8-Hydroxyadenine (7,8-dihydro-8-oxoadenine) induces misincorporation in in vitro DNA synthesis and mutations in NIH 3T3 cells.

An oligodeoxyribonucleotide containing 8-hydroxyadenine (OH8Ade) was chemically synthesized and single- and double-stranded c-Ha-ras gene fragments with OH8Ade at the second position of codon 61 were prepared. The single-stranded ras gene fragment was used as a template for in vitro DNA synthesis with the Klenow fragment of Escherichia coli DNA polymerase I, Taq DNA polymerase, rat DNA polymerase beta and mouse DNA polymerase alpha. The former two enzymes exclusively incorporated dTMP opposite OH8Ade. The DNA polymerases alpha and beta misinserted dGMP, and dAMP and dGMP, respectively. The c-Ha-ras gene was constructed using the double-stranded ras gene fragment containing OH8Ade and was transfected into NIH 3T3 cells. The gene with OH8Ade induced focus formation, indicating that OH8Ade elicited point mutations in cells. When c-Ha-ras genes present in transformed cells were analyzed, an A-->G transition and an A-->C transversion were detected. These results indicate that OH8Ade induced misincorporation in in vitro DNA synthesis and mutations in mammalian cells.     

Chenodeoxycholic acid stabilization of LDL receptor mRNA depends on 3'-untranslated region and AU-rich element-binding protein

Human low-density lipoprotein receptor (LDLR) mRNA is unstable and contains four AU-rich elements (AREs) in the 3′-untranslated region (3′-UTR). The aim of this study was to verify the involvement of the 3′-UTR in the rapid degradation of LDLR mRNA. This study revealed that the 3′-UTR is necessary and sufficient for the degradation, and that the 1st ARE (ARE1) close to the stop codon associates with cytoplasmic proteins, and is primarily responsible for the degradation. Chenodeoxycholic acid (CDCA) treatment stabilized chimeric GFP-LDLR 3′-UTR mRNA and accompanied mitogen-activated protein kinase (MAPK) activation. The UV cross-linking assays showed that a protein of 80 kDa increasingly binds to the region including the ARE1 in response to CDCA-mediated MAPK activation.     

Quantitative trait loci for body weight in the intercross between SM/J and A/J mice.

We performed a genome-wide quantitative trait locus (QTL) analysis of body weight at 10 weeks of age in a population of 321 intercross offspring from SM/J and A/J mice, progenitor strains of SMXA recombinant inbred strains. Interval mapping revealed two significant QTLs, Bwq3 (body weight QTL3) and Bwq4, on Chromosomes (Chrs) 8 and 18 respectively, and five suggestive QTLs on Chrs 2, 6, 7, 15 and 19. Bwq3 and Bwq4 explained 6% of the phenotypic variance. The SM/J alleles at both QTLs increased body weight, though the SM/J mouse was smaller than the A/J mouse. On the other hand, four of the five suggestive QTLs detected had male-specific effects on body weight and the remainder was female-specific. These suggestive QTLs explained 5-6% of the phenotypic variance and all the SM/J alleles decreased body weight.     

Identification of a novel autoantigen UACA in patients with panuveitis

To identify the target autoantigens in Vogt-Koyanagi-Harada disease, we made use of an immunoscreening of a bovine uveal cDNA expression library with serum samples obtained from patients with Vogt-Koyanagi-Harada disease. We identified a novel bovine antigen and homologous human autoantigen and designated it as UACA (uveal autoantigen with coiled coil domains and ankyrin repeats). mRNA of human UACA is expressed most abundantly in skeletal muscles and in various human tissues, including choroid, retina, and epidermal melanocytes. IgG autoantibodies were quantitated in an ELISA, using recombinant C-terminal 18.0% fragment of human UACA. The prevalence of IgG anti-UACA autoantibodies in patients with panuveitis (Vogt-Koyanagi-Harada disease, Beh?et's disease, sarcoidosis) was significantly higher than that in healthy controls (19.6-28.1% vs 0%, P < 0.05) indicating that autoimmunity directed against UACA is a common phenomenon in these diseases.     

