Regulation of the mitochondrial proton gradient by cytosolic Ca2+ signals

Mitochondria convert the energy stored in carbohydrate and fat into ATP molecules that power enzymatic reactions within cells, and this process influences cellular calcium signals in several ways. By providing ATP to calcium pumps at the plasma and intracellular membranes, mitochondria power the calcium gradients that drive the release of Ca2+ from stores and the entry of Ca2+ across plasma membrane channels. By taking up and subsequently releasing calcium ions, mitochondria determine the spatiotemporal profile of cellular Ca2+ signals and the activity of Ca2+-regulated proteins, including Ca2+ entry channels that are themselves part of the Ca2+ circuitry. Ca2+ elevations in the mitochondrial matrix, in turn, activate Ca2+-dependent enzymes that boost the respiratory chain, increasing the ability of mitochondria to buffer calcium ions. Mitochondria are able to encode and decode Ca2+ signals because the respiratory chain generates an electrochemical gradient for protons across the inner mitochondrial membrane. This proton motive force (Δp) drives the activity of the ATP synthase and has both an electrical component, the mitochondrial membrane potential (ΔΨ m ), and a chemical component, the mitochondrial proton gradient (ΔpH m ). ΔΨ m contributes about 190mV to Δp and drives the entry of Ca2+ across a recently identified Ca2+-selective channel known as the mitochondrial Ca2+ uniporter. ΔpH m contributes ~30mV to Δp and is usually ignored or considered a minor component of mitochondria respiratory state. However, the mitochondrial proton gradient is an essential component of the chemiosmotic theory formulated by Peter Mitchell in 1961 as ΔpH m sustains the entry of substrates and metabolites required for the activity of the respiratory chain and drives the activity of electroneutral ion exchangers that allow mitochondria to maintain their osmolarity and volume. In this review, we summarize the mechanisms that regulate the mitochondrial proton gradient and discuss how thermodynamic concepts derived from measurements in purified mitochondria can be reconciled with our recent findings that mitochondria have high proton permeability in situ and that ΔpH m decreases during mitochondrial Ca2+ elevation

Соціальний захист населення як показник результативності соціального партнерства

У статті на основі аналізу трудових відносин, охорони праці, експертиз умов праці, соціального обслуговування у Волинській області проаналізовані результати діяльності ГУ ПСЗН в рамках завдань, поставлених обласною радою соціального партнерства, а також рекомендовані пріоритетні напрямки його діяльності на найближчу перспективу. On the basis o f analysis o f labor relations, labor, examinations o f term s o f labor protection, social service at the Volyn region the results o f the GU PCZH activity within the framework of tasks o f put by the regional soviet o f social partnership, are analyzed at the article and also the recommended priority directions o f its activity on the nearest prospect

Innovative Development of the Transport System of Ukraine

Дослідження складових транспортного комплексу (залізничний транспорт, автомобільний, водний) дало змогу стверджувати, що транспортна система є важливим сегментом для економіки країни, стабільне функціонування якого забезпечує необхідні умови цілісності держави та підвищення рівня життя населення. Проаналізовано стан транспортної системи України та розглянуто перспективи її розвитку в контексті кризових змін. Визначено негативні тенденції у перевезеннях. Відзначено зменшення вантажообігу залізничного, автомобільного, водного, трубопровідного й авіаційного транспорту. Ринок транспортних послуг задовольняє тільки базові потреби економіки України та населення у перевезеннях. Виявлено, що нерозвинена система управління транспортною системою, стан виробничо-технічної бази і технологічний рівень організації перевезень за багатьма параметрами не відповідають зростаючим потребам суспільства, що негативно впливає на транспортну систему країни. Перспективним для держави визначено інноваційні перетворення в напрямку удосконалення структури міжнародних транспортно-логістичних систем, запровадження інноваційних інформаційно-управлінських технологій, побудованих на моделюванні інтегрованих транспортних систем, розвиток мультимодальних перевезень та пришвидшене інтегрування національної транспортної системи.Transport is considered to be one of the leading branches of material production; it participates in the formation of the sectoral structure of national production, in the allocation of production capacities and the formation of prices. Through economic channels, the transport industry affects all indicators of quality of life, creating the benefits of economic growth in the region and the country, developing a national infrastructure. The quality of functioning of the transport system depends on the quality of the formation of the social environment. Greater mobility and time-saving as the most important resource of the present create wider opportunities for human development. Therefore, the purpose of the work is to analyze the innovation activity of transport enterprises, determine the role of financing in the innovative development of the industry, and propose measures to increase innovation activity. The authors have analysed the main directions and practical results of introducing innovations in the transport and road sector of Ukraine, identifies the factors that hamper innovation processes in the transport sector, substantiates the priority directions of the state policy to activate innovative transformations in the transport sector with the aim of modernizing the transport and road complex. The object of research is the transport and road complex of Ukraine. Nowadays, the innovative activity of transport enterprises is characterized by negative dynamics, namely: the number of scientific organizations, scientists, innovative-active transport enterprises. Innovative development of the industry is hampered by a lack of financing for the introduction of innovations at the enterprises of the transport and road complex and the slow pace of implementing structural reforms in the sphere of the transport and road complex. There is an objective need for a balanced state policy for the development of a transport and road complex on an innovative basis

