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Sample records for intracellular ph compartmentation

  1. Evolution of intracellular compartmentalization.

    PubMed

    Diekmann, Yoan; Pereira-Leal, José B

    2013-01-15

    Cells compartmentalize their biochemical functions in a variety of ways, notably by creating physical barriers that separate a compartment via membranes or proteins. Eukaryotes have a wide diversity of membrane-based compartments, many that are lineage- or tissue-specific. In recent years, it has become increasingly evident that membrane-based compartmentalization of the cytosolic space is observed in multiple prokaryotic lineages, giving rise to several types of distinct prokaryotic organelles. Endosymbionts, previously believed to be a hallmark of eukaryotes, have been described in several bacteria. Protein-based compartments, frequent in bacteria, are also found in eukaryotes. In the present review, we focus on selected intracellular compartments from each of these three categories, membrane-based, endosymbiotic and protein-based, in both prokaryotes and eukaryotes. We review their diversity and the current theories and controversies regarding the evolutionary origins. Furthermore, we discuss the evolutionary processes acting on the genetic basis of intracellular compartments and how those differ across the domains of life. We conclude that the distinction between eukaryotes and prokaryotes no longer lies in the existence of a compartmentalized cell plan, but rather in its complexity.

  2. Mapping intracellular diffusion distribution using single quantum dot tracking: compartmentalized diffusion defined by endoplasmic reticulum.

    PubMed

    Li, Hui; Dou, Shuo-Xing; Liu, Yu-Ru; Li, Wei; Xie, Ping; Wang, Wei-Chi; Wang, Peng-Ye

    2015-01-14

    The crowded intracellular environment influences the diffusion-mediated cellular processes, such as metabolism, signaling, and transport. The hindered diffusion of macromolecules in heterogeneous cytoplasm has been studied over years, but the detailed diffusion distribution and its origin still remain unclear. Here, we introduce a novel method to map rapidly the diffusion distribution in single cells based on single-particle tracking (SPT) of quantum dots (QDs). The diffusion map reveals the heterogeneous intracellular environment and, more importantly, an unreported compartmentalization of QD diffusions in cytoplasm. Simultaneous observations of QD motion and green fluorescent protein-tagged endoplasmic reticulum (ER) dynamics provide direct evidence that the compartmentalization results from micron-scale domains defined by ER tubules, and ER cisternae form perinuclear areas that restrict QDs to enter. The same phenomenon was observed using fluorescein isothiocyanate-dextrans, further confirming the compartmentalized diffusion. These results shed new light on the diffusive movements of macromolecules in the cell, and the mapping of intracellular diffusion distribution may be used to develop strategies for nanoparticle-based drug deliveries and therapeutics.

  3. Patterns of intracellular compartmentalization, trafficking and acidification of 5'-fluorescein labeled phosphodiester and phosphorothioate oligodeoxynucleotides in HL60 cells.

    PubMed Central

    Tonkinson, J L; Stein, C A

    1994-01-01

    We have examined the intracellular compartmentalization and trafficking of fluorescein labeled (F) phosphodiester (PO) and phosphorothioate (PS) oligodeoxynucleotides (oligos) in HL60 cells. A series of F-oligos (PO and PS) were incubated for 6 hrs. with HL60 cells and the mean intracellular fluorescence determined by flow cytometry. The F signal was normalized by the addition of the ionophore monensin. An increase in signal intensity following addition of monensin indicated that the oligo was resident in an acidic intracellular environment. F-PS, but not F-PO oligos were found to reside in an acidic environment. An exception was a PO homopolymer of 15 cytidine bases (FOdC15) which was acidified. Using two different methods, the average resident intracellular pH of F-PS oligos and F-OdC15 was shown to be approximately 1 pH unit lower than that of F-PO oligos. Acidification of F-PS oligos could be blocked by the antibiotic bafilomycin, indicating that acidification was occurring in endosomes or vacuoles. F-PO and F-PS oligos were effluxed from HL60 cells from two intracellular compartments. However, approximately 60% of internalized F-PO oligo resided in a 'shallow' compartment that was turned over rapidly (t1/2 = 5-10 min.) whereas only 20% of F-PS oligo resided in this compartment. Conversely, approximately 80% of the internalized F-PS oligo but only 40% of F-PO oligo resided in a 'deep' compartment that turned over with t1/2 = 2-5 hrs. This report is the first quantitative demonstration that PO and PS oligos, and PO oligos of different sequences are trafficked differently by HL60 cells. Images PMID:7937155

  4. Intracellular pH in sperm physiology.

    PubMed

    Nishigaki, Takuya; José, Omar; González-Cota, Ana Laura; Romero, Francisco; Treviño, Claudia L; Darszon, Alberto

    2014-08-01

    Intracellular pH (pHi) regulation is essential for cell function. Notably, several unique sperm ion transporters and enzymes whose elimination causes infertility are either pHi dependent or somehow related to pHi regulation. Amongst them are: CatSper, a Ca(2+) channel; Slo3, a K(+) channel; the sperm-specific Na(+)/H(+) exchanger and the soluble adenylyl cyclase. It is thus clear that pHi regulation is of the utmost importance for sperm physiology. This review briefly summarizes the key components involved in pHi regulation, their characteristics and participation in fundamental sperm functions such as motility, maturation and the acrosome reaction.

  5. [Measurement of intracellular pH].

    PubMed

    Hanaoka, K; Imai, M; Yoshitomi, K

    1992-09-01

    Since various cellular processes depend on changes in pH, the regulation of intracellular pH (pHi) is important both for the individual cell and for the organism. The mechanisms of the regulation of pHi can be investigated by monitoring pHi. In this report, we discuss the four major techniques available for measuring pHi, which are 1) Distribution of weak acids and bases, 2) pH-sensitive microelectrodes, 3) pH-sensitive dyes, and 4) Nuclear magnetic resonance. Among four techniques, the advantage of the microelectrode approach is that it can monitor membrane potential at the same time and be applied to a single cell. The dye technique is a relative new developing technique, which has lots of advantages. It is easy to use, and is capable of monitoring rapid pHi changes, and being applied to a smaller cell, or a single cell.

  6. Intracellular Redox Compartmentation and ROS-Related Communication in Regulation and Signaling1[OPEN

    PubMed Central

    2016-01-01

    Recent years have witnessed enormous progress in understanding redox signaling related to reactive oxygen species (ROS) in plants. The consensus view is that such signaling is intrinsic to many developmental processes and responses to the environment. ROS-related redox signaling is tightly wedded to compartmentation. Because membranes function as barriers, highly redox-active powerhouses such as chloroplasts, peroxisomes, and mitochondria may elicit specific signaling responses. However, transporter functions allow membranes also to act as bridges between compartments, and so regulated capacity to transmit redox changes across membranes influences the outcome of triggers produced at different locations. As well as ROS and other oxidizing species, antioxidants are key players that determine the extent of ROS accumulation at different sites and that may themselves act as signal transmitters. Like ROS, antioxidants can be transported across membranes. In addition, the intracellular distribution of antioxidative enzymes may be modulated to regulate or facilitate redox signaling appropriate to the conditions. Finally, there is substantial plasticity in organellar shape, with extensions such as stromules, peroxules, and matrixules playing potentially crucial roles in organelle-organelle communication. We provide an overview of the advances in subcellular compartmentation, identifying the gaps in our knowledge and discussing future developments in the area. PMID:27208308

  7. Kinetics and intracellular compartmentalization of HTLV-1 gene expression: nuclear retention of HBZ mRNAs

    PubMed Central

    Rende, Francesca; Cavallari, Ilaria; Corradin, Alberto; Silic-Benussi, Micol; Toulza, Frederic; Toffolo, Gianna M.; Tanaka, Yuetsu; Jacobson, Steven; Taylor, Graham P.; D'Agostino, Donna M.; Bangham, Charles R. M.

    2011-01-01

    Human T-cell leukemia virus type 1 (HTLV-1) codes for 9 alternatively spliced transcripts and 2 major regulatory proteins named Tax and Rex that function at the transcriptional and posttranscriptional levels, respectively. We investigated the temporal sequence of HTLV-1 gene expression in primary cells from infected patients using splice site-specific quantitative RT-PCR. The results indicated a two-phase kinetics with the tax/rex mRNA preceding expression of other viral transcripts. Analysis of mRNA compartmentalization in cells transfected with HTLV-1 molecular clones demonstrated the strict Rex-dependency of the two-phase kinetics and revealed strong nuclear retention of HBZ mRNAs, supporting their function as noncoding transcripts. Mathematical modeling underscored the importance of a delay between the functions of Tax and Rex, which was supported by experimental evidence of the longer half-life of Rex. These data provide evidence for a temporal pattern of HTLV-1 expression and reveal major differences in the intracellular compartmentalization of HTLV-1 transcripts. PMID:21398577

  8. Intracellular pH in Sperm Physiology

    PubMed Central

    Nishigaki, Takuya; José, Omar; González-Cota, Ana Laura; Romero, Francisco; Treviño, Claudia L.; Darszon, Alberto

    2014-01-01

    Intracellular pH (pHi) regulation is essential for cell function. Notably, several unique sperm ion transporters and enzymes whose elimination causes infertility are either pHi dependent or somehow related to pHi regulation. Amongst them are: CatSper, a Ca2+ channel; Slo3, a K+ channel; the sperm-specific Na+/H+ exchanger and the soluble adenylyl cyclase. It is thus clear that pHi regulation is of the utmost importance for sperm physiology. This review briefly summarizes the key components involved in pHi regulation, their characteristics and participation in fundamental sperm functions such as motility, maturation and the acrosome reaction. PMID:24887564

  9. Compartmentation of malic acid in mesophyll cells of Kalanchoee daigremontiana: indications of a intracellular cytosolic vesicle transport mechanism

    SciTech Connect

    Balsamo, R.A.; Uribe, E.G.

    1987-04-01

    Leaf tissue was harvested over a 24hr period at one to three hour intervals. The malic acid levels in the tissue were assayed spectrophotometrically and the percent cell volume occupied by cytosolic vesicles in replicate samples was determined. The total volume of the cytosolic vesicles fluctuated throughout the photoperiod concommitantly with malic acid concentrations present in the tissue. An intact leaf tissue section (10.2cm/sup 2/) was radiolabeled with /sup 14/CO/sub 2/ seven hours into the dark period for thirty minutes. Two dimensional thin layer chromatography and electrophoresis of the tissue determined that 96% of the label was incorporated into malic acid. A freeze substitution procedure was initiated followed by microautoradiography (Fisher 1971) which allowed for the tracing of intracellular malic acid migration and compartmentation within the mesophyll cells. The results and interpretation of this experiment will be presented.

  10. The Listeria monocytogenes hemolysin has an acidic pH optimum to compartmentalize activity and prevent damage to infected host cells.

    PubMed

    Glomski, Ian J; Gedde, Margaret M; Tsang, Albert W; Swanson, Joel A; Portnoy, Daniel A

    2002-03-18

    Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from a phagosome and grows in the host cell cytosol. The pore-forming cholesterol-dependent cytolysin, listeriolysin O (LLO), mediates bacterial escape from vesicles and is approximately 10-fold more active at an acidic than neutral pH. By swapping dissimilar residues from a pH-insensitive orthologue, perfringolysin O (PFO), we identified leucine 461 as unique to pathogenic Listeria and responsible for the acidic pH optimum of LLO. Conversion of leucine 461 to the threonine present in PFO increased the hemolytic activity of LLO almost 10-fold at a neutral pH. L. monocytogenes synthesizing LLO L461T, expressed from its endogenous site on the bacterial chromosome, resulted in a 100-fold virulence defect in the mouse listeriosis model. These bacteria escaped from acidic phagosomes and initially grew normally in cells and spread cell to cell, but prematurely permeabilized the host membrane and killed the cell. These data show that the acidic pH optimum of LLO results from an adaptive mutation that acts to limit cytolytic activity to acidic vesicles and prevent damage in the host cytosol, a strategy also used by host cells to compartmentalize lysosomal hydrolases.

  11. Live cell imaging of the intracellular compartmentalization of the contaminate benzo[a]pyrene.

    PubMed

    Ali, Rizwan; Trump, Saskia; Lehmann, Irina; Hanke, Thomas

    2015-05-01

    This study investigates the cellular response of murine hepatoma cells to the polycyclic aromatic hydrocarbon benzo[a]pyrene (B[a]P) using two-photon and confocal laser scanning microscopy. The intracellular distribution of B[a]P and the B[a]P/AhR complex was visualized time- and concentration-dependent for up to 48 h of exposure. B[a]P was predominantly found in lipid droplets, endoplasmic reticulum and lysosomes, where B[a]P is collected and forms large aggregates. Changes in mitochondrial membrane potential and bleb formation due to high B[a]P concentrations were observed. The imaging data presented in this study provide new insights into the systemic cellular regulation following B[a]P exposure.

  12. Hyperpolarised organic phosphates as NMR reporters of compartmental pH.

    PubMed

    Jensen, Pernille Rose; Meier, Sebastian

    2016-02-07

    Organic phosphate metabolites contain functional groups with pKa values near the physiologic pH range, yielding pH-dependent (13)C chemical shift changes of adjacent quaternary carbon sites. When formed in defined cellular compartments from exogenous hyperpolarised (13)C substrates, metabolites can thus yield localised pH values and correlations of organelle pH and catalytic activity.

  13. Intracellular pH responses in the industrially important fungus Trichoderma reesei.

    PubMed

    Valkonen, Mari; Penttilä, Merja; Benčina, Mojca

    2014-09-01

    Preserving an optimal intracellular pH is critical for cell fitness and productivity. The pH homeostasis of the industrially important filamentous fungus Trichoderma reesei (Hypocrea jecorina) is largely unexplored. We analyzed the impact of growth conditions on regulation of intracellular pH of the strain Rut-C30 and the strain M106 derived from the Rut-C30 that accumulates L-galactonic acid-from provided galacturonic acid-as a consequence of L-galactonate dehydratase deletion. For live-cell measurements of intracellular pH, we used the genetically encoded ratiometric pH-sensitive fluorescent protein RaVC. Glucose and lactose, used as carbon sources, had specific effects on intracellular pH of T. reesei. The growth in lactose-containing medium extensively acidified cytosol, while intracellular pH of hyphae cultured in a medium with glucose remained at a higher level. The strain M106 maintained higher intracellular pH in the presence of D-galacturonic acid than its parental strain Rut-C30. Acidic external pH caused significant acidification of cytosol. Altogether, the pH homeostasis of T. reesei Rut-C30 strain is sensitive to extracellular pH and the degree of acidification depends on carbon source.

  14. Regulation of Intracellular pH in Lungs and Other Tissues During Hypercapnia

    DTIC Science & Technology

    1978-03-10

    was observed in terms of "percent pH regula- sumed to equal venous Pco 2. tion." As shown in Fig. 1, the pH of kidney, lung, and Intracellular pH was...buffering. The value, "percent pH 80 Z70regulation" (19), calculated as (Alog HCO3/log Pco 2) - x 100 is also used to quantitate pH regulation. This...42: 2080-2093, 1964. 6. FENN, W. 0. Carbon dioxide and intracellular homeostasis . 19. SCHAEFER, K. E., M. HASSON, AND H. NIEMOELLER. Effect of Ann. NY

  15. Determination of Glucose Utilization Rates in Cultured Astrocytes and Neurons with [(14)C]deoxyglucose: Progress, Pitfalls, and Discovery of Intracellular Glucose Compartmentation.

    PubMed

    Dienel, Gerald A; Cruz, Nancy F; Sokoloff, Louis; Driscoll, Bernard F

    2017-01-01

    2-Deoxy-D-[(14)C]glucose ([(14)C]DG) is commonly used to determine local glucose utilization rates (CMRglc) in living brain and to estimate CMRglc in cultured brain cells as rates of [(14)C]DG phosphorylation. Phosphorylation rates of [(14)C]DG and its metabolizable fluorescent analog, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), however, do not take into account differences in the kinetics of transport and metabolism of [(14)C]DG or 2-NBDG and glucose in neuronal and astrocytic cells in cultures or in single cells in brain tissue, and conclusions drawn from these data may, therefore, not be correct. As a first step toward the goal of quantitative determination of CMRglc in astrocytes and neurons in cultures, the steady-state intracellular-to-extracellular concentration ratios (distribution spaces) for glucose and [(14)C]DG were determined in cultured striatal neurons and astrocytes as functions of extracellular glucose concentration. Unexpectedly, the glucose distribution spaces rose during extreme hypoglycemia, exceeding 1.0 in astrocytes, whereas the [(14)C]DG distribution space fell at the lowest glucose levels. Calculated CMRglc was greatly overestimated in hypoglycemic and normoglycemic cells because the intracellular glucose concentrations were too high. Determination of the distribution space for [(14)C]glucose revealed compartmentation of intracellular glucose in astrocytes, and probably, also in neurons. A smaller metabolic pool is readily accessible to hexokinase and communicates with extracellular glucose, whereas the larger pool is sequestered from hexokinase activity. A new experimental approach using double-labeled assays with DG and glucose is suggested to avoid the limitations imposed by glucose compartmentation on metabolic assays.

  16. Intracellular pH and the Control of Multidrug Resistance

    NASA Astrophysics Data System (ADS)

    Simon, Sanford; Roy, Deborshi; Schindler, Melvin

    1994-02-01

    Many anticancer drugs are classified as either weak bases or molecules whose binding to cellular structures is pH dependent. Accumulation of these drugs within tumor cells should be affected by transmembrane pH gradients. Indeed, development of multidrug resistance (MDR) in tumor cells has been correlated with an alkaline shift of cytosolic pH. To examine the role of pH in drug partitioning, the distribution of two drugs, doxorubicin and daunomycin, was monitored in fibroblasts and myeloma cells. In both cell types the drugs rapidly accumulated within the cells. The highest concentrations were measured in the most acidic compartments-e.g., lysosomes. Modifying the cellular pH in drug-sensitive cells to mimic reported shifts in MDR caused an immediate change in the cellular drug concentration. Drug accumulation was enhanced by acidic shifts and reversed by alkaline shifts. All of these effects were rapid and reversible. These results demonstrate that the alkaline shift observed in MDR is sufficient to prevent the accumulation of chemotherapeutic drugs independent of active drug efflux.

  17. Intracellular pH and the control of multidrug resistance.

    PubMed Central

    Simon, S; Roy, D; Schindler, M

    1994-01-01

    Many anticancer drugs are classified as either weak bases or molecules whose binding to cellular structures is pH dependent. Accumulation of these drugs within tumor cells should be affected by transmembrane pH gradients. Indeed, development of multidrug resistance (MDR) in tumor cells has been correlated with an alkaline shift of cytosolic pH. To examine the role of pH in drug partitioning, the distribution of two drugs, doxorubicin and daunomycin, was monitored in fibroblasts and myeloma cells. In both cell types the drugs rapidly accumulated within the cells. The highest concentrations were measured in the most acidic compartments--e.g., lysosomes. Modifying the cellular pH in drug-sensitive cells to mimic reported shifts in MDR caused an immediate change in the cellular drug concentration. Drug accumulation was enhanced by acidic shifts and reversed by alkaline shifts. All of these effects were rapid and reversible. These results demonstrate that the alkaline shift observed in MDR is sufficient to prevent the accumulation of chemotherapeutic drugs independent of active drug efflux. Images PMID:8302842

  18. Interrelationship between growth factor-induced pH changes and intracellular Ca/sup 2 +/

    SciTech Connect

    Ives, H.E.; Daniel, T.O.

    1987-04-01

    Many mitogens cause rapid changes in intracellular pH and Ca/sup 2 +/. The authors studied the patterns of pH and Ca/sup 2 +/ changes after exposure of murine fibroblasts to platelet-derived growth factor (PDGF), bombesin, phorbol 12-myristate 13-acetate (PMA), and the vasoactive peptide bradykinin. Intracellular pH and Ca/sup 2 +/ were measured by using the fluorescent dyes 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and fura-2. Three distinct patterns of intracellular pH change were observed. (i) PDGF and bombesin caused a rapid cytoplasmic acidification of 0.03 pH unit followed by a slower alkalinization of approx. = 0.11 pH unit above the resting pH of 6.88. (ii) PMA caused alkalinization without causing the early acidification. (iii) Bradykinin caused rapid acidification without the slower net alkalinization. All acidification responses were amiloride resistant. Patterns of intracellular Ca/sup 2 +/ response were also determined for each agent. In Ca/sup 2 +/-buffered cells, PDGF, bombesin, bradykinin, and ionomycin failed to induce cellular acidification, but alkalinization responses to PDGF, bombesin, and PMA persisted. They propose that the transient acidification seen with PDGF, bombesin, and other agents is the result of increased intracellular Ca/sup 2 +/. However, growth factor-induced alkalinization via the Na/sup +//H/sup +/ exchanger is independent of changes in Ca/sup 2 +/.

  19. Regulation of intracellular pH in cnidarians: response to acidosis in Anemonia viridis.

    PubMed

    Laurent, Julien; Venn, Alexander; Tambutté, Éric; Ganot, Philippe; Allemand, Denis; Tambutté, Sylvie

    2014-02-01

    The regulation of intracellular pH (pHi) is a fundamental aspect of cell physiology that has received little attention in studies of the phylum Cnidaria, which includes ecologically important sea anemones and reef-building corals. Like all organisms, cnidarians must maintain pH homeostasis to counterbalance reductions in pHi, which can arise because of changes in either intrinsic or extrinsic parameters. Corals and sea anemones face natural daily changes in internal fluids, where the extracellular pH can range from 8.9 during the day to 7.4 at night. Furthermore, cnidarians are likely to experience future CO₂-driven declines in seawater pH, a process known as ocean acidification. Here, we carried out the first mechanistic investigation to determine how cnidarian pHi regulation responds to decreases in extracellular and intracellular pH. Using the anemone Anemonia viridis, we employed confocal live cell imaging and a pH-sensitive dye to track the dynamics of pHi after intracellular acidosis induced by acute exposure to decreases in seawater pH and NH₄Cl prepulses. The investigation was conducted on cells that contained intracellular symbiotic algae (Symbiodinium sp.) and on symbiont-free endoderm cells. Experiments using inhibitors and Na⁺-free seawater indicate a potential role of Na⁺/H⁺ plasma membrane exchangers (NHEs) in mediating pHi recovery following intracellular acidosis in both cell types. We also measured the buffering capacity of cells, and obtained values between 20.8 and 43.8 mM per pH unit, which are comparable to those in other invertebrates. Our findings provide the first steps towards a better understanding of acid-base regulation in these basal metazoans, for which information on cell physiology is extremely limited.

  20. Application of fluorescence lifetime imaging of enhanced green fluorescent protein to intracellular pH measurements.

    PubMed

    Nakabayashi, Takakazu; Wang, Hui-Ping; Kinjo, Masataka; Ohta, Nobuhiro

    2008-06-01

    We have shown that the intracellular pH of a single HeLa cell expressing the enhanced green fluorescent protein (EGFP) can be imaged using the fluorescence lifetime of EGFP, which can be interpreted in terms of the pH-dependent ionic equilibrium of the p-hydroxybenzylidene-imidazolidinone structure of the chromophore of EGFP.

  1. Measurement of individual intracellular pH and membrane potential values in living cells

    NASA Astrophysics Data System (ADS)

    Slavik, Jan; Lanz, Edvard; Cimprich, Petr

    1999-07-01

    It was assumed that each cell is a homogeneous suspension may have a slightly different pH and membrane potential. A wide range of pH-sensitive fluorescent dyes BCECF, SNARF, FITC, carboxyfluorescein, fluorescein and pyranine have been carefully tested for the accuracy and reliability of their pH-response inside living cells. The intracellular milieu was simulated by a series of mineral buffers with addition of proteins. The pH values have been determined from the excitation ratios 490/435 nm for BCECF, FITC, carboxyfluorescein and fluorescein, and 450/400 nm for pyranine, emission ratios 518/529 nm for BCECF and 635/590 nm for SNARF. The spectrally determined values were then compared with the pH values of buffers measured by a glass electrode. Using the data from the calibration procedure, we evaluated individual intracellular pH values of a large number of cells within one cell population. The confocal ratio fluorescence microscopy revealed pH maps from which both cytoplasmic and vacuolar pH values could be determine, flow cytometry gave enormous amount of average intracellular pH values of individual cells of a whole cell population. Each cell population exhibited significant differences in both cytoplasmic pH values among individual cells. The pH distribution of a typical cell population appeared to fit a Gaussian curve. In yeast it was a Gaussian curve with half- width values around 0.4 pH unit. The men pH values depended on the growth phase, H-ATPase activity and external pH values. The preliminary result with the new membrane potential dye tetramethylrhodamine methyl ester indicated that similarly to pH values, there is a heterogeneity in membrane potential values among cell sin one cell population. The data presented above suggest that each ell behaves as an individual with an individual set up of its metabolism. This 'fine tuning' of the metabolism result in slightly higher or lower pH or membrane potential values that can be detected by fluorescence

  2. Effect of altitude on brain intracellular pH and inorganic phosphate levels

    PubMed Central

    Shi, Xian-Feng; Carlson, Paul J.; Kim, Tae-Suk; Sung, Young-Hoon; Hellem, Tracy L.; Fiedler, Kristen K.; Kim, Seong-Eun; Glaeser, Breanna; Wang, Kristina; Zuo, Chun S.; Jeong, Eun-Kee; Renshaw, Perry F.; Kondo, Douglas G.

    2015-01-01

    Normal brain activity is associated with task-related pH changes. Although central nervous system syndromes associated with significant acidosis and alkalosis are well understood, the effects of less dramatic and chronic changes in brain pH are uncertain. One environmental factor known to alter brain pH is the extreme, acute change in altitude encountered by mountaineers. However, the effect of long-term exposure to moderate altitude has not been studied. The aim of this two-site study was to measure brain intracellular pH and phosphate-bearing metabolite levels at two altitudes in healthy volunteers, using phosphorus-31 magnetic resonance spectroscopy (31P-MRS). Increased brain pH and reduced inorganic phosphate (Pi) levels were found in healthy subjects who were long-term residents of Salt Lake City, UT (4720 ft/1438 m), compared with residents of Belmont, MA (20 ft/6 m). Brain intracellular pH at the altitude of 4720 ft was more alkaline than that observed near sea level. In addition, the ratio of inorganic phosphate to total phosphate signal also shifted toward lower values in the Salt Lake City region compared with the Belmont area. These results suggest that long-term residence at moderate altitude is associated with brain chemical changes. PMID:24768210

  3. Effect of altitude on brain intracellular pH and inorganic phosphate levels.

    PubMed

    Shi, Xian-Feng; Carlson, Paul J; Kim, Tae-Suk; Sung, Young-Hoon; Hellem, Tracy L; Fiedler, Kristen K; Kim, Seong-Eun; Glaeser, Breanna; Wang, Kristina; Zuo, Chun S; Jeong, Eun-Kee; Renshaw, Perry F; Kondo, Douglas G

    2014-06-30

    Normal brain activity is associated with task-related pH changes. Although central nervous system syndromes associated with significant acidosis and alkalosis are well understood, the effects of less dramatic and chronic changes in brain pH are uncertain. One environmental factor known to alter brain pH is the extreme, acute change in altitude encountered by mountaineers. However, the effect of long-term exposure to moderate altitude has not been studied. The aim of this two-site study was to measure brain intracellular pH and phosphate-bearing metabolite levels at two altitudes in healthy volunteers, using phosphorus-31 magnetic resonance spectroscopy ((31)P-MRS). Increased brain pH and reduced inorganic phosphate (Pi) levels were found in healthy subjects who were long-term residents of Salt Lake City, UT (4720ft/1438m), compared with residents of Belmont, MA (20ft/6m). Brain intracellular pH at the altitude of 4720ft was more alkaline than that observed near sea level. In addition, the ratio of inorganic phosphate to total phosphate signal also shifted toward lower values in the Salt Lake City region compared with the Belmont area. These results suggest that long-term residence at moderate altitude is associated with brain chemical changes.

  4. Photosensory transduction in ciliates. Role of intracellular pH and comparison between Stentor coeruleus and Blepharisma japonicum.

    PubMed

    Fabczak, H; Fabczak, S; Song, P S; Checcucci, G; Ghetti, F; Lenci, F

    1993-11-01

    To test the hypothesis that light signal transduction in the unicellular ciliates Stentor coeruleus and Blepharisma japonicum involves a change in intracellular pH as an initial signal following photoexcitation, we studied the dependence of the photophobic responses of the cells to changes in extracellular pH and to reagents that specifically affect intracellular pH. The extracellular pH can modify not only the intracellular pH, but can even reverse the sign of the pH gradient across the cell membrane. Thus, as predicted by the hypothesis, low extracellular pH reversibly inhibited the photophobic response of the ciliates. The intracellular pH-modulating reagents tested included ammonium chloride, a membrane-permeable weak acid that lowers the intracellular pH, and the protonophores carbonylcyanide m-chlorophenyl-hydrazone (CCCP) and carbonylcyanide p-(trifluoromethoxy)-phenyl-hydrazone (FCCP), which collapse the pH gradient across the cell membrane. The low pH and protonophore treatments caused a gradual inhibition of the photophobic responses in both ciliates. The observed reduction of the responsiveness of the cells to visible light can be attributed to the alteration of the intracellular pH, which is suggested to play a specific role in the photosensory transduction in both Stentor coeruleus and Blepharisma japonicum.

  5. Simultaneous intracellular chloride and pH measurements using a GFP-based sensor.

    PubMed

    Arosio, Daniele; Ricci, Fernanda; Marchetti, Laura; Gualdani, Roberta; Albertazzi, Lorenzo; Beltram, Fabio

    2010-07-01

    Chloride and protons perform important closely related roles in many cellular responses. Here we developed a ratiometric biosensor, ClopHensor, based on a highly chloride-sensitive Aequorea victoria GFP variant that is suited for the combined real-time optical detection of pH changes and chloride fluxes in live cells. We detected high chloride concentration in large dense-core exocytosis granules by targeting ClopHensor to these intracellular compartments.

  6. Effects of acute hypoxia/acidosis on intracellular pH in differentiating neural progenitor cells.

    PubMed

    Nordström, Tommy; Jansson, Linda C; Louhivuori, Lauri M; Akerman, Karl E O

    2012-06-21

    The response of differentiating mouse neural progenitor cells, migrating out from neurospheres, to conditions simulating ischemia (hypoxia and extracellular or intracellular acidosis) was studied. We show here, by using BCECF and single cell imaging to monitor intracellular pH (pH(i)), that two main populations can be distinguished by exposing migrating neural progenitor cells to low extracellular pH or by performing an acidifying ammonium prepulse. The cells dominating at the periphery of the neurosphere culture, which were positive for neuron specific markers MAP-2, calbindin and NeuN had lower initial resting pH(i) and could also easily be further acidified by lowering the extracellular pH. Moreover, in this population, a more profound acidification was seen when the cells were acidified using the ammonium prepulse technique. However, when the cell population was exposed to depolarizing potassium concentrations no alterations in pH(i) took place in this population. In contrast, depolarization caused an increase in pH(i) (by 0.5 pH units) in the cell population closer to the neurosphere body, which region was positive for the radial cell marker (GLAST). This cell population, having higher resting pH(i) (pH 6.9-7.1) also responded to acute hypoxia. During hypoxic treatment the resting pH(i) decreased by 0.1 pH units and recovered rapidly after reoxygenation. Our results show that migrating neural progenitor cells are highly sensitive to extracellular acidosis and that irreversible damage becomes evident at pH 6.2. Moreover, our results show that a response to acidosis clearly distinguishes two individual cell populations probably representing neuronal and radial cells.

  7. Effects of phorbol ester on contraction, intracellular pH and intracellular Ca2+ in isolated mammalian ventricular myocytes.

    PubMed Central

    MacLeod, K T; Harding, S E

    1991-01-01

    1. We have investigated the actions of certain phorbol esters on the intracellular pH, intracellular Ca2+ and contractility of isolated rat and guinea-pig cardiac myocytes. Intracellular pH was measured using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and intracellular Ca2+ was measured using Fura-2. 2. Application of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (also called phorbol 12-myristate 13-acetate) (TPA) (which activates protein kinase C) to rat cardiac myocytes significantly increased cell shortening by 116 +/- 34% (n = 8) (p less than 0.02). The rate of change of cell length during contraction (i.e. +dL/dt) increased from 67.2 +/- 8.7 microns/s to 127.7 +/- 14.1 microns/s (n = 7). The rate of change of cell length during relaxation (-dL/dt) increased from 55.8 +/- 7.4 microns/s to 118.9 +/- 12.1 microns/s (n = 7). Time to peak shortening was unchanged. 3. Application of 4 alpha-phorbol 12,13-didecanoate, which does not activate protein kinase C, did not affect rat myocyte contractility. An insignificant decrease in contractility (by 7.5 +/- 7.5%) was observed (n = 5). The positive inotropic effect of TPA may therefore be evoked through an activation of protein kinase C. 4. In rat myocytes we have measured the changes of pHi and contractility (cell shortening) during an alkalosis and acidosis induced by exposure to and subsequent removal of NH4Cl both in the presence and absence of TPA. Recovery times from an acid load were significantly (p less than 0.05) enhanced by 15.1 +/- 6.9% (n = 13) in the presence of TPA. Recovery times of cell shortening were also more rapid (p less than 0.05) by an average of 59.1 +/- 10.6% (n = 5) in the presence of TPA. Recovery times were unchanged in the presence of 4-phorbol 12,13-didecanoate (which does not activate protein kinase C). 5. Since pHi recovery of an isolated myocyte from an acid load is partially inhibited by the presence of 1 mM-amiloride and inhibited by removing extracellular Na

  8. Intracellular pH of brown adipose tissue increases during norepinephrine stimulation of thermogenesis

    SciTech Connect

    Horwitz, B.A.; Hamilton, J.S.

    1986-03-01

    Norepinephrine (NE) activation of brown fat (BAT) thermogenesis appears to involve dissociation of purine nucleotides from the mitochondrial uncoupling protein, resulting in release of normal respiratory control and enhanced substrate oxidation. Since the affinity of the uncoupling protein for purine nucleotides decreases significantly with increasing pH, the authors wished to determine if NE administration shifted the intracellular pH of BAT. To examine this question under in vivo conditions, they positioned a nuclear magnetic resonance (NMR) surface coil over the interscapular BAT of anesthetized male Syrian hamsters. The underlying and surrounding musculature was shielded to minimize their contribution to the /sup 31/P spectra. The hamster was placed in a Nicolet 200 Mhz spectrometer, operating in the Fourier Transform mode and tuned to /sup 31/P. Scans taken during infusion of ascorbate buffer (vehicle for NE) were compared to those taken during NE infusion (8 ng/g x min). During this infusion, BAT temperature increased 3.7 +/- 0.5/sup 0/C, confirming that BAT thermogenesis was activated. There also occurred a statistically significant PPM (parts per million) shift, averaging 0.070 +/- 0.022 (n = 22) and corresponding to an increase of approximately 0.07 pH units. This shift in intracellular pH from 7.32 to 7.39, although small, would facilitate the maintenance of loosely coupled brown fat mitochondria.

  9. Novel pH-sensitive probes with a ratiometric detection for intracellular pH

    NASA Astrophysics Data System (ADS)

    Ipuy, Martin; Billon, Cyrielle; Micouin, Guillaume; Samarut, Jacques; Andraud, Chantal; Bretonnière, Yann

    2014-08-01

    The development of new pH-sensitive fluorescent probes based on a push-pull architecture is presented with a 2- dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofurane as strong electron acceptor group. With a small structural change, it is possible to obtain a large range of phenolic pKa from 4.8 to 8.6 with some close to neutrality, underlining the role of the electron density modulation on the acidic properties. Remarkable changes in the optical properties (both absorption and fluorescence) were observed as a function of the pH. Ratiometric imaging of intracellular pH was carried out with the most promising probes and highlighted the possibility to distinguish near-neutral minor pH fluctuations in cells.

  10. Intracellular pH regulates basolateral K+ and Cl- conductances in colonic epithelial cells by modulating Ca2+ activation

    PubMed Central

    1991-01-01

    The role of intracellular pH as a modulator of basolateral K+ and Cl- conductances in epithelial cells was studied using digitonin- permeabilized colonic cell layers so that cytosolic pH could be clamped at specific values, while basolateral K+ and Cl- conductances were activated by stepwise increases in intracellular free Ca2+. Increasing the intracellular pH from 6.6 to 8.0 enhanced the sensitivity of both ionic conductances to intracellular Ca2+, but changing extracellular pH had no effect. Maximal K+ and Cl- currents activated by Ca2+ were not affected by changes in intracellular pH, suggesting that protons do not alter the conduction properties of the channels. Hill analysis of the Ca2+ activation process revealed that raising the cytosolic pH from 6.6 to 8.0 reduced the K1/2 for Ca2+ activation. In the absence of Ca2+, changes in intracellular pH did not have a significant effect on the basolateral K+ and Cl- conductances. These results are consistent with the notion that changes in cytosolic pH can modulate basolateral conductances by modifying the action of calcium, perhaps by acting at or near the activation site to provide a mechanism of variable "gain control." PMID:1719125

  11. Epidermal growth factor elevates intracellular pH in chicken granulosa cells.

    PubMed

    Li, M; Morley, P; Asem, E K; Tsang, B K

    1991-08-01

    Many bioregulators, such as epidermal growth factor (EGF), induce intracellular alkalinization by activating a membrane bound Na+/H+ antiporter. The present studies were designed to examine the influence of EGF on intracellular pH (pHi) in chicken granulosa cells. pHi in granulosa cells from the two largest preovulatory follicles of hens was determined spectrofluorometrically using the dye 2',7'-bis(carboxyethyl-5(6)-carboxyfluorescein. The resting pHi was 6.81 +/- 0.006 (n = 30) when the extracellular pH and sodium concentration (Na+o) were 7.3 and 144 mM, respectively. EGF (5-100 ng/ml) induced a concentration-dependent increase in pHi, which reached a maximum of 0.217 +/- 0.009 pH units at a concentration of 100 ng/ml EGF. Cytosolic alkalinization was observed within 10 min of the addition of EGF and lasted over the 60 min observation period. The increase in pHi was dependent upon the presence of Na+o, since the EGF effect was attenuated when Na+o was substituted with equimolar concentrations of nonpermeant choline chloride. The EGF-induced pHi change was also inhibited by amiloride, dimethyl amiloride, and ethylisopropyl amiloride, inhibitors of the Na+/H+ antiporter. The alkalinization effect of EGF was mimicked by transforming growth factor-alpha but not by insulin, insulin-like growth factor-I, or transforming growth factor-beta. These studies suggest for the first time that intracellular alkalinization resulting from activation of the Na+/H+ antiporter may be a part of the transmembrane signaling pathway in the action of EGF on chicken granulosa cells.

  12. Live-Cell imaging and measurement of intracellular pH in filamentous fungi using a genetically encoded ratiometric probe.

    PubMed

    Bagar, Tanja; Altenbach, Kirsten; Read, Nick D; Bencina, Mojca

    2009-05-01

    A novel, genetically encoded, ratiometric pH probe (RaVC) was constructed to image and measure intracellular pH in living hyphae of Aspergillus niger. RaVC is a chimeric protein based on the pH-sensitive probe pHluorin, which was partially codon optimized for expression in Aspergillus. Intracellular pH imaging and measurement was performed by simultaneous, dual-excitation confocal ratio imaging. The mean cytoplasmic pH measured was 7.4 to 7.7 based on calibrating RaVC in situ within nigericin-treated hyphae. Pronounced, longitudinal cytoplasmic pH gradients were not observed in the apical 20 microm of actively growing hyphae at the periphery of 18-h-old colonies. The cytoplasmic pH remained unchanged after prolonged growth in buffered medium with pH values between 2.5 or 9.5. Sudden changes in external pH significantly changed cytoplasmic pH by <1.3 pH units, but it returned to its original value within 20 min following treatment. The weak acid and antifungal food preservative sorbic acid caused prolonged, concentration-dependent intracellular acidification. The inhibition of ATPases with N-ethylmaleimide, dicychlohexylcarbodimide, or sodium azide caused the cytoplasmic pH to decrease by <1 pH unit. Treatment with the protonophore carbonyl cyanide m-chlorophenylhydrazone or cyanide p-(trifluoromethoxy) phenylhydrazone reduced the cytoplasmic pH by <1 pH unit. In older hyphae from 32-h-old cultures, RaVC became sequestered within large vacuoles, which were shown to have pH values between 6.2 and 6.5. Overall, our study demonstrates that RaVC is an excellent probe for visualizing and quantifying intracellular pH in living fungal hyphae.

  13. Intracellular pH Response to Weak Acid Stress in Individual Vegetative Bacillus subtilis Cells.

    PubMed

    Pandey, Rachna; Vischer, Norbert O E; Smelt, Jan P P M; van Beilen, Johan W A; Ter Beek, Alexander; De Vos, Winnok H; Brul, Stanley; Manders, Erik M M

    2016-11-01

    Intracellular pH (pHi) critically affects bacterial cell physiology. Hence, a variety of food preservation strategies are aimed at perturbing pHi homeostasis. Unfortunately, accurate pHi quantification with existing methods is suboptimal, since measurements are averages across populations of cells, not taking into account interindividual heterogeneity. Yet, physiological heterogeneity in isogenic populations is well known to be responsible for differences in growth and division kinetics of cells in response to external stressors. To assess in this context the behavior of intracellular acidity, we have developed a robust method to quantify pHi at single-cell levels in Bacillus subtilis Bacilli spoil food, cause disease, and are well known for their ability to form highly stress-resistant spores. Using an improved version of the genetically encoded ratiometric pHluorin (IpHluorin), we have quantified pHi in individual B. subtilis cells, cultured at an external pH of 6.4, in the absence or presence of weak acid stresses. In the presence of 3 mM potassium sorbate, a decrease in pHi and an increase in the generation time of growing cells were observed. Similar effects were observed when cells were stressed with 25 mM potassium acetate. Time-resolved analysis of individual bacteria in growing colonies shows that after a transient pH decrease, long-term pH evolution is highly cell dependent. The heterogeneity at the single-cell level shows the existence of subpopulations that might be more resistant and contribute to population survival. Our approach contributes to an understanding of pHi regulation in individual bacteria and may help scrutinizing effects of existing and novel food preservation strategies.

  14. Control of intracellular pH and growth by fibronectin in capillary endothelial cells

    NASA Technical Reports Server (NTRS)

    Ingber, D. E.; Prusty, D.; Frangioni, J. V.; Cragoe, E. J. Jr; Lechene, C.; Schwartz, M. A.

    1990-01-01

    The aim of this work was to analyze the mechanism by which fibronectin (FN) regulates capillary endothelial cell proliferation. Endothelial cell growth can be controlled in chemically-defined medium by varying the density of FN coated on the substratum (Ingber, D. E., and J. Folkman. J. Cell Biol. 1989. 109:317-330). In this system, DNA synthetic rates are stimulated by FN in direct proportion to its effect on cell extension (projected cell areas) both in the presence and absence of saturating amounts of basic FGF. To investigate direct growth signaling by FN, we carried out microfluorometric measurements of intracellular pH (pHi), a cytoplasmic signal that is commonly influenced by soluble mitogens. pHi increased 0.18 pH units as FN coating densities were raised and cells progressed from round to spread. Intracellular alkalinization induced by attachment to FN was rapid and followed the time course of cell spreading. When measured in the presence and absence of FGF, the effects of FN and FGF on pHi were found to be independent and additive. Furthermore, DNA synthesis correlated with pHi for all combinations of FGF and FN. Ethylisopropylamiloride, a specific inhibitor of the plasma membrane Na+/H+ antiporter, completely suppressed the effects of FN on both pHi and DNA synthesis. However, cytoplasmic pH per se did not appear to be a critical determinant of growth since DNA synthesis was not significantly inhibited when pHi was lowered over the physiological range by varying the pH of the medium. We conclude that FN and FGF exert their growth-modulating effects in part through activation of the Na+/H+ exchanger, although they appear to trigger this system via separate pathways.

  15. Hydroxylated near-infrared BODIPY fluorophores as intracellular pH sensors.

    PubMed

    Salim, Mohamed M; Owens, Eric A; Gao, Tielong; Lee, Jeong Heon; Hyun, Hoon; Choi, Hak Soo; Henary, Maged

    2014-10-07

    In this study, a series of new, highly sensitive BF2-chelated tetraarylazadipyrromethane dyes are synthesized and analyzed to be suitable as on/off photo-induced electron transfer modulated fluorescent sensors for determination of intracellular pH. The ethanolic solutions of the new indicators feature absorption maxima in the range of 696-700 nm and a fluorescence emission maximum at 720 nm. Molar absorptivity and fluorescence quantum yield data were determined for the studied set of aza-BODIPY indicators. These indicators have high molar absorption coefficients of ∼80,000 M(-1) cm(-1) and quantum yields (up to 18%). Corresponding pKa values of indicators are determined from absorbance and fluorescence measurements and range from 9.1 to 10.8, depending on the selective positioning of electron-donating functionalities. The excellent photostability of the aza-BODIPY indicators makes them particularly suitable for long duration measurements. The in vitro cellular staining of living tissues in PC3 cells based on the isosbestic point at pH 7.8 and pH 9.3 has been employed which shows an increase in fluorescence intensity at 800 nm with increase in pH for certain compounds and fluorescence intensity decreases at 700 nm. Therefore, the new indicators are suitable for exploitation and adaptation in a diverse range of analytical applications.

  16. Lactate modulates the intracellular pH sensitivity of human TREK1 channels.

    PubMed

    Ghatak, Swagata; Sikdar, Sujit Kumar

    2016-05-01

    Tissue acidosis and high lactate concentrations are associated with cerebral ischaemia. The degree of acidosis is dependent on circulating glucose concentration, hyperglycaemia being associated with increased acidosis. Among other agents, lactate and protons have been shown to activate the leak potassium channel; TREK1 (TWIK related potassium channel 1) from the intracellular side and its increased activity is implicated in tolerance towards ischaemic cell damage. In the present study, we show that ischaemic concentrations of lactate (30 mM) at pH 7.0 and 6.5, commonly observed during ischemia, cause robust potentiation of human TREK1 (hTREK1) activity at single-channel level in cell-free inside-out membrane patches, while 30 mM lactate at pH 6.0 to 5.5, commonly observed during hyperglycaemic ischemia, reduces hTREK1 channel activity significantly. The biphasic effect of 30 mM lactate (ischaemic concentrations) on modulation of hTREK1 by varying pH conditions is specific since basal concentrations of lactate (3 mM) and 30 mM pyruvate at pH 7.0 and 5.5 failed to show similar effect as lactate. Experiments with deletion and point mutants of hTREK1 channel suggest that lactate changes the pH modulation of hTREK1 by interacting differently with the histidine residue at 328th position (H328) above and below its pKa (∼6.0) in the intracellular carboxyl-terminal domain of TREK1. This lactate-induced pH modulation of hTREK1 is absent in C-terminal deletion mutant, CTDΔ100, and is similar in E321A-hTREK1 mutant as in wild-type hTREK1 suggesting that it is independent of pH-sensitive glutamate residue at 321st position. Such a differential pH-dependent effect of lactate on an ion channel function has not been reported earlier and has important implications in different stages of ischaemia.

  17. The effect of aminosulfonate buffers on the light responses and intracellular pH of goldfish retinal horizontal cells.

    PubMed

    Trenholm, Stuart; Baldridge, William H

    2010-10-01

    Retinal horizontal cell feedback acts as a gain control at the first synapse in the visual system and generates center-surround receptive fields in the outer retina. One model of feedback proposes that elevation of protons in the photoreceptor synaptic cleft produces feedback. Most evidence supporting the proton model has depended on the effect of proton buffers, in particular aminosulfonates, but these agents could potentially have effects other than external pH regulation. We therefore determined if the effects of aminosulfonates on horizontal cell rollback, an indicator of feedback, were consistent with external proton buffering. Intracellular recording from horizontal cells in isolated goldfish retina revealed that rollback was blocked only by aminosulfonates with an acid dissociation constant suited for buffering at the pH (7.5) of the Ringer's solution. In isolated goldfish horizontal cells, aminosulfonates, even those that did not block rollback, altered intracellular pH. This suggests that the effect of aminosulfonates on rollback is not because of changing intracellular pH. Measures of both intracellular and extracellular pH revealed that treatment with either glutamate or kainate resulted in acidification. As glutamate produced both internal and external acidification, intracellular and extracellular horizontal cell pH would be expected to increase in response to light, a change consistent with the proton model of feedback.

  18. Intracellular Temperature Sensing: An Ultra-bright Luminescent Nanothermometer with Non-sensitivity to pH and Ionic Strength

    PubMed Central

    Liu, Helin; Fan, Yanyan; Wang, Jianhai; Song, Zhongsen; Shi, Hao; Han, Rongcheng; Sha, Yinlin; Jiang, Yuqiang

    2015-01-01

    Luminescence thermometry usually suffer from cellular complexity of the biochemical environment (such as pH and ionic strength), and thus the accuracy and reliability of the determined intracellular temperature are directly affected. Herein, a photoluminescent nanothermometer composed of polymer encapsulated quantum dots (P-QD) has been developed. And the prepared nanothermometer exhibits some advantages: such as non-sensitivity to pH and ionic strength, as well as high detection sensitivity and ultrahigh reversibility. The intracellular temperature was accurately determined under physiological conditions with different pH and ionic strength, and direct measurement of thermogenesis in individual cells has been achieved. PMID:26445905

  19. Enhanced sensitivity of pHluorin-based monitoring of intracellular pH changes achieved through synchronously scanned fluorescence spectra.

    PubMed

    Plášek, Jaromír; Melcrová, Adéla; Gášková, Dana

    2015-10-06

    Since its introduction in 1998, genetically encoded pH-sensitive sensor ratiometric pHluorin proved to be a valuable tool for cell physiology studies. Here, we show how the sensitivity of pHluorin-based monitoring of intracellular pH changes performed with cell suspensions can be enhanced by using synchronously scanned fluorescence spectroscopy. In the suspensions of S. cerevisiae cells subjected to varying extracellular pH values, we have been able to measure statistically significant changes in intracellular pH of less than 0.1 unit, which were not detectable using a standard ratiometric approach.

  20. Cellular chloride and bicarbonate retention alters intracellular pH regulation in Cftr KO crypt epithelium

    PubMed Central

    Walker, Nancy M.; Liu, Jinghua; Stein, Sydney R.; Stefanski, Casey D.; Strubberg, Ashlee M.

    2015-01-01

    Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), an anion channel providing a major pathway for Cl− and HCO3− efflux across the apical membrane of the epithelium. In the intestine, CF manifests as obstructive syndromes, dysbiosis, inflammation, and an increased risk for gastrointestinal cancer. Cftr knockout (KO) mice recapitulate CF intestinal disease, including intestinal hyperproliferation. Previous studies using Cftr KO intestinal organoids (enteroids) indicate that crypt epithelium maintains an alkaline intracellular pH (pHi). We hypothesized that Cftr has a cell-autonomous role in downregulating pHi that is incompletely compensated by acid-base regulation in its absence. Here, 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein microfluorimetry of enteroids showed that Cftr KO crypt epithelium sustains an alkaline pHi and resistance to cell acidification relative to wild-type. Quantitative real-time PCR revealed that Cftr KO enteroids exhibit downregulated transcription of base (HCO3−)-loading proteins and upregulation of the basolateral membrane HCO3−-unloader anion exchanger 2 (Ae2). Although Cftr KO crypt epithelium had increased Ae2 expression and Ae2-mediated Cl−/HCO3− exchange with maximized gradients, it also had increased intracellular Cl− concentration relative to wild-type. Pharmacological reduction of intracellular Cl− concentration in Cftr KO crypt epithelium normalized pHi, which was largely Ae2-dependent. We conclude that Cftr KO crypt epithelium maintains an alkaline pHi as a consequence of losing both Cl− and HCO3− efflux, which impairs pHi regulation by Ae2. Retention of Cl− and an alkaline pHi in crypt epithelium may alter several cellular processes in the proliferative compartment of Cftr KO intestine. PMID:26542396

  1. Cellular chloride and bicarbonate retention alters intracellular pH regulation in Cftr KO crypt epithelium.

    PubMed

    Walker, Nancy M; Liu, Jinghua; Stein, Sydney R; Stefanski, Casey D; Strubberg, Ashlee M; Clarke, Lane L

    2016-01-15

    Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), an anion channel providing a major pathway for Cl(-) and HCO3 (-) efflux across the apical membrane of the epithelium. In the intestine, CF manifests as obstructive syndromes, dysbiosis, inflammation, and an increased risk for gastrointestinal cancer. Cftr knockout (KO) mice recapitulate CF intestinal disease, including intestinal hyperproliferation. Previous studies using Cftr KO intestinal organoids (enteroids) indicate that crypt epithelium maintains an alkaline intracellular pH (pHi). We hypothesized that Cftr has a cell-autonomous role in downregulating pHi that is incompletely compensated by acid-base regulation in its absence. Here, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein microfluorimetry of enteroids showed that Cftr KO crypt epithelium sustains an alkaline pHi and resistance to cell acidification relative to wild-type. Quantitative real-time PCR revealed that Cftr KO enteroids exhibit downregulated transcription of base (HCO3 (-))-loading proteins and upregulation of the basolateral membrane HCO3 (-)-unloader anion exchanger 2 (Ae2). Although Cftr KO crypt epithelium had increased Ae2 expression and Ae2-mediated Cl(-)/HCO3 (-) exchange with maximized gradients, it also had increased intracellular Cl(-) concentration relative to wild-type. Pharmacological reduction of intracellular Cl(-) concentration in Cftr KO crypt epithelium normalized pHi, which was largely Ae2-dependent. We conclude that Cftr KO crypt epithelium maintains an alkaline pHi as a consequence of losing both Cl(-) and HCO3 (-) efflux, which impairs pHi regulation by Ae2. Retention of Cl(-) and an alkaline pHi in crypt epithelium may alter several cellular processes in the proliferative compartment of Cftr KO intestine.

  2. A volatile inhibitor immobilizes sea urchin sperm in semen by depressing the intracellular pH

    SciTech Connect

    Johnson, C.H.; Clapper, D.L.; Winkler, M.M.; Lee, H.C.; Epel, D.

    1983-08-01

    Sea urchin spermatozoa are normally immotile in semen, but motility can be initiated by increasing gas flow over the semen--for example, by blowing N2 gas over a thin layer of semen. This result indicates that sperm motility is not O2 limited and suggests that seminal fluid contains a volatile inhibitor of motility which is responsible for the paralysis of sperm in semen. This inhibitor might be carbon dioxide, which reversibly immobilizes sperm. /sup 31/P-NMR measurements of pH show that the sperm intracellular pH (pHi) increases by 0.36 pH unit upon dilution of semen into seawater. Since previous studies have shown that this magnitude of pH increase is sufficient to trigger sperm motility, we suggest that the volatile inhibitor is inhibiting sperm motility in semen by depressing the pHi. A simple hypothesis that explains these observations is that the volatile motility inhibitor is CO/sub 2/, which could acidify pHi as a diffusable weak acid. In this regard, sperm diluted into seawater release acid, and this acid release is related to the pHi increase and motility initiation. In fact, nearly half of the acid released by sperm upon dilution is volatile and may therefore be due to CO/sub 2/ efflux. Most of the acid, however, cannot be attributed to CO/sub 2/ release because it is not volatile. Thus, when sperm are diluted into seawater, they raise their pHi by releasing CO/sub 2/ and protons from the cytoplasm into the surrounding seawater.

  3. Intracellular pH in prolonged hypoxia: effect of a CO/sub 2/ load

    SciTech Connect

    Gonzalez, N.C.; Clancy, R.L.

    1986-03-05

    The authors have previously shown that rats exposed to 0.5 atmosphere for three weeks (3WHx) have a great extracellular pH regulation during acute hypercapnia than normoxic (Nx) rats; this is due, in part, to a more effective renal compensation of hypercapnia in 3WHx. The present experiments were performed to study the intracellular pH (pHi) regulation of heart and of skeletal muscle (SKM) of 3WHx intact, conscious rats exposed acutely to an increase in inspired PCO/sub 2/. pHi was determined with the 5,5-dimethyl, 2-4-oxazolidine dione method. In the absence of inspired CO/sub 2/, there was no significant difference in pHe or pHi of heart or SKM between 3WHx and Nx. Hypercapnia resulted in a smaller decrease in SKM pHi in 3WHx than in Nx rats: intracellular non bicarbonate buffer value of various SKM was 5 to 15 times higher in 3WHx than in Nx. Heart pHi showed similar changes in both 3WHx and Nx. When PCO/sub 2/ was increased in nephrectomized rats, the difference in SKM pHi regulation between 3WHx and Nx either disappeared or was attenuated, depending on the muscle. Nephrectomy did not alter the effect of hypercapnia on heart pHi. The data show that SKM pHi regulation during a CO/sub 2/ load is improved in prolonged hypoxia, and that this appears to be due in part to a more effective renal compensation.

  4. Capacity for intracellular pH compensation during hypercapnia in white sturgeon primary liver cells.

    PubMed

    Huynh, Khuong Tuyen; Baker, Daniel W; Harris, Robert; Church, John; Brauner, Colin J

    2011-10-01

    Fish, exposed to elevated water CO(2), experience a rapid elevation in blood CO(2) (hypercapnia), resulting in acidification of both intra- and extra-cellular compartments. White sturgeon, Acipenser transmontanus, are exceptionally CO(2) tolerant and can regulate tissue intracellular pH (pH(i)) in the presence of a pronounced hypercapnic acidosis (preferential pH(i) regulation). In this study, pH(i) regulatory capacity of sturgeon liver cells in primary culture was examined to assess the suitability of employing this in vitro system to understand in vivo CO(2) tolerance in sturgeon. Using the pH-sensitive fluoroprobe BCECF, real-time changes in resting pH(i) and rates of pH(i) recovery were investigated during exposure to hypercapnia (3 and 6% CO(2)) in the absence and presence of additional acid loads induced by (20 mM) ammonium prepulse. During short-term (10 min) exposure to hypercapnia (3 and 6% CO(2)), sturgeon cells were acidified and no pH(i) compensation was observed. However, when exposure to 6% CO(2) was extended to over 19 h, the CO(2)-induced intracellular acidosis was partially compensated by a pH(i) increase of over 0.2 pH unit despite the sustained extracellular acidosis, indicative of a capacity for preferential pH(i) regulation in vitro. Since this capacity in sturgeon liver is present both in vivo and in vitro, the transmembrane transporters involved may be the same. Therefore, cell culture may be a suitable tool to identify the transporters (i.e., the cellular mechanisms underlying in vivo CO(2) tolerance) in white sturgeon and possibly in other hypercapnia-tolerant species.

  5. Intracellular pH and oxygen chemoreception in the cat carotid body in vitro.

    PubMed

    Iturriaga, R; Rumsey, W L; Lahiri, S; Spergel, D; Wilson, D F

    1992-06-01

    To test the hypothesis that O2 chemoreception in the carotid body (CB) is mediated by cellular acidosis, we simultaneously measured responses of the chemosensory and intracellular pH (pHi) to agents that are known to change pHi and studied the effects of hypoxia and ischemia on these variables in the cat CB. The CB was perfused and superfused in vitro with a modified Tyrode's solution at 36.0 +/- 0.5 degrees C with or without CO2-HCO3- (pH 7.40) and equilibrated at a given PO2. Chemosensory discharges were recorded from the whole carotid sinus nerve. To measure pHi changes, the CB was loaded with the pH-sensitive indicator 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, and the fluorescence (excitation 420-490 nm, emission greater than 515 nm) was detected by an intensified charged coupled device camera with an epifluorescence macroscope. Boluses of Tyrode's solution (0.5 ml, free of CO2-HCO3-) containing sodium acetate or NH4Cl prolonged perfusion of acid Tyrode's solution (pH 7.20-6.50), and boluses of Tyrode's solution with CO2-HCO3- were used. A decrease of fluorescence indicated pHi turning acid, and an increase of fluorescence indicated a change in alkaline pHi. Chemosensory activity varied inversely with the fluorescence change after application of these agents. Interruption of perfusate flow or application of hypoxic perfusate resulted in large increases in chemosensory discharge without any change in the fluorescence. The results indicated that chemosensory responses to brief ischemia and hypoxia were not mediated by a fall of pHi of CB cells, whereas those to CO2 and extracellular acidity were associated with decreases in pHi.

  6. High intracellular pH reversibly prevents gating-charge immobilization in squid axons.

    PubMed Central

    Wanke, E; Testa, P L; Prestipino, G; Carbone, E

    1983-01-01

    Squid giant axons were used to study the reversible effects of high intracellular pH (pHi) on gating currents. Under depolarization, when Na channels are activated, internal solutions buffered at high pHi (10.2) affect considerably the time course of gating charge associated with channel closing, QOFF, with almost no alteration of QON records. In particular, at pHi 10.2 the charge corresponding to the fast phase of IgOFF, measured after long depolarizing pulses (7.7 ms), was consistently larger than that recorded at physiological pHi (7.2). This suggests that high pH prevents immobilization of gating charges induced by Na inactivation. In this respect, the present data agree reasonably well with previous observations, which show that pHi greater than 7.2 reversibly removes the fast Na inactivation with little effects on activation kinetics (Carbone, E., P. L. Testa, and E. Wanke, 1981, Biophys. J., 35:393-413; Brodwick, M.S., and D. C. Eaton, 1978, Science [Wash. DC], 200:1494-1496). Unexpectedly, high pH increases the amount of charge associated with the slow phase of IgOFF. In our opinion, this might be the result of either an increment of the net charge produced by the exposure to high pHi or that gating charges that return to the closed state might experience a larger fraction of the potential drop across the membrane (Neumcke, B., W. Schwarz, and R. Stampfli, 1980, Biophys. J., 31:325-332). PMID:6652218

  7. Accurate Quantitative Sensing of Intracellular pH based on Self-ratiometric Upconversion Luminescent Nanoprobe

    NASA Astrophysics Data System (ADS)

    Li, Cuixia; Zuo, Jing; Zhang, Li; Chang, Yulei; Zhang, Youlin; Tu, Langping; Liu, Xiaomin; Xue, Bin; Li, Qiqing; Zhao, Huiying; Zhang, Hong; Kong, Xianggui

    2016-12-01

    Accurate quantitation of intracellular pH (pHi) is of great importance in revealing the cellular activities and early warning of diseases. A series of fluorescence-based nano-bioprobes composed of different nanoparticles or/and dye pairs have already been developed for pHi sensing. Till now, biological auto-fluorescence background upon UV-Vis excitation and severe photo-bleaching of dyes are the two main factors impeding the accurate quantitative detection of pHi. Herein, we have developed a self-ratiometric luminescence nanoprobe based on förster resonant energy transfer (FRET) for probing pHi, in which pH-sensitive fluorescein isothiocyanate (FITC) and upconversion nanoparticles (UCNPs) were served as energy acceptor and donor, respectively. Under 980 nm excitation, upconversion emission bands at 475 nm and 645 nm of NaYF4:Yb3+, Tm3+ UCNPs were used as pHi response and self-ratiometric reference signal, respectively. This direct quantitative sensing approach has circumvented the traditional software-based subsequent processing of images which may lead to relatively large uncertainty of the results. Due to efficient FRET and fluorescence background free, a highly-sensitive and accurate sensing has been achieved, featured by 3.56 per unit change in pHi value 3.0–7.0 with deviation less than 0.43. This approach shall facilitate the researches in pHi related areas and development of the intracellular drug delivery systems.

  8. Effects of Oxygen Availability on Acetic Acid Tolerance and Intracellular pH in Dekkera bruxellensis

    PubMed Central

    Capusoni, Claudia; Arioli, Stefania; Zambelli, Paolo; Moktaduzzaman, M.; Mora, Diego

    2016-01-01

    ABSTRACT The yeast Dekkera bruxellensis, associated with wine and beer production, has recently received attention, because its high ethanol and acid tolerance enables it to compete with Saccharomyces cerevisiae in distilleries that produce fuel ethanol. We investigated how different cultivation conditions affect the acetic acid tolerance of D. bruxellensis. We analyzed the ability of two strains (CBS 98 and CBS 4482) exhibiting different degrees of tolerance to grow in the presence of acetic acid under aerobic and oxygen-limited conditions. We found that the concomitant presence of acetic acid and oxygen had a negative effect on D. bruxellensis growth. In contrast, incubation under oxygen-limited conditions resulted in reproducible growth kinetics that exhibited a shorter adaptive phase and higher growth rates than those with cultivation under aerobic conditions. This positive effect was more pronounced in CBS 98, the more-sensitive strain. Cultivation of CBS 98 cells under oxygen-limited conditions improved their ability to restore their intracellular pH upon acetic acid exposure and to reduce the oxidative damage to intracellular macromolecules caused by the presence of acetic acid. This study reveals an important role of oxidative stress in acetic acid tolerance in D. bruxellensis, indicating that reduced oxygen availability can protect against the damage caused by the presence of acetic acid. This aspect is important for optimizing industrial processes performed in the presence of acetic acid. IMPORTANCE This study reveals an important role of oxidative stress in acetic acid tolerance in D. bruxellensis, indicating that reduced oxygen availability can have a protective role against the damage caused by the presence of acetic acid. This aspect is important for the optimization of industrial processes performed in the presence of acetic acid. PMID:27235432

  9. Accurate Quantitative Sensing of Intracellular pH based on Self-ratiometric Upconversion Luminescent Nanoprobe

    PubMed Central

    Li, Cuixia; Zuo, Jing; Zhang, Li; Chang, Yulei; Zhang, Youlin; Tu, Langping; Liu, Xiaomin; Xue, Bin; Li, Qiqing; Zhao, Huiying; Zhang, Hong; Kong, Xianggui

    2016-01-01

    Accurate quantitation of intracellular pH (pHi) is of great importance in revealing the cellular activities and early warning of diseases. A series of fluorescence-based nano-bioprobes composed of different nanoparticles or/and dye pairs have already been developed for pHi sensing. Till now, biological auto-fluorescence background upon UV-Vis excitation and severe photo-bleaching of dyes are the two main factors impeding the accurate quantitative detection of pHi. Herein, we have developed a self-ratiometric luminescence nanoprobe based on förster resonant energy transfer (FRET) for probing pHi, in which pH-sensitive fluorescein isothiocyanate (FITC) and upconversion nanoparticles (UCNPs) were served as energy acceptor and donor, respectively. Under 980 nm excitation, upconversion emission bands at 475 nm and 645 nm of NaYF4:Yb3+, Tm3+ UCNPs were used as pHi response and self-ratiometric reference signal, respectively. This direct quantitative sensing approach has circumvented the traditional software-based subsequent processing of images which may lead to relatively large uncertainty of the results. Due to efficient FRET and fluorescence background free, a highly-sensitive and accurate sensing has been achieved, featured by 3.56 per unit change in pHi value 3.0–7.0 with deviation less than 0.43. This approach shall facilitate the researches in pHi related areas and development of the intracellular drug delivery systems. PMID:27934889

  10. NHE1 is the sodium-hydrogen exchanger active in acute intracellular pH regulation in preimplantation mouse embryos.

    PubMed

    Siyanov, Violetta; Baltz, Jay M

    2013-06-01

    Sodium-hydrogen exchangers (NHE) of the Slc9 gene family are the major regulators of intracellular pH against acidosis in mammalian cells. Of five plasma membrane NHE isoforms, mouse oocytes and preimplantation embryos express mRNAs encoding NHE1 (SLC9A1), NHE3 (SLC9A3), and NHE4 (SLC9A4), with higher mRNA levels for each in oocytes through one-cell stage embryos and lower levels after the two-cell stage. NHE2 (SLC9A2) and NHE5 (SLC9A5) are not expressed. Measurements of intracellular pH during recovery from induced acidosis indicated that recovery occurred via NHE activity at all preimplantation stages assessed (one-cell, two-cell, eight-cell and morula). Recovery from acidosis at each stage was entirely inhibited by cariporide, which is very highly selective for NHE1. In contrast, the moderately NHE3-selective inhibitor S3226 did not preferentially block recovery, nor did adding S3226 increase inhibition over cariporide alone, indicating that NHE3 did not play a role. There was no indication of NHE4 activity. Another regulator of intracellular pH against acidosis, the sodium-dependent bicarbonate/chloride exchanger (NDBCE; SLC4A8), had low or absent activity in two-cell embryos. Thus, NHE1 appears to be the only significant regulator of intracellular pH in preimplantation mouse embryos. Culturing embryos from the one-cell or two-cell stages in acidotic medium inhibited their development. Unexpectedly, inhibition of NHE1 with cariporide, NDBCE with DIDS, or both together did not affect embryo development to the blastocyst stage more substantially under conditions of chronic acidosis than at normal pH. Preimplantation mouse embryos thus appear to have limited capacity to resist chronic acidosis using intracellular pH regulatory mechanisms.

  11. Intracellular pH homeostasis in Candida glabrata in infection-associated conditions.

    PubMed

    Ullah, Azmat; Lopes, Maria Inês; Brul, Stanley; Smits, Gertien J

    2013-04-01

    Candida glabrata is an opportunistic fungal pathogen which is a growing concern for immunocompromised patients. It is ranked as the second most common cause of candidiasis after Candida albicans. For pathogenic yeasts, intracellular pH (pHi) has been implicated in proliferation, dimorphic switching and virulence. We expressed the pH-sensitive green fluorescent protein variant ratiometric pHluorin in the cytosol of C. glabrata to study pHi dynamics in living cells. We evaluated the response of pHi to the various growth and stress conditions encountered during interaction with the host and during antifungal treatment. C. glabrata maintained a pHi higher than that of Saccharomyces cerevisiae in all growth conditions. The pHi of S. cerevisiae cells appeared better controlled than the pHi in C. glabrata when the cells were exposed to food and fermentation-associated conditions. C. glabrata in turn maintained its pHi better when exposed to host-associated conditions.

  12. Intracellular pH and ionic channels in the Loligo vulgaris giant axon.

    PubMed Central

    Carbone, E; Testa, P L; Wanke, E

    1981-01-01

    Squid giant axons were used to investigate the reversible effects of intracellular pH(pHi) on the kinetic properties of ionic channels. The pharmacologically separated K+ and Na+ currents were measured under: (a) internal perfusion, (b) enzymatic Pronase treatment, and (c) continuous estimate of periaxonal ion accumulation. Variation of internal pH from 4.8 to 11 resulted in: (a) a decrease of steady-state sodium inactivation at positive potentials similar to the effect of the proteolytic enzyme Pronase, (b) a shift of the h infinity (E) curve toward depolarizing voltages, and (c) a decrease of the time constant of inactivation for potentials below -20 mV (an increase above). A plot of the steady-state sodium conductance at E = +40 mV as a function of pHi suggests that two groups with pKa 10.4 and 5.6 affect respectively the inactivation gate and the rate constants for the transition from the inactivated to the second open state (h2) (Chandler and Meves, 1970b). The voltage shifts of the kinetic parameters predicted by the Gouy-Chapman-Stern theory are well satisfied at high pHi and less at low. Once corrected for voltage shifts, the forward rate constants for channel opening were found to be slowed with the acidity of the internal or external solution. PMID:6268214

  13. Direct measurement of intracellular pH changes in Xenopus eggs at fertilization and cleavage

    PubMed Central

    1981-01-01

    We have used Thomas-type recessed-tip pH-sensitive microelectrodes to measure the intracellular pH (pHi) in Xenopus eggs during both fertilization and ionophore activation. The average pHi in unfertilized eggs is 7.33 +/- 0.11 (SD; n = 21) with a resting membrane potential of -10.1 +/- 3.5 (SD; n = 38) mV. Within 2 min after the onset of the fertilization potential, there is a slight, transient pHi decrease of 0.03 +/- (SD, n = 8), followed by a distinct, permanent pHi increase of 0.31 +/- 0.11 (SD; n = 7) beginning approximately 10 min after the start of the fertilization potential and becoming complete approximately 1 h later. The pHi remains near this level of 7.67 +/- 0.13 (SD, n = 10) through at least 10 cleavage cycles, but it is possible to discern pHi oscillations with a mean amplitude of 0.03 +/- 0.02 (SD, n = 38). Eggs perfused for at least 2 h in Na+-free solution with 1 mM amiloride exhibited all of these pHi changes, so these changes do not require extracellular Na+. Similar cytoplasmic alkalinizations that accompany the activation of metabolism and the cell cycle in a wide variety of cell types are discussed. PMID:6796594

  14. In Situ Determination of the Intracellular pH of Lactococcus lactis and Lactobacillus plantarum during Pressure Treatment

    PubMed Central

    Molina-Gutierrez, Adriana; Stippl, Volker; Delgado, Antonio; Gänzle, Michael G.; Vogel, Rudi F.

    2002-01-01

    Hydrostatic pressure may affect the intracellular pH of microorganisms by (i) enhancing the dissociation of weak organic acids and (ii) increasing the permeability of the cytoplasmic membrane and inactivation of enzymes required for pH homeostasis. The internal pHs of Lactococcus lactis and Lactobacillus plantarum during and after pressure treatment at 200 and 300 MPa and at pH values ranging from 4.0 to 6.5 were determined. Pressure treatment at 200 MPa for up to 20 min did not reduce the viability of either strain at pH 6.5. Pressure treatment at pH 6.5 and 300 MPa reduced viable cell counts of Lactococcus lactis and Lactobacillus plantarum by 5 log after 20 and 120 min, respectively. Pressure inactivation was faster at pH 5 or 4. At ambient pressure, both strains maintained a transmembrane pH gradient of 1 pH unit at neutral pH and about 2 pH units at pH 4.0. During pressure treatment at 200 and 300 MPa, the internal pH of L. lactis was decreased to the value of the extracellular pH during compression. The same result was observed during treatment of Lactobacillus plantarum at 300 MPa. Lactobacillus plantarum was unable to restore the internal pH after a compression-decompression cycle at 300 MPa and pH 6.5. Lactococcus lactis lost the ability to restore its internal pH after 20 and 4 min of pressure treatment at 200 and 300 MPa, respectively. As a consequence, pressure-mediated stress reactions and cell death may be considered secondary effects promoted by pH and other environmental conditions. PMID:12200293

  15. Continuous direct measurement of intracellular chloride and pH in frog skeletal muscle

    PubMed Central

    Bolton, T. B.; Vaughan-Jones, R. D.

    1977-01-01

    1. Ion-sensitive electrodes (made with a chloride-sensitive ion-exchange resin) were used to measure the internal chloride activity (aiCl) of frog sartorius fibres at 25° C. 2. The internal pH (pHi) of other sartorius fibres was measured with a recessed tip pH-sensitive electrode (made with pH-sensitive glass). 3. In normal bicarbonate-free solution (containing 2·5 mM potassium), the average chloride equilibrium potential, ECl (calculated from aiCl and the measured chloride activity of the external solution (aoCl) was 87·7 ± 1·7 mV (mean ± S.E.; n = 16) in fibres where the average membrane potential, Em, was 88·3 ± 1·5 mV (mean ± S.E.; n = 16). In experiments where aiCl was varied between about 1 and 10 mM (which corresponds to values of Em between about -105 and -50 mV) ECl was within 1-3 mV of Em at equilibrium. These measurements of aiCl were obtained from the potential difference between the chloride-sensitive electrode and an intracellular indifferent micro-electrode filled with potassium chloride. If a potassium sulphate-filled indifferent micro-electrode was used, then values of aiCl below about 5 mM were erroneously high, probably due to interference from other sarcoplasmic ions at the indifferent electrode. 4. In solutions containing 15 mM bicarbonate and gassed with 5% CO2, pHi was 6·9, corresponding to an internal bicarbonate concentration of 7·6 mM. ECl measured in this solution was some 4 mV positive to Em. Most of the difference between ECl and Em could be ascribed to interference by sarcoplasmic bicarbonate on the basis of selectivity measurements of chloride against bicarbonate made on the ion-exchange resin in the relevant range of aCl. 5. If bicarbonate/CO2 in the external solution was replaced by HEPES/pure O2 at constant pH, then pHi rose from 6·88 ± 0·02 (mean ± S.E.) to 7·05 ± 0·02. A change in external pH of 1 unit caused pHi to change by about 0·02 unit and the intracellular buffering power was calculated to be about 35

  16. Intracellular pH and calcium signaling as molecular targets of diclofenac-induced apoptosis against colon cancer.

    PubMed

    Kaur, Jasmeet; Sanyal, Sankar Nath

    2011-07-01

    The role of intracellular pH and Ca2+ and their association with mitochondrial dysfunction and intracellular reactive oxygen species (ROS) are explored in the chemoprevention of colon cancer. 1,2-dimethylhydrazine dihydrochloride (DMH), a potent procarcinogen with selectivity for the colon, at a dose of 30 mg/kg body weight was used to induce initial stages of colon cancer when administered for 6 weeks in male Sprague-Dawley rats. Diclofenac, a preferential cyclooxygenase-2 inhibitor, was used at the anti-inflammatory dose (8 mg/kg body weight) for chemoprevention. The control group was administered vehicles for both DMH and diclofenac. A diclofenac-alone group with the same dose was also run simultaneously. Intracellular pH values as determined by biscarboxyethyl carboxyfluorescein fluorescence assay showed an alkaline pH in colonocytes from the DMH-treated group as compared with the control group. Moreover, the level of intracellular Ca2+ was also found to be decreased with DMH treatment, as shown by the fura-2 acetoxymethyl study and chlortetracycline assay. Apoptosis was studied by comet assay and Apaf-1 immunofluorescent expression and was found to be markedly decreased in this group, indicating that disturbances in pH and Ca2+ homeostasis promoted proliferation in colon and inhibited apoptosis. Changes in mitochondrial membrane potential and ROS levels were analyzed in isolated colonocytes by rhodamine 123 and 2,7-dichlorofluorescein diacetate labeling, respectively. DMH treatment promoted a higher mitochondrial membrane potential while reducing ROS levels. These parameters are known to be associated with pH and Ca2+ changes intracellularly and hence can be suggested to be linked with them in this study also. Diclofenac promoted apoptosis in colonocytes when coadministered with DMH and also ameliorated the changes observed in the above parameters, confirming these mechanisms as early events for the onset of apoptosis in cancer cells.

  17. Participation of intracellular and extracellular pH changes in photosynthetic response development induced by variation potential in pumpkin seedlings.

    PubMed

    Sherstneva, O N; Vodeneev, V A; Katicheva, L A; Surova, L M; Sukhov, V S

    2015-06-01

    Electrical signals presented in plants by action potential and by variation potential (VP) can induce a reversible inactivation of photosynthesis. Changes in the intracellular and extracellular pH during VP generation are a potential mechanism of photosynthetic response induction; however, this hypothesis requires additional experimental investigation. The purpose of the present work was to analyze the influence of pH changes on induction of the photosynthetic response in pumpkin. It was shown that a burning of the cotyledon induced VP propagation into true leaves of pumpkin seedlings inducing a decrease in the photosynthetic CO2 assimilation and an increase in non-photochemical quenching of fluorescence, whereas respiration was activated insignificantly. The photosynthetic response magnitude depended linearly on the VP amplitude. The intracellular and extracellular concentrations of protons were analyzed using pH-sensitive fluorescent probes, and the VP generation was shown to be accompanied by apoplast alkalization (0.4 pH unit) and cytoplasm acidification (0.3 pH unit). The influence of changes in the incubation medium pH on the non-photochemical quenching of fluorescence of isolated chloroplasts was also investigated. It was found that acidification of the medium stimulated the non-photochemical quenching, and the magnitude of this increase depended on the decrease in pH. Our results confirm the contribution of changes in intracellular and extracellular pH to induction of the photosynthetic response caused by VP. Possible mechanisms of the influence of pH changes on photosynthesis are discussed.

