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At least 271 records · Page 15Linked to original sources
PubMed1997

Combined transgenic expression of alpha-galactosidase and alpha1,2-fucosyltransferase leads to optimal reduction in the major xenoepitope Galalpha(1,3)Gal.

Hyperacute rejection of pig organs by humans involves the interaction of Galalpha(1,3)Gal with antibodies and complement. Strategies to reduce the amount of xenoantigen Galalpha(1,3)Gal were investigated by overexpression of human lysosomal alpha-galactosidase in cultured porcine cells and transgenic mice. The overexpression of human alpha-galactosidase in cultured porcine endothelial cells and COS cells resulted in a 30-fold reduction of cell surface Galalpha(1,3)Gal and a 10-fold reduction in cell reactivity with natural human antibodies. Splenocytes from transgenic mice overexpressing human alpha-galactosidase showed only a 15-25% reduction in binding to natural human anti-Galalpha(1,3)Gal antibodies; however, this decrease was functionally significant as demonstrated by reduced susceptibility to human antibody-mediated lysis. However, because there is residual Galalpha(1,3)Gal and degalactosylation results in the exposure of N-acetyllactosamine residues and potential new xenoepitopes, using alpha-galactosidase alone is unlikely to overcome hyperacute rejection. We previously reported that mice overexpressing human alpha1,2-fucosyltransferase as a transgene had approximately 90% reduced Galalpha(1,3)Gal levels due to masking of the xenoantigen by fucosylation; we evaluated the effect of overexpressing alpha-galactosidase and alpha1,2-fucosyltransferase on Galalpha(1,3)Gal levels. Galalpha(1, 3)Gal-positive COS cells expressing alpha1,3-galactosyltransferase, alpha1,2-fucosyltransferase, and alpha-galactosidase showed negligible cell surface staining and were not susceptible to lysis by human serum containing antibody and complement. Thus, alpha1, 2-fucosyltransferase and alpha-galactosidase effectively reduced the expression of Galalpha(1,3)Gal on the cell surface and could be used to produce transgenic pigs with negligible levels of cell surface Galalpha(1,3)Gal, thereby having no reactivity with human serum and improving graft survival.

Animals↗
PubMed1995

Pan-neurotrophin 1: a genetically engineered neurotrophic factor displaying multiple specificities in peripheral neurons in vitro and in vivo.

Pan-neurotrophin 1 (PNT-1) is a synthetic trophic factor engineered by combining active domains of the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT-3) into an NT-3 backbone. This molecule was produced in transiently transfected COS cells or in baculovirus-infected insect cells transfected COS cells or in baculovirus-infected insect cells and subsequently purified to homogeneity. Saturation binding in embryonic spinal sensory neurons demonstrated a greater number of high-affinity binding sites for PNT-1 than for its parental molecule NT-3. PNT-1 was shown to efficiently block the chemical crosslinking of NGF, BDNF, and NT-3 to their cognate Trk receptors and to the low-affintiy NGF receptor expressed on neuronal and nonneuronal cells. PNT-1 stimulated survival and proliferation of MG87 fibroblasts expressing either TrkA, TrkB, or TrkC. PNT-1 also promoted survival of a greater number of embryonic dorsal root ganglion neurons than any of the other neurotrophins alone, and its effects were equivalent to a combination of NGF, BDNF, and NT-3. Analysis of receptor-specific neurotrophic activities demonstrated that PNT-1 efficiently rescued TrkA mRNA-containing sympathetic neurons and TrkB and TrkC mRNA-containing sensory neurons from the dorsal root and nodose ganglia. Finally, PNT-1 showed robust retrograde transport to DRG neurons in vivo after injection into the sciatic nerve. Radiolabeled PNT-1 accumulated in small-, medium-, and large-sized neurons. Coinjection with different unlabeled neurotrophins inhibited PNT-1 transport in distinct subpopulations of neurons of different sizes, suggesting that this molecule affects sensory neurons of different modalities. These results indicate that PNT-1 is a potent and multispecific neurotrophic factor that may be useful in the treatment of peripheral neurophathies and nerve damage.

