bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism for normal splenic T lymphocyte functions in proestrus females after trauma: enhanced local synthesis of 17β-estradiol
Authors: Samy T. S. Anantha , Zheng Rui , Matsutani Takeshi , Rue Loring W. , Bland Kirby I. , Chaudry Irshad H.
Published: May 2013

Trauma-hemorrhage and resuscitation (TH) produces profound immunodepression and enhances susceptibility to sepsis in males but not in proestrus females, suggesting gender dimorphism in the immune responses. However, the mechanism responsible for the maintenance of immune functions in proestrus females after TH is unclear. Splenic T lymphocytes express receptors for estrogen (ER), contain enzymes involved in estrogen metabolism, and are the major source of cytokine production; the metabolism of 17β-estradiol was assessed in the splenic T lymphocytes of proestrus and ovariectomized mice by using appropriate substrates after TH. Analysis for aromatase and 17β-hydroxysteroid dehydrogenases indicated increased 17β-estradiol synthesis and low conversion into estrone in T lymphocytes of proestrus but not of ovariectomized mice. The effect of 17β-estradiol on T lymphocyte cytokine release was reliant on ER expressions. This was apparent in the differences of ER expression, especially that of ER-β, and an association between increased 17β-estradiol synthesis and sustained release of IL-2 and IL-6 in T lymphocytes of proestrus females after TH. Because 17β-estradiol is able to regulate cytokine genes, and the splenic T lymphocyte cytokine releases is altered after TH, continued synthesis of 17β-estradiol in proestrus females appears to be responsible for the maintenance of T lymphocyte cytokine release associated with the protection of immune functions after TH.
System y+ localizes to different membrane subdomains in the basolateral plasma membrane of epithelial cells
Authors: Kizhatil Krishnakumar , Albritton Lorraine M.
Published: May 2013

We report here that the system y+ cationic amino acid transporter ATRC1 localized to clusters within the basolateral membrane of polarized Madin-Darby canine kidney and human embryonic kidney (HEK) cells, suggesting that the transporters are restricted to discrete membrane microdomains in epithelial cells. Based on solubility in nonionic detergents, two populations of ATRC1 molecules existed: approximately half of the total ATRC1 in HEK cells associated with the actin membrane cytoskeleton, whereas another one-fourth resided in detergent-resistant membranes (DRM). In agreement with these findings, cytochalasin D reduced the amount of ATRC1 associated with the actin membrane cytoskeleton. Although some ATRC1 clusters in HEK cells colocalized with caveolin, the majority of ATRC1 did not colocalize with this marker protein for a type of DRM called caveolae. This distribution of ATRC1 is somewhat different from that reported for pulmonary artery endothelial cells in which transporters cluster predominantly in caveolae, suggesting that differences in the proportion of ATRC1 in specific membrane microdomains correlate with differences in the physiological role of the transporter in polarized kidney epithelial vs. vascular endothelial cells.
An ecdysone and tetracycline dual regulatory expression system for studies on Rac1 small GTPase-mediated signaling
Authors: Lai Jen-Feng , Juang Shin-Hun , Hung Yi-Mei , Cheng Hsin-Yuan , Cheng Tzu-Ling , Mostov Keith E. , Jou Tzuu-Shuh
Published: May 2013

Regulated expression systems are invaluable for studying gene function, offer advantages of dosage-dependent and temporally defined gene expression, and limit possible clonal variation when toxic or pleiotropic genes are overexpressed. Previously, establishment of inducible expression systems, such as tetracycline- and ecdysone-inducible systems, required assessment of the inducible characteristics of individual clones by tedious luciferase assays. Taking advantage of a green fluorescent protein (GFP) reporter controlled by tetracycline- or ecdysone-responsive element and fluorescence-activated cell sorting, we propose a simple and efficient strategy to select highly inducible cell lines according to their fluorescence profiles after transiently transfecting the candidate cell pools with a surrogate GFP reporter. We have demonstrated that tetracycline- and ecdysone-inducible systems could be set up in Madin-Darby canine kidney and HEK-293 cells by employing this selection scheme. Importantly, this dual regulatory expression system is applied in studying the complex interplay between two Ras-related small GTPases, Cdc42 and Rac1, on detachment-induced apoptosis. Furthermore, establishment of two tightly regulated expression systems in one target cell line could be of great advantage for dissecting small GTPase Rac1-transduced signaling pathways by using global gene expression approaches such as proteomic assays.
Regulation of the mammalian cell cycle: a model of the G1-to-S transition
Authors: Qu Zhilin , Weiss James N. , MacLellan W. Robb
Published: May 2013

We have formulated a mathematical model for regulation of the G1-to-S transition of the mammalian cell cycle. This mathematical model incorporates the key molecules and interactions that have been identified experimentally. By subdividing these critical molecules into modules, we have been able to systematically analyze the contribution of each to dynamics of the G1-to-S transition. The primary module, which includes the interactions between cyclin E (CycE), cyclin-dependent kinase 2 (CDK2), and protein phosphatase CDC25A, exhibits dynamics such as limit cycle, bistability, and excitable transient. The positive feedback between CycE and transcription factor E2F causes bistability, provided that the total E2F is constant and the retinoblastoma protein (Rb) can be hyperphosphorylated. The positive feedback between active CDK2 and cyclin-dependent kinase inhibitor (CKI) generates a limit cycle. When combined with the primary module, the E2F/Rb and CKI modules potentiate or attenuate the dynamics generated by the primary module. In addition, we found that multisite phosphorylation of CDC25A, Rb, and CKI was critical for the generation of dynamics required for cell cycle progression.
Ca2+ antagonist-insensitive coronary smooth muscle contraction involves activation of ϵ-protein kinase C-dependent pathway
Authors: Dallas Andrea , Khalil Raouf A.
Published: May 2013

