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S Santi

Publications and source records attributed to S Santi.

At least 19 recordsLinked to original sources

Nuclear changes in a case of X-linked Emery-Dreifuss muscular dystrophy.

Ultrastructural alterations in the nuclear architecture were found in skeletal muscle and skin cultured cells from a patient affected by X-linked Emery-Dreifuss muscular dystrophy (EMD) carrying a null mutation. The molecular defect of X-linked EMD is the absence of emerin, a nuclear envelope-associated protein which is considered a component of the nuclear lamina. The nuclear changes were present in skeletal muscle and skin cultured cells with a frequency of about 10% and 18%, respectively. The main structures of the nuclear periphery were involved: lamina and nuclear envelope-associated heterochromatin were affected, whereas the cisterna and the pore complexes appeared preserved, and the cytoplasm of the same cells appeared normal. Analogous localized defects were detectable by immunolabeling with antilamin A/C and B2 antibodies, as well as by selective propidium iodide chromatin staining. The lesions we describe could be the result of anomalous nuclear lamina organization in the absence of emerin.

Cell Nucleus

The novel structural protein of human cytomegalovirus, pUL25, is localized in the viral tegument.

Human cytomegalovirus UL25 codes for a structural phosphoprotein of 85 kDa (C. J. Baldick and T. Shenk, J. Virol. 70:6097-6105, 1996; M. C. Battista et al., J. Virol. 73:3800-3809, 1999). In this study we analyzed the intracellular and intraviral localization of pUL25 by confocal and immunoelectron microscopy and found that pUL25 is a component of the viral tegument and the dense body matrix, acquired during the late cytoplasmic phase of virus maturation.

Cell Line

The concept of 'glomerulonephritis'. the fascinating history of evolution and emergence of a specialist's nosology focus on Italy and Torino.

Though the term 'nephritis' first appeared in the 19th century, this word did not bear the same meaning as it does today; indeed, for many years it was used to indicate 'renal diseases' (in the sense of Bright's disease) in a larger sense. This review summarizes the long gestation of the concept of 'glomerulonephritis' from the prehistory of medicine up to the beginning of the second half of the 20th century with emphasis on Italy and, in particular, on Torino, which was the capital of the Kingdom of Italy from 1861 to 1865. To the best of our kowledge, this is the first study reporting an epidemiology survey of Bright's disease in Italy from 1880 up to 1960. Towards the end of the 19th century, Bright's disease accounted for 26 deaths/year/10(5) population (in comparison with more than 200 from tuberculosis) in Italy, roughly paralleling that reported in the USA. At the beginning of the 20th century, Bright's disease was the seventh cause of death (almost 1% of total deaths) in Italy. Furthermore, in Italy, as elsewhere, autopsy studies showed a higher percentage of deaths attributed to Bright's disease (5-7%) in comparison with those obtained from vital statistics. In 1960, just before the beginning of renal replacement therapy, Bright's disease accounted for 15.7 deaths/year/10(5) population (= 1.46% of all deaths), roughly paralleling that reported in the United Kingdom (13.8/10(5) population = 1.25% of deaths). Probably, it was difficult to recognize the real incidence of chronic renal diseases leading to death in the 1960s, and vital statistics were able to furnish only approximate estimates. However, noteworthy is the fact that these values were very close to those estimated as being the annual need for renal replacement therapy (10-20 cases/year/10(5) population).

Glomerulonephritis

Correlation between cytomegalovirus infection and Raynaud's phenomenon in lupus nephritis.

