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41.
Theileriosis is an infectious disease in tropical countries and in the Mediterranean area. It is caused by Theileria, a haemoprotozoan, transmitted by vectors belonging to the Ixodidae. In Southern Italy and in Sicily the infection is due mainly to T. annulata, but in some cases other species are involved in the disease. The authors describe a method to identify theileriosis in cattle blood samples, using PCR and hybridization techniques. Different primer sets were used to amplify different DNA target sequences, both genus and species specific. Blood samples from cattle were collected in Sicily. The DNA extracted from blood samples was employed first to detect the presence of the 18S ribosomal subunit gene specific for Theileria genus. Successively the positive samples were analysed to identify the species, T. annulata or T. buffeli/orientalis, using as target sequences for amplification respectively a fragment of the TAMS-1 and p33/34 antigens gene. Here the authors describe for the first time the presence of T. buffeli/orientalis infection in Sicilian herds. In fact 66% of positive blood samples were T. buffeli/orientalis infected. 相似文献
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How to overcome (and exploit) tumor hypoxia for targeted gene therapy 总被引:17,自引:0,他引:17
44.
Complement-independent, peroxide-induced antibody lysis of platelets in HIV-1-related immune thrombocytopenia 总被引:8,自引:0,他引:8
Immunologic thrombocytopenia is seen commonly in HIV-1 infection. The pathogenesis of this problem has been unclear, but it is associated with circulating immune complexes that contain platelet membrane components and anti-platelet membrane GPIIIa49-66 IgG antibodies. These antibodies cause acute thrombocytopenia when injected into mice. We now show that purified anti-GPIIIa49-66 causes platelet fragmentation, in vitro in the absence of complement, and in vivo in wild-type and C3-deficient mice. The mechanism of complement-independent platelet lysis is shown to be caused by the antibody-induced generation of H202, as indicated by in vitro experiments with inhibitors of reactive oxygen species, and in vivo studies carried out with p47phox-deficient mice. Thus, a novel mechanism of immunologic platelet clearance is described in which an anti-platelet IgG causes platelet fragmentation via the induction of reactive oxygen species. 相似文献
45.
Mechanisms of cytotoxicity induced by horseradish peroxidase/indole-3-acetic acid gene therapy 总被引:7,自引:0,他引:7
We have previously proposed the horseradish peroxidase (HRP) and the non-toxic plant hormone indole-3-acetic acid (IAA) as a novel system for gene-directed enzyme/prodrug therapy (GDEPT). The cytotoxic potential of HRP/IAA GDEPT and the induction of a bystander effect were demonstrated in vitro under normoxic as well as hypoxic tumour conditions. To date, the chemical agents and the cellular targets involved in HRP/IAA-mediated toxicity have not been identified. In the present work, some of the molecular and morphological features of the cells treated with HRP/IAA gene therapy were analysed. Human T24 bladder carcinoma cells transiently transfected with the HRP cDNA and exposed to the prodrug IAA showed chromatin condensation, formation of apoptotic bodies, DNA fragmentation, and Annexin V binding. Similar effects were observed when the cells were incubated with the apoptotic agent cisplatin. Caspases appeared to be involved as effectors in HRP/IAA-mediated apoptosis, since treatment with a general caspase inhibitor decreased the fraction of cells with micronuclei (MN) by 30%, with fragmented DNA by 50%, and with condensed chromatin by 60%. However, very little degradation of one of the downstream targets of caspase-3, PARP, could be detected, and apoptosis alone did not appear to account for the killing levels measured with a clonogenic assay. The effect of HRP/IAA treatment on cell cycle progression was also investigated, and a rapid cytostatic effect, equally affecting all phases of the division cycle, was observed. 相似文献
46.
Activation tagging using the En-I maize transposon system in Arabidopsis 总被引:21,自引:0,他引:21
Marsch-Martinez N Greco R Van Arkel G Herrera-Estrella L Pereira A 《Plant physiology》2002,129(4):1544-1556
A method for the generation of stable activation tag inserts was developed in Arabidopsis using the maize (Zea mays) En-I transposon system. The method employs greenhouse selectable marker genes that are useful to efficiently generate large populations of insertions. A population of about 8,300 independent stable activation tag inserts has been produced. Greenhouse-based screens for mutants in a group of plants containing about 2,900 insertions revealed about 31 dominant mutants, suggesting a dominant mutant frequency of about 1%. From the first batch of about 400 stable insertions screened in the greenhouse, four gain-in-function, dominant activation-tagged, morphological mutants were identified. A novel gain-in-function mutant called thread is described, in which the target gene belongs to the same family as the YUCCA flavin-mono-oxygenase that was identified by T-DNA activation tagging. The high frequency of identified gain-in-function mutants in the population suggests that the En-I system described here is an efficient strategy to saturate plant genomes with activation tag inserts. Because only a small number of primary transformants are required to generate an activation tag population, the En-I system appears to be an attractive alternative to study plant species where the present transformation methods have low efficiencies. 相似文献
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48.