Certain angina and coronary artery disease forms do not respond to Ca2+ channel blockers, and a role for vasoactive eicosanoids such as PGF2α in Ca2+ antagonist-insensitive coronary vasospasm is suggested; however, the signaling mechanisms are unclear. We investigated whether PGF2α-induced coronary smooth muscle contraction is Ca2+ antagonist insensitive and involves activation of a PKC-dependent pathway. We measured contraction in single porcine coronary artery smooth muscle cells and intracellular free Ca2+ concentration ([Ca2+]i) in fura 2-loaded cells and examined cytosolic and particulate fractions for PKC activity and reactivity with isoform-specific PKC antibodies. In Hanks' solution (1 mM Ca2+), PGF2α (10-5 M) caused transient [Ca2+]i increase followed by maintained [Ca2+]i increase and 34% cell contraction. Ca2+ channel blockers verapamil and diltiazem (10-6 M) abolished maintained PGF2α-induced [Ca2+]i increase but only partially inhibited PGF2α-induced cell contraction to 17%. Verapamil-insensitive PGF2α contraction was inhibited by PKC inhibitors GF-109203X, calphostin C, and ϵ-PKC V1-2. PGF2α caused Ca2+-dependent α-PKC and Ca2+-independent ϵ-PKC translocation from cytosolic to particulate fractions that was inhibited by calphostin C. Verapamil abolished PGF2α-induced α-but not ϵ-PKC translocation. PMA (10-6 M), a direct activator of PKC, caused 21% contraction with no significant [Ca2+]i increase and ϵ-PKC translocation that were inhibited by calphostin C but not verapamil. Membrane depolarization by 51 mM KCl, which stimulates Ca2+ influx, caused 36% cell contraction and [Ca2+]i increase that were inhibited by verapamil but not GF-109203X or calphostin C and did not cause α- or ϵ-PKC translocation. Thus a significant component of PGF2α-induced contraction of coronary smooth muscle is Ca2+ antagonist insensitive, involves Ca2+-independent ϵ-PKC activation and translocation, and may represent a signaling mechanism of Ca2+ antagonist-resistant coronary vasospasm.
Fundamental step size in single cardiac and skeletal sarcomeres
Authors: Yakovenko Olga , Blyakhman Felix , Pollack Gerald H.
Published: May 2013

In attempting to deduce the size of the elementary molecular translation step, recent experiments using single myosin molecules translating over actin filaments have shown a consistent step size of 5.4 nm (10, 21). We have carried out parallel measurements on single myofibrils from rabbit cardiac muscle and bumblebee flight muscle. Activated specimens were released or stretched with a motor-imposed ramp, and the time course of length of individual sarcomeres was measured by projecting the image of the striations onto a linear photodiode array and tracking the spacing between A-band centroids. We confirmed the 5.4-nm step. With subnanometer precision, however, we find that this value is two times that of a more fundamental step size of 2.7 nm. Step sizes were always integer multiples of 2.7 nm, whether the length change was positive or negative. This value is equal to the linear repeat of actin monomers along the thin filament, a result that ties dynamic events to molecular structure and places narrow constraints on any proposed molecular mechanism.
In vitro and in vivo characterization of the minimal promoter region of the human thiamin transporter SLC19A2
Authors: Reidling Jack C. , Said Hamid M.
Published: May 2013

The molecular mechanisms involved in the regulation of thiamin transport in mammalian cells are poorly understood. Previous studies established that a human thiamin transporter, SLC19A2, plays a role in thiamin uptake in human tissues. We cloned the 5′ regulatory region of the SLC19A2 gene, identified the minimal promoter required for basal activity, and located multiple putative cis elements. To further characterize the SLC19A2 promoter, we investigated, in the present study, the role of the putative cis elements in regulating the activity of the SLC19A2 promoter in vitro and confirmed the activity of the SLC19A2 promoter in vivo. In vitro studies demonstrated that mutation of specific cis elements in the SLC19A2 minimal promoter [Gut-enriched Krupple-like factor (GKLF), nuclear factor-1 (NF-1), and stimulating protein-1 (SP-1)] led to a decrease in activity. Using electrophoretic mobility shift assays, four specific DNA/protein complexes were identified. The interacting factors were determined by oligonucleotide competition and antibody supershift analysis and shown to be GKLF, NF-1, and SP-1. Cotransfection studies of the SLC19A2 promoter with an SP-1 containing vector in Drosophila SL2 cells further confirmed a role for SP-1 in regulating SLC19A2 promoter activity. In vivo studies using transgenic mice established the functionality of the full-length and minimal SLC19A2 promoters. Furthermore, our studies revealed that the pattern of expression of the SLC19A2 promoter-Luciferase constructs in transgenic mice was similar to the reported SLC19A2 RNA expression pattern in native human tissues. The results demonstrate the importance of GKLF, NF-1, and SP-1 in regulating the activity of the SLC19A2 promoter and provide direct in vivo confirmation of promoter activity.
Regulation of C2C12 myogenic terminal differentiation by MKK3/p38α pathway
Authors: Cabane Candice , Englaro Walter , Yeow Karen , Ragno Michel , Dérijard Benoît
Published: May 2013

