Cloning of a Na+-driven Cl/HCO3 exchanger from squid giant fiber lobe

2003 ◽  
Vol 285 (4) ◽  
pp. C771-C780 ◽  
Author(s):  
Leila V. Virkki ◽  
Inyeong Choi ◽  
Bruce A. Davis ◽  
Walter F. Boron
Keyword(s):  
Northern Blot Analysis ◽  
Stellate Ganglion ◽  
Disulfonic Acid ◽  
Giant Fiber ◽  
Slow Recovery ◽  
Degenerate Primers ◽  
Bicarbonate Transporter ◽  
Loligo Pealei ◽  
Full Length Clone

We extracted RNA from the giant fiber lobe (GFL) of the squid Loligo pealei and performed PCR with degenerate primers that were based on highly conserved regions of Na+-coupled HCO3- transporters. This approach yielded a novel, 290-bp sequence related to the bicarbonate transporter superfamily. Using an L. opalescens library, we extended the initial fragment in the 3′ and 5′ directions by a combination of library screening and PCR and obtained the full-length clone (1,198 amino acids) by PCR from L. pealei GFL. The amino acid sequence is 46% identical to mammalian electrogenic and electroneutral Na-HCO3 cotransporters and 33% identical to the anion exchanger AE1. Northern blot analysis showed strong signals in L. pealei GFL, optic lobe, and heart and weaker signals in gill and stellate ganglion. To assess function, we injected in vitro-transcribed cRNA into Xenopus oocytes and subsequently used microelectrodes to monitor intracellular pH (pHi) and membrane voltage ( Vm). Superfusing these oocytes with 5% CO2-33 mM HCO3- caused a CO2-induced fall in pHi, followed by a slow recovery. The absence of a rapid HCO3--induced hyperpolarization indicates that the pHi recovery mechanism is electroneutral. Ion substitutions showed that Na+ and Cl- are required on opposite sides of the membrane. Transport was blocked by 50 μM 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). The characteristics of our novel clone fit those of a Na+-driven Cl/HCO3 exchanger (NDCBE).

Over-expression of GbRac1 gene in transgenic tobacco enhances disease resistance to Alternaria alternata in vitro

2005 ◽  
Vol 2 (2) ◽  
pp. 73-77 ◽  
Author(s):  
Li Wei-Min ◽  
Wang Zhi-Xing ◽  
Jia Shi-Rong
Keyword(s):  
Disease Resistance ◽  
Transgenic Plants ◽  
Alternaria Alternata ◽  
Northern Blot Analysis ◽  
Challenge Test ◽  
Constitutive Expression ◽  
Degenerate Primers ◽  
Over Expression ◽  
Detached Leaves

AbstractGbRac1 gene was cloned from Gossypium barbadense with degenerate primers and 3′-RACE. Northern blot analysis indicated that GbRac1 mRNA was expressed abundantly in G. barbadense seedlings inoculated with Verticillium dehliae compared with mock-inoculated plants. A plant constitutive expression vector pRac harbouring GbRac1 gene was constructed and leaf discs of tobacco (Nicotiana tabacum L. cv. NC89) were transformed with pRac by Agrobacterium-mediated transformation. Disease challenge test of detached leaves of the transgenic plants by inoculation with Alternaria alternata showed that resistance was enhanced dramatically compared with the non-transgenic plants. Results suggest that GbRac1 gene might have potential application in the genetic engineering of plants with enhanced disease resistance.

Functional characterization of human NBC4 as an electrogenic Na+-HCO 3 − cotransporter (NBCe2)

2002 ◽  
Vol 282 (6) ◽  
pp. C1278-C1289 ◽  
Author(s):  
Leila V. Virkki ◽  
Darren A. Wilson ◽  
Richard D. Vaughan-Jones ◽  
Walter F. Boron
Keyword(s):  
Human Heart ◽  
Functional Characterization ◽  
Reversal Potential ◽  
Regulatory Function ◽  
Disulfonic Acid ◽  
Xenopus Laevis Oocytes ◽  
Slow Recovery ◽  
Bicarbonate Transporter ◽  
Electrode Voltage

