Activities of Various FLP Recombinases Expressed by Adenovirus Vectors in Mammalian Cells

FLP, like Cre, is a frequently employed site-specific recombinase. Because wild-type FLP (wtFLP) is thermolabile, a thermostable FLP mutant (FLPe) has been developed for efficient recombination of FLP in studies using mammalian cells and animals. FLPe and wtFLP have been compared in multiple assays in vitro and in vivo, and in mouse genetics, FLPe has been shown to be very effective like Cre. Here we show an adenovirus vector (AdV) system to be valuable for quantitative measurements of the enzyme activity in mammalian cells and, using this system, precisely compare activities of wtFLP and FLPe. Unexpectedly, we found that the recombination efficiency of FLPe enzyme was lower on a molar basis than that of wtFLP even at 37 °C and, consequently, that the higher recombination yield per transduced AdV genome expressing FLPe compared to wtFLP was due not to inherently higher enzyme activity, but rather to higher steady-state levels of FLPe by its thermostability. Therefore, trying to increase FLPe levels further, we generated a “humanized” FLPe (hFLPe) gene with codon usage optimized for mammals. hFLPe produced about 10-fold more FLPe enzyme in transfection experiments than FLPe, as expected. However, hFLPe-expressing AdV was unstable and could not be prepared without deletion, suggesting that a subtle deleterious effect of FLP on 293 cells may exist. With hFLPe-expressing AdV thus unavailable, of the AdV constructs tested, AdV-expressing FLPe yielded the most recombined targets, despite the lower recombination efficiency of FLPe per enzyme molecule compared with that of wtFLP. We found hFLPe to be valuable for plasmid transfection, and its properties are probably suitable for experiments involving cell lines and transgenic mice.     

Transient proteasome inhibition as a strategy to enhance lentiviral transduction of hematopoietic CD34(+) cells and T lymphocytes: implications for the use of low viral doses and large-size vectors

The proteasome system restricts lentiviral transduction of stem cells. We exploited proteasome inhibition as a strategy to enhance transduction of both hematopoietic stem cells (HSC) and T lymphocytes with low dose or large-size lentiviral vectors (LV). HSC showed higher transduction efficiency if transiently exposed to proteasome inhibitor MG132 (41.8% vs 10.7%, p < 0.0001). Treatment with MG132 (0.5 μM) retained its beneficial effect with 3 different LV of increasing size up to 10.9 Kb (p < 0.01). We extended, for the first time, the application of proteasome inhibition to the transduction of T lymphocytes. A transient exposure to MG132 significantly improved lentiviral T-cell transduction. The mean percentage of transduced T cells progressively increased from 13.5% of untreated cells, to 21% (p = 0.3), 30% (p = 0.03) and 37% (p = 0.01) of T lymphocytes that were pre-treated with MG132 at 0.1, 0.5 and 1 μM, respectively. MG132 did not affect viability or functionality of HSC or T cells, nor significantly increased the number of integrated vector copies. Transient proteasome inhibition appears as a new procedure to safely enhance lentiviral transduction of HSC and T lymphocytes with low viral doses. This approach could be useful in settings where the use of large size vectors may impair optimal viral production.     

Beta2-adrenergic receptor regulates cardiac fibroblast autophagy and collagen degradation

Autophagy is a physiological degradative process key to cell survival during nutrient deprivation, cell differentiation and development. It plays a major role in the turnover of damaged macromolecules and organelles, and it has been involved in the pathogenesis of different cardiovascular diseases. Activation of the adrenergic system is commonly associated with cardiac fibrosis and remodeling, and cardiac fibroblasts are key players in these processes. Whether adrenergic stimulation modulates cardiac fibroblast autophagy remains unexplored. In the present study, we aimed at this question and evaluated the effects of b₂-adrenergic stimulation upon autophagy. Cultured adult rat cardiac fibroblasts were treated with agonists or antagonists of beta-adrenergic receptors (b-AR), and autophagy was assessed by electron microscopy, GFP-LC3 subcellular distribution, and immunowesternblot of endogenous LC3. The predominant expression of b₂-ARs was determined and characterized by radioligand binding assays using [³H]dihydroalprenolol. Both, isoproterenol and norepinephrine (non-selective b-AR agonists), as well as salbutamol (selective b₂-AR agonist) increased autophagic flux, and these effects were blocked by propanolol (b-AR antagonist), ICI-118,551 (selective b₂-AR antagonist), 3-methyladenine but not by atenolol (selective b₁-AR antagonist). The increase in autophagy was correlated with an enhanced degradation of collagen, and this effect was abrogated by the inhibition of autophagic flux. Overall, our data suggest that b₂-adrenergic stimulation triggers autophagy in cardiac fibroblasts, and that this response could contribute to reduce the deleterious effects of high adrenergic stimulation upon cardiac fibrosis.     

