Complement-independent, peroxide-induced antibody lysis of platelets in HIV-1-related immune thrombocytopenia

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.     

Mechanisms of cytotoxicity induced by horseradish peroxidase/indole-3-acetic acid gene therapy

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.     

Iron and hydrogen peroxide detoxification properties of DNA-binding protein from starved cells. A ferritin-like DNA-binding protein of Escherichia coli

The DNA-binding proteins from starved cells (Dps) are a family of proteins induced in microorganisms by oxidative or nutritional stress. Escherichia coli Dps, a structural analog of the 12-subunit Listeria innocua ferritin, binds and protects DNA against oxidative damage mediated by H(2)O(2). Dps is shown to be a Fe-binding and storage protein where Fe(II) oxidation is most effectively accomplished by H(2)O(2) rather than by O(2) as in ferritins. Two Fe(2+) ions bind at each of the 12 putative dinuclear ferroxidase sites (P(Z)) in the protein according to the equation, 2Fe(2+) + P(Z) --> [(Fe(II)(2)-P](FS)(Z+2) + 2H(+). The ferroxidase site (FS) bound iron is then oxidized according to the equation, [(Fe(II)(2)-P](FS)(Z+2) + H(2)O(2) + H(2)O --> [Fe(III)(2)O(2)(OH)-P](FS)(Z-1) + 3H(+), where two Fe(II) are oxidized per H(2)O(2) reduced, thus avoiding hydroxyl radical production through Fenton chemistry. Dps acquires a ferric core of approximately 500 Fe(III) according to the mineralization equation, 2Fe(2+) + H(2)O(2) + 2H(2)O --> 2Fe(III)OOH((core)) + 4H(+), again with a 2 Fe(II)/H(2)O(2) stoichiometry. The protein forms a similar ferric core with O(2) as the oxidant, albeit at a slower rate. In the absence of H(2)O(2) and O(2), Dps forms a ferrous core of approximately 400 Fe(II) by the reaction Fe(2+) + H(2)O + Cl(-) --> Fe(II)OHCl((core)) + H(+). The ferrous core also undergoes oxidation with a stoichiometry of 2 Fe(II)/H(2)O(2). Spin trapping experiments demonstrate that Dps greatly attenuates hydroxyl radical production during Fe(II) oxidation by H(2)O(2). These results and in vitro DNA damage assays indicate that the protective effect of Dps on DNA most likely is exerted through a dual action, the physical association with DNA and the ability to nullify the toxic combination of Fe(II) and H(2)O(2). In the latter process a hydrous ferric oxide mineral core is produced within the protein, thus avoiding oxidative damage mediated by Fenton chemistry.     

Activation tagging using the En-I maize transposon system in Arabidopsis

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.     

Cyclophilin A peptidyl-prolyl isomerase activity promotes ZPR1 nuclear export

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.     

CD44v10: an antimetastatic membrane glycoprotein for pancreatic cancer

AIMS: The aims of this study were 1) to investigate the mRNA pattern of CD44 variants in three primary (MIA PaCa 2, PANC-1, PSN-1) and two metastatic (CAPAN-1, SUIT-2) pancreatic cancer (PC) cell lines; 2) to ascertain whether the genetic transfer of CD44s and CD44v10 modifies the adhesion of PC cells to the extracellular matrix (ECM) in vitro and their metastatic behavior in vivo. METHODS: CD44 mRNA analysis was done by means of RT-PCR. Adhesion to ECM the was assessed using coated microtiter plates. For the study of CD44v10 insertion in the CAPAN-1 line, liposome-mediated DNA transfer was used. SCID mice were employed for in vivo experiments. RESULTS: CD44v10 mRNA was not expressed by the CAPAN-1 nor by four of the six SUIT-2-derived clones. The stable expression of CD44v10 by modified CAPAN-1 significantly enhanced fibronectin adhesion. Mice without either liver or pancreatic metastases were more frequently found among the animals injected with modified (CD44v10 expressing) than with non-modified CAPAN-1. CONCLUSIONS: 1) It is possible to differentiate between metastatic and non-metastatic PC cells on the basis of CD44v10 expression; 2) CD44v10 seems to be involved in mediating fibronectin adhesion in vitro and in counteracting metastases in vivo.     

Determinants of human immunodeficiency virus (HIV) prevalence in homosexual and bisexual men screened for admission to a cohort study of HIV negatives in Belo Horizonte,Brazil: Project Horizonte

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.     

Disturbances in purinergic [Ca2+]i signaling pathways in a transformed rat thyroid cell line

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.