Eutectic formation analysis of amino acid mixtures using molecular dynamics simulations

The mechanism of eutectic formation was investigated via computer-aided molecular dynamics techniques based on experimental results. The CBZ group mixtures CBZ-l-Asp/d-AlaNH2 x HCl/methanol, CBZ-l-Asp/l-PheOMe x HCl/methanol, and CBZ-l-Tyr/l-ArgNH2 x 2HCl/methanol formed transparent eutectic melts. The non-CBZ group mixtures l-Asp/d-AlaNH2 x HCl/methanol, l-Asp/l-PheOMe x HCl/methanol, and l-Tyr/l-ArgNH2 x 2HCl/methanol did not form eutectic melts. According to molecular dynamics simulation results, increase in the kinetic energy values of eutectic forming mixtures was much larger than the increase in potential energy over a temperature shift from 298 to 333 K. However, the results for non-eutectic forming mixtures were reversed. The Coulomb interaction energies of eutectic forming mixtures significantly decreased, because eutectic melting can increase the mobility of molecules in the mixtures. The enhancement of molecular mobility was confirmed by increased self-diffusion constant values, and the change of solid-to-liquid phase was detected by radial distribution function results. The periodic boundary conditions for calculation of molecular dynamics were found to be reliable.     

Reactivity of alkynyl Pd(II) azido complexes toward organic isocyanides, isothiocyanates, and nitriles

Alkynyl Pd(II) azido complexes of the type [Pd(N₃)(CCR)L₂] (1-3) were obtained by reactions of aqueous NaN₃ with [Pd(Cl)(CCR)L₂] (R = Ph or C(O)OMe). Treating compounds 1-3 with organic isocyanides (R-NC) afforded novel complexes, trans-[Pd(CCPh)(NCNR)(PMe₃)₂] (R = 2,6-Me₂C₆H₃ (4) or 2,6-Et₂C₆H₃ (5)) and trans-[Pd(CCR)(CN₄-t-Bu)L₂] (6: L = PMe₃, R = Ph; 7: L = PEt₃, R = C(O)OMe; 8: L = PMe₃, R = C(O)OMe), which contain either a carbodiimido or a C-coordinated tetrazolato group. Reactions of compounds 1 and 2 with R-NCS (R = 2,6-Me₂C₆H₃ or CH₂CH₃) and 1,4-phenylene diisothiocyanate (C₆H₄(NCS)₂) smoothly proceeded to give tetrazole-thiolato complexes, trans-[Pd(CCPh)(SCN₄-R)L₂] (L = PMe₃, R = Et (9) or 2,6-Me₂C₆H₃ (10); L = PEt₃, R = 2,6-Me₂C₆H₃ (11)), and a phenylene-bridged dinuclear Pd(II) tetrazole-thiolato complex, [(PEt₃)₂(CCPh)Pd(SCN₄-(μ-C₆H₄)-SCN₄)Pd(CCPh)(PEt₃)₂] (12), respectively. Complexes 9-12 contain the Pd-S bond that is formed by the dipolar cycloaddition of the organic isothiocyanate to the Pd-azido bond. In contrast, the corresponding reactions of compounds 1and 2 with C₆F₅CN and Me₃SiCN (organic nitriles, R-CN) gave an N-coordinated Pd(II)-tetrazolato compound {trans-[Pd(CCPh)(N₄C-C₆F₅)(PMe₃)₂] (13)} and a mixture of Pd(II)-cyano complexes {trans-[Pd(CCPh)(CN)(PEt₃)₂] (14) and [Pd(CN)₂(PEt₃)₂] (15)}, respectively. Bis(phosphine) bis(cyano) complexes of Pd and Ni, [M(CN)₂L₂] (L = PEt₃, PMe₃; L₂ = DEPE), could be obtained independently by the reactions of [M(N₃)₂L₂] with excess Me₃SiCN in organic solvents.     

H-Ras-specific activation of Rac-MKK3/6-p38 pathway: its critical role in invasion and migration of breast epithelial cells

