12 research outputs found
Transgenic Mice for a Tamoxifen-Induced, Conditional Expression of the Cre Recombinase in Osteoclasts
Background: Studies on osteoclasts, the bone resorbing cells, have remained limited due to the lack of transgenic mice allowing the conditional knockout of genes in osteoclasts at any time during development or adulthood. Methodology/Principal Finding: We report here on the generation of transgenic mice which specifically express a tamoxifen-inducible Cre recombinase in osteoclasts. These mice, generated on C57BL/6 and FVB background, express a fusion Cre recombinase-ERT2 protein whose expression is driven by the promoter of cathepsin K (CtsK), a gene highly expressed in osteoclasts. We tested the cellular specificity of Cre activity in CtsKCreERT2 strains by breeding with Rosa26LacZ reporter mice. PCR and histological analyses of the CtsKCreERT2LacZ positive adult mice and E17.5 embryos show that Cre activity is restricted largely to bone tissue. In vitro, primary osteoclasts derived from the bone marrow of CtsKCreERT2+/2LacZ+/2 adult mice show a Cre-dependent b-galactosidase activity after tamoxifen stimulation
RAGE-Dependent Effect of Exogenous Methylglyoxal Intake on Lung Biomechanics in Mice
Methylglyoxal (MG) is a known highly reactive dicarbonyl and precursor to free radicals and advanced glycation end-products (AGEs). It is discussed to be involved in tissue aging and in the pathogenesis of different degenerative diseases. The effect of long-term oral administration of MG, simulating dietary MG intake, on the lung biomechanics of wild type (WT) and receptor for advanced glycation end-products knockout (RAGE-KO) mice was studied using an ex vivo ventilation system starting at the age of 6 months and after feeding for 6 and 12 months with MG. Our results showed that MG was taken up in the circulation and efficiently excreted with urine. The amount of free urinary MG measured after 12 months of feeding was lowered. After 12 months feeding, a significant airway resistance increase accompanied by a decrease of the maximal inspiratory airflow was observed in WT animals. No effect of MG in lung function of RAGE-KO mice could be detected. Despite the evidence that MG entered the systemic circulation, no MG-derived AGE accumulation was detected in the lung lysates in dependency on MG-feeding. Our data indicate that the short-term feeding of MG has little effect in vivo. Only after long-term treatment was MG secretion reduced, leading to tissue impairment
Borrelia burgdorferi Organisms Lacking Plasmids 25 and 28-1 Are Internalized by Human Blood Phagocytes at a Rate Identical to That of the Wild-Type Strain
Lyme borreliosis caused by Borrelia burgdorferi is a persistent infection capable of withstanding the host's vigorous immune response. Several reports have shown that the spirochete's linear plasmids 25 and 28-1 are essential for its infectivity. In this context, it was proposed that Borrelia burgdorferi organisms control their uptake by macrophages and polymorphonuclear leukocytes (PMNs) through plasmid-encoded proteins and that this mechanism confers resistance to phagocytosis. To investigate this proposal, a precise flow-cytometry-based method with human blood was used to study the impact of the plasmids 25 and 28-1 on B. burgdorferi clearance over 150 min and to investigate whether low-passage organisms are more resistant to phagocytosis than high-passage B. burgdorferi. Exposure of human blood PMNs or blood monocytes to fluorescein isothiocyanate-labeled B. burgdorferi B31 organisms lacking the linear plasmids 25, 28-1, or both revealed that all spirochete populations were internalized at the same rate as the wild-type borrelia parent strain B31. Moreover, no differences in phagocytosis kinetics were detected when low- or high-passage wild-type B. burgdorferi B31 or N40 were cocultured with blood cells. Plasmid loss and probable associated surface protein changes due to serial in vitro propagation of B. burgdorferi do not affect the resistance of these organisms to internalization by phagocytic cells. In particular, we found no evidence for a plasmid-controlled (lp25 and lp28-1) resistance of B. burgdorferi to phagocytosis by leukocytes of the host's innate immune system
The Effect of Resveratrol on Mitochondrial Function in Myoblasts of Patients with the Common m.3243A>G Mutation
Mitochondrial function is essential for ATP-supply, especially in response to different cellular stressors. Increased mitochondrial biogenesis resulting from caloric restriction (CR) has been reported. Resveratrol (RSV) is believed to mimic the physiological effects of CR mainly via a sirtuin (SIRT) 1-dependent pathway. The effect of RSV on the physiological function of mitochondrial respiratory complexes was evaluated using a Seahorse XF96. Myoblasts of five patients harboring the m.3243A>G mutation and five controls were analyzed. The relative expression of several genes involved in mitochondrial biogenesis was evaluated for a better understanding of the coherent mechanisms. Additionally, media-dependent effects of nutritional compounds and hormonal restrictions (R) on myoblasts from patients and controls in the presence or absence of RSV were investigated. Culturing of myoblasts under these conditions led to an upregulation of almost all the investigated genes compared to normal nutrition. Under normal conditions, there was no positive effect of RSV on mitochondrial respiration in patients and controls. However, under restricted conditions, the respiratory factors measured by Seahorse were improved in the presence of RSV. Further studies are necessary to clarify the involved mechanisms and elucidate the controversial effects of resveratrol on SIRT1 and SIRT3 expression
Development of standards of internal financial control of the enterprise
В статті аналізується стан законодавства, що регулює здійснення внутрішнього контролю на підприємствах. Обґрунтовується створення внутрішніх стандартів контролю для підвищення ефективності організації внутрішнього контролю на підприємствах.The article analyzes the state law governing the internal control in enterprises. Substantiated creation of internal control standards to improve the efficiency of internal control in enterprises
Expression of Cre and β-galactosidase in adult mouse tissues.
<p>β-galactosidase and Cre expression in organs and bones of CtsKCreERT2+/−LacZ+/− (strain #4).Total RNAs were isolated from various tissues and expression was detected by semi quantitative (A) and quantitative (B) PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037592#s3" target="_blank">materials and methods</a>. GAPDH was used as internal control and normalization.</p
Tissue distribution of functional Cre recombinase in CtsKCreERT2LacZ E17.5 embryos.
<p>CtsKCreERT2LacZ and wild-type females were mated with ROSA26 males. Pregnant females were treated with tamoxifen as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037592#s3" target="_blank">materials and methods</a>. At day E17.5, the embryos were collected and analyzed for β-galactosidase activity (X-gal staining) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037592#s3" target="_blank">materials and methods</a> using embryos with various genotypes.</p
CtsKCreERT2 construct and <i>In vitro</i> functionality.
<p><b>A</b>: Schematic diagram of transgenic CtsKCreERT2 expression construct used to generate transgenic mice, which includes the full sequence of the CtsK promoter, the CreERT2 fusion sequence, and the SV40 polyA signal. <b>B.</b> Schematic representation of the floxed neomycin construct before and after Cre-mediated recombination and expected size of PCR products. <b>C</b>: Cre-mediated recombination in Hela cells (upper panel). Hela cells transfected with the floxed neomycin plasmid only (lane 1) or co-transfected with CtsKCreERT2 and floxed nemycin constructs and treated (lane 2) or not (lane 3) with 4-OHT. Untrasfected Hela cells are shown in lane 4. The recombination resulted in a 200 bp PCR fragment. Cre expression was also monitored in the same samples by PCR (lower panel). M: molecular weight markers.</p