bluuebunny/crossref_metadata_2025 huggingface dataset:
Stimulation of Na,K-ATPase by low potassium requires reactive oxygen species
Authors: Zhou Xiaoming , Yin Wu , Doi Sonia Q. , Robinson Shawn W. , Takeyasu Kunio , Fan Xuetao
Published: May 2013

The signaling pathway that transduces the stimulatory effect of low K+ on the biosynthesis of Na,K-ATPase remains largely unknown. The present study was undertaken to examine whether reactive oxygen species (ROS) mediated the effect of low K+ in Madin-Darby canine kidney (MDCK) cells. Low K+ increased ROS activity in a time- and dose-dependent manner, and this effect was abrogated by catalase and N-acetylcysteine (NAC). To determine the role of ROS in low-K+-induced gene expression, the cells were first stably transfected with expression constructs in which the reporter gene chloramphenicol acetyl transferase (CAT) was under the control of the avian Na,K-ATPase α-subunit 1.9 kb and 900-bp 5′-flanking regions that have a negative regulatory element. Low K+ increased the CAT expression in both constructs. Catalase or NAC inhibited the effect of low K+. To determine whether the increased CAT activity was mediated through releasing the repressive effect or a direct stimulation of the promoter, the cells were transfected with a CAT expression construct directed by a 96-bp promoter fragment that has no negative regulatory element. Low K+ also augmented the CAT activity expressed by this construct. More importantly, both catalase and NAC abolished the effect of low K+. Moreover, catalase and NAC also inhibited low-K+-induced increases in the Na,K-ATPase α1- and β1-subunit protein abundance and ouabain binding sites. The antioxidants had no significant effect on the basal levels of CAT activity, protein abundance, or ouabain binding sites. In conclusion, low K+ enhances the Na,K-ATPase gene expression by a direct stimulation of the promoter activity, and ROS mediate this stimulation and also low-K+-induced increases in the Na,K-ATPase protein contents and cell surface molecules.
Cyclosporin A-induced hair growth in mice is associated with inhibition of calcineurin-dependent activation of NFAT in follicular keratinocytes
Authors: Gafter-Gvili Anat , Sredni Benjamin , Gal Rivka , Gafter Uzi , Kalechman Yona
Published: May 2013

One of the most common side effects of treatment with cyclosporin A (CsA) is hypertrichosis. This study shows that calcineurin activity is associated with hair keratinocyte differentiation in vivo, affecting nuclear factor of activated T cells (NFAT1) activity in these cells. Treatment of nude or C57BL/6 depilated normal mice with CsA inhibited the expression of keratinocyte terminal differentiation markers associated with catagen, along with the inhibition of calcineurin and NFAT1 nuclear translocation. This was associated with induction of hair growth in nude mice and retardation of spontaneous catagen induction in depilated normal mice. Furthermore, calcineurin inhibition blocked the expression of p21waf/cip1 and p27kip1, which are usually induced with differentiation. This was also associated with an increase in interleukin-1α expression (nude mice), a decrease in transforming growth factor-β (nude and normal mice), and no change in keratinocyte growth factor expression in the skin. Retardation of catagen in CsA-treated mice was accompanied by significant alterations in apoptosis-related gene product expression in hair follicle keratinocytes. The ratio of the anti-apoptotic Bcl-2 to proapoptotic Bax expression increased, and expression of p53 and interleukin-1β converting enzyme activity decreased. These data provide the first evidence that calcineurin is functionally active in follicular keratinocytes and that inhibition of the calcineurin-NFAT1 pathway in these cells in vivo by CsA enhances hair growth.
Rab5a and rab11a mediate agonist-induced trafficking of protease-activated receptor 2
Authors: Roosterman Dirk , Schmidlin Fabien , Bunnett Nigel W.
Published: May 2013

We evaluated the contribution of rab5a and rab11a to trafficking and signaling of protease-activated receptor 2 (PAR2), a receptor for trypsin and tryptase. Agonists stimulated internalization of PAR2 into early endosomes containing rab5a. Dominant negative rab5aS34N disrupted early endosomes and inhibited agonist-stimulated endocytosis of PAR2. Internalized PAR2 was sorted to lysosomes, and rab5a remained in early endosomes. Rab5a promoted and rab5aS34N impeded resensitization of trypsin-induced calcium mobilization. Rab11a was detected in the Golgi apparatus with PAR2, and PAR2 agonists stimulated redistribution of rab11a into vesicles containing PAR2 that migrated to the cell surface. Dominant negative rab11aS25N was mostly confined to the Golgi apparatus. Although expression of rab11aS25N caused retention of PAR2 in the Golgi apparatus, it did not abolish trafficking of PAR2 to the cell surface. However, expression of wild-type rab11a accelerated both recovery of PAR2 at the cell surface and resensitization of PAR2 signaling. Thus rab5a is required for PAR2 endocytosis and resensitization, whereas rab11a contributes to trafficking of PAR2 from the Golgi apparatus to the plasma membrane.
Alternative splicing yields novel BMAL2 variants: tissue distribution and functional characterization
Authors: Schoenhard John A. , Eren Mesut , Johnson Carl H. , Vaughan Douglas E.
Published: May 2013

