Mitochondria and hypoxia-induced gene expression mediated by hypoxia-inducible factors.

The influence of mitochondrial activity on gene expression programs, particularly those involved in neuroprotection and repair, is likely to play an important role in the pathophysiology of neurodegenerative diseases. One such gene expression program is activated by the cellular pathway that senses a decrease in optimal oxygen levels and leads to activation of a family of transcriptional activators called hypoxia-inducible factors (HIFs). HIFs are members of the bHLH-PAS family of transcription factors and are heterodimers composed of HIF-alpha and HIF-beta (also known as aryl hydrocarbon receptor nuclear translocator) subunits that bind to canonical DNA sequences (hypoxia-regulated elements) in the promoters or enhancers of target genes. HIFs activate the expression of more than a hundred genes encoding proteins that regulate cell metabolism, survival, angiogenesis, vascular tone, hematopoiesis, and other functions. There is considerable evidence showing a bidirectional crosstalk between mitochondrial signals and HIF activity. For instance, mitochondrial reactive oxygen species and metabolic substrates from the tricarboxylic acid cycle are implicated in the regulation of the HIF pathway. Conversely, HIF activity leads to the expression of target genes that influence mitochondrial function. In this chapter we will review the complex interactions between mitochondria and the HIF pathway and we will discuss the relevance of this interaction for metabolic adaptation to hypoxia.

Intracellular antigens released from Balb/c mice kidneys under chemically induced apoptosis and/or necrosis.

The present investigation assesses the possible role of apoptosis and necrosis in intracellular antigen exposure of kidneys from Balb/c mice. Renal tissues were cultured and treated with chemicals to induce apoptosis and /or necrosis. The expression of intracellular antigens Sm, RNP, Ro and La were monitored with antibodies against these antigens. Main results confirm that renal intracellular antigens are released and exposed onto the surface of apoptotic and necrotic cells, therefore these antigens become an easy target of autoantibodies. This mechanism may be important in the lupus nephritis pathogenesis.

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.

Small-subunit rRNA processome proteins are translationally regulated during differentiation of Trypanosoma cruzi.

We used differential display to select genes differentially expressed during differentiation of epimastigotes into metacyclic trypomastigotes in the protozoan parasite Trypanosoma cruzi. One of the selected clones had a sequence similar to that of the small-subunit (SSU) processome protein Sof1p, which is involved in rRNA processing. The corresponding T. cruzi protein, TcSof1, displayed a nuclear localization and is downregulated during metacyclogenesis. Heterologous RNA interference assays showed that depletion of this protein impaired growth but did not affect progression through the cell cycle, suggesting that ribosome synthesis regulation and the cell cycle are uncoupled in this parasite. Quantitative PCR (qPCR) assays of several SSU processome-specific genes in T. cruzi also showed that most of them were regulated posttranscriptionally. This process involves the accumulation of mRNA in the polysome fraction of metacyclic trypomastigotes, where TcSof1 cannot be detected. Metacyclic trypomastigote polysomes were purified and separated by sucrose gradient sedimentation. Northern blot analysis of the sucrose gradient fractions showed the association of TcSof1 mRNA with polysomes, confirming the qPCR data. The results suggest that the mechanism of regulation involves the blocking of translation elongation and/or termination.

Clinical and prognostic value of autoantibodies in puerto Ricans with systemic lupus erythematosus.

The aim of this study was to determine the association between lupus autoantibodies and the clinical manifestations and outcome in a cohort of Puerto Ricans patients with systemic lupus erythematosus (SLE). All patients fulfilled the American College of Rheumatology classification criteria for SLE. Demographic parameters, clinical manifestations over time and damage accrual were obtained at the last study visit. Disease damage was assessed with the Systemic Lupus International Collaborating Clinics Damage Index (SDI). ANA, ANA pattern, and anti-dsDNA, anti-Smith, anti-Ro (SSA), anti-La (SSB) and anti-snRNP antibodies were measured at the time of SLE diagnosis. Chi-square test, Fisher exact test, ANOVA, logistic regression and general lineal model analyses were used to evaluate these associations. Ninety-six percent of patients were females. The cohort had a mean age of 40.2 +/- 12.0 years and mean disease duration of 9.6 +/- 7.0 years. Patients with elevated anti-dsDNA antibodies were more likely to have vasculitis, pericardial effusion, renal involvement, anaemia, leukopenia, lymphopenia and thrombocytopenia. Anti-Smith antibodies were positively associated with skin ulcerations, elevated liver enzymes, renal involvement and thrombocytopenia. Anti-Ro antibodies were related with the presence of discoid lupus, serositis, pneumonitis, elevated liver enzymes, hemolytic anaemia, leukopenia and lymphopenia. No positive associations were found for anti-snRNP or anti-La antibodies. The presence of anti-dsDNA, anti-Smith and anti-Ro antibodies was associated with higher SDI scores. In conclusion, anti-dsDNA, anti-Smith and anti-Ro antibodies are associated with several clinical manifestations and more damage accrual in Puerto Ricans with SLE. These findings provide valuable clinical and prognostic information for this ethnic population.

