Proteomics analysis of plasma membrane from liver sinusoidal endothelial cells after partial hepatectomy by an improved two-dimensional electrophoresis

Liver regeneration is an angiogenesis-associated phenomenon. To identify key plasma membrane (PM) proteins of endothelial cells involved in the initiation of angiogenesis during liver regeneration, the PM of liver sinusoidal endothelial cells (LSEC) at 72 h after partial hepatectomy was enriched by an established in vivo membrane density perturbation method. The differentially expressed membrane proteins compared to those from sham operation were quantified using an improved two-dimensional 16-BAC/SDS-PAGE and identified by LC-MS/MS. Several proteins were further confirmed by cICAT labeling quantitative strategy. A total of 47 proteins were identified including known and novel proteins involved in angiogenesis or liver regeneration, such as inducible nitric oxide synthase, type IV collagen, and integrin beta3. Our results indicated that the combination of the membrane density perturbation strategy and the improved two-dimensional electrophoresis (2-DE) method are useful for investigating the endothelial dysfunctions in vivo.     

An internal signal sequence directs intramembrane proteolysis of a cellular immunoglobulin domain protein

Precursor proteolysis is a crucial mechanism for regulating protein structure and function. Signal peptidase (SP) is an enzyme with a well defined role in cleaving N-terminal signal sequences but no demonstrated function in the proteolysis of cellular precursor proteins. We provide evidence that SP mediates intraprotein cleavage of IgSF1, a large cellular Ig domain protein that is processed into two separate Ig domain proteins. In addition, our results suggest the involvement of signal peptide peptidase (SPP), an intramembrane protease, which acts on substrates that have been previously cleaved by SP. We show that IgSF1 is processed through sequential proteolysis by SP and SPP. Cleavage is directed by an internal signal sequence and generates two separate Ig domain proteins from a polytopic precursor. Our findings suggest that SP and SPP function are not restricted to N-terminal signal sequence cleavage but also contribute to the processing of cellular transmembrane proteins.     

Poly(ADP-Ribose) polymerase inhibition improves endothelial dysfunction induced by hypochlorite

Reactive oxygen species, such as myeloperoxidase-derived hypochlorite, induce oxidative stress and DNA injury. The subsequent activation of the DNA-damage-poly(ADP-ribose) polymerase (PARP) pathway has been implicated in the pathogenesis of various diseases, including ischemia-reperfusion injury, circulatory shock, diabetic complications, and atherosclerosis. We investigated the effect of PARP inhibition on the impaired endothelium-dependent vasorelaxation induced by hypochlorite. In organ bath experiments for isometric tension, we investigated the endothelium-dependent and endothelium-independent vasorelaxation of isolated rat aortic rings using cumulative concentrations of acetylcholine and sodium nitro-prusside. Endothelial dysfunction was induced by exposing rings to hypochlorite (100-400 microM). In the treatment group, rings were preincubated with the PARP inhibitor INO-1001. DNA strand breaks were assessed by the TUNEL method. Immunohistochemistry was performed for 4-hydroxynonenal (a marker of lipid peroxidation), nitrotyrosine (a marker of nitrosative stress), and poly(ADP-ribose) (an enzymatic product of PARP). Exposure to hypochlorite resulted in a dose-dependent impairment of endothelium-dependent vasorelaxation of aortic rings, which was significantly improved by PARP inhibition, whereas the endothelium-independent vasorelaxation remained unaffected. In the hypochlorite groups we found increased DNA breakage, lipidperoxidation, and enhanced nitrotyrosine formation. The hypochloride-induced activation of PARP was prevented by INO-1001. Our results demonstrate that PARP activation contributes to the pathogenesis of hypochlorite-induced endothelial dysfunction, which can be prevented by PARP inhibitors.     

Vasodilator action of docosahexaenoic acid (DHA) in human coronary arteries in vitro

Coronary arterial tissues obtained from mammalian hearts are known to develop spontaneous phasic contractions. The aim of the present study was to investigate the vasodilatory effects of docosahexaenoic acid (DHA) on the rhythmic contractions of isolated human coronary arterial (HCA) preparations obtained from the recipient hearts of patients undergoing cardiac transplantation. Results from 8 hearts show that: (i) most HCA tissues displayed spontaneous rhythmic phasic contractions with a cycle length around 10 min in the absence or presence of PGF2alpha or elevated [K+]0 (20 mM); (ii) the rhythmic activity could be suppressed by a free fatty acid DHA (30 microM); (iii) high [K+]0 (20 and 80 mM) could induce sustained tonic contraction in addition to phasic contractions in HCA tissues, the tonic contraction could be antagonized by L-type Ca(2+) channel blockers or by DHA (depending on [K+]0); (iv) a digitalis substance ouabain also could induce tonic contraction and suppress phasic contraction; (v) in isolated HCA vascular smooth muscle cells, DHA increased the magnitude of outward voltage-gated K+ (IKV) currents and the inwardly rectifying IK1 currents. Enhancement of K+ currents could be related to vasorelaxation induced by DHA in HCA preparations. Further studies on the effects of DHA on various ionic currents and intracellular Ca(2+) transient are needed to clarify the Ca(2+)-dependent and the Ca(2+)-independent actions of DHA in HCA.     