  18. Regulation of intracellular pH in rat lactotrophs: involvement of anionic exchangers.

    PubMed

    Garcia, L; Boué-Grabot, E; Garret, M; Sartor, P

    1997-10-01

    Regulation of the intracellular pH (pHi) of normal rat lactotrophs was studied. As this cell type, cultured with 10% FCS, can achieve a relatively alkaline pHi (7.3-7.5), we investigated the presence of a mechanism based on Cl-/HCO3- exchange. Using the pHi-sensitive probe SNARF-1 (seminaphtorodafluor) in its permeant form, SNARF-1/AM, we studied pHi recovery after acidic loading in individual cells with a microspectrofluorometric approach. We showed the involvement of anionic exchange in lactotroph cell pHi regulation. Acute CO2-bicarbonate cell acidic loading combined with external Cl- depletion induces the activation of a Cl-/HCO3- exchange. This exchange is 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid sensitive and corresponds to the type 3 anionic exchanger (AE3). However, after nigericin acidification, Na+/H+ exchange can also participate in recovery. In addition, incubation experiments strongly suggest that a 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-insensitive anionic exchanger (type 2 anionic exchanger or AE2) is present in rat lactotrophs. The presence and involvement of carbonic anhydrase in pHi regulation have been demonstrated. Finally, using Northern blot and reverse transcription-PCR techniques, messenger RNAs for both AE2 and AE3 were identified in anterior pituitary cell extracts. We concluded that in normal rat lactotrophs, pHi regulation is achieved by a complex system in which Cl-/HCO3- exchange has a pivotal role.

  19. Intracellular pH in lizard Dipsosaurus dorsalis in relation to changing body temperatures.

    PubMed

    Bickler, P E

    1982-12-01

    Mean whole-body and tissue-specific intracellular pH values (pHi) were measured in Dipsosaurus dorsalis by the dimethyloxazolidinedione technique. pHi was measured in lizards at constant body temperatures (Tb) (18, 25, 35, and 42 degrees C) and in lizards undergoing changes in Tb between 18 and 42 degrees C. Constant Tb between 18 and 42 degrees C maintained for 24 h or more produced a delta pH/delta Tb of -0.015 for the mean whole-body, -0.012 for venous blood, -0.0104 for cardiac muscle, and -0.0098 for skeletal muscle. Within the preferred range of Tb values (35-42 degrees C), the delta pH/delta Tb patterns were closer to that expected to achieve constant dissociation of protein imidazole (approximately -0.017): mean whole-body -0.020, cardiac muscle -0.016, and skeletal muscle -0.018. Tissue water contents were independent of Tb. Whole-body pHi during gradual warming and cooling (approximately 2 h elapsed time for each direction) closely corresponded to steady-state values. Upon cooling to 18 degrees C, tissue-specific and whole-body pHi often fell 0.1-0.2 unit below that expected; in each case this was correlated with an extracellular acidosis. A gradual recovery of pHi occurred with the recovery of the extracellular acidosis. Over the normally experienced Tb range, adjustments in pHi apparently rapidly achieve steady-state values and are in accord with the imidazole alphastat hypothesis. These patterns are discussed in terms of the thermal ecology of Dipsosaurus.

  20. Ratio imaging: practical considerations for measuring intracellular Ca2+ and pH in living cells.

    PubMed

    O'Connor, Nathan; Silver, Randi B

    2013-01-01

    This chapter discusses the use of ratiometric fluorescent probes for measuring intracellular pH (pHi) and Cai(2+) concentration at the single cell level. The development of sensitive and stable probes for monitoring pHi and Cai(2+) in living cells has provided the scientists with invaluable tools for studying a multitude of cellular processes. These probes afford a noninvasive and semiquantitative assessment of pHi and Cai(2+), eliminating the need to impale cells with microelectrodes. The development and availability of membrane permeant Cai(2+)- and pH-specific fluorescent probes coupled to major advances in the technology and design of low-light-level charge-coupled devices geared toward biological applications, and improved microscope optics, have made it possible to visualize a two-dimensional fluorescence signal that is related to Cai(2+) and pHi. The chapter describes the basis for using dual excitation ratio imaging and tries to provide a framework for understanding and developing the technique for investigating the roles of Cai(2+) and pHi in cellular processes. The technique of quantitative ratio imaging for the measurement of pHi and Cai(2+) has revolutionized the field of cell physiology. Using the proper equipment and choosing the right dyes for the experimental needs should provide reliable and reproducible results. More importantly, the amount of data produced from each experiment, when analyzing pHi and Cai(2+) on an individual cell basis, yields valuable information on the heterogeneity of cellular responses.

  1. Monitoring the intracellular pH of Zygosaccharomyces bailii by green fluorescent protein.

    PubMed

    Dang, T D T; De Maeseneire, S L; Zhang, B Y; De Vos, W H; Rajkovic, A; Vermeulen, A; Van Impe, J F; Devlieghere, F

    2012-06-01

    It is generally known that intracellular pH (pH(i)) plays a vital role in cell physiology and that pH(i) homeostasis is essential for normal cellular functions. Therefore, it is desirable to know the pH(i) during cell life cycle or under various growth conditions. Different methods to measure pH(i) have been developed and among these methods, the use of pH-sensitive green fluorescent protein (GFP) as a pH(i) indicator is a promising technique. By using this approach, not only can more accurate pH(i) results be obtained but also long-term experiments on pH(i) can be performed. In this study, the wild type Zygosaccharomyces bailii, a notorious food spoilage yeast, was transformed with a plasmid encoding a pH-sensitive GFP (i.e. pHluorin), enabling the pH(i) of the yeast to be determined based on cellular fluorescent signals. After the transformation, growth and pH(i) of the yeast were investigated in four different acidic conditions at 22°C during 26days. From the experimental results, the transformation effectiveness was verified and a good correlation between yeast growth and pH(i) was noticed. Particularly, it was observed that the yeast has an ability to tolerate a significant pH(i) drop during exponential phase and a subsequent pH(i) recovery in stationary phase, which may underlie the exceptional acid resistance of the yeast. This was the first time that a GFP-based approach for pH(i) measurement was applied in Z. bailii and that the pH(i) of the yeast was monitored during such a long period (26days). It can be expected that greater understanding of the physiological properties and mechanisms behind the special acid resistance of the yeast will be obtained from further studies on this new yeast strain.

  2. Proliferation and intracellular pH in cultured proximal tubular cells

    SciTech Connect

    Larsson, S.H.; Fukuda, Y.; Koelare, S.A.; Aperia, A. )

    1990-03-01

    Renal proximal tubule (PT) cells from adult rats will maintain much of their functional characteristics in short-term primary culture. This study examines the growth regulation of these highly differentiated cells with particular reference to cell density, intracellular pH (pHi), and the expression of the Na(+)-H+ exchanger. PT cells were obtained from young adult rats and studied after 48 h in culture. The mitotic rate was determined as the labeling index (LI) after (3H)thymidine autoradiography, and pHi was determined by 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein quantitative fluorescence microscopy in single cells. Cells were grown either continuously in serum (S) or were serum deprived after 24 h (D). The cells were nonconfluent and grew in colonies. We defined the two peripheral layers of cells in a colony as peripheral (P) cells and the remaining cells as central (C). In C cells LI/h and pHi were in the range of what has been observed under in vivo conditions. In S condition LI/h was 2.2 +/- 0.3% and in D condition was 0.3 +/- 0.1%. LI was significantly higher in P than in C cells both under S (2.5 +/- 0.4-fold) and D conditions (5.6 +/- 0.8-fold). The rapidly growing P cells had a significantly lower pHi than the growth-retarded C cells both under S (7.25 +/- 0.02 vs. 7.30 +/- 0.01, P less than 0.05) and D conditions (7.21 +/- 0.02 vs. 7.28 +/- 0.01, P less than 0.05).

  3. Muscle fatigue in frog semitendinosus: role of intracellular pH

    NASA Technical Reports Server (NTRS)

    Thompson, L. V.; Balog, E. M.; Fitts, R. H.

    1992-01-01

    The purpose of this study was to utilize glass microelectrodes to characterize the intracellular pH (pHi) before and during recovery from fatigue in the frog semitendinosus (ST) muscle. A second objective was to evaluate the relationship between pHi and contractile function. The frog ST muscle (22 degrees C) was fatigued by direct electrical stimulation with 100-ms 150-Hz trains at 1/s for 5 min. Peak tetanic force (Po) was reduced to 8.5% of initial force and recovered in a biphasic manner, returning to the resting value by 40 min. Resting pHi was 7.00 +/- 0.02 (n = 37) and declined with fatigue to an average value of 6.42 at 3 min of recovery. During recovery pHi significantly increased and by 25 min had returned to the prefatigue value. The pHi recovery was highly correlated to the slow phase of Po recovery (r = 0.98, P less than 0.001). The mean resting membrane potential was -78 +/- 1.0 mV (n = 42) and at 3 min of recovery was depolarized to -67 +/- 4 mV. Both the peak rate of twitch force development (+dP/dt) (r = 0.99, P less than 0.001) and decline (-dP/dt) (r = 0.94, P less than 0.014) were highly correlated to pHi during the slow phase of recovery. Contraction time (CT) and one-half relaxation time (1/2RT) increased significantly and recovered exponentially. The recovery of CT and 1/2RT were both significantly correlated to pHi (r = -0.93, P less than 0.001 and r = -0.86, P less than 0.001 for CT and 1/2RT, respectively).(ABSTRACT TRUNCATED AT 250 WORDS).

  4. Anaerobic phosphate release from activated sludge with enhanced biological phosphorus removal. A possible mechanism of intracellular pH control

    SciTech Connect

    Bond, P.L.; Keller, J.; Blackall, L.L.

    1999-06-05

    The biochemical mechanisms of the wastewater treatment process known as enhanced biological phosphorus removal (EBPR) are presently described in a metabolic model. The authors investigated details of the EBPR model to determine the nature of the anaerobic phosphate release and how this may be metabolically associated with polyhydroxyalkanoate (PHA) formation. Iodoacetate, an inhibitor of glycolysis, was found to inhibit the anaerobic formation of PHA and phosphate release, supporting the pathways proposed in the EBPR metabolic model. In the metabolic model, it is proposed that polyphosphate degradation provides energy for the microorganisms in anaerobic regions of these treatment systems. Other investigations have shown that anaerobic phosphate release depends on the extracellular pH. The authors observed that when the intracellular pH of EBPR sludge was raised, substantial anaerobic phosphate release was caused without volatile fatty acid (VFA) uptake. Acidification of the sludge inhibited anaerobic phosphate release even in the presence of VFA. from these observations, the authors postulate that an additional possible role of anaerobic polyphosphate degradation in EBPR is for intracellular pH control. Intracellular pH control may be a metabolic feature of EBPR, not previously considered, that could have some use in the control and optimization of EBPR.

  5. Effect of ammonia production on intracellular pH: Consequent effect on adenovirus vector production.

    PubMed

    Ferreira, T B; Carrondo, M J T; Alves, P M

    2007-05-01

    Recombinant adenoviral vectors (AdV) have proven to be highly efficient for the delivery and expression of foreign genes in a broad spectrum of cell types and species both for vaccination and gene therapy in a number of specific applications. In this study, the effect of ammonia production on intracellular pH (pH(i)) and consequently inhibition of AdV production at high cell densities is assessed. Different specific ammonia production rates were obtained for 293 cells adapted to grow in glutamate supplemented medium (non-ammoniagenic medium) as compared with 293 cells growing in glutamine supplemented medium (ammoniagenic medium); pH(i) was observed to be lower during cell growth and AdV production at both high and low CCI in the ammoniagenic medium, where the specific ammonia production rate is higher. In addition, after infection at CCI of 3x10(6)cell/ml, the cell viability decreased significantly in the ammoniagenic medium, attributed to the activation of an acidic pathway of apoptosis. Furthermore, AdV DNA was observed to be degraded at the observed pH(i) in the ammoniagenic medium, decreasing significantly the amount of AdV DNA available for encapsulation. To elucidate the pH(i) effect upon AdV production, 293 cells were infected at a CCI of 1 x 10(6)cell/ml in the non-ammoniagenic medium with a manipulated pH(i) as observed at the time of infection at CCI of 3 x 10(6)cell/ml in the ammoniagenic (pH(i) 7.0) and non-ammoniagenic (pH(i) 7.3) media; AdV volumetric productivities were observed to be lower when the cells were exposed to the lower pH(i). Thus, the importance of controlling all the factors contributing to pH(i) on AdV production, such as ammonia production, has been established.

  6. Preferential intracellular pH regulation represents a general pattern of pH homeostasis during acid-base disturbances in the armoured catfish, Pterygoplichthys pardalis.

    PubMed

    Harter, T S; Shartau, R B; Baker, D W; Jackson, D C; Val, A L; Brauner, C J

    2014-08-01

    Preferential intracellular pH (pHi) regulation, where pHi is tightly regulated in the face of a blood acidosis, has been observed in a few species of fish, but only during elevated blood PCO2. To determine whether preferential pHi regulation may represent a general pattern for acid-base regulation during other pH disturbances we challenged the armoured catfish, Pterygoplichthys pardalis, with anoxia and exhaustive exercise, to induce a metabolic acidosis, and bicarbonate injections to induce a metabolic alkalosis. Fish were terminally sampled 2-3 h following the respective treatments and extracellular blood pH, pHi of red blood cells (RBC), brain, heart, liver and white muscle, and plasma lactate and total CO2 were measured. All treatments resulted in significant changes in extracellular pH and RBC pHi that likely cover a large portion of the pH tolerance limits of this species (pH 7.15-7.86). In all tissues other than RBC, pHi remained tightly regulated and did not differ significantly from control values, with the exception of a decrease in white muscle pHi after anoxia and an increase in liver pHi following a metabolic alkalosis. Thus preferential pHi regulation appears to be a general pattern for acid-base homeostasis in the armoured catfish and may be a common response in Amazonian fishes.

  7. Does low intensity He-Ne laser radiation affect the intracellular pH of intact Escherichia coli cells?

    NASA Astrophysics Data System (ADS)

    Quickenden, Terence I.; Daniels, Lillian L. L.; Byrne, Lyndsay T.

    1995-05-01

    Claims that low levels of He-Ne laser light (cw, (lambda) equals 632.8 nm) can provide clinical benefits and can enhance in vitro cellular growth are still controversial (T.I. Quickenden and L.L. Daniels, 1993, Photochem. Photobiol. 57, 272-278; L.L. Daniels and T.I. Quickenden, 1994, Photochem. Photobiol., 60, 481-485). The present study tests the suggestion (T.I. Karu, 1988, Lasers life Sci. 2, 53-74; H. Friedmann, R. Lubart, I. Laulicht and S. Rochkink, 1991, J. Photochem. Photobiol. B: Biol. 11, 87-91) that red light stimulates mitosis by raising intracellular pH via absorption by chromophores in the respiratory chain. In order to search for photoinduced changes in intracellular pH, the effect of 5 mW He-Ne laser irradiation on cultures of E. coli was examined using a 300 MHz Nuclear Magnetic Resonance (NMR) spectrometer. The pH difference between the intracellular and extracellular fluid was monitored in the presence and absence of radiation by determining the difference in chemical shift for 31P resonances arising from the H2PO4- ⇔ HPO42- + H+ equilibrium in the two environments.

  8. Simultaneous imaging of intracellular [Ca2+] and pH in single MDCK and glomerular epithelial cells.

    PubMed

    Wiegmann, T B; Welling, L W; Beatty, D M; Howard, D E; Vamos, S; Morris, S J

    1993-10-01

    The interrelationships between changes in intracellular calcium concentration ([Ca2+]i) and intracellular pH in Madin-Darby canine kidney cells and kidney glomerular epithelial cells exposed to various stimuli were analyzed simultaneously using a new design of a fluorescence video microscope. Cells were double labeled with indo 1 and SNARF 1 dyes and were excited simultaneously at 350 and 540 nm. Images at four emission wavelengths were captured simultaneously at 405, 475, 575, and 640 nm at 30 frames/s for the two ratio dyes. SNARF sensitivity to pH between 6.5 and 8.0 was unchanged by [Ca2+]i. The SNARF ratio maps were used to correct the pH-dependent changes in the calculation of local cell calcium. NH4Cl loading produced the expected alkalinization and a concurrent rise in [Ca2+]i. When the NH4Cl was removed and the cells became acidic, a second rise in [Ca2+]i was recorded. Both changes in [Ca2+]i were from intracellular stores since they persisted in the absence of extracellular calcium. The findings demonstrate the need for pH correction of indo 1 recordings.

  9. Influence of extracellular pH on growth, viability, cell size, acidification activity, and intracellular pH of Lactococcus lactis in batch fermentations.

    PubMed

    Hansen, Gunda; Johansen, Claus Lindvald; Marten, Gunvor; Wilmes, Jacqueline; Jespersen, Lene; Arneborg, Nils

    2016-07-01

    In this study, we investigated the influence of three extracellular pH (pHex) values (i.e., 5.5, 6.5, and 7.5) on the growth, viability, cell size, acidification activity in milk, and intracellular pH (pHi) of Lactococcus lactis subsp. lactis DGCC1212 during pH-controlled batch fermentations. A universal parameter (e.g., linked to pHi) for the description or prediction of viability, specific acidification activity, or growth behavior at a given pHex was not identified. We found viability as determined by flow cytometry to remain high during all growth phases and irrespectively of the pH set point. Furthermore, regardless of the pHex, the acidification activity per cell decreased over time which seemed to be linked to cell shrinkage. Flow cytometric pHi determination demonstrated an increase of the averaged pHi level for higher pH set points, while the pH gradient (pHi-pHex) and the extent of pHi heterogeneity decreased. Cells maintained positive pH gradients at a low pHex of 5.5 and even during substrate limitation at the more widely used pHex 6.5. Moreover, the strain proved able to grow despite small negative or even absent pH gradients at a high pHex of 7.5. The larger pHi heterogeneity at pHex 5.5 and 6.5 was associated with more stressful conditions resulting, e.g., from higher concentrations of non-dissociated lactic acid, while the low pHi heterogeneity at pHex 7.5 most probably corresponded to lower concentrations of non-dissociated lactic acid which facilitated the cells to reach the highest maximum active cell counts of the three pH set points.

  10. Alterations in mouse embryo intracellular pH by DMO during culture impair implantation and fetal growth.

    PubMed

    Zander-Fox, Deirdre L; Mitchell, Megan; Thompson, Jeremy G; Lane, Michelle

    2010-08-01

    The preimplantation embryo is highly susceptible to in-vitro stress, and although this does not necessarily perturb blastocyst development, it can significantly affect embryo physiology and the ability to form a viable pregnancy. This study determined that the preimplantation mouse embryo is highly sensitive to a small decrease in intracellular pH (<0.2 pH units). Embryos cultured in media containing a weak acid (5,5-dimethyl-2,4-oxazolidinedione; DMO) formed blastocysts with decreased cell number and inner cell mass number, as well as increased apoptosis, even though blastocyst development and morphology were unchanged. Interestingly, the effects were similar regardless of whether the pH stress was present for a short-term 'acute' exposure (during the zygote to 2-cell, or 2-cell to 8-cell division) or an extended 'chronic' period of time (continually from the zygote to the blastocyst stage). Exposure to DMO during the first cleavage division did not alter implantation; however, fetal weight and crown-rump length were significantly decreased (P<0.05). In contrast, continuous exposure to DMO throughout preimplantation development reduced not only implantation but also fetal weight and crown-rump length. This study highlights the importance of correct intracellular pH and demonstrates that slight deviations can significantly impact embryo development and viability.

  11. Intracellular pH regulation in unstimulated Calliphora salivary glands is Na+ dependent and requires V-ATPase activity.

    PubMed

    Schewe, Bettina; Blenau, Wolfgang; Walz, Bernd

    2012-04-15

    Salivary gland cells of the blowfly Calliphora vicina have a vacuolar-type H(+)-ATPase (V-ATPase) that lies in their apical membrane and energizes the secretion of a KCl-rich primary saliva upon stimulation with serotonin (5-hydroxytryptamine). Whether and to what extent V-ATPase contributes to intracellular pH (pH(i)) regulation in unstimulated gland cells is unknown. We used the fluorescent dye BCECF to study intracellular pH(i) regulation microfluorometrically and show that: (1) under resting conditions, the application of Na(+)-free physiological saline induces an intracellular alkalinization attributable to the inhibition of the activity of a Na(+)-dependent glutamate transporter; (2) the maintenance of resting pH(i) is Na(+), Cl(-), concanamycin A and DIDS sensitive; (3) recovery from an intracellular acid load is Na(+) sensitive and requires V-ATPase activity; (4) the Na(+)/H(+) antiporter is not involved in pH(i) recovery after a NH(4)Cl prepulse; and (5) at least one Na(+)-dependent transporter and the V-ATPase maintain recovery from an intracellular acid load. Thus, under resting conditions, the V-ATPase and at least one Na(+)-dependent transporter maintain normal pH(i) values of pH 7.5. We have also detected the presence of a Na(+)-dependent glutamate transporter, which seems to act as an acid loader. Despite this not being a common pH(i)-regulating transporter, its activity affects steady-state pH(i) in C. vicina salivary gland cells.

  12. Effect of cationic side-chains on intracellular delivery and cytotoxicity of pH sensitive polymer-doxorubicin nanocarriers

    NASA Astrophysics Data System (ADS)

    Fang, Chen; Kievit, Forrest M.; Cho, Yong-Chan; Mok, Hyejung; Press, Oliver W.; Zhang, Miqin

    2012-10-01

    Fine-tuning the design of polymer-doxorubicin conjugates permits optimization of an efficient nanocarrier to greatly increase intracellular uptake and cytotoxicity. Here, we report synthesis of a family of self-assembled polymer-doxorubicin nanoparticles and an evaluation of the effects of various types of side-chains on intracellular uptake and cytotoxicity of the nanocarriers for lymphoma cells. Monomers with three different cationic side-chains (CA) and pKa's, i.e., a guanidinium group (Ag), an imidazole group (Im), and a tertiary amine group (Dm), were comparatively investigated. The cationic monomer, poly(ethylene glycol) (PEG), and doxorubicin (Dox) were reacted with 1,4-(butanediol) diacrylate (BUDA) to prepare a poly(β-amino ester) (PBAE) polymer via Michael addition. All three polymer-Dox conjugates spontaneously formed nanoparticles (NP) through hydrophobic interactions between doxorubicin in aqueous solution, resulting in NP-Im/Dox, NP-Ag/Dox, and NP-Dm/Dox, with hydrodynamic sizes below 80 nm. Doxorubicin was linked to all 3 types of NPs with a hydrazone bond to assure selective release of doxorubicin only at acidic pH, as it occurs in the tumor microenvironment. Both NP-Im/Dox and NP-Ag/Dox exhibited much higher intracellular uptake by Ramos cells (Burkitt's lymphoma) than NP-Dm/Dox, suggesting that the type of side chain in the NPs determines the extent of intracellular uptake. As a result, NP-Im/Dox and NP-Ag/Dox showed cytotoxicity that was comparable to free Dox in vitro. Our findings suggest that the nature of surface cationic group on nanocarriers may profoundly influence their intracellular trafficking and resulting therapeutic efficacy. Thus, it is a crucial factor to be considered in the design of novel carriers for intracellular drug delivery.

  13. Modulation of a sustained calcium current by intracellular pH in horizontal cells of fish retina

    PubMed Central

    1993-01-01

    A sustained high voltage-activated (HVA), nifedipine- and cadmium- sensitive calcium current and a sustained calcium action potential (AP) were recorded from horizontal cells isolated from catfish retina. pH indicator dyes showed that superfusion with NH4Cl alkalinized these cells and that washout of NH4Cl or superfusion with Na-acetate acidified them. HVA current was slightly enhanced during superfusion of NH4Cl but was suppressed upon NH4Cl washout or application of Na- acetate. When 25 mM HEPES was added to the patch pipette to increase intracellular pH buffering, the effects of NH4Cl and Na-acetate on HVA current were reduced. These results indicated that intracellular acidification reduces HVA calcium current and alkalinization increases it. Sustained APs, recorded with high resistance, small diameter microelectrodes, were blocked by cobalt and cadmium and their magnitude varied with extracellular calcium concentration. These results provide confirmatory evidence that the HVA current is a major component of the AP and indicate that the AP can be used as a measure of how the HVA current can be modified in intact, undialyzed cells. The duration of APs was increased by superfusion with NH4Cl and reduced by washout of NH4Cl or superfusion with Na-acetate. The Na-acetate and NH4Cl washout- dependent shortening of the APs was observed in the presence of intracellular BAPTA, a calcium chelator, IBMX, a phosphodiesterase inhibitor, and in Na-free or TEA-enriched saline. These findings provide supportive evidence that intracellular acidification may directly suppress the HVA calcium current in intact cells. Intracellular pH changes would thereby be expected to modulate not only the resting membrane potential of these cells in darkness, but calcium- dependent release of neurotransmitter from these cells as well. Furthermore, this acidification-dependent suppression of calcium current could serve a protective role by reducing calcium entry during retinal ischemia, which

  14. Embryonic common snapping turtles (Chelydra serpentina) preferentially regulate intracellular tissue pH during acid-base challenges.

    PubMed

    Shartau, Ryan B; Crossley, Dane A; Kohl, Zachary F; Brauner, Colin J

    2016-07-01

    The nests of embryonic turtles naturally experience elevated CO2 (hypercarbia), which leads to increased blood PCO2  and a respiratory acidosis, resulting in reduced blood pH [extracellular pH (pHe)]. Some fishes preferentially regulate tissue pH [intracellular pH (pHi)] against changes in pHe; this has been proposed to be associated with exceptional CO2 tolerance and has never been identified in amniotes. As embryonic turtles may be CO2 tolerant based on nesting strategy, we hypothesized that they preferentially regulate pHi, conferring tolerance to severe acute acid-base challenges. This hypothesis was tested by investigating pH regulation in common snapping turtles (Chelydra serpentina) reared in normoxia then exposed to hypercarbia (13 kPa PCO2 ) for 1 h at three developmental ages: 70% and 90% of incubation, and yearlings. Hypercarbia reduced pHe but not pHi, at all developmental ages. At 70% of incubation, pHe was depressed by 0.324 pH units while pHi of brain, white muscle and lung increased; heart, liver and kidney pHi remained unchanged. At 90% of incubation, pHe was depressed by 0.352 pH units but heart pHi increased with no change in pHi of other tissues. Yearlings exhibited a pHe reduction of 0.235 pH units but had no changes in pHi of any tissues. The results indicate common snapping turtles preferentially regulate pHi during development, but the degree of response is reduced throughout development. This is the first time preferential pHi regulation has been identified in an amniote. These findings may provide insight into the evolution of acid-base homeostasis during development of amniotes, and vertebrates in general.

  15. Regulation of intracellular pH values in higher plant cells. Carbon-13 and phosphorus-31 nuclear magnetic resonance studies.

    PubMed

    Gout, E; Bligny, R; Douce, R

    1992-07-15

    The regulation of the cytoplasmic and vacuolar pH values (pHc and pHv) in sycamore (Acer pseudoplatanus L.) cells was analyzed using 31P and 13C nuclear magnetic resonance spectroscopy. Suspension-cultured cells were compressed in the NMR tube and perfused with the help of an original arrangement enabling a tight control of the pH (external pH, pHe) of the carefully oxygenated circulating nutrient medium. Intracellular pH values were measured from the chemical shifts of: CH2-linked carboxyl groups of citric acid below pH 5.7; orthophosphate between pH 5.7 and 8.0; 13C-enriched bicarbonate over pH 8.0. pHc and pHv were independent of pHe over the range 4.5-7.5. In contrast intracellular pH values decreased rapidly below pHe 4.5 and increased progressively at pHe over 7.5. There was an acceleration in the rate of O2 consumption accompanied with a decrease in cytoplasmic ATP concentration as pHe decreased. When the rate of O2 consumption was approaching the uncoupled O2 uptake rate, a loss of pHc control was observed. It is concluded that as pHe decreased, the plasma membrane ATPase consumed more and more ATP to reject the invading H+ ions in order to maintain pHc at a constant value. Below pHe 4.5 the efficiency of the H+ pump to react to back leakage of H+ ions became insufficient, leading to an acidification of pHc and to an alkalinization of pHe. On the other hand, over pHe 7.5 a passive influx of OH- ions was observed, and pHc increased proportionally to the increase of pHe. Simultaneously appreciable amounts of organic acids (malate and citrate) were synthesized by cells during the course of the alkalinization of the cytoplasmic compartment. The synthesis of organic acids which partially counteract the alkalinization of the cytoplasmic compartment may result from a marked activation of the cytoplasmic phosphoenolpyruvate carboxylase induced by an increase in cytoplasmic bicarbonate concentration. The fluctuations of pHv followed a similar course to that of p

  16. The synthesis of new fluorescent bichromophoric compounds as ratiometric pH probes for intracellular measurements.

    PubMed

    Saura, A Vanessa; Marín, María J; Burguete, M Isabel; Russell, David A; Galindo, Francisco; Luis, Santiago V

    2015-07-28

    Three different bichromophoric compounds (1-3) containing an aminomethyl anthracene moiety linked to a second chromophore (pyrene, 4-nitrobenzo-2-oxa-1,3-diazole (NBD) and dansyl) through a valine-derived pseudopeptidic spacer have been prepared and their fluorescent properties studied. The results obtained show that upon irradiation the photophysical behavior of these probes involves electronic energy transfer from the excited anthracene to the second chromophore and also intramolecular photoinduced electron transfer. The X-ray structure obtained for 3 reveals that the folding associated with the pseudopeptidic spacer favours a close proximity of the two chromophores. The emissive response of 3 is clearly dependent on the pH of the medium, hence this bichromophoric compound was shown to be an excellent ratiometric pH fluorescent sensor. The emission intensity due to the anthracene moiety exhibits a decrease at neutral-basic pH values that is concomitant with an increase in the intensity arising from the dansyl fluorophore. These properties make this compound a good candidate for biological pH sensing as has been confirmed by preliminary studies with RAW 264.7 macrophage cells imaged by means of confocal fluorescence microscopy with an average pH estimation of 5.4-5.8 for acidic organelles.

  17. Compartmentalization in proteinoid microspheres

    NASA Technical Reports Server (NTRS)

    Brooke, S.; Fox, S. W.

    1977-01-01

    Proteinoid microspheres with stable internal compartments and internal structure are made from acidic proteinoid and basic proteinoid with calcium. The populations of microspheres are characterized by a wide diversity of structure. A model of primitive intracellular communication is suggested by the observed movement of internal particles between compartments of a multicompartmentalized unit. Differential response to pH change and to temperature change has been demonstrated within one population and suggests one mode of adaptive selection among primordial cell populations.

  18. Modeling the effects of sodium chloride, acetic acid, and intracellular pH on survival of Escherichia coli O157:H7.

    PubMed

    Hosein, Althea M; Breidt, Frederick; Smith, Charles E

    2011-02-01

    Microbiological safety has been a critical issue for acid and acidified foods since it became clear that acid-tolerant pathogens such as Escherichia coli O157:H7 can survive (even though they are unable to grow) in a pH range of 3 to 4, which is typical for these classes of food products. The primary antimicrobial compounds in these products are acetic acid and NaCl, which can alter the intracellular physiology of E. coli O157:H7, leading to cell death. For combinations of acetic acid and NaCl at pH 3.2 (a pH value typical for non-heat-processed acidified vegetables), survival curves were described by using a Weibull model. The data revealed a protective effect of NaCl concentration on cell survival for selected acetic acid concentrations. The intracellular pH of an E. coli O157:H7 strain exposed to acetic acid concentrations of up to 40 mM and NaCl concentrations between 2 and 4% was determined. A reduction in the intracellular pH was observed for increasing acetic acid concentrations with an external pH of 3.2. Comparing intracellular pH with Weibull model predictions showed that decreases in intracellular pH were significantly correlated with the corresponding times required to achieve a 5-log reduction in the number of bacteria.

  19. Complete intracellular pH protection during extracellular pH depression is associated with hypercarbia tolerance in white sturgeon, Acipenser transmontanus.

    PubMed

    Baker, D W; Matey, V; Huynh, K T; Wilson, J M; Morgan, J D; Brauner, C J

    2009-06-01

    Sturgeons are among the most CO2 tolerant of fishes investigated to date. However, the basis of this exceptional CO2 tolerance is unknown. Here, white sturgeon, Acipenser transmontanus, were exposed to elevated CO2 to investigate the mechanisms associated with short-term hypercarbia tolerance. During exposure to 1.5 kPa Pco2, transient blood pH [extracellular pH (pHe)] depression was compensated within 24 h and associated with net plasma HCO3- accumulation and equimolar Cl- loss, and changes in gill morphology, such as a decrease in apical surface area of mitochondrial-rich cells. These findings indicate that pHe recovery at this level of hypercarbia is accomplished in a manner similar to most freshwater teleost species studied to date, although branchial mechanisms involved may differ. White sturgeon exposed to more severe hypercarbia (3 and 6 kPa Pco2) for 48 h exhibited incomplete pH compensation in blood and red blood cells. Despite pHe depression, intracellular pH (pHi) of white muscle, heart, brain, and liver did not decrease during a transient (6 h of 1.5 kPa Pco2) or prolonged (48 h at 3 and 6 kPa Pco2 blood acidosis. This pHi protection was not due to high intrinsic buffering in tissues. Such tight active cellular regulation of pHi in the absence of pHe compensation represents a unique pattern for non-air-breathing fishes, and we hypothesize that it is the basis for the exceptional CO2 tolerance of white sturgeon and, likely, other CO2 tolerant fishes. Further research to elucidate the specific mechanisms responsible for this tremendous pH regulatory capacity in tissues of white sturgeon is warranted.

  20. Effects of 2-methoxyethanol on fetal development, postnatal behavior, and embryonic intracellular pH of rats.

    PubMed

    Nelson, B K; Vorhees, C V; Scott, W J; Hastings, L

    1989-01-01

    The industrial solvent 2-methoxyethanol (2ME) is a reproductive and developmental toxicant when administered by inhalation, gavage, and IP injection. The present research established that this solvent can produce teratogenicity in rats when administered in liquid diet. Groups of 10 Sprague-Dawley rats were given various percentages of 2ME in liquid diet on gestation days 7-18. Day 20 fetuses were examined for visceral or skeletal malformations. Concentrations above 0.025% 2ME (approximately 73 mg/kg/day) produced total embryo-mortality. Cardiovascular malformations were produced at lower levels. The teratogenic no-effect level was 0.006% 2ME (16 mg/kg). In a second experiment, groups of 12 Sprague-Dawley rats were given 0, 0.006 and 0.012% of 2ME as above. Litters were culled to 8 pups, and tested for auditory and tactile startle and conditioned lick suppression, and for performance in figure-8 activity and the Cincinnati water maze on postnatal days 48-65. The high dose of 2ME produced approximately 50% mortality in the offspring and increased the number of errors in the Cincinnati maze. No other behavioral effects were observed at either dose. An interaction study was conducted to determine if simultaneous exposure to 2ME and ethanol would reduce the teratogenicity of 2ME, but no reduction was observed. The hypothesis that 2ME acts by altering embryonic intracellular pH was tested by injecting 0.33 ml/kg of 2ME into rats on gestation day 13, and determining embryonic intracellular pH at 2, 4, 8, and 24 hours thereafter. There was an increase in pH at 4 hours, but not at later time points. Another group of rats was given 2ME along with amiloride, which blocks the sodium/hydrogen antiporter. The combined 2ME-amiloride exposure produced an incidence of cardiovascular malformations in fetuses twice that of 2ME alone. These studies confirmed the structural teratogenicity of 2ME even when given in liquid diet, as it was given for the first time in the present study. At

  1. Effects of fatigue and reduced intracellular pH on segment dynamics in 'isometric' relaxation of frog muscle fibres.

    PubMed Central

    Curtin, N A; Edman, K A

    1989-01-01

    1. Longitudinal movements of marked segments of single fibres from the anterior tibialis muscle were recorded during tetanus and relaxation under isometric (fixed-end) conditions. 2. During relaxation, shortening and lengthening of different segments occurred simultaneously, starting at about the same time as the end of the linear fall of force (shoulder on the force record). 3. Variations in intracellular pH, measured with pH-sensitive microelectrodes, along the length of fibres were not statistically significant, and are unlikely to be responsible for the non-uniform behaviour of different segments. 4. As expected from earlier studies, both fatigue (produced by increasing tetanus duration or decreasing the time between tetani) and intracellular acidification (produced by raised extracellular CO2), reduced the tetanus force and prolonged the linear phase of force decline in relaxation. Each treatment delayed the start and markedly reduced the amount of segment movement in relaxation. 5. Fatigue and intracellular acidification have a smaller effect on force during stretching than on force produced under isometric conditions. This may contribute to making the segments behave in a more uniform way during relaxation under these conditions. 6. Changes in the Ca2+ uptake mechanisms are also discussed as possible causes for the changes in segment behaviour in relaxation. PMID:2600846

  2. Effect of Tris-Hydroxymethyl Aminomethane on intracellular pH depends on the extracellular non-bicarbonate buffering capacity.

    PubMed

    Giunti, Carine; Priouzeau, Fabrice; Allemand, Denis; Levraut, Jacques

    2007-12-01

    The effect of Tris-Hydroxymethyl Aminomethane (THAM) on intracellular pH (pHi) is unknown. We previously demonstrated that the effect of sodium bicarbonate on pHi depends on the non-bicarbonate buffering system. First, human hepatocytes from hepatocytes cell culture (HepG2) were perfused with an acidotic artificial medium containing 5-mmol/L (H5) or 30-mmol/L (H30) concentrations of 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid (HEPES), a non-bicarbonate buffer. We studied the effect of THAM on the pHi in both conditions. We repeated the same protocol using an acidotic human blood with a 5% or 40% hematocrit. The pHi was measured with the pH-sensitive fluorescent dye bis-carboxyethyl carboxy-fluorescein (BCECF). Gas analysis was performed before and during the alkaline infusion. The results showed that THAM caused an intracellular alkalization that was higher when the non-bicarbonate buffer concentration was low (0.45 +/- 0.21 and 0.22 +/- 0.14 pH units with H5 and H30, respectively). A significant relationship was found between changes in pHi and changes in PCO(2). Similar results were obtained with the human blood. In conclusion, the intracellular alkalizing effect of THAM is caused by the induced decrease of PCO(2) linked to the extracellular non-bicarbonate buffer capacity: The smaller the concentration of extracellular non-bicarbonate buffer, the higher the PCO(2) decrease caused by THAM.

  3. Intracellular carbonic anhydrase activity sensitizes cancer cell pH signaling to dynamic changes in CO2 partial pressure.

    PubMed

    Hulikova, Alzbeta; Aveyard, Nicholas; Harris, Adrian L; Vaughan-Jones, Richard D; Swietach, Pawel

    2014-09-12

    Carbonic anhydrase (CA) enzymes catalyze the chemical equilibration among CO2, HCO3(-) and H(+). Intracellular CA (CAi) isoforms are present in certain types of cancer, and growing evidence suggests that low levels correlate with disease severity. However, their physiological role remains unclear. Cancer cell CAi activity, measured as cytoplasmic CO2 hydration rate (kf), ranged from high in colorectal HCT116 (∼2 s(-1)), bladder RT112 and colorectal HT29, moderate in fibrosarcoma HT1080 to negligible (i.e. spontaneous kf = 0.18 s(-1)) in cervical HeLa and breast MDA-MB-468 cells. CAi activity in cells correlated with CAII immunoreactivity and enzymatic activity in membrane-free lysates, suggesting that soluble CAII is an important intracellular isoform. CAi catalysis was not obligatory for supporting acid extrusion by H(+) efflux or HCO3(-) influx, nor for maintaining intracellular pH (pHi) uniformity. However, in the absence of CAi activity, acid loading from a highly alkaline pHi was rate-limited by HCO3(-) supply from spontaneous CO2 hydration. In solid tumors, time-dependence of blood flow can result in fluctuations of CO2 partial pressure (pCO2) that disturb cytoplasmic CO2-HCO3(-)-H(+) equilibrium. In cancer cells with high CAi activity, extracellular pCO2 fluctuations evoked faster and larger pHi oscillations. Functionally, these resulted in larger pH-dependent intracellular [Ca(2+)] oscillations and stronger inhibition of the mTORC1 pathway reported by S6 kinase phosphorylation. In contrast, the pHi of cells with low CAi activity was less responsive to pCO2 fluctuations. Such low pass filtering would "buffer" cancer cell pHi from non-steady-state extracellular pCO2. Thus, CAi activity determines the coupling between pCO2 (a function of tumor perfusion) and pHi (a potent modulator of cancer cell physiology).

  4. Effects of intracellular pH on ATP-sensitive K+ channels in mouse pancreatic beta-cells.

    PubMed Central

    Proks, P; Takano, M; Ashcroft, F M

    1994-01-01

    1. The effects of intracellular pH (pHi) on the ATP-sensitive K+ channel (K+ATP channel) from mouse pancreatic beta-cells were examined in inside-out patches exposed to symmetrical 140 mM K+ solutions. 2. The relationship between channel activity and pHi was described by the Hill equation with half-maximal inhibition (Ki) at pHi 6.25 and a Hill coefficient of 3.7. 3. Following exposure to pHi < 6.8, channel activity did not recover to its original level. Subsequent application of trypsin to the intracellular membrane surface restored channel activity to its initial level or above. 4. At -60 mV the relationship between pHi and the single-channel current amplitude was described by a modified Hill equation with a Hill coefficient of 2.1, half-maximal inhibition at pHi 6.48 and a maximum inhibition of 18.5%. 5. A decrease in pHi reduced the extent of channel inhibition by ATP: Ki was 18 microM at pH 7.2 and 33 microM at pH 6.4. The Hill coefficient was also reduced, being 1.65 at pH 7.2 and 1.17 at pH 6.4. 6. When channel activity was plotted as a function of ATP4- (rather than total ATP) there was no effect of pHi on the relationship. This suggests that ATP4- is the inhibitory ion species and that the effects of reducing pHi are due to the lowered concentration of ATP4-. 7. Changes in external pH had little effect on either single-channel or whole-cell K+ATP currents. 8. The effects of pHi do not support a role for H+ in linking glucose metabolism to K+ATP channel inhibition in pancreatic beta-cells. PMID:8189391

  5. Intracellular pH and its regulation in isolated type I carotid body cells of the neonatal rat.

    PubMed Central

    Buckler, K J; Vaughan-Jones, R D; Peers, C; Nye, P C

    1991-01-01

    1. The dual-emission pH-sensitive fluoroprobe carboxy-SNARF-1 (carboxy-seminaptharhodofluor) was used to measure pHi in type I cells enzymically dispersed from the neonatal rat carotid body. 2. Steady-state pHi in cells bathed in a HEPES-buffered Tyrode solution (pH 7.4) was found to be remarkably alkaline (pHi = 7.77) whereas cells bathed in a CO2-HCO3(-)-buffered Tyrode solution (pH 7.4) had a more 'normal' pHi (pHi = 7.28). These observations were further substantiated by using an independent nullpoint test method to determine pHi. 3. Intracellular intrinsic buffering (beta, determined by acidifying the cell using an NH4Cl pre-pulse) was in the range 7-20 mM per pH unit and appeared to be dependent upon pHi with beta increasing as pHi decreased. 4. In cells bathed in a HEPES-buffered Tyrode solution, pHi recovery from an induced intracellular acid load (10 mM-NH4Cl pre-pulse) was inhibited by the Na(+)-H+ exchange inhibitor ethyl isopropyl amiloride (EIPA; 150 microM) or substitution of Nao+ with N-methyl-D-glucamine (NMG). Both EIPA and Nao+ removal also caused a rapid intracellular acidification, which in the case of Nao+ removal, was readily reversible. The rate of this acidification was similar for both Nao+ removal and EIPA addition. 5. Transferring cells from a HEPES-buffered Tyrode solution to one buffered with 5% CO2-HCO3- resulted in an intracellular acidification which was partially, or wholly, sustained. The rate of acidification upon transfer to CO2-HCO3- was considerably slowed by the membrane permeant carbonic anhydrase inhibitor, acetazolamide, thus indicating the presence of the enzyme in these cells. 6. In CO2-HCO3(-)-buffered Tyrode solution, pHi recovery from an intracellular acidosis (NH4+ pre-pulse) was only partially inhibited by EIPA or amiloride whereas Nao+ removal completely inhibited the recovery. The stilbene DIDS (4,4-diisothiocyanatostilbenedisulphonic acid, 200 microM) also partially inhibited pHi recovery following an induced

  6. Intracellular pH in Gastric and Rectal Tissue Post Cardiac Arrest

    NASA Astrophysics Data System (ADS)

    Fisher, Elaine M.; Steiner, Richard P.; LaManna, Joseph C.

    We directly measured pHi using the pH sensitive dye, neutral red. We defined pHi for rectal and gastric tissue in whole tissue and by layer under control and arrest conditions. Fifteen minutes of arrest was not sufficient time to alter the pHi at the rectal or gastric site. On initial inspection, the stomach may be more sensitive to ischemic changes than the rectum. Understanding the mechanism by which PCO2 generation is used to track clinical changes is vital to the early detection of tissue dysoxia in order to effectively treat and manage critically ill patients.

  7. Proton/l-Glutamate Symport and the Regulation of Intracellular pH in Isolated Mesophyll Cells 1

    PubMed Central

    Snedden, Wayne A.; Chung, Induk; Pauls, Randy H.; Bown, Alan W.

    1992-01-01

    Addition of l-[U-14C]glutamate to a suspension of mechanically isolated asparagus (Asparagus sprengeri Regel) mesophyll cells results in (a) alkalinization of the medium, (b) uptake of l-[U-14C]glutamate, and (c) efflux of [14C]4-aminobutyrate, a product of glutamate decarboxylation. All three phenomena were eliminated by treatment with 1 millimolar aminooxyacetate. In vitro glutamate decarboxylase (GAD) assays showed that (a) 2 millimolar aminooxyacetate eliminated enzyme activity, (b) activity was pyridoxal phosphate-dependent, and (c) activity exhibited a sharp pH optimum at 6.0 that decreased to 20% of optimal activity at pH 5.0 and 7.0. Addition of 1.5 millimolar sodium butyrate or sodium acetate to cell suspensions caused immediate alkalinization of the medium followed by a resumption of acidification of the medium at a rate approximately double the initial rate. The data indicate that (a) continued H+/l-glutamate contransport is dependent upon GAD activity, (b) the pH-dependent properties of GAD are consistent with a role in a metabolic pH-stat, and (c) the regulation of intracellular pH during H+/l-Glu symport may involve both H+ consumption during 4-aminobutyrate production and ATP-driven H+ efflux. PMID:16668938

  8. Construction of pH-Sensitive "Submarine" Based on Gold Nanoparticles with Double Insurance for Intracellular pH Mapping, Quantifying of Whole Cells and in Vivo Applications.

    PubMed

    Yu, Kang-Kang; Li, Kun; Qin, Hui-Huan; Zhou, Qian; Qian, Chen-Hui; Liu, Yan-Hong; Yu, Xiao-Qi

    2016-09-07

    A series of "submarines", which composed of gold nanoparticles and modified with rhodamine and fluorescein derivatives, were presented. With dual sensitive units for both acidic and basic environment, these "gold nano-submarines" not only allow efficient intracellular pH mapping but also provide more accurate quantitative detection of pH alteration under different stimuli with distinct pH quantification range. Moreover, they even have the ability to pass through the blood brain barrier (BBB).

  9. Influence of sodium-hydrogen exchange on intracellular pH, sodium and tension in sheep cardiac Purkinje fibres.

    PubMed Central

    Kaila, K; Vaughan-Jones, R D

    1987-01-01

    1. The influence of sarcolemmal Na+-H+ exchange upon intracellular Na+ activity (aiNa), intracellular pH (pHi), extracellular surface pH (pHs) and tonic tension was investigated in sheep cardiac Purkinje fibres. Intracellular ion activities were measured with liquid sensor ion-selective micro-electrodes. A two-micro-electrode voltage-clamp was also used to control membrane potential while simultaneously recording tonic tension. 2. Inhibition of the sarcolemmal Na+-K+ pump by strophanthidin (10 mumol/l) produced a rise in aiNa, an increase in [Ca2+]i as evidenced by a rise in tonic tension, and a fall in pHi of 0.1-0.3 units. The intracellular acidosis has been shown previously to be linked to the rise in [Ca2+]i (Vaughan-Jones, Lederer & Eisner, 1983). 3. Amiloride (1-2 mmol/l), an inhibitor of Na+-H+ exchange, produced a small reversible decrease in pHi and aiNa. Both effects became more pronounced in strophanthidin-exposed fibres. In addition, pHi decreased during application of strophanthidin and this decrease was reversibly inhibited by amiloride. It is concluded that sarcolemmal Na+-H+ exchange is stimulated following inhibition of the Na+-K+ pump. 4. In strophanthidin-exposed fibres, a rise in [Ca2+]i resulted in an intracellular acidosis which could still be observed in the presence of amiloride (1 mmol/l). This suggests that the fall in pHi was not caused by a modulatory effect of [Ca2+]i on sarcolemmal Na+-H+ exchange. 5. Tetrodotoxin (TTX) produced a small fall in aiNa (ca. 0.5 mmol/l) which was not augmented in the presence of strophanthidin. Furthermore, the effects on aiNa of TTX and amiloride were additive. Thus the influence of amiloride on aiNa does not involve blockade of voltage-gated Na+ channels. 6. The stoicheiometry of Na+-H+ exchange, estimated from the rates of change of pHi and aiNa in amiloride, appeared to be electroneutral (1:1). The stoicheiometry was unaffected by changes in pHi. 7. In strophanthidin-exposed fibres (i.e. aiNa is

  10. The role of carbonic anhydrase 9 in regulating extracellular and intracellular ph in three-dimensional tumor cell growths.

    PubMed

    Swietach, Pawel; Patiar, Shalini; Supuran, Claudiu T; Harris, Adrian L; Vaughan-Jones, Richard D

    2009-07-24

    We have studied the role of carbonic anhydrase 9 (CA9), a cancer-associated extracellular isoform of the enzyme carbonic anhydrase in multicellular spheroid growths (radius of approximately 300 microm) of human colon carcinoma HCT116 cells. Spheroids were transfected with CA9 (or empty vector) and imaged confocally (using fluorescent dyes) for both intracellular pH (pH(i)) and pH in the restricted extracellular spaces (pH(e)). With no CA9 expression, spheroids developed very low pH(i) (approximately 6.3) and reduced pH(e) (approximately 6.9) at their core, associated with a diminishing gradient of acidity extending out to the periphery. With CA9 expression, core intracellular acidity was less prominent (pH(i) = approximately 6.6), whereas extracellular acidity was enhanced (pH(e) = approximately 6.6), so that radial pH(i) gradients were smaller and radial pH(e) gradients were larger. These effects were reversed by eliminating CA9 activity with membrane-impermeant CA inhibitors. The observation that CA9 activity reversibly reduces pH(e) indicates the enzyme is facilitating CO(2) excretion from cells (by converting vented CO(2) to extracellular H(+)), rather than facilitating membrane H(+) transport (such as H(+) associated with metabolically generated lactic acid). This latter process requires titration of exported H(+) ions with extracellular HCO(3)(-), which would reduce rather than increase extracellular acidity. In a multicellular structure, the net effect of CA9 on pH(e) will depend on the cellular CO(2)/lactic acid emission ratio (set by local oxygenation and membrane HCO(3)(-) uptake). Our results suggest that CO(2)-producing tumors may express CA9 to facilitate CO(2) excretion, thus raising pH(i) and reducing pH(e), which promotes tumor proliferation and survival. The results suggest a possible basis for attenuating tumor development through inhibiting CA9 activity.

  11. An Na(+)-independent short-chain fatty acid transporter contributes to intracellular pH regulation in murine colonocytes

    PubMed Central

    1995-01-01

    Short-chain fatty acids (SCFAs) are the major anions in the colonic lumen. Experiments studied how intracellular pH (pHi) of isolated colonocytes was affected by exposure to SCFAs normally found in the colon. Isolated crypt fragments were loaded with SNARF-1 (a fluorescent dye with pH-sensitive excitation and emission spectra) and studied in a digital imaging microscope. Intracellular pH was measured in individual colonocytes as the ratio of fluorescence intensity in response to alternating excitation wavelengths (575/505 nm). After exposure to 65 mM acetate, propionate, n-butyrate, or iso-butyrate in isosmotic Na(+)- free media (substituted with tetramethylammonia), all colonocytes acidified rapidly and then > 90% demonstrated a pHi alkalinization (Na(+)-independent pHi recovery). Upon subsequent removal of the SCFA, pHi alkalinized beyond the starting pHi (a pHi overshoot). Using propionate as a test SCFA, experiments demonstrate that the acidification and pHi overshoot are explained by transmembrane influx and efflux of nonionized SCFA, respectively. The basis for the pHi overshoot is shown to be accumulation of propionate during pHi alkalinization. The Na(+)-independent pHi recovery (a) demonstrates saturable propionate activation kinetics; (b) demonstrates substrate specificity for unmodified aliphatic carbon chains; (c) occurs after exposure to SCFAs of widely different metabolic activity, (d) is electroneutral; and (e) is not inhibited by changes in the K+ gradient, Cl- gradient or addition of the anion transport inhibitors DIDS (1 mM), SITS (1 mM), alpha-cyano-4-hydroxycinnamate (4 mM), or probenicid (1 mM). Results suggest that most mouse colonocytes have a previously unreported SCFA transporter which mediates Na(+)-independent pHi recovery. PMID:7658194

  12. The effect of carbon dioxide on the intracellular pH and buffering power of snail neurones.

    PubMed Central

    Thomas, R C

    1976-01-01

    1. Intracellular pH (pHi) was measured using pH-sensitive glass micro-electrodes. The effects on pHi of CO2 applied externally and HCO3-, H+ and NH4+ injected iontophoretically, were investigated. 2. The transport numbers for iontophoretic injection into aqueous micro-droples were found by potentiometric titration to be 0-3 for HCO3- and 0-94 for H+. 3. Exposure to Ringer, pH 7-5, equilibrated with 2-2% CO2 caused a rapid, but only transient, fall in pHi. Within 1 or 2 min pHi began to return exponentially to normal, with a time constant of about 5 min. 4. When external CO2 was removed, pHi rapidly increased, and then slowly returned to normal. The pHi changes with CO2 application or removal gave a calculated intracellular buffer value of about 30 m-equiv H+/pH unit per litre. 5. Injection of HCO3- caused a rise in pHi very similar to that seen on removal of external CO2. 6. The pHi responses to CO2 application, CO2 removal and HCO3- injection were slowed by the carbonic anhydrase inhibitor acetazolamide. 7. H+ injection caused a transient fall in pHi. In CO2 Ringer pHi fell less and recovered faster than in CO2-free Ringer. Calculation of the internal buffer value from the pHi responses to H+ and HCO3- injection gave very similar values. 8. The internal buffer value (measured by H+ injection) was greatly increased by exposure to CO2 Ringer. Acetazolamide reduced this effect of CO2, suggesting that the function of intracellular carbonic anhydrase may be to maximize the internal buffering power in CO2. 9. It was concluded that the internal HCO3- was determined primarily by the CO2 level and pHi, that internal HCO3- made a large contribution to the buffering power, and that after internal acidfication pHi was restored to normal by active transport of H+, OH- or HCO3- across the cell membrane. The active transport was much faster in CO2 than in CO2-free Ringer. PMID:4614

  13. Vcx1 and ESCRT components regulate intracellular pH homeostasis in the response of yeast cells to calcium stress.

    PubMed

    Papouskova, Klara; Jiang, Linghuo; Sychrova, Hana

    2015-03-01

    Endosomal sorting complexes required for transport (ESCRTs) are involved in the formation of multivesicular bodies and sorting of targeted proteins to the yeast vacuole. The deletion of seven genes encoding components of the ESCRT machinery render Saccharomyces cerevisiae cells sensitive to high extracellular CaCl2 concentrations as well as to low pH in media. In this work, we focused on intracellular pH (pHin) homeostasis of these mutants. None of the studied ESCRT mutants exhibited an altered pHin level compared to the wild type under standard growth conditions. Nevertheless, 60 min of CaCl2 treatment resulted in a more significant drop in pHin levels in these mutants than in the wild type, suggesting that pHin homeostasis is affected in ESCRT mutants upon the addition of calcium. Similarly, CaCl2 treatment caused a bigger pHin decrease in cells lacking the vacuolar Ca(2+)/H(+) antiporter Vcx1 which indicates a role for this protein in the maintenance of proper pHin homeostasis when cells need to cope with a high CaCl2 concentration in media. Importantly, ESCRT gene deletions in the vcx1Δ strain did not result in an increase in the CaCl2-invoked drop in the pHin levels of cells, which demonstrates a genetic interaction between VCX1 and studied ESCRT genes.

  14. Glucose Uptake and Intracellular pH in a Mouse Model of Ductal Carcinoma In situ (DCIS) Suggests Metabolic Heterogeneity

    PubMed Central

    Lobo, Rebecca C.; Hubbard, Neil E.; Damonte, Patrizia; Mori, Hidetoshi; Pénzváltó, Zsófia; Pham, Cindy; Koehne, Amanda L.; Go, Aiza C.; Anderson, Steve E.; Cala, Peter M.; Borowsky, Alexander D.

    2016-01-01

    Mechanisms for the progression of ductal carcinoma in situ (DCIS) to invasive breast carcinoma remain unclear. Previously we showed that the transition to invasiveness in the mammary intraepithelial neoplastic outgrowth (MINO) model of DCIS does not correlate with its serial acquisition of genetic mutations. We hypothesized instead that progression to invasiveness depends on a change in the microenvironment and that precancer cells might create a more tumor-permissive microenvironment secondary to changes in glucose uptake and metabolism. Immunostaining for glucose transporter 1 (GLUT1) and the hypoxia marker carbonic anhydrase 9 (CAIX) in tumor, normal mammary gland and MINO (precancer) tissue showed differences in expression. The uptake of the fluorescent glucose analog dye, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG), reflected differences in the cellular distributions of glucose uptake in normal mammary epithelial cells (nMEC), MINO, and Met1 cancer cells, with a broad distribution in the MINO population. The intracellular pH (pHi) measured using the fluorescent ratio dye 2′,7′-bis(2-carboxyethyl)-5(6)-155 carboxyfluorescein (BCECF) revealed expected differences between normal and cancer cells (low and high, respectively), and a mixed distribution in the MINO cells, with a subset of cells in the MINO having an increased rate of acidification when proton efflux was inhibited. Invasive tumor cells had a more alkaline baseline pHi with high rates of proton production coupled with higher rates of proton export, compared with nMEC. MINO cells displayed considerable variation in baseline pHi that separated into two distinct populations: MINO high and MINO low. MINO high had a noticeably higher mean acidification rate compared with nMEC, but relatively high baseline pHi similar to tumor cells. MINO low cells also had an increased acidification rate compared with nMEC, but with a more acidic pHi similar to nMEC. These findings

  15. Role of Sodium Bicarbonate Cotransporters in Intracellular pH Regulation and Their Regulatory Mechanisms in Human Submandibular Glands.

    PubMed

    Namkoong, Eun; Shin, Yong-Hwan; Bae, Jun-Seok; Choi, Seulki; Kim, Minkyoung; Kim, Nahyun; Hwang, Sung-Min; Park, Kyungpyo

    2015-01-01

    Sodium bicarbonate cotransporters (NBCs) are involved in the pH regulation of salivary glands. However, the roles and regulatory mechanisms among different NBC isotypes have not been rigorously evaluated. We investigated the roles of two different types of NBCs, electroneutral (NBCn1) and electrogenic NBC (NBCe1), with respect to pH regulation and regulatory mechanisms using human submandibular glands (hSMGs) and HSG cells. Intracellular pH (pHi) was measured and the pHi recovery rate from cell acidification induced by an NH4Cl pulse was recorded. Subcellular localization and protein phosphorylation were determined using immunohistochemistry and co-immunoprecipitation techniques. We determined that NBCn1 is expressed on the basolateral side of acinar cells and the apical side of duct cells, while NBCe1 is exclusively expressed on the apical membrane of duct cells. The pHi recovery rate in hSMG acinar cells, which only express NBCn1, was not affected by pre-incubation with 5 μM PP2, an Src tyrosine kinase inhibitor. However, in HSG cells, which express both NBCe1 and NBCn1, the pHi recovery rate was inhibited by PP2. The apparent difference in regulatory mechanisms for NBCn1 and NBCe1 was evaluated by artificial overexpression of NBCn1 or NBCe1 in HSG cells, which revealed that the pHi recovery rate was only inhibited by PP2 in cells overexpressing NBCe1. Furthermore, only NBCe1 was significantly phosphorylated and translocated by NH4Cl, which was inhibited by PP2. Our results suggest that both NBCn1 and NBCe1 play a role in pHi regulation in hSMG acinar cells, and also that Src kinase does not regulate the activity of NBCn1.

  16. Effects of micro electric current load during cooling of plant tissues on intracellular ice crystal formation behavior and pH.

    PubMed

    Ninagawa, Takako; Kawamura, Yukio; Konishi, Tadashi; Narumi, Akira

    2016-08-01

    Cryopreservation techniques are expected to evolve further to preserve biomaterials and foods in a fresh state for extended periods of time. Long-term cryopreservation of living materials such as food and biological tissue is generally achieved by freezing; thus, intracellular freezing occurs. Intracellular freezing injures the cells and leads to cell death. Therefore, a dream cryopreservation technique would preserve the living materials without internal ice crystal formation at a temperature low enough to prevent bacterial activity. This study was performed to investigate the effect of micro electrical current loading during cooling as a new cryopreservation technique. The behavior of intracellular ice crystal formation in plant tissues with or without an electric current load was evaluated using the degree of supercooling, degree of cell deformation, and grain size and growing rate of intracellular ice crystal. Moreover, the transition of intracellular pH during plant tissue cooling with or without electric current loading was also examined using the fluorescence intensity ratio to comprehend cell activity at lower temperatures. The results indicated that micro electric current load did not only decrease the degree of cell deformation and grain size of intracellular ice crystal but also reduced the decline in intracellular pH due to temperature lowering, compared with tissues subjected to the same cooling rate without an electric current load. Thus, the effect of electric current load on cryopreservation and the potential of a new cryopreservation technique using electric current load were discussed based on these results.

  17. Decrease of intracellular pH as possible mechanism of embryotoxicity of glycol ether alkoxyacetic acid metabolites.

    PubMed

    Louisse, Jochem; Bai, Yanqing; Verwei, Miriam; van de Sandt, Johannes J M; Blaauboer, Bas J; Rietjens, Ivonne M C M

    2010-06-01

    Embryotoxicity of glycol ethers is caused by their alkoxyacetic acid metabolites, but the mechanism underlying the embryotoxicity of these acid metabolites is so far not known. The present study investigates a possible mechanism underlying the embryotoxicity of glycol ether alkoxyacetic acid metabolites using the methoxyacetic acid (MAA) metabolite of ethylene glycol monomethyl ether as the model compound. The results obtained demonstrate an MAA-induced decrease of the intracellular pH (pH(i)) of embryonic BALB/c-3T3 cells as well as of embryonic stem (ES)-D3 cells, at concentrations that affect ES-D3 cell differentiation. These results suggest a mechanism for MAA-mediated embryotoxicity similar to the mechanism of embryotoxicity of the drugs valproic acid and acetazolamide (ACZ), known to decrease the pH(i)in vivo, and therefore used as positive controls. The embryotoxic alkoxyacetic acid metabolites ethoxyacetic acid, butoxyacetic acid and phenoxyacetic acid also caused an intracellular acidification of BALB/c-3T3 cells at concentrations that are known to inhibit ES-D3 cell differentiation. Two other embryotoxic compounds, all-trans-retinoic acid and 5-fluorouracil, did not decrease the pH(i) of embryonic cells at concentrations that affect ES-D3 cell differentiation, pointing at a different mechanism of embryotoxicity of these compounds. MAA and ACZ induced a concentration-dependent inhibition of ES-D3 cell differentiation, which was enhanced by amiloride, an inhibitor of the Na(+)/H(+)-antiporter, corroborating an important role of the pH(i) in the embryotoxic mechanism of both compounds. Together, the results presented indicate that a decrease of the pH(i) may be the mechanism of embryotoxicity of the alkoxyacetic acid metabolites of the glycol ethers.

  18. Intracellular pH changes in human aortic smooth muscle cells in response to fluid shear stress

    NASA Technical Reports Server (NTRS)

    Stamatas, G. N.; Patrick, C. W. Jr; McIntire, L. V.

    1997-01-01

    The smooth muscle cell (SMC) layers of human arteries may be exposed to blood flow after endothelium denudation, for example, following balloon angioplasty treatment. These SMCs are also constantly subjected to pressure driven transmural fluid flow. Flow-induced shear stress can alter SMC growth and metabolism. Signal transduction mechanisms involved in these flow effects on SMCs are still poorly understood. In this work, the hypothesis that shear stress alters the intracellular pH (pHi) of SMC is examined. When exposed to venous and arterial levels of shear stress, human aortic smooth muscle cells (hASMC) undergo alkalinization. The alkalinization plateau persisted even after 20 min of cell exposure to flow. Addition of amiloride (10 micromoles) or its 5-(N-ethyl-N-isopropyl) analog (EIPA, 10 micromoles), both Na+/H+ exchanger inhibitors, attenuated intracellular alkalinization, suggesting the involvement of the Na+/H+ exchanger in this response. The same concentrations of these inhibitors did not show an effect on pHi of hASMCs in static culture. 4-Acetamido-4'-isothio-cyanatostilbene-2,2'-disulfonic acid (SITS, 1 mM), a Cl-/HCO3- exchange inhibitor, affected the pHi of hASMCs both in static and flow conditions. Our results suggest that flow may perturb the Na+/H+ exchanger leading to an alkalinization of hASMCs, a different response from the flow-induced acidification seen with endothelial cells at the same levels of shear stress. Understanding the flow-induced signal transduction pathways in the vascular cells is of great importance in the tissue engineering of vascular grafts. In the case of SMCs, the involvement of pHi changes in nitric oxide production and proliferation regulation highlights further the significance of such studies.

  19. Effect of electroneutral luminal and basolateral lactate transport on intracellular pH in salamander proximal tubules

    PubMed Central

    1987-01-01

    We used microelectrodes to examine the effects of organic substrates, particularly lactate (Lac-), on the intracellular pH (pHi) and basolateral membrane potential (Vbl) in isolated, perfused proximal tubules of the tiger salamander. Exposure of the luminal and basolateral membranes to 3.6 mM Lac- caused pHi to increase by approximately 0.2, opposite to the decrease expected from nonionic diffusion of lactic acid (HLac) into the cell. Addition of Lac- to only the lumen also caused alkalinization, but only if Na+ was present. This alkalinization was not accompanied by immediate Vbl changes, which suggests that it involves luminal, electroneutral Na/Lac cotransport. Addition of Lac- to only the basolateral solution caused pHi to decrease by approximately 0.08. The initial rate of this acidification was a saturable function of [Lac-], was not affected by removal of Na+, and was reversibly reduced by alpha-cyano-4-hydroxycinnamate (CHC). Thus, the pHi decrease induced by basolateral Lac- appears to be due to the basolateral entry of H+ and Lac-, mediated by an H/Lac cotransporter (or a Lac-base exchanger). Our data suggest that this transporter is electroneutral and is not present at the luminal membrane. A key question is how the addition of Lac- to the lumen increases pHi. We found that inhibition of basolateral H/Lac cotransport by basolateral CHC reduced the initial rate of pHi increase caused by luminal Lac-. On the other hand, luminal CHC had no effect on the luminal Lac(-)-induced alkalinization. These data suggest that when Lac- is present in the lumen, it enters the cell from the lumen via electroneutral Na/Lac cotransport and then exists with H+ across the basolateral membrane via electroneutral H/Lac cotransport. The net effect is transepithelial Lac- reabsorption, basolateral acid extrusion, and intracellular alkalinization. PMID:3440860

  20. Decrease of intracellular pH as possible mechanism of embryotoxicity of glycol ether alkoxyacetic acid metabolites

    SciTech Connect

    Louisse, Jochem; Verwei, Miriam; Sandt, Johannes J.M. van de; Rietjens, Ivonne M.C.M.

    2010-06-01

    Embryotoxicity of glycol ethers is caused by their alkoxyacetic acid metabolites, but the mechanism underlying the embryotoxicity of these acid metabolites is so far not known. The present study investigates a possible mechanism underlying the embryotoxicity of glycol ether alkoxyacetic acid metabolites using the methoxyacetic acid (MAA) metabolite of ethylene glycol monomethyl ether as the model compound. The results obtained demonstrate an MAA-induced decrease of the intracellular pH (pH{sub i}) of embryonic BALB/c-3T3 cells as well as of embryonic stem (ES)-D3 cells, at concentrations that affect ES-D3 cell differentiation. These results suggest a mechanism for MAA-mediated embryotoxicity similar to the mechanism of embryotoxicity of the drugs valproic acid and acetazolamide (ACZ), known to decrease the pH{sub i}in vivo, and therefore used as positive controls. The embryotoxic alkoxyacetic acid metabolites ethoxyacetic acid, butoxyacetic acid and phenoxyacetic acid also caused an intracellular acidification of BALB/c-3T3 cells at concentrations that are known to inhibit ES-D3 cell differentiation. Two other embryotoxic compounds, all-trans-retinoic acid and 5-fluorouracil, did not decrease the pH{sub i} of embryonic cells at concentrations that affect ES-D3 cell differentiation, pointing at a different mechanism of embryotoxicity of these compounds. MAA and ACZ induced a concentration-dependent inhibition of ES-D3 cell differentiation, which was enhanced by amiloride, an inhibitor of the Na{sup +}/H{sup +}-antiporter, corroborating an important role of the pH{sub i} in the embryotoxic mechanism of both compounds. Together, the results presented indicate that a decrease of the pH{sub i} may be the mechanism of embryotoxicity of the alkoxyacetic acid metabolites of the glycol ethers.

  1. Carbonic anhydrase inhibitors modify intracellular pH transients and contractions of rat middle cerebral arteries during CO2/HCO3(-) fluctuations.

    PubMed

    Rasmussen, Jacob K; Boedtkjer, Ebbe

    2017-01-01

    The CO2/HCO3(-) buffer minimizes pH changes in response to acid-base loads, HCO3(-) provides substrate for Na(+),HCO3(-)-cotransporters and Cl(-)/HCO3(-)-exchangers, and H(+) and HCO3(-) modify vasomotor responses during acid-base disturbances. We show here that rat middle cerebral arteries express cytosolic, mitochondrial, extracellular, and secreted carbonic anhydrase isoforms that catalyze equilibration of the CO2/HCO3(-) buffer. Switching from CO2/HCO3(-)-free to CO2/HCO3(-)-containing extracellular solution results in initial intracellular acidification due to hydration of CO2 followed by gradual alkalinization due to cellular HCO3(-) uptake. Carbonic anhydrase inhibition decelerates the initial acidification and attenuates the associated transient vasoconstriction without affecting intracellular pH or artery tone at steady-state. Na(+),HCO3(-)-cotransport and Na(+)/H(+)-exchange activity after NH4(+)-prepulse-induced intracellular acidification are unaffected by carbonic anhydrase inhibition. Extracellular surface pH transients induced by transmembrane NH3 flux are evident under CO2/HCO3(-)-free conditions but absent when the buffer capacity and apparent H(+) mobility increase in the presence of CO2/HCO3(-) even after the inhibition of carbonic anhydrases. We conclude that (a) intracellular carbonic anhydrase activity accentuates pH transients and vasoconstriction in response to acute elevations of pCO2, (b) CO2/HCO3(-) minimizes extracellular surface pH transients without requiring carbonic anhydrase activity, and

  2. Role of H(+)-pyrophosphatase activity in the regulation of intracellular pH in a scuticociliate parasite of turbot: Physiological effects.

    PubMed

    Mallo, Natalia; Lamas, Jesús; de Felipe, Ana-Paula; Sueiro, Rosa-Ana; Fontenla, Francisco; Leiro, José-Manuel

    2016-10-01

    The scuticociliatosis is a very serious disease that affects the cultured turbot, and whose causal agent is the anphizoic and marine euryhaline ciliate Philasterides dicentrarchi. Several protozoans possess acidic organelles that contain high concentrations of pyrophosphate (PPi), Ca(2+) and other elements with essential roles in vesicular trafficking, pH homeostasis and osmoregulation. P. dicentrarchi possesses a pyrophosphatase (H(+)-PPase) that pumps H(+) through the membranes of vacuolar and alveolar sacs. These compartments share common features with the acidocalcisomes described in other parasitic protozoa (e.g. acid content and Ca(2+) storage). We evaluated the effects of Ca(2+) and ATP on H (+)-PPase activity in this ciliate and analyzed their role in maintaining intracellular pH homeostasis and osmoregulation, by the addition of PPi and inorganic molecules that affect osmolarity. Addition of PPi led to acidification of the intracellular compartments, while the addition of ATP, CaCl2 and bisphosphonates analogous of PPi and Ca(2+) metabolism regulators led to alkalinization and a decrease in H(+)-PPase expression in trophozoites. Addition of NaCl led to proton release, intracellular Ca(2+) accumulation and downregulation of H(+)-PPase expression. We conclude that the regulation of the acidification of intracellular compartments may be essential for maintaining the intracellular pH homeostasis necessary for survival of ciliates and their adaptation to salt stress, which they will presumably face during the endoparasitic phase, in which the salinity levels are lower than in their natural environment.

  3. Metabolic regulation of neutrophil spreading, membrane tubulovesicular extensions (cytonemes) formation and intracellular pH upon adhesion to fibronectin.

    PubMed

    Galkina, Svetlana I; Sud'ina, Galina F; Klein, Thomas

    2006-08-01

    Circulating leukocytes have a round cell shape and roll along vessel walls. However, metabolic disorders can lead them to adhere to the endothelium and spread (flatten). We studied the metabolic regulation of adhesion, spreading and intracellular pH (pHi) of neutrophils (polymorphonuclear leukocytes) upon adhesion to fibronectin-coated substrata. Resting neutrophils adhered and spread on fibronectin. An increase in pHi accompanied neutrophil spreading. Inhibition of oxidative phosphorylation or inhibition of P- and F-type ATPases affected neither neutrophil spreading nor pHi. Inhibition of glucose metabolism or V-ATPase impaired neutrophil spreading, blocked the increase in the pHi and induced extrusion of membrane tubulovesicular extensions (cytonemes), anchoring cells to substrata. Omission of extracellular Na(+) and inhibition of chloride channels caused a similar effect. We propose that these tubulovesicular extensions represent protrusions of exocytotic trafficking, supplying the plasma membrane of neutrophils with ion exchange mechanisms and additional membrane for spreading. Glucose metabolism and V-type ATPase could affect fusion of exocytotic trafficking with the plasma membrane, thus controlling neutrophil adhesive state and pHi. Cl(-) efflux through chloride channels and Na(+) influx seem to be involved in the regulation of the V-ATPase by carrying out charge compensation for the proton-pumping activity and through V-ATPase in regulation of neutrophil spreading and pHi.

  4. Metabolic regulation of neutrophil spreading, membrane tubulovesicular extensions (cytonemes) formation and intracellular pH upon adhesion to fibronectin

    SciTech Connect

    Galkina, Svetlana I. . E-mail: galkina@genebee.msu.su; Sud'ina, Galina F.; Klein, Thomas

    2006-08-01

    Circulating leukocytes have a round cell shape and roll along vessel walls. However, metabolic disorders can lead them to adhere to the endothelium and spread (flatten). We studied the metabolic regulation of adhesion, spreading and intracellular pH (pHi) of neutrophils (polymorphonuclear leukocytes) upon adhesion to fibronectin-coated substrata. Resting neutrophils adhered and spread on fibronectin. An increase in pHi accompanied neutrophil spreading. Inhibition of oxidative phosphorylation or inhibition of P- and F-type ATPases affected neither neutrophil spreading nor pHi. Inhibition of glucose metabolism or V-ATPase impaired neutrophil spreading, blocked the increase in the pHi and induced extrusion of membrane tubulovesicular extensions (cytonemes), anchoring cells to substrata. Omission of extracellular Na{sup +} and inhibition of chloride channels caused a similar effect. We propose that these tubulovesicular extensions represent protrusions of exocytotic trafficking, supplying the plasma membrane of neutrophils with ion exchange mechanisms and additional membrane for spreading. Glucose metabolism and V-type ATPase could affect fusion of exocytotic trafficking with the plasma membrane, thus controlling neutrophil adhesive state and pHi. Cl{sup -} efflux through chloride channels and Na{sup +} influx seem to be involved in the regulation of the V-ATPase by carrying out charge compensation for the proton-pumping activity and through V-ATPase in regulation of neutrophil spreading and pHi.

  5. Intracellular pH of Mycobacterium avium subsp. paratuberculosis following exposure to antimicrobial compounds monitored at the single cell level.

    PubMed

    Gaggìa, Francesca; Nielsen, Dennis Sandris; Biavati, Bruno; Siegumfeldt, Henrik

    2010-07-31

    Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne's disease; moreover, it seems to be implicated in the development of Crohn's disease in humans. In the present study, fluorescence ratio imaging microscopy (FRIM) was used to assess changes in intracellular pH (pH(i)) of one strain of MAP after exposure to nisin and neutralized cell-free supernatants (NCSs) from five bacteriocin-producing lactic acid bacteria (LAB) with known probiotic properties. The evaluation of pH(i) by FRIM provides information about the physiological state of bacterial cells, bypassing the long and problematic incubations needed for methods relying upon growth of MAP such as determination of colony forming units. The FRIM results showed that both nisin and the cell-free supernatant from Lactobacillus plantarum PCA 236 affected the pH(i) of MAP within a few hours. However, monitoring the population for 24h revealed the presence of a subpopulation of cells probably resistant to the antimicrobial compounds tested. Use of nisin and bacteriocin-producing LAB strains could lead to new intervention strategies for the control of MAP based on in vivo application of probiotic cultures as feed additives at farm level.