The BMAL2 gene encodes a member of the basic helix-loop-helix PER-ARNT-SIM family of transcription factors, which control diverse physiological processes including circadian rhythms. We identified four novel human BMAL2 transcripts that differ by alternative splicing within their NH2-terminal regions. Divergent expression of these and previously reported transcripts was observed among human tissues. The functional consequences of alternative splicing for transcriptional activation by CLOCK:BMAL2 heterodimers were assessed using luciferase reporter gene constructs that contained one of three diurnally regulated promoters, namely, those of the mouse period1, mouse vasopressin, and human plasminogen activator inhibitor-1 genes. These studies revealed that alternative splicing generates BMAL2 isoforms possessing high, medium, low, or no transcriptional activity. Similar results were obtained with each promoter, suggesting that alternative splicing may influence the amplitudes of both central and peripheral oscillators. Indeed, alternative splicing of BMAL2 may provide tissues with a rheostat capable of regulating CLOCK:BMAL2 heterodimer function across a broad continuum of potential transcriptional activities to accommodate varied metabolic demands and physiological roles.
Molecular cloning and characterization of a rat sensory nerve Ca2+-sensing receptor
Authors: Wang Yanlin , Awumey Emmanuel K. , Chatterjee Pradeep K. , Somasundaram Chandra , Bian Ka , Rogers Kimberly V. , Dunn Christine , Bukoski Richard D.
Published: May 2013

A full-length cDNA encoding a Ca2+-sensing receptor (CaSR) expressed in rat dorsal root ganglia (DRG) was identified using rapid amplification of 5′-cDNA ends and primer extension and then cloned into the plasmid vector pCR3.1. The DNA sequence of the DRG CaSR was 99.9% homologous with published rat kidney CaSR in the coding region and 247 bp upstream of the start site but showed little homology 5′ to this site, which maps to exonic junction I/II, supporting the hypothesis that CaSR message arises as a splice variant and showing tissue-to-tissue heterogeneity. Western blot revealed a doublet of 140 and 160 kDa in a thyroparathyroid preparation and a single 140-kDa band in DRG. Deglycosylation using N-glycanase increased the mobility of CaSR protein from both DRG and thyroparathyroid, whereas endo-H was without effect, indicating that the DGR CaSR is a mature form of the receptor. A DRG CaSR-pEGFP fusion product was constructed, and when transfected into HEK-293 cells, it was distributed at the cell membrane and resulted in extracellular Ca2+ (0.5–3 mM)-evoked increases in intracellular Ca2+, which in some instances exhibited oscillatory behavior. We conclude that DRG CaSR cDNA arises from tissue-specific alternative splicing of a single gene, that the amino acid sequence of DRG CaSR is homologous to other known CaSRs, and that the DRG CaSR undergoes differential posttranslational processing relative to the thyroparathyroid CaSR and is functionally active when transfected into a human-derived cell line.
A sulfated proteoglycan is necessary for storage of exocrine secretory proteins in the rat parotid gland
Authors: Venkatesh S. G. , Gorr S.-U.
Published: May 2013

Sulfated proteoglycans have been proposed to play a role in the sorting and storage of secretory proteins in exocrine secretory granules. Rat parotid acinar cells expressed a 40- to 60-kDa proteoglycan that was stored in secretory granules. Treatment of the tissue with the proteoglycan synthesis inhibitor paranitrophenyl xyloside resulted in the complete abrogation of the sulfated proteoglycan. Pulse-chase experiments in the presence of the xyloside analog showed a significant reduction in the stimulated secretion and granule storage of the newly synthesized regulated secretory proteins amylase and parotid secretory protein. Inhibition of proteoglycan sulfation by chlorate did not affect the sorting of these proteins. The effect of proteoglycan synthesis inhibition on protein sorting was completely reversed upon treatment with a weak acid. These results suggest that the sulfated proteoglycan is necessary for sorting and storage of regulated secretory proteins in the exocrine parotid gland. Preliminary evidence suggests that the mechanism involves the modulation of granule pH by the proteoglycan rather than a direct interaction with other granule components.
The effect of changes in ambient oxygen concentration on the bioelectric properties of middle ear mucosa
Authors: Mandell David L. , Devor Daniel C. , Madia Joseph V. , Lo Chia-Yee , Hake Hilary , Hebda Patricia A.
Published: May 2013

The purpose of the present study was to compare the effect of 24 h of exposure to 7% O2 (normal middle ear physiological conditions) vs. 21% O2 (found in the middle ear after ventilation tube placement) on transepithelial Na+ absorption and Cl– secretion in cultured gerbil middle ear epithelial cell monolayers. Although no difference in apical Na+ absorption was identified, the UTP-induced stimulation of apical Cl– secretion in the presence of apical Na+ channel blockade with amiloride was significantly enhanced after exposure to 21% O2 compared with 7% O2 exposure. In the presence of a calcium-activated Cl– channel inhibitor, DIDS, UTP-induced stimulation of Cl– secretion after 21% O2 exposure was decreased, suggesting a role for calcium-activated Cl– channels in middle ear Cl– secretion in response to relative hyperoxia.
Three-dimensional tissue structure affects sensitivity of fibroblasts to TGF-β1
Authors: Kunz-Schughart Leoni A. , Wenninger Sabine , Neumeier Thomas , Seidl Paula , Knuechel Ruth
Published: May 2013

Transforming growth factor-β (TGF-β) is known to induce α-smooth muscle actin (α-SMA) in fibroblasts and is supposed to play a role in myofibroblast differentiation and tumor desmoplasia. Our objective was to elucidate the impact of TGF-β1 on α-SMA expression in fibroblasts in a three-dimensional (3-D) vs. two-dimensional (2-D) environment. In monolayer culture, all fibroblast cultures responded in a similar fashion to TGF-β1 with regard to α-SMA expression. In fibroblast spheroids, α-SMA expression was reduced and induction by TGF-β1 was highly variable. This difference correlated with a differential regulation in the TGF-β receptor (TGFβR) expression, in particular with a reduction in TGF-βRII in part of the fibroblast types. Our data indicate that 1) sensitivity to TGF-β1-induced α-SMA expression in a 3-D environment is fibroblast-type specific, 2) fibroblast type-independent regulatory mechanisms, such as a general reduction/loss in TGF-βRIII, contribute to an altered TGFβR expression profile in spheroid compared with monolayer culture, and 3) fibroblast type-specific alterations in TGFβR types I and II determine the sensitivity to TGF-β1-induced α-SMA expression in the 3-D setting. We suggest that fibroblasts that can be induced by TGF-β1 to produce α-SMA in spheroid culture reflect a “premyofibroblastic” phenotype.
Differential expression and distribution of Kir5.1 and Kir4.1 inwardly rectifying K+ channels in retina
Authors: Ishii Masaru , Fujita Akikazu , Iwai Kaori , Kusaka Shunji , Higashi Kayoko , Inanobe Atsushi , Hibino Hiroshi , Kurachi Yoshihisa
Published: May 2013