Human tumors frequently exhibit constitutively activated Ras signaling, which contributes to the malignant phenotype. Mounting evidence suggests unique roles of the Ras family members, H-Ras, N-Ras and K-Ras, in normal and pathological conditions. In an effort to dissect distinct Ras isoform-specific functions in malignant phenotypic changes, we previously established H-Ras- and N-Ras-activated MCF10A human breast epithelial cell lines. Using these, we showed that p38 kinase is a key signaling molecule differentially regulated between H-Ras and N-Ras, leading to H-Ras-specific induction of invasive and migrative phenotypes. The present study is to further investigate H-Ras- and N-Ras-mediated signaling pathways and to unveil how these pathways are integrated for regulation of invasive/migrative phenotypic conversion of human breast epithelial cells. Here we report that the Rac-MAPK kinase (MKK)3/6-p38 pathway is a unique signaling pathway activated by H-Ras, leading to the invasive/migrative phenotype. In contrast, Raf-MEK-ERK and phosphatidylinositol 3-kinase-Akt pathways, which are fundamental to proliferation and differentiation, are activated by both H-Ras and N-Ras. A significant role for p38 in cell invasion is further supported by the observation that p38 activation by MKK6 transfection is sufficient to induce invasive and migrative phenotypes in MCF10A cells. Activation of the MKK6-p38 pathway results in a marked induction of matrix metalloproteinase (MMP)-2, whereas it had little effect on MMP-9, suggesting MMP-2 up-regulation by MKK6-p38 pathway as a key step for H-Ras-induced invasion and migration. We also provide evidence for cross-talk among the Rac, Raf, and phosphatidylinositol 3-kinase pathways critical for regulation of MMP-2 and MMP-9 expression and invasive phenotype. Taken together, the present study elucidated the role of the Rac-MKK3/6-p38 pathway leading to H-Ras-specific induction of malignant progression in breast epithelial cells, providing implications for developing therapeutic strategies for mammary carcinoma to target Ras downstream signaling molecules required for malignant cancer cell behavior but less critical for normal cell functions.     

Role of protein phosphatase 2A in mGluR5-regulated MEK/ERK phosphorylation in neurons

The regulation of protein phosphorylation requires coordinated interaction between protein kinases and protein phosphatases (PPs). Recent evidence has shown that the Galphaq-protein-coupled metabotropic glutamate receptor (mGluR) 5 up-regulates phosphorylation of MAPK/ERK1/2. However, signaling mechanisms linking mGluR5 to ERK are poorly understood. In this study, roles of a major serine/threonine PP, PP2A, in this event were evaluated in cultured neurons. We found that the PP1/2A inhibitors okadaic acid and calyculin A mimicked the effect of the mGluR5 agonists (RS)-3,5-dihydroxyphenylglycine and (RS)-2-chloro-5-hydroxyphenylglycine in facilitating phosphorylation of ERK1/2 and its upstream kinase, MEK1/2, in a PP2A-dependent but not PP1-dependent manner. Co-administration of either inhibitor with an mGluR5 agonist produced additive phosphorylation of ERK1/2. Enzymatic assays showed a basal level of phosphatase activity of PP2A under normal conditions, and activation of mGluR5 selectively inhibited PP2A, but not PP1, activity. In addition, a physical association of the cytoplasmic C terminus of mGluR5 with PP2A was observed, and ligand activation of mGluR5 reduced mGluR5-PP2A binding. Additional mechanistic studies revealed that mGluR5 activation increased tyrosine (Tyr307) phosphorylation of PP2A, which was dependent on activation of a p60c-Src family tyrosine kinase, but not the epidermal growth factor receptor tyrosine kinase and resulted in dissociation of PP2A from mGluR5 and reduced PP2A activity. Together, we have identified a novel, mGluR5-triggered signaling mechanism involving use- and Src-dependent inactivation of PP2A, which contributes to mGluR5 activation of MEK1/2 and ERK1/2.     

Inactivation of organellar glutamyl- and seryl-tRNA synthetases leads to developmental arrest of chloroplasts and mitochondria in higher plants

Aminoacyl-tRNA synthetases (ARSs) are key enzymes involved in protein translation, and both cytosolic and organellar forms are present in the genomes of eukaryotes. In this study, we investigated cellular effects of depletion of organellar forms of ARS using virus-induced gene silencing (VIGS) in Nicotiana benthamiana. VIGS of NbERS and NbSRS, which encode organellar GluRS and SerRS, respectively, resulted in a severe leaf-yellowing phenotype. The NbERS and NbSRS genes were ubiquitously expressed in plant tissues, and induced in response to light. Green fluorescent protein (GFP) fusion proteins of the full-length glutamyl-tRNA synthetase (ERS) and seryl-tRNA synthetase (SRS) of Arabidopsis and GFP fusions to the N-terminal extension of these proteins were all dualtargeted to chloroplasts and mitochondria. At the cell level, depletion of NbERS and NbSRS resulted in dramatically reduced numbers of chloroplasts with reduced sizes and chlorophyll content. The numbers and/or physiology of mitochondria were also severely affected. The abnormal chloroplasts lacked most of the thylakoid membranes and appeared to be degenerating, whereas some of them showed doublet morphology, indicating defective chloroplast division. Pulse-field gel electrophoresis analyses demonstrated that chloroplast DNA in subgenomic sizes is the predominant form in the abnormal chloroplasts. Interestingly, despite severe abnormalities in chloroplasts and mitochondria, expression of many nuclear genes encoding chloroplastor mitochondria-targeted proteins, and chlorophyll biosynthesis genes remained unchanged in the ERS and SRS VIGS lines. This is the first report to analyze the effect of ARS disruption on organelle development in plants.     

