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At least 253 records · Page 14Linked to original sources
PubMed1997

Inwardly rectifying potassium channels expressed by gene transfection into the green Monkey kidney cell line COS-1.

1. cDNA encoding a functional inwardly rectifying K+ (IRK1) channel was transfected into COS-1 cells (a Green Monkey kidney cell line) using the liposome method, and voltage clamp experiments were done after 48-72 h. 2. Transfected cells showed inward rectification under whole-cell recording. The unitary current-voltage relationships in the inside-out configuration were almost linear in the absence of internal Mg2+ and polyamines, and the channel conductance averaged 34.1 +/- 2.0 pS (n = 15) at 23-26 degrees C. 3. Internal Mg2+ (2-10 microM) induced sublevels in the outward current with one-third and two-thirds of the unitary amplitude as in native channels. 4. To determine the subunit stoichiometry, we constructed tandem multimeric cDNAs consisting of the coding sequences of the IRK1 gene linked in a head-to-tail fashion. Cells transfected with tandem homomultimers up to octamers showed similar inwardly rectifying K+ channels. 5. A mutation (E138Q) eliminated the ionic conductance of the channel. Channels expressed by dimeric constructs containing a single mutant have a conductance ranging between 5 and 35 pS. 6. The E138Q mutant cotransfected with a wild-type dimeric, trimeric or tetrameric construct did not alter the channel conductance. The results do not support the notion that IRK1 channel proteins consist of four subunits.

Animals↗
PubMed1994

'Liver-type' 11 beta-hydroxysteroid dehydrogenase cDNA encodes reductase but not dehydrogenase activity in intact mammalian COS-7 cells.

11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) catalyses the metabolism of corticosterone to inert 11-dehydrocorticosterone, thus preventing glucocorticoid access to otherwise non-selective renal mineralocorticoid receptors (MRs), producing aldosterone selectivity in vivo. At least two isoforms of 11 beta-HSD exist. One isoform (11 beta-HSD1) has been purified from rat liver and an encoding cDNA cloned from a rat liver library. Transfection of rat 11 beta-HSD1 cDNA into amphibian cells with a mineralocorticoid phenotype encodes 11 beta-reductase activity (activation of inert 11-dehydrocorticosterone) suggesting that 11 beta-HSD1 does not have the necessary properties to protect renal MRs from exposure to glucocorticoids. This function is likely to reside in a second 11 beta-HSD isoform. 11 beta-HSD1 is co-localized with glucocorticoid receptors (GRs) and may modulate glucocorticoid access to this receptor type. To examine the predominant direction of 11 beta-HSD1 activity in intact mammalian cells, and the possible role of 11 beta-HSD in regulating glucocorticoid access to GRs, we transfected rat 11 beta-HSD1 cDNA into a mammalian kidney-derived cell system (COS-7) which has little endogenous 11 beta-HSD activity or mRNA expression. Homogenates of COS-7 cells transfected with increasing amounts of 11 beta-HSD cDNA exhibited a dose-related increase in 11 beta-dehydrogenase activity. In contrast, intact cells did not convert corticosterone to 11-dehydrocorticosterone over 24 h, but showed a clear dose-related 11 beta-reductase activity, apparent within 4 h of addition of 11-dehydrocorticosterone to the medium.(ABSTRACT TRUNCATED AT 250 WORDS)

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1996

Down-regulation of secretion of human complement component C2 by the product of an alternatively spliced C2 messenger RNA.

