Overexpression of hnRNPA2/B1 in bronchoscopic specimens: a potential early detection marker in lung cancer.

BACKGROUND: Overexpression of heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 has recently been suggested to be a promising marker for early detection of lung cancer. The aim of this study was to determine the utility of its detection in bronchoscopic specimens. PATIENTS AND METHODS: Brushing and biopsy specimens were obtained from 61 patients suspected of having lung cancer, as well as from 30 healthy subjects (controls), who underwent bronchoscopy. hnRNPA2/B1 expression levels were evaluated by immunoblotting. RESULTS: Specificity of hnRNP A2/B1 overexpression was 75.9% in brushing and 78.3% in biopsy. Sensitivity in non-small cell lung cancer was 84.8% in brushing and 80.8% in biopsies, while in small cell lung cancer it was 66.7 % and 75%, respectively. Overexpression of hnRNPA2/B1 was also detected in bronchoscopic specimens of nine patients initially undiagnosed. The follow-up of these patients 2 years later showed that seven of them had developed lung cancer. CONCLUSION: Overexpression of hnRNPA2/B1 was significantly higher in patients suffering from lung cancer and may be useful in the early detection of lung cancer.

Clinical, immunological, and immunogenetic aspects of autoantibody production against Ro/SSA, La/SSB and their linear epitopes in primary Sjögren's syndrome (pSS): a European multicentre study.

OBJECTIVES: To investigate the clinical and immunogenetic aspects of antibody formation against Ro/SSA and La/SSB as well as their linear B cell epitopes in patients with primary Sjögren's syndrome (pSS) from different European countries. PATIENTS AND METHODS: Ninety patients with pSS from six European centres were studied. Serum samples from all patients were tested in a control laboratory for anti-Ro/SSA and anti-La/SSB autoantibodies by RNA precipitation assay and autoantibodies to the previously reported B cell linear epitopes of Ro 60 kDa (p169-190aa and p211-232aa) and La/SSB (p147-154aa, p291-302aa, p301-318aa, and p349-364aa). DNA from 88 patients was used for the determination of HLA-DRB1, -DQA1, and -DQB1 genotypes. Analysis of the results was performed in the 88 patients who were genotyped and tested also for antipeptide antibodies. RESULTS: Antibodies to B cell epitopes of Ro 60 kDa were detected at a low frequency (range 10-37%). In contrast, B cell epitopes of La/SSB were detected frequently (range 58-86%) among the anti-La/SSB positive sera. Autoantibodies to the La/SSB epitope, p349-364aa, were significantly positively associated with longer disease duration (p<0.05), recurrent or permanent parotid gland enlargement (p<0.005), and a higher proportion of non-exocrine manifestations (p<0.005), compared with patients without autoantibodies. The presence of anti-Ro/SSA and anti-La/SSB autoantibodies was significantly associated with the presence of HLA-DRB1*03 and DQB1*02 (p=0.038 and p=0.034, respectively). This association was even more prominent and extended to HLA-DQA1*0501 when patients were stratified according the presence of autoantibodies to discrete La/SSB B cell epitopes in comparison with autoantibody negative patients (p<0.01). They were found also to be highly associated with the alleles HLA-DQB1*02 and HLA-DQA1*0501 as well as the presence of a shared amino acid motif in the region 59-69aa of DQB1 first domain (p<0.01, respectively). CONCLUSIONS: Autoantibodies against La/SSB, binding to four synthetic peptides, derived from the sequence of the La protein were identified with increased frequency in sera of patients with pSS. The formation of autoantibodies against B cell epitope analogues of La/SSB in European patients with pSS may be dependent on the presence of a permissive HLA-DQ heterodimer, most prominently represented by the HLA-DQA1*0501/DQB1*0201 heterodimer, suggesting that a model of HLA restricted presentation of La/SSB peptide determinants is crucial for the autoimmune response against La/SSB.

The 72/74-kDa polypeptides of the 70-110 S large heterogeneous nuclear ribonucleoprotein complex (LH-nRNP) represent a discrete subset of the hnRNP M protein family.

