Biomedical subjects

N Setterblad

Publications and source records attributed to N Setterblad.

7 recordsLinked to original sources

PubMed1998

The role of protein kinase C signaling in activated DRA transcription.

Expression of human MHC HLA-DRA class II gene can be up-regulated in B cells by Ig cross-linking as well as by phorbol esters such as 12-O-tetradecanoyl phorbol 13-acetate (TPA). Induced DRA expression involves activation of restricted protein kinase C (PKC) isoforms, resulting in activated activator protein-1-dependent transcription. In this report expression profiles and activation of PKC were analyzed in human Raji B lymphoblastoid cells. Transient transfection analysis with target plasmids containing either DRA promoter (wild-type or mutated) or TPA response elements demonstrated that pretreatment with the selective PKC inhibitor GF 109203X repressed TPA-mediated activation. Western analysis performed on cellular fractions of resting cells and of TPA-activated cells revealed abundant expression of classical PKC-alpha (cPKC-alpha), cPKC-betaII, and atypical PKC-zeta isoforms and identified a sustained translocation of cPKC-alpha and cPKC-betaII from the cytosolic compartment to membranes. As expected, the distribution of atypical PKC-zeta was unaffected by TPA treatment and displayed an even distribution between cytosol and membranes. This finding was confirmed by immunofluorescence microscopy. The TPA-mediated translocation of cPKC-alpha and cPKC-betaII was not influenced by pretreatment with GF 109203X. Finally, functional activation and translocation of PKC were investigated with a selective in vitro kinase assay. Together, these results show that activated HLA-DRA expression in response to TPA treatment is strictly dependent on PKC activation acting on the X2 box of the DRA promoter and that selective inhibition of PKC enzymatic activity does not influence subcellular localization of expressed PKC isoenzymes. Thus, the translocation event per se occurs independently of PKC activation in these cells.

B-Lymphocytes↗

PubMed1998

Retroelements in the human MHC class II region.

Molecular genetic studies of the human major histocompatibility complex (MHC) have led to the identification of more than 200 genes. Besides the large number of genes in the MHC, densely clustered areas of retroelements have been identified. These include short and long interspersed elements (SINEs and LINEs), and human endogenous retroviruses (HERVs). The presence of retroelements in the MHC provides a clear example of how these elements affect the genome plasticity of the host. Comparative analyses of these retroelements have proven highly useful in evolutionary studies of the MHC. Recently, HERV-encoded superantigens have been implicated as candidate autoimmune genes in type I diabetes and multiple sclerosis. In addition, genetic analyses have revealed that autoimmune diseases show strong associations with MHC class II genes. The intriguing correlations between retroviral encoded antigens, MHC class II genes and the development of autoimmune disease merit intense future investigations of retroelements, in particular those endogenous retroviruses located in the MHC class II region proper.

Antigens↗

PubMed1998

The beta-gal interferon assay: a new, precise and sensitive method.

Cells of a human glioblastoma line were stably transfected with a glial fibrillary acidic protein (GFAP) promoter sequence/lacZ reporter gene. Following this modification, they produced Escherichia coli beta-galactosidase constitutively in amounts that could be measured through their conversion of an added fluorophore into a product readily estimated by fluorimetry. Human interferons (IFN) selectively and in a dose-dependent manner reduce the formation of beta-galactosidase in this system. We have used it as the basis for a novel assay that is sensitive (4-40 pg/ml), precise, completed in 30 h, and applicable to both type I and type II human IFNs. Statistical analysis showed interassay relative standard deviations ranging from 5% to 11%, and most individual assays revealed potencies with limits of error within 85%-115%. Neither partially trypsin-digested IFN nor the other cytokines and mitogens we tested reacted in this system, except for tumor necrosis factor-alpha (TNF-alpha). The high selectivity was further shown by the loss of response to IFN in the presence of the appropriate specific anti-IFN or anti-IFN-gamma receptor antibodies.

Biological Assay↗

PubMed1997

Role of the X2 box in activated transcription from the DRA promoter in B cells.

We investigated the function of the evolutionary conserved X2 box in the promoter of the HLA-DRA gene from the human major histocompatibility complex (MHC) in resting and activated B cells. NF-X2, which contains members of the AP-1/ATF/CREB families of transcription factors, interacts with the X2 box (5'-TGCGTCA-3') from positions -97 to -91 in the DRA promoter. In resting Raji cells, little to no binding to the X2 box was observed. In sharp contrast, in B cells treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), strong interactions between the X2 box and NF-X2 containing c-Fos were observed. As determined by transient expression and RNA analyses, the activation of protein kinase C (PKC) also increased rates of transcription from the wild-type DRA promoter but not from a DRA promoter bearing clustered point mutations in the X2 box. Since the co-expression with a dominant negative c-Fos abolished the responsiveness to TPA, we conclude that activated transcription of the DRA gene depends on interactions between the X2 box and NF-X2, which contains c-Fos.

