Transfection of Babesia bovis by Double Selection with WR99210 and Blasticidin-S and Its Application for Functional Analysis of Thioredoxin Peroxidase-1

Genetic manipulation is an essential technique to analyze gene function; however, limited methods are available for Babesia bovis, a causative pathogen of the globally important cattle disease, bovine babesiosis. To date, two stable transfection systems have been developed for B. bovis, using selectable markers blasticidin-S deaminase (bsd) or human dihydrofolate reductase (hdhfr). In this work, we combine these two selectable markers in a sequential transfection system. Specifically, a parent transgenic B. bovis line which episomally expresses green fluorescent protein (GFP) and human dihydrofolate reductase (hDHFR), was transfected with a plasmid encoding a fusion protein consisting of red fluorescent protein (RFP) and blasticidin-S deaminase (BSD). Selection with WR99210 and blasticidin-S resulted in the emergence of parasites double positive for GFP and RFP. We then applied this method to complement gene function in a parasite line in which thioredoxin peroxidase-1 (Bbtpx-1) gene was knocked out using hDHFR as a selectable marker. A plasmid was constructed harboring both RFP-BSD and Bbtpx-1 expression cassettes, and transfected into a Bbtpx-1 knockout (KO) parasite. Transfectants were independently obtained by two transfection methods, episomal transfection and genome integration. Complementation of Bbtpx-1 resulted in full recovery of resistance to nitrosative stress, via the nitric oxide donor sodium nitroprusside, which was impaired in the Bbtpx-1 KO parasites. In conclusion, we developed a sequential transfection method in B. bovis and subsequently applied this technique in a gene complementation study. This method will enable broader genetic manipulation of Babesia toward enhancing our understanding of the biology of this parasite.     

Apicomplexan parasites and subversion of the host cell microRNA pathway

RNA silencing plays a major role in innate antiviral and antibacterial defenses in plants, insects, and animals through the action of microRNAs (miRNAs). miRNAs can act in favor of the microorganism, either when it is pathogen-encoded or when the microorganism subverts host miRNAs to its benefit. Recent data point to the possibility that apicomplexan parasites have developed tactics to interfere with host miRNA populations in a parasite-specific manner, thereby identifying the RNA-silencing pathway as a new means to reshape their cellular environment. This review highlights the current understanding and new insights concerning the mechanisms that could be involved and the potential roles of the host microRNome (miRNome) in apicomplexan infection.     

Overexpression of the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) in skeletal muscle repatterns energy metabolism in the mouse

Transgenic mice, containing a chimeric gene in which the cDNA for phosphoenolpyruvate carboxykinase (GTP) (PEPCK-C) (EC 4.1.1.32) was linked to the alpha-skeletal actin gene promoter, express PEPCK-C in skeletal muscle (1-3 units/g). Breeding two founder lines together produced mice with an activity of PEPCK-C of 9 units/g of muscle (PEPCK-C(mus) mice). These mice were seven times more active in their cages than controls. On a mouse treadmill, PEPCK-C(mus) mice ran up to 6 km at a speed of 20 m/min, whereas controls stopped at 0.2 km. PEPCK-C(mus) mice had an enhanced exercise capacity, with a VO(2max) of 156 +/- 8.0 ml/kg/min, a maximal respiratory exchange ratio of 0.91 +/- 0.03, and a blood lactate concentration of 3.7 +/- 1.0 mm after running for 32 min at a 25 degrees grade; the values for control animals were 112 +/- 21 ml/kg/min, 0.99 +/- 0.08, and 8.1 +/- 5.0 mm respectively. The PEPCK-C(mus) mice ate 60% more than controls but had half the body weight and 10% the body fat as determined by magnetic resonance imaging. In addition, the number of mitochondria and the content of triglyceride in the skeletal muscle of PEPCK-C(mus) mice were greatly increased as compared with controls. PEPCK-C(mus) mice had an extended life span relative to control animals; mice up to an age of 2.5 years ran twice as fast as 6-12-month-old control animals. We conclude that overexpression of PEPCK-C repatterns energy metabolism and leads to greater longevity.     

Functional interplay between histone demethylase and deacetylase enzymes

Histone deacetylase (HDAC) inhibitors are a promising class of anticancer agents for the treatment of solid and hematological malignancies. The precise mechanism by which HDAC inhibitors mediate their effects on tumor cell growth, differentiation, and/or apoptosis is the subject of intense research. Previously we described a family of multiprotein complexes that contain histone deacetylase 1/2 (HDAC1/2) and the histone demethylase BHC110 (LSD1). Here we show that HDAC inhibitors diminish histone H3 lysine 4 (H3K4) demethylation by BHC110 in vitro. In vivo analysis revealed an increased H3K4 methylation concomitant with inhibition of nucleosomal deacetylation by HDAC inhibitors. Reconstitution of recombinant complexes revealed a functional connection between HDAC1 and BHC110 only when nucleosomal substrates were used. Importantly, while the enzymatic activity of BHC110 is required to achieve optimal deacetylation in vitro, in vivo analysis following ectopic expression of an enzymatically dead mutant of BHC110 (K661A) confirmed the functional cross talk between the demethylase and deacetylase enzymes. Our studies not only reveal an intimate link between the histone demethylase and deacetylase enzymes but also identify histone demethylation as a secondary target of HDAC inhibitors.     