Pre-mRNA processing in eukaryotes is thought to take place on a multitude of nuclear ribonucleoprotein (RNP) complexes, the most abundant of them being the heterogeneous nuclear (hn) RNP complexes. The identification in mammalian nuclear extracts of a novel, less-abundant 70-110 S heterogeneous RNP, named large heterogeneous nuclear RNP (LH-nRNP), has previously been reported by Aidinis, Sekeris and Guialis (1995) Nucleic Acids Res. 23, 2742-2753. The structural composition of the LH-nRNP complex has been determined following the production of polyclonal antibodies against the major protein constituents of the complex, the pair of the 72/74-kDa polypeptides. In the present study evidence is shown to prove that the 72/74-kDa proteins are members of the hnRNP M protein family, hereafter referred to as 72/74(M) polypeptides. The extensive application of two-dimensional gel electrophoresis, combined with specific immunoprecipitation and immunoblotting assays, has allowed the assignment of the 72/74(M) proteins to a subset of the hnRNP M family, characteristic of the presence of the LH-nRNP complex and distinct from the hnRNP-associated M1-M4 components. Moreover, the immunoselection of the LH-nRNP complex from [(32)P]orthophosphate-labelled HeLa cells, with the parallel application of UV irradiation, has permitted the identification of the 72/74(M) polypeptides as the sole protein constituents of the complex in direct contact with the RNA. It is proposed that LH-nRNP constitutes a discrete subset of hnRNP complexes, having a possible role in establishing specific interactions between hnRNP and nuclear-matrix protein components.

Molecular characterization of a murine, major A/B type hnRNP protein: mBx.

We have previously identified a discrete hnRNP polypeptide of the A/B type, named mBx, as an abundant protein species in murine cells. The molecular characterization of this protein is now accomplished. From all evidence provided, mBx polypeptide represents a new gene product, distinct from the known members of the A/B family A1 and A2/B1. It is, instead, mostly related to a still hypothetical human protein of A/B type, as well as to the Xenopus hnRNPA3 protein species.

No evidence of epitope spreading after immunization with the major Sm epitope P-P-G-M-R-P-P anchored to sequential oligopeptide carriers (SOCs).

The sequence Pro-Pro-Gly-Meth-Arg-Pro-Pro (PPGMRPP) is the major B-cell epitope of the Sm autoantigen. The aim of the present study was to evaluate the immune response against the native forms of Sm and U1RNP and immune mediated tissue injury after immunization with the sequence PPGMRPP anchored in five copies to a new type helicoid sequential oligopeptide carrier (SOC) formed by the repetitive Lys-Aib-Gly moiety, [(PPGMRPP)(5)SOC(5)]. Rabbits (n=3) were immunized with 0.5 mg of (PPGMRPP)(5)SOC(5)in complete Freud's adjuvant and boosted at days 26, 53, 99; control rabbits were immunized with the PPGMRPP alone (n=3), phosphate buffered saline (PBS) (n=1), SOC(5)alone (n=1), a peptide at aminoacid (aa) position 158-177 of myelin basic protein (MBP aa 158-147) (n=1) and three La/SSB autoantigen B-cell epitopes (n=3). Antibodies to (PPGMRPP)(5)SOC(5)were determined by enzyme linked immunosorbent assay (ELISA); precipitating anti-Sm and anti-U1RNP antibodies were detected by RNA precipitation and western blot on HeLa total cellular and nuclear extract and 12s sucrose gradient fraction of rat liver extracts. High titres of anti-(PPGMRPP)(5)SOC(5)antibodies not recognizing the native forms of Sm or U1RNP antigens were detected in the (PPGMRPP)(5)SOC(5)immunized but not in the control animals. The sera of two (PPGMRPP)(5)SOC(5)immunized but not of the control rabbits recognized a 67 kDa protein in HeLa nuclear extract, distinct from the 70 kDa U1RNP antigen. Diffuse and segmental increase of mesangeal matrix and cells, crescent formation, segmental glomerular necrosis, rarely massive subendothelial deposits occluding the lumen and C3 complement component in the mesangeal area were seen in the kidneys of one (PPGMRPP)(5)SOC(5)immunized, but not of the remaining animals. In conclusion, the immune response induced by (PPGMRPP)(5)SOC(5)was specific for the immunizing epitope but not for the native forms of Sm and U1RNP antigens, but it was associated with immune mediated kidney injury.

Activation of embryonic genome in chick.

