Intracellular distribution of Tankyrases as detected by multicolor immunofluorescence techniques.

Poly(ADP-ribose) polymerases are a family of enzymes that catalyze the conversion of NAD+ into ADP-ribose. Among them, Tankyrases have been found to bind to centrosome, mitotic spindle and microsome proteins, in the cytoplasm, and to telomeres in the nucleus, where they play a relevant role in telomere metabolism. However, their precise intracellular localization during interphase has not been so far fully elucidated. We investigated this aspect in situ by double immunofluorescence experiments using antibodies recognizing Tankyrases 1-2 or other proteins residing in specific organelles (Golgi apparatus, mitochondria, lysosomes, endoplasmic reticulum). We used HeLa cells as a model system in vitro, before and after treatment with either actinomycin D or etoposide, to also investigate the possible relocation of Tankyrases during apoptosis. We observed that Tankyrases are distributed both in the nucleus and in the cytoplasm; in this latter compartment, they were found to colocate with the Golgi apparatus but never with the mitochondria; a pool of Tankyrases also colocates with the endoplasmic reticulum and lysosomes. Interestingly, in cells with clear signs of apoptosis, Tankyrases were detectable in the cytoplasmic blebs: this suggests that they are not massively cleaved during apoptosis and persist in the largely heterogeneous apoptotic remnants which are known to contain components of cytoplasmic and nuclear origin.

Multicentric evaluation of a second generation assay to detect antiviral citrullinated peptide antibodies: a collaborative study by the Forum Interdisciplinare per la Ricerca nelle Malattie Autoimmuni.

AIMS: A novel immunoenzymatic assay using viral citrullinated peptides derived from Epstein-Barr virus-encoded proteins (viral citrullinated peptide 2 (VCP2)) has been developed and evaluated by means of a multicentre collaborative study. METHODS: Three hundred nine sera from patients with established rheumatoid arthritis (RA), 36 with early arthritis, 12 with juvenile arthritis and 453 controls were tested for VCP2 and cyclic citrullinated peptide (CCP) antibodies. RESULTS: The VCP2 assay showed 78.3% sensitivity and 97.1% specificity. VCP2 and CCP had a high concordance rate in patients with RA (88%) and controls (97%). However, 36 RA sera were positive in the CCP assay but negative on VCP2, and two RA sera reacted only on VCP2. CONCLUSIONS: The new VCP2 assay is endowed with high sensitivity and specificity. VCP2-positive RA sera are mostly but not completely contained in the CCP-positive population. Studies are in progress to establish whether the VCP2 assay can detect clinically distinct subsets of patients with RA.

Prevalence of undiagnosed autoimmune rheumatic diseases in the first trimester of pregnancy. Results of a two-steps strategy using a self-administered questionnaire and autoantibody testing.

OBJECTIVE: To evaluate the prevalence of undiagnosed rheumatic diseases in the first trimester of pregnancy. DESIGN: We screened for rheumatic diseases in 1210 consecutive pregnant women during the first trimester of pregnancy using a 10-item questionnaire. SETTING: A university hospital in northern Italy. POPULATION: One hundred and thirty-seven (11.3%) women who answered positively to at least one question constituted the cases and were compared with 107 negative controls. METHODS: Cases and controls were tested for rheumatic autoantibodies (antinuclear antibody, anti-double-stranded DNA, anti-extractable nuclear antigen, anticardiolipin antibody, anti-beta2-glycoprotein I antibodies and lupus anticoagulant) and were evaluated by a rheumatologist for a definite diagnosis of rheumatic disease. MAIN OUTCOME MEASURES: Prevalence of undiagnosed rheumatic disease in the first trimester of pregnancy. RESULTS: The overall rate of positivity to the antibodies tested was 43.1% (59/137) among cases and 9.3% (10/107) in the controls (P < 0.001). A definitive diagnosis of rheumatic disease was made in 35 cases (25.5%) and in none of the controls (P <0.001). In stepwise logistic regression analysis, photosensitivity (adjusted OR 5.72; 95% CI 2.38-13.8), erythema or malar rash (adjusted OR 3.91; 95% CI 1.53-10) and history of two or more miscarriages (adjusted OR 5.6; 95% CI 1.55-20.6) were independent predictors of a definitive diagnosis of rheumatic disease (area under receiving operator curve = 0.814; 95% CI 0.76-0.86). Birthweight was lower (3180 g +/- 475 compared with 3340 g +/- 452, P= 0.008), and overall serious pregnancy complications (miscarriage, fetal growth restriction, delivery before 34 weeks of pregnancy and severe pre-eclampsia) were higher among cases (12/137) than controls (2/107) (adjusted OR 5.60; 95% CI 1.29-24.3; P= 0.021). CONCLUSIONS: A two-step screening process with a self-administered questionnaire proved to be a useful method to screen for undiagnosed rheumatic diseases during the first trimester of pregnancy.

Enzyme-assisted photosensitization with rose Bengal acetate induces structural and functional alteration of mitochondria in HeLa cells.

Rose Bengal acetate (RB-Ac) can be used as a fluorogenic substrate for photosensitization of cells both in vivo and in vitro: once inside the cells, RB-Ac is converted into photoactive rose Bengal (RB) molecules which redistribute dynamically in the cytoplasm and, upon irradiation by visible green light, can damage organelles such as the endoplasmic reticulum, the Golgi apparatus, and the cytoskeleton. Recently, evidence has been provided that mitochondria may also be affected. The aims of the present study were to describe RB-induced photodamage of mitochondria in single HeLa cells and to define, on a quantitative basis, the effects of photosensitization on their morphofunctional features. HeLa cell cultures were exposed to 10(-5) M RB-Ac for 60 min and then irradiated with a light emitting diode at 530 nm (total light dose, 1.6 J/cm2). After irradiation, the cells were transferred to a drug-free complete medium and allowed to grow for 24-72 h. Using conventional and confocal fluorescence microscopy, transmission electron microscopy, and flow cytometry, we demonstrate that, in photosensitized cells, mitochondria undergo structural and functional alterations which can lead cells to apoptosis. Interestingly, in our system some cells were able to survive 72 h post-treatment and to recover, exhibiting the same mitochondrial structure, distribution and inner membrane potential as those in untreated controls. Taking into account that the photoactive molecules redistribute dynamically inside the cell upon RB-Ac administration, it may be hypothesized that cells can be differently affected by irradiation, depending on the relative amount and organelle location of the photosensitizer.

Nuclear localization of phosphorylated c-Myc protein in human tumor cells.

Using immunocytochemical techniques at light and electron microscopy, we analysed the distribution of phosphorylated c-Myc in actively proliferating human HeLa cells. The distribution pattern of c-Myc was also compared with those of other ribonucleoprotein (RNP)-containing components (PANA, hnRNP-core proteins, fibrillarin) or RNP-associated nuclear proteins (SC-35 splicing factor). Our results provide the first evidence that phosphorylated c-Myc accumulates in the nucleus of tumor cells, where it colocalizes with fibrillarin, both in the nucleolus and in extranucleolar structures.