A transgenic class I antigen is recognized as self and functions as a restriction element

The function of a transgenic Dd class I molecule in the induction of immunologic tolerance to major histocompatibility complex antigens and in directing major histocompatibility complex restriction in C57BL/6 mice were investigated. All of the transgenic Dd mouse strains were found to be tolerant for the Dd antigen. Spleen cells from transgenic mice were immunocompetent but consistently failed to generate an anti-Dd cytotoxic T lymphocyte response in vitro, and skin grafts between transgenic Dd mice were not rejected. These data suggests that the Dd antigen was recognized as a self molecule. In addition, the transgenic Dd mice generated antigen-specific Dd-restricted cytotoxic T lymphocyte, indicating that the Dd antigen also functioned as a restriction element for antigen recognition. These observations demonstrate the usefulness of the transgenic mouse system for studying class I antigen expression and function.     

Regulating gene expression in transgenic animals.

Regardless of the field of application, the raison d'etre of transgenic animals is to study gene regulation and function. With increasing frequency, mammalian genes are being isolated with no concomitant knowledge of their function. The human genome mapping initiative will undoubtedly produce a cornucopia of such genes. While the merit of taking a transgenic route to study genes of unknown function is axiomatic, the choices of strategies for gene regulation in vivo may not be fully appreciated. This review will address two main points: first, the targeted and regulated expression of genes, and second, the structural and functional ablation of genes.     

Stabilization of the prostate‐specific tumor suppressor NKX3.1 by the oncogenic protein kinase Pim‐1 in prostate cancer cells

Loss of NKX3.1 is an early and consistent event in prostate cancer and is associated with increased proliferation of prostate epithelial cells and poor prognosis. NKX3.1 stability is regulated post‐translationally through phosphorylation at multiple sites by several protein kinases. Here, we report the paradoxical stabilization of the prostate‐specific tumor suppressor NKX3.1 by the oncogenic protein kinase Pim‐1 in prostate cancer cells. Pharmacologic Pim‐1 inhibition using the small molecule inhibitor CX‐6258 decreased steady state levels and half‐life of NKX3.1 protein but mRNA was not affected. This effect was reversed by inhibition of the 26S‐proteasome, demonstrating that Pim‐1 protects NKX3.1 from proteasome‐mediated degradation. Mass spectrometric analyses revealed Thr89, Ser185, Ser186, Ser195, and Ser196 as Pim‐1 phospho‐acceptor sites on NKX3.1. Through mutational analysis, we determined that NKX3.1 phosphorylation at Ser185, Ser186, and within the N‐terminal PEST domain is essential for Pim‐1‐mediated stabilization. Further, we also identified Lys182 as a critical residue for NKX3.1 stabilization by Pim‐1. Pim‐1‐mediated NKX3.1 stabilization may be important in maintaining normal cellular homeostasis in normal prostate epithelial cells, and may maintain basal NKX3.1 protein levels in prostate cancer cells. J. Cell. Biochem. 114: 1050–1057, 2013. © 2012 Wiley Periodicals, Inc.     

Regulation of apoptosis during neuronal differentiation by ceramide and b-series complex gangliosides

Lipid analysis of gestational day E14.5 mouse brain revealed elevation of ceramide to a tissue concentration that induced apoptosis when added to the medium of neuroprogenitor cells grown in cell culture. Elevation of ceramide was coincident with the first appearance of b-series complex gangliosides (BCGs). Expression of BCGs by stable transfection of murine neuroblastoma (F-11) cells with sialyltransferase-II (ST2) resulted in a 70% reduction of ceramide-induced apoptosis. This was most likely due to an 80% reduced expression of prostate apoptosis response-4 (PAR-4). PAR-4 expression and apoptosis were restored by preincubation of ST2-transfected cells with N-butyl deoxinojirimycin (NB-DNJ) or PD98059, two inhibitors of ganglioside biosynthesis or p42/44 mitogen-activated protein (MAPK) kinase, respectively. In sections of day E14.5 mouse brain, the intermediate zone showed intensive staining for complex gangliosides, but only low staining for apoptosis (TUNEL) and PAR-4. Apoptosis and PAR-4 expression, however, were elevated in the ventricular zone which only weakly stained for complex gangliosides. Whole cell patch clamping revealed a 2-fold increased calcium influx in ST2-transfected cells, the blocking of which with nifedipine restored apoptosis to the level of untransfected cells. In serum-free culture, supplementation of the medium with IGF-1 was required to maintain MAPK phosphorylation and the anti-apoptotic effect of BCG expression. BCG-enhanced calcium influx and the presence of insulin-like growth factor-1 may thus activate a cell survival mechanism that selectively protects developing neurons against ceramide-induced apoptosis by up-regulation of MAPK and reduction of PAR-4 expression.     