The earliest stages of development in most animals are under the control of maternally inherited information. The initiation of embryonic gene expression has been reported at the mid-blastula in amphibians and the mid-2-cell stage to the early morula in mammals. In chick embryos, embryonic gene expression was detectable at stage X (morula) and showed marked activation at stage XIII (blastula) with a gradual increase thereafter. Synthesis of rRNA and tRNA was low at stage X and was already the major class of RNA at stage XIII in chick embryos. The observed upregulation of RNA synthesis seems to coincide with a period of extensive fine structural differentiation when the first major cellular migrations start and signal the formation of the primitive streak in the chick embryo.

Structural/functional properties of a mammalian multi-component structure containing all major spliceosomal small nuclear ribonucleoprotein particles.

An approx. 40 S multi-component structure, consisting of all major spliceosomal small nuclear ribonucleoprotein particles (snRNP) (U1, U2, U4/U6 and U5) in stable association with a large number of polypeptides, mainly in the range 50-210 kDa, has been reported to exist within rat liver nuclear extracts [Guialis, Moraitou, Patrinou-Georgoula and Dangli (1991) Nucleic Acids Res. 19, 287-296]. Using a new polyclonal antibody recognizing a 63 kDa protein component of the complex, this multi-snRNP assembly was detected within rat liver nuclear extracts as efficiently as with the antibody for the U2 snRNP-specific B' polypeptide. The 63 kDa protein was found to correspond to the 66 kDa subunit of the splicing factor SF3a, a known integral component of the HeLa 17 S U2 snRNP. Anti-2,2,7-trimethylguanosine affinity chromatography was an easy and efficient way of purifying the multi-snRNP complex from rat liver 40 S heterogeneous nuclear ribonucleoprotein particle (hnRNP)-containing sucrose gradient fractions. By subsequent glycerol-gradient sedimentation, all known snRNP forms active in RNA splicing were identified among its constituents. A complex structurally similar to the rat multi-snRNP was also identified in HeLa nuclear extracts. Preservation of hnRNP-snRNP interactions was observed within HeLa 40 S fractions. Moreover, these fractions were capable of restoring splicing activity when applied in reconstitution studies to supplement a micrococcal nuclease-treated splicing extract.

Non-organ specific autoantibodies against cellular components in HIV-1 infected individuals.

OBJECTIVE: To detect the presence of autoantibodies against cellular components in HIV-1 infected individuals. METHODS: Serum samples from 87 patients with HIV-1 infection and from 17 healthy HIV-seronegative controls were tested using sensitive and specific techniques (immunoblot, RNA precipitation), as well as counterimmunoelectrophoresis and indirect immunofluorescence. RESULTS: Four sera (4.6%) showed reactivity in immunoblot against various unidentified cytoplasmic autoantigens. The four positive sera were obtained from patients belonging in equal numbers to the non-AIDS and AIDS groups. CONCLUSION: It appears that the production of antibodies against cellular components rarely occurs during the clinical course of HIV infection.

Recognition of subsets of the mammalian A/B-type core heterogeneous nuclear ribonucleoprotein polypeptides by novel autoantibodies.

The structurally related A/B-type core heterogeneous nuclear ribonucleoprotein (hnRNP) polypeptides of 34-39 kDa (A1, A2, B1 and B2) belong to a family of RNA-binding proteins that are major components of 40 S hnRNP complexes. By two-dimensional gel electrophoresis and peptide mapping analysis we compared each member of the A/B-type core proteins in the human and rat liver cells. This comparison revealed the unique presence in rat cells of major protein species, referred to as mBx polypeptides, that appeared as three charge isoforms at a position corresponding to the minor HeLa B1b protein spot. In addition, clear differences in the ratios of the A1 polypeptide to the A1b isoform were observed. The detection, in sera of patients with rheumatic autoimmune diseases, of two novel autoantibody specificities, one recognizing solely B2 protein and the second both the B2 and mBx polypeptides, helped to identify mBx proteins as new A/B-type hnRNP components, immunologically related to B2 protein. A common immunoreactive V8 protease peptide of approx. 17 kDa has been identified in B2 and mBx hnRNP polypeptides. mBx protein species are identified in cells of murine origin, and have a ubiquitous tissue distribution and developmental appearance.

Predominance of IgM anti-U1RNP antibodies in patients with systemic lupus erythematosus.

