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W J Habets

Publications and source records attributed to W J Habets.

30 records · Page 2Linked to original sources
PubMed1985

Further characterization and subcellular localization of Sm and U1 ribonucleoprotein antigens.

Sera from patients with systemic autoimmune diseases often contain antibodies against small nuclear ribonucleoprotein (snRNP) particles. Anti-Sm antibodies react with the entire set of U1, U2, U4, U5 and U6 (U1-U6) RNP particles whereas anti-(U1)RNP sera specifically recognize particles containing U1 RNA. Here we performed semi-quantitative immunoblotting using 16 human anti-Sm, 15 human anti-(U1)RNP sera and two mouse monoclonal antibodies to establish which snRNA-associated proteins carry antigenic determinants. Almost every (15/16) human anti-Sm sera recognized epitopes present on a 28-kDa (B/B') protein doublet and on a 16-kDa (D) polypeptide. Nine anti-(U1)RNP sera also recognized the B/B' doublet, but in all cases a much stronger reaction was observed with one or more of the specifically U1 RNA-associated 70 kDa, A or C antigens. With affinity-purified antibody fractions eluted from individual antigen bands on nitrocellulose blots it is shown that the anti-Sm-reactive polypeptides B/B' and D contain common epitopes. We also report the finding of one human anti-Sm serum with exclusive specificity for the B/B' doublet and a mouse monoclonal anti-Sm antibody recognizing only the D protein, indicating that these antigens also carry unique epitopes. In immunoprecipitation assays, purified anti-B/B' and -D antibodies react with (U1-U6) RNP while purified anti-70 kDa, anti-A and anti-C antibodies precipitate exclusively U1 RNP particles. Finally, we established the subcellular localization of Sm and U1 RNP antigens using a biochemical cell fractionation procedure. Part of the 70 kDa and B/B' antigens were found in a nuclease and high salt-resistant nuclear substructure, usually referred to as nuclear matrix, while the A and D antigens could be extracted completely from HeLa nuclei by ribonuclease treatment and subsequent high salt extraction.

Antibody Specificity↗
PubMed1985

Scl-86, a marker antigen for diffuse scleroderma.

More than 300 sera from patients with a connective tissue disease were analyzed with the immunoblotting technique. The presence of autoantibodies against an 86,000-mol wt marker antigen for diffuse scleroderma (Scl-86) was found in 14 out of 33 patients with scleroderma. The presence of anti-Scl-86 antibodies seemed to correlate with the diagnosis of diffuse scleroderma since they were found in 13 out of 22 diffuse scleroderma patients and in only one out of 11 patients with limited scleroderma. All scleroderma sera (33 patients' sera and 13 reference sera) were also tested for the presence of anti-Scl-70 antibodies. It was found that all anti-Scl-70 positive sera (n = 25) contained anti-Scl-86 antibody as well, suggesting a relationship between these two antigens. However, the Scl-86 antigen was shown to be an extremely insoluble nonchromosomal protein, resistant to boiling in sodium dodecyl sulfate. This contrasts with the Scl-70 antigen, which has been described as a thermolabile, soluble antigen present in the chromatin fraction. Together, our results are consistent with the idea that Scl-70 is a degradation product of Scl-86. The Scl-86 antigen is present in freshly prepared rabbit thymus, spleen, and liver nuclei as well as in nuclei from various cultured cell lines, but is not detectable in extractable nuclear antigen from rabbit thymus. In a limited retrospective study, the anti-Scl-86 antibodies were found in two sera from patients with Raynaud's phenomenon before the development of diffuse scleroderma. Therefore, it is possible that screening of patients' serum for this antibody might predict the development of diffuse scleroderma.

Animals↗
PubMed1985

Quantitation of anti-RNP and anti-Sm antibodies in MCTD and SLE patients by immunoblotting.

A quantitative immunoblotting assay (QIBA) for the determination of specific antibody titres in human autoimmune sera is described. In this assay, a total HeLa nuclear protein fraction, immobilized on nitrocellulose blot strips, was used as source of antigens and immunoreactive species of autoantibodies were quantitated by an enzyme linked second antibody procedure. Besides being more discriminative, QIBA appeared to be up to 500 times more sensitive than immunodiffusion or immunoelectrophoresis. In this study we used 21 sera from patients with SLE or MCTD for a quantitative analysis of their specific autoantibody content. Within this group, a very diverse spectrum of antibody populations was observed; anti-RNP sera appeared to contain, among others, high tired antibody versus 70K and 31K polypeptides while all (n = 6) anti-Sm sera recognized a 25kD protein doublet. In a follow-up study of two MCTD patients significant flares in specific antibody content could be observed.

Adult↗
PubMed1983

Characterization of the SS-B (La) antigen in adenovirus-infected and uninfected HeLa cells.

The molecular composition and subcellular localization of the antigens recognized by anti-SS-B (La or Ha) antibodies was investigated. Ten anti-SS-B sera were selected by indirect immunofluorescence and by their immunological identity in counter-immunoelectrophoresis (CIE) with an anti-SS-B reference serum. All sera precipitated virus-associated (VA) RNA from cellular extracts of adenovirus-infected HeLa cells. Earlier results had shown that in adenovirus-infected HeLa cells a cellular 50 000 mol. wt. protein was tightly associated with VA RNA in situ. Our present results indicate that this 50 000 protein is the only SS-B antigen present in adenovirus-infected as well as in uninfected cells. A major part (greater than 80%) of the SS-B antigen is present in a readily extractable, soluble form. The rest is found in an insoluble form tightly associated with an internal nuclear structure that is mostly referred to as the nuclear matrix. Both forms are very susceptible to proteolytic degradation resulting in at least two distinct breakdown products of mol. wts. 40 000 and 25 000. The cellular 50 000 polypeptide is present in extracts of various types of cells and tissues, indicating that this antigen is very well conserved during evolution. The association of the 50 000 mol. wt. antigen with host- as well as viral-coded RNA polymerase III products also suggests an important function for this protein in the metabolism of these small RNAs.

