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1.
The Na,K-ATPase is a major ion transport protein found in higher eukaryotic cells. The enzyme is composed of two subunits,
α and β, and tissue-specific isoforms exist for each of these, α1, α2 and α3 and β1, β2 and β3. We have proposed that an additional
α isoform, α4, exists based on genomic and cDNA cloning. The mRNA for this gene is expressed in rats and humans, exclusively
in the testis, however the expression of a corresponding protein has not been demonstrated. In the current study, the putative
α4 isoform has been functionally characterized as a novel isoform of the Na,K-ATPase in both rat testis and in α4 isoform
cDNA transfected 3T3 cells. Using an α4 isoform-specific polyclonal antibody, the protein for this novel isoform is detected
for the first time in both rat testis and in transfected cell lines. Ouabain binding competition assays reveal the presence
of high affinity ouabain receptors in both rat testis and in transfected cell lines that have identical K
D
values. Further studies of this high affinity ouabain receptor show that it also has high affinities for both Na+ and K+. The results from these experiments definitively demonstrate the presence of a novel isoform of the Na,K-ATPase in testis.
Received: 4 December 1998/Revised: 1 February 1999 相似文献
2.
Effect of calbindin-D28K on cyclosporine toxicity in cultured renal proximal tubular cells 总被引:3,自引:0,他引:3
Cyclosporine A (CsA) is known to have direct toxicity to renal tubular cells. Its toxicity may be mediated by intracellular calcium because CsA increases intracellular calcium concentration and enhances the activities of calcium-dependent calpains and caspases. Calbindin-D28k, a cytosolic calcium binding protein, has been used as an intracellular Ca2+ buffer to reduce calcium-mediated cytotoxicity in non-renal cells such as neuronal cells. We investigated the effects of gene transfer of calbindin-D28k cDNA on CsA cytotoxicity and intracellular calcium concentration ([Ca2+]i) in cultured murine proximal tubular (MCT) cells. A plasmid containing calbindin-D28k cDNA under the control of CMV promoter was transfected to MCT cells with liposomes. Cytotoxicity was assessed by LDH release and cell viability assay, and [Ca2+]i was measured ratiometrically with fura-2. Compared with MCT cells, cells transfected with calbindin-D28k cDNA showed a reduction in LDH release by 27, 30, 32, 33, and 19% (all P < 0.05), respectively, after 24 h exposure to 1, 2.5, 5, 10, and 25 microM CsA. Cell viability after CsA treatment was also significantly higher in CB cells. A mock transfection using plasmid without calbindin-D28k cDNA insert did not affect the LDH release or cell viability after CsA treatment. CsA treatment did not affect the protein and mRNA abundance of transfected calbindin-D28k cDNA. The expression of calbindin-D28k did not affect the baseline [Ca2+]i, but significantly suppressed CsA-induced elevation in [Ca2+]i. The expression of calbindin-D28k in renal tubular cells provides cytoprotective effects against CsA toxicity, probably through its buffering effects on [Ca2+]i. 相似文献
3.
Unidirectional, ouabain-insensitive K+ influx rose steeply with warming at temperatures above 37°C in guinea pig erythrocytes incubated in isotonic medium. The
only component of ouabain-insensitive K+ influx to show the same steep rise was K-Cl cotransport (Q10 of 10 between 37 and 41°C); Na-K-Cl cotransport remained constant or declined and residual K+ influx in hypertonic medium with ouabain and bumetanide rose only gradually. Similar results were obtained for unidirectional
K+ efflux.
Thermal activation of K-Cl cotransport-mediated K+ influx was fully dependent on the presence of chloride in the medium; none occurred with nitrate replacing chloride. The
increase of K+ influx through K-Cl cotransport from 37 to 41°C was blocked by calyculin A, a phosphatase inhibitor.
