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11.
Daniela Nicole Filoni Rossana Pesi Maria Giovanna Careddu Marcella Camici Simone Allegrini Anita Collavoli 《Nucleosides, nucleotides & nucleic acids》2013,32(12):1155-1160
IMP preferring cytosolic 5 ′-nucleotidase II (cN-II) is a widespread enzyme whose amino acid sequence is highly conserved among vertebrates. Fluctuations of its activity have been reported in some pathological conditions and its mRNA levels have been proposed as a prognostic factor for poor outcome in patients with adult acute myeloid leukemia. As a member of the oxypurine cycle, cN-II is involved in the regulation of intracellular concentration of 5′-inosine monophosphate (IMP), 5′-guanosine monophosphate (GMP), and also 5-phosphoribose 1-pyrophosphate (PRPP) and is therefore involved in the regulation of purine and pyrimidine de novo and salvage synthesis. In addition, several studies demonstrated the involvement of cN-II in pro-drug metabolism. Notwithstanding some publications indicating that cN-II is essential for the survival of several cell types, its role in cell metabolism remains uncertain. To address this issue, we built two eucaryotic cellular models characterized by different cN-II expression levels: a constitutive cN-II knockdown in the astrocytoma cell line (ADF) by short hairpin RNA (shRNA) strategy and a cN-II expression in the diploid strain RS112 of Saccharomyces cerevisiae. Preliminary results suggest that cN-II is essential for cell viability, probably because it is directly involved in the regulation of nucleotide pools. These two experimental approaches could be very useful for the design of a personalized chemotherapy. 相似文献
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Although Epstein-Barr virus (EBV) is an orally transmitted virus, viral transmission through the oropharyngeal mucosal epithelium is not well understood. In this study, we investigated how EBV traverses polarized human oral epithelial cells without causing productive infection. We found that EBV may be transcytosed through oral epithelial cells bidirectionally, from both the apical to the basolateral membranes and the basolateral to the apical membranes. Apical to basolateral EBV transcytosis was substantially reduced by amiloride, an inhibitor of macropinocytosis. Electron microscopy showed that virions were surrounded by apical surface protrusions and that virus was present in subapical vesicles. Inactivation of signaling molecules critical for macropinocytosis, including phosphatidylinositol 3-kinases, myosin light-chain kinase, Ras-related C3 botulinum toxin substrate 1, p21-activated kinase 1, ADP-ribosylation factor 6, and cell division control protein 42 homolog, led to significant reduction in EBV apical to basolateral transcytosis. In contrast, basolateral to apical EBV transcytosis was substantially reduced by nystatin, an inhibitor of caveolin-mediated virus entry. Caveolae were detected in the basolateral membranes of polarized human oral epithelial cells, and virions were detected in caveosome-like endosomes. Methyl β-cyclodextrin, an inhibitor of caveola formation, reduced EBV basolateral entry. EBV virions transcytosed in either direction were able to infect B lymphocytes. Together, these data show that EBV transmigrates across oral epithelial cells by (i) apical to basolateral transcytosis, potentially contributing to initial EBV penetration that leads to systemic infection, and (ii) basolateral to apical transcytosis, which may enable EBV secretion into saliva in EBV-infected individuals. 相似文献
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Use of the fluorescent timer DsRED-E5 as reporter to monitor dynamics of gene activity in plants 总被引:1,自引:0,他引:1 下载免费PDF全文
Fluorescent proteins, such as green fluorescent protein and red fluorescent protein (DsRED), have become frequently used reporters in plant biology. However, their potential to monitor dynamic gene regulation is limited by their high stability. The recently made DsRED-E5 variant overcame this problem. DsRED-E5 changes its emission spectrum over time from green to red in a concentration independent manner. Therefore, the green to red fluorescence ratio indicates the age of the protein and can be used as a fluorescent timer to monitor dynamics of gene expression. Here, we analyzed the potential of DsRED-E5 as reporter in plant cells. We showed that in cowpea (Vigna unguiculata) mesophyll protoplasts, DsRED-E5 changes its fluorescence in a way similar to animal cells. Moreover, the timing of this shift is suitable to study developmental processes in plants. To test whether DsRed-E5 can be used to monitor gene regulation in plant organs, we placed DsRED-E5 under the control of promoters that are either up- or down-regulated (MtACT4 and LeEXT1 promoters) or constitutively expressed (MtACT2 promoter) during root hair development in Medicago truncatula. Analysis of the fluorescence ratios clearly provided more accurate insight into the timing of promoter activity. 相似文献
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Serra PA Rocchitta G Delogu MR Migheli R Taras MG Mura MP Esposito G Miele E Desole MS Miele M 《Journal of neurochemistry》2003,86(6):1403-1413