Osteoblast-derived oxysterol is a migration-inducing factor for human breast cancer cells

Bone metastasis is the major reason for death caused by breast cancer. We used human breast cancer (MCF-7) cells that are poorly metastatic but show highly inducible migration to determine bone-derived factors that induce migration of initially non-disseminating breast cancer cells. We have found that a lipid fraction from human osteoblast-like MG63 cell-conditioned medium (MG63CM) contains a migration-inducing factor for MCF-7 cells. In this fraction, we have identified oxysterol (OS) as a lipid mediator for tumor cell migration. In MCF-7 cells, insulin-like growth factor 1 elevates the expression of OS-binding protein-related protein 7. Binding of OS to OS-binding protein or OS-binding protein-related protein is known to trigger elevation of sphingomyelin, a sphingolipid that organizes lipid microdomains in the cell membrane. In MCF-7 cells, OS increases the intracellular concentration of sphingomyelin and other phospholipids and induces the translocation of the small GTPase p21Ras to GM1- and cholesterol-rich membrane areas. The induction of migration by MG63CM is prevented by incubation of MG63 cells with mevinolin, a statin-type cholesterol biosynthesis inhibitor that depletes the conditioned medium of OS. Osteoblast-derived OS may, thus, be a yet unrecognized lipid mediator for bone metastasis of breast cancer and a new target for anti-metastasis chemotherapy with statins.     

Treatment with apolipoprotein A-1 mimetic peptide reduces lupus-like manifestations in a murine lupus model of accelerated atherosclerosis

Introduction

The purpose of this study was to evaluate the effects of L-4F, an apolipoprotein A-1 mimetic peptide, alone or with pravastatin, in apoE^-/-Fas^-/-C57BL/6 mice that spontaneously develop immunoglobulin G (IgG) autoantibodies, glomerulonephritis, osteopenia, and atherosclerotic lesions on a normal chow diet.

Methods

Female mice, starting at eight to nine weeks of age, were treated for 27 weeks with 1) pravastatin, 2) L-4F, 3) L-4F plus pravastatin, or 4) vehicle control, followed by disease phenotype assessment.

Results

In preliminary studies, dysfunctional, proinflammatory high-density lipoproteins (piHDL) were decreased six hours after a single L-4F, but not scrambled L-4F, injection in eight- to nine-week old mice. After 35 weeks, L-4F-treated mice, in the absence/presence of pravastatin, had significantly smaller lymph nodes and glomerular tufts (P_{L, LP} < 0.05), lower serum levels of IgG antibodies to double stranded DNA (dsDNA) (P_L < 0.05) and oxidized phospholipids (oxPLs) (P_{L, LP} < 0.005), and elevated total and vertebral bone mineral density (P_{L, LP} < 0.01) compared to vehicle controls. Although all treatment groups presented larger aortic root lesions compared to vehicle controls, enlarged atheromas in combination treatment mice had significantly less infiltrated CD68⁺ macrophages (P_LP < 0.01), significantly increased mean α-actin stained area (P_LP < 0.05), and significantly lower levels of circulating markers for atherosclerosis progression, CCL19 (P_{L, LP} < 0.0005) and VCAM-1 (P_L < 0.0002).

Conclusions

L-4F treatment, alone or with pravastatin, significantly reduced IgG anti-dsDNA and IgG anti-oxPLs, proteinuria, glomerulonephritis, and osteopenia in a murine lupus model of accelerated atherosclerosis. Despite enlarged aortic lesions, increased smooth muscle content, decreased macrophage infiltration, and decreased pro-atherogenic chemokines in L-4F plus pravastatin treated mice suggest protective mechanisms not only on lupus-like disease, but also on potential plaque remodeling in a murine model of systemic lupus erythematosus (SLE) and accelerated atherosclerosis.     