Relationships between viruses and autoimmune diseases such as systemic lupus erythematosus (SLE) are still elusive. Recent reports demonstrated the association of some viral infections with peculiar clinical events in the general population, such as cytomegalovirus (CMV) with arterial damage and Parvovirus B19 (PV-B19) with hematologic abnormalities. We planned to look for this kind of viral imprinting in SLE, hypothesizing that traces of specific features of some viral infections might be found in some subsets of seropositive SLE patients. In 60 SLE patients recruited at our nephrologic center, serology for CMV, PV-B19, Epstein-Barr virus viral capsid antigen (EBV-VCA), Epstein-Barr nuclear antigen (EBNA) and Epstein-Barr virus early antigen (EBV-EA) was performed. chi2 and ANOVA were employed to compare the frequency and titers of antiviral antibodies in SLE patients with groups of transplant, hemodialysis and blood donor subjects. chi2, Fisher's test, Bonferroni and Scheffe's test were employed to compare the different biochemical/clinical features between seropositive and seronegative SLE patients. Univariate and multivariate analysis (logistic regression models) were employed to evaluate the odds ratio (OR) of different risk factors for vascular events (including Raynaud's phenomenon, deep venous thrombosis) and hematologic abnormalities (including severe anemia, leukopenia and thrombocytopenia). Anti-CMV (82%), anti-PV-B19 (60%), anti-EBV-VCA (92%) and EBV-EA (45%) IgG antibodies were frequent in SLE, with higher prevalence in comparison with the blood donor group and higher titers in comparison with transplant and hemodialysis groups. CMV seropositivity was a highly significant risk factor for Raynaud's phenomenon (OR +alpha in univariate and multivariate analysis = 13.51 using a correction of 0.5 in case of a zero event), but not for venous vascular events (OR = 1.31). An increased though not significant risk factor was found for antiphospholipid antibodies (OR = 2.71, p = 0.19), while the presence of nephrotic syndrome during the follow-up was a significant protective factor (OR = 0.15, p = 0.035). There was no significantly increased OR for PV-B19 seropositivity in cases with severe anemia (OR = 2.09, p = 0. 29). No significant associations were found with the status of EBV reactivation. In conclusion, our results support the hypothesis that viral infection may imprint the course of SLE leading to specific clinical subsets (i.e. CMV and 'vascular' SLE, with more frequent Raynaud's phenomenon and a less frequent typical histological renal picture responsible for nephrotic syndrome). Further prospective studies are justified to validate these correlations, mainly dealing with associations between acute viral infections and vascular events, thus eventually leading to a better understanding of mutual relationships between viruses and SLE.

Adult

Phosphatidylinositol 3-kinase translocation to the nucleus is induced by interleukin 1 and prevented by mutation of interleukin 1 receptor in human osteosarcoma Saos-2 cells.

Although interleukin 1 (IL-1) functions have been extensively characterized, the mechanisms by which IL-1 signals are transduced from the plasma membrane to the nucleus are less known. Recent evidence indicates that phosphatidylinositol 3-kinase (PI3-kinase) could be activated by a direct association with the activated IL-1 receptor. In this study we analyzed the effects of IL-1 on the intracellular distribution of PI3-kinase in wild-type Saos-2 human osteosarcoma cells, and in cell clones overexpressing type I IL-1 receptor (IL-1RI). PI3-kinase intracellular distribution displays two distinct patterns. In quiescent cells, PI3-kinase is distributed through the cytoplasm, although a portion is present in the nucleus; following stimulation with IL-1, PI3-kinase is redistributed, increasing in the nuclear compartment. Both immunoblotting and immunofluorescence data indicate that IL-1 causes a rapid and transient translocation of PI3-kinase from the cytoplasm to the nucleus. This phenomenon is prevented by PI3-kinase inhibitors, suggesting that the maintenance of PI3-kinase activity is essential for IL-1-induced translocation. Indeed, in cell clones stably transfected with Y479F receptor mutant, in which the binding of the enzyme to the activated receptor is blocked, IL-1-induced PI3-kinase translocation to the nucleus is completely prevented. These data suggest that PI3-kinase translocation to the nucleus upon IL-1R activation is an early event in IL-1 signaling mechanism, and may be involved in transcriptional activation.

Base Sequence

Constitutive expression of interleukin-1beta (IL-1beta) in rat oligodendrocytes.

