Highly stereoselective synthesis and biological properties of nucleoside analogues bearing a spiro inserted oxirane ring

Starting from 2',5'-di-O-TBDMS-3'-ketouridine 1 or its thymine analogue 2, both xylo (3-10) and ribo (20) epimers of a series of 3"-substituted 3'-spironucleosides have been obtained in good yields and with a total stereoselectivity. Most new compounds were moderately cytotoxic with in some cases slightly selective antiproliferative activities. None of these compounds was active against HIV, but some other antiviral activities against HSV-2, CMV, EBV, or VZV, in the micromolar range, were noted in specific cases.     

Cadherin-directed actin assembly: E-cadherin physically associates with the Arp2/3 complex to direct actin assembly in nascent adhesive contacts

Cadherin cell adhesion molecules are major determinants of tissue patterning which function in cooperation with the actin cytoskeleton. In the context of stable adhesion, cadherin/catenin complexes are often envisaged to passively scaffold onto cortical actin filaments. However, cadherins also form dynamic adhesive contacts during wound healing and morphogenesis. Here actin polymerization has been proposed to drive cell surfaces together, although F-actin reorganization also occurs as cell contacts mature. The interaction between cadherins and actin is therefore likely to depend on the functional state of adhesion. We sought to analyze the relationship between cadherin homophilic binding and cytoskeletal activity during early cadherin adhesive contacts. Dissecting the specific effect of cadherin ligation alone on actin regulation is difficult in native cell-cell contacts, due to the range of juxtacrine signals that can arise when two cell surfaces adhere. We therefore activated homophilic ligation using a specific functional recombinant protein. We report the first evidence that E-cadherin associates with the Arp2/3 complex actin nucleator and demonstrate that cadherin binding can exert an active, instructive influence on cells to mark sites for actin assembly at the cell surface.     

Regulation of mesangial cell Na+/Ca2+ exchanger isoforms

An isoform of the Na(+)/Ca(2+) exchanger (SDNCX1.10) was cloned from mesangial cells of Sprague-Dawley rat. Regulation of this isoform was compared to two other clones that were derived from the Dahl/Rapp salt sensitive (SNCX) and salt resistant rat (RNCX). All isoforms differ at the alternative splice site and at amino acid 218 for SNCX. PKC activates RNCX but not SNCX while SDNCX1.10 was also activated by PKC. Regulation of exchanger activities by intracellular calcium ([Ca(2+)](i)), pH, and kinases was assessed using Na-dependent (45)Ca(2+) uptake assays in OK-PTH cells expressing the vector, RNCX, SNCX, or SDNCX1.10. [Ca(2+)](i) was elevated from 50 to 125 nM (n = 4) with thapsigargin (40 nM) and reduced from 50 to 29 nM (n = 4) and 18 nM (n = 4) with 10 or 20 microM BAPTA, respectively. RNCX was active at all three [Ca(2+)](i) while SNCX and SDNCX1.10 were only active at lower [Ca(2+)](i). Varying extracellular pH (pH(e), without nigericin) or pH(e) and intracellular pH (pH(i), with 10 microM nigericin) from pH 7.4 to 6.2, 6.8, or 8.0 showed that SNCX activity was attenuated at both low and high pHs. SDNCX1.10 activity was attenuated only at pH 6.2 and 6.8 (with or without nigericin) while RNCX activity was attenuated at pH 6.2 (with or without nigericin) and pH 6.8 (with nigericin). Finally, only SDNCX1.10 activity was stimulated by 250 microM CPT-cAMP or 250 microM DB-cGMP treatment. Thus the differential regulation of [Ca(2+)](i) by these exchangers is dependent upon the pattern of cellular Na(+)/Ca(2+) exchanger isoform expression.     