The signal transduction pathways connecting cell surface receptors to the activation of muscle-specific promoters and leading to myogenesis are still largely unknown. Recently, a contribution of the p38 mitogen-activated protein kinase (MAPK) pathway to this process was evoked through the use of pharmacological inhibitors. We used several mutants of the kinases composing this pathway to modulate the activity of the muscle-specific myosin light chain and myogenin promoters in C2C12 cells by transient transfections. In addition, we show for the first time, using a stable C2C12 cell line expressing a dominant-negative form of the p38 activator MAPK kinase (MKK)3, that a functional p38 MAPK pathway is indeed required for terminal muscle cell differentiation. The most obvious phenotype of this cell line, besides the inhibition of the activation of p38, is its inability to undergo terminal differentiation. This phenotype is accompanied by a drastic inhibition of cell cycle and myogenesis markers such as p21, p27, MyoD, and troponin T, as well as a profound disorganization of the cytoskeleton.
Identification of an apical \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Cl}^{-}{/}\mathrm{HCO}_{3}^{-}\) \end{document} exchanger in rat kidney proximal tubule
Authors: Petrovic Snezana , Ma Liyun , Wang Zhaohui , Soleimani Manoocher
Published: May 2013

SLC26A6 (or putative anion transporter 1, PAT1) is located on the apical membrane of mouse kidney proximal tubule and mediates [Formula: see text] exchange in in vitro expression systems. We hypothesized that PAT1 along with a [Formula: see text] exchange is present in apical membranes of rat kidney proximal tubules. Northern hybridizations indicated the exclusive expression of SLC26A6 (PAT1 or CFEX) in rat kidney cortex, and immunocytochemical staining localized SLC26A6 on the apical membrane of proximal tubules, with complete prevention of the labeling with the preadsorbed serum. To examine the functional presence of apical [Formula: see text] exchanger, proximal tubules were isolated, microperfused, loaded with the pH-sensitive dye BCPCF-AM, and examined by digital ratiometric imaging. The pH of the perfusate and bath was kept at 7.4. Buffering capacity was measured, and transport rates were calculated as equivalent base flux. The results showed that in the presence of basolateral DIDS (to inhibit [Formula: see text] cotransporter 1) and apical EIPA (to inhibit Na+/H+ exchanger 3), the magnitude of cell acidification in response to addition of luminal Cl– was ∼5.0-fold higher in the presence than in the absence of [Formula: see text]. The Cl–-dependent base transport was inhibited by ∼61% in the presence of 0.5 mM luminal DIDS. The presence of physiological concentrations of oxalate in the lumen (200 μM) did not affect the [Formula: see text] exchange activity. These results are consistent with the presence of SLC26A6 (PAT1) and [Formula: see text] exchanger activity in the apical membrane of rat kidney proximal tubule. We propose that SLC26A6 is likely responsible for the apical [Formula: see text] (and Cl–/OH–) exchanger activities in kidney proximal tubule.
Glucose regulates the cortical actin network through modulation of Cdc42 cycling to stimulate insulin secretion
Authors: Nevins Angela K. , Thurmond Debbie C.
Published: May 2013

Glucose-stimulated insulin granule exocytosis in pancreatic β-cells involves cortical actin remodeling that results in the transient disruption of the interaction between polymerized actin with the plasma membrane t-SNARE (target membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex. To examine the mechanism underlying the initiation of cortical actin remodeling, we have used the actin nucleating/stabilizing agent jasplakinolide to show that remodeling is initiated at a step proximal to the ATP-sensitive K+ channels in the stimulus-secretion pathway. Confocal immunofluorescent microscopy revealed that cortical actin remodeling was required for glucose-stimulated insulin secretion. Furthermore, glucose was found to mediate the endogenous activation state of the Rho family GTPase Cdc42, a positive proximal effector of actin polymerization, resulting in a net decrease of Cdc42-GTP within 5 min of stimulation. Intriguingly, glucose stimulation resulted in the rapid and reversible glucosylation of Cdc42, suggesting that glucose inactivated Cdc42 by selective glucosylation to induce cortical actin rearrangement. Moreover, expression of the constitutively active form of Cdc42 (Q61L) inhibited glucose-stimulated insulin secretion, whereas the dominant negative form (T17N) was without effect, suggesting that glucose-stimulated insulin secretion requires Cdc42 cycling to the GDP-bound state. In contrast, KCl-stimulated insulin secretion was unaffected by the expression of dominant negative or constitutively active Cdc42 and ceased to modulate endogenous Cdc42 activation, consistent with glucose-dependent cortical actin remodeling. These findings reveal that glucose regulates the cortical actin network through modulation of Cdc42 cycling to induce insulin secretion in pancreatic β-cells.
Phosphorous metabolites and steady-state energetics of transformed fibroblasts during three-dimensional growth
Authors: Kunz-Schughart Leoni A. , Freyer James P.
Published: May 2013

Rat1-T1 and MR1 spheroids represent separate transformed phenotypes originated from the same rat fibroblasts that differ in three-dimensional (3D) growth kinetics, histological structure, and oxygenation status. In the present study,31P-NMR spectroscopy of perfused spheroid suspensions was used to investigate cellular energetics relative to 3D growth, development of necrosis, and cell cycle distribution. Both spheroid types were characterized by a remarkably low amount of free (inorganic) phosphate (Pi) and a low phosphocreatine peak. The ratio of nucleoside triphosphate (NTP) to Pi ranged between 1.5 and 2.0. Intracellular pH, NTP-to-Pi ratio, and NTP/cell remained constant throughout spheroid growth, being unaffected by the emergence of oxygen deficiency, cell quiescence, and necrosis. However, a 50% decrease in the ratio of the lipid precursors phosphorylcholine and phosphorylethanolamine (PC/PE) was observed with increasing spheroid size and was correlated with an increased G1/G0 phase cell fraction. In addition, the ratio of the phospholipid degradation products glycerophosphorylcholine and glycerophosphorylethanolamine (GPC/GPE) increased with spheroid diameter in Rat1-T1 aggregates. We conclude that changes in phospholipid metabolism, rather than alterations in energy-rich phosphates, reflect cell quiescence in spheroid cultures, because cells in the inner oxygen-deficient zones seem to adapt their energy metabolism to the environmental conditions before necrotic cell destruction.
Upregulation of Ca2+ removal in human skeletal muscle: a possible role for Ca2+-dependent priming of mitochondrial ATP synthesis
Authors: Koopman Werner J. H. , Renders Michel , Oosterhof Arie , van Kuppevelt Toin H. , van Engelen Baziel G. M. , Willems Peter H. G. M.
Published: May 2013

