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Biomedical subjects

M Renz

Publications and source records attributed to M Renz.

At least 19 recordsLinked to original sources
PubMed1995

The major dermatomyositis-specific Mi-2 autoantigen is a presumed helicase involved in transcriptional activation.

OBJECTIVE: To characterize the complementary DNA (cDNA) and protein sequences of autoantigens recognized by anti-Mi-2 antibodies, using recombinant Mi-2 proteins for improved autoantibody detection. METHODS: A cDNA expression library was immunoscreened, and cDNA isolation, alignment, and sequence analysis were performed. Northern blotting and in situ hybridization techniques were used. A recombinant protein (rMi-2) was synthesized. Immunoprecipitation of 35S-methionine-labeled HEp-2 cell proteins and immunoblotting of rMi-2 and natural nuclear proteins were performed. Immunofluorescence studies were done with anti-Mi-2 positive sera of dermatomyositis (DM) patients, and with human or rabbit antibodies specific for rMi-2. Antibody screening of systemic lupus erythematosus, rheumatoid arthritis, DM, and antinuclear antibody-positive human sera was performed using an rMi-2 protein enzyme-linked immunosorbent assay (ELISA). RESULTS: A major antigen recognized by anti-Mi-2 positive sera of DM patients was found to constitute a 218-kd nuclear protein (218-kd Mi-2) encoded on chromosome 12 and to belong to the SNF2/RAD 54 helicase family. Human and rabbit antibodies that were affinity purified using the recombinant protein reacted with and precipitated a nuclear protein of similar size, which was also recognized by anti-Mi-2 sera. Anti-218-kd Mi-2 antibodies detected by rMi-2 protein ELISA seemed to be mainly restricted to sera from patients with DM. CONCLUSION: The molecular characterization of the 218-kd Mi-2 antigen may contribute to our understanding of autoimmune phenomena in DM. The use of immunoreactive recombinant proteins allows structural and functional studies of the helicase and the development of sensitive and accurate antibody screening tests.

Adenosine Triphosphatases↗
PubMed1994

Interferon-gamma-inducible protein p16. A new target of antinuclear antibodies in patients with systemic lupus erythematosus.

OBJECTIVE: To determine the cellular expression and localization of interferon-gamma (IFN gamma)-inducible protein p16, a new antigen specificity of antinuclear antibodies (ANA), and to evaluate the prevalence of anti-p16, particularly in SLE patients. METHODS: Serum levels of anti-p16 were determined by immunoblotting with recombinant p16 and cellular p16 messenger RNA (mRNA) by Northern blotting. We also utilized immunoprecipitation of 35S-methionine-labeled proteins, immunostaining of blotted proteins of subcellular fractions, and immunofluorescence studies with affinity-purified rabbit and human anti-recombinant p16. RESULTS: Protein p16 was localized within the nucleolus and nucleoplasm and constitutively expressed in Raji, HeLa, HEp-2, K562, and HL-60 cells. Synthesis of mRNA and protein was increased in the presence of IFN gamma. The prevalence of anti-p16 was 29% in 374 SLE patients (35% in those positive for anti-double-stranded DNA) and 0% in 188 healthy individuals. Anti-p16 was always accompanied by positive findings on indirect immunofluorescence for ANA. CONCLUSION: Anti-p16 represents a main ANA specificity. Anti-p16 may help to elucidate IFN gamma-dependent nuclear processes and to link autoantibody production to states of IFN gamma activation.

Animals↗
PubMed1994

Molecular genetic analyses of a 376-kilodalton Golgi complex membrane protein (giantin)