We have functionally characterized Na+-driven bicarbonate transporter (NBC)4, originally cloned from human heart by Pushkin et al. (Pushkin A, Abuladze N, Newman D, Lee I, Xu G, and Kurtz I. Biochem Biophys Acta 1493: 215–218, 2000). Of the four NBC4 variants currently present in GenBank, our own cloning efforts yielded only variant c. We expressed NBC4c (GenBank accession no. AF293337 ) in Xenopus laevis oocytes and assayed membrane potential ( V m) and pH regulatory function with microelectrodes. Exposing an NBC4c-expressing oocyte to a solution containing 5% CO2 and 33 mM HCO[Formula: see text]elicited a large hyperpolarization, indicating that the transporter is electrogenic. The initial CO2-induced decrease in intracellular pH (pHi) was followed by a slow recovery that was reversed by removing external Na+. Two-electrode voltage clamp of NBC4c-expressing oocytes revealed large HCO[Formula: see text]- and Na+-dependent currents. When we voltage clamped V m far from NBC4c's estimated reversal potential ( E rev), the pHirecovery rate increased substantially. Both the currents and pHi recovery were blocked by 200 μM 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). We estimated the transporter's HCO[Formula: see text]:Na+ stoichiometry by measuring E rev at different extracellular Na+ concentration ([Na+]o) values. A plot of E rev against log[Na+]o was linear, with a slope of 54.8 mV/log[Na+]o. This observation, as well as the absolute E rev values, are consistent with a 2:1 stoichiometry. In conclusion, the behavior of NBC4c, which we propose to call NBCe2-c, is similar to that of NBCe1, the first electrogenic NBC.

Human primary fibroblasts in vitro express a purinergic P2X7 receptor coupled to ion fluxes, microvesicle formation and IL-6 release

1999 ◽  
Vol 112 (3) ◽  
pp. 297-305
Author(s):  
A. Solini ◽  
P. Chiozzi ◽  
A. Morelli ◽  
R. Fellin ◽  
F. Di Virgilio
Keyword(s):  
Purinergic Receptor ◽  
Morphological Changes ◽  
Human Fibroblasts ◽  
Ion Fluxes ◽  
Disulfonic Acid ◽  
Triggered Release ◽  
Primary Fibroblasts ◽  
Purinergic P2x7 Receptor ◽  
Skin Biopsies

We have investigated reponses to extracellular ATP in human fibroblasts obtained by skin biopsies. Our data show that these cells express a P2X7 purinergic receptor, as judged by (1) RT-PCR with specific primers, (2) reactivity with a specific anti-P2X7 antiserum, (3) activation by the selective P2X agonist benzoylbenzoylATP and (4) stimulation of transmembrane ion fluxes. Stimulation with benzoylbenzoylATP, and to a lesser extent with ATP, also caused striking morphological changes and increased formation of cytoplasmic microvesicles. These changes were fully reversible upon nucleotide removal. Two known blockers of P2X receptors, oxidised ATP and pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid, inhibited the morphological changes fully and the ion fluxes partially. The residual rise in intracellular Ca2+ levels and membrane depolarization observed in the presence of the inhibitors were dependent upon activation of a P2Y-type receptor exhibiting a peculiar pharmacological profile, in that CTP was the preferred agonist. ATP stimulation triggered release of the pro-inflammatory cytokine IL-6 in fibroblasts pre-treated with PMA and bacterial endotoxin. These observations reveal a novel pathway for fibroblast activation and for their recruitment in the inflammatory response.

scholarly journals Regulation of erythropoietin production in a human hepatoblastoma cell line

Blood ◽  
1987 ◽  
Vol 70 (6) ◽  
pp. 1904-1909
Author(s):  
OJ Nielsen ◽  
SJ Schuster ◽  
R Kaufman ◽  
AJ Erslev ◽  
J Caro
Keyword(s):  
Cell Line ◽  
Northern Blot ◽  
Proliferative Activity ◽  
Northern Blot Analysis ◽  
Cobalt Chloride ◽  
Biologically Active ◽  
Erythropoietin Production ◽  
Rna Probe ◽  
Hepatoblastoma Cell Line

Production of immuno and biologically active erythropoietin was documented to occur in the human hepatoblastoma cell line HepG-2. The expression of the erythropoietin gene was further verified by Northern blot analysis using a single stranded RNA probe. In vitro studies showed that erythropoietin production by these cells was not stimulated by hypoxia or cobalt chloride, but was related to the proliferative activity of the cells in culture. In addition it was found that the secretion of erythropoietin was almost completely abrogated by tunicamycin, an inhibitor of N-linked glycosylation. This effect of tunicamycin was also observed in a permanently transfected cell line that secretes erythropoietin in large quantities.