We have previously described alternatively spliced transcripts of the human C2 gene. Among those, C2delta(17), in which exon 17 has been spliced out, encodes a polypeptide that contains the C4b binding and the CIs cleavage sites of C2, but lacks the serine protease active center. To study the possible function of this variant, we constructed C2delta(17) cDNA by deletional mutagenesis and expressed it transiently in COS cells. Transfected COS cells secreted only trace amounts of the C2delta(17) polypeptide, which had no detectable hemolytic activity and could not be cleaved by CIs. Pulse-chase experiments using [35S]methionine demonstrated that the majority of the 88-kDa C2delta(17) remained intracellular. Control wild-type (wt) C2 in the intracellular compartment consisted of two bands of 93 and 99 kDa, the latter corresponding to mature secreted C2. Intracellular C2delta(17) and only the 93-kDa wt C2 were sensitive to endoglycosidase H, a marker for transport from the endoplasmic reticulum (ER) to the Golgi. Experiments using brefeldin A and double label immunofluorescence staining indicated that C2delta(17) exhibited a typical ER distribution pattern, while wt C2 accumulated in the ER-Golgi intermediate compartment. Secretion of C2 by COS cells cotransfected with wt C2 and C2delta(17) cDNA was significantly decreased compared with that by cells transfected with wt C2 alone. These combined results indicate that C2delta(17) is retained in the ER probably because it is incorrectly folded and that it could down-regulate the expression of the wt C2 gene.

Alternative Splicing↗
PubMed1996

The effect of overexpression of the protein tyrosine phosphatase PTPMEG on cell growth and on colony formation in soft agar in COS-7 cells.

We established stable COS-7 cell lines overexpressing recombinant PTPMEG and an inactive mutant form in which the active site cysteine is mutated to serine (PTPMEGCS). We found that both endogenous and recombinant enzyme were primarily located in the membrane and cytoskeletal fractions of COS-7 cells. Endogenous PTPMEG accounts for only 1/3000th of the total tyrosine phosphatase activity in COS-7 cells and transfected cells expressed 2- to 7-fold higher levels of the enzyme. These levels of overexpression did not result in detectable changes in either total tyrosine phosphatase activity or the state of protein tyrosine phosphorylation as determined by immunoblotting of cell homogenates with anti-phosphotyrosine antibodies. Despite the low levels of activity for PTPMEG, we found that overexpressing cells grew slower and reached confluence at a lower density than vector transfected cells. Surprisingly, PTPMEGCS-transfected cells also reach confluence at a lower density than vector-transfected cells, although they grow to higher density than PTPMEG-transfected cells. Both constructs inhibited the ability of COS-7 cells to form colonies in soft agar, with the native PTPMEG having a greater effect (30-fold) than PTPMEGCS (10-fold). These results indicate that in COS-7 cells both PTPMEG and PTPMEGCS inhibit cell proliferation, reduce the saturation density, and block the ability of these cells to grow without adhering to a solid matrix.

Animals↗
PubMed1997

Caspase-dependent apoptosis of COS-7 cells induced by Bax overexpression: differential effects of Bcl-2 and Bcl-xL on Bax-induced caspase activation and apoptosis.

Bcl-2 family proteins and ICE/CED-3 family proteases (caspases) are regarded as the basic regulators of apoptotic cell death. They are evolutionarily conserved and implicated in a variety of apoptosis. However, the precise mechanism by which these two families interact to regulate cell death is not yet known. In this study, we found that the overexpression of the Bcl-2 family member Bax induced apoptotic cell death in COS-7 cells through the activation of CPP32 (caspase-3)-like proteases that cleaved the DEVD tetrapeptide. This apoptotic cell death was suppressed by the viral proteins CrmA and p35, as well as by the chemically synthesized caspase inhibitors Z-Asp-CH2-DCB and zVAD-fmk. We also found that the Bax-induced apoptosis of COS-7 cells was suppressed by Bcl-xL and Bcl-2, though both Bcl-xL and Bcl-2 similarly prevented etoposide-induced apoptosis in COS-7 cells. In addition, Bcl-xL inhibited the activation of caspase-3-like proteases accompanying Bax-induced COS-7 cell death but Bcl-2 did not. These results indicate that the caspase activation is essential for Bax-induced apoptosis, and that the ability of Bcl-2 and Bcl-xL to prevent the Bax-induced caspase activation and apoptosis in COS-7 cells could be differentially regulated. Our results also suggest that Bcl-2 family proteins function upstream of caspase activation and control apoptosis through the regulation of caspase activity.