The amyloid percursor protein of Alzheimer disease is expressed as a 130 kDa polypeptide in various cultured cell types

The vascular and parenchymal amyloid deposits in Alzheimer disease (AD), normal aging and Down syndrome are mainly composed of a 4 kDa polypeptide (A4), which derives from a larger precursor protein (APP). There is evidence that APP is a transmembrane glycoprotein present in most tissues, but the characteristics of APP in intact cells are not yet known. In order to investigate this issue, we examined the immunoreactivity of fibroblasts of human and nonhuman cell lines with antisera raised to synthetic peptides corresponding to A4 and to two other domains of the APP. All three antisera recognized a 130 kDa polypeptide (APP-130) in immunoblots from all cell lines. In fibroblasts, an additional polypeptide of 228 kDa (APP-228) was recognized by the antiserum to A4. In immunoblots of two dimensional gels, APP-130 showed a pI of 6.2, while APP-228 failed to focus in the pH range of 4.7-7.0. Sequential extractions of cells with buffer and with Triton X-100 indicate that APP-130 is extractable with nonionic detergents at high ionic strength, whereas 228 kDa APP is a cystolic component. Immunofluorescence staining is consistent with an intracellular perinuclear and plasma membrane localization. It is concluded that APP-130 and APP-228 are two forms of the APP which result from extensive posttranslational modifications of a smaller original gene product. It is likely that APP undergoes similar posttranslational modifications in different cell types.     

Heavy metal cations permeate the TRPV6 epithelial cation channel

TRPV6 belongs to the vanilloid family of the transient receptor potential channel (TRP) superfamily. This calcium-selective channel is highly expressed in the duodenum and the placenta, being responsible for calcium absorption in the body and fetus. Previous observations have suggested that TRPV6 is not only permeable to calcium but also to other divalent cations in epithelial tissues. In this study, we tested whether TRPV6 is indeed also permeable to cations such as zinc and cadmium. We found that the basal intracellular calcium concentration was higher in HEK293 cells transfected with hTRPV6 than in non-transfected cells, and that this difference almost disappeared in nominally calcium-free solution. Live cell imaging experiments with Fura-2 and NewPort Green DCF showed that overexpression of human TRPV6 increased the permeability for Ca(2+), Ba(2+), Sr(2+), Mn(2+), Zn(2+), Cd(2+), and interestingly also for La(3+) and Gd(3+). These results were confirmed using the patch clamp technique. (45)Ca uptake experiments showed that cadmium, lanthanum and gadolinium were also highly efficient inhibitors of TRPV6-mediated calcium influx at higher micromolar concentrations. Our results suggest that TRPV6 is not only involved in calcium transport but also in the transport of other divalent cations, including heavy metal ions, which may have toxicological implications.     

Epigenetic activation of SOX11 in lymphoid neoplasms by histone modifications

Recent studies have shown aberrant expression of SOX11 in various types of aggressive B-cell neoplasms. To elucidate the molecular mechanisms leading to such deregulation, we performed a comprehensive SOX11 gene expression and epigenetic study in stem cells, normal hematopoietic cells and different lymphoid neoplasms. We observed that SOX11 expression is associated with unmethylated DNA and presence of activating histone marks (H3K9/14Ac and H3K4me3) in embryonic stem cells and some aggressive B-cell neoplasms. In contrast, adult stem cells, normal hematopoietic cells and other lymphoid neoplasms do not express SOX11. Such repression was associated with silencing histone marks H3K9me2 and H3K27me3. The SOX11 promoter of non-malignant cells was consistently unmethylated whereas lymphoid neoplasms with silenced SOX11 tended to acquire DNA hypermethylation. SOX11 silencing in cell lines was reversed by the histone deacetylase inhibitor SAHA but not by the DNA methyltransferase inhibitor AZA. These data indicate that, although DNA hypermethylation of SOX11 is frequent in lymphoid neoplasms, it seems to be functionally inert, as SOX11 is already silenced in the hematopoietic system. In contrast, the pathogenic role of SOX11 is associated with its de novo expression in some aggressive lymphoid malignancies, which is mediated by a shift from inactivating to activating histone modifications.     

Lineage-specific restraint of pituitary gonadotroph cell adenoma growth

Although pituitary adenomas are usually benign, unique trophic mechanisms restraining cell proliferation are unclear. As GH-secreting adenomas are associated with p53/p21-dependent senescence, we tested mechanisms constraining non-functioning pituitary adenoma growth. Thirty six gonadotroph-derived non-functioning pituitary adenomas all exhibited DNA damage, but undetectable p21 expression. However, these adenomas all expressed p16, and >90% abundantly expressed cytoplasmic clusterin associated with induction of the Cdk inhibitor p15 in 70% of gonadotroph and in 26% of somatotroph lineage adenomas (p = 0.006). Murine LβT2 and αT3 gonadotroph pituitary cells, and αGSU.PTTG transgenic mice with targeted gonadotroph cell adenomas also abundantly expressed clusterin and exhibited features of oncogene-induced senescence as evidenced by C/EBPβ and C/EBPδ induction. In turn, C/EBPs activated the clusterin promoter ～5 fold, and elevated clusterin subsequently elicited p15 and p16 expression, acting to arrest murine gonadotroph cell proliferation. In contrast, specific clusterin suppression by RNAis enhanced gonadotroph proliferation. FOXL2, a tissue-specific gonadotroph lineage factor, also induced the clusterin promoter ～3 fold in αT3 pituitary cells. As nine of 12 pituitary carcinomas were devoid of clusterin expression, this protein may limit proliferation of benign adenomatous pituitary cells. These results point to lineage-specific pathways restricting uncontrolled murine and human pituitary gonadotroph adenoma cell growth.     

