scholarly journals Human Rhesus B and Rhesus C glycoproteins: properties of facilitated ammonium transport in recombinant kidney cells

2005 ◽  
Vol 391 (1) ◽  
pp. 33-40 ◽  
Author(s):  
Nedjma Zidi-Yahiaoui ◽  
Isabelle Mouro-Chanteloup ◽  
Anne-Marie D'Ambrosio ◽  
Claude Lopez ◽  
Pierre Gane ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Ammonium Transport ◽  
Kidney Cells ◽  
Wild Type ◽  
Kinetic Rate Constant ◽  
Kinetic Rate ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
Ph Variation ◽  
Rh Glycoproteins

The mammalian Rh (Rhesus) protein family belongs to the Amt/Mep (ammonia transporter/methylammonium permease)/Rh superfamily of ammonium transporters. Whereas RhCE, RhD and RhAG are erythroid specific, RhBG and RhCG are expressed in key organs associated with ammonium transport and metabolism. We have investigated the ammonium transport function of human RhBG and RhCG by comparing intracellular pH variation in wild-type and transfected HEK-293 (human embryonic kidney) cells and MDCK (Madin–Darby canine kidney) cells in the presence of ammonium (NH4+/NH3) gradients. Stopped-flow spectrofluorimetry analysis, using BCECF [2′,7′-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein] as a pH-sensitive probe, revealed that all cells submitted to inwardly or outwardly directed ammonium gradients exhibited rapid alkalinization or acidification phases respectively, which account for ammonium movements in transfected and native cells. However, as compared with wild-type cells known to have high NH3 lipid permeability, RhBG- and RhCG-expressing cells exhibited ammonium transport characterized by: (i) a five to six times greater kinetic rate-constant; (ii) a weak temperature-dependence; and (iii) reversible inhibition by mercuric chloride (IC50: 52 μM). Similarly, when subjected to a methylammonium gradient, RhBG- and RhCG-expressing cells exhibited kinetic rate constants greater than those of native cells. However, these constants were five times higher for RhBG as compared with RhCG, suggesting a difference in substrate accessibility. These results, indicating that RhBG and RhCG facilitate rapid and low-energy-dependent bi-directional ammonium movement across the plasma membrane, favour the hypothesis that these Rh glycoproteins, together with their erythroid homologue RhAG [Ripoche, Bertrand, Gane, Birkenmeier, Colin and Cartron (2005) Proc. Natl. Acad. Sci. U.S.A. 101, 17222–17227] constitute a family of NH3 channels in mammalian cells.

Cloning, characterization, and gene organization of K-Cl cotransporter from pig and human kidney and C. elegans

1998 ◽  
Vol 275 (4) ◽  
pp. F550-F564 ◽  
Author(s):  
Eli J. Holtzman ◽  
Sumit Kumar ◽  
Carol A. Faaland ◽  
Fern Warner ◽  
Paul J. Logue ◽  
...  
Keyword(s):  
Amino Acids ◽  
Human Kidney ◽  
Gene Organization ◽  
Kidney Cells ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
C Elegans ◽  
Transmembrane Segments ◽  
Extracellular Loops ◽  
Stimulation Of

We isolated and characterized the cDNAs for the human, pig, and Caenorhabditis elegansK-Cl cotransporters. The pig and human homologs are 94% identical and contain 1,085 and 1,086 amino acids, respectively. The deduced protein of the C. elegans K-Cl cotransporter clone (CE-KCC1) contains 1,003 amino acids. The mammalian K-Cl cotransporters share ∼45% similarity with CE-KCC1. Hydropathy analyses of the three clones indicate typical KCC topology patterns with 12 transmembrane segments, large extracellular loops between transmembrane domains 5 and 6 (unique to KCC), and large COOH-terminal domains. Human KCC1 is widely expressed among various tissues. This KCC1 gene spans 23 kb and is organized in 24 exons, whereas the CE-KCC1 gene spans 3.5 kb and contains 10 exons. Transiently and stably transfected human embryonic kidney cells (HEK-293) expressing the human, pig, and C. elegans K-Cl cotransporter fulfilled two (pig) or five (human and C. elegans) criteria for increased expression of the K-Cl cotransporter. The criteria employed were basal K-Cl cotransport; stimulation of cotransport by swelling, N-ethylmaleimide, staurosporine, and reduced cell Mg concentration; and secondary stimulation of Na-K-Cl cotransport.