Mechanism of bicarbonate exit across basolateral membrane of rabbit proximal straight tubule

1987 ◽  
Vol 252 (1) ◽  
pp. F11-F18 ◽  
Author(s):  
S. Sasaki ◽  
T. Shiigai ◽  
N. Yoshiyama ◽  
J. Takeuchi
Keyword(s):  
Negative Charge ◽  
Driving Forces ◽  
Basolateral Membrane ◽  
Disulfonic Acid ◽  
Exit Mechanism ◽  
Total Replacement ◽  
Straight Tubules ◽  
Proximal Straight Tubule ◽  
Basolateral Membrane Potential

To clarify the mechanism(s) of HCO3- (or related base) transport across the basolateral membrane, rabbit proximal straight tubules were perfused in vitro, and intracellular pH (pHi) and Na+ activity (aiNa) were measured by double-barreled ion-selective microelectrodes. Lowering bath HCO3- from 25 to 5 mM at constant PCO2 depolarized basolateral membrane potential (Vbl), and reduced pHi. Most of these changes were inhibited by adding 1 mM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) to the bath. Total replacement of bath Na+ with choline also depolarized Vbl and reduced pHi, and these changes were also inhibited by SITS. Reduction in aiNa was observed when bath HCO3- was lowered. Taken together, these findings suggest that HCO3- exists the basolateral membrane with Na+ and negative charge. Calculation of the electrochemical driving forces suggests that the stoichiometry of HCO3-/Na+ must be larger than two for maintaining HCO3- efflux. Total replacement of bath Cl- with isethionate depolarized Vbl gradually and increased pHi slightly, implying the existence of a Cl(-)-related HCO3- exit mechanism. The rate of decrease in pHi induced by lowering bath HCO3- was slightly reduced (20%) by the absence of bath Cl-. Therefore, the importance of Cl(-)-related HCO3- transport is small relative to total basolateral HCO3- exit. Accordingly, these data suggest that most of HCO3- exits the basolateral membrane through the rheogenic Na+/HCO3- cotransport mechanism with a stoichiometry of HCO3-/Na+ of more than two.

Impaired sarcoplasmic reticulum Ca2+ release rate after fatiguing stimulation in rat skeletal muscle

2000 ◽  
Vol 89 (1) ◽  
pp. 210-217 ◽  
Author(s):  
Niels Ørtenblad ◽  
Gisela Sjøgaard ◽  
Klavs Madsen
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Release Rate ◽  
Slow Phase ◽  
Release Mechanism ◽  
Rat Skeletal Muscle ◽  
Slow Recovery ◽  
Tetanic Force ◽  
Longus Muscle ◽  
Two Phases

The purpose of the study was to characterize the sarcoplasmic reticulum (SR) function and contractile properties before and during recovery from fatigue in the rat extensor digitorum longus muscle. Fatiguing contractions (60 Hz, 150 ms/s for 4 min) induced a reduction of the SR Ca2+release rate to 66% that persisted for 1 h, followed by a gradual recovery to 87% of prefatigue release rate at 3 h recovery. Tetanic force and rate of force development (+dF/d t) and relaxation (−dF/d t) were depressed by ∼80% after stimulation. Recovery occurred in two phases: an initial phase, in which during the first 0.5–1 h the metabolic state recovered to resting levels, and a slow phase from 1–3 h characterized by a rather slow recovery of the mechanical properties. The recovery of SR Ca2+ release rate was closely correlated to +dF/d t during the slow phase of recovery ( r 2 = 0.51; P < 0.05). Despite a slowing of the relaxation rate, we did not find any significant alterations in the SR Ca2+ uptake function. These data demonstrate that the Ca2+ release mechanism of SR is sensitive to repetitive in vitro muscle contraction. Moreover, the results indicate that +dF/d t to some extent depends on the rate of Ca2+ release during the slow phase of recovery.

