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Biomedical subjects

U Krawinkel

Publications and source records attributed to U Krawinkel.

At least 19 recordsLinked to original sources
PubMed1995

Human ribosomal protein L7 inhibits cell-free translation in reticulocyte lysates and affects the expression of nuclear proteins upon stable transfection into Jurkat T-lymphoma cells.

Eucaryotic ribosomal protein L7 carries a 'Basic-Region-Leucine-Zipper (BZIP)'-like region which mediates high-affinity binding to mRNA and 28S rRNA and formation of homodimers [P. Hemmerich et al. (1993) Nucleic Acids Res. 21, 223-231). Its biological function is unknown as yet and no direct L7-equivalent in procaryotes has been found. In this report we show that eucaryotic L7 specifically inhibits the cell-free translation of reporter mRNAs. The interaction of L7 with mRNA is an essential step in this reaction which is inhibitable by antibodies directed against the BZIP-like region of L7, and by competitors of mRNA binding. L7-mediated inhibition of cell-free translation of polyA+ RNA from Jurkat T-lymphoma cells is selective in that the synthesis of a major 46 kD protein is suppressed. Upon stable transfection of L7 cDNA into Jurkat lymphoma cells two major proteins disappear, namely one nuclear protein and one which associates with the nucleus. Our data suggest a regulatory role of protein L7 in the eucaryotic translation apparatus.

Amino Acid Sequence↗
PubMed1995

T cell lines recognizing the 70-kD protein of U1 small nuclear ribonucleoprotein (U1snRNP).

In sera of patients with mixed connective tissue disease (MCTD) high titres of IgG autoantibodies to U1snRNP-specific proteins (70 kD, A, C) are found, suggesting an antigen-driven and T-cell-dependent process. In order to establish U1snRNP-specific T cell lines we cultured under various culture conditions mononuclear cells from MCTD patients and healthy donors with a highly purified UsnRNP preparation from HeLa cells. Nine T cell lines were established by limiting dilution cloning from two MCTD patients and five T cell lines from a healthy individual. All T cell lines expressed the TCR alpha beta/CD3 complex. Surprisingly, most of the T cells lines exhibited the CD8 phenotype. Irrespective of this phenotype, all T cell lines showed a proliferative response to an N-terminal part (aa 51-195) of recombinant U1-specific 70-kD protein. One CD8+ T cell clone exhibited cytotoxic activity against an autologous B cell line pulsed with snRNP or recombinant fragments (aa 51-95 and aa 51-88). Interestingly, two T cell lines proliferated in response to four recombinant polypeptides representing different parts of the U1snRNP 70-kD protein. Since regions of sequence homology are distributed over the 70-kD molecule, it is suggested that conserved motifs may be recognized by the T cell lines.

Adult↗
PubMed1995

Autoantibodies against eukaryotic protein L7 in patients suffering from systemic lupus erythematosus and progressive systemic sclerosis: frequency and correlation with clinical, serological and genetic parameters. The SLE Study Group.

Recent studies have shown that sera of patients suffering from systemic autoimmune diseases contain autoantibodies directed against the eukaryotic ribosomal protein L7 [1]. In the present study we screened a large panel of sera from patients with systemic lupus erythematosus (SLE) for the presence of anti-L7 autoantibodies and their relationship to clinical, serological and genetic parameters of SLE. By means of an ELISA employing recombinant protein L7 as antigen we detected anti-L7 autoantobodies in 172 of 506 SLE sera (34%). Negative correlations were observed between the presence of anti-L7 autoantibodies, serum IgG levels and proteinuria; a potentially positive relationship existed with lung fibrosis. In order to analyse further this possibility we screened sera of 129 patients suffering from progressive systemic sclerosis (PSS) for anti-L7 reactivity; 45 of these patients had lung fibrosis. Of the PSS patients, 41% exhibited anti-L7 autoantibodies, but positive reactions were evenly distributed among patients with and without lung fibrosis. Protein L7 thus represents a major autoantigen of systemic autoimmune diseases, but does not so far define a distinct subpopulation of patients.

Autoantibodies↗
PubMed1995

Autoantigenic epitopes on eukaryotic L7.