Molecular genetic analyses of a 376-kDa Golgi complex (GC) membrane protein (giantin) are described. The immunoglobulin G fraction of a human serum containing antibodies against GC antigens as revealed by indirect immunofluorescence microscopy with Hep-2 cells was used to screen a HeLa cDNA expression library, yielding four overlapping cross-hybridizing clones. Additional cDNA clones were retrieved from a lambda gt11 human thyroid cDNA library or generated by reverse transcriptase-mediated PCR from HeLa cell mRNA. Alignment of the clones resulted in a consensus cDNA of 10,300 bp encoding a protein of 376 kDa. The corresponding mRNA with a size of about 10 kb was detected by Northern (RNA) blotting of HeLa, Hep-G2, and Jurkat cell RNA. Sequence analyses of the protein revealed an extraordinarily high content of heptad repeats with the probability of forming coiled coils similar to the proteins of the myosin family. Five overlapping recombinant proteins covering the entire sequence were synthesized and used for antibody production in rabbits and for affinity purification of human and rabbit antibodies. Indirect immunofluorescence experiments also done with brefeldin A-treated Hep-2 and Pt K1 cells revealed an identical GC staining of both the affinity-purified human and rabbit antibodies. Double labeling experiments with antibodies against the GC marker mannosidase II as well as immunoelectron microscopic studies confirmed the localization of the protein within the GC. A corresponding endogenous large-molecular-mass protein of about 390 kDa was found in [35S]methionine-labeled Hep-2 cell lysates as well as in GC-enriched subcellular fractions from rat liver. The protein as well as the recently described proteins golgin-95 and golgin-160 (M. J. Fritzler, J. C. Hamel, R. L. Ochs, and E. K. L. Chan, J. Exp. Med. 178:49-62, 1993) may belong to a new group of Golgi proteins with a high content of heptad repeats which may exert functions in scaffold formation or vesicle transport. As far as can be concluded from immunological and personally communicated partial cDNA sequence data, the protein seems to be identical with a 400-kDa Golgi protein (giantin) recently described (A. D. Linstedt and H. P. Hauri, Mol. Biol. Cell 4:679-693, 1993). Therefore, we agreed to adopt the name giantin.

Amino Acid Sequence↗
PubMed1992

MHC associations of autoantibodies against recombinant Ro and La proteins in systemic lupus erythematosus. Results of a multicenter study. SLE Study Group.

Antibodies against recombinant 52 kD-Ro, recombinant 60 kD-Ro and recombinant La protein were determined by ELISA in over 300 central European patients with systemic lupus erythematosus (SLE). A strong association with HLA-DR3 was found for antibodies against 52 kD-Ro and La, but not for recombinant 60 kD-Ro antibodies in the absence of antibodies against 52 kD-Ro or La. Ro/La negative SLE patients still showed an increased frequency of HLA-DR3 as compared to healthy controls. These results indicated that the preferential formation of Ro and La antibodies was not due to an unspecific stimulatory effect of HLA-DR3 but that the antibody response to certain defined proteins (52 kD-Ro and La) was influenced by MHC genes in SLE. Furthermore, the association of SLE with HLA-DR3 was independent of the effects of DR3 on the formation of 52 kD-Ro and La antibodies.

Antibody Specificity↗
PubMed1991

A recombinant 70K protein ELISA. Screening for antibodies against U1snRNP proteins in human sera.

Antibodies to uridylic acid rich small nuclear ribonucleoprotein particles (UsnRNP) are mainly detected in patients with systemic lupus erythematosus (SLE) or mixed connective tissue disease (MCTD). Particularly those directed against epitopes of the 70K protein of U1snRNP serve as important markers for the diagnosis of MCTD. To establish an ELISA for determination of anti-70K protein antibodies in patients' sera a 1239 bp long cDNA insert coding for the epitopes of the 70K protein was ligated into a fusion expression vector. The bacterially expressed fusion protein was purified by chromatography on DEAE cellulose. Microtiter plates were coated with the fusion protein as well as with partially purified calf thymus extract (CTE) containing all natural UsnRNP antigens and RNase digested calf thymus extract (CTERNase) in which the natural 70K antigen was destroyed by the nuclease treatment. 10,888 sera of patients with suspected or overt rheumatic disease were analyzed for antibodies against these antigens simultaneously. Antibodies against CTE or CTERNase were not detected in 9123 sera, none of these showed reactivity with the 70K protein indicating a high degree of specificity of the assay. Positive results in each the 70K protein, CTE as well as the CTERNase ELISAs were obtained with 474 sera. 319 sera were only positive with CTE and 70K protein. Of these 793 anti-70K protein ELISA positive sera, 79% could be confirmed by immunoblot. Of 967 sera reacting with CTE and CTERNase but not with the recombinant 70K protein, 31% contained antibodies against various other UsnRNP proteins as shown by immunoblotting. 2.4% of these sera revealed also antibodies against the 70K protein. The use of the recombinant 70K protein as antigen meets the criterion for a simple and specific assay to detect anti-U1snRNP antibodies. Nevertheless, the sole use of this recombinant protein for anti-U1snRNP antibody screening may not be appropriate, because antibodies against other frequently occurring U1snRNP proteins (A, C) cannot be detected with this test. Therefore it should be used together with a natural UsnRNP antigen until further studies in patients with well established diagnoses will show whether natural antigens may be omitted.