scholarly journals Murine protein tyrosine phosphatase-PEST, a stable cytosolic protein tyrosine phosphatase

1995 ◽  
Vol 308 (2) ◽  
pp. 425-432 ◽  
Author(s):  
A Charest ◽  
J Wagner ◽  
S H Shen ◽  
M L Tremblay
Keyword(s):  
Protein Tyrosine Phosphatase ◽  
Northern Blot Analysis ◽  
Catalytic Domain ◽  
Tyrosine Phosphatase ◽  
Glutathione S Transferase ◽  
Epitope Tag ◽  
Reading Frame ◽  
Protein Tyrosine ◽  
Cytosolic Protein

We have isolated the murine cDNA homologue of the human protein tyrosine phosphatase PTP-PEST (MPTP-PEST) from an 18.5-day mouse embryonic kidney library. The cDNA isolated has a single open reading frame predicting a protein of 775 amino acids. When expressed in vitro as a glutathione S-transferase fusion protein, the catalytic domain (residues 1-453) shows intrinsic phosphatase activity. Reverse transcriptase PCR and Northern-blot analysis show that MPTP-PEST mRNA is expressed throughout murine development. Indirect immunofluorescence in COS-1 cells against a heterologous epitope tag attached to the N-terminus of MPTP-PEST, together with cellular fractionation and Western-blot experiments from different murine cell lines, indicate that MPTP-PEST is a free cytosolic protein of 112 kDa. Finally, sequence analysis indicates that the C-terminal portion of the protein contains four regions rich in proline, glutamate, serine and threonine, otherwise known as PEST sequences. These are characteristic of proteins that display very short intracellular half-lives. Despite the presence of these motifs, pulse-chase labelling experiments demonstrate that MPTP-PEST has a half-life of more than 4 h.

scholarly journals Regioselectivity of glucosylation of caffeic acid by a UDP-glucose:glucosyltransferase is maintained in planta1

2003 ◽  
Vol 373 (3) ◽  
pp. 987-992 ◽  
Author(s):  
Eng-Kiat LIM ◽  
Gillian S. HIGGINS ◽  
Yi LI ◽  
Dianna J. BOWLES
Keyword(s):  
Enzyme Activity ◽  
Caffeic Acid ◽  
Northern Blot Analysis ◽  
Transgenic Arabidopsis ◽  
Structural Features ◽  
Cauliflower Mosaic Virus ◽  
Leaf Extracts ◽  
Transgenic Lines ◽  
First Time

Caffeic acid is a phenylpropanoid playing an important role in the pathways leading to lignin synthesis and the production of a wide variety of secondary metabolites. The compound is also an antioxidant and has potential utility as a general protectant against free radicals. Three glucosylated forms of caffeic acid are known to exist: the 3-O- and 4-O-glucosides and the glucose ester. This study describes for the first time a glucosyltransferase [UDP-glucose:glucosyltransferase (UGT)] that is specific for the 3-hydroxyl, and not the 4-hydroxyl, position of caffeic acid. The UGT sequence of Arabidopsis, UGT71C1, has been expressed as a recombinant fusion protein in Escherichia coli, purified and assayed against a range of substrates in vitro. The assay confirmed that caffeic acid as the preferred substrate when compared with other hydroxycinnamates, although UGT71C1 also exhibited substantial activity towards flavonoid substrates, known to have structural features that can be recognized by many different UGTs. The expression of UGT71C1 in transgenic Arabidopsis was driven by the constitutive cauliflower mosaic virus 35 S (CaMV35S) promoter. Nine independent transgenic lines were taken to homozygosity and characterized by Northern-blot analysis, assay of enzyme activity in leaf extracts and HPLC analysis of the glucosides. The level of expression of UGT71C1 was enhanced considerably in several lines, leading to a higher level of the corresponding enzyme activity and a higher level of caffeoyl-3-O-glucoside. The data are discussed in the context of the utility of UGTs for natural product biotransformations.

