Mercury-induced anti-nucleolar autoantibodies can transgress the membrane of living cells in vivo and in vitro.

Treatment with HgCl2 induces a systemic autoimmune disease in certain mice and rats. The major characteristic of this disease in mice with H-2s genotype is the production of anti-nucleolar autoantibodies (ANoIA). The exact mechanism(s) for the production and the functional role of mercury-induced ANoIA are not known. We have studied the ability of mercury-induced ANoIA to enter the living cells in vivo and in vitro. We found that in highly susceptible mice, treatment with mercury induced ANoIA capable of localizing in the nucleoli of kidney and liver cells in vivo. No detectable nucleoli localization of ANoIA were found in the cells of the heart, stomach, intestine and spleen. Consistent with the in vivo studies, mercury-induced ANoIA were also able to enter and translocate in the nucleoli of certain cells in vitro. The highest degree of antibody penetration was found in A-498 cells (a human kidney cell line) followed by 3T3 cells (a mouse fibroblast cell line), whereas the cells of lymphoid origin exhibited a very low degree of antibody penetration. Penetrated ANoIA could be recovered from the nucleoli of live 3T3 cells previously treated with ANoIA. The in vitro nucleolar translocation by ANoIA did not affect the DNA synthesis, but was found to be an active process dependent on time and temperature. Furthermore, pre-treatment of living cells with trypsin markedly inhibited both cell entry and nucleolar accumulation of ANoIA. Thus, mercury-induced ANoIA have a unique ability to transgress the membrane of certain living cells in vivo and in vitro, and to localize in the nucleoli.     

Post-mitotic assembly of the nucleolus. I. The internal matrix network is a recruitment site for processing nucleolar components in prenucleolar bodies

The aim of this work was to investigate whether the nuclear matrix could provide the nucleation sites for dispersed parental nucleolar components to form post-mitotic prenucleolar bodies (PNBs). For this purpose, nuclear matrices from asynchronous populations of onion cells were fractionated, and the distribution of the insoluble components of the nucleolar processing complexes in the matrices were analysed by fibrillarin immunolabelling. The ultrastructural organization of the nuclear matrix of cells from late telophase to late G₁, corresponding to the period of nucleolar reassembly and activation, was also analysed. Our results demonstrate that PNBs are structural components of the telophasic nuclear matrix and that this structure provides recruitment and assembly sites for the components of the nucleolar processing machinery, and suggests that the telophasic matrix network is involved in the early steps of post-mitotic nucleologenesis.accepted for publication by D. L. Spector     

Xenobiotic metal-induced autoimmunity: mercury and silver differentially induce antinucleolar autoantibody production in susceptible H-2s,H-2q and H-2f mice

Xenobiotic-metals such as mercury (Hg) and silver (Ag) induce an H-2 linked antinucleolar autoantibody (ANolA) production in susceptible mice. The mechanism for induction of ANolA synthesis is not well understood. However, it has been suggested that both metals interact with nucleolar proteins and reveal cryptic self-peptides to nontolerant autoreactive T cells, which in turn stimulate specific autoreactive B cells. In this study, we considered this suggestion and asked if mercury and silver display, if not identical, similar cryptic self-peptides, they would induce comparable ANolA responses in H-2 susceptible mice. We analysed the development of ANolA production in mercury- and/or silver-treated mice of H-2s, H-2q and H-2f genotypes. We found that while mercury stimulated ANolA synthesis in all strains tested, silver induced ANolA responses of lower magnitudes in only H-2s and H-2q mice, but not in H-2f mice. Resistance to silver in H-2f mice was independent of the dosage/time-period of silver-treatment and non-H-2 genes. Further studies showed that F1 hybrid crosses between silver-susceptible A.SW (H-2s) and -resistant A.CA (H-2f) mice were resistant to silver, but not mercury with regard to ANolA production. Additionally, the magnitudes of mercury-induced ANolA responses in the F1 hybrids were lower than those of their parental strains. The above differential ANolA responses to mercury and silver can be explained by various factors, including the different display of nucleolar cryptic peptides by these xenobiotics, determinant capture and coexistence of different MHC molecules. Our findings also suggest that the ability of a xenobiotic metal merely to create cryptic self-peptides may not be sufficient for the induction of an ANolA response.     

A novel mutation at the N-terminal of SMN Tudor domain inhibits its interaction with target proteins

Abstract Although most patients with spinal muscular atrophy (SMA) are homozygous for deletion of the SMN1 gene, some patients bear one SMN1 copy with a subtle mutation. Detection of such an intragenic mutation may be helpful not only in confirming diagnosis but also in elucidating functional domains of the SMN protein. In this study, we identified a novel mutation in SMN1 of two Japanese patients with type I SMA. DHPLC and sequencing analysis revealed that they harbored a point mutation in SMN1 exon 3, 275G > C, leading to tryptophan-to-serine substitution at amino acid 92 (W92S) at the Nterminal of SMN Tudor domain. In-vitro protein binding assays showed that the mutation severely reduced interaction of the domain with SmB protein and fibrillarin, suggesting that it impairs the critical function of SMN. In conclusion, we reported here that a novel mutation, W92S, in the Tudor domain affects the interaction of SMN with the target proteins.     

