Enzyme activities of human erythrocyte ghosts: effects of various treatments

Summary Ghosts of human erythrocytes prepared by hypotonic hemolysis were assayed for aldolase, triosephosphate isomerase, glyceraldehyde phosphate dehydrogenase, phosphoglycerate kinase, pyruvate kinase, lactate dehydrogenase, and glutathione peroxidase and reductase. Cryptic activity of the enzymes was demonstrated by an increase in activity on dilution with water, which caused fragmentation of the ghosts. Aldolase and glyceraldehyde phosphate dehydrogenase were classed as firmly bound; phosphoglycerate kinase was intermediate; the others were loosely bound. Triton X-100 increased the activities of aldolase, glyceraldehyde phosphate dehydrogenase, and phosphoglycerate kinase. The pH of the medium had little effect upon the firmly bound enzymes but it markedly affected the retention of hemoglobin and the activities of the loosely bound enzymes. The presence of Mg or Ca ions enhanced the retention of hemoglobin and the activity of lactate dehydrogenase and pyruvate kinase, with little effect on aldolase and glyceraldehyde phosphate dehydrogenase. Ghosts diluted in water disintegrated into fragments and tubules or vesicles; Mg or Ca at 1mm afforded protection against this. When ghosts were treated with Triton X-100 and adenosine triphosphate, they contracted to about one-seventh of their volume. The shrunken ghosts had lost a considerable proportion of their cholesterol and protein to the medium.     

Site-specific cassette exchange and germline transmission with mouse ES cells expressing phiC31 integrase

Currently two site-specific recombinases are available for engineering the mouse genome: Cre from P1 phage and Flp from yeast. Both enzymes catalyze recombination between two 34-base pair recognition sites, lox and FRT, respectively, resulting in excision, inversion, or translocation of DNA sequences depending upon the location and the orientation of the recognition sites. Furthermore, strategies have been designed to achieve site-specific insertion or cassette exchange. The problem with both recombinase systems is that when they insert a circular DNA into the genome (trans event), two cis-positioned recognition sites are created, which are immediate substrates for excision. To stabilize the trans event, functional mutant recognition sites had to be identified. None of the systems, however, allowed efficient selection-free identification of insertion or cassette exchange. Recently, an integrase from Streptomyces phage phiC31 has been shown to function in Schizosaccharomyces pombe and mammalian cells. This enzyme recombines between two heterotypic sites: attB and attP. The product sites of the recombination event (attL and attR) are not substrates for the integrase. Therefore, the phiC31 integrase is ideal to facilitate site-specific insertions into the mammalian genome.     

Conditional and inducible transgene expression in mice through the combinatorial use of Cre-mediated recombination and tetracycline induction

Here we describe a triple transgenic mouse system, which combines the tissue specificity of any Cre-transgenic line with the inducibility of the reverse tetracycline transactivator (rtTA)/tetracycline-responsive element (tet-O)-driven transgenes. To ensure reliable rtTA expression in a broad range of cell types, we have targeted the rtTA transgene into the ROSA26 locus. The rtTA expression, however, is conditional to a Cre recombinase-mediated excision of a STOP region from the ROSA26 locus. We demonstrate the utility of this technology through the inducible expression of the vascular endothelial growth factor (VEGF-A) during embryonic development and postnatally in adult mice. Our results of adult induction recapitulate several different hepatic and immune cell pathological phenotypes associated with increased systemic VEGF-A protein levels. This system will be useful for studying genes in which temporal control of expression is necessary for the discovery of the full spectrum of functions. The presented approach abrogates the need to generate tissue-specific rtTA transgenes for tissues where well-characterized Cre lines already exist.     

Cre recombinase specificity defined by the tau locus

We generated a transgenic mouse line (tau::Cre) by targeting the Cre to the tau locus (Mapt). Based on previous reports on the expression of Tau during development, we expected the Cre recombinase to be expressed in a neuron-specific and pan-neuronal manner. However, intercrosses between the tau::Cre and the Cre-activatable reporter animals resulted in offspring with recombination either restricted to the nervous system or throughout the entire conceptus, indicating expression of Tau early in development. The percentage of neuron-specific excision was dependent on the Cre reporter used representing different Cre target sites in the mouse genome. In spite of the observed variability, our data suggest that the tau::Cre mouse line can be used for pan-neuronal recombination of floxed alleles when it is used with caution.     

Disruption of paraoxonase 3 impairs proliferation and antioxidant defenses in human A549 cells and causes embryonic lethality in mice

We had shown previously that paraoxonase 3 (PON3), a putative circulating antioxidant, was systemically upregulated in late-gestation rat, sheep, and human fetuses. Our overarching hypothesis is that preterm human infants are delivered with low levels of PON3 and that this contributes to a state of oxidative stress. We sought to determine whether absence of Pon3 was associated with reduced neonatal viability in mice and studied the offspring from crosses between Pon3(+/-) mice. The number of Pon3(-/-) animals at E10.5 and E17.5 was significantly lower than the expected 25% (9.3 and 7.9% respectively, P < 0.001). On the first day of postnatal life, this was reduced further (2.4%, significantly less than the proportion in fetal life, P = 0.04). Pon3(+/-) animals had lower body and placental weights than wild-type littermates at E17.5, an effect that was independent of the parent of origin of the mutant allele. We then studied the effect of PON3 knockdown in a human cell line, A549. Stable knockdown of PON3 using short-hairpin RNA reduced cell proliferation in 21% oxygen. We then studied the effect of transient knockdown of PON3 using short interfering RNA (siRNA) in the same cell line in low (2%) or ambient (21%) oxygen. Knockdown of PON3 using siRNA reduced total antioxidant capacity in 21% (P = 0.008) but not 2% oxygen. We conclude that the absence of Pon3 in mice resulted in increased rates of early fetal and neonatal death. Knockdown of PON3 in human cells reduced cell proliferation and total antioxidant capacity.