  6. Measurement of intracellular pH in pancreatic duct cells: a new method for calibrating the fluorescence data.

    PubMed

    Hegyi, Péter; Rakonczay, Zoltán; Gray, Mike A; Argent, Barry E

    2004-05-01

    Pancreatic duct cells secrete the bicarbonate ions found in pancreatic juice. Impairment of ductal bicarbonate secretion, as occurs in cystic fibrosis, has serious consequences for pancreatic function and for the structural integrity of the gland. As bicarbonate is a buffer ion, the accurate measurement of intracellular pH (pHi) in duct cells is an important technique for studying the mechanisms of bicarbonate transport. Commonly, pHi is measured using the fluorescent dye biscarboxyethylcarboxyfluorescein (BCECF). The purpose of this study was to develop a new technique for the accurate calibration of BCECF fluorescent signals. Our results indicate that BCECF fluorescence is not only dependent on pHi but also on the total fluorescence intensity of the detected area (which may be influenced by dye loading, dye leakage, and the shutter size on the photomultiplier). The outcome is that one calibration curve is not sufficient for accurate determination of pHi. In fact, an appropriate calibration curve must be selected for each individual experiment. Moreover, the calibration plot is only linear over a narrow range of pHi values. In conclusion, we have developed a new technique that should be applicable to all cell types for the accurate calibration of fluorescent signals from the pH-sensitive dye BCECF.

  7. A phosphorus nuclear magnetic resonance study of metabolites and intracellular pH in rabbit vascular smooth muscle.

    PubMed Central

    Spurway, N C; Wray, S

    1987-01-01

    1. 31P nuclear magnetic resonance (n.m.r.) spectroscopy was used to investigate metabolites, intracellular pH (pHi) and the effects of pHi on tone in rabbit blood vessels. The vessels were bathed in mammalian Ringer solution and maintained at 20 degrees C while inside the spectrometer. 2. Vascular spectra showed relatively low phosphocreatine (PCr) concentrations compared to skeletal muscle. The [PCr]/[ATP] ratio was only 1.32 +/- 0.09 (n = 7). There was also a prominent phosphomonoester (PME) peak. Similar features have been reported for other smooth muscles examined by 31P n.m.r. 3. The [PCr] was higher and the inorganic phosphate (Pi) concentration lower than values deduced from chemical analysis of arterial extracts. However, the [PCr] value fell within the range obtained for other smooth muscles when studied by 31P n.m.r. 4. Measurement of pHi under control conditions (external pH 7.25) gave a mean value of 7.19 +/- 0.03 at 20 degrees C (n = 5). Metabolic inhibition brought about by 0.5 mM-cyanide and 0.2 mM-fluoride did not significantly alter pHi. At higher inhibitor concentrations (3 and 1 mM respectively) there was a significant acidosis. 5. The effects of NH4Cl upon pH were investigated in metabolically inhibited preparations. During 10 min applications of 30 mM-NH4Cl (isosmotically substituted for NaCl) the pHi rose; during subsequent NH4Cl removal it fell below control values. In the least inhibited tissues the total pHi excursion between NH4Cl applications and removals was 0.5 unit. 6. Rabbit ear vessels have been found to increase vascular tone during manoeuvres which were expected to decrease pHi. From the direct measurement of pHi reported in this study, it is concluded that the vascular tone changes brought about by NH4Cl application and withdrawal may be attributed to the alteration of pHi. PMID:3446806

  8. The impact of pH inhomogeneities on CHO cell physiology and fed-batch process performance - two-compartment scale-down modelling and intracellular pH excursion.

    PubMed

    Brunner, Matthias; Braun, Philipp; Doppler, Philipp; Posch, Christoph; Behrens, Dirk; Herwig, Christoph; Fricke, Jens

    2017-01-12

    Due to high mixing times and base addition from top of the vessel, pH inhomogeneities are most likely to occur during large-scale mammalian processes. The goal of this study was to set-up a scale-down model of a 10-12 m(3) stirred tank bioreactor and to investigate the effect of pH perturbations on CHO cell physiology and process performance. Short-term changes in extracellular pH are hypothesized to affect intracellular pH and thus cell physiology. Therefore, batch fermentations, including pH shifts to 9.0 and 7.8, in regular one-compartment systems are conducted. The short-term adaption of the cells intracellular pH are showed an immediate increase due to elevated extracellular pH. With this basis of fundamental knowledge, a two-compartment system is established which is capable of simulating defined pH inhomogeneities. In contrast to state-of-the-art literature, the scale-down model is included parameters (e.g. volume of the inhomogeneous zone) as they might occur during large-scale processes. pH inhomogeneity studies in the two-compartment system are performed with simulation of temporary pH zones of pH 9.0. The specific growth rate especially during the exponential growth phase is strongly affected resulting in a decreased maximum viable cell density and final product titer. The gathered results indicate that even short-term exposure of cells to elevated pH values during large-scale processes can affect cell physiology and overall process performance. In particular, it could be shown for the first time that pH perturbations, which might occur during the early process phase, have to be considered in scale-down models of mammalian processes.

  9. Noninvasive high-throughput single-cell analysis of the intracellular pH of Saccharomyces cerevisiae by ratiometric flow cytometry.

    PubMed

    Valkonen, Mari; Mojzita, Dominik; Penttilä, Merja; Bencina, Mojca

    2013-12-01

    The ability of cells to maintain pH homeostasis in response to environmental changes has elicited interest in basic and applied research and has prompted the development of methods for intracellular pH measurements. Many traditional methods provide information at population level and thus the average values of the studied cell physiological phenomena, excluding the fact that cell cultures are very heterogeneous. Single-cell analysis, on the other hand, offers more detailed insight into population variability, thereby facilitating a considerably deeper understanding of cell physiology. Although microscopy methods can address this issue, they suffer from limitations in terms of the small number of individual cells that can be studied and complicated image processing. We developed a noninvasive high-throughput method that employs flow cytometry to analyze large populations of cells that express pHluorin, a genetically encoded ratiometric fluorescent probe that is sensitive to pH. The method described here enables measurement of the intracellular pH of single cells with high sensitivity and speed, which is a clear improvement compared to previously published methods that either require pretreatment of the cells, measure cell populations, or require complex data analysis. The ratios of fluorescence intensities, which correlate to the intracellular pH, are independent of the expression levels of the pH probe, making the use of transiently or extrachromosomally expressed probes possible. We conducted an experiment on the kinetics of the pH homeostasis of Saccharomyces cerevisiae cultures grown to a stationary phase after ethanol or glucose addition and after exposure to weak acid stress and glucose pulse. Minor populations with pH homeostasis behaving differently upon treatments were identified.

  10. Regulation of intracellular pH in the smooth muscle of guinea-pig ureter: Na+ dependence.

    PubMed Central

    Aickin, C C

    1994-01-01

    1. Mechanisms involved in the regulation of intracellular pH (pHi) in smooth muscle cells of guinea-pig ureter have been investigated using double-barrelled pH-sensitive microelectrodes in isolated strips of tissue. 2. Removal of CO2-HCO3- from the superfusing solution caused a fall in the steady-state pHi except in a few cells which had been excised from the animal for many hours (usually > 24 h). The pHi value was 7.22 +/- 0.09 (n = 89; mean +/- S.D. of an observation) in solution buffered with 5% CO2-21 mM HCO3-, compared with 6.92 +/- 0.24 (n = 67) in the nominal absence of CO2-HCO3-. Recovery from experimentally induced acidosis was faster in the presence, rather than nominal absence, of CO2-HCO3- (mean half-times of 2.7 +/- 0.7 min, n = 41, and 4.6 +/- 1.3 min, n = 12, respectively). These results suggest the presence of both HCO(3-)-dependent and -independent mechanisms for the effective extrusion of acid equivalents. 3. Recovery from acidosis was dependent on external Na+ (Na+o) in both the presence and nominal absence of CO2-HCO3-, with an apparent half-maximal activation at approximately 4 and 20 mM Na+o, respectively. Removal of Na+o in the steady state caused a fall in pHi which proceeded at a faster rate in the presence rather than in the nominal absence of CO2-HCO3-. 4. Amiloride (100 microM-1 mM) reversibly inhibited the recovery from acidosis and caused a fall in the steady-state pHi when applied in the nominal absence of CO2-HCO3-, but had no measurable effect on either the recovery from acidosis or steady-state pHi in the presence of CO2-HCO3-. These results suggest that Na(+)-H+ exchange was responsible for extrusion of acid equivalents in the nominal absence of CO2 and HCO3-, but that it played little part under more physiological conditions. 5. Although Na(+)-H+ exchange appeared to be activated below a pHi of about 7.2, it was incapable of maintaining a 'normal' pHi in the nominal absence of CO2-HCO3- in freshly excised cells, where values

  11. In vivo intracellular pH measurements in tobacco and Arabidopsis reveal an unexpected pH gradient in the endomembrane system.

    PubMed

    Martinière, Alexandre; Bassil, Elias; Jublanc, Elodie; Alcon, Carine; Reguera, Maria; Sentenac, Hervé; Blumwald, Eduardo; Paris, Nadine

    2013-10-01

    The pH homeostasis of endomembranes is essential for cellular functions. In order to provide direct pH measurements in the endomembrane system lumen, we targeted genetically encoded ratiometric pH sensors to the cytosol, the endoplasmic reticulum, and the trans-Golgi, or the compartments labeled by the vacuolar sorting receptor (VSR), which includes the trans-Golgi network and prevacuoles. Using noninvasive live-cell imaging to measure pH, we show that a gradual acidification from the endoplasmic reticulum to the lytic vacuole exists, in both tobacco (Nicotiana tabacum) epidermal (ΔpH -1.5) and Arabidopsis thaliana root cells (ΔpH -2.1). The average pH in VSR compartments was intermediate between that of the trans-Golgi and the vacuole. Combining pH measurements with in vivo colocalization experiments, we found that the trans-Golgi network had an acidic pH of 6.1, while the prevacuole and late prevacuole were both more alkaline, with pH of 6.6 and 7.1, respectively. We also showed that endosomal pH, and subsequently vacuolar trafficking of soluble proteins, requires both vacuolar-type H(+) ATPase-dependent acidification as well as proton efflux mediated at least by the activity of endosomal sodium/proton NHX-type antiporters.

  12. Subcellular compartmentation of ascorbate and its variation in disease states.

    PubMed

    Bánhegyi, Gábor; Benedetti, Angelo; Margittai, Eva; Marcolongo, Paola; Fulceri, Rosella; Németh, Csilla E; Szarka, András

    2014-09-01

    Beyond its general role as antioxidant, specific functions of ascorbate are compartmentalized within the eukaryotic cell. The list of organelle-specific functions of ascorbate has been recently expanded with the epigenetic role exerted as a cofactor for DNA and histone demethylases in the nucleus. Compartmentation necessitates the transport through intracellular membranes; members of the GLUT family and sodium-vitamin C cotransporters mediate the permeation of dehydroascorbic acid and ascorbate, respectively. Recent observations show that increased consumption and/or hindered entrance of ascorbate in/to a compartment results in pathological alterations partially resembling to scurvy, thus diseases of ascorbate compartmentation can exist. The review focuses on the reactions and transporters that can modulate ascorbate concentration and redox state in three compartments: endoplasmic reticulum, mitochondria and nucleus. By introducing the relevant experimental and clinical findings we make an attempt to coin the term of ascorbate compartmentation disease.

  13. The V-ATPase is expressed in the choroid plexus and mediates cAMP-induced intracellular pH alterations.

    PubMed

    Christensen, Henriette L; Păunescu, Teodor G; Matchkov, Vladimir; Barbuskaite, Dagne; Brown, Dennis; Damkier, Helle H; Praetorius, Jeppe

    2017-01-01

    The cerebrospinal fluid (CSF) pH influences brain interstitial pH and, therefore, brain function. We hypothesized that the choroid plexus epithelium (CPE) expresses the vacuolar H(+)-ATPase (V-ATPase) as an acid extrusion mechanism in the luminal membrane to counteract detrimental elevations in CSF pH. The expression of mRNA corresponding to several V-ATPase subunits was demonstrated by RT-PCR analysis of CPE cells (CPECs) isolated by fluorescence-activated cell sorting. Immunofluorescence and electron microscopy localized the V-ATPase primarily in intracellular vesicles with only a minor fraction in the luminal microvillus area. The vesicles did not translocate to the luminal membrane in two in vivo models of hypocapnia-induced alkalosis. The Na(+)-independent intracellular pH (pHi) recovery from acidification was studied in freshly isolated clusters of CPECs. At extracellular pH (pHo) 7.4, the cells failed to display significant concanamycin A-sensitive pHi recovery (i.e., V-ATPase activity). The recovery rate in the absence of Na(+) amounted to <10% of the pHi recovery rate observed in the presence of Na(+) Recovery of pHi was faster at pHo 7.8 and was abolished at pHo 7.0. The concanamycin A-sensitive pHi recovery was stimulated by cAMP at pH 7.4 in vitro, but intraventricular infusion of the membrane-permeant cAMP analog 8-CPT-cAMP did not result in trafficking of the V-ATPase. In conclusion, we find evidence for the expression of a minor fraction of V-ATPase in the luminal membrane of CPECs. This fraction does not contribute to enhanced acid extrusion at high extracellular pH, but seems to be activated by cAMP in a trafficking-independent manner.

  14. Induction of intracellular Ca2+ and pH changes in Sf9 insect cells by rhodojaponin-III, a natural botanic insecticide isolated from Rhododendron molle.

    PubMed

    Cheng, Xing-An; Xie, Jian-Jun; Hu, Mei-Ying; Zhang, Yan-Bo; Huang, Jing-Fei

    2011-04-15

    Many studies on intracellular calcium ([Ca2+](i)) and intracellular pH (pH(i)) have been carried out due to their importance in regulation of different cellular functions. However, most of the previous studies are focused on human or mammalian cells. The purpose of the present study was to characterize the effect of Rhodojaponin-III (R-III) on [Ca2+](i) and pH(i) and the proliferation of Sf9 cells. R-III strongly inhibited Sf9 cells proliferation with a time- and dose-dependent manner. Flow cytometry established that R-III interfered with Sf9 cells division and arrested them in G2/M. By using confocal scanning technique, effects of R-III on intracellular free calcium ([Ca2+](i)) and intracellular pH (pH(i)) in Sf9 cells were determined. R-III induced a significant dose-dependent (1, 10, 100, 200 μg/mL) increase in [Ca2+](i) and pH(i) of Sf9 cells in presence of Ca2+-containing solution (Hanks) and an irreversible decrease in the absence of extra cellular Ca2+. We also found that both extra cellular Ca2+ and intracellular Ca2+ stores contributed to the increase of [Ca2+](i), because completely treating Sf9 cells with CdCl(2) (5 mM), a Ca2+ channels blocker, R-III (100 μg/mL) induced a transient elevation of [Ca2+](i) in case of cells either in presence of Ca2+ containing or Ca2+ free solution. In these conditions, pH(i) showed similar changes with that of [Ca2+](i) on the whole. Accordingly, we supposed that there was a certain linkage for change of [Ca2+](i), cell cycle arrest, proliferation inhibition in Sf9 cells induced by R-III.

  15. Zonation of gluconeogenesis, ketogenesis and intracellular pH in livers from normal and diabetic ketoacidotic rats: evidence for intralobular redistribution of metabolic events in ketoacidosis.

    PubMed Central

    Burns, S P; Cohen, R D; Iles, R A; Bailey, R A; Desai, M; Germain, J P; Going, T C

    1999-01-01

    The intralobular distribution of metabolism was examined in the livers from rats with severe diabetic ketoacidosis (DKA), perfused at pH 6.8, and compared with that in livers from normal starved animals perfused at either pH 7.4 or 6.8. With lactate and palmitate as substrates, the perivenous uptake of periportally synthesized glucose seen in normal livers at pH 7.4 was abolished during DKA; indeed, gluconeogenesis was most active in the perivenous region. Whereas in normal livers perfused at pH 7.4 the periportal region showed a markedly elevated intracellular pH (pH(i)) compared with the perivenous zone, this distribution of pH(i) was reversed in DKA, with an intermediate distribution in normal livers perfused at pH 6. 8. 3-Hydroxybutyrate was generated throughout the lobule. Some acetoacetate generated periportally was converted to 3-hydroxybutyrate more perivenously. A steep gradient of oxygen uptake along the radius of the lobule was apparent in all three groups; oxygen uptake was greatly decreased perivenously despite adequate oxygen supply. These findings are consistent with our previous observations of the lobular co-location of high pH(i) and gluconeogenesis, and might offer an explanation of how high gluconeogenic rates can continue in spite of severe systemic acidosis in DKA. The findings provide direct evidence for a marked redistribution of intralobular metabolism in DKA. PMID:10493939

  16. Intracellular pH and its response to CO2-driven seawater acidification in symbiotic versus non-symbiotic coral cells.

    PubMed

    Gibbin, Emma M; Putnam, Hollie M; Davy, Simon K; Gates, Ruth D

    2014-06-01

    Regulating intracellular pH (pHi) is critical for optimising the metabolic activity of corals, yet the mechanisms involved in pH regulation and the buffering capacity within coral cells are not well understood. Our study investigated how the presence of symbiotic dinoflagellates affects the response of pHi to PCO2-driven seawater acidification in cells isolated from Pocillopora damicornis. Using the fluorescent dye BCECF-AM, in conjunction with confocal microscopy, we simultaneously characterised the pHi response in host coral cells and their dinoflagellate symbionts, in symbiotic and non-symbiotic states under saturating light, with and without the photosynthetic inhibitor DCMU. Each treatment was run under control (pH 7.8) and CO2-acidified seawater conditions (decreasing pH from 7.8 to 6.8). After 105 min of CO2 addition, by which time the external pH (pHe) had declined to 6.8, the dinoflagellate symbionts had increased their pHi by 0.5 pH units above control levels when in the absence of DCMU. In contrast, in both symbiotic and non-symbiotic host coral cells, 15 min of CO2 addition (0.2 pH unit drop in pHe) led to cytoplasmic acidosis equivalent to 0.3-0.4 pH units irrespective of whether DCMU was present. Despite further seawater acidification over the duration of the experiment, the pHi of non-symbiotic coral cells did not change, though in host cells containing a symbiont cell the pHi recovered to control levels when photsynthesis was not inhibited. This recovery was negated when cells were incubated with DCMU. Our results reveal that photosynthetic activity of the endosymbiont is tightly coupled with the ability of the host cell to recover from cellular acidosis after exposure to high CO2/low pH.

  17. Effect of Ca2+ and K+ on the intracellular pH of an Escherichia coli L-form.

    PubMed

    Onoda, T; Oshima, A; Fukunaga, N; Nakatani, A

    1992-06-01

    The L-form NC7, derived from Escherichia coli K12, grew in a complex medium containing 0.2 M-CaCl2 as osmotic stabilizer, but not at pH values above 7.8. The cessation of growth at alkaline pH was not due to cell death. In complex media containing K+ or Na+, the L-form grew ove a wide pH range. Growth at alkaline pH was inhibited by 1 mM-amiloride, indicating that Na+/H+ antiport activity was required for growth at alkaline pH. The internal pH (pHi) of the L-form in media containing K+, Na+ or Ca2+ was constant at about 7.8 to 8.0 at external pH (pHo) values of 7.2 and 8.2. The rates of O2 consumption by intact cells, lactate oxidation by membrane vesicles from cells grown in Ca(2+)-containing medium, and cell division were all strongly repressed under alkaline conditions.

  18. Modelling the extra and intracellular uptake and discharge of heavy metals in Fontinalis antipyretica transplanted along a heavy metal and pH contamination gradient.

    PubMed

    Fernández, J A; Vázquez, M D; López, J; Carballeira, A

    2006-01-01

    Samples of the aquatic bryophyte Fontinalis antipyretica Hedw. were transplanted to different sites with the aim of characterizing the kinetics of the uptake and discharge of heavy metals in the extra and intracellular compartments. The accumulation of metals in extracellular compartments, characterized by an initial rapid accumulation, then a gradual slowing down over time, fitted perfectly to a Michaelis-Menten model. The discharge of metals from the same compartment followed an inverse linear model or an inverse Michaelis-Menten model, depending on the metal. In intracellular sites both uptake and discharge occurred more slowly and progressively, following a linear model. We also observed that the acidity of the environment greatly affected metal accumulation in extracellular sites, even when the metals were present at relatively high concentrations, whereas the uptake of metals within cells was much less affected by pH.

  19. In vivo measurement of cytosolic and mitochondrial pH using a pH-sensitive GFP derivative in Saccharomyces cerevisiae reveals a relation between intracellular pH and growth.

    PubMed

    Orij, Rick; Postmus, Jarne; Ter Beek, Alex; Brul, Stanley; Smits, Gertien J

    2009-01-01

    The specific pH values of cellular compartments affect virtually all biochemical processes, including enzyme activity, protein folding and redox state. Accurate, sensitive and compartment-specific measurements of intracellular pH (pHi) dynamics in living cells are therefore crucial to the understanding of stress response and adaptation. We used the pH-sensitive GFP derivative 'ratiometric pHluorin' expressed in the cytosol and in the mitochondrial matrix of growing Saccharomyces cerevisiae to assess the variation in cytosolic pH (pHcyt) and mitochondrial pH (pHmit) in response to nutrient availability, respiratory chain activity, shifts in environmental pH and stress induced by addition of sorbic acid. The in vivo measurement allowed accurate determination of organelle-specific pH, determining a constant pHcyt of 7.2 and a constant pHmit of 7.5 in cells exponentially growing on glucose. We show that pHcyt and pHmit are differentially regulated by carbon source and respiratory chain inhibitors. Upon glucose starvation or sorbic acid stress, pHi decrease coincided with growth stasis. Additionally, pHi and growth coincided similarly in recovery after addition of glucose to glucose-starved cultures or after recovery from a sorbic acid pulse. We suggest a relation between pHi and cellular energy generation, and therefore a relation between pHi and growth.

  20. Listeria monocytogenes varies among strains to maintain intracellular pH homeostasis under stresses by different acids as analyzed by a high-throughput microplate-based fluorometry.

    PubMed

    Cheng, Changyong; Yang, Yongchun; Dong, Zhimei; Wang, Xiaowen; Fang, Chun; Yang, Menghua; Sun, Jing; Xiao, Liya; Fang, Weihuan; Song, Houhui

    2015-01-01

    Listeria monocytogenes, a food-borne pathogen, has the capacity to maintain intracellular pH (pHi) homeostasis in acidic environments, but the underlying mechanisms remain elusive. Here, we report a simple microplate-based fluorescent method to determine pHi of listerial cells that were prelabeled with the fluorescent dye carboxyfluorescein diacetate N-succinimidyl ester and subjected to acid stress. We found that L. monocytogenes responds differently among strains toward organic and inorganic acids to maintain pHi homeostasis. The capacity of L. monocytogenes to maintain pHi at extracellular pH 4.5 (pHex) was compromised in the presence of acetic acid and lactic acid, but not by hydrochloric acid and citric acid. Organic acids exhibited more inhibitory effects than hydrochloric acid at certain pH conditions. Furthermore, the virulent stains L. monocytogenes EGDe, 850658 and 10403S was more resistant to acidic stress than the avirulent M7 which showed a defect in maintaining pHi homeostasis. Deletion of sigB, a stress-responsive alternative sigma factor from 10403S, markedly altered intracellular pHi homeostasis, and showed a significant growth and survival defect under acidic conditions. Thus, this work provides new insights into bacterial survival mechanism to acidic stresses.

  1. Listeria monocytogenes varies among strains to maintain intracellular pH homeostasis under stresses by different acids as analyzed by a high-throughput microplate-based fluorometry

    PubMed Central

    Cheng, Changyong; Yang, Yongchun; Dong, Zhimei; Wang, Xiaowen; Fang, Chun; Yang, Menghua; Sun, Jing; Xiao, Liya; Fang, Weihuan; Song, Houhui

    2015-01-01

    Listeria monocytogenes, a food-borne pathogen, has the capacity to maintain intracellular pH (pHi) homeostasis in acidic environments, but the underlying mechanisms remain elusive. Here, we report a simple microplate-based fluorescent method to determine pHi of listerial cells that were prelabeled with the fluorescent dye carboxyfluorescein diacetate N-succinimidyl ester and subjected to acid stress. We found that L. monocytogenes responds differently among strains toward organic and inorganic acids to maintain pHi homeostasis. The capacity of L. monocytogenes to maintain pHi at extracellular pH 4.5 (pHex) was compromised in the presence of acetic acid and lactic acid, but not by hydrochloric acid and citric acid. Organic acids exhibited more inhibitory effects than hydrochloric acid at certain pH conditions. Furthermore, the virulent stains L. monocytogenes EGDe, 850658 and 10403S was more resistant to acidic stress than the avirulent M7 which showed a defect in maintaining pHi homeostasis. Deletion of sigB, a stress-responsive alternative sigma factor from 10403S, markedly altered intracellular pHi homeostasis, and showed a significant growth and survival defect under acidic conditions. Thus, this work provides new insights into bacterial survival mechanism to acidic stresses. PMID:25667585

  2. Quantum-dot/dopamine bioconjugates function as redox coupled assemblies for in vitro and intracellular pH sensing

    NASA Astrophysics Data System (ADS)

    Medintz, Igor L.; Stewart, Michael H.; Trammell, Scott A.; Susumu, Kimihiro; Delehanty, James B.; Mei, Bing C.; Melinger, Joseph S.; Blanco-Canosa, Juan B.; Dawson, Philip E.; Mattoussi, Hedi

    2010-08-01

    The use of semiconductor quantum dots (QDs) for bioimaging and sensing has progressively matured over the past decade. QDs are highly sensitive to charge-transfer processes, which can alter their optical properties. Here, we demonstrate that QD-dopamine-peptide bioconjugates can function as charge-transfer coupled pH sensors. Dopamine is normally characterized by two intrinsic redox properties: a Nernstian dependence of formal potential on pH and oxidation of hydroquinone to quinone by O2 at basic pH. We show that the latter quinone can function as an electron acceptor quenching QD photoluminescence in a manner that depends directly on pH. We characterize the pH-dependent QD quenching using both electrochemistry and spectroscopy. QD-dopamine conjugates were also used as pH sensors that measured changes in cytoplasmic pH as cells underwent drug-induced alkalosis. A detailed mechanism describing the QD quenching processes that is consistent with dopamine's inherent redox chemistry is presented.

  3. Compartmentalization of Decay in Trees.

    ERIC Educational Resources Information Center

    Shigo, Alex L.

    1985-01-01

    Unlike animals, which heal, trees compartmentalize by setting boundaries that resist the spread of invading microorganisms. Discusses the creation of new walls by anatomical and chemical means in response to death of a branch or pruning. Points out that genetic control of compartmentalization has resulted from evolution of resistant species. (DH)

  4. Optical nanosensors for chemical analysis inside single living cells. 2. Sensors for pH and calcium and the intracellular application of PEBBLE sensors.

    PubMed

    Clark, H A; Kopelman, R; Tjalkens, R; Philbert, M A

    1999-11-01

    Optical nanosensors, or PEBBLEs (probes encapsulated by biologically localized embedding), have been produced for intracellular measurements of pH and calcium. Five varieties of pH-sensitive sensors and three different calcium-selective sensors are presented and discussed. Each sensor combines an ion-selective fluorescent indicator and an ion-insensitive internal standard entrapped within an acrylamide polymeric matrix. Calibrations and linear ranges are presented for each sensor. The photobleaching of dyes incorporated into PEBBLEs is comparable to that of the respective free dye that is incorporated within the matrix. These PEBBLE sensors are fully reversible over many measurements. The leaching of fluorescent indicator from the polymer is less than 50% over a 48-h period (note that a typical application time is only a few hours). The PEBBLE sensors have also been applied to intracellular analysis of the calcium flux in the cytoplasm of neural cells during the mitochondrial permeability transition. Specifically, a distinct difference is noted between cells of different types (astrocyte vs neuron-derived cells) with respect to their response to the toxicant m-dinitrobenzene (DNB). Use of PEBBLE sensors permits the quantitative discrimination of subtle differences between the ability of human SY5Y neuroblastoma and C6 glioma to respond to challenge with DNB. Specifically, measurement of intracellular calcium, the precursor to cell death, has been achieved.

  5. pH-dependent fluorescence of a heterologously expressed Aequorea green fluorescent protein mutant: in situ spectral characteristics and applicability to intracellular pH estimation.

    PubMed

    Robey, R B; Ruiz, O; Santos, A V; Ma, J; Kear, F; Wang, L J; Li, C J; Bernardo, A A; Arruda, J A

    1998-07-14

    The green fluorescent protein of Aequorea victoria (GFP) is a natural peptide chromophore without substrate or cofactor requirements for fluorescence. In vitro, a recombinant F64L/S65T GFP mutant (GFPmut1) exhibited pH sensitive fluorescence within the physiologic range. When heterologously expressed in BS-C-1 cells or rabbit proximal tubule cells, uniform cytosolic and nuclear fluorescence was observed. Cytosolic fluorescence constituted over 80% of the total. Excitation scanning of transfected cells revealed two GFPmut1-specific regions that were pH-sensitive over the physiologic range, and each region exhibited a unique pH "bias" in fluorescence emission. Excitation at or near the expected maximum of 488 nm (region II) uniformly resulted in fluorescence that was preferentially altered at acidic pH. In contrast, a novel "wild-type" excitation peak at 400 nm (region I) resulted in alkaline-biased fluorescence similar to that described for the wild-type chromophore in vitro, suggesting that wild-type spectral features disrupted in vitro by mutagenesis may be recovered in intact cells. Calibration of intracellular pH (pHi) with in situ fluorescence following excitation in either region revealed a semilogarithmic relationship between fluorescence intensity and pH within the physiologic range. We therefore measured pHi changes attributable to altered Na/HCO3 cotransport (NBC) activity both in GFPmut1-expressing cells and in paired untransfected cells loaded with BCECF. Basal NBC activity was the same in each group, as was the stimulation of activity by 10% CO2, thus validating the utility of GFPmut1 as a fluorescent probe for pHi and establishing a novel, useful, and practical application for GFPmut1 in monitoring pHi in real time.

  6. Lactate supply as a determinant of the distribution of intracellular pH within the hepatic lobule.

    PubMed Central

    Burns, S P; Murphy, H C; Iles, R A; Cohen, R D

    2001-01-01

    When isolated livers from starved rats are perfused with lactate at constant perfusate pH and P(co(2)), there is a marked gradient of cell pH (pH(i)) along the length of the lobular radius, with periportal cells being substantially more alkaline than perivenous cells. In the present studies, the perivenous 21% of the lobular volume was destroyed by retrograde digitonin perfusion, and antegrade perfusion restored. pH(i) was determined by (31)P-NMR. The remaining periportal cells, the site of gluconeogenesis from lactate, had a substantially higher mean pH(i) (7.42) than did the intact liver (7.23). When lactate was removed from the perfusate, mean pH(i) decreased to 7.25. The corresponding concentration of cell bicarbonate fell with a half-time of approximately 5 min. When lactate was re-introduced mean pH(i) rose to 7.34. We conclude that a major contributor to periportal alkalinity under these conditions is proton consumption during gluconeogenesis from lactate ions. PMID:11535120

  7. Apical membrane Na+/H+ exchange in Necturus gallbladder epithelium. Its dependence on extracellular and intracellular pH and on external Na+ concentration

    PubMed Central

    1990-01-01

    Intracellular microelectrode techniques and extracellular pH measurements were used to study the dependence of apical Na+/H+ exchange on mucosal and intracellular pH and on mucosal solution Na+ concentration ([Na+]o). When mucosal solution pH (pHo) was decreased in gallbladders bathed in Na(+)-containing solutions, aNai fell. The effect of pHo is consistent with titration of a single site with an apparent pK of 6.29. In Na(+)-depleted tissues, increasing [Na+]o from 0 to values ranging from 2.5 to 110 mM increased aNai; the relationship was well described by Michaelis-Menten kinetics. The apparent Km was 15 mM at pHo 7.5 and increased to 134 mM at pHo 6.5, without change in Vmax. In Na(+)-depleted gallbladders, elevating [Na+]o from 0 to 25 mM increased aNai and pHi and caused acidification of a poorly buffered mucosal solution upon stopping the superfusion; lowering pHo inhibited both apical Na+ entry and mucosal solution acidification. Both effects can be ascribed to titration of a single site; the apparent pK's were 7.2 and 7.4, respectively. Diethylpyrocarbonate (DEPC), a histidine- specific reagent, reduced mucosal acidification by 58 +/- 4 or 39 +/- 6% when exposure to the drug was at pHo 7.5 or 6.5, respectively. Amiloride (1 mM) did not protect against the DEPC inhibition, but reduced both apical Na+ entry and mucosal acidification by 63 +/- 5 and 65 +/- 9%, respectively. In the Na(+)-depleted tissues mean pHi was 6.7. Cells were alkalinized by exposure to mucosal solutions containing high concentrations of nicotine or methylamine. Estimates of apical Na+ entry at varying pHi, upon increasing [Na+]o from 0 to 25 mM, indicate that Na+/H+ exchange is active at pHi 7.4. Intracellular H+ stimulated apical Na+ entry by titration of more than one site (apparent pK 7.1, Hill coefficient 1.7). The results suggest that external Na+ and H+ interact with one site of the Na+/H+ exchanger and that cytoplasmic H+ acts on at least two sites. The external titratable group

  8. Intracellular pH regulation by HCO3-/Cl- exchange is activated during early mouse zygote development.

    PubMed

    Phillips, K P; Baltz, J M

    1999-04-15

    We report here that at least one major pHi-regulatory mechanism, the HCO3-/Cl- exchanger, is quiescent in unfertilized mouse eggs but becomes fully activated during early development following fertilization. Zygotes (8-12 h postfertilization) exhibited a marked intracellular alkalinization upon external Cl- removal, which is indicative of active HCO3-/Cl- exchangers, in contrast to the very small response observed in eggs. In addition, efflux of Cl- from eggs upon external Cl- removal was much slower than that from zygotes, indicating additional pathways for Cl- to cross the plasma membrane in zygotes. Furthermore, while zygotes quickly recovered from an induced alkalosis, eggs exhibited only a slow, incomplete recovery. Following in vitro fertilization (IVF), increased HCO3-/Cl- exchanger activity was first detectable about 4 h postfertilization and reached the maximal level after about 8 h. The upregulation of HCO3-/Cl- exchanger activity after fertilization appeared to occur by activation of existing, inactive exchangers rather than by synthesis or transport of new exchangers, as the increase in activity following IVF was unaffected by inhibition of protein synthesis or by disruption of the Golgi apparatus or the cytoskeleton. This activation may depend on the Ca2+ transients which follow fertilization, as suppression of these transients, using the Ca2+ chelator BAPTA, reduced subsequent upregulation of HCO3-/Cl- exchanger activity by about 50%. Activation of pHi-regulatory systems may be a widespread feature of the earliest period of embryonic development, not restricted to species such as marine invertebrates as previously believed.

  9. The evolution of Root effect hemoglobins in the absence of intracellular pH protection of the red blood cell: insights from primitive fishes.

    PubMed

    Regan, Matthew D; Brauner, Colin J

    2010-06-01

    The Root effect, a reduction in blood oxygen (O(2)) carrying capacity at low pH, is used by many fish species to maximize O(2) delivery to the eye and swimbladder. It is believed to have evolved in the basal actinopterygian lineage of fishes, species that lack the intracellular pH (pH(i)) protection mechanism of more derived species' red blood cells (i.e., adrenergically activated Na(+)/H(+) exchangers; betaNHE). These basal actinopterygians may consequently experience a reduction in blood O(2) carrying capacity, and thus O(2) uptake at the gills, during hypoxia- and exercise-induced generalized blood acidoses. We analyzed the hemoglobins (Hbs) of seven species within this group [American paddlefish (Polyodon spathula), white sturgeon (Acipenser transmontanus), spotted gar (Lepisosteus oculatus), alligator gar (Atractosteus spatula), bowfin (Amia calva), mooneye (Hiodon tergisus), and pirarucu (Arapaima gigas)] for their Root effect characteristics so as to test the hypothesis of the Root effect onset pH value being lower than those pH values expected during a generalized acidosis in vivo. Analysis of the haemolysates revealed that, although each of the seven species displayed Root effects (ranging from 7.3 to 40.5% desaturation of Hb with O(2), i.e., Hb O(2) desaturation), the Root effect onset pH values of all species are considerably lower (ranging from pH 5.94 to 7.04) than the maximum blood acidoses that would be expected following hypoxia or exercise (pH(i) 7.15-7.3). Thus, although these primitive fishes possess Hbs with large Root effects and lack any significant red blood cell betaNHE activity, it is unlikely that the possession of a Root effect would impair O(2) uptake at the gills following a generalized acidosis of the blood. As well, it was shown that both maximal Root effect and Root effect onset pH values increased significantly in bowfin over those of the more basal species, toward values of similar magnitude to those of most of the more derived

  10. Individual cells of Saccharomyces cerevisiae and Zygosaccharomyces bailii exhibit different short-term intracellular pH responses to acetic acid.

    PubMed

    Arneborg, N; Jespersen, L; Jakobsen, M

    2000-01-01

    The effects of perfusion with 2.7 and 26 mM undissociated acetic acid in the absence or presence of glucose on short-term intracellular pH (pH(i)) changes in individual Saccharormyces cerevisiae and Zygosaccharomyces bailii cells were studied using fluorescence-ratio-imaging microscopy and a perfusion system. In the S. cerevisiae cells, perfusion with acetic acid induced strong short-term pH(i) responses, which were dependent on the undissociated acetic acid concentration and the presence of glucose in the perfusion solutions. In the Z. bailii cells, perfusion with acetic acid induced only very weak short-term pH(i) responses, which were neither dependent on the undissociated acetic acid concentration nor on the presence of glucose in the perfusion solutions. These results clearly show that Z. bailii is more resistant than S. cerevisiae to short-term pH(i) changes caused by acetic acid.

  11. Zn(2+) induces hyperpolarization by activation of a K(+) channel and increases intracellular Ca(2+) and pH in sea urchin spermatozoa.

    PubMed

    Beltrán, Carmen; Rodríguez-Miranda, Esmeralda; Granados-González, Gisela; García de De la Torre, Lucia; Nishigaki, Takuya; Darszon, Alberto

    2014-10-01

    Zinc (Zn(2+)) has been recently recognized as a crucial element for male gamete function in many species although its detailed mechanism of action is poorly understood. In sea urchin spermatozoa, Zn(2+) was reported as an essential trace ion for efficient sperm motility initiation and the acrosome reaction by modulating intracellular pH (pHi). In this study we found that submicromolar concentrations of free Zn(2+) change membrane potential (Em) and increase the concentration of intracellular Ca(2+) ([Ca(2+)]i) and cAMP in Lytechinus pictus sperm. Our results indicate that the Zn(2+) response in sperm of this species mainly involves an Em hyperpolarization caused by K(+) channel activation. The pharmacological profile of the Zn(2+)-induced hyperpolarization indicates that the cGMP-gated K(+) selective channel (tetraKCNG/CNGK), which is crucial for speract signaling, is likely a main target for Zn(2+). Considering that Zn(2+) also induces [Ca(2+)]i fluctuations, our observations suggest that Zn(2+) activates the signaling cascade of speract, except for an increase in cGMP, and facilitates sperm motility initiation upon spawning. These findings provide new insights about the role of Zn(2+) in male gamete function.

  12. Neutral pH hydrogen-enriched electrolyzed water achieves tumor-preferential clonal growth inhibition over normal cells and tumor invasion inhibition concurrently with intracellular oxidant repression.

    PubMed

    Saitoh, Yasukazu; Okayasu, Hajime; Xiao, Li; Harata, Yoshikazu; Miwa, Nobuhiko

    2008-01-01

    The properties and effects of neutral pH hydrogen-enriched electrolyzed water (NHE water) on tumor cells were examined. NHE water diminished hydroxyl radicals as demonstrated by ESR in a cell-free system. Human tongue carcinoma cells HSC-4 were inhibited for either colony formation efficiencies or colony sizes by NHE water without significant inhibition to normal human tongue epithelial-like cells DOK. Furthermore, NHE water caused growth inhibition, cell degeneration, and inhibition of invasion through the reconstituted basement membrane to human fibrosarcoma cells HT-1080. Intracellular oxidants such as hydroperoxides and hydrogen peroxides were scavenged in HSC-4 or HT-1080 cells by NHE water. In the human oral cavity, a dissolved hydrogen concentrations (DH) of NHE water was drastically declined from 1.1 to 0.5 ppm, but settled to 0.3-0.4 ppm until 180 s, upon static holding without gargling. Thus, NHE water was shown to achieve tumor-preferential growth inhibition and tumor invasion together with scavenging of intracellular oxidants, and is expected as a preventive material against tumor progression and invasion.

  13. Encapsulation as a Strategy for the Design of Biological Compartmentalization.

    PubMed

    Giessen, Tobias W; Silver, Pamela A

    2016-02-27

    Compartmentalization is one of the defining features of life. Through intracellular spatial control, cells are able to organize and regulate their metabolism. One of the most broadly used organizational principles in nature is encapsulation. Cellular processes can be encapsulated within either membrane-bound organelles or proteinaceous compartments that create distinct microenvironments optimized for a given task. Further challenges addressed through intracellular compartmentalization are toxic or volatile pathway intermediates, slow turnover rates and competing side reactions. This review highlights a selection of naturally occurring membrane- and protein-based encapsulation systems in microbes and their recent applications and emerging opportunities in synthetic biology. We focus on examples that use engineered cellular organization to control metabolic pathway flux for the production of useful compounds and materials.

  14. Kinetic activity, membrane mitochondrial potential, lipid peroxidation, intracellular pH and calcium of frozen/thawed bovine spermatozoa treated with metabolic enhancers.

    PubMed

    Boni, R; Gallo, A; Cecchini, S

    2017-01-01

    Owing to the progressive decline of sperm motility during storage there is a need to find substances capable of enhancing sperm energy metabolism and motility and/or preserving it from oxidative damage. The aim of this study was to evaluate in frozen/thawed bovine spermatozoa the effect of several compounds, such as myo-inositol, pentoxifylline, penicillamine + hypotaurine + epinephrine mixture (PHE), caffeine and coenzyme Q10+ zinc + d-aspartate mixture (CZA), on either kinetic or metabolic parameters. Sperm kinetics was evaluated by Sperm Class Analyser whereas specific fluorochromes were used to evaluated mitochondrial membrane potential (MMP), intracellular pH, intracellular calcium concentration and lipid peroxidation. Lipid peroxidation was also evaluated by TBARS analysis. Treatments significantly affected total and progressive motility with different dynamics in relation to the incubation time. After the first hour of incubation, CZA treatment produced the best performance in total and progressive sperm motility as well as in curvilinear velocity, average path velocity and amplitude of head displacement, whereas pentoxifylline stimulated the highest straight-line velocity. MMP showed higher values (p < 0.01) after treatment with pentoxifylline and PHE. Intracytoplasmic calcium concentration and lipid peroxidation were significantly (p < 0.05) affected by the incubation time rather than the treatments. Intracellular pH varied significantly (p < 0.01) in relation to either the incubation time or treatments. In particular, it showed a progressive increase throughout incubation with values in control group significantly higher than in myo-inositol, PHE, caffeine, pentoxifylline and CZA groups (7.37 ± 0.03 vs. 7.29 ± 0.03, 7.28 ± 0.03, 7.26 ± 0.03, 7.22 ± 0.03 and 7.00 ± 0.03, respectively; p < 0.01).; however, among treatments, CZA displayed the lowest values. Significant correlations were found between sperm kinetic and metabolic

  15. New applications of pHluorin--measuring intracellular pH of prototrophic yeasts and determining changes in the buffering capacity of strains with affected potassium homeostasis.

    PubMed

    Maresová, Lydie; Hosková, Barbora; Urbánková, Eva; Chaloupka, Roman; Sychrová, Hana

    2010-06-01

    pHluorin is a pH-sensitive variant of green fluorescent protein for measuring intracellular pH (pH(in)) in living cells. We constructed a new pHluorin plasmid with the dominant selection marker KanMX. This plasmid allows pH measurements in cells without auxotrophic mutations and/or grown in chemically indefinite media. We observed differing values of pH(in) for three prototrophic wild-types. The new construct was also used to determine the pH(in) in strains differing in the activity of the plasma membrane Pma1 H(+)-ATPase and the influence of glucose on pH(in). We describe in detail pHluorin measurements performed in a microplate reader, which require much less hands-on time and much lower cell culture volumes compared to standard cuvettes measurements. We also utilized pHluorin in a new method of measuring the buffering capacity of yeast cell cytosol in vivo, shown to be ca. 52 mM/pH for wild-type yeast and moderately decreased in mutants with affected potassium transport.

  16. 31P and 1H MRS of DB-1 Melanoma Xenografts: Lonidamine Selectively Decreases Tumor Intracellular pH and Energy Status and Sensitizes Tumors to Melphalan

    PubMed Central

    Nath, Kavindra; Nelson, David S.; Ho, Andrew; Lee, Seung-Cheol; Darpolor, Moses M.; Pickup, Stephen; Zhou, Rong; Heitjan, Daniel F.; Leeper, Dennis B.; Glickson, Jerry D.

    2012-01-01

    In vivo 31P MRS demonstrates that human melanoma xenografts in immunosuppressed mice treated with lonidamine (LND, 100 mg/kg, i.p.) exhibit a decrease in intracellular pH (pHi) from 6.90 ± 0.05 to 6.33 ± 0.10 (p < 0.001), a slight decrease in extracellular pH (pHe) from 7.00 ± 0.04 to 6.80 ± 0.07 (p > 0.05), and a monotonic decline in bioenergetics (NTP/Pi) by 66.8 ± 5.7% (p < 0.001) relative to the baseline level. Both bioenergetics and pHi decreases were sustained for at least 3 hr following LND treatment. Liver exhibited a transient intracellular acidification by 0.2 ± 0.1 pH units (p > 0.05) at 20 min post-LND with no significant change in pHe and a small transient decrease in bioenergetics, 32.9 ± 10.6 % (p > 0.05), at 40 min post-LND. No changes in pHi or ATP/Pi were detected in the brain (pHi, bioenergetics; p > 0.1) or skeletal muscle (pHi, pHe, bioenergetics; p > 0.1) for at least 120 min post-LND. Steady-state tumor lactate monitored by 1H MRS with a selective multiquantum pulse sequence with Hadamard localization increased ~3-fold (p = 0.009). Treatment with LND increased systemic melanoma response to melphalan (LPAM; 7.5 mg/kg, i.v.) producing a growth delay of 19.9 ± 2.0 d (tumor doubling time = 6.15 ± 0.31d, log10 cell-kill = 0.975 ± 0.110, cell-kill = 89.4 ± 2.2%) compared to LND alone of 1.1 ± 0.1 d and LPAM alone of 4.0 ± 0.0 d. The study demonstrates that the effects of LND on tumor pHi and bioenergetics may sensitize melanoma to pH-dependent therapeutics such as chemotherapy with alkylating agents or hyperthermia. PMID:22745015

  17. Dual role of CO2/HCO3(-) buffer in the regulation of intracellular pH of three-dimensional tumor growths.

    PubMed

    Hulikova, Alzbeta; Vaughan-Jones, Richard D; Swietach, Pawel

    2011-04-22

    Intracellular pH (pH(i)), a major modulator of cell function, is regulated by acid/base transport across membranes. Excess intracellular H(+) ions (e.g. produced by respiration) are extruded by transporters such as Na(+)/H(+) exchange, or neutralized by HCO(3)(-) taken up by carriers such as Na(+)-HCO(3)(-) cotransport. Using fluorescence pH(i) imaging, we show that cancer-derived cell lines (colorectal HCT116 and HT29, breast MDA-MB-468, pancreatic MiaPaca2, and cervical HeLa) extrude acid by H(+) efflux and HCO(3)(-) influx, largely sensitive to dimethylamiloride and 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS), respectively. The magnitude of HCO(3)(-) influx was comparable among the cell lines and may represent a constitutive element of tumor pH(i) regulation. In contrast, H(+) efflux varied considerably (MDA-MB-468 > HCT116 > HT29 > MiaPaca2 > HeLa). When HCO(3)(-) flux was pharmacologically inhibited, acid extrusion in multicellular HT29 and HCT116 spheroids (∼10,000 cells) was highly non-uniform and produced low pH(i) at the core. With depth, acid extrusion became relatively more DIDS-sensitive because the low extracellular pH at the spheroid core inhibits H(+) flux more than HCO(3)(-) flux. HCO(3)(-) flux inhibition also decelerated HCT116 spheroid growth. In the absence of CO(2)/HCO(3)(-), acid extrusion by H(+) flux in HCT116 and MDA-MB-468 spheroids became highly non-uniform and inadequate at the core. This is because H(+) transporters require extracellular mobile pH buffers, such as CO(2)/HCO(3)(-), to overcome low H(+) ion mobility and chaperone H(+) ions away from cells. CO(2)/HCO(3)(-) exerts a dual effect: as substrate for membrane-bound HCO(3)(-) transporters and as a mobile buffer for facilitating extracellular diffusion of H(+) ions extruded from cells. These processes can be augmented by carbonic anhydrase activity. We conclude that CO(2)/HCO(3)(-) is important for maintaining uniformly alkaline pH(i) in small, non-vascularized tumor

  18. Cellular compartmentalization of secondary metabolism

    PubMed Central

    Kistler, H. Corby; Broz, Karen

    2015-01-01

    Fungal secondary metabolism is often considered apart from the essential housekeeping functions of the cell. However, there are clear links between fundamental cellular metabolism and the biochemical pathways leading to secondary metabolite synthesis. Besides utilizing key biochemical precursors shared with the most essential processes of the cell (e.g., amino acids, acetyl CoA, NADPH), enzymes for secondary metabolite synthesis are compartmentalized at conserved subcellular sites that position pathway enzymes to use these common biochemical precursors. Co-compartmentalization of secondary metabolism pathway enzymes also may function to channel precursors, promote pathway efficiency and sequester pathway intermediates and products from the rest of the cell. In this review we discuss the compartmentalization of three well-studied fungal secondary metabolite biosynthetic pathways for penicillin G, aflatoxin and deoxynivalenol, and summarize evidence used to infer subcellular localization. We also discuss how these metabolites potentially are trafficked within the cell and may be exported. PMID:25709603

  19. Effects of angiotensin II on intracellular Ca2+ and pH in isolated beating rabbit hearts and myocytes loaded with the indicator indo-1.

    PubMed Central

    Ikenouchi, H; Barry, W H; Bridge, J H; Weinberg, E O; Apstein, C S; Lorell, B H

    1994-01-01

    1. Angiotensin II increases myocardial contractility in several species, including the rabbit and man. However, it is controversial whether the predominant mechanism is an increase in free cytosolic [Ca2+]i or a change in myofilament Ca2+ sensitivity. To address this question, we infused angiotensin II in isolated perfused rabbit hearts loaded with the Ca2+ indicator indo-1 AM and measured changes in beat-to-beat surface transients of the Ca2+i-sensitive 400:500 nm ratio and left ventricular contractility. The effects of angiotensin II were compared with the response to a Ca(2+)-dependent increase in the inotropic state produced by a change in the perfusate [Ca2+] from 0.9 to 3.6 nM. 2. In the isolated beating heart, an increase in perfusate [Ca2+] caused an increase in left ventricular pressure +dP/dt in association with an increase in peak systolic [Ca2+]i. Angiotensin II perfusion caused a similar increase in left ventricular +dP/dt in the absence of any increase in peak systolic [Ca2+]i. 3. To exclude any contribution of non-myocyte sources of Ca(2+)-sensitive fluorescence which may be present in the intact heart, we also compared the effects of angiotensin II and a change in superfusate [Ca2+] in collagenase-dissociated paced adult rabbit ventricular myocytes loaded with indo-1 AM. In the isolated rabbit myocytes a change in perfusate [Ca2+] from 0.9 to 3.6 mM caused an increase in peak systolic cell shortening coincident with an increase in peak systolic [Ca2+]i. In contrast, angiotensin II caused a similar increase in peak systolic cell shortening whereas there was no increase in peak systolic [Ca2+]i. There was also no change in inward Ca2+ current (ICa) in response to angiotensin II. 4. To investigate further the mechanism of the positive inotropic action of angiotensin II, its effects on intracellular pH were studied in isolated rabbit myocytes loaded with the fluorescent H+ probe SNARF 1. These experiments demonstrated that angiotensin II induced a 0.2 pH

  20. A validation of intracellular pH measurements in fish exposed to hypercarbia: the effect of duration of tissue storage and efficacy of the metabolic inhibitor tissue homogenate method.

    PubMed

    Baker, D W; May, C; Brauner, C J

    2009-07-01

    This study assessed the effect of tissue storage duration and accuracy of the metabolic inhibitor tissue homogenate (MITH) method on intracellular pH (pHi) values of tissues of white sturgeon Acipenser transmontanus during hypercarbia. No effect of storage in liquid nitrogen of up to 30 days was observed and MITH appears appropriate for measurement of pH in fish exposed to up to 10% CO2 (10000 Pa pCO2).

  1. The metabolic interaction of cancer cells and fibroblasts – coupling between NAD(P)H and FAD, intracellular pH and hydrogen peroxide

    PubMed Central

    Druzhkova, Irina N.; Shirmanova, Marina V.; Lukina, Maria M.; Dudenkova, Varvara V.; Mishina, Nataliya M.; Zagaynova, Elena V.

    2016-01-01

    ABSTRACT Alteration in the cellular energy metabolism is a principal feature of tumors. An important role in modifying cancer cell metabolism belongs to the cancer-associated fibroblasts. However, the regulation of their interaction has been poorly studied to date. In this study we monitored the metabolic status of both cell types by using the optical redox ratio and the fluorescence lifetimes of the metabolic co-factors NAD(P)H and FAD, in addition to the intracellular pH and the hydrogen peroxide levels in the cancer cells, using genetically encoded sensors. In the co-culture of human cervical carcinoma cells HeLa and human fibroblasts we observed a metabolic shift from oxidative phosphorylation toward glycolysis in cancer cells, and from glycolysis toward OXPHOS in fibroblasts, starting from Day 2 of co-culturing. The metabolic switch was accompanied by hydrogen peroxide production and slight acidification of the cytosol in the cancer cells in comparison with that of the corresponding monoculture. Therefore, our HeLa-huFb system demonstrated metabolic behavior similar to Warburg type tumors. To our knowledge, this is the first time that these 3 parameters have been investigated together in a model of tumor-stroma co-evolution. We propose that determination of the start-point of the metabolic alterations and understanding of the mechanisms of their realization can open a new ways for cancer treatment. PMID:26986068

  2. Intracellular pH regulation by Na⁺/H⁺ exchanger-1 (NHE1) is required for growth factor-induced mammary branching morphogenesis.

    PubMed

    Jenkins, Edmund C; Debnath, Shawon; Gundry, Stephen; Gundry, Sajini; Uyar, Umit; Fata, Jimmie E

    2012-05-01

    Regulation of intracellular pH (pHi) and protection against cytosolic acidification is primarily a function of the ubiquitous plasma membrane Na+/H+exchanger-1 (NHE1), which uses a highly conserved process to transfer cytosolic hydrogen ions (H+) across plasma membranes in exchange for extracellular sodium ions (Na+). Growth factors, which are essential regulators of morphogenesis, have also been found to be key activators of NHE1 exchanger activity; however, the crosstalk between both has not been fully evaluated during organ development. Here we report that mammary branching morphogenesis induced by transforming growth factor-alpha (TGFα) requires PI3K-dependent NHE1-activation and subsequent pHi alkalization. Inhibiting NHE1 activity after TGFα stimulation with 10 μM of the NHE1-specific inhibitor N-Methyl-N-isobutyl Amiloride (MIA) dramatically disrupted branching morphogenesis, induced extensive proliferation, ectopic expression of the epithelial hyper-proliferative marker Keratin-6 and sustained activation of MAPK. Together these findings indicate a novel developmental signaling cascade involving TGFα>PI3K>NHE1>pHi alkalization, which leads to a permissible environment for MAPK negative feedback inhibition and thus regulated mammary branching morphogenesis.

  3. Different rate-limiting activities of intracellular pH regulators for HCO3(-) secretion stimulated by forskolin and carbachol in rat parotid intralobular ducts.

    PubMed

    Ueno, Kaori; Hirono, Chikara; Kitagawa, Michinori; Shiba, Yoshiki; Sugita, Makoto

    2016-11-01

    Intracellular pH (pHi) regulation fundamentally participates in maintaining HCO3(-) release from HCO3(-)-secreting epithelia. We used parotid intralobular ducts loaded with BCECF to investigate the contributions of a carbonic anhydrase (CA), anion channels and a Na(+)-H(+) exchanger (NHE) to pHi regulation for HCO3(-) secretion by cAMP and Ca(2+) signals. Resting pHi was dispersed between 7.4 and 7.9. Forskolin consistently decreased pHi showing the dominance of pHi-lowering activities, but carbachol gathered pHi around 7.6. CA inhibition suppressed the forskolin-induced decrease in pHi, while it allowed carbachol to consistently increase pHi by revealing that carbachol prominently activated NHE via Ca(2+)-calmodulin. Under NHE inhibition, forskolin and carbachol induced the remarkable decreases in pHi, which were slowed predominantly by CA inhibition and by CA or anion channel inhibition, respectively. Our results suggest that forskolin and carbachol primarily activate the pHi-lowering CA and pHi-raising NHE, respectively, to regulate pHi for HCO3(-) secretion.

  4. Overexpression of the cystic fibrosis transmembrane conductance regulator in NIH 3T3 cells lowers membrane potential and intracellular pH and confers a multidrug resistance phenotype.

    PubMed Central

    Wei, L Y; Stutts, M J; Hoffman, M M; Roepe, P D

    1995-01-01

    Because of the similarities between the cystic fibrosis transmembrane conductance regulator (CFTR) and multidrug resistance (MDR) proteins, recent observations of decreased plasma membrane electrical potential (delta psi) in cells overexpressing either MDR protein or the CFTR, and the effects of delta psi on passive diffusion of chemotherapeutic drugs, we have analyzed chemotherapeutic drug resistance for NIH 3T3 cells overexpressing different levels of functional CFTR. Three separate clones not previously exposed to chemotherapeutic drugs exhibit resistance to doxorubicin, vincristine, and colchicine that is similar to MDR transfectants not previously exposed to chemotherapeutic drugs. Two other clones expressing lower levels of CFTR are less resistant. As shown previously these clones exhibit decreased plasma membrane delta psi similar to MDR transfectants, but four of five exhibit mildly acidified intracellular pH in contrast to MDR transfectants, which are in general alkaline. Thus the MDR protein and CFTR-mediated MDR phenotypes are distinctly different. Selection of two separate CFTR clones on either doxorubicin or vincristine substantially increases the observed MDR and leads to increased CFTR (but not measurable MDR or MRP) mRNA expression. CFTR overexpressors also exhibit a decreased rate of 3H -vinblastine uptake. These data reveal a new and previously unrecognized consequence of CFTR expression, and are consistent with the hypothesis that membrane depolarization is an important determinant of tumor cell MDR. Images FIGURE 1 FIGURE 3 FIGURE 6 PMID:8519988

  5. Elevated intracellular pH appears in aged oocytes and causes oocyte aneuploidy associated with the loss of cohesion in mice.

    PubMed

    Cheng, Jin-Mei; Li, Jian; Tang, Ji-Xin; Chen, Su-Ren; Deng, Shou-Long; Jin, Cheng; Zhang, Yan; Wang, Xiu-Xia; Zhou, Chen-Xi; Liu, Yi-Xun

    2016-09-16

    Increases in the aneuploidy rate caused by the deterioration of cohesion with increasing maternal age have been well documented. However, the molecular mechanism for the loss of cohesion in aged oocytes remains unknown. In this study, we found that intracellular pH (pHi) was elevated in aged oocytes, which might disturb the structure of the cohesin ring to induce aneuploidy. We observed for the first time that full-grown germinal vesicle (GV) oocytes displayed an increase in pHi with advancing age in CD1 mice. Furthermore, during the in vitro oocyte maturation process, the pHi was maintained at a high level, up to ∼7.6, in 12-month-old mice. Normal pHi is necessary to maintain protein localization and function. Thus, we put forward a hypothesis that the elevated oocyte pHi might be related to the loss of cohesion and the increased aneuploidy in aged mice. Through the in vitro alkalinization treatment of young oocytes, we observed that the increased pHi caused an increase in the aneuploidy rate and the sister inter-kinetochore (iKT) distance associated with the strength of cohesion and caused a decline in the cohesin subunit SMC3 protein level. Young oocytes with elevated pHi exhibited substantially the increase in chromosome misalignment.

  6. Heterologous expression of tyrosinase recapitulates the misprocessing and mistrafficking in oculocutaneous albinism type 2: effects of altering intracellular pH and pink-eyed dilution gene expression.

    PubMed

    Ni-Komatsu, Li; Orlow, Seth J

    2006-03-01

    The processing and trafficking of tyrosinase, a melanosomal protein essential for pigmentation, was investigated in a human epithelial 293 cell line that stably expresses the protein. The effects of the pink-eyed dilution (p) gene product, in which mutations result in oculocutaneous albinism type 2 (OCA2), on the processing and trafficking of tyrosinase in this cell line were studied. The majority of tyrosinase was retained in the endoplasmic reticulum-Golgi intermediate compartment and the early Golgi compartment in the 293 cells expressing the protein. Coexpression of p could partially correct the mistrafficking of tyrosinase in 293 cells. Tyrosinase was targeted to the late endosomal and lysosomal compartments after treatment of the cells with compounds that correct the tyrosinase mistrafficking in albino melanocytes, most likely through altering intracellular pH, while the substrate tyrosine had no effect on the processing of tyrosinase. Remarkably, this heterologous expression system recapitulates the defective processing and mistrafficking of tyrosinase observed in OCA2 albino melanocytes and certain amelanotic melanoma cells. Coexpression of other melanosomal proteins in this heterologous system may further aid our understanding of the details of normal and pathologic processing of melanosomal proteins.

  7. Using extracellular action potential recordings to constrain compartmental models.

    PubMed

    Gold, Carl; Henze, Darrell A; Koch, Christof

    2007-08-01

    We investigate the use of extracellular action potential (EAP) recordings for biophysically faithful compartmental models. We ask whether constraining a model to fit the EAP is superior to matching the intracellular action potential (IAP). In agreement with previous studies, we find that the IAP method under-constrains the parameters. As a result, significantly different sets of parameters can have virtually identical IAP's. In contrast, the EAP method results in a much tighter constraint. We find that the distinguishing characteristics of the waveform--but not its amplitude-resulting from the distribution of active conductances are fairly invariant to changes of electrode position and detailed cellular morphology. Based on these results, we conclude that EAP recordings are an excellent source of data for the purpose of constraining compartmental models.

  8. Phosphorylation of ribosomal proteins during meiotic maturation and following activation in starfish oocytes: its relationship with changes of intracellular pH.

    PubMed

    Peaucellier, G; Picard, A; Robert, J J; Capony, J P; Labbe, J C; Doree, M

    1988-01-01

    An increased phosphorylation of ribosomal protein S6 has been shown to be correlated with an increase of intracellular pH (pHi) and with stimulation of protein synthesis in many systems. In this research changes in ribosome phosphorylation following hormone-induced meiotic maturation and fertilization or activation by ionophore A23187 were investigated in starfish oocytes. The hormone was found to stimulate, even in the absence of external Na+, the phosphorylation on serine residues of an Mr 31,000 protein identified as S6, as well as that of an acidic Mr 47,000 protein, presumably S1, on threonine residues. Phosphorylation of ribosomes was an early consequence of hormonal stimulation and did not decrease after completion of meiotic maturation. Fertilization or activation by ionophore of prophase-arrested oocytes also stimulated ribosome phosphorylation. Only S6 was labeled in this case, but to a lesser extent than upon hormone-induced meiotic maturation. Changes in pHi were monitored with ion-specific microelectrodes throughout meiotic maturation and following either fertilization or activation. The pHi did not change before germinal vesicle breakdown (GVBD) following hormone addition, but it increased before first polar body emission. It also increased following fertilization or activation by ionophore or the microinjection of Ca-EGTA. In all cases, alkalinization did not depend on activation of an amiloride-sensitive Na+/H+ exchanger. Microinjection of an alkaline Hepes buffer or external application of ammonia, both of which increased pHi, prevented unfertilized oocytes from arresting after formation of the female pronucleus and induced chromosome cycling. Phosphorylation of S6 was still observed following fertilization or induction of maturation when pHi was decreased by external application of acetate, a treatment which suppressed the emission of polar bodies. Protein synthesis increased in prophase-arrested oocytes after fertilization or activation. It also

  9. Intracellular pH regulation in isolated trout gill mitochondrion-rich (MR) cell subtypes: evidence for Na+/H+ activity.

    PubMed

    Parks, Scott K; Tresguerres, Martin; Galvez, Fernando; Goss, Greg G

    2010-02-01

    We have studied intracellular pH (pH(i)) recovery in isolated trout gill mitochondrion-rich (MR) cells following acidification by the NH(4)Cl pre-pulse technique. Within a mixed MR cell population, one cell type displayed Na(+)-independent pH(i) recovery while the other cell type lacked a Na(+)-independent pH(i) recovery. Cells displaying Na(+) independent recovery exhibited a significantly higher buffering capacity compared to cells lacking Na(+)-independent pH(i) recovery. Cells displaying Na(+) independent recovery were identified as PNA(+) (peanut lectin agluttinin binding) MR cells while those unable to recover were identified as PNA(-) (non-peanut lectin agluttinin binding) MR cells. Therefore, recovery from acidification in the absence of Na(+) provides a direct functional marker for PNA(+) and PNA(-) MR cells. Re-addition of Na(+) to acidified cells resulted in a transient pH(i) recovery in both cell types. This event was abolished by amiloride (500 microM) but it was insensitive to phenamil (50 microM). The phorbol ester PMA (1 microM) potentiated the Na(+) induced pH(i) recovery suggesting that activation by PKC is required for continuous Na(+)/H(+) exchanger activity in trout gill MR cells. This study is the first functional description of pH(i) recovery in lectin-identified trout gill MR cells and provides insight into a putative cellular signaling mechanism that may control pH(i) regulation in the gill epithelium.

  10. The effect of bacteriocin-producing Lactobacillus plantarum strains on the intracellular pH of sessile and planktonic Listeria monocytogenes single cells.

    PubMed

    Nielsen, Dennis S; Cho, Gyu-Sung; Hanak, Alexander; Huch, Melanie; Franz, Charles M A P; Arneborg, Nils

    2010-07-31

    A wide range of lactic acid bacteria (LAB) produce bacteriocins mainly active against other closely related LAB, but some bacteriocins are also active against the food-borne pathogen Listeria monocytogenes. With the aim of increasing food safety it has thus been considered to utilise bacteriocins and/or bacteriocin-producing LAB as "natural" food preservatives in foods such as cheese, meat and ready-to-eat products. Some strains of Lactobacillus plantarum produce bacteriocins termed plantaricins. Using a single-cell based approach, the effect on the intracellular pH as a measure of the physiological state of sessile and planktonic L. monocytogenes (strains EGDe and N53-1) during co-culturing with plantaricin-producing L. plantarum (strains BFE 5092 and PCS 20) was investigated using fluorescence ratio imaging microscopy (FRIM). Mono-cultures of L. monocytogenes were used as control. Expression levels of plantaricin-encoding genes by sessile and planktonic L. plantarum were determined using qRT-PCR. L.plantarum BFE 5092 possesses the genes for plantaricin EF, JK and N, while L. plantarum PCS 20 contains the genes for plantaricin EF, although determination of the nucleotide sequence of the PCS 20 plantaricin E gene showed that this peptide is probably non-functional. When cultured as mono-culture, both L. monocytogenes strains maintained pH(i) at a constant level around 7.2-7.6 throughout the experiment, independently of the matrix. On a solid surface, L. plantarum BFE 5092 strongly affected pH(i) of L. monocytogenes N53-1 with only 20% of the cells being able to maintain pH(i) in the physiological optimal range with pH>7 and 52% of the cells with pH(i) approximately pH(ex,) showing that the cells had no proton gradient towards the environment. The effect on L. monocytogenes EGDe was less pronounced, but still notable. L.plantarum PCS 20 left both strains of L. monocytogenes virtually unaffected when co-cultured on a solid surface. In liquid, both L. plantarum

  11. Myristoylated and non-myristoylated forms of the pH sensor protein hisactophilin II: intracellular shuttling to plasma membrane and nucleus monitored in real time by a fusion with green fluorescent protein.

    PubMed Central

    Hanakam, F; Albrecht, R; Eckerskorn, C; Matzner, M; Gerisch, G

    1996-01-01

    Hisactophilins are myristoylated proteins that are rich in histidine residues and known to exist in Dictyostelium cells in a plasma membrane-bound and a soluble cytoplasmic state. Intracellular translocation of these proteins in response to pH changes was monitored using hisactophilin fusions with green fluorescent protein (GFP) and confocal laser scanning microscopy. Both the normal and a mutated non-myristoylated fusion protein shuffled within the cells in a pH-dependent manner. After lowering the pH, these proteins translocated within minutes between the cytoplasm, the plasma membrane and the nucleus. The role of histidine clusters on the surface of hisactophilin molecules in binding of the proteins to the plasma membrane and in their transfer to the nucleus is discussed on the basis of a pH switch mechanism. Images PMID:8670794

  12. Current Ideas about Prebiological Compartmentalization

    PubMed Central

    Monnard, Pierre-Alain; Walde, Peter

    2015-01-01

    Contemporary biological cells are highly sophisticated dynamic compartment systems which separate an internal volume from the external medium through a boundary, which controls, in complex ways, the exchange of matter and energy between the cell’s interior and the environment. Since such compartmentalization is a fundamental principle of all forms of life, scenarios have been elaborated about the emergence of prebiological compartments on early Earth, in particular about their likely structural characteristics and dynamic features. Chemical systems that consist of potentially prebiological compartments and chemical reaction networks have been designed to model pre-cellular systems. These systems are often referred to as “protocells”. Past and current protocell model systems are presented and compared. Since the prebiotic formation of cell-like compartments is directly linked to the prebiotic availability of compartment building blocks, a few aspects on the likely chemical inventory on the early Earth are also summarized. PMID:25867709

  13. Intracellular pH (pHin) and cytosolic calcium ([Ca2+]cyt) regulation via ATPases: studies in cell populations, single cells, and subcellular compartments

    NASA Astrophysics Data System (ADS)

    Rojas, Jose D.; Sanka, Shankar C.; Gyorke, Sandor; Wesson, Donald E.; Minta, Akwasi; Martinez-Zaguilan, Raul

    1999-07-01

    Changes in pHin and (Ca2+)cyt are important in the signal transduction mechanisms leading to many physiological responses including cell growth, motility, secretion/exocytosis, etc. The concentrations of these ions are regulated via primary and secondary ion transporting mechanisms. In diabetes, specific pH and Ca2+ regulatory mechanism might be altered. To study these ions, we employ fluorescence spectroscopy, and cell imagin spectroscopy/confocal microscopy. pH and Ca2+ indicators are loaded in the cytosol with acetoxymethyl ester forms of dyes, and in endosomal/lysosomal (E/L) compartments by overnight incubation of cells with dextran- conjugated ion fluorescent probes. We focus on specific pH and Ca2+ regulatory systems: plasmalemmal vacuolar- type H+-ATPases (pm V-ATPases) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA). As experimental models, we employ vascular smooth muscle (VSM) and microvascular endothelial cells. We have chosen these cells because they are important in blood flow regulation and in angiogenesis. These processes are altered in diabetes. In many cell types, ion transport processes are dependent on metabolism of glucose for maximal activity. Our main findings are: (a) glycolysis coupling the activity of SERCA is required for cytosolic Ca2+ homeostasis in both VSM and microvascular endothelial cells; (b) E/L compartments are important for pH and Ca2+ regulation via H+-ATPases and SERCA, respectively; and (c) pm-V- ATPases are important for pHin regulation in microvascular endothelial cells.

  14. Modeling the effects of sodium chloride, acetic acid and intracellular pH on the survival of Escherichia coli O157:H7

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microbiological safety has been a critical issue for acid and acidified foods since it became clear that acid-tolerant pathogens such as Escherichia coli O157:H7 can survive (even though they are unable to grow) in a pH range of 3 to 4, which is typical for these classes of food products. The primar...

  15. Compartmentalization of mammalian pantothenate kinases.

    PubMed

    Alfonso-Pecchio, Adolfo; Garcia, Matthew; Leonardi, Roberta; Jackowski, Suzanne

    2012-01-01

    The pantothenate kinases (PanK) catalyze the first and the rate-limiting step in coenzyme A (CoA) biosynthesis and regulate the amount of CoA in tissues by differential isoform expression and allosteric interaction with metabolic ligands. The four human and mouse PanK proteins share a homologous carboxy-terminal catalytic domain, but differ in their amino-termini. These unique termini direct the isoforms to different subcellular compartments. PanK1α isoforms were exclusively nuclear, with preferential association with the granular component of the nucleolus during interphase. PanK1α also associated with the perichromosomal region in condensing chromosomes during mitosis. The PanK1β and PanK3 isoforms were cytosolic, with a portion of PanK1β associated with clathrin-associated vesicles and recycling endosomes. Human PanK2, known to associate with mitochondria, was specifically localized to the intermembrane space. Human PanK2 was also detected in the nucleus, and functional nuclear localization and export signals were identified and experimentally confirmed. Nuclear PanK2 trafficked from the nucleus to the mitochondria, but not in the other direction, and was absent from the nucleus during G2 phase of the cell cycle. The localization of human PanK2 in these two compartments was in sharp contrast to mouse PanK2, which was exclusively cytosolic. These data demonstrate that PanK isoforms are differentially compartmentalized allowing them to sense CoA homeostasis in different cellular compartments and enable interaction with regulatory ligands produced in these same locations.

  16. Compartmentation of photosynthesis in cells and tissues of C(4) plants.

    PubMed

    Edwards, G E; Franceschi, V R; Ku, M S; Voznesenskaya, E V; Pyankov, V I; Andreo, C S

    2001-04-01

    Critical to defining photosynthesis in C(4) plants is understanding the intercellular and intracellular compartmentation of enzymes between mesophyll and bundle sheath cells in the leaf. This includes enzymes of the C(4) cycle (including three subtypes), the C(3) pathway and photorespiration. The current state of knowledge of this compartmentation is a consequence of the development and application of different techniques over the past three decades. Initial studies led to some alternative hypotheses on the mechanism of C(4) photosynthesis, and some controversy over the compartmentation of enzymes. The development of methods for separating mesophyll and bundle sheath cells provided convincing evidence on intercellular compartmentation of the key components of the C(4) pathway. Studies on the intracellular compartmentation of enzymes between organelles and the cytosol were facilitated by the isolation of mesophyll and bundle sheath protoplasts, which can be fractionated gently while maintaining organelle integrity. Now, the ability to determine localization of photosynthetic enzymes conclusively, through in situ immunolocalization by confocal light microscopy and transmission electron microscopy, is providing further insight into the mechanism of C(4) photosynthesis and its evolution. Currently, immunological, ultrastructural and cytochemical studies are revealing relationships between anatomical arrangements and photosynthetic mechanisms which are probably related to environmental factors associated with evolution of these plants. This includes interesting variations in the C(4) syndrome in leaves and cotyledons of species in the tribe Salsoleae of the family Chenopodiaceae, in relation to evolution and ecology. Thus, analysis of structure-function relationships using modern techniques is a very powerful approach to understanding evolution and regulation of the photosynthetic carbon reduction mechanisms.

  17. Positive roles of compartmentalization in internal reactions.

    PubMed

    Ichihashi, Norikazu; Yomo, Tetsuya

    2014-10-01

    Recently, many researchers have attempted to construct artificial cell models using a bottom-up approach in which various biochemical reactions that involve a defined set of molecules are reconstructed in cell-like compartments, such as liposomes and water-in-oil droplets. In many of these studies, the cell-like compartments have acted only as containers for the encapsulated biochemical reactions, whereas other studies have indicated that compartmentalization improves the rates and yields of these reactions. Here, we introduce two ways in which compartmentalization can improve internal reactions: the isolation effect and the condensation effect. These positive effects of compartmentalization might have played an important role in the genesis of the first primitive cell on early Earth.

  18. H⁺-activated Na⁺ influx in the ventricular myocyte couples Ca²⁺-signalling to intracellular pH.

    PubMed

    Garciarena, Carolina D; Youm, Jae Boum; Swietach, Pawel; Vaughan-Jones, Richard D

    2013-08-01

    Acid extrusion on Na(+)-coupled pH-regulatory proteins (pH-transporters), Na(+)/H(+) exchange (NHE1) and Na(+)-HCO3(-) co-transport (NBC), drives Na(+) influx into the ventricular myocyte. This H(+)-activated Na(+)-influx is acutely up-regulated at pHi<7.2, greatly exceeding Na(+)-efflux on the Na(+)/K(+) ATPase. It is spatially heterogeneous, due to the co-localisation of NHE1 protein (the dominant pH-transporter) with gap-junctions at intercalated discs. Overall Na(+)-influx via NBC is considerably lower, but much is co-localised with L-type Ca(2+)-channels in transverse-tubules. Through a functional coupling with Na(+)/Ca(2+) exchange (NCX), H(+)-activated Na(+)-influx increases sarcoplasmic-reticular Ca(2+)-loading and release during intracellular acidosis. This raises Ca(2+)-transient amplitude, rescuing it from direct H(+)-inhibition. Functional coupling is biochemically regulated and linked to membrane receptors, through effects on NHE1 and NBC. It requires adequate cytoplasmic Na(+)-mobility, as NHE1 and NCX are spatially separated (up to 60μm). The relevant functional NCX activity must be close to dyads, as it exerts no effect on bulk diastolic Ca(2+). H(+)-activated Na(+)-influx is up-regulated during ischaemia-reperfusion and some forms of maladaptive hypertrophy and heart failure. It is thus an attractive system for therapeutic manipulation. This article is part of a Special Issue entitled "Na(+) Regulation in Cardiac Myocytes".