Kir5.1 is an inwardly rectifying K+ channel subunit whose functional role has not been fully elucidated. Expression and distribution of Kir5.1 in retina were examined with a specific polyclonal antibody. Kir5.1 immunoreactivity was detected in glial Müller cells and in some retinal neurons. In the Kir5.1-positive neurons the expression of glutamic acid decarboxylase (GAD65) was detected, suggesting that they may be GABAergic-amacrine cells. In Müller cells, spots of Kir5.1 immunoreactivity distributed diffusely at the cell body and in the distal portions, where Kir4.1 immunoreactivity largely overlapped. In addition, Kir4.1 immunoreactivity without Kir5.1 was strongly concentrated at the endfoot of Müller cells facing the vitreous surface or in the processes surrounding vessels. The immunoprecipitant obtained from retina with anti-Kir4.1 antibody contained Kir5.1. These results suggest that heterotetrameric Kir4.1/Kir5.1 channels may exist in the cell body and distal portion of Müller cells, whereas homomeric Kir4.1 channels are clustered in the endfeet and surrounding vessels. It is possible that homomeric Kir4.1 and heteromeric Kir4.1/Kir5.1 channels play different functional roles in the K+-buffering action of Müller cells.
Partial inhibition of SERCA is responsible for extracellular Ca2+ dependence of AlF–4-induced [Ca2+]i oscillations in rat pancreatic
Authors: Chong Seon Ah , Hong Soo Young , Moon Seok Jun , Park Jee Won , Hong Jeong-Hee , An Jeong Mi , Lee Syng-Ill , Shin Dong Min , Seo Jeong Taeg
Published: May 2013

AlF4-is known to generate oscillations in intracellular Ca2+ concentration ([Ca2+]i) by activating G proteins in many cell types. However, in rat pancreatic acinar cells, AlF4--evoked [Ca2+]i oscillations were reported to be dependent on extracellular Ca2+, which contrasts with the [Ca2+]i oscillations induced by cholecystokinin (CCK). Therefore, we investigated the mechanisms by which AlF4- generates extracellular Ca2+-dependent [Ca2+]i oscillations in rat pancreatic acinar cells. AlF4--induced [Ca2+]i oscillations were stopped rapidly by the removal of extracellular Ca2+ and were abolished on the addition of 20 mM caffeine and 2 μM thapsigargin, indicating that Ca2+ influx plays a crucial role in maintenance of the oscillations and that an inositol 1,4,5-trisphosphate-sensitive Ca2+ store is also required. The amount of Ca2+ in the intracellular Ca2+ store was decreased as the AlF4--induced [Ca2+]i oscillations continued. Measurement of 45Ca2+ influx into isolated microsomes revealed that AlF4-directly inhibited sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). The activity of plasma membrane Ca2+-ATPase during AlF4- stimulation was not significantly different from that during CCK stimulation. After partial inhibition of SERCA with 1 nM thapsigargin, 20 pM CCK-evoked [Ca2+]i oscillations were dependent on extracellular Ca2+. This study shows that AlF4- induces [Ca2+]i oscillations, probably by inositol 1,4,5-trisphosphate production via G protein activation but that these oscillations are strongly dependent on extracellular Ca2+ as a result of the partial inhibition of SERCA.
MHC-based fiber type and E-C coupling characteristics in mechanically skinned muscle fibers of the rat
Authors: Goodman Craig , Patterson Michael , Stephenson Gabriela
Published: May 2013

In this study, we investigated whether the previously established differences between fast- and slow-twitch single skeletal muscle fibers of the rat, in terms of myosin heavy chain (MHC) isoform composition and contractile function, are also detectable in excitation-contraction (E-C) coupling. We compared the contractile responsiveness of electrophoretically typed, mechanically skinned single fibers from the soleus (Sol), the extensor digitorum longus (EDL), and the white region of the sternomastoid (SM) muscle to t-system depolarization-induced activation. The quantitative parameters assessed were the amplitude of the maximum depolarization-induced force response (DIFRmax; normalized to the maximum Ca2+-activated force in that fiber) and the number of responses elicited until the force declined by 75% of DIFRmax (R-D75%). The mean DIFRmaxvalues for type IIB EDL and type IIB SM fibers were not statistically different, and both were greater than the mean DIFRmax for type I Sol fibers. The mean R-D75% for type IIB EDL fibers was greater than that for type I Sol fibers as well as type IIB SM fibers. These data suggest that E-C coupling characteristics of mechanically skinned rat single muscle fibers are related to MHC-based fiber type and the muscle of origin.
Expression of fatty acid binding proteins inhibits lipid accumulation and alters toxicity in L cell fibroblasts
Authors: Atshaves Barbara P. , Storey Stephen M. , Petrescu Anca , Greenberg Cynthia C. , Lyuksyutova Olga I. , Smith Roger , Schroeder Friedhelm
Published: May 2013

