Forced expression of the cell cycle inhibitor p57Kip2 in cardiomyocytes attenuates ischemia-reperfusion injury in the mouse heart

<p>Abstract</p> <p>Background</p> <p>Myocardial hypoxic-ischemic injury is the cause of significant morbidity and mortality worldwide. The cardiomyocyte response to hypoxic-ischemic injury is known to include changes in cell cycle regulators. The cyclin-dependent kinase inhibitor <it>p57</it>^{<it>Kip</it>2}is involved in cell cycle control, differentiation, stress signaling and apoptosis. In contrast to other cyclin-dependent kinase inhibitors, p57^Kip2expression diminishes during postnatal life and is reactivated in the adult heart under conditions of cardiac stress. Overexpression of <it>p57</it>^{<it>Kip</it>2}has been previously shown to prevent apoptotic cell death <it>in vitro </it>by inhibiting stress-activated kinases. Therefore, we hypothesized that <it>p57</it>^{<it>Kip</it>2}has a protective role in cardiomyocytes under hypoxic conditions. To investigate this hypothesis, we created a transgenic mouse (<it>R26loxpTA-p57</it>^<it>k</it>/+) that expresses p57^Kip2specifically in cardiac tissue under the ventricular cardiomyocyte promoter <it>Mlc2v</it>.</p> <p>Results</p> <p>Transgenic mice with cardiac specific overexpression of <it>p57</it>^{<it>Kip</it>2}are viable, fertile and normally active and their hearts are morphologically indistinguishable from the control hearts and have similar heart weight/body weight ratio. The baseline functional parameters, including left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), LVdp/dt_max, heart rate (HR) and rate pressure product (RPR) were not significantly different between the different groups as assessed by the Langendorff perfused heart preparation. However, after subjecting the heart <it>ex vivo </it>to 30 minutes of ischemia-reperfusion injury, the <it>p57</it>^{<it>Kip</it>2}overexpressing hearts demonstrated preserved cardiac function compared to control mice with higher left ventricular developed pressure (63 ± 15 vs 30 ± 6 mmHg, p = 0.05), rate pressure product (22.8 ± 4.86 vs 10.4 ± 2.1 × 10³bpm × mmHg, p < 0.05) and coronary flow (3.5 ± 0.5 vs 2.38 ± 0.24 ml/min, p <0.05).</p> <p>Conclusion</p> <p>These data suggest that forced cardiac expression of p57^Kip2does not affect myocardial growth, differentiation and baseline function but attenuates injury from ischemia-reperfusion in the adult mouse heart.</p

Ligand Binding Study of Human PEBP1/RKIP: Interaction with Nucleotides and Raf-1 Peptides Evidenced by NMR and Mass Spectrometry

Background Human Phosphatidylethanolamine binding protein 1 (hPEBP1) also known as Raf kinase inhibitory protein (RKIP), affects various cellular processes, and is implicated in metastasis formation and Alzheimer's disease. Human PEBP1 has also been shown to inhibit the Raf/MEK/ERK pathway. Numerous reports concern various mammalian PEBP1 binding ligands. However, since PEBP1 proteins from many different species were investigated, drawing general conclusions regarding human PEBP1 binding properties is rather difficult. Moreover, the binding site of Raf-1 on hPEBP1 is still unknown. Methods/Findings In the present study, we investigated human PEBP1 by NMR to determine the binding site of four different ligands: GTP, FMN, and one Raf-1 peptide in tri-phosphorylated and non-phosphorylated forms. The study was carried out by NMR in near physiological conditions, allowing for the identification of the binding site and the determination of the affinity constants KD for different ligands. Native mass spectrometry was used as an alternative method for measuring KD values. Conclusions/Significance Our study demonstrates and/or confirms the binding of hPEBP1 to the four studied ligands. All of them bind to the same region centered on the conserved ligand-binding pocket of hPEBP1. Although the affinities for GTP and FMN decrease as pH, salt concentration and temperature increase from pH 6.5/NaCl 0 mM/20°C to pH 7.5/NaCl 100 mM/30°C, both ligands clearly do bind under conditions similar to what is found in cells regarding pH, salt concentration and temperature. In addition, our work confirms that residues in the vicinity of the pocket rather than those within the pocket seem to be required for interaction with Raf-1.METASU

CpG methylation potentiates pixantrone and doxorubicin-induced DNA damage and is a marker of drug sensitivity

DNA methylation is an epigenetic modification of the mammalian genome that occurs predominantly at cytosine residues of the CpG dinucleotide. Following formaldehyde activation, pixantrone alkylates DNA and particularly favours the CpG motif. Aberrations in CpG methylation patterns are a feature of most cancer types, a characteristic that may determine their susceptibility to specific drug treatments. Given their common target, DNA methylation may modulate the DNA damage induced by formaldehyde-activated pixantrone. In vitro transcription, mass spectrometry and oligonucleotide band shift assays were utilized to establish that pixantrone–DNA adduct formation was consistently enhanced 2–5-fold at discrete methylated CpG doublets. The methylation-mediated enhancement was exquisitely sensitive to the position of the methyl substituent since methylation at neighboring cytosine residues failed to confer an increase in pixantrone–DNA alkylation. Covalent modification of DNA by formaldehyde-activated doxorubicin, but not cisplatin, was augmented by neighbouring CpG methylation, indicating that modulation of binding by CpG methylation is not a general feature of all alkylators. HCT116 colon cancer cells vastly deficient in CpG methylation were 12- and 10-fold more resistant to pixantrone and doxorubicin relative to the wild-type line, suggesting that these drugs may selectively recognize the aberrant CpG methylation profiles characteristic of most tumour types

