miR-200b downregulates CFTR during hypoxia in human lung epithelial cells

Abstract Background Hypoxic conditions induce the expression of hypoxia-inducible factors (HIFs) that allow cells to adapt to the changing conditions and alter the expression of a number of genes including the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is a low abundance mRNA in airway epithelial cells even during normoxic conditions, but during hypoxia its mRNA expression decreases even further. Methods In the current studies, we examined the kinetics of hypoxia-induced changes in CFTR mRNA and protein levels in two human airway epithelial cell lines, Calu-3 and 16HBE14o-, and in normal primary bronchial epithelial cells. Our goal was to examine the posttranscriptional modifications that affected CFTR expression during hypoxia. We utilized in silico predictive protocols to establish potential miRNAs that could potentially regulate CFTR message stability and identified miR-200b as a candidate molecule. Results Analysis of each of the epithelial cell types during prolonged hypoxia revealed that CFTR expression decreased after 12 h during a time when miR-200b was continuously upregulated. Furthermore, manipulation of the miRNA levels during normoxia and hypoxia using miR-200b mimics and antagomirs decreased and increased CFTR mRNA levels, respectively, and thus established that miR-200b downregulates CFTR message levels during hypoxic conditions. Conclusion The data suggest that miR-200b may be a suitable target for modulating CFTR levels in vivo

Pkhd1

Autosomal-recessive polycystic kidney disease (ARPKD; MIM #263200) is a severe, hereditary, hepato-renal fibrocystic disorder that causes early childhood morbidity and mortality. Mutations in the polycystic kidney and hepatic disease 1 (PKHD1) gene, which encodes the protein fibrocystin/polyductin complex (FPC), cause all typical forms of ARPKD. Several mouse lines carrying diverse, genetically engineered disruptions in the orthologous Pkhd1 gene have been generated, but none expresses the classic ARPKD renal phenotype. In the current study, we characterized a spontaneous mouse Pkhd1 mutation that is transmitted as a recessive trait and causes cysticliver (cyli), similar to the hepato-biliary disease in ARPKD, but which is exacerbated by age, sex, and parity. We mapped the mutation to Chromosome 1 and determined that an insertion/deletion mutation causes a frameshift within Pkhd1 exon 48, which is predicted to result in a premature termination codon (UGA). Pkhd

Suppression of CFTR-mediated Cl- Secretion of Airway Epithelium in Vitamin C-deficient Mice

Hyperoxic ventilation induces detrimental effects on the respiratory system, and ambient oxygen may be harmful unless compensated by physiological anti-oxidants, such as vitamin C. Here we investigate the changes in electrolyte transport of airway epithelium in mice exposed to normobaric hyperoxia and in gulonolacton oxidase knock-out (gulo[-/-]) mice without vitamin C (Vit-C) supplementation. Short-circuit current (Isc) of tracheal epithelium was measured using Ussing chamber technique. After confirming amiloride-sensitive Na+ absorption (ΔIsc,amil), cAMP-dependent Cl- secretion (ΔIsc,forsk) was induced by forskolin. To evaluate Ca2+-dependent Cl- secretion, ATP was applied to the luminal side (ΔIsc,ATP). In mice exposed to 98% PO2 for 36 hr, ΔIsc,forsk decreased, ΔIsc,amil and ΔIsc,ATP was not affected. In gulo(-/-) mice, both ΔIsc,forsk and ΔIsc,ATP decreased from three weeks after Vit-C deprivation, while both were unchanged with Vit-C supplementation. At the fourth week, tissue resistance and all electrolyte transport activities were decreased. An immunofluorescence study showed that the expression of cystic fibrosis conductance regulator (CFTR) was decreased in gulo(-/-) mice, whereas the expression of KCNQ1 K+ channel was preserved. Taken together, the CFTR-mediated Cl- secretion of airway epithelium is susceptible to oxidative stress, which suggests that supplementation of the antioxidant might be beneficial for the maintenance of airway surface liquid

Serum and glucocorticoid-inducible kinase1 increases plasma membrane wt-CFTR in human airway epithelial cells by inhibiting its endocytic retrieval