  19. Compartmental models for apical efflux by P-glycoprotein. Part 1. Evaluation of model complexity

    PubMed Central

    Nagar, Swati; Tucker, Jalia; Weiskircher, Erica A.; Bhoopathy, Siddhartha; Hidalgo, Ismael J.; Korzekwa, Ken

    2013-01-01

    Purpose With the goal of quantifying P-gp transport kinetics, Part 1 of these manuscripts evaluates different compartmental models and Part 2 applies these models to kinetic data. Methods Models were developed to simulate the effect of apical efflux transporters on intracellular concentrations of six drugs. The effect of experimental variability on model predictions was evaluated. Several models were evaluated, and characteristics including membrane configuration, lipid content, and apical surface area (asa) were varied. Results Passive permeabilities from MDCK-MDR1 cells in the presence of cyclosporine gave lower model errors than from MDCK control cells. Consistent with the results in Part 2, model configuration had little impact on calculated model errors. The 5-compartment model was the simplest model that reproduced experimental lag times. Lipid content and asa had minimal effect on model errors, predicted lag times, and intracellular concentrations. Including endogenous basolateral uptake activity can decrease model errors. Models with and without explicit membrane barriers differed markedly in their predicted intracellular concentrations for basolateral drug exposure. Single point data resulted in clearances similar to time course data. Conclusions Compartmental models are useful to evaluate the impact of efflux transporters on intracellular concentrations. Whereas a 3-compartment model may be sufficient to predict the impact of transporters that efflux drugs from the cell, a 5-compartment model with explicit membranes may be required to predict intracellular concentrations when efflux occurs from the membrane. More complex models including additional compartments may be unnecessary. PMID:24019023

  20. Compartmentalized storage tank for electrochemical cell system

    NASA Technical Reports Server (NTRS)

    Piecuch, Benjamin Michael (Inventor); Dalton, Luke Thomas (Inventor)

    2010-01-01

    A compartmentalized storage tank is disclosed. The compartmentalized storage tank includes a housing, a first fluid storage section disposed within the housing, a second fluid storage section disposed within the housing, the first and second fluid storage sections being separated by a movable divider, and a constant force spring. The constant force spring is disposed between the housing and the movable divider to exert a constant force on the movable divider to cause a pressure P1 in the first fluid storage section to be greater than a pressure P2 in the second fluid storage section, thereby defining a pressure differential.

  1. Compartmentalized Platforms for Neuro-pharmacological Research

    PubMed Central

    Jadhav, Amol D.; Wei, Li; Shi, Peng

    2016-01-01

    Dissociated primary neuronal cell culture remains an indispensable approach for neurobiology research in order to investigate basic mechanisms underlying diverse neuronal functions, drug screening and pharmacological investigation. Compartmentalization, a widely adopted technique since its emergence in 1970s enables spatial segregation of neuronal segments and detailed investigation that is otherwise limited with traditional culture methods. Although these compartmental chambers (e.g. Campenot chamber) have been proven valuable for the investigation of Peripheral Nervous System (PNS) neurons and to some extent within Central Nervous System (CNS) neurons, their utility has remained limited given the arduous manufacturing process, incompatibility with high-resolution optical imaging and limited throughput. The development in the area of microfabrication and microfluidics has enabled creation of next generation compartmentalized devices that are cheap, easy to manufacture, require reduced sample volumes, enable precise control over the cellular microenvironment both spatially as well as temporally, and permit highthroughput testing. In this review we briefly evaluate the various compartmentalization tools used for neurobiological research, and highlight application of the emerging microfluidic platforms towards in vitro single cell neurobiology. PMID:26813122

  2. Compartmentalized Platforms for Neuro-Pharmacological Research.

    PubMed

    Jadhav, Amol D; Wei, Li; Shi, Peng

    2016-01-01

    Dissociated primary neuronal cell culture remains an indispensable approach for neurobiology research in order to investigate basic mechanisms underlying diverse neuronal functions, drug screening and pharmacological investigation. Compartmentalization, a widely adopted technique since its emergence in 1970s enables spatial segregation of neuronal segments and detailed investigation that is otherwise limited with traditional culture methods. Although these compartmental chambers (e.g. Campenot chamber) have been proven valuable for the investigation of Peripheral Nervous System (PNS) neurons and to some extent within Central Nervous System (CNS) neurons, their utility has remained limited given the arduous manufacturing process, incompatibility with high-resolution optical imaging and limited throughput. The development in the area of microfabrication and microfluidics has enabled creation of next generation compartmentalized devices that are cheap, easy to manufacture, require reduced sample volumes, enable precise control over the cellular microenvironment both spatially as well as temporally, and permit highthroughput testing. In this review we briefly evaluate the various compartmentalization tools used for neurobiological research, and highlight application of the emerging microfluidic platforms towards in vitro single cell neurobiology.

  3. COMPARTMENTAL MODEL OF NITRATE RETENTION IN STREAMS

    EPA Science Inventory

    A compartmental modeling approach is presented to route nitrate retention along a cascade of stream reach sections. A process transfer function is used for transient storage equations with first order reaction terms to represent nitrate uptake in the free stream, and denitrifica...

  4. Photoacoustic "nanobombs" fight against undesirable vesicular compartmentalization of anticancer drugs.

    PubMed

    Chen, Aiping; Xu, Chun; Li, Min; Zhang, Hailin; Wang, Diancheng; Xia, Mao; Meng, Gang; Kang, Bin; Chen, Hongyuan; Wei, Jiwu

    2015-10-20

    Undesirable intracellular vesicular compartmentalization of anticancer drugs in cancer cells is a common cause of chemoresistance. Strategies aimed at circumventing this problem may improve chemotherapeutic efficacy. We report a novel photophysical strategy for controlled-disruption of vesicular sequestration of the anticancer drug doxorubicin (DOX). Single-walled carbon nanotubes (SWCNTs), modified with folate, were trapped in acidic vesicles after entering lung cancer cells. Upon irradiation by near-infrared pulsed laser, these vesicles were massively broken by the resulting photoacoustic shockwave, and the vesicle-sequestered contents were released, leading to redistribution of DOX from cytoplasm to the target-containing nucleus. Redistribution resulted in 12-fold decrease of the EC50 of DOX in lung cancer cells, and enhanced antitumor efficacy of low-dose DOX in tumor-bearing mice. Side effects were not observed. These findings provide insights of using nanotechnology to improve cancer chemotherapy, i.e. not only for drug delivery, but also for overcoming intracellular drug-transport hurdles.

  5. Intracellular proteoglycans.

    PubMed Central

    Kolset, Svein Olav; Prydz, Kristian; Pejler, Gunnar

    2004-01-01

    Proteoglycans (PGs) are proteins with glycosaminoglycan chains, are ubiquitously expressed and have a wide range of functions. PGs in the extracellular matrix and on the cell surface have been the subject of extensive structural and functional studies. Less attention has so far been given to PGs located in intracellular compartments, although several reports suggest that these have biological functions in storage granules, the nucleus and other intracellular organelles. The purpose of this review is, therefore, to present some of these studies and to discuss possible functions linked to PGs located in different intracellular compartments. Reference will be made to publications relevant for the topics we present. It is beyond the scope of this review to cover all publications on PGs in intracellular locations. PMID:14759226

  6. Surface interactions with compartmentalized cellular phosphates explain rare earth oxide nanoparticle hazard and provide opportunities for safer design.

    PubMed

    Li, Ruibin; Ji, Zhaoxia; Chang, Chong Hyun; Dunphy, Darren R; Cai, Xiaoming; Meng, Huan; Zhang, Haiyuan; Sun, Bingbing; Wang, Xiang; Dong, Juyao; Lin, Sijie; Wang, Meiying; Liao, Yu-Pei; Brinker, C Jeffrey; Nel, Andre; Xia, Tian

    2014-02-25

    Growing international exploitation of rare earth oxides (REOs) for commercial and biological use has increased the possibility of human exposure and adverse health effects. Occupational exposure to rare earth materials in miners and polishers leads to a severe form of pneumoconiosis, while gadolinium-containing MRI contrast agents cause nephrogenic systemic fibrosis in patients with renal impairment. The mechanisms for inducing these adverse pro-fibrogenic effects are of considerable importance for the safety assessment of REO particles as well as presenting opportunities for safer design. In this study, using a well-prepared REO library, we obtained a mechanistic understanding of how REOs induce cellular and pulmonary damage by a compartmentalized intracellular biotransformation process in lysosomes that results in pro-fibrogenic growth factor production and lung fibrosis. We demonstrate that rare earth oxide ion shedding in acidifying macrophage lysosomes leads to biotic phosphate complexation that results in organelle damage due to stripping of phosphates from the surrounding lipid bilayer. This results in nanoparticle biotransformation into urchin shaped structures and setting in motion a series of events that trigger NLRP3 inflammasome activation, IL-1β release, TGF-β1 and PDGF-AA production. However, pretreatment of REO nanoparticles with phosphate in a neutral pH environment prevents biological transformation and pro-fibrogenic effects. This can be used as a safer design principle for producing rare earth nanoparticles for biological use.

  7. Transport, Compartmentation, and Metabolism of Homoserine in Higher Plant Cells

    PubMed Central

    Aubert, Serge; Curien, Gilles; Bligny, Richard; Gout, Elisabeth; Douce, Roland

    1998-01-01

    The transport, compartmentation, and metabolism of homoserine was characterized in two strains of meristematic higher plant cells, the dicotyledonous sycamore (Acer pseudoplatanus) and the monocotyledonous weed Echinochloa colonum. Homoserine is an intermediate in the synthesis of the aspartate-derived amino acids methionine, threonine (Thr), and isoleucine. Using 13C-nuclear magnetic resonance, we showed that homoserine actively entered the cells via a high-affinity proton-symport carrier (Km approximately 50–60 μm) at the maximum rate of 8 ± 0.5 μmol h−1 g−1 cell wet weight, and in competition with serine or Thr. We could visualize the compartmentation of homoserine, and observed that it accumulated at a concentration 4 to 5 times higher in the cytoplasm than in the large vacuolar compartment. 31P-nuclear magnetic resonance permitted us to analyze the phosphorylation of homoserine. When sycamore cells were incubated with 100 μm homoserine, phosphohomoserine steadily accumulated in the cytoplasmic compartment over 24 h at the constant rate of 0.7 μmol h−1 g−1 cell wet weight, indicating that homoserine kinase was not inhibited in vivo by its product, phosphohomoserine. The rate of metabolism of phosphohomoserine was much lower (0.06 μmol h−1 g−1 cell wet weight) and essentially sustained Thr accumulation. Similarly, homoserine was actively incorporated by E. colonum cells. However, in contrast to what was seen in sycamore cells, large accumulations of Thr were observed, whereas the intracellular concentration of homoserine remained low, and phosphohomoserine did not accumulate. These differences with sycamore cells were attributed to the presence of a higher Thr synthase activity in this strain of monocot cells. PMID:9490758

  8. Intracellular delivery and trafficking dynamics of a lymphoma-targeting antibody-polymer conjugate.

    PubMed

    Berguig, Geoffrey Y; Convertine, Anthony J; Shi, Julie; Palanca-Wessels, Maria Corinna; Duvall, Craig L; Pun, Suzie H; Press, Oliver W; Stayton, Patrick S

    2012-12-03

    Ratiometric fluorescence and cellular fractionation studies were employed to characterize the intracellular trafficking dynamics of antibody-poly(propylacrylic acid) (PPAA) conjugates in CD22+ RAMOS-AW cells. The HD39 monoclonal antibody (mAb) directs CD22-dependent, receptor-mediated uptake in human B-cell lymphoma cells, where it is rapidly trafficked to the lysosomal compartment. To characterize the intracellular-release dynamics of the polymer-mAb conjugates, HD39-streptavidin (HD39/SA) was dual-labeled with pH-insensitive Alexa Fluor 488 and pH-sensitive pHrodo fluorophores. The subcellular pH distribution of the HD39/SA-polymer conjugates was quantified as a function of time by live-cell fluorescence microscopy, and the average intracellular pH value experienced by the conjugates was also characterized as a function of time by flow cytometry. PPAA was shown to alter the intracellular trafficking kinetics strongly relative to HD39/SA alone or HD39/SA conjugates with a control polymer, poly(methacryclic acid) (PMAA). Subcellular trafficking studies revealed that after 6 h, only 11% of the HD39/SA-PPAA conjugates had been trafficked to acidic lysosomal compartments with values at or below pH 5.6. In contrast, the average intracellular pH of HD39/SA alone dropped from 6.7 ± 0.2 at 1 h to 5.6 ± 0.5 after 3 h and 4.7 ± 0.6 after 6 h. Conjugation of the control polymer PMAA to HD39/SA showed an average pH drop similar to that of HD39/SA. Subcellular fractionation studies with tritium-labeled HD39/SA demonstrated that after 6 h, 89% of HD39/SA was associated with endosomes (Rab5+) and lysosomes (Lamp2+), while 45% of HD39/SA-PPAA was translocated to the cytosol (lactate dehydrogenase+). These results demonstrate the endosomal-releasing properties of PPAA with antibody-polymer conjugates and detail their intracellular trafficking dynamics and subcellular compartmental distributions over time.

  9. Unchanged mitochondrial organization and compartmentation of high-energy phosphates in creatine-deficient GAMT−/− mouse hearts

    PubMed Central

    Branovets, Jelena; Sepp, Mervi; Kotlyarova, Svetlana; Jepihhina, Natalja; Sokolova, Niina; Aksentijevic, Dunja; Lygate, Craig A.; Neubauer, Stefan; Birkedal, Rikke

    2013-01-01

    Disruption of the creatine kinase (CK) system in hearts of CK-deficient mice leads to changes in the ultrastructure and regulation of mitochondrial respiration. We expected to see similar changes in creatine-deficient mice, which lack the enzyme guanidinoacetate methyltransferase (GAMT) to produce creatine. The aim of this study was to characterize the changes in cardiomyocyte mitochondrial organization, regulation of respiration, and intracellular compartmentation associated with GAMT deficiency. Three-dimensional mitochondrial organization was assessed by confocal microscopy. On populations of permeabilized cardiomyocytes, we recorded ADP and ATP kinetics of respiration, competition between mitochondria and pyruvate kinase for ADP produced by ATPases, ADP kinetics of endogenous pyruvate kinase, and ATP kinetics of ATPases. These data were analyzed by mathematical models to estimate intracellular compartmentation. Quantitative analysis of morphological and kinetic data as well as derived model fits showed no difference between GAMT-deficient and wild-type mice. We conclude that inactivation of the CK system by GAMT deficiency does not alter mitochondrial organization and intracellular compartmentation in relaxed cardiomyocytes. Thus, our results suggest that the healthy heart is able to preserve cardiac function at a basal level in the absence of CK-facilitated energy transfer without compromising intracellular organization and the regulation of mitochondrial energy homeostasis. This raises questions on the importance of the CK system as a spatial energy buffer in unstressed cardiomyocytes. PMID:23792673

  10. Unchanged mitochondrial organization and compartmentation of high-energy phosphates in creatine-deficient GAMT-/- mouse hearts.

    PubMed

    Branovets, Jelena; Sepp, Mervi; Kotlyarova, Svetlana; Jepihhina, Natalja; Sokolova, Niina; Aksentijevic, Dunja; Lygate, Craig A; Neubauer, Stefan; Vendelin, Marko; Birkedal, Rikke

    2013-08-15

    Disruption of the creatine kinase (CK) system in hearts of CK-deficient mice leads to changes in the ultrastructure and regulation of mitochondrial respiration. We expected to see similar changes in creatine-deficient mice, which lack the enzyme guanidinoacetate methyltransferase (GAMT) to produce creatine. The aim of this study was to characterize the changes in cardiomyocyte mitochondrial organization, regulation of respiration, and intracellular compartmentation associated with GAMT deficiency. Three-dimensional mitochondrial organization was assessed by confocal microscopy. On populations of permeabilized cardiomyocytes, we recorded ADP and ATP kinetics of respiration, competition between mitochondria and pyruvate kinase for ADP produced by ATPases, ADP kinetics of endogenous pyruvate kinase, and ATP kinetics of ATPases. These data were analyzed by mathematical models to estimate intracellular compartmentation. Quantitative analysis of morphological and kinetic data as well as derived model fits showed no difference between GAMT-deficient and wild-type mice. We conclude that inactivation of the CK system by GAMT deficiency does not alter mitochondrial organization and intracellular compartmentation in relaxed cardiomyocytes. Thus, our results suggest that the healthy heart is able to preserve cardiac function at a basal level in the absence of CK-facilitated energy transfer without compromising intracellular organization and the regulation of mitochondrial energy homeostasis. This raises questions on the importance of the CK system as a spatial energy buffer in unstressed cardiomyocytes.

  11. Compartmental modelling for magnetic resonance renography.

    PubMed

    Sourbron, Steven

    2010-01-01

    A basic formalism is presented for generating and interpreting compartmental models for dynamic contrast-enhanced MRI data in the kidney. A graphical convention is introduced to represent and design compartmental models in a transparent and physically intuitive manner. A systematic system of notations and a simple set of rules allows direct translation of the graphical representation into a mathematical solution. The rules are derived from the physical principle of mass conservation, and the interpretation provided by the general tracer-kinetic theory of linear and stationary systems. The power of the formalism is illustrated using examples of models that have been proposed in the literature on perfusion MRI, and by generating a number of advanced models that may be of use in the kidney.

  12. Compartmentalization of proteinases and amylases in Nauphoeta cinerea midgut.

    PubMed

    Elpidina, E N; Vinokurov, K S; Gromenko, V A; Rudenskaya, Y A; Dunaevsky, Y E; Zhuzhikov, D P

    2001-12-01

    Compartmentalization of proteinases, amylases, and pH in the midgut of Nauphoeta cinerea Oliv. (Blattoptera:Blaberidae) was studied in order to understand the organization of protein and starch digestion. Total proteolytic activity measured with azocasein was maximal at pH 11.5 both in anterior (AM) and posterior (PM) halves of the midgut, but the bulk of activity (67%) was found in PM. Total AM and PM preparations were fractionated on a Sephadex G-50 column and further analysed by means of activity electrophoresis and specific inhibitors and activators. The major activity in PM was classified as an unusual SH-dependent proteinase with M(r) 24,000 and pH optimum with synthetic substrate BApNA at 10.0. The enzyme was 43-fold activated in the presence of 1 mM DTT, insensitive to synthetic inhibitors of serine (PMSF, TLCK, TPCK) and cysteine (IAA, E-64) proteinases, strongly inhibited by STI, and displayed four active bands on zymograms. In PM, activities of trypsin-like, chymotrypsin-like, subtilisin-like, and cysteine proteinases were observed. Aspartic and metalloproteinases were not detected. In AM, activity of unusual SH-dependent proteinase also dominated and activity of chymotrypsin-like proteinase was observed, but their levels were much lower than in PM. Distribution of amylase activity, exhibiting an optimum at pH 6.0, was quite the opposite. The major part of it (67%) was located in AM. Treatment of amylase preparation with proteinases from AM and PM reduced amylase activity twofold. pH of the midgut contents was 6.0-7.2 in AM, 6.4-7.6 in the first and 8.8-9.3 in the second halves of PM. Thus, pH in AM is in good agreement with the optimal pH of amylase, located in this compartment, but the activity of proteinases, including the ability to degrade amylase, in such an environment is low. Active proteolysis takes place in the second half of PM, where pH of the gut is close to the optimal pH of proteinases.

  13. Time-dependent activity of Na+/H+ exchanger isoform 1 and homeostasis of intracellular pH in astrocytes exposed to CoCl2 treatment.

    PubMed

    Wang, Peng; Li, Ling; Zhang, Zhenxiang; Kan, Quancheng; Gao, Feng; Chen, Suyan

    2016-05-01

    Hypoxia causes injury to the central nervous system during stroke and has significant effects on pH homeostasis. Na+/H+ exchanger isoform 1 (NHE1) is important in the mechanisms of hypoxia and intracellular pH (pHi) homeostasis. As a well-established hypoxia-mimetic agent, CoCl2 stabilizes and increases the expression of hypoxia inducible factor‑1α (HIF-1α), which regulates several genes involved in pH balance, including NHE1. However, it is not fully understood whether NHE1 is activated in astrocytes under CoCl2 treatment. In the current study, pHi and NHE activity were analyzed using the pHi‑sensitive dye BCECF‑AM. Using cariporide (an NHE1‑specific inhibitor) and EIPA (an NHE nonspecific inhibitor), the current study demonstrated that it was NHE1, not the other NHE isoforms, that was important in regulating pHi homeostasis in astrocytes during CoCl2 treatment. Additionally, the present study observed that, during the early period of CoCl2 treatment (the first 2 h), NHE1 activity and pHi dropped immediately, and NHE1 mRNA expression was reduced compared with control levels, whereas expression levels of the NHE1 protein had not yet changed. In the later period of CoCl2 treatment, NHE1 activity and pHi significantly increased compared with the control levels, as did the mRNA and protein expression levels of NHE1. Furthermore, the cell viability and injury of astrocytes was not changed during the initial 8 h of CoCl2 treatment; their deterioration was associated with the higher levels of pHi and NHE1 activity. The current study concluded that NHE1 activity and pHi homeostasis are regulated by CoCl2 treatment in a time-dependent manner in astrocytes, and may be responsible for the changes in cell viability and injury observed under hypoxia-mimetic conditions induced by CoCl2 treatment.

  14. Phosphodiesterases and subcellular compartmentalized cAMP signaling in the cardiovascular system.

    PubMed

    Stangherlin, Alessandra; Zaccolo, Manuela

    2012-01-01

    Phosphodiesterases are key enzymes in the cAMP signaling cascade. They convert cAMP in its inactive form 5'-AMP and critically regulate the intensity and the duration of cAMP-mediated signals. Multiple isoforms exist that possess different intracellular distributions, different affinities for cAMP, and different catalytic and regulatory properties. This complex repertoire of enzymes provides a multiplicity of ways to modulate cAMP levels, to integrate more signaling pathways, and to respond to the specific needs of the cell within distinct subcellular domains. In this review we summarize key findings on phosphodiesterase compartmentalization in the cardiovascular system.

  15. Compartmentation of cellular tetrahydrofolate pools in rat liver

    SciTech Connect

    Barlowe, C.K.; Appling, D.R.

    1987-05-01

    Folate coenzymes are essential in shuttling one carbon units between a number of cellular processes. Serine, glycine, histidine and formate may serve as one-carbon sources by donation of their one-carbon unit to tetrahydrofolate (THF) which may then be utilized in the biosynthesis of nucleic acids and amino acids or oxidized to carbon dioxide (CO/sub 2/) via formyl-THF dehydrogenase. Nitrous Oxide (N/sub 2/O) has been observed to inactivate methionine synthase causing an increase in the cellular concentration of methyl-THF at the expense of other folate forms, specifically THF. They are interested in determining if THF dependent pathways that utilize serine, formate and histidine are equally effected by N/sub 2/O. Radiometric methods indicate that oxidation of the ..beta..-carbon of serine to CO/sub 2/ is uneffected by N/sub 2/O exposure, but oxidation of formate and the 2-ring carbon of histidine to CO/sub 2/ are reduced by about 50% and 95% of control levels, respectively. In contrast, the use of these one carbon sources for purine biosynthesis are all reduced roughly 50% upon exposure to N/sub 2/O. These data suggest an intracellular compartmentation of THF pools available to different sources of one-carbon units.

  16. Reading the Evolution of Compartmentalization in the Ribosome Assembly Toolbox: The YRG Protein Family.

    PubMed

    Mier, Pablo; Pérez-Pulido, Antonio J; Reynaud, Emmanuel G; Andrade-Navarro, Miguel A

    2017-01-01

    Reconstructing the transition from a single compartment bacterium to a highly compartmentalized eukaryotic cell is one of the most studied problems of evolutionary cell biology. However, timing and details of the establishment of compartmentalization are unclear and difficult to assess. Here, we propose the use of molecular markers specific to cellular compartments to set up a framework to advance the understanding of this complex intracellular process. Specifically, we use a protein family related to ribosome biogenesis, YRG (YlqF related GTPases), whose evolution is linked to the establishment of cellular compartments, leveraging the current genomic data. We analyzed orthologous proteins of the YRG family in a set of 171 proteomes for a total of 370 proteins. We identified ten YRG protein subfamilies that can be associated to six subcellular compartments (nuclear bodies, nucleolus, nucleus, cytosol, mitochondria, and chloroplast), and which were found in archaeal, bacterial and eukaryotic proteomes. Our analysis reveals organism streamlining related events in specific taxonomic groups such as Fungi. We conclude that the YRG family could be used as a compartmentalization marker, which could help to trace the evolutionary path relating cellular compartments with ribosome biogenesis.

  17. Reading the Evolution of Compartmentalization in the Ribosome Assembly Toolbox: The YRG Protein Family

    PubMed Central

    Pérez-Pulido, Antonio J.; Reynaud, Emmanuel G.; Andrade-Navarro, Miguel A.

    2017-01-01

    Reconstructing the transition from a single compartment bacterium to a highly compartmentalized eukaryotic cell is one of the most studied problems of evolutionary cell biology. However, timing and details of the establishment of compartmentalization are unclear and difficult to assess. Here, we propose the use of molecular markers specific to cellular compartments to set up a framework to advance the understanding of this complex intracellular process. Specifically, we use a protein family related to ribosome biogenesis, YRG (YlqF related GTPases), whose evolution is linked to the establishment of cellular compartments, leveraging the current genomic data. We analyzed orthologous proteins of the YRG family in a set of 171 proteomes for a total of 370 proteins. We identified ten YRG protein subfamilies that can be associated to six subcellular compartments (nuclear bodies, nucleolus, nucleus, cytosol, mitochondria, and chloroplast), and which were found in archaeal, bacterial and eukaryotic proteomes. Our analysis reveals organism streamlining related events in specific taxonomic groups such as Fungi. We conclude that the YRG family could be used as a compartmentalization marker, which could help to trace the evolutionary path relating cellular compartments with ribosome biogenesis. PMID:28072865

  18. Ras trafficking, localization and compartmentalized signalling.

    PubMed

    Prior, Ian A; Hancock, John F

    2012-04-01

    Ras proteins are proto-oncogenes that are frequently mutated in human cancers. Three closely related isoforms, HRAS, KRAS and NRAS, are expressed in all cells and have overlapping but distinctive functions. Recent work has revealed how differences between the Ras isoforms in their trafficking, localization and protein-membrane orientation enable signalling specificity to be determined. We review the various strategies used to characterize compartmentalized Ras localization and signalling. Localization is an important contextual modifier of signalling networks and insights from the Ras system are of widespread relevance for researchers interested in signalling initiated from membranes.

  19. Passive Noise Filtering by Cellular Compartmentalization.

    PubMed

    Stoeger, Thomas; Battich, Nico; Pelkmans, Lucas

    2016-03-10

    Chemical reactions contain an inherent element of randomness, which presents itself as noise that interferes with cellular processes and communication. Here we discuss the ability of the spatial partitioning of molecular systems to filter and, thus, remove noise, while preserving regulated and predictable differences between single living cells. In contrast to active noise filtering by network motifs, cellular compartmentalization is highly effective and easily scales to numerous systems without requiring a substantial usage of cellular energy. We will use passive noise filtering by the eukaryotic cell nucleus as an example of how this increases predictability of transcriptional output, with possible implications for the evolution of complex multicellularity.

  20. Reactive oxygen species and redox compartmentalization.

    PubMed

    Kaludercic, Nina; Deshwal, Soni; Di Lisa, Fabio

    2014-01-01

    Reactive oxygen species (ROS) formation and signaling are of major importance and regulate a number of processes in physiological conditions. A disruption in redox status regulation, however, has been associated with numerous pathological conditions. In recent years it has become increasingly clear that oxidative and reductive modifications are confined in a spatio-temporal manner. This makes ROS signaling similar to that of Ca(2+) or other second messengers. Some subcellular compartments are more oxidizing (such as lysosomes or peroxisomes) whereas others are more reducing (mitochondria, nuclei). Moreover, although more reducing, mitochondria are especially susceptible to oxidation, most likely due to the high number of exposed thiols present in that compartment. Recent advances in the development of redox probes allow specific measurement of defined ROS in different cellular compartments in intact living cells or organisms. The availability of these tools now allows simultaneous spatio-temporal measurements and correlation between ROS generation and organelle and/or cellular function. The study of ROS compartmentalization and microdomains will help elucidate their role in physiology and disease. Here we will examine redox probes currently available and how ROS generation may vary between subcellular compartments. Furthermore, we will discuss ROS compartmentalization in physiological and pathological conditions focusing our attention on mitochondria, since their vulnerability to oxidative stress is likely at the basis of several diseases.

  1. Compartmental model of 18F-choline

    NASA Astrophysics Data System (ADS)

    Janzen, T.; Tavola, F.; Giussani, A.; Cantone, M. C.; Uusijärvi, H.; Mattsson, S.; Zankl, M.; Petoussi-Henß, N.; Hoeschen, C.

    2010-03-01

    The MADEIRA Project (Minimizing Activity and Dose with Enhanced Image quality by Radiopharmaceutical Administrations), aims to improve the efficacy and safety of 3D functional imaging by optimizing, among others, the knowledge of the temporal variation of the radiopharmaceuticals' uptake in and clearance from tumor and healthy tissues. With the help of compartmental modeling it is intended to optimize the time schedule for data collection and improve the evaluation of the organ doses to the patients. Administration of 18F-choline to screen for recurrence or the occurrence of metastases in prostate cancer patients is one of the diagnostic applications under consideration in the frame of the project. PET and CT images have been acquired up to four hours after injection of 18F-choline. Additionally blood and urine samples have been collected and measured in a gamma counter. The radioactivity concentration in different organs and data of plasma clearance and elimination into urine were used to set-up a compartmental model of the biokinetics of the radiopharmaceutical. It features a central compartment (blood) exchanging with organs. The structure describes explicitly liver, kidneys, spleen, plasma and bladder as separate units with a forcing function approach. The model is presented together with an evaluation of the individual and population kinetic parameters, and a revised time schedule for data collection is proposed. This optimized time schedule will be validated in a further set of patient studies.

  2. Compartmentalization and molecular traffic in secondary metabolism: a new understanding of established cellular processes

    PubMed Central

    Roze, Ludmila V.; Chanda, Anindya; Linz, John E.

    2010-01-01

    Great progress has been made in understanding the regulation of expression of genes involved in secondary metabolism. Less is known about the mechanisms that govern the spatial distribution of the enzymes, cofactors, and substrates that mediate catalysis of secondary metabolites within the cell. Filamentous fungi in the genus Aspergillus synthesize an array of secondary metabolites and provide useful systems to analyze the mechanisms that mediate the temporal and spatial regulation of secondary metabolism in eukaryotes. For example, aflatoxin biosynthesis in A. parasiticus has been studied intensively because this mycotoxin is highly toxic, mutagenic, and carcinogenic in humans and animals. Using aflatoxin synthesis to illustrate key concepts, this review focuses on the mechanisms by which sub-cellular compartmentalization and intra-cellular molecular traffic contribute to the initiation and completion of secondary metabolism within the cell. We discuss the recent discovery of aflatoxisomes, specialized trafficking vesicles that participate in the compartmentalization of aflatoxin synthesis and export of the toxin to the cell exterior; this work provides a new and clearer understanding of how cells integrate secondary metabolism into basic cellular metabolism via the intracellular trafficking machinery. PMID:20519149

  3. Fractional kinetics in multi-compartmental systems.

    PubMed

    Dokoumetzidis, Aristides; Magin, Richard; Macheras, Panos

    2010-10-01

    Fractional calculus, the branch of calculus dealing with derivatives of non-integer order (e.g., the half-derivative) allows the formulation of fractional differential equations (FDEs), which have recently been applied to pharmacokinetics (PK) for one-compartment models. In this work we extend that theory to multi-compartmental models. Unlike systems defined by a single ordinary differential equation (ODE), considering fractional multi-compartmental models is not as simple as changing the order of the ordinary derivatives of the left-hand side of the ODEs to fractional orders. The latter may produce inconsistent systems which violate mass balance. We present a rationale for fractionalization of ODEs, which produces consistent systems and allows processes of different fractional orders in the same system. We also apply a method of solving such systems based on a numerical inverse Laplace transform algorithm, which we demonstrate that is consistent with analytical solutions when these are available. As examples of our approach, we consider two cases of a basic two-compartment PK model with a single IV dose and multiple oral dosing, where the transfer from the peripheral to the central compartment is of fractional order α < 1, accounting for anomalous kinetics and deep tissue trapping, while all other processes are of the usual order 1. Simulations with the studied systems are performed using the numerical inverse Laplace transform method. It is shown that the presence of a transfer rate of fractional order produces a non-exponential terminal phase, while multiple dose and constant infusion systems never reach steady state and drug accumulation carries on indefinitely. The IV fractional system is also fitted to PK data and parameter values are estimated. In conclusion, our approach allows the formulation of systems of FDEs, mixing different fractional orders, in a consistent manner and also provides a method for the numerical solution of these systems.

  4. Regulation and compartmentalization of β‐lactam biosynthesis

    PubMed Central

    Martín, Juan F.; Ullán, Ricardo V.; García‐Estrada, Carlos

    2010-01-01

    Summary Penicillins and cephalosporins are β‐lactam antibiotics widely used in human medicine. The biosynthesis of these compounds starts by the condensation of the amino acids l‐α‐aminoadipic acid, l‐cysteine and l‐valine to form the tripeptide δ‐l‐α‐aminoadipyl‐l‐cysteinyl‐d‐valine catalysed by the non‐ribosomal peptide ‘ACV synthetase’. Subsequently, this tripeptide is cyclized to isopenicillin N that in Penicillium is converted to hydrophobic penicillins, e.g. benzylpenicillin. In Acremonium and in streptomycetes, isopenicillin N is later isomerized to penicillin N and finally converted to cephalosporin. Expression of genes of the penicillin (pcbAB, pcbC, pendDE) and cephalosporin clusters (pcbAB, pcbC, cefD1, cefD2, cefEF, cefG) is controlled by pleitropic regulators including LaeA, a methylase involved in heterochromatin rearrangement. The enzymes catalysing the last two steps of penicillin biosynthesis (phenylacetyl‐CoA ligase and isopenicillin N acyltransferase) are located in microbodies, as shown by immunoelectron microscopy and microbodies proteome analyses. Similarly, the Acremonium two‐component CefD1–CefD2 epimerization system is also located in microbodies. This compartmentalization implies intracellular transport of isopenicillin N (in the penicillin pathway) or isopenicillin N and penicillin N in the cephalosporin route. Two transporters of the MFS family cefT and cefM are involved in transport of intermediates and/or secretion of cephalosporins. However, there is no known transporter of benzylpenicillin despite its large production in industrial strains. PMID:21255328

  5. Stoichiometry and compartmentation of NADH metabolism in Saccharomyces cerevisiae.

    PubMed

    Bakker, B M; Overkamp, K M; van Maris AJ; Kötter, P; Luttik, M A; van Dijken JP; Pronk, J T

    2001-01-01

    In Saccharomyces cerevisiae, reduction of NAD(+) to NADH occurs in dissimilatory as well as in assimilatory reactions. This review discusses mechanisms for reoxidation of NADH in this yeast, with special emphasis on the metabolic compartmentation that occurs as a consequence of the impermeability of the mitochondrial inner membrane for NADH and NAD(+). At least five mechanisms of NADH reoxidation exist in S. cerevisiae. These are: (1) alcoholic fermentation; (2) glycerol production; (3) respiration of cytosolic NADH via external mitochondrial NADH dehydrogenases; (4) respiration of cytosolic NADH via the glycerol-3-phosphate shuttle; and (5) oxidation of intramitochondrial NADH via a mitochondrial 'internal' NADH dehydrogenase. Furthermore, in vivo evidence indicates that NADH redox equivalents can be shuttled across the mitochondrial inner membrane by an ethanol-acetaldehyde shuttle. Several other redox-shuttle mechanisms might occur in S. cerevisiae, including a malate-oxaloacetate shuttle, a malate-aspartate shuttle and a malate-pyruvate shuttle. Although key enzymes and transporters for these shuttles are present, there is as yet no consistent evidence for their in vivo activity. Activity of several other shuttles, including the malate-citrate and fatty acid shuttles, can be ruled out based on the absence of key enzymes or transporters. Quantitative physiological analysis of defined mutants has been important in identifying several parallel pathways for reoxidation of cytosolic and intramitochondrial NADH. The major challenge that lies ahead is to elucidate the physiological function of parallel pathways for NADH oxidation in wild-type cells, both under steady-state and transient-state conditions. This requires the development of techniques for accurate measurement of intracellular metabolite concentrations in separate metabolic compartments.

  6. Field Testing of Compartmentalization Methods for Multifamily Construction

    SciTech Connect

    Ueno, K.; Lstiburek, J. W.

    2015-03-01

    The 2012 International Energy Conservation Code (IECC) has an airtightness requirement of 3 air changes per hour at 50 Pascals test pressure (3 ACH50) for single-family and multifamily construction (in climate zones 3–8). The Leadership in Energy & Environmental Design certification program and ASHRAE Standard 189 have comparable compartmentalization requirements. ASHRAE Standard 62.2 will soon be responsible for all multifamily ventilation requirements (low rise and high rise); it has an exceptionally stringent compartmentalization requirement. These code and program requirements are driving the need for easier and more effective methods of compartmentalization in multifamily buildings.

  7. Compartmental analysis of dynamic nuclear medicine data: models and identifiability

    NASA Astrophysics Data System (ADS)

    Delbary, Fabrice; Garbarino, Sara; Vivaldi, Valentina

    2016-12-01

    Compartmental models based on tracer mass balance are extensively used in clinical and pre-clinical nuclear medicine in order to obtain quantitative information on tracer metabolism in the biological tissue. This paper is the first of a series of two that deal with the problem of tracer coefficient estimation via compartmental modelling in an inverse problem framework. Specifically, here we discuss the identifiability problem for a general n-dimension compartmental system and provide uniqueness results in the case of two-compartment and three-compartment compartmental models. The second paper will utilize this framework in order to show how nonlinear regularization schemes can be applied to obtain numerical estimates of the tracer coefficients in the case of nuclear medicine data corresponding to brain, liver and kidney physiology.

  8. Calcium dynamics and compartmentalization in leech neurons.

    PubMed

    Andjelic, Sofija; Torre, Vincent

    2005-12-01

    Calcium dynamics in leech neurons were studied using a fast CCD camera. Fluorescence changes (DeltaF/F) of the membrane impermeable calcium indicator Oregon Green were measured. The dye was pressure injected into the soma of neurons under investigation. DeltaF/F caused by a single action potential (AP) in mechanosensory neurons had approximately the same amplitude and time course in the soma and in distal processes. By contrast, in other neurons such as the Anterior Pagoda neuron, the Annulus Erector motoneuron, the L motoneuron, and other motoneurons, APs evoked by passing depolarizing current in the soma produced much larger fluorescence changes in distal processes than in the soma. When APs were evoked by stimulating one distal axon through the root, DeltaF/F was large in all distal processes but very small in the soma. Our results show a clear compartmentalization of calcium dynamics in most leech neurons in which the soma does not give propagating action potentials. In such cells, the soma, while not excitable, can affect information processing by modulating the sites of origin and conduction of AP propagation in distal excitable processes.

  9. Compartmentalization and Transport in Synthetic Vesicles

    PubMed Central

    Schmitt, Christine; Lippert, Anna H.; Bonakdar, Navid; Sandoghdar, Vahid; Voll, Lars M.

    2016-01-01

    Nanoscale vesicles have become a popular tool in life sciences. Besides liposomes that are generated from phospholipids of natural origin, polymersomes fabricated of synthetic block copolymers enjoy increasing popularity, as they represent more versatile membrane building blocks that can be selected based on their specific physicochemical properties, such as permeability, stability, or chemical reactivity. In this review, we focus on the application of simple and nested artificial vesicles in synthetic biology. First, we provide an introduction into the utilization of multicompartmented vesosomes as compartmentalized nanoscale bioreactors. In the bottom-up development of protocells from vesicular nanoreactors, the specific exchange of pathway intermediates across compartment boundaries represents a bottleneck for future studies. To date, most compartmented bioreactors rely on unspecific exchange of substrates and products. This is either based on changes in permeability of the coblock polymer shell by physicochemical triggers or by the incorporation of unspecific porin proteins into the vesicle membrane. Since the incorporation of membrane transport proteins into simple and nested artificial vesicles offers the potential for specific exchange of substances between subcompartments, it opens new vistas in the design of protocells. Therefore, we devote the main part of the review to summarize the technical advances in the use of phospholipids and block copolymers for the reconstitution of membrane proteins. PMID:26973834

  10. Compartmentalized Cytokine Responses in Hidradenitis Suppurativa

    PubMed Central

    Savva, Athina; Kersten, Brigit; Pistiki, Aikaterini; van de Veerdonk, Frank L.; Netea, Mihai G.; van der Meer, Jos W.; Giamarellos-Bourboulis, Evangelos J.

    2015-01-01

    Background Favorable treatment outcomes with TNF blockade led us to explore cytokine responses in hidradenitis suppurativa (HS). Methods Blood monocytes of 120 patients and 24 healthy volunteers were subtyped by flow cytometry. Isolated blood mononuclear cells (PBMCs) were stimulated for cytokine production; this was repeated in 13 severe patients during treatment with etanercept. Cytokines in pus were measured. Results CD14brightCD16dim inflammatory monocytes and patrolling monocytes were increased in Hurley III patients. Cytokine production by stimulated PBMCs was low compared to controls but the cytokine gene copies did not differ, indicating post-translational inhibition. The low production of IL-17 was restored, when cells were incubated with adalimumab. In pus, high concentrations of pro-inflammatory cytokines were detected. Based on the patterns, six different cytokine profiles were discerned, which are potentially relevant for the choice of treatment. Clinical improvement with etanercept was predicted by increased production of IL-1β and IL-17 by PBMCs at week 8. Conclusions Findings indicate compartmentalized cytokine expression in HS; high in pus but suppressed in PBMCs. This is modulated through blockade of TNF. PMID:26091259

  11. Regulation of NAD+ metabolism, signaling and compartmentalization in the yeast Saccharomyces cerevisiae.

    PubMed

    Kato, Michiko; Lin, Su-Ju

    2014-11-01

    Pyridine nucleotides are essential coenzymes in many cellular redox reactions in all living systems. In addition to functioning as a redox carrier, NAD(+) is also a required co-substrate for the conserved sirtuin deacetylases. Sirtuins regulate transcription, genome maintenance and metabolism and function as molecular links between cells and their environment. Maintaining NAD(+) homeostasis is essential for proper cellular function and aberrant NAD(+) metabolism has been implicated in a number of metabolic- and age-associated diseases. Recently, NAD(+) metabolism has been linked to the phosphate-responsive signaling pathway (PHO pathway) in the budding yeast Saccharomyces cerevisiae. Activation of the PHO pathway is associated with the production and mobilization of the NAD(+) metabolite nicotinamide riboside (NR), which is mediated in part by PHO-regulated nucleotidases. Cross-regulation between NAD(+) metabolism and the PHO pathway has also been reported; however, detailed mechanisms remain to be elucidated. The PHO pathway also appears to modulate the activities of common downstream effectors of multiple nutrient-sensing pathways (Ras-PKA, TOR, Sch9/AKT). These signaling pathways were suggested to play a role in calorie restriction-mediated beneficial effects, which have also been linked to Sir2 function and NAD(+) metabolism. Here, we discuss the interactions of these pathways and their potential roles in regulating NAD(+) metabolism. In eukaryotic cells, intracellular compartmentalization facilitates the regulation of enzymatic functions and also concentrates or sequesters specific metabolites. Various NAD(+)-mediated cellular functions such as mitochondrial oxidative phosphorylation are compartmentalized. Therefore, we also discuss several key players functioning in mitochondrial, cytosolic and vacuolar compartmentalization of NAD(+) intermediates, and their potential roles in NAD(+) homeostasis. To date, it remains unclear how NAD(+) and NAD(+) intermediates

  12. Estimating the electrotonic structure of neurons with compartmental models.

    PubMed

    Holmes, W R; Rall, W

    1992-10-01

    1. A procedure based on compartmental modeling called the "constrained inverse computation" was developed for estimating the electrotonic structure of neurons. With the constrained inverse computation, a set of N electrotonic parameters are estimated iteratively with use of a Newton-Raphson algorithm given values of N parameters that can be measured or estimated from experimental data. 2. The constrained inverse computation is illustrated by several applications to the basic example of a neuron represented as one cylinder coupled to a soma. The number of unknown parameters estimated was different (ranging from 2 to 6) when different sets of constraints were chosen. The unknowns were chosen from the following: dendritic membrane resistivity Rmd, soma membrane resistivity Rms, intracellular resistivity Ri, membrane capacity Cm, dendritic membrane area AD, soma membrane area As, electrotonic length L, and resistivity-free length, rfl (rfl = 2l/d1/2 where l and d are length and diameter of the cylinder). The values of the unknown parameters were estimated from the values of an equal number of known parameters, which were chosen from the following: the time constants and coefficients of a voltage transient tau 0, tau 1, ..., C0, C1, ..., voltage-clamp time constants tau vc1, tau vc2, ..., and input resistance RN. Note that initially, morphological data were treated as unknown, rather than known. 3. When complete morphology was not known, parameters from voltage and current transients, combined with the input resistance were not sufficient to completely specify the electrotonic structure of the neuron. For a neuron represented as a cylinder coupled to a soma, there were an infinite number of combinations of Rmd, Rms, Ri, Cm, AS, AD, and L that could be fitted to the same voltage and current transients and input resistance. 4. One reason for the nonuniqueness when complete morphology was not specified is that the Ri estimate is intrinsically bound to the morphology. Ri

  13. Towards repurposing the yeast peroxisome for compartmentalizing heterologous metabolic pathways.

    PubMed

    DeLoache, William C; Russ, Zachary N; Dueber, John E

    2016-03-30

    Compartmentalization of enzymes into organelles is a promising strategy for limiting metabolic crosstalk and improving pathway efficiency, but improved tools and design rules are needed to make this strategy available to more engineered pathways. Here we focus on the Saccharomyces cerevisiae peroxisome and develop a sensitive high-throughput assay for peroxisomal cargo import. We identify an enhanced peroxisomal targeting signal type 1 (PTS1) for rapidly sequestering non-native cargo proteins. Additionally, we perform the first systematic in vivo measurements of nonspecific metabolite permeability across the peroxisomal membrane using a polymer exclusion assay. Finally, we apply these new insights to compartmentalize a two-enzyme pathway in the peroxisome and characterize the expression regimes where compartmentalization leads to improved product titre. This work builds a foundation for using the peroxisome as a synthetic organelle, highlighting both promise and future challenges on the way to realizing this goal.

  14. Compartmentalization of Incompatible Catalytic Transformations for Tandem Catalysis.

    PubMed

    Lu, Jie; Dimroth, Jonas; Weck, Marcus

    2015-10-14

    In Nature, incompatible catalytic transformations are being carried out simultaneously through compartmentalization that allows for the combination of incompatible catalysts in tandem reactions. Herein, we take the compartmentalization concept to the synthetic realm and present an approach that allows two incompatible transition metal catalyzed transformations to proceed in one pot in tandem. The key is the site isolation of both catalysts through compartmentalization using a core-shell micellar support in an aqueous environment. The support is based on amphiphilic triblock copolymers of poly(2-oxazoline)s with orthogonal functional groups on the side chain that can be used to cross-link covalently the micelle and to conjugate two metal catalysts in different domains of the micelle. The micelle core and shell provide different microenvironments for the transformations: Co-catalyzed hydration of an alkyne proceeds in the hydrophobic core, while the Rh-catalyzed asymmetric transfer hydrogenation of the intermediate ketone into a chiral alcohol occurs in the hydrophilic shell.

  15. Towards repurposing the yeast peroxisome for compartmentalizing heterologous metabolic pathways

    SciTech Connect

    DeLoache, William C.; Russ, Zachary N.; Dueber, John E.

    2016-03-30

    Compartmentalization of enzymes into organelles is a promising strategy for limiting metabolic crosstalk and improving pathway efficiency, but improved tools and design rules are needed to make this strategy available to more engineered pathways. Here we focus on the Saccharomyces cerevisiae peroxisome and develop a sensitive high-throughput assay for peroxisomal cargo import. We identify an enhanced peroxisomal targeting signal type 1 (PTS1) for rapidly sequestering non-native cargo proteins. Additionally, we perform the first systematic in vivo measurements of nonspecific metabolite permeability across the peroxisomal membrane using a polymer exclusion assay. Finally, we apply these new insights to compartmentalize a two-enzyme pathway in the peroxisome and characterize the expression regimes where compartmentalization leads to improved product titre. Lastly, this work builds a foundation for using the peroxisome as a synthetic organelle, highlighting both promise and future challenges on the way to realizing this goal.

  16. Towards repurposing the yeast peroxisome for compartmentalizing heterologous metabolic pathways

    DOE PAGES

    DeLoache, William C.; Russ, Zachary N.; Dueber, John E.

    2016-03-30

    Compartmentalization of enzymes into organelles is a promising strategy for limiting metabolic crosstalk and improving pathway efficiency, but improved tools and design rules are needed to make this strategy available to more engineered pathways. Here we focus on the Saccharomyces cerevisiae peroxisome and develop a sensitive high-throughput assay for peroxisomal cargo import. We identify an enhanced peroxisomal targeting signal type 1 (PTS1) for rapidly sequestering non-native cargo proteins. Additionally, we perform the first systematic in vivo measurements of nonspecific metabolite permeability across the peroxisomal membrane using a polymer exclusion assay. Finally, we apply these new insights to compartmentalize a two-enzymemore » pathway in the peroxisome and characterize the expression regimes where compartmentalization leads to improved product titre. Lastly, this work builds a foundation for using the peroxisome as a synthetic organelle, highlighting both promise and future challenges on the way to realizing this goal.« less

  17. Phosphorylation-mediated RNA/peptide complex coacervation as a model for intracellular liquid organelles

    NASA Astrophysics Data System (ADS)

    Aumiller, William M.; Keating, Christine D.

    2016-02-01

    Biological cells are highly organized, with numerous subcellular compartments. Phosphorylation has been hypothesized as a means to control the assembly/disassembly of liquid-like RNA- and protein-rich intracellular bodies, or liquid organelles, that lack delimiting membranes. Here, we demonstrate that charge-mediated phase separation, or complex coacervation, of RNAs with cationic peptides can generate simple model liquid organelles capable of reversibly compartmentalizing biomolecules. Formation and dissolution of these liquid bodies was controlled by changes in peptide phosphorylation state using a kinase/phosphatase enzyme pair. The droplet-generating phase transition responded to modification of even a single serine residue. Electrostatic interactions between the short cationic peptides and the much longer polyanionic RNAs drove phase separation. Coacervates were also formed on silica beads, a primitive model for localization at specific intracellular sites. This work supports phosphoregulation of complex coacervation as a viable mechanism for dynamic intracellular compartmentalization in membraneless organelles.

  18. A compartmentalized solute transport model for redox zones in contaminated aquifers--2, Field-scale simulations

    USGS Publications Warehouse

    Abrams , Robert H.; Loague, Keith

    2000-01-01

    This paper, the second of two parts [see Abrams and Loague, this issue], reports the field-scale application of COMPTRAN (compartmentalized solute transport model) for simulating the development of redox zones. COMPTRAN is fully developed and described in the companion paper. Redox zones, which are often delineated by the relative concentrations of dissolved oxygen, have been observed around the globe. The distribution of other redox-sensitive species is affected by redox zonation. At the U.S. Geological Survey's Cape Cod research site, an anoxic zone containing high concentrations of dissolved iron has been observed. Field data were abstracted from the Cape Cod site for the one-dimensional and two-dimensional COMPTRAN simulations reported in this paper. The purpose of the concept-development simulations was to demonstrate that the compartmentalized approach reported by Abrams et al. [1998] can be linked with a solute transport model to simulate field-scale phenomena. The results presented in this paper show that COMPTRAN successfully simulated the development of redox zones at the field scale, including trends in pH and alkalinity. Thermodynamic constraints were used to prevent lower-energy redox reactions from occurring under infeasible geochemical conditions without imposing equilibrium among all redox species. Empirical methods of reaction inhibition were not needed for the simulations conducted for this study. COMPTRAN can be extended easily to include additional compartments and reactions and is capable of handling complex velocity fields in more than one dimension.

  19. A compartmentalized solute transport model for redox zones in contaminated aquifers: 2. Field-scale simulations

    NASA Astrophysics Data System (ADS)

    Abrams, Robert H.; Loague, Keith

    2000-08-01

    This paper, the second of two parts [see Abrams and Loague, this issue], reports the field-scale application of COMPTRAN (compartmentalized solute transport model) for simulating the development of redox zones. COMPTRAN is fully developed and described in the companion paper. Redox zones, which are often delineated by the relative concentrations of dissolved oxygen, have been observed around the globe. The distribution of other redox-sensitive species is affected by redox zonation. At the U.S. Geological Survey's Cape Cod research site, an anoxic zone containing high concentrations of dissolved iron has been observed. Field data were abstracted from the Cape Cod site for the one-dimensional and two-dimensional COMPTRAN simulations reported in this paper. The purpose of the concept-development simulations was to demonstrate that the compartmentalized approach reported by Abrams et al. [1998] can be linked with a solute transport model to simulate field-scale phenomena. The results presented in this paper show that COMPTRAN successfully simulated the development of redox zones at the field scale, including trends in pH and alkalinity. Thermodynamic constraints were used to prevent lower-energy redox reactions from occurring under infeasible geochemical conditions without imposing equilibrium among all redox species. Empirical methods of reaction inhibition were not needed for the simulations conducted for this study. COMPTRAN can be extended easily to include additional compartments and reactions and is capable of handling complex velocity fields in more than one dimension.

  20. The role of carbon dioxide (and intracellular pH) in the pathomechanism of several mental disorders. Are the diseases of civilization caused by learnt behaviour, not the stress itself?

    PubMed

    Sikter, András; Faludi, Gábor; Rihmer, Zoltán

    2009-09-01

    The role of carbon dioxide (CO2) is underestimated in the pathomechanism of neuropsychiatric disorders, though it is an important link between psyche and corpus. The actual spiritual status also influences respiration (we start breathing rarely, frequently, irregularly, etc.) causing pH alteration in the organism; on the other hand the actual cytosolic pH of neurons is one of the main modifiers of Ca2+-conductance, hence breathing directly, quickly, and effectively influences the second messenger system through Ca2+-currents. (Decreasing pCO2 turns pH into alkalic direction, augments psychic arousal, while increasing pCO2 turns pH acidic, diminishes arousal.) One of the most important homeostatic function is to maintain or restore the permanence of H+-concentration, hence the alteration of CO2 level starts cascades of contraregulation. However it can be proved that there is no perfect compensation, therefore compensational mechanisms may generate psychosomatic disorders causing secondary alterations in the "milieu interieur". Authors discuss the special physico-chemical features of CO2, the laws of interweaving alterations of pCO2 and catecholamine levels (their feedback mechanism), the role of acute and chronic hypocapnia in several hyperarousal disorders (delirium, panic disorder, hyperventilation syndrome, generalized anxiety disorder, bipolar disorder), the role of "locus minoris resistentiae" in the pathomechanism of psychosomatic disorders. It is supposed that the diseases of civilization are caused not by the stress itself but the lack of human instinctive reaction to it, and this would cause long-lasting CO2 alteration. Increased brain-pCO2, acidic cytosol pH and/or increased basal cytosolic Ca2+ level diminish inward Ca2+-current into cytosol, decrease arousal--they may cause dysthymia or depression. This state usually co-exists with ATP-deficiency and decreased cytosolic Mg2+ content. This energetical- and ion-constellation is also typical of ageing

  1. Constraining compartmental models using multiple voltage recordings and genetic algorithms.

    PubMed

    Keren, Naomi; Peled, Noam; Korngreen, Alon

    2005-12-01

    Compartmental models with many nonlinearly and nonhomogeneous distributions of voltage-gated conductances are routinely used to investigate the physiology of complex neurons. However, the number of loosely constrained parameters makes manually constructing the desired model a daunting if not impossible task. Recently, progress has been made using automated parameter search methods, such as genetic algorithms (GAs). However, these methods have been applied to somatically recorded action potentials using relatively simple target functions. Using a genetic minimization algorithm and a reduced compartmental model based on a previously published model of layer 5 neocortical pyramidal neurons we compared the efficacy of five cost functions (based on the waveform of the membrane potential, the interspike interval, trajectory density, and their combinations) to constrain the model. When the model was constrained using somatic recordings only, a combined cost function was found to be the most effective. This combined cost function was then applied to investigate the contribution of dendritic and axonal recordings to the ability of the GA to constrain the model. The more recording locations from the dendrite and the axon that were added to the data set the better was the genetic minimization algorithm able to constrain the compartmental model. Based on these simulations we propose an experimental scheme that, in combination with a genetic minimization algorithm, may be used to constrain compartmental models of neurons.

  2. Shared and Distinct Mechanisms of Compartmentalized and Cytosolic Ciliogenesis.

    PubMed

    Avidor-Reiss, Tomer; Leroux, Michel R

    2015-12-07

    Most motile and all non-motile (also known as primary) eukaryotic cilia possess microtubule-based axonemes that are assembled at the cell surface to form hair-like or more elaborate compartments endowed with motility and/or signaling functions. Such compartmentalized ciliogenesis depends on the core intraflagellar transport (IFT) machinery and the associated Bardet-Biedl syndrome complex (BBSome) for dynamic delivery of ciliary components. The transition zone (TZ), an ultrastructurally complex barrier or 'gate' at the base of cilia, also contributes to the formation of compartmentalized cilia. Yet, some ciliated protists do not have IFT components and, like some metazoan spermatozoa, use IFT-independent mechanisms to build axonemes exposed to the cytosol. Moreover, various ciliated protists lack TZ components, whereas Drosophila sperm surprisingly requires the activity of dynamically localized TZ proteins for cytosolic ciliogenesis. Here, we discuss the various ways eukaryotes use IFT and/or TZ proteins to generate the wide assortment of compartmentalized and cytosolic cilia observed in nature. Consideration of the different ciliogenesis pathways allows us to propose how three types of cytosol-exposed cilia (primary, secondary and tertiary), including cilia found in the human sperm proximal segment, are likely generated by evolutionary derivations of compartmentalized ciliogenesis.

  3. Compartmental models for continuous flow reactors derived from CFD simulations.

    PubMed

    Gresch, Markus; Brügger, Raphael; Meyer, Alain; Gujer, Willi

    2009-04-01

    Reactor modeling is of major interest in environmental technology. In this context, new contaminants with higher degradation requirements increase the importance of reactor hydraulics. CFD (Computational Fluid Dynamics) may meet this challenge but is expensive for everyday use. In this paper, we provide research and practice with a methodology designed to automatically reduce the complexity of such a high-dimensional flow model to a compartmental model. The derivation is based on the concentration field of a reacting species which is included in the steady state CFD simulation. While still capturing the most important flow features, the compartmental model is fast, easy to use, and open for process modeling with yet unknown compounds. The inherent overestimation of diffusion by compartmental models has been corrected by locally adjusting turbulent fluxes. We successfully applied the methodology to the ozonation process and experimentally verified it with tracer experiments. The loss of information was quantified as a deviation from CFD performance prediction for different reactions. With increasing discretisation of the compartmental model, these deviations diminish. General advice on the necessary discretisation is given.

  4. Kinetic compartmental analysis of carnitine metabolism in the dog

    SciTech Connect

    Rebouche, C.J.; Engel, A.G.

    1983-01-01

    This study was undertaken to quantitate the dynamic parameters of carnitine metabolism in the dog. Six mongrel dogs were given intravenous injections of L-(methyl-3H)carnitine and the specific radioactivity of carnitine was followed in plasma and urine for 19-28 days. The data were analyzed by kinetic compartmental analysis. A three-compartment, open-system model ((a) extracellular fluid, (b) cardiac and skeletal muscle, (c) other tissues, particularly liver and kidney) was adopted and kinetic parameters (carnitine flux, pool sizes, kinetic constants) were derived. In four of six dogs the size of the muscle carnitine pool obtained by kinetic compartmental analysis agreed (+/- 5%) with estimates based on measurement of carnitine concentrations in different muscles. In three of six dogs carnitine excretion rates derived from kinetic compartmental analysis agreed (+/- 9%) with experimentally measured values, but in three dogs the rates by kinetic compartmental analysis were significantly higher than the corresponding rates measured directly. Appropriate chromatographic analyses revealed no radioactive metabolites in muscle or urine of any of the dogs. Turnover times for carnitine were (mean +/- SEM): 0.44 +/- 0.05 h for extracellular fluid, 232 +/- 22 h for muscle, and 7.9 +/- 1.1 h for other tissues. The estimated flux of carnitine in muscle was 210 pmol/min/g of tissue. Whole-body turnover time for carnitine was 62.9 +/- 5.6 days (mean +/- SEM). Estimated carnitine biosynthesis ranged from 2.9 to 28 mumol/kg body wt/day. Results of this study indicate that kinetic compartmental analysis may be applicable to study of human carnitine metabolism.

  5. Compartmentalization of Gd liposomes: the quenching effect explained.

    PubMed

    Guenoun, Jamal; Doeswijk, Gabriela N; Krestin, Gabriel P; Bernsen, Monique R

    2016-01-01

    Cationic liposomes carrying high [Gd] can be used as efficient cell-labeling agents. In a compartmentalized state, Gd can cause signal loss (relaxivity quenching). The contributions of liposomal [Gd], size and compartmentalization state to relaxivity quenching were assessed. The dependency of signal intensity (SI) on intraliposomal [Gd] was assessed comparing three different [Gd] (0.3, 0.6 and 1.0 M Gd) in both small (80 nm) and large (120 nm) cationic liposomes. In addition, five compartmentalization states were compared: free Gd, intact Gd liposomes, ruptured Gd liposomes, Gd liposomes in intact cells and Gd liposomes in ruptured cells (simulating cell death). Gd also causes R2 effects, which is often overlooked. Therefore, both R1 and R2 relaxation rates of a dilution range were measured by T1 and T2 mapping on a 7 T clinical scanner. Less is more. As the unidirectional water efflux rate (outbound across the liposome membrane, κle) is proportional to the surface:volume ratio, smaller liposomes yielded a consistently higher R1 than larger liposomes. For equal voxel [Gd] less concentrated liposomes (0.3 M Gd) yielded higher R1/R2 ratio because of the higher extraliposomal water fraction (vl ). Gd exhibits a dualistic behavior: from hypointensity to hyperintensity to hypointensity, with decreasing [Gd]. Regarding compartmentalization, fewer membrane barriers means a higher R1 /R2 ratio. Gd liposomes exhibit a versatile contrast behavior, dependent on the compartmentalization state, liposomal size, intraliposomal [Gd] and liposome number. Both R1 and R2 effects contribute to this. The versatility allows one to tailor the optimal liposomal formulation to desired goals in cell labeling and tracking.

  6. Alkaline pH sensor molecules.

    PubMed

    Murayama, Takashi; Maruyama, Ichiro N

    2015-11-01

    Animals can survive only within a narrow pH range. This requires continual monitoring of environmental and body-fluid pH. Although a variety of acidic pH sensor molecules have been reported, alkaline pH sensor function is not well understood. This Review describes neuronal alkaline pH sensors, grouped according to whether they monitor extracellular or intracellular alkaline pH. Extracellular sensors include the receptor-type guanylyl cyclase, the insulin receptor-related receptor, ligand-gated Cl- channels, connexin hemichannels, two-pore-domain K+ channels, and transient receptor potential (TRP) channels. Intracellular sensors include TRP channels and gap junction channels. Identification of molecular mechanisms underlying alkaline pH sensing is crucial for understanding how animals respond to environmental alkaline pH and how body-fluid pH is maintained within a narrow range.

  7. Compartmentation of fluorescent tracers injected into the epidermal cells of Egeria densa leaves.

    PubMed

    Goodwin, P B; Shepherd, V; Erwee, M G

    1990-04-01

    We have compared the movement of a series of fluorescent tracers of increasing molecular weight injected into the cytoplasm in the epidermal cells of leaves of Egeria densa Planch. In general, the tracers showed major movement into three cellular compartments: first, to the cytoplasm of adjacent cells; secondly, from the cytoplasm, to the vacuole (irreversible); and thirdly, from the cytoplasm to the nucleus (reversible). No visible accumulation in chloroplasts or mitochondria, or loss across the plasmalemma was observed. No evidence for metabolic breakdown was found in extracts from injected leaves. The time course of accumulation of the dye in the three major compartments (cytoplasm, nucleus, vacuole) was monitored using fluorescence microscopy. The rate measurements and the quantified geometry of the cells were used to generate a model of compartmentation during intercellular transport. Permeability coefficients were calculated and related to the molecular sizes of the tracers. The coefficients for the tonoplast and nuclear envelope were independent of the molecular sizes of the tracers, and were in the range 2.4·10(-6)-4.1· 10(-6) cm·s(-1) for the tonoplast, and 2.6·10(-5)-9.4.10(-5) cm· s(-1) for the nuclear envelope. For intercellular movement, permeabilities were strongly dependent on molecular size, and ranged from 1.1·10(-4) cm·s(-1) for 6-carboxyfluorescein (376 daltons (Da)) to 9·10(-9) cm·s(-1) for fluorescein leucyldiglutamylleucine (874 Da). Thus, the differences in cell-to-cell movement of these tracers are based upon their differing ability to cross the intercellular walls, not upon differences in their intracellular compartmentation.

  8. Compartmentalized PDE4A5 Signaling Impairs Hippocampal Synaptic Plasticity and Long-Term Memory

    PubMed Central

    Park, Alan J.; Tolentino, Rosa E.; Bruinenberg, Vibeke M.; Tudor, Jennifer C.; Lee, Yool; Hansen, Rolf T.; Guercio, Leonardo A.; Linton, Edward; Neves-Zaph, Susana R.; Meerlo, Peter; Baillie, George S.; Houslay, Miles D.

    2016-01-01

    Alterations in cAMP signaling are thought to contribute to neurocognitive and neuropsychiatric disorders. Members of the cAMP-specific phosphodiesterase 4 (PDE4) family, which contains >25 different isoforms, play a key role in determining spatial cAMP degradation so as to orchestrate compartmentalized cAMP signaling in cells. Each isoform binds to a different set of protein complexes through its unique N-terminal domain, thereby leading to targeted degradation of cAMP in specific intracellular compartments. However, the functional role of specific compartmentalized PDE4 isoforms has not been examined in vivo. Here, we show that increasing protein levels of the PDE4A5 isoform in mouse hippocampal excitatory neurons impairs a long-lasting form of hippocampal synaptic plasticity and attenuates hippocampus-dependent long-term memories without affecting anxiety. In contrast, viral expression of a truncated version of PDE4A5, which lacks the unique N-terminal targeting domain, does not affect long-term memory. Further, overexpression of the PDE4A1 isoform, which targets a different subset of signalosomes, leaves memory undisturbed. Fluorescence resonance energy transfer sensor-based cAMP measurements reveal that the full-length PDE4A5, in contrast to the truncated form, hampers forskolin-mediated increases in neuronal cAMP levels. Our study indicates that the unique N-terminal localization domain of PDE4A5 is essential for the targeting of specific cAMP-dependent signaling underlying synaptic plasticity and memory. The development of compounds to disrupt the compartmentalization of individual PDE4 isoforms by targeting their unique N-terminal domains may provide a fruitful approach to prevent cognitive deficits in neuropsychiatric and neurocognitive disorders that are associated with alterations in cAMP signaling. SIGNIFICANCE STATEMENT Neurons exhibit localized signaling processes that enable biochemical cascades to be activated selectively in specific subcellular

  9. Fractional two-compartmental model for articaine serum levels

    NASA Astrophysics Data System (ADS)

    Petronijevic, Branislava; Sarcev, Ivan; Zorica, Dusan; Janev, Marko; Atanackovic, Teodor M.

    2016-06-01

    Two fractional two-compartmental models are applied to the pharmacokinetics of articaine. Integer order derivatives are replaced by fractional derivatives, either of different, or of same orders. Models are formulated so that the mass balance is preserved. Explicit forms of the solutions are obtained in terms of the Mittag-Leffler functions. Pharmacokinetic parameters are determined by the use of the evolutionary algorithm and trust regions optimization to recover the experimental data.

  10. Digital PCR on an integrated self-priming compartmentalization chip.

    PubMed

    Zhu, Qiangyuan; Qiu, Lin; Yu, Bingwen; Xu, Yanan; Gao, Yibo; Pan, Tingting; Tian, Qingchang; Song, Qi; Jin, Wei; Jin, Qinhan; Mu, Ying

    2014-03-21

    An integrated on-chip valve-free and power-free microfluidic digital PCR device is for the first time developed by making use of a novel self-priming compartmentalization and simple dehydration control to realize 'divide and conquer' for single DNA molecule detection. The high gas solubility of PDMS is exploited to provide the built-in power of self-priming so that the sample and oil are sequentially sucked into the device to realize sample self-compartmentalization based on surface tension. The lifespan of its self-priming capability was about two weeks tested using an air-tight packaging bottle sealed with a small amount of petroleum jelly, which is significant for a practical platform. The SPC chip contains 5120 independent 5 nL microchambers, allowing the samples to be compartmentalized completely. Using this platform, three different abundances of lung cancer related genes are detected to demonstrate the feasibility and flexibility of the microchip for amplifying a single nucleic acid molecule. For maximal accuracy, within less than 5% of the measurement deviation, the optimal number of positive chambers is between 400 and 1250 evaluated by the Poisson distribution, which means one panel can detect an average of 480 to 4804 template molecules. This device without world-to-chip connections eliminates the constraint of the complex pipeline control, and is an integrated on-chip platform, which would be a significant improvement to digital PCR automation and more user-friendly.

  11. Rewiring and regulation of cross-compartmentalized metabolism in protists.

    PubMed

    Ginger, Michael L; McFadden, Geoffrey I; Michels, Paul A M

    2010-03-12

    Plastid acquisition, endosymbiotic associations, lateral gene transfer, organelle degeneracy or even organelle loss influence metabolic capabilities in many different protists. Thus, metabolic diversity is sculpted through the gain of new metabolic functions and moderation or loss of pathways that are often essential in the majority of eukaryotes. What is perhaps less apparent to the casual observer is that the sub-compartmentalization of ubiquitous pathways has been repeatedly remodelled during eukaryotic evolution, and the textbook pictures of intermediary metabolism established for animals, yeast and plants are not conserved in many protists. Moreover, metabolic remodelling can strongly influence the regulatory mechanisms that control carbon flux through the major metabolic pathways. Here, we provide an overview of how core metabolism has been reorganized in various unicellular eukaryotes, focusing in particular on one near universal catabolic pathway (glycolysis) and one ancient anabolic pathway (isoprenoid biosynthesis). For the example of isoprenoid biosynthesis, the compartmentalization of this process in protists often appears to have been influenced by plastid acquisition and loss, whereas for glycolysis several unexpected modes of compartmentalization have emerged. Significantly, the example of trypanosomatid glycolysis illustrates nicely how mathematical modelling and systems biology can be used to uncover or understand novel modes of pathway regulation.

  12. Rewiring and regulation of cross-compartmentalized metabolism in protists

    PubMed Central

    Ginger, Michael L.; McFadden, Geoffrey I.; Michels, Paul A. M.

    2010-01-01

    Plastid acquisition, endosymbiotic associations, lateral gene transfer, organelle degeneracy or even organelle loss influence metabolic capabilities in many different protists. Thus, metabolic diversity is sculpted through the gain of new metabolic functions and moderation or loss of pathways that are often essential in the majority of eukaryotes. What is perhaps less apparent to the casual observer is that the sub-compartmentalization of ubiquitous pathways has been repeatedly remodelled during eukaryotic evolution, and the textbook pictures of intermediary metabolism established for animals, yeast and plants are not conserved in many protists. Moreover, metabolic remodelling can strongly influence the regulatory mechanisms that control carbon flux through the major metabolic pathways. Here, we provide an overview of how core metabolism has been reorganized in various unicellular eukaryotes, focusing in particular on one near universal catabolic pathway (glycolysis) and one ancient anabolic pathway (isoprenoid biosynthesis). For the example of isoprenoid biosynthesis, the compartmentalization of this process in protists often appears to have been influenced by plastid acquisition and loss, whereas for glycolysis several unexpected modes of compartmentalization have emerged. Significantly, the example of trypanosomatid glycolysis illustrates nicely how mathematical modelling and systems biology can be used to uncover or understand novel modes of pathway regulation. PMID:20124348

  13. Cross-membranes orchestrate compartmentalization and morphogenesis in Streptomyces

    PubMed Central

    Celler, Katherine; Koning, Roman I.; Willemse, Joost; Koster, Abraham J.; van Wezel, Gilles P.

    2016-01-01

    Far from being simple unicellular entities, bacteria have complex social behaviour and organization, living in large populations, and some even as coherent, multicellular entities. The filamentous streptomycetes epitomize such multicellularity, growing as a syncytial mycelium with physiologically distinct hyphal compartments separated by infrequent cross-walls. The viability of mutants devoid of cell division, which can be propagated from fragments, suggests the presence of a different form of compartmentalization in the mycelium. Here we show that complex membranes, visualized by cryo-correlative light microscopy and electron tomography, fulfil this role. Membranes form small assemblies between the cell wall and cytoplasmic membrane, or, as evidenced by FRAP experiments, large protein-impermeable cross-membrane structures, which compartmentalize the multinucleoid mycelium. All areas containing cross-membrane structures are nucleoid-restricted zones, suggesting that the membrane assemblies may also act to protect nucleoids from cell-wall restructuring events. Our work reveals a novel mechanism of controlling compartmentalization and development in multicellular bacteria. PMID:27291257

  14. Steady-state compartmentalization of lipid membranes by active proteins.

    PubMed Central

    Sabra, M C; Mouritsen, O G

    1998-01-01

    Using a simple microscopic model of lipid-protein interactions, based on the hydrophobic matching principle, we study some generic aspects of lipid-membrane compartmentalization controlled by a dispersion of active integral membrane proteins. The activity of the proteins is simulated by conformational excitations governed by an external drive, and the deexcitation is controlled by interaction of the protein with its lipid surroundings. In response to the flux of energy into the proteins from the environment and the subsequent dissipation of energy into the lipid bilayer, the lipid-protein assembly reorganizes into a steady-state structure with a typical length scale determined by the strength of the external drive. In the specific case of a mixed dimyristoylphosphatidylcholine-distearoylphosphatidylcholine bilayer in the gel-fluid coexistence region, it is shown explicitly by computer simulation that the activity of an integral membrane protein can lead to a compartmentalization of the lipid-bilayer membrane. The compartmentalization is related to the dynamical process of phase separation and lipid domain formation. PMID:9533687

  15. Field Testing of Compartmentalization Methods for Multifamily Construction

    SciTech Connect

    Ueno, K.; Lstiburek, J.

    2015-03-01

    The 2012 IECC has an airtightness requirement of 3 air changes per hour at 50 Pascals test pressure for both single-family and multifamily construction in Climate Zones 3-8. Other programs (LEED, ASHRAE 189, ASHRAE 62.2) have similar or tighter compartmentalization requirements, driving the need for easier and more effective methods of compartmentalization in multifamily buildings. Builders and practitioners have found that fire-resistance rated wall assemblies are a major source of difficulty in air sealing/compartmentalization, particularly in townhouse construction. This problem is exacerbated when garages are “tucked in” to the units and living space is located over the garages. In this project, Building Science Corporation examined the taping of exterior sheathing details to improve air sealing results in townhouse and multifamily construction, when coupled with a better understanding of air leakage pathways. Current approaches are cumbersome, expensive, time consuming, and ineffective; these details were proposed as a more effective and efficient method. The effectiveness of these air sealing methods was tested with blower door testing, including “nulled” or “guarded” testing (adjacent units run at equal test pressure to null out inter-unit air leakage, or “pressure neutralization”). Pressure diagnostics were used to evaluate unit-to-unit connections and series leakage pathways (i.e., air leakage from exterior, into the fire-resistance rated wall assembly, and to the interior).

  16. Intracellular Parasite Invasion Strategies

    NASA Astrophysics Data System (ADS)

    Sibley, L. D.

    2004-04-01

    Intracellular parasites use various strategies to invade cells and to subvert cellular signaling pathways and, thus, to gain a foothold against host defenses. Efficient cell entry, ability to exploit intracellular niches, and persistence make these parasites treacherous pathogens. Most intracellular parasites gain entry via host-mediated processes, but apicomplexans use a system of adhesion-based motility called ``gliding'' to actively penetrate host cells. Actin polymerization-dependent motility facilitates parasite migration across cellular barriers, enables dissemination within tissues, and powers invasion of host cells. Efficient invasion has brought widespread success to this group, which includes Toxoplasma, Plasmodium, and Cryptosporidium.

  17. Compartmental microfluidic system for studying muscle-neuron communication and neuromuscular junction maintenance.

    PubMed

    Ionescu, Ariel; Zahavi, Eitan Erez; Gradus, Tal; Ben-Yaakov, Keren; Perlson, Eran

    2016-02-01

    Molecular communication between the motoneuron and the muscle is vital for neuromuscular junction (NMJ) formation and maintenance. Disruption in the structure and function of NMJs is a hallmark of various neurodegenerative processes during both development and pathological events. Still due to the complexity of this process, it is very difficult to elucidate the cellular mechanisms underlying it, generating a keen interest for developing better tools for investigating it. Here we describe a simplified method to study mechanisms of NMJs formation, maintenance and disruption. A spinal cord explant from mice expressing the Hb9::GFP motoneuron marker is plated on one side of a compartmental chamber, and myotubes derived from muscle satellite progenitor cells are plated on the other. The GFP labeled motoneurons extend their axons via microgrooves in the chamber to innervate the muscle cells and to form functional in-vitro NMJs. Next we provide procedures to measure axon growth and to reliably quantify NMJ activity using imaging of both muscle contractions and fast intracellular calcium changes. This platform allows precise control, monitoring and manipulation of subcellular microenvironments. Specifically, it enables to distinguish local from retrograde signaling mechanisms and allows restricted experimental intervention in local compartments along the muscle-neuron route.

  18. Functional Compartmentalization of the Human Superficial Masseter Muscle

    PubMed Central

    Guzmán-Venegas, Rodrigo A.; Biotti Picand, Jorge L.; de la Rosa, Francisco J. Berral

    2015-01-01

    Some muscles have demonstrated a differential recruitment of their motor units in relation to their location and the nature of the motor task performed; this involves functional compartmentalization. There is little evidence that demonstrates the presence of a compartmentalization of the superficial masseter muscle during biting. The aim of this study was to describe the topographic distribution of the activity of the superficial masseter (SM) muscle’s motor units using high-density surface electromyography (EMGs) at different bite force levels. Twenty healthy natural dentate participants (men: 4; women: 16; age 20±2 years; mass: 60±12 kg, height: 163±7 cm) were selected from 316 volunteers and included in this study. Using a gnathodynamometer, bites from 20 to 100% maximum voluntary bite force (MVBF) were randomly requested. Using a two-dimensional grid (four columns, six electrodes) located on the dominant SM, EMGs in the anterior, middle-anterior, middle-posterior and posterior portions were simultaneously recorded. In bite ranges from 20 to 60% MVBF, the EMG activity was higher in the anterior than in the posterior portion (p-value = 0.001).The center of mass of the EMG activity was displaced towards the posterior part when bite force increased (p-value = 0.001). The topographic distribution of EMGs was more homogeneous at high levels of MVBF (p-value = 0.001). The results of this study show that the superficial masseter is organized into three functional compartments: an anterior, a middle and a posterior compartment. However, this compartmentalization is only seen at low levels of bite force (20–60% MVBF). PMID:25692977

  19. Functional compartmentalization of the human superficial masseter muscle.

    PubMed

    Guzmán-Venegas, Rodrigo A; Biotti Picand, Jorge L; de la Rosa, Francisco J Berral

    2015-01-01

    Some muscles have demonstrated a differential recruitment of their motor units in relation to their location and the nature of the motor task performed; this involves functional compartmentalization. There is little evidence that demonstrates the presence of a compartmentalization of the superficial masseter muscle during biting. The aim of this study was to describe the topographic distribution of the activity of the superficial masseter (SM) muscle's motor units using high-density surface electromyography (EMGs) at different bite force levels. Twenty healthy natural dentate participants (men: 4; women: 16; age 20±2 years; mass: 60±12 kg, height: 163±7 cm) were selected from 316 volunteers and included in this study. Using a gnathodynamometer, bites from 20 to 100% maximum voluntary bite force (MVBF) were randomly requested. Using a two-dimensional grid (four columns, six electrodes) located on the dominant SM, EMGs in the anterior, middle-anterior, middle-posterior and posterior portions were simultaneously recorded. In bite ranges from 20 to 60% MVBF, the EMG activity was higher in the anterior than in the posterior portion (p-value = 0.001).The center of mass of the EMG activity was displaced towards the posterior part when bite force increased (p-value = 0.001). The topographic distribution of EMGs was more homogeneous at high levels of MVBF (p-value = 0.001). The results of this study show that the superficial masseter is organized into three functional compartments: an anterior, a middle and a posterior compartment. However, this compartmentalization is only seen at low levels of bite force (20-60% MVBF).