scholarly journals Replication Past the -Radiation-Induced Guanine-Thymine Cross-Link G[8,5-Me]T by Human and Yeast DNA Polymerase

2010 ◽  
Vol 2010 ◽  
pp. 1-10 ◽  
Author(s):  
Paromita Raychaudhury ◽  
Ashis K. Basu
Keyword(s):  
Dna Polymerase ◽  
Mammalian Cells ◽  
Translesion Synthesis ◽  
Kidney Cells ◽  
Equal Frequency ◽  
Cross Link ◽  
Dominant Mutation ◽  
Human Embryonic Kidney Cells ◽  
Radiation Induced

-Radiation-induced intrastrand guanine-thymine cross-link, G[8,5-Me]T, hinders replicationin vitroand is mutagenic in mammalian cells. Herein we reportin vitrotranslesion synthesis of G[8,5-Me]T by human and yeast DNA polymerase (hPol and yPol ). dAMP misincorporation opposite the cross-linked G by yPol was preferred over correct incorporation of dCMP, but further extension was 100-fold less efficient for :A compared to :C. For hPol , both incorporation and extension were more efficient with the correct nucleotides. To evaluate translesion synthesis in the presence of all four dNTPs, we have developed a plasmid-based DNA sequencing assay, which showed that yPol was more error-prone. Mutational frequencies of yPol and hPol were 36% and 14%, respectively. Targeted was the dominant mutation by both DNA polymerases. But yPol induced targeted in 23% frequency relative to 4% by hPol . For yPol , targeted and constituted 83% of the mutations. By contrast, with hPol , semi-targeted mutations (7.2%), that is, mutations at bases near the lesion, occurred at equal frequency as the targeted mutations (6.9%). The kind of mutations detected with hPol showed significant similarities with the mutational spectrum of G[8,5-Me]T in human embryonic kidney cells.

scholarly journals Multidrug resistance-associated protein 9 (ABCC12) is present in mouse and boar sperm

2007 ◽  
Vol 406 (1) ◽  
pp. 31-40 ◽  
Author(s):  
Nobuhito Ono ◽  
Ingrid Van der Heijden ◽  
George L. Scheffer ◽  
Koen Van de Wetering ◽  
Elizabeth Van Deemter ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Kidney Cells ◽  
Cell Fractionation ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
Hek 293 Cells ◽  
Drug Conjugates ◽  
293 Cells ◽  
Boar Sperm ◽  
Alternatively Spliced

The human and murine genes for MRP9 (multidrug resistance-associated protein 9; ABCC12) yield many alternatively spliced RNAs. Using a panel of monoclonal antibodies, we detected full-length Mrp9 only in testicular germ cells and mouse sperm; we obtained no evidence for the existence of the truncated 100 kDa MRP9 protein reported previously. In contrast with other MRPs, neither murine Mrp9 nor the human MRP9 produced in MRP9-transfected HEK-293 cells (human embryonic kidney cells) appears to contain N-linked carbohydrates. In mouse and boar sperm, Mrp9 localizes to the midpiece, a structure containing all sperm mitochondria. However, immunolocalization microscopy and cell fractionation studies with transfected HEK-293 cells and mouse testis show that MRP9/Mrp9 does not localize to mitochondria. In HEK-293 cells, it is predominantly localized in the endoplasmic reticulum. We have been unable to demonstrate transport by MRP9 of substrates transported by other MRPs, such as drug conjugates and other organic anions.

Extracellular signal-regulated kinase activation by parathyroid hormone in distal tubule cells

2007 ◽  
Vol 292 (3) ◽  
pp. F1028-F1034 ◽  
Author(s):  
W. Bruce Sneddon ◽  
Yanmei Yang ◽  
Jianming Ba ◽  
Lisa M. Harinstein ◽  
Peter A. Friedman
Keyword(s):  
Conditioned Media ◽  
Kidney Cells ◽  
Wild Type ◽  
Egfr Transactivation ◽  
Hek 293 ◽  
Erk Activation ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Extracellular Signal