High levels of saturated, branched-chain fatty acids are deleterious to cells and animals, resulting in lipid accumulation and cytotoxicity. Although fatty acid binding proteins (FABPs) are thought to be protective, this hypothesis has not previously been examined. Phytanic acid (branched chain, 16-carbon backbone) induced lipid accumulation in L cell fibroblasts similar to that observed with palmitic acid (unbranched, C16): triacylglycerol ≫ free fatty acid > cholesterol > cholesteryl ester ≫ phospholipid. Although expression of sterol carrier protein (SCP)-2, SCP-x, or liver FABP (L-FABP) in transfected L cells reduced [3H]phytanic acid uptake (57–87%) and lipid accumulation (21–27%), nevertheless [3H]phytanic acid oxidation was inhibited (74–100%) and phytanic acid toxicity was enhanced in the order L-FABP ≫ SCP-x > SCP-2. These effects differed markedly from those of [3H]palmitic acid, whose uptake, oxidation, and induction of lipid accumulation were not reduced by L-FABP, SCP-2, or SCP-x expression. Furthermore, these proteins did not enhance the cytotoxicity of palmitic acid. In summary, intracellular FABPs reduce lipid accumulation induced by high levels of branched-chain but not straight-chain saturated fatty acids. These beneficial effects were offset by inhibition of branched-chain fatty acid oxidation that correlated with the enhanced toxicity of high levels of branched-chain fatty acid.
Control of ascorbic acid efflux in rat luteal cells: role of intracellular calcium and oxygen radicals
Authors: Pepperell John R. , Porterfield D. Marshall , Keefe David L. , Behrman Harold R. , Smith Peter J. S.
Published: May 2013

In luteal cells, prostaglandin (PG)F2a mobilizes intracellular calcium concentration ([Ca]i), generates reactive oxygen species (ROS), depletes ascorbic acid (AA) levels, inhibits steroidogenesis, and ultimately induces cell death. We investigated the hypothesis that [Ca]i mobilization stimulates ROS, which results in depletion of cellular AA in rat luteal cells. We used a self-referencing AA-selective electrode that noninvasively measures AA flux at the extended boundary layer of single cells and fluorescence microscopy with fura 2 and dichlorofluorescein diacetate (DCF-DA) to measure [Ca]i and ROS, respectively. Menadione, a generator of intracellular superoxide radical ([Formula: see text]), PGF2a, and calcium ionophore were shown to increase [Ca]i and stimulate intracellular ROS. With calcium ionophore and PGF2a, but not menadione, the generation of ROS was dependent on extracellular calcium influx. In unstimulated cells there was a net efflux of AA of 121.5 ± 20.3 fmol · cm–1 · s–1 (mean ± SE, n = 8), but in the absence of extracellular calcium the efflux was significantly reduced (10.3 ± 4.9 fmol · cm–1 · s–1; n = 5, P < 0.05). PGF2a and menadione stimulated AA efflux, but calcium ionophore had no significant effect. These data suggest two AA regulatory mechanisms: Under basal conditions, AA efflux is calcium dependent and may represent recycling and maintenance of an antioxidant AA gradient at the plasma membrane. Under luteolytic hormone and/or oxidative stress, AA efflux is stimulated that is independent of extracellular calcium influx or generation of ROS. Although site-specific mobilization of calcium pools and ROS cannot be ruled out, the release of AA by PGF2a-stimulated luteal cells may occur through other signaling pathways.
Human nongastric H+-K+-ATPase: transport properties of ATP1al1 assembled with different β-subunits
Authors: Crambert Gilles , Horisberger Jean-Daniel , Modyanov Nikolai N. , Geering Käthi
Published: May 2013

To investigate whether nongastric H+-K+-ATPases transport Na+ in exchange for K+ and whether different β-isoforms influence their transport properties, we compared the functional properties of the catalytic subunit of human nongastric H+-K+-ATPase, ATP1al1 (AL1), and of the Na+-K+-ATPase α1-subunit (α1) expressed in Xenopus oocytes, with different β-subunits. Our results show that βHK and β1-NK can produce functional AL1/β complexes at the oocyte cell surface that, in contrast to α1/β1 NK and α1/βHK complexes, exhibit a similar apparent K+ affinity. Similar to Na+-K+-ATPase, AL1/β complexes are able to decrease intracellular Na+ concentrations in Na+-loaded oocytes, and their K+ transport depends on intra- and extracellular Na+ concentrations. Finally, controlled trypsinolysis reveals that β-isoforms influence the protease sensitivity of AL1 and α1 and that AL1/β complexes, similar to the Na+-K+-ATPase, can undergo distinct K+-Na+- and ouabain-dependent conformational changes. These results provide new evidence that the human nongastric H+-K+-ATPase interacts with and transports Na+ in exchange for K+ and that β-isoforms have a distinct effect on the overall structural integrity of AL1 but influence its transport properties less than those of the Na+-K+-ATPase α-subunit.
Cytochrome c activates K+ channels before inducing apoptosis
Authors: Platoshyn Oleksandr , Zhang Shen , McDaniel Sharon S. , Yuan Jason X.-J.
Published: May 2013

Cell shrinkage is an early prerequisite for apoptosis. The apoptotic volume decrease is due primarily to loss of cytoplasmic ions. Increased outward K+ currents have indeed been implicated in the early stage of apoptosis in many cell types. We found that cytoplasmic dialysis of cytochrome c(cyt- c), a mitochondria-dependent apoptotic inducer, increases K+ currents before inducing nuclear condensation and breakage in pulmonary vascular smooth muscle cells. The cyt- c-mediated increase in K+ currents took place rapidly and was not affected by treatment with a specific inhibitor of caspase-9. Cytoplasmic dialysis of recombinant (active) caspase-9 negligibly affected the K+ currents. Furthermore, treatment of the cells with staurosporine (ST), an apoptosis inducer that mediates translocation of cyt- c from mitochondria to the cytosol, also increased K+ currents, caused cell shrinkage, and induced apoptosis (determined by apoptotic nuclear morphology and TdT-UTP nick end labeling assay). The staurosporine-induced increase in K+ currents concurred to the volume decrease but preceded the activation of apoptosis (nuclear condensation and breakage). These results suggest that the cyt- c-induced activation of K+ channels and the resultant K+ loss play an important role in initiating the apoptotic volume decrease when cells undergo apoptosis.
Ion channels in secretory granules of the pancreas and their role in exocytosis and release of secretory proteins
Authors: Thévenod Frank
Published: May 2013