  20. Transit times and mean ages for nonautonomous and autonomous compartmental systems

    SciTech Connect

    Rasmussen, Martin; Hastings, Alan; Smith, Matthew J.; Agusto, Folashade B.; Chen-Charpentier, Benito M.; Hoffman, Forrest M.; Jiang, Jiang; Todd-Brown, Katherine E. O.; Wang, Ying; Wang, Ying-Ping; Luo, Yiqi

    2016-04-01

    We develop a theory for residence times and mean ages for nonautonomous compartmental systems. Using the McKendrick–von Forster equation, we show that the mean ages of mass in a compartmental system satisfy a linear nonautonomous ordinary differential equation that is exponentially stable. We then define a nonautonomous version of residence time as the mean age of mass leaving the compartmental system at a particular time and show that our nonautonomous theory is consistent with the autonomous case. We apply these results to study a nine-dimensional nonautonomous compartmental system modeling the carbon cycle, which is a simplified version of the Carnegie–Ames–Stanford approach (CASA) model.

  1. Small Molecule AKAP-Protein Kinase A (PKA) Interaction Disruptors That Activate PKA Interfere with Compartmentalized cAMP Signaling in Cardiac Myocytes*

    PubMed Central

    Christian, Frank; Szaszák, Márta; Friedl, Sabine; Drewianka, Stephan; Lorenz, Dorothea; Goncalves, Andrey; Furkert, Jens; Vargas, Carolyn; Schmieder, Peter; Götz, Frank; Zühlke, Kerstin; Moutty, Marie; Göttert, Hendrikje; Joshi, Mangesh; Reif, Bernd; Haase, Hannelore; Morano, Ingo; Grossmann, Solveig; Klukovits, Anna; Verli, Judit; Gáspár, Róbert; Noack, Claudia; Bergmann, Martin; Kass, Robert; Hampel, Kornelia; Kashin, Dmitry; Genieser, Hans-Gottfried; Herberg, Friedrich W.; Willoughby, Debbie; Cooper, Dermot M. F.; Baillie, George S.; Houslay, Miles D.; von Kries, Jens Peter; Zimmermann, Bastian; Rosenthal, Walter; Klussmann, Enno

    2011-01-01

    A-kinase anchoring proteins (AKAPs) tether protein kinase A (PKA) and other signaling proteins to defined intracellular sites, thereby establishing compartmentalized cAMP signaling. AKAP-PKA interactions play key roles in various cellular processes, including the regulation of cardiac myocyte contractility. We discovered small molecules, 3,3′-diamino-4,4′-dihydroxydiphenylmethane (FMP-API-1) and its derivatives, which inhibit AKAP-PKA interactions in vitro and in cultured cardiac myocytes. The molecules bind to an allosteric site of regulatory subunits of PKA identifying a hitherto unrecognized region that controls AKAP-PKA interactions. FMP-API-1 also activates PKA. The net effect of FMP-API-1 is a selective interference with compartmentalized cAMP signaling. In cardiac myocytes, FMP-API-1 reveals a novel mechanism involved in terminating β-adrenoreceptor-induced cAMP synthesis. In addition, FMP-API-1 leads to an increase in contractility of cultured rat cardiac myocytes and intact hearts. Thus, FMP-API-1 represents not only a novel means to study compartmentalized cAMP/PKA signaling but, due to its effects on cardiac myocytes and intact hearts, provides the basis for a new concept in the treatment of chronic heart failure. PMID:21177871

  2. Small molecule AKAP-protein kinase A (PKA) interaction disruptors that activate PKA interfere with compartmentalized cAMP signaling in cardiac myocytes.

    PubMed

    Christian, Frank; Szaszák, Márta; Friedl, Sabine; Drewianka, Stephan; Lorenz, Dorothea; Goncalves, Andrey; Furkert, Jens; Vargas, Carolyn; Schmieder, Peter; Götz, Frank; Zühlke, Kerstin; Moutty, Marie; Göttert, Hendrikje; Joshi, Mangesh; Reif, Bernd; Haase, Hannelore; Morano, Ingo; Grossmann, Solveig; Klukovits, Anna; Verli, Judit; Gáspár, Róbert; Noack, Claudia; Bergmann, Martin; Kass, Robert; Hampel, Kornelia; Kashin, Dmitry; Genieser, Hans-Gottfried; Herberg, Friedrich W; Willoughby, Debbie; Cooper, Dermot M F; Baillie, George S; Houslay, Miles D; von Kries, Jens Peter; Zimmermann, Bastian; Rosenthal, Walter; Klussmann, Enno

    2011-03-18

    A-kinase anchoring proteins (AKAPs) tether protein kinase A (PKA) and other signaling proteins to defined intracellular sites, thereby establishing compartmentalized cAMP signaling. AKAP-PKA interactions play key roles in various cellular processes, including the regulation of cardiac myocyte contractility. We discovered small molecules, 3,3'-diamino-4,4'-dihydroxydiphenylmethane (FMP-API-1) and its derivatives, which inhibit AKAP-PKA interactions in vitro and in cultured cardiac myocytes. The molecules bind to an allosteric site of regulatory subunits of PKA identifying a hitherto unrecognized region that controls AKAP-PKA interactions. FMP-API-1 also activates PKA. The net effect of FMP-API-1 is a selective interference with compartmentalized cAMP signaling. In cardiac myocytes, FMP-API-1 reveals a novel mechanism involved in terminating β-adrenoreceptor-induced cAMP synthesis. In addition, FMP-API-1 leads to an increase in contractility of cultured rat cardiac myocytes and intact hearts. Thus, FMP-API-1 represents not only a novel means to study compartmentalized cAMP/PKA signaling but, due to its effects on cardiac myocytes and intact hearts, provides the basis for a new concept in the treatment of chronic heart failure.

  3. Septins and the lateral compartmentalization of eukaryotic membranes.

    PubMed

    Caudron, Fabrice; Barral, Yves

    2009-04-01

    Eukaryotic cells from neurons and epithelial cells to unicellular fungi frequently rely on cellular appendages such as axons, dendritic spines, cilia, and buds for their biology. The emergence and differentiation of these appendages depend on the formation of lateral diffusion barriers at their bases to insulate their membranes from the rest of the cell. Here, we review recent progress regarding the molecular mechanisms and functions of such barriers. This overview underlines the importance and conservation of septin-dependent diffusion barriers, which coordinately compartmentalize both plasmatic and internal membranes. We discuss their role in memory establishment and the control of cellular aging.

  4. Analytical properties of a three-compartmental dynamical demographic model.

    PubMed

    Postnikov, E B

    2015-07-01

    The three-compartmental demographic model by Korotaeyv-Malkov-Khaltourina, connecting population size, economic surplus, and education level, is considered from the point of view of dynamical systems theory. It is shown that there exist two integrals of motion, which enables the system to be reduced to one nonlinear ordinary differential equation. The study of its structure provides analytical criteria for the dominance ranges of the dynamics of Malthus and Kremer. Additionally, the particular ranges of parameters enable the derived general ordinary differential equations to be reduced to the models of Gompertz and Thoularis-Wallace.

  5. Analytical properties of a three-compartmental dynamical demographic model

    NASA Astrophysics Data System (ADS)

    Postnikov, E. B.

    2015-07-01

    The three-compartmental demographic model by Korotaeyv-Malkov-Khaltourina, connecting population size, economic surplus, and education level, is considered from the point of view of dynamical systems theory. It is shown that there exist two integrals of motion, which enables the system to be reduced to one nonlinear ordinary differential equation. The study of its structure provides analytical criteria for the dominance ranges of the dynamics of Malthus and Kremer. Additionally, the particular ranges of parameters enable the derived general ordinary differential equations to be reduced to the models of Gompertz and Thoularis-Wallace.

  6. Dynamic behaviors of a class of HIV compartmental models

    NASA Astrophysics Data System (ADS)

    Chen, Xiaoyan; Huang, Lihong; Yu, Pei

    2015-06-01

    Based on heterogeneities in drug efficacy (either spatial or phenotypic), two HIV compartmental models were proposed in Callaway and Perelson (2002) to study the HIV virus dynamics under drug treatment. In this paper, we provide a global analysis on the two models, including the positivity and boundedness of solutions and the global stability of equilibrium solutions. In particular, we show that when the basic reproduction number R0 ⩽ 1 (for which the infection equilibrium does not exist), the infection-free equilibrium is globally asymptotically stable; while when R0 > 1 (for which the infection equilibrium exists), the infection equilibrium is globally asymptotically stable.

  7. Controls of Intracellular Communication Mediated by Gap Junctions

    NASA Technical Reports Server (NTRS)

    Bennett, M. V. L.

    1983-01-01

    Experiments were done using aequorin and no increase in aequorin luminescence during acidification adequate to uncouple cells was seen. The pH sensitivity of the conductance of the perfused membrane was essentially the same as that observed with intracellular pH microelectrodes.

  8. Analysis of Ras/ERK Compartmentalization by Subcellular Fractionation.

    PubMed

    Agudo-Ibañez, Lorena; Crespo, Piero; Casar, Berta

    2017-01-01

    A vast number of stimuli use the Ras/Raf/MEK/ERK signaling cascade to transmit signals from their cognate receptors, in order to regulate multiple cellular functions, including key processes such as proliferation, cell cycle progression, differentiation, and survival. The duration, intensity and specificity of the responses are, in part, controlled by the compartmentalization/subcellular localization of the signaling intermediaries. Ras proteins are found in different plasma membrane microdomains and endomembranes. At these localizations, Ras is subject to site-specific regulatory mechanisms, distinctively engaging effector pathways and switching-on diverse genetic programs to generate a multitude of biological responses. The Ras effector pathway leading to ERKs activation is also subject to space-related regulatory processes. About half of ERK1/2 substrates are found in the nucleus and function mainly as transcription factors. The other half resides in the cytosol and other cellular organelles. Such subcellular distribution enhances the complexity of the Ras/ERK cascade and constitutes an essential mechanism to endow variability to its signals, which enables their participation in the regulation of a broad variety of functions. Thus, analyzing the subcellular compartmentalization of the members of the Ras/ERK cascade constitutes an important factor to be taken into account when studying specific biological responses evoked by Ras/ERK signals. Herein, we describe methods for such purpose.

  9. Super-resolution Microscopy Reveals Compartmentalization of Peroxisomal Membrane Proteins*

    PubMed Central

    Galiani, Silvia; Waithe, Dominic; Reglinski, Katharina; Cruz-Zaragoza, Luis Daniel; Garcia, Esther; Clausen, Mathias P.; Schliebs, Wolfgang; Erdmann, Ralf; Eggeling, Christian

    2016-01-01

    Membrane-associated events during peroxisomal protein import processes play an essential role in peroxisome functionality. Many details of these processes are not known due to missing spatial resolution of technologies capable of investigating peroxisomes directly in the cell. Here, we present the use of super-resolution optical stimulated emission depletion microscopy to investigate with sub-60-nm resolution the heterogeneous spatial organization of the peroxisomal proteins PEX5, PEX14, and PEX11 around actively importing peroxisomes, showing distinct differences between these peroxins. Moreover, imported protein sterol carrier protein 2 (SCP2) occupies only a subregion of larger peroxisomes, highlighting the heterogeneous distribution of proteins even within the peroxisome. Finally, our data reveal subpopulations of peroxisomes showing only weak colocalization between PEX14 and PEX5 or PEX11 but at the same time a clear compartmentalized organization. This compartmentalization, which was less evident in cases of strong colocalization, indicates dynamic protein reorganization linked to changes occurring in the peroxisomes. Through the use of multicolor stimulated emission depletion microscopy, we have been able to characterize peroxisomes and their constituents to a yet unseen level of detail while maintaining a highly statistical approach, paving the way for equally complex biological studies in the future. PMID:27311714

  10. Super-resolution Microscopy Reveals Compartmentalization of Peroxisomal Membrane Proteins.

    PubMed

    Galiani, Silvia; Waithe, Dominic; Reglinski, Katharina; Cruz-Zaragoza, Luis Daniel; Garcia, Esther; Clausen, Mathias P; Schliebs, Wolfgang; Erdmann, Ralf; Eggeling, Christian

    2016-08-12

    Membrane-associated events during peroxisomal protein import processes play an essential role in peroxisome functionality. Many details of these processes are not known due to missing spatial resolution of technologies capable of investigating peroxisomes directly in the cell. Here, we present the use of super-resolution optical stimulated emission depletion microscopy to investigate with sub-60-nm resolution the heterogeneous spatial organization of the peroxisomal proteins PEX5, PEX14, and PEX11 around actively importing peroxisomes, showing distinct differences between these peroxins. Moreover, imported protein sterol carrier protein 2 (SCP2) occupies only a subregion of larger peroxisomes, highlighting the heterogeneous distribution of proteins even within the peroxisome. Finally, our data reveal subpopulations of peroxisomes showing only weak colocalization between PEX14 and PEX5 or PEX11 but at the same time a clear compartmentalized organization. This compartmentalization, which was less evident in cases of strong colocalization, indicates dynamic protein reorganization linked to changes occurring in the peroxisomes. Through the use of multicolor stimulated emission depletion microscopy, we have been able to characterize peroxisomes and their constituents to a yet unseen level of detail while maintaining a highly statistical approach, paving the way for equally complex biological studies in the future.

  11. MDCT of hand and wrist infections: emphasis on compartmental anatomy.

    PubMed

    Ahlawat, S; Corl, F M; LaPorte, D M; Fishman, E K; Fayad, L M

    2017-04-01

    Hand and wrist infections can present with a spectrum of manifestations ranging from cellulitis to deep-space collections. The various infectious processes can be categorised as superficial or deep infections based on their respective locations relative to the tendons. Superficial hand infections are located superficial to the tendons and are comprised of cellulitis, lymphangitis, paronychia, pulp-space infections, herpetic whitlow, and include volar as well as dorsal subcutaneous abscesses. Deep hand infections are located deep to the tendon sheaths and include synovial space infections, such as infectious tenosynovitis, deep fascial space infections, septic arthritis, necrotising fasciitis, and osteomyelitis. Knowledge of hand and wrist compartmental anatomy is essential for the accurate diagnosis and management of hand infections. Although early and superficial infections of the hand may respond to non-surgical management, most hand infections are surgical emergencies. Multidetector computed tomography (MDCT), with its muliplanar reformation (MPR) and three-dimensional (3D) capabilities, is a powerful tool in the emergency setting for the evaluation of acute hand and wrist pathology. The clinical and imaging features of hand and wrist infections as evident on MDCT will be reviewed with emphasis on contiguous and closed synovial and deep fascial spaces. Knowledge of hand compartmental anatomy enables accurate characterisation of the infectious process and localise the extent of disease in the acute setting.

  12. Multiple compartmentalization of sodium conferred salt tolerance in Salicornia europaea.

    PubMed

    Lv, Sulian; Jiang, Ping; Chen, Xianyang; Fan, Pengxiang; Wang, Xuchu; Li, Yinxin

    2012-02-01

    Euhalophyte Salicornia europaea L., one of the most salt-tolerant plant species in the world, can tolerate more than 1000 mM NaCl. To study the salt tolerance mechanism of this plant, the effects of different NaCl concentrations on plant growth, as well as Na(+) accumulation and distribution at organ, tissue, and subcellular levels, were investigated. Optimal growth and an improved photosynthetic rate were observed with the plant treated with 200-400 mM NaCl. The Na(+) content in the shoots was considerably higher than that in the roots of S. europaea. The Na(+) in S. europaea cells may act as an effective osmotic adjuster to maintain cell turgor, promoting photosynthetic competence and plant growth. The results from the SEM-X-ray and TEM-X-ray microanalyses demonstrate that Na(+) was compartmentalized predominantly into the cell vacuoles of shoot endodermis tissues. Accordingly, the transcript amounts of SeNHX1, SeVHA-A, and SeVP1 increased significantly with increased NaCl concentration, suggesting their important roles in Na(+) sequestration into the vacuoles. Therefore, a multiple sodium compartmentalization mechanism is proposed to enhance further the salt tolerance of S. europaea.

  13. Detachment, compartmentalization, and schizophrenia: linking dissociation and psychosis by subtype.

    PubMed

    Vogel, Matthias; Braungardt, Tanja; Grabe, Hans Jörgen; Schneider, Wolfgang; Klauer, Thomas

    2013-01-01

    To explain the phenomenological overlap between dissociation and schizophrenia, a dissociative subtype of schizophrenia has been proposed as a possibility. Dissociation is often believed to be organized on a continuum, although 2 qualitatively different phenomena can be distinguished in theory, research, and clinical practice: (a) states of separation from self or environment (detachment dissociation) and (b) inaccessibility of normally accessible mental contents (compartmentalization dissociation). This study used the Positive and Negative Syndrome Scale (PANSS) and the Association for Methodology and Documentation in Psychiatry module for the interview assessment of dissociation to investigate the relationships between PANSS subscales, detachment dissociation, and compartmentalization dissociation in a sample of 72 patients with schizophrenia. A confirmatory factor analysis sustained the bipartite model, yielding factors that grouped dissociative items around amnesia and depersonalization/derealization. The latter factor also contained identity disturbances and was therefore not entirely consistent with the theoretical formulations of detachment dissociation. It is important to note that the structure of those factors may be influenced by the symptoms of schizophrenia to which they were specifically linked: The factor containing depersonalization/derealization was connected to the positive symptoms subscale of the PANSS, whereas the factor containing amnesia was associated with the negative subscale. Hence, a dichotomy of dissociation is confirmed inasmuch as its subtypes are as distinguishable as PANSS subscales. This has implications on theoretical and clinical levels.

  14. Single-cell intracellular nano-pH probes.

    PubMed

    Özel, Rıfat Emrah; Lohith, Akshar; Mak, Wai Han; Pourmand, Nader

    2015-01-01

    Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular pH of individual cells within a cell population is challenging with existing technologies, and there is a need to engineer new methodologies. In this paper, we discuss the use of nanopipette technology to overcome the limitations of intracellular pH measurements at the single-cell level. We have developed a nano-pH probe through physisorption of chitosan onto hydroxylated quartz nanopipettes with extremely small pore sizes (~100 nm). The dynamic pH range of the nano-pH probe was from 2.6 to 10.7 with a sensitivity of 0.09 units. We have performed single-cell intracellular pH measurements using non-cancerous and cancerous cell lines, including human fibroblasts, HeLa, MDA-MB-231 and MCF-7, with the pH nanoprobe. We have further demonstrated the real-time continuous single-cell pH measurement capability of the sensor, showing the cellular pH response to pharmaceutical manipulations. These findings suggest that the chitosan-functionalized nanopore is a powerful nano-tool for pH sensing at the single-cell level with high temporal and spatial resolution.

  15. Quantum dot photoluminescence lifetime-based pH nanosensor.

    PubMed

    Ruedas-Rama, Maria J; Orte, Angel; Hall, Elizabeth A H; Alvarez-Pez, Jose M; Talavera, Eva M

    2011-03-14

    The first CdSe/ZnS quantum dot photoluminescence lifetime-based pH nanosensor has been developed. The average lifetime of mercaptopropionic acid-capped QD nanosensors showed a linear response in the pH range of 5.2-6.9. These nanosensors have been satisfactorily applied for pH estimation in simulated intracellular media, with high sensitivity and high selectivity toward most of the intracellular components.

  16. Building America Case Study: Field Testing of Compartmentalization Methods for Multifamily Construction (Fact Sheet)

    SciTech Connect

    Not Available

    2015-01-01

    The 2012 IECC has an airtightness requirement of 3 air changes per hour at 50 Pascals test pressure for both single family and multifamily construction in Climate Zones 3-8. Other programs (LEED, ASHRAE 189, ASHRAE 62.2) have similar or tighter compartmentalization requirements, thus driving the need for easier and more effective methods of compartmentalization in multifamily buildings.

  17. Non-stationary 13C metabolic flux analysis of Chinese hamster ovary cells in batch culture using extracellular labeling highlights metabolic reversibility and compartmentation

    PubMed Central

    2014-01-01

    Background Mapping the intracellular fluxes for established mammalian cell lines becomes increasingly important for scientific and economic reasons. However, this is being hampered by the high complexity of metabolic networks, particularly concerning compartmentation. Results Intracellular fluxes of the CHO-K1 cell line central carbon metabolism were successfully determined for a complex network using non-stationary 13C metabolic flux analysis. Mass isotopomers of extracellular metabolites were determined using [U-13C6] glucose as labeled substrate. Metabolic compartmentation and extracellular transport reversibility proved essential to successfully reproduce the dynamics of the labeling patterns. Alanine and pyruvate reversibility changed dynamically even if their net production fluxes remained constant. Cataplerotic fluxes of cytosolic phosphoenolpyruvate carboxykinase and mitochondrial malic enzyme and pyruvate carboxylase were successfully determined. Glycolytic pyruvate channeling to lactate was modeled by including a separate pyruvate pool. In the exponential growth phase, alanine, glycine and glutamate were excreted, and glutamine, aspartate, asparagine and serine were taken up; however, all these amino acids except asparagine were exchanged reversibly with the media. High fluxes were determined in the pentose phosphate pathway and the TCA cycle. The latter was fueled mainly by glucose but also by amino acid catabolism. Conclusions The CHO-K1 central metabolism in controlled batch culture proves to be robust. It has the main purpose to ensure fast growth on a mixture of substrates and also to mitigate oxidative stress. It achieves this by using compartmentation to control NADPH and NADH availability and by simultaneous synthesis and catabolism of amino acids. PMID:24773761

  18. Compartmental and Data-Based Modeling of Cerebral Hemodynamics: Linear Analysis

    PubMed Central

    Henley, B.C.; Shin, D.C.; Zhang, R.; Marmarelis, V.Z.

    2015-01-01

    Compartmental and data-based modeling of cerebral hemodynamics are alternative approaches that utilize distinct model forms and have been employed in the quantitative study of cerebral hemodynamics. This paper examines the relation between a compartmental equivalent-circuit and a data-based input-output model of dynamic cerebral autoregulation (DCA) and CO2-vasomotor reactivity (DVR). The compartmental model is constructed as an equivalent-circuit utilizing putative first principles and previously proposed hypothesis-based models. The linear input-output dynamics of this compartmental model are compared with data-based estimates of the DCA-DVR process. This comparative study indicates that there are some qualitative similarities between the two-input compartmental model and experimental results. PMID:26900535

  19. Förster Resonance Energy Transfer - An approach to visualize the spatiotemporal regulation of macromolecular complex formation and compartmentalized cell signaling

    PubMed Central

    Sinha, Chandrima; Arora, Kavisha; Moon, Chang Suk; Yarlagadda, Sunitha; Woodrooffe, Koryse; Naren, Anjaparavanda P.

    2014-01-01

    Background Signaling messengers and effector proteins provide an orchestrated molecular machinery to relay extracellular signals to the inside of cells and thereby facilitate distinct cellular behaviors. Formations of intracellular macromolecular complexes and segregation of signaling cascades dynamically regulate the flow of a biological process. Scope of Review In this review, we provide an overview of the development and application of FRET technology in monitoring cyclic nucleotide-dependent signalings and protein complexes associated with these signalings in real time and space with brief mention of other important signaling messengers and effector proteins involved in compartmentalized signaling. Major Conclusion The preciseness, rapidity and specificity of cellular responses indicate restricted alterations of signaling messengers, particularly in subcellular compartments rather than globally. Not only the physical confinement and selective depletion, but also the intra- and inter-molecular interactions of signaling effectors modulate the direction of signal transduction in a compartmentalized fashion. To understand the finer details of various intracellular signaling cascades and crosstalk between proteins and other effectors, it is important to visualize these processes in live cells. Förster Resonance Energy Transfer (FRET) has been established as a useful tool to do this, even with its inherent limitations. General Significance FRET technology remains as an effective tool for unraveling the complex organization and distribution of various endogenous signaling proteins, as well as the spatiotemporal dynamics of second messengers inside a single cell to distinguish the heterogeneity of cell signaling under normal physiological conditions and during pathological events. PMID:25086255

  20. Compartmentalization in penicillin G biosynthesis by Penicillium chrysogenum PQ-96.

    PubMed

    Kurzątkowski, Wiesław; Staniszewska, Monika; Bondaryk, Małgorzata; Gębska-Kuczerowska, Anita

    2014-01-01

    The arrangement of organelles in the sub-apical productive non-growing vacuolated hyphal cells of the high- and the low-penicillin-pro- ducing strains Penicillium chrysogenum was compared using transmission electron microscopy. In the productive cells of the high-yielding strain the endoplasmic reticulum and the polyribosomes with associated peroxisomes are frequently arranged at the periphery of the cytoplasm and around the vacuoles. At the high activity of penicillin G biosynthesis the immuno-label of the cytosolic isopenicillin N synthase is concentrated at the polyribosomes arranged in the peripheral cytoplasm and along the tonoplast as well as around the peroxisomes. On the basis of the obtained results the compartmentalization of the pathway of penicillin G biosymthesis is discussed. The obtained results support the phenylacetic acid detoxification hypothesis of penicillin G biosynthesis.

  1. Compartmentalization of NO signaling cascade in skeletal muscles

    SciTech Connect

    Buchwalow, Igor B. . E-mail: buchwalo@uni-muenster.de; Minin, Evgeny A.; Samoilova, Vera E.; Boecker, Werner; Wellner, Maren; Schmitz, Wilhelm; Neumann, Joachim

    2005-05-06

    Skeletal muscle functions regulated by NO are now firmly established. However, the literature on the compartmentalization of NO signaling in myocytes is highly controversial. To address this issue, we examined localization of enzymes engaged in L-arginine-NO-cGMP signaling in the rat quadriceps muscle. Employing immunocytochemical labeling complemented with tyramide signal amplification and electron microscopy, we found NO synthase expressed not only in the sarcolemma, but also along contractile fibers, in the sarcoplasmic reticulum and mitochondria. The expression pattern of NO synthase in myocytes showed striking parallels with the enzymes engaged in L-arginine-NO-cGMP signaling (arginase, phosphodiesterase, and soluble guanylyl cyclase). Our findings are indicative of an autocrine fashion of NO signaling in skeletal muscles at both cellular and subcellular levels, and challenge the notion that the NO generation is restricted to the sarcolemma.

  2. Partitioning Variability of a Compartmentalized In Vitro Transcriptional Thresholding Circuit.

    PubMed

    Kapsner, Korbinian; Simmel, Friedrich C

    2015-10-16

    Encapsulation of in vitro biochemical reaction circuits into small, cell-sized compartments can result in considerable variations in the dynamical properties of the circuits. As a model system, we here investigate a simple in vitro transcriptional reaction circuit, which generates an ultrasensitive fluorescence response when the concentration of an RNA transcript reaches a preset threshold. The reaction circuit is compartmentalized into spherical water-in-oil microemulsion droplets, and the reaction progress is monitored by fluorescence microscopy. A quantitative statistical analysis of thousands of individual droplets ranging in size from a few up to 20 μm reveals a strong variability in effective RNA production rates, which by computational modeling is traced back to a larger-than-Poisson variability in RNAP activities in the droplets. The noise level in terms of the noise strength (the Fano factor) is strongly dependent on the ratio between transcription templates and polymerases, and increases for higher template concentrations.

  3. Compartmentalization of vertebrate optic neuroephithelium: external cues and transcription factors.

    PubMed

    Kim, Hyoung-Tai; Kim, Jin Woo

    2012-04-01

    The vertebrate eye is a laterally extended structure of the forebrain. It develops through a series of events, including specification and regionalization of the anterior neural plate, evagination of the optic vesicle (OV), and development of three distinct optic structures: the neural retina (NR), optic stalk (OS), and retinal pigment epithelium (RPE). Various external signals that act on the optic neuroepithelium in a spatial- and temporal-specific manner control the fates of OV subdomains by inducing localized expression of key transcription factors. Investigating the mechanisms underlying compartmentalization of these distinct optic neuroepithelium-derived tissues is therefore not only important from the standpoint of accounting for vertebrate eye morphogenesis, it is also helpful for understanding the fundamental basis of fate determination of other neuroectoderm- derived tissues. This review focuses on the molecular signatures of OV subdomains and the external factors that direct the development of tissues originating from the OV.

  4. Physiological Consequences of Compartmentalized Acyl-CoA Metabolism*

    PubMed Central

    Cooper, Daniel E.; Young, Pamela A.; Klett, Eric L.; Coleman, Rosalind A.

    2015-01-01

    Meeting the complex physiological demands of mammalian life requires strict control of the metabolism of long-chain fatty acyl-CoAs because of the multiplicity of their cellular functions. Acyl-CoAs are substrates for energy production; stored within lipid droplets as triacylglycerol, cholesterol esters, and retinol esters; esterified to form membrane phospholipids; or used to activate transcriptional and signaling pathways. Indirect evidence suggests that acyl-CoAs do not wander freely within cells, but instead, are channeled into specific pathways. In this review, we will discuss the evidence for acyl-CoA compartmentalization, highlight the key modes of acyl-CoA regulation, and diagram potential mechanisms for controlling acyl-CoA partitioning. PMID:26124277

  5. The human NAD metabolome: Functions, metabolism and compartmentalization

    PubMed Central

    Nikiforov, Andrey; Kulikova, Veronika; Ziegler, Mathias

    2015-01-01

    Abstract The metabolism of NAD has emerged as a key regulator of cellular and organismal homeostasis. Being a major component of both bioenergetic and signaling pathways, the molecule is ideally suited to regulate metabolism and major cellular events. In humans, NAD is synthesized from vitamin B3 precursors, most prominently from nicotinamide, which is the degradation product of all NAD-dependent signaling reactions. The scope of NAD-mediated regulatory processes is wide including enzyme regulation, control of gene expression and health span, DNA repair, cell cycle regulation and calcium signaling. In these processes, nicotinamide is cleaved from NAD+ and the remaining ADP-ribosyl moiety used to modify proteins (deacetylation by sirtuins or ADP-ribosylation) or to generate calcium-mobilizing agents such as cyclic ADP-ribose. This review will also emphasize the role of the intermediates in the NAD metabolome, their intra- and extra-cellular conversions and potential contributions to subcellular compartmentalization of NAD pools. PMID:25837229

  6. Compartmentation of Metabolites in Regulating Epigenomes of Cancer

    PubMed Central

    Zhao, Zhiqiang; Wang, Li; Di, Li-jun

    2016-01-01

    Covalent modifications of DNA and histones are important epigenetic events and the genomewide reshaping of epigenetic markers is common in cancer. Epigenetic markers are produced by enzymatic reactions, and some of these reactions require the presence of metabolites, specifically Epigenetic Enzyme Required Metabolites (EERMs), as cofactors. Recent studies found that the abundance of these EERMs correlates with epigenetic enzyme activities. Also, the subcellular compartmentation, especially the nuclear localization of these EERMs, may play a role in regulating the activities of epigenetic enzymes. Moreover, gene-specific recruitment of enzymes that produce the EERMs in the proximity of the epigenetic modification events accompanying the regulation of gene expression, were proposed. Therefore, it is important to summarize findings of EERMs in regulating epigenetic modifications at both the DNA and histone levels, and to understand how EERMs contribute to cancer development by addressing their global versus local distribution. PMID:27258652

  7. Compartmentalized vesicular traffic around the hair cell cuticular plate.

    PubMed

    Kachar, B; Battaglia, A; Fex, J

    1997-05-01

    Through thin-section and freeze-fracture electron microscopy, we identify structural correlates of an intense vesicular traffic in a narrow band of cytoplasm around the cuticular plate of the bullfrog vestibular hair cells. Myriads of coated and uncoated vesicles associated with longitudinally oriented microtubules populate the narrow cytoplasmic region between the cuticular plate and the actin network of the apical junctional belt. If microtubules in the sensory hair cells, like those in axons, are pathways for organelle transport, then the characteristic distribution of microtubules around the cuticular plate represents transport pathways across the apical region of the hair cells. This compartmentalized membrane traffic system appears to support an intense vesicular release and uptake along a band of apical plasma membrane near the cell border. Functions of this transport system may include membrane recycling as well as exocytotic and endocytotic exchange between the hair cell cytoplasm and the endolymphatic compartment.

  8. Hydrological Compartmentalization: A Grand Challenge in the Critical Zone

    NASA Astrophysics Data System (ADS)

    McDonnell, J.; Evaristo, J. A.; Orlowski, N.; Jasechko, S.

    2014-12-01

    Current terrestrial biosphere models assume that plant transpiration, groundwater and streamflow are all sourced and mediated by the same well mixed soil reservoir. Recent stable water isotope data from Oregon and Mexico and now global meta-analysis and remote sensing measurements have all shown evidence of hydrological compartmentalization: a mobile compartment that forms groundwater and streamflow and a poorly mobile compartment that supplies plant transpiration. The way we now measure and model this poorly mobile water is a grand challenge for understanding subsurface mixing, water residence time, and its interaction and feedback to ecosystem processes. Here we review the latest results from this work and outline some of the future research challenges for understanding soil-plant-atmospheric interactions between the bedrock and the boundary layer.

  9. Hydrological Compartmentalization: A Grand Challenge in the Critical Zone

    NASA Astrophysics Data System (ADS)

    McDonnell, J.; Evaristo, J. A.; Orlowski, N.; Jasechko, S.

    2015-12-01

    Current terrestrial biosphere models assume that plant transpiration, groundwater and streamflow are all sourced and mediated by the same well mixed soil reservoir. Recent stable water isotope data from Oregon and Mexico and now global meta-analysis and remote sensing measurements have all shown evidence of hydrological compartmentalization: a mobile compartment that forms groundwater and streamflow and a poorly mobile compartment that supplies plant transpiration. The way we now measure and model this poorly mobile water is a grand challenge for understanding subsurface mixing, water residence time, and its interaction and feedback to ecosystem processes. Here we review the latest results from this work and outline some of the future research challenges for understanding soil-plant-atmospheric interactions between the bedrock and the boundary layer.

  10. The human NAD metabolome: Functions, metabolism and compartmentalization.

    PubMed

    Nikiforov, Andrey; Kulikova, Veronika; Ziegler, Mathias

    2015-01-01

    The metabolism of NAD has emerged as a key regulator of cellular and organismal homeostasis. Being a major component of both bioenergetic and signaling pathways, the molecule is ideally suited to regulate metabolism and major cellular events. In humans, NAD is synthesized from vitamin B3 precursors, most prominently from nicotinamide, which is the degradation product of all NAD-dependent signaling reactions. The scope of NAD-mediated regulatory processes is wide including enzyme regulation, control of gene expression and health span, DNA repair, cell cycle regulation and calcium signaling. In these processes, nicotinamide is cleaved from NAD(+) and the remaining ADP-ribosyl moiety used to modify proteins (deacetylation by sirtuins or ADP-ribosylation) or to generate calcium-mobilizing agents such as cyclic ADP-ribose. This review will also emphasize the role of the intermediates in the NAD metabolome, their intra- and extra-cellular conversions and potential contributions to subcellular compartmentalization of NAD pools.

  11. Apartment Compartmentalization With an Aerosol-Based Sealing Process

    SciTech Connect

    Maxwell, S.; Berger, D.; Harrington, C.

    2015-03-01

    Air sealing of building enclosures is a difficult and time-consuming process. Current methods in new construction require laborers to physically locate small and sometimes large holes in multiple assemblies and then manually seal each of them. The innovation demonstrated under this research study was the automated air sealing and compartmentalization of buildings through the use of an aerosolized sealant, developed by the Western Cooling Efficiency Center at University of California Davis. CARB sought to demonstrate this new technology application in a multifamily building in Queens, NY. The effectiveness of the sealing process was evaluated by three methods: air leakage testing of overall apartment before and after sealing, point-source testing of individual leaks, and pressure measurements in the walls of the target apartment during sealing.

  12. Heart mitochondrial TTP synthesis and the compartmentalization of TMP.

    PubMed

    Kamath, Vasudeva G; Hsiung, Chia-Heng; Lizenby, Zachary J; McKee, Edward E

    2015-01-23

    The primary pathway of TTP synthesis in the heart requires thymidine salvage by mitochondrial thymidine kinase 2 (TK2). However, the compartmentalization of this pathway and the transport of thymidine nucleotides are not well understood. We investigated the metabolism of [(3)H]thymidine or [(3)H]TMP as precursors of [(3)H]TTP in isolated intact or broken mitochondria from the rat heart. The results demonstrated that [(3)H]thymidine was readily metabolized by the mitochondrial salvage enzymes to TTP in intact mitochondria. The equivalent addition of [(3)H]TMP produced far less [(3)H]TTP than the amount observed with [(3)H]thymidine as the precursor. Using zidovudine to inhibit TK2, the synthesis of [(3)H]TTP from [(3)H]TMP was effectively blocked, demonstrating that synthesis of [(3)H]TTP from [(3)H]TMP arose solely from the dephosphorysynthase pathway that includes deoxyuridine triphosphatelation of [(3)H]TMP to [(3)H]thymidine. To determine the role of the membrane in TMP metabolism, mitochondrial membranes were disrupted by freezing and thawing. In broken mitochondria, [(3)H]thymidine was readily converted to [(3)H]TMP, but further phosphorylation was prevented even though the energy charge was well maintained by addition of oligomycin A, phosphocreatine, and creatine phosphokinase. The failure to synthesize TTP in broken mitochondria was not related to a loss of membrane potential or inhibition of the electron transport chain, as confirmed by addition of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone and potassium cyanide, respectively, in intact mitochondria. In summary, these data, taken together, suggest that the thymidine salvage pathway is compartmentalized so that TMP kinase prefers TMP synthesized by TK2 over medium TMP and that this is disrupted in broken mitochondria.

  13. Tissue Level Compartmentation of (R)-Amygdalin and Amygdalin Hydrolase Prevents Large-Scale Cyanogenesis in Undamaged Prunus Seeds.

    PubMed Central

    Poulton, J. E.; Li, C. P.

    1994-01-01

    Plum (Prunus domestica) seeds, which contain the cyanogenic diglucoside (R)-amygdalin and lesser amounts of the corresponding monoglucoside (R)-prunasin, release the respiratory toxin HCN upon tissue disruption. Amygdalin hydrolase (AH) and prunasin hydrolase (PH), two specific [beta]-glucosidases responsible for hydrolysis of these glucosides, were purified to near homogeneity by concanavalin A-Sepharose 4B and carboxymethyl-cellulose chromatography. Both proteins appear as polypeptides with molecular masses of 60 kD upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but they exhibit different isoelectric points (PH, 5.6-6.0; AH, 7.8-8.2). AH and PH were localized within mature plum seeds by tissue printing, histochemistry, and silver-enhanced immunogold labeling. As was previously shown in black cherry (Prunus serotina) seeds (E.Swain, C.P. Li, J.E. Poulton [1992] Plant Physiol 100: 291-300), AH and PH are restricted to protein bodies of specific procambial cells and are absent from the cotyledonary parenchyma, bundle sheath, and endosperm cells. In contrast, the cyanogenic glycosides in both plum and black cherry seeds, which were detected by tissue printing, occur solely in the cotyledonary parenchyma and are absent from the procambium and endosperm. It is concluded that tissue level compartmentation prevents large-scale cyanoglycoside hydrolysis in intact Prunus seeds. PMID:12232058

  14. Synapse-specific compartmentalization of signaling cascades for LTP induction in CA3 interneurons.

    PubMed

    Galván, E J; Pérez-Rosello, T; Gómez-Lira, G; Lara, E; Gutiérrez, R; Barrionuevo, G

    2015-04-02

    Inhibitory interneurons with somata in strata radiatum and lacunosum-molecular (SR/L-M) of hippocampal area CA3 receive excitatory input from pyramidal cells via the recurrent collaterals (RCs), and the dentate gyrus granule cells via the mossy fibers (MFs). Here we demonstrate that Hebbian long-term potentiation (LTP) at RC synapses on SR/L-M interneurons requires the concomitant activation of calcium-impermeable AMPARs (CI-AMPARs) and N-methyl-d-aspartate receptors (NMDARs). RC LTP was prevented by voltage clamping the postsynaptic cell during high-frequency stimulation (HFS; 3 trains of 100 pulses delivered at 100 Hz every 10s), with intracellular injections of the Ca(2+) chelator BAPTA (20mM), and with the NMDAR antagonist D-AP5. In separate experiments, RC and MF inputs converging onto the same interneuron were sequentially activated. We found that RC LTP induction was blocked by inhibitors of the calcium/calmodulin-dependent protein kinase II (CaMKII; KN-62, 10 μM or KN-93, 10 μM) but MF LTP was CaMKII independent. Conversely, the application of the protein kinase A (PKA) activators forskolin/IBMX (50 μM/25 μM) potentiated MF EPSPs but not RC EPSPs. Together these data indicate that the aspiny dendrites of SR/L-M interneurons compartmentalize synapse-specific Ca(2+) signaling required for LTP induction at RC and MF synapses. We also show that the two signal transduction cascades converge to activate a common effector, protein kinase C (PKC). Specifically, LTP at RC and MF synapses on the same SR/LM interneuron was blocked by postsynaptic injections of chelerythrine (10 μM). These data indicate that both forms of LTP share a common mechanism involving PKC-dependent signaling modulation.

  15. Synapse-specific compartmentalization of signaling cascades for LTP induction in CA3 interneurons

    PubMed Central

    Galván, Emilio J; Pérez-Rosello, Tamara; Gómez-Lira, Gisela; Lara, Erika; Gutiérrez, Rafael; Barrionuevo, Germán

    2015-01-01

    Inhibitory interneurons with somata in strata radiatum and lacunosun-moleculare (SR/L-M) of hippocampal area CA3 receive excitatory input from pyramidal cells via the recurrent collaterals (RC), and the dentate gyrus granule cells via the mossy fibers (MFs). Here we demonstrate that Hebbian long-term potentiation (LTP) at RC synapses on SR/L-M interneurons requires the concomitant activation of calcium-impermeable AMPARs (CI- AMPARs) and NMDARs. RC LTP was prevented by voltage clamping the postsynaptic cell during high-frequency stimulation (HFS; 3 trains of 100 pulses delivered at 100 Hz every 10 s), with intracellular injections of the Ca2+ chelator BAPTA (20 mM), and with the N-methyl-D-aspartate receptor (NMDAR) antagonist D-AP5. In separate experiments, RC and MF inputs converging onto the same interneuron were sequentially activated. We found that RC LTP induction was blocked by inhibitors of the calcium/calmodulin-dependent protein kinase II (CaMKII; KN-62, 10 μM or KN-93, 10 μM) but MF LTP was CaMKII independent. Conversely, the application of the protein kinase A (PKA) activators forskolin/IBMX(50 μM/25 μM) potentiated MF EPSPs but not RC EPSPs. Together these data indicate that the aspiny dendrites of SR/L-M interneurons compartmentalize synaptic-specific Ca2+ signaling required for LTP induction at RC and MF synapses. We also show that the two signal transduction cascades converge to activate a common effector, protein kinase C (PKC). Specifically, LTP at RC and MF synapses on the same SR/LM interneuron was blocked by postsynaptic injections of chelerythrine (10 μM). These data indicate that both forms of LTP share a common mechanism involving PKC-dependent signaling modulation. PMID:25637803

  16. Differential compartmentalization of Streptococcus pyogenes virulence factors and host protein binding properties as a mechanism for host adaptation.

    PubMed

    Kilsgård, Ola; Karlsson, Christofer; Malmström, Erik; Malmström, Johan

    2016-11-01

    Streptococcus pyogenes is an important human pathogen responsible for substantial morbidity and mortality worldwide. Although S. pyogenes is a strictly human pathogen with no other known animal reservoir, several murine infection models exist to explore different aspects of the bacterial pathogenesis. Inoculating mice with wild-type S. pyogenes strains can result in the generation of new bacterial phenotypes that are hypervirulent compared to the original inoculum. In this study, we used a serial mass spectrometry based proteomics strategy to investigate if these hypervirulent strains have an altered distribution of virulence proteins across the intracellular, surface associated and secreted bacterial compartments and if any change in compartmentalization can alter the protein-protein interaction network between bacteria and host proteins. Quantitative analysis of the S. pyogenes surface and secreted proteomes revealed that animal passaged strains are associated with significantly higher amount of virulence factors on the bacterial surface and in the media. This altered virulence factor compartmentalization results in increased binding of several mouse plasma proteins to the bacterial surface, a trend that was consistent for mouse plasma from several different mouse strains. In general, both the wild-type strain and animal passaged strain were capable of binding high amounts of human plasma proteins. However, compared to the non-passaged strains, the animal passaged strains displayed an increased ability to bind mouse plasma proteins, in particular for M protein binders, indicating that the increased affinity for mouse blood plasma proteins is a consequence of host adaptation of this pathogen to a new host. In conclusion, plotting the total amount of virulence factors against the total amount of plasma proteins associated to the bacterial surface could clearly separate out animal passaged strains from wild type strains indicating a virulence model that could

  17. Twenty years of fluorescence imaging of intracellular chloride

    PubMed Central

    Arosio, Daniele; Ratto, Gian Michele

    2014-01-01

    Chloride homeostasis has a pivotal role in controlling neuronal excitability in the adult brain and during development. The intracellular concentration of chloride is regulated by the dynamic equilibrium between passive fluxes through membrane conductances and the active transport mediated by importers and exporters. In cortical neurons, chloride fluxes are coupled to network activity by the opening of the ionotropic GABAA receptors that provides a direct link between the activity of interneurons and chloride fluxes. These molecular mechanisms are not evenly distributed and regulated over the neuron surface and this fact can lead to a compartmentalized control of the intracellular concentration of chloride. The inhibitory drive provided by the activity of the GABAA receptors depends on the direction and strength of the associated currents, which are ultimately dictated by the gradient of chloride, the main charge carrier flowing through the GABAA channel. Thus, the intracellular distribution of chloride determines the local strength of ionotropic inhibition and influences the interaction between converging excitation and inhibition. The importance of chloride regulation is also underlined by its involvement in several brain pathologies, including epilepsy and disorders of the autistic spectra. The full comprehension of the physiological meaning of GABAergic activity on neurons requires the measurement of the spatiotemporal dynamics of chloride fluxes across the membrane. Nowadays, there are several available tools for the task, and both synthetic and genetically encoded indicators have been successfully used for chloride imaging. Here, we will review the available sensors analyzing their properties and outlining desirable future developments. PMID:25221475

  18. Intracellularly Swollen Polypeptide Nanogel Assists Hepatoma Chemotherapy

    PubMed Central

    Shi, Bo; Huang, Kexin; Ding, Jianxun; Xu, Weiguo; Yang, Yu; Liu, Haiyan; Yan, Lesan; Chen, Xuesi

    2017-01-01

    Nowadays, chemotherapy is one of the principal modes of treatment for tumor patients. However, the traditional formulations of small molecule drugs show short circulation time, low tumor selectivity, and high toxicity to normal tissues. To address these problems, a facilely prepared, and pH and reduction dual-responsive polypeptide nanogel was prepared for selectively intracellular delivery of chemotherapy drug. As a model drug, doxorubicin (DOX) was loaded into the nanogel through a sequential dispersion and dialysis technique, resulting in a high drug loading efficiency (DLE) of 96.7 wt.%. The loading nanogel, defined as NG/DOX, exhibited a uniform spherical morphology with a mean hydrodynamic radius of 58.8 nm, pH and reduction dual-triggered DOX release, efficient cell uptake, and cell proliferation inhibition in vitro. Moreover, NG/DOX exhibited improved antitumor efficacy toward H22 hepatoma-bearing BALB/c mouse model compared with free DOX·HCl. Histopathological and immunohistochemical analyses were implemented to further confirm the tumor suppression activity of NG/DOX. Furthermore, the variations of body weight, histopathological morphology, bone marrow cell micronucleus rate, and white blood cell count verified that NG/DOX showed excellent safety in vivo. With these excellent properties in vitro and in vivo, the pH and reduction dual-responsive polypeptide nanogel exhibits great potential for on-demand intracellular delivery of antitumor drug, and holds good prospect for future clinical application. PMID:28255361

  19. Transit times and mean ages for nonautonomous and autonomous compartmental systems

    SciTech Connect

    Rasmussen, Martin; Hastings, Alan; Smith, Matthew J.; Agusto, Folashade B.; Chen-Charpentier, Benito M.; Hoffman, Forrest M.; Jiang, Jiang; Todd-Brown, Katherine E. O.; Wang, Ying; Wang, Ying -Ping; Luo, Yiqi

    2016-04-01

    In this study, we develop a theory for transit times and mean ages for nonautonomous compartmental systems. Using the McKendrick–von Förster equation, we show that the mean ages of mass in a compartmental system satisfy a linear nonautonomous ordinary differential equation that is exponentially stable. We then define a nonautonomous version of transit time as the mean age of mass leaving the compartmental system at a particular time and show that our nonautonomous theory generalises the autonomous case. We apply these results to study a nine-dimensional nonautonomous compartmental system modeling the terrestrial carbon cycle, which is a modification of the Carnegie–Ames–Stanford approach model, and we demonstrate that the nonautonomous versions of transit time and mean age differ significantly from the autonomous quantities when calculated for that model.

  20. Transit times and mean ages for nonautonomous and autonomous compartmental systems

    DOE PAGES

    Rasmussen, Martin; Hastings, Alan; Smith, Matthew J.; ...

    2016-04-01

    In this study, we develop a theory for transit times and mean ages for nonautonomous compartmental systems. Using the McKendrick–von Förster equation, we show that the mean ages of mass in a compartmental system satisfy a linear nonautonomous ordinary differential equation that is exponentially stable. We then define a nonautonomous version of transit time as the mean age of mass leaving the compartmental system at a particular time and show that our nonautonomous theory generalises the autonomous case. We apply these results to study a nine-dimensional nonautonomous compartmental system modeling the terrestrial carbon cycle, which is a modification of themore » Carnegie–Ames–Stanford approach model, and we demonstrate that the nonautonomous versions of transit time and mean age differ significantly from the autonomous quantities when calculated for that model.« less

  1. MULTIMEDIA ENVIRONMENTAL DISTRIBUTION OF TOXICS (MEND-TOX): PART I, HYBRID COMPARTMENTAL-SPATIAL MODELING FRAMEWORK

    EPA Science Inventory

    An integrated hybrid spatial-compartmental modeling approach is presented for analyzing the dynamic distribution of chemicals in the multimedia environment. Information obtained from such analysis, which includes temporal chemical concentration profiles in various media, mass ...

  2. Compartmentalization of the endoplasmic reticulum in the early C. elegans embryos

    PubMed Central

    Lee, Zuo Yen; Prouteau, Manoël

    2016-01-01

    The one-cell Caenorhabditis elegans embryo is polarized to partition fate determinants between the cell lineages generated during its first division. Using fluorescence loss in photobleaching, we find that the endoplasmic reticulum (ER) of the C. elegans embryo is physically continuous throughout the cell, but its membrane is compartmentalized shortly before nuclear envelope breakdown into an anterior and a posterior domain, indicating that a diffusion barrier forms in the ER membrane between these two domains. Using mutants with disorganized ER, we show that ER compartmentalization is independent of the morphological transition that the ER undergoes in mitosis. In contrast, compartmentalization takes place at the position of the future cleavage plane in a par-3–dependent manner. Together, our data indicate that the ER membrane is compartmentalized in cells as diverse as budding yeast, mouse neural stem cells, and the early C. elegans embryo. PMID:27597753

  3. Nuclear Pore-Like Structures in a Compartmentalized Bacterium

    PubMed Central

    Sagulenko, Evgeny; Green, Kathryn; Yee, Benjamin; Morgan, Garry; Leis, Andrew; Lee, Kuo-Chang; Butler, Margaret K.; Chia, Nicholas; Pham, Uyen Thi Phuong; Lindgreen, Stinus; Catchpole, Ryan; Poole, Anthony M.; Fuerst, John A.

    2017-01-01

    Planctomycetes are distinguished from other Bacteria by compartmentalization of cells via internal membranes, interpretation of which has been subject to recent debate regarding potential relations to Gram-negative cell structure. In our interpretation of the available data, the planctomycete Gemmata obscuriglobus contains a nuclear body compartment, and thus possesses a type of cell organization with parallels to the eukaryote nucleus. Here we show that pore-like structures occur in internal membranes of G.obscuriglobus and that they have elements structurally similar to eukaryote nuclear pores, including a basket, ring-spoke structure, and eight-fold rotational symmetry. Bioinformatic analysis of proteomic data reveals that some of the G. obscuriglobus proteins associated with pore-containing membranes possess structural domains found in eukaryote nuclear pore complexes. Moreover, immunogold labelling demonstrates localization of one such protein, containing a β-propeller domain, specifically to the G. obscuriglobus pore-like structures. Finding bacterial pores within internal cell membranes and with structural similarities to eukaryote nuclear pore complexes raises the dual possibilities of either hitherto undetected homology or stunning evolutionary convergence. PMID:28146565

  4. Development-based compartmentalization of the Drosophila central brain.

    PubMed

    Pereanu, Wayne; Kumar, Abilasha; Jennett, Arnim; Reichert, Heinrich; Hartenstein, Volker

    2010-08-01

    The neuropile of the Drosophila brain is subdivided into anatomically discrete compartments. Compartments are rich in terminal neurite branching and synapses; they are the neuropile domains in which signal processing takes place. Compartment boundaries are defined by more or less dense layers of glial cells as well as long neurite fascicles. These fascicles are formed during the larval period, when the approximately 100 neuronal lineages that constitute the Drosophila central brain differentiate. Each lineage forms an axon tract with a characteristic trajectory in the neuropile; groups of spatially related tracts congregate into the brain fascicles that can be followed from the larva throughout metamorphosis into the adult stage. Here we provide a map of the adult brain compartments and the relevant fascicles defining compartmental boundaries. We have identified the neuronal lineages contributing to each fascicle, which allowed us to compare compartments of the larval and adult brain directly. Most adult compartments can be recognized already in the early larval brain, where they form a "protomap" of the later adult compartments. Our analysis highlights the morphogenetic changes shaping the Drosophila brain; the data will be important for studies that link early-acting genetic mechanisms to the adult neuronal structures and circuits controlled by these mechanisms.

  5. Shape control and compartmentalization in active colloidal cells

    PubMed Central

    Spellings, Matthew; Engel, Michael; Klotsa, Daphne; Sabrina, Syeda; Drews, Aaron M.; Nguyen, Nguyen H. P.; Bishop, Kyle J. M.; Glotzer, Sharon C.

    2015-01-01

    Small autonomous machines like biological cells or soft robots can convert energy input into control of function and form. It is desired that this behavior emerges spontaneously and can be easily switched over time. For this purpose we introduce an active matter system that is loosely inspired by biology and which we term an active colloidal cell. The active colloidal cell consists of a boundary and a fluid interior, both of which are built from identical rotating spinners whose activity creates convective flows. Similarly to biological cell motility, which is driven by cytoskeletal components spread throughout the entire volume of the cell, active colloidal cells are characterized by highly distributed energy conversion. We demonstrate that we can control the shape of the active colloidal cell and drive compartmentalization by varying the details of the boundary (hard vs. flexible) and the character of the spinners (passive vs. active). We report buckling of the boundary controlled by the pattern of boundary activity, as well as formation of core–shell and inverted Janus phase-separated configurations within the active cell interior. As the cell size is increased, the inverted Janus configuration spontaneously breaks its mirror symmetry. The result is a bubble–crescent configuration, which alternates between two degenerate states over time and exhibits collective migration of the fluid along the boundary. Our results are obtained using microscopic, non–momentum-conserving Langevin dynamics simulations and verified via a phase-field continuum model coupled to a Navier–Stokes equation. PMID:26253763

  6. Limb, Respiratory and Masticatory Muscle Compartmentalization: Developmental and Hormonal Considerations

    PubMed Central

    Widmer, C. G.; Morris-Wiman, J.

    2011-01-01

    Neuromuscular compartments are subvolumes of muscle that have unique biomechanical actions and can be activated singly or in groups to perform the necessary task. Beside unique biomechanical actions, other evidence that supports the neuromuscular compartmentalization of muscles includes segmental reflexes that preferentially excite motoneurons from the same compartment, proportions of motor unit types that differ among compartments and a central partitioning of motoneurons that innervate each compartment. The current knowledge regarding neuromuscular compartments in representative muscles involved in locomotion, respiration and mastication is presented to compare and contrast these different motor systems. Developmental features of neuromuscular compartment formation in these three motor systems are reviewed to identify when these compartments are formed, their innervation patterns and the process of refinement to achieve the adult phenotype. Finally, the role of androgen modulation of neuromuscular compartment maturation in representative muscles of these motor systems is reviewed and the impact of testosterone on specific myosin heavy chain fiber types is discussed based on recent data. In summary, neuromuscular compartments are pre-patterned output elements in muscle that undergo refinement of compartment boundaries and muscle fiber phenotype during maturation. Further studies are needed to understand how these output elements are selectively controlled during locomotion, respiration and mastication. PMID:21111201

  7. Adaptive and neuroadaptive control for nonnegative and compartmental dynamical systems

    NASA Astrophysics Data System (ADS)

    Volyanskyy, Kostyantyn Y.

    Neural networks have been extensively used for adaptive system identification as well as adaptive and neuroadaptive control of highly uncertain systems. The goal of adaptive and neuroadaptive control is to achieve system performance without excessive reliance on system models. To improve robustness and the speed of adaptation of adaptive and neuroadaptive controllers several controller architectures have been proposed in the literature. In this dissertation, we develop a new neuroadaptive control architecture for nonlinear uncertain dynamical systems. The proposed framework involves a novel controller architecture with additional terms in the update laws that are constructed using a moving window of the integrated system uncertainty. These terms can be used to identify the ideal system weights of the neural network as well as effectively suppress system uncertainty. Linear and nonlinear parameterizations of the system uncertainty are considered and state and output feedback neuroadaptive controllers are developed. Furthermore, we extend the developed framework to discrete-time dynamical systems. To illustrate the efficacy of the proposed approach we apply our results to an aircraft model with wing rock dynamics, a spacecraft model with unknown moment of inertia, and an unmanned combat aerial vehicle undergoing actuator failures, and compare our results with standard neuroadaptive control methods. Nonnegative systems are essential in capturing the behavior of a wide range of dynamical systems involving dynamic states whose values are nonnegative. A sub-class of nonnegative dynamical systems are compartmental systems. These systems are derived from mass and energy balance considerations and are comprised of homogeneous interconnected microscopic subsystems or compartments which exchange variable quantities of material via intercompartmental flow laws. In this dissertation, we develop direct adaptive and neuroadaptive control framework for stabilization, disturbance

  8. Shape control and compartmentalization in active colloidal cells.

    PubMed

    Spellings, Matthew; Engel, Michael; Klotsa, Daphne; Sabrina, Syeda; Drews, Aaron M; Nguyen, Nguyen H P; Bishop, Kyle J M; Glotzer, Sharon C

    2015-08-25

    Small autonomous machines like biological cells or soft robots can convert energy input into control of function and form. It is desired that this behavior emerges spontaneously and can be easily switched over time. For this purpose we introduce an active matter system that is loosely inspired by biology and which we term an active colloidal cell. The active colloidal cell consists of a boundary and a fluid interior, both of which are built from identical rotating spinners whose activity creates convective flows. Similarly to biological cell motility, which is driven by cytoskeletal components spread throughout the entire volume of the cell, active colloidal cells are characterized by highly distributed energy conversion. We demonstrate that we can control the shape of the active colloidal cell and drive compartmentalization by varying the details of the boundary (hard vs. flexible) and the character of the spinners (passive vs. active). We report buckling of the boundary controlled by the pattern of boundary activity, as well as formation of core-shell and inverted Janus phase-separated configurations within the active cell interior. As the cell size is increased, the inverted Janus configuration spontaneously breaks its mirror symmetry. The result is a bubble-crescent configuration, which alternates between two degenerate states over time and exhibits collective migration of the fluid along the boundary. Our results are obtained using microscopic, non-momentum-conserving Langevin dynamics simulations and verified via a phase-field continuum model coupled to a Navier-Stokes equation.

  9. Compartmentalized nitric oxide signaling in the resistance vasculature

    PubMed Central

    Mutchler, Stephanie M.; Straub, Adam C.

    2015-01-01

    Nitric oxide (NO) was first described as a bioactive molecule through its ability to stimulate soluble guanylate cyclase, but the revelation that NO was the endothelium derived relaxation factor drove the field to its modern state. The wealth of research conducted over the past 30 years has provided us with a picture of how diverse NO signaling can be within the vascular wall, going beyond simple vasodilation to include such roles as signaling through protein S-nitrosation. This expanded view of NO’s actions requires highly regulated and compartmentalized production. Importantly, resistance arteries house multiple proteins involved in the production and transduction of NO allowing for efficient movement of the molecule to regulate vascular tone and reactivity. In this review, we focus on the many mechanisms regulating NO production and signaling action in the vascular wall, with a focus on the control of endothelial nitric oxide synthase (eNOS), the enzyme responsible for synthesizing most of the NO within these confines. We also explore how cross talk between the endothelium and smooth muscle in the microcirculation can modulate NO signaling, illustrating that this one small molecule has the capability to produce a plethora of responses. PMID:26028569

  10. Nanovehicular Intracellular Delivery Systems

    PubMed Central

    PROKOP, ALES; DAVIDSON, JEFFREY M.

    2013-01-01

    This article provides an overview of principles and barriers relevant to intracellular drug and gene transport, accumulation and retention (collectively called as drug delivery) by means of nanovehicles (NV). The aim is to deliver a cargo to a particular intracellular site, if possible, to exert a local action. Some of the principles discussed in this article apply to noncolloidal drugs that are not permeable to the plasma membrane or to the blood–brain barrier. NV are defined as a wide range of nanosized particles leading to colloidal objects which are capable of entering cells and tissues and delivering a cargo intracelullarly. Different localization and targeting means are discussed. Limited discussion on pharmacokinetics and pharmacodynamics is also presented. NVs are contrasted to micro-delivery and current nanotechnologies which are already in commercial use. Newer developments in NV technologies are outlined and future applications are stressed. We also briefly review the existing modeling tools and approaches to quantitatively describe the behavior of targeted NV within the vascular and tumor compartments, an area of particular importance. While we list “elementary” phenomena related to different level of complexity of delivery to cancer, we also stress importance of multi-scale modeling and bottom-up systems biology approach. PMID:18200527

  11. Compartmentalization of hepatitis C virus variants in patients with hepatocellular carcinoma.

    PubMed

    Pérez, Paula S; Di Lello, Federico A; Mullen, Eduardo G; Galdame, Omar A; Livellara, Beatriz I; Gadano, Adrián C; Campos, Rodolfo H; Flichman, Diego M

    2017-02-01

    Chronic Hepatitis C Virus (HCV) infection is a major risk for hepatocellular carcinoma (HCC) development. HCV Core protein has been associated with the modulation of potentially oncogenic cellular processes and E2 protein has been useful in evolutive studies to analyze the diversity of HCV. Thus, the aim of this study was to evaluate HCV compartmentalization in tumoral, non-tumoral liver tissue and serum and to identify viral mutations potentially involved in carcinogenesis. Samples were obtained from four patients with HCC who underwent liver transplantation. Core and E2 were amplified, cloned and sequenced. Phylogenies and BaTS Test were performed to analyze viral compartmentalization and a signature sequence analysis was conducted by VESPA. The likelihood and Bayesian phylogenies showed a wide degree of compartmentalization in the different patients, ranging from total clustering to a more scattered pattern with small groups. Nevertheless, the association test showed compartmentalization for the three compartments and both viral regions tested in all the patients. Signature amino acid pattern supported the compartmentalization in three of the cases for E2 protein and in two of them for Core. Changes observed in Core included polymorphism R70Q/H previously associated with HCC. In conclusion, evidence of HCV compartmentalization in the liver of HCC patients was provided and further biological characterization of these variants may contribute to the understanding of carcinogenesis mediated by HCV infection. © 2016 Wiley Periodicals, Inc.

  12. Imaging and controlling intracellular reactions: Lysosome transport as a function of diameter and the intracellular synthesis of conducting polymers

    NASA Astrophysics Data System (ADS)

    Payne, Christine

    2014-03-01

    Eukaryotic cells are the ultimate complex environment with intracellular chemical reactions regulated by the local cellular environment. For example, reactants are sequestered into specific organelles to control local concentration and pH, motor proteins transport reactants within the cell, and intracellular vesicles undergo fusion to bring reactants together. Current research in the Payne Lab in the School of Chemistry and Biochemistry at Georgia Tech is aimed at understanding and utilizing this complex environment to control intracellular chemical reactions. This will be illustrated using two examples, intracellular transport as a function of organelle diameter and the intracellular synthesis of conducting polymers. Using single particle tracking fluorescence microscopy, we measured the intracellular transport of lysosomes, membrane-bound organelles, as a function of diameter as they underwent transport in living cells. Both ATP-dependent active transport and diffusion were examined. As expected, diffusion scales with the diameter of the lysosome. However, active transport is unaffected suggesting that motor proteins are insensitive to cytosolic drag. In a second example, we utilize intracellular complexity, specifically the distinct micro-environments of different organelles, to carry out chemical reactions. We show that catalase, found in the peroxisomes of cells, can be used to catalyze the polymerization of the conducting polymer PEDOT:PSS. More importantly, we have found that a range of iron-containing biomolecules are suitable catalysts with different iron-containing biomolecules leading to different polymer properties. These experiments illustrate the advantage of intracellular complexity for the synthesis of novel materials.

  13. An intracellular anion channel critical for pigmentation.

    PubMed

    Bellono, Nicholas W; Escobar, Iliana E; Lefkovith, Ariel J; Marks, Michael S; Oancea, Elena

    2014-12-16

    Intracellular ion channels are essential regulators of organellar and cellular function, yet the molecular identity and physiological role of many of these channels remains elusive. In particular, no ion channel has been characterized in melanosomes, organelles that produce and store the major mammalian pigment melanin. Defects in melanosome function cause albinism, characterized by vision and pigmentation deficits, impaired retinal development, and increased susceptibility to skin and eye cancers. The most common form of albinism is caused by mutations in oculocutaneous albinism II (OCA2), a melanosome-specific transmembrane protein with unknown function. Here we used direct patch-clamp of skin and eye melanosomes to identify a novel chloride-selective anion conductance mediated by OCA2 and required for melanin production. Expression of OCA2 increases organelle pH, suggesting that the chloride channel might regulate melanin synthesis by modulating melanosome pH. Thus, a melanosomal anion channel that requires OCA2 is essential for skin and eye pigmentation.

  14. Efficient Vaccine Distribution Based on a Hybrid Compartmental Model

    PubMed Central

    Yu, Zhiwen; Liu, Jiming; Wang, Xiaowei; Zhu, Xianjun; Wang, Daxing; Han, Guoqiang

    2016-01-01

    To effectively and efficiently reduce the morbidity and mortality that may be caused by outbreaks of emerging infectious diseases, it is very important for public health agencies to make informed decisions for controlling the spread of the disease. Such decisions must incorporate various kinds of intervention strategies, such as vaccinations, school closures and border restrictions. Recently, researchers have paid increased attention to searching for effective vaccine distribution strategies for reducing the effects of pandemic outbreaks when resources are limited. Most of the existing research work has been focused on how to design an effective age-structured epidemic model and to select a suitable vaccine distribution strategy to prevent the propagation of an infectious virus. Models that evaluate age structure effects are common, but models that additionally evaluate geographical effects are less common. In this paper, we propose a new SEIR (susceptible—exposed—infectious šC recovered) model, named the hybrid SEIR-V model (HSEIR-V), which considers not only the dynamics of infection prevalence in several age-specific host populations, but also seeks to characterize the dynamics by which a virus spreads in various geographic districts. Several vaccination strategies such as different kinds of vaccine coverage, different vaccine releasing times and different vaccine deployment methods are incorporated into the HSEIR-V compartmental model. We also design four hybrid vaccination distribution strategies (based on population size, contact pattern matrix, infection rate and infectious risk) for controlling the spread of viral infections. Based on data from the 2009–2010 H1N1 influenza epidemic, we evaluate the effectiveness of our proposed HSEIR-V model and study the effects of different types of human behaviour in responding to epidemics. PMID:27233015

  15. Deciphering Neuron-Glia Compartmentalization in Cortical Energy Metabolism

    PubMed Central

    Jolivet, Renaud; Magistretti, Pierre J.; Weber, Bruno

    2009-01-01

    Energy demand is an important constraint on neural signaling. Several methods have been proposed to assess the energy budget of the brain based on a bottom-up approach in which the energy demand of individual biophysical processes are first estimated independently and then summed up to compute the brain's total energy budget. Here, we address this question using a novel approach that makes use of published datasets that reported average cerebral glucose and oxygen utilization in humans and rodents during different activation states. Our approach allows us (1) to decipher neuron-glia compartmentalization in energy metabolism and (2) to compute a precise state-dependent energy budget for the brain. Under the assumption that the fraction of energy used for signaling is proportional to the cycling of neurotransmitters, we find that in the activated state, most of the energy (∼80%) is oxidatively produced and consumed by neurons to support neuron-to-neuron signaling. Glial cells, while only contributing for a small fraction to energy production (∼6%), actually take up a significant fraction of glucose (50% or more) from the blood and provide neurons with glucose-derived energy substrates. Our results suggest that glycolysis occurs for a significant part in astrocytes whereas most of the oxygen is utilized in neurons. As a consequence, a transfer of glucose-derived metabolites from glial cells to neurons has to take place. Furthermore, we find that the amplitude of this transfer is correlated to (1) the activity level of the brain; the larger the activity, the more metabolites are shuttled from glia to neurons and (2) the oxidative activity in astrocytes; with higher glial pyruvate metabolism, less metabolites are shuttled from glia to neurons. While some of the details of a bottom-up biophysical approach have to be simplified, our method allows for a straightforward assessment of the brain's energy budget from macroscopic measurements with minimal underlying

  16. Compartmentalized immune response reflects clinical severity of beryllium disease.

    PubMed

    Newman, L S; Bobka, C; Schumacher, B; Daniloff, E; Zhen, B; Mroz, M M; King, T E

    1994-07-01

    Although beryllium disease has been associated with a bronchoalveolar lavage (BAL) lymphocytosis and T cell-mediated immune response, we do not know if either the BAL cellular profile or the compartmentalized pulmonary response to the antigen reflect the severity of the disease. We studied 110 subjects divided into three groups of subjects: beryllium disease patients (n = 55), beryllium-sensitized patients without disease (n = 8), and control subjects (n = 47). Evaluation included completion of a respiratory symptom questionnaire, clinical examination, chest radiograph, spirometry, body plethysmographic lung volumes, and diffusing capacity (DLCO). In the patient groups, we performed maximal exercise testing with an indwelling arterial line. In addition, we examined BAL and performed blood and BAL beryllium lymphocyte transformation tests (BeLT) as measures of the beryllium-specific T cell-mediated response in these two compartments. In beryllium disease patients we correlated the BAL cellular constituents with clinical parameters indicative of disease severity. Beryllium disease patients exhibited elevated numbers of white cells and lymphocytes in BAL compared with both other groups; however, this lymphocytic alveolitis was significantly obscured in smokers. The BAL cellular constituents correlated with BAL BeLT but not with the blood BeLT. BAL cellular constituents also correlated with the radiographic profusion of small opacities, FEV1/FVC, DLCO, maximal achievable work load, VO2max, and measures of gas exchange at rest and at maximum exercise. We conclude that the lymphocyte-predominant pulmonary inflammatory response in beryllium disease is related to the magnitude of the localized response to antigen and that BAL cellularity, differential cell count, and BeLT reflect beryllium disease clinical severity.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. Mechanisms of salt tolerance in habanero pepper plants (Capsicum chinense Jacq.): Proline accumulation, ions dynamics and sodium root-shoot partition and compartmentation

    PubMed Central

    Bojórquez-Quintal, Emanuel; Velarde-Buendía, Ana; Ku-González, Ángela; Carillo-Pech, Mildred; Ortega-Camacho, Daniela; Echevarría-Machado, Ileana; Pottosin, Igor; Martínez-Estévez, Manuel

    2014-01-01

    Despite its economic relevance, little is known about salt tolerance mechanisms in pepper plants. To address this question, we compared differences in responses to NaCl in two Capsicum chinense varieties: Rex (tolerant) and Chichen-Itza (sensitive). Under salt stress (150 mM NaCl over 7 days) roots of Rex variety accumulated 50 times more compatible solutes such as proline compared to Chichen-Itza. Mineral analysis indicated that Na+ is restricted to roots by preventing its transport to leaves. Fluorescence analysis suggested an efficient Na+ compartmentalization in vacuole-like structures and in small intracellular compartments in roots of Rex variety. At the same time, Na+ in Chichen-Itza plants was compartmentalized in the apoplast, suggesting substantial Na+ extrusion. Rex variety was found to retain more K+ in its roots under salt stress according to a mineral analysis and microelectrode ion flux estimation (MIFE). Vanadate-sensitive H+ efflux was higher in Chichen-Itza variety plants, suggesting a higher activity of the plasma membrane H+-ATPase, which fuels the extrusion of Na+, and, possibly, also the re-uptake of K+. Our results suggest a combination of stress tolerance mechanisms, in order to alleviate the salt-induced injury. Furthermore, Na+ extrusion to apoplast does not appear to be an efficient strategy for salt tolerance in pepper plants. PMID:25429292

  18. Mechanisms of salt tolerance in habanero pepper plants (Capsicum chinense Jacq.): Proline accumulation, ions dynamics and sodium root-shoot partition and compartmentation.

    PubMed

    Bojórquez-Quintal, Emanuel; Velarde-Buendía, Ana; Ku-González, Angela; Carillo-Pech, Mildred; Ortega-Camacho, Daniela; Echevarría-Machado, Ileana; Pottosin, Igor; Martínez-Estévez, Manuel

    2014-01-01

    Despite its economic relevance, little is known about salt tolerance mechanisms in pepper plants. To address this question, we compared differences in responses to NaCl in two Capsicum chinense varieties: Rex (tolerant) and Chichen-Itza (sensitive). Under salt stress (150 mM NaCl over 7 days) roots of Rex variety accumulated 50 times more compatible solutes such as proline compared to Chichen-Itza. Mineral analysis indicated that Na(+) is restricted to roots by preventing its transport to leaves. Fluorescence analysis suggested an efficient Na(+) compartmentalization in vacuole-like structures and in small intracellular compartments in roots of Rex variety. At the same time, Na(+) in Chichen-Itza plants was compartmentalized in the apoplast, suggesting substantial Na(+) extrusion. Rex variety was found to retain more K(+) in its roots under salt stress according to a mineral analysis and microelectrode ion flux estimation (MIFE). Vanadate-sensitive H(+) efflux was higher in Chichen-Itza variety plants, suggesting a higher activity of the plasma membrane H(+)-ATPase, which fuels the extrusion of Na(+), and, possibly, also the re-uptake of K(+). Our results suggest a combination of stress tolerance mechanisms, in order to alleviate the salt-induced injury. Furthermore, Na(+) extrusion to apoplast does not appear to be an efficient strategy for salt tolerance in pepper plants.

  19. A protein–dye hybrid system as a narrow range tunable intracellular pH sensor† †Electronic supplementary information (ESI) available: Figures depicting various photophysical properties, cytotoxicity studies and confocal fluorescence images. See DOI: 10.1039/c6sc02659a Click here for additional data file.

    PubMed Central

    Anees, Palapuravan; Sudheesh, Karivachery V.; Jayamurthy, Purushothaman; Chandrika, Arunkumar R.; Omkumar, Ramakrishnapillai V.

    2016-01-01

    Accurate monitoring of pH variations inside cells is important for the early diagnosis of diseases such as cancer. Even though a variety of different pH sensors are available, construction of a custom-made sensor array for measuring minute variations in a narrow biological pH window, using easily available constituents, is a challenge. Here we report two-component hybrid sensors derived from a protein and organic dye nanoparticles whose sensitivity range can be tuned by choosing different ratios of the components, to monitor the minute pH variations in a given system. The dye interacts noncovalently with the protein at lower pH and covalently at higher pH, triggering two distinguishable fluorescent signals at 700 and 480 nm, respectively. The pH sensitivity region of the probe can be tuned for every unit of the pH window resulting in custom-made pH sensors. These narrow range tunable pH sensors have been used to monitor pH variations in HeLa cells using the fluorescence imaging technique. PMID:28042467

  20. Characterization of intracellular pteroylpolyglutamate hydrolase (PPH) from human intestinal mucosa

    SciTech Connect

    Wang, T.T.Y.; Chandler, C.J.; Halsted, C.H.

    1986-03-01

    There are two forms of pteroylpolyglutamate hydrolase (PPH) in the human intestinal mucosa, one in the brush border membrane and the other intracellular; brush border PPH is an exopeptidase with optimal activity at pH 6.5 and a requirement for zinc. The presence study characterized human intracellular PPH and compared its properties to those of brush border PPH. Intracellular PPH was purified 30-fold. The enzyme had a MW of 75,000 by gel filtration, was optimally active at pH 4.5, and had an isoelectric point at pH 8.0. In contrast to brush border PPH, intracellular PPH was unstable at increasing temperatures, was unaffected by dialysis against chelating agents and showed no requirement for Zn/sup 2 +/. Using PteGlu/sub 2/(/sup 14/C)Glu as substrate, they demonstrated a K/sub m/ of 1.2 ..mu..M and increasing affinity for folates with longer glutamate chains. Intracellular PPH required the complete folic acid (PteGlu) moiety and a ..gamma..-glutamyl linkage for activity. Using ion exchange chromatography and an HPLC method to determine the hydrolytic products of the reaction, they found intracellular PPH could cleave both internal and terminal ..gamma..-glutamyl linkages, with PteGlu as an end product. After subcellular fractionation of the mucosa, PPH was found in the lysosomes. In summary, the distinct characteristics of brush border and intracellular PPH suggest that the two hydrolases serve different roles in folate metabolism.