The peptidyl-prolyl isomerase (PPIase) cyclophilin A (Cpr1p) is conserved from eubacteria to mammals, yet its biological function has resisted elucidation. Unable to identify a phenotype that is suggestive of Cpr1p's function in a cpr1Delta Saccharomyces cerevisiae strain, we screened for CPR1-dependent strains. In all cases, dependence was conferred by mutations in ZPR1, a gene encoding an essential zinc finger protein. CPR1 dependence was suppressed by overexpression of EF1alpha (a translation factor that binds Zpr1p), Cpr6p (another cyclophilin), or Fpr1p (a structurally unrelated PPIase). Suppression by a panel of cyclophilin A mutants correlated with PPIase activity, confirming the relevance of this activity in CPR1-dependent strains. In CPR1(+) cells, wild-type Zpr1p was distributed equally between the nucleus and cytoplasm. In contrast, proteins encoded by CPR1-dependent alleles of ZPR1 accumulated in the nucleus, as did wild-type Zpr1p in cpr1Delta cells. Transport kinetic studies indicated that nuclear export of Zpr1p was defective in cpr1Delta cells, and rescue of this defect correlated with PPIase activity. Our results demonstrate a functional interaction between Cpr1p, Zpr1p, and EF1alpha, a role for Cpr1p in Zpr1p nuclear export, and a biological function for Cpr1p PPIase activity. 相似文献
49.
Carneiro M Cardoso FA Greco M Oliveira E Andrade J Greco DB Antunes CM 《Memórias do Instituto Oswaldo Cruz》2003,98(3):325-329
Project Horizonte, an open cohort of homosexual and bisexual human immunodeficiency virus (HIV-1) negative men, is a component of the AIDS Vaccine Program, in Belo Horizonte, Minas Gerais, Brazil. The objective of this study was to compare volunteers testing HIV positive at cohort entry with a sample of those who tested HIV negative in order to identify risk factors for prevalent HIV infection, in a population being screened for enrollment at Project Horizonte. A nested case-control study was conducted. HIV positive volunteers at entry (cases) were matched by age and admission date to three HIV negative controls each. Selected variables used for the current analysis included demographic factors, sexual behavior and other risk factors for HIV infection. During the study period (1994-2001), among the 621 volunteers screened, 61 tested positive for HIV. Cases were matched to 183 HIV negative control subjects. After adjustments, the main risk factors associated with HIV infection were unprotected sex with an occasional partners, OR = 3.7 (CI 95% 1.3-10.6), receptive anal intercourse with an occasional partner, OR = 2.8 (95% CI 0.9-8.9) and belonging to the negro racial group, OR = 3.4 (CI 95% 1.1-11.9). These variables were associated with an increase in the risk of HIV infection among men who have sex with men at the screening for admission to an open HIV negative cohort. 相似文献
50.
It was previously shown that in rat thyroid PC-Cl3 cell line, a purinergic P2Y receptor increases the concentration of free cytosolic Ca(2+) ([Ca(2+)](i)) via phospholipase C activation. We here studied whether in a transformed cell line (PC-E1Araf) derived from parental PC-Cl3 cells, ATP is still able to transduce the [Ca(2+)](i)-based intracellular signal.We demonstrate the expression of mRNA for P2Y2 in both PC-Cl3 and PC-E1Araf cells; mRNAs for P2Y1, P2Y4, P2Y6 and P2Y11 were absent. In both cell lines activation of P2Y2 receptor provokes a transient increase in [Ca(2+)](i) followed by a lower sustained phase persisting for over 5min in PC-Cl3 and only 1.5 min in PC-E1Araf cells. In both cell lines the [Ca(2+)](i) reached a plateau level significantly higher than the basal [Ca(2+)](i) level persisting for over 10 min. Removal of extracellular Ca(2+) reduced the initial transient response to ATP in PC-Cl3, but not in PC-E1Araf cells, and completely abolished the plateau phase in both cell lines.In the presence of extracellular Ca(2+) thapsigargin (TG) caused a rise in [Ca(2+)](i) significantly higher in PC-Cl3 than transformed PC-E1Araf cells, while in Ca(2+)-free medium the effect of TG was similar in both cell lines. The capacitative Ca(2+)-entry in PC-Cl3 resulted significantly higher than in PC-E1Araf cells.Further studies were performed in order to investigate whether the different effects of ATP on [Ca(2+)](i) was due to variation in divalent cation plasma membrane permeability. PC-E1Araf cells showed a much lower permeability to Ca(2+), Ba(2+), Sr(2+), Mn(2+), and Co(2+) that may be responsible for the differences in purinergic Ca(2+) signaling pathway with respect to parental PC-Cl3 cells. 相似文献