Association of autoantibodies against small nuclear ribonucleoproteins (snRNPs) with symptomatic Toxocara canis infestation

Several studies have demonstrated the occurrence of autoantibodies in the course of infestations with helminth parasites and a number of target proteins have been identified. Sera from patients suffering from toxocarosis, a disease caused by the parasitic roundworm Toxocara canis, and from healthy individuals were tested for autoantibodies by immunofluorescence and immunoblot assays using HEp-2 cells as antigen. A considerable proportion of the sera from patients with toxocarosis-associated symptoms were autoantibody-positive, with a speckled staining pattern in the immunofluorescence test (62%) and with anti-snRNP reactivity in the immunoblot assay (98%). In contrast, significantly fewer sera from asymptomatic individuals scored positive in these assays (18% in the immunofluorescence test, P < 0.005; 24% in the immunoblot, P < 0.005). Although the causative link between Toxocara infestation and the occurrence of autoantibodies is still unclear, our results show that increased amounts of autoantibodies are associated with clinical symptoms of inflammation. Thus a serum test for autoantibodies in toxocarosis patients might be a valuable gatekeeper assay for the decision for or against anti-inflammatory treatment.     

Autoantigenic epitopes of the B polypeptide of Sm small nuclear RNP particles. Identification of regions accessible only within the U1 small nuclear RNP.

OBJECTIVE. To analyze the autoantigenic epitopes of the Sm B polypeptide of the U1 small nuclear RNP (snRNP) using complementary DNA (cDNA) clones. METHODS. Expression of Sm B fusion proteins in lambda phage vectors, immunoblots, immunoprecipitations, and affinity purification of antibodies. RESULTS. Immunoblots using antibodies affinity-purified from B fusion proteins demonstrated that there were cross-reactive epitopes between the B'/B and A polypeptides of the U1 snRNP. Immunoprecipitation assays suggested that there were at least 3 different autoantigenic epitopes on the B polypeptide that could be classified into 2 general groups based upon autoantibody reactivity. The first group of autoantibodies, which bound 2 separate autoantigenic epitopes (BU1-1, BU1-2), participated in immunoprecipitation of the U1 snRNP alone. The second group, which bound the third type of autoantigenic epitope (BSm-1), immunoprecipitated all the abundant Sm snRNPs. CONCLUSION. There is at least 1 region on the B proteins that is accessible to antibodies only within the structure of the U1 snRNP, as well as a region that is accessible on all Sm snRNPs. These data support the notion that the native U1 RNP, perhaps containing B proteins in a different conformation than those found on other Sm snRNPs, may drive the humoral immune response in systemic lupus erythematosus.     

U1 and U2 small nuclear RNAs are present in nuclear speckles.

The localization of U1 and U2 small nuclear RNAs (snRNAs) has been examined by in situ hybridization using 2'-O-alkyl oligonucleotide probes. We have found that these snRNAs, which are essential for pre-mRNA splicing, localize in a speckled distribution, in addition to being present in three of four foci, in HeLa cell nuclei. However, in cells of defined passage, such as Detroit 551 and WI-38 fibroblasts, these snRNAs are concentrated in nuclear speckles, and foci are not observed. The speckled distribution of U1 and U2 snRNAs is coincident with the speckled regions enriched in small nuclear ribonucleoprotein particle (snRNP) proteins and the essential non-snRNP splicing factor SC-35. The localization of these key components of the pre-mRNA splicing machinery to speckled nuclear regions suggests that these regions may be involved in pre-mRNA splicing.     

Detection and localization of small nuclear ribonucleoproteins (snRNPs) in trypanosomatids using anti-m3G antibodies

This work reports for the first time the identification and immunolocalization, by confocal and conventional indirect immunofluorescence, of m3G epitopes present in ribonucleoproteins of the following trypanosomatids: Trypanosoma cruzi epimastigotes of three different strains, Blastocrithidia ssp., and Leishmania major promastigotes. The identity of these epitopes and hence the specificity of the anti-m3G monoclonal antibody were ascertained through competition reaction with 7-methylguanosine that blocks the Ig binding sites, abolishing the fluorescence in all the parasites tested and showing a specific perinuclear localization of the snRNPs, which suggests their nuclear reimport in the parasites. Using an immunoprecipitation technique, it was also possible to confirm the presence of the trimethylguanosine epitopes in trypanosomatids.     