Background: Chloride (Cl) secretion by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) located in the apical membrane of respiratory epithelial cells plays a critical role in maintenance of the airway surface liquid and mucociliary clearance of pathogens. Previously, we and others have shown that the serum and glucocorticoid-inducible kinase-1 (SGK1) increases wild type CFTR (wt-CFTR) mediated Cl transport in Xenopus oocytes by increasing the amount of wt-CFTR protein in the plasma membrane. However, the effect of SGK1 on the membrane abundance of wt-CFTR in airway epithelial cells has not been examined, and the mechanism whereby SGK1 increases membrane wt-CFTR has also not been examined. Thus, the goal of this study was to elucidate the mechanism whereby SGK1 regulates the membrane abundance of wt-CFTR in human airway epithelial cells. Methods and Results: We report that elevated levels of SGK1, induced by dexamethasone, increase plasma membrane abundance of wt-CFTR. Reduction of SGK1 expression by siRNA (siSGK1) and inhibition of SGK1 activity by the SGK inhibitor GSK 650394 abrogated the ability of dexamethasone to increase plasma membrane wt-CFTR. Overexpression of a constitutively active SGK1 (SGK1-S422D) increased plasma membrane abundance of wt-CFTR. To understand the mechanism whereby SGK1 increased plasma membrane wt-CFTR, we examined the effects of siSGK1 and SGK1-S442D on the endocytic retrieval of wt-CFTR. While siSGK1 increased wt-CFTR endocytosis, SGK1-S442D inhibited CFTR endocytosis. Neither siSGK1 nor SGK1-S442D altered the recycling of endocytosed wt-CFTR back to the plasma membrane. By contrast, SGK1 increased the endocytosis of the epidermal growth factor receptor (EGFR). Conclusion: This study demonstrates for the first time that SGK1 selectively increases wt-CFTR in the plasma membrane of human airway epithelia cells by inhibiting its endocytic retrieval from the membrane. © 2014 Bomberger et al

Long-Distance Delivery of Bacterial Virulence Factors by Pseudomonas aeruginosa Outer Membrane Vesicles

Bacteria use a variety of secreted virulence factors to manipulate host cells, thereby causing significant morbidity and mortality. We report a mechanism for the long-distance delivery of multiple bacterial virulence factors, simultaneously and directly into the host cell cytoplasm, thus obviating the need for direct interaction of the pathogen with the host cell to cause cytotoxicity. We show that outer membrane–derived vesicles (OMV) secreted by the opportunistic human pathogen Pseudomonas aeruginosa deliver multiple virulence factors, including β-lactamase, alkaline phosphatase, hemolytic phospholipase C, and Cif, directly into the host cytoplasm via fusion of OMV with lipid rafts in the host plasma membrane. These virulence factors enter the cytoplasm of the host cell via N-WASP–mediated actin trafficking, where they rapidly distribute to specific subcellular locations to affect host cell biology. We propose that secreted virulence factors are not released individually as naked proteins into the surrounding milieu where they may randomly contact the surface of the host cell, but instead bacterial derived OMV deliver multiple virulence factors simultaneously and directly into the host cell cytoplasm in a coordinated manner

ATP-binding cassette (ABC) transporters in normal and pathological lung

ATP-binding cassette (ABC) transporters are a family of transmembrane proteins that can transport a wide variety of substrates across biological membranes in an energy-dependent manner. Many ABC transporters such as P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1) and breast cancer resistance protein (BCRP) are highly expressed in bronchial epithelium. This review aims to give new insights in the possible functions of ABC molecules in the lung in view of their expression in different cell types. Furthermore, their role in protection against noxious compounds, e.g. air pollutants and cigarette smoke components, will be discussed as well as the (mal)function in normal and pathological lung. Several pulmonary drugs are substrates for ABC transporters and therefore, the delivery of these drugs to the site of action may be highly dependent on the presence and activity of many ABC transporters in several cell types. Three ABC transporters are known to play an important role in lung functioning. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene can cause cystic fibrosis, and mutations in ABCA1 and ABCA3 are responsible for respectively Tangier disease and fatal surfactant deficiency. The role of altered function of ABC transporters in highly prevalent pulmonary diseases such as asthma or chronic obstructive pulmonary disease (COPD) have hardly been investigated so far. We especially focused on polymorphisms, knock-out mice models and in vitro results of pulmonary research. Insight in the function of ABC transporters in the lung may open new ways to facilitate treatment of lung diseases

Analisis faktor-faktor yang mempengaruhi persepsi mahasiswa akuntansi dalam pemilihan karir akuntan publik (Studi Kasus pada Mahasiswa Jurusan Akuntansi UKWMS)

