Wild type p53 gene causes reorganization of cytoskeleton and,therefore, the impaired deformability and difficult migration of murine erythroleukemia cells

We studied the role of p53 gene in the biophysics and biology in murine erythroleukemia cell line (MEL), with the goal of understanding the influence of this tumor suppressor gene on the deformability and metastasis of tumor cells. Experiments were performed on MEL and p53-transfected MEL (MEL-M with mutant p53 gene and MEL-W with wild-type p53 gene). The cell growth curves indicated that the over-expression of wild-type p53 gene significantly suppressed the growth of MEL, with G(0)-G(1) arrest and apoptosis shown by flow cytometric assays. Confocal laser scanning microscopy revealed that the MEL-W had a more compact organization of the F-Actin cytoskeleton than MEL and MEL-M. Fluorescence polarization measurement indicated a higher membrane fluidity of MEL-W than the other two groups. Fourier transform infrared spectroscopy (FT-IR) showed changes in the composition and/or structure of membrane lipids in MEL-W, with decreases in secondary structures of proteins such as alpha-helix, turns and bends and random coil, in comparison to MEL and MEL-M. The osmotic fragility curves indicated that MEL-W was more fragile and micropipette experiments showed that they had increased elasticity and reduced deformability in comparison to MEL and MEL-M. The adhesion assay with the use of the flow chamber revealed a lower adhesion rate of MEL-W to endothelial cells at high shear stress. The present study on the molecular biology with biophysics of MEL cells contributes to our knowledge on the tumor suppressor gene p53.     

Hybrids of dendritic cells and tumor cells generated by electrofusion simultaneously present immunodominant epitopes from multiple human tumor-associated antigens in the context of MHC class I and class II molecules

Hybrid cells generated by fusing dendritic cells with tumor cells (DC-TC) are currently being evaluated as cancer vaccines in preclinical models and human immunization trials. In this study, we evaluated the production of human DC-TC hybrids using an electrofusion protocol previously defined for murine cells. Human DCs were electrically fused with allogeneic melanoma cells (888mel) and were subsequently analyzed for coexpression of unique DC and TC markers using FACS and fluorescence microscopy. Dually fluorescent cells were clearly observed using both techniques after staining with Abs against distinct surface molecules suggesting that true cell fusion had occurred. We also evaluated the ability of human DC-TC hybrids to present tumor-associated epitopes in the context of both MHC class I and class II molecules. Allogeneic DCs expressing HLA-A*0201, HLA-DR beta 1*0401, and HLA-DR beta 1*0701 were fused with 888mel cells that do not express any of these MHC molecules, but do express multiple melanoma-associated Ags. DC-888mel hybrids efficiently presented HLA-A*0201-restricted epitopes from the melanoma Ags MART-1, gp100, tyrosinase, and tyrosinase-related protein 2 as evaluated by specific cytokine secretion from six distinct CTL lines. In contrast, DCs could not cross-present MHC class I-restricted epitopes after exogenously loading with gp100 protein. DC-888mel hybrids also presented HLA-DR beta 1*0401- and HLA-DR beta 1*0701-restricted peptides from gp100 to CD4(+) T cell populations. Therefore, fusions of DCs and tumor cells express both MHC class I- and class II-restricted tumor-associated epitopes and may be useful for the induction of tumor-reactive CD8(+) and CD4(+) T cells in vitro and in human vaccination trials.     

Identification of uteroglobin-related protein 1 and macrophage scavenger receptor with collagenous structure as a lung-specific ligand-receptor pair

High in normal (HIN)-1 is a secreted protein highly expressed in normal breast epithelium and down-regulated in breast carcinomas. By searching GenBank expressed sequence tag databases, we identified HIN-2, a protein homologous to HIN-1. HIN-2 is identical with a recently identified protein called uteroglobin-related protein 1 (UGRP1). Northern blot analysis demonstrated that UGRP1 is specifically expressed by lung, but not by the other tissues examined. By in situ hybridization experiments, UGRP1 was shown to be expressed by lung Clara-like cells in the bronchial epithelium and to be up-regulated in cystic fibrosis. In a mammalian expression system, secreted recombinant UGRP1 was copurified with apolipoprotein A-I. Using a retroviral vector-mediated expression cloning approach, we identified macrophage scavenger receptor with collagenous structure (MARCO) as a receptor for UGRP1. Northern blot and in situ hybridization experiments indicated that MARCO is expressed by alveolar macrophages in the lung. UGRP1 also bound to bacteria and yeast. LPS, a previously identified MARCO ligand, competed with UGRP1 for binding to MARCO and bacteria. Our findings suggest that UGRP1-MARCO is a ligand-receptor pair that is probably involved in inflammation and pathogen clearance in the lung.     

