hNRAGE, a human neurotrophin receptor interacting MAGE homologue, regulates p53 transcriptional activity and inhibits cell proliferation

hNRAGE, a neurotrophin receptor p75 interacting MAGE homologue, is cloned from a human placenta cDNA library. hNRAGE can inhibit the colony formation of and arrest cell proliferation at the G1/S and G2/M stages in hNRAGE overexpressing cells. Interestingly, hNRAGE also increases the p53 protein level as well as its phosphorylation (Ser392). Further studies demonstrated that hNRAGE does not affect the proliferation of mouse p53-/- embryonic fibroblasts, suggesting that p53 function is required for hNRAGE induced cell cycle arrest. Moreover, the cell cycle inhibiting protein p21(WAF) is induced by hNRAGE in a p53 dependent manner. The data provide original evidence that hNRAGE arrests cell growth through a p53 dependent pathway.     

Intragenic spatial patterns of codon usage bias in prokaryotic and eukaryotic genomes

To study the roles of translational accuracy, translational efficiency, and the Hill-Robertson effect in codon usage bias, we studied the intragenic spatial distribution of synonymous codon usage bias in four prokaryotic (Escherichia coli, Bacillus subtilis, Sulfolobus tokodaii, and Thermotoga maritima) and two eukaryotic (Saccharomyces cerevisiae and Drosophila melanogaster) genomes. We generated supersequences at each codon position across genes in a genome and computed the overall bias at each codon position. By quantitatively evaluating the trend of spatial patterns using isotonic regression, we show that in yeast and prokaryotic genomes, codon usage bias increases along translational direction, which is consistent with purifying selection against nonsense errors. Fruit fly genes show a nearly symmetric M-shaped spatial pattern of codon usage bias, with less bias in the middle and both ends. The low codon usage bias in the middle region is best explained by interference (the Hill-Robertson effect) between selections at different codon positions. In both yeast and fruit fly, spatial patterns of codon usage bias are characteristically different from patterns of GC-content variations. Effect of expression level on the strength of codon usage bias is more conspicuous than its effect on the shape of the spatial distribution.     

Apolipoprotein E4 domain interaction occurs in living neuronal cells as determined by fluorescence resonance energy transfer

Apolipoprotein (apo) E4 is a major risk factor for Alzheimer disease. Although the mechanisms remain to be determined, the detrimental effects of apoE4 in neurobiology must be based on its unique structural and biophysical properties. One such property is domain interaction mediated by a salt bridge between Arg-61 in the N-terminal domain and Glu-255 in the C-terminal domain of apoE4. This interaction, which does not occur in apoE3 or apoE2, causes apoE4 to bind preferentially to certain lipoprotein particles in vitro and in vivo. Here we used fluorescence resonance energy transfer (FRET) to determine whether apoE4 domain interaction occurs in living neuronal cells. Neuro-2a cells were transfected with constructs encoding apoE3 or apoE4 in which yellow fluorescent protein (YFP) was fused to the N terminus, and cyan fluorescent protein (CFP) was fused to the C terminus. To generate a FRET signal that can be detected by spectrum confocal microscopy, the labeled N and C termini must be in close proximity (<100 A). FRET signals occurred in cells transfected with YFP-apoE4-CFP but not in those transfected with YFP-apoE3-CFP, suggesting that the N and C termini of apoE4 are in close proximity in living cells and that those of apoE3 are not. FRET signals did not occur in cells cotransfected with YFP-apoE4 and apoE4-CFP, suggesting that the FRET in YFP-apoE4-CFP-transfected cells was intramolecular. Mutation of Arg-61 to Thr or Glu-255 to Ala in apoE4, which disrupts domain interaction, abolished FRET in Neuro-2a cells, strongly suggesting that the FRET in YFP-apoE4-CFP cells was caused by domain interaction. ApoE4-producing cells secreted less phospholipid than apoE3-producing cells, but after disruption of domain interaction in apoE4, phospholipid secretion increased to the levels seen with apoE3, suggesting that domain interaction decreases the phospholipid-binding capacity of apoE4. Thus, apoE4 domain interaction occurs in living neuronal cells and may be a molecular basis for apoE4-related neurodegeneration.     