Anti-U1RNP antibodies occur in patients with mixed connective tissue disease (MCTD), systemic sclerosis (SSc), systemic lupus erythematosus (SLE) and other ill-defined connective tissue diseases. To associate the isotypes of anti-U1RNP antibodies with the diagnosis of the disease, namely SLE or MCTD, sequential sera of patients positive for anti-U1RNP antibodies by counterimmunoelectrophoresis (CIE) (32 with SLE, 35 with MCTD) were tested for IgG and IgM anti-U1RNP antibodies by enzyme-linked immunosorbent assay (ELISA) using affinity-purified U1snRNP complexes. Results from ELISA were confirmed by RNA precipitation. IgG RNA precipitation of HeLa cellular extracts was performed using the bulk of the IgG fraction removed from each serum after binding to protein A-Sepharose beads. IgM RNA precipitation was carried out on the IgM fraction of the serum bound to protein A-Sepharose-rabbit anti-human IgM immune complexes. RNAs were electrophoresed in 10.5% acrylamide-7 M urea gels and detected with the silver stain. ELISA showed that all sera were positive to IgG anti-U1RNP, while 12 of the 35 MCTD and 21 of the 32 SLE patients possessed IgM anti-U1RNP (P < 0.025). IgM anti-U1RNP reactivity was found during the follow-up in 20% of 44 sera from 17 MCTD patients and 68% of 112 sera from 23 SLE patients (P < 0.0001). IgG from all the sera precipitated U1RNPs. Eight of the MCTD sera also precipitated U2RNPs and 14 of the SLE sera U2 and/or U4/U6, U5 RNPs. IgM from MCTD sera did not precipitate URNPs, while IgM from SLE sera precipitated predominantly U1RNPs. These data suggest that IgM anti-U1RNP antibodies occur predominantly in patients with SLE. The occurrence of IgG anti-U1RNP without IgM is more frequent in MCTD.

Detection of human-specific anti-la(SSB) antibodies in patients with rheumatoid arthritis.

Sera from anti-Ro(SSA) positive and negative patients with rheumatoid arthritis (RA) were compared with those from patients with primary Sjogren's syndrome (pSS) and systemic lupus erythematosus (SLE) with regard to anti-La(SSB) antibodies. Several assays for anti-La(SSB) (RNA precipitation, counterimmunoelectrophoresis, and immunoblotting) demonstrated the presence of such antibodies in selected anti-Ro(SSA) positive (10/19 in RA and 18/37 in pSS and SLE) but not in anti-Ro(SSA) negative sera. In agreement with previous reports, anti-La(SSB) antibodies from pSS and SLE patients uniformly reacted with various cellular extracts used as sources of La(SSB) antigen, including extracts from human cultured cells (HeLa), calf thymus and rabbit thymus. In contrast, while all 10 anti-La(SSB) sera from RA patients reacted with the human HeLa cell extracts, only three of them also reacted with calf and rabbit thymus extracts. These results were further supported by the analysis of a cohort of 70 consecutively selected sera (displaying monospecific anti-Ro(SSA) reactivity against calf thymus extract) from patients with various rheumatic disorders, where human-specific anti-La(SSB) were detected in two out of the five of RA patients studied.

Two immunologically related polypeptides of 72/74 kDa specify a novel 70-100S heterogeneous nuclear RNP.

Evidence suggesting the presence in rat liver nuclear extracts of a new RNP complex of 70-110S has been provided [Hatzoglou, M., Adamtziki, E., Margaritis, L. and Sekeris, C.E (1985) Exp. Cell. Res. 157, 227-241]. Biochemical features unique to this RNP were its stability to salt and RNase digestion and the presence of a pair of polypeptides of 72/74 kDa. By producing antibodies against the 72/74 kDa polypeptides these proteins have been defined as integral components of the 70-110S RNP complex. They comprise two immunologically related polypeptides with an exclusively nucleoplasmic localization, giving a speckled pattern in a diffuse background, similar, but not identical, to the Sm antigen. The 70-110S RNP complex, referred to as large heterogeneous nuclear RNP (LH-nRNP), has a simple protein pattern that includes, in addition to the 72/74 kDa proteins, three stably associated polypeptides of apparent molecular size 110, 61 and 59 kDa. The bulk of its RNA component represents a discrete RNA population of 10-20S, belonging to a subset of the RNA detected within immunopurified HeLa hnRNP complexes. These RNA species are RNA polymerase II transcripts of greater stability relative to the bulk of hnRNA, containing oligo(A) or poly(A) sequences. Immunodepletion and/or antibody addition studies in HeLa splicing extracts using antibodies with specificity for the 72/74 kDa proteins revealed a rather strong inhibition of splicing activity, suggesting participation of the LH-nRNP complex in in vitro splicing.

