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Results for “Exosome Multienzyme Ribonuclease Complex”

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PubMed2004

Patients with antibodies to both PmScl and dsDNA.

OBJECTIVE: To determine the significance of dsDNA antibodies in patients with antibodies to PmScl. METHODS: All patients testing positive for PmScl and/or dsDNA antibodies at an academic medical center between 1977 and 2002 were identified. Charts for the PmScl-positive patients were reviewed for manifestations of lupus, scleroderma, or polymyositis/dermatomyositis. Patients with antibodies to dsDNA were matched to each of the double-positive PmScl+/dsDNA+ patients on the basis of sex, race, age, and date of autoantibody testing. Standard classification criteria for lupus, scleroderma, and myositis were used (excluding dsDNA, PmScl, or antinuclear antibodies as criteria), and the number of subjects meeting classification criteria was recorded. RESULTS: Records were available for 38 out of 47 patients who were identified as PmScl-positive. The prevalence of dsDNA antibodies in this group was 42% (16/38). Patients with PmScl and dsDNA antibodies had a higher prevalence of systemic lupus erythematosus (8/16 vs 2/22; p = 0.008) and a lower rate of scleroderma or myositis (1/16 vs 9/22; p = 0.025) than dsDNA-negative patients with PmScl antibodies. The prevalence of systemic lupus erythematosus, myositis, and scleroderma in patients with PmScl and dsDNA antibodies was not different from the prevalences of these diseases in a matched cohort of patients who were dsDNA-positive. CONCLUSION: Antibodies to PmScl are associated with scleroderma and myositis when dsDNA antibodies are not present. In the presence of dsDNA antibodies, PmScl antibodies do not appear to have clinical relevance.

Adult↗
PubMed2005

Construction and expression of dendritic cell nucleic acid vaccine containing CML28 gene in human dendritic cells.

This study was aimed to construct nucleic acid vaccine containing the coding region of the CML28 gene and to express it in human dendritic cells. The full length of CML28 cDNA was amplified from K562 by RT-PCR and subcloned into pGEM-T vector. The CML28 fragment was digested and subsequently inserted into the EcoRI-Xba I sites of pcDNA3.1HisA to construct the recombinant expression vector pcDNA3.1HisA-CML28, which was identified by restrition analysis and sequencing. Human dendritic cells (DC) were separated from peripheral blood mononuclear cells (PBMC) by culture with rhGM-CSF, rhIL-4 and assessed by flow cytometry. The constructed plasmid pcDNA3.1 HisA-CML28 was transfected into DC by electroporation. Western blot was used to detect the expression of fusion protein His-CML28. The results showed that recombinant plasmid pcDNA3.1HisA-CML28 contained the correct full CML28 cDNA identified by restriction analysis and sequencing, and can express the fusion protein His-CML28 in DCs. It is concluded that nucleic acid vaccine containing CML28 gene was constructed and expressed in DC successfully.

Antigens, Neoplasm↗
PubMed2005

[Monitoring CML28 mRNA levels in patients before and after HSCT by real-time quantitative RT-PCR].

The purpose of this study was to establish a SYBR Green I real-time quantitative RT-PCR method for investigating the correlation between CML28 mRNA expression levels and relapse of leukemia after allo-hematopoietic stem cell transplantation (HSCT). pcDNA3.1HisA-CML28 plasmid had been constructed as the standard template. Serial monitoring of CML28 mRNA levels by SYBR Green I real-time quantitative RT-PCR technique was performed in 14 patients, including 10 patients with CML and 3 patients with AML, 1 patient with Ph(+) ALL. The results showed that the sensitivity of the established method was at 10(-4) (0.05 ng) level, with interassay variation and intraassay variation of standard samples both below 10%. The CML28 was highly expressed in AML and CML-BP or AP. CML28 level in newly diagnosed group was (6.58 +/- 2.34) x 10(-2), in pre-conditioning regimen group was (2.19 +/- 0.32) x 10(-2), in group that 1 month after HSCT was (1.35 +/- 1.28) x 10(-2), in group that 3 months after HSCT was (4.57 +/- 6.39) x 10(-3). CML28 can be detected 3 months after HSCT in 1 patient with CML-CP and 3 patients with CML-AP or BC. 2 of them with low level (<2 x 10(-2)) survived without relapse, the other 2 case with high level (>2 x 10(-2)) relapsed within one year, 1 case died and 1 case received the second time HSCT, CML28 level decreased rapidly after HSCT, but still higher than 2 x 10(-2) and relapse has taken place. The conclusions was made that CML28 mRNA level is obviously correlated with the development of leukemia. Serial quantification of CML28 mRNA levels are necessary for HSCT recipients, and more informative than a single detection. Using of this assay to evaluate MRD in the patients received HSCT is helpful for prediction of relapse.

