Natriuretic Peptides and Nitric Oxide Stimulate cGMP Synthesis in Different Cellular Compartments

Cyclic nucleotide-gated (CNG) channels are a family of ion channels activated by the binding of cyclic nucleotides. Endogenous channels have been used to measure cyclic nucleotide signals in photoreceptor outer segments and olfactory cilia for decades. Here we have investigated the subcellular localization of cGMP signals by monitoring CNG channel activity in response to agonists that activate either particulate or soluble guanylyl cyclase. CNG channels were heterologously expressed in either human embryonic kidney (HEK)-293 cells that stably overexpress a particulate guanylyl cyclase (HEK-NPRA cells), or cultured vascular smooth muscle cells (VSMCs). Atrial natriuretic peptide (ANP) was used to activate the particulate guanylyl cyclase and the nitric oxide donor S-nitroso-n-acetylpenicillamine (SNAP) was used to activate the soluble guanylyl cyclase. CNG channel activity was monitored by measuring Ca2+ or Mn2+ influx through the channels using the fluorescent dye, fura-2. We found that in HEK-NPRA cells, ANP-induced increases in cGMP levels activated CNG channels in a dose-dependent manner (0.05–10 nM), whereas SNAP (0.01–100 μM) induced increases in cGMP levels triggered little or no activation of CNG channels (P < 0.01). After pretreatment with 100 μM 3-isobutyl-1-methylxanthine (IBMX), a nonspecific phosphodiesterase inhibitor, ANP-induced Mn2+ influx through CNG channels was significantly enhanced, while SNAP-induced Mn2+ influx remained small. In contrast, we found that in the presence of IBMX, both 1 nM ANP and 100 μM SNAP triggered similar increases in total cGMP levels. We next sought to determine if cGMP signals are compartmentalized in VSMCs, which endogenously express particulate and soluble guanylyl cyclase. We found that 10 nM ANP induced activation of CNG channels more readily than 100 μM SNAP; whereas 100 μM SNAP triggered higher levels of total cellular cGMP accumulation. These results suggest that cGMP signals are spatially segregated within cells, and that the functional compartmentalization of cGMP signals may underlie the unique actions of ANP and nitric oxide

Functioning and factors of the formation of the regional tourist market of Ukraine in conditions of decentralization of economic relations

У статті розглянуто особливості функціонування регіонального туристичного ринку. Проаналізовано економічні засади дослідження туристичних ринків у роботах вітчизняних вчених. Досліджено моделі економічної співпраці на регіональному туристичному ринку Відзначено, напрямки формування туристичного ринку, через головні регулюючі фактори попиту та пропозиції туристичних послуг в регіоні. Відмічено роль децентралізації в розвитку соціально - економічних відносин на регіональному туристичному ринку.В статье рассмотрены особенности функционирования регионального туристического рынка. Проанализированы экономические аспекты исследования туристических рынков в работах отечественных ученых. Исследована модель экономического сотрудничества на региональном туристическом рынке. Отмечены, направления формирования туристического рынка через главные регулирующие факторы спроса и предложения туристических услуг в регионе. Выделена роль децентрализации в развитии социально- экономических отношений на региональном туристическом рынке.The article deals with the peculiarities of the functioning of the regional tourism market. The economic principles of research of tourist markets in the works of domestic scientists are analyzed. The models of economic cooperation on the regional tourist market are investigated. The directions of the formation of the tourist market, through the main regulatory factors of demand and offer of tourist services in the region are noted. The role of decentralization in the development of socio - economic relations in the regional tourism market is noted

Description of tourism activity for the purposes of accounting

Розглянуто основні особливості діяльності підприємств туристичного бізнесу для потреб формування обліково-аналітичної інформації в умовах застосування МСФЗ та фінансової кризи. Охарактеризовано особливості ведення бухгалтерського обліку туристичної діяльності. Проаналізовано порядок обліку операцій туристичного бізнесу.Рассмотрены основные особенности деятельности предприятий туристического бизнеса для нужд формирования учетно-аналитической информации в условиях применения МСФО и финансового кризиса. Охарактеризованы особенности ведения бухгалтерского учета туристической деятельности. Проанализирован порядок учета операций туристического бизнеса.The main features of tourism business enterprises for the purpose of forming accounting and analytical information in terms of IFRS and financial crisis. The features of accounting of tourism. Analyzed the accounting operations of tourism

The actual problems of the tourist industry of Ukraine and the ways of its decision on the conditions of socioeconomic Environmental transformation