Increased fibrillarin expression is associated with tumor progression and an unfavorable prognosis in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the sixth most common cancer and third most common cause of cancer-associated mortality worldwide. Hepatectomy and liver transplantation are the main treatments for early HCC. Immunotherapy and targeted therapy for advanced HCC have become increasingly popular; however, their clinical benefits are limited. Thus, identification of novel therapeutic targets for advanced HCC remains essential. Fibrillarin (FBL) is an essential nucleolar protein that catalyzes the 2′-O-methylation of ribosomal RNAs. Recently, experimental data have suggested that FBL can influence breast-cancer progression. However, the association between FBL expression and HCC remains known. In the present study, the UALCAN database was used to assess FBL mRNA expression in HCC. Immunohistochemistry analysis was performed to detect FBL protein expression in 139 patients with HCC. In addition, bioinformatic analysis was performed using the UALCAN, the Database for Annotation, Visualization and Integrated Discovery, cBioportal and TargetScan databases. Data were analyzed using Kaplan-Meier curves and the log-rank test, and a Cox proportional hazards regression model. The results demonstrated that FBL expression was significantly higher in tumor tissues compared with para-tumor tissues. Furthermore, high FBL expression was significantly associated with tumor diameter and advanced TNM stage in HCC. High FBL expression also predicted a shorter overall survival time and disease-free survival time in patients with HCC. Bioinformatics analysis demonstrated that FBL may be regulated by methylation modification. In addition, analyses of functional annotations using the Gene Ontology database indicated that FBL-related genes were predominantly enriched in DNA repair and proliferation-related cell-signaling pathways. Notably, high FBL expression signified larger tumor diameter, advanced tumor stage and a poor prognosis. Taken together, the results of the present study suggest that FBL may be a potential target for HCC treatment.     

Selective induction of anti-fibrillarin autoantibodies by silver nitrate in mice.

Female SJL (H-2s) mice developed serum IgG anti-nucleolar antibodies (ANoA) after 5 weeks treatment with 0.05% or 0.01% silver nitrate (AgNO3) in drinking water. Five more weeks of treatment increased the ANoA titre to 3410 +/- 853 and 640 +/- 175 (reciprocal mean +/- s.e.m.), respectively. Controls receiving ordinary tap water and mice given 0.002% AgNO3 showed no antinucleolar antibodies. The high-titre ANoA targeted a 34-kD nucleolar protein identified as fibrillarin, the major autoantigen in murine mercury-induced autoimmunity and in a fraction of patients with systemic scleroderma. Serum autoantibodies to chromatin or histones, kidney, spleen, stomach, thyroid, or skin antigens (except the nucleolus) were not found in any of the mice. There was no consistent significant increase of serum IgG1, IgG2a, IgG2b, or IgG3 concentrations after AgNO3 treatment compared with controls. Mice treated with 0.05% AgNO3 for 10 weeks showed a slight decrease in serum IgG1, IgG2b and IgG3 concentrations. These mice also showed a small but statistically significant increase in renal, mesangial IgM deposits, which was not accompanied by any increase in C3c deposits, whereas mice given lower doses of silver nitrate showed no significant increase in mesangial immunoglobulin immune deposits. Systemic vessel wall immune deposits were not found in any of the mice. In mice given 0.05% silver nitrate, the kidney showed the highest concentration of silver (12.2 +/- 0.09 micrograms Ag/g wet weight; mean +/- s.e.m.), followed by the spleen (8.7 +/- 1.3), and the liver (3.9 +/- 0.4). Treatment with 0.01% silver nitrate caused a different distribution of silver, with the highest concentration in the spleen (2.1 +/- 0.16 micrograms Ag/g), followed by the kidney (0.63 +/- 0.037), and the liver (< 0.29 micrograms Ag/g; mean). Silver seems to be a more specific inducer of antinucleolar/anti-fibrillarin autoantibodies than mercury and gold, lacks the general immune stimulating potential of mercury, and has only a weak tendency to induce renal immune deposits. These observations suggest that the autoimmune sequelae induced in mice by metals is dependent, not only upon the genetic haplotype of the murine strain, but also on the metal under investigation.     

Fibrillarin and other snoRNP proteins are targets of autoantibodies in xenobiotic-induced autoimmunity

Exposure of SJL/J mice to mercury induces an anti-nucleolar autoantibody response. The predominant target is fibrillarin, a 34-kDa component of the small nucleolar ribonucleoprotein particles (snoRNP), but other proteins are also recognized. To characterize these proteins, monoclonal IgG anti-nucleolar antibodies were produced from HgC12-treated SJL/J mice. One monoclonal, 17C12, recognized fibrillarin, while two others, 7G3 and 6G10, were found to immunoprecipitate snoRNP particles but not fibrillarin. Antibody 6G10 gave a nucleolar immunofluorescence pattern in human, murine, and amphibian cells, but was negative in immunoblot. The 7G3 monoclone reacted with a 60-kDa protein conserved in human and murine, but not amphibian, cell lines. The 7G3 and 6G10 antigens and fibrillarin colocalized to the nucleolus and Cajal bodies in interphase cells and decorated metaphase chromosomes. These studies suggest that the mercury-induced anti-nucleolar antibody response targets other protein components of the snoRNP particles in addition to fibrillarin.