Ribosomal protein L7 has been established recently as a novel autoantigen representing a frequent target for autoantibodies from patients with systemic autoimmune diseases. Up to 75% of systemic lupus erythematosus (SLE) patients and 50% of mixed connective tissue disease (MCTD) and progressive systemic sclerosis (PSS) patients produce antibodies in vitro translated L7 and form immunoprecipitable complexes. In this study the B cell response to protein L7 was investigated with respect to the immunogenic determinants recognized by autoantibodies. Eighteen truncated fragments of protein L7 were generated as recombinant fusions with glutathione-S-transferase and examined by immunoblotting for their reactivity with sera from patients suffering from systemic rheumatic diseases. Anti-L7 antibodies target three major nonoverlapping autoepitopes. Two epitopes reside in the highly conserved C-terminal part of the protein, whereas the N-terminal autoepitope is not conserved during evolution. The N-terminal epitope comprises 24 amino acid residues. Ten amino acid resides of this epitope are shared with the BZIP-like RNA binding domain of protein L7. Autoantibodies recognizing this epitope crossreact with the corresponding region of a L7 homologue, namely ribosomal protein L7 (RPL7) from Dictyostelium discoideum. This indicates that amino acid residues 14VPE...KKR22, which are conserved between humans and fungi, contribute essentially to the formation of autoantibody-autoantigen complexes.

Amino Acid Sequence↗
PubMed1994

Oligoclonality and skewed T cell receptor V beta gene segment expression in in vivo activated human intestinal intraepithelial T lymphocytes.

Intraepithelial intestinal T lymphocytes (IEL) bearing alpha beta or gamma delta T cell receptors (TCR) are positioned to serve as a first line of defense against enteric pathogens. To investigate whether intestinal IEL are subject to antigenic selective forces distinct from that influencing (xp T cells in the peripheral blood (PBL), we performed a comparative analysis of V beta gene segment usage in IEL and PBL of immunologically normal donors by quantitative PCR. Primers for 22 different human TCR V beta gene segments of V beta gene segments families were used to analyze the repertoire of TCR beta chain transcripts in colonic IEL (c-IEL), in corresponding colonic lamina propria lymphocytes (c-LPL), and in peripheral blood lymphocytes. In each of the three individuals examined, a limited number (1-4 out of 22) of TCR V beta families predominated and accounted for more than 50% of the total beta chain transcripts from c-IEL, whereas in PBL and c-LPL a more even distribution of V beta gene families was observed. The dominating V beta gene families were V beta 2, V beta 3, V beta 6, V beta 8 and V beta 14. In one individual, V beta 3 comprised more than 40% of the entire repertoire of c-IEL beta chain transcripts. Sequence analysis of the predominant V beta 3 family in this individual revealed identical sequences in 13 of 17 clones analyzed. Human alpha beta TCR+ c-IEL could not be driven to proliferate or exhibit cytotoxic function in vitro however, PCR analysis for detection of lymphokine mRNA revealed constitutive production of several lymphokines known to exert trophic effects on intestinal epithelial cells and pro-inflammatory activities. Taken together, the striking degree of oligoclonality may indicate that the intraepithelial intestinal immune system is targeted to a limited set of hitherto unknown self- or foreign antigens present in the intestine and orchestrates intramucosal inflammatory and regenerative processes.

Base Sequence↗
PubMed1994

Characterization of eukaryotic protein L7 as a novel autoantigen which frequently elicits an immune response in patients suffering from systemic autoimmune disease.

Autoantibodies targeted against cellular proteins and nucleic acids are a common feature of autoimmune diseases. In this study, we show that ribosomal protein L7 is a novel autoantigen in patients suffering from systemic lupus erythematosus (SLE) and other connective tissue diseases. From 24 patients diagnosed as having SLE, 18 produce antibodies which precipitate in vitro translated L7 protein. The anti-L7 titer appears to correlate with the active state of the disease. Anti-L7 autoantibodies were also detected in 7 of 13 patients with mixed connective tissue disease (MCTD), 2 of 7 patients with rheumatoid arthritis (RA), 1 of 4 patients with Sjögren's syndrome (SS) and in 1 patient with progressive systemic sclerosis (PSS). Anti-L7 autoantibodies belong to the IgG-class and detect specifically at least two epitopes on the L7 molecule, as shown by immunoprecipitation and immunoblotting. The epitope(s) of the highly conserved C-terminal region are preferentially recognized. Utilizing rabbit anti-L7 serum, autoimmune sera and affinity-purified anti-L7 autoantibodies in immunoblotting, and rabbit and chicken anti-L7 antibodies in indirect immunofluorescence, we detect L7 protein in the nuclei and in the cytoplasm of various cell-lines. Yet unlike most integral structural components of ribosomes, L7 is absent from nucleoli.