The Q10 of K-Cl cotransport fully activated by hydroxylamine and hypotonicity was about 2. The time course of K+ entry showed an immediate transition to a higher rate when cells were instantly warmed from 37 to 41°C, but there was a 7-min
time lag in returning to a lower rate when cells were cooled from 41 to 37°C. These results indicate that the steepness of
the response of K-Cl cotransport to mild warming is due to altered regulation of the transporter.
Total unidirectional K+ influx was equal to total unidirectional K+ efflux at 37–45°C, but K+ influx exceeded K+ efflux at 41°C when K-Cl cotransport was inhibited by calyculin or prevented by hypertonic incubation. The net loss of K+ that results from the thermal activation of isosomotic K-Cl cotransport reported here would offset a tendency for cell swelling
that could arise with warming through an imbalance of pump and leak for Na+ or for K+.
Received: 1 November 1997/Revised: 5 March 1998 相似文献
4.
R. Toriano P. Ford V. Rivarola B.K. Tamarappoo A.S. Verkman M. Parisi 《The Journal of membrane biology》1998,161(2):141-149
Transepithelial water permeability was measured in LLC-PK1 cells stably transfected with aquaporins (AQPs): AQP1, AQP2, and
a chimera of AQP1 and AQP2 containing 41 amino acids of the C-terminus of AQP2. Transepithelial water fluxes (Jw) were not
previously reported in cells transfected with aquaporins. Jw were now recorded each minute using a specially developed experimental
device. A significant increase in Posm after forskolin (FK) plus vasopressin (VP) was found in AQP2 transfected cells (39.9
± 8.2 vs. 12.5 ± 3.3 cm · sec−1· 10−3), but not in cells transfected with AQP1 (15.3 ± 3.6 vs. 13.4 ± 3.6 cm · sec−1· 10−3). In the case of the AQP1/2 cells (chimera) the FK plus VP induced Posm was smaller than in AQP2 cells but significantly
higher than in mock cells at rest (18.1 ± 4.8 vs. 6.7 ± 1.0 cm · sec−1· 10−3). The increases in Posm values were not paralleled by increases in 14C-Mannitol permeability. HgCl2 inhibited the hydrosmotic response to FK plus VP in AQP2 transfected epithelia. Results were comparable to those observed,
in parallel experiments, in a native ADH-sensitive water channel containing epithelial barrier (the toad urinary bladder).
Electron microscopy showed confluent LLC-PK1 cells with microvilli at the mucosal border. The presence of spherical or elongated
intracellular vacuoles was observed in AQP2 transfected cells, specially after FK plus VP stimulus and under an osmotic gradient.
These results demonstrate regulated transepithelial water permeability in epithelial cells transfected with AQP2.
Received: 24 June 1997/Revised: 16 September 1997 相似文献
5.
M Kobayashi M Sugibayashi T Sasaoka K Egawa Y Shigeta M Tamaki E Nakamura H Teraoka 《Biochemical and biophysical research communications》1990,167(3):1073-1078
To study whether the G----T point mutation of insulin proreceptors at the cleavage site which changed -Arg-Lys-Arg-Arg- to -Arg-Lys-Arg-Ser- caused unprocessed insulin receptors with decreased insulin binding affinity, we performed transfection of cDNA with the mutation in COS 7 cells and examined the expressed insulin receptors. After site-directed mutagenesis, an expression vector pGEM3SV was used to make a plasmid which contained full-length HIRcDNA behind SV40 early promoter. Transfection of normal HIR cDNA produced normal insulin receptors on the plasma membranes in COS 7 cells. However, transfection of cDNA with the mutation resulted in the presence of 210K proreceptors in the plasma membranes with decreased insulin binding ability (35% of normal). These results suggest that the mutation, not the defect of converting enzyme, was the cause for unprocessed insulin proreceptors in the patients with insulin resistance. 相似文献
6.
