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J Y Chou

Publications and source records attributed to J Y Chou.

At least 91 records · Page 5Linked to original sources
PubMed1991

Mutation of a single amino acid converts germ cell alkaline phosphatase to placental alkaline phosphatase.

Human placental and germ cell alkaline phosphatases (PLAP and GCAP, respectively), are characterized by their differential sensitivities to inhibition by L-leucine, EDTA, and heat. Yet, they differ by only 7 amino acids at positions 15, 67, 68, 84, 241, 254, and 429 within their respective 484 residues. To determine the structural basis and the amino acid(s) involved in these physicochemical differences, we constructed three GCAP mutants by site-directed mutagenesis and six GCAP/PLAP chimeras and then expressed these alkaline phosphatase mutants in COS-1 cells. We report that the differential reactivity of PLAP and GCAP depends critically on a single amino acid at position 429. GCAP with Gly-429 is strongly inhibited by L-leucine, EDTA, and heat, whereas PLAP with Glu-429 is resistant. By substituting Gly-429 of GCAP with a series of amino acids, we demonstrate that the relative sensitivities of these mutants to L-leucine, EDTA, and heat inhibition are, in general, parallel. Mutants in the order of resistance to these treatments are: Glu (most resistant), Asp/Ile/Leu, Gln/Val/Lys, Ser/His, and Arg/Thr/Met/Cys/Phe/Trp/Tyr/Pro/Asn/Ala/Gly (least resistant). However, the Ser-429 and His-429 mutants were more resistant to EDTA and heat inhibition than the wild-type GCAP, but were equally sensitive to L-leucine inhibition. Structural analysis of mammalian alkaline phosphatase modeled on the refined crystal structure of Escherichia coli alkaline phosphatase indicates that the negative charge of Glu-429 of PLAP, which simultaneously stabilizes the protein as a whole and the metal binding specifically, probably acts through interactions with the metal ligand His-320 (His-331 in E. coli alkaline phosphatase). Replacement of codon 429 with Gly in GCAP leads to destabilization and loosening of the metal binding. The data suggest that the natural binding site for L-leucine may be near position 429, with the amino and carboxyl groups of L-leucine interacting with bound phosphate and His-432 (His-412 in E. coli alkaline phosphatase), respectively.

Alkaline Phosphatase↗
PubMed1991

Cloning and expression of a new pregnancy-specific beta 1-glycoprotein member.

Human pregnancy-specific beta 1-glycoproteins (PSGs) are a group of closely related placental glycoproteins and members of the immunoglobulin superfamily. To study possible functional roles of PSG, three type 1 cDNA clones which encode PSG in a N-A1-A2-B2-C domain arrangement have been characterized in this laboratory. Now, we report the cloning of a type II cDNA (PSG9) which is 1512 bp and encodes a PSG of 332 amino acids in a N-A1-B2-C domain arrangement. In vitro expression experiments demonstrate that PSG9 encodes a 36 kDa nonglycosylated protein which is processed to a glycosylated protein of 46 kDa. Ribonuclease protection analyses show that PSG mRNAs that share sequence similarity with the PSG9 transcript are expressed in both human placenta and placental fibroblasts.

Amino Acid Sequence↗
PubMed1991

Transcriptional regulation and the effects of sodium butyrate and glycosylation on catalytic activity of human germ cell alkaline phosphatase.

Human choriocarcinoma cells, the malignant trophoblasts, synthesize germ cell alkaline phosphatase (GCAP) which shares 98% sequence identity with the placental alkaline phosphatase (AP). The two isozymes are immunologically similar but react differentially toward inhibition by L-leucine or EDTA. Administration of sodium butyrate to choriocarcinoma cells greatly increased the transcription rate of the GCAP gene, resulting in an increase in mRNA expression and enzyme biosynthesis. The butyrate-modulated AP induction was blocked by cycloheximide, suggesting that a mediator protein may be involved. Protein sequence deduced from complementary DNA analysis suggests that GCAP contains two potential sites for asparagine (N)-linked glycosylation. The marked increase in GCAP expression by butyrate in choriocarcinoma cells allowed us to study the extent of N-linked glycosylation and its role on GCAP enzyme activity. After limited tunicamycin treatment, Mr 65,000 (fully processed), Mr 58,000 (nonglycosylated), and Mr 62,000 polypeptides were synthesized by these cells in the presence of butyrate. This suggests that the Mr 62,000 product may be the singly glycosylated GCAP monomer and that both sites are glycosylated in this phosphatase. The glycosylated and nonglycosylated GCAPs, synthesized by butyrate-treated choriocarcinoma cells in the absence or presence of tunicamycin, respectively, were similarly inhibited by L-leucine or EDTA. Moreover, the specific enzyme activity of glycosylated and nonglycosylated GCAP remained unchanged, indicating that AP lacking N-linked oligosaccharide side chains was catalytically active. This is supported by the finding that nonglycosylated GCAP incorporated inorganic phosphate which binds to the active site of AP. Since the active form of AP is a homodimer, our data indicate that the glycan moieties are not required for the dimerization and catalytic activity of GCAP.

Alkaline Phosphatase↗
PubMed1991

Isolation and characterization of mouse hepatocyte lines carrying a lethal albino deletion.