The PTH receptor (PTH1R) activates multiple signaling pathways, including extracellular signal-regulated kinases 1 and 2 (ERK1/2). The role of epidermal growth factor receptor (EGFR) transactivation in ERK1/2 activation by PTH in distal kidney cells, a primary site of PTH action, was characterized. ERK1/2 phosphorylation was stimulated by PTH and blocked by the EGFR inhibitor, AG1478. Upon PTH stimulation, metalloprotease cleavage of membrane-bound heparin-binding fragment (HB-EGF) induced EGFR transactivation of ERK. Conditioned media from PTH-treated distal kidney cells activated ERK in HEK-293 cells. AG1478 added to HEK-293 cells ablated transactivation by conditioned media. HB-EGF directly activated ERK1/2 in HEK-293 cells. Pretreatment of distal kidney cells with the metalloprotease inhibitor GM-6001 abolished transactivation of ERK1/2 by PTH. The role of the PTH1R COOH terminus in PTX-sensitive ERK1/2 activation was characterized in HEK-293 cells transfected with wild-type PTH1R, with a PTH1R mutated at its COOH terminus, or with PTH1R truncated at position 480. PTH stimulated ERK by wild-type, mutated and truncated PTH1Rs 21-, 27- and 57-fold, respectively. Thus, the PTH1R COOH terminus exerts an inhibitory effect on ERK activation. EBP50, a scaffolding protein that binds to the PDZ recognition domain of the PTH1R, impaired PTH but not isoproterenol or calcitonin-induced ERK activation. Pertussis toxin inhibited PTH-stimulated ERK1/2 by mutated and truncated PTH1Rs and abolished ERK1/2 activation by wild-type PTH1R. We conclude that ERK phosphorylation in distal kidney cells by PTH requires PTH1R activation of Gi, which leads to stimulation of metalloprotease-mediated cleavage of HB-EGF and transactivation of the EGFR and is regulated by EBP50.

scholarly journals Caffeine induces Ca2+ release by reducing the threshold for luminal Ca2+ activation of the ryanodine receptor

2008 ◽  
Vol 414 (3) ◽  
pp. 441-452 ◽  
Author(s):  
Huihui Kong ◽  
Peter P. Jones ◽  
Andrea Koop ◽  
Lin Zhang ◽  
Henry J. Duff ◽  
...  
Keyword(s):  
Ryanodine Receptor ◽  
Cardiac Arrhythmias ◽  
Single Channel ◽  
Kidney Cells ◽  
Human Embryonic Kidney ◽  
Activation Threshold ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
293 Cells ◽  
High Concentrations

Caffeine has long been used as a pharmacological probe for studying RyR (ryanodine receptor)-mediated Ca2+ release and cardiac arrhythmias. However, the precise mechanism by which caffeine activates RyRs is elusive. In the present study, we investigated the effects of caffeine on spontaneous Ca2+ release and on the response of single RyR2 (cardiac RyR) channels to luminal or cytosolic Ca2+. We found that HEK-293 cells (human embryonic kidney cells) expressing RyR2 displayed partial or ‘quantal’ Ca2+ release in response to repetitive additions of submaximal concentrations of caffeine. This quantal Ca2+ release was abolished by ryanodine. Monitoring of endoplasmic reticulum luminal Ca2+ revealed that caffeine reduced the luminal Ca2+ threshold at which spontaneous Ca2+ release occurs. Interestingly, spontaneous Ca2+ release in the form of Ca2+ oscillations persisted in the presence of 10 mM caffeine, and was diminished by ryanodine, demonstrating that unlike ryanodine, caffeine, even at high concentrations, does not hold the channel open. At the single-channel level, caffeine markedly reduced the threshold for luminal Ca2+ activation, but had little effect on the threshold for cytosolic Ca2+ activation, indicating that the major action of caffeine is to reduce the luminal, but not the cytosolic, Ca2+ activation threshold. Furthermore, as with caffeine, the clinically relevant, pro-arrhythmic methylxanthines aminophylline and theophylline potentiated luminal Ca2+ activation of RyR2, and increased the propensity for spontaneous Ca2+ release, mimicking the effects of disease-linked RyR2 mutations. Collectively, our results demonstrate that caffeine triggers Ca2+ release by reducing the threshold for luminal Ca2+ activation of RyR2, and suggest that disease-linked RyR2 mutations and RyR2-interacting pro-arrhythmic agents may share the same arrhythmogenic mechanism.

scholarly journals Evaluation of the effect of montelukast on cadmium induced toxicity in human embryonic kidney cells (HEK-293)