Regulated secretion in exocrine and neuroendocrine cells occurs through exocytosis of secretory granules and the subsequent release of stored small molecules and proteins. The introduction of biophysical techniques with high temporal and spatial resolution, and the identification of Ca2+-dependent and -independent “docking” and “fusion” proteins, has greatly enhanced our understanding of exocytosis. The cloning of families of ion channel proteins, including intracellular ion channels, has also revived interest in the role of secretory granule ion channels in exocytotic secretion. Thus secretory granules of pancreatic acinar cell express a ClC-2 Cl−channel, a HCO[Formula: see text]-permeable member of the CLCA Ca2+-dependent anion channel family, and a KCNQ1 K+ channel. Evidence suggests that these channels may facilitate the release of digestive enzymes and/or prevent exocytosed granules from collapsing during “kiss and run” recycling. In pancreatic β-cells, a granular ClC-3 Cl− channel provides a shunt pathway for a vacuolar-type H+-ATPase. Acidification “primes” the granules for Ca2+-dependent exocytosis and release of insulin. In summary, secretory granules are equipped with specific sets of ion channels, which modulate regulated exocytosis and the release of macromolecules. These channels could represent excellent targets for therapeutic interventions to control exocytotic secretion in relevant diseases, such as pancreatitis, cystic fibrosis, or diabetes mellitus.
Kinetic evidence that the Na-PO4 cotransporter is the molecular mechanism for Na/Li exchange in human red blood cells
Authors: Elmariah Sammy , Gunn Robert B.
Published: May 2013

The molecular basis for Na/Li exchange is unknown. Li can be transported by the Na pump, anion exchanger (AE1), a background leak, and the Na/Li exchanger. In vivo the intraerythrocyte concentration of Li results from the balance of passive entry, mostly on AE1, and the active extrusion on the Na/Li exchanger. Here we show that erythrocytes have Li-activated PO4 transport that behaves as if it is mediated by the Na-PO4 cotransporter (hBNP1) and provide evidence that this Na/Li-PO4 cotransporter is also the mechanism for Na/Li exchange. First, external Li (>20 mM) activated PO4 influx severalfold. Li activation of PO4 influx was potentiated by the presence of external Na. Second, the ouabain-insensitive 22Na efflux was stimulated by external Li and then inhibited by external PO4. Third, phloretin inhibited Na- and Li-activated PO4 flux with the same Ki, 0.25 mM. Fourth, external PO4 (0.1–1.0 mM) inhibited ouabain-insensitive Li efflux only if external Na was present. Fifth, arsenate, a phosphate congener, inhibited both Na-PO4 cotransport and Li-activated PO4 flux with similar kinetics when Na or Li concentration was high but did not inhibit Liout/Nain exchange when Liout concentration was low. The collective results suggest that both Na and Li are substrates for at least two sites on the same PO4 cotransporter and that Na/Li exchange behaves as if it is mediated by this Na/Li-PO4 cotransporter when only one cation is bound. Plasma and intracellular PO4 concentrations may be important regulators of Li transport and its therapeutic effects.
Intrinsic gating mechanisms of epithelial sodium channels
Authors: Ji Hong-Long , Fuller Catherine M. , Benos Dale J.
Published: May 2013

The hypothesis that there is a highly conserved, positively charged region distal to the second transmembrane domain in α-ENaC (epithelial sodium channel) that acts as a putative receptor site for the negatively charged COOH-terminal β- and γ-ENaC tails was tested in mutagenesis experiments. After expression in Xenopus oocytes, α-ENaC constructs in which positively charged arginine residues were converted into negatively charged glutamic acids could not be inhibited by blocking peptides. These observations provide insight into the gating machinery of ENaC.
IL-1β-induced production of metalloproteinases by synovial cells depends on gap junction conductance
Authors: Kolomytkin Oleg V. , Marino Andrew A. , Waddell David D. , Mathis J. Michael , Wolf Robert E. , Sadasivan Kalia K. , Albright James A.
Published: May 2013

Synovial cells can form networks connected by gap junctions. The purpose of this study was to obtain evidence for a necessary role of gap junction intercellular communication in protein secretion by synovial cells. We developed a novel assay to measure the enzymatic activity of metalloproteinases (MMPs) produced by synovial cells in response to interleukin-1β (IL-1β) and employed the assay to explore the biological function of gap junctions. IL-1β produced a dose-dependent increase in MMP activity that was blocked by exposure to the gap junction inhibitors 18α-glycyrrhetinic acid and octanol for as few as 50 min. The inhibitors produced an immediate and marked reduction in intercellular communication, as assessed by transient current analysis using the nystatin perforated-patch method. These observations suggest that communication through gap junctions early in IL-1β signal transduction is critical to the process of cytokine-regulated secretion of MMPs by synovial cells.
Dependence of Na+-K+ pump current-voltage relationship on intracellular Na+, K+, and Cs+ in rabbit cardiac myocytes
Authors: Hansen Peter S. , Buhagiar Kerrie A. , Kong Benjamin Y. , Clarke Ronald J. , Gray David F. , Rasmussen Helge H.
Published: May 2013