Під час написання були використані такі методи дослідження: аналіз, порівняльний, статистичний, системний та ін. В результаті дослідження було розглянуто актуальні проблеми туристичної галузі України. Запропоновано способи вирішення наявних проблем. Узагальнено основні проблеми України в галузі туризму та запропоновано деякі рекомендації щодо усунення наявних проблем. Україна – надзвичайно перспективна країна для розвитку туризму, визначивши основні проблеми та шляхи для їх вирішення, можна значно просунути Україну на світовому туристичному ринку. Тим самим, це призведе до значного розвитку економіки країни.При написании были использованы следующие методы исследования: анализ, сравнительный, статистический, системный и др. В результате исследования были рассмотрены актуальные проблемы туристической отрасли Украины. Предложены способы решения имеющихся проблем. Обобщены основные проблемы Украины в области туризма и предложены рекомендации по устранению имеющихся проблем. Украина - очень перспективная страна для развития туризма, определив основные проблемы и пути для их решения, можно значительно продвинуть Украину на мировом туристическом рынке. Тем самым, это приведет к развитию экономики страны.During the research there were used such methods as analysis: analysis, comparative analysis, statistical analysis, system analysis, etc. As a result of the study, actual problems of the tourism industry in Ukraine were considered. Suggested ways to solve existing problems. An overview of the main problems of Ukraine in the field of tourism and recommendations on how to eliminate the existing problems. Ukraine is a very promising country for the development of tourism. Having identified the main problems and methods for solving them, Ukraine can be significantly promoted in the global tourism market. Thus, this will lead to the development of the country's economy

Strategies of tourism development in the black sea region

Під час написання були використані такі методи дослідження: аналіз, порівняльний, статистичний, системний та ін. В результаті дослідження було розглянуто сучасні стратегії розвитку Причорноморського регіону та попередні стратегії і їхні результати. Охарактеризовано етапи формування загальної стратегії державного управління. Пропонуються рекомендації щодо формування стратегії розвитку державного управління в туристичній індустрії та охарактеризовано вже існуючі стратегії. Україна – надзвичайно перспективна країна для розвитку туризму, завдяки різноманітним стратегіям, ми розвиваємо нашу країну в різних галузях. Необхідно удосконалювати вже існуючі стратегії та створювати нові, тому що це самий дієвий спосіб розвитку туризму в нашій країні.При написании были использованы следующие методы исследования: анализ, сравнительный, статистический, системный и др. В результате исследования были рассмотрены современные стратегии развития Причерноморского региона и предыдущие стратегии и их результаты. Охарактеризованы этапы формирования общей стратегии государственного управления. Предложены рекомендации по формированию стратегии развития государственного управления в туристической индустрии и охарактеризованы уже существующие стратегии. Украина - очень перспективная страна для развития туризма, благодаря разнообразным стратегиям, мы развиваем нашу страну в различных отраслях. Необходимо совершенствовать уже существующие стратегии и создавать новые, потому что это самый действенный способ развития туризма в нашей стране.During the research there were used such methods as analysis: comparative analysis , statistical analysis , systemic analysis, etc. In a result of the study there were considering modern strategies of development of the Black Sea region, previous strategies and their results. Was described it’s stages of formation of the general strategy of governance. Were offered recommendations about the formation of the strategy of development of public administration in the tourism industry and described existing strategies. Ukraine is an extremely promising country for the development of tourism. Thanks to diverse strategies, we can develop our country in various fields. It is necessary to improve already existing strategies and create new ones, because this is the most effective way of developing tourism in our country

Inducible expression of beta defensins by human respiratory epithelial cells exposed to Aspergillus fumigatus organisms

