Antitumor progression potential of caffeic acid phenethyl ester involving p75NTR in C6 glioma cells

The previous data showed that caffeic acid phenethyl ester (CAPE), a component of propolis, possesses inducing cell cycle arrest and antiproliferation effect on C6 glioma cells in vitro and in vivo. In the present study, C6 glioma cells treated with CAPE resulted in morphological changes to an astrocytic phenotype and increased the expression of glial differentiation marker proteins including glial fibrillary acidic protein (GFAP) and S-100β. In addition, with scratch assay and Boyden chamber assay, CAPE exhibited inhibitory effects on the motility and invasion of C6 glioma cells. Furthermore, CAPE induced the expression of nerve growth factor (NGF) and p75 neurotrophin receptor (p75^NTR), which were involved in neural cell differentiation. CAPE could also inhibit the activity of matrix metalloproteinases (MMPs) and induce the expression of RhoB, a tumor suppressor. To examine the involvement of p75^NTR in the anti-invasive property of CAPE, Western blotting and Boyden Chamber assay were performed by addition of an anti-p75^NTR antibody in C6 cells. The results showed that blocking p75^NTR could decrease the CAPE-induced expression of RhoB and the inactivation of MMP-2, -9 as well as the anti-invasion effect in C6 glioma cells. Furthermore, CAPE suppressed IκB-α phosphorylation which was down stream of p75^NTR. Finally, the effect of CAPE on metastasis by lung colonization of the tumor cell in nude mice was also evaluated. It was found that the groups of nude mice injected with CAPE-pretreated cells could decrease both lung size and weight as compared to the positive control group which did not receive CAPE treatment. In addition, histological examination of the mouse lung sections showed that the CAPE-treated group inhibited the metastasis of C6 glioma cells. These data suggest CAPE possesses antitumor progression potential.     

Aberrant Cdk5 activation by p25 triggers pathological events leading to neurodegeneration and neurofibrillary tangles

Cyclin-dependent kinase 5 (Cdk5) and its regulatory subunit p35 are integral players in the proper development of the mammalian central nervous system. Proteolytic cleavage of p35 generates p25, leading to aberrant Cdk5 activation. The accumulation of p25 is implicated in several neurodegenerative diseases. In primary neurons, p25 causes apoptosis and tau hyperphosphorylation. Current mouse models expressing p25, however, fail to rigorously recapitulate these phenotypes in vivo. Here, we generated inducible transgenic mouse lines overexpressing p25 in the postnatal forebrain. Induction of p25 preferentially directed Cdk5 to pathological substrates. These animals exhibited neuronal loss in the cortex and hippocampus, accompanied by forebrain atrophy, astrogliosis, and caspase-3 activation. Endogenous tau was hyperphosphorylated at many epitopes, aggregated tau accumulated, and neurofibrillary pathology developed progressively in these animals. Our cumulative findings provide compelling evidence that in vivo deregulation of Cdk5 by p25 plays a causative role in neurodegeneration and the development of neurofibrillary pathology.     

DNA Demethylation by DNMT3A and DNMT3B in vitro and of Methylated Episomal DNA in Transiently Transfected Cells

The DNA methylation program in vertebrates is an essential part of the epigenetic regulatory cascade of development, cell differentiation, and progression of diseases including cancer. While the DNA methyltransferases (DNMTs) are responsible for the in vivo conversion of cytosine (C) to methylated cytosine (5mC), demethylation of 5mC on cellular DNA could be accomplished by the combined action of the ten-eleven translocation (TET) enzymes and DNA repair. Surprisingly, the mammalian DNMTs also possess active DNA demethylation activity in vitro in a Ca²⁺- and redox conditions-dependent manner, although little is known about its molecular mechanisms and occurrence in a cellular context. In this study, we have used LC-MS/MS to track down the fate of the methyl group removed from 5mC on DNA by mouse DNMT3B in vitro and found that it becomes covalently linked to the DNA methylation catalytic cysteine of the enzyme. We also show that Ca²⁺ homeostasis-dependent but TET1/TET2/TET3/TDG-independent demethylation of methylated episomal DNA by mouse DNMT3A or DNMT3B can occur in transfected human HEK 293 and mouse embryonic stem (ES) cells. Based on these results, we present a tentative working model of Ca²⁺ and redox conditions-dependent active DNA demethylation by DNMTs. Our study substantiates the potential roles of the vertebrate DNMTs as double-edged swords in DNA methylation-demethylation during Ca²⁺-dependent physiological processes.     