The RT-PCR analysis of RNA from progenitor and differentiated primary rat oligodendrocytes, and from the oligodendrocyte CG-4 cell line, shows the presence of the IL-1beta mRNA, the type I IL-1beta receptor and the IL-1 receptor accessory protein in these cells. In situ hybridization of a rat IL-1beta probe to primary progenitor and differentiated rat oligodendrocytes results in a positive signal. The double hybridization of the IL-1beta probe, together with an oligodendrocyte-specific differentiation marker, to sections of postnatal rat brain at different stages of differentiation is also positive. The double immuno-labelling technique utilized indicates coincidence of the signals on the brain slices. The results show that IL-1beta mRNA is constitutively expressed in rat brain oligodendrocytes from 1 day after birth onward. In agreement with this observation, CG-4 cells, primary progenitor and differentiated rat oligodendrocytes are positively stained by antibodies against IL-1beta. Postnatal brain slices from 1 and 4 day old and adult rats, labelled with a double immunofluorescence technique, are also stained by antibodies against IL-1beta. This signal coincides with that of antibodies against oligodendrocyte-specific surface markers. We conclude that IL-1beta is constitutively expressed in rat brain progenitor and differentiated oligodendrocytes.

Animals

Catastrophic antiphospholipid syndromes in systemic lupus erythematosus.

The objective of this study was to look for the occurrence of catastrophic antiphospholipid syndromes (APS) and to try to detect discriminating factors for predicting a worse prognosis, related to Lupus anticoagulant (LA) and antiphospholipid antibodies (aPL), in systemic lupus erythematosus (SLE) with main renal involvement. Regression, recursive partition and logistic regression analyses were applied to our 80 SLE patients prospectively followed up since 1980. Immunologic and other laboratory parameters including beta 2-glycoprotein 1 dependence, resistance to activated protein C caused by a substitution on the coagulation factor V gene, induction of monocyte procoagulant activity. Regression studies demonstrated an overall worse prognosis in term of both thrombosis and death for the group of LA/aPL positive patients (33/80). However, recursive partition analysis was able to isolate a small high risk-subgroup (8/33) characterized by persistent LA/aPL antibodies positive result, widespread signs of noninflammatory vasculopathy (skin, brain, kidney) and renal pathology mimicking that of thrombotic microangiopathy or arteriolosclerosis, also in the absence of classic SLE-nephritis. Only in this subset, three catastrophic APS were recorded, while, in traditional SLE nephritis, even persistent LA/aPL positive results (sometimes after one previous thrombosis) did not seem to imply a particularly severe prognosis. All serologic criteria employed are unable to identify high-risk patients. We conclude that catastrophic APS is a rare event in renal SLE. Before more predictive serologic markers become available, a simple algorithm, dealing with clinical data and renal histologic patterns, may help physicians to identify putatively high risk-LA/aPL antibodies in SLE patients with main renal involvement. This ominous subset does not belong to the group of classic SLE-nephritis.

Adolescent

[Correlations between viral serology and different clinical characteristics in patients with lupus nephropathy].

BACKGROUND: Relationships between viral infections and autoimmune diseases are complex and debated: on the one hand, patients with LES are particularly prone to develop viral infections, on the other, some virus are thought to play a role both in triggering the onset of the immunologic disease and in conferring peculiar clinical features to flare-ups. METHODS: This study has drawn an epidemiologic profile of viral pathology from Epstein Barr virus, Parvovirus B 19 and Cytomegalovirus in 60 patients with LES followed-up for a period ranging from 13 to 340 months (on average 158 months). RESULTS: Cytomegalovirus seropositivity has turned out to be a strong, statistically significant risk factor for vascular accidents and especially for peripheral ones, such as Raynaud phenomenon, ulcers and distal necrosis (OR = 6.5 IC = 0.7-7.9* p = 0.037). CONCLUSIONS: Although Cytomegalovirus seropositivity is associated with an increased frequency of LAC/ACA, its relationship with vascular events does not seem to be mediated through such antibodies, apparently acting as an independent risk factor which also works in LAC/ACA negative patients. Parvovirus B 19 seropositivity has turned out to be the only increased risk factor for the development of anemia, although not reaching statistical significance, whereas Epstein Barr seropositivity does not appear to influence clinical features significantly.