Animals↗
PubMed1996

Structure-function relationship of lipoprotein lipase-mediated enhancement of very low density lipoprotein binding and catabolism by the low density lipoprotein receptor. Functional importance of a properly folded surface loop covering the catalytic center.

We examined the structure-function relationship of human lipoprotein lipase (hLPL) in its ability to enhance the binding and catabolism of very low density lipoproteins (VLDL) in COS cells. Untransfected COS cells did not bind to or catabolize normal VLDL. Expression of wild-type hLPL by transient transfection enhanced binding, uptake, and degradation of the VLDL (a property of LPL that we call bridge function). Heparin pretreatment and a monoclonal antibody ID7 that blocks LDL receptor-binding domain of apoE both inhibited binding, and apoE2/E2 VLDL from a Type III hyperlipidemic subject did not bind. However, LDL did not reduce 125I-VLDL binding to the hLPL-expressing cells, whereas rabbit beta-VLDL was an effective competitor. By contrast, LDL reduced uptake and degradation of 125I-VLDL to the same extent as excess unlabeled VLDL or beta-VLDL. These data suggest that binding occurs by direct interaction of VLDL with LPL but the subsequent catabolism of the VLDL is mediated by the LDL receptor. Mutant hLPLs that were catalytically inactive, S132A, S132D, as well as the partially active mutant, S251T, and S172G, gave normal enhancement of VLDL binding and catabolism, whereas the partially active mutant S172D had markedly impaired capacity for the process; thus, there is no correlation between bridge function and lipolytic activity. A naturally occurring genetic variant hLPL, S447-->Ter, has normal bridge function. The catalytic center of LPL is covered by a 21-amino acid loop that must be repositioned before a lipid substrate can gain access to the active site for catalysis. We studied three hLPL loop mutants (LPL-cH, an enzymatically active mutant with the loop replaced by a hepatic lipase loop; LPL-cP, an enzymatically inactive mutant with the loop replaced by a pancreatic lipase loop; and C216S/C239S, an enzymatically inactive mutant with the pair of Cys residues delimiting the loop substituted by Ser residues) and a control double Cys mutant, C418S/C438S. Two of the loop mutants (LPL-cH and LPL-cP) and the control double Cys mutant C418S/C438S gave normal enhancement of VLDL binding and catabolism, whereas the third loop mutant, C216S/C239S, was completely inactive. We conclude that although catalytic activity and the actual primary sequence of the loop of LPL are relatively unimportant (wild-type LPL loop and pancreatic lipase loops have little sequence similarity), the intact folding of the loop, flanked by disulfide bonds, must be maintained for LPL to express its bridge function.

Animals↗
PubMed1989

A characterization of the elements comprising the promoter of the mouse ribosomal protein gene RPS16.

The elements comprising the mouse rpS16 promoter were characterized by transfection experiments with mutant genes in which various portions of the 5' flanking region and exon I were removed or substituted with extraneous DNA sequence. These experiments were carried out with otherwise intact rpS16 genes transfected into monkey kidney (COS) cells and also with chimeric rpS16-CAT gene constructs transfected into mouse plasmacytoma cells and COS cells. The locations of the functionally important elements were generally correlated with the locations of binding sites for specific nuclear factors, which were identified by gel-mobility shift analyses and methylation interference footprints. The most upstream element, which is located approximately 165 bp from the cap site, binds the Sp1 transcription factor and augments the promoter activity by 2 to 2.5-fold. In addition, there is a complex bipartite element in the -83 to -59 region, an element in the -37 to -12 region and an element in the +9 to +29 region of exon I, all of which are essential for rpS16 expression. The rpS16 promoter has a general architecture that resembles other mouse rp promoters; however, it also possesses some distinctive characteristics.

Animals↗
PubMed1993

Analysis of Lewis fucosyltransferase genes from the human gastric mucosa of Lewis-positive and -negative individuals.