Droplet Digital PCR Shows the D-Loop to be an Error Prone Locus for Mitochondrial DNA Copy Number Determination

Absolute quantification of mitochondrial DNA copy number (mCN) provides important insights in many fields of research including cancer, cardiovascular and reproductive health. Droplet digital PCR (ddPCR) natively reports absolute copy number, and we have developed a single-dye, multiplex assay to measure rat mCN that is accurate, precise and affordable. We demonstrate simple methods to optimize this assay and to determine nuclear reference pseudogene copy number to extend the range of mCN that can be measured with this assay. We evaluated two commonly used mitochondrial DNA reference loci to determine mCN, the ND1 gene and the D-Loop. Harnessing the absolute measures of ddPCR, we found that the D-Loop amplifies with a copy number of ~1.0–1.5 relative to other sites on the mitochondrial genome. This anomalous copy number varied significantly between rats and tissues (aorta, brain, heart, liver, soleus muscle). We advocate for avoiding the D-Loop as a mitochondrial reference in future studies of mCN. Further, we report a novel approach to quantifying immunolabelled mitochondrial DNA that provides single-cell estimates of mCN that closely agree with the population analyses by ddPCR. The combination of these assays represents a cost-effective and powerful suite of tools to study mCN

Problems of small business in Ukraine and ways for their overcoming

Досліджено сутність малого бізнесу та критерії зарахування суб’єктів господарювання до малих підприємств і до мікропідприємств як за підходом вітчизняного законодавства, так і за європейськими нормами. Досліджено місце малого бізнесу в економіці держави та його роль у її розвитку. Сформульовано фактори, які формують середовище для малого бізнесу в Україні та перешкоджають його ефективному функціонуванню. Зокрема, юридичні потужності малого бізнесу та упереджене ставлення з боку правоохоронних органів до малих підприємств перманентно здійснюють тиск на малі підприємства й обтяжують процес їх діяльності. Проаналізовано й досліджено широкий спектр гальмівних чинників та факторів, з якими стикається малий бізнес в Україні. Виділено ключові напрямки, що найбільш гостро потребують реформування, проведено їх детальний та поглиблений аналіз. У результаті сформовано та згруповано перелік проблем малого бізнесу в Україні. Серед перешкод з боку податкової системи – відмова в реєстрації податкових накладних з незрозумілих причин, затримання розблокування податкових накладних та реєстрації податковими органами накладних за рішенням суду, а також необґрунтованість підстав, за якими платників податку включають до ризикових і відсутність чітких вказівок для виключення з переліку ризикових. З боку правоохоронних органів – безпідставні кримінальні провадження, гальмування процесів повернення суб’єктів підприємництва до нормальної роботи. З боку трудових відносин – складність та негнучкість взаємодії працівника й роботодавця і процедури звільнення працівників; ведення роботодавцем документального адміністрування праці у паперовій формі; значна розпорошеність правового регулювання перевірок з питань праці. З боку доступності фінансування – невідповідність вимогам банківського кредитування та відсутність інформування про міжнародні програми, гранти та донорське фінансування за участі європейських організацій, а також про державні програми підтримки бізнесу. На основі окреслених проблем сформовано ряд шляхів їх подолання, що дозволить вивести малий бізнес в Україні на новий рівень та укріпити економічне становище державиThe essence of small business and the criteria for enrollment of business entities to small enterprises and micro-enterprises, both according to the approach of domestic legislation and European standards are investigated in this paper The place of small business in the state economy and its role in its development is studied. Factors that shape the environment for small business in Ukraine and hinder its effective functioning are formulated. In particular, the legal capacity of small businesses and the bi ased attitude of law enforcement agencies towards small businesses permanently put pressure on small businesses and complicate the process of their activities. A wide range of inhibitory factors and factors faced by small business in Ukraine are analyzed and studied. The key areas that are most urgently in need of reform are identified, and their detailed and in-depth analysis is carried out. As the result, a list of small business problems in Ukraine is formed and grouped. Obstacles on the part of the tax system include refusal to register tax invoices for unknown reasons, delays in unblocking tax invoices and registration of invoices by tax authorities, as well as unfounded grounds on which taxpayers are included in risky and lack of clear instructions for exclusion from the list of risky. On the part of law enforcement agencies – unfounded criminal proceedings, slowing down the process of returning business entities to normal operation. On the part of labor relations are the complexity and inflexibility of the interaction between employee and employer and the procedure for dismissal of employees; conducting by the employer of documentary administration of labor in paper form; significant dispersion of legal regulation of labor inspections. In terms of availability of funding are non-compliance with the requirements of bank lending and lack of information about international programs, grants and donor funding with the participation of European organizations, as well as government programs to support business. Based on the outlined problems, a number of ways to overcome them, which will bring small business in Ukraine to a new level and strengthen the economic position of the state are forme