Molecular architecture of the ribosome‐bound Hepatitis C Virus internal ribosomal entry site RNA

Internal ribosomal entry sites (IRESs) are structured cis‐acting RNAs that drive an alternative, cap‐independent translation initiation pathway. They are used by many viruses to hijack the translational machinery of the host cell. IRESs facilitate translation initiation by recruiting and actively manipulating the eukaryotic ribosome using only a subset of canonical initiation factor and IRES transacting factors. Here we present cryo‐EM reconstructions of the ribosome 80S‐ and 40S‐bound Hepatitis C Virus (HCV) IRES. The presence of four subpopulations for the 80S•HCV IRES complex reveals dynamic conformational modes of the complex. At a global resolution of 3.9 Å for the most stable complex, a derived atomic model reveals a complex fold of the IRES RNA and molecular details of its interaction with the ribosome. The comparison of obtained structures explains how a modular architecture facilitates mRNA loading and tRNA binding to the P‐site. This information provides the structural foundation for understanding the mechanism of HCV IRES RNA‐driven translation initiation.     

Selective apoptosis of pluripotent mouse and human stem cells by novel ceramide analogues prevents teratoma formation and enriches for neural precursors in ES cell-derived neural transplants

The formation of stem cell-derived tumors (teratomas) is observed when engrafting undifferentiated embryonic stem (ES) cells, embryoid body-derived cells (EBCs), or mammalian embryos and is a significant obstacle to stem cell therapy. We show that in tumors formed after engraftment of EBCs into mouse brain, expression of the pluripotency marker Oct-4 colocalized with that of prostate apoptosis response-4 (PAR-4), a protein mediating ceramide-induced apoptosis during neural differentiation of ES cells. We tested the ability of the novel ceramide analogue N-oleoyl serinol (S18) to eliminate mouse and human Oct-4(+)/PAR-4(+) cells and to increase the proportion of nestin(+) neuroprogenitors in EBC-derived cell cultures and grafts. S18-treated EBCs persisted in the hippocampal area and showed neuronal lineage differentiation as indicated by the expression of beta-tubulin III. However, untreated cells formed numerous teratomas that contained derivatives of endoderm, mesoderm, and ectoderm. Our results show for the first time that ceramide-induced apoptosis eliminates residual, pluripotent EBCs, prevents teratoma formation, and enriches the EBCs for cells that undergo neural differentiation after transplantation.     

Isolation of a cytosolic beta-glucosidase from calf liver and characterization of its active site with alkyl glucosides and basic glycosyl derivatives

A beta-glucosidase/beta-galactosidase with Mr 52,500 was isolated from calf liver cytosol by a four-step procedure incorporating affinity chromatography on N-(9-carboxynonyl)-deoxynojirimycin-AH-Sepharose. Its pH optimum was at 5.8 with half-maximal activity at pH 3.5 and 8.6. Affinity for gluco compounds expressed by Km or Ki of substrates and inhibitors was 2- to 10-fold higher than for the corresponding galacto compounds. Alkyl glucosides were hydrolyzed with lower Vmax than p-nitrophenyl and 4-methylumbelliferyl glucosides, but due to their higher affinity the alkyl glucosides displayed values for kcat/Km of the same magnitude of the aryl glucosides when the alkyl chains were longer than octyl. Glucosylsphingosine was bound with Ki (= Km) 2.2 microM and hydrolyzed with a Vmax that was 50-fold lower than the Vmax for 4-methylumbelliferyl beta-glucoside. The product sphingosine was inhibitory with Ki 0.30 microM. A systematic study with alkyl glucosides and glucosylamines defined the aglycon site as a narrow, strongly hydrophobic cleft able to accommodate up to 10 methylene groups. Each CH2 group contributed 3.1 kJ/mol to the standard free energy of binding. The inhibition by gluco- and galactosylamine and by 1-deoxynojirimycin and its D-galacto analog was approximately 200-fold better than by corresponding nonbasic compounds. pH dependence of the inhibition and comparison with permanently cationic glycosyl derivatives showed that the nonprotonated form was the inhibiting species. This feature puts the cytosolic beta-glucosidase in the large class of glycoside hydrolases which strongly bind basic glycosyl derivatives by their protonation at the active site and formation of a shielded ion pair with the carboxylate of an aspartic or glutamic side chain.