<p>Abstract</p> <p>Background</p> <p><it>Aspergillus fumigatus</it>, a saprophytic mould, is responsible for life-threatening, invasive pulmonary diseases in immunocompromised hosts. The role of the airway epithelium involves a complex interaction with the inhaled pathogen. Antimicrobial peptides with direct antifungal and chemotactic activities may boost antifungal immune response.</p> <p>Results</p> <p>The inducible expression of defensins by human bronchial epithelial 16HBE cells and A549 pneumocyte cells exposed to <it>A. fumigatus </it>was investigated. Using RT-PCR and real time PCR, we showed an activation of hBD2 and hBD9 defensin genes: the expression was higher in cells exposed to swollen conidia (SC), compared to resting conidia (RC) or hyphal fragments (HF). The kinetics of defensin expression was different for each one, evoking a putative distinct function for each investigated defensin. The decrease of defensin expression in the presence of heat-inactivated serum indicated a possible link between defensins and the proteins of the host complement system. The presence of defensin peptide hBD2 was revealed using immunofluorescence that showed a punctual cytoplasmic and perinuclear staining. Quantification of the cells stained with anti hBD2 antibody demonstrated that SC induced a greater number of cells that synthesized hBD2, compared to RC or HF. Labelling of the cells with anti-hBD-2 antibody showed a positive immunofluorescence signal around RC or SC in contrast to HF. This suggests co-localisation of hBD2 and digested conidia. The HBD2 level was highest in the supernatants of cells exposed to SC, as was determined by sandwich ELISA. Experiments using neutralising anti-interleukine-1β antibody reflect the autocrine mechanism of defensin expression induced by SC. Investigation of defensin expression at transcriptional and post-transcriptional levels demonstrated the requirement of transcription as well as new protein synthesis during <it>A. fumigatus </it>defensin induction. Finally, induced defensin expression in primary culture of human respiratory cells exposed to <it>A. fumigatus </it>points to the biological significance of described phenomena.</p> <p>Conclusion</p> <p>Our findings provide evidence that respiratory epithelium might play an important role in the immune response during <it>Aspergillus </it>infection. Understanding the mechanisms of regulation of defensin expression may thus lead to new approaches that could enhance expression of antimicrobial peptides for potential therapeutic use during aspergillosis treatment.</p

Rotavirus Disrupts Calcium Homeostasis by NSP4 Viroporin Activity

Many viruses alter intracellular calcium homeostasis. The rotavirus nonstructural protein 4 (NSP4), an endoplasmic reticulum (ER) transmembrane glycoprotein, increases intracellular levels of cytoplasmic Ca2+ ([Ca2+]cyto) through a phospholipase C-independent pathway, which is required for virus replication and morphogenesis. However, the NSP4 domain and mechanism that increases [Ca2+]cyto are unknown. We identified an NSP4 domain (amino acids [aa] 47 to 90) that inserts into membranes and has structural characteristics of viroporins, a class of small hydrophobic viral proteins that disrupt membrane integrity and ion homeostasis to facilitate virus entry, assembly, or release. Mutational analysis showed that NSP4 viroporin activity was mediated by an amphipathic α-helical domain downstream of a conserved lysine cluster. The lysine cluster directed integral membrane insertion of the viroporin domain and was critical for viroporin activity. In epithelial cells, expression of wild-type NSP4 increased the levels of free cytoplasmic Ca2+ by 3.7-fold, but NSP4 viroporin mutants maintained low levels of [Ca2+]cyto, were retained in the ER, and failed to form cytoplasmic vesicular structures, called puncta, which surround viral replication and assembly sites in rotavirus-infected cells. When [Ca2+]cyto was increased pharmacologically with thapsigargin, viroporin mutants formed puncta, showing that elevation of calcium levels and puncta formation are distinct functions of NSP4 and indicating that NSP4 directly or indirectly responds to elevated cytoplasmic calcium levels. NSP4 viroporin activity establishes the mechanism for NSP4-mediated elevation of [Ca2+]cyto, a critical event that regulates rotavirus replication and virion assembly

Staphylococcus aureus seroproteomes discriminate ruminant isolates causing mild or severe mastitis

Staphylococcus aureus is a major cause of mastitis in ruminants. In ewe mastitis, symptoms range from subclinical to gangrenous mastitis. S. aureus factors or host-factors contributing to the different outcomes are not completely elucidated. In this study, experimental mastitis was induced on primiparous ewes using two S. aureus strains, isolated from gangrenous (strain O11) or subclinical (strain O46) mastitis. Strains induced drastically distinct clinical symptoms when tested in ewe and mice experimental mastitis. Notably, they reproduced mild (O46) or severe (O11) mastitis in ewes. Ewe sera were used to identify staphylococcal immunoreactive proteins commonly or differentially produced during infections of variable severity and to define core and accessory seroproteomes. Such SERological Proteome Analysis (SERPA) allowed the identification of 89 immunoreactive proteins, of which only 52 (58.4%) were previously identified as immunogenic proteins in other staphylococcal infections. Among the 89 proteins identified, 74 appear to constitute the core seroproteome. Among the 15 remaining proteins defining the accessory seroproteome, 12 were specific for strain O11, 3 were specific for O46. Distribution of one protein specific for each mastitis severity was investigated in ten other strains isolated from subclinical or clinical mastitis. We report here for the first time the identification of staphylococcal immunogenic proteins common or specific to S. aureus strains responsible for mild or severe mastitis. These findings open avenues in S. aureus mastitis studies as some of these proteins, expressed in vivo, are likely to account for the success of S. aureus as a pathogen of the ruminant mammary gland