Human Apolipoprotein B Transgenic Mice Generated with 207- and 145-Kilobase Pair Bacterial Artificial Chromosomes. Evidence that a distant 5'-element confers appropriate transgene expression in the intestine

We reported previously that ~80-kilobase pair (kb) P1 bacteriophage clones spanning either the human or mouse apoB gene (clones p158 and p649, respectively) confer apoB expression in the liver of transgenic mice, but not in the intestine. We hypothesized that the absence of intestinal expression was due to the fact that these clones lacked a distant DNA element controlling intestinal expression. To test this possibility, transgenic mice were generated with 145- and 207-kb bacterial artificial chromosomes (BACs) that contained the human apoB gene and more extensive 5'- and 3'-flanking sequences. RNase protection, in situ hybridization, immunohistochemical, and genetic complementation studies revealed that the BAC transgenic mice manifested appropriate apoB gene expression in both the intestine and the liver, indicating that both BACs contained the distant intestinal element. To determine whether the regulatory element was located 5' or 3' to the apoB gene, transgenic mice were generated by co-microinjecting embryos with p158 and either the 5'- or 3'-sequences from the 145-kb BAC. Analysis of these mice indicated that the apoB gene's intestinal element is located 5' to the structural gene. Cumulatively, the transgenic mouse studies suggest that the intestinal element is located between -33 and -70 kb 5' to the apoB gene

Cooperative regulation of extracellular signal-regulated kinase activation and cell shape change by filamin A and beta-arrestins.: FLNA AND ßarr COOPERATE TO REGULATE ERK AND CELL SHAPE

14 pagesbeta-Arrestins (betaarr) are multifunctional adaptor proteins that can act as scaffolds for G protein-coupled receptor activation of mitogen-activated protein kinases (MAPK). Here, we identify the actin-binding and scaffolding protein filamin A (FLNA) as a betaarr-binding partner using Son of sevenless recruitment system screening, a classical yeast two-hybrid system, coimmunoprecipitation analyses, and direct binding in vitro. In FLNA, the betaarr-binding site involves tandem repeat 22 in the carboxyl terminus. betaarr binds FLNA through both its N- and C-terminal domains, indicating the presence of multiple binding sites. We demonstrate that betaarr and FLNA act cooperatively to activate the MAPK extracellular signal-regulated kinase (ERK) downstream of activated muscarinic M1 (M1MR) and angiotensin II type 1a (AT1AR) receptors and provide experimental evidence indicating that this phenomenon is due to the facilitation of betaarr-ERK2 complex formation by FLNA. In Hep2 cells, stimulation of M1MR or AT1AR results in the colocalization of receptor, betaarr, FLNA, and active ERK in membrane ruffles. Reduction of endogenous levels of betaarr or FLNA and a catalytically inactive dominant negative MEK1, which prevents ERK activation, inhibit membrane ruffle formation, indicating the functional requirement for betaarr, FLNA, and active ERK in this process. Our results indicate that betaarr and FLNA cooperate to regulate ERK activation and actin cytoskeleton reorganization

Androgen modulation of pro-inflammatory and antiinflammatory cytokines during preadipocyte differentiation

