Identification of cryptochrome DASH from vertebrates

A new type of cryptochrome, CRY-DASH, has been recently identified. The CRY-DASH proteins constitute the fifth subfamily of the photolyase/cryptochrome family. CRY-DASHs have been identified from Synechocystis sp. PCC 6803, Vibrio cholerae, and Arabidopsis thaliana. The Synechocystis CRY-DASH was the first cryptochrome identified from bacteria, and its biochemical features and tertiary structure have been extensively investigated. To determine how broadly the subfamily is distributed within living organisms, we searched for new CRY-DASH candidates within several databases. We found five sequences as new CRY-DASH candidates, which are derived from four marine bacteria and Neurospora crassa. We also found many CRY-DASH candidates from the EST databases, which included sequences from fish and amphibians. We cloned and sequenced the cDNAs of the zebrafish and Xenopus laevis candidates, based on the EST sequences. The proteins encoded by the two genes were purified and characterized. Both proteins contained folate and flavin cofactors, and have a weak DNA photolyase activity. A phylogenetic analysis revealed that the seven candidates actually belong to the new type of cryptochrome subfamily. This is the first report of the CRY-DASH members from vertebrates and fungi.     

CD27/CD70 interaction directly drives B cell IgG and IgM synthesis

CD27 is a T cell activation antigen expressed on a majority of peripheral blood T cells. CD27 is also expressed on a subpopulation of human B cells, and it is reported that CD27⁺ B cells secrete both IgG and IgM. CD70, a ligand for CD27, is expressed on activated T and B cells, suggesting an interaction between T and B cells via CD27/CD70 ligation. Here, we analyze B cell immunoglobulin synthesis using a CD70 transfectant and present functional data showing that B cells secrete large amounts of IgG and IgM as a result of the CD27/CD70 interaction. A flow cytometric analysis showed that CD27 expression was increased and CD70 was expressed on tonsillar and peripheral blood B cells after activation with Staphylococcus aureus Cowan strain (SAC) plus interleukin (IL-2). In addition, the proliferation of B cells was enhanced mildly by the addition of CD70 transfectant, and its proliferation was blocked by anti-CD70 mAb. More importantly, the CD70 transfectant enhanced IgG and IgM production by purified B cells greatly in the presence of SAC plus IL-2. The enhancement was completely blocked by the addition of either anti-CD70 mAb or anti-CD27 mAb. Strongly suggesting that the interaction of CD27 with its ligand, CD70, on B cells plays an important role in B cell growth and differentiation to produce IgG and IgM.     

Association of genetic variation of the RIL gene,encoding a PDZ-LIM domain protein and localized in 5q31.1, with low bone mineral density in adult Japanese women

Twin and family studies had shown that genetic factors are important determinants of bone mass. Multiple genes might be involved. One candidate gene, the reversion-induced LIM gene (RIL), is a PDZ and LIM-domain-containing protein and has been localized within the cytokine cluster of chromosome 5 (5q31.1). In a genetic study of 370 adult Japanese women, we investigated the correlation between radial bone mineral density (BMD) and a genetic variation (−3333T→C) of the 5'-flanking region of RIL gene. A significant association was identified between the RIL variation −3333T→C and radial BMD (r=0.15, P=0.003). The variation of the RIL locus may be an important determinant of osteoporosis.     

Immunoelectron microscopic localization of carcinoembryonic antigen in gastric adenocarcinoma cell lines

The distribution of carcinoembryonic antigen (CEA) in human gastric adenocarcinoma cell lines (HPE-GAC-3 cells and HPE-GAC-2 cells) was determined immunohistochemically by indirect peroxidase-labeled antibody method at the light and electron microscopic levels. In GAC-3 cells that proliferated as non-adherent single cells, CEA was located in the perinuclear spaces, the endoplasmic reticulum, Golgi apparatus, vesicles, multivesicular body (MVB) and entire plasma membrane. Membrane CEA was shown to be internalized into MVB in GAC-3 cells. In GAC-2 cells that form an acinus, CEA was predominantly present along the microvilli of the lumina) surface and in glycocalyceal bodies, the vesicles which bud from the microvilli into the lumen. These results suggest that in poorly differentiated cancer cells CEA is transported over the entire cell surface, retained on the membrane and accumulated Into the cell by way of the MVB, but in well differentiated cancer cells the newly synthesized CEA is rapidly and predominantly transported to the luminal surface and rapidly released from the membrane into the lumen by way of the glycocalyceal body.     