The expression of Lewis fucosyltransferase (FT) mRNA was examined in gastric mucosa from two Lewis-positive [Le(+)] and two Lewis-negative [Le(-)] individuals. Northern blot analysis demonstrated that levels of mRNA were similar in both Le(+) and Le(-) gastric mucosa. We isolated the protein-coding region of the Lewis FT cDNA from Le(+) and Le(-) gastric mucosa by polymerase chain reaction (PCR) amplification. The sequence of cDNA from the Le(-) gastric mucosa shows two single-base substitutions of G for T at position 59 and of A for G at position 508 from the A of the initiation codon of cDNA. These substitutions may be the cause of changes in two amino acid residues, Arg for Leu at position 20 and Ser for Gly at position 170 from the N-terminal. To determine whether either or both of these base substitutions is responsible for the Le(-) gene, we constructed chimera cDNAs and expressed them in COS cells. Those COS cells transfected with a chimera cDNA containing a mutation of the 508th nucleotide did not express Lewis antigen, whereas those cells transfected with a chimera cDNA containing the 59th nucleotide mutation expressed Lewis antigen, indicating that a single-base change from G to A at position 508 is responsible for the Le(-) gene. The G to A transition at position 508 created a new site for PvuII endonuclease. The digestion by PvuII endonuclease of PCR products between the 386th and 612th nucleotides of Lewis FT cDNA from one of the Le(-) individuals proved to be homozygous for the PvuII site. However, the other Le(-) individual was heterozygous for the PvuII site, suggesting the presence of other Le(-) allele(s). Thus, we isolated one of the silent Lewis genes (le).

Amino Acid Sequence↗
PubMed2004

[Expression of GBD gene of Streptococcus mutans glucan binding protein A in mammalian cells].

OBJECTIVE: To evaluate the expression of recombinant plasmid pcDNA3.1/GBD of glucan binding protein of Streptococcus mutans in mammalian cells COS-7. METHODS: Eukaryotic plasmid carrying encoding gene of GBD of Streptococcus mutans gbpA was constructed and the plasmid was introduced into COS-7 cells by Lipofectamine reagent. The transient expressed protein in COS-7 cells was detected by immunochemistry technique. RESULTS: The positive expression was detected in plasma of the cells which were transfected with recombinant plasmid pcDNA3.1/GBD. The cells which were transfected with pcDNA3.1 were negative. CONCLUSION: GBD can translate and express in COS-7 cells after transfected with recombinant plasmid pcDNA3.1/GBD. The expressed protein locates in the plasma and the protein is able to combine with anti-GbpA antibody. The expressed protein has the antigenicity and is a candidate gene vaccine.

Animals↗
PubMed2002

Analysis of heme oxygenase isomers in rat.

AIM: To purify and identify heme oxygenase (HO) isomers which exist in rat liver, spleen and brain treated with hematin and phenylhydrazine and in untreated rat liver and to investigate the characteristics of HO isomers, to isolate and confirm the rat HO-1 cDNA that actually encodes HO-1 by expressing cDNA in monkey kidney cells (COS-1 cells), to prepare the rat heme oxygenase-1 (HO-1) mutant and to detect inhibition of HO-1 mutated enzyme. METHODS: First, rat liver, spleen and brain microsomal fractions were purified by DEAE-Sephacel and hydroxylapatite. The characteristics including activity, immunity and inducibility of two isomers (HO-1 and HO-2), and their apparent molecular weight were measured by detecting enzymatic activities, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis, respectively. Second, plasmid pcDNA3HO1 containing native rat HO-1 cDNA and pcDNA3HO1D25 carrying mutated rat HO-1 cDNA (His25Ala) were constructed by site-directed mutagenesis. COS-1 cells transfected with pcDNA3HO1 and pcDNA3HO1D25 were collected and disrupted by sonication, the microsomes were prepared by ultracentrifugation. Third, the inhibition of rat HO-1 mutant was analyzed. RESULTS: Two isomers were purified and identified in treated rat liver, spleen, brain and untreated rat liver. HO-1 was the predominant form with a ratio of 2.0:1 and 3.2:1 of HO-1 and HO-2 in liver and spleen, respectively, but only the activity of HO-2 in the brain and untreated liver could be detected. The apparent molecular weights of HO-1 and HO-2 were about M(r)30 000 and M(r) 36 000 under reducing conditions, respectively. The antiserum against liver HO-2 was employed in Western blotting analysis, the reactivity of HO-1 in the liver was not observed. The plasmid pcDNA3HO1 was highly expressed in endoplasmic reticulum of transfected COS-1 cells. The specific activity was -5-fold higher than that of the control. However, the enzyme activity of mutated HO-1 declined. While an equal amount of mutant was added to the enzyme reaction system, the levels of bilirubin decreased 42 %. CONCLUSION: The studies suggest that HO-1 and HO-2 exist in the hematin and phenylhydrazine treated rat liver and spleen, but only HO-2 in the brain and untreated liver. Two constitutive forms are different in molecular weight, inducibility and immunochemical properties. The activity of expressed HO-1 in COS-1 cells is higher than that of purified enzyme from rat spleen tissue. It suggests that this clone has an insert of 1030 base-pairs encodes HO-1. His25Ala mutant reduced the formation of bilirubin and it suggests that the mutant could completely bind the heme with native enzyme.