In muscle, ATP is required for the powerstroke of the myosin head, the detachment of actin and myosin filaments, and the reuptake of Ca2+ into the sarcoplasmic reticulum. During contraction-relaxation, large amounts of ATP are consumed at the sites of action of the myosin-ATPase and sarcoplasmic reticulum Ca2+-ATPase. The present study addresses the consequences of a reduction in mitochondrial ATP production capacity on sarcoplasmic Ca2+ handling. To this end, myotubes were cultured from patient quadriceps with a biochemically defined decrease in the maximal rate of mitochondrial ATP production and were loaded with indo 1 for imaging of sarcoplasmic Ca2+ changes in real time by confocal microscopy. Myotubes were field-stimulated with 10-ms pulses of 16 V to evoke transient rises in sarcoplasmic Ca2+ concentration ([Ca2+]S). Three single pulses, two pulse trains (1 Hz), and one single pulse were applied in succession to mimic changing workloads. Control myotubes displayed [Ca2+]S transients with an amplitude that was independent of the strength of the stimulus. Intriguingly, the rate of sarcoplasmic Ca2+ removal (CRR) was significantly upregulated during the second and subsequent transients. In myotubes with a reduced mitochondrial ATP production capacity, the amplitude of the [Ca2+]S transients was markedly increased at higher stimulus intensities. Moreover, upregulation of the CRR was significantly decreased compared with control. Taken together, these results are in good agreement with a tight coupling between mitochondrial ATP production and sarcoplasmic Ca2+ handling. Moreover, they support the existence of a relatively long-lasting mitochondrial memory for sarcoplasmic [Ca2+] rises. This memory, which manifested itself as an increase in CRR upon recurrent stimulation, was impaired in patient myotubes with a reduced mitochondrial ATP production capacity.
Functional and molecular identification of ERG channels in murine portal vein myocytes
Authors: Ohya Susumu , Horowitz Burton , Greenwood Iain A.
Published: May 2013

Ion channels encoded by ether-à-go-go-related genes (ERG) have been implicated in repolarization of the cardiac action potential and also as components of the resting membrane conductance in various cells. The aim of the present study was to determine whether ERG channels were expressed in smooth muscle cells isolated from portal vein. RT-PCR demonstrated the expression of murine ERG (mERG), and real-time quantitative PCR showed that the mERG1b isoform predominated over the mERG1a, mERG2, and mERG3 in portal vein. Single myocytes from portal vein displayed membrane staining with an ERG1-specific antibody. Whole cell voltage-clamp experiments were performed to determine whether portal vein myocytes expressed functional ERG channels. Large inward currents with distinctive kinetics were elicited that were inhibited rapidly by E-4031 (mean amplitude of the E-4031-sensitive current at −120 mV was −205 ± 24 pA; n = 14). Deactivation of the E-4031-sensitive current was voltage dependent (mean time constants at −80 and −120 mV were 103 ± 9 and 33 ± 2 ms, respectively; n = 13). Because of the rapid kinetics of mERG currents at more negative potentials, there was a substantial noninactivating “window” current that reached a maximum of −66 ± 10 pA at −70 mV. Complete portal veins exhibited spontaneous contractile activity in isometric tension experiments, and this activity was modified significantly by E-4031. These data show that ERG channels are expressed in murine portal vein myocytes that may contribute to the resting membrane conductance.
Slow intracellular trafficking of catalase nanoparticles targeted to ICAM-1 protects endothelial cells from oxidative stress
Authors: Muro Silvia , Cui Xiumin , Gajewski Christine , Murciano Juan-Carlos , Muzykantov Vladimir R. , Koval Michael
Published: May 2013

Nanotechnologies promise new means for drug delivery. ICAM-1 is a good target for vascular immunotargeting of nanoparticles to the perturbed endothelium, although endothelial cells do not internalize monomeric anti-ICAM-1 antibodies. However, coupling ICAM-1 antibodies to nanoparticles creates multivalent ligands that enter cells via an amiloride-sensitive endocytic pathway that does not require clathrin or caveolin. Fluorescence microscopy revealed that internalized anti-ICAM nanoparticles are retained in a stable form in early endosomes for an unusually long time (1-2 h) and subsequently were degraded following slow transport to lysosomes. Inhibition of lysosome acidification by chloroquine delayed degradation without affecting anti-ICAM trafficking. Also, the microtubule disrupting agent nocodazole delayed degradation by inhibiting anti-ICAM nanoparticle trafficking to lysosomes. Addition of catalase to create anti-ICAM nanoparticles with antioxidant activity did not affect the mechanisms of nanoparticle uptake or trafficking. Intracellular anti-ICAM/catalase nanoparticles were active, because endothelial cells were resistant to H2O2-induced oxidative injury for 1-2 h after nanoparticle uptake. Chloroquine and nocodazole increased the duration of antioxidant protection by decreasing the extent of anti-ICAM/catalase degradation. Therefore, the unique trafficking pathway followed by internalized anti-ICAM nanoparticles seems well suited for targeted delivery of therapeutic enzymes to endothelial cells and may provide a basis for treatment of acute vascular oxidative stress.
Muscle electrotransfer as a tool for studying muscle fiber-specific and nerve-dependent activity of promoters
Authors: Bertrand Anne , Ngô-Muller Valérie , Hentzen Danièle , Concordet Jean-Paul , Daegelen Dominique , Tuil David
Published: May 2013