Animals↗
PubMed2005

Phagocytic signaling molecules in lipid rafts of COS-1 cells transfected with FcgammaRIIA.

COS-1 cells bearing FcgammaRIIA were used as a model to demonstrate co-localization of several enzymes previously shown to regulate neutrophil phagocytosis. In COS-1 cells, phospholipase D (PLD) in the membrane fraction was activated during phagocytosis. PLD was found almost exclusively in lipid rafts, along with RhoA and ARF1. Protein kinase C-delta (PKCdelta) and Raf-1 translocated to lipid rafts. In neutrophils, ceramide levels increase during phagocytosis, indicating that FcgammaRIIA engagement initiates ceramide generation. Applying this model, we transfected COS-1 cells with FcgammaRIIA that had been mutated in the ITAM region, rendering them unable to ingest particles. When the mutant receptors were engaged, ceramide was generated and MAPK was activated normally, thus these processes did not require actual ingestion of particles. These results indicate that signaling proteins for phagocytosis are either constitutively present in, or are recruited to, lipid rafts where they are readily available to activate one another.

Animals↗
PubMed2006

Third strand-mediated psoralen-induced correction of the sickle cell mutation on a plasmid transfected into COS-7 cells.

A significant level of correction of the mutation responsible for sickle cell anemia has been achieved in monkey COS-7 cells on a plasmid containing a beta-globin gene fragment. The plasmid was treated in vitro with a nucleic acid 'third strand' bearing a terminal photoreactive psoralen moiety that binds immediately adjacent to the mutant base pair. Following covalent attachment of the psoralen by monoadduct or diadduct formation to the mutant T-residue on the coding strand, the treated plasmid was transfected into the cells, which were then incubated for 48 h to allow the cellular DNA repair mechanisms to remove the photoadducts. Upon re-isolation and amplification of the transfected plasmid, sickle cell mutation correction, as determined by sequence analysis of both complementary strands, was established in a full 1%. This result encourages extension of the approach to correct the mutation directly on the chromosome.

Anemia, Sickle Cell↗
PubMed1996

Desensitization of melanotropin receptors in COS-7 cells.

Non-transfected COS-7 cells have been found to possess functional melanotropin receptors on their cell surface. These receptors, and the properties of the melanocyte stimulating hormone (MSH) peptides can be characterized by measuring melanotropin stimulation of cAMP accumulation in the cells. In these cells we studied the ultra-long lasting super agonist [Nle(4)-D-Phe(7)]-alpha-MSH (NDP-alpha-MSH), and compared it with the endogenous MSH peptides with respect to potency, maximal activity, duration of action, and rate of desensitization. Surprisingly, NDP-alpha-MSH did not act as a full agonist in COS-7 cells. In multiple experiments, it could stimulate cAMP accumulation to approximately 50% of the level of alpha-MSH, beta-MSH and adrenocorticotropic hormone (ACTH). The MSH receptor mediating this activity is unknown. The time course of cAMP accumulation, and the duration of receptor activation was also investigated. In contrast to other systems NDP-alpha-MSH did not induce prolonged activity, with respect to cAMP accumulation, in COS-7 cells. The MSH receptors present in COS-7 were found to desensitize rapidly subsequent to pretreatment by any of the MSH peptides. As expected for a partial agonist, the activity of NDP-alpha-MSH desensitized more rapidly than any of the full agonists. Surprisingly, desensitization induced by pretreatment with NDP-alpha-MSH also occurred more rapidly than desensitization induced by the other MSH analogs.

Animals↗
PubMed1997

Hereditary hemochromatosis: effects of C282Y and H63D mutations on association with beta2-microglobulin, intracellular processing, and cell surface expression of the HFE protein in COS-7 cells.