2019 ◽  
Vol 24 (4) ◽  
pp. 125-137
Author(s):  
Farnoosh Kaviani ◽  
Seyedeh Missagh Jalali ◽  
Elham Hoveizi ◽  
Javad Jamshidian ◽  
Masoomeh Ahmadizadeh ◽  
...  
Keyword(s):  
Kidney Cells ◽  
Human Embryonic Kidney ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells

Non-Sense-Mediated mRNA Decay in ADAMTS13 Gene Caused by 29 Nucleotide Deletion.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1061-1061
Author(s):  
Isabella Garagiola ◽  
Silvia Lavoretano ◽  
Hale Oren ◽  
Martina Bohm ◽  
Pier Mannuccio Mannucci ◽  
...  
Keyword(s):  
Hemolytic Anemia ◽  
Mammalian Cells ◽  
Expression Vector ◽  
Mrna Level ◽  
Gene Mutations ◽  
Wild Type ◽  
Hek 293 ◽  
Decay Mechanism ◽  
Nucleotide Deletion ◽  
Cell Supernatant

Abstract Genetic or acquired deficiency of ADAMTS13 causes thrombotic thrombocytopenic purpura (TTP), a condition characterized by thrombocytopenia and hemolytic anemia with microvascular platelet thrombi. Several mutations spread across the ADAMTS13 gene have been identified in patients with congenital TTP. We analyzed the ADAMTS13 gene in two Turkish brothers affected by hereditary TTP. One of them is a 23 years old male, who experienced two acute TTP episodes and is currently in prophylactic treatment with plasma infusion once a month. His sister has mild thrombocytopenia and a mild hemolytic anemia but never had any acute TTP episode.Their ADAMTS13 antigen level and activity in plasma were found to be <1% and <6% respectively. Genetic analysis revealed that both patients are double heterozygote for a 29 nucleotide deletion in exon 3 (291–319del) leading to a premature stop at codon 368 in the metalloprotease domain (Peyvandi,2004) and a single base insertion in exon 29 (4143–4144insA) in the second CUB domain resulting in the loss of the 49 amino acids (Schneppenheim,2003). To evaluate the molecular mechanism of these mutations, we cloned the ADAMTS13 cDNA into pcDNA3.1 expression vector and introduced each mutation using the wild type cDNA as template (pcDNA3.1ADAMTS13insA and pcDNA3.1ADAMTS13–29del). These constructs were transiently transfected in HEK-293 cells. Expression studies demonstrated that the 4143–4144insA mutation leads to a secretion defect resulting in a reduced protease activity, measured by CBA in cell supernatant (14% of wild type activity), as reported by Pimanda (2004).The intracellular accumulation was demonstrated by WB analysis and confirmed by immunofluorescence studies showing that rADAMTS13insA was present throughout the cytoplasm while the rADAMTS13WT was mainly localized in the perinuclear area.Expression studies of 29bp deletion showed the absence of recombinant protein in cell supernatant analyzed by WB. Previous studies showed that in mammalian cells exists a regulatory mechanism (PTC-mediated decay mechanism) that triggers the destruction of PTC-bearing mRNAs and a reduction in m-RNA levels, and this decay mechanism is intron-dependent (Zhang, 1998). The 29bp deletion leads to PTC in the metalloprotease domain. To verify whether the PTC introduced by 29bp deletion could lead to degradation and decrease of mRNA level, we have constructed a minigene extending from exon 4 to exon 6 of ADAMTS13 gene including introns 4 and 5. Subsequently this fragment was subcloned in frame into pcDNA3.1ADAMTS13–29del expression vector. Reverse transcription PCR was performed on total mRNA isolated from cells transfected with ADAMTS13-WT and ADAMTS13–29del constructs.The band obtained by RT-PCR of ADAMTS13–29del mRNA was fainter than that ADAMTS13WT (roughly 10%) suggesting that the presence of PTC due to the 29bp deletion triggers probably a decay process leading to a reduction of ADAMTS13 mRNA level. In conclusion, our work shows how the association of two different ADAMTS13 gene mutations could lead to a severe ADAMTS13 deficiency, with effects on two different levels: a partial impairment of mRNA synthesis due to 29bp deletion, and an alteration of the secretion pathway caused by 4143–4144insA.

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