Tat peptide as an efficient molecule to translocate gold nanoparticles into the cell nucleus

The labeling of targeting molecules with nanoparticles has revolutionized the visualization of cellular or tissue components by electron microscopy. A particularly desirable target is the nucleus, because the genetic information is there. To date, utilizing nanoparticles for nuclear targeting has not proved very successful due to the impermeable nature of the plasma and nuclear membranes; thus nanoparticle design and synthesis is a critical factor. We report in this article the synthesis of water-soluble gold nanoparticles functionalized with a Tat protein-derived peptide sequence by a straightforward and economical methodology. The particles were subsequently tested in vitro with a human fibroblast cell line by optical and transmission electron microscopy to determine the biocompatibility of these nanoparticles and whether the functionalization with the translocation peptide allowed particles to transfer across the cell membrane and locate in the nucleus.     

The heterospecific habitat copying hypothesis: can competitors indicate habitat quality?

According to the "habitat copying" hypothesis, animals use thereproductive performance of conspecifics to assess habitat suitabilityand choose their future breeding site. This is because conspecificsshare ecological needs and thus indicate habitat suitability.Here, we propose the "heterospecific habitat copying" hypothesis,which states that animals should use public information (i.e.,information derived from the performance of others) from con-and heterospecifics sharing ecological needs. In a correlationalapproach we test some assumptions and predictions of this hypothesiswith a data set from two sympatric bird populations, rollers(Coracias garrulus) and kestrels (Falco tinnunculus), usingthe same nest-boxes and exploiting similar food resources. Sincekestrels are residents and breed earlier, we assumed that theyare dominant over rollers for nest-box acquisition. The environmentappears to be patchy for both species and temporally predictablefor kestrels only. Two results suggest that the use of heterospecificpublic information in breeding habitat selection may be at work:(1) an increase in the reoccupancy probability by kestrels ofprevious roller nests with increasing nest success, and (2)an increase in roller breeding population with increasing localkestrel success. Most of the other observed patterns could beexplained by alternative mechanisms such as natal philopatry,breeding fidelity, conspecific attraction, intraspecific habitatcopying, and the effect of interspecific competition.     

High performance liquid chromatography analysis of a 4-anilinoquinazoline derivative (PD153035), a specific inhibitor of the epidermal growth factor receptor tyrosine kinase, in rat plasma

The quinazoline derivative, 4-N-(3'-bromo-phenyl)amino-6,7-dimethoxyquinazoline (PD153035), has recently been identified as a potential drug for the treatment of proliferative disease. Here, we report a sensitive high performance liquid chromatography (HPLC)-based quantitative detection method for measurement of PD153035 levels in rat plasma. Sample pretreatment involved a two-step extraction with chloroform. The analytes were separated on a column packed with OmniSpher C18 material and eluted with acetonitrile-0.1 M ammonium acetate, pH 7.2 (70:30, v/v). The column effluent was monitored by UV detection at 330 nm. A linear response was achieved over the concentration range 0.50-100.00 microM using multilevel calibration with an internal standard. The analytical method inter- and intra-run accuracy and precision were better than +/-15%. The lower limit of quantification was 0.50 microM. The method has been applied to study the preclinical pharmacokinetics of this compound in rats.     

PrP(106-126) activates neuronal intracellular kinases and Egr1 synthesis through activation of NADPH-oxidase independently of PrPc

Prion diseases are characterised by severe neural lesions linked to the presence of an abnormal protease-resistant isoform of cellular prion protein (PrPc). The peptide PrP(106-126) is widely used as a model of neurotoxicity in prion diseases. Here, we examine in detail the intracellular signalling cascades induced by PrP(106-126) in cortical neurons and the participation of PrPc. We show that PrP(106-126) induces the activation of subsets of intracellular kinases (e.g., ERK1/2), early growth response 1 synthesis and induces caspase-3 activity, all of which are mediated by nicotinamide adenine dinucleotide phosphate hydrogen-oxidase activity and oxidative stress. However, cells lacking PrPc are similarly affected after peptide exposure, and this questions the involvement of PrPc in these effects.     

Importance of the GP dipeptide of the antiporter motif and other membrane-embedded proline and glycine residues in tetracycline efflux protein Tet(L)