Muscle electrotransfer has recently become a promising tool for efficient delivery of plasmids and transgene expression in skeletal muscle. This technology has been mainly applied to use of muscle as a bioreactor for production of therapeutic proteins. However, it remains to be determined whether muscle electrotransfer may also be accurately used as an alternative tool to transgenesis for studying aspects of muscle-specific gene control that must be explored in fully mature muscle fibers in vivo, such as fiber specificity and nerve dependence. It was also not known to what extent the initial electrical stimulations alter muscle physiology and gene expression. Therefore, optimized conditions of skeletal muscle electroporation were first tested for their effects on muscles of transgenic mice harboring a pM310-CAT transgene in which the CAT reporter gene was under control of the fast IIB fiber-specific and nerve-dependent aldolase A pM promoter. Surprisingly, electrostimulation led to a drastic but transient shutdown of pM310-CAT transgene expression concomitant with very transient activation of MyoD and, mostly, with activation of myogenin, suggesting profound alterations in transcriptional status of the electroporated muscle. Return to a normal transcriptional state was observed 7-10 days after electroporation. Therefore, we investigated whether a reporter construct placed under control of pM could exhibit fiber-specific expression 10 days after electrotransfer in either fast tibialis anterior or slow soleus muscle. We show that not only fiber specificity, but also nerve dependence, of a pM-driven construct can be reproduced. However, after electrotransfer, pM displayed a less tight control than previously observed for the same promoter when integrated in a chromatin context.
Opposing effects of PKCα and PKCε on basolateral membrane dynamics in intestinal epithelia
Authors: Song Jaekyung Cecilia , Rangachari Patangi K. , Matthews Jeffrey B.
Published: May 2013

PKC is a critical effector of plasma membrane dynamics, yet the mechanism and isoform-specific role of PKC are poorly understood. We recently showed that the phorbol ester PMA (100 nM) induces prompt activation of the novel isoform PKCε followed by late activation of the conventional isoform PKCα in T84 intestinal epithelia. PMA also elicited biphasic effects on endocytosis, characterized by an initial stimulatory phase followed by an inhibitory phase. Activation of PKCε was shown to be responsible for stimulation of basolateral endocytosis, but the role of PKCα was not defined. Here, we used detailed time-course analysis as well as selective activators and inhibitors of PKC isoforms to infer the action of PKCα on basolateral endocytosis. Inhibition of PKCα by the selective conventional PKC inhibitor Gö-6976 (5 μM) completely blocked the late inhibitory phase and markedly prolonged the stimulatory phase of endocytosis measured by FITC-dextran uptake. The PKCε-selective agonist carbachol (100 μM) induced prolonged stimulation of endocytosis devoid of an inhibitory phase. Actin disassembly caused by PMA was completely blocked by Gö-6850 but not by Gö-6976, implicating PKCε as the key isoform responsible for actin disruption. The Ca2+ agonist thapsigargin (5 μM) induced early activation of PKCα when added simultaneously with PMA. This early activation of PKCα blocked the ability of PMA to remodel basolateral F-actin and abolished the stimulatory phase of basolateral endocytosis. Activation of PKCα stabilizes F-actin and thereby opposes the effect of PKCε on membrane remodeling in T84 cells.
Gene expression profile of endothelin-1-induced growth in glomerular mesangial cells
Authors: Mishra Rangnath , Leahy Patrick , Simonson Michael S.
Published: May 2013

Endothelin (ET)-1 is a vasoconstrictor and mitogen involved in vascular remodeling. Changes in gene expression that underlie control of cell growth by ET-1 remain poorly characterized. To identify pathways of growth control we used microarrays to analyze ET-1-regulated gene expression in human mesangial cells, an important ET-1 vascular target cell in vivo. Statistical assessment of differential expression (significance analysis of microarrays) revealed upregulated transcripts for growth factors [heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), fibroblast growth factor (FGF), interleukin (IL)-6] and downregulated transcripts for genes that inhibit growth (BAX, p27KIP1, DAD1). Consistent with the gene expression profile, quantitative RT-PCR and Western blotting confirmed induction of HB-EGF by ET-1. To test a functional role for HB-EGF in ET-1 signaling, we showed that exogenous HB-EGF stimulated phosphorylation of ErbB1 and growth of mesangial cells. ET-1-induced proliferation was blocked by an ErbB1 receptor-selective kinase inhibitor and by a specific ErbB1 receptor-neutralizing antibody. Proliferation in response to ET-1 was also inhibited by neutralizing antisera against human HB-EGF. Together, these results provide data for modeling ET-1 pathways for growth control and suggest a specific role for HB-EGF gene induction in mesangial cell growth in response to ET-1.
Inhibition of muscle carbonic anhydrase slows the Ca2+ transient in rat skeletal muscle fibers
Authors: Wetzel Petra , Kleinke Tanja , Papadopoulos Simon , Gros Gerolf
Published: May 2013