Background: Macrophages and adipocytes contribute to release of cytokines resulting in the chronic inflammatory profile of the metabolic syndrome. The local increase of proinflammatory cytokines impairs adipogenesis, resulting in formation of dysfunctional adipocytes that are unable to store and handle lipids. The altered lipid fluxes in/from adipocytes affect whole-body metabolism. We investigated the role of androgens on adipocyte-derived proinflammatory and anti-inflammatory cytokines during preadipocyte differentiation. Materials and methods: Various differentiation methods were used to obtain full conversion of 3T3-L1 into mature adipocytes. The degree of adipocyte conversion in the presence/absence of dihydrotestosterone (DHT) was analyzed by measuring intracellular triglycerides (Oil Red O staining). The effects of DHT administration on interleukin 1Β (IL-1Β), IL-2, IL-6, IL-10, IL-12, interferon γ (IFNΓ) and tumor necrosis factor α (TNFα) secretion was measured at days 0, 4, 6 and 8 of differentiation using the SearchLight multiplex protein array. Results: DHT regulates a number of cytokines in committed and mature 3T3-L1 adipocytes. IL-1Β and TNFα were readily suppressed at the very early stages of differentiation. IFNΓ release was inhibited at day 4, but the effect was no longer detectable on day 8. IL-6 and IL-12 were significantly reduced at day 8 of differentiation. Conversely, the differentiation-dependent increase of IL-2 and IL-10 was further stimulated by DHT since day 0. Conclusions: We provide evidence that androgens promote an anti-inflammatory profile that parallels the acquisition of a functional adipocyte phenotype. The crosstalk between androgens, adipocyte-derived mediators of inflammation and intracellular lipid fluxes could have profound implications on metabolism of men with obesity and metabolic syndrome. © 2010, by Walter de Gruyter Berlin New York. All rights reserved

Comparative health technology assessment of robotic-assisted, direct manual laparoscopic and open surgery:a prospective study

Background: Despite many publications reporting on the increased hospital cost of robotic-assisted surgery (RAS) compared to direct manual laparoscopic surgery (DMLS) and open surgery (OS), the reported health economic studies lack details on clinical outcome, precluding valid health technology assessment (HTA). Methods: The present prospective study reports total cost analysis on 699 patients undergoing general surgical, gynecological and thoracic operations between 2011 and 2014 in the Italian Public Health Service, during which period eight major teaching hospitals treated the patients. The study compared total healthcare costs of RAS, DMLS and OS based on prospectively collected data on patient outcome in addition to healthcare costs incurred by the three approaches. Results: The cost of RAS operations was significantly higher than that of OS and DMLS for both gynecological and thoracic operations (p < 0.001). The study showed no significant difference in total costs between OS and DMLS. Total costs of general surgery RAS were significantly higher than those of OS (p < 0.001), but not against DMLS general surgery. Indirect costs were significantly lower in RAS compared to both DMLS general surgery and OS gynecological surgery due to the shorter length of hospital stay of RAS approach (p < 0.001). Additionally, in all specialties compared to OS, patients treated by RAS experienced a quicker recovery and significantly less pain during the hospitalization and after discharge. Conclusions: The present HTA while confirming higher total healthcare costs for RAS operations identified significant clinical benefits which may justify the increased expenditure incurred by this approach

Differential nucleocytoplasmic shuttling of beta-arrestins. Characterization of a leucine-rich nuclear export signal in beta-arrestin2

International audiencebeta-arrestins (betaarrs) are two highly homologous proteins that uncouple G protein-coupled receptors from their cognate G proteins, serve as adaptor molecules linking G protein-coupled receptors to clathrin-coat components (AP-2 complex and clathrin), and act as scaffolding proteins for ERK1/2 and JNK3 cascades. A striking difference between the two betaarrs (betaarr1 and betaarr2) is that betaarr1 is evenly distributed throughout the cell, whereas betaarr2 shows an apparent cytoplasmic localization at steady state. Here, we investigate the molecular determinants underlying this differential distribution. betaarr2 is constitutively excluded from the nucleus by a leptomycin B-sensitive pathway because of the presence of a classical leucine-rich nuclear export signal in its C terminus (L395/L397) that is absent in betaarr1. In addition, using a nuclear import assay in yeast we showed that betaarr2 is actively imported into the nucleus, suggesting that betaarr2 undergoes constitutive nucleocytoplasmic shuttling. In cells expressing betaarr2, JNK3 is mostly cytosolic. A point mutation of the nuclear export signal (L395A) in betaarr2, which was sufficient to redistribute betaarr2 from the cytosol to the nucleus, also caused the nuclear relocalization of JNK3. These data indicate that the nucleocytoplasmic shuttling of betaarr2 controls the subcellular distribution of JNK3