Synthesis of 3-(1H-benzimidazol-2-yl)-5-isoquinolin-4-ylpyrazolo[1,2-b]pyridine, a potent cyclin dependent kinase 1 (CDK1) inhibitor

The novel compound 3-(1H-benzimidazol-2-yl)-5-isoquinolin-4-ylpyrazolo[1,2-b]pyridine was discovered to be a potent CDK1 inhibitor. Described here is the chemistry for its synthesis, including Pd(II) catalyzed Stille coupling reaction and sulfur(0) induced benzimidazole formation. Its effects on VEGFR-2 kinase activity and tumour cell proliferation are also described.     

Establishment of the active catalytic domain of human PDGFRbeta tyrosine kinase-based ELISA assay for inhibitor screening

Tyrosine kinases are emerging as frequent targets of primary oncogenic events and therefore represent an optimal focus of therapeutic intervention. In an effort towards therapeutic PDGFR inactivation, we expressed the catalytic domain of PDGFRbeta as a soluble active kinase using Bac-to-Bac expression system, and studied the correlations between PDGFRbeta activity and enzyme concentration, ATP concentration, substrate concentration and divalent cation type. And a convenient, effective and non-radioactive ELISA screening model is then established for identification of the potential inhibitors targeting PDGFRbeta kinase. Of 500 RTK target-based compounds, TKI-30 was identified as a small molecule potential inhibitor of PDGFRbeta (IC(50)=0.34 microM). Further studies indicated that TKI-30 blocked PDGF-BB-induced autophosphorylation of PDGFRbeta in a dose-dependent manner in Swiss 3T3 cells and human umbilical vein smooth muscle cells (HUVSMCs). Moreover, it dose-dependently suppressed the PDGF-BB-induced proliferation in HUVSMCs and tube formation of HUVEC. Our data collectively indicated that PDGFRbeta-based ELISA assay is a new method available for screening inhibitors targeting PDGFRbeta kinase and TKI-30 is a potential novel anti-cancer agent worthy of being further investigated.     

The Arabidopsis Nitrate Transporter NRT1.7, Expressed in Phloem,Is Responsible for Source-to-Sink Remobilization of Nitrate

Several quantitative trait locus analyses have suggested that grain yield and nitrogen use efficiency are well correlated with nitrate storage capacity and efficient remobilization. This study of the Arabidopsis thaliana nitrate transporter NRT1.7 provides new insights into nitrate remobilization. Immunoblots, quantitative RT-PCR, β-glucuronidase reporter analysis, and immunolocalization indicated that NRT1.7 is expressed in the phloem of the leaf minor vein and that its expression levels increase coincidentally with the source strength of the leaf. In nrt1.7 mutants, more nitrate was present in the older leaves, less ¹⁵NO₃⁻ spotted on old leaves was remobilized into N-demanding tissues, and less nitrate was detected in the phloem exudates of old leaves. These data indicate that NRT1.7 is responsible for phloem loading of nitrate in the source leaf to allow nitrate transport out of older leaves and into younger leaves. Interestingly, nrt1.7 mutants showed growth retardation when external nitrogen was depleted. We conclude that (1) nitrate itself, in addition to organic forms of nitrogen, is remobilized, (2) nitrate remobilization is important to sustain vigorous growth during nitrogen deficiency, and (3) source-to-sink remobilization of nitrate is mediated by phloem.     

Synthesis and biological evaluation of a novel 99mTc nitrido radiopharmaceutical with deoxyglucose dithiocarbamate,showing tumor uptake

The deoxyglucose dithiocarbamate (DGDTC) was synthesized and radiolabelled with [^99mTcN]²⁺ intermediate to form the ^99mTcN–DGDTC complex. The radiochemical purity of the ^99mTcN–DGDTC complex was over 90%, as measured by TLC and by HPLC, without any notable decomposition at room temperature over a period of 6 h. The partition coefficient and electrophoresis results indicated that this complex was hydrophilic and neutral. The biodistribution of ^99mTcN–DGDTC in mice bearing S 180 tumor showed that the complex accumulated in the tumor with high uptake and good retention. The tumor/blood and tumor/muscle ratios increased with time and reached 2.32 and 1.68 at 4 h post-injection, suggesting it would be a promising candidate for tumor imaging.     

Biomarker discovery for arsenic exposure using functional data. Analysis and feature learning of mass spectrometry proteomic data

Plasma biomarkers of exposure to environmental contaminants play an important role in early detection of disease. The emerging field of proteomics presents an attractive opportunity for candidate biomarker discovery, as it simultaneously measures and analyzes a large number of proteins. This article presents a case study for measuring arsenic concentrations in a population residing in an As-endemic region of Bangladesh using plasma protein expressions measured by SELDI-TOF mass spectrometry. We analyze the data using a unified statistical method based on functional learning to preprocess mass spectra and extract mass spectrometry (MS) features and to associate the selected MS features with arsenic exposure measurements. The task is challenging due to several factors, the high dimensionality of mass spectrometry data, complicated error structures, and a multiple comparison problem. We use nonparametric functional regression techniques for MS modeling, peak detection based on the significant zero-downcrossing method, and peak alignment using a warping algorithm. Our results show significant associations of arsenic exposure to either under- or overexpressions of 20 proteins.