A countertransport of H+ is coupled to Ca2+ transport across the sarcoplasmic reticulum (SR) membrane. We propose that SR carbonic anhydrase (CA) accelerates the CO2-HCO[Formula: see text]reaction so that H+ ions, which are exchanged for Ca2+ ions, are produced or buffered in the SR at sufficient rates. Inhibition of this SR-CA is expected to reduce the rate of H+ fluxes, which then will retard the kinetics of Ca2+ transport. Fura 2 signals and isometric force were simultaneously recorded in fiber bundles of the soleus (SOL) and extensor digitorum longus (EDL) from rats in the absence and presence of the lipophilic CA inhibitors L-645151, chlorzolamide (CLZ), and ethoxzolamide (ETZ), as well as the hydrophilic inhibitor acetazolamide (ACTZ). Fura 2 and force signals were analyzed for time to peak (TTP), 50% decay time ( t 50), and their amplitudes. L-645151, CLZ, and ETZ significantly increased TTP of fura 2 by 10–25 ms in SOL and by 5–7 ms in EDL and TTP of force by 6–30 ms in both muscles. L-645151 and ETZ significantly prolonged t 50 of fura 2 and force by 20–55 and 40–160 ms, respectively, in SOL and EDL. L-645151, CLZ, and ETZ also increased peak force of single twitches and amplitudes of fura fluorescence ratio (R340/380) at an excitation wavelength of 340 to 380 nm. All effects of CA inhibitors on fura 2 and force signals could be reversed. ACTZ did not affect TTP, t 50, and amplitudes of fura 2 signals or force. L-645151, CLZ, and ETZ had no effects on myosin-, Ca2+-, and Na+-K+-ATPase activities, nor did they affect the amplitude and half-width of action potentials. We conclude that inhibition of SR-CA by impairing H+ countertransport is responsible for deceleration of intracellular Ca2+transients and contraction times.
Exposure to UV light causes increased biotinylation of histones in Jurkat cells
Authors: Peters Dorothea M. , Griffin Jacob B. , Stanley J. Steven , Beck Mary M. , Zempleni Janos
Published: May 2013

Biotin in breakdown products of biotinylated carboxylases serves as substrate for biotinylation of histones by biotinidase. Here we determined whether biotinylation of histones might play a role in repair of damaged DNA and in apoptosis. Jurkat cells were exposed to UV light to induce DNA damage. Abundance of thymine dimers increased about three times in response to UV exposure, consistent with DNA damage. Biotin-containing carboxylases were degraded in response to UV exposure, as judged by Western blot analysis and carboxylase activities. Mitochondrial integrity decreased in response to UV exposure (as judged by confocal microscopy), facilitating the release of breakdown products of carboxylases from mitochondria. Biotinylation of histones increased in response to UV exposure; biotinylation of histones did not occur specifically at sites of newly repaired DNA. UV exposure triggered apoptosis, as judged by caspase-3 activity and analysis by confocal microscopy. In summary, this study provided evidence that increased biotinylation of histones in DNA-damaged cells might either be a side product of carboxylase degradation or a step during apoptosis.
Preconditioning of cortical neurons by oxygen-glucose deprivation: tolerance induction through abbreviated neurotoxic signaling
Authors: Tauskela Joseph S. , Brunette Eric , Monette Robert , Comas Tanya , Morley Paul
Published: May 2013

Transient exposure of rat cortical cultures to nonlethal oxygen-glucose deprivation (OGD preconditioning) induces tolerance to otherwise lethal oxygen-glucose deprivation (OGD) or N-methyl-d-aspartate 24 h later. This study evaluates the role of cytosolic and mitochondrial Ca2+-dependent cellular signaling. Mechanistic findings are placed in context with other models of ischemic preconditioning or known neurotoxic pathways within cortical neurons. Tolerance to otherwise lethal OGD is suppressed by performing OGD preconditioning in the presence of the broad-scope catalytic antioxidants Mn(III)tetra(4-carboxyphenyl)porphyrin (MnTBAP) or Zn(II)tetra(4-carboxyphenyl)porphyrin [Zn(II)TBAP], but not by a less active analog, Mn(III)tetra(4-sulfonatophenyl)porphyrin, or a potent superoxide scavenger, Mn(III)tetra( N-ethyl-2-pyridyl)porphyrin chloride. Inhibitors of adenosine A1 receptors, nitric oxide synthase, mitogen-activated protein kinase, and poly(ADP-ribose) polymerase fail to suppress OGD preconditioning despite possible links with reactive oxygen species in other models of ischemic preconditioning. Preconditioning is suppressed by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), which has been ascribed elsewhere to inhibition of superoxide transport to the cytosol through mitochondrial anion channels. However, although it induces mitochondrial Ca2+ uptake, neuronal preconditioning is largely insensitive to mitochondrial uncoupling with carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone or 2,4-dinitrophenol. Un-couplers will prevent production of mitochondrial reactive oxygen species, implying nonmitochondrial targets by MnTBAP, Zn(II)TBAP, and DIDS. Emphasizing the importance of an increase in cytosolic Ca2+ during preconditioning, a Ca2+/calmodulin-dependent protein kinase II inhibitor, KN-62, suppresses development of subsequent tolerance. Summarizing, only those cellular transduction pathways that have the potential to be neurotoxic may be activated by preconditioning in cortical neurons. Finally, a marked decrease in extracellular glutamate is observed during otherwise lethal OGD in preconditioned cultures, suggesting that this end effector may represent a point of convergence across different preconditioning models.
MAP kinases contribute to IL-8 secretion by intestinal epithelial cells via a posttranscriptional mechanism
Authors: Jijon Humberto B. , Panenka William J. , Madsen Karen L. , Parsons Howard G.
Published: May 2013

The intracellular pathways that regulate intestinal epithelial gene expression are poorly understood. In this study we examined the roles of extracellular signal-regulated kinase (ERK) and p38 in the expression of interleukin-8 (IL-8) and intercellular adhesion molecule-1 (ICAM-1) using the human intestinal cell line HT-29. HT-29 cells were treated with tumor necrosis factor-α (TNF-α) in the presence or absence of ERK and p38 pathway inhibitors. TNF-α treatment resulted in increased IL-8 and ICAM-1 protein and mRNA synthesis, increased ERK and p38 activity, and activation of the transcription factors activator protein-1 (AP-1) and nuclear factor-κB (NF-κB). Inhibition of the ERK and p38 pathways attenuated IL-8 secretion but did not alter ICAM-1 expression. Furthermore, AP-1 and NF-κB DNA binding was not affected by ERK and p38 inhibition. In contrast, ERK and p38 inhibition resulted in the accelerated degradation of the IL-8 mRNA, suggesting that in HT-29 cells, p38 and ERK contribute to TNF-α-stimulated IL-8 secretion by intestinal epithelial cells via a posttranscriptional mechanism that involves stabilization of the IL-8 transcript.
Lyn- and ERK-mediated vs. Ca2+-mediated neutrophil O 2 − responses with thermal injury
Authors: Fazal Nadeem , Al-Ghoul Walid M. , Schmidt Megan J. , Choudhry Mashkoor A. , Sayeed Mohammed M.
Published: May 2013