Transcriptional and translational expression of calbindin-D9k in the duodenum, kidney and uterus of a female canine model

Calbindin-D9k (CaBP-9k) is a cytosolic calcium-binding protein expressed in tissues in the intestine, uterus, placenta, kidney, pituitary gland and bone. Its exact function is unknown, but it is considered to regulate intracytoplasmic concentration and transport of free ions (Ca2+). CaBP-9k protein is involved in intestinal calcium absorption in the intestine and in the regulation of myometrial activity by intracellular calcium in the uterus. Renal CaBP-9k protein is expressed at the site of calcium re-absorption in the kidney and expressed in distal convoluted tubules, where it is thought to facilitate calcium re-absorption. Expression of the CaBP-9k gene has been explored in most mammalians except in a canine model. Presently, we elucidated the expression of CaBP-9k mRNA and protein in the duodenum, kidney and uterus in a canine model involving two adult (2.5-year-old) female beagles. To collect tissues, the dogs were euthanized and then the abdominal cavity was exposed by midline incision. The proximal duodenum, cortex of kidney and uterine horn were collected. Expression of CaBP-9k mRNA was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. CaBP-9k protein expression and localization were ascertained by Western blot analysis and immunohistochemistry, respectively. CaBP-9k mRNA was detected in the duodenum, but not in the kidney and uterus. Its protein was expressed only in the enterocytes of the duodenum. Taken together, the results indicate that CaBP-9k mRNA and protein are highly expressed in the enterocytes of the duodenum of a canine model, consistent with findings in other mammalian species

Reverse mode Na+/Ca2+ exchange mediated by STIM1 contributes to Ca2+ influx in airway smooth muscle following agonist stimulation

<p>Abstract</p> <p>Background</p> <p>Agonist stimulation of airway smooth muscle (ASM) results in IP₃mediated Ca²⁺release from the sarcoplasmic reticulum followed by the activation of store operated and receptor operated non-selective cation channels. Activation of these non-selective channels also results in a Na⁺influx. This localised increase in Na⁺levels can potentially switch the Na⁺/Ca²⁺exchanger into reverse mode and so result in a further influx of Ca²⁺. The aim of this study was to characterise the expression and physiological function of the Na⁺/Ca²⁺exchanger in cultured human bronchial smooth muscle cells and determine its contribution to agonist induced Ca²⁺influx into these cells.</p> <p>Methods</p> <p>The expression profile of NCX (which encodes the Na⁺/Ca²⁺exchanger) homologues in cultured human bronchial smooth muscle cells was determined by reverse transcriptase PCR. The functional activity of reverse mode NCX was investigated using a combination of whole cell patch clamp, intracellular Ca²⁺measurements and porcine airway contractile analyses. KB-R7943 (an antagonist for reverse mode NCX) and target specific siRNA were utilised as tools to inhibit NCX function.</p> <p>Results</p> <p>NCX1 protein was detected in cultured human bronchial smooth muscle cells (HBSMC) cells and NCX1.3 was the only mRNA transcript variant detected. A combination of intracellular Na⁺loading and addition of extracellular Ca²⁺induced an outwardly rectifying current which was augmented following stimulation with histamine. This outwardly rectifying current was inhibited by 10 μM KB-R7943 (an antagonist of reverse mode NCX1) and was reduced in cells incubated with siRNA against NCX1. Interestingly, this outwardly rectifying current was also inhibited following knockdown of STIM1, suggesting for the first time a link between store operated cation entry and NCX1 activation. In addition, 10 μM KB-R7943 inhibited agonist induced changes in cytosolic Ca²⁺and induced relaxation of porcine peripheral airways.</p> <p>Conclusions</p> <p>Taken together, these data demonstrate a potentially important role for NCX1 in control of Ca²⁺homeostasis and link store depletion via STIM1 directly with NCX activation.</p