  1. Spatial Phosphoprotein Profiling Reveals a Compartmentalized Extracellular Signal-regulated Kinase Switch Governing Neurite Growth and Retraction

    SciTech Connect

    Wang, Yingchun; Yang, Feng; Fu, Yi; Huang, Xiahe; Wang, Wei; Jiang, Xining; Gritsenko, Marina A.; Zhao, Rui; Monroe, Matthew E.; Pertz, Olivier C.; Purvine, Samuel O.; Orton, Daniel J.; Jacobs, Jon M.; Camp, David G.; Smith, Richard D.; Klemke, Richard L.

    2011-05-20

    Abstract - Brain development and spinal cord regeneration require neurite sprouting and growth cone navigation in response to extension and collapsing factors present in the extracellular environment. These external guidance cues control neurite growth cone extension and retraction processes through intracellular protein phosphorylation of numerous cytoskeletal, adhesion, and polarity complex signaling proteins. However, the complex kinase/substrate signaling networks that mediate neuritogenesis have not been investigated. Here, we compare the neurite phosphoproteome under growth and retraction conditions using neurite purification methodology combined with mass spectrometry. More than 4000 non-redundant phosphorylation sites from 1883 proteins have been annotated and mapped to signaling pathways that control kinase/phosphatase networks, cytoskeleton remodeling, and axon/dendrite specification. Comprehensive informatics and functional studies revealed a compartmentalized ERK activation/deactivation cytoskeletal switch that governs neurite growth and retraction, respectively. Our findings provide the first system-wide analysis of the phosphoprotein signaling networks that enable neurite growth and retraction and reveal an important molecular switch that governs neuritogenesis.

  2. The role of extracellular conductivity profiles in compartmental models for neurons: particulars for layer 5 pyramidal cells.

    PubMed

    Wang, Kai; Riera, Jorge; Enjieu-Kadji, Herve; Kawashima, Ryuta

    2013-07-01

    With the rapid increase in the number of technologies aimed at observing electric activity inside the brain, scientists have felt the urge to create proper links between intracellular- and extracellular-based experimental approaches. Biophysical models at both physical scales have been formalized under assumptions that impede the creation of such links. In this work, we address this issue by proposing a multicompartment model that allows the introduction of complex extracellular and intracellular resistivity profiles. This model accounts for the geometrical and electrotonic properties of any type of neuron through the combination of four devices: the integrator, the propagator, the 3D connector, and the collector. In particular, we applied this framework to model the tufted pyramidal cells of layer 5 (PCL5) in the neocortex. Our model was able to reproduce the decay and delay curves of backpropagating action potentials (APs) in this type of cell with better agreement with experimental data. We used the voltage drops of the extracellular resistances at each compartment to approximate the local field potentials generated by a PCL5 located in close proximity to linear microelectrode arrays. Based on the voltage drops produced by backpropagating APs, we were able to estimate the current multipolar moments generated by a PCL5. By adding external current sources in parallel to the extracellular resistances, we were able to create a sensitivity profile of PCL5 to electric current injections from nearby microelectrodes. In our model for PCL5, the kinetics and spatial profile of each ionic current were determined based on a literature survey, and the geometrical properties of these cells were evaluated experimentally. We concluded that the inclusion of the extracellular space in the compartmental models of neurons as an extra electrotonic medium is crucial for the accurate simulation of both the propagation of the electric potentials along the neuronal dendrites and the

  3. Natural isotopic signatures of variations in body nitrogen fluxes: a compartmental model analysis.

    PubMed

    Poupin, Nathalie; Mariotti, François; Huneau, Jean-François; Hermier, Dominique; Fouillet, Hélène

    2014-10-01

    Body tissues are generally 15N-enriched over the diet, with a discrimination factor (Δ15N) that varies among tissues and individuals as a function of their nutritional and physiopathological condition. However, both 15N bioaccumulation and intra- and inter-individual Δ15N variations are still poorly understood, so that theoretical models are required to understand their underlying mechanisms. Using experimental Δ15N measurements in rats, we developed a multi-compartmental model that provides the first detailed representation of the complex functioning of the body's Δ15N system, by explicitly linking the sizes and Δ15N values of 21 nitrogen pools to the rates and isotope effects of 49 nitrogen metabolic fluxes. We have shown that (i) besides urea production, several metabolic pathways (e.g., protein synthesis, amino acid intracellular metabolism, urea recycling and intestinal absorption or secretion) are most probably associated with isotope fractionation and together contribute to 15N accumulation in tissues, (ii) the Δ15N of a tissue at steady-state is not affected by variations of its P turnover rate, but can vary according to the relative orientation of tissue free amino acids towards oxidation vs. protein synthesis, (iii) at the whole-body level, Δ15N variations result from variations in the body partitioning of nitrogen fluxes (e.g., urea production, urea recycling and amino acid exchanges), with or without changes in nitrogen balance, (iv) any deviation from the optimal amino acid intake, in terms of both quality and quantity, causes a global rise in tissue Δ15N, and (v) Δ15N variations differ between tissues depending on the metabolic changes involved, which can therefore be identified using simultaneous multi-tissue Δ15N measurements. This work provides proof of concept that Δ15N measurements constitute a new promising tool to investigate how metabolic fluxes are nutritionally or physiopathologically reorganized or altered. The existence of such

  4. Natural Isotopic Signatures of Variations in Body Nitrogen Fluxes: A Compartmental Model Analysis

    PubMed Central

    Poupin, Nathalie; Mariotti, François; Huneau, Jean-François; Hermier, Dominique; Fouillet, Hélène

    2014-01-01

    Body tissues are generally 15N-enriched over the diet, with a discrimination factor (Δ15N) that varies among tissues and individuals as a function of their nutritional and physiopathological condition. However, both 15N bioaccumulation and intra- and inter-individual Δ15N variations are still poorly understood, so that theoretical models are required to understand their underlying mechanisms. Using experimental Δ15N measurements in rats, we developed a multi-compartmental model that provides the first detailed representation of the complex functioning of the body's Δ15N system, by explicitly linking the sizes and Δ15N values of 21 nitrogen pools to the rates and isotope effects of 49 nitrogen metabolic fluxes. We have shown that (i) besides urea production, several metabolic pathways (e.g., protein synthesis, amino acid intracellular metabolism, urea recycling and intestinal absorption or secretion) are most probably associated with isotope fractionation and together contribute to 15N accumulation in tissues, (ii) the Δ15N of a tissue at steady-state is not affected by variations of its P turnover rate, but can vary according to the relative orientation of tissue free amino acids towards oxidation vs. protein synthesis, (iii) at the whole-body level, Δ15N variations result from variations in the body partitioning of nitrogen fluxes (e.g., urea production, urea recycling and amino acid exchanges), with or without changes in nitrogen balance, (iv) any deviation from the optimal amino acid intake, in terms of both quality and quantity, causes a global rise in tissue Δ15N, and (v) Δ15N variations differ between tissues depending on the metabolic changes involved, which can therefore be identified using simultaneous multi-tissue Δ15N measurements. This work provides proof of concept that Δ15N measurements constitute a new promising tool to investigate how metabolic fluxes are nutritionally or physiopathologically reorganized or altered. The existence of such

  5. Intracellular Sterol Dynamics

    PubMed Central

    Mesmin, Bruno; Maxfield, Frederick R.

    2009-01-01

    We review the cellular mechanisms implicated in cholesterol trafficking and distribution. Recent studies have provided new information about the distribution of sterols within cells, including analysis of its transbilayer distribution. The cholesterol interaction with other lipids and its engagement in various trafficking processes will determine its proper level in a specific membrane; making the cholesterol distribution uneven among the various intracellular organelles. The cholesterol content is important since cholesterol plays an essential role in membranes by controlling their physicochemical properties as well as key cellular events such as signal transduction and protein trafficking. Cholesterol movement between cellular organelles is highly dynamic, and can be achieved by vesicular and non-vesicular processes. Various studies have analyzed the proteins that play a significant role in these processes, giving us new information about the relative importance of these two trafficking pathways in cholesterol transport. Although still poorly characterized in many trafficking routes, several potential sterol transport proteins have been described in detail; as a result, molecular mechanisms for sterol transport among membranes start to be appreciated. PMID:19286471

  6. Compartmentalized Intrapulmonary Pharmacokinetics of Amphotericin B and Its Lipid Formulations

    PubMed Central

    Groll, Andreas H.; Lyman, Caron A.; Petraitis, Vidmantas; Petraitiene, Ruta; Armstrong, Derek; Mickiene, Diana; Alfaro, Raul M.; Schaufele, Robert L.; Sein, Tin; Bacher, John; Walsh, Thomas J.

    2006-01-01

    We investigated the compartmentalized intrapulmonary pharmacokinetics of amphotericin B and its lipid formulations in healthy rabbits. Cohorts of three to seven noninfected, catheterized rabbits received 1 mg of amphotericin B deoxycholate (DAMB) per kg of body weight or 5 mg of either amphotericin B colloidal dispersion (ABCD), amphotericin B lipid complex (ABLC), or liposomal amphotericin B (LAMB) per kg once daily for a total of 8 days. Following sparse serial plasma sampling, rabbits were sacrificed 24 h after the last dose, and epithelial lining fluid (ELF), pulmonary alveolar macrophages (PAM), and lung tissue were obtained. Pharmacokinetic parameters in plasma were derived by model-independent techniques, and concentrations in ELF and PAM were calculated based on the urea dilution method and macrophage cell volume, respectively. Mean amphotericin B concentrations ± standard deviations (SD) in lung tissue and PAM were highest in ABLC-treated animals, exceeding concurrent plasma levels by 70- and 375-fold, respectively (in lung tissue, 16.24 ± 1.62 versus 2.71 ± 1.22, 6.29 ± 1.17, and 6.32 ± 0.57 μg/g for DAMB-, ABCD-, and LAMB-treated animals, respectively [P = 0.0029]; in PAM, 89.1 ± 37.0 versus 8.92 ± 2.89, 5.43 ± 1.75, and 7.52 ± 2.50 μg/ml for DAMB-, ABCD-, and LAMB-treated animals, respectively [P = 0.0246]). By comparison, drug concentrations in ELF were much lower than those achieved in lung tissue and PAM. Among the different cohorts, the highest ELF concentrations were found in LAMB-treated animals (2.28 ± 1.43 versus 0.44 ± 0.13, 0.68 ± 0.27, and 0.90 ± 0.28 μg/ml in DAMB-, ABCD-, and ABLC-treated animals, respectively [P = 0.0070]). In conclusion, amphotericin B and its lipid formulations displayed strikingly different patterns of disposition in lungs 24 h after dosing. Whereas the disposition of ABCD was overall not fundamentally different from that of DAMB, ABLC showed prominent accumulation in lung tissue and PAM, while LAMB

  7. B cells undergo unique compartmentalized redistribution in multiple sclerosis.

    PubMed

    Haas, Jürgen; Bekeredjian-Ding, Isabelle; Milkova, Miriam; Balint, Bettina; Schwarz, Alexander; Korporal, Mirjam; Jarius, Sven; Fritz, Brigitte; Lorenz, Hanns-Martin; Wildemann, Brigitte

    2011-12-01

    Increasing evidence fosters the role of B cells (BC) in multiple sclerosis (MS). The compartmentalized distribution of BC in blood and cerebrospinal fluid (CSF) is incompletely understood. In this study, we analyzed BC-patterns and BC-immunoreactivity at these sites during active and during stable disease and the impact of disease modifying drugs (DMD) on peripheral BC-homeostasis. For this purpose we assessed BC-subsets in blood and CSF from patients with clinically isolated syndrome (CIS), relapsing remitting MS (RRMS), rheumatoid arthritis (RA), and healthy controls (HC) by flow cytometric detection of whole (W-BC), naïve, transitional (TN-BC), class-switched memory (CSM-BC), unswitched memory (USM-BC), double-negative memory (DNM-BC) BC-phenotypes, plasma blasts (PB), and plasma cells (PC). FACS-data were correlated with BC-specific chemotactic activities in CSF, intrathecal CXCL13-levels, and immunoreactivity of peripheral W-BC. Our study revealed that frequencies of systemic CSM-BC/USM-BC became contracted in active CIS/MS while proportions of naive BC, TN-BC and DNM-BC were reciprocally expanded. Moreover, the shifted BC-composition promoted reduced immunoreactivity of W-BC and resolved during remission. Cross-over changes in CSF included privileged accumulation of CSM-BC linked to intrathecal CXCL13-concentrations and expansion of PB/PC. Treatment with interferon-beta and natalizumab evoked distinct though differing redistribution of circulating BC-subsets. We conclude that symptomatic CIS and MS are accompanied by distinctive changes in peripheral and CSF BC-homeostasis. The privileged reciprocal distribution between naïve versus CSM-phenotypes in both compartments together with the marked chemotactic driving force towards BC prompted by CSF supernatants renders it likely that CSF BC are mainly recruited from peripheral blood during active CIS/MS, whereas constantly low percentages of circulating PB/PC and their failure to respond to migratory stimuli

  8. Treatment of Mycobacterium intracellulare Infected Mice with Walter Reed Compound H

    DTIC Science & Technology

    1980-09-25

    including Mycobacterium tuberculosis, Mycobacterium leprae , Staphylococcusaureus, Pseudomonas aeruginosa, Escherichia coli, enterococ----ci, Neissr•~n...97 SAD "Treatment of Mycobacterium intracellulare Infected Mice with Walter Reed Compound H" Final Comprehensive Report J. Kenneth McClatchy, Ph.D...REPORT & PERIOD COVERED "TREATMENT OF MYCOBACTERIUM INTRACELLULARE - Final Comprehensive Report INFECTED MICE WITH WALTER REED COMPOUND H"li G

  9. Na+ compartmentalization related to salinity stress tolerance in upland cotton (Gossypium hirsutum) seedlings

    PubMed Central

    Peng, Zhen; He, Shoupu; Sun, Junling; Pan, Zhaoe; Gong, Wenfang; Lu, Yanli; Du, Xiongming

    2016-01-01

    The capacity for ion compartmentalization among different tissues and cells is the key mechanism regulating salt tolerance in plants. In this study, we investigated the ion compartmentalization capacity of two upland cotton genotypes with different salt tolerances under salt shock at the tissue, cell and molecular levels. We found that the leaf glandular trichome could secrete more salt ions in the salt-tolerant genotype than in the sensitive genotype, demonstrating the excretion of ions from tissue may be a new mechanism to respond to short-term salt shock. Furthermore, an investigation of the ion distribution demonstrated that the ion content was significantly lower in critical tissues and cells of the salt-tolerant genotype, indicating the salt-tolerant genotype had a greater capacity for ion compartmentalization in the shoot. By comparing the membrane H+-ATPase activity and the expression of ion transportation-related genes, we found that the H+-ATPase activity and Na+/H+ antiporter are the key factors determining the capacity for ion compartmentalization in leaves, which might further determine the salt tolerance of cotton. The novel function of the glandular trichome and the comparison of Na+ compartmentalization between two cotton genotypes with contrasting salt tolerances provide a new understanding of the salt tolerance mechanism in cotton. PMID:27698468

  10. Dissociation energies of PH and PH+.

    NASA Astrophysics Data System (ADS)

    Reddy, R. R.; Nazeer Ahammed, Y.; Srinivasa Rao, A.; Rao, T. V. R.

    1995-12-01

    Dissociation energies for the ground electronic states of diatomic PH and PH+ are determined by fitting empirical potential functions to the respective RKRV curves using correlation coefficients. The estimated ground state dissociation energies of PH and PH+ are 3.10 and 3.20 eV respectively by the curve fitting procedure using the Lippincott potential function. The computed values are in good agreement with experimental values.

  11. Preventing age-related decline of gut compartmentalization limits microbiota dysbiosis and extends lifespan

    PubMed Central

    Li, Hongjie; Qi, Yanyan; Jasper, Heinrich

    2016-01-01

    Summary Compartmentalization of the gastrointestinal (GI) tract of metazoans is critical for health. GI compartments contain specific microbiota, and microbiota dysbiosis is associated with intestinal dysfunction. Dysbiosis develops in aging intestines, yet how this relates to changes in GI compartmentalization remains unclear. The Drosophila GI tract is an accessible model to address this question. Here we show that the stomach-like copper cell region (CCR) in the middle midgut controls distribution and composition of the microbiota. We find that chronic activation of JAK/Stat signaling in the aging gut induces a metaplasia of the gastric epithelium, CCR decline, and subsequent commensal dysbiosis and epithelial dysplasia along the GI tract. Accordingly, inhibition of JAK/Stat signaling in the CCR specifically prevents age-related metaplasia, commensal dysbiosis and functional decline in old guts, and extends lifespan. Our results establish a mechanism by which age-related chronic inflammation causes the decline of intestinal compartmentalization and microbiota dysbiosis, limiting lifespan. PMID:26867182

  12. Global identifiability of linear compartmental models--a computer algebra algorithm.

    PubMed

    Audoly, S; D'Angiò, L; Saccomani, M P; Cobelli, C

    1998-01-01

    A priori global identifiability deals with the uniqueness of the solution for the unknown parameters of a model and is, thus, a prerequisite for parameter estimation of biological dynamic models. Global identifiability is however difficult to test, since it requires solving a system of algebraic nonlinear equations which increases both in nonlinearity degree and number of terms and unknowns with increasing model order. In this paper, a computer algebra tool, GLOBI (GLOBal Identifiability) is presented, which combines the topological transfer function method with the Buchberger algorithm, to test global identifiability of linear compartmental models. GLOBI allows for the automatic testing of a priori global identifiability of general structure compartmental models from general multi input-multi output experiments. Examples of usage of GLOBI to analyze a priori global identifiability of some complex biological compartmental models are provided.

  13. Association of efferent neurons to the compartmental architecture of the superior colliculus.

    PubMed Central

    Illing, R B

    1992-01-01

    The superior colliculus is a layered structure in the mammalian midbrain serving multimodal sensorimotor integration. Its intermediate layers are characterized by a compartmental architecture. These compartments are apparent through the clustering of terminals of major collicular afferents, which in many instances match the heterogeneous distribution of tissue components such as acetylcholinesterase, choline acetyltransferase, substance P, and parvalbumin. The present study was undertaken to determine whether efferent cells observe this compartmental architecture. It was found that subpopulations of both descending and ascending collicular efferents originate from perikarya situated in characteristic positions relative to the collicular compartments defined by elevated acetylcholinesterase activity and that their dendrites appear to be specifically coordinated with the heterogeneous environment. With the specific interlocking of afferent and efferent neurons through spatially distinguished neural networks, the compartmental architecture apparently constitutes an essential element for the determination of information flow in the superior colliculus. Images PMID:1438296

  14. Compartmentalization of incompatible reagents within Pickering emulsion droplets for one-pot cascade reactions.

    PubMed

    Yang, Hengquan; Fu, Luman; Wei, Lijuan; Liang, Jifen; Binks, Bernard P

    2015-01-28

    It is a dream that future synthetic chemistry can mimic living systems to process multistep cascade reactions in a one-pot fashion. One of the key challenges is the mutual destruction of incompatible or opposing reagents, for example, acid and base, oxidants and reductants. A conceptually novel strategy is developed here to address this challenge. This strategy is based on a layered Pickering emulsion system, which is obtained through lamination of Pickering emulsions. In this working Pickering emulsion, the dispersed phase can separately compartmentalize the incompatible reagents to avoid their mutual destruction, while the continuous phase allows other reagent molecules to diffuse freely to access the compartmentalized reagents for chemical reactions. The compartmentalization effects and molecular transport ability of the Pickering emulsion were investigated. The deacetalization-reduction, deacetalization-Knoevenagel, deacetalization-Henry and diazotization-iodization cascade reactions demonstrate well the versatility and flexibility of our strategy in processing the one-pot cascade reactions involving mutually destructive reagents.

  15. Preventing Age-Related Decline of Gut Compartmentalization Limits Microbiota Dysbiosis and Extends Lifespan.

    PubMed

    Li, Hongjie; Qi, Yanyan; Jasper, Heinrich

    2016-02-10

    Compartmentalization of the gastrointestinal (GI) tract of metazoans is critical for health. GI compartments contain specific microbiota, and microbiota dysbiosis is associated with intestinal dysfunction. Dysbiosis develops in aging intestines, yet how this relates to changes in GI compartmentalization remains unclear. The Drosophila GI tract is an accessible model to address this question. Here we show that the stomach-like copper cell region (CCR) in the middle midgut controls distribution and composition of the microbiota. We find that chronic activation of JAK/Stat signaling in the aging gut induces a metaplasia of the gastric epithelium, CCR decline, and subsequent commensal dysbiosis and epithelial dysplasia along the GI tract. Accordingly, inhibition of JAK/Stat signaling in the CCR specifically prevents age-related metaplasia, commensal dysbiosis and functional decline in old guts, and extends lifespan. Our results establish a mechanism by which age-related chronic inflammation causes the decline of intestinal compartmentalization and microbiota dysbiosis, limiting lifespan.

  16. Compartmentalization of cyclic nucleotide signaling: A question of when, where, and why?

    PubMed Central

    Arora, Kavisha; Sinha, Chandrima; Zhang, Weiqiang; Ren, Aixia; Moon, Chang Suk; Yarlagadda, Sunitha; Naren, Anjaparavanda P.

    2013-01-01

    Preciseness of cellular behavior depends upon how an extracellular cue mobilizes a correct orchestra of cellular messengers and effector proteins spatially and temporally. This concept, termed compartmentalization of cellular signaling, is now known to form the molecular basis of many aspects of cellular behavior in health and disease. The cyclic nucleotides cAMP and cGMP are ubiquitous cellular messengers that can be compartmentalized in three ways: first, by their physical containment; second, by formation of multiple protein signaling complexes; and third, by their selective depletion. Compartmentalized cyclic nucleotide signaling is a very prevalent response among all cell types. In order to understand how it becomes relevant to cellular behavior, it is important to know how it is executed in cells to regulate physiological responses and, also, how its execution or dysregulation can lead to a pathophysiological condition, which forms the current scope of the presented review. PMID:23604972

  17. Compartmental models of rat cerebellar Purkinje cells based on simultaneous somatic and dendritic patch-clamp recordings

    PubMed Central

    Roth, Arnd; Häusser, Michael

    2001-01-01

    Simultaneous dendritic and somatic patch-clamp recordings were made from Purkinje cells in cerebellar slices from 12- to 21-day-old rats. Voltage responses to current impulses injected via either the dendritic or the somatic pipette were obtained in the presence of the selective Ih blocker ZD 7288 and blockers of spontaneous synaptic input. Neurons were filled with biocytin for subsequent morphological reconstruction. Four neurons were reconstructed and converted into detailed compartmental models. The specific membrane capacitance (Cm), specific membrane resistance (Rm) and intracellular resistivity (Ri) were optimized by direct fitting of the model responses to the electrophysiological data from the same cell. Mean values were: Cm, 0.77 ± 0.17 μF cm−2 (mean ±s.d.; range, 0.64-1.00 μF cm−2), Rm, 122 ± 18 kΩ cm2 (98-141 kΩ cm2) and Ri, 115 ± 20 Ω cm (93-142 Ω cm). The steady-state electrotonic architecture of these cells was compact under the experimental conditions used. However, somatic voltage-clamp recordings of parallel fibre and climbing fibre synaptic currents were substantially filtered and attenuated. The detailed models were compared with a two-compartment model of Purkinje cells. The range of synaptic current kinetics that can be faithfully recorded using somatic voltage clamp is predicted fairly well by the two-compartment model, even though some of its underlying assumptions are violated. A model of Ih was constructed based on voltage-clamp data, and inserted into the passive compartmental models. Somatic EPSP amplitude was substantially attenuated compared to the amplitude of dendritic EPSPs at their site of generation. However, synaptic efficacy of the same quantal synaptic conductance, as measured by the somatic EPSP amplitude, was only weakly dependent on synaptic location on spiny branchlets. The passive electrotonic structure of Purkinje cells is unusual in that the steady-state architecture is very compact, while voltage transients

  18. A Computational Modeling and Simulation Approach to Investigate Mechanisms of Subcellular cAMP Compartmentation.

    PubMed

    Yang, Pei-Chi; Boras, Britton W; Jeng, Mao-Tsuen; Docken, Steffen S; Lewis, Timothy J; McCulloch, Andrew D; Harvey, Robert D; Clancy, Colleen E

    2016-07-01

    Subcellular compartmentation of the ubiquitous second messenger cAMP has been widely proposed as a mechanism to explain unique receptor-dependent functional responses. How exactly compartmentation is achieved, however, has remained a mystery for more than 40 years. In this study, we developed computational and mathematical models to represent a subcellular sarcomeric space in a cardiac myocyte with varying detail. We then used these models to predict the contributions of various mechanisms that establish subcellular cAMP microdomains. We used the models to test the hypothesis that phosphodiesterases act as functional barriers to diffusion, creating discrete cAMP signaling domains. We also used the models to predict the effect of a range of experimentally measured diffusion rates on cAMP compartmentation. Finally, we modeled the anatomical structures in a cardiac myocyte diad, to predict the effects of anatomical diffusion barriers on cAMP compartmentation. When we incorporated experimentally informed model parameters to reconstruct an in silico subcellular sarcomeric space with spatially distinct cAMP production sites linked to caveloar domains, the models predict that under realistic conditions phosphodiesterases alone were insufficient to generate significant cAMP gradients. This prediction persisted even when combined with slow cAMP diffusion. When we additionally considered the effects of anatomic barriers to diffusion that are expected in the cardiac myocyte dyadic space, cAMP compartmentation did occur, but only when diffusion was slow. Our model simulations suggest that additional mechanisms likely contribute to cAMP gradients occurring in submicroscopic domains. The difference between the physiological and pathological effects resulting from the production of cAMP may be a function of appropriate compartmentation of cAMP signaling. Therefore, understanding the contribution of factors that are responsible for coordinating the spatial and temporal

  19. A Computational Modeling and Simulation Approach to Investigate Mechanisms of Subcellular cAMP Compartmentation

    PubMed Central

    Yang, Pei-Chi; Boras, Britton W.; Jeng, Mao-Tsuen; Lewis, Timothy J.; McCulloch, Andrew D.; Harvey, Robert D.; Clancy, Colleen E.

    2016-01-01

    Subcellular compartmentation of the ubiquitous second messenger cAMP has been widely proposed as a mechanism to explain unique receptor-dependent functional responses. How exactly compartmentation is achieved, however, has remained a mystery for more than 40 years. In this study, we developed computational and mathematical models to represent a subcellular sarcomeric space in a cardiac myocyte with varying detail. We then used these models to predict the contributions of various mechanisms that establish subcellular cAMP microdomains. We used the models to test the hypothesis that phosphodiesterases act as functional barriers to diffusion, creating discrete cAMP signaling domains. We also used the models to predict the effect of a range of experimentally measured diffusion rates on cAMP compartmentation. Finally, we modeled the anatomical structures in a cardiac myocyte diad, to predict the effects of anatomical diffusion barriers on cAMP compartmentation. When we incorporated experimentally informed model parameters to reconstruct an in silico subcellular sarcomeric space with spatially distinct cAMP production sites linked to caveloar domains, the models predict that under realistic conditions phosphodiesterases alone were insufficient to generate significant cAMP gradients. This prediction persisted even when combined with slow cAMP diffusion. When we additionally considered the effects of anatomic barriers to diffusion that are expected in the cardiac myocyte dyadic space, cAMP compartmentation did occur, but only when diffusion was slow. Our model simulations suggest that additional mechanisms likely contribute to cAMP gradients occurring in submicroscopic domains. The difference between the physiological and pathological effects resulting from the production of cAMP may be a function of appropriate compartmentation of cAMP signaling. Therefore, understanding the contribution of factors that are responsible for coordinating the spatial and temporal

  20. Compartmentation of cAMP signalling in cardiomyocytes in health and disease.

    PubMed

    Perera, R K; Nikolaev, V O

    2013-04-01

    3',5'-cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger critically involved in the regulation of heart function. It has been shown to act in discrete subcellular signalling compartments formed by differentially localized receptors, phosphodiesterases and protein kinases. Cardiac diseases such as hypertrophy or heart failure are associated with structural and functional remodelling of these microdomains which leads to changes in cAMP compartmentation. In this review, we will discuss recent key findings which provided new insights into cAMP compartmentation in cardiomyocytes with a particular focus on its alterations in heart disease.

  1. Fully Automated Enhanced Tumor Compartmentalization: Man vs. Machine Reloaded

    PubMed Central

    Meier, Raphael; Verma, Rajeev; Jilch, Astrid; Fichtner, Jens; Knecht, Urspeter; Radina, Christian; Schucht, Philippe; Beck, Jürgen; Raabe, Andreas; Slotboom, Johannes; Reyes, Mauricio; Wiest, Roland

    2016-01-01

    Objective Comparison of a fully-automated segmentation method that uses compartmental volume information to a semi-automatic user-guided and FDA-approved segmentation technique. Methods Nineteen patients with a recently diagnosed and histologically confirmed glioblastoma (GBM) were included and MR images were acquired with a 1.5 T MR scanner. Manual segmentation for volumetric analyses was performed using the open source software 3D Slicer version 4.2.2.3 (www.slicer.org). Semi-automatic segmentation was done by four independent neurosurgeons and neuroradiologists using the computer-assisted segmentation tool SmartBrush® (referred to as SB), a semi-automatic user-guided and FDA-approved tumor-outlining program that uses contour expansion. Fully automatic segmentations were performed with the Brain Tumor Image Analysis (BraTumIA, referred to as BT) software. We compared manual (ground truth, referred to as GT), computer-assisted (SB) and fully-automated (BT) segmentations with regard to: (1) products of two maximum diameters for 2D measurements, (2) the Dice coefficient, (3) the positive predictive value, (4) the sensitivity and (5) the volume error. Results Segmentations by the four expert raters resulted in a mean Dice coefficient between 0.72 and 0.77 using SB. BT achieved a mean Dice coefficient of 0.68. Significant differences were found for intermodal (BT vs. SB) and for intramodal (four SB expert raters) performances. The BT and SB segmentations of the contrast-enhancing volumes achieved a high correlation with the GT. Pearson correlation was 0.8 for BT; however, there were a few discrepancies between raters (BT and SB 1 only). Additional non-enhancing tumor tissue extending the SB volumes was found with BT in 16/19 cases. The clinically motivated sum of products of diameters measure (SPD) revealed neither significant intermodal nor intramodal variations. The analysis time for the four expert raters was faster (1 minute and 47 seconds to 3 minutes and 39

  2. Uptake and intracellular activity of fluconazole in human polymorphonuclear leukocytes.

    PubMed Central

    Pascual, A; García, I; Conejo, C; Perea, E J

    1993-01-01

    The penetration of fluconazole into human polymorphonuclear leukocytes (PMNs) and tissue culture epithelial cells (McCoy) was evaluated. At different extracellular concentrations (0.5 to 10 mg/liter), fluconazole reached cell-associated concentrations greater than the extracellular ones in either human PMNs (intracellular concentration to extracellular concentration ratio, > or = 2.2) or McCoy cells (intracellular concentration to extracellular concentration ratio, > or = 1.3). The uptake of fluconazole by PMNs was rapid and reversible but was not energy dependent. The intracellular penetration of fluconazole was not affected by environmental pH or temperature. Ingestion of opsonized zymosan and opsonized Candida albicans did not significantly increase the amount of PMN-associated fluconazole. At therapeutic extracellular concentrations, the intracellular activity of fluconazole against C. albicans in PMNs was significantly lower than that of amphotericin B. It was concluded that fluconazole reaches high intracellular concentrations within PMNs but shows moderate activity against intracellular C. albicans in vitro. PMID:8452347

  3. 21 CFR 888.3535 - Knee joint femorotibial (uni-compartmental) metal/polymer porous-coated uncemented prosthesis.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    .../polymer porous-coated uncemented prosthesis. 888.3535 Section 888.3535 Food and Drugs FOOD AND DRUG... Devices § 888.3535 Knee joint femorotibial (uni-compartmental) metal/polymer porous-coated uncemented prosthesis. (a) Identification. A knee joint femorotibial (uni-compartmental) metal/polymer...

  4. 21 CFR 888.3535 - Knee joint femorotibial (uni-compartmental) metal/polymer porous-coated uncemented prosthesis.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    .../polymer porous-coated uncemented prosthesis. 888.3535 Section 888.3535 Food and Drugs FOOD AND DRUG... Devices § 888.3535 Knee joint femorotibial (uni-compartmental) metal/polymer porous-coated uncemented prosthesis. (a) Identification. A knee joint femorotibial (uni-compartmental) metal/polymer...

  5. INTRACELLULAR SIGNALING AND DEVELOPMENTAL NEUROTOXICITY.

    EPA Science Inventory

    A book chapter in ?Molecular Toxicology: Transcriptional Targets? reviewed the role of intracellular signaling in the developmental neurotoxicity of environmental chemicals. This chapter covered a number of aspects including the development of the nervous system, role of intrace...

  6. Functional genomics of intracellular bacteria.

    PubMed

    de Barsy, Marie; Greub, Gilbert

    2013-07-01

    During the genomic era, a large amount of whole-genome sequences accumulated, which identified many hypothetical proteins of unknown function. Rapidly, functional genomics, which is the research domain that assign a function to a given gene product, has thus been developed. Functional genomics of intracellular pathogenic bacteria exhibit specific peculiarities due to the fastidious growth of most of these intracellular micro-organisms, due to the close interaction with the host cell, due to the risk of contamination of experiments with host cell proteins and, for some strict intracellular bacteria such as Chlamydia, due to the absence of simple genetic system to manipulate the bacterial genome. To identify virulence factors of intracellular pathogenic bacteria, functional genomics often rely on bioinformatic analyses compared with model organisms such as Escherichia coli and Bacillus subtilis. The use of heterologous expression is another common approach. Given the intracellular lifestyle and the many effectors that are used by the intracellular bacteria to corrupt host cell functions, functional genomics is also often targeting the identification of new effectors such as those of the T4SS of Brucella and Legionella.

  7. Exogenous control over intracellular acidification: Enhancement via proton caged compounds coupled to gold nanoparticles.

    PubMed

    Carbone, Marilena; Sabbatella, Gianfranco; Antonaroli, Simonetta; Remita, Hynd; Orlando, Viviana; Biagioni, Stefano; Nucara, Alessandro

    2015-11-01

    The pH regulation has a fundamental role in several intracellular processes and its variation via exogenous compounds is a potential tool for intervening in the intracellular processes. Proton caged compounds (PPCs) release protons upon UV irradiation and may efficiently provoke intracellular on-command acidification. Here, we explore the intracellular pH variation, when purposely synthesized PCCs are coupled to gold nanoparticles (AuNPs) and dosed to HEK-293 cells. We detected the acidification process caused by the UV irradiation by monitoring the intensity of the asymmetric stretching mode of the CO(2) molecule at 2343 cm(-1). The comparison between free and AuNPs functionalized proton caged compound demonstrates a highly enhanced CO(2) yield, hence pH variation, in the latter case. Finally, PCC functionalized AuNPs were marked with a purposely synthesized fluorescent marker and dosed to HEK-293 cells. The corresponding fluorescence optical images show green grains throughout the whole cytoplasm.

  8. Water Diffusion, T2, and Compartmentation in Frog Sciatic Nerve

    PubMed Central

    Peled, Sharon; Cory, David G.; Raymond, Stephen A.; Kirschner, Daniel A.; Jolesz, Ferenc A.

    2010-01-01

    A potential relationship between structural compartments in neural tissue and NMR parameters may increase the specificity of MRI in diagnosing diseases. Nevertheless, our understanding of MR of nerves and white matter is limited, particularly the influence of various water compartments on the MR signal is not known. In this study, components of the 1H transverse relaxation decay curve in frog peripheral nerve were correlated with the diffusion characteristics of the water in the nerve. Three T2 values were identified with nerve. Water mobility was found to be unrestricted on the timescale of 100 msec in the component of the signal with the intermediate T2 time, suggesting some contribution from the interstitial space to this T2 component. Restricted diffusion was observed in the component with the longest T2 time, supporting the assignment of at least part of the spins contributing to this component to an intracellular compartment. The observed nonexponential behavior of the diffusion attenuation curves was investigated and shown to be potentially caused by the wide range of axon sizes in the nerve. PMID:10542350

  9. Dengue virus compartmentalization during antibody-enhanced infection

    PubMed Central

    Ong, Eugenia Z.; Zhang, Summer L.; Tan, Hwee Cheng; Gan, Esther S.; Chan, Kuan Rong; Ooi, Eng Eong

    2017-01-01

    Secondary infection with a heterologous dengue virus (DENV) serotype increases the risk of severe dengue, through a process termed antibody-dependent enhancement (ADE). During ADE, DENV is opsonized with non- or sub-neutralizing antibody levels that augment entry into monocytes and dendritic cells through Fc-gamma receptors (FcγRs). We previously reported that co-ligation of leukocyte immunoglobulin-like receptor-B1 (LILRB1) by antibody-opsonized DENV led to recruitment of SH2 domain-containing phosphatase-1 (SHP-1) to dephosphorylate spleen tyrosine kinase (Syk) and reduce interferon stimulated gene induction. Here, we show that LILRB1 also signals through SHP-1 to attenuate the otherwise rapid acidification for lysosomal enzyme activation following FcγR-mediated uptake of DENV. Reduced or slower trafficking of antibody-opsonized DENV to lytic phagolysosomal compartments, demonstrates how co-ligation of LILRB1 also permits DENV to overcome a cell-autonomous immune response, enhancing intracellular survival of DENV. Our findings provide insights on how antiviral drugs that modify phagosome acidification should be used for viruses such as DENV. PMID:28084461

  10. An actomyosin-based barrier inhibits cell mixing at compartmental boundaries in Drosophila embryos.

    PubMed

    Monier, Bruno; Pélissier-Monier, Anne; Brand, Andrea H; Sanson, Bénédicte

    2010-01-01

    Partitioning tissues into compartments that do not intermix is essential for the correct morphogenesis of animal embryos and organs. Several hypotheses have been proposed to explain compartmental cell sorting, mainly differential adhesion, but also regulation of the cytoskeleton or of cell proliferation. Nevertheless, the molecular and cellular mechanisms that keep cells apart at boundaries remain unclear. Here we demonstrate, in early Drosophila melanogaster embryos, that actomyosin-based barriers stop cells from invading neighbouring compartments. Our analysis shows that cells can transiently invade neighbouring compartments, especially when they divide, but are then pushed back into their compartment of origin. Actomyosin cytoskeletal components are enriched at compartmental boundaries, forming cable-like structures when the epidermis is mitotically active. When MyoII (non-muscle myosin II) function is inhibited, including locally at the cable by chromophore-assisted laser inactivation (CALI), in live embryos, dividing cells are no longer pushed back, leading to compartmental cell mixing. We propose that local regulation of actomyosin contractibility, rather than differential adhesion, is the primary mechanism sorting cells at compartmental boundaries.

  11. A compartmentalized phosphorylation/dephosphorylation system that regulates U snRNA export from the nucleus.

    PubMed

    Kitao, Saori; Segref, Alexandra; Kast, Juergen; Wilm, Matthias; Mattaj, Iain W; Ohno, Mutsuhito

    2008-01-01

    PHAX (phosphorylated adaptor for RNA export) is the key regulator of U snRNA nuclear export in metazoa. Our previous work revealed that PHAX is phosphorylated in the nucleus and is exported as a component of the U snRNA export complex to the cytoplasm, where it is dephosphorylated (M. Ohno, A. Segref, A. Bachi, M. Wilm, and I. W. Mattaj, Cell 101:187-198, 2000). PHAX phosphorylation is essential for export complex assembly, whereas its dephosphorylation causes export complex disassembly. Thus, PHAX is subject to a compartmentalized phosphorylation/dephosphorylation cycle that contributes to transport directionality. However, neither essential PHAX phosphorylation sites nor the modifying enzymes that contribute to the compartmentalized system have been identified. Here, we identify PHAX phosphorylation sites that are necessary and sufficient for U snRNA export. Mutation of the phosphorylation sites inhibited U snRNA export in a dominant-negative way. We also show, by both biochemical and RNA interference knockdown experiments, that the nuclear kinase and the cytoplasmic phosphatase for PHAX are CK2 kinase and protein phosphatase 2A, respectively. Our results reveal the composition of the compartmentalized phosphorylation/dephosphorylation system that regulates U snRNA export. This finding was surprising in that such a specific system for U snRNA export regulation is composed of two such universal regulators, suggesting that this compartmentalized system is used more broadly for gene expression regulation.

  12. Krebs cycle metabolon formation: metabolite concentration gradient enhanced compartmentation of sequential enzymes.

    PubMed

    Wu, Fei; Pelster, Lindsey N; Minteer, Shelley D

    2015-01-25

    Dynamics of metabolon formation in mitochondria was probed by studying diffusional motion of two sequential Krebs cycle enzymes in a microfluidic channel. Enhanced directional co-diffusion of both enzymes against a substrate concentration gradient was observed in the presence of intermediate generation. This reveals a metabolite directed compartmentation of metabolic pathways.

  13. Beyond Compartmentalization: A Relational Approach towards Agency and Vulnerability of Young Migrants

    ERIC Educational Resources Information Center

    Huijsmans, Roy

    2012-01-01

    Based on fieldwork material from Lao People's Democratic Republic, this paper introduces an analytical framework that transcends compartmentalized approaches towards migration involving young people. The notions of fluid and institutionalized forms of migration illuminate key differences and commonalities in the relational fabric underpinning…

  14. Analysis of a compartmental model of amyloid beta production, irreversible loss and exchange in humans.

    PubMed

    Elbert, Donald L; Patterson, Bruce W; Bateman, Randall J

    2015-03-01

    Amyloid beta (Aβ) peptides, and in particular Aβ42, are found in senile plaques associated with Alzheimer's disease. A compartmental model of Aβ production, exchange and irreversible loss was recently developed to explain the kinetics of isotope-labeling of Aβ peptides collected in cerebrospinal fluid (CSF) following infusion of stable isotope-labeled leucine in humans. The compartmental model allowed calculation of the rates of production, irreversible loss (or turnover) and short-term exchange of Aβ peptides. Exchange of Aβ42 was particularly pronounced in amyloid plaque-bearing participants. In the current work, we describe in much greater detail the characteristics of the compartmental model to two distinct audiences: physician-scientists and biokineticists. For physician-scientists, we describe through examples the types of questions the model can and cannot answer, as well as correct some misunderstandings of previous kinetic analyses applied to this type of isotope labeling data. For biokineticists, we perform a system identifiability analysis and a sensitivity analysis of the kinetic model to explore the global and local properties of the model. Combined, these analyses motivate simplifications from a more comprehensive physiological model to the final model that was previously presented. The analyses clearly demonstrate that the current dataset and compartmental model allow determination with confidence a single 'turnover' parameter, a single 'exchange' parameter and a single 'delay' parameter. When combined with CSF concentration data for the Aβ peptides, production rates may also be obtained.

  15. A Compartmentalized Out-of-Equilibrium Enzymatic Reaction Network for Sustained Autonomous Movement

    PubMed Central

    2016-01-01

    Every living cell is a compartmentalized out-of-equilibrium system exquisitely able to convert chemical energy into function. In order to maintain homeostasis, the flux of metabolites is tightly controlled by regulatory enzymatic networks. A crucial prerequisite for the development of lifelike materials is the construction of synthetic systems with compartmentalized reaction networks that maintain out-of-equilibrium function. Here, we aim for autonomous movement as an example of the conversion of feedstock molecules into function. The flux of the conversion is regulated by a rationally designed enzymatic reaction network with multiple feedforward loops. By compartmentalizing the network into bowl-shaped nanocapsules the output of the network is harvested as kinetic energy. The entire system shows sustained and tunable microscopic motion resulting from the conversion of multiple external substrates. The successful compartmentalization of an out-of-equilibrium reaction network is a major first step in harnessing the design principles of life for construction of adaptive and internally regulated lifelike systems. PMID:27924313

  16. Origin of reservoir compartmentalization in Lower Ordovician Karstic Dolostones, Ellenburger Group, West Texas

    SciTech Connect

    Kerans, C.

    1988-01-01

    Ellenburger Group reservoirs constitute a major play in the Permian basin of west Texas, with over 1.4 billion bbl cumulative production through 1985. These reservoirs typically have been developed by assuming homogeneous fracture-related pore system. Examination of core, log, and production data demonstrates that most Ellenburger reservoirs are characterized by pronounced vertical and lateral heterogeneities created by post-Ellenburger karst development. Vertical reservoir compartmentalization in the Ellenburger evolved from development of a laterally extensive cave system between 100 and 300 ft beneath the original land surface. Caves were filled by relatively impermeable siliciclastics from the overlying Simpson Group, effectively isolating permeable cave-roof breccias (uppermost Ellenburger) from collapse breccias deposited on cave floors prior to shale infill. Lateral compartmentalization of Ellenburger reservoirs originated by localized collapse of the cave system both during karst formation and after burial. In the Shafter Lake field, lateral compartmentalization is the result of a 200-ft vertical collapse during deposition of Simpson Group sands. Abrupt lateral discontinuities in the Big Lake and Glasco fields may represent similar collapse-related features, such as are spectacularly displayed in Ellenburger-equivalent outcrops of the Franklin Mountains. An estimated 750 million bbl of remaining mobile oil, in addition to conventional reserves, occurs in this mature but complexly compartmentalized play. Considering this paleokarst model will aid in further exploitation of Ellenburger reservoirs.

  17. A-kinase anchoring proteins: cAMP compartmentalization in neurodegenerative and obstructive pulmonary diseases

    PubMed Central

    Poppinga, W J; Muñoz-Llancao, P; González-Billault, C; Schmidt, M

    2014-01-01

    The universal second messenger cAMP is generated upon stimulation of Gs protein-coupled receptors, such as the β2-adreneoceptor, and leads to the activation of PKA, the major cAMP effector protein. PKA oscillates between an on and off state and thereby regulates a plethora of distinct biological responses. The broad activation pattern of PKA and its contribution to several distinct cellular functions lead to the introduction of the concept of compartmentalization of cAMP. A-kinase anchoring proteins (AKAPs) are of central importance due to their unique ability to directly and/or indirectly interact with proteins that either determine the cellular content of cAMP, such as β2-adrenoceptors, ACs and PDEs, or are regulated by cAMP such as the exchange protein directly activated by cAMP. We report on lessons learned from neurons indicating that maintenance of cAMP compartmentalization by AKAP5 is linked to neurotransmission, learning and memory. Disturbance of cAMP compartments seem to be linked to neurodegenerative disease including Alzheimer's disease. We translate this knowledge to compartmentalized cAMP signalling in the lung. Next to AKAP5, we focus here on AKAP12 and Ezrin (AKAP78). These topics will be highlighted in the context of the development of novel pharmacological interventions to tackle AKAP-dependent compartmentalization. PMID:25132049

  18. Technology Solutions Case Study: Field Testing of Compartmentalization Methods for Multifamily Construction

    SciTech Connect

    2015-01-01

    Fire-resistance rated (or area separation) wall assemblies present a great difficulty in air sealing/compartmentalization, particularly in townhouse construction. To address this challenge, Building Science Corporation partnered with builder K. Hovnanian Homes to determine whether taping exterior sheathing details improves air sealing in townhouse and multifamily construction, and to better understand air leakage pathways.

  19. SYNCHROTRON X-RAY ABSORPTION-EDGE COMPUTED MICROTOMOGRAPHY IMAGING OF THALLIUM COMPARTMENTALIZATION IN IBERIS INTERMEDIA

    EPA Science Inventory

    Thallium (TI) is an extremely toxic metal which, due to its similarities to K, is readily taken up by plants. Thallium is efficiently hyperaccumulated in Iberis intermedia as TI(I). Distribution and compartmentalization of TI in I. intermedia is highes...

  20. Proton Fall or Bicarbonate Rise: GLYCOLYTIC RATE IN MOUSE ASTROCYTES IS PAVED BY INTRACELLULAR ALKALINIZATION.

    PubMed

    Theparambil, Shefeeq M; Weber, Tobias; Schmälzle, Jana; Ruminot, Ivàn; Deitmer, Joachim W

    2016-09-02

    Glycolysis is the primary step for major energy production in the cell. There is strong evidence suggesting that glucose consumption and rate of glycolysis are highly modulated by cytosolic pH/[H(+)], but those can also be stimulated by an increase in the intracellular [HCO3 (-)]. Because proton and bicarbonate shift concomitantly, it remained unclear whether enhanced glucose consumption and glycolytic rate were mediated by the changes in intracellular [H(+)] or [HCO3 (-)]. We have asked whether glucose metabolism is enhanced by either a fall in intracellular [H(+)] or a rise in intracellular [HCO3 (-)], or by both, in mammalian astrocytes. We have recorded intracellular glucose in mouse astrocytes using a FRET-based nanosensor, while imposing different intracellular [H(+)] and [CO2]/[HCO3 (-)]. Glucose consumption and glycolytic rate were augmented by a fall in intracellular [H(+)], irrespective of a concomitant rise or fall in intracellular [HCO3 (-)]. Transport of HCO3 (-) into and out of astrocytes by the electrogenic sodium bicarbonate cotransporter (NBCe1) played a crucial role in causing changes in intracellular pH and [HCO3 (-)], but was not obligatory for the pH-dependent changes in glucose metabolism. Our results clearly show that it is the cytosolic pH that modulates glucose metabolism in cortical astrocytes, and possibly also in other cell types.

  1. Intracellular Energetic Units regulate metabolism in cardiac cells.

    PubMed

    Saks, Valdur; Kuznetsov, Andrey V; Gonzalez-Granillo, Marcela; Tepp, Kersti; Timohhina, Natalja; Karu-Varikmaa, Minna; Kaambre, Tuuli; Dos Santos, Pierre; Boucher, François; Guzun, Rita

    2012-02-01

    This review describes developments in historical perspective as well as recent results of investigations of cellular mechanisms of regulation of energy fluxes and mitochondrial respiration by cardiac work - the metabolic aspect of the Frank-Starling law of the heart. A Systems Biology solution to this problem needs the integration of physiological and biochemical mechanisms that take into account intracellular interactions of mitochondria with other cellular systems, in particular with cytoskeleton components. Recent data show that different tubulin isotypes are involved in the regular arrangement exhibited by mitochondria and ATP-consuming systems into Intracellular Energetic Units (ICEUs). Beta II tubulin association with the mitochondrial outer membrane, when co-expressed with mitochondrial creatine kinase (MtCK) specifically limits the permeability of voltage-dependent anion channel for adenine nucleotides. In the MtCK reaction this interaction changes the regulatory kinetics of respiration through a decrease in the affinity for adenine nucleotides and an increase in the affinity for creatine. Metabolic Control Analysis of the coupled MtCK-ATP Synthasome in permeabilized cardiomyocytes showed a significant increase in flux control by steps involved in ADP recycling. Mathematical modeling of compartmentalized energy transfer represented by ICEUs shows that cyclic changes in local ADP, Pi, phosphocreatine and creatine concentrations during contraction cycle represent effective metabolic feedback signals when amplified in the coupled non-equilibrium MtCK-ATP Synthasome reactions in mitochondria. This mechanism explains the regulation of respiration on beat to beat basis during workload changes under conditions of metabolic stability. This article is part of a Special Issue entitled "Local Signaling in Myocytes."

  2. Transverse Relaxation and Magnetization Transfer in Skeletal Muscle: Effect of pH

    PubMed Central

    Louie, Elizabeth A.; Gochberg, Daniel F.; Does, Mark D.; Damon, Bruce M.

    2008-01-01

    Exercise increases the intracellular T2 (T2,i) of contracting muscles. The mechanism(s) for the T2,i increase have not been fully described, and may include increased intracellular free water and acidification. These changes may alter chemical exchange processes between intracellular free water and proteins. In this study, the hypotheses were tested that 1) pH changes T2,i by affecting the rate of magnetization transfer (MT) between free intracellular water and intracellular proteins and 2) the magnitude of the T2,i effect depends on acquisition mode (localized or non-localized) and echo spacing. Frog gastrocnemius muscles were excised and their intracellular pH was either kept at physiological pH (7.0) or modified to model exercising muscle (pH 6.5). The intracellular transverse relaxation rate (R2,i =1/T2,i) always decreased in the acidic muscles, but the changes were greater when measured using more rapid refocusing rates. The MT rate from the macromolecular proton pool to the free water proton pool, its reverse rate, and the spin-lattice relaxation rate of water decreased in acidic muscles. It is concluded that intracellular acidification alters the R2,i of muscle water in a refocusing rate-dependent manner and that the R2,i changes are correlated with changes in the MT rate between macromolecules and free intracellular water. PMID:19097244

  3. Differential distribution of NCX1 contributes to spine–dendrite compartmentalization in CA1 pyramidal cells

    PubMed Central

    Lőrincz, Andrea; Rózsa, Balázs; Katona, Gergely; Vizi, E. Sylvester; Tamás, Gábor

    2007-01-01

    Compartmentalization of Ca2+ between dendritic spines and shafts is governed by diffusion barriers and a range of Ca2+ extrusion mechanisms. The distinct contribution of different Ca2+ clearance systems to Ca2+ compartmentalization in dendritic spines versus shafts remains elusive. We applied a combination of ultrastructural and functional imaging methods to assess the subcellular distribution and role of NCX1 in rat CA1 pyramidal cells. Quantitative electron microscopic analysis of preembedding immunogold reactions revealed uniform densities of NCX1 along the shafts of apical and basal dendrites, but densities in dendritic shafts were approximately seven times higher than in dendritic spines. In line with these results, two-photon imaging of synaptically activated Ca2+ transients during NCX blockade showed preferential action localized to the dendritic shafts for NCXs in regulating spine–dendrite coupling. PMID:17215351

  4. Cell-free selection of domain antibodies by in vitro compartmentalization.

    PubMed

    Sepp, Armin; Griffiths, Andrew

    2012-01-01

    Efficient identification of antibodies, or any fragments thereof, displaying desired specificity and affinity is critical for the development of novel immunotherapeutics. Here we describe the adaptation of in vitro compartmentalization for the cell-free selection of Vκ and VH domain antibodies (dAbs™) from large combinatorial libraries. The dAbs™ are in vitro expressed in fusion to the N-terminus of single-chain variant of phage P22 Arc repressor DNA-binding domain that links the compartmentally expressed protein molecules to their encoding PCR fragment-based genes via cognate operator sites present on the DNA. Libraries of up to 10(10) in size can be rapidly assembled and selected for improved affinity in equilibrium and off-rate conditions.

  5. Structurally controlled and aligned tight gas reservoir compartmentalization in the San Juan and Piceance Basins

    SciTech Connect

    Decker, A.D.; Kuuskraa, V.A.; Klawitter, A.L.

    1995-10-01

    Recurrent basement faulting is the primary controlling mechanism for aligning and compartmentalizing upper Cretaceous aged tight gas reservoirs of the San Juan and Piceance Basins. Northwest trending structural lineaments that formed in conjunction with the Uncompahgre Highlands have profoundly influenced sedimentation trends and created boundaries for gas migration; sealing and compartmentalizing sedimentary packages in both basins. Fractures which formed over the structural lineaments provide permeability pathways which allowing gas recovery from otherwise tight gas reservoirs. Structural alignments and associated reservoir compartments have been accurately targeted by integrating advanced remote sensing imagery, high resolution aeromagnetics, seismic interpretation, stratigraphic mapping and dynamic structural modelling. This unifying methodology is a powerful tool for exploration geologists and is also a systematic approach to tight gas resource assessment in frontier basins.

  6. Subcellular compartmentalization of saccharide moieties in cultured normal and malignant cells

    PubMed Central

    1980-01-01

    We studied subcellular localization of saccharide moieties in cultured normal and malignant cells fixed in paraformaldehyde and treated with a nonionic detergent, using lectins specific for various surgar residues as probes in fluorescence microscopy. In normal cells, concanavalin A and Lens culinaris agglutinin, specific for mannose-rich carbohydrate cores in glycoproteins, labeled the endoplasmic reticulum as a wide perinuclear region. Other lectins, on the other hand, stained the Golgi apparatus as a juxtanuclear reticular structure. A similar compartmentalization was also seen in all malignant cells studied, although the Golgi apparatus in these cells was distinctly vesicular in appearance. Our results indicate that saccharide moieties in both normal and malignant cells are similarly compartmentalized, and thus speak in favor of a unidirectional subcellular flow for both membrane and secreted glycoconjugates. PMID:7372714

  7. Subcellular compartmentalization of saccharide moieties in cultured normal and malignant cells.

    PubMed

    Virtanen, I; Ekblom, P; Laurila, P

    1980-05-01

    We studied subcellular localization of saccharide moieties in cultured normal and malignant cells fixed in paraformaldehyde and treated with a nonionic detergent, using lectins specific for various surgar residues as probes in fluorescence microscopy. In normal cells, concanavalin A and Lens culinaris agglutinin, specific for mannose-rich carbohydrate cores in glycoproteins, labeled the endoplasmic reticulum as a wide perinuclear region. Other lectins, on the other hand, stained the Golgi apparatus as a juxtanuclear reticular structure. A similar compartmentalization was also seen in all malignant cells studied, although the Golgi apparatus in these cells was distinctly vesicular in appearance. Our results indicate that saccharide moieties in both normal and malignant cells are similarly compartmentalized, and thus speak in favor of a unidirectional subcellular flow for both membrane and secreted glycoconjugates.

  8. Diversity in the dynamical behaviour of a compartmentalized programmable biochemical oscillator

    NASA Astrophysics Data System (ADS)

    Weitz, Maximilian; Kim, Jongmin; Kapsner, Korbinian; Winfree, Erik; Franco, Elisa; Simmel, Friedrich C.

    2014-04-01

    In vitro compartmentalization of biochemical reaction networks is a crucial step towards engineering artificial cell-scale devices and systems. At this scale the dynamics of molecular systems becomes stochastic, which introduces several engineering challenges and opportunities. Here we study a programmable transcriptional oscillator system that is compartmentalized into microemulsion droplets with volumes between 33 fl and 16 pl. Simultaneous measurement of large populations of droplets reveals major variations in the amplitude, frequency and damping of the oscillations. Variability increases for smaller droplets and depends on the operating point of the oscillator. Rather than reflecting the stochastic kinetics of the chemical reaction network itself, the variability can be attributed to the statistical variation of reactant concentrations created during their partitioning into droplets. We anticipate that robustness to partitioning variability will be a critical challenge for engineering cell-scale systems, and that highly parallel time-series acquisition from microemulsion droplets will become a key tool for characterization of stochastic circuit function.

  9. Use of a simulated annealing algorithm to fit compartmental models with an application to fractal pharmacokinetics.

    PubMed

    Marsh, Rebeccah E; Riauka, Terence A; McQuarrie, Steve A

    2007-01-01

    Increasingly, fractals are being incorporated into pharmacokinetic models to describe transport and chemical kinetic processes occurring in confined and heterogeneous spaces. However, fractal compartmental models lead to differential equations with power-law time-dependent kinetic rate coefficients that currently are not accommodated by common commercial software programs. This paper describes a parameter optimization method for fitting individual pharmacokinetic curves based on a simulated annealing (SA) algorithm, which always converged towards the global minimum and was independent of the initial parameter values and parameter bounds. In a comparison using a classical compartmental model, similar fits by the Gauss-Newton and Nelder-Mead simplex algorithms required stringent initial estimates and ranges for the model parameters. The SA algorithm is ideal for fitting a wide variety of pharmacokinetic models to clinical data, especially those for which there is weak prior knowledge of the parameter values, such as the fractal models.

  10. Aspects of astrocyte energy metabolism, amino acid neurotransmitter homoeostasis and metabolic compartmentation.

    PubMed

    Kreft, Marko; Bak, Lasse K; Waagepetersen, Helle S; Schousboe, Arne

    2012-04-27

    Astrocytes are key players in brain function; they are intimately involved in neuronal signalling processes and their metabolism is tightly coupled to that of neurons. In the present review, we will be concerned with a discussion of aspects of astrocyte metabolism, including energy-generating pathways and amino acid homoeostasis. A discussion of the impact that uptake of neurotransmitter glutamate may have on these pathways is included along with a section on metabolic compartmentation.

  11. Simulation of Drug Uptake in a Two Compartmental Fractional Model for a Biological System.

    PubMed

    Petráš, Ivo; Magin, Richard L

    2011-12-01

    This paper presents a very effective numerical method for the solution of the two-compartmental pharmacokinetic model for oral drug administration. This model consists of a set of two fractional order differential equations which connect the two compartments. The first compartment represents the gut while the second compartment corresponds to the drug concentration in the target tissue. For ease of computation, the numerical solution is also created as a Matlab function.

  12. Self-priming compartmentalization digital LAMP for point-of-care.

    PubMed

    Zhu, Qiangyuan; Gao, Yibo; Yu, Bingwen; Ren, Hao; Qiu, Lin; Han, Sihai; Jin, Wei; Jin, Qinhan; Mu, Ying

    2012-11-21

    Digital nucleic acid amplification provides unprecedented opportunities for absolute nucleic acid quantification by counting of single molecules. This technique is useful for molecular genetic analysis in cancer, stem cell, bacterial, non-invasive prenatal diagnosis in which many biologists are interested. This paper describes a self-priming compartmentalization (SPC) microfluidic chip platform for performing digital loop-mediated amplification (LAMP). The energy for the pumping is pre-stored in the degassed bulk PDMS by exploiting the high gas solubility of PDMS; therefore, no additional structures other than channels and reservoirs are required. The sample and oil are sequentially sucked into the channels, and the pressure difference of gas dissolved in PDMS allows sample self-compartmentalization without the need for further chip manipulation such as with pneumatic microvalves and control systems, and so on. The SPC digital LAMP chip can be used like a 384-well plate, so, the world-to-chip fluidic interconnections are avoided. The microfluidic chip contains 4 separate panels, each panel contains 1200 independent 6 nL chambers and can be used to detect 4 samples simultaneously. Digital LAMP on the microfluidic chip was tested quantitatively by using β-actin DNA from humans. The self-priming compartmentalization behavior is roughly predictable using a two-dimensional model. The uniformity of compartmentalization was analyzed by fluorescent intensity and fraction of volume. The results showed that the feasibility and flexibility of the microfluidic chip platform for amplifying single nucleic acid molecules in different chambers made by diluting and distributing sample solutions. The SPC chip has the potential to meet the requirements of a general laboratory: power-free, valve-free, operating at isothermal temperature, inexpensive, sensitive, economizing labour time and reagents. The disposable analytical devices with appropriate air-tight packaging should be

  13. Bioinspired genotype–phenotype linkages: mimicking cellular compartmentalization for the engineering of functional proteins

    PubMed Central

    van Vliet, Liisa D.; Colin, Pierre-Yves; Hollfelder, Florian

    2015-01-01

    The idea of compartmentalization of genotype and phenotype in cells is key for enabling Darwinian evolution. This contribution describes bioinspired systems that use in vitro compartments—water-in-oil droplets and gel-shell beads—for the directed evolution of functional proteins. Technologies based on these principles promise to provide easier access to protein-based therapeutics, reagents for processes involving enzyme catalysis, parts for synthetic biology and materials with biological components. PMID:26464791

  14. A compartmentalized solute transport model for redox zones in contaminated aquifers--1, Theory and development

    USGS Publications Warehouse

    Abrams , Robert H.; Loague, Keith

    2000-01-01

    This paper, the first of two parts [see Abrams and Loague, this issue], takes the compartmentalized approach for the geochemical evolution of redox zones presented by Abrams et al. [1998] and embeds it within a solute transport framework. In this paper the compartmentalized approach is generalized to facilitate the description of its incorporation into a solute transport simulator. An equivalent formulation is developed which removes any discontinuities that may occur when switching compartments. Rate-limited redox reactions are modeled with a modified Monod relationship that allows either the organic substrate or the electron acceptor to be the rate-limiting reactant. Thermodynamic constraints are used to inhibit lower-energy redox reactions from occurring under infeasible geochemical conditions without imposing equilibrium on the lower-energy reactions. The procedure used allows any redox reaction to be simulated as being kinetically limited or thermodynamically limited, depending on local geochemical conditions. Empirical reaction inhibition methods are not needed. The sequential iteration approach (SIA), a technique which allows the number of solute transport equations to be reduced, is adopted to solve the coupled geochemical/solute transport problem. When the compartmentalized approach is embedded within the SIA, with the total analytical concentration of each component as the dependent variable in the transport equation, it is possible to reduce the number of transport equations even further than with the unmodified SIA. A one-dimensional, coupled geochemical/solute transport simulation is presented in which redox zones evolve dynamically in time and space. The compartmentalized solute transport (COMPTRAN) model described in this paper enables the development of redox zones to be simulated under both kinetic and thermodynamic constraints. The modular design of COMPTRAN facilitates the use of many different, preexisting solute transport and geochemical codes

  15. A Compartmental Model for Computing Cell Numbers in CFSE-based Lymphocyte Proliferation Assays

    DTIC Science & Technology

    2012-01-31

    in Scientific Computation and Center for Quantitative Sciences in Biomedicine North Carolina State University, Raleigh, NC 27695-8212 Cristina...exactly. The values of the function ni−1(t, x), though already computed , will only be available at discrete points (t(m), x(k)) and thus (13) must be...operator for the compartmental model. In practice, because the transformed model solution ñ(t, z) is computed only at discrete points (tj , z j k), we

  16. Aspects of astrocyte energy metabolism, amino acid neurotransmitter homoeostasis and metabolic compartmentation

    PubMed Central

    Kreft, Marko; Bak, Lasse K; Waagepetersen, Helle S; Schousboe, Arne

    2012-01-01

    Astrocytes are key players in brain function; they are intimately involved in neuronal signalling processes and their metabolism is tightly coupled to that of neurons. In the present review, we will be concerned with a discussion of aspects of astrocyte metabolism, including energy-generating pathways and amino acid homoeostasis. A discussion of the impact that uptake of neurotransmitter glutamate may have on these pathways is included along with a section on metabolic compartmentation. PMID:22435484

  17. A compartmentalized solute transport model for redox zones in contaminated aquifers: 1: Theory and development

    NASA Astrophysics Data System (ADS)

    Abrams, Robert H.; Loague, Keith

    2000-08-01

    This paper, the first of two parts [see Abrams and Loague, this issue], takes the compartmentalized approach for the geochemical evolution of redox zones presented by Abrams et al. [1998] and embeds it within a solute transport framework. In this paper the compartmentalized approach is generalized to facilitate the description of its incorporation into a solute transport simulator. An equivalent formulation is developed which removes any discontinuities that may occur when switching compartments. Rate-limited redox reactions are modeled with a modified Monod relationship that allows either the organic substrate or the electron acceptor to be the rate-limiting reactant. Thermodynamic constraints are used to inhibit lower-energy redox reactions from occurring under infeasible geochemical conditions without imposing equilibrium on the lower-energy reactions. The procedure used allows any redox reaction to be simulated as being kinetically limited or thermodynamically limited, depending on local geochemical conditions. Empirical reaction inhibition methods are not needed. The sequential iteration approach (SIA), a technique which allows the number of solute transport equations to be reduced, is adopted to solve the coupled geochemical/solute transport problem. When the compartmentalized approach is embedded within the SIA, with the total analytical concentration of each component as the dependent variable in the transport equation, it is possible to reduce the number of transport equations even further than with the unmodified SIA. A one-dimensional, coupled geochemical/solute transport simulation is presented in which redox zones evolve dynamically in time and space. The compartmentalized solute transport (COMPTRAN) model described in this paper enables the development of redox zones to be simulated under both kinetic and thermodynamic constraints. The modular design of COMPTRAN facilitates the use of many different, preexisting solute transport and geochemical codes

  18. Verification of Compartmental Epidemiological Models using Metamorphic Testing, Model Checking and Visual Analytics

    SciTech Connect

    Ramanathan, Arvind; Steed, Chad A; Pullum, Laura L

    2012-01-01

    Compartmental models in epidemiology are widely used as a means to model disease spread mechanisms and understand how one can best control the disease in case an outbreak of a widespread epidemic occurs. However, a significant challenge within the community is in the development of approaches that can be used to rigorously verify and validate these models. In this paper, we present an approach to rigorously examine and verify the behavioral properties of compartmen- tal epidemiological models under several common modeling scenarios including birth/death rates and multi-host/pathogen species. Using metamorphic testing, a novel visualization tool and model checking, we build a workflow that provides insights into the functionality of compartmental epidemiological models. Our initial results indicate that metamorphic testing can be used to verify the implementation of these models and provide insights into special conditions where these mathematical models may fail. The visualization front-end allows the end-user to scan through a variety of parameters commonly used in these models to elucidate the conditions under which an epidemic can occur. Further, specifying these models using a process algebra allows one to automatically construct behavioral properties that can be rigorously verified using model checking. Taken together, our approach allows for detecting implementation errors as well as handling conditions under which compartmental epidemiological models may fail to provide insights into disease spread dynamics.

  19. Human Lsg1 defines a family of essential GTPases that correlates with the evolution of compartmentalization

    PubMed Central

    Reynaud, Emmanuel G; Andrade, Miguel A; Bonneau, Fabien; Ly, Thi Bach Nga; Knop, Michael; Scheffzek, Klaus; Pepperkok, Rainer

    2005-01-01

    Background Compartmentalization is a key feature of eukaryotic cells, but its evolution remains poorly understood. GTPases are the oldest enzymes that use nucleotides as substrates and they participate in a wide range of cellular processes. Therefore, they are ideal tools for comparative genomic studies aimed at understanding how aspects of biological complexity such as cellular compartmentalization evolved. Results We describe the identification and characterization of a unique family of circularly permuted GTPases represented by the human orthologue of yeast Lsg1p. We placed the members of this family in the phylogenetic context of the YlqF Related GTPase (YRG) family, which are present in Eukarya, Bacteria and Archea and include the stem cell regulator Nucleostemin. To extend the computational analysis, we showed that hLsg1 is an essential GTPase predominantly located in the endoplasmic reticulum and, in some cells, in Cajal bodies in the nucleus. Comparison of localization and siRNA datasets suggests that all members of the family are essential GTPases that have increased in number as the compartmentalization of the eukaryotic cell and the ribosome biogenesis pathway have evolved. Conclusion We propose a scenario, consistent with our data, for the evolution of this family: cytoplasmic components were first acquired, followed by nuclear components, and finally the mitochondrial and chloroplast elements were derived from different bacterial species, in parallel with the formation of the nucleolus and the specialization of nuclear components. PMID:16209721

  20. Compartmentalization Approaches in Soft Matter Science: From Nanoreactor Development to Organelle Mimics.

    PubMed

    Schoonen, Lise; van Hest, Jan C M

    2016-02-10

    Compartmentalization is an essential feature found in living cells to ensure that biological processes occur without being affected by undesired external influences. Over the years many scientists have designed self-assembled soft matter structures that mimic these natural catalytic compartments. The rationale behind this research is threefold. First of all, compartmentalization leads to the creation of a secluded environment for the catalytic species, which solves compatibility issues and which can improve catalyst efficiency and selectivity. Secondly, nano- and micro-compartments are constructed with the aim to obtain microenvironments that more closely mimic the cellular architecture. These biomimetic platforms are used to attain a better understanding of how cellular processes are executed. Thirdly, natural design rules are applied to create biomolecular assemblies with unusual functionality, which for example are used as artificial organelles. Here, recent developments will be discussed regarding these compartmentalized catalytic systems, with a selected number of illustrative examples to demonstrate which strategies have been followed, and to show to what extent the ambitious goals of this field of science have been reached. The focus here is on the field of soft matter science, covering the wide spectrum from polymeric assemblies to protein nanocages.

  1. GM130 is required for compartmental organization of dendritic Golgi outposts

    PubMed Central

    Zhou, Wei; Chang, Jin; Wang, Xin; Savelieff, Masha G.; Zhao, Yinyin; Ke, Shanshan; Ye, Bing

    2014-01-01

    SUMMARY Golgi complexes (Golgi) play important roles in the development and function of neurons [1–3]. Not only are Golgi present in the neuronal soma (somal Golgi) but they also exist in the dendrites as Golgi outposts [4–7]. Previous studies have shown that Golgi outposts serve as local microtubule organizing centers [8] and secretory stations in dendrites [6, 9]. It is unknown whether the structure and function of Golgi outposts differ from those of somal Golgi. Here we show in Drosophila that, unlike somal Golgi, the biochemically distinct cis, medial, and trans compartments of Golgi are often disconnected in dendrites in vivo. The Golgi structural protein GM130 is responsible for connecting distinct Golgi compartments in soma and dendritic branch points, and specific distribution of GM130 determines the compartmental organization of dendritic Golgi in dendritic shafts. We further show that compartmental organization regulates the role of Golgi in acentrosomal microtubule growth in dendrites and in dendritic branching. Our study provides insights into the structure and function of dendritic Golgi outposts as well as the regulation of compartmental organization of Golgi in general. PMID:24835455

  2. Analysis of the Compartmentalized Metabolome – A Validation of the Non-Aqueous Fractionation Technique

    PubMed Central

    Klie, Sebastian; Krueger, Stephan; Krall, Leonard; Giavalisco, Patrick; Flügge, Ulf-Ingo; Willmitzer, Lothar; Steinhauser, Dirk

    2011-01-01

    With the development of high-throughput metabolic technologies, a plethora of primary and secondary compounds have been detected in the plant cell. However, there are still major gaps in our understanding of the plant metabolome. This is especially true with regards to the compartmental localization of these identified metabolites. Non-aqueous fractionation (NAF) is a powerful technique for the determination of subcellular metabolite distributions in eukaryotic cells, and it has become the method of choice to analyze the distribution of a large number of metabolites concurrently. However, the NAF technique produces a continuous gradient of metabolite distributions, not discrete assignments. Resolution of these distributions requires computational analyses based on marker molecules to resolve compartmental localizations. In this article we focus on expanding the computational analysis of data derived from NAF. Along with an experimental workflow, we describe the critical steps in NAF experiments and how computational approaches can aid in assessing the quality and robustness of the derived data. For this, we have developed and provide a new version (v1.2) of the BestFit command line tool for calculation and evaluation of subcellular metabolite distributions. Furthermore, using both simulated and experimental data we show the influence on estimated subcellular distributions by modulating important parameters, such as the number of fractions taken or which marker molecule is selected. Finally, we discuss caveats and benefits of NAF analysis in the context of the compartmentalized metabolome. PMID:22645541

  3. Lipid transfer proteins and the tuning of compartmental identity in the Golgi apparatus.

    PubMed

    McDermott, Mark I; Mousley, Carl J

    2016-10-01

    The Golgi complex constitutes a central way station of the eukaryotic endomembrane system, an intricate network of organelles engaged in control of membrane trafficking and the processing of various cellular components. Previous ideas of compartmental stability within this network are gradually being reshaped by concepts describing a biochemical continuum of hybrid organelles whose constitution is regulated by compartmental maturation. Membrane lipid composition and lipid signaling processes make fundamental contributions to compartmentalization strategies that are themselves critical for organizing cellular architecture and biochemical activities. Phosphatidylinositol transfer proteins (PITPs) are increasingly recognized as key regulators of membrane trafficking through the secretory pathway. They do so by coordinating lipid metabolism with lipid signaling, translating this information to core protein components of the membrane trafficking machinery. In this capacity, PITPs can be viewed as regulators of an essential lipid-protein interface of cisternal maturation. It is also now becoming appreciated, for the first time, that such an interface plays important roles in larger systems processes that link secretory pathway function with cell proliferation.

  4. Diminished viral replication and compartmentalization of hepatitis C virus in hepatocellular carcinoma tissue

    PubMed Central

    Harouaka, Djamila; Engle, Ronald E.; Wollenberg, Kurt; Diaz, Giacomo; Tice, Ashley B.; Zamboni, Fausto; Govindarajan, Sugantha; Alter, Harvey; Kleiner, David E.; Farci, Patrizia

    2016-01-01

    Analysis of hepatitis C virus (HCV) replication and quasispecies distribution within the tumor of patients with HCV-associated hepatocellular carcinoma (HCC) can provide insight into the role of HCV in hepatocarcinogenesis and, conversely, the effect of HCC on the HCV lifecycle. In a comprehensive study of serum and multiple liver specimens from patients with HCC who underwent liver transplantation, we found a sharp and significant decrease in HCV RNA in the tumor compared with surrounding nontumorous tissues, but found no differences in multiple areas of control non-HCC cirrhotic livers. Diminished HCV replication was not associated with changes in miR-122 expression. HCV genetic diversity was significantly higher in livers containing HCC compared with control non-HCC cirrhotic livers. Tracking of individual variants demonstrated changes in the viral population between tumorous and nontumorous areas, the extent of which correlated with the decline in HCV RNA, suggesting HCV compartmentalization within the tumor. In contrast, compartmentalization was not observed between nontumorous areas and serum, or in controls between different areas of the cirrhotic liver or between liver and serum. Our findings indicate that HCV replication within the tumor is restricted and compartmentalized, suggesting segregation of specific viral variants in malignant hepatocytes. PMID:26787866

  5. The mechanics of cellular compartmentalization as a model for tumor spreading

    NASA Astrophysics Data System (ADS)

    Fritsch, Anatol; Pawlizak, Steve; Zink, Mareike; Kaes, Josef A.

    2012-02-01

    Based on a recently developed surgical method of Michael H"ockel, which makes use of cellular confinement to compartments in the human body, we study the mechanics of the process of cell segregation. Compartmentalization is a fundamental process of cellular organization and occurs during embryonic development. A simple model system can demonstrate the process of compartmentalization: When two populations of suspended cells are mixed, this mixture will eventually segregate into two phases, whereas mixtures of the same cell type will not. In the 1960s, Malcolm S. Steinberg formulated the so-called differential adhesion hypothesis which explains the segregation in the model system and the process of compartmentalization by differences in surface tension and adhesiveness of the interacting cells. We are interested in to which extend the same physical principles affect tumor growth and spreading between compartments. For our studies, we use healthy and cancerous breast cell lines of different malignancy as well as primary cells from human cervix carcinoma. We apply a set of techniques to study their mechanical properties and interactions. The Optical Stretcher is used for whole cell rheology, while Cell-cell-adhesion forces are directly measured with a modified AFM. In combination with 3D segregation experiments in droplet cultures we try to clarify the role of surface tension in tumor spreading.

  6. Cell-free compartmentalized protein synthesis inside double emulsion templated liposomes with in vitro synthesized and assembled ribosomes.

    PubMed

    Caschera, Filippo; Lee, Jin Woo; Ho, Kenneth K Y; Liu, Allen P; Jewett, Michael C

    2016-04-07

    A cell-free expression platform for making bacterial ribosomes encapsulated within giant liposomes was capable of synthesizing sfGFP. The liposomes were prepared using a double emulsion template, and compartmentalized in vitro protein synthesis was analysed using spinning disk confocal microscopy. Two different liposome phospholipid formulations were investigated to characterize their effects on the compartmentalized reaction kinetics. This study was performed as a necessary step towards the synthesis of minimal cells.