Animals↗
PubMed1994

Phospholipase C activation and Ca2+ mobilization by cloned human somatostatin receptor subtypes 1-5, in transfected COS-7 cells.

We transfected the COS-7 cells with cDNAs encoding different human somatostatin receptor (hSSTR) subtypes, and found that hSSTR subtypes mediate not only the inhibition of forskolin-induced cAMP accumulation but also the stimulation of phospholipase C (PLC) and Ca2+ mobilization. Activation of PLC by 1 microM somatostatin (SRIF) was in the order of: hSSTR5 > hSSTR2 > hSSTR3 > hSSTR4 >> hSSTR1. Pertussis toxin (PTX) treatment completely or partially reversed the PLC activation. 1 nM SRIF was equally effective for adenylate cyclase (AC) inhibition in a PTX-sensitive manner, in all the cells expressing different hSSTRs, except for hSSTR1. Nevertheless, SRIF stimulated AC even in the presence of forskolin at higher doses of SRIF in PTX-treated hSSTR5-expressing cells. We conclude that the cloned hSSTRs differentially couple to PTX-sensitive and -insensitive G-proteins to modulate PLC, Ca2+ mobilization and AC.

Calcium↗
PubMed2006

Molecular cloning and initial characterization of African green monkey (Cercopithecus aethiops) corticotropin releasing factor receptor type 1 (CRF1) from COS-7 cells.

We report the expression of endogenous CRF1 in COS-7 cells (African green monkey origin). Cloning of the coding region of CRF1 gene identified three alternatively spliced isoforms with nucleotide and predicted amino acid sequences corresponding to the membrane bound alpha and c and soluble e isoforms. DNA sequencing of the main isoform CRF1alpha showed homologies of 99%, 97% and 91% with the rhesus monkey, human and rodent genes, respectively; the deduced protein sequence differed in only one amino acid with rhesus monkey and human. Western blot analysis with antibodies against human CRF1 demonstrated immunoreactive proteins with MW of 37, 52, 70 and 80-85 in crude membrane or cytoplasm preparation; two additional species of 40 and 60 kDa were detected only in the cytoplasmic fraction. On immunocytochemistry CRF1 was localized to both the cell surface and intracellularly. The receptor was functional, e.g., addition of CRF to COS-7 cells inhibited cell proliferation and stimulated release of arachidonic acid; nevertheless, it was poorly coupled to cAMP production (its stimulation was minimal in native cells). In conclusion, COS cells that are routinely used for the study of transfected CRF receptors do express endogenous CRF1 mRNA with splicing behavior similar to that reported in human and rodent cells, and translated into functional CRF1 receptors.

Alternative Splicing↗
PubMed2000

Very long-chain acyl-CoA synthetases. Human "bubblegum" represents a new family of proteins capable of activating very long-chain fatty acids.