Selective Ion Changes during Spontaneous Mitochondrial Transients in Intact Astrocytes

The bioenergetic status of cells is tightly regulated by the activity of cytosolic enzymes and mitochondrial ATP production. To adapt their metabolism to cellular energy needs, mitochondria have been shown to exhibit changes in their ionic composition as the result of changes in cytosolic ion concentrations. Individual mitochondria also exhibit spontaneous changes in their electrical potential without altering those of neighboring mitochondria. We recently reported that individual mitochondria of intact astrocytes exhibit spontaneous transient increases in their Na+ concentration. Here, we investigated whether the concentration of other ionic species were involved during mitochondrial transients. By combining fluorescence imaging methods, we performed a multiparameter study of spontaneous mitochondrial transients in intact resting astrocytes. We show that mitochondria exhibit coincident changes in their Na+ concentration, electrical potential, matrix pH and mitochondrial reactive oxygen species production during a mitochondrial transient without involving detectable changes in their Ca2+ concentration. Using widefield and total internal reflection fluorescence imaging, we found evidence for localized transient decreases in the free Mg2+ concentration accompanying mitochondrial Na+ spikes that could indicate an associated local and transient enrichment in the ATP concentration. Therefore, we propose a sequential model for mitochondrial transients involving a localized ATP microdomain that triggers a Na+-mediated mitochondrial depolarization, transiently enhancing the activity of the mitochondrial respiratory chain. Our work provides a model describing ionic changes that could support a bidirectional cytosol-to-mitochondria ionic communication

Transit of H2O2 across the endoplasmic reticulum membrane is not sluggish

Cellular metabolism provides various sources of hydrogen peroxide (H2O2) in different organelles and compartments. The suitability of H2O2 as an intracellular signaling molecule therefore also depends on its ability to pass cellular membranes. The propensity of the membranous boundary of the endoplasmic reticulum (ER) to let pass H2O2 has been discussed controversially. In this essay, we challenge the recent proposal that the ER membrane constitutes a simple barrier for H2O2 diffusion and support earlier data showing that (i) ample H2O2 permeability of the ER membrane is a prerequisite for signal transduction, (ii) aquaporin channels are crucially involved in the facilitation of H2O2 permeation, and (iii) a proper experimental framework not prone to artifacts is necessary to further unravel the role of H2O2 permeation in signal transduction and organelle biology. © 2016 Elsevier Inc

Different patterns of Ca2+ signals are induced by low compared to high concentrations of P2Y agonists in microglia

Brain-resident macrophages (microglia) are key cellular elements in the preservation of tissue integrity. On the other hand, they can also contribute to the development of pathological events by causing an extensive and inappropriate inflammatory response. A growing number of reports indicate the involvement of nucleotides in the control of microglial functions. With this study on P2Y receptors in rat microglia, we want to contribute to the definition of their expression profile and to the characterisation of their signalling mechanisms leading to Ca2+ movements. Endogenous nucleotides, when applied at a concentration of 100 μM, elicited robust Ca2+ transients, thanks to a panel of metabotropic receptors comprising mainly P2Y2, P2Y6 and P2Y12 subtypes. The involvement of P2Y12 receptors in Ca2+ responses induced by adenine nucleotides was confirmed by the pharmacological and pertussis toxin sensitivity of the response induced by adenosine diphosphate (ADP). Beside the G protein involved, Gi and Gq respectively, adenine and uracil nucleotides differed also for induction by the latter of a capacitative Ca2+ plateau. Moreover, when applied at low (sub-micromolar) concentrations with a long-lasting challenge, uracil nucleotides elicited oscillatory Ca2+ changes with low frequency of occurrence (≤ 1 min−), sometimes superimposed to an extracellular Ca2+-dependent sustained Ca2+ rise. We conclude that different patterns of Ca2+ transients are induced by low (i.e., oscillatory Ca2+ activity) compared to high (i.e., fast release followed by sustained raise) concentrations of nucleotides, which can suggest different roles played by receptor stimulation depending not only on the type but also on the concentration of nucleotides