Bending the MDCK Cell Primary Cilium Increases Intracellular Calcium 总被引:29,自引:0,他引:29
We tested the hypothesis that the primary cilium of renal epithelia is mechanically sensitive and serves as a flow sensor
in MDCK cells using differential interference contrast and fluorescence microscopy. Bending the cilium, either by suction
with a micropipette or by increasing the flow rate of perfusate, causes intracellular calcium to substantially increase as
indicated by the fluorescent indicator, Fluo-4. This calcium signal is initiated by Ca2+-influx through mechanically sensitive channels that probably reside in the cilium or its base. The influx is followed by
calcium release from IP3-sensitive stores. The calcium signal then spreads as a wave from the perturbed cell to its neighbors by diffusion of a second
messenger through gap junctions. This spreading of the calcium wave points to flow sensing as a coordinated event within the
tissue, rather than an isolated phenomenon in a single cell. Measurement of the membrane potential difference by microelectrode
during perfusate flow reveals a profound hyperpolarization during the period of elevated intracellular calcium. We conclude
that the primary cilium in MDCK cells is mechanically sensitive and responds to flow by greatly increasing intracellular calcium.
Received: 4 April 2001/Revised: 28 June 2001 相似文献
7.
Subjecting Saccharomyces cerevisiae cells to a hypotonic downshift by transferring cells from YPD medium containing 0.8 m sorbitol to YPD medium without sorbitol induces a transient rapid influx of Ca2+ and other divalent cations into the cell. For cells grown in YPD at 37°C, this hypotonic downshift increases Ca2+ accumulation 6.7-fold. Hypotonic downshift-induced Ca2+ accumulation and steady-state Ca2+ accumulation in isotonic YPD medium are differentially affected by dodecylamine and Mg2+. The Ca2+-influx pathway responsible for hypotonic-induced Ca2+ influx may account for about 10–35% of Ca2+ accumulation by cells growing in YPD. Ca2+ influx is not required for cells to survive a hypotonic downshift. Hypotonic downshift greatly reduces the ability of S. cerevisiae cells to survive a 5-min exposure to 10 mm Cd2+ suggesting that mutants resistant to acute Cd2+ exposure may help identify genes required for hypotonic downshift-induced divalent cation influx.
Received: 14 January 1997/Revised: 20 June 1997 相似文献
8.
9.
Although expression vectors using viral and mammalian promoters constitutively express genes of interest in adherent cells, few studies have examined whether the function of these vectors in suspended cells, such as in over-agar or soft agar assay (an in vitro cell transformation assay), is as robust as when they are in adherent cells. The selection of appropriate expression vector to optimally express genes in suspended cells would be useful in determining whether these genes play a critical role in maintaining colony formation or cell transformation. To compare promoter-driven expression vector function in adherent versus suspension cells, we performed transient transfection assays using viral (simian virus 40 [SV40] and cytomegalovirus [CMV]) and mammalian (beta-actin) promoters fused to luciferase or beta-galactosidase reporter gene. Over-agar assay was used to suspend cells on top of agar, which allowed cell retrieval and analysis. We found that beta-actin and SV40 promoters exhibited suppressed gene expression of 70 and 56%, respectively, in cells suspended on agar compared with those attached on plates. The suppressed response by the exogenous beta-actin promoter in suspension was consistent with the response of the endogenous beta-actin promoter activity because the steady-state level of beta-actin messenger ribonucleic acid in suspended cells was significantly reduced by 50% relative to that expressed in attached cells. In contrast to SV40 promoter, CMV promoter activity was not decreased in cells suspended in over-agar when compared with adherent cells. These studies show that regardless of mammalian or viral vectors, one cannot assume that all expression vectors behave similarly in both suspension and adherent state. 相似文献
10.