p38 Mitogen-activated protein kinase activation in endothelial cell is implicated in cell alignment and elongation induced by fluid shear stress.

Fluid shear stress is thought to be important in maintaining the phenotype of endothelial cells (ECs) in vivo. The purpose of the study was to determine the effect of varying levels of laminar shear stress on EC elongation and alignment and the role of p38 mitogen-activated protein kinase (MAPK) on the morphologic change induced by shear stress. Cultured bovine aortic ECs were subjected to 1, 4, 7, 14, or 20 dyne/cm(2) laminar steady shear stress. On morphometric analysis of static ECs, the average orientation angle was 41 degrees , whereas after 24 h shear stress at 1, 4, 7, 14, and 20 dyne/cm(2) the angles were 34 degrees, 33 degrees, 16 degrees, 11 degrees, and 10 degrees, respectively. The shape index of static ECs was 0.76, whereas the indexes of ECs exposed to shear stress were 0.72, 0.72, 0.65, 0.50, and 0.47, respectively. The time and the magnitude of activation of p38 MAPK were dependent on the level of shear stress. The results indicate that a minimum shear stress of 7 to 14 dynes/cm(2) is necessary for cell alignment and elongation and this correlates with activity of p38 MAPK. ECs exposed to shear stress in the presence of the p38 MAPK inhibitor SB-203580 did not orient in any manner and the shape index was similar to the static cells.     

HIGH-GRADE SURFACE OSTEOSARCOMA OF THE LEFT ILIUM A Case Report and Review of the Literature

A rare case of high-grade surface osteosarcoma of the left ilum is reported. Trephine biopsy performed on a 31-year-old woman suffering from a huge tumor of the left buttock revealed high-grade osteosarcoma, and hemipel-vectomy was undertaken. The surgical specimen showed that the tumor was present on the surface of the left ilium. Ten months after the operation, the patient died of brain metastasis. From these results, we considered this case to be one of high-grade surface osteosarcoma. ACTA PATHOL JPN 38: 235 -240, 1988.     

Long-lived polymer radicals, 2. An ESR study on the reactions of the propagating polymer radicals of N-methylacrylamide and N-methylmethacrylamide with vinyl monomers at room temperature

Acrylamide (AAm), methacrylamide (MAm), N-methylacrylamide (NMAAm) and N-methylmethacrylamide (NMMAm) were found to yield long-lived propagating polymer radicals in the photo-sensitized polymerizations in 1,4-dioxane or benzene. The concentration of poly(NMAAm) radicals reached 1.10^?3 mol/l. Some post-effect was observed at room temperature in the photo-sensitized polymerization of AAm with di-tert-butyl peroxide in 1,4-dioxane, while no post-polymerization proceeded at room temperature in the polymerization of NMMAm in benzene. The reactions of poly(NMAAm) and poly(NMMAm) radicals with various vinyl monomers were found to produce long-lived propagating polymer radicals of the second monomers at room temperature. Polymer radicals of non-homopolymerizable monomers such as α-methylstyrene and 1,1-diphenylethylene were easily formed in such a block-copolymerization matrix. The formation of the propagating polymer radicals of the vinyl monomers was investigated by means of ESR spectroscopy.     