Antibody Specificity↗
PubMed1991

[Hepatitis C virus: sequence homology of a European isolate and divergence from the prototype].

The polymerase chain reaction (PCR) detected specific hepatitis C viral (HCV) RNA sequences in liver biopsies from two patients with chronic hepatitis, in the tissue of a liver implantate, in plasma from four chronic non-A, non-B hepatitis (NANBH) patients and, for the first time, in an infectious anti-D-immunoglobulin preparation. A comparison of the viral sequences coding for a region for the nonstructural NS3 protein from the liver tissues revealed only a very small degree of sequence divergence on the cDNA as well as on the amino acid level (between 0 and 5%). The sequence similarities of the RNA isolated from plasma of the four chronic NANBH patients and the anti-D-immunoglobulin preparation were partly somewhat lower but altogether also high (between 90 and 100%). In contrast, all eight cDNA and amino acid sequences exhibited a significantly higher degree of divergence in comparison with the HCV prototype sequence (between 29 and 32%) than among themselves (between 0 and 10%). This unexpected high sequence similarity of the eight European isolates and their low homology to the Northamerican prototype sequence is indicative for the existence of different types of HCV. This will be important not only for epidemiological studies but also for the development of effective diagnostic procedures and vaccines. Concerning the pathogenesis of NANBH, a double infection or a helper mechanism has to be considered: in addition to the C virus, sequences of an other virus particle were found in the infectious IgG preparation as well as in the liver biopsies.

Amino Acid Sequence↗
PubMed1991

[Rapid diagnosis of tuberculosis by the polymerase chain reaction (PCR)].

The polymerase chain reaction (PCR) was used for the detection of Mycobacterium tuberculosis DNA. More than 2000 different clinical specimens were analyzed by this assay. The efficiency of two different methods for processing the DNA from biological material was analyzed. DNA amplification was done according to standard protocols by amplifying a segment of 402 bp of the 65 kD mycobacterial gene, electrophoretic separation of the amplification product followed by Southern transfer and hybridization with a Mycobacterium tuberculosis-specific probe or by a semi-nested amplification procedure in which the initial amplification product was reamplified by a second round with a Mycobacterium tuberculosis-specific primer. The specificity of primers and probe for mycobacterial DNA was proven by testing 70 of class-I microorganisms, as well as 20 different strains and own isolates of Mycobacterium tuberculosis and 67 strains of 25 different MOTTs. Some of the amplification products were sequenced. The clinical relevance of the results and the sensitivity of the PCR method were confirmed by simultaneous quantitative bacterial culture from the same clinical specimens. The results of conventional culture method received after 8 to 10 weeks culture time correlated with the results from PCR obtained within 12 hours in 95.4% in the semi-nested amplification procedure. The discrepancy of 4.6% was caused by positive results of PCR and negative cultures which might be due to the higher sensitivity of PCR compared to culture technique. The results show that PCR may be used for detection of Mycobacterium tuberculosis in clinical specimens. The specificity can be regarded as largely proven, advantages are velocity and sensitivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1990

[The demonstration of hepatitis B virus DNA with the polymerase chain reaction].