Anti-5S RNA/protein (RNP) antibody levels correlate with disease activity in a patient with systemic lupus erythematosus (SLE) nephritis.

A patient with systemic lupus erythematosus (SLE) and nephritis without antibodies to dsDNA but with antibodies to a 5S RNA/protein (RNP) complex is presented. Combined RNA precipitation and Western blotting experiments strongly suggested that these newly identified autoantibodies recognized a distinct epitope on the L5 ribosomal protein of the L5/5S RNP complex first described by Steitz et al. [1]. Quantification of the anti-5S RNP antibody levels was done by hybridizing Northern blots of immunoprecipitated RNA from serial serum samples with a 32P-labelled oligoprobe specific for the 5S ribosomal RNA. These studies revealed a strong association between anti-5S RNP autoantibody titre and severity of SLE nephritis over a 3-year prospective study. Our results indicate that the L5/5S RNP can be a target of autoimmune response, and and may serve, in some cases, as marker of SLE severity and response to therapy.

Detection of anti-Ro(SSA) antibodies in autoimmune diseases: comparison of five methods.

Counterimmunoelectrophoresis (CIE), RNA precipitation, ELISA and immunoblotting against cytoplasmic HeLa cell extract (IB-HeLa) and erythrocyte extract (IB-RBC) were applied to detect anti-Ro(SSA) antibodies in 93 sera selected from patients with various autoimmune diseases [47 were anti-Ro(SSA) positive by CIE]. The RNA precipitation assay, which demonstrated the highest sensitivity was selected as the reference method. CIE was found to be reliable with a specificity of 100% and a sensitivity of 89%. ELISA showed a comparable specificity (95%) but somewhat lower sensitivity (72%). Antibodies to 52 or 60 kDa Ro(SSA) proteins by IB-HeLa demonstrated a high specificity (95 and 97% respectively) but a low overall sensitivity (36 and 17% respectively). Anti-Ro(SSA) antibodies to 52, 54 and 60 kDa erythrocyte proteins by IB-RBC, had a variable overall specificity (95, 97 and 57%) and sensitivity (51, 13 and 34%). The anti-52 kDa antibodies detected by IB-HeLa correlated to those found by IB-RBC (P < 0.001) and occurred predominantly in primary Sjögren's syndrome (P < 0.001, sensitivity: 71 and 77%) as well as in sera with anti-Ro(SSA) and anti-La(SSB) antibodies (P < 0.001). These findings confirm that RNA precipitation assay has the highest sensitivity and specificity for anti-Ro(SSA) antibody detection. However, until a more sensitive ELISA is available, CIE because of its reliability appears to be the method of choice. Finally IB-RBC was found to be more sensitive than IB-HeLa for the detection of anti-Ro52 kDa antibodies.

A novel 40S multi-snRNP complex isolated from rat liver nuclei.

Two structurally distinct RNP complexes (MI and MII), each with a sedimentation value of approx. 40S, were isolated from rat liver nuclear extracts by sucrose gradient centrifugation and subsequent native gel electrophoresis of the 40S hnRNP-containing fractions. MII RNP contained the bulk of hnRNA and hnRNP proteins (i.e. the 32-45KD core proteins and polypeptides of 60-80 and 110-130KD). MI RNP was characterized by the exclusive presence of U-snRNAs (U1, U2, U4, U5 and U6), their well known snRNP polypeptides and a number of Sm-associated proteins in the range of 50-210KD. Immunoselection experiments employing a monoclonal antibody with an established specificity for the U2-snRNP-specific B" polypeptide proved that the RNA and protein components characteristic of MI were part of a single multi-snRNP unit. The prominent 200/210KD protein doublet of MI was identified immunochemically as the rat homologue of the yeast PRP8 protein, a known U5-associated splicing component. Based on the major biochemical and immunochemical features of MI and MII RNP complexes, we conclude that MII represents the monomeric 40S hnRNP structure, whereas MI defines a novel multi-snRNP entity.