  7. Intracellular Signalling in Retinal Ischemia

    DTIC Science & Technology

    1990-07-01

    36) However, vascularization of the RPE is not known to occur in human diseases of photoreceptor degeneration, such as retinitis pigmentosa ...A.C. (1986) Retinitis pigmentosa and retinal neovascularization. Ophthalmology 91, 1599- 1603. Figure la: Control rat retina, 8 weeks of age, central...TITLE (Include Security Classification) Intracellular Signalling in Retinal Ischemia 12. PERSONAL AUTHOR(S) Burns, Margaret Sue; Bellhorn, Roy William

  8. Direct Measurement of Intracellular Pressure

    PubMed Central

    Petrie, Ryan J.; Koo, Hyun

    2014-01-01

    A method to directly measure the intracellular pressure of adherent, migrating cells is described in the Basic Protocol. This approach is based on the servo-null method where a microelectrode is introduced into the cell to directly measure the physical pressure of the cytoplasm. We also describe the initial calibration of the microelectrode as well as the application of the method to cells migrating inside three-dimensional (3D) extracellular matrix (ECM). PMID:24894836

  9. Revisiting intracellular calcium signaling semantics.

    PubMed

    Haiech, Jacques; Audran, Emilie; Fève, Marie; Ranjeva, Raoul; Kilhoffer, Marie-Claude

    2011-12-01

    Cells use intracellular free calcium concentration changes for signaling. Signal encoding occurs through both spatial and temporal modulation of the free calcium concentration. The encoded message is detected by an ensemble of intracellular sensors forming the family of calcium-binding proteins (CaBPs) which must faithfully translate the message using a new syntax that is recognized by the cell. The cell is home to a significant although limited number of genes coding for proteins involved in the signal encoding and decoding processes. In a cell, only a subset of this ensemble of genes is expressed, leading to a genetic regulation of the calcium signal pathways. Calmodulin (CaM), the most ubiquitous expressed intracellular calcium-binding protein, plays a major role in calcium signal translation. Similar to a hub, it is central to a large and finely tuned network, receiving information, integrating it and dispatching the cognate response. In this review, we examine the different steps starting with an external stimulus up to a cellular response, with special emphasis on CaM and the mechanism by which it decodes calcium signals and translates it into exquisitely coordinated cellular events. By this means, we will revisit the calcium signaling semantics, hoping that we will ease communication between scientists dealing with calcium signals in different biological systems and different domains.

  10. Stochastic models of intracellular transport

    NASA Astrophysics Data System (ADS)

    Bressloff, Paul C.; Newby, Jay M.

    2013-01-01

    The interior of a living cell is a crowded, heterogenuous, fluctuating environment. Hence, a major challenge in modeling intracellular transport is to analyze stochastic processes within complex environments. Broadly speaking, there are two basic mechanisms for intracellular transport: passive diffusion and motor-driven active transport. Diffusive transport can be formulated in terms of the motion of an overdamped Brownian particle. On the other hand, active transport requires chemical energy, usually in the form of adenosine triphosphate hydrolysis, and can be direction specific, allowing biomolecules to be transported long distances; this is particularly important in neurons due to their complex geometry. In this review a wide range of analytical methods and models of intracellular transport is presented. In the case of diffusive transport, narrow escape problems, diffusion to a small target, confined and single-file diffusion, homogenization theory, and fractional diffusion are considered. In the case of active transport, Brownian ratchets, random walk models, exclusion processes, random intermittent search processes, quasi-steady-state reduction methods, and mean-field approximations are considered. Applications include receptor trafficking, axonal transport, membrane diffusion, nuclear transport, protein-DNA interactions, virus trafficking, and the self-organization of subcellular structures.

  11. New Insight into the Mechanism of Accumulation and Intraerythrocytic Compartmentation of Albitiazolium, a New Type of Antimalarial

    PubMed Central

    Tran Van Ba, Christophe; Maynadier, Marjorie; Bordat, Yann; Perez, Julie; Peyrottes, Suzanne; Fraisse, Laurent

    2014-01-01

    Bis-thiazolium salts constitute a new class of antihematozoan drugs that inhibit parasite phosphatidylcholine biosynthesis. They specifically accumulate in Plasmodium- and Babesia-infected red blood cells (IRBC). Here, we provide new insight into the choline analogue albitiazolium, which is currently being clinically tested against severe malaria. Concentration-dependent accumulation in P. falciparum-infected erythrocytes reached steady state after 90 to 120 min and was massive throughout the blood cycle, with cellular accumulation ratios of up to 1,000. This could not occur through a lysosomotropic effect, and the extent did not depend on the food vacuole pH, which was the case for the weak base chloroquine. Analysis of albitiazolium accumulation in P. falciparum IRBC revealed a high-affinity component that was restricted to mature stages and suppressed by pepstatin A treatment, and thus likely related to drug accumulation in the parasite food vacuole. Albitiazolium also accumulated in a second high-capacity component present throughout the blood cycle that was likely not related to the food vacuole and also observed with Babesia divergens-infected erythrocytes. Accumulation was strictly glucose dependent, drastically inhibited by H+/K+ and Na+ ionophores upon collapse of ionic gradients, and appeared to be energized by the proton-motive force across the erythrocyte plasma membrane, indicating the importance of transport steps for this permanently charged new type of antimalarial agent. This specific, massive, and irreversible accumulation allows albitiazolium to restrict its toxicity to hematozoa-infected erythrocytes. The intraparasitic compartmentation of albitiazolium corroborates a dual mechanism of action, which could make this new type of antimalarial agent resistant to parasite resistance. PMID:25001307

  12. Polycaprolactone/maltodextrin nanocarrier for intracellular drug delivery: formulation, uptake mechanism, internalization kinetics, and subcellular localization

    PubMed Central

    Korang-Yeboah, Maxwell; Gorantla, Yamini; Paulos, Simon A; Sharma, Pankaj; Chaudhary, Jaideep; Palaniappan, Ravi

    2015-01-01

    Prostate cancer (PCa) disease progression is associated with significant changes in intracellular and extracellular proteins, intracellular signaling mechanism, and cancer cell phenotype. These changes may have direct impact on the cellular interactions with nanocarriers; hence, there is the need for a much-detailed understanding, as nanocarrier cellular internalization and intracellular sorting mechanism correlate directly with bioavailability and clinical efficacy. In this study, we report the differences in the rate and mechanism of cellular internalization of a biocompatible polycaprolactone (PCL)/maltodextrin (MD) nanocarrier system for intracellular drug delivery in LNCaP, PC3, and DU145 PCa cell lines. PCL/MD nanocarriers were designed and characterized. PCL/MD nanocarriers significantly increased the intracellular concentration of coumarin-6 and fluorescein isothiocyanate-labeled bovine serum albumin, a model hydrophobic and large molecule, respectively. Fluorescence microscopy and flow cytometry analysis revealed rapid internalization of the nanocarrier. The extent of nanocarrier cellular internalization correlated directly with cell line aggressiveness. PCL/MD internalization was highest in PC3 followed by DU145 and LNCaP, respectively. Uptake in all PCa cell lines was metabolically dependent. Extraction of endogenous cholesterol by methyl-β-cyclodextrin reduced uptake by 75%±4.53% in PC3, 64%±6.01% in LNCaP, and 50%±4.50% in DU145, indicating the involvement of endogenous cholesterol in cellular internalization. Internalization of the nanocarrier in LNCaP was mediated mainly by macropinocytosis and clathrin-independent pathways, while internalization in PC3 and DU145 involved clathrin-mediated endocytosis, clathrin-independent pathways, and macropinocytosis. Fluorescence microscopy showed a very diffused and non-compartmentalized subcellular localization of the PCL/MD nanocarriers with possible intranuclear localization and minor colocalization in

  13. Polycaprolactone/maltodextrin nanocarrier for intracellular drug delivery: formulation, uptake mechanism, internalization kinetics, and subcellular localization.

    PubMed

    Korang-Yeboah, Maxwell; Gorantla, Yamini; Paulos, Simon A; Sharma, Pankaj; Chaudhary, Jaideep; Palaniappan, Ravi

    2015-01-01

    Prostate cancer (PCa) disease progression is associated with significant changes in intracellular and extracellular proteins, intracellular signaling mechanism, and cancer cell phenotype. These changes may have direct impact on the cellular interactions with nanocarriers; hence, there is the need for a much-detailed understanding, as nanocarrier cellular internalization and intracellular sorting mechanism correlate directly with bioavailability and clinical efficacy. In this study, we report the differences in the rate and mechanism of cellular internalization of a biocompatible polycaprolactone (PCL)/maltodextrin (MD) nanocarrier system for intracellular drug delivery in LNCaP, PC3, and DU145 PCa cell lines. PCL/MD nanocarriers were designed and characterized. PCL/MD nanocarriers significantly increased the intracellular concentration of coumarin-6 and fluorescein isothiocyanate-labeled bovine serum albumin, a model hydrophobic and large molecule, respectively. Fluorescence microscopy and flow cytometry analysis revealed rapid internalization of the nanocarrier. The extent of nanocarrier cellular internalization correlated directly with cell line aggressiveness. PCL/MD internalization was highest in PC3 followed by DU145 and LNCaP, respectively. Uptake in all PCa cell lines was metabolically dependent. Extraction of endogenous cholesterol by methyl-β-cyclodextrin reduced uptake by 75%±4.53% in PC3, 64%±6.01% in LNCaP, and 50%±4.50% in DU145, indicating the involvement of endogenous cholesterol in cellular internalization. Internalization of the nanocarrier in LNCaP was mediated mainly by macropinocytosis and clathrin-independent pathways, while internalization in PC3 and DU145 involved clathrin-mediated endocytosis, clathrin-independent pathways, and macropinocytosis. Fluorescence microscopy showed a very diffused and non-compartmentalized subcellular localization of the PCL/MD nanocarriers with possible intranuclear localization and minor colocalization in

  14. Intracellular targeting with engineered proteins

    PubMed Central

    Miersch, Shane; Sidhu, Sachdev S.

    2016-01-01

    If the isolation, production, and clinical use of insulin marked the inception of the age of biologics as therapeutics, the convergence of molecular biology and combinatorial engineering techniques marked its coming of age. The first wave of recombinant protein-based drugs in the 1980s demonstrated emphatically that proteins could be engineered, formulated, and employed for clinical advantage. Yet despite the successes of protein-based drugs such as antibodies, enzymes, and cytokines, the druggable target space for biologics is currently restricted to targets outside the cell. Insofar as estimates place the number of proteins either secreted or with extracellular domains in the range of 8000 to 9000, this represents only one-third of the proteome and circumscribes the pathways that can be targeted for therapeutic intervention. Clearly, a major objective for this field to reach maturity is to access, interrogate, and modulate the majority of proteins found inside the cell. However, owing to the large size, complex architecture, and general cellular impermeability of existing protein-based drugs, this poses a daunting challenge. In recent years, though, advances on the two related fronts of protein engineering and drug delivery are beginning to bring this goal within reach. First, prompted by the restrictions that limit the applicability of antibodies, intense efforts have been applied to identifying and engineering smaller alternative protein scaffolds for the modulation of intracellular targets. In parallel, innovative solutions for delivering proteins to the intracellular space while maintaining their stability and functional activity have begun to yield successes. This review provides an overview of bioactive intrabodies and alternative protein scaffolds amenable to engineering for intracellular targeting and also outlines advances in protein engineering and formulation for delivery of functional proteins to the interior of the cell to achieve therapeutic action

  15. Pharmacology of intracellular signalling pathways

    PubMed Central

    Nahorski, Stefan R

    2006-01-01

    This article provides a brief and somewhat personalized review of the dramatic developments that have occurred over the last 45 years in our understanding of intracellular signalling pathways associated with G-protein-coupled receptor activation. Signalling via cyclic AMP, the phosphoinositides and Ca2+ is emphasized and these systems have already been revealed as new pharmacological targets. The therapeutic benefits of most of such targets are, however, yet to be realized, but it is certain that the discipline of pharmacology needs to widen its boundaries to meet these challenges in the future. PMID:16402119

  16. Organelle pH in the Arabidopsis endomembrane system.

    PubMed

    Shen, Jinbo; Zeng, Yonglun; Zhuang, Xiaohong; Sun, Lei; Yao, Xiaoqiang; Pimpl, Peter; Jiang, Liwen

    2013-09-01

    The pH of intracellular compartments is essential for the viability of cells. Despite its relevance, little is known about the pH of these compartments. To measure pH in vivo, we have first generated two pH sensors by combining the improved-solubility feature of solubility-modified green fluorescent protein (GFP) (smGFP) with the pH-sensing capability of the pHluorins and codon optimized for expression in Arabidopsis. PEpHluorin (plant-solubility-modified ecliptic pHluorin) gradually loses fluorescence as pH is lowered with fluorescence vanishing at pH 6.2 and PRpHluorin (plant-solubility-modified ratiomatric pHluorin), a dual-excitation sensor, allowing for precise measurements. Compartment-specific sensors were generated by further fusing specific sorting signals to PEpHluorin and PRpHluorin. Our results show that the pH of cytosol and nucleus is similar (pH 7.3 and 7.2), while peroxisomes, mitochondrial matrix, and plastidial stroma have alkaline pH. Compartments of the secretory pathway reveal a gradual acidification, spanning from pH 7.1 in the endoplasmic reticulum (ER) to pH 5.2 in the vacuole. Surprisingly, pH in the trans-Golgi network (TGN) and multivesicular body (MVB) is, with pH 6.3 and 6.2, quite similar. The inhibition of vacuolar-type H(+)-ATPase (V-ATPase) with concanamycin A (ConcA) caused drastic increase in pH in TGN and vacuole. Overall, the PEpHluorin and PRpHluorin are excellent pH sensors for visualization and quantification of pH in vivo, respectively.

  17. The Role of Cell Compartmentalization and Cell Differentiation in Cyanobacterial Excavation of Miineral Carbonates

    NASA Astrophysics Data System (ADS)

    Garcia-Pichel, F.; Guida, B. S.; Couradeau, E.

    2015-12-01

    The bioerosion of coastal limestones and biogenic carbonates by boring filamentous or pseudo-filamentous cyanobacteria is not only a geomicrobial phenomenon of global proportions, but also plays an important role in the demise of coral reefs, and affects significantly human enterprises like bivalve fisheries. In spite of its importance, the mechanism by which cyanobacteria excavate carbonates constitutes an apparent paradox, in that their metabolism will tend to precipitate carbonates, not dissolved them. We have previously advanced, and obtained evidence for, a mechanism of excavation that relies on the uptake of Ca2+ by cells at the boring front, its trans-cellular transport along the filaments, and its eventual active excretion at the solid/liquid interface. It was postulated that the mechanism involved the strategically organized deployment of Ca2+ transport enzymes like P-type Ca2+ ATPases and Ca2+ channels. Here we present evidence that confirms this basic mechanism, but also reveals that it is based on an unexpected level of cellular complexity. The model organism Mastigocoleus testarum BC008, transports Ca2+ from the mineral to the external medium using a repetitive, polar arrangement of Ca2+ ATPases, localized preferentially on one cellular pole, in a ring conformation on the cell membrane adjacent to the trans-cellular septum, pumping Ca2+ locally towards the periplasmic space, from which it passively enters the next cell. This strain also develops specialized groups of cells, which we named calcicytes, often but not exclusively located at the ends of filaments, that accumulate large concentrations of Ca2+, some 40-fold higher than typical in microbes, and seem to act as sinks or capacitors in the trans-cellular Ca2+ transport. Calcicytes are also characterized by a lack of photosynthetic pigments, and a very high intracellular pH. These cellular adaptations can also be found in evolutionary distant euendoliths such as the pseudofilamentous Hyella sp.

  18. Shape-directed compartmentalized delivery of a nanoparticle-conjugated small-molecule activator of an epigenetic enzyme in the brain.

    PubMed

    Chaturbedy, Piyush; Kumar, Manoj; Salikolimi, Krishnachary; Das, Sadhan; Sinha, Sarmistha Halder; Chatterjee, Snehajyoti; Suma, B S; Kundu, Tapas K; Eswaramoorthy, Muthusamy

    2015-11-10

    Targeted drug delivery to specific subcellular compartments of brain cells is challenging despite their importance in the treatment of several brain-related diseases. Herein, we report on shape-directed intracellular compartmentalization of nanoparticles in brain cells and their ability to deliver therapeutic molecules to specific organelles. Iron oxide (Fe3O4) nanoparticles with different morphologies (spheres, spindles, biconcaves, and nanotubes) were synthesized and coated with a fluorescent carbon layer derived from glucose (Fe3O4@C). In vivo studies showed that the Fe3O4@C nanoparticles with biconcave geometry localized predominantly in the nuclei of the brain cells, whereas those with nanotube geometry were contained mostly in the cytoplasm. Remarkably, a small-molecule activator of histone acetyltransferases delivered into the nuclei of the brain cells using nanoparticles with biconcave geometry showed enhancement in enzymatic activity by a factor of three and resulted in specific gene expression (transcription) compared with that of the molecule delivered to the cytoplasm using nanotube geometry.

  19. Development and testing of a compartmentalized reaction network model for redox zones in contaminated aquifers

    USGS Publications Warehouse

    Abrams, R.H.; Loague, K.; Kent, D.B.

    1998-01-01

    The work reported here is the first part of a larger effort focused on efficient numerical simulation of redox zone development in contaminated aquifers. The sequential use of various electron acceptors, which is governed by the energy yield of each reaction, gives rise to redox zones. The large difference in energy yields between the various redox reactions leads to systems of equations that are extremely ill-conditioned. These equations are very difficult to solve, especially in the context of coupled fluid flow, solute transport, and geochemical simulations. We have developed a general, rational method to solve such systems where we focus on the dominant reactions, compartmentalizing them in a manner that is analogous to the redox zones that are often observed in the field. The compartmentalized approach allows us to easily solve a complex geochemical system as a function of time and energy yield, laying the foundation for our ongoing work in which we couple the reaction network, for the development of redox zones, to a model of subsurface fluid flow and solute transport. Our method (1) solves the numerical system without evoking a redox parameter, (2) improves the numerical stability of redox systems by choosing which compartment and thus which reaction network to use based upon the concentration ratios of key constituents, (3) simulates the development of redox zones as a function of time without the use of inhibition factors or switching functions, and (4) can reduce the number of transport equations that need to be solved in space and time. We show through the use of various model performance evaluation statistics that the appropriate compartment choice under different geochemical conditions leads to numerical solutions without significant error. The compartmentalized approach described here facilitates the next phase of this effort where we couple the redox zone reaction network to models of fluid flow and solute transport.

  20. Review: Intracardiac intracellular angiotensin system in diabetes

    PubMed Central

    Kumar, Rajesh; Yong, Qian Chen; Thomas, Candice M.

    2012-01-01

    The renin-angiotensin system (RAS) has mainly been categorized as a circulating and a local tissue RAS. A new component of the local system, known as the intracellular RAS, has recently been described. The intracellular RAS is defined as synthesis and action of ANG II intracellularly. This RAS appears to differ from the circulating and the local RAS, in terms of components and the mechanism of action. These differences may alter treatment strategies that target the RAS in several pathological conditions. Recent work from our laboratory has demonstrated significant upregulation of the cardiac, intracellular RAS in diabetes, which is associated with cardiac dysfunction. Here, we have reviewed evidence supporting an intracellular RAS in different cell types, ANG II's actions in cardiac cells, and its mechanism of action, focusing on the intracellular cardiac RAS in diabetes. We have discussed the significance of an intracellular RAS in cardiac pathophysiology and implications for potential therapies. PMID:22170614

  1. Balancing Type I and Type II error concerns in fMRI through compartmentalized analysis.

    PubMed

    Cunningham, William A; Koscik, Timothy R

    2017-03-13

    We seek to balance the need to minimize false positives with the need to maximize power. We propose a compartmentalized series of analyses that a priori selects regions of voxels that have different degrees of predicted involvement. Alpha thresholds are allocated based on the strength of expected theoretical relationships. For example, confirmatory studies might allocate most of the error to the regions predicted from the literature and thus use a relatively more liberal threshold on these voxels. Simulations reveal that this technique increases power for hypothesized regions, while maintaining a constant false-positive rate and allowing exploratory analysis.

  2. Nuclear matrix and structural and functional compartmentalization of the eucaryotic cell nucleus.

    PubMed

    Razin, S V; Borunova, V V; Iarovaia, O V; Vassetzky, Y S

    2014-07-01

    Becoming popular at the end of the 20th century, the concept of the nuclear matrix implies the existence of a nuclear skeleton that organizes functional elements in the cell nucleus. This review presents a critical analysis of the results obtained in the study of nuclear matrix in the light of current views on the organization of the cell nucleus. Numerous studies of nuclear matrix have failed to provide evidence of the existence of such a structure. Moreover, the existence of a filamentous structure that supports the nuclear compartmentalization appears to be unnecessary, since this function is performed by the folded genome itself.

  3. Nilpotent singularities and dynamics in an SIR type of compartmental model with hospital resources

    NASA Astrophysics Data System (ADS)

    Shan, Chunhua; Yi, Yingfei; Zhu, Huaiping

    2016-03-01

    An SIR type of compartmental model with a standard incidence rate and a nonlinear recovery rate was formulated to study the impact of available resources of public health system especially the number of hospital beds. Cusp, focus and elliptic type of nilpotent singularities of codimension 3 are discovered and analyzed in this three dimensional model. Complex dynamics of disease transmission including multi-steady states and multi-periodicity are revealed by bifurcation analysis. Large-amplitude oscillations found in our model provide a more reasonable explanation for disease recurrence. With clinical data, our studies have practical implications for the prevention and control of infectious diseases.

  4. Local intracellular ion measurements with luminescent indicators using confocal laser scanning microscopy

    NASA Astrophysics Data System (ADS)

    Opitz, N.; Merten, E.; Acker, H.

    1995-09-01

    Ion sensitive fluoroprobes such as BCECF (pH) and FURA-II (Ca2+) are frequently used indicators for determination of ion activities in single cells and subcellular compartments, e.g. by video enhanced or video intensified microscopy. Moreover, using confocal laser scanning microscopy (CLSM) with its inherent potential for noninvasive optical sectioning of cells and tissues and subsequent 3D image reconstruction, intracellular ion topographies can be monitored via pseudocolor encoded ratio imaging from pixel to pixel enabling in vivo measurements of dynamic intracellular processes. Regardless of the degree of spatial resolution, reliable qualtitative determinations essentially depend on accurate calibration of the intracellularly entrapped fluoroprobe. Calibration is either established on the basis of a whole cell or within a more or less extended subcellular compartment and the characteristics are displayed as concentration encoded pseudocolor bar within the image frame. This calibration is assumed to be valid for other cellular compartments and, in case of ion imaging, it is even thought to be valid for every single pixel of the complete pixel field. However, the assumption of a topographically invariant intracellular calibration requires a reliable behavior of the intracellularly applied indicator. This intracellular integrity of the dyes often does not seem to exist since intracellular calibration curves considerably deviate from in vitro calibration characteristics. Deviations may be due to intracellular interactions of indicator molecules with cytoplasmic macromolecules, e.g. proteins, resulting in spectral distortions and/or sensitivity deficits as demonstrated by the indicators BCECF and FURA-RED (a FURA-II analogue) or to intracellular redistribution of the indicator as exemplified by pH measurements using carboxy-SNARF-1. Consequences of these investigations as well as further potential interferences are discussed with special respect to ion imaging

  5. The influence of intracellular lactate and H+ on cell volume in amphibian skeletal muscle

    PubMed Central

    Usher-Smith, Juliet A; Fraser, James A; Bailey, Peter S J; Griffin, Julian L; Huang, Christopher L-H

    2006-01-01

    The combined effects of intracellular lactate and proton accumulation on cell volume, Vc, were investigated in resting Rana temporaria striated muscle fibres. Intracellular lactate and H+ concentrations were simultaneously increased by exposing resting muscle fibres to extracellular solutions that contained 20–80 mm sodium lactate. Cellular H+ and lactate entry was confirmed using pH-sensitive electrodes and 1H-NMR, respectively, and effects on Vc were measured using confocal microscope xz-scanning. Exposure to extracellular lactate up to 80 mm produced significant changes in pH and intracellular lactate (from a pH of 7.24 ± 0.03, n = 8, and 4.65 ± 1.07 mm, n = 6, respectively, in control fibres, to 6.59 ± 0.03, n = 4, and 26.41 ± 0.92 mm, n = 3, respectively) that were comparable to those observed following fatiguing stimulation (6.30–6.70 and 18.04 ± 1.78 mm, n = 6, respectively). Yet, the increase in intracellular osmolarity expected from such an increase in intracellular lactate did not significantly alter Vc. Simulation of these experimental results, modified from the charge difference model of Fraser & Huang, demonstrated that such experimental manoeuvres produced changes in intracellular [H+] and [lactate] comparable to those observed during muscle fatigue, and accounted for this paradoxical conservation of Vc through balancing negative osmotic effects resulting from the net cation efflux that would follow a titration of intracellular membrane-impermeant anions by the intracellular accumulation of protons. It demonstrated that with established physiological values for intracellular buffering capacity and the permeability ratio of lactic acid and anionic lactate, PLacH: PLac−, this would provide a mechanism that precisely balanced any effect on cell volume resulting from lactate accumulation during exercise. PMID:16613877

  6. Distinct patterns of compartmentalization and proteolytic stability of PDE6C mutants linked to achromatopsia.

    PubMed

    Cheguru, Pallavi; Majumder, Anurima; Artemyev, Nikolai O

    2015-01-01

    Phosphodiesterase-6 (PDE6) is an essential effector enzyme in vertebrate photoreceptor cells. Mutations in rod and cone PDE6 cause recessive retinitis pigmentosa and achromatopsia, respectively. The mechanisms of missense PDE6 mutations underlying severe visual disorders are poorly understood. To probe these mechanisms, we expressed seven known missense mutants of cone PDE6C in rods of transgenic Xenopus laevis and examined their stability and compartmentalization. PDE6C proteins with mutations in the catalytic domain, H602L and E790K, displayed modestly reduced proteolytic stability, but they were properly targeted to the outer segment of photoreceptor cells. Mutations in the regulatory GAF domains, R104W, Y323N, and P391L led to a proteolytic degradation of the proteins involving a cleavage in the GAFb domain. Lastly, the R29W and M455V mutations residing outside the conserved PDE6 domains produced a pattern of subcellular compartmentalization different from that of PDE6C. Thus, our results suggest a spectrum of mechanisms of missense PDE6C mutations in achromatopsia including catalytic defects, protein mislocalization, or a specific sequence of proteolytic degradation.

  7. Software for compartmental translation analysis and virtual three-dimensional visualization of the pivot shift phenomenon.

    PubMed

    Suero, Eduardo M; Citak, Musa; Choi, Daniel; Bosscher, Marianne Roberta Frederiek; Citak, Mustafa; Pearle, Andrew D; Plaskos, Christopher

    2011-01-01

    Anterior cruciate ligament (ACL) injury may cause knee instability and may result in damage to the menisci and the articular cartilage. The pivot shift test is commonly used to identify rotational instability of the knee following injury to the ACL. The magnitude of lateral compartment translation correlates well with the grade of the pivot shift. However, commonly used navigation systems do not readily provide individualized compartmental translation. We aimed to develop software to (a) quantify individual medial and lateral compartmental translation in the knee during the pivot shift test, and (b) generate animated three-dimensional renderings of recorded pivot shift examinations. Twelve paired cadaveric knees were used to test the software. Three mechanized pivot shift tests were performed on each knee with the ACL intact and again after sectioning the ACL. Using the Pivot Shift Processor, we successfully analyzed the data recorded using the navigation system. After sectioning the ACL, there was a greater increase in tibiofemoral translation in the lateral compartment compared to the medial compartment. The Pivot Shift Visualizer successfully produced a 3D rendering of the knee joint and the recorded pivot shift maneuvers. This virtual representation of the pivot shift phenomenon from multiple points of view allows for efficient side-by-side comparison of tibiofemoral motion tracking across conditions, which is not possible in the in vivo / in vitro settings. This, in turn, could lead to a better understanding of the kinematics in play during the pivot shift phenomenon.

  8. Modulation in Wistar rats of blood corticosterone compartmentation by sex and a cafeteria diet.

    PubMed

    Romero, María del Mar; Holmgren-Holm, Fredrik; Grasa, Maria del Mar; Esteve, Montserrat; Remesar, Xavier; Fernández-López, José Antonio; Alemany, Marià

    2013-01-01

    In the metabolic syndrome, glucocorticoid activity is increased, but circulating levels show little change. Most of blood glucocorticoids are bound to corticosteroid-binding globulin (CBG), which liver expression and circulating levels are higher in females than in males. Since blood hormones are also bound to blood cells, and the size of this compartment is considerable for androgens and estrogens, we analyzed whether sex or eating a cafeteria diet altered the compartmentation of corticosterone in rat blood. The main corticosterone compartment in rat blood is that specifically bound to plasma proteins, with smaller compartments bound to blood cells or free. Cafeteria diet increased the expression of liver CBG gene, binding plasma capacity and the proportion of blood cell-bound corticosterone. There were marked sex differences in blood corticosterone compartmentation in rats, which were unrelated to testosterone. The use of a monoclonal antibody ELISA and a polyclonal Western blot for plasma CBG compared with both specific plasma binding of corticosterone and CBG gene expression suggested the existence of different forms of CBG, with varying affinities for corticosterone in males and females, since ELISA data showed higher plasma CBG for males, but binding and Western blot analyses (plus liver gene expression) and higher physiological effectiveness for females. Good cross-reactivity to the antigen for polyclonal CBG antibody suggests that in all cases we were measuring CBG. The different immunoreactivity and binding affinity may help explain the marked sex-related differences in plasma hormone binding as sex-linked different proportions of CBG forms.

  9. Nuclear Compartmentalization Contributes to Stage-Specific Gene Expression Control in Trypanosoma cruzi

    PubMed Central

    Pastro, Lucía; Smircich, Pablo; Di Paolo, Andrés; Becco, Lorena; Duhagon, María A.; Sotelo-Silveira, José; Garat, Beatriz

    2017-01-01

    In the protozoan parasite Trypanosoma cruzi, as in other trypanosomatids, transcription of protein coding genes occurs in a constitutive fashion, producing large polycistronic transcription units. These units are composed of non-functionally related genes which are pervasively processed to yield each mRNA. Therefore, post-transcriptional processes are crucial to regulate gene expression. Considering that nuclear compartmentalization could contribute to gene expression regulation, we comparatively studied the nuclear, cytoplasmic and whole cell transcriptomes of the non-infective epimastigote stage of T. cruzi, using RNA-Seq. We found that the cytoplasmic transcriptome tightly correlates with the whole cell transcriptome and both equally correlate with the proteome. Nonetheless, 1,200 transcripts showed differential abundance between the nuclear and cytoplasmic fractions. For the genes with transcript content augmented in the nucleus, significant structural and compositional differences were found. The analysis of the reported epimastigote translatome and proteome, revealed scarce ribosome footprints and encoded proteins for them. Ontology analyses unveiled that many of these genes are distinctive of other parasite life-cycle stages. Finally, the relocalization of transcript abundance in the metacyclic trypomastigote infective stage was confirmed for specific genes. While gene expression is strongly dependent on transcript steady-state level, we here highlight the importance of the distribution of transcripts abundance between compartments in T. cruzi. Particularly, we show that nuclear compartmentation is playing an active role in the developmental stage determination preventing off-stage expression. PMID:28243589

  10. Compartmentalized Fluid Flow In The Nevado Del Ruiz Volcano Hydrothermal System(S)

    NASA Astrophysics Data System (ADS)

    Zuluaga, C. A.; Mejia, E.

    2011-12-01

    Combination of several extensive and compressive fault/fracture systems with different lithologic units compartmentalized the hydrothermal system(s) in the vicinity of the Nevado del Ruiz volcano. Three main fault/fracture systems are observed in the Ruiz volcano area, a N10°-20°E system (San Jerónimo and Palestina faults), a N40°-60°W system (Villamaría-Termales, San Ramón, Nereidas, Río Claro, San Eugenio and Campoalegrito faults), and a N60°-80°E system (Santa Rosa fault). The NW trend system act as the main path for fluid circulation, location of faults and fractures belonging to this system and their intersections with other fault systems and/or with lithologic contacts control hot springs location. The observed fault location and hot spring location pattern allow to subdivide the hydrothermal system(s) in at least five blocks. In the southernmost block, hot springs are mostly located in one of the four quadrants originated by fault intersections suggesting that there is a compartmentalization into higher and lower permeability quadrants. It is still unknown if all blocks belong to the same hydrothermal system or if there is more than one hydrothermal system.

  11. Detecting compartmental non-Gaussian diffusion with symmetrized double-PFG MRI.

    PubMed

    Paulsen, Jeffrey L; Özarslan, Evren; Komlosh, Michal E; Basser, Peter J; Song, Yi-Qiao

    2015-11-01

    Diffusion in tissue and porous media is known to be non-Gaussian and has been used for clinical indications of stroke and other tissue pathologies. However, when conventional NMR techniques are applied to biological tissues and other heterogeneous materials, the presence of multiple compartments (pores) with different Gaussian diffusivities will also contribute to the measurement of non-Gaussian behavior. Here we present symmetrized double PFG (sd-PFG), which can separate these two contributions to non-Gaussian signal decay as having distinct angular modulation frequencies. In contrast to prior angular d-PFG methods, sd-PFG can unambiguously extract kurtosis as an oscillation from samples with isotropic or uniformly oriented anisotropic pores, and can generally extract a combination of compartmental anisotropy and kurtosis. The method further fixes its sensitivity with respect to the time dependence of the apparent diffusion coefficient. We experimentally demonstrate the measurement of the fourth cumulant (kurtosis) of diffusion and find it consistent with theoretical predictions. By enabling the unambiguous identification of contributions of compartmental kurtosis to the signal, sd-PFG has the potential to help identify the underlying micro-structural changes corresponding to current kurtosis based diagnostics, and act as a novel source of contrast to better resolve tissue micro-structure.

  12. A compartmental-spatial system dynamics approach to ground water modeling.

    PubMed

    Roach, Jesse; Tidwell, Vince

    2009-01-01

    High-resolution, spatially distributed ground water flow models can prove unsuitable for the rapid, interactive analysis that is increasingly demanded to support a participatory decision environment. To address this shortcoming, we extend the idea of multiple cell (Bear 1979) and compartmental (Campana and Simpson 1984) ground water models developed within the context of spatial system dynamics (Ahmad and Simonovic 2004) for rapid scenario analysis. We term this approach compartmental-spatial system dynamics (CSSD). The goal is to balance spatial aggregation necessary to achieve a real-time integrative and interactive decision environment while maintaining sufficient model complexity to yield a meaningful representation of the regional ground water system. As a test case, a 51-compartment CSSD model was built and calibrated from a 100,0001 cell MODFLOW (McDonald and Harbaugh 1988) model of the Albuquerque Basin in central New Mexico (McAda and Barroll 2002). Seventy-seven percent of historical drawdowns predicted by the MODFLOW model were within 1 m of the corresponding CSSD estimates, and in 80% of the historical model run years the CSSD model estimates of river leakage, reservoir leakage, ground water flow to agricultural drains, and riparian evapotranspiration were within 30% of the corresponding estimates from McAda and Barroll (2002), with improved model agreement during the scenario period. Comparisons of model results demonstrate both advantages and limitations of the CCSD model approach.

  13. Modeling the Kinetics of a Memory-Associated Immediate Early Gene's Compartmental Expression After Sensory Experience

    NASA Astrophysics Data System (ADS)

    Willats, Adam; Ivanova, Tamara; Prinz, Astrid; Liu, Robert

    2015-03-01

    Immediate Early Genes (IEGs) are rapidly and transiently transcribed in neurons after a sensory experience. Some of these genes act as effector IEGs, which mediate specific effects on cellular function. Arc is one such effector IEG that is essential for synaptic plasticity and memory consolidation in hippocampus and cortex. The expression of Arc in neurons has previously been examined using an imaging method known as Compartmental Analysis of Temporal Fluorescent In-Situ Hybridization. Previous work found that the time course of Arc expression within the nuclear and perinuclear cytoplasmic compartments of a neuron is altered by prior sensory experience. We explore a simple model of the kinetics of IEG transcription and nuclear export, with the aim of eventually uncovering possible mechanisms for how experience alters expression kinetics. Thus far, we characterize our compartmental model using phase-plane analysis and validate it against several IEG expression data sets, including one where prior experience with vocalizing mice alters the time course of call-induced Arc expression in the auditory cortex of a listening mouse. Our model provides a framework to explore why Arc expression may change depending on a receiver's past sound experience and internal state. Adam Willats was supported by NIH Training Grant 5T90DA032466. This research was also supported by NIDCD R01 DC8343.

  14. Distribution and compartmentalization of human circulating and tissue-resident memory T cell subsets

    PubMed Central

    Sathaliyawala, Taheri; Kubota, Masaru; Yudanin, Naomi; Turner, Damian; Camp, Philip; Thome, Joseph J. C.; Bickham, Kara L.; Lerner, Harvey; Goldstein, Michael; Sykes, Megan; Kato, Tomoaki; Farber, Donna L.

    2012-01-01

    SUMMARY Knowledge of human T cells derives chiefly from studies of peripheral blood, whereas their distribution and function in tissues remains largely unknown. Here, we present a unique analysis of human T cells in lymphoid and mucosal tissues obtained from individual organ donors, revealing tissue-intrinsic compartmentalization of naive, effector and memory subsets conserved between diverse individuals. Effector-memory CD4+ T cells producing IL-2 predominated in mucosal tissues and accumulated as central-memory subsets in lymphoid tissue, whereas CD8+ T cells were maintained as naïve subsets in lymphoid tissues and IFN-γ-producing effector-memory CD8+ T cells in mucosal sites. The T cell activation marker, CD69, was constitutively expressed by memory T cells in all tissues, distinguishing them from circulating subsets, with mucosal memory T cells exhibiting additional distinct phenotypic and functional properties. Our results provide an assessment of human T cell compartmentalization as a new baseline for understanding human adaptive immunity. PMID:23260195

  15. 4D maximum a posteriori reconstruction in dynamic SPECT using a compartmental model-based prior.

    PubMed

    Kadrmas, D J; Gullberg, G T

    2001-05-01

    A 4D ordered-subsets maximum a posteriori (OSMAP) algorithm for dynamic SPECT is described which uses a temporal prior that constrains each voxel's behaviour in time to conform to a compartmental model. No a priori limitations on kinetic parameters are applied; rather, the parameter estimates evolve as the algorithm iterates to a solution. The estimated parameters and time-activity curves are used within the reconstruction algorithm to model changes in the activity distribution as the camera rotates, avoiding artefacts due to inconsistencies of data between projection views. This potentially allows for fewer, longer-duration scans to be used and may have implications for noise reduction. The algorithm was evaluated qualitatively using dynamic 99mTc-teboroxime SPECT scans in two patients, and quantitatively using a series of simulated phantom experiments. The OSMAP algorithm resulted in images with better myocardial uniformity and definition, gave time-activity curves with reduced noise variations, and provided wash-in parameter estimates with better accuracy and lower statistical uncertainty than those obtained from conventional ordered-subsets expectation-maximization (OSEM) processing followed by compartmental modelling. The new algorithm effectively removed the bias in k21 estimates due to inconsistent projections for sampling schedules as slow as 60 s per timeframe, but no improvement in wash-out parameter estimates was observed in this work. The proposed dynamic OSMAP algorithm provides a flexible framework which may benefit a variety of dynamic tomographic imaging applications.

  16. Reconstruction of Arabidopsis metabolic network models accounting for subcellular compartmentalization and tissue-specificity.

    PubMed

    Mintz-Oron, Shira; Meir, Sagit; Malitsky, Sergey; Ruppin, Eytan; Aharoni, Asaph; Shlomi, Tomer

    2012-01-03

    Plant metabolic engineering is commonly used in the production of functional foods and quality trait improvement. However, to date, computational model-based approaches have only been scarcely used in this important endeavor, in marked contrast to their prominent success in microbial metabolic engineering. In this study we present a computational pipeline for the reconstruction of fully compartmentalized tissue-specific models of Arabidopsis thaliana on a genome scale. This reconstruction involves automatic extraction of known biochemical reactions in Arabidopsis for both primary and secondary metabolism, automatic gap-filling, and the implementation of methods for determining subcellular localization and tissue assignment of enzymes. The reconstructed tissue models are amenable for constraint-based modeling analysis, and significantly extend upon previous model reconstructions. A set of computational validations (i.e., cross-validation tests, simulations of known metabolic functionalities) and experimental validations (comparison with experimental metabolomics datasets under various compartments and tissues) strongly testify to the predictive ability of the models. The utility of the derived models was demonstrated in the prediction of measured fluxes in metabolically engineered seed strains and the design of genetic manipulations that are expected to increase vitamin E content, a significant nutrient for human health. Overall, the reconstructed tissue models are expected to lay down the foundations for computational-based rational design of plant metabolic engineering. The reconstructed compartmentalized Arabidopsis tissue models are MIRIAM-compliant and are available upon request.

  17. Directed evolution of an extremely fast phosphotriesterase by in vitro compartmentalization

    PubMed Central

    Griffiths, Andrew D.; Tawfik, Dan S.

    2003-01-01

    We describe the selection of a phosphotriesterase with a very fast kcat (over 105 s–1), 63 times higher than the already very efficient wild-type enzyme. The enzyme was selected from a library of 3.4 × 107 mutated phosphotriesterase genes using a novel strategy based on linking genotype and phenotype by in vitro compartmentalization (IVC) using water-in-oil emulsions. First, microbeads, each displaying a single gene and multiple copies of the encoded protein, are formed by compartmentalized in vitro translation. These microbeads can then be selected for catalysis or binding. To select for catalysis the microbeads are re-emulsified in a reaction buffer of choice with a soluble substrate. The product and any unreacted substrate are coupled to the beads when the reaction is finished. Product-coated beads, displaying active enzymes and the genes that encode them, are detected with anti-product antibodies and selected using flow cytometry. This completely in vitro process selects for all enzymatic features simultaneously (substrate recognition, product formation, rate acceleration and turnover) and single enzyme molecules can be detected. PMID:12505981

  18. [Compartmentalization of the cell nucleus and spatial organization of the genome].

    PubMed

    Gavrilov, A A; Razin, S V

    2015-01-01

    The eukaryotic cell nucleus is one of the most complex cell organelles. Despite the absence of membranes, the nuclear space is divided into numerous compartments where different processes in- volved in the genome activity take place. The most important nuclear compartments include nucleoli, nuclear speckles, PML bodies, Cajal bodies, histone locus bodies, Polycomb bodies, insulator bodies, transcription and replication factories. The structural basis for the nuclear compartmentalization is provided by genomic DNA that occupies most of the nuclear volume. Nuclear compartments, in turn, guide the chromosome folding by providing a platform for the spatial interaction of individual genomic loci. In this review, we discuss fundamental principles of higher order genome organization with a focus on chromosome territories and chromosome domains, as well as consider the structure and function of the key nuclear compartments. We show that the func- tional compartmentalization of the cell nucleus and genome spatial organization are tightly interconnected, and that this form of organization is highly dynamic and is based on stochastic processes.

  19. Anomalous dynamics in intracellular transport

    NASA Astrophysics Data System (ADS)

    Dinner, Aaron

    2013-03-01

    This talk will describe quantitative analyses of particle tracking data for systems with cytoskeletally associated molecular motors to better understand the motions contributing to intracellular transport and, more generally, means for characterizing systems far from equilibrium. In particular, we have studied the motions of insulin-containing vesicles (granules) in a pancreatic beta cell line. We find subdiffusive behavior with correlations in both space and time. These data can be modeled by subordinating an ergodic random walk process to a non-ergodic one. We relate the dynamics to the underlying microtubule structure by imaging in the presence of the drug vinblastine. Our results provide a simple physical mechanism for how diverse pools of insulin granules and, in turn, biphasic secretion could arise. Time permitting, these dynamics will be compared with those of actomyosin assemblies.

  20. THE ALTERATION OF INTRACELLULAR ENZYMES

    PubMed Central

    Kaplan, J. Gordin

    1954-01-01

    1. The ability of homologous series of alcohols, ketones, and aldehydes to cause alteration of intracellular catalase increases approximately threefold for each methylene group added, thus following Traube's rule. Equiactive concentrations of alcohols (methanol to octanol) varied over a 4,000-fold range, yet the average corresponding surface tension was 42 ± 2 dynes/cm., that for ketones 43 ± 2, and for aldehydes (above C1) 41 ± 3. 2. Above C8 the altering activity of alcohols ceased to follow Traube's rule, and at C18 was nil. Yet the surface activities of alcohols from nonanol to dodecanol did follow Traube's rule. These two facts show that the interface which is being affected by these agents is not the cell surface, for if it were, altering activity should not fall off between C9 and C12 where surface activity is undiminished; they show also that micelle formation by short range association of hydrocarbon "tails," usually invoked to explain decrease in biological activity of compounds above C8, is not responsible for this effect in these experiments, in which permeability of the cell membrane probably is involved. 3. The most soluble alcohols and aldehydes (alcohols C1 to C8; aldehydes C1, C2), but not ketones, cause, above optimal concentration, an irreversible inhibition of yeast catalase. 4. The critical concentration of altering agent (i.e., that concentration just sufficient to cause doubling of the catalase activity of the yeast suspension) was independent of the concentration of the yeast cells. 5. Viability studies show that the number of yeast cells killed by the altering agents was not related to the degree of activation of the catalase produced. While all the cells were invariably killed by concentrations of altering agent which produced complete activation, all the cells had been killed by concentrations which were insufficient to cause more than 50 per cent maximal activation. Further, the evidence suggested that the catalase may be partially

  1. Insider trading: Extracellular matrix proteins and their non-canonical intracellular roles.

    PubMed

    Hellewell, Andrew L; Adams, Josephine C

    2016-01-01

    In metazoans, the extracellular matrix (ECM) provides a dynamic, heterogeneous microenvironment that has important supportive and instructive roles. Although the primary site of action of ECM proteins is extracellular, evidence is emerging for non-canonical intracellular roles. Examples include osteopontin, thrombospondins, IGF-binding protein 3 and biglycan, and relate to roles in transcription, cell-stress responses, autophagy and cancer. These findings pose conceptual problems on how proteins signalled for secretion can be routed to the cytosol or nucleus, or can function in environments with diverse redox, pH and ionic conditions. We review evidence for intracellular locations and functions of ECM proteins, and current knowledge of the mechanisms by which they may enter intracellular compartments. We evaluate the experimental methods that are appropriate to obtain rigorous evidence for intracellular localisation and function. Better insight into this under-researched topic is needed to decipher the complete spectrum of physiological and pathological roles of ECM proteins.

  2. Intracellular dehydroascorbic acid inhibits SVCT2-dependent transport of ascorbic acid in mitochondria.

    PubMed

    Fiorani, Mara; Azzolini, Catia; Guidarelli, Andrea; Cerioni, Liana; Scotti, Maddalena; Cantoni, Orazio

    2015-09-01

    Exposure of U937 cells to low concentrations of L-ascorbic acid (AA) is associated with a prompt cellular uptake and a further mitochondrial accumulation of the vitamin. Under the same conditions, dehydroascorbic acid (DHA) uptake was followed by rapid reduction and accumulation of identical intracellular levels of AA, however, in the absence of significant mitochondrial uptake. This event was instead observed after exposure to remarkably greater concentrations of DHA. Furthermore, experiments performed in isolated mitochondria revealed that DHA transport through hexose transporters and Na(+) -dependent transport of AA were very similar. These results suggest that the different subcellular compartmentalization of the vitamin is mediated by events promoting inhibition of mitochondrial AA transport, possibly triggered by low levels of DHA. We obtained results in line with this notion in intact cells, and more direct evidence in isolated mitochondria. This inhibitory effect was promptly reversible after DHA removal and comparable with that mediated by established inhibitors, as quercetin. The results presented collectively indicate that low intracellular concentrations of DHA, because of its rapid reduction back to AA, are a poor substrate for direct mitochondrial uptake. DHA concentrations, however, appear sufficiently high to mediate inhibition of mitochondrial transport of AA/DHA-derived AA.

  3. Apoptosis in an interleukin-2-dependent cytotoxic T lymphocyte cell line is associated with intracellular acidification. Role of the Na(+)/H(+)-antiport.

    PubMed

    Li, J; Eastman, A

    1995-02-17

    Apoptosis is a form of cell death associated with DNA fragmentation and chromatin condensation. We recently established that intracellular acidification occurred during apoptosis following cytotoxic insult. The current studies were designed to determine whether intracellular acidification was more generally associated with apoptosis, specifically in a model of growth factor withdrawal. Upon withdrawal of interleukin-2, CTLL-2 cells accumulated in the G1 phase of the cell cycle and started to fragment their DNA around 12 h concurrent with both decreased pH and increased Ca2. Chelation of Ca2+ did not inhibit DNA digestion, whereas incubation with a calcium ionophore prevented both acidification and DNA digestion. Hence, acidification rather than increased Ca2+ was associated with apoptosis. The acidified cells represented a discrete population up to 0.7 pH units below normal. The extent of acidification depended upon the extracellular pH; above pH 6.3, intracellular pH was significantly below extracellular pH, whereas below pH 6.3, the cells still regulated their pH. Inhibition of the Na+/H(+)-antiport prevented the apoptotic cells from regulating their intracellular pH under these acidic conditions. These intracellular pH under these acidic conditions. These results demonstrate that apoptotic cells retain a functional antiport but that its set-point has changed. Many survival factors are known to phosphorylate and activate the antiport, hence apoptosis is likely to be associated with dephosphorylation. Although acidification always occurred during apoptosis, maintaining intracellular pH above 7.2 did not prevent apoptosis, suggesting that an acid pH is not essential for apoptosis. We hypothesize that other critical regulators of apoptosis must be subject to dephosphorylation.

  4. Plant Habitat (PH)

    NASA Technical Reports Server (NTRS)

    Onate, Bryan

    2016-01-01

    The International Space Station (ISS) will soon have a platform for conducting fundamental research of Large Plants. Plant Habitat (PH) is designed to be a fully controllable environment for high-quality plant physiological research. PH will control light quality, level, and timing, temperature, CO2, relative humidity, and irrigation, while scrubbing ethylene. Additional capabilities include leaf temperature and root zone moisture and oxygen sensing. The light cap will have red (630 nm), blue (450 nm), green (525 nm), far red (730 nm) and broad spectrum white LEDs. There will be several internal cameras (visible and IR) to monitor and record plant growth and operations.

  5. Intracellular Organisms as Placental Invaders

    PubMed Central

    Vigliani, Marguerite B.; Bakardjiev, Anna I.

    2015-01-01

    In this article we present a novel model for how the human placenta might get infected via the hematogenous route. We present a list of diverse placental pathogens, like Listeria monocytogenes or Cytomegalovirus, which are familiar to most obstetricians, but others, like Salmonella typhi, have only been reported in case studies or small case series. Remarkably, all of these organisms on this list are either obligate or facultative intracellular organisms. These pathogens are able to enter and survive inside host immune cells for at least a portion of their life cycle. We suggest that many blood-borne pathogens might arrive at the placenta via transportation inside of maternal leukocytes that enter the decidua in early pregnancy. We discuss mechanisms by which extravillous trophoblasts could get infected in the decidua and spread infection to other layers in the placenta. We hope to raise awareness among OB/GYN clinicians that organisms not typically associated with the TORCH list might cause placental infections and pregnancy complications. PMID:27695204

  6. Secretome of obligate intracellular Rickettsia

    PubMed Central

    Gillespie, Joseph J.; Kaur, Simran J.; Rahman, M. Sayeedur; Rennoll-Bankert, Kristen; Sears, Khandra T.; Beier-Sexton, Magda; Azad, Abdu F.

    2014-01-01

    The genus Rickettsia (Alphaproteobacteria, Rickettsiales, Rickettsiaceae) is comprised of obligate intracellular parasites, with virulent species of interest both as causes of emerging infectious diseases and for their potential deployment as bioterrorism agents. Currently, there are no effective commercially available vaccines, with treatment limited primarily to tetracycline antibiotics, although others (e.g. josamycin, ciprofloxacin, chloramphenicol, and azithromycin) are also effective. Much of the recent research geared toward understanding mechanisms underlying rickettsial pathogenicity has centered on characterization of secreted proteins that directly engage eukaryotic cells. Herein, we review all aspects of the Rickettsia secretome, including six secretion systems, 19 characterized secretory proteins, and potential moonlighting proteins identified on surfaces of multiple Rickettsia species. Employing bioinformatics and phylogenomics, we present novel structural and functional insight on each secretion system. Unexpectedly, our investigation revealed that the majority of characterized secretory proteins have not been assigned to their cognate secretion pathways. Furthermore, for most secretion pathways, the requisite signal sequences mediating translocation are poorly understood. As a blueprint for all known routes of protein translocation into host cells, this resource will assist research aimed at uniting characterized secreted proteins with their apposite secretion pathways. Furthermore, our work will help in the identification of novel secreted proteins involved in rickettsial ‘life on the inside’. PMID:25168200

  7. Intracellular alkalization causes pain sensation through activation of TRPA1 in mice

    PubMed Central

    Fujita, Fumitaka; Uchida, Kunitoshi; Moriyama, Tomoko; Shima, Asako; Shibasaki, Koji; Inada, Hitoshi; Sokabe, Takaaki; Tominaga, Makoto

    2008-01-01

    Vertebrate cells require a very narrow pH range for survival. Cells accordingly possess sensory and defense mechanisms for situations where the pH deviates from the viable range. Although the monitoring of acidic pH by sensory neurons has been attributed to several ion channels, including transient receptor potential vanilloid 1 channel (TRPV1) and acid-sensing ion channels (ASICs), the mechanisms by which these cells detect alkaline pH are not well understood. Here, using Ca2+ imaging and patch-clamp recording, we showed that alkaline pH activated transient receptor potential cation channel, subfamily A, member 1 (TRPA1) and that activation of this ion channel was involved in nociception. In addition, intracellular alkalization activated TRPA1 at the whole-cell level, and single-channel openings were observed in the inside-out configuration, indicating that alkaline pH activated TRPA1 from the inside. Analyses of mutants suggested that the two N-terminal cysteine residues in TRPA1 were involved in activation by intracellular alkalization. Furthermore, intraplantar injection of ammonium chloride into the mouse hind paw caused pain-related behaviors that were not observed in TRPA1-deficient mice. These results suggest that alkaline pH causes pain sensation through activation of TRPA1 and may provide a molecular explanation for some of the human alkaline pH–related sensory disorders whose mechanisms are largely unknown. PMID:19033673

  8. Hydrophilic fluorescent nanogel thermometer for intracellular thermometry.

    PubMed

    Gota, Chie; Okabe, Kohki; Funatsu, Takashi; Harada, Yoshie; Uchiyama, Seiichi

    2009-03-04

    The first methodology to measure intracellular temperature is described. A highly hydrophilic fluorescent nanogel thermometer developed for this purpose stays in the cytoplasm and emits stronger fluorescence at a higher temperature. Thus, intracellular temperature variations associated with biological processes can be monitored by this novel thermometer with a temperature resolution of better than 0.5 degrees C.

  9. pH Basics

    ERIC Educational Resources Information Center

    Lunelli, Bruno; Scagnolari, Francesco

    2009-01-01

    The exposition of the pervasive concept of pH, of its foundations and implementation as a meaningful quantitative measurement, in nonspecialist university texts is often not easy to follow because too many of its theoretical and operative underpinnings are neglected. To help the inquiring student we provide a concise introduction to the depth just…

  10. pH optrode

    DOEpatents

    Northrup, M. Allen; Langry, Kevin C.

    1993-01-01

    A process is provided for forming a long-lasting, stable, pH-sensitive dye-acrylamide copolymer useful as a pH-sensitive material for use in an optrode or other device sensitive to pH. An optrode may be made by mechanically attaching the copolymer to a sensing device such as an optical fiber.

  11. Ph.D. shortage

    NASA Astrophysics Data System (ADS)

    The late 1990s will see a shortage of Ph.D. graduates, according to the Association of American Universities, Washington, D.C. AAU's new comprehensive study, “The Ph.D. Shortage: The Federal Role,” reports that competition for new Ph.D.s is already intense and can only intensify because demand is greater than supply in both academic and nonacademic markets.Doctoral education plays an increasingly important role in U.S. research and development programs. Students have a pivotal part in doing research and enriching it with new ideas. The AAU report says that graduate students are “major determinants of the creativity and productivity of U.S. academic research, the source of more than 50% of the nation's basic research.’ The market for doctoral education extends beyond the university. In 1985, about 43% of all Ph.D.s employed in this country were working outside higher education; the demand for doctorate recipients in nonacademic sectors continues to grow.

  12. Dynamic of CSF and serum biomarkers in HIV-1 subtype C encephalitis with CNS genetic compartmentalization-case study.

    PubMed

    de Almeida, Sergio M; Rotta, Indianara; Ribeiro, Clea E; Oliveira, Michelli F; Chaillon, Antoine; de Pereira, Ana Paula; Cunha, Ana Paula; Zonta, Marise; Bents, Joao França; Raboni, Sonia M; Smith, Davey; Letendre, Scott; Ellis, Ronald J

    2017-02-28

    Despite the effective suppression of viremia with antiretroviral therapy, HIV can still replicate in the central nervous system (CNS). This was a longitudinal study of the cerebrospinal fluid (CSF) and serum dynamics of several biomarkers related to inflammation, the blood-brain barrier, neuronal injury, and IgG intrathecal synthesis in serial samples of CSF and serum from a patient infected with HIV-1 subtype C with CNS compartmentalization.The phylogenetic analyses of plasma and CSF samples in an acute phase using next-generation sequencing and F-statistics analysis of C2-V3 haplotypes revealed distinct compartmentalized CSF viruses in paired CSF and peripheral blood mononuclear cell samples. The CSF biomarker analysis in this patient showed that symptomatic CSF escape is accompanied by CNS inflammation, high levels of cell and humoral immune biomarkers, CNS barrier dysfunction, and an increase in neuronal injury biomarkers with demyelization. Independent and isolated HIV replication can occur in the CNS, even in HIV-1 subtype C, leading to compartmentalization and development of quasispecies distinct from the peripheral plasma. These immunological aspects of the HIV CNS escape have not been described previously. To our knowledge, this is the first report of CNS HIV escape and compartmentalization in HIV-1 subtype C.

  13. A Compartmentalized Phosphorylation/Dephosphorylation System That Regulates U snRNA Export from the Nucleus▿ †

    PubMed Central

    Kitao, Saori; Segref, Alexandra; Kast, Juergen; Wilm, Matthias; Mattaj, Iain W.; Ohno, Mutsuhito

    2008-01-01

    PHAX (phosphorylated adaptor for RNA export) is the key regulator of U snRNA nuclear export in metazoa. Our previous work revealed that PHAX is phosphorylated in the nucleus and is exported as a component of the U snRNA export complex to the cytoplasm, where it is dephosphorylated (M. Ohno, A. Segref, A. Bachi, M. Wilm, and I. W. Mattaj, Cell 101:187-198, 2000). PHAX phosphorylation is essential for export complex assembly, whereas its dephosphorylation causes export complex disassembly. Thus, PHAX is subject to a compartmentalized phosphorylation/dephosphorylation cycle that contributes to transport directionality. However, neither essential PHAX phosphorylation sites nor the modifying enzymes that contribute to the compartmentalized system have been identified. Here, we identify PHAX phosphorylation sites that are necessary and sufficient for U snRNA export. Mutation of the phosphorylation sites inhibited U snRNA export in a dominant-negative way. We also show, by both biochemical and RNA interference knockdown experiments, that the nuclear kinase and the cytoplasmic phosphatase for PHAX are CK2 kinase and protein phosphatase 2A, respectively. Our results reveal the composition of the compartmentalized phosphorylation/dephosphorylation system that regulates U snRNA export. This finding was surprising in that such a specific system for U snRNA export regulation is composed of two such universal regulators, suggesting that this compartmentalized system is used more broadly for gene expression regulation. PMID:17967890

  14. Sequence Stratigraphy of the Dakota Sandstone, Eastern San Juan Basin, New Mexico, and its Relationship to Reservoir Compartmentalization

    SciTech Connect

    Varney, Peter J.

    2002-04-23

    This research established the Dakota-outcrop sequence stratigraphy in part of the eastern San Juan Basin, New Mexico, and relates reservoir quality lithologies in depositional sequences to structure and reservoir compartmentalization in the South Lindrith Field area. The result was a predictive tool that will help guide further exploration and development.

  15. Lipid droplet-associated proteins (LDAPs) are required for the dynamic regulation of neutral lipid compartmentation in plant cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Eukaryotic cells compartmentalize neutral lipids into organelles called lipid droplets (LDs), and while much is known about the role of LDs in storing triacylglycerols (TAGs) in seeds, their biogenesis and function in non-seed tissues is poorly understood. Recently, we identified a class of plant-sp...

  16. Nucleolar structure across evolution: the transition between bi- and tri-compartmentalized nucleoli lies within the class Reptilia.

    PubMed

    Lamaye, Françoise; Galliot, Sonia; Alibardi, Lorenzo; Lafontaine, Denis L J; Thiry, Marc

    2011-05-01

    Two types of nucleolus can be distinguished among eukaryotic cells: a tri-compartmentalized nucleolus in amniotes and a bi-compartmentalized nucleolus in all the others. However, though the nucleolus' ultrastructure is well characterized in mammals and birds, it has been so far much less studied in reptiles. In this work, we examined the ultrastructural organization of the nucleolus in various tissues from different reptilian species (three turtles, three lizards, two crocodiles, and three snakes). Using cytochemical and immunocytological methods, we showed that in reptiles both types of nucleolus are present: a bi-compartmentalized nucleolus in turtles and a tri-compartmentalized nucleolus in the other species examined in this study. Furthermore, in a given species, the same type of nucleolus is present in all the tissues, however, the importance and the repartition of those nucleolar components could vary from one tissue to another. We also reveal that, contrary to the mammalian nucleolus, the reptilian fibrillar centers contain small clumps of condensed chromatin and that their surrounding dense fibrillar component is thicker. Finally, we also report that Cajal bodies are detected in reptiles. Altogether, we believe that these results have profound evolutionarily implications since they indicate that the point of transition between bipartite and tripartite nucleoli lies at the emergence of the amniotes within the class Reptilia.

  17. Deriving effective vaccine allocation strategies for pandemic influenza: Comparison of an agent-based simulation and a compartmental model

    PubMed Central

    Dalgıç, Özden O.; Özaltın, Osman Y.; Ciccotelli, William A.; Erenay, Fatih S.

    2017-01-01

    Individuals are prioritized based on their risk profiles when allocating limited vaccine stocks during an influenza pandemic. Computationally expensive but realistic agent-based simulations and fast but stylized compartmental models are typically used to derive effective vaccine allocation strategies. A detailed comparison of these two approaches, however, is often omitted. We derive age-specific vaccine allocation strategies to mitigate a pandemic influenza outbreak in Seattle by applying derivative-free optimization to an agent-based simulation and also to a compartmental model. We compare the strategies derived by these two approaches under various infection aggressiveness and vaccine coverage scenarios. We observe that both approaches primarily vaccinate school children, however they may allocate the remaining vaccines in different ways. The vaccine allocation strategies derived by using the agent-based simulation are associated with up to 70% decrease in total cost and 34% reduction in the number of infections compared to the strategies derived by using the compartmental model. Nevertheless, the latter approach may still be competitive for very low and/or very high infection aggressiveness. Our results provide insights about potential differences between the vaccine allocation strategies derived by using agent-based simulations and those derived by using compartmental models. PMID:28222123

  18. Plasma Membrane is Compartmentalized by a Self-Similar Cortical Actin Meshwork

    NASA Astrophysics Data System (ADS)

    Sadegh, Sanaz; Higgins, Jenny L.; Mannion, Patrick C.; Tamkun, Michael M.; Krapf, Diego

    2017-01-01

    A broad range of membrane proteins display anomalous diffusion on the cell surface. Different methods provide evidence for obstructed subdiffusion and diffusion on a fractal space, but the underlying structure inducing anomalous diffusion has never been visualized because of experimental challenges. We addressed this problem by imaging the cortical actin at high resolution while simultaneously tracking individual membrane proteins in live mammalian cells. Our data confirm that actin introduces barriers leading to compartmentalization of the plasma membrane and that membrane proteins are transiently confined within actin fences. Furthermore, superresolution imaging shows that the cortical actin is organized into a self-similar meshwork. These results present a hierarchical nanoscale picture of the plasma membrane.

  19. Savannah River Laboratory DOSTOMAN code: a compartmental pathways computer model of contaminant transport

    SciTech Connect

    King, C M; Wilhite, E L; Root, Jr, R W; Fauth, D J; Routt, K R; Emslie, R H; Beckmeyer, R R; Fjeld, R A; Hutto, G A; Vandeven, J A

    1985-01-01

    The Savannah River Laboratory DOSTOMAN code has been used since 1978 for environmental pathway analysis of potential migration of radionuclides and hazardous chemicals. The DOSTOMAN work is reviewed including a summary of historical use of compartmental models, the mathematical basis for the DOSTOMAN code, examples of exact analytical solutions for simple matrices, methods for numerical solution of complex matrices, and mathematical validation/calibration of the SRL code. The review includes the methodology for application to nuclear and hazardous chemical waste disposal, examples of use of the model in contaminant transport and pathway analysis, a user's guide for computer implementation, peer review of the code, and use of DOSTOMAN at other Department of Energy sites. 22 refs., 3 figs.

  20. Compartmentalization of gypsum and halite associated with cyanobacteria in saline soil crusts.

    PubMed

    Canfora, Loredana; Vendramin, Elisa; Vittori Antisari, Livia; Lo Papa, Giuseppe; Dazzi, Carmelo; Benedetti, Anna; Iavazzo, Pietro; Adamo, Paola; Jungblut, Anne D; Pinzari, Flavia

    2016-06-01

    The interface between biological and geochemical components in the surface crust of a saline soil was investigated using X-ray diffraction, and variable pressure scanning electron microscopy in combination with energy dispersive X-ray spectrometry. Mineral compounds such as halite and gypsum were identified crystallized around filaments of cyanobacteria. A total of 92 genera were identified from the bacterial community based on 16S gene pyrosequencing analysis. The occurrence of the gypsum crystals, their shapes and compartmentalization suggested that they separated NaCl from the immediate microenvironment of the cyanobacteria, and that some cyanobacteria and communities of sulfur bacteria may had a physical control over the distinctive halite and gypsum structures produced. This suggests that cyanobacteria might directly or indirectly promote the formation of a protective envelope made of calcium and sulfur-based compounds.

  1. Copper accumulation and compartmentalization in mouse fibroblast lacking metallothionein and copper chaperone, Atox1

    SciTech Connect

    Miyayama, Takamitsu; Suzuki, Kazuo T.; Ogra, Yasumitsu

    2009-06-01

    Copper (Cu) is the active center of some enzymes because of its redox-active property, although that property could have harmful effects. Because of this, cells have strict regulation/detoxification systems for this metal. In this study, multi-disciplinary approaches, such as speciation and elemental imaging of Cu, were applied to reveal the detoxification mechanisms for Cu in cells bearing a defect in Cu-regulating genes. Although Cu concentration in metallothionein (MT)-knockout cells was increased by the knockdown of the Cu chaperone, Atox1, the concentrations of the Cu influx pump, Ctr1, and another Cu chaperone, Ccs, were paradoxically increased; namely, the cells responded to the Cu deficiency despite the fact that cellular Cu concentration was actually increased. Cu imaging showed that the elevated Cu was compartmentalized in cytoplasmic vesicles. Together, the results point to the novel roles of MT and cytoplasmic vesicles in the detoxification of Cu in mammalian cells.

  2. Estimation of pharmacokinetic parameters from non-compartmental variables using Microsoft Excel.

    PubMed

    Dansirikul, Chantaratsamon; Choi, Malcolm; Duffull, Stephen B

    2005-06-01

    This study was conducted to develop a method, termed 'back analysis (BA)', for converting non-compartmental variables to compartment model dependent pharmacokinetic parameters for both one- and two-compartment models. A Microsoft Excel spreadsheet was implemented with the use of Solver and visual basic functions. The performance of the BA method in estimating pharmacokinetic parameter values was evaluated by comparing the parameter values obtained to a standard modelling software program, NONMEM, using simulated data. The results show that the BA method was reasonably precise and provided low bias in estimating fixed and random effect parameters for both one- and two-compartment models. The pharmacokinetic parameters estimated from the BA method were similar to those of NONMEM estimation.

  3. Mitochondrial Dynamics is a Distinguishing Feature of Skeletal Muscle Fiber Types and Regulates Organellar Compartmentalization.

    PubMed

    Mishra, Prashant; Varuzhanyan, Grigor; Pham, Anh H; Chan, David C

    2015-12-01

    Skeletal muscle fibers differentiate into specific fiber types with distinct metabolic properties determined by their reliance on oxidative phosphorylation (OXPHOS). Using in vivo approaches, we find that OXPHOS-dependent fibers, compared to glycolytic fibers, contain elongated mitochondrial networks with higher fusion rates that are dependent on the mitofusins Mfn1 and Mfn2. Switching of a glycolytic fiber to an oxidative IIA type is associated with elongation of mitochondria, suggesting that mitochondrial fusion is linked to metabolic state. Furthermore, we reveal that mitochondrial proteins are compartmentalized to discrete domains centered around their nuclei of origin. The domain dimensions are dependent on fiber type and are regulated by the mitochondrial dynamics proteins Mfn1, Mfn2, and Mff. Our results indicate that mitochondrial dynamics is tailored to fiber type physiology and provides a rationale for the segmental defects characteristic of aged and diseased muscle fibers.

  4. Host–parasite oscillation dynamics and evolution in a compartmentalized RNA replication system

    PubMed Central

    Bansho, Yohsuke; Furubayashi, Taro; Ichihashi, Norikazu; Yomo, Tetsuya

    2016-01-01

    To date, various cellular functions have been reconstituted in vitro such as self-replication systems using DNA, RNA, and proteins. The next important challenges include the reconstitution of the interactive networks of self-replicating species and investigating how such interactions generate complex ecological behaviors observed in nature. Here, we synthesized a simple replication system composed of two self-replicating host and parasitic RNA species. We found that the parasitic RNA eradicates the host RNA under bulk conditions; however, when the system is compartmentalized, a continuous oscillation pattern in the population dynamics of the two RNAs emerges. The oscillation pattern changed as replication proceeded mainly owing to the evolution of the host RNA. These results demonstrate that a cell-like compartment plays an important role in host–parasite ecological dynamics and suggest that the origin of the host–parasite coevolution might date back to the very early stages of the evolution of life. PMID:27035976

  5. Subcellular Compartmentalization and Trafficking of the Biosynthetic Machinery for Fungal Melanin.

    PubMed

    Upadhyay, Srijana; Xu, Xinping; Lowry, David; Jackson, Jennifer C; Roberson, Robert W; Lin, Xiaorong

    2016-03-22

    Protection by melanin depends on its subcellular location. Although most filamentous fungi synthesize melanin via a polyketide synthase pathway, where and how melanin biosynthesis occurs and how it is deposited as extracellular granules remain elusive. Using a forward genetic screen in the pathogen Aspergillus fumigatus, we find that mutations in an endosomal sorting nexin abolish melanin cell-wall deposition. We find that all enzymes involved in the early steps of melanin biosynthesis are recruited to endosomes through a non-conventional secretory pathway. In contrast, late melanin enzymes accumulate in the cell wall. Such subcellular compartmentalization of the melanin biosynthetic machinery occurs in both A. fumigatus and A. nidulans. Thus, fungal melanin biosynthesis appears to be initiated in endosomes with exocytosis leading to melanin extracellular deposition, much like the synthesis and trafficking of mammalian melanin in endosomally derived melanosomes.

  6. Spatial Compartmentalization Specializes the Function of Aurora A and Aurora B.

    PubMed

    Li, Si; Deng, Zhaoxuan; Fu, Jingyan; Xu, Caiyue; Xin, Guangwei; Wu, Zhige; Luo, Jia; Wang, Gang; Zhang, Shuli; Zhang, Boyan; Zou, Fangdong; Jiang, Qing; Zhang, Chuanmao

    2015-07-10

    Aurora kinase A and B share great similarity in sequences, structures, and phosphorylation motif, yet they show different localizations and play distinct crucial roles. The factors that determine such differences are largely unknown. Here we targeted Aurora A to the localization of Aurora B and found that Aurora A phosphorylates the substrate of Aurora B and substitutes its function in spindle checkpoint. In return, the centrosome targeting of Aurora B substitutes the function of Aurora A in the mitotic entry. Expressing the chimera proteins of the Auroras with exchanged N termini in cells indicates that the divergent N termini are also important for their spatiotemporal localizations and functions. Collectively, we demonstrate that functional divergence of Aurora kinases is determined by spatial compartmentalization, and their divergent N termini also contribute to their spatial and functional differentiation.

  7. Spatial Compartmentalization Specializes the Function of Aurora A and Aurora B*

    PubMed Central

    Li, Si; Deng, Zhaoxuan; Fu, Jingyan; Xu, Caiyue; Xin, Guangwei; Wu, Zhige; Luo, Jia; Wang, Gang; Zhang, Shuli; Zhang, Boyan; Zou, Fangdong; Jiang, Qing; Zhang, Chuanmao

    2015-01-01

    Aurora kinase A and B share great similarity in sequences, structures, and phosphorylation motif, yet they show different localizations and play distinct crucial roles. The factors that determine such differences are largely unknown. Here we targeted Aurora A to the localization of Aurora B and found that Aurora A phosphorylates the substrate of Aurora B and substitutes its function in spindle checkpoint. In return, the centrosome targeting of Aurora B substitutes the function of Aurora A in the mitotic entry. Expressing the chimera proteins of the Auroras with exchanged N termini in cells indicates that the divergent N termini are also important for their spatiotemporal localizations and functions. Collectively, we demonstrate that functional divergence of Aurora kinases is determined by spatial compartmentalization, and their divergent N termini also contribute to their spatial and functional differentiation. PMID:25987563

  8. The construction of next-generation matrices for compartmental epidemic models.

    PubMed

    Diekmann, O; Heesterbeek, J A P; Roberts, M G

    2010-06-06

    The basic reproduction number (0) is arguably the most important quantity in infectious disease epidemiology. The next-generation matrix (NGM) is the natural basis for the definition and calculation of (0) where finitely many different categories of individuals are recognized. We clear up confusion that has been around in the literature concerning the construction of this matrix, specifically for the most frequently used so-called compartmental models. We present a detailed easy recipe for the construction of the NGM from basic ingredients derived directly from the specifications of the model. We show that two related matrices exist which we define to be the NGM with large domain and the NGM with small domain. The three matrices together reflect the range of possibilities encountered in the literature for the characterization of (0). We show how they are connected and how their construction follows from the basic model ingredients, and establish that they have the same non-zero eigenvalues, the largest of which is the basic reproduction number (0). Although we present formal recipes based on linear algebra, we encourage the construction of the NGM by way of direct epidemiological reasoning, using the clear interpretation of the elements of the NGM and of the model ingredients. We present a selection of examples as a practical guide to our methods. In the appendix we present an elementary but complete proof that (0) defined as the dominant eigenvalue of the NGM for compartmental systems and the Malthusian parameter r, the real-time exponential growth rate in the early phase of an outbreak, are connected by the properties that (0) > 1 if and only if r > 0, and (0) = 1 if and only if r = 0.

  9. The role of the bi-compartmental stem cell niche in delaying cancer

    NASA Astrophysics Data System (ADS)

    Shahriyari, Leili; Komarova, Natalia L.

    2015-10-01

    In recent years, by using modern imaging techniques, scientists have found evidence of collaboration between different types of stem cells (SCs), and proposed a bi-compartmental organization of the SC niche. Here we create a class of stochastic models to simulate the dynamics of such a heterogeneous SC niche. We consider two SC groups: the border compartment, S1, is in direct contact with transit-amplifying (TA) cells, and the central compartment, S2, is hierarchically upstream from S1. The S1 SCs differentiate or divide asymmetrically when the tissue needs TA cells. Both groups proliferate when the tissue requires SCs (thus maintaining homeostasis). There is an influx of S2 cells into the border compartment, either by migration, or by proliferation. We examine this model in the context of double-hit mutant generation, which is a rate-limiting step in the development of many cancers. We discover that this type of a cooperative pattern in the stem niche with two compartments leads to a significantly smaller rate of double-hit mutant production compared with a homogeneous, one-compartmental SC niche. Furthermore, the minimum probability of double-hit mutant generation corresponds to purely symmetric division of SCs, consistent with the literature. Finally, the optimal architecture (which minimizes the rate of double-hit mutant production) requires a large proliferation rate of S1 cells along with a small, but non-zero, proliferation rate of S2 cells. This result is remarkably similar to the niche structure described recently by several authors, where one of the two SC compartments was found more actively engaged in tissue homeostasis and turnover, while the other was characterized by higher levels of quiescence (but contributed strongly to injury recovery). Both numerical and analytical results are presented.

  10. Pathway Compartmentalization in Peroxisome of Saccharomyces cerevisiae to Produce Versatile Medium Chain Fatty Alcohols.

    PubMed

    Sheng, Jiayuan; Stevens, Joseph; Feng, Xueyang

    2016-05-27

    Fatty alcohols are value-added chemicals and important components of a variety of industries, which have a >3 billion-dollar global market annually. Long chain fatty alcohols (>C12) are mainly used in surfactants, lubricants, detergents, pharmaceuticals and cosmetics while medium chain fatty alcohols (C6-C12) could be used as diesel-like biofuels. Microbial production of fatty alcohols from renewable feedstock stands as a promising strategy to enable sustainable supply of fatty alcohols. In this study, we report, for the first time, that medium chain fatty alcohols could be produced in yeast via targeted expression of a fatty acyl-CoA reductase (TaFAR) in the peroxisome of Saccharomyces cerevisiae. By tagging TaFAR enzyme with peroxisomal targeting signal peptides, the TaFAR could be compartmentalized into the matrix of the peroxisome to hijack the medium chain fatty acyl-CoA generated from the beta-oxidation pathway and convert them to versatile medium chain fatty alcohols (C10 &C12). The overexpression of genes encoding PEX7 and acetyl-CoA carboxylase further improved fatty alcohol production by 1.4-fold. After medium optimization in fed-batch fermentation using glucose as the sole carbon source, fatty alcohols were produced at 1.3 g/L, including 6.9% 1-decanol, 27.5% 1-dodecanol, 2.9% 1-tetradecanol and 62.7% 1-hexadecanol. This work revealed that peroxisome could be engineered as a compartmentalized organelle for producing fatty acid-derived chemicals in S. cerevisiae.