Hereditary hemochromatosis (HH) is the most common autosomal recessive disorder known in humans. A candidate gene for HH called HFE has recently been cloned that encodes a novel member of the major histocompatibility complex class I family. Most HH patients are homozygous for a Cys-282-->Tyr (C282Y) mutation in HFE gene, which has been shown to disrupt interaction with beta2-microglobulin; a second mutation, His-63-->Asp (H63D), is enriched in HH patients who are heterozygous for C282Y mutation. The aims of this study were to determine the effects of the C282Y and H63D mutations on the cellular trafficking and degradation of the HFE protein in transfected COS-7 cells. The results indicate that, while the wild-type and H63D HFE proteins associate with beta2-microglobulin and are expressed on the cell surface of COS-7 cells, these capabilities are lost by the C282Y HFE protein. We present biochemical and immunofluorescence data that indicate that the C282Y mutant protein: (i) is retained in the endoplasmic reticulum and middle Golgi compartment, (ii) fails to undergo late Golgi processing, and (iii) is subject to accelerated degradation. The block in intracellular transport, accelerated turnover, and failure of the C282Y protein to be presented normally on the cell surface provide a possible basis for impaired function of this mutant protein in HH.

Animals↗
PubMed1997

PKA-mediated phosphorylation and inhibition of Na(+)-K(+)-ATPase in response to beta-adrenergic hormone.

The activity of Na(+)-K(+)-ATPase can be regulated by hormones that activate adenosine 3',5'-cyclic monophosphate-dependent protein kinase (PKA). Here, using a site-directed phosphorylation state-specific antibody, we show that hormonal regulation of Na(+)-K(+)-ATPase can occur via phosphorylation of Ser-943 on its alpha-subunit. cDNAs coding for wild-type rat Na(+)-K(+)-ATPase and Na(+)-K(+)-ATPase in which the PKA phosphorylation site Ser-943 was mutated to Ala were stably and transiently transfected into COS cells. In COS cells expressing wild-type Na(+)-K(+)-ATPase the beta-adrenergic agonist isoproterenol (1 microM) significantly increased the level of phosphorylation of the alpha-subunit. Phosphorylation was accompanied by a significant inhibition of the enzyme activity, as reflected by a decrease in ATP hydrolysis and 86Rb+ transport. The effect of isoproterenol was reproduced by the PKA activator forskolin used in combination with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine and was abolished by the specific PKA inhibitor H-89. Okadaic acid, an inhibitor of protein phosphatases 1 and 2A, enhanced phosphorylation and inhibition of Na(+)-K(+)-ATPase induced by isoproterenol. The changes in activity of Na(+)-K(+)-ATPase linearly correlated with the extent of the alpha-subunit of Na(+)-K(+)-ATPase being phosphorylated. When Ser-943 was replaced by alanine, stimulation of the phosphorylation and inhibition of the activity of Na(+)-K(+)-ATPase induced by isoproterenol, alone or in combination with okadaic acid, were not observed. These results indicate that, in intact cells, modulation of the activity of Na(+)-K(+)-ATPase can be achieved by regulation of the state of phosphorylation of Ser-943. Moreover, they provide a biochemical mechanism by which beta-adrenergic agonists can regulate Na(+)-K(+)-ATPase activity.

1-Methyl-3-isobutylxanthine↗
PubMed1996

Functional expression of the cDNA encoded by the human ATP1AL1 gene.