Association of U2, U4, U5, and U6 small nuclear ribonucleoproteins in a spliceosome-type complex in absence of precursor RNA.

Small nuclear ribonucleoprotein particles (snRNPs) associate to form multi-snRNP complexes during splicing of mRNA precursors. A vast majority of the three snRNPs U4, U5, and U6 are present in a nuclear extract in a single complex, while U1 and U2 snRNPs exist as separate particles. Under conditions optimal for splicing in vitro the U4-U5-U6 (U4/5/6) complex dissociates to release free snRNPs, suggesting that the interactions between its components are dynamic. Several forms of splicing complexes assemble on precursor RNA during splicing in vitro. One of these forms, spliceosome B, contains U2, U4, U5, and U6 snRNPs bound to the precursor RNA. This same set of snRNPs associates efficiently in the absence of precursor RNA during incubation of the extract at high salt concentration. Formation of this U2-U4-U5-U6 (U2/4/5/6) complex, the pseudospliceosome, suggests that the basic structure of the spliceosome is specified by snRNP-snRNP interactions.     

Human autoantibodies against the 70-kd polypeptide of U1 small nuclear RNP are associated with HLA-DR4 among connective tissue disease patients

Serum samples from patients with connective tissue disease (CTD) were characterized using a recently developed enzyme-linked immunosorbent assay for reactivity with individual specific polypeptides of U1 small nuclear ribonucleoproteins, including the U1-70-kd protein. The distribution of HLA antigens was compared in CTD patients with and in those without anti-U1-70-kd autoantibodies and in normal controls. The frequencies of HLA-DR4 and HLA-DRw53 were increased among the anti-U1-70-kd autoantibody positive CTD group compared with the frequencies in anti-U1-70-kd autoantibody negative systemic lupus erythematosus patients and compared with normal controls. We conclude that the presence of autoantibodies reactive with the anti-U1-70-kd protein antigen is associated with HLA-DR4 and HLA-DRw53.     

High-grade atrioventricular heart block in an adult with systemic lupus erythematosus: the association of nuclear RNP (U1 RNP) antibodies, a case report, and review of the literature

High-grade atrioventricular heart block (HGAVB) occurring as a manifestation of systemic lupus erythematosus is an exceedingly rare event. We describe a patient with HGAVB who had myositis and antibodies to nuclear RNP (previously associated with myocarditis). A review of the literature on HGAVB associated with systemic lupus erythematosus is also presented.     

Functioning of the Drosophila integral U1/U2 protein Snf independent of U1 and U2 small nuclear ribonucleoprotein particles is revealed by snf(+) gene dose effects

Snf, encoded by sans fille, is the Drosophila homolog of mammalian U1A and U2B" and is an integral component of U1 and U2 small nuclear ribonucleoprotein particles (snRNPs). Surprisingly, changes in the level of this housekeeping protein can specifically affect autoregulatory activity of the RNA-binding protein Sex-lethal (Sxl) in an action that we infer must be physically separate from Snf's functioning within snRNPs. Sxl is a master switch gene that controls its own pre-mRNA splicing as well as splicing for subordinate switch genes that regulate sex determination and dosage compensation. Exploiting an unusual new set of mutant Sxl alleles in an in vivo assay, we show that Snf is rate-limiting for Sxl autoregulation when Sxl levels are low. In such situations, increasing either maternal or zygotic snf(+) dose enhances the positive autoregulatory activity of Sxl for Sxl somatic pre-mRNA splicing without affecting Sxl activities toward its other RNA targets. In contrast, increasing the dose of genes encoding either the integral U1 snRNP protein U1-70k, or the integral U2 snRNP protein SF3a(60), has no effect. Increased snf(+) enhances Sxl autoregulation even when U1-70k and SF3a(60) are reduced by mutation to levels that, in the case of SF3a(60), demonstrably interfere with Sxl autoregulation. The observation that increased snf(+) does not suppress other phenotypes associated with mutations that reduce U1-70k or SF3a(60) is additional evidence that snf(+) dose effects are not caused by increased snRNP levels. Mammalian U1A protein, like Snf, has a snRNP-independent function.     