Adolescent↗
PubMed2006

Childhood onset systemic sclerosis: classification, clinical and serologic features, and survival in comparison with adult onset disease.

OBJECTIVE: To describe the differences between patients with systemic sclerosis (SSc) having childhood versus adult onset evaluated at a single medical center. METHODS: Patients were divided into those with childhood onset (first SSc symptom or finding before age 16 yrs) and those with early adult and late adult onset. The 3 groups were compared with respect to disease classification, clinical, laboratory and serologic data, and survival. RESULTS: One hundred eleven childhood onset SSc cases seen between 1960 and 2003 were compared with 2559 adult onset SSc cases (1087 with onset age 16-40 and 1472 with onset after age 40 yrs) first evaluated between 1972 and 2001. Age distribution at onset was unimodal, suggesting that childhood disease is part of the spectrum of adult onset SSc. A significantly greater proportion of childhood onset patients had overlap syndromes, most frequently with polymyositis-dermatomyositis (PM-DM), and skeletal muscle involvement. Children with diffuse cutaneous (dc) SSc had significantly lower maximum mean total skin thickness scores than adult patients with dcSSc. Renal involvement was uncommon in childhood onset cases, and the frequency increased with age of onset. Serum anti-PM-Scl and anti-U1RNP antibodies were detected significantly more frequently in childhood than in adult onset cases. In contrast, anti-RNA polymerase III and anticentromere antibodies were found significantly more frequently in adults. Survival was significantly better among childhood than all adult onset cases combined, but similar to survival in young adult onset SSc cases. Scleroderma heart disease was a frequent cause of death among children with SSc. CONCLUSION: Patients with juvenile onset SSc more frequently have an overlap syndrome with PM-DM, higher frequency of skeletal muscle involvement, serum anti-PM-Scl and anti-U1RNP antibody, fatal cardiac disease, and improved survival compared with adult onset SSc cases.

Adult↗
PubMed1996

Dis3, implicated in mitotic control, binds directly to Ran and enhances the GEF activity of RCC1.

Using the two-hybrid method, we isolated a Saccharomyces cerevisiae cDNA encoding a protein homologous to Schizosaccharomyces pombe protein Dis3sp, using as bait, human GTPase Ran. The DIS3 gene is essential for viability and complements S.pombe mutant dis3-54 which is defective in mitosis. Although Dis3sc has no homology to RanBP1, it bound directly to Ran and the S.cerevisiae Ran homologue Cnr1, but not to the S.cerevisiae RCC1 homologue Srm1. Upon binding to Ran with a 1:1 molar ratio, Dis3sc enhanced a nucleotide-releasing activity of RCC1 on Ran. In the presence of Dis3sc, the K(m) of RCC1 on Ran decreased by half, while the kcat was unchanged. In vivo, Dis3sp was present as oligomers of M(r) 670-200 kDa as previously reported, and the 200 kDa oligomer of Dis3sp was found to include Spi1 and Pim1, the S.pombe homologues of Ran and RCC1, respectively. Although the biological function of the heterotrimeric oligomer consisting of Dis3, Spi1 and Pim1 is unknown, our results indicate that Dis3 is a component of the RCC1-Ran pathway.

Amino Acid Sequence↗
PubMed1997

Isolation of murine and human homologues of the fission-yeast dis3+ gene encoding a mitotic-control protein and its overexpression in cancer cells with progressive phenotype.

To investigate genes involved in metastasis, we used a differential display method to compare the levels of gene expression in three cell lines derived from murine colon-adenocarcinoma 26 that show different metastatic potentials. The results, and subsequent Northern analyses, confirmed that one gene was expressed most strongly in NL17, the cell line with the highest experimentally metastatic potential to the lung; strongly in NL22, the line with moderately metastatic potential; and very weakly in NL4, which has no metastatic potential in recipient mice. Using this fragment as a probe, we isolated the murine cDNA as well as its human homologue and determined their DNA sequences. The cDNA sequences from both species contained open reading frames of 2874 nucleotides, encoding peptides of 958 amino acids with calculated molecular weights of approximately 109,000; the murine and human nucleotide sequences were 90% identical. The deduced amino acid sequences of these cDNAs revealed significant homology (45% identity) to the dis3+ gene product of Schizosaccharomyces pombe, a protein thought to be essential for mitotic control in the yeast. We therefore termed the murine and human genes hmc (homologue to the mitotic-control gene) and HMC, respectively. In 7 of 13 patients with colorectal cancers and liver metastases, expression of HMC was increased up to 38-fold in primary tumors and metastatic foci as compared to adjacent normal colorectal mucosa. An increase in expression of HMC, its novel product likely to belong to a structurally distinct family of mitotic-control proteins, may be associated with malignant phenotypes of some colorectal cancers.