Method for large-scale isolation and purification of R-phycoerythrin from red alga Polysiphonia urceolata Grev

R-phycoerythrin was isolated and purified from a red alga, Polysiphonia urceolata Grev, using Streamline column combined with ion-exchange chromatography or hydroxyapatite chromatography. The purity of R-phycoerythrin isolated by Streamline column was up to 1.66 and the yield of R-phycoerythrin could be as high as 0.68 mg/g frozen P. urceolata. All the eluates from Streamline column were divided into two equivalent parts, respectively. One part was pumped into the ion-exchange column loaded with Q-Sepharose and the other was applied to the adsorption column loaded with hydroxyapatite. The purities of R-phycoerythrin purified using these two methods were both up to 3.26, more than 3.2 the commonly accepted criterion. The yield of purified R-phycoerythrin from the ion-exchange chromatography was 0.40 mg/g frozen P. urceolata and that from the hydroxyapatite chromatography could reach 0.34 mg/g frozen P. urceolata. The purified protein had three absorption peaks at 498, 535, and 565 nm and displayed a fluorescence maximum at 580 nm, which was consistent with the typical spectrum of R-phycoerythrin. The purified R-PE was also identified with electrophoresis. Only one single protein band appeared on native-PAGE with silver staining. SDS-PAGE demonstrated the presence of one 20 kDa major subunit, and one low intensity band corresponding to 33 kDa subunit. The results indicate that using the expanded bed adsorption combined with ion-exchange chromatography or hydroxyapatite chromatography, R-phycoerythrin can be purified from frozen P. urceolata on large scale.     

Severe acute respiratory syndrome coronavirus 7a accessory protein is a viral structural protein

Severe acute respiratory syndrome coronavirus (SCoV) 7a protein is one of the viral accessory proteins. In expressing cells, 7a protein exhibits a variety of biological activities, including induction of apoptosis, activation of the mitogen-activated protein kinase signaling pathway, inhibition of host protein translation, and suppression of cell growth progression. Analysis of SCoV particles that were purified by either sucrose gradient equilibrium centrifugation or a virus capture assay, in which intact SCoV particles were specifically immunoprecipitated by anti-S protein monoclonal antibody, demonstrated that 7a protein was associated with purified SCoV particles. Coexpression of 7a protein with SCoV S, M, N, and E proteins resulted in production of virus-like particles (VLPs) carrying 7a protein, while 7a protein was not released from cells expressing 7a protein alone. Although interaction between 7a protein and another SCoV accessory protein, 3a, has been reported, 3a protein was dispensable for assembly of 7a protein into VLPs. S protein was not required for the 7a protein incorporation into VLPs, and yet 7a protein interacted with S protein in coexpressing cells. These data established that, in addition to 3a protein, 7a protein was a SCoV accessory protein identified as a SCoV structural protein.     

Quantitative measurements of vancomycin binding to self-assembled peptide monolayers on chips by quartz crystal microbalance

This paper describes direct binding of a small vancomycin to peptide ligands immobilized on a sensor chip using quartz crystal microbalance. In this study, the binding ligands were composed of three components: a molecular recognition element (peptide), a conformationally flexible and hydrophilic linker, and a long-chain alkanethiol. These peptide ligands were used to establish the well-packed, self-assembled monolayers on quartz chips and could be readily synthesized using conventional organic chemistry protocols. Results of quartz crystal microbalance measurements showed that vancomycin specifically associated with the d-Ala-d-Ala-containing peptide with an affinity of 3.2+/-0.3 microM and was, as expected, completely inactive to the self-assembled monolayer presenting l-Ala-l-Ala peptide. The dissociation constant obtained correlated well with values reported in literature and was further confirmed by surface plasmon resonance measurement (2.7+/-0.7 microM). The technique used in this study should be applicable to both peptidyl and nonpeptidyl ligands of greater complexity than that used here. This method is practical, it provides quantitative binding information, and complicated analysis is avoided.     

Innate Immune Dysfunctions in Aged Mice Facilitate the Systemic Dissemination of Methicillin-Resistant S. aureus

Elderly humans show increased susceptibility to invasive staphylococcal disease after skin and soft tissue infection. However, it is not understood how host immunity changes with aging, and how that predisposes to invasive disease. In a model of severe skin infection, we showed that aged mice (16- to 20-month-old) exhibit dramatic bacterial dissemination compared with young adult mice (2-month-old). Bacterial dissemination was associated with significant reductions of CXCL1 (KC), polymorphonuclear cells (PMNs), and extracellular DNA traps (NETs) at the infection site. PMNs and primary skin fibroblasts isolated from aged mice showed decreased secretion of CXCL2 (MIP-2) and KC in response to MRSA, and in vitro analyses of mitochondrial functions revealed that the mitochondrial electron transport chain complex I plays a significant role in induction of chemokines in the cells isolated from young but not old mice. Additionally, PMNs isolated from aged mice have reduced ability to form NETs and to kill MRSA. Expression of nuclease by S. aureus led to increased bacterial systemic dissemination in young but not old mice, suggesting that defective NETs formation in elderly mice permitted nuclease and non-nuclease expressing S. aureus to disseminate equally well. Overall, these findings suggest that gross impairment of both skin barrier function and innate immunity contributes to the propensity for MRSA to disseminate in aged mice. Furthermore, the study indicates that contribution of bacterial factors to pathogenicity may vary with host age.