Perusahaan membutuhkan dana untuk menjalankan operasinya yang berasal dari investor dan kreditor. Laporan keuangan dibutuhkan sebagai penyampaian informasi mengenai pertanggungjawaban pengelolaan dana perusahaan. Kebutuhan perusahaan akan laporan keuangan sejalan dengan kebutuhan akan tenaga akuntan. Bidang pekerjaan akuntan yang bisa digeluti oleh lulusan akuntansi yaitu akuntan publik dan akuntan non publik oleh karena itu penelitian ini bertujuan untuk menguji dan menganalisis pengaruh gaji, lingkungan kerja, nilai-nilai sosial, pertimbangan pasar kerja, pelatihan profesional, pengakuan profesional, dan personalitas terhadap persepsi mahasiswa dalam pemilihan karir akuntan publik bagi mahasiswa akuntansi. Desain penelitian merupakan penelitian kuantitatif dengan hipotesis. Gaji, lingkungan kerja, nilai-nilai sosial, pertimbangan pasar kerja, pelatihan profesional, pengakuan profesional, dan personalitas diukur dengan pertanyaan dari kuesioner Stolle (1976). Objek penelitian adalah mahasiswa jurusan akuntansi Universitas Katolik Widya Mandala angkatan 2008 dan 2009. Sumber data diperoleh dengan menyebarkan kuesioner secara langsung kepada mahasiswa jurusan akuntansi Universitas Katolik Widya Mandala. Analisis data menggunakan regresi logistik. Hasil analisis menunjukan bahwa gaji, nilai-nilai sosial, pertimbangan pasar kerja, pelatihan profesional dan personalitas berpengaruh positif terhadap persepsi mahasiswa akuntansi dalam pemilihan karir akuntan publik. Lingkungan kerja tidak berpengaruh terhadap persepsi mahasiswa akuntansi dalam pemilihan karir akuntan publik karena sebagian besar responden penelitian adalah mahasiswi yang tidak menyukai lingkungan kerja akuntan publik yang penuh tantangan dan tekanan. Pengakuan profesional juga tidak berpengaruh terhadap persepsi mahasiswa akuntansi dalam pemilihan karir akuntan publik karena mahasiswa menganggap untuk mendapat pengakuan atas prestasi kerja dalam karir akuntan publik dibutuhkan keahlian tertentu, waktu yang tidak sebentar dan jenjang karir yang panjang

Perbedaan kecerdasan seksual remaja yang tinggal di panti asuhan dan remaja yang tinggal dengan orang tua

Kecerdasan seksual merupakan elemen penting untuk menggambarkan perilaku seksual pada seorang remaja. Seorang remaja pada dasarnya harus memperoleh pengetahuan dan pendidikan seks dari orangtua, sehingga dapat berperilaku sesuai dengan norma yang berlaku di masyarakat. Sedangkan remaja yang berada di panti asuhan hanya memiliki pengasuh yang belum tentu memiliki kelekatan dengan setiap anak. Oleh karena itu, tujuan penelitian ini adalah untuk mengetahui apakah ada perbedaan kecerdasan seksual remaja yang tinggal di panti asuhan dan remaja yang tinggal dengan orangtua. Subjek penelitian (N=44) adalah remaja di Panti Asuhan Undaan, Panti Asuhan Lydia dan remaja di Kelurahan Keputran yang berusia 15-18 tahun. Pengambilan sampel dilakukan dengan cara Total Population Study dan Insidental Quota Sampling, sedangkan pengumpulan data dilakukan dengan menggunakan Skala Kecerdasan Seksual. Data yang diperoleh dianalisis dengan teknik ANAVA (Analisis Varians Satu Arah). Hasil analisis Independent Sample test, diperoleh nilai t = 5,623 dan sig = 0,000 (p < 0,05). Hal ini berarti hipotesis diterima yaitu ada perbedaan kecerdasan seksual antara remaja yang tinggal dipanti asuhan dan remaja yang tinggal dengan orangtua. Selain itu, dapat diketahui bahwa mean kelompok remaja yang tinggal di panti asuhan sebesar 113,74 sementara remaja yang tinggal bersama orangtua sebesar 88,91. Hal ini menunjukkan bahwa kecerdasan seksual yang dimiliki remaja yang tinggal di panti asuhan lebih tinggi daripada remaja yang tinggal bersama orangtua