whereas chlorothiazide had a weak positive effect on this enzymatic activity. The production of macrophage colony-stimulating factor (M-CSF) was stimulated in the presence of 1,25(OH)2D3 (50 nM), TNF-alpha (2 ng/ml) or both in MG-63 cells. HCTZ (25 microM, 24 hr of preincubation) did not modify basal M-CSF production and did not reduce the response to 1,25(OH)2D3 alone. In contrast, HCTZ inhibited the response to TNF-alpha alone (P < 0.05), and also reduced the response to a combination of 1,25(OH)2D3 and TNF-alpha (P < 0.01). In conclusion, these results indicate that thiazide diuretics show a selective inhibition of osteocalcin secretion and M-CSF production by MG-63 cells unlike structurally related drugs. Therefore, these features may explain, in part, the positive effect of thiazides on bone mineral density.

Current evidence suggests that the mixed lineage kinase family member dual leucine zipper-bearing kinase (DLK) might play a significant role in the regulation of cell growth and differentiation, particularly during the process of tissue remodeling. To further explore this working model, we have investigated the regulation of host and recombinant DLK in NIH3T3 and COS-1 cells undergoing apoptosis. Using calphostin C, a potent and selective inhibitor of protein kinase C and a recognized apoptosis inducer for various cell types, we demonstrate, by immunoblot analysis, that DLK protein levels are rapidly and dramatically down-regulated during the early phases of apoptosis. Down-regulation in calphostin C-treated cells was also accompanied by the appearance of SDS-and mercaptoethanol-resistant high molecular weight DLK immunoreactive oligomers. Experiments aimed at elucidating the mechanism(s) underlying DLK oligomerization revealed that the tissue transglutaminase (tTG) inhibitor monodansylcadaverine antagonized the effects of calphostin C almost completely, thereby suggesting the involvement of a tTGcatalyzed reaction as the root cause of DLK downregulation and accumulation as high molecular weight species. In support of this notion, we also show that DLK can serve as a substrate for tTG-dependent cross-linking in vitro and that this covalent post-translational modification leads to the functional inactivation of DLK. Taken together, these observations suggest that transglutamination and oligomerization may constitute a relevant physiological mechanism for the regulation of DLK activity.

Previous reports have suggested the involvement of voltage-activated calcium (Ca 2؉) channels in bone metabolism and in particular on the secretion of osteocalcin by osteoblast-like cells.(1) We now report that potassium (K

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The pattern of nucleosomal histones poly(ADP-ribosy1)ation is changed under conditions which affect the poly(ADP-ribosy1)ation state of the enzyme. At low NAD concentrations the enzyme can poly(ADP-ribosy1)ate histones H1 and H1 O, H2A, A2A, and H2B. However at NAD concentrations above 10 yM the enzyme preferentially poly(ADP-ribosy1)ates histone H1 to a hyper ADP-ribosylated form. Furthermore we have observed hyper ADP-ribosylation of histone H2B at NAD concentrations of 10 yM suggesting that histone H2B can undergo the same type of ADP-ribosylation pattern as histone H1. Also at higher NAD concentrations an elongation of the polymer attached to the enzyme and other nuclear proteins takes place.There is growing evidence that poly(ADP-ribosy1)ation of nucleosomal proteins, which is a post-translational modification, might be related to the alteration of the chromatin structure during the cellular process of DNA repair and replication [l -31. The reaction is catalyzed by the nuclear enzyme poly(ADP4bose) polymerase [4 -61 which can act not only as a catalyst but also as the acceptor of the product of its own activity [7-91. Amongst the histone proteins, it has been shown that histones HI, H2B, A24 and to a lesser extent histones H3 and H2A could be poly(ADP-ribosy1)ated in nuclei as well as in nucleosomes [lo-121. Histone HI has been described to be the major histone acceptor in trout testis nuclei [13] and pancreatic nuclei and nucleosomes [14, 151 at NAD concentrations above 100 pM; whereas in rat liver and HeLa cells histones H1 and H2B have been shown to be the major histone protein acceptors of poly(ADP-ribose) [12,16].The biological role of the polymer has been ascribed to different nuclear events such as: DNA replication [17], DNA repair [18, 191, cell growth and differentiation [20, 211. Furthermore it has been shown that there is an increase in the poly(ADP-ribosy1)ation of histone HI during DNA repair in permeabilized cells [22]. Also studies in vivo have shown increased poly(ADP-ribosy1)ation of histone H1 and core histones [23] during DNA repair. Cell cycle studies have indicated increased poly(ADP-ribosy1)ation of histone H1 during S phase [24]. It has been suggested recently by Poirier et al. [25] that poly(ADP-ribosy1)ation of histone H1 could alter the chromatin structure and be one of the mechanisms by which such nuclear events could be facilitated [25]. Indeed, they observed an opening of the chromatin structure, that is a relaxation, following the poly(ADP-ribosy1)ation of histone H1 both upon the addition of exogenous poly(ADP-ribose) polymerase [25] or by the intrinsic enzymatic activity of the nucleosomes [26].Abbreviations. Poly(ADP-ribose), polymer of adenosine 5'-diphosphate ribose; LDS, lithium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis.Enzyme. Poly(ADP-ribose)polymerase (EC 2.4.99. -).In order to have a better understanding of the interaction between poly(ADP-ribose) polymerase and nucleosomal proteins during poly(ADP-ribosyl)ation, we have performed acceptor studies und...

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