The human ATP1AL1 gene encodes a protein expressed in brain, kidney, and skin and that is highly homologous to the recently cloned nongastric isoforms of H-K-adenosinetriphosphatase H-K-ATPase). We have generated polyclonal antibodies against the protein encoded by ATP1AL1 and used them to monitor the protein's expression and distribution in transfection studies. The protein was retained in the endplasmic reticulum when it was transiently expressed alone in COS cells. In COS cells cotransfected with ATP1AL1 plus gastric H-K-ATPase beta-subunit cDNAs (ATP1AL1-gH-K beta), both proteins reached the surface. Stably transfected lines of HEK 293 cells expressing both of these proteins demonstrate a 86Rb+ uptake activity sensitive to both 2-methyl,8-(phenylmeoxy)imidazo(1,2-a)pyridine 3-acetonitrile (SCH-28080) and ouabain (inhibitory constants of approximately 131 and 42 microM, respectively). Outward proton fluxes were measured in the same cells as the spontaneous intracellular pH (pHi) recovery in Cells loaded with a pH-sensitive dye [2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein] and subjected to acid loading through an NH4Cl pulse. The cells expressing both the ATP1AL1-encoded protein and the gastric H-K-ATPase beta-subunit possess a net acid extrusion activity that can be inhibited by 1 mM ouabain. Comparison of the 86Rb+ influx and proton efflux, however, does not support equal H+/Rb+ exchange mediated by this pump under the conditions of pHi-monitoring experiments. Moreover, whereas the acid extrusion activity mediated by the pump shows a marked pH dependence, the 86Rb+ uptake activity present in the cells expressing the ATP1AL1-gH-K beta complex cannot be stimulated by acute lowering of pHi. These data suggest that the ATP1AL1-encoded protein is the catalytic alpha-subunit of a human K(+)-dependent ATPase. The possible implications of the discrepancy between 86Rb+ uptake and pHi monitoring data are discussed.

Acids↗
PubMed2004

Protective effect of Blue-M1 against oxidative stress on COS-1 cells.

Blue-M1 is a blue pigment formed from xylose and glycine in the Maillard reaction. Previous work revealed that Blue-M1 scavenged hydroxyl radicals, and prevented the autoxidation of linoleic acid in vitro. We investigated the protective effect of Blue-M1 for 2,2'-azobis(2-amidino-propane)dihydrochloride (AAPH)-induced toxicity in COS-1 cells. COS-1 cells were cultured in AAPH containing DMEM medium with or without Blue-M1 at 37 degrees C for 24 h. Blue-M1 decreased the AAPH-induced toxicity in COS-1 cells, and this effect was dose-dependent. Furthermore, COS-1 cells were treated with diphenyl-1-pyrenylphosphine (DPPP), as a reagent for the detection of lipid peroxide, and then were cultured in AAPH containing DMEM medium with or without Blue-M1 at 37 degrees C for 6 h. Blue-M1 prevented the AAPH-induced peroxidation of cell membrane on COS-1 cells, and this effect was also dose-dependent. These results suggest that Blue-M1 prevents the oxidative cell injury. Therefore, Blue-M1 will be an antioxidant, which protect against the oxidative stress in living systems.

Amidines↗
PubMed1999

Inhibition of volume-regulated anion channels by expression of the cystic fibrosis transmembrane conductance regulator.

1. To investigate whether the cystic fibrosis transmembrane conductance regulator (CFTR) interacts with volume regulated anion channels (VRACs), we measured the volume-activated chloride current (ICl,swell) using the whole-cell patch-clamp technique in calf pulmonary artery endothelial (CPAE) cells and in COS cells transiently transfected with wild-type (WT) CFTR and the deletion mutant DeltaF508 CFTR. 2. ICl,swell was significantly reduced in CPAE cells expressing WT CFTR to 66.5 +/- 8.8 % (n = 13; mean +/- s. e.m.) of the control value (n = 11). This reduction was independent of activation of the CFTR channel. 3. Expression of DeltaF508 CFTR resulted in two groups of CPAE cells. In the first group IBMX and forskolin could activate a Cl- current. In these cells ICl,swell was reduced to 52.7 +/- 18.8 % (n = 5) of the control value (n = 21). In the second group IBMX and forskolin could not activate a current. The amplitude of ICl,swell in these cells was not significantly different from the control value (112.4 +/- 13.7 %, n = 11; 21 control cells). 4. Using the same method we showed that expression of WT CFTR in COS cells reduced ICl,swell to 62.1 +/- 11.9 % (n = 14) of the control value (n = 12) without any changes in the kinetics of the current. Non-stationary noise analysis suggested that there is no significant difference in the single channel conductance of VRAC between CFTR expressing and non-expressing COS cells. 5. We conclude that expression of WT CFTR down-regulates ICl, swell in CPAE and COS cells, suggesting an interaction between CFTR and VRAC independent of activation of CFTR.