Adult

P-glycoprotein subcellular localization and cell morphotype in MDR1 gene-transfected human osteosarcoma cells.

Efflux of chemotherapy agents by P-glycoprotein at the plasma membrane is thought to be a major cause of cancer multidrug-resistance (MDR). However, the mechanism underlying the cellular accumulation and distribution of cytotoxic drugs is still poorly defined. We have recently found that P-glycoprotein is expressed also in the nucleus of MDR cell lines selected in doxorubicin (DXR), suggesting the possible involvement of this protein in the direct extrusion of the drug from the nucleus of resistant cells. In this study, we analyzed the subcellular localization of P-glycoprotein, in a series of U-2 OS osteosarcoma cell clones transfected with MDR1 gene in order to verify whether the nucleus is a constant site for the localization and functional activity of P-glycoprotein, and in which way some aspects of cell morphology related to MDR depend on the subcellular P-glycoprotein localization rather than on the exposure to the selective drug. Our results indicate that to achieve a subcellular drug distribution prevailing in the cytoplasm but not in the nucleus, a significant increase in the expression of P-glycoprotein at the different cellular compartments, including the plasma membrane, the cytoplasm, and the nucleus, is needed, although the in vitro drug resistance appears to be mainly dependent on the expression of P-glycoprotein at the cell surface. With regard to the morphological characteristics of MDR cells involving the cell surface and the chromatin arrangement, the influence of DXR appears to be prevalent, although P-glycoprotein overexpression cannot be excluded.

ATP Binding Cassette Transporter, Subfamily B, Mem

CD38 functions are regulated through an internalization step.

The endocytosis of the human CD38 molecule has been investigated in normal lymphocytes and in a number of leukemia- and lymphoma-derived cell lines. CD38 internalization was followed using radioiodinated Abs in an acidic elution endocytosis assay to monitor the effects of cross-linking on internalization processes and to quantify the ratio of the internalized molecule. Second, conventional, confocal, and electron microscopies were used to evaluate the morphologic effects induced by ligation of the molecule with Abs mimicking the natural ligand(s). The results demonstrated that internalization is a reproducible phenomenon following CD38 ligation with both agonistic and nonagonistic specific Abs and involving only a fraction of the entire amount of the surface molecule. It is independent from signal transduction as can be inferred by the observation that 1) both agonistic and non agonistic Abs are effective and 2) the dynamic of internalization is much slower than that of cellular signaling. Morphologic studies demonstrated that endocytosis induced as a result of CD38 ligation presents a very specific pathway consisting of subcellular organelles fundamental to the processing of the complex. Our data indicate that down-regulation by endocytosis may be, in parallel with shedding, a regulatory element in activation and adhesion processes mediated by CD38. However, internalization seems not to be a key step in triggering intracellular signaling; more likely, it is a negative feedback control mechanism which interrupts signal transduction or cell-cell cross-talks mediated by membrane CD38.

ADP-ribosyl Cyclase

A multiple technical approach to the study of apoptotic cell micronuclei.

Apoptotic micronuclei have been studied, in different cell types, from a morphologic and functional point of view. Conventional electron microscopy, in various staining conditions, selective cytochemistry for DNA, and freeze fracture for the analysis of chromatin fiber organization and size were performed. In situ TdT and Pol I immunofluorescent techniques were carried out to detect double- and single-strand DNA breaking points by confocal laser scanning microscopy. Apoptotic cell ultrathin cryosections were also performed and were analysed by field emission in lens scanning electron microscopy. Double/single strand massively cleaved DNA was detected in micronuclei, with a highly supercoiled, uniformly packed, very dense arrangement.

Animals

Phospholipid rearrangement of apoptotic membrane does not depend on nuclear activity.