To examine effects of cytosolic Na+, K+, and Cs+ on the voltage dependence of the Na+-K+ pump, we measured Na+-K+ pump current ( I p) of ventricular myocytes voltage-clamped at potentials ( V m) from −100 to +60 mV. Superfusates were designed to eliminate voltage dependence at extracellular pump sites. The cytosolic compartment of myocytes was perfused with patch pipette solutions with a Na+ concentration ([Na]pip) of 80 mM and a K+ concentration from 0 to 80 mM or with solutions containing Na+ in concentrations from 0.1 to 100 mM and K+ in a concentration of either 0 or 80 mM. When [Na]pip was 80 mM, K+ in pipette solutions had a voltage-dependent inhibitory effect on I pand induced a negative slope of the I p- V m relationship. Cs+ in pipette solutions had an effect on I p qualitatively similar to that of K+. Increases in I p with increases in [Na]pip were voltage dependent. The dielectric coefficient derived from [Na]pip- I p relationships at the different test potentials was 0.15 when pipette solutions included 80 mM K+ and 0.06 when pipette solutions were K+free.
Regulation of arterial tone by smooth muscle myosin type II
Authors: Löhn Matthias , Kämpf Dietmar , Gui-Xuan Chai , Haller Hermann , Luft Friedrich C. , Gollasch Maik
Published: May 2013

The initiation of contractile force in arterial smooth muscle (SM) is believed to be regulated by the intracellular Ca2+concentration and SM myosin type II phosphorylation. We tested the hypothesis that SM myosin type II operates as a molecular motor protein in electromechanical, but not in protein kinase C (PKC)-induced, contraction of small resistance-sized cerebral arteries. We utilized a SM type II myosin heavy chain (MHC) knockout mouse model and measured arterial wall Ca2+ concentration ([Ca2+]i) and the diameter of pressurized cerebral arteries (30–100 μm) by means of digital fluorescence video imaging. Intravasal pressure elevation caused a graded [Ca2+]i increase and constricted cerebral arteries of neonatal wild-type mice by 20–30%. In contrast, intravasal pressure elevation caused a graded increase of [Ca2+]i without constriction in (−/−) MHC-deficient arteries. KCl (60 mM) induced a further [Ca2+]i increase but failed to induce vasoconstriction of (−/−) MHC-deficient cerebral arteries. Activation of PKC by phorbol ester (phorbol 12-myristate 13-acetate, 100 nM) induced a strong, sustained constriction of (−/−) MHC-deficient cerebral arteries without changing [Ca2+]i. These results demonstrate a major role for SM type II myosin in the development of myogenic tone and Ca2+-dependent constriction of resistance-sized cerebral arteries. In contrast, the sustained contractile response did not depend on myosin and intracellular Ca2+ but instead depended on PKC. We suggest that SM myosin type II operates as a molecular motor protein in the development of myogenic tone but not in pharmacomechanical coupling by PKC in cerebral arteries. Thus PKC-dependent phosphorylation of cytoskeletal proteins may be responsible for sustained contraction in vascular SM.
Growth factor regulation of uncoupling protein-1 mRNA expression in brown adipocytes
Authors: García Bibian , Obregón Maria-Jesús
Published: May 2013

To study the effect of the mitogens epidermal growth factor (EGF), acidic and basic fibroblast growth factors (aFGF and bFGF), and vasopressin on brown adipocyte differentiation, we analyzed the expression of uncoupling protein-1 (UCP-1) mRNA. Quiescent brown preadipocytes express high levels of UCP-1 mRNA in response to triiodothyronine (T3) and norepinephrine (NE). The addition of serum or the mitogenic condition aFGF + vasopressin + NE or EGF + vasopressin + NE decreases UCP-1 mRNA. A second addition of mitogens further decreases UCP-1 mRNA. Treatment with aFGF or bFGF alone increases UCP-1 mRNA, whereas the addition of EGF or vasopressin dramatically reduces UCP-1 mRNA levels. The continuous presence of T3 increases UCP-1 mRNA levels in cells treated with EGF, aFGF, or bFGF. The effect of T3 on the stimulation of DNA synthesis also was tested. T3 inhibits the mitogenic activity of aFGF and bFGF. In conclusion, mitogens like aFGF or bFGF allow brown adipocyte differentiation, whereas EGF and vasopressin inhibit the differentiation process. T3 behaves as an important hormone that regulates both brown adipocyte proliferation and differentiation.
Adaptive β-cell proliferation increases early in high-fat feeding in mice, concurrent with metabolic changes, with induction of islet cyclin D2 expression
Authors: Stamateris Rachel E. , Sharma Rohit B. , Hollern Douglas A. , Alonso Laura C.
Published: May 2013

Type 2 diabetes (T2D) is caused by relative insulin deficiency, due in part to reduced β-cell mass ( 11 , 62 ). Therapies aimed at expanding β-cell mass may be useful to treat T2D ( 14 ). Although feeding rodents a high-fat diet (HFD) for an extended period (3–6 mo) increases β-cell mass by inducing β-cell proliferation ( 16 , 20 , 53 , 54 ), evidence suggests that adult human β-cells may not meaningfully proliferate in response to obesity. The timing and identity of the earliest initiators of the rodent compensatory growth response, possible therapeutic targets to drive proliferation in refractory human β-cells, are not known. To develop a model to identify early drivers of β-cell proliferation, we studied mice during the first week of HFD exposure, determining the onset of proliferation in the context of diet-related physiological changes. Within the first week of HFD, mice consumed more kilocalories, gained weight and fat mass, and developed hyperglycemia, hyperinsulinemia, and glucose intolerance due to impaired insulin secretion. The β-cell proliferative response also began within the first week of HFD feeding. Intriguingly, β-cell proliferation increased before insulin resistance was detected. Cyclin D2 protein expression was increased in islets by day 7, suggesting it may be an early effector driving compensatory β-cell proliferation in mice. This study defines the time frame and physiology to identify novel upstream regulatory signals driving mouse β-cell mass expansion, in order to explore their efficacy, or reasons for inefficacy, in initiating human β-cell proliferation.
Vitamin A deficiency inhibits intestinal adaptation by modulating apoptosis, proliferation, and enterocyte migration
Authors: Swartz-Basile Deborah A. , Wang Lihua , Tang Yuzhu , Pitt Henry A. , Rubin Deborah C. , Levin Marc S.
Published: May 2013

