Efficacy of antibacterial-loaded coating in an in vivo model of acutely highly contaminated implant.

PURPOSE: The purpose of this study was to test the ability of DAC®, a fast resorbable, antibacterial-loaded hydrogel coating, to prevent acute bacterial colonization in an in vivo model of an intra-operatively highly contaminated implant. METHODS: A histocompatibility study was performed in 10 adult New Zealand rabbits. Then, methicillin-resistant Staph. aureus were inoculated in the femur of 30 adult New Zealand rabbits at the time of intra-medullary nailing; vancomycin-loaded DAC® coated nails were compared to controls regarding local and systemic infection development. RESULTS: Histocompatibility study showed no detrimental effect of DAC® hydrogel on bone tissue after 12 weeks from implant. After seven days from implant, none of the rabbits receiving vancomycin-loaded DAC® nail showed positive blood cultures, compared to all the controls; vancomycin-loaded DAC® coating was associated with local bacterial load reduction ranging from 72 to 99 %, compared to controls. CONCLUSIONS: Vancomycin-loaded DAC® coating is able to significantly reduce bacterial colonization in an animal model of an intra-operatively highly contaminated implant, without local or general side effect.

Acetylation of UBF changes during the cell cycle and regulates the interaction of UBF with RNA polymerase I.

The upstream binding factor UBF, an activator of RNA polymerase I transcription, is posttranslationally modified by phosphorylation and acetylation. We found that in NIH3T3 cells, UBF is acetylated in S-phase but not in G1-phase. To assess the role of acetylation in regulation of UBF activity, we have established an NIH3T3 cell line that inducibly overexpresses HDAC1. Both in vivo and in vitro, HDAC1 efficiently hypoacetylates UBF. Immunoprecipitation with antibodies against the Pol I-associated factor PAF53 co-precipitated UBF in mock cells but not in cells overexpressing HDAC1. Pull-down experiments showed that acetylation of UBF augments the interaction with Pol I. Consistent with acetylation of UBF being important for association of PAF53 and recruitment of Pol I, the level of Pol I associated with rDNA and pre-rRNA synthesis were reduced in cells overexpressing HDAC1. The results suggest that acetylation and deacetylation of UBF regulate rRNA synthesis during cell cycle progression.

Succinate synthesis and excretion by Penicillium simplicissimum under aerobic and anaerobic conditions.

Succinate is an interesting chemical for industries producing food and pharmaceutical products, surfactants, detergents and biodegradable plastics. Succinate is produced mainly by a mixed-acid fermentation process using anaerobically growing bacteria. However, succinate excretion is also widespread among fungi. In this article we report results on the intracellular concentration and the excretion of succinate by Penicillium simplicissimum under aerobic and anaerobic conditions. The intracellular concentration of succinate increased slightly with the specific growth rate and strongly if the respiratory chain was inhibited by sodium azide or anaerobic conditions (N(2)). A strong increase of succinate excretion was observed if the respiratory chain was inhibited. It is suggested that succinate synthesis under functional (sodium azide) or environmental (N(2)) anaerobic conditions occurs via the reductive part of the tricarboxylic acid cycle. Succinate is then excreted because the oxidative part of the tricarboxylic acid cycle is inactive. A possible role of succinate synthesis in the regeneration of NAD ('fumarate respiration') is discussed.

In vitro phosphorylation and acetylation of the murine pocket protein Rb2/p130.

The retinoblastoma protein (pRb) and the related proteins Rb2/p130 and 107 represent the "pocket protein" family of cell cycle regulators. A key function of these proteins is the cell cycle dependent modulation of E2F-regulated genes. The biological activity of these proteins is controlled by acetylation and phosphorylation in a cell cycle dependent manner. In this study we attempted to investigate the interdependence of acetylation and phosphorylation of Rb2/p130 in vitro. After having identified the acetyltransferase p300 among several acetyltransferases to be associated with Rb2/p130 during S-phase in NIH3T3 cells in vivo, we used this enzyme and the CDK4 protein kinase for in vitro modification of a variety of full length Rb2/p130 and truncated versions with mutations in the acetylatable lysine residues 1079, 128 and 130. Mutation of these residues results in the complete loss of Rb2/p130 acetylation. Replacement of lysines by arginines strongly inhibits phosphorylation of Rb2/p130 by CDK4; the inhibitory effect of replacement by glutamines is less pronounced. Preacetylation of Rb2/p130 strongly enhances CDK4-catalyzed phosphorylation, whereas deacetylation completely abolishes in vitro phosphorylation. In contrast, phosphorylation completely inhibits acetylation of Rb2/p130 by p300. These results suggest a mutual interdependence of modifications in a way that acetylation primes Rb2/p130 for phosphorylation and only dephosphorylated Rb2/p130 can be subject to acetylation. Human papillomavirus 16-E7 protein, which increases acetylation of Rb2/p130 by p300 strongly reduces phosphorylation of this protein by CDK4. This suggests that the balance between phosphorylation and acetylation of Rb2/p130 is essential for its biological function in cell cycle control.

Cell cycle dependent role of HDAC1 for proliferation control through modulating ribosomal DNA transcription.

Ribosome biogenesis and ribosomal DNA transcription are closely correlated with the growth and proliferation of cells, with these processes being under tight epigenetic control. We have investigated the effect of ectopically expressed murine HDAC1 in reporter assays, on ribosomal DNA transcription, cell cycle progression and proliferation in transfected mammalian cells. Ectopically expressed mHDAC1 represses transcription in ribosomal reporter assays driven by ribosomal promoter elements in NIH3T3 cells as well as Cos-7 cells. Following stable transfection of NIH3T3 cells, flag-tagged HDAC1 is assembled into functional, enzymatically active HDAC-complexes that display correct nuclear localization. Induction of flag-HDAC1 expression in NIH3T3 cells caused a cell-cycle phase specific reduction in the initiation of endogenous rDNA transcription, reflected in a reduction of nascent rRNA as well as a marked depression of proliferation due to prolongation of G2-phase. This was substantiated by FACS analysis and cyclin B1 expression analysis. However, prolongation of the G2-phase in HDAC1-overexpressing cells finally led to overcompensation and thus to an increase in total ribosomal RNA. The transient downregulation of rRNA synthesis after induction of HDAC1 overexpression led to a prolongation of G2-phase. These observations were most likely a consequence of HDAC1-mediated deacetylation of upstream binding factor (UBF).

Exploring the connection unit in the HDAC inhibitor pharmacophore model: novel uracil-based hydroxamates.

Starting from the pharmacophore model for HDAC inhibitor design, a novel series of hydroxamates bearing a uracil moiety as connecting unit (CU) has been prepared and tested. Almost all compounds exhibited HDAC inhibiting activity at low nanomolar concentrations, the N-hydroxy-6-(3,4-dihydro-4-oxo-6-benzyl- and -6-phenyl-2-pyrimidinylthio)hexanamides 1d and 1l being more potent than SAHA in enzymatic assays. Such compounds also caused hyperacetylation in NIH3T3 cell core histones and were endowed with interesting antiproliferative and cytodifferentiating effects in human leukemia (HL-60) cells.