In vitro microdialysis was used to investigate the mechanism of nitric oxide (NO) donor-induced changes in dopamine (DA) secretion from PC12 cells. Infusion of the NO-donor S-nitroso-N-acetylpenicillamine (SNAP, 1.0 mm) induced a long-lasting increase in DA and 3-methoxytyramine (3-MT) dialysate concentrations. SNAP-induced increases were inhibited either by pre-infusion of the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4] oxadiazolo[4,3]quinoxalin-1-one (ODQ, 0.1 mm) or by Ca2+ omission. Ca2+ re-introduction restored SNAP effects. SNAP-induced increases in DA + 3-MT were unaffected by co-infusion of the l-type Ca2+ channel inhibitor nifedipine. The NO-donor (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR-3, 1.0 mm) induced a short-lasting decrease in dialysate DA + 3-MT. Ascorbic acid (0.2 mm) co-infusion allowed NOR-3 to increase dialysate DA + 3-MT. ODQ pre-infusion inhibited NOR-3 + ascorbic acid-induced DA + 3-MT increases. Infusion of high K+ (75 mm) induced a 2.5-fold increase in dialysate DA + 3-MT. The increase was abolished by NOR-3 co-infusion. Conversely, co-infusion of ascorbic acid (0.2 mm) with NOR-3 + high K+ restored high K+ effects. Co-infusion of nifedipine inhibited high K+-induced DA + 3-MT increases. These results suggest that activation of the NO/sGC/cyclic GMP pathway may be the underlying mechanism of extracellular Ca2+-dependent effects of exogenous NO on DA secretion from PC12 cells. Extracellular Ca2+ entry may occur through nifedipine-insensitive channels. NO effects and DA concentrations in dialysates largely depend on both the timing of NO generation and the extracellular environment in which NO is generated. 相似文献
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Puntoni R Filiberti R Cerrano PG Neri M Andreatta R Bonassi S 《Mutation research》2003,544(2-3):385-396
The carcinogenic effect of asbestos has been reported in the literature since 40 years, and early studies describing the epidemic occurrence of malignant mesothelioma (MM) in asbestos workers, have become a paradigm of occupational cancer research. Research on MM was abandoned for many years since MM was considered as an asbestos-related disease, interesting only from a perspective of disease control and preventive policies. The introduction of new biological endpoints in the epidemiological studies has boosted research in the field, providing new tools for the study of emerging priority in cancer research and in public health. This approach, known as molecular epidemiology has a great potential in the study of MM, contributing to the understanding of susceptibility factors, to the evaluation of cancer risk in people occupationally or environmentally exposed to carcinogens, and to the enhancement of diagnosis and therapy. A comprehensive approach based on the use of banks of biological samples is presented and its advantages discussed here. The application of innovative endpoints, such as oncoproteins in biologic fluids, genetic polimorphisms, or gene function is discussed, and relevant literature reviewed. 相似文献
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Three innovative and complementary morphological approaches were employed to study the T cell/antigen presenting cell (APC) interaction: (i) high resolution three-dimensional confocal microscopy of the T cell-APC contact site; (ii) time lapse video recording in living T cells of [Ca2+]I and changes in distribution of various GFP fusion proteins with TCR/CD3-zeta complex associated- and other signaling components; (iii) measurement of lateral TCR mobility and that of recruited signaling components using techniques based on fluorescence recovery after photo-bleaching. These approaches were combined with biochemical and functional experiments to investigate two principal issues: (A) Recruitment and the three-dimensional arrangement of receptors and signaling components at the contact site between human CD4+ T lymphocytes and APCs, (B) Structure of the immunological synapse formed at the contact site between cytotoxic T lymphocytes (CTLs) and target cells. We discuss evidence indicating that TCR engagement and triggering can occur in the absence of large-scale molecular segregation into the T cell-APC contact site. Taken together our results indicate that although not required for TCR engagement and triggering, formation of the IS is important to reinforce TCR-mediated signal transduction and achieve full T cell activation. 相似文献
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Ríos G Lossow A Hertel B Breuer F Schaefer S Broich M Kleinow T Jásik J Winter J Ferrando A Farrás R Panicot M Henriques R Mariaux JB Oberschall A Molnár G Berendzen K Shukla V Lafos M Koncz Z Rédei GP Schell J Koncz C 《The Plant journal : for cell and molecular biology》2002,32(2):243-253
To assist in the analysis of plant gene functions we have generated a new Arabidopsis insertion mutant collection of 90 000 lines that carry the T-DNA of Agrobacterium gene fusion vector pPCV6NFHyg. Segregation analysis indicates that the average frequency of insertion sites is 1.29 per line, predicting about 116 100 independent tagged loci in the collection. The average T-DNA copy number estimated by Southern DNA hybridization is 2.4, as over 50% of the insertion loci contain tandem T-DNA copies. The collection is pooled in two arrays providing 40 PCR templates, each containing DNA from either 4000 or 5000 individual plants. A rapid and sensitive PCR technique using high-quality template DNA accelerates the identification of T-DNA tagged genes without DNA hybridization. The PCR screening is performed by agarose gel electrophoresis followed by isolation and direct sequencing of DNA fragments of amplified T-DNA insert junctions. To estimate the mutation recovery rate, 39 700 lines have been screened for T-DNA tags in 154 genes yielding 87 confirmed mutations in 73 target genes. Screening the whole collection with both T-DNA border primers requires 170 PCR reactions that are expected to detect a mutation in a gene with at least twofold redundancy and an estimated probability of 77%. Using this technique, an M2 family segregating a characterized gene mutation can be identified within 4 weeks. 相似文献