scholarly journals VDAC1 is essential for neurite maintenance and the inhibition of its oligomerization protects spinal cord from demyelination and facilitates locomotor function recovery after spinal cord injury

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Vera Paschon ◽  
Beatriz Cintra Morena ◽  
Felipe Fernandes Correia ◽  
Giovanna Rossi Beltrame ◽  
Gustavo Bispo dos Santos ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Cell Death ◽  
Conformational Changes ◽  
Secondary Response ◽  
Disulfonic Acid ◽  
Function Recovery ◽  
Cord Injury

Abstract During the progression of the neurodegenerative process, mitochondria participates in several intercellular signaling pathways. Voltage-dependent anion-selective channel 1 (VDAC1) is a mitochondrial porin involved in the cellular metabolism and apoptosis intrinsic pathway in many neuropathological processes. In spinal cord injury (SCI), after the primary cell death, a secondary response that comprises the release of pro-inflammatory molecules triggers apoptosis, inflammation, and demyelination, often leading to the loss of motor functions. Here, we investigated the functional role of VDAC1 in the neurodegeneration triggered by SCI. We first determined that in vitro targeted ablation of VDAC1 by specific morpholino antisense nucleotides (MOs) clearly promotes neurite retraction, whereas a pharmacological blocker of VDAC1 oligomerization (4, 4′-diisothiocyanatostilbene-2, 2′-disulfonic acid, DIDS), does not cause this effect. We next determined that, after SCI, VDAC1 undergoes conformational changes, including oligomerization and N-terminal exposition, which are important steps in the triggering of apoptotic signaling. Considering this, we investigated the effects of DIDS in vivo application after SCI. Interestingly, blockade of VDAC1 oligomerization decreases the number of apoptotic cells without interfering in the neuroinflammatory response. DIDS attenuates the massive oligodendrocyte cell death, subserving undisputable motor function recovery. Taken together, our results suggest that the prevention of VDAC1 oligomerization might be beneficial for the clinical treatment of SCI.

Electroneutral H+ secretion in distal tubule of Amphiuma

1987 ◽  
Vol 252 (4) ◽  
pp. F691-F699 ◽  
Author(s):  
B. Stanton ◽  
A. Omerovic ◽  
B. Koeppen ◽  
G. Giebisch
Keyword(s):  
Basolateral Membrane ◽  
Apical Cell ◽  
Distal Tubule ◽  
Cell Types ◽  
Disulfonic Acid ◽  
Type I ◽  
Cellular Mechanisms ◽  
Apical Cell Membrane ◽  
Membrane Type

This study examines the cellular mechanisms of acid secretion by the in vitro perfused late distal tubule of Amphiuma kidney. Acidification of tubule fluid occurred against an electrochemical gradient of 16 mV; thus H+ secretion was active. Amiloride (1 mM) or a reduction of sodium in the perfusion fluid (from 83.7 to 7.7 mM) partially reduced acidification. Amiloride, in the presence of low sodium, completely inhibited acidification. Furthermore, acetazolamide and ouabain in the bath solution (0.1 mM) also inhibited acidification. Conductive properties of the epithelium and of individual cell membranes were determined by means of cable analysis of the tubule and intracellular voltage recordings. The transepithelial voltage and resistance averaged -0.4 +/- 0.4 mV, lumen negative, and 7,147 +/- 845 omega X cm, respectively. Two functionally different cell types were identified by intracellular microelectrodes. Type I cells had a basolateral membrane voltage (Vbl) of -67.7 mV. As determined by ion substitution experiments, the basolateral membrane was conductive to K+ and Cl-. This cell also had a 4-acetamido-4'-isothiocyanostilbene-2-2'-disulfonic acid (SITS)-sensitive Na+-dependent HCO3- exit pathway in the basolateral membrane. Type II cells had a Vbl of -76.1 mV (P less than 0.05 vs. type I) and the basolateral membrane was conductive to K+ and Cl- but not to HCO3-. HCO3- movement across the basolateral membrane in this cell may occur by electroneutral Cl- -HCO3- exchange. The apical cell membrane of both cell types did not contain measurable ionic conductances, as evidenced by a high value of apical membrane fractional resistance (0.98 +/- 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

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