Clinical correlations with HLA type in Japanese patients with connective tissue disease and anti–U1 small nuclear RNP antibodies

Objective. To elucidate the roles of HLA genes in the clinical presentation of patients with connective tissue disease and serum anti–U1 small nuclear RNP antibody. Methods. HLA class I antigens and HLA class II alleles were determined in 43 Japanese patients with anti–U1 RNP antibody alone, by microcytotoxicity testing and DNA typing, respectively. Prospectively recorded clinical and laboratory features were analyzed in relation to HLA class I and class II types. Results. DQB1*0303 was associated with lupusrelated symptoms including fever, malar rash, oral ulcers, hypocomplementemia, and high-titer anti–double-stranded DNA antibody. Other HLA–clinical associations included DR2 with pleuritis, DR4 with hand swelling, and DRB1*0405 with arthritis. Conclusion. These HLA–clinical associations explain, in part, the heterogeneous clinical presentation in patients with anti–U1 RNP antibody.     

Electron microscopy of small nuclear ribonucleoprotein (snRNP) particles U2 and U5: evidence for a common structure-determining principle in the major U snRNP family.

We have studied by electron microscopy the structures of native small nuclear ribonucleoprotein (snRNP) particles U2 and U5 from HeLa cells. The structure of native U2 snRNP is characterized by a main body 8 nm in diameter with one additional domain about 4 nm long and 6 nm wide. Electron micrographs show that the 20S U5 snRNP, which contains at least seven U5-specific proteins in addition to the common proteins, has an elongated structure measuring 20-23 nm in length and 11-14 nm in width. Two main structural domains can be distinguished: a small head and a large elongated body about twice the size of the head. In addition to the head, the body of the 20S U5 snRNP possesses three short protuberances. The U2 and U5 core RNP particles--that is, of the snRNPs U2 and U5 without the snRNP-specific proteins, look much simpler and smaller under the electron microscope. They both are round in shape with a diameter of approximately 8 nm. With respect to their size, appearance, and fine structure, the U2 and U5 snRNP cores not only closely resemble each other but also share these properties with the core domain of U1 snRNP. We propose that the characteristic shape of each of the major snRNP species U1, U2, U4/U6, and U5 is determined by (i) a core domain containing the proteins that are common to all members of this family, which has the same shape for each member, and (ii) peripheral structures, which for snRNPs U1, U2, and U5 arise from the specific proteins, that give each of these snRNP species its characteristic shape.     

Protein binding sites are conserved in U1 small nuclear RNA from insects and mammals.

To gain insight into the ribonucleoprotein (RNP) structure of small nuclear RNAs, HeLa cell poly(A)+ mRNA was translated in a reticulocyte lysate, and the in vitro binding of 35S-labeled proteins to individual small nuclear RNA species was examined by using human autoimmune antibodies. A Mr 32,000 protein binds to U1 RNA but not to U2, U4, U5, or U6. The resulting U1 RNP complex is recognized both by Sm and RNP antibodies. U2 RNA also forms a complex with protein, which is recognized by Sm antibody. Thus, the lack of binding of the Mr 32,000 protein to U2 RNA is not due to a failure of U2 to bind specific proteins in the in vitro system. Similar translation-assembly experiments with Drosophila poly(A)+ mRNA reveal that a Mr 26,000 protein identified previously in Drosophila U1 RNP [Wieben, E. D. & Pederson, T. (1982) Mol. Cell. Biol. 2, 914-920] also binds to U1 RNA in vitro. When the translation products of HeLa or Drosophila mRNA are presented with U1 RNA of the other species, the Mrs 32,000 and 26,000 proteins recognize binding sites on the heterologous U1 and, in both cases, form complexes recognized by RNP antibody. These results establish that a Mr 32,000 protein is unique to U1 RNA in human cells and that the U1 RNA binding sites for this and a Mr 26,000 homologue have been highly conserved in evolution. These sites may be the identical 13 nucleotides at the 5' ends of human and Drosophila U1 RNA or a highly conserved aspect of U1 secondary structure.     

Elevated glucose 6-phosphate levels are associated with plasmid mutations in vivo.