Activation by thioesterification to coenzyme A is a prerequisite for most reactions involving fatty acids. Enzymes catalyzing activation, acyl-CoA synthetases, have been classified by their chain length specificities. The most recently identified family is the very long-chain acyl-CoA synthetases (VLCS). Although several members of this group are capable of activating very long-chain fatty acids (VLCFA), one is a bile acid-CoA synthetase, and others have been characterized as fatty acid transport proteins. It was reported that the Drosophila melanogaster mutant bubblegum (BGM) had elevated VLCFA and that the product of the defective gene had sequence homology to acyl-CoA synthetases. Therefore, we cloned full-length cDNA for a human homolog of BGM, and we investigated the properties of its protein product, hsBG, to determine whether it had VLCS activity. Northern blot analysis showed that hsBG is expressed primarily in brain. Compared with vector-transfected cells, COS-1 cells expressing hsBG had increased acyl-CoA synthetase activity with either long-chain fatty acid (2.4-fold) or VLCFA (2.6-fold) substrates. Despite this increased VLCFA activation, hsBG-expressing cells did not have increased rates of VLCFA degradation. Confocal microscopy showed that hsBG had a cytoplasmic localization in some COS-1 cells expressing the protein, whereas it appeared to associate with plasma membrane in others. Fractionation of these cells revealed that most of the hsBG-dependent acyl-CoA synthetase activity was soluble and not membrane-bound. Immunoaffinity-purified hsBG from transfected COS-1 cells was enzymatically active. hsBG and hsVLCS are only 15% identical, and comparison with sequences of two conserved motifs from all known families of acyl-CoA synthetases revealed that hsBG along with the D. melanogaster and murine homologs comprise a new family of acyl-CoA synthetases. Thus, two protein families are now known that contain enzymes capable of activating VLCFA. Because hsBG is expressed in brain but previously described VLCSs were not highly expressed in this organ, hsBG may play a central role in brain VLCFA metabolism and myelinogenesis.

Amino Acid Sequence↗
PubMed1996

Hypotonic stimulation induced Ca2+ release from IP3-sensitive internal stores in a green monkey kidney cell line.

1. Hypotonic stimulation (180 +/- 5 mosmol l-1) increased [Ca2+]i in fura-2-loaded Green monkey kidney cells (COS-7 cells) and depolarized the membrane. 2. COS-7 cells were depolarized up to -3.5 +/- 4.4 mV from a resting membrane potential of -35.2 +/- 2.3 mV in response to hypotonic stimulation, when the patch electrode was filled with a 160 mM KCl-0.5 mM EGTA-based intracellular medium. 3. The increase in [Ca2+]i induced by hypotonic stimulation was divided into two phases. One was transient and oscillatory, and observed in Ca(2+)-free medium; the other was persistent, blocked by 100 microM La3+, and observed only in Ca(2+)-containing medium. 4. The increase in [Ca2+]i in Ca(2+)-free medium was blocked by pretreatment with 10 microM thapsigargin. The increase in [Ca2+]i induced by 10 microM thapsigargin was reduced after hypotonic stimulation which induced an increase in [Ca2+]i in Ca(2+)-free medium. 5. The increase in [Ca2+]i in Ca(2+)-free medium was not affected by treatment with 5 mM caffeine or 1-10 microM ryanodine. Neither caffeine nor ryanodine induced an increase in [Ca2+]i. 6. Adenosine 5'-O-2-thiodiphosphate (ADP-beta-S; a P2Y receptor agonist) induced an increase in [Ca2+]i in Ca(2+)-free medium and caused phosphoinositide breakdown in COS-7 cells. Exposure to 10 microM ADP-beta-S blocked the increase in [Ca2+]i induced in the Ca(2+)-free medium by hypotonic stimulation. The results of summary points 4, 5, and 6 suggest that the increase in [Ca2+]i induced by hypotonic stimulation is due to Ca2+ release from inositol 1,4,5-trisphosphate (IP3)-sensitive internal stores. 7. The hypotonic stimulation-activated hydrolysis of phosphoinositides was decreased by pertussis toxin (PTX) in a dose-dependent manner. 8. These observations strongly suggest that hypotonic stimulation induced an increase in [Ca2+]i in Ca(2+)-free medium through activation of cascades using PTX-sensitive guanine nucleotide binding protein (G protein) and IP3.

Animals↗
PubMed1997

Production of bioactive salmon calcitonin from the nonendocrine cell lines COS-7 and CHO.

To produce bioactive salmon calcitonin from the conventional nonendocrine cell lines, COS-7 and CHO, we devised a salmon calcitonin expression vector by combining the amino-terminus of human calcitonin precursor with a salmon calcitonin sequence, inserting the efficient furin-cleavable processing sequence Arg-X-Arg-X-Lys-Arg before salmon calcitonin, and deleting the carboxyl-terminal extension peptide. This chimeric calcitonin precursor terminates at glycine to easily receive an amidation reaction. COS-7 and CHO produced a high level of bioactive calcitonin by the resorption pit formation assay. Although amidating activity is highly expressed in CHO, but only a little in COS-7 cells, both cells produced a similar level of bioactive calcitonin. Thus, the engineered salmon calcitonin expression vector enables nonendocrine cells even with low amidation activity to produce bioactive calcitonin.