Longitudinal fluctuation of antibodies to extractable nuclear antigens in systemic lupus erythematosus.

OBJECTIVE: To examine the appearance, persistence, and disappearance of anti-extractable nuclear antigen (ENA, Sm, U1-RNP, Ro/SSA, and La/SSB) and anti-native DNA (dsDNA) antibodies during systemic lupus erythematosus (SLE) followup. METHODS: One hundred and thirty patients who fulfilled American College of Rheumatology classification criteria for SLE with at least 5 yearly anti-ENA and dsDNA tests between 1987-2002 were retrospectively selected. Four longitudinal antibody data patterns were considered for each antibody: always absent, always present, absent at diagnosis with positive seroconversion, and present at diagnosis with negative seroconversion. RESULTS: Antibodies to Ro/SSA were present in 47%, U1-RNP in 36%, DNA in 32%, Sm in 23%, and La/SSB in 7% of patients. Among patients ever positive for a given autoantibody, the frequency of the "always present" pattern was 52% for anti-Ro/SSA, 38% for U1-RNP, 17% for Sm, 11% for La/SSB, and 9% for DNA antibodies; the frequency of positive seroconversion was 56% for anti-La/SSB, 33% for DNA, 26% for Sm, 21% for U1-RNP, and 15% for Ro/SSA. Time to positive seroconversion varied from 1 to 8 years after diagnosis. Among patients with a positive test at diagnosis the frequency of those remaining positive between the 2nd and 4th year of followup decreased to 39-78%, depending upon autoantibody specificity; between the 5th and 10th years this rate was 20-75%. Antibody data pattern frequency differed significantly among autoantibody specificities except between anti-U1-RNP and Ro/SSA (p = 0.15) and between anti-DNA and Sm antibodies (p = 0.29). CONCLUSION: The high frequency of longitudinal fluctuation in anti-ENA antibodies suggests that a periodic reappraisal may be appropriate in seronegative patients with a suspect diagnosis of SLE. The clinical significance of such fluctuation deserves future study.

Characterization of Saccharomyces cerevisiae Nop17p, a novel Nop58p-interacting protein that is involved in Pre-rRNA processing.

In eukaryotes, pre-rRNA processing depends on cis-acting elements and on a large number of non-ribosomal trans-acting factors, including endonucleases and exonucleases, RNA helicases, rRNA modifying enzymes and components of snoRNPs. The exosome is a conserved eukaryotic protein complex containing multiple 3'-5' exonucleases, which has been implicated in pre-rRNA, snoRNA and snRNA processing, as well as in mRNA degradation. In order to identify new proteins involved in rRNA processing, we have screened a yeast two-hybrid cDNA library, to isolate proteins interacting with the exosome subunit Rrp43p. In this screen, a novel nucleolar protein, Nop17p, was identified which also interacts with the box C/D snoRNP protein Nop58p. The NOP17 gene is not essential for cell viability but its deletion causes a temperature-sensitive phenotype. Pre-rRNA processing analyses revealed that rRNA formation is affected in the Deltanop17 strain subjected to the non-permissive temperature, although it is not blocked completely. In addition, primer extension analyses of RNA isolated from Nop17p-depleted cells subjected to the non-permissive temperature indicates that the pre-rRNA is undergoing different modification or degradation processes in these cells as compared to the parental strain. Nop17p was recently described in the same complex as Nop58p and, interestingly, its depletion leads to mislocalization of Nop1p, Nop56p, Nop58p and Snu13p, which are the core proteins of the box C/D ribonucleoprotein (snoRNP), indicating that Nop17p function is required either for nucleolar retention or for the proper assembly of the box C/D snoRNP.