In a prior study, vitamin A-deficient rats subjected to submassive small bowel resections did not mount a normal intestinal adaptive response by 10 days postoperatively, although adaptive increases in crypt cell proliferation were not attenuated and there were no differences in apoptotic indexes. The present study was designed to address the mechanisms by which vitamin A status effects adaptation by analyzing proliferation, apoptosis, and enterocyte migration in the early postoperative period (16 and 48 h) in vitamin A-sufficient, -deficient, and partially replenished sham-resected and resected rats. At 16 h postresection, apoptosis was significantly greater in the remnant ileum of resected vitamin A-deficient rats compared with the sufficient controls. Crypt cell proliferation was increased by resection in all dietary groups at both timepoints. However, at 48 h postresection, proliferation was significantly decreased in the vitamin A-deficient and partially replenished rats. By 48 h after resection, vitamin A deficiency also reduced enterocyte migration rates by 44%. This occurred in conjunction with decreased immunoreactive collagen IV at 48 h and 10 days postoperation. Laminin expression was also reduced by deficiency at 10 days postresection, whereas fibronectin and pancadherin were unchanged at 48 h and 10 days. These studies indicate that vitamin A deficiency inhibits intestinal adaptation following partial small bowel resection by reducing crypt cell proliferation, by enhancing early crypt cell apoptosis, and by markedly reducing enterocyte migration rates, which may be related to changes in the expression of collagen IV and other extracellular matrix components.
Analysis of Hysteresis-Free Creep of the Stack Piezoelectric Actuator
Authors: Zhao Xueliang , Zhang Chengjin , Liu Hongbo , Zhang Guilin , Li Kang
Published: May 2013

A modified log-type creep model without hysteresis of the stack piezoelectric actuator is presented. For high-speed micro-/nanopositioning system, the time scale should be less than one second for creep modeling and control in the stack piezoelectric actuator. But creep effect was studied in the frame of minutes in previous works. Meanwhile, parameters of the classical creep models are hard to be determined. By the proposed model, the hysteresis and the creep effect can be separated. A series of experiments have been performed, where different staircase voltages have been applied to the actuator. There are two clear rules to follow in small duration and different heights to determine parameters. Firstly,L0starts from fixed point either in ascending stage or in descending stage and rotates clockwise. Secondly,γconverges to a small vicinity of a constant when the duration is small enough.
Differentiation of Mesenchymal Stem Cells from Human Umbilical Cord Tissue into Odontoblast-Like Cells Using the Conditioned Medium of Tooth Germ CellsIn Vitro
Authors: Li Tian Xia , Yuan Jie , Chen Yan , Pan Li Jie , Song Chun , Bi Liang Jia , Jiao Xiao Hui
Published: May 2013

The easily accessible mesenchymal stem cells in the Wharton's jelly of human umbilical cord tissue (hUCMSCs) have excellent proliferation and differentiation potential, but it remains unclear whether hUCMSCs can differentiate into odontoblasts. In this study, mesenchymal stem cells were isolated from the Wharton's jelly of human umbilical cord tissue using the simple method of tissue blocks culture attachment. UCMSC surface marker expression was then evaluated for the isolated cells using flow cytometry. The third-passage hUCMSCs induced by conditioned medium from developing tooth germ cells (TGC-CM) displayed high alkaline phosphatase (ALP) levels (P<0.001), an enhanced ability to proliferate (P<0.05), and the presence of mineralized nodules. These effects were not observed in cells treated with regular medium. After induction of hUCMSCs, the results of reverse transcriptional polymerase chain reaction (PCR) indicated that the dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP1) genes were significantly tested. Additionally, dentin sialoprotein (DSP) and DMP1 demonstrated significant levels of staining in an immunofluorescence analysis. In contrast, the control cells failed to display the characteristics of odontoblasts. Taken together, these results suggest that hUCMSCs can be induced to differentiate into odontoblast-like cells with TGC-CM and provide a novel strategy for tooth regeneration research.
High Efficient Numerical Methods for Viscous and Nonviscous Wave Problems
Authors: Lv Xiujie , Qin Jinggang , Wang Tongke
Published: May 2013

This paper is concerned with accurate and efficient numerical methods for solving viscous and nonviscous wave problems. The paper first introduces a new second-order PR-ADI like scheme. For an efficient simulation, the scheme is also extended to a high-order compact PRADI like method. Both of them have the advantages of unconditional stability, less impact of the perturbing terms on the accuracy, and being convenient to compute the boundary values of the intermediates. Besides this, the compact scheme has high-order accuracy and costs less in computational time. Numerical results are presented to show the accuracy and efficiency of the new algorithms.
The Importance of Gestational Sac Size of Ectopic Pregnancy in Response to Single-Dose Methotrexate
Authors: Kimiaei Parichehr , Khani Zahra , Marefian Azadeh , Gholampour Ghavamabadi Maryam , Salimnejad Maryam
Published: May 2013