Mechanisms of chromosome banding

A series of biochemical investigations were undertaken to determine the mechanism of Q-banding. The results were as follows: 1. In agreement with previous studies, highly AT-rich DNA, such as poly(dA)-poly(dT), markedly enhanced quinacrine fluorescence while GC containing DNA quenched fluorescence. These effects persisted at DNA concentrations comparable to those in the metaphase chromosome. 2. Studies of quinacrine-DNA complexes in regard to the hypochromism of quinacrine, DNA Tm, DNA viscosity, and equilibrium dialysis, indicated the quinacrine was bound by intercalation with relatively little side binding. 3. Single or double stranded nucleotide polymers, in the form of complete or partial helices, were 1000-fold more effective in quenching than solutions of single nucleotides, suggesting that base stacking is required for quenching. 4. Studies of polymers in the A conformation, such as transfer RNA and DNA-RNA hybrids, indicated that marked base tilting does not affect the ability of nucleic acids to cause quenching or enhancement of quinacrine fluorescence. 5. Salts inhibit the binding of quinacrine to DNA. 6. Spermine, polylysine and polyarginine, which bind in the small groove of DNA, inhibited quinacrine binding and quenching, while histones, which probably bind in the large groove, had little effect. This correlated with the observation that removal of histones with acid has no effect on Q-banding. 7. Mouse liver chromatin was separated into five fractions. At concentrations of quinacrine from 2×10^?6 to 2×10^?5 M all fractions inhibited to varying degrees the ability of the chromatin DNA to bind quinacrine and quench quinacrine fluorescence. At saturating levels of quinacrine two fractions, the 400 g pellet (rich in heterochromatin) and a dispersed euchromatin supernatant fraction, showed a decreased number of binding sites for quinacrine. These two fractions were also the richest in non-histone proteins. 8. DNA isolated from the different fractions all showed identical quenching of quinacrine fluorescence. 9. Mouse GC-rich, mid-band, AT-rich, and satellite DNA, isolated by CsCl and Cs₂SO₄-Ag⁺ centrifugation all showed identical quenching of quinacrine fluorescence, indicating that within a given organism, except for very AT or GC-rich satellites, the variation in base composition is not adequate to explain Q-banding. — We interpret these results to indicate that: (a) quinacrine binds to chromatin by intercalation of the three planar rings with the large group at position 9 lying in the small groove of DNA, (b) most pale staining regions are due to a decrease binding of quinacrine, and (c) this inhibition of binding is predominately due to non-histone proteins.     

Reduced food intake and body weight in mice deficient for the G protein-coupled receptor GPR82

G protein-coupled receptors (GPCR) are involved in the regulation of numerous physiological functions. Therefore, GPCR variants may have conferred important selective advantages during periods of human evolution. Indeed, several genomic loci with signatures of recent selection in humans contain GPCR genes among them the X-chromosomally located gene for GPR82. This gene encodes a so-called orphan GPCR with unknown function. To address the functional relevance of GPR82 gene-deficient mice were characterized. GPR82-deficient mice were viable, reproduced normally, and showed no gross anatomical abnormalities. However, GPR82-deficient mice have a reduced body weight and body fat content associated with a lower food intake. Moreover, GPR82-deficient mice showed decreased serum triacylglyceride levels, increased insulin sensitivity and glucose tolerance, most pronounced under Western diet. Because there were no differences in respiratory and metabolic rates between wild-type and GPR82-deficient mice our data suggest that GPR82 function influences food intake and, therefore, energy and body weight balance. GPR82 may represent a thrifty gene most probably representing an advantage during human expansion into new environments.