Presence of autoantibodies against HeLa small nuclear ribonucleoproteins in chagasic and non-chagasic cardiac patients

We detected anti-human small nuclear ribonucleoprotein (snRNP) autoantibodies in chagasic patients by different immunological methods using HeLa snRNPs. ELISA with Trypanosoma cruzi total lysate antigen or HeLa human U small nuclear ribonucleoproteins (UsnRNPs) followed by incubation with sera from chronic chagasic and non-chagasic cardiac patients was used to screen and compare serum reactivity. Western blot analysis using a T. cruzi total cell extract was also performed in order to select some sera for Western blot and immunoprecipitation assays with HeLa nuclear extract. ELISA showed that 73 and 95 percent of chronic chagasic sera reacted with HeLa UsnRNPs and T. cruzi antigens, respectively. The Western blot assay demonstrated that non-chagasic cardiac sera reacted with high molecular weight proteins present in T. cruzi total extract, probably explaining the 31 percent reactivity found by ELISA. However, these sera reacted weakly with HeLa UsnRNPs, in contrast to the chagasic sera, which showed autoantibodies with human Sm (from Stefanie Smith, the first patient in whom this activity was identified) proteins (B/B', D1, D2, D3, E, F, and G UsnRNP). Immunoprecipitation reactions using HeLa nuclear extracts confirmed the reactivity of chagasic sera and human UsnRNA/RNPs, while the other sera reacted weakly only with U1snRNP. These findings agree with previously reported data, thus supporting the idea of the presence of autoimmune antibodies in chagasic patients. Interestingly, non-chagasic cardiac sera also showed reactivity with T. cruzi antigen and HeLa UsnRNPs, which suggests that individuals with heart disease of unknown etiology may develop autoimmune antibodies at any time. The detection of UsnRNP autoantibodies in chagasic patients might contribute to our understanding of how they develop upon initial T. cruzi infection.

Presence of autoantibodies against HeLa small nuclear ribonucleoproteins in chagasic and non-chagasic cardiac patients.

We detected anti-human small nuclear ribonucleoprotein (snRNP) autoantibodies in chagasic patients by different immunological methods using HeLa snRNPs. ELISA with Trypanosoma cruzi total lysate antigen or HeLa human U small nuclear ribonucleoproteins (UsnRNPs) followed by incubation with sera from chronic chagasic and non-chagasic cardiac patients was used to screen and compare serum reactivity. Western blot analysis using a T. cruzi total cell extract was also performed in order to select some sera for Western blot and immunoprecipitation assays with HeLa nuclear extract. ELISA showed that 73 and 95% of chronic chagasic sera reacted with HeLa UsnRNPs and T. cruzi antigens, respectively. The Western blot assay demonstrated that non-chagasic cardiac sera reacted with high molecular weight proteins present in T. cruzi total extract, probably explaining the 31% reactivity found by ELISA. However, these sera reacted weakly with HeLa UsnRNPs, in contrast to the chagasic sera, which showed autoantibodies with human Sm (from Stefanie Smith, the first patient in whom this activity was identified) proteins (B/B', D1, D2, D3, E, F, and G UsnRNP). Immunoprecipitation reactions using HeLa nuclear extracts confirmed the reactivity of chagasic sera and human UsnRNA/RNPs, while the other sera reacted weakly only with U1snRNP. These findings agree with previously reported data, thus supporting the idea of the presence of autoimmune antibodies in chagasic patients. Interestingly, non-chagasic cardiac sera also showed reactivity with T. cruzi antigen and HeLa UsnRNPs, which suggests that individuals with heart disease of unknown etiology may develop autoimmune antibodies at any time. The detection of UsnRNP autoantibodies in chagasic patients might contribute to our understanding of how they develop upon initial T. cruzi infection.

Trypanosoma cruzi: identification and characterization of a novel ribosomal protein L27 (TcrL27) that cross-reacts with an affinity-purified anti-Sm antibody.

Small nuclear ribonucleoproteins (snRNPs) are involved in trans-splicing processing of pre-mRNA in Trypanosoma cruzi. To clone T. cruzi snRNPs we screened an epimastigote cDNA library with a purified antibody raised against the Sm-binding site of a yeast sequence. A clone was obtained containing a 507 bp-insert with an ORF of 399 bp and coding for a protein of 133 amino acids. Sequence analysis revealed high identity with the L27 ribosomal proteins from different species including: Canis familiaris, Homo sapiens, Schizosaccharomyces pombe and Saccharomyces cerevisiae. This protein has not been previously described in the literature and seems to be a new ribosomal protein in T. cruzi and was given the code TcrL27. To express this recombinant T. cruzi L27 ribosomal protein in E. coli, the insert was subcloned into the pET32a vector and a 26 kDa recombinant protein was purified. Immunoblotting studies demonstrated that this purified recombinant protein was recognized by the same anti-Sm serum used in the library screening as well as by chagasic and systemic lupus erythemathosus (SLE) sera. Our results suggest that the T. cruzi L27 ribosomal protein may be involved in autoimmunity of Chagas disease.