The behaviour of plasma membrane was studied in UV-treated cells to investigate its involvement in apoptosis. It was studied in HL60 cells, in which DNA oligonucleosomic cleavage occurs, and in Molt-4 cells, which are characterised by a different fragmentation pattern. During the early stages of apoptosis, a membrane lipid rearrangement occurs, which involves phosphatidylserine translocation from the inner to the outer leaflet. This molecular alteration was investigated by annexin V-FITC binding, analysed by flow cytometry and confocal microscopy. It was correlated with transmission electron microscopy, subdiploid peak appearance and DNA fragmentation. Our data indicate that the plasma membrane represents an early apoptotic target, even if its alterations are not detectable by ultrastructural analysis, which indicates its good preservation until late apoptotic stages. In addition, the study of apoptotic cells with absent or inactivated endonuclease demonstrates the independence of this membrane mechanism from nuclear activity.

Apoptosis

Ataxin-3 is transported into the nucleus and associates with the nuclear matrix.

It has been reported that the ataxin-3 protein containing a polyglutamine sequence in the pathological range (61-84Q) is localized within the nucleus of neuronal cells, whereas ataxin-3 with a normal repeat length (12-37Q) is predominantly a cytoplasmic protein. In this study, the subcellular localization of the full-length ataxin-3 protein with a glutamine sequence in the normal range (Q3KQ22) was analysed in two mammalian cell lines. Using two affinity-purified polyclonal antibodies raised against the N- or C-terminal portion of ataxin-3, the protein was detected predominantly, but not exclusively, in the nucleus of COS-7 as well as neuroblastoma cells by immunofluorescence and confocal laser scanning microscopy (CLSM). The distribution of the protein in these cellular compartments was confirmed by biochemical subcellular fractionations. Furthermore, CLSM revealed that the ataxin-3 protein present in the nucleus of neuroblastoma cells is associated with the inner nuclear matrix. Our results taken together with the finding of a nuclear localization signal in ataxin-3 indicate that the ataxin-3 protein per se translocates to the nucleus and that an expanded glutamine repeat is not essential for this transport.

Animals

Cytochemistry of the functional domains of the nucleus in normal and in pathologic conditions.

By means of ultrastructural cytochemistry significant advances have been made in understanding the functional roles of many nuclear domains. This review gives schematic information about the main nuclear domains involved in replication, transcription, processing and transport of the transcripts in normal and in pathologic conditions. Particular attention is paid to a functional domain that appears to be involved in signal transduction. Data are reported on the intranuclear specific localization of key elements of the polyphosphoinositide signal transduction system in different cell types including human osteosarcoma cell lines. Compared with the compartmentalization of the cytoplasm, the nucleus has long been considered as relatively unstructured. On the other hand, fundamental nuclear functions, such as DNA replication and RNA transcription, can be molecularly characterized also in cell-free systems, suggesting that supramolecular organization is not so strictly required as for other cell functions occurring within intact cytoplasmic organelles. Nevertheless, a stringent organization is required for packing about 200 cm of DNA in the about 30 micron 3 of the nucleus. In the absence of membrane-delimited organelles, the nuclear organization is based on functional compartments, or domains, whose spatial localization involves the nuclear matrix, which shares many properties with the cytoskeleton. The nuclear domains are defined as structural compartments, not necessarily stable but dynamically variable, which perform specific metabolic functions through the partitioning of molecular complexes. Their identification has been made possible in the last few years by the development of specific nuclear probes for confocal and electron microscope immunocytochemistry. Therefore, the complex network of structures and enzymatic functions that make up the nucleus is in several cases yielding to molecular analysis, but a large part remains unknown (Strouboulis and Wolffe, 1996; Laemmli and Tjian, 1996). Rapid advances in understanding the functional role of the nuclear domains have been made recently: in particular, of the nuclear envelope, of the nucleolus, and of RNA splicing. In other cases, e.g. the precise localization of the nuclear domains involved in signal transduction, much remains to be clarified (Forbes and Johnson, 1997). It is conceivable that in the near future unexpected new nuclear domains will come to light and new nuclear functions may emerge, especially in field of post-transcriptional processing and transport of RNAs, and in the relationships between the nucleo-skeleton and enzymic fixed sites involved in replication, transcription and signal transduction. The aim of this review is to provide information about the morphological characteristics, the associated functions and the molecular composition of the main nuclear domains found to date. To simplify the exposition, the main data on each nuclear domain are reported in Tables, together with the principal references on the subject. Figures refer to original findings on some aspects of nuclear domain organization.

