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1.
Filippo Genovese Stefania Ferrari Giambattista Guaitoli Monica Caselli M. Paola Costi Glauco Ponterini 《Protein science : a publication of the Protein Society》2010,19(5):1023-1030
An ad hoc bioconjugation/fluorescence resonance energy transfer (FRET) assay has been designed to spectroscopically monitor the quaternary state of human thymidylate synthase dimeric protein. The approach enables the chemoselective engineering of allosteric residues while preserving the native protein functions through reversible masking of residues within the catalytic site, and is therefore suitable for activity/oligomerization dual assay screenings. It is applied to tag the two subunits of human thymidylate synthase at cysteines 43 and 43′ with an excitation energy donor/acceptor pair. The dimer–monomer equilibrium of the enzyme is then characterized through steady‐state fluorescence determination of the intersubunit resonance energy transfer efficiency. 相似文献
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Cristina Cereda Emanuela Leoni Pamela Milani Orietta Pansarasa Giuliano Mazzini Stefania Guareschi Elena Alvisi Andrea Ghiroldi Luca Diamanti Stefano Bernuzzi Mauro Ceroni Emanuela Cova 《PloS one》2013,8(10)
Several lines of evidence support the hypothesis of a toxic role played by wild type SOD1 (WT-SOD1) in the pathogenesis of sporadic amyotrophic lateral sclerosis (SALS). In this study we investigated both distribution and expression profile of WT-SOD1 in leukocytes from 19 SALS patients and 17 healthy individuals. Immunofluorescence experiments by confocal microscopy showed that SOD1 accumulates in the nuclear compartment in a group of SALS subjects. These results were also confirmed by western blot carried out on soluble nuclear and cytoplasmic fractions, with increased nuclear SOD1 level (p<0.05). In addition, we observed the presence of cytoplasmic SOD1 aggregates in agreement with an increased amount of the protein recovered by the insoluble fraction. A further confirmation of the overall increased level of SOD1 has been obtained from single cells analysis using flow cytometry as cells from SALS patients showed an higher SOD1 protein content (p<0.05). These findings add further evidence to the hypothesis of an altered WT-SOD1 expression profile in peripheral blood mononuclear cells (PBMCs) from patients with ALS suggesting that WT-SOD1 species with different degrees of solubility could be involved in the pathogenesis of the disease. 相似文献
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Calcium/calmodulin regulation of ATPase activity and endogenous phosphorylation of mammalian brain actomyosin 总被引:1,自引:0,他引:1
Rabbit brain actomyosin showed several fold stimulation of the MgATPase activity by Ca2+ alone and by Ca2+/calmodulin. The calmodulin-binding drug, fluphenazine, abolished the stimulated activity. In the presence of Ca2+, exogenous calmodulin had a biphasic effect on ATPase activity at low concentrations (less than 0.15 microM) and activated the ATPase activity by 60-70% at about 1 microM. Tropomyosin-troponin complex from skeletal muscle did not stimulate the ATPase activity of brain actomyosin, but conferred Ca2+ sensitivity to a skeletal muscle myosin/brain actomyosin mixture. These results indicate the presence of myosin-linked, calmodulin-dependent, Ca2+-regulatory system for brain actomyosin. Heavy and light chains of brain myosin were found to be rapidly phosphorylated by endogenous Ca2+-dependent protein kinase(s). Ca2+-independent phosphorylation of one of the light chains was also observed. 相似文献
6.