Prevalence of 13 autoantibodies and of the 16/6 and related pathogenic idiotypes in 465 patients with systemic lupus erythematosus and their relationship with disease activity.

With a cross sectional study of 465 consecutive systemic lupus erythematosus (SLE) patients tested for 13 autoantibodies (Aab) and two idiotypes we determined the prevalence of Aab according to disease activity, both general and at particular organ systems. Seventy seven percent of SLE sera had at least one Aab and 56% had it at high titres. Pathogenic idiotypes had a prevalence of less than 10% and 166 sera had Aab to 5 or more antigens and 9 sera had Aab against all 13 antigens tested. Patients with active disease had increased prevalence of Aab to DNP, ssDNA, ENA, mitochondria and histones when considered at 5 s.d. above the mean of normal controls. The higher positivity of Aab in patients with active disease was confirmed in logistic regression analysis adjusted by age, disease duration, and intensity of treatment. A trend was observed of increased prevalence and titres of Aab from inactive disease without treatment, to inactive disease but still being treated, to active disease. Only 22% of patients with active disease had no Aab and the higher the number of Aab the higher the frequency of active disease. Patients with active arthritis, and to a lesser degree those with active mucocutaneous involvement, had higher prevalence and titres of most autoantibodies than patients with disease activity at other organ systems. Active renal disease associated only with anti-dsDNA, whereas active CNS disease associated with anti-mitochondrial Aab. Our findings support the vision of SLE as an immune dysregulation leading to polyclonal B cell activation with resulting production of multiple Aab. Their profiles seem influenced by genetical, hormonal and environmental factors and, in turn, they contribute to the clinical picture in each patient. Disease activity influences the presence of some, but not all, Aab and some of them may remain present in some patients, even in remission.

Utility of anti-Sm, anti-RNP, anti-Ro/SS-A, and anti-La/SS-B (extractable nuclear antigens) detected by enzyme-linked immunosorbent assay for the diagnosis of systemic lupus erythematosus.

OBJECTIVE: To determine the utility of anti-extractable nuclear antigen (anti-ENA) antibodies detected by enzyme-linked immunosorbent assay as a predictor for the diagnosis of systemic lupus erythematosus (SLE). METHODS: Among 2,185 serum samples sent for testing for antinuclear antibodies (ANA) by indirect immunofluorescence, 259 consecutive patients with positive ANA were identified. Medical charts of these patients were reviewed to assess the clinical diagnosis, with the reviewer having no knowledge of the anti-ENA result. Clinical data were abstracted for all patients, and diagnoses established using American College of Rheumatology criteria. The utility of ENA antibodies in the diagnosis of SLE was determined by univariate and multivariate analysis among all patients who were positive for ANA, patients who were positive for ANA and for anti-double-stranded DNA (anti-dsDNA), and patients who were positive for ANA and negative for anti-dsDNA. Clinical differences between SLE patients with and those without anti-ENA antibodies were assessed. RESULTS: Anti-ENA antibodies, especially anti-Ro/SS-A, showed strong predictive diagnostic value among ANA+/anti-dsDNA- patients, but were of no utility among ANA+/anti-dsDNA+ patients. The only clinical manifestations that were more common among anti-ENA+ SLE patients were pleuritis and the use of hydroxychloroquine. CONCLUSION: The presence of anti-ENA antibodies, especially anti-Ro/SS-A, is a useful predictor for the diagnosis of SLE, primarily among patients attending a referral rheumatology center who are positive for ANA and negative for anti-dsDNA. No major clinical differences were noted among ANA+ SLE patients with versus those without ENA.

Nuclear Sm antigens in the sperm of different organisms.

Immunoblot analysis of sperm protein from several species revealed the presence of polypeptides recognised by anti-Sm sera obtained from patients with systemic lupus erythematosus. Immunoreactive polypeptides in human, bull, mouse and rat sperm were identified as protein B', B and D as compared with the Sm polypeptides of HeLa cells. In the sperm of rooster, the teleost fish Cyprinus carpio and the mussel Choromytilus chorus, the immunoreactive polypeptide profile was more complex. To ascertain the sperm origin of the Sm antigens, immunolocalisation with anti-Sm serum was carried out. The results demonstrated that in all the species studied staining was confined to the sperm nucleus, confirming that some polypeptides of the small nuclear ribonucleoprotein complex are present in the gamete.