Animals↗
PubMed2002

Geldanamycin inhibits trichostatin A-induced cell death and histone H4 hyperacetylation in COS-7 cells.

As widely believed treating cells with trichostatin A (TSA), an inhibitor of histone deacetylase, results in histone H4 hyperacetylation and cell cycle arrest. This compound is often compared with other potential anticancer drugs in cell cycle, proliferation and differentiation research. Furthermore, geldanamycin (GA), a 90-kDa heat shock protein (HSP90) specific inhibitor, is a well-known potential anticancer agent. This study examines whether GA can affect the cellular functions induced by TSA. When using TSA treatment, although caused COS-7 cell death, pretreatment of 0.5 microg/ml GA for 30 min and an addition of 50 ng/ml TSA (GA + TSA) apparently averted cell death. Our results indicated that the cell survival rate was only approximately 20% when prolonged treatment was undertaken with 50 ng/ml TSA (TSA) alone for 24 h. In contrast, the cell survival rate was enhanced by two folds when treating with GA + TSA. Furthermore, DNA fragmentation assay revealed that fragmented DNA was produced 8 h after prolonged treatment with TSA alone. Within 16 h, the apoptotic percentages of TSA-treated cells were between 15-25%. In contrast, the other treatments did not exceed 6%. Furthermore, GA inhibited TSA-induced histone H4 hyperacetylation. Western blotting analysis further demonstrated that the HSP70 levels did not significantly increase in TSA-treated cells. However, the accumulated 70-kDa heat shock protein (HSP70) markedly increased up to 2 to 3 folds at 8 h in GA- and GA + TSA-treated cells, and the maximum amount up to 5 to 7 folds at 20 h. Conversely, HSP90 did not markedly increase in all treatments. Based on the results in this study, we suggest that apoptosis induced by TSA can be prevented by GA-induced increment of heat shock proteins, particularly HSP70.

Acetylation↗
PubMed1999

Expression and functional interaction of the catalytic and regulatory subunits of human methionine adenosyltransferase in mammalian cells.

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (AdoMet). The mammalian MAT II isozyme consists of catalytic alpha(2) and regulatory beta subunits. The aim of this study was to investigate the interaction and kinetic behavior of the human MAT II subunit proteins in mammalian cells. COS-1 cells were transiently transfected with pTargeT vector harboring full-length cDNA that encodes for the MAT II alpha(2) or beta subunits. Expression of the His-tagged recombinant alpha(2) (ralpha(2)) subunit in COS-1 cells markedly increased MAT II activity and resulted in a shift in the K(m) for L-methionine (L-Met) from 15 microM (endogenous MAT II) to 75 microM (ralpha(2)), and with the apparent existence of two kinetic forms of MAT in the transfected COS-1 cell extracts. By contrast, expression of the recombinant beta (rbeta) subunit had no effect on the K(m) for L-Met of the endogenous MAT II, while it did cause an increase in both the V(max) and the specific activity of endogenous MAT. Co-expression of both ralpha(2) and rbeta subunits resulted in a significant increase of MAT specific activity with the appearance of a single kinetic form of MAT (K(m) = 20 microM). The recombinant MAT II alpha(2) and rbeta subunit associated spontaneously either in cell-free system or in COS-1 cells co-expressing both subunits. Analysis of nickel-agarose-purified His-tagged ralpha(2) subunit from COS-1 cell extracts showed that the beta subunit co-purified with the alpha(2) subunit. Furthermore, the alpha(2) and beta subunits co-migrated in native polyacrylamide gels. Together, the data provide evidence for alpha(2) and beta MAT subunit association. In addition, the beta subunit regulated MAT II activity by reducing its K(m) for L-Met and by rendering the enzyme more susceptible to feedback inhibition by AdoMet. We believe that the previously described differential expression of MAT II beta subunit may be an important mechanism by which MAT activity can be modulated to provide different levels of AdoMet that may be required at different stages of cell growth and differentiation.