Avian Podocytes,Which Lack Nephrin,Use Adherens Junction Proteins at Intercellular Junctions

Nephrin, a major intercellular junction (ICJ) molecule of mammalian podocytes in the renal glomerulus, is absent in the avian genome. We hypothesized that birds use ICJ molecules other than nephrin in their podocytes. Therefore, in the present study, we examined the possible involvement of adherens junction (AJ) proteins in the ICJs of avian podocytes. We found the AJ proteins N-cadherin and α- and β-catenins in podocytes of quail and chickens but not in those of rats, pigs or humans. The AJ proteins were prominent in avian glomerulus-rich fractions in immunoblot analyses, and in immunofluorescence microscopy analyses, they were localized along glomerular capillary walls appearing in at least two staining patterns: weakly diffuse and distinctly granular. Immunoelectron microscopy demonstrated that the significant accumulation of immunogold particles for the AJ proteins were especially evident in avian slit diaphragms and AJs. Furthermore, N-cadherin was found to be expressed in all nephron cells in the early developmental stage but became confined to podocytes during maturation. These results indicate that avian slit diaphragms clearly express AJ proteins as compared with that in the mammal—where AJ proteins are suppressed to an extremely low level—and that avian podocytes are interconnected by AJs per se in addition to slit diaphragms.     

Isolation of Biopterin-α-glucoside from Spirulina (Arthrospira) platensis and Its Physiologic Function

A fluorescent substance was isolated from the cyanobacterium with a yield of 4.5 mg per 10 g of dried Spirulina (Arthrospira) platensis cells by gentle extraction and ethanol fractionation followed by column chromatography. The fluorescent substance, which has absorption maxima at 256 nm and 362 nm (pH 8.4), was identified as biopterin-α-glucoside by spectrophotometry and nuclear magnetic resonance spectroscopy. Biopterin-α-glucoside prevented decolorization of the photosynthetic pigments, chlorophyll a, phycocyanin, and carotenoids in photosynthetic vesicles of Spirulina platensis cells, by ultraviolet irradiation. Received June 23, 1998; accepted September 10, 1998.     

Efficient transfer of a tumor antigen-reactive TCR to human peripheral blood lymphocytes confers anti-tumor reactivity.

The tumor-associated-Ag MART-1 is expressed by most human melanomas. The genes encoding an alphabeta TCR from a MART-1-specific, HLA-A2-restricted, human T cell clone have been efficiently transferred and expressed in human PBL. These retrovirally transduced PBL cultures were MART-1 peptide reactive, and most cultures recognized HLA-A2+ melanoma lines. Limiting dilution clones were generated from three bulk transduced PBL cultures to investigate the function of individual clones within the transduced cultures. Twenty-nine of 29 CD8+ clones specifically secreted IFN-gamma in response to T2 cells pulsed with MART-1(27-35) peptide, and 23 of 29 specifically secreted IFN-gamma in response to HLA-A2+ melanoma lines. Additionally, 23 of 29 CD8+ clones lysed T2 cells pulsed with the MART-1(27-35) peptide and 15 of 29 lysed the HLA-A2+ melanoma line 888. CD4+ clones specifically secreted IFN-gamma in response to T2 cells pulsed with the MART-1(27-35) peptide. TCR gene transfer to patient PBL can produce CTL with anti-tumor reactivity in vitro and could potentially offer a treatment for patients with metastatic melanoma. This approach could also be applied to the treatment of other tumors and viral infections. Additionally, TCR gene transfer offers unique opportunities to study the fate of adoptively transferred T cells in vivo.     

Regulatory role of peritoneal NK1.1+ alpha beta T cells in IL-12 production during Salmonella infection.

NK1.1+ alpha beta T cells emerge in the peritoneal cavity after an i.p. infection with Salmonella choleraesuis in mice. To elucidate the role of the NK1.1+ alpha beta T cells during murine salmonellosis, mice lacking NK1.1+ alpha beta T cells by disruption of TCR beta (TCR beta-/-), beta 2m (beta 2m-/-), or J alpha 281 (J alpha 281-/-) gene were i.p. inoculated with S. choleraesuis. The peritoneal exudate T cells in wild type (wt) mice on day 3 after infection produced IL-4 upon TCR alpha beta stimulation, whereas those in TCR beta-/-, beta 2m-/-, or J alpha 281-/- mice showed no IL-4 production upon the stimulation, indicating that NK1.1+ alpha beta T cells are the main source of IL-4 production at the early phase of Salmonella infection. Neutralization of endogenous IL-4 by administration of anti-IL-4 mAb to wt mice reduced the number of Salmonella accompanied by increased IL-12 production by macrophages after Salmonella infection. The IL-12 production by the peritoneal macrophages was significantly augmented in mice lacking NK1.1+ alpha beta T cells after Salmonella infection accompanied by increased serum IFN-gamma level. The aberrantly increased IL-12 production in infected TCR beta-/- or J alpha 281-/- mice was suppressed by adoptive transfer of T cells containing NK1.1+ alpha beta T cells but not by the transfer of T cells depleted of NK1.1+ alpha beta T cells or T cells from J alpha 281-/- mice. Taken together, it is suggested that NK1. 1+ alpha beta T cells eliciting IL-4 have a regulatory function in the IL-12 production by macrophages at the early phase of Salmonella infection.