Giuseppe Familiari M.D. Stefania A. Nottola Antonio Familiari Pietro M. Motta 《Cell and tissue research》1989,257(2):247-253
Summary The present study provides further details on the fine-structural three-dimensional architecture of the zona pellucida (ZP) in growing and atretic follicles of mice by use of ruthenium red in combination with the detergents Triton X100 and saponin. These detergents were used for extraction of the soluble fraction of the zonal proteins in an attempt to expose the structural zonal glycoproteins, which in turn can be viewed as minute three-dimensional networks upon transmission- and scanning electron-microscopic examination. By use of these methods, the ZP of growing follicles appeared to be formed by interconnected filaments which also bind to globular structures building up a three-dimensional lattice. In contrast, the ZP of stage I as well as other (II and III) stages of atretic follicles showed a structure characterized by the presence of closely packed granules connected with short filaments to form a close-mesh reticulum. This structural change of the ZP, which in the present study is also associated with the disappearance of gap junctions within the granulosa and cumulus cell population, might represent one of the early events involved in the onset of atresia. These changes, most probably depending on an altered secretory activity of both oocytes and follicle cells, might lead to a degradation of the ZP network structure and to its subsequent increased density (condensation). All these morphodynamic events eventually contribute to a sequestration of the oocyte in the early stage of atresia. 相似文献
7.
Tissue plasminogen activator inhibitor in patients with systemic lupus erythematosus and thrombosis.
F Violi D Ferro G Valesini C Quintarelli M Saliola M A Grandilli F Balsano 《BMJ (Clinical research ed.)》1990,300(6732):1099-1102
OBJECTIVE--To examine the relations among tissue plasminogen activator antigen, plasminogen activator inhibitor, the lupus anticoagulant, and anticardiolipin antibodies in patients with systemic lupus erythematosus. DESIGN--Prospective study of blood samples (a) from selected patients with systemic lupus erythematosus whose disease was and was not complicated by a history of thrombosis or recurrent abortions, or both, and (b) from a series of healthy controls with a similar age and sex distribution. SETTING--University based medical clinic. SUBJECTS--23 Patients with definite systemic lupus erythematosus (American Rheumatism Association criteria), of whom 11 (eight women) aged 26-51 had a history of thrombosis or recurrent abortions, or both, and 12 (10 women) aged 23-53 had no such history. 15 Healthy subjects (10 women) aged 25-58 served as controls. MAIN OUTCOME MEASURES--Tissue plasminogen activator concentrations, plasminogen activator inhibitor activities, detection of the lupus anticoagulant, and values of anticardiolipin antibodies in the two groups of patients and in the patients with a history of thrombosis or abortions compared with controls. Other measurements included concentrations of proteins that are known to change during the acute phase of systemic lupus erythematosus--namely, fibrinogen, C3 and C4, and C reactive protein. RESULTS--Patients with a history of thrombosis or abortions, or both, had significantly higher values of tissue plasminogen activator and plasminogen activator inhibitor than patients with no such history. A significant correlation between tissue plasminogen activator and plasminogen activator inhibitor (r = 0.80) was found only in the patients with a history of complications of their disease. The lupus anticoagulant was detected in six of the 11 patients with a history of thrombosis or abortions when tested by measuring the activated partial thromboplastin time but was found in all 11 patients when tested by measuring the diluted activated partial thromboplastin time. Nine of these 11 patients had raised values of anticardiolipin antibodies. The findings showed no relation to the activity of the disease. CONCLUSIONS--A significant correlation between tissue plasminogen activator concentrations and plasminogen activator inhibitor activities was found only in patients whose systemic lupus erythematosus was complicated by a history of thrombosis or recurrent abortions. The findings show that these patients have raised plasminogen activator inhibitor activities, and the frequent association between these raised activities and the presence of the lupus anticoagulant suggests that the two may be linked. 相似文献
8.
Elvira D'Alessandro Corinna De Matteis Vaccarella Maria Luisa Lo Re Francesco Cappa Angela D'Alfonso Stefania Discepoli Maria Rosa Della Penna Giuseppe Del Porto 《Human genetics》1988,80(2):203-204
Summary Pericentric inversion of chromosome 19 has been found in several members of three unrelated families from a restricted geographical region. In one of the families, an additional pericentric inversion of chromosome 9 was observed. Reproductive problems, multiple abortions in two families and a neonatal death in the third, were present. A review of previously described cases is included, and the genetic risk connected with this type of rearrangement is also discussed. 相似文献
9.