Pleckstrin homology domains of phospholipase C-gamma1 directly interact with beta-tubulin for activation of phospholipase C-gamma1 and reciprocal modulation of beta-tubulin function in microtubule assembly

Phosphoinositide-specific phospholipase C-gamma1 (PLC-gamma1) has two pleckstrin homology (PH) domains, an N-terminal domain and a split PH domain. Here we show that pull down of NIH3T3 cell extracts with PLC-gamma1 PH domain-glutathione S-transferase fusion proteins, followed by matrix-assisted laser desorption ionization-time of flight-mass spectrometry, identified beta-tubulin as a binding protein of both PLC-gamma1 PH domains. Tubulin is a main component of microtubules and mitotic spindle fibers, which are composed of alpha- and beta-tubulin heterodimers in all eukaryotic cells. PLC-gamma1 and beta-tubulin colocalized in the perinuclear region in COS-7 cells and cotranslocated to the plasma membrane upon agonist stimulation. Membrane-targeted translocation of depolymerized tubulin by agonist stimulation was also supported by immunoprecipitation analyses. The phosphatidylinositol 4,5-bisphosphate (PIP(2)) hydrolyzing activity of PLC-gamma1 was substantially increased in the presence of purified tubulin in vitro, whereas the activity was not promoted by bovine serum albumin, suggesting that beta-tubulin activates PLC-gamma1. Furthermore, indirect immunofluorescent microscopy showed that PLC-gamma1 was highly concentrated in mitotic spindle fibers, suggesting that PLC-gamma1 is involved in spindle fiber formation. The effect of PLC-gamma1 in microtubule formation was assessed by overexpression and silencing PLC-gamma1 in COS-7 cells, which resulted in altered microtubule dynamics in vivo. Cells overexpressing PLC-gamma1 showed higher microtubule densities than controls, whereas PLC-gamma1 silencing with small interfering RNAs led to decreased microtubule network densities as compared with control cells. Taken together, our results suggest that PLC-gamma1 and beta-tubulin transmodulate each other, i.e. that PLC-gamma1 modulates microtubule assembly by beta-tubulin, and beta-tubulin promotes PLC-gamma1 activity.     

A functional analysis of mouse models of cardiac disease through metabolic profiling

Since the completion of the human and mouse genomes, the focus in mammalian biology has been on assessing gene function. Tools are needed for assessing the phenotypes of the many mouse models that are now being generated, where genes have been "knocked out," "knocked in," or mutated, so that gene expression can be understood in its biological context. Metabolic profiling of cardiac tissue through high resolution NMR spectroscopy in conjunction with multivariate statistics has been used to classify mouse models of cardiac disease. The data sets included metabolic profiles from mouse models of Duchenne muscular dystrophy, two models of cardiac arrhythmia, and one of cardiac hypertrophy. The metabolic profiles demonstrate that the strain background is an important component of the global metabolic phenotype of a mouse, providing insight into how a given gene deletion may result in very different responses in diverse populations. Despite these differences associated with strain, multivariate statistics were capable of separating each mouse model from its control strain, demonstrating that metabolic profiles could be generated for each disease. Thus, this approach is a rapid method of phenotyping mouse models of disease.     

Clostridium difficile toxin A regulates inducible cyclooxygenase-2 and prostaglandin E2 synthesis in colonocytes via reactive oxygen species and activation of p38 MAPK

Clostridium difficile toxin A induces acute colitis with neutrophil infiltration and up-regulation of numerous pro-inflammatory mediators, but the contribution of cyclooxygenase-2 (COX-2) induction in this infection is unknown. We report here that toxin A induces expression of COX-2 and secretion of prostaglandin E2 (PGE2) in a dose- and time-dependent manner in cultured NCM460 human colonocytes and in human intestinal xenografts. This induction was blocked by SB203580, a p38 MAPK inhibitor, which also decreased the phosphorylation of MSK-1, CREB/ATF-1, and COX-2 promoter activity following toxin A stimulation. Gel shift assays indicated that CREB/ATF-1 was the major proteins binding to the COX-2-CRE. Moreover, colonocytes exposed to toxin A produced reactive oxygen species (ROS), which activated p38 MAPK, MSK-1, and CREB/ATF-1, leading to subsequent COX-2 induction and PGE2 secretion. In intact mice, blockage of p38 MAPK inhibited toxin A-mediated induction of COX-2 in enterocytes as well as lamina propria cells, and significantly blocked the toxin A-induced ileal secretion of fluid and PGE2. Furthermore, a selective COX-2 inhibitor also diminished toxin A-associated ileal fluid and PGE2 secretion. The main signaling pathway for toxin A induction of human COX-2 involves ROS-mediated activation of p38 MAPK, MSK-1, CREB, and ATF-1. Toxin A triggers ileal inflammation and secretion of fluid via COX-2 induction and release of PGE2.