Cutting edge: SR-PSOX/CXC chemokine ligand 16 mediates bacterial phagocytosis by APCs through its chemokine domain

SR-PSOX and CXC chemokine ligand (CXCL)16, which were originally identified as a scavenger receptor and a transmembrane-type chemokine, respectively, are indicated to be identical. In this study, we demonstrate that membrane-bound SR-PSOX/CXCL16 mediates adhesion and phagocytosis of both Gram-negative and Gram-positive bacteria. Importantly, our prepared anti-SR-PSOX mAb, which suppressed chemotactic activity of SR-PSOX, significantly inhibited bacterial phagocytosis by human APCs including dendritic cells. Various scavenger receptor ligands inhibited the bacterial phagocytosis of SR-PSOX. In addition, the recognition specificity for bacteria was determined by only the chemokine domain of SR-PSOX/CXCL16. Thus, SR-PSOX/CXCL16 may play an important role in facilitating uptake of various pathogens and chemotaxis of T and NKT cells by APCs through its chemokine domain.     

The role of p53 deacetylation in p21Waf1 regulation by laminar flow

Laminar flow arrests vascular endothelial cells at the G0/G1 phase with concurrent increase in p53 and p21Waf1. We investigated the molecular mechanism by which laminar flow activates p53 and p21Waf1 in endothelial cells. The application of a laminar flow (12 dyn/cm2) increased the deacetylation at Lys-320 and Lys-373 of p53 and the acetylation at Lys-382 in human umbilical vein endothelial cells. Laminar flow increased the activity of histone deacetylase (HDAC) and the association of p53 with HDAC1. Treating human umbilical vein endothelial cells with trichostatin A (TSA), an HDAC inhibitor, abolished the flow-induced p53 deacetylation at Lys-320 and Lys-373. To investigate the role of the HDAC-deacetylated p53 in the flow activation of p21Waf1, we found that TSA inhibited the activation at both the mRNA and protein levels. Deletion and mutation analyses of the p21Waf1 promoter revealed that flow activated p21Waf1 through p53 and TSA abrogated this p53-dependent activation. The expression plasmid encoding the p53 mutant, with Lys-320 and Lys-373 replaced by Arg, increased the activity of the co-transfected p21Waf1 promoter, which demonstrates that HDAC-deacetylated p53 can transactivate the p21Waf1 gene. The regulation of the p53-p21Waf1 pathway by laminar flow was further supported by observations that flow caused an increase of p21Waf1 level in the wild-type HCT116 (p53+/+) cells but not in the p53-null HCT116 cells.     

Src phosphorylates Grb2-associated binder 1 upon hepatocyte growth factor stimulation

Grb2-associated binder 1 (Gab1) is known to play an important role in hepatocyte growth factor (HGF) signaling, which rapidly becomes tyrosine-phosphorylated upon HGF stimulation. In this study, we found that the tyrosine phosphorylation of Gab1 in the cells derived from Src/Yes/Fyn null mouse embryos was approximately 40% lower than that in their wild type counterparts upon HGF stimulation. Increased expression of wild-type Src enhanced HGF-induced phosphorylation of Gab1, and, in contrast, expression of the Src kinase-deficient mutant or treatment of the specific Src inhibitor PP1 suppressed it. Expression of a constitutively active Src mutant (Y527F) or oncogenic v-Src led to a prominent increase in Gab1 phosphorylation independent of HGF stimulation. Moreover, Src interacted with Gab1 via both its Src homology 2 and 3 domains and was capable of phosphorylating purified Gab1 in vitro. Finally, the increased phosphorylation of Gab1 by Src selectively potentiated HGF-induced activation of ERK and AKT. Taken together, our results establish a new role for Src in HGF-induced Gab1 phosphorylation.     