We evaluated the dependency of neutrophil O[Formula: see text] production on PTK-Lyn and MAPK-ERK1/2 in rats after thermal injury. Activation of PTK-Lyn was assessed by immunoprecipitation. Phosphorylation of ERK1/2 was assessed by Western blot analysis. O[Formula: see text] production was measured by isoluminol-enhanced luminometry. Imaging technique was employed to measure neutrophil [Ca2+]i in individual cells. Thermal injury caused marked upregulation of Lyn and ERK1/2 accompanying enhanced neutrophil O[Formula: see text] production. Treatment of rats with PTK blocker (AG556) or MAPK blocker (AG1478) before burn injury caused complete inhibition of the respective kinase activation. Both AG556 and AG1478 produced an ∼66% inhibition in O[Formula: see text] production. Treatment with diltiazem (DZ) produced an ∼37% inhibition of O[Formula: see text] production without affecting Lyn or ERK1/2 activation with burn injury. Ca2+mobilization was upregulated with burn injury but not affected by treatment of burn rats with AG556. Unlike the partial inhibition of burn-induced O[Formula: see text] production by AG556, AG1478, or DZ, platelet-activating factor antagonist (PAFa) treatment of burn rats produced near complete inhibition of O[Formula: see text] production. PAFa treatment also blocked activation of Lyn. The findings suggest that the near complete inhibition of O[Formula: see text] production by PAFa was a result of blockade of PTK as well as Ca2+signaling. Overall, our studies show that enhanced neutrophil O[Formula: see text] production after thermal injury is a result of potentiation of Ca2+-linked and -independent signaling triggered by inflammatory agents such as PAF.
Changes in Na+-K+-ATPase activity influence cell attachment to fibronectin
Authors: Belusa Roger , Aizman Oleg , Andersson Ronnie M. , Aperia Anita
Published: May 2013

Most vital cellular functions are dependent on a fine-tuned regulation of intracellular ion homeostasis. Here we have demonstrated, using COS cells that were untransfected or transfected with wild-type rat ouabain-resistant Na+-K+-ATPase, that partial inhibition of Na+-K+-ATPase has a dramatic influence on cell attachment to fibronectin. Ouabain dose-dependently decreased attachment in untransfected cells and in cells expressing wild-type Na+-K+-ATPase, but not in cells expressing ouabain-insensitive Na+-K+-ATPase, whereas inhibition of Na+-K+-ATPase by lowering extracellular K+ concentration decreased attachment in all three cell types. Thirty percent inhibition of Na+-K+-ATPase significantly attenuated attachment. Na+-K+-ATPase inhibition caused a sustained increase in the intracellular Ca2+ concentration that obscured Ca2+ transients observed in untreated cells during attachment. Inhibitors of Ca2+ transporters significantly decreased attachment, but inhibition of Na+/H+ exchanger did not. Ouabain reduced focal adhesion kinase autophosphorylation but had no effect on cell surface integrin expression. These results suggest that the level of Na+-K+-ATPase activity strongly influences cell attachment, possibly by an effect on intracellular Ca2+.
Potentiation of nitric oxide-induced apoptosis in p53−/− vascular smooth muscle cells
Authors: Kibbe Melina R , Li Jianrong , Nie Suhua , Choi Byung Min , Kovesdi Imre , Lizonova Alena , Billiar Timothy R , Tzeng Edith
Published: May 2013

The functional role of p53 in nitric oxide (NO)-mediated vascular smooth muscle cell (VSMC) apoptosis remains unknown. In this study, VSMC from p53−/− and p53+/+ murine aortas were exposed to exogenous or endogenous sources of NO. Unexpectedly, p53−/− VSMC were much more sensitive to the proapoptotic effects of NO than were p53+/+ VSMC. Furthermore, this paradox appeared to be specific to NO, because other proapoptotic agents did not demonstrate this differential effect on p53−/− cells. NO-induced apoptosis in p53−/− VSMC occurred independently of cGMP generation. However, mitogen-activated protein kinase (MAPK) pathways appeared to play a significant role. Treatment of the p53−/− VSMC with S-nitroso- N-acetylpenicillamine resulted in a marked activation of p38 MAPK and, to a lesser extent, of c-Jun NH2-terminal kinase, mitogen-activated protein kinase kinase (MEK) 1/2, and p42/44 (extracellular signal-regulated kinase, ERK). Furthermore, basal activity of the MEK-p42/44 (ERK) pathway was increased in the p53+/+ VSMC. Inhibition of p38 MAPK with SB-203580 or of MEK1/2 with PD-98059 blocked NO-induced apoptosis. Therefore, p53 may protect VSMC against NO-mediated apoptosis, in part, through differential regulation of MAPK pathways.
Key role of PLC-γ in EGF protection of epithelial barrier against iNOS upregulation and F-actin nitration and disassembly
Authors: Banan A. , Zhang L. J. , Shaikh M. , Fields J. Z. , Farhadi A. , Keshavarzian A.
Published: May 2013