Adolescent↗
PubMed1994

Characterization of an HLA-DR4-restricted T cell clone recognizing a protein moiety of small nuclear ribonucleoproteins (UsnRNP).

In sera of patients with mixed connective tissue disease (MCTD, Sharp Syndrome) high titres of IgG autoantibodies to U1snRNP-specific proteins are found. The isolated occurrence of these autoantibodies is highly associated with the HLA-DR4 haplotype. snRNP-specific T cells are supposed to be involved in this autoantibody production. To address this question we cultured mononuclear cells from MCTD patients and healthy donors with a highly purified UsnRNP preparation from HeLa cells using bulk or limiting dilution cultures. Secondary responses to snRNP were detected only rarely with T cell lines from two patients and two controls, and turned out to be unstable during further expansion. One T cell line derived from a healthy individual retained its snRNP reactivity upon limiting dilution cloning and could be characterized in detail. The CD4+ T cell clone recognized native snRNP particles presented by monocytes in an HLA-DR4 (B1*0401)-restricted manner. Separation of the protein and RNA moieties of snRNP particles revealed that the T cell clone responded specifically to the protein fraction, but not to RNA and diverse control antigens. Sequencing of the T cell receptor alpha and beta chain cDNAs revealed that the clone used the V alpha 14.2 and V beta 14 elements. Upon antigen-specific and mitogenic stimulation the T cell clone showed a Th1-specific cytokine pattern, and did not provide helper activity for in vitro immunoglobulin production. This study demonstrate the presence of self-reactive snRNP-specific T cells in a healthy donor. The T cell clone may not represent a helper T cell for the formation of U1snRNP-specific autoantibodies.

Amino Acid Sequence↗
PubMed1993

Analysis of T cell receptor V beta regions expressed by rheumatoid synovial T lymphocytes.

The T cell receptor (TCR) V beta gene segment repertoire of T lymphocytes derived from peripheral blood of two healthy individuals and synovial tissue, synovial fluid and peripheral blood of three rheumatoid arthritis (RA) patients was analyzed. A sensitive assay based on the amplification of cDNA by the polymerase chain reaction (PCR) was used to analyze the levels of expression of 20 TCR V beta gene segment families. The relative expression of V beta gene segments in lymphocytes derived from peripheral blood, synovial tissue and synovial fluid was conserved over 155 days in one patient. V beta 9 transcripts were undetectable in the cells of this individual. In the two other patients the frequency of V beta 2 transcripts in synovial T cells of affected joints was significantly higher than in their peripheral blood lymphocytes. Dominance of distinct rearrangements among the V beta 2 transcripts from the synovial cells of these patients support the idea that the synovial T cell response is driven by antigen.

Amino Acid Sequence↗
PubMed1992

Characterization of germ-line genes of the VGAM3.8 VH gene family from BALB/c mice.

Five germ-line genes of the VGAM3.8 VH family in BALB/c mice have been isolated from genomic libraries and sequenced. The genes are functional and three are expressed in antibodies of different specificities. Overall nucleotide sequence homologies within the family are greater than 90%, whereas homologies with other VH families are less than 70%. Southern blot hybridization and sequencing indicate a minimum family size of six genes. Differences in the coding regions are mostly confined to CDR, where there is a high replacement/silent substitution ratio, indicative of positive selection for diversification associated with Ag binding. VHVGAM3.8 sequences are highly conserved, and polymorphism in the coding regions appears to be very limited. Evidence is presented that the family has evolved, and been homogenized, by recombinatorial events.

Amino Acid Sequence↗
PubMed1992

Unusual sequence of immunoglobulin L-chain rearrangements in a gamma heavy chain disease patient.