Animals↗

PubMed1996

Evolutionary relationship between human major histocompatibility complex HLA-DR haplotypes.

HLA-DR haplotypes of the human major histocompatibility complex are organized in five different groups. They can be identified based on the serological specificity expressed by the polymorphic DRB1 locus and by the presence of a characteristic set of DRB genes. The nucleotide sequences of introns 4 and 5 of the two DRB genes (DRB1(*)01 and DRB6(*)01 ) from a DR1 haplotype and the three DRB genes (DRB1(*)15, DRB6(*)15 , and DRB5(*)15 ), from a DR51 haplotype were determined. This study identified endogenous retroviral long terminal repeat elements (ERV9 LTR) located at identical positions in intron 5 of the DRB1 genes in both the DR1 and DR51 haplotypes. Phylogenetic analyses revealed a close evolutionary relationship between these two haplotypes. The DRB5 gene, unique for the DR51 haplotype, may have been lost by a recent gene deletion event creating the DR1 haplotype. A model for the evolution of the human DR haplotypes involving separate duplication and contraction events is presented.

Base Sequence↗

PubMed1995

Primate DRB genes from the DR3 and DR8 haplotypes contain ERV9 LTR elements at identical positions.

The HLA-DRB genes of the human major histocompatibility complex constitute a multigene family with a varying number of DRB genes in different haplotypes. To gain further knowledge concerning the evolutionary relationship, the complete nucleotide sequence was determined for a region spanning introns 4 and 5 of the three DRB genes (DRB1*0301, DRB2, and DRB3*0101) from a DR52 haplotype and the single DRB gene (DRB1*08021) in the DR8 haplotype. These analyses identified as endogenous retroviral long terminal repeat element (ERV9 LTR3), inserted at identical positions in intron 5 of the functional DRB genes in these two haplotypes. Comparison of the nucleotide sequence from introns 4 and 5 including the ERV9 LTR elements revealed a strong similarity between the three expressed DRB genes. The DRB3*0101 and DRB1*08021 genes were most similar in this comparison. These findings provide further evidence for a separate duplication in a primordial DR52 haplotype followed by a gene contraction event in the DR8 haplotype. A homologous element was found in a chimpanzee DRB gene from a DR52 haplotype. This represents the first characterized ERV9 LTR element in a nonhuman species. The corresponding introns of the DRB genes in the DR4 haplotype contain no ERV9 LTRs. In contrast, these genes have insertions of distinct Alu repeats, implying distinct evolutionary histories of DR52 and DR53 haplotypes, respectively. Phylogenetic analyses of DRB introns from DR52, DR53, and DR8 haplotypes showed a close relationship between the DRB2 and DRB4 genes. Thus, the ancestral DR haplotype that evolved to generate the DR52 and DR53 haplotypes most likely shared a primordial common DRB gene.

Animals↗

PubMedDate not supplied

Signalling via MHC class II molecules modifies the composition of GEMs in APC.

Major histocompatibility complex (MHC) class II molecules are responsible for peptide presentation to helper T lymphocytes and as such play an essential role in the immune response. These molecules transmit intracellular signals leading to diverse consequences in B lymphocytes including proliferation and apoptosis. Recent studies have revealed that glycolipid enriched membrane microdomains (GEMs) behave as signalling platforms for a variety of lymphocyte receptors. We have quantified human leucocyte antigen (HLA)-DR molecules localized in GEMs in human B lymphocytes. Use of a model imitating the interaction of HLA-DR with a T-cell receptor (TCR) modified the constituents of the HLA-DR-enriched GEMs. Confocal microscopy demonstrated a recruitment of HLA-DR and the ganglioside GM1 at the site of HLA-DR interaction with the stimulating ligand. Moreover, cholesterol depletion efficiently impaired this recruitment. Co-localizing proteins detected in HLA-DR-enriched GEMs include protein kinase C (PKC)-delta and actin. These data reveal that MHC class II antigens are localized in GEMs in mature human B lymphocytes and indicates that the formation of the immunological synapse regulates the composition of HLA-DR enriched GEMs in the antigen presenting cell (APC).

Actins↗