Proline and glycine residues are well represented among functionally important residues in hydrophobic domains of membrane transport proteins, and several critical roles have been suggested for them. Here, the effects of mutational changes in membrane-embedded proline and glycine residues of Tet(L) were examined, with a focus on the conserved GP(155,156) dipeptide of motif C, a putative "antiporter motif". Mutation of Gly155 to cysteine resulted in a mutant Tet(L) that bound its tetracycline-divalent metal (Tc-Me2+) substrate but did not catalyze efflux or exchange of Tc-Me2+ or catalyze uptake or exchange of Rb+ which was used to monitor the coupling ion. These results support suggestions that this region is involved in the conformational changes required for translocation. Mutations in Pro156 resulted in reduction (P156G) or loss (P156A or P156C) of Tc-Me2+ efflux capacity. All three Pro156 mutants exhibited a K+ leak (monitored by 86Rb+ fluxes) that was not observed in wild-type Tet(L). A similar leak was observed in a mutant in a membrane-embedded proline residue elsewhere in the Tet(L) protein (P175C) as well as in a P156C mutant of related antiporter Tet(K). These findings are consistent with roles proposed for membrane-embedded prolines in tight helix packing. Patterns of Tc resistance conferred by additional Tet(L) mutants indicate important roles for another GP dipeptide in transmembrane segment (TMS) X as well as for membrane-embedded glycine residues in TMS XIII.     

Human DNA methyltransferase 1 is required for maintenance of the histone H3 modification pattern

DNA methyltransferase 1 (DNMT1) plays an essential role in murine development and is thought to be the enzyme primarily responsible for maintenance of the global methylation status of genomic DNA. However, loss of DNMT1 in human cancer cells affects only the methylation status of a limited number of pericentromeric sequences. Here we show that human cancer cells lacking DNMT1 display at least two important differences with respect to wild type cells: a profound disorganization of nuclear architecture, and an altered pattern of histone H3 modification that results in an increase in the acetylation and a decrease in the dimethylation and trimethylation of lysine 9. Additionally, this phenotype is associated with a loss of interaction of histone deacetylases (HDACs) and HP1 (heterochromatin protein 1) with histone H3 and pericentromeric repetitive sequences (satellite 2). Our data indicate that DNMT1 activity, via maintenance of the appropriate histone H3 modifications, contributes to the preservation of the correct organization of large heterochromatic regions.     

Beta7 integrin deficiency suppresses B cell homing and attenuates chronic ileitis in SAMP1/YitFc mice

Lymphocyte recruitment to intestinal tissues depends on β(7) integrins. In this study, we studied disease severity and lymphocyte recruitment into the small intestine in SAMP1/YitFc mice, which develop chronic ileitis with similarity to human Crohn's disease. To assess the role of β(7) integrins in chronic ileitis, we generated SAMP1/YitFc lacking β(7) integrins (SAMP1/YitFc Itgb7(-/-)) using a congenic strain developed via marker-assisted selection. We analyzed ileal inflammation in SAMP1/YitFc and SAMP1/YitFc Itgb7(-/-) mice by histopathology and the distribution of T and B lymphocytes in the mesenteric lymph nodes (MLNs) by flow cytometry. Short-term (18 h) adoptive transfer experiments were used to study the in vivo homing capacity of T and B lymphocytes. In both young (<20 wk) and old (20-50 wk) SAMP1/YitFc Itgb7(-/-) mice, ileitis was reduced by 30-50% compared with SAMP1/YitFc mice. SAMP1/YitFc Itgb7(-/-) mice showed a dramatic 67% reduction in the size of their MLNs, which was caused by a 85% reduction in lymphocyte numbers and reduced short-term B cell homing. Flow cytometric analysis revealed a highly significant decrease in the percentage of B cells in MLNs of SAMP1/YitFc Itgb7(-/-) mice. Cotransfer of SAMP1/YitFc MLN B cells but not SAMP1/YitFc Itgb7(-/-) MLN B cells along with CD4(+) T cells resulted in exacerbated ileitis severity in SCID mice. Our findings suggest that β(7) integrins play an essential role in spontaneous chronic ileitis in vivo by promoting homing of disease-exacerbating B cells to MLNs and other intestinal tissues.     

New functions of XPC in the protection of human skin cells from oxidative damage

Xeroderma pigmentosum (XP) C is involved in the recognition of a variety of bulky DNA-distorting lesions in nucleotide excision repair. Here, we show that XPC plays an unexpected and multifaceted role in cell protection from oxidative DNA damage. XP-C primary keratinocytes and fibroblasts are hypersensitive to the killing effects of DNA-oxidizing agents and this effect is reverted by expression of wild-type XPC. Upon oxidant exposure, XP-C primary keratinocytes and fibroblasts accumulate 8,5'-cyclopurine 2'-deoxynucleosides in their DNA, indicating that XPC is involved in their removal. In the absence of XPC, a decrease in the repair rate of 8-hydroxyguanine (8-OH-Gua) is also observed. We demonstrate that XPC-HR23B complex acts as cofactor in base excision repair of 8-OH-Gua, by stimulating the activity of its specific DNA glycosylase OGG1. In vitro experiments suggest that the mechanism involved is a combination of increased loading and turnover of OGG1 by XPC-HR23B complex. The accumulation of endogenous oxidative DNA damage might contribute to increased skin cancer risk and account for internal cancers reported for XP-C patients.