T cell epitopes of type II collagen in HLA-DRB1*0101 or DRB1*0405-positive Japanese patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is a T cell-mediated autoimmune disease, but target antigens (autoantigens) responsible for T cell activation remain unclear. Type II collagen (CII) is a candidate autoantigen that is largely confined to the articular cartilage. To investigate whether CII is an important antigen in patients with RA, we examined peripheral blood T cell reactivity to CII in HLA-DRB1*0101 and DRB1*0405-positive RA patients. Reactivities to candidate T cell epitopes of CII were also examined. Peripheral blood T cell reactivity to CII and CII peptides (256-271, 429-442, 593-610, 1064-1081) were detected by measurement of IL-2, IFN-gamma, and IL-4 in culture supernatant of PBMC after in vitro antigen stimulation. Cytokine concentration was measured by ELISA. In DRB1*0101-positive patients, T cell reactivity to CII as detected by measurement of IL-2 production in culture supernatant, was present in 4 out of 9 patients. IL-2 production upon stimulation with CII 256-271 peptide was found in all of these 4 patients. In DRB1*0405-positive patients, high frequency of positive T cell response to CII was detected in 9 out of 11 patients. IFN-gamma production was also detected in 4 out of 6 patients producing IL-2 by stimulation with CII. T cell response to CII 256-271 and/or CII 1064-1081 was detected in these patients. In DRB1*0101-positive RA patients, CII 256-271 peptide might function as a T cell epitope, whereas either CII 256-271 or CII 1064-1081 peptide may be a major T cell epitope in DRB1*0405-positive RA patients. In DRB1*0405-positive RA patients, CII reactive T cells might play a crucial role in the development of RA through IFN-gamma production.     

Three-dimensional analysis of alveolar structure in usual interstitial pneumonia

The etiology of usual interstitial pneumonia (UIP), a progressive lung disease, remains unclear. We examined alveolar structure in UIP three-dimensionally. Lung biopsy specimens from five patients with idiopathic pulmonary fibrosis were used. Sections 150-microm thick were stained with elastica solution for elastic fibers, with alpha-smooth muscle actin antibody for myofibroblasts, with anti-Thomsen-Friedenreich antibody for type-II pneumocytes and with anti-CD34 antibody for blood vessels. We examined them three-dimensionally using a laser confocal microscope or light microscope. In the fibrotic lesions, the thick elastic fibers forming the alveolar framework were not particularly dense considering the reduction in alveolar volume. Near the fibrotic lesions, some of the thin elastic fibers in the alveolar wall were slightly sinuous and ended with rounded tips. Type-II pneumocytes had proliferated and were distributed uniformly over the alveolar surface. Smooth muscle actin filaments were detected only around the alveolar orifice. These findings show that in UIP destruction of the elastic fiber framework of the alveoli may lead to irreversible focal alveolar collapse after damage to the alveolar epithelial cells, and proliferation of type-II pneumocytes may be involved with this elastolysis.     

Analysis of intestinal HLA-DR bound peptides and dysregulated immune responses to enteric flora in the pathogenesis of inflammatory bowel disease

Isolation of antigenic peptides from the MHC-groove has contributed to the understanding of T cell responses. However, these MHC-associated peptides have been isolated from various murine and human cell lines. The specific antigen responsible for the pathogenesis of inflammatory bowel disease is unknown. We examined antigenic peptides bound to the class II major histocompatibility complex (MHC) groove in human intestine by ion-trap tandem mass spectrometry equipped with online reverse-phase high performance liquid chromatography. We detected 55 parent proteins from 4 controls, 9 patients with ulcerative colitis, and 9 patients with Crohn's disease. The calculated molecular masses (m/z) of these peptides ranged from 874.4 to 2727.4, representing 10-26 amino acid residues. Fifty-one of these 55 parent proteins were exogenous proteins. Escherichia coli-, Saccharomyces cerevisiae-, and Caenorhabditis elegans-derived peptides were found frequently in patients with inflammatory bowel disease. The present results suggest that in vivo antigen processing by antigen-presenting cells and T lymphocytes in human intestine participate with exogenous antigen presentation. Increased immune responses against E. coli, S. cerevisiae and C. elegans found in patients with inflammatory bowel may participate as dysregulated immune responses to enteric flora in the pathogenesis of inflammatory bowel disease.