Plasmids supplying the Q-qut-controlled gam function permit growth of lambda red- gam- (Fec-) bacteriophages on recA- hosts

A plasmid, pgam, has been constructed which expresses the phage lambda gene, gam, under the control of the lambda late promoter, p'R, contained in a form of a p'R-qut-t'R1 module. Lambda red- gam-, which normally do not grow on recA- hosts, are able to grow on recA- hosts containing pgam, because their Q function can turn on the gam gene expression. This facilitates cloning with lambda red- gam- vectors in recA- hosts.     

Eimeria tenella: construction of a recombinant fowlpox virus expressing rhomboid gene and its protective efficacy against homologous infection

A recombinant fowlpox virus (rFPV) expressing the Eimeria tenella rhomboid gene was constructed and its protective efficacy against homologous infection in chickens determined. Three-day-old-specific pathogen free (SPF) chickens were immunized s.c. with 10(2) plaque forming units (PFU), 10(4) PFU, or 10(6) PFU of rFPV-rhomboid, and challenged with 5x10(4) homologous sporulated oocysts 14 days post-immunization (p.i.). The specific antibody response and lymphocyte proliferation were measured 1, 2, 3 and 4 weeks p.i. Oocyst output, body weight gains and lesion scores were measured to evaluate the protective effects of immunization. rFPV-rhomboid elicited a specific humoral immune response and stimulated proliferation of peripheral blood lymphocytes. The lesion scores in groups vaccinated with rFPV-rhomboid were significantly higher than in other groups. At the same time, rFPV-rhomboid improved body weight significantly compared with other groups. Immunization with rFPV-rhomboid reduced oocyst shedding significantly, resulting in a protection rate of 39.6%, 41.1% or 41.7% given a dose of 10(2) PFU, 10(4) PFU, or 10(6) PFU of rFPV-rhomboid, respectively. These results indicated that rFPV can induce immune responses and offer partial protection of chickens against E. tenella challenge.     

Beneficial effects of population bottlenecks in an RNA virus evolving at increased error rate

RNA viruses replicate their genomes with a very high error rate and constitute highly heterogeneous mutant distributions similar to the molecular quasispecies introduced to explain the evolution of prebiotic replicators. The genetic information included in a quasispecies can only be faithfully transmitted below a critical error rate. When the error threshold is crossed, the population structure disorganizes, and it is substituted by a randomly distributed mutant spectrum. For viral quasispecies, the increase in error rate is associated with a decrease in specific infectivity that can lead to the extinction of the population. In contrast, a strong resistance to extinction has been observed in populations subjected to bottleneck events despite the increased accumulation of mutations. In the present study, we show that the mutagenic nucleoside analogue 5-azacytidine (AZC) is a potent mutagen for bacteriophage Qβ. We have evaluated the effect of the increase in the replication error rate in populations of the bacteriophage Qβ evolving either in liquid medium or during development of clonal populations in semisolid agar. Populations evolving in liquid medium in the presence of AZC were extinguished, while during plaque development in the presence of AZC, the virus experienced a significant increase in the replicative ability. Individual viruses isolated from preextinction populations could withstand high error rates during a number of plaque-to-plaque transfers. The response to mutagenesis is interpreted in the light of features of plaque development versus infections by free-moving virus particles and the distance to a mutation-selection equilibrium. The results suggest that clonal bacteriophage populations away from equilibrium derive replicative benefits from increased mutation rates. This is relevant to the application of lethal mutagenesis in vivo, in the case of viruses that encounter changing environments and are transmitted from cell to cell under conditions of limited diffusion that mimic the events taking place during plaque development.     

REDOR NMR characterization of DNA packaging in bacteriophage T4

Bacteriophage T4 is a large-tailed Escherichia coli virus whose capsid is 120 × 86 nm. ATP-driven DNA packaging of the T4 capsid results in the loading of a 171-kb genome in less than 5 min during viral infection. We have isolated 50-mg quantities of uniform ¹⁵N- and [ε-¹⁵N]lysine-labeled bacteriophage T4. We have also introduced ¹⁵NH₄⁺ into filled, unlabeled capsids from synthetic medium by exchange. We have examined lyo- and cryoprotected lyophilized T4 using ¹⁵N{³¹P} and ³¹P{¹⁵N} rotational-echo double resonance. The results of these experiments have shown that (i) packaged DNA is in an unperturbed duplex B-form conformation; (ii) the DNA phosphate negative charge is balanced by lysyl amines (3.2%), polyamines (5.8%), and monovalent cations (40%); and (iii) 11% of lysyl amines, 40% of -NH₂ groups of polyamines, and 80% of monovalent cations within the lyophilized T4 capsid are involved in the DNA charge balance. The NMR evidence suggests that DNA enters the T4 capsid in a charge-unbalanced state. We propose that electrostatic interactions may provide free energy to supplement the nanomotor-driven T4 DNA packaging.