Adenocarcinoma↗
PubMed2005

Structural basis of 3' end RNA recognition and exoribonucleolytic cleavage by an exosome RNase PH core.

The exosome is a macromolecular complex that plays fundamental roles in the biogenesis and turnover of a large number of RNA species. Here we report the crystal structures of the Rrp41-Rrp42 core complex of the S. solfataricus exosome bound to short single-stranded RNAs and to ADP. The RNA binding cleft recognizes four nucleotides in a sequence-unspecific manner, mainly by electrostatic interactions with the phosphate groups. Interactions at the 2' hydroxyls of the sugars provide specificity for RNA over DNA. The structures show both the bound substrate and the cleaved product of the reaction, suggesting a catalytic mechanism for the 3'-5' phosphorolytic activity of the exosome.

Archaeal Proteins↗
PubMed2005

Structural framework for the mechanism of archaeal exosomes in RNA processing.

Exosomes emerge as central 3'-->5' RNA processing and degradation machineries in eukaryotes and archaea. We determined crystal structures of two 230 kDa nine subunit archaeal exosome isoforms. Both exosome isoforms contain a hexameric ring of RNase phosphorolytic (PH) domain subunits with a central chamber. Tungstate soaks identified three phosphorolytic active sites in this processing chamber. A trimer of Csl4 or Rrp4 subunits forms a multidomain macromolecular interaction surface on the RNase-PH domain ring with central S1 domains and peripheral KH and zinc-ribbon domains. Structural and mutational analyses suggest that the S1 domains and a subsequent neck in the RNase-PH domain ring form an RNA entry pore to the processing chamber that only allows access of unstructured RNA. This structural framework can mechanistically unify observed features of exosomes, including processive degradation of unstructured RNA, the requirement for regulatory factors to degrade structured RNA, and left-over tails in rRNA trimming.

Archaeal Proteins↗
PubMed2001

Prediction of the archaeal exosome and its connections with the proteasome and the translation and transcription machineries by a comparative-genomic approach.

By comparing the gene order in the completely sequenced archaeal genomes complemented by sequence profile analysis, we predict the existence and protein composition of the archaeal counterpart of the eukaryotic exosome, a complex of RNAses, RNA-binding proteins, and helicases that mediates processing and 3'->5' degradation of a variety of RNA species. The majority of the predicted archaeal exosome subunits are encoded in what appears to be a previously undetected superoperon. In Methanobacterium thermoautotrophicum, this predicted superoperon consists of 15 genes; in the Crenarchaea, Sulfolobus solfataricus and Aeropyrum pernix, one and two of the genes from the superoperon, respectively, are relocated in the genome, whereas in other Euryarchaeota, the superoperon is split into a variable number of predicted operons and solitary genes. Methanococcus jannaschii partially retains the superoperon, but lacks the three core exosome subunits, and in Halobacterium sp., the superoperon is divided into two predicted operons, with the same three exosome subunits missing. This suggests concerted gene loss and an alteration of the structure and function of the predicted exosome in the Methanococcus and Halobacterium lineages. Additional potential components of the exosome are encoded by partially conserved predicted small operons. Along with the orthologs of eukaryotic exosome subunits, namely an RNase PH and two RNA-binding proteins, the predicted archaeal exosomal superoperon also encodes orthologs of two protein subunits of RNase P. This suggests a functional and possibly a physical interaction between RNase P and the postulated archaeal exosome, a connection that has not been reported in eukaryotes. In a pattern of apparent gene loss complementary to that seen in Methanococcus and Halobacterium, Thermoplasma acidophilum lacks the RNase P subunits. Unexpectedly, the identified exosomal superoperon, in addition to the predicted exosome components, encodes the catalytic subunits of the archaeal proteasome, two ribosomal proteins and a DNA-directed RNA polymerase subunit. These observations suggest that in archaea, a tight functional coupling exists between translation, RNA processing and degradation, (apparently mediated by the predicted exosome) and protein degradation (mediated by the proteasome), and may have implications for cross-talk between these processes in eukaryotes.

Adenosine Triphosphatases↗