Adenoviruses, Human↗
PubMed1983

Antibodies against distinct nuclear matrix proteins are characteristic for mixed connective tissue disease.

Specific nuclear proteins, separated according to their molecular weight (mol. wt) by polyacrylamide gel electrophoresis (PAGE) and subsequently transferred to nitrocellulose sheets, are able to bind antibodies in sera from patients suffering from different types of connective tissue diseases. Antibodies against a characteristic set of nuclear protein antigens are found in sera from patients with mixed connective tissue disease (MCTD). Screening of 21 MCTD sera revealed a typical immunoblot pattern with major protein antigens of mol. wt 70,000 (20/21) (not identical with the Scl-70 antigen characteristic for scleroderma), mol. wt 31,000 (17/21), two proteins around mol. wt 23,000 (15/21) and two around mol. wt 19,000 (10/21). The 70,000, 23,000 and 19,000 antigens appeared to be rather insoluble nuclear proteins (i.e. components of the nuclear matrix). On behalf of their structural character they were present in nuclei from several types of cells but only in low amounts detectable in salt extracts of thymus acetone powder. The presence of antibodies directed against the mol. wt 70,000 antigen correlated strongly with the diagnosis of MCTD. This 70,000 antigen is not identical with the RNP antigen, a soluble ribonuclease sensitive ribonucleoprotein, since antibodies against nuclear RNP can be separated from anti-nuclear matrix antibodies by affinity chromatography using immobilized thymus salt extract. The distinct character of soluble nuclear RNP and structural nuclear matrix antigens is further supported by the fact that from 14 other anti-RNP sera obtained from patients with systemic lupus erythematosus (SLE), only three contained antibodies against the mol. wt 70,000 protein. Since the immunoblot pattern obtained with MCTD sera mostly was clearly distinguishable from the patterns obtained with sera from patients with related connective tissue diseases our results suggest that the immunoblotting technique might be useful as a diagnostic tool and support the concept of MCTD as a distinct entity.

Antibodies, Antinuclear↗
PubMed1982

Anti-nuclear matrix antibodies in mixed connective tissue disease.

Purified serum antibodies of patients suffering from mixed connective tissue disease were tested for their immunological specificity against nuclear constituents of HeLa S3 cells. In the indirect immunofluorescent staining technique, using cells and nuclei as targets, a typical speckled intranuclear staining pattern was obtained, that persisted after degradation and extraction of all nucleic acids and their associated proteins. This treatment of nuclei with detergents, DNase, RNase and high salt concentrations leave intact only the so-called nuclear matrix which is an intranuclear proteinaceous network. Further proof that nuclear matrix proteins were targets of the autoimmune reaction was obtained after separation of these proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrophoretic transfer to nitrocellulose (blotting). A specific number of blot-transferred matrix proteins reacted with purified serum antibodies of 10 patients with mixed connective tissue disease, whereas this reaction was negative with normal healthy individuals. IgG preparations of 7 patients with systemic lupus erythematosus showed a weak, if any, reaction with matrix constituents. Obviously, in some connective tissue diseases serum antibodies are expressed which are directed to specific nuclear matrix antigens.

Autoantibodies↗
PubMed1982

Nitrogen control in Pseudomonas aeruginosa: mutants affected in the synthesis of glutamine synthetase, urease, and NADP-dependent glutamate dehydrogenase.

Mutants were isolated from Pseudomonas aeruginosa that were impaired in the utilization of a number of nitrogen sources. In contrast to the wild-type strain, these mutants appeared to be unable to derepress the formation of glutamine synthetase and urease under nitrogen-limited growth conditions, whereas NADP-dependent glutamate dehydrogenase became derepressed. This GlnR- phenotype appeared to be caused by a mutation located in the early region of the P. aeruginosa PAO chromosomal map, close to hisIV59. Partial suppression of the GlnR- phenotype due to a mutation located close to hisII4 was observed. These revertants were different from both the wild-type strain and the GlnR- mutant with respect to the regulation of the synthesis of glutamine synthetase, urease, and NADP-dependent glutamate dehydrogenase (GlnRc phenotype). Also the regulation of glutamine synthetase activity by adenylylation/deadenylylation was altered in the revertants. The results suggest the presence of a regulatory gene that plays a role in the regulation of enzyme formation in response to the availability of ammonia.

Chromosomes, Bacterial↗
PubMedDate not supplied

Anti-56K: a novel, frequently occurring autoantibody specificity in connective tissue disease.

A novel frequently occurring autoantibody specificity in serum from connective tissue disease patients is described. The autoantibodies as detected by immunoblotting are directed against a 56,000 Dalton (56K) antigen, that after biochemical fractionation predominantly is found in the cytoplasmic fraction of various cell types and tissues. Attempts to localize the antigen more precisely were unsuccessful primarily because these antibodies do not produce a positive immunofluorescence pattern. The antigen in its native form is not associated with DNA or one of the common cytoplasmic or nuclear RNAs in the cell. Immunoprecipitation studies also showed that the protein is not closely associated with other proteins in a multi-component complex. Anti-56K antibodies are found in about 8% of patients with connective tissue disease, most commonly in patients with Sjögren's syndrome (14%). It is not found in patients with mixed connective tissue disease, polymyositis/dermatomyositis or scleroderma and rarely (less than 1%) in healthy control subjects. The fact that 22% of the anti-56K sera also contain La/SS-B antibodies support the idea that this antibody specificity might be characteristic for a subclass of Sjögren's syndrome patients.

Adolescent↗