Results with the polymerase chain reaction and conventional DNA hybridizing technique (dot-blot) for the detection of hepatitis B virus (HBV) DNA were compared for 439 patients. In 261 patients who were positive for HBs antigen (Ag), HBV DNA was demonstrated with the polymerase reaction in all of the 69 HBe-Ag positive sera (dot-blot 56%), as well as in 81 (62%) of 131 anti-HBe positive sera (dot-blot 5%), in 29 (48%) of 61 HBe marker negatives (dot-blot 3%) and in six (29%) of 21 delta positive sera. Among HBs-Ag negative patients, HBV DNA was detected in six (22%) of 27 anti-HBc positive sera, but not in sera (n = 50) which were both anti-HBc and anti-HBs positive, as well as in HBV marker negative patients with liver disease (n = 30) and healthy controls (n = 50). If infectiousness is to be checked or in case of double infection, the more sensitive polymerase chain reaction is to be recommended for the detection of HBV DNA. HBe-Ag positive patients must be considered as infectious: tests for HBV DNA can in general be omitted.

Antigens, Viral↗
PubMed1990

[Autoantibodies against U1-n-RNP (ENA)--detection using a recombinant 70-kDa protein].

An ELISA for the demonstration of antibodies to RNP antigens (ENA) in sera of patients with systemic rheumatic diseases was developed using the recombinant 70-kDa protein, a marker antigen of U1-n-RNP. The specificity and sensitivity of the method was evaluated with 3588 patients' sera. The results were compared with those obtained by natural antigens isolated from calf thymus and Western blot analysis using HeLa-cell nuclear extracts. The test was found to be specific and sensitive and to be superior in routine laboratory screening than the other tests commonly used.

Autoantibodies↗
PubMed1989

Protein-DNA conjugates produced by UV irradiation and their use as probes for hybridization.

Several proteins have been crosslinked to DNA by low dose uv irradiation. The principle of the method is based on an efficient and fast radiation induced reaction of amino acid residues with DNA at low pH. The method seems to be of general applicability for crosslinking proteins to DNA in a very simple one step procedure. Some of such DNA-protein conjugates have been used as probes for hybridization experiments. DNA-protein A probes were found to be most useful.

Blotting, Southern↗
PubMed1987

Chromatin association and DNA binding properties of the c-fos proto-oncogene product.

As a first step in the analysis of the molecular function of the nuclear c-fos proto-oncogene product we have studied its subnuclear localization in serum-stimulated mouse fibroblasts where it forms a non-covalent, apparently monodisperse complex with another nuclear protein, p39. The c-fos/p39 complex is almost quantitatively released from intact nuclei by DNasel or micrococcus nuclease treatment under conditions where only a minor fraction of DNA and nuclear proteins is released. In gel filtration experiments, c-fos/p39 comigrates with chromatin and seems to be associated with regions of increased DNasel accessibility. c-fos/p39 is bound to chromatin by electrostatic forces of moderate strength since greater than 90% of the complex can be eluted from nuclei at 0.4 M NaCl. In vitro, the c-fos/p39 complex in nuclear extracts binds to double- and single-stranded calf thymus DNA, suggesting that the association of c-fos/p39 with chromatin is at least in part due to its interaction with DNA. In agreement with this conclusion, c-fos/p39 is released from nuclei by incubation with tRNA, presumably due to competition for binding sites. Our observations are compatible with the hypothesis that c-fos may play a role in the regulation of gene expression.

Animals↗
PubMed1987

Different types of modification in c-fos and its associated protein p39: modulation of DNA binding by phosphorylation.

We have studied the biosynthesis and biochemical properties of the c-fos gene product and its associated protein (p39) in growth factor-stimulated fibroblasts. c-fos is a markedly acidic protein that is extensively post-translationally modified by phosphorylation and another type of modification not changing its relative molecular mass (Mr). More than 10 different forms of c-fos protein can be identified by two-dimensional gel analysis. In c-fos-transformed cells, however, most of the highly modified forms are missing. The affinity for DNA of less phosphorylated c-fos-protein complexes is higher than that of the highly modified ones. The transforming potential of c-fos protein and its affinity for DNA thus seems to be inversely correlated with the extent of its phosphorylation. In contrast to c-fos, p39 is a basic protein that is rendered even more basic by post-translational modification. Two other forms of p39 differing in specific domains of the protein (p41 and p43) were also found to be complexed with c-fos.