Animals↗
PubMed1999

The distribution of circulating receptors on COS 7 cells.

Rapidly circulating receptors on the surfaces of migrating or spreading cells are often concentrated toward the cells' leading edges. This polar distribution is the principal evidence that circulating membrane is returned to the surface of these cells at their fronts, where it is proposed to assist the forward extension of the cell. However, when low-density lipoprotein (LDL) receptors are transiently expressed in COS cells-cells which actively spread-their distribution is random, indicating that COS cells do not behave like other motile cells. This anomaly is examined here. I find that COS cell lines expressing either transferrin or LDL receptors do, in fact, usually have polar distributions of both receptors. The transient expression of LDL receptors interferes, in an unknown way, with the polar properties of COS cells. Furthermore, it is shown that whether receptors are located toward the cell's edge or are randomly distributed depends both on the ability of the receptor to circulate and on the state of the particular cell at that time.

Animals↗
PubMed1993

A monoclonal antibody reactive with terminal lactotriaosyl residue-containing oligosaccharides and its application to characterizing cell surface expression of the glyco-epitopes in COS-1 cells.

A new monoclonal antibody (MoAb), designated JF12, reactive strongly with Lc3 (GlcNAc beta 1-->3Gal beta 1-->4Glc beta 1-->Cer) but only slightly with nLc5 (GlcNAc beta 1-->3Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4Glc beta 1-->Cer) has been prepared after immunization of Balb/c mice with Lc3. By the flowcytometrical analyses of COS-1 cells, which was strongly stained with JF12, the reactivity completely disappeared in the confluent condition upon harvesting by the protease treatments of the cell surface. In the sparse condition, however, the cells still retained JF12 reactivity in spite of the protease treatments. This strongly suggests that the expression of terminal GlcNAc-containing glycoconjugates on the surface of COS-1 cells may be dramatically modulated by protease-sensitive membranous components being dependent upon the cell density.

Animals↗
PubMed1994

Expression and characterization of a functional rat liver Na+ bile acid cotransport system in COS-7 cells.

A cDNA for the rat liver sodium-dependent bile acid cotransporter was expressed in COS-7 cells to study the functional properties of the translated protein in a mammalian cell line. A 1.2-kb insert was ligated into a pMAMneo vector and transiently transfected using electroporation. After optimal conditions were established, the transiently transfected COS cells were screened with fluorescent-conjugated labeled bile acids for evidence of expression of the cotransporter after 48 h. The uptake of [3H]taurocholate ([3H]TC) was then determined in cells transfected with or without the bile acid insert. Progressive uptake of [3H]TC (0.45 microM) was observed for 30 min in the presence of sodium. In contrast, no uptake of [3H]TC was observed in the absence of sodium, in nontransfected COS cells, or in COS cells transfected with the empty plasmid. Kinetic studies revealed a Michaelis constant (Km) of 29 microM, essentially identical to the Km of this cotransporter described in intact rat hepatocytes and membrane vesicles. Uptake of [3H]TC (5.0 microM) at 5 min (n = 3-6) was inhibited by 100 microM taurochenodeoxycholic acid (81%), tauroursodeoxycholic acid (77%), cholic acid (55%), chenodeoxycholic acid (74%), and ursodeoxycholic acid (56%) but not by 100 microM taurodehydrocholate, 1 mM probenecid, or 100 microM bilirubin. In contrast, bumetanide (500 microM) inhibited [3H]TC uptake by 52%. These studies indicate that the isolated cDNA codes for a physiological bile acid transporter present in rat hepatocytes and that posttranslational factors present in mammalian cells may not be as important in defining properties of this cotransport system.

Animals↗
PubMed1992

Expression and transport of rabbit surfactant protein A in COS-1 cells.