Antioxidant effects of American ginseng berry extract in cardiomyocytes exposed to acute oxidant stress

It is postulated that antioxidant properties of American ginseng root mediate its cardioprotective actions. The antioxidant capabilities of the American ginseng root have been demonstrated previously, however, the berry of the American ginseng has not yet been evaluated. In this study, we tested the American ginseng berry extract (AGBE) for its antioxidant effects in cell-free chemical systems using H(2)O(2)/FeSO(4) to generate hydroxyl radicals which were measured by a fluorescent probe, 2', 7'-dichlorofluorescin diacetate (DCFH/DA). Xanthine/xanthine oxidase was used to generate superoxide anion, which was measured by a fluorescent probe dihydroethidium (DHE). We found that AGBE decreased fluorescence significantly, suggesting that AGBE scavenges oxygen free radicals. We further tested whether AGBE (0.1-1 mg/ml) can protect cardiomyocytes from oxidative injury induced by exogenous or endogenous oxidants. Cells were exposed to either H(2)O(2) or antimycin A (a mitochondrial electron transport chain site III inhibitor that augments mitochondrial oxidant production). The resulting oxidant stress was measured using DCFH/DA and the cell death was assessed using propidium iodide staining. Pretreatment with AGBE (1 mg/ml) significantly attenuated DCF fluorescence by 49% or 85% and reduced cell death by 59% or 63% in cells exposed to H(2)O(2) or antimycin A, respectively. When the effects of extracts from berry and root of American ginseng were compared in cardiomyocytes exposed to antimycin A, we observed that AGBE conferred greater antioxidant protection at the same dose. We conclude that AGBE is a potent antioxidant that protects cardiomyocytes against oxidant-mediated injury and this protection is partly mediated by its free radical scavenging properties.     

Modulation of human 5-lipoxygenase activity by membrane lipids

Mammalian 5-lipoxygenase (5-LO) catalyzes the conversion of arachidonic acid (AA) to leukotrienes, potent inflammatory mediators. 5-LO is activated by a Ca(2+)-mediated translocation to membranes, and demonstrates the characteristic features of interfacially activated enzymes, yet the mechanism of membrane binding of 5-LO is not well understood. In an attempt to understand the mechanism of lipid-mediated activation of 5-LO, we have studied the effects of a large set of lipids on human recombinant 5-LO activity, as well as mutual structural effects of 5-LO and membranes. In the presence of 0.35 mM phosphatidylcholine (PC) and 0.2 mM Ca(2+), there was substrate inhibition at >100 microM AA. Data analysis at low AA concentrations yielded the following: K(m) approximately 103 microM and k(cat) approximately 56 s(-1). 5-LO activity was supported by PC more than by any other lipid tested except for a cationic lipid, which was more stimulatory than PC. Binding of 5-LO to zwitterionic and acidic membranes was relatively weak; the extent of binding increased 4-8 times in the presence of Ca(2+), whereas binding to cationic membranes was stronger and essentially Ca(2+)-independent. Polarized attenuated total reflection infrared experiments implied that 5-LO binds to membranes at a defined orientation with the symmetry axis of the putative N-terminal beta-barrel tilted approximately 45 degrees from the membrane normal. Furthermore, membrane binding of 5-LO resulted in dehydration of the membrane surface and was paralleled with stabilization of the structures of both 5-LO and the membrane. Our results provide insight into the understanding of the effects of membrane surface properties on 5-LO-membrane interactions and the interfacial activation of 5-LO.     

Casein kinase 1alpha interacts with retinoid X receptor and interferes with agonist-induced apoptosis

Agonists of retinoid X receptors (RXRs), which include the natural 9-cis-retinoic acid and synthetic analogs, are potent inducers of growth arrest and apoptosis in some cancer cells. As such, they are being used in clinical trials for the treatment and prevention of solid tumors and are used to treat cutaneous T cell lymphoma. However, the molecular mechanisms that underlie the anti-cancer effects of RXR agonists remain unclear. Here, we show that a novel pro-apoptotic pathway that is induced by RXR agonist is negatively regulated by casein kinase 1alpha (CK1alpha). CK1alpha associates with RXR in an agonist-dependent manner and phosphorylates RXR. The ability of an RXR agonist to recruit CK1alpha to a complex with RXR in cells correlates inversely with its ability to inhibit growth. Remarkably, depletion of CK1alpha in resistant cells renders them susceptible to RXR agonist-induced growth inhibition and apoptosis. Our study shows that CK1alpha can promote cell survival by interfering with RXR agonist-induced apoptosis. Inhibition of CK1alpha may enhance the anti-cancer effects of RXR agonists.     

Is mRNA and protein level of CD46 altered in measles virus vaccine strain S191-infected cells?

Previous research showed that the expression of measles virus receptor CD46 was downregulated after expression of measles virus hemagglutinin protein on the surface of the virus infected cell or triggered by infected cell-to-cell contact. We reported here that the mRNA level of CD46 in MV infected cells was not changed which was tested by real-time quantitative PCR. To further analyse the surface expression alteration of CD46 after MV infection, flow cytometric analysis and indirect immunofluorescence were used to detect the protein level of CD46. Altogether, our results provided a demonstration that the expression of CD46 was not downregulated by the infection of MV strain S191 both on mRNA level and cellular surface protein level. Previous results reported that the "downregulation" of CD46 expression on the cell surface may take place because H protein masks the antibody recognition site on CD46 which results in "downregulation" of the expression of CD46.