Downregulation of RKIP Is Associated with Poor Outcome and Malignant Progression in Gliomas

Malignant gliomas are highly infiltrative and invasive tumors, which precludes the few treatment options available. Therefore, there is an urgent need to elucidate the molecular mechanisms underlying gliomas aggressive phenotype and poor prognosis. The Raf Kinase Inhibitory protein (RKIP), besides regulating important intracellular signaling cascades, was described to be associated with progression, metastasis and prognosis in several human neoplasms. Its role in the prognosis and tumourigenesis of gliomas remains unclear

Evapotranspiration estimation considering anthropogenic heat based on remote sensing in urban area

Urbanization influences hydrologic cycle significantly on local, regional even global scale. With urbanization the water resources demand for dense population sharpened, thus it is a great challenge to ensure water supply for some metropolises such as Beijing. Urban area is traditionally considered as the area with lower evapotranspiration (ET) on account of the impervious surface and the lower wind speed. For most remote sensing models, the ET, defined as latent heat in energy budget, is estimated as the difference between net radiation and sensible heat. The sensible heat is generally higher in urban area due to the high surface temperature caused by heat island, therefore the latent heat (i.e. the ET) in urban area is lower than that in other region. We estimated water consumption from 2003 to 2012 in Beijing based on water balance method and found that the annual mean ET in urban area was about 654 mm. However, using Surface Energy Balance System (SEBS) model, the annual mean ET in urban area was only 348 mm. We attributed this inconsistence to the impact of anthropogenic heat and quantified this impact on the basis of the night-light maps. Therefore, a new model SEBS-Urban, coupling SEBS model and anthropogenic heat was developed to estimate the ET in urban area. The ET in urban area of Beijing estimated by SEBS-Urban showed a good agreement with the ET from water balance method. The findings from this study highlighted that anthropogenic heat should be included in the surface energy budget for a highly urbanized area

Identification of Protein Targets of Reactive Metabolites of Tienilic Acid in Human Hepatocytes

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Chemical Research in Toxicology, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://pubs.acs.org/doi/abs/10.1021/tx300103jTienilic acid (TA) is a uricosuric diuretic that was withdrawn from the market only months after its introduction because of reports of serious incidents of drug-induced liver injury including some fatalities. Its hepatotoxicity is considered to be primarily immunoallergic in nature. Like other thiophene compounds, TA undergoes biotransformation to a S-oxide metabolite which then reacts covalently with cellular proteins. To identify protein targets of TA metabolites, we incubated [14C]-TA with human hepatocytes, separated cellular proteins by 2D gel electrophoresis, and analyzed proteins in 36 radioactive spots by tryptic digestion followed by LC-MS/MS. Thirty one spots contained at least one identifiable protein. Sixteen spots contained only one of 14 non-redundant proteins which were thus considered to be targets of TA metabolites. Six of the 14 were also found in other radioactive spots that contained from 1 to 3 additional proteins. Eight of the 14 had not been reported to be targets for any reactive metabolite other than TA. The other 15 spots each contained from 2–4 identifiable proteins, many of which are known targets of other chemically reactive metabolites, but since adducted peptides were not observed, the identity of the adducted protein(s) in these spots is ambiguous. Interestingly, all the radioactive spots corresponded to proteins of low abundance, while many highly abundant proteins in the mixture showed no radioactivity. Furthermore, of approximately 16 previously reported protein targets of TA in rat liver (Methogo, R., Dansette, P. and Klarskov, K. (2007) Int. J. Mass Spectrom., 268, 284–295), only one (fumarylacetoacetase) is among the 14 targets identified in this work. One reason for this difference may be statistical, given that each study identified a small number of targets from among thousands present in hepatocytes. Another may be the species difference (i.e. rat vs. human), and still another may be the method of detection of adducted proteins (i.e. Western blot vs. C-14). Knowledge of human target proteins is very limited. Of more than 350 known protein targets of reactive metabolites, only 42 are known from human and only 21 of these are known to be targets for more than one chemical. Nevertheless, the demonstration that human target proteins can be identified using isolated hepatocytes in vitro should enable the question of species differences to be addressed more fully in the future

Parallel-stranded DNA under topological stress: rearrangement of (dA)15.(dT)15 to a d(A.A.T)n triplex.

DNA oligonucleotides with appropriate sequences can form a stable duplex in which the two strands are paired in a parallel orientation instead of as the conventional antiparallel double helix of B-DNA. In parallel-stranded DNA (ps-DNA) base pairing is noncanonical with the glycosidic bonds in a trans orientation. The two grooves are equivalent. We have synthesized DNA duplexes consisting of a central parallel-stranded (dA)15.(dT)15 tract flanked by normal antiparallel regions, and ligated them into the pUC18 plasmid. The effect of negative supercoiling on the covalently closed circular molecules was studied by two-dimensional agarose gel electrophoresis and by chemical modification with OsO4-pyridine (Os,py) and diethylpyrocarbonate (DEPC). The following results were obtained: (i) The ps insert, and by inference ps-DNA in general, adopts a right handed helical form. (ii) Upon increasing the negative superhelix density (-sigma) to greater than 0.03 the 15 bp ps insert undergoes a major transition leading to a relaxation corresponding to a reduction in twist of approximately 2.5 helical turns. The transition free surgery is approximately kcal/mol. (iii) The chemical modification pattern of the resulting structure suggests that the purine strand folds back and associates with the pyrimidine strand, forming a novel intramolecular triplex structure consisting of d(A.A.T) base triplets. A model for the triplex conformation is proposed and its thermodynamic properties are analyzed by statistical mechanics