Autoantibodies to the core proteins of hnRNPs.

A novel autoantibody reacting the the core polypeptides of hnRNP particles has been detected in the serum of a patient with systemic lupus erythematosus (SLE) and Sjögren's syndrome manifestations. Immunoblot analysis, using either rat liver or HeLa nuclear extracts as the antigen source, demonstrated that the autoantibody interacts with a specific subgroup of the core polypeptides of hnRNP particles, namely A2, B1 and B2, but not with A1, C1 and C2.

Distribution of snRNP complexes in rat liver nuclear extracts: biochemical and immunochemical analysis.

Rat liver nuclei were extracted with 0.14 M NaCl and the extracts submitted to sucrose gradient fractionation. Aliquots of the nuclear residue remaining after the 0.14 M NaCl extraction were also extracted either with 0.3 M NaCl or 1 M urea, and the extracts similarly submitted to sucrose gradient fractionation. Thereafter, both the presence and relative distribution of individual U-snRNA (U1-U6) species was followed. Results showed an extensive association of all U-snRNAs to RNP structures of greater than or equal to 40 S. However, characteristic differences in the association of mostly U1 and U5--which were the major identifiable species in the extracts--to these structures were observed. Only a small fraction of U1 appeared complexed to less than or equal to 40 S RNP structures, while most of it sedimenting in the greater than 20 S region of the gradient. In contrast, U5-snRNA had a tight and almost exclusive association to 40 S RNP structures. No pool of 10-12 S U5-snRNP complexes was detected. Combined immunoprecipitation and immunoblotting experiments on nuclear 0.14 M NaCl extracts using anti-Sm and/or anti-RNP antisera showed that all snRNA species, whether recovered as 10-12 S complexes or segregated with greater than or equal to 40 S RNP components, existed as snRNP structures bearing at least their Sm-antigenic polypeptides. These and our previous results [Guialis, A., Arvanitopoulou, A, Patrinou-Georgoula, M. and Sekeris, C.E. (1983) FEBS Lett. 151, 127-133], support the existence of snRNP-enriched RNP structures of greater than or equal to 40 S. In such structures the core polypeptides (Mr = 32,000-45,000) of 40 S monoparticles are not obligatory components.

Structural relationship of the heterogeneous nuclear ribonucleoprotein core polypeptides from rat liver nuclei.

The individual species of the core polypeptide family of 30-50 S hnRNP resolved on two-dimensional electrophoresis (nonequilibrium pH gradient gels combined with sodium dodecyl sulfate polyacrylamide gels) have been subjected to the enzymatic cleavage procedure of D.W. Cleveland, S.G. Fischer, M.W. Kirschner, and U.K. Laemmli (1977, J. Biol. Chem. 252, 1102-1106). This allowed direct and extensive structural analysis of almost every member of the core polypeptide family by comparison of their overall peptide maps. Thus, the over 20 protein species, resolved on two-dimensional gels, from the four major bands (A, B, C, and D) on one-dimensional sodium dodecyl sulfate-polyacrylamide gels, belong mainly to three distinct protein groups, and each species represents the product of extensive post-translational modification. Furthermore, their inability to bind the lectin concanavalin A makes it unlikely that the modifications of these proteins represent glycosylations. Therefore, the core polypeptides cannot be glycoproteins of the general class with affinity for concanavalin A.

Identification of two discrete ribonucleoprotein particles within the monomer population of rat liver nuclear RNPs.

30-50 S RNP particles (monoparticles) isolated from rat liver nuclei were submitted to electrophoresis in native 0.5% agarose gels. Two RNP fractions were thus separated, a minor one remaining closer to the top of the gel (MI) and a more abundant one migrating further into the gel (MII). SDS-polyacrylamide gel electrophoresis revealed that MII contains the major monoparticle (Mr 30 000-40 000 or 'core') polypeptides and higher molecular weight proteins, whereas MI contains several minor proteins of Mr greater than 40 000. Some proteins are common to both particle classes. Urea-acrylamide gel electrophoresis revealed that HnRNA is mainly present in MII, whereas snRNA is confined to the MI particle class.