Cell Nucleus

IMP dehydrogenase inhibitor, tiazofurin, induces apoptosis in K562 human erythroleukemia cells.

Tiazofurin, an anticancer drug which inhibits IMP dehydrogenase, decreases cellular GTP concentration, induces differentiation and down-regulates ras and myc oncogene expression, caused apoptosis of K562 cells in a time- and dose-dependent fashion. Apoptotic cells were detected by (1) flow cytometry, (2) electron microscopy, and (3) fluorescence in situ nick translation and confocal microscopy, while the DNA ladder was not detectable. The induced apoptosis was abrogated by guanosine which replenishes GTP pools through the guanosine salvage pathways, while it was enhanced by hypoxanthine, a competitive inhibitor of GPRT. The tiazofurin-mediated apoptosis may therefore be linked with the decrease of GTP and the consequent impairment of specific signal transduction pathways. Tiazofurin induced apoptosis also in lymphoblastic MOLT-4 cells, suggesting that this action is not confined to cells of the myeloid lineage, where the differentiating effects of the drug are more pronounced.

Apoptosis

Intramembrane protein distribution in cell cultures is affected by 50 Hz pulsed magnetic fields.

Intramembrane proteins (IMP) represent a class of proteins located in the lipid bilayer of the cell membrane which function as ion channels, enzymes or receptors. Since it has been argued that biological effects of extremely low frequency (ELF) electromagnetic fields are mediated by plasma membrane. this work was designed to study the possible effects of 50 Hz pulsed magnetic fields (PMF) of the type used to stimulate bone repair, on the distribution of IMP in the plasma membrane of Swiss NIH 3T3 fibroblasts. Evaluations were based on the calculation of a distribution factor, which allows discrimination between random, regular and clustered distribution of IMP, in electron microscope images of freeze-fractured membranes. The results indicate that cells exposed to PMF for more than two hours have a significant clustering of the IMP distribution compared to control unexposed cells.

3T3 Cells

Enhanced resolution of specific chromosome and nuclear regions by reflectance laser scanning confocal microscopy.

DNA sequences digested by HaeIII and reconstructed by in situ nick translation employing digoxigenin-labelled nucleotides are usually revealed either by horseradish peroxidase or FITC fluorescence. To obtain a significant improvement in terms of resolution, sensitivity and specificity, colloidal gold has been used instead of FITC (as the reporter molecule) to reveal the labelled DNA. Colloidal gold and propidium iodide were visualised by employing the reflectance mode and the 488-nm laser line of a confocal laser scanning microscope. In chromosomes, the fluorescent reaction pattern showed diffuse areas of labelling in which it was impossible to identify any specific kind of banding along the arms. In some chromosomes and, in particular, 1 and 9, a C-negative banding due to the negativity of the centromeric areas was seen. A more accurate localisation on chromosomes, including telomeric regions, often organised in spot pairs that resembled an R-like banding, was detected using 1-nm colloidal gold. A fine labelling was also demonstrated in nuclei, especially at their peripheral heterochromatin. The non-fading properties of colloidal gold combined with visualisation by reflectance confocal laser scanning microscopy demonstrated the possibility of obtaining a higher spatial resolution than when using conventional fluorophores or higher laser wavelength. This improved way to study the localization of HaeIII digestion sites in single chromosomes and in interphase nuclei made the reaction a valuable tool for the detection of antigens or of specific DNA sequences in biological preparations.

Cell Nucleus