Animals↗
PubMed1997

Expression of bacteriorhodopsin in Sf9 and COS-1 cells.

We report studies on the expression of the archaebacterial membrane protein bacteriorhodopsin in Sf9 insect cells and in COS-1 mammalian cells. In both cell systems, the apoprotein bacterio-opsin was expressed at levels of approximately 1 microgram/10(6) cells. Immunofluorescence studies showed that the expressed protein was accumulated in the endoplasmic reticulum. However, upon addition of all-trans retinal to membranes isolated from either Sf9 or COS-1 cells expressing bacterio-opsin, the characteristic bacteriorhodopsin chromophore (lambda max at approximately 560 nm) was rapidly generated. This is in contrast to bacterio-opsin expressed in E. coli, which cannot be functionally reconstituted with retinal unless it is first denatured, and then renatured in vitro. These studies demonstrate that the bacterio-opsin expressed is correctly folded and show that localization of a heterologously expressed membrane protein in the endoplasmic reticulum does not necessarily imply that it is misfolded.

Animals↗
PubMed1999

Comparison of SERCA1 and SERCA2a expressed in COS-1 cells and cardiac myocytes.

Cultured COS-1 cells, as well as chicken embryonic and neonatal rat cardiac myocytes, were infected with recombinant adenovirus vectors to define limiting factors in the expression and Ca2+ transport function of recombinant sarcoplasmic-endoplasmic reticulum Ca(2+) (SERCA) isoforms. Titration experiments showed that all COS-1 cells and myocytes in culture could be infected by an adenovirus titer of 10 plaque-forming units (pfu) per seeded cell. Raising the adenovirus titer further yielded higher protein expression up to an asymptotic limit for functional, membrane-bound SERCA protein. The asymptotic behavior of SERCA expression was not transcription related but was due to posttranscriptional events. The minimal (-268) cardiac troponin T (cTnT) promoter was a convenient size for adenovirus vector construction and manifested tight muscle specificity. However, its efficiency was lower than that of the nonspecific cytomegalovirus (CMV) promoter. At any rate, identical maximal levels of SERCA expression were obtained with the CMV and the cTnT promoter, as long as the viral titer was adjusted to compensate for transcription efficiency. A maximal threefold increase of total SERCA protein expression over the level of the endogenous SERCA of control myocytes was reached (a sevenfold increase compared with the endogenous SERCA of the same infected myocytes due to reduction of endogenous SERCA after infection). In contrast with previous reports [Ji et al. Am. J. Physiol. 276 (Heart Circ. Physiol. 45): H89-H97, 1999], a higher kinetic turnover was demonstrated for the SERCA1 compared with the SERCA2a isoform as shown by a 5.0- versus 2.6-fold increase in calcium uptake rate accompanying maximal expression of recombinant SERCA1 or SERCA2a, respectively. This information is deemed necessary for studies attempting to modify myocardial cell function by manipulation of SERCA expression.

Adenoviridae↗
PubMed1994

Substitution mutations affecting a small region of the Moloney murine leukemia virus MA gag protein block assembly and release of virion particles.

A series of substitution mutations affecting the Moloney murine leukemia virus MA protein were introduced into a cloned proviral DNA, and the mutant DNAs were tested for their biological activity in NIH/3T3 cells and COS cells. Many of the mutant viruses were viable and replicated with kinetics indistinguishable from the wild type. Seven mutants with alterations in a small region (residues 7-14 from the amino terminus) were replication-defective. These mutants were blocked in assembly and release of the virion particles in NIH/3T3 cells and were defective in both gag and gag-pol gene function. The results suggest that this very small region near the amino terminus of both proteins is required for their membrane targeting or self-association. Three of the defective mutant DNAs were able to induce virion particle formation when present at high copy number in COS cells.

3T3 Cells↗