Santa Spaggiare Michael J. Wallach Joseph T. Tupper 《Journal of cellular physiology》1976,89(3):403-416
The components of unidirectional K influx and efflux have been investigated in the 3T3 cell and the SV40 transformed 3T3 cell in exponential and stationary growth phase. Over the cell densities used for transport experiments the 3T3 cell goes from exponential growth to density dependent inhibition of growth (4 × 104 to 4 × 105 cell cm?2) whereas the SV40 3T3 maintains exponential or near exponential growth (4 × 104 to 1 × 106 cell cm?2). In agreement with previous observations, volume per cell and mg protein per cell decrease with increasing cell density. Thus, transport measurements have been expressed on a per volume basis. Total unidirectional K influx and efflux in the 3T3 cell is approximately double that of the SV40 3T3 cell at all cell densities investigated. Both cell types have similar volumes initially and show similar decreases with increasing cell density. Thus, in this clone of the 3T3 cell SV40 transformation specifically decreases unidirectional K flux. The magnitude of the total K flux does not change substantially for either cell line during transition from sparse to dense cultures. However, the components of the K transport undergo distinct changes. Both cell lines possess a ouabain sensitive component of K influx, presumably representing the active inward K pump. Both also possess components of K influx and efflux sensitive to furosemide. The data suggest this component represents a one-for-one K exchange mechanism. The fraction of K influx mediated by the ouabain sensitive component is reduced to one half its value when exponential versus density inhibited 3T3 cells are compared (63% versus 31% of total influx). No comparable drop occurs in the SV40 3T3 cell at equivalent cell densities (64% versus 56% of total influx). Thus, the pump mediated component of K influx would appear to be correlated with growth. In contrast, the furosemide sensitive component represents approximately 20% of the total unidirectional K influx and efflux in both cell lines in sparse culture. At high cell densities, where growth inhibition occurs in the 3T3 cell but not the SV40 3T3, the furosemide sensitive component doubles in both cell lines. Thus, the apparent K-K exchange mechanism is density dependent rather than growth dependent. 相似文献
11.
Effect of ouabain upon diuretic-sensitive K+ transport in cultured cells. Evidence for separate modes of operation of the transporter 总被引:3,自引:0,他引:3
(1) Unidirectional K+ (86Rb) influx and efflux were measured in subconfluent layers of MDCK renal epithelial cells and HeLa carcinoma cells. (2) In both MDCK and HeLa cells, the furosemide-inhibitable and chloride-dependent component of K+ influx/efflux was stimulated 2-fold by a 30 min incubation in 1 . 10(-3) M ouabain. (3) Measurements of net K+ loss and Na+ gain in ouabain-treated cells at 1 h failed to show any diuretic sensitive component, confirming the exchange character of the diuretic-sensitive fluxes. (4) Prolonged incubations for 2.5 h in ouabain revealed a furosemide- and anion-dependent K+ (Cl-) outward net flux uncoupled from net Na+ movement. Net K+ (Cl-) outward flux was half-maximally inhibited by 2 microM furosemide. (5) After 2.5 h ouabain treatment, the anion and cation dependence of the diuretic-sensitive K+ influx/efflux were essentially unchanged when compared to untreated controls. 相似文献
12.
13.
《Journal of biotechnology》1988,9(1):61-70
Chinese hamster ovary (CHO) cells were transfected with a human interleukin 4 (IL-4) expression plasmid in which human IL-4 cDNA is linked downstream of the human cytomegalovirus/human immunodeficiency virus chimeric promoter. The plasmid also contained a mouse dihydrofolate reductase (dhfr) gene, expression of which is directed by the SV40 early promoter. The resulting methotrexate-resistant, transformed cells constitutively secreted a high level of human IL-4. CHO cells producing human IL-4 were cultured on microcarriers in a perfusion cell culture system containing 1 l of culture medium, and a high level of human IL-4 (5 × 104U ml−1) was produced at a high cell density (1 × 107cells per ml). Serum-free culture was also examined. 相似文献
14.