SV40-transformed green monkey kidney (COS-1) cells were transfected with expression plasmids that contained either the structural gene or cDNA for surfactant protein A (SP-A), a major protein of rabbit lung surfactant. The transfected COS-1 cells synthesized several isoforms of SP-A that were found to be less acidic than those produced in rabbit lung tissue. SP-A species with apparent molecular weight (M(r)) approximately equal to 29,000-33,000 were detected in the transfected cells, whereas glycosylated forms with apparent M(r) approximately equal to 33,000-38,000 were detectable only in the culture medium. Analysis of transfected cells by indirect immunofluorescence revealed that SP-A was localized in punctate bodies throughout the cytoplasm. Expressed SP-A was not detectable on the cell surface nor was there evidence that secreted SP-A was endocytosed by COS-1 cells. After subcellular fractionation of the transfected COS-1 cells, SP-A was found to be localized predominantly in the 5,000- and 18,000-g pellet fractions; little or no immunoreactive SP-A was detectable in cytosolic fractions. Treatment of transfected cells with the glycosylation inhibitor tunicamycin prevented secretion of SP-A into the medium, suggesting a role of glycosylation in secretion of SP-A. On the other hand, treatment of transfected cells with inhibitors of proline hydroxylation, which may cause destabilization of the collagen-like domain of SP-A, reduced but did not prevent secretion of SP-A into the culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

A mammalian host-vector system that regulates expression and amplification of transfected genes by temperature induction.

SV40-transformed simian cells that permit temperature-dependent regulation of vector DNA replication were isolated and characterized. These cell lines (ts COS cells) produce high levels of thermolabile large T antigen under the transcriptional control of the Rous sarcoma virus long terminal repeat. The ts COS cell lines can complement SV40 A gene mutants and support replication of SV40-origin containing vectors at 33 degrees C but not at 40 degrees C. It should now be possible to regulate the copy number of transfected plasmid DNA's and also maintain selectable vector sequences either as integrated DNA or as autonomously replicating episomes by modulating T antigen activity in ts COS cells.

Animals↗
PubMed1999

Human egasyn binds beta-glucuronidase but neither the esterase active site of egasyn nor the C terminus of beta-glucuronidase is involved in their interaction.

Lysosomal beta-glucuronidase shows a dual localization in mouse liver, where a significant fraction is retained in the endoplasmic reticulum (ER) by interaction with an ER-resident carboxyl esterase called egasyn. This interaction of mouse egasyn (mEg) with murine beta-glucuronidase (mGUSB) involves binding of the C-terminal 8 residues of the mGUSB to the carboxylesterase active site of the mEg. We isolated the recombinant human homologue of the mouse egasyn cDNA and found that it too binds human beta-glucuronidase (hGUSB). However, the binding appears not to involve the active site of the human egasyn (hEg) and does not involve the C-terminal 18 amino acids of hGUSB. The full-length cDNA encoding hEg was isolated from a human liver cDNA library using full-length mEg cDNA as a probe. The 1941-bp cDNA differs by only a few bases from two previously reported cDNAs for human liver carboxylesterase, allowing the anti-human carboxylesterase antiserum to be used for immunoprecipitation of human egasyn. The cDNA expressed bis-p-nitrophenyl phosphate (BPNP)-inhibitable esterase activity in COS cells. When expressed in COS cells, it is localized to the ER. The intracellular hEg coimmunoprecipitated with full-length hGUSB and with a truncated hGUSB missing the C-terminal 18-amino-acid residue when extracts of COS cells expressing both proteins were treated with anti-hGUSB antibody. It did not coimmunoprecipitate with mGUSB from extracts of coexpressing COS cells. Unlike mEg, hEg was not released from the hEg-GUSB complex with BPNP. Thus, hEg resembles mEg in that it binds hGUSB. However, it differs from mEg in that (i) it does not appear to use the esterase active site for binding since treatment with BPNP did not release hEg from hGUSB and (ii) it does not use the C terminus of GUSB for binding, since a C-terminal truncated hGUSB (the C-terminal 18 amino acids are removed) bound as well as nontruncated hGUSB. Evidence is presented that an internal segment of 51 amino acids between 228 and 279 residues contributes to binding of hGUSB by hEg.

Amino Acid Sequence↗