Maria Di Bello Valeria Lucchini Stefania Chiari Roberto Colleoni Nicoletta Colombo Alberto Mantovani Paola Allavena 《Cancer immunology, immunotherapy : CII》1988,27(1):63-68
Summary Expression of HLA-DR antigens by purified preparations of human ovarian carcinoma cells freshly isolated from surgical specimens was examined in parallel with the capacity of tumor cells to elicit a blastogenic response from autologous lymphocytes in mixed lymphocyte-tumor culture (MLTC) assay. Of 21 tumor preparations, 11 (52%) reacted with monoclonal antibodies 279 and/or 949 specific for a monomorphic determinant of HLA-DR antigens, with heterogeneous positivity, ranging between 30% and 95%. In this series of patients positive MLTC occurred in 8/21 individual experiments. The HLA-DR expression was proportionally similar in tumors giving positive MLTC (4/8=50%) and negative MLTC (7/13=53%). The lack of correlation between DR expression on tumor cells and stimulatory activity in autologous MLTC and the fact that DR-negative tumors could induce lymphocyte stimulation, support the hypothesis that blastogenesis occurs upon recognition of tumor-associated antigens, different from DR molecules, possibly tumor-specific antigens. 相似文献
10.
Pierpaolo Correale Antonio Procopio Luigi Celio Michele Caraglia Geppino Genua Vincenzo Coppola Stefano Pepe Nicola Normanno Immacolata Vecchio Giovannella Palmieri Stefania Montagnani Pierosandro Tagliaferri Angelo Raffaele Bianco 《Cancer immunology, immunotherapy : CII》1992,34(4):272-278
Summary Human melanoma cells are sensitive to the lytic activity of natural killer (NK) and lymphokine-activated killer (LAK) cells in vitro. The events resulting in tumour cell killing by lymphocytic effectors have not been completely clarified, and the same target cell determinants regulating responsiveness to immune cytolysis have not yet been identified. Indeed, changes in the differentiative status of leukemia cells as well as in the expression of major histocompatibility complex (MHC) antigens have been described to modulate sensitivity to cytotoxic effectors; moreover surface expression of adhesion factors or extracellular matrix proteins by the cancer cells can promote the activation of the cytolytic effectors and has been described to correlate with tumour cell sensitivity to cytolytic cells. We reasoned that treatment with differentiation inducers could modulate melanoma cell sensitivity to NK and LAK cells. The present study demonstrates that human melanoma GLL-19 cells, when treated with the phorbol diester phorbol 12-myristate 13-acetate (PMA) in vitro, undergo growth inhibition and neuron-like differentiation. Moreover PMA treatment induces an evident inhibition of GLL-19 cell sensitivity to NK- and LAK-mediated cytotoxicity. GLL-19 cells express constitutively MHC class I antigens. PMA treatment, however, does not modify the expression of MHC class I and class II DR antigens in human melanoma GLL-19 cells. We have finally evaluated the effects of PMA on the expression at the cell surface of adhesion factors such as ICAM-1, and extracellular matrix proteins such as collagen IV, laminin and fibronectin; we have also studied the expression of the integrin vitronectin receptor, a membrane receptor for adhesive proteins. While adhesion factors and extracellular matrix proteins appear to play an important role in the interaction between immune effector and tumour target, it can be supposed that the modulation of such membrane-associated proteins or glycoproteins induces NK and LAK resistance in cancer cells. We indeed found that PMA treatment induced in GLL-19 a marked reduction of membrane expression of collagen IV and ICAM-1; moreover PMA reduced the cell membrane expression of the integrin vitronectin receptor. On the other hand, membrane expression of fibronectin and laminin was not affected by PMA. These data indicate that the acquisition of a NK- and LAK-resistant phenotype by GLL-19 cells occurs together with cell differentiation, down-regulation of membrane expression of collagen IV, ICAM-1 and vitronectin receptor, but in the absence of changes in MHC antigens.This work has been supported by the Italian Association for Cancer Research (A. I. R. C.) and by Istituto Superiore di Sanità, Italy-USA joint program on New Therapies on Neoplasia. 相似文献