Alkaline phosphatase as a reporter enzyme 总被引:3,自引:0,他引:3
This study examines the use of alkaline phosphatase (AP) as a reporter enzyme. We constructed a plasmid containing the cDNA which encodes the bone/liver/kidney rat AP under the control of the simian virus 40 (SV40) early promoter and used it to transfect Chinese hamster ovary, SV40-transformed African Green Monkey kidney 7, and rat osteosarcoma 25/1 mammalian cells. AP activity in these cells, measured three days later, was 40-400-fold above background. When AP and chloramphenicol acetyltransferase (CAT) plasmids were cotransfected, the detection of AP activity by a simple spectrophotometric assay was at least as sensitive as the detection of CAT activity using a radioactive substrate. Moreover, since mammalian AP is a membrane-bound ectoenzyme, transfected cells can be visualized by histochemical staining. This approach was used to estimate transfection efficiency. The convenient methods for AP detection should make it a useful reporter enzyme. 相似文献
15.
Transfection efficiency in reporter gene assays is usually determined by cotransfection of a reference reporter gene under the control of a constitutively active strong promoter and determination of the reference enzyme activity. The SV40 promoter-driven beta-galactosidase reporter plasmid is frequently used as the reference reporter plasmid. Here we show that the beta-galactosidase expression in different cell lines does not correctly reflect the amount of plasmid taken up by cells and thus is not an accurate measure of transfection efficiency. The direct determination of introduced plasmid concentration in lysates of transfected cells is suitable for monitoring the transfection efficiency in reporter gene assays even if different cell lines are compared. 相似文献
16.
The effect of interferon on the expression of the vesicular stomatitis virus glycoprotein G gene was examined in simian COS cells transfected with the expression vector pSVGL containing the G gene under the control of the SV40 late promoter. When COS cells were treated with interferon 24 h after transfection, the synthesis of vesicular stomatitis virus G protein was inhibited by about 80% as compared to that in untreated controls. By contrast, under the same conditions, neither the plasmid copy number nor the G gene mRNA levels were detectably affected by interferon treatment. Likewise, the synthesis of simian virus 40 large T-antigen was not inhibited by interferon treatment of transfected COS cells even though the synthesis of vesicular stomatitis virus G protein was markedly inhibited. The residual G protein synthesized in transfected, interferon-treated COS cells appeared to be normally glycosylated. 相似文献
17.
E. Garcia-Valenzuela R. Rayanade J. C. Perales C. A. Davidson R. W. Hanson S. C. Sharma 《Developmental neurobiology》1997,32(1):111-112
Modification of the intracellular functions of mature neurons through specific gene transfer has many potential applications. Here we present a new methodology for the successful transfection of retinal ganglion cells by administration of plasmid at the cut end of the optic nerve, or at their intact axon terminals; the latter is significantly more efficient. Plasmids contained either the SV40 promoter linked to the luciferase gene, or the CMV or RSV promoter linked to the lacZ gene. Assays for both reporter genes demonstrated significant expression of exogenous DNA in the retina for at least 10 days after retrograde transport. Duration of expression was extended to 20 days or more (duration of the experiment) when plasmid DNA was condensed with poly(L-lysine). β-Galactosidase analysis revealed transfection of ganglion cells in high numbers. Such an approach for gene delivery to specific subpopulations of neurons might be useful in studies of molecular functions in vivo and as an experimental therapeutic strategy to extend survival and restore their function. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 111–122, 1997. 相似文献
18.