Upregulation of inducible nitric oxide synthase (iNOS) is key to oxidant-induced disruption of intestinal (Caco-2) monolayer barrier, and EGF protects against this disruption by stabilizing the cytoskeleton. PLC-γ appears to be essential for monolayer integrity. We thus hypothesized that PLC-γ activation is essential in EGF protection against iNOS upregulation and the consequent cytoskeletal oxidation and disarray and monolayer disruption. Intestinal cells were transfected to stably overexpress PLC-γ or to inhibit its activation and were then pretreated with EGF ± oxidant (H2O2). Wild-type (WT) intestinal cells were treated similarly. Relative to WT monolayers exposed to oxidant, pretreatment with EGF protected monolayers by: increasing native PLC-γ activity; decreasing six iNOS-related variables (iNOS activity/protein, NO levels, oxidative stress, actin oxidation/nitration); increasing stable F-actin; maintaining actin stability; and enhancing barrier integrity. Relative to WT cells exposed to oxidant, transfected monolayers overexpressing PLC-γ (+2.3-fold) were protected, as indicated by decreases in all measures of iNOS-driven pathway and enhanced actin and barrier integrity. Overexpression-induced inhibition of iNOS was potentiated by low doses of EGF. Stable inhibition of PLC-γ prevented all measures of EGF protection against iNOS upregulation. We conclude that 1) EGF protects against oxidative stress disruption of intestinal barrier by stabilizing F-Actin, largely through the activation of PLC-γ and downregulation of iNOS pathway; 2) activation of PLC-γ is by itself essential for cellular protection against oxidative stress of iNOS; and 3) the ability to suppress iNOS-driven reactions and cytoskeletal oxidation and disassembly is a novel mechanism not previously attributed to the PLC family of isoforms.
cAMP-dependent protein kinase and proliferation differ in normal and polycystic kidney epithelia
Authors: Marfella-Scivittaro Carmela , Quiñones Andrea , Orellana Stephanie A.
Published: May 2013

Developmental control of cell proliferation is crucial, and abnormal principal cell proliferation may contribute to cystogenesis in polycystic kidney disease. This study investigates roles of cAMP and its primary effector, cAMP-dependent protein kinase (protein kinase A; PKA), in control of cell proliferation in filter-grown noncystic (NC) and cystic (CY)-derived principal cell cultures. These cultures had similar cAMP pathway characteristics upstream of PKA subunit distribution but differed in predicted PKA subtype distribution. Functionally, cultures were proliferative before polarization, with constitutively higher proliferation in CY cultures. NC cultures achieved levels similar to those of CY cultures on pharmacological manipulation of cAMP production or PKA activation or inhibition of PKA subtype I activity. Inhibition of overall PKA activity, or of PKA subtype II anchoring, diminished cAMP/PKA-mediated proliferation in NC cultures but had no effect on CY cultures. Polarized CY monolayers remained proliferative, but NC monolayers lost responsiveness. No large proliferation changes resulted from treatments of polarized cultures; however, polarized NC and CY cultures differed in poststimulation handling of PKA catalytic and type IIα regulatory subunits. Our results support PKA subtype regulation of prepolarization proliferation in NC principal cells and altered regulation of PKA in CY cells and suggest that differences at or downstream of PKA can contribute to altered proliferation in a developmental renal disease.
Role of calcium in metabolic signaling between cardiac sarcoplasmic reticulum and mitochondria in vitro
Authors: Balaban Robert S. , Bose Salil , French Stephanie A. , Territo Paul R.
Published: May 2013

The role of Ca2+ as a cytosolic signaling molecule between porcine cardiac sarcoplasmic reticulum (SR) ATPase and mitochondrial ATP production was evaluated in vitro. The Ca2+ sensitivity of these processes was determined individually and in a reconstituted system with SR and mitochondria in a 0.5:1 protein-to-cytochrome aa 3 ratio. The half-maximal concentration ( K 1/2) of SR ATPase was 335 nM Ca2+. The ATP synthesis dependence was similar with a K 1/2 of 243 nM for dehydrogenases and 114 nM for overall ATP production. In the reconstituted system, Ca2+ increased thapsigargin-sensitive ATP production (maximum ∼5-fold) with minimal changes in mitochondrial reduced nicotinamide adenine dinucleotide (NADH). NADH concentration remained stable despite graded increases in NADH turnover induced over a wide range of Ca2+ concentrations (0 to ∼500 nM). These data are consistent with a balanced activation of SR ATPase and mitochondrial ATP synthesis by Ca2+ that contributes to a homeostasis of energy metabolism metabolites. It is suggested that this balanced activation by cytosolic Ca2+ is partially responsible for the minimal alteration in energy metabolism intermediates that occurs with changes in cardiac workload in vivo.
Hydrogen peroxide stimulates ubiquitin-conjugating activity and expression of genes for specific E2 and E3 proteins in skeletal muscle myotubes
Authors: Li Yi-Ping , Chen Yuling , Li Andrew S. , Reid Michael B.
Published: May 2013

Reactive oxygen species (ROS) are thought to promote muscle atrophy in chronic wasting diseases, but the underlying mechanism has not been determined. Here we show that H2O2 stimulates ubiquitin conjugation to muscle proteins through transcriptional regulation of the enzymes (E2 and E3 proteins) that conjugate ubiquitin to muscle proteins. Incubation of C2C12 myotubes with 100 μM H2O2 increased the rate of 125I-labeled ubiquitin conjugation to muscle proteins in whole cell extracts. This response required at least 4-h exposure to H2O2 and persisted for at least 24 h. Preincubating myotubes with cycloheximide or actinomycin D blocked H2O2 stimulation of ubiquitin-conjugating activity, suggesting that gene transcription is required. Northern blot analyses revealed that H2O2 upregulates expression of specific E3 and E2 proteins that are thought to regulate muscle catabolism, including atrogin1/MAFbx, MuRF1, and E214k. These results suggest that ROS stimulate protein catabolism in skeletal muscle by upregulating the ubiquitin conjugation system.