The incubation in vitro of plasmid pBR322 DNA with glucose 6-phosphate (Glc-6-P) has been shown to have a mutagenic effect when the plasmid was transformed into wild-type Escherichia coli. To further investigate the modifications of DNA by the reducing sugar Glc-6-P, we have developed an in vivo model to monitor plasmid DNA mutations. E. coli strains that are defective for phosphoglucose isomerase (strain DF40) alone or phosphoglucose isomerase and glucose-6-phosphate dehydrogenase (strain DF2000) accumulate Glc-6-P when grown in gluconate minimal medium in the presence of glucose. These strains and the control strain K10 were transformed with pAM006, a plasmid that carries the genes for ampicillin resistance and beta-galactosidase production, and grown for 24 hr under conditions that prompted the accumulation of Glc-6-P. An increase in plasmid mutations was observed (7- and 13-fold) that was associated with the increased intracellular levels of Glc-6-P (20- and 30-fold) present in the DF40 and DF2000 E. coli strains, respectively. Growth of the mutant bacteria in gluconate minimal medium does not increase the intracellular levels of Glc-6-P or the rate of plasmid mutations over background. Further characterization of the mutated plasmid DNA showed that insertions, deletions, and point mutations were responsible for the loss of beta-galactosidase production. The increase in plasmid mutations as a function of increased intracellular Glc-6-P levels suggests that the accumulation of adducts formed by Glc-6-P and other reducing sugars may contribute to DNA damage.     

Insulin-like growth factor 2 (IGF2 ) and IGF-binding protein 1 (IGFBP1) gene variants are associated with overfeeding-induced metabolic changes

Aims/hypothesis. The aim of this study was to investigate the role of insulin-like growth factor 1 (IGF1), IGF2, IGF binding protein 1 (IGFBP1) and IGFBP3 gene variants on the metabolic changes observed in response to a 100-day overfeeding protocol conducted with 12 pairs of monozygotic twins. Methods: Genotyping was done by PCR-RFLP and DNA sequencer methods. Body fat measurements included hydrodensitometry and abdominal fat from computed tomography. Plasma glucose and insulin during fasting and in response to an OGTT were assayed. Plasma lipids were measured enzymatically. Results: In response to caloric surplus, fasting plasma insulin (p < 0.05) and OGTT insulin (p = 0.004) but not glucose area, increased more among the subjects with IGF2 Apa I GG (n = 12) than those with AA + AG (n = 12). The changes were independent of changes in total fatness. The subjects with IGFBP1 Bgl II AA (n = 8) showed greater increases in abdominal visceral fat (p < 0.01), OGTT insulin area (p = 0.05) and total cholesterol (p < 0.03) with overfeeding than the subjects with AG + GG (n = 16). IGFBP3 Nde I and the IGF1 (CT)_n markers were not associated with responsiveness to overfeeding. Conclusion/interpretation: Insulin sensitivity decreased in the subjects with IGF2 Apa I GG and the subjects with IGFBP1 Bgl II AA showed an accumulation of abdominal visceral fat and the early symptoms of the metabolic syndrome after long-term caloric surplus. Genetic variation at the IGF2 and IGFBP1 loci could be among the factors responsible for the inter-individual differences observed in the response to long-term alterations in energy balance and should be further investigated in larger cohorts. [Diabetologia (2001) 44: 2231–2236] Received: 26 March 2001 and in revised form: 31 August 2001     

Nucleotide sequence of a bean (Phaseolus vulgaris) U1 small nuclear RNA gene: implications for plant pre-mRNA splicing.

We have isolated a U1 small nuclear RNA (snRNA) gene from common bean (Phaseolus vulgaris). The haploid bean genome contains only one or a few copies of the U1 snRNA gene. The bean and human U1 snRNA genes are 65% homologous but share no significant similarity in the 5' or 3' flanking regions. The predicted secondary structure of bean U1 snRNA is identical to that of other U1 snRNAs, and the sequences of the single-stranded regions have been highly conserved. Thus, both the sequence of the single-stranded regions and the secondary structure of U1 snRNA appear to be important for its function. The role of U1 snRNA in pre-mRNA splicing is probably similar in plants and animals.     

Polymorphisms of peroxiredoxin 1, 2 and 6 are not associated with esophageal cancer

Purpose  

Peroxiredoxins, which reduced intracellular peroxides as a noval kind of antioxidant protein, were extensively expressed in various types of cancers and were thought as a biomarker of cancer cells. In this work, we performed genotyping analyses for tag SNP of Prdx 1, 2 and 6, and then evaluated the association with susceptibility and clinic stage of esophageal squamous cell carcinoma (ESCC) in a case–control study.     

Immunization of mice with purified U1 small nuclear ribonucleoprotein (RNP) induces a pattern of antibody specificities characteristic of the anti-Sm and anti-RNP autoimmune response of patients with lupus erythematosus, as measured by monoclonal antibodies.