Sequence requirements for plasmid nuclear import 总被引:8,自引:0,他引:8
The nuclear envelope is a major barrier for nuclear uptake of plasmids and represents one of the most significant unsolved problems of nonviral gene delivery. We have previously shown that the nuclear entry of plasmid DNA is sequence-specific, requiring a 366-bp fragment containing the SV40 origin of replication and early promoter. In this report, we show that, although fragments throughout this region can support varying degrees of nuclear import, the 72-bp repeats of the SV40 enhancer facilitate maximal transport. The functions of the promoter and the origin of replication are not needed for nuclear localization of plasmid DNA. In contrast to the import activity of the SV40 enhancer, two other strong promoter and enhancer sequences, the human cytomegalovirus (CMV) immediate-early promoter and the Rous sarcoma virus LTR, were unable to direct nuclear localization of plasmids. The inability of the CMV promoter to mediate plasmid nuclear import was confirmed by measurement of the CMV promoter-driven expression of green fluorescent protein (GFP) in microinjected cells. At times before cell division, as few as 3 to 10 copies per cell of cytoplasmically injected plasmids containing the SV40 enhancer gave significant GFP expression, while no expression was obtained with more than 1000 copies per cell of plasmids lacking the SV40 sequence. However, the levels of expression were the same for both plasmids after cell division in cytoplasmically injected cells and at all times in nuclear injected cells. Thus, the inclusion this SV40 sequence in nonviral vectors may greatly increase their ability to be transported into the nucleus, especially in nondividing cells. 相似文献
19.
cDNA encoding the full-length hKv1.3 lymphocyte channel and a C-terminal truncated (Δ459-523) form that lacks the putative
PKA Ser468 phosphorylation site were stably transfected in human embryonic kidney (HEK) 293 cells. Immunostaining of the transfected
cells revealed a distribution at the plasma membrane that was uniform in the case of the full-length channel whereas clustering
was observed in the case of the truncated channel. Some staining within the cell cytoplasm was found in both instances, suggesting
an active process of biosynthesis. Analyses of the K+ current by the patch-clamp technique in the whole cell configuration showed that depolarizing steps to 40 mV from a holding
potential (HP) of −80 mV elicited an outward current of 2 to 10 nA. The current threshold was positive to −40 mV and the current
amplitude increased in a voltage-dependent manner. The parameters of activation were −5.7 and −9.9 mV (slope factor) and −35
mV (half activation, V
0.5) in the case of the full-length and truncated channels, respectively. The characteristics of the inactivation were 14.2 and
24.6 mV (slope factor) and −17.3 and −39.0 mV (V
0.5) for the full-length and truncated channels, respectively. The activation time constant of the full-length channel for potentials
ranging from −30 to 40 mV decreased from 18 to 12 msec whereas the inactivation time constant decreased from 6600 msec at
−30 mV to 1800 msec at 40 mV. The unit current amplitude measured in cells bathing in 140 mm KCl was 1.3 ± 0.1 pA at 40 mV, the unit conductance, 34.5 pS and the zero current voltage, 0 mV. Both forms of the channels
were inhibited by TEA, 4-AP, Ni2+ and charybdotoxin. In contrast to the native (Jurkat) lymphocyte Kv1.3 channel that is fully inhibited by PKA and PKC, the
addition of TPA resulted in 34.6 ± 7.3% and 38.7 ± 9.4% inhibition of the full-length and the truncated channels, respectively.
8-BrcAMP induced a 39.4 ± 5.4% inhibition of the full-length channel but had no effect (8.6 ± 8.3%) on the truncated channel.
Cell dialysis with alkaline phosphatase had no effects, suggesting that the decreased sensitivity of the transfected channels
to PKA and PKC was not due to an already phosphorylated channel. Patch extract experiments suggested that the hKv1.3 channel
was partially sensitive to PKA and PKC. Cotransfecting the Kvβ1.2 subunit resulted in a decrease in the value of the time
constant of inactivation of the full-length channel but did not modify its sensitivity to PKA and PKC. The cotransfected Kvβ2
subunit had no effects. Our results indicate that the hKv1.3 lymphocyte channel retains its electrophysiological characteristics
when transfected in the Kvβ-negative HEK 293 cell line but its sensitivity to modulation by PKA and PKC is significantly reduced.
Received: 18 June 1997/Revised: 7 October 1997 相似文献