This retrospective cohort study was designed in a selective group of 185 patients diagnosed with and treated for ectopic pregnancy. Intramuscular administration of a single dose of methotrexate (50 mg/m2) was performed to measure predictors of failure or resistance to treatment necessitating surgical intervention. During the time of treatment with a single dose of MTX, 20 patients (10.8%) failed to response, in which 6 of 20 (30%) indicated side effects to MTX and rupture of the ectopic pregnancy. Remaining cases () showed resistance to the drug; the level of β-hCG did not fall at least 15% during 7 days after treatment and necessitated laparotomy. In backward-step analysis by multiple logistic regressions of various types of predictor factors, size of gestational sac (coefficient = 1.91, OR = 6.78, 95% confidence interval = 3.18–8.22) and baseline level β-hCG (coefficient = 1.60, OR = 5.0, 95% confidence interval = 4.26–6.72) had significant correlation with leading EP patients failing to response to MTX. This study suggests that further investigation for finding relative contraindications of MTX treatment in EP women should be considered on the gestational sac size because other variables are in the causal pathway of this variable.
New Polymeric Admixture for Cement Based on Hyperbranched Poly Amide-Ester with Pentaerythritol Core
Authors: Amin Ibrahim Amal , El-Sayed Abdel-Megied Ahmed , Sayed Selim Mohamed , Hassenen Darweesh Hassan , Mohamed Ayoub Magdy
Published: May 2013

Hyperbranched poly amide-ester (HBPAE) was synthesized by a solution condensation polymerization through one-step process using pentaerythritol as a central core and AB2 prepolymerized monomer which was rapidly prepared at room temperature (25°C) using commercially available maleic anhydride (MA) and diethanolamine (DEA) monomers in the presence of p-toluene sulfonic acid as a catalyst. The prepared polymer was characterized by GPC, IR, 1H-NMR, and thermal analysis (TGA and DSC). The influence of the polymer addition to the cement paste was investigated by measuring the effect of 1, 3 and 5 wt% HBPAE solutions on the properties of ordinary portland cement (OPC). Accordingly, several parameters were investigated such as water of consistency, setting times, bulk density, apparent porosity, compressive strength, and combined water content of the polymer/cement pastes. The SEM images proved that the incorporation of HBPAE with the OPC cement did not affect the chemical composition of hydrates, but it only affected the physical state, shape, size, morphology, and crystallinity of the formed hydrates. The results showed that the polymer addition to OPC pastes improved the physicomechanical properties of cement.
Dopant Effect and Cell-Configuration-Dependent Dielectric Properties of Nematic Liquid Crystals
Authors: Lin Feng-Ching , Wu Po-Chang , Jian Bo-Ru , Lee Wei
Published: May 2013

The dielectric polarizations induced by impurity ions in both high- and low-resistivity nematic liquid crystals (NLCs) were investigated. Upon adding carbon nanotubes (CNTs) as a dopant, the former showed no distinct change in the low-frequency (< 102 Hz) dielectric spectrum, whereas the latter exhibited dramatic decrease in dielectric constant in that the CNTs remarkably trapped the impurity ions in the NLC of high ionic content. Consequently, the dopant raised the voltage holding ratio by 26% and prolonged the lifetime of the cell. Also investigated were ionic behaviors in the low-resistivity NLC confined in cells with three different configurations. The diffusion constant of the ions in homeotropic cells was found to be the greatest, and the dc conductivity, determined by the diffusion constant, was also higher in the homeotropic ones.
Development and Validation of High Performance Liquid Chromatographic Analysis of Residual N,N-Dimethylformamide in Spent Medium after Biodegradation by Paracoccus denitrificans SD1
Authors: Sanganal Sanjeevkumar , Kulkarni Guruprasad B. , Karegoudar Timmanagouda B.
Published: May 2013

N,N-Dimethylformamide (DMF) is a toxic organic solvent commonly found in the textile and pharmaceutical industrial effluents. The DMF degradation was successfully archived by bacterial strain Paracoccus denitrificans SD1. The study demonstrates the high performance liquid chromatographic (HPLC) approach for the estimation of residual DMF in liquid medium. The investigation mainly focuses on the method development for the detection and quantification of DMF. The bacterium is capable of utilizing DMF (1% v/v) as the sole source of carbon and nitrogen. Utilization of DMF by the bacterium was investigated at regular intervals of time to check the complete degradation at a particular period. The method was validated based on the precision, accuracy, limit of detection, and limit of quantification. Herein, the method was executed in liquid chromatographic condition which enables direct analysis of aqueous samples from the spent medium avoiding the extraction and prederivatization. This improved method allows estimation of residual DMF from the aqueous medium in adequate ranges of precision and accuracy with 99.17% and 99.43% recovery, respectively. The method was validated by investigating the limit of detection (LOD) and limit of quantification (LOQ) of 0.2 and 0.40 mg/l, respectively. The method was found to be precise for detection of DMF by using liquid chromatography.
Roles of Fatty Acid Oversupply and Impaired Oxidation in Lipid Accumulation in Tissues of Obese Rats
Authors: Oakes Nicholas D. , Kjellstedt Ann , Thalén Pia , Ljung Bengt , Turner Nigel
Published: May 2013

To test the roles of lipid oversupply versus oxidation in causing tissue lipid accumulation associated with insulin resistance/obesity, we studiedin vivofatty acid (FA) metabolism in obese (Obese) and lean (Lean) Zucker rats. Indices of local FA utilization and storage were calculated using the partially metabolizable [9,10-3H]-(R)-2-bromopalmitate (3H-R-BrP) and [U-14C]-palmitate (14C-P) FA tracers, respectively. Whole-body FA appearance (Ra) was estimated from plasma14C-P kinetics. Whole-body FA oxidation rate (Rox) was assessed using3H2O production from3H-palmitate infusion, and tissue FA oxidative capacity was evaluatedex vivo. In the basal fasting state Obese had markedly elevated FA levels andRa, associated with elevated FA utilization and storage in most tissues. Estimated rates of muscle FA oxidation were not lower in obese rats and were similarly enhanced by contraction in both lean and obese groups. At comparable levels of FA availability, achieved by nicotinic acid,Roxwas lower in Obese than Lean. In Obese rats, FA oxidative capacity was 35% higher than that in Lean in skeletal muscle, 67% lower in brown fat and comparable in other organs. In conclusion, lipid accumulation in non-adipose tissues of obese Zucker rats appears to result largely from systemic FA oversupply.