Association study between interleukin-1beta gene (IL-1beta) and schizophrenia

An increasing amount of evidence suggests that the pathophysiology of schizophrenia is associated with the abnormal immune system, and cytokines may be important in schizophrenia. Among these cytokines, interleukin-1beta may play a role in the pathogenesis of the disease. In the present study, we investigated the genetic association between a TaqI polymorphism in interleukin-1beta gene (IL-1beta) and schizophrenia by restriction fragment length polymorphism (RFLP) analysis among 132 Chinese families of Han descent. The transmission disequilibrium test (TDT) did not demonstrate an allelic association with schizophrenia. Our results suggested that the TaqI polymorphism in IL-1beta gene might not confer increased susceptibility for schizophrenia.     

A fusion protein of conotoxin MVIIA and thioredoxin expressed in Escherichia coli has significant analgesic activity

omega-Conotoxin MVIIA (CTX MVIIA) is a potent and selective blocker of the N-type voltage-sensitive calcium channel in neurons. Its analgesic and neuroprotective effects may prove useful in treatment of severe pains and ischemia. In this paper, we report that a fusion form of CTX MVIIA with thioredoxin (Trx) has analgesic function. The DNA fragments were chemically synthesized and ligated to form the DNA sequence encoding CTX MVIIA. The synthetic gene was then cloned into the expression vector pET-32a(+) and the fusion protein Trx-CTX MVIIA containing 6x His-tag was purified by one-step metal chelated affinity chromatography (MCAC). The purity of final product was over 95% determined by HPLC and the yield of the fusion protein was approximately 40 mg/L. The analgesic function was detected by using mouse hot-plate assay. After intracranially administering fusion protein with the dose of 0.6 mg/kg, marked analgesia was observed. The analgesic effects (elevated pain thresholds) were dose-dependent and the biological half-life of the fusion toxin was approximately 1.6 h.     

Lipopolysaccharide down-regulates inducible nitric oxide synthase expression in swine heart in vivo

Studies of the regulation of iNOS expression have provided many contradictory results. Comparing iNOS expression profile between cell types or organs of the same animal under the same experimental conditions may provide an explanation for these conflicting results. We have examined iNOS mRNA and protein expression in heart and liver of the same group of pigs. We found that there is a sharp difference in iNOS expression between heart and liver. The iNOS mRNA and protein was constitutively expressed in the heart at high level, but was not detectable in the liver of the same control animal. Lipopolysaccharide (LPS, 100 microg/kg, i.v.) caused a marked iNOS induction in the liver, but significantly down-regulated iNOS expression in the heart. This differential iNOS expression appears to be physiologically relevant, since LPS and the iNOS inhibitor, S-methylisothiourea, exerted different effects on hepatic and myocardial blood flow. Our data demonstrate a fundamental difference in iNOS regulation in the heart and liver of swine, and may explain the contradictory data on the regulation of iNOS expression.     

The footprint of antibody bound to pig cells: evidence of complex surface topology

The disaccharide Gal(alpha)1-3Gal is found on more than 45 different molecules on the endothelium of porcine cells and has recently attracted considerable interest, being the major target recognized by xenoreactive antibodies. In this study, the distribution and topology of Gal(alpha)1-3Gal on porcine endothelial cells was examined to access whether some Gal(alpha)1-3Gal-containing molecules might be preferentially recognized by antibodies binding to Gal(alpha)1-3Gal. Thirteen percent of the Gal(alpha)1-3Gal was found on glycolipid and 87% on glycoproteins. Of all the glycoproteins and glycolipids containing Gal(alpha)1-3Gal, two molecules, fibronectin and the integrin beta1 subunit, were most intensely labeled by galactose oxidase, suggesting that these molecules may be preferentially exposed on the apical surface of the endothelium. Binding of anti-Gal(alpha)1-3Gal antibodies to endothelial cell surfaces significantly diminished labeling of fibronectin and the integrin beta1 subunit by galactose oxidase, indicating that these glycoproteins are targets for the antibodies when binding to intact porcine cells.