  11. Caveolin-3 regulates compartmentation of cardiomyocyte beta2-adrenergic receptor-mediated cAMP signaling.

    PubMed

    Wright, Peter T; Nikolaev, Viacheslav O; O'Hara, Thomas; Diakonov, Ivan; Bhargava, Anamika; Tokar, Sergiy; Schobesberger, Sophie; Shevchuk, Andrew I; Sikkel, Markus B; Wilkinson, Ross; Trayanova, Natalia A; Lyon, Alexander R; Harding, Sian E; Gorelik, Julia

    2014-02-01

    The purpose of this study was to investigate whether caveolin-3 (Cav3) regulates localization of β2-adrenergic receptor (β2AR) and its cAMP signaling in healthy or failing cardiomyocytes. We co-expressed wildtype Cav3 or its dominant-negative mutant (Cav3DN) together with the Förster resonance energy transfer (FRET)-based cAMP sensor Epac2-camps in adult rat ventricular myocytes (ARVMs). FRET and scanning ion conductance microscopy were used to locally stimulate β2AR and to measure cytosolic cAMP. Cav3 overexpression increased the number of caveolae and decreased the magnitude of β2AR-cAMP signal. Conversely, Cav3DN expression resulted in an increased β2AR-cAMP response without altering the whole-cell L-type calcium current. Following local stimulation of Cav3DN-expressing ARVMs, β2AR response could only be generated in T-tubules. However, the normally compartmentalized β2AR-cAMP signal became diffuse, similar to the situation observed in heart failure. Finally, overexpression of Cav3 in failing myocytes led to partial β2AR redistribution back into the T-tubules. In conclusion, Cav3 plays a crucial role for the localization of β2AR and compartmentation of β2AR-cAMP signaling to the T-tubules of healthy ARVMs, and overexpression of Cav3 in failing myocytes can partially restore the disrupted localization of these receptors.

  12. Target-Dependent Compartmentalization of the Corelease of Glutamate and GABA from the Mossy Fibers.

    PubMed

    Galván, Emilio J; Gutiérrez, Rafael

    2017-01-18

    The mossy fibers (MFs) corelease glutamate and GABA onto pyramidal cells of CA3 during development, until the end of the third postnatal week. However, the major target cells of the MF are the interneurons of CA3. Therefore, it has been shown that the interneurons of the hilus and stratum lucidum receive this dual monosynaptic input on MF stimulation. Because the plasticity of glutamatergic transmission from the different terminals of the MF is target specific, we here asked whether the corelease of glutamate and GABA was also subjected to a target-dependent compartmentalization. We analyzed the occurrence and plasticity of MF simultaneous glutamatergic-GABAergic signaling onto interneurons of the different strata of CA3 in rats during the third postnatal week. We show the coexistence of time-locked, glutamate receptor and GABA receptor-mediated mono synaptic responses evoked by MF stimulation in interneurons from stratum lucidum and stratum radiatum, but not in interneurons from stratum lacunosum-moleculare. As expected from the transmission of MF origin, MF GABAergic responses were depressed by the activation of metabotropic glutamate receptors. Strikingly, while MF glutamatergic responses underwent LTD, the simultaneous MF GABAergic responses of stratum lucidum interneurons, but not of stratum radiatum interneurons, displayed a Hebbian form of LTP that was mimicked by PKC activation. PKA activation potentiated MF glutamatergic responses of stratum radiatum interneurons, whereas in stratum lucidum interneurons only GABAergic responses were potentiated. We here disclose that the corelease of glutamate and GABA, as well as their plasticity are compartmentalized in a target-dependent manner, showing counterbalanced compensatory plasticity of two neurotransmitters released by different terminals of the same pathway.

  13. Pathway Compartmentalization in Peroxisome of Saccharomyces cerevisiae to Produce Versatile Medium Chain Fatty Alcohols

    PubMed Central

    Sheng, Jiayuan; Stevens, Joseph; Feng, Xueyang

    2016-01-01

    Fatty alcohols are value-added chemicals and important components of a variety of industries, which have a >3 billion-dollar global market annually. Long chain fatty alcohols (>C12) are mainly used in surfactants, lubricants, detergents, pharmaceuticals and cosmetics while medium chain fatty alcohols (C6–C12) could be used as diesel-like biofuels. Microbial production of fatty alcohols from renewable feedstock stands as a promising strategy to enable sustainable supply of fatty alcohols. In this study, we report, for the first time, that medium chain fatty alcohols could be produced in yeast via targeted expression of a fatty acyl-CoA reductase (TaFAR) in the peroxisome of Saccharomyces cerevisiae. By tagging TaFAR enzyme with peroxisomal targeting signal peptides, the TaFAR could be compartmentalized into the matrix of the peroxisome to hijack the medium chain fatty acyl-CoA generated from the beta-oxidation pathway and convert them to versatile medium chain fatty alcohols (C10 & C12). The overexpression of genes encoding PEX7 and acetyl-CoA carboxylase further improved fatty alcohol production by 1.4-fold. After medium optimization in fed-batch fermentation using glucose as the sole carbon source, fatty alcohols were produced at 1.3 g/L, including 6.9% 1-decanol, 27.5% 1-dodecanol, 2.9% 1-tetradecanol and 62.7% 1-hexadecanol. This work revealed that peroxisome could be engineered as a compartmentalized organelle for producing fatty acid-derived chemicals in S. cerevisiae. PMID:27230732

  14. Compartmentalization of oxidative stress and antioxidant defense in the larval gut of Spodoptera littoralis.

    PubMed

    Krishnan, Natraj; Sehnal, Frantisek

    2006-09-01

    Allelochemicals play important roles in the plant defense against herbivorous insects. They act as feeding deterrents, interfere with digestion and nutrient absorption, and cause production of potentially dangerous oxidative radicals. This study demonstrates that the distributions of oxidative radicals and of the antioxidant enzymes that eliminate them are compartmentalized in the digestive tract of Spodoptera littoralis larvae. Feeding on diets supplemented with the tannic acid (TA), alpha-solanine, and demissidine, respectively, did not affect the rate of food passage through the digestive tract of larvae but 1.25, 2.5, and 5% TA evoked a strong oxidative response. The amount of the superoxide anion in the foregut tissue and content increased up to 70-fold and the titer of total peroxides in the foregut content about 3-fold. This oxidative stress was associated with enhanced carbonyl content in the foregut tissue proteins, indicative of certain tissue deterioration. Extensive foregut damage was probably prevented by elevated activity of the glutathione S-transferase peroxidase. A complex antioxidant response was elicited in the midgut. The activities of superoxide dismutase and catalase increased significantly in the midgut tissue and content, and the activity of ascorbate peroxidase rose in the midgut tissue. The enzymes apparently eliminated oxidative radicals passing to midgut from the foregut with the food bolus and thereby prevented carbonylation of the midgut proteins. We postulate that the generation of oxidative radicals in the foregut and the induction of antioxidant defense in the midgut are controlled processes and that their compartmentalization is an important functional feature of the digestive tract. The glycoalkaloid alpha-solanine and the aglycone demissidine applied at 0.05 and 0.1% concentrations had no effect on any of the examined parameters.

  15. Consequences of plant invasions on compartmentalization and species' roles in plant-pollinator networks.

    PubMed

    Albrecht, Matthias; Padrón, Benigno; Bartomeus, Ignasi; Traveset, Anna

    2014-08-07

    Compartmentalization-the organization of ecological interaction networks into subsets of species that do not interact with other subsets (true compartments) or interact more frequently among themselves than with other species (modules)-has been identified as a key property for the functioning, stability and evolution of ecological communities. Invasions by entomophilous invasive plants may profoundly alter the way interaction networks are compartmentalized. We analysed a comprehensive dataset of 40 paired plant-pollinator networks (invaded versus uninvaded) to test this hypothesis. We show that invasive plants have higher generalization levels with respect to their pollinators than natives. The consequences for network topology are that-rather than displacing native species from the network-plant invaders attracting pollinators into invaded modules tend to play new important topological roles (i.e. network hubs, module hubs and connectors) and cause role shifts in native species, creating larger modules that are more connected among each other. While the number of true compartments was lower in invaded compared with uninvaded networks, the effect of invasion on modularity was contingent on the study system. Interestingly, the generalization level of the invasive plants partially explains this pattern, with more generalized invaders contributing to a lower modularity. Our findings indicate that the altered interaction structure of invaded networks makes them more robust against simulated random secondary species extinctions, but more vulnerable when the typically highly connected invasive plants go extinct first. The consequences and pathways by which biological invasions alter the interaction structure of plant-pollinator communities highlighted in this study may have important dynamical and functional implications, for example, by influencing multi-species reciprocal selection regimes and coevolutionary processes.

  16. Compartmental distribution of GABAB receptor-mediated currents along the somatodendritic axis of hippocampal principal cells.

    PubMed

    Degro, Claudius E; Kulik, Akos; Booker, Sam A; Vida, Imre

    2015-01-01

    Activity of cortical principal cells is controlled by the GABAergic system providing inhibition in a compartmentalized manner along their somatodendritic axis. While GABAAR-mediated inhibitory synaptic transmission has been extensively characterized in hippocampal principal cells, little is known about the distribution of postsynaptic effects of GABABRs. In the present study, we have investigated the functional localization of GABABRs and their effector inwardly rectifying potassium (Kir3) channels by combining electrophysiological recordings in acute rat hippocampal slices, high-resolution immunoelectron microscopic analysis and single cell simulations. Pharmacologically isolated slow inhibitory postsynaptic currents were elicited in the three major hippocampal principal cell types by endogenous GABA released by electrical stimulation, photolysis of caged-GABA, as well as the canonical agonist baclofen, with the highest amplitudes observed in the CA3. Spatially restricted currents were assessed along the axis of principal cells by uncaging GABA in the different hippocampal layers. GABABR-mediated currents were present along the entire somatodendritic axis of principal cells, but non-uniformly distributed: largest currents and the highest conductance densities determined in the simulations were consistently found on the distal apical dendrites. Finally, immunocytochemical localization of GABABRs and Kir3 channels showed that distributions overlap but their densities diverge, particularly on the basal dendrites of pyramidal cells. GABABRs current amplitudes and the conductance densities correlated better with Kir3 density, suggesting a bottlenecking effect defined by the effector channel. These data demonstrate a compartmentalized distribution of the GABABR-Kir3 signaling cascade and suggest differential control of synaptic transmission, dendritic integration and synaptic plasticity at afferent pathways onto hippocampal principal cells.

  17. The Golgi apparatus regulates cGMP-dependent protein kinase I compartmentation and proteolysis.

    PubMed

    Kato, Shin; Chen, Jingsi; Cornog, Katherine H; Zhang, Huili; Roberts, Jesse D

    2015-06-01

    cGMP-dependent protein kinase I (PKGI) is an important effector of cGMP signaling that regulates vascular smooth muscle cell (SMC) phenotype and proliferation. PKGI has been detected in the perinuclear region of cells, and recent data indicate that proprotein convertases (PCs) typically resident in the Golgi apparatus (GA) can stimulate PKGI proteolysis and generate a kinase fragment that localizes to the nucleus and regulates gene expression. However, the role of the endomembrane system in PKGI compartmentation and processing is unknown. Here, we demonstrate that PKGI colocalizes with endoplasmic reticulum (ER), ER-Golgi intermediate compartment, GA cisterna, and trans-Golgi network proteins in pulmonary artery SMC and cell lines. Moreover, PKGI localizes with furin, a trans-Golgi network-resident PC known to cleave PKGI. ER protein transport influences PKGI localization because overexpression of a constitutively inactive Sar1 transgene caused PKGI retention in the ER. Additionally, PKGI appears to reside within the GA because PKGI immunoreactivity was determined to be resistant to cytosolic proteinase K treatment in live cells. The GA appears to play a role in PKGI proteolysis because overexpression of inositol 1,4,5-trisphosphate receptor-associated cGMP kinase substrate, not only tethered heterologous PKGI-β to the ER and decreased its localization to the GA, but also diminished PKGI proteolysis and nuclear translocation. Also, inhibiting intra-GA protein transport with monensin was observed to decrease PKGI cleavage. These studies detail a role for the endomembrane system in regulating PKGI compartmentation and proteolysis. Moreover, they support the investigation of mechanisms regulating PKGI-dependent nuclear cGMP signaling in the pulmonary vasculature with Golgi dysfunction.

  18. Bioaccessibility of selenium after human ingestion in relation to its chemical species and compartmentalization in maize.

    PubMed

    Mombo, Stéphane; Schreck, Eva; Dumat, Camille; Laplanche, Christophe; Pierart, Antoine; Longchamp, Mélanie; Besson, Philippe; Castrec-Rouelle, Maryse

    2016-06-01

    Selenium is a micronutrient needed by all living organisms including humans, but often present in low concentration in food with possible deficiency. From another side, at higher concentrations in soils as observed in seleniferous regions of the world, and in function of its chemical species, Se can also induce (eco)toxicity. Root Se uptake was therefore studied in function of its initial form for maize (Zea mays L.), a plant widely cultivated for human and animal food over the world. Se phytotoxicity and compartmentalization were studied in different aerial plant tissues. For the first time, Se oral human bioaccessibility after ingestion was assessed for the main Se species (Se(IV) and Se(VI)) with the BARGE ex vivo test in maize seeds (consumed by humans), and in stems and leaves consumed by animals. Corn seedlings were cultivated in hydroponic conditions supplemented with 1 mg L(-1) of selenium (Se(IV), Se(VI), Control) for 4 months. Biomass, Se concentration, and bioaccessibility were measured on harvested plants. A reduction in plant biomass was observed under Se treatments compared to control, suggesting its phytotoxicity. This plant biomass reduction was higher for selenite species than selenate, and seed was the main affected compartment compared to control. Selenium compartmentalization study showed that for selenate species, a preferential accumulation was observed in leaves, whereas selenite translocation was very limited toward maize aerial parts, except in the seeds where selenite concentrations are generally high. Selenium oral bioaccessibility after ingestion fluctuated from 49 to 89 % according to the considered plant tissue and Se species. Whatever the tissue, selenate appeared as the most human bioaccessible form. A potential Se toxicity was highlighted for people living in seleniferous regions, this risk being enhanced by the high Se bioaccessibility.

  19. Evaluation of a compartmental model for estimating tumor hypoxia via FMISO dynamic PET imaging

    NASA Astrophysics Data System (ADS)

    Wang, Wenli; Georgi, Jens-Christoph; Nehmeh, Sadek A.; Narayanan, Manoj; Paulus, Timo; Bal, Matthieu; O'Donoghue, Joseph; Zanzonico, Pat B.; Schmidtlein, C. Ross; Lee, Nancy Y.; Humm, John L.

    2009-05-01

    This paper systematically evaluates a pharmacokinetic compartmental model for identifying tumor hypoxia using dynamic positron emission tomography (PET) imaging with 18F-fluoromisonidazole (FMISO). A generic irreversible one-plasma two-tissue compartmental model was used. A dynamic PET image dataset was simulated with three tumor regions—normoxic, hypoxic and necrotic—embedded in a normal-tissue background, and with an image-based arterial input function. Each voxelized tissue's time activity curve (TAC) was simulated with typical values of kinetic parameters, as deduced from FMISO-PET data from nine head-and-neck cancer patients. The dynamic dataset was first produced without any statistical noise to ensure that correct kinetic parameters were reproducible. Next, to investigate the stability of kinetic parameter estimation in the presence of noise, 1000 noisy samples of the dynamic dataset were generated, from which 1000 noisy estimates of kinetic parameters were calculated and used to estimate the sample mean and covariance matrix. It is found that a more peaked input function gave less variation in various kinetic parameters, and the variation of kinetic parameters could also be reduced by two region-of-interest averaging techniques. To further investigate how bias in the arterial input function affected the kinetic parameter estimation, a shift error was introduced in the peak amplitude and peak location of the input TAC, and the bias of various kinetic parameters calculated. In summary, mathematical phantom studies have been used to determine the statistical accuracy and precision of model-based kinetic analysis, which helps to validate this analysis and provides guidance in planning clinical dynamic FMISO-PET studies.

  20. Interaction of copper(II) complex of compartmental Schiff base ligand N,N'-bis(3-hydroxysalicylidene)ethylenediamine with bovine serum albumin.

    PubMed

    Boghaei, Davar M; Farvid, Shokouh S; Gharagozlou, Mehrnaz

    2007-03-01

    Circular dichroism (CD) spectroscopy, cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used to investigate the interaction between copper(II) complex of compartmental Schiff base ligand (L), N,N'-bis(3-hydroxysalicylidene)ethylenediamine, and bovine serum albumin (BSA) in 0.1 mol dm(-3) phosphate buffer solution adjusted to physiological pH 7.0 containing 20% (w/w) dimethylsulfoxide at room temperature. CD spectra show that the interaction of the copper(II) complex with BSA leads to changes in the alpha-helical content of BSA and therefore changes in secondary structure of the protein with the slight red shift (2 nm) in CD spectra. From the voltammetric data, i.e. changes in limiting current with addition of BSA, the binding constant (K) of the interaction of copper(II) complex with BSA was found to be 1.96 x 10(4)dm(3)mol(-1). From the shifts in potential with the addition of BSA, the equilibrium constant ratio (K(2)/K(1)) for the binding of the oxidized Cu(II)L (K(1)) and reduced Cu(I)L (K(2)) species to BSA was found to be 3.77, which shows that the reduced form Cu(I)L is bound more strongly to BSA than the oxidized form Cu(II)L.

  1. Ratiometric Imaging of Extracellular pH in Dental Biofilms.

    PubMed

    Schlafer, Sebastian; Dige, Irene

    2016-03-09

    The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites are not distributed evenly in dental biofilms. A complex interplay of sorption to and reaction with organic matter in the biofilm reduces the diffusion paths of solutes and creates steep gradients of reactive molecules, including organic acids, across the biofilm. Quantitative fluorescent microscopic methods, such as fluorescence life time imaging or pH ratiometry, can be employed to visualize pH in different microenvironments of dental biofilms. pH ratiometry exploits a pH-dependent shift in the fluorescent emission of pH-sensitive dyes. Calculation of the emission ratio at two different wavelengths allows determining local pH in microscopic images, irrespective of the concentration of the dye. Contrary to microelectrodes the technique allows monitoring both vertical and horizontal pH gradients in real-time without mechanically disturbing the biofilm. However, care must be taken to differentiate accurately between extra- and intracellular compartments of the biofilm. Here, the ratiometric dye, seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) is employed to monitor extracellular pH in in vivo grown dental biofilms of unknown species composition. Upon exposure to glucose the dye is up-concentrated inside all bacterial cells in the biofilms; it is thus used both as a universal bacterial stain and as a marker of extracellular pH. After confocal microscopic image acquisition, the bacterial biomass is removed from all pictures using digital image analysis software, which permits to exclusively calculate extracellular pH. pH ratiometry with the ratiometric dye is well-suited to study extracellular pH in thin biofilms of up to 75 µm thickness, but is limited to the pH range between 4.5 and 7.0.

  2. Detection of intracellular lactate with localized diffusion { 1H- 13C}-spectroscopy in rat glioma in vivo

    NASA Astrophysics Data System (ADS)

    Pfeuffer, Josef; Lin, Joseph C.; DelaBarre, Lance; Ugurbil, Kamil; Garwood, Michael

    2005-11-01

    The aim of this study was to compare the diffusion characteristic of lactate and alanine in a brain tumor model to that of normal brain metabolites known to be mainly intracellular such as N-acetylaspartate or creatine. The diffusion of 13C-labeled metabolites was measured in vivo with localized NMR spectroscopy at 9.4 T (400 MHz) using a previously described localization and editing pulse sequence known as ACED-STEAM ('adiabatic carbon editing and decoupling'). 13C-labeled glucose was administered and the apparent diffusion coefficients of the glycolytic products, { 1H- 13C}-lactate and { 1H- 13C}-alanine, were determined in rat intracerebral 9L glioma. To obtain insights into { 1H- 13C}-lactate compartmentation (intra- versus extracellular), the pulse sequence used very large diffusion weighting (50 ms/μm 2). Multi-exponential diffusion attenuation of the lactate metabolite signals was observed. The persistence of a lactate signal at very large diffusion weighting provided direct experimental evidence of significant intracellular lactate concentration. To investigate the spatial distribution of lactate and other metabolites, 1H spectroscopic images were also acquired. Lactate and choline-containing compounds were consistently elevated in tumor tissue, but not in necrotic regions and surrounding normal-appearing brain. Overall, these findings suggest that lactate is mainly associated with tumor tissue and that within the time-frame of these experiments at least some of the glycolytic product ([ 13C] lactate) originates from an intracellular compartment.

  3. Tollip, an intracellular trafficking protein, is a novel modulator of the transforming growth factor-β signaling pathway.

    PubMed

    Zhu, Lu; Wang, Lingdi; Luo, Xiaolin; Zhang, Yongxian; Ding, Qiurong; Jiang, Xiaomeng; Wang, Xiao; Pan, Yi; Chen, Yan

    2012-11-16

    Upon activation, TGF-β type I receptor (TβRI) undergoes active ubiquitination via recruitment of E3 ligases to the receptor complex by Smad7. However, how ubiquitination of TβRI is coupled to intracellular trafficking, and protein degradation remains unclear. We report here that Tollip, an adaptor protein that contains both ubiquitin-associated domains and endosome-targeting domain, plays an important role in modulating trafficking and degradation of TβRI. Tollip was previously demonstrated to possess a functional role in modulating the signaling of interleukin-1 and Toll-like receptors. We identify here that Tollip interacts with Smad7, a major modulatory protein involved in the negative regulation of TGF-β signaling. Overexpression of Tollip antagonizes TGF-β-stimulated transcriptional response, Smad2 phosphorylation, and epithelial-mesenchymal transition. Tollip also interacts with ubiquitinated TβRI, and such interaction requires ubiquitin-associated domains of Tollip. The interaction and intracellular colocalization of Tollip with TβRI is enhanced by Smad7. Overexpression of Tollip accelerates protein degradation of activated TβRI. In addition, Tollip alters subcellular compartmentalization and endosomal trafficking of activated TβRI. Collectively, our studies reveal that Tollip cooperates with Smad7 to modulate intracellular trafficking and degradation of ubiquitinated TβRI, whereby negatively regulates TGF-β signaling pathway.

  4. Measuring Phagosomal pH by Fluorescence Microscopy.

    PubMed

    Canton, Johnathan; Grinstein, Sergio

    2017-01-01

    Dual wavelength ratiometric imaging has become a powerful tool for the study of pH in intracellular compartments. It allows for the dynamic imaging of live cells while accounting for changes in the focal plane, differential loading of the fluorescent probe, and photobleaching caused by repeated image acquisitions. Ratiometric microscopic imaging has the added advantage over whole population methods of being able to resolve individual cells and even individual organelles. In this chapter we provide a detailed discussion of the basic principles of ratiometric imaging and its application to the measurement of phagosomal pH, including probe selection, the necessary instrumentation, and calibration methods.

  5. Effects of ammonium and bicarbonate-CO2 on intracellular chloride levels in Aplysia neurons.

    PubMed Central

    Russell, J M

    1978-01-01

    The level of intracellular free chloride in Aplysia giant neurons can be made to decline by pretreatment with 50 mM NH4+ solution followed by washing with 10 mM HCO3-/0.4% CO2-containing fluids. This effect can be completely blocked by the anion flux inhibitor, 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonic acid (SITS). The net change of free chloride in the cell cannot be explained by changes in the electrochemical gradient of chloride. These results support the hypothesis that at least one mechanism for intracellular pH regulation involves a Cl-/HCO-3 exchange process. PMID:25096

  6. Effect of extracellular pH on growth and proton motive force of Bacteroides succinogenes, a cellulolytic ruminal bacterium.

    PubMed Central

    Russell, J B

    1987-01-01

    The utilization of cellulose or cellobiose by Bacteroides succinogenes S85 was severely inhibited at pH values of less than 5.7. Since low pH inhibited the utilization of both cellobiose and cellulose, changes in cellulase activity could not explain the effect. At an extracellular pH of 6.9, the pH gradient (delta pH) across the cell membrane was only 0.07 U. As extracellular pH declined from 6.9 to 5.7, intracellular pH decreased to a smaller extent than extracellular pH and delta pH increased. Below pH 5.7, there was a linear and nearly proportional decrease in intracellular pH. B. succinogenes took up the lipophilic cation tetraphenylphosphonium ion (TPP+) in the presence of cellobiose, and uptake was sensitive to the ionophore valinomycin. As pH was decreased with phosphoric acid, the cells lost TPP+ and electrical potential, delta psi, decreased. From extracellular pH 6.9 to 5.7, the decrease in delta psi was compensated for by an increase in delta pH, and the proton motive force ranged from 152 to 158 mV. At a pH of less than 5.7, there was a large decrease in proton motive force, and this decrease corresponded to the inhibition of cellobiose utilization. PMID:2827568

  7. Inorganic Carbon Accumulation and Photosynthesis in a Blue-green Alga as a Function of External pH 1

    PubMed Central

    Coleman, John R.; Colman, Brian

    1981-01-01

    The blue-green alga Coccochloris peniocystis photosynthesizes optimally over the pH range of 7.0 to 10.0, but the O2-evolution rate is inhibited below pH 7.0 and ceases below pH 5.25. Measurement of the inorganic carbon pool in this alga in the light, using the silicone-fluid filtration technique demonstrated that the rate of accumulation of dissolved inorganic carbon remained relatively constant over a wide pH range. At external dissolved inorganic carbon concentrations of 0.56 to 0.89 millimolar the internal concentration after 30 seconds illumination was greater than 3.5 millimolar over the entire pH range. Intracellular pH measured in the light using [14C]5,5-dimethyloxazolidine-2,4-dione and [14C]methylamine dropped from pH 7.6 at an external pH of 7.0 to pH 6.6 at an external pH of 5.25. Above an external pH of 7.0 the intracellular pH rose gradually to pH 7.9 at an external pH 10.0. Ribulose-1,5-bisphosphate carboxylase activity of cell-free algal extracts exhibited optimal activity at pH 7.5 to 7.8 but was inactive below pH 6.5. It is suggested that the inability of Coccochloris to maintain its intracellular pH when in an acidic environment restricts its photosynthetic capacity by a direct pH effect on the principal CO2 fixing enzyme. PMID:16661792

  8. Vinyl acetate induces intracellular acidification in mouse oral buccal epithelial cells.

    PubMed

    Nakamoto, Tetsuji; Wagner, Mark; Melvin, James E; Bogdanffy, Matthew S

    2005-08-14

    Vinyl acetate exposure in drinking water has been associated with tumor formation in the upper gastrointestinal tract of rats and mice. One potential mechanism for inducing carcinogenesis involves acidification of the intracellular environment due to the metabolism of vinyl acetate to acetic acid. Prolonged intracellular acidification is thought to produce cytotoxic and/or mitogenic responses that are the sentinel pharmacodynamic steps toward cancer. To determine whether exposure to vinyl acetate affects the intracellular pH of intact oral cavity tissue, isolated mouse oral buccal epithelium was loaded with the pH-sensitive dye BCECF, and then exposed to vinyl acetate concentrations ranging from 10 to 1000 microM for up to 4 min. Extracellular vinyl acetate exposure induced a progressive intracellular acidification that was reversible upon removal of the vinyl acetate. The rate of the acidification was concentration-dependent and increased exponentially within the concentration range tested. The magnitude of the vinyl acetate-induced acidification was inhibited by pretreatment with the carboxylesterase inhibitor bis(p-nitrophenyl)phosphate. These results are consistent with the hypothesis that vinyl acetate contributes to the generation and progression of oral cavity tumors via a process of intracellular acidification. Such a process has been proposed to have practical dose-response thresholds below which the intracellular environment can be maintained within homeostatic bounds and the contribution of exposure to carcinogenic risk is negligible.

  9. Intracellularly induced cyclophilins play an important role in stress adaptation and virulence of Brucella abortus.

    PubMed

    Roset, Mara S; García Fernández, Lucía; DelVecchio, Vito G; Briones, Gabriel

    2013-02-01

    Brucella is an intracellular bacterial pathogen that causes the worldwide zoonotic disease brucellosis. Brucella virulence relies on its ability to transition to an intracellular lifestyle within host cells. Thus, this pathogen must sense its intracellular localization and then reprogram gene expression for survival within the host cell. A comparative proteomic investigation was performed to identify differentially expressed proteins potentially relevant for Brucella intracellular adaptation. Two proteins identified as cyclophilins (CypA and CypB) were overexpressed in the intracellular environment of the host cell in comparison to laboratory-grown Brucella. To define the potential role of cyclophilins in Brucella virulence, a double-deletion mutant was constructed and its resulting phenotype was characterized. The Brucella abortus ΔcypAB mutant displayed increased sensitivity to environmental stressors, such as oxidative stress, pH, and detergents. In addition, the B. abortus ΔcypAB mutant strain had a reduced growth rate at lower temperature, a phenotype associated with defective expression of cyclophilins in other microorganisms. The B. abortus ΔcypAB mutant also displays reduced virulence in BALB/c mice and defective intracellular survival in HeLa cells. These findings suggest that cyclophilins are important for Brucella virulence and survival in the host cells.

  10. The effects of pH changes on human and ferret detrusor muscle function.

    PubMed Central

    Liston, T G; Palfrey, E L; Raimbach, S J; Fry, C H

    1991-01-01

    1. The effects of altering extracellular pH on the electrically evoked contractions of ferret and human bladder (detrusor) smooth muscle have been investigated. pH was varied by changing superfusate PCO2 or NaHCO3 concentration. Acidosis increased force when superfusate PCO2 was raised but decreased force when the NaHCO3 concentration was reduced. 2. Intracellular pH (pHi) in isolated ferret detrusor cells was measured separately by epifluorescence microscopy. Extracellular pH changes caused by altering superfusate PCO2 were accompanied by similar changes of pHi, whereas variation of the NaHCO3 concentration had smaller effects on pHi. 3. It was proposed that intracellular acidosis increased contraction but extracellular acidosis depressed contraction. 4. Other interventions, such as addition and removal of NH4Cl, Cl- replacement, and NaHCO3 replacement with HEPES, changed pHi and had predictable effects on force. It was possible to describe unique relationships between tension and either intracellular or extracellular pH regardless of the means whereby pH changes were brought about. 5. Resting tension was reduced whether brought about by either intracellular or extracellular acidosis. K+ contractures were similarly affected by acidosis. Ferret preparations showed low levels of spontaneous activity, which was reduced by acidosis and enhanced by alkalosis. PMID:1653318

  11. 17-4 PH and 15-5 PH

    NASA Technical Reports Server (NTRS)

    Johnson, Howard T.

    1995-01-01

    17-4 PH and 15-5 PH are extremely useful and versatile precipitation-hardening stainless steels. Armco 17-4 PH is well suited for the magnetic particle inspection requirements of Aerospace Material Specification. Armco 15-5 PH and 17-4 PH are produced in billet, plate, bar, and wire. Also, 15-5 PH is able to meet the stringent mechanical properties required in the aerospace and nuclear industries. Both products are easy to heat treat and machine, making them very useful in many applications.

  12. The Semen pH Affects Sperm Motility and Capacitation.

    PubMed

    Zhou, Ji; Chen, Li; Li, Jie; Li, Hongjun; Hong, Zhiwei; Xie, Min; Chen, Shengrong; Yao, Bing

    2015-01-01

    As the chemical environment of semen can have a profound effect on sperm quality, we examined the effect of pH on the motility, viability and capacitation of human sperm. The sperm in this study was collected from healthy males to avoid interference from other factors. The spermatozoa cultured in sperm nutrition solution at pH 5.2, 6.2, 7.2 and 8.2 were analyzed for sperm total motility, progressive motility (PR), hypo-osmotic swelling (HOS) rate, and sperm penetration. Our results showed that these parameters were similar in pH 7.2 and 8.2 sperm nutrition solutions, but decreased in pH 5.2 and 6.2 solutions. The HOS rate exhibited positive correlation with the sperm total motility and PR. In addition, the sperm Na(+)/K(+)-ATPase activity at different pHs was measured, and the enzyme activity was significantly lower in pH 5.2 and 6.2 media, comparing with that in pH 8.2 and pH 7.2 solutions. Using flow cytometry (FCM) and laser confocal scanning microscopy (LCSM) analysis, the intracellular Ca2(+ )concentrations of sperm cultured in sperm capacitation solution at pH 5.2, 6.2, 7.2 and 8.2 were determined. Compared with that at pH 7.2, the mean fluorescence intensity of sperm in pH 5.2 and 6.2 media decreased significantly, while that of pH 8.2 group showed no difference. Our results suggested that the declined Na(+)/K(+)-ATPase activity at acidic pHs result in decreased sperm movement and capacitation, which could be one of the mechanisms of male infertility.

  13. Intracellular sequestration of zinc, cadmium and silver in Hebeloma mesophaeum and characterization of its metallothionein genes.

    PubMed

    Sácký, Jan; Leonhardt, Tereza; Borovička, Jan; Gryndler, Milan; Briksí, Aleš; Kotrba, Pavel

    2014-06-01

    Sequestration of intracellular heavy metals in eukaryotes involves compartmentalization and binding with cytosolic, cysteine-rich metallothionein (MT) peptides. We examined the roles of these processes in handling of zinc (Zn), cadmium (Cd) and silver (Ag) in sporocarps and a metal-exposed extraradical mycelium of Hebeloma mesophaeum, the Zn-accumulating ectomycorrhizal (EM) species frequently associated with metal disturbed sites. Size exclusion chromatography revealed that the majority of Zn and Cd in the sporocarps and mycelium was contained in a low molecular mass fraction attributable to compartmentalized metal. The staining of hyphal cells with the Zn-specific Zinquin and Cd-specific Leadmium fluorescent tracers labeled Zn and Cd in small, punctuated vesicles and vacuoles, respectively. By contrast, the sporocarp and mycelium Ag was associated with cysteine-rich, 5-kDa peptides. The peptides of the same size were also identified in minor Zn and Cd complexes from the metal-exposed mycelium. We have further isolated and characterized HmMT1, HmMT2 and HmMT3 genes coding for different 5-kDa MTs of H. mesophaeum collected at a lead smelter site. Heterologous complementation assays in metal-sensitive yeast mutants indicated that HmMTs encode functional, metal-specific peptides: only HmMT1 was able to complement sensitivity to Zn; HmMT1 conferred higher tolerance to Cd and Cu than HmMT2 or HmMT3; and both HmMT2 and HmMT3, but not HmMT1, conferred increased tolerance to Ag. The presence of HmMT1 and HmMT3, but not HmMT2, was also confirmed in a H. mesophaeum isolate from an unpolluted site. Gene expression analysis in the extraradical mycelium of this isolate revealed that the transcription of HmMT1 was preferentially induced in the presence of Zn and Cd, while Ag was a stronger inducer of HmMT3. Altogether, these results improve our understanding of the handling of intracellular Zn, Cd and Ag in Hebeloma and represent the first evidence suggesting involvement of MTs

  14. Intracellular minerals and metal deposits in prokaryotes.

    PubMed

    Edwards, K J; Bazylinski, D A

    2008-06-01

    Thanks to the work of Terrance J. Beveridge and other pioneers in the field of metal-microbe interactions, prokaryotes are well known to sequester metals and other ions intracellularly in various forms. These forms range from poorly ordered deposits of metals to well-ordered mineral crystals. Studies on well-ordered crystalline structures have generally focused on intracellular organelles produced by magnetotactic bacteria that are ubiquitous in terrestrial and marine environments that precipitate Fe(3)O(4) or Fe(3)S(4), Fe-bearing minerals that have magnetic properties and are enclosed in intracellular membranes. In contrast, studies on less-well ordered minerals have focused on Fe-, As-, Mn-, Au-, Se- and Cd-precipitates that occur intracellularly. The biological and environmental function of these particles remains a matter of debate.

  15. Nanoparticles for intracellular-targeted drug delivery

    NASA Astrophysics Data System (ADS)

    Paulo, Cristiana S. O.; Pires das Neves, Ricardo; Ferreira, Lino S.

    2011-12-01

    Nanoparticles (NPs) are very promising for the intracellular delivery of anticancer and immunomodulatory drugs, stem cell differentiation biomolecules and cell activity modulators. Although initial studies in the area of intracellular drug delivery have been performed in the delivery of DNA, there is an increasing interest in the use of other molecules to modulate cell activity. Herein, we review the latest advances in the intracellular-targeted delivery of short interference RNA, proteins and small molecules using NPs. In most cases, the drugs act at different cellular organelles and therefore the drug-containing NPs should be directed to precise locations within the cell. This will lead to the desired magnitude and duration of the drug effects. The spatial control in the intracellular delivery might open new avenues to modulate cell activity while avoiding side-effects.

  16. Intracellular Protein Delivery for Treating Breast Cancer

    DTIC Science & Technology

    2014-08-01

    nanocapsules with specific cancer cell targeting ligands; Task 3. Preparing and testing of MMP activatable cell penetrating peptides (ACCPs)-coupled...AD_________________ Award Number: W81XWH-11-1-0371 TITLE: Intracellular Protein Delivery for Treating Breast Cancer PRINCIPAL INVESTIGATOR: Dr...SUBTITLE Intracellular Protein Delivery for Treating Breast Cancer 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-11-1-0371 5c. PROGRAM ELEMENT NUMBER 6

  17. Imaging pH with hyperpolarized 13C.

    PubMed

    Gallagher, Ferdia A; Kettunen, Mikko I; Brindle, Kevin M

    2011-10-01

    pH is a fundamental physiological parameter that is tightly controlled by endogenous buffers. The acid-base balance is altered in many disease states, such as inflammation, ischemia and cancer. Despite the importance of pH, there are currently no routine methods for imaging the spatial distribution of pH in humans. The enormous gain in sensitivity afforded by dynamic nuclear polarization (DNP) has provided a novel way in which to image tissue pH using MR, which has the potential to be translated into the clinic. This review explores the advantages and disadvantages of current pH imaging techniques and how they compare with DNP-based approaches for the measurement and imaging of pH with hyperpolarized (13)C. Intravenous injection of hyperpolarized (13)C-labeled bicarbonate results in the rapid production of hyperpolarized (13)CO(2) in the reaction catalyzed by carbonic anhydrase. As this reaction is close to equilibrium in the body and is pH dependent, the ratio of the (13)C signal intensities from H(13)CO(3)(-) and (13)CO(2), measured using MRS, can be used to calculate pH in vivo. The application of this technique to a murine tumor model demonstrated that it measured predominantly extracellular pH and could be mapped in the animal using spectroscopic imaging techniques. A second approach has been to use the production of hyperpolarized (13)CO(2) from hyperpolarized [1-(13)C]pyruvate to measure predominantly intracellular pH. In tissues with a high aerobic capacity, such as the heart, the hyperpolarized [1-(13)C]pyruvate undergoes rapid oxidative decarboxylation, catalyzed by intramitochondrial pyruvate dehydrogenase. Provided that there is sufficient carbonic anhydrase present to catalyze the rapid equilibration of the hyperpolarized (13)C label between CO(2) and bicarbonate, the ratio of their resonance intensities may again be used to estimate pH, which, in this case, is predominantly intracellular. As both pyruvate and bicarbonate are endogenous molecules they

  18. Nitric oxide and pH modulation in gynaecological cancer.

    PubMed

    Sanhueza, Carlos; Araos, Joaquín; Naranjo, Luciano; Barros, Eric; Subiabre, Mario; Toledo, Fernando; Gutiérrez, Jaime; Chiarello, Delia I; Pardo, Fabián; Leiva, Andrea; Sobrevia, Luis

    2016-12-01

    Nitric oxide plays several roles in cellular physiology, including control of the vascular tone and defence against pathogen infection. Neuronal, inducible and endothelial nitric oxide synthase (NOS) isoforms synthesize nitric oxide. Cells generate acid and base equivalents, whose physiological intracellular concentrations are kept due to membrane transport systems, including Na(+) /H(+) exchangers and Na(+) /HCO3(-) transporters, thus maintaining a physiological pH at the intracellular (~7.0) and extracellular (~7.4) medium. In several pathologies, including cancer, cells are exposed to an extracellular acidic microenvironment, and the role for these membrane transport mechanisms in this phenomenon is likely. As altered NOS expression and activity is seen in cancer cells and because this gas promotes a glycolytic phenotype leading to extracellular acidosis in gynaecological cancer cells, a pro-inflammatory microenvironment increasing inducible NOS expression in this cell type is feasible. However, whether abnormal control of intracellular and extracellular pH by cancer cells regards with their ability to synthesize or respond to nitric oxide is unknown. We, here, discuss a potential link between pH alterations, pH controlling membrane transport systems and NOS function. We propose a potential association between inducible NOS induction and Na(+) /H(+) exchanger expression and activity in human ovary cancer. A potentiation between nitric oxide generation and the maintenance of a low extracellular pH (i.e. acidic) is proposed to establish a sequence of events in ovarian cancer cells, thus preserving a pro-proliferative acidic tumour extracellular microenvironment. We suggest that pharmacological therapeutic targeting of Na(+) /H(+) exchangers and inducible NOS may have benefits in human epithelial ovarian cancer.

  19. Engineering nanostructured polymer blends with controlled nanoparticle location for excellent microwave absorption: a compartmentalized approach.

    PubMed

    Biswas, Sourav; Kar, Goutam Prasanna; Bose, Suryasarathi

    2015-07-14

    In order to obtain better materials, control over the precise location of nanoparticles is indispensable. It is shown here that ordered arrangements of nanoparticles, possessing different characteristics (electrical/magnetic dipoles), in the blend structure can result in excellent microwave absorption. This is manifested from a high reflection loss of ca. -67 dB for the best blend structure designed here. To attenuate electromagnetic radiation, the key parameters of high electrical conductivity and large dielectric/magnetic loss are targeted here by including a conductive material [multiwall carbon nanotubes, MWNTs], ferroelectric nanostructured material with associated relaxations in the GHz frequency [barium titanate, BT] and lossy ferromagnetic nanoparticles [nickel ferrite, NF]. In this study, bi-continuous structures were designed using 50/50 (by wt) blends of polycarbonate (PC) and polyvinylidene fluoride (PVDF). The MWNTs were modified using an electron acceptor molecule, a derivative of perylenediimide, which facilitates π-π stacking with the nanotubes and stimulates efficient charge transport in the blends. The nanoscopic materials have specific affinity towards the PVDF phase. Hence, by introducing surface-active groups, an ordered arrangement can be tailored. To accomplish this, both BT and NF were first hydroxylated followed by the introduction of amine-terminal groups on the surface. The latter facilitated nucleophilic substitution reactions with PC and resulted in their precise location. In this study, we have shown for the first time that by a compartmentalized approach, superior EM attenuation can be achieved. For instance, when the nanoparticles were localized exclusively in the PVDF phase or in both the phases, the minimum reflection losses were ca. -18 dB (for the MWNT/BT mixture) and -29 dB (for the MWNT/NF mixture), and the shielding occurred primarily through reflection. Interestingly, by adopting the compartmentalized approach wherein the

  20. Compartmentalization of endocannabinoids into lipid rafts in a microglial cell line devoid of caveorrlin-1

    PubMed Central

    Rimmerman, Neta; Bradshaw, Heather B; Kozela, Ewa; Levy, Rivka; Juknat, Ana; Vogel, Zvi

    2012-01-01

    BACKGROUND AND PURPOSE N-acyl ethanolamines (NAEs) and 2-arachidonoyl glycerol (2-AG) are endogenous cannabinoids and along with related lipids are synthesized on demand from membrane phospholipids. Here, we have studied the compartmentalization of NAEs and 2-AG into lipid raft fractions isolated from the caveolin-1-lacking microglial cell line BV-2, following vehicle or cannabidiol (CBD) treatment. Results were compared with those from the caveolin-1-positive F-11 cell line. EXPERIMENTAL APPROACH BV-2 cells were incubated with CBD or vehicle. Cells were fractionated using a detergent-free continuous OptiPrep density gradient. Lipids in fractions were quantified using HPLC/MS/MS. Proteins were measured using Western blot. KEY RESULTS BV-2 cells were devoid of caveolin-1. Lipid rafts were isolated from BV-2 cells as confirmed by co-localization with flotillin-1 and sphingomyelin. Small amounts of cannabinoid CB1 receptors were found in lipid raft fractions. After incubation with CBD, levels and distribution in lipid rafts of 2-AG, N-arachidonoyl ethanolamine (AEA), and N-oleoyl ethanolamine (OEA) were not changed. Conversely, the levels of the saturated N-stearoyl ethanolamine (SEA) and N-palmitoyl ethanolamine (PEA) were elevated in lipid raft fractions. In whole cells with growth medium, CBD treatment increased AEA and OEA time-dependently, while levels of 2-AG, PEA and SEA did not change. CONCLUSIONS AND IMPLICATIONS Whereas levels of 2-AG were not affected by CBD treatment, the distribution and levels of NAEs showed significant changes. Among the NAEs, the degree of acyl chain saturation predicted the compartmentalization after CBD treatment suggesting a shift in cell signalling activity. LINKED ARTICLES This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10

  1. A microcomputer algorithm for solving first-order compartmental models involving recycling.

    PubMed

    Birchall, A; James, A C

    1989-06-01

    A general algorithm for solving first-order compartmental models including recycling systems has been developed and its implementation on a microcomputer is described. Matrix algebra is used to obtain for any compartmental model an analytical solution, which is expressed as the exponential of a matrix of rate constants. A special technique is used in the algorithm to enable this exponential to be evaluated with a rapidly converging series. Truncation errors incurred in this process are estimated automatically. Thus, in an extreme case, where these errors may be significant, the appropriate action can be taken. Given a particular model, the user enters the model parameters into a rate matrix according to a simple rule. The algorithm then uses this matrix to solve the model, and thus no specialized mathematical knowledge is needed. The algorithm is given in a short BASIC program (60 lines) listed in an appendix. No additional software is required. By running this program on a standard microcomputer, the user can solve models of any complexity: those up to 15 compartments in seconds and those up to 30 compartments within a minute. The algorithm is thus ideally suited to solve kinetic models describing the transport of radionuclides in the environment or the translocation of elements in biological systems such as the metabolic models recommended by the International Commission on Radiological Protection (ICRP). Given the initial amount of material in each compartment at time t = 0, together with its radioactive decay constant, the algorithm gives both the amount in each compartment at any future time t and the number of disintegrations that will have occurred in each compartment up to time t. The computer program, shown in an appendix, could easily be used to calculate disintegrations over any time interval of interest, or to predict the quantities or fractions of an intake expected to be present in any in vivo or excretion compartments of interest. Thus, the algorithm

  2. Umami changes intracellular Ca2+ levels using intracellular and extracellular sources in mouse taste receptor cells.

    PubMed

    Narukawa, Masataka; Mori, Tomohiko; Hayashi, Yukako

    2006-11-01

    Recently, candidates for umami receptors have been identified in taste cells, but the precise transduction mechanisms of the downstream receptor remain unknown. To investigate how intracellular Ca(2+) increases in the umami transduction pathway, we measured changes in intracellular Ca(2+) levels in response to umami stimuli monosodium glutamate (MSG), IMP, and MSG + IMP in mouse taste receptor cells (TRCs) by Ca(2+) imaging. Even when extracellular Ca(2+) was absent, 1/3 of umami-responsive TRCs exhibited increased intracellular Ca(2+) levels. When intracellular Ca(2+) was depleted, half of the TRCs retained their response to umami. These results suggest that umami-responsive TRCs increase their intracellular Ca(2+) levels through two pathways: by releasing Ca(2+) from intracellular stores and by an influx of Ca(2+) from extracellular sources. We conclude that the Ca(2+) influx from extracellular source might play an important role in the synergistic effect between MSG and IMP.

  3. Silica nanoparticles for cell imaging and intracellular sensing

    NASA Astrophysics Data System (ADS)

    Korzeniowska, B.; Nooney, R.; Wencel, D.; McDonagh, C.

    2013-11-01

    There is increasing interest in the use of nanoparticles (NPs) for biomedical applications. In particular, nanobiophotonic approaches using fluorescence offers the potential of high sensitivity and selectivity in applications such as cell imaging and intracellular sensing. In this review, we focus primarily on the use of fluorescent silica NPs for these applications and, in so doing, aim to enhance and complement the key recent review articles on these topics. We summarize the main synthetic approaches, namely the Stöber and microemulsion processes, and, in this context, we deal with issues in relation to both covalent and physical incorporation of different types of dyes in the particles. The important issue of NP functionalization for conjugation to biomolecules is discussed and strategies published in the recent literature are highlighted and evaluated. We cite recent examples of the use of fluorescent silica NPs for cell imaging in the areas of cancer, stem cell and infectious disease research, and we review the current literature on the use of silica NPs for intracellular sensing of oxygen, pH and ionic species. We include a short final section which seeks to identify the main challenges and obstacles in relation to the potential widespread use of these particles for in vivo diagnostics and therapeutics.

  4. Staining of intracellular deposits of uranium in cultured murine macrophages.

    PubMed

    Kalinich, J F; McClain, D E

    2001-01-01

    In our studies of the health effects of internalized depleted uranium, we developed a simple and rapid light microscopic method to stain specifically intracellular uranium deposits. Using J774 cells, a mouse macrophage line, treated with uranyl nitrate and the pyridylazo dye 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol, uranium uptake by the cells was followed. Specificity of the stain for uranium was accomplished by using masking agents to prevent the interaction of the stain with other metals. Prestaining wash consisting of a mixture of sodium citrate and ethylenediaminetetraacetic acid eliminated staining of metals other than uranium. The staining solution consisted of the pyridylazo dye in borate buffer along with a quaternary ammonium salt, ethylhexadecyldimethylammonium bromide, and the aforementioned sodium citrate/ethylenediaminetetraacetic acid mixture. The buffer was essential for maintaining the pH within the optimum range of 8 to 12, and the quaternary ammonium salt prevented precipitation of the dye. Staining was conducted at room temperature and was complete in 30 min. Staining intensity correlated with both uranyl nitrate concentration and incubation time. Our method provides a simple procedure for detecting intracellular uranium deposits in macrophages.

  5. Hybrid micro-/nanogels for optical sensing and intracellular imaging

    PubMed Central

    Wu, Weitai; Zhou, Shuiqin

    2010-01-01

    Hybrid micro-/nanogels are playing an increasing important part in a diverse range of applications, due to their tunable dimensions, large surface area, stable interior network structure, and a very short response time. We review recent advances and challenges in the developments of hybrid micro-/nanogels toward applications for optical sensing of pH, temperature, glucose, ions, and other species as well as for intracellular imaging. Due to their unique advantages, hybrid micro-/nanogels as optical probes are attracting substantial interests for continuous monitoring of chemical parameters in complex samples such as blood and bioreactor fluids, in chemical research and industry, and in food quality control. In particular, their intracellular probing ability enables the monitoring of the biochemistry and biophysics of live cells over time and space, thus contributing to the explanation of intricate biological processes and the development of novel diagnoses. Unlike most other probes, hybrid micro-/nanogels could also combine other multiple functions into a single probe. The rational design of hybrid micro-/nanogels will not only improve the probing applications as desirable, but also implement their applications in new arenas. With ongoing rapid advances in bionanotechnology, the well-designed hybrid micro-/nanogel probes will be able to provide simultaneous sensing, imaging diagnosis, and therapy toward clinical applications. PMID:22110866

  6. Synthesis of Monodisperse Bi-Compartmentalized Amphiphilic Janus Microparticles for Tailored Assembly at the Oil-Water Interface.

    PubMed

    Kim, Jeong Won; Cho, Jangwoo; Cho, Jaehong; Park, Bum Jun; Kim, Yong-Jin; Choi, Kyung-Ho; Kim, Jin Woong

    2016-03-24

    Janus particles endowed with controlled anisotropies represent promising building blocks and assembly materials because of their asymmetric functionalities. Herein, we show that using the seeded monomer swelling and polymerization technique allows us to obtain bi-compartmentalized Janus microparticles that are generated depending on the phase miscibility of the poly (alkyl acrylate) chains against the polystyrene seed, thus minimizing the interfacial free energy. When tetradecyl acrylate is used, complete compartmentalization into two distinct bulbs can be achieved, while tuning the relative dimension ratio of compartmented bulb against the whole particle. Finally, we have demonstrated that selectively patching the silica nanoparticles onto one of the bulb surfaces gives amphiphilicity to the particles that can assemble at the oil-water interface with a designated level of adhesion, thus leading to development of a highly stable Pickering emulsion system.

  7. Cadherins in cerebellar development: translation of embryonic patterning into mature functional compartmentalization.

    PubMed

    Redies, Christoph; Neudert, Franziska; Lin, Juntang

    2011-09-01

    Cadherins are cell adhesion molecules with multiple morphogenic functions in brain development, for example, in neuroblast migration and aggregation, axon navigation, neural circuit formation, and synaptogenesis. More than 100 members of the cadherin superfamily are expressed in the developing and mature brain. Most of the cadherins investigated, in particular classic cadherins and δ-protocadherins, are expressed in the cerebellum. For several cadherin subtypes, expression begins at early embryonic stages and persists until mature stages of cerebellar development. At intermediate stages, distinct Purkinje cell clusters exhibit unique rostrocaudal and mediolateral expression profiles for each cadherin. In the chicken, mouse, and other species, the Purkinje cell clusters are separated by intervening raphes of migrating granule cells. This pattern of Purkinje cell clusters/raphes is, at least in part, continuous with the parasagittal striping pattern that is apparent in the mature cerebellar cortex, for example, for zebrin II/aldolase C. Moreover, subregions of the deep cerebellar nuclei, vestibular nuclei and the olivary complex also express cadherins differentially. Neuroanatomical evidence suggests that the nuclear subregions and cortical domains that express the same cadherin subtype are connected to each other, to form neural subcircuits of the cerebellar system. Cadherins thus provide a molecular code that specifies not only embryonic structures but also functional cerebellar compartmentalization. By following the implementation of this code, it can be revealed how mature functional architecture emerges from embryonic patterning during cerebellar development. Dysfunction of some cadherins is associated with psychiatric diseases and developmental impairments and may also affect cerebellar function.

  8. Plasma Membrane Polarity and Compartmentalization are Established Before Cellularization in the Fly Embryo

    PubMed Central

    Mavrakis, Manos; Rikhy, Richa; Lippincott-Schwartz, Jennifer

    2009-01-01

    Summary Patterning in the Drosophila embryo requires local activation and dynamics of proteins in the plasma membrane (PM). We used in vivo fluorescence imaging to characterize the organization and diffusional properties of the PM in the early embryonic syncytium. Before cellularization, the PM is polarized into discrete domains having epithelial-like characteristics. One domain resides above individual nuclei and has apical-like characteristics, while the other domain is lateral to nuclei and contains markers associated with basolateral membranes and junctions. Pulse-chase photoconversion experiments show that molecules can diffuse within each domain but do not exchange between PM regions above adjacent nuclei. Drug-induced F-actin depolymerization disrupted both the apicobasal-like polarity and the diffusion barriers within the syncytial PM. These events correlated with perturbations in the spatial pattern of dorsoventral Toll signaling. We propose that epithelial-like properties and an intact F-actin network compartmentalize the PM and shape morphogen gradients in the syncytial embryo. PMID:19154721

  9. Local Microenvironment Controls the Compartmentalization of NK Cell Responses during Systemic Inflammation in Mice.

    PubMed

    Rasid, Orhan; Ciulean, Ioana Sonya; Fitting, Catherine; Doyen, Noelle; Cavaillon, Jean-Marc

    2016-09-15

    Systemic inflammatory response syndrome is a whole-body reaction to a triggering insult that often results in life-threatening illness. Contributing to the development of this inflammatory cascade are numerous cellular partners, among which NK cells were shown to play a key role. Accumulating evidence points to organ-specific properties of systemic inflammation and NK cells. However, little is known about compartment-specific activation of NK cells during systemic inflammatory response syndrome or the relative contribution of NK cell-intrinsic properties and microenvironmental cues. In this study, we undertook a sequential characterization of NK responses in the spleen, lungs, bone marrow, peritoneum, and blood using a mouse model of endotoxemia. We report that, despite similar systemic dynamics of NK cell responses, expression of activation markers (CD69 and CD25) and effector molecules (IFN-γ, granzyme B, and IL-10) display organ-specific thresholds of maximum activation. Using adoptive transfers of spleen and lung NK cells, we found that these cells have the capacity to quickly adapt to a new environment and adjust their response levels to that of resident NK cells. This functional adaptation occurs without significant alterations in phenotype and independently of subpopulation-specific trafficking. Thus, using a dynamic in vivo-transfer system, to our knowledge our study is the first to report the compartmentalization of NK cells responses during systemic inflammation and to show that NK cell-intrinsic properties and microenvironmental cues are involved in this process, in a sequential manner.

  10. Reservoir compartmentalization and management strategies: Lessons learned in the Illinois basin

    SciTech Connect

    Grube, J.P.; Crockett, J.E.; Huff, B.G.

    1997-08-01

    A research project jointly sponsored by the US Department of Energy and the Illinois State Geological Survey focused on the Cypress and Aux Vases Formations (Mississippian), major clastic reservoirs in the Illinois Basin. Results from the research showed that understanding the nature and distribution of reservoir compartments, and using effective reservoir management strategies, can significantly improve recovery efficiencies from oil fields in this mature basin. Compartments can be most effectively drained where they are geologically well defined and reservoir management practices are coordinated through unified, compartment-wide, development programs. Our studies showed that the Cypress and Aux Vases reservoirs contain lateral and vertical permeability barriers forming compartments that range in size from isolated, interlaminated sandstone and shale beds to sandstone bodies tens of feet in thickness and more than a mile in length. Stacked or shingled, genetically similar sandstone bodies are commonly separated by thin impermeable intervals that can be difficult to distinguish on logs and can, therefore, cause correlation problems, even between wells drilled on spacing of less than ten acres. Lateral separation of sandstone bodies causes similar problems. Reservoir compartmentalization reduces primary and particularly secondary recovery by trapping pockets of by-passed or banked oil. Compartments can be detected by comparing recovery factors of genetically similar sandstone bodies within a field; using packers to separate commingled intervals and analyzing fluid recoveries and pressures; making detailed core-to-log calibrations that identify compartment boundaries; and analyzing pressure data from waterflood programs.

  11. Immunometabolism of obesity and diabetes: microbiota link compartmentalized immunity in the gut to metabolic tissue inflammation.

    PubMed

    McPhee, Joseph B; Schertzer, Jonathan D

    2015-12-01

    The bacteria that inhabit us have emerged as factors linking immunity and metabolism. Changes in our microbiota can modify obesity and the immune underpinnings of metabolic diseases such as Type 2 diabetes. Obesity coincides with a low-level systemic inflammation, which also manifests within metabolic tissues such as adipose tissue and liver. This metabolic inflammation can promote insulin resistance and dysglycaemia. However, the obesity and metabolic disease-related immune responses that are compartmentalized in the intestinal environment do not necessarily parallel the inflammatory status of metabolic tissues that control blood glucose. In fact, a permissive immune environment in the gut can exacerbate metabolic tissue inflammation. Unravelling these discordant immune responses in different parts of the body and establishing a connection between nutrients, immunity and the microbiota in the gut is a complex challenge. Recent evidence positions the relationship between host gut barrier function, intestinal T cell responses and specific microbes at the crossroads of obesity and inflammation in metabolic disease. A key problem to be addressed is understanding how metabolite, immune or bacterial signals from the gut are relayed and transferred into systemic or metabolic tissue inflammation that can impair insulin action preceding Type 2 diabetes.

  12. Nucleolus-like compartmentalization of the transcription machinery in fast-growing bacterial cells.

    PubMed

    Jin, Ding Jun; Mata Martin, Carmen; Sun, Zhe; Cagliero, Cedric; Zhou, Yan Ning

    2017-02-01

    We have learned a great deal about RNA polymerase (RNA Pol), transcription factors, and the transcriptional regulation mechanisms in prokaryotes for specific genes, operons, or transcriptomes. However, we have only begun to understand how the transcription machinery is three-dimensionally (3D) organized into bacterial chromosome territories to orchestrate the transcription process and to maintain harmony with the replication machinery in the cell. Much progress has been made recently in our understanding of the spatial organization of the transcription machinery in fast-growing Escherichia coli cells using state-of-the-art superresolution imaging techniques. Co-imaging of RNA polymerase (RNA Pol) with DNA and transcription elongation factors involved in ribosomal RNA (rRNA) synthesis, and ribosome biogenesis has revealed similarities between bacteria and eukaryotes in the spatial organization of the transcription machinery for growth genes, most of which are rRNA genes. Evidence supports the notion that RNA Pol molecules are concentrated, forming foci at the clustering of rRNA operons resembling the eukaryotic nucleolus. RNA Pol foci are proposed to be active transcription factories for both rRNA genes expression and ribosome biogenesis to support maximal growth in optimal growing conditions. Thus, in fast-growing bacterial cells, RNA Pol foci mimic eukaryotic Pol I activity, and transcription factories resemble nucleolus-like compartmentation. In addition, the transcription and replication machineries are mostly segregated in space to avoid the conflict between the two major cellular functions in fast-growing cells.

  13. The anomalous kinetics of coupled aspartate aminotransferase and malate dehydrogenase. Evidence for compartmentation of oxaloacetate.

    PubMed Central

    Bryce, C F; Williams, D C; John, R A; Fasella, P

    1976-01-01

    Cytoplasmic aspartate aminotransferase and malate dehydrogenase were purified from pig heart. Kinetic parameters were determined for the separate reaction catalysed by each enzyme and used to predict the course of the coupled reaction: (see article). Although a lag phase should have been easily seen, none was detected. The same coupled reaction was also carried out by using radioactive aspartate in the presence of unlabelled oxaloacetate. The reaction was quenched with HClO4 after 70 ms and the specific radioactivity of the malate produced in this system was found to be essentially the same as that of the original aspartate. These results show that oxaloacetate produced by the aspartate aminotransferase is converted into malate by malate dehydrogenase before it equilibrates with the pool of unlabelled oxaloacetate and are consistent with a proposal that the enzymes are associated in a complex. However, no physical evidence of the existence of a complex could be found. An alternative means of compartmentation of the intermediate as an unstable isomer is considered. Images Fig. 2. Fig. 3. PMID:942372

  14. Microfluidic device for the formation of optically excitable, three-dimensional, compartmentalized motor units

    PubMed Central

    Uzel, Sebastien G. M.; Platt, Randall J.; Subramanian, Vidya; Pearl, Taylor M.; Rowlands, Christopher J.; Chan, Vincent; Boyer, Laurie A.; So, Peter T. C.; Kamm, Roger D.

    2016-01-01

    Motor units are the fundamental elements responsible for muscle movement. They are formed by lower motor neurons and their muscle targets, synapsed via neuromuscular junctions (NMJs). The loss of NMJs in neurodegenerative disorders (such as amyotrophic lateral sclerosis or spinal muscle atrophy) or as a result of traumatic injuries affects millions of lives each year. Developing in vitro assays that closely recapitulate the physiology of neuromuscular tissues is crucial to understand the formation and maturation of NMJs, as well as to help unravel the mechanisms leading to their degeneration and repair. We present a microfluidic platform designed to coculture myoblast-derived muscle strips and motor neurons differentiated from mouse embryonic stem cells (ESCs) within a three-dimensional (3D) hydrogel. The device geometry mimics the spinal cord–limb physical separation by compartmentalizing the two cell types, which also facilitates the observation of 3D neurite outgrowth and remote muscle innervation. Moreover, the use of compliant pillars as anchors for muscle strips provides a quantitative functional readout of force generation. Finally, photosensitizing the ESC provides a pool of source cells that can be differentiated into optically excitable motor neurons, allowing for spatiodynamic, versatile, and noninvasive in vitro control of the motor units. PMID:27493991

  15. Kinase suppressor of Ras1 compartmentalizes hippocampal signal transduction and subserves synaptic plasticity and memory formation.

    PubMed

    Shalin, Sara C; Hernandez, Caterina M; Dougherty, Michele K; Morrison, Deborah K; Sweatt, J David

    2006-06-01

    The ERK/MAP kinase cascade is important for long-term memory formation and synaptic plasticity, with a myriad of upstream signals converging upon ERK activation. Despite this convergence of signaling, neurons routinely activate appropriate biological responses to different stimuli. Scaffolding proteins represent a mechanism to achieve compartmentalization of signaling and the appropriate targeting of ERK-dependent processes. We report that kinase suppressor of Ras (KSR1) functions biochemically in the hippocampus to scaffold the components of the ERK cascade, specifically regulating the cascade when a membrane fraction of ERK is activated via a PKC-dependent pathway but not via a cAMP/PKA-dependent pathway. Specificity of KSR1-dependent signaling also extends to specific downstream targets of ERK. Behaviorally and physiologically, we found that the absence of KSR1 leads to deficits in associative learning and theta burst stimulation-induced LTP. Our report provides novel insight into the endogenous scaffolding role of KSR1 in controlling kinase activation within the nervous system.

  16. Integrated compartmental model for describing the transport of solute in a fractured porous medium. [FRACPORT

    SciTech Connect

    DeAngelis, D.L.; Yeh, G.T.; Huff, D.D.

    1984-10-01

    This report documents a model, FRACPORT, that simulates the transport of a solute through a fractured porous matrix. The model should be useful in analyzing the possible transport of radionuclides from shallow-land burial sites in humid environments. The use of the model is restricted to transport through saturated zones. The report first discusses the general modeling approach used, which is based on the Integrated Compartmental Method. The basic equations of solute transport are then presented. The model, which assumes a known water velocity field, solves these equations on two different time scales; one related to rapid transport of solute along fractures and the other related to slower transport through the porous matrix. FRACPORT is validated by application to a simple example of fractured porous medium transport that has previously been analyzed by other methods. Then its utility is demonstrated in analyzing more complex cases of pulses of solute into a fractured matrix. The report serves as a user's guide to FRACPORT. A detailed description of data input, along with a listing of input for a sample problem, is provided. 16 references, 18 figures, 3 tables.

  17. Proposing a Compartmental Model for Leprosy and Parameterizing Using Regional Incidence in Brazil

    PubMed Central

    2016-01-01

    Hansen’s disease (HD), or leprosy, is still considered a public health risk in much of Brazil. Understanding the dynamics of the infection at a regional level can aid in identification of targets to improve control. A compartmental continuous-time model for leprosy dynamics was designed based on understanding of the biology of the infection. The transmission coefficients for the model and the rate of detection were fit for each region using Approximate Bayesian Computation applied to paucibacillary and multibacillary incidence data over the period of 2000 to 2010, and model fit was validated on incidence data from 2011 to 2012. Regional variation was noted in detection rate, with cases in the Midwest estimated to be infectious for 10 years prior to detection compared to 5 years for most other regions. Posterior predictions for the model estimated that elimination of leprosy as a public health risk would require, on average, 44–45 years in the three regions with the highest prevalence. The model is easily adaptable to other settings, and can be studied to determine the efficacy of improved case finding on leprosy control. PMID:27532862

  18. Some Formal Approaches to the Analysis of Kinetic Data in Terms of Linear Compartmental Systems

    PubMed Central

    Berman, Mones; Shahn, Ezra; Weiss, Marjory F.

    1962-01-01

    A formal approach to the routine analysis of kinetic data in terms of linear compartmental systems is presented. The methods of analysis are general in that they include much of the theory in common use, such as direct solution of differential equations, integral equations, transfer functions, fitting of data to sums of exponentials, matrix solutions, etc. The key to the formalism presented lies in the fact that a basic operational unit—called “compartment”—has been defined, in terms of which physical and mathematical models as well as input and output functions can be expressed. Additional features for calculating linear combinations of functions and for setting linear dependence relations between parameters add to the versatility of this method. The actual computations for the values of model parameters to yield a least squares fit of the data are performed on a digital computer. A general computer program was developed that permits the routine fitting of data and the evolution of models. PMID:13867976

  19. Systems infection biology: a compartmentalized immune network of pig spleen challenged with Haemophilus parasuis

    PubMed Central

    2013-01-01

    Background Network biology (systems biology) approaches are useful tools for elucidating the host infection processes that often accompany complex immune networks. Although many studies have recently focused on Haemophilus parasuis, a model of Gram-negative bacterium, little attention has been paid to the host's immune response to infection. In this article, we use network biology to investigate infection with Haemophilus parasuis in an in vivo pig model. Results By targeting the spleen immunogenome, we established an expression signature indicative of H. parasuis infection using a PCA/GSEA combined method. We reconstructed the immune network and estimated the network topology parameters that characterize the immunogene expressions in response to H. parasuis infection. The results showed that the immune network of H. parasuis infection is compartmentalized (not globally linked). Statistical analysis revealed that the reconstructed network is scale-free but not small-world. Based on the quantitative topological prioritization, we inferred that the C1R-centered clique might play a vital role in responding to H. parasuis infection. Conclusions Here, we provide the first report of reconstruction of the immune network in H. parasuis-infected porcine spleen. The distinguishing feature of our work is the focus on utilizing the immunogenome for a network biology-oriented analysis. Our findings complement and extend the frontiers of knowledge of host infection biology for H. parasuis and also provide a new clue for systems infection biology of Gram-negative bacilli in mammals. PMID:23339624

  20. Magnetic resonance imaging demonstrates compartmental muscle mechanisms of human vertical fusional vergence

    PubMed Central

    Clark, Robert A.

    2015-01-01

    Vertical fusional vergence (VFV) normally compensates for slight vertical heterophorias. We employed magnetic resonance imaging to clarify extraocular muscle contributions to VFV induced by monocular two-prism diopter (1.15°) base-up prism in 14 normal adults. Fusion during prism viewing requires monocular infraduction. Scans were repeated without prism, and with prism shifted contralaterally. Contractility indicated by morphometric indexes was separately analyzed in medial and lateral vertical rectus and superior oblique (SO) putative compartments, and superior and inferior horizontal rectus extraocular muscle putative compartments, but in the whole inferior oblique (IO). Images confirmed appropriate VFV that was implemented by the inferior rectus (IR) medial compartment contracting ipsilateral and relaxing contralateral to prism. There was no significant contractility in the IR lateral compartment. The superior but not inferior lateral rectus (LR) compartment contracted significantly in the prism viewing eye, but not contralateral to prism. The IO contracted ipsilateral but not contralateral to the prism. In the infraducting eye, the SO medial compartment relaxed significantly, while the lateral compartment was unchanged; contralateral to prism, the SO lateral compartment contracted, while the medial compartment was unchanged. There was no contractility in the superior or medial rectus muscles in either eye. There was no globe retraction. We conclude that the vertical component of VFV is primarily implemented by IR medial compartment contraction. Since appropriate vertical rotation is not directly implemented, or is opposed, by associated differential LR and SO compartmental activity, and IO contraction, these actions probably implement a torsional component of VFV. PMID:25589593

  1. Origin and evolution of metabolic sub-cellular compartmentalization in eukaryotes.

    PubMed

    Gabaldón, Toni; Pittis, Alexandros A

    2015-12-01

    A high level of subcellular compartmentalization is a hallmark of eukaryotic cells. This intricate internal organization was present already in the common ancestor of all extant eukaryotes, and the determination of the origins and early evolution of the different organelles remains largely elusive. Organellar proteomes are determined through regulated pathways that target proteins produced in the cytosol to their final subcellular destinations. This internal sorting of proteins can vary across different physiological conditions, cell types and lineages. Evolutionary retargeting - the alteration of a subcellular localization of a protein in the course of evolution - has been rampant in eukaryotes and involves any possible combination of organelles. This fact adds another layer of difficulty to the reconstruction of the origins and evolution of organelles. In this review we discuss current themes in relation to the origin and evolution of organellar proteomes. Throughout the text, a special focus is set on the evolution of mitochondrial and peroxisomal proteomes, which are two organelles for which extensive proteomic and evolutionary studies have been performed.

  2. A Compartmental Model for Zika Virus with Dynamic Human and Vector Populations

    PubMed Central

    Lee, Eva K; Liu, Yifan; Pietz, Ferdinand H

    2016-01-01

    The Zika virus (ZIKV) outbreak in South American countries and its potential association with microcephaly in newborns and Guillain-Barré Syndrome led the World Health Organization to declare a Public Health Emergency of International Concern. To understand the ZIKV disease dynamics and evaluate the effectiveness of different containment strategies, we propose a compartmental model with a vector-host structure for ZIKV. The model utilizes logistic growth in human population and dynamic growth in vector population. Using this model, we derive the basic reproduction number to gain insight on containment strategies. We contrast the impact and influence of different parameters on the virus trend and outbreak spread. We also evaluate different containment strategies and their combination effects to achieve early containment by minimizing total infections. This result can help decision makers select and invest in the strategies most effective to combat the infection spread. The decision-support tool demonstrates the importance of “digital disease surveillance” in response to waves of epidemics including ZIKV, Dengue, Ebola and cholera. PMID:28269870

  3. The SPECT/CT Evaluation of Compartmental Changes after Open Wedge High Tibial Osteotomy

    PubMed Central

    Kim, Tae Won; Kim, Byung Kag; Kim, Dong Whan; Sim, Jae Ang; Lee, Beom Koo; Lee, Yong Seuk

    2016-01-01

    Purpose The purpose of this study was to evaluate compartmental changes using combined single-photon emission computerized tomography and conventional computerized tomography (SPECT/CT) after open wedge high tibial osteotomy (OWHTO) for providing clinical guidance for proper correction. Materials and Methods Analysis was performed using SPECT/CT from around 1 year after surgery on 22 patients who underwent OWHTO. Postoperative mechanical axis was measured and classified into 3 groups: group I (varus), group II (0°–3° valgus), and group III (>3° valgus). Patella location was evaluated using Blackburne-Peel (BP) ratio. On SPECT/CT, the knee joint was divided into medial, lateral, and patellofemoral compartments and the brighter signal was marked as a positive signal. Results Increased signal activity in the medial compartment was observed in 12 cases. No correlation was observed between postoperative mechanical axis and medial signal increase. Lateral increased signal activity was observed in 3 cases, and as valgus degree increased, lateral compartment’s signal activity increased. Increased signal activity of the patellofemoral joint was observed in 7 cases, and significant correlation was observed between changes in BP ratio and increased signal activity. Conclusions For the treatment of medial osteoarthritis, OWHTO requires overcorrection that does not exceed 3 valgus. In addition, the possibility of a patellofemoral joint problem after OWHTO should be kept in mind. PMID:27894172

  4. Dynamin regulates metaphase furrow formation and plasma membrane compartmentalization in the syncytial Drosophila embryo

    PubMed Central

    Rikhy, Richa; Mavrakis, Manos; Lippincott-Schwartz, Jennifer

    2015-01-01

    ABSTRACT The successive nuclear division cycles in the syncytial Drosophila embryo are accompanied by ingression and regression of plasma membrane furrows, which surround individual nuclei at the embryo periphery, playing a central role in embryo compartmentalization prior to cellularization. Here, we demonstrate that cell cycle changes in dynamin localization and activity at the plasma membrane (PM) regulate metaphase furrow formation and PM organization in the syncytial embryo. Dynamin was localized on short PM furrows during interphase, mediating endocytosis of PM components. Dynamin redistributed off ingressed PM furrows in metaphase, correlating with stabilized PM components and the associated actin regulatory machinery on long furrows. Acute inhibition of dynamin in the temperature sensitive shibire mutant embryo resulted in morphogenetic consequences in the syncytial division cycle. These included inhibition of metaphase furrow ingression, randomization of proteins normally polarized to intercap PM and disruption of the diffusion barrier separating PM domains above nuclei. Based on these findings, we propose that cell cycle changes in dynamin orchestrate recruitment of actin regulatory machinery for PM furrow dynamics during the early mitotic cycles in the Drosophila embryo. PMID:25661871

  5. Modeling Heterogeneity in Direct Infectious Disease Transmission in a Compartmental Model

    PubMed Central

    Kong, Lingcai; Wang, Jinfeng; Han, Weiguo; Cao, Zhidong

    2016-01-01

    Mathematical models have been used to understand the transmission dynamics of infectious diseases and to assess the impact of intervention strategies. Traditional mathematical models usually assume a homogeneous mixing in the population, which is rarely the case in reality. Here, we construct a new transmission function by using as the probability density function a negative binomial distribution, and we develop a compartmental model using it to model the heterogeneity of contact rates in the population. We explore the transmission dynamics of the developed model using numerical simulations with different parameter settings, which characterize different levels of heterogeneity. The results show that when the reproductive number, R0, is larger than one, a low level of heterogeneity results in dynamics similar to those predicted by the homogeneous mixing model. As the level of heterogeneity increases, the dynamics become more different. As a test case, we calibrated the model with the case incidence data for severe acute respiratory syndrome (SARS) in Beijing in 2003, and the estimated parameters demonstrated the effectiveness of the control measures taken during that period. PMID:26927140

  6. Fluvial architecture and reservoir compartmentalization in the Oligocene middle Frio Formation of south Texas

    SciTech Connect

    Kerr, D.R.; Jirik, L.A. )

    1990-09-01

    Seeligson, Stratton, and Agua Dulce fields are being studied as part of a Gas Research Institute/Department of Energy/State of Texas cosponsored program designed to develop and test methodologies and technologies for gas reserve growth in conventional reservoirs in mature gas fields. Over the last four decades, each field has produced approximately 2 tcf of gas from middle Frio reservoirs alone. Recent drilling and workover results and reservoir pressure data, however, point to the possibility of additional reserves. Stratigraphic and sedimentologic studies based on well logs and cores indicate that middle Frio reservoirs are architecturally complex. Deposition on an aggrading coastal plain resulted in a continuum of architectural styles that has important implications for reservoir compartmentalization. The middle Frio is composed of sand-rich channel-fill and splay deposits interstratified with floodplain mudstones, all forming part of the Gueydan fluvial system. Relatively slow aggradation resulted in laterally stacked channel systems; whereas more rapid aggradation resulted in vertically stacked channel systems. Laterally stacked sandstone bodies predominate at Seeligson field, leading to separate but potentially leaky reservoir compartments. By contrast, vertically stacked sandstone bodies predominate at Stratton and Agua Dulce fields, favoring more isolated reservoir compartments. Thus, a high potential for reserve growth through the identification of untapped compartments, poorly drained acreage, and bypassed zones exists for each of these fields, but differences in reservoir architecture must be taken into account as part of exploitation strategies.

  7. A Compartmental Model for Zika Virus with Dynamic Human and Vector Populations.

    PubMed

    Lee, Eva K; Liu, Yifan; Pietz, Ferdinand H

    2016-01-01

    The Zika virus (ZIKV) outbreak in South American countries and its potential association with microcephaly in newborns and Guillain-Barré Syndrome led the World Health Organization to declare a Public Health Emergency of International Concern. To understand the ZIKV disease dynamics and evaluate the effectiveness of different containment strategies, we propose a compartmental model with a vector-host structure for ZIKV. The model utilizes logistic growth in human population and dynamic growth in vector population. Using this model, we derive the basic reproduction number to gain insight on containment strategies. We contrast the impact and influence of different parameters on the virus trend and outbreak spread. We also evaluate different containment strategies and their combination effects to achieve early containment by minimizing total infections. This result can help decision makers select and invest in the strategies most effective to combat the infection spread. The decision-support tool demonstrates the importance of "digital disease surveillance" in response to waves of epidemics including ZIKV, Dengue, Ebola and cholera.

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