Animals↗
PubMed1985

Regulation of c-fos transcription in mouse fibroblasts: identification of DNase I-hypersensitive sites and regulatory upstream sequences.

In quiescent mouse fibroblasts, the c-fos gene is expressed at very low levels, but is rapidly and transiently inducible by peptide growth factors. In this study, we have identified in quiescent cells five DNase I-hypersensitive sites located -1700, -290, +10, +240 and +700 bp relative to the 5' cap site. After serum stimulation, the distinct nuclease hypersensitive site at position +10 rapidly disappeared, and instead a broad region of DNase I accessibility between positions 0 and +250 occurred. Nucleotide sequence analysis of the 5'-flanking region of the mouse c-fos gene showed that the hypersensitive site around position -290 is located in a region that is highly conserved between mouse and human, and that contains an enhancer-like structure. When the mouse c-fos promoter and 351 bp of 5'-flanking sequences were linked to the bacterial chloramphenicol acetyl transferase (CAT) gene and transfected into NIH3T3 cells efficient, constitutive expression of CAT activity was observed, even in unstimulated, quiescent cells. However, removal of a 256-bp stretch upstream from position -95 completely abolished CAT expression, indicating that sequences within a region of approximately 350 bp upstream from the cap site are indispensable for c-fos transcription. In addition, our findings point to the existence of other sequence elements that exert negative regulation in the absence of growth factor stimulation. Such sites may be found around the growth factor-responsive nuclease hypersensitive sites in the vicinity of the cap site.

Acetyltransferases↗
PubMed1985

[Diagnostic significance of the detection of hepatitis B virus DNA in acute and chronic hepatitis].

1065 sera from patients with acute and chronic hepatitis-B-virus-(HBV-) infections, double infections (HBV, HAV, nonA-nonB, delta-Ag) as well as patients with HBs-Ag-negative hepatitis (HAV, nonA-nonB) and healthy subjects were investigated for the presence of hepatitis-B-virus-DNA using molecular hybridisation. The sensitivity of the method was 0.1 pg HBV-DNA/100 microliters. HBV-DNA could be detected in 62% of cases of HBs-Ag-positive sera with HBe-Ag, in 8.9% with anti-HBe and in 11% of e-marker free sera. In acute hepatitis HBV-DNA was present in 44%, in chronic hepatitis in 71% of HBe-Ag-positive sera. In HBs-Ag-negative sera containing only anti-HBc, HBV-DNA, depending on the anti-HBc-titre, was present in 13-24% of cases. HBV-DNA could not be detected in patients with HBV infections (anti-HBc and anti-HBs positive) in the past or in HBV-marker-negative hepatitis. Follow-up investigations on acute and chronic HBV-infections showed that the disappearance of HBV-DNA generally preceded the disappearance of HBe-antigen by about 2-3 weeks. In chronic hepatitis the time interval can amount to several months or years. Double infections with other hepatotropic viruses (nonA-nonB and delta-virus) can lead to a temporary suppression of HBV-DNA replication.

DNA, Viral↗
PubMed1984

Simple sequences are ubiquitous repetitive components of eukaryotic genomes.

Simple sequences are stretches of DNA which consist of only one, or a few tandemly repeated nucleotides, for example poly (dA) X poly (dT) or poly (dG-dT) X poly (dC-dA). These two types of simple sequence have been shown to be repetitive and interspersed in many eukaryotic genomes. Several other types have been found by sequencing eukaryotic DNA. In this report we have undertaken a systematical survey for simple sequences. We hybridized synthetical simple sequence DNA to genome blots of phylogenetically different organisms. We found that many, probably even all possible types of simple sequence are repetitive components of eukaryotic genomes. We propose therefore that they arise by common mechanisms namely slippage replication and unequal crossover and that they might have no general function with regards to gene expression. This latter inference is supported by the fact that we have detected simple sequences only in the metabolically inactive micronucleus of the protozoan Stylonychia, but not in the metabolically active macronucleus which is derived from the micronucleus by chromosome diminution.

Animals↗