Der Einflu? der Mauser auf Blutparameter beim StarSturnus vulgaris

Moulting Starlings (n=9) show increased plasma values of glucose: 314,2±24,96 mg/dl; the corresponding values were 277,5±28,8 for non-moulting birds (mean value over all periods: 295,6±32,2 mg/dl). In all other blood values (e. g. blood cells, hemoglobin, protein-concentration, osmotic pressure, hematocrit) no significant differences between moulting and non-moulting birds could be observed (Prinzinger &Hakimi 1996); e. g. plasma-cholesterin was 195,6±22,6 mg/dl (moulting) and 197,5±43,8 (non-moulting), respectively.     

Reduced immunogenicity and improved pharmacokinetics of humanized anti-Tac in cynomolgus monkeys

The anti-Tac mAb has been shown to bind to the p55 chain of the IL-2R, block IL-2 binding and inhibit T cell proliferation. A humanized form of anti-Tac (HAT) has been constructed that retains the binding properties of murine anti-Tac (MAT). These two mAb were evaluated in cynomolgus monkeys to compare relative immunogenicity and pharmacokinetic properties. Monkeys treated with HAT daily for 14 days exhibited anti-HAT antibody titers which were 5- to 10-fold lower than their MAT-treated counterparts and these antibodies developed later than in the MAT-treated monkeys. Two of four monkeys receiving a single injection of MAT developed anti-MAT antibodies, whereas none of four monkeys developed antibodies after a single treatment with HAT. In monkeys injected with either HAT or MAT daily for 14 days, the anti-antibody titers induced were inversely related to the amount of anti-Tac administered. Antibodies that developed against MAT were both anti-isotypic and anti-idiotypic, whereas those developed against HAT appeared to be predominantly anti-idiotypic. The pharmacokinetic properties, that is the half-life and area under the curve values, of HAT were also significantly different from those of MAT. The area under the curve values for HAT in naive monkeys were approximately twofold more than those for MAT, and the mean serum half-life of HAT was 214 h, approximately four- to fivefold more than MAT. These pharmacokinetic values were reduced in monkeys previously sensitized with HAT or MAT suggesting that the presence of anti-antibodies altered these parameters.     

Murine epidermal side population possesses unique angiogenic properties

Total epidermal keratinocytes are a heterogeneous population of cells, including undifferentiated stem/progenitor cells (EpSPs) and their more differentiated progeny (Non-SP cells). Our previous in vivo data showed that EpSPs enhanced blood flow restoration when injected into an ischemic limb, whereas Non-SP cells had no significant effect on in vivo blood flow restoration. However, the cellular and molecular mechanisms of this observation remain largely unknown. Therefore, the aim of this study was to investigate the angiogenic properties of different epidermal subpopulations in vitro and the mechanism by which EpSPs enhanced blood flow in vivo. Using migration assay and capillary network formation, we show that EpSPs secrete higher levels of pro-angiogenic molecules compared to Non-SP cells, unsorted keratinocytes and fibroblasts in vitro. Secretion of vascular endothelial growth factor (VEGF) was detected at higher levels in EpSP conditioned medium than the medium conditioned by other epidermal subpopulations and fibroblasts. Also, RT-PCR analyses revealed a unique angiogenic gene profile for EpSPs. Finally, gene array data indicate significant changes in angiogenic gene expression six days after cell injection in murine ischemic limbs. Therefore, we conclude that EpSPs possess unique angiogenic properties and that these cells may be indirectly responsible for the angiogenic response previously observed in our ischemic limb model.     

The motor protein kinesin-1 links neurofibromin and merlin in a common cellular pathway of neurofibromatosis

Mutations in either of the two tumor suppressor genes NF1 (neurofibromin) and NF2 (merlin) result in Neurofibromatosis, a condition predisposing individuals to developing a variety of benign and malignant tumors of the central and peripheral nervous systems. Here we report the identification of two distinct NF1-containing complexes, one in the soluble and the other in the particulate fraction of HeLa extract. We show that the soluble NF1 complex delineates a large holo-NF1 complex (2 MDa) encompassing the components of a smaller particulate core-NF1 complex (400 kDa). Purification of the core-NF1 complex followed by mass spectrometric analysis revealed the motor protein, kinesin-1 heavy chain (HsuKHC/KIF5B), as a catalytic subunit of both NF-1-containing complexes. Importantly, although NF1 and NF2 are not in a stable association, NF2 is also a component of a distinct kinesin-1-containing complex. These results point to kinesin-1 as a common denominator between NF1 and NF2.