Patients with gamma heavy chain disease (gamma-HCD) generally produce incomplete immunoglobulin (Ig) gamma-heavy chains (gamma-HCD protein) which cannot associate with light chains (IgL). In most patients Bence Jones proteins (BJP) are not observed. However, in the 61-year-old patient WIN we found gamma l-HCD proteins and lambda BJP in serum and urine. WIN gamma l-HCD protein does not carry the Ig Fd region, has a molecular weight of 33.5 kDa, and the seven N-terminal amino acid residues are not translated from any of the known immunoglobulin heavy chain (IgH) gene sequences. These residues are followed by the C gamma l-hinge region. In DNA from peripheral blood lymphocytes of patient WIN we found bands representing dominant rearrangements in one of the two alleles of the IgH, Ig kappa and Ig lambda locus. Taken together, the data from protein and DNA analysis strongly suggest, albeit do not formally prove, that one dominant B-cell clone which carries a rearranged and a non-rearranged allele of each Ig locus produces gamma-HCD protein and lambda BJP. The productive lambda-gene rearrangement in this clone thus has not been preceded by abortive rearrangements in both kappa-locus alleles. Lymphocytes with an unusual sequence of IgL-chain gene activation seem to be involved in the case of gamma-HCD described here.

Amino Acid Sequence↗
PubMed1991

Biased T cell receptor V alpha region repertoire in the synovial fluid of rheumatoid arthritis patients.

Synovial T lymphocytes seem to contribute to the pathogenesis of rheumatoid arthritis (RA). Since very little is known about the structural heterogeneity of their T cell antigen receptors (TcR), we analyzed TcR alpha chain mRNA of synovial fluid T cells from two RA patients. TcR alpha chain cDNA was amplified by the polymerase chain reaction with single-sided specificity for the alpha chain constant (C alpha) gene segment, and the nucleotide sequences of 51 functionally rearranged cDNA clones were determined. Twenty different V alpha genes and 26 different J alpha gene segments were utilized in these cDNA clones. Three of the V alpha gene segments which are frequently (8%-17% total) expressed in synovial fluid T cells have rarely been found in the TcR repertoire of peripheral blood T cells from healthy individuals. The T cell responses in the rheumatic synovia analyzed here are not oligoclonal, but the usage of TcR V alpha genes is biased.

Antibody Diversity↗
PubMed1991

[Gamma-1 heavy chain disease with the demonstration of Bence-Jones proteins].

We describe a 61-year-old patient suffering from gamma-1-heavy-chain disease (gamma 1-HCD) associated with Bence-Jones-lambda proteinemia and proteinuria. The analysis of the patients gamma 1-HCD protein (WIN) shows a deletion of the complete Fd fragment. The N-terminal seven amino-acid residue does not resemble any of the known immunoglobulin-heavy-chain variable regions. Unexpectedly, in PBL-DNA and in DNA from EBV-immortalized cells we found in addition to the expected predominantly rearranged Ig-lambda-light-chain gene a predominant rearrangement of an Ig-kappa gene. These findings show that the gamma-1-heavy-chain disease of the patient involves a defective regulation of Ig-light-chain-gene activation as well.

Amino Acid Sequence↗
PubMed1989

Physical linkage of variable, diversity and joining gene segments in the immunoglobulin heavy chain locus of the mouse.

The variable region of antibody heavy chains is encoded by multiple variable (V), diversity (D) and joining (J) gene segments which seem to be assembled in a programmed developmental pathway during the maturation of B lymphocytes. A physical map linking V, D and J gene segments in the a haplotype of the murine Igh locus is presented in this report. The V gene located next to the known D and J gene segments maps 80 kb upstream of the JH locus and is a member of the VGAM3.8 V gene family. There is evidence for as yet unknown D gene segments located 5' to this V gene.

Animals↗
PubMed1989

Recombination between VH pseudogenes.

The peculiar pattern of nucleotide differences between germline VH genes V104A/VAR104 and 122B could be the result of an exchange of genetic information through gene conversion. A palindromic sequence in one of the genes seems to have been the target site for the break that initiated recombination.

Animals↗