Sera of patients with systemic lupus erythematosus and related autoimmune diseases often possess anti-Sm and anti-ribonucleoprotein (RNP) autoantibodies that recognize antigenic sites on small nuclear (sn) RNPs containing the snRNAs U1, U2, U4, U5, and U6. Although the major immunoreactive Sm polypeptides B', B, and D are present in snRNPs U1-U6, the RNP-antigenic proteins termed 70 kDa, A, and C are found only in U1 snRNP particles. We have immunized genetically nonautoimmune C57BL/6 mice with isolated human U1 snRNP particles and found that the major RNP and Sm antigenic polypeptides are also primarily immunogenic in the experimental immune response. Monoclonal antibodies (mAbs) derived from the mice thus immunized reacted specifically with polypeptides 70 kDa or A or recognized the Sm polypeptides B', B, and D simultaneously. One mAb reacted with an epitope shared by the U1 RNP polypeptide A and the U2 RNP polypeptide B". Competitive binding studies with the mAbs and anti-RNP/Sm patient sera indicated that the mAbs recognize the same regions on the antigenic snRNP polypeptides as the autoantibodies. This shows that B cells capable of producing autoantibodies against snRNPs are in principle present in the normal immune system. The striking similarity that we observed between the antibody pattern produced by the mice against exogenous U1 snRNP particles and that of systemic lupus erythematosus patients suggests the possibility that the anti-Sm and anti-RNP autoimmune response may be triggered by the endogenous U snRNPs rather than by a crossreacting viral or bacterial neoantigen. The possibility of eliciting anti-RNP and -Sm-type autoantibodies against exogenous snRNPs in normal mice should also allow potential pathogenic effects of such circulating antibodies to be investigated.     

The use of immunoblotting and immunoprecipitation of (U) small nuclear ribonucleoproteins in the analysis of sera of patients with mixed connective tissue disease and systemic lupus erythematosus. A cross-sectional, longitudinal study

Antibodies to ribonucleoproteins (RNP) and to the Sm antigen in sera from patients with mixed connective tissue disease (MCTD) and systemic lupus erythematosus were studied using the techniques of immunoblotting and immunoprecipitation of U small nuclear RNPs. A cross-sectional study indicated that antibodies reacting with a 68K protein were associated with anti-RNP specificity in MCTD, but rarely occurred in systemic lupus erythematosus patients' sera. A longitudinal study demonstrated the persistence of MCTD blotting patterns over many years, and the subsequent disappearance of those specificities in sera from patients who were in prolonged remission.     

Common variants in glutamine:fructose-6-phosphate amidotransferase 2 (GFPT2) gene are associated with type 2 diabetes, diabetic nephropathy, and increased GFPT2 mRNA levels

Increased flux of glucose through the hexosamine biosynthetic pathway has been implicated in insulin resistance, altered insulin secretion, and diabetic nephropathy. Glutamine:fructose-6-phosphate amidotransferase (GFPT), the rate limiting enzyme in hexosamine biosynthesis, is encoded by the unlinked but highly homologous genes GFPT1 and GFPT2. We tested the hypothesis that GFPT2 sequence variation contributed to the susceptibility to type 2 diabetes mellitus (T2DM) and diabetic nephropathy in Caucasian and African-American individuals. We identified 11 single nucleotide polymorphisms (SNPs), of which seven were common. A single variant in exon 14, I471V, altered the amino acid sequence, is conserved between human and mouse genes, and was associated with T2DM among Caucasians (P = 0.05). A trend to an association was noted with diabetic nephropathy among African-American individuals (P = 0.15). Several variants in the 3' untranslated region (UTR) and exon 18 were also associated with T2DM in Caucasian individuals (P < 0.05), and the SNP in the 3' UTR was associated with diabetic nephropathy in African-American subjects (P = 0.047). GFPT2 mRNA levels in transformed lymphocytes from study subjects were significantly increased among African-American subjects compared with Caucasian individuals, regardless of diagnosis. Furthermore, the associated allele of the 3' UTR SNP was approximately 2-fold overexpressed. We propose that the 3' UTR variant results in increased GFPT2 mRNA levels with resultant increased hexosamine flux. The I471V variant may contribute to altered protein function or may simply be in linkage disequilibrium with the 3' UTR.