Mice homozygous for chromosomal deletions at or around the albino locus on chromosome 7 express reduced levels of a group of liver genes, including tyrosine aminotransferase (TAT) and phosphoenolpyruvate carboxykinase (PEPCK), and generally die perinatally. Sequences within the deleted region are thought to encode a regulatory factor(s) that affects expression of these genes in trans. To facilitate study of the putative factors, we immortalized hepatocytes derived from newborn cch wild-type and c14CoS deletion homozygous mice as well as cch/c14CoS heterozygous mice using a SV40 temperature-sensitive A255 mutant virus. Three c14CoS deletion homozygous hepatocyte lines were characterized and compared with the homozygous wild-type and heterozygous lines. The SV40 tsA255 mutant-transformed hepatocyte lines were temperature-sensitive for maintenance of transformation and expressed many liver-specific genes. In agreement with in vivo studies, hepatocyte lines derived from mice homozygous for the deletion expressed reduced mRNA levels of a number of liver genes including TAT, PEPCK, X1, X2, and X7 in comparison with heterozygous and wild-type cell lines. Similar mRNA levels of transferrin and albumin, genes whose expression is unaffected by the mutation in vivo, were observed in all cell lines. The expression of two genes, X5 and metallothionein, reported to be reduced in newborn mutant mice, did not differ appreciably among cell lines. TAT and PEPCK have been shown to respond poorly to glucocorticoids and cAMP in newborn mutant mice. Interestingly, all affected liver genes tested were responsive to glucocorticoids and dibutyryl cAMP in deletion homozygous cell lines as well as in wild-type and heterozygote-derived cell lines. This may suggest that effects of the deletion on expression of liver-specific genes do not cause loss of responsiveness to glucocorticoids and cAMP. These immortalized hepatocyte lines, which express most, if not all, liver-specific genes, should provide a useful means for further investigation of the effects of the albino lethal deletion.

Albinism↗
PubMed1991

Regulation of the uteroferrin gene promoter in endometrial cells: interactions among estrogen, progesterone, and prolactin.

Expression of the gene for the porcine transplacental iron transport protein uteroferrin (UF) is largely restricted to the uterus, where it is differentially regulated by estrogen (E) and progesterone (P). To study the regulatory mechanisms subserving these effects, a 2-kilobase genomic fragment corresponding to -2005 to 48 nucleotides of the UF gene was ligated up-stream to the reporter gene chloramphenicol acetyltransferase (CAT). This construct (UF-CAT) was transiently transfected into rabbit endometrial (HRE-H9), mouse fibroblastic (AKR-2B), and human choriocarcinoma (JEG-3) cells. The basal gene promoter activity of UF-CAT was exhibited in H9 cells, but not in AKR-2B or JEG-3 cells. In contrast, a simian virus-40 early promoter (SV2) was functional in all three cell lines. The H9 cells were used to examine steroid regulation of the UF gene promoter. The CAT expression in H9 cells primed with E and PRL, but not with E or PRL alone, was stimulated by P. In contrast, basal activity of SV2 in these cells was unaffected by hormones, singly or in combination. To examine the basis for the E/PRL-dependent response to P, levels of P and E receptors in H9 cells were quantified. PRL and E plus PRL increased the number of high affinity sites for P, but had little effect on levels of high affinity sites for E in treated vs. untreated H9 cells. In vivo administration of PRL to cyclic gilts had no effect on levels of endometrial UF mRNA and secreted UF protein; however, E- plus PRL-treated gilts had higher (P less than 0.05) levels of endometrial UF mRNA and luminal UF than PRL-treated gilts. These results demonstrate in vitro functional activity of the UF gene promoter and associated 5' flanking region and suggest that sequences within this region may mediate tissue-specific and steroid hormone-regulated expression of the UF gene. Moreover, interactions among E, PRL, and P modulate UF gene expression in vivo and in vitro.

Acid Phosphatase↗
PubMed1991

Isolation and characterization of a human amnion epithelial cell line that expresses the pregnancy-specific beta 1-glycoprotein gene.

Human pregnancy-specific beta 1-glycoprotein (PSG) is a family of closely related glycoproteins of 72K, 64K, 62K, and 54K. Together with the carcinoembryonic antigen, they form new members of the immunoglobulin superfamily. To study the molecular mechanisms that regulate expression of the PSG gene, we established a human amnion cell line, HAA58OD-8C, immortalized with an origin-defective simian virus-40 (SV40) temperature-sensitive A58 mutant virus. HAA58OD-8C cells were temperature sensitive for maintenance of transformation and expressed genes encoding PSG and the alpha- and beta-subunits of hCG. At the permissive temperature (33 C; transformed phenotype), they expressed low levels of PSG, hCG alpha, and hCG beta mRNAs and synthesized low levels of a 48K PSG polypeptide. At the nonpermissive temperature (39.5 C), HAA58OD-8C cells exhibited a differentiated phenotype, expressed increased levels of PSG, hCG alpha, and hCG beta mRNAs, and produced high levels of PSG polypeptides of 72K and 48K. Sodium butyrate induced PSG mRNA expression, and in the presence of butyrate, HAA58OD-8C cells produced high amounts of PSG polypeptides of 72K, 62K, and 48K. Ribonuclease protection analysis indicated that similar PSG transcripts were expressed by HAA58OD-8C cells and human term placenta. However, these amnion cells expressed selectively a certain population of PSG transcripts. Our results show that this amnion cell line provides a suitable model for studies of PSG gene expression and regulation.

Amnion↗
PubMed1991

Characterization of endocrine cell lines immortalized by a temperature-sensitive mutant SV40.

An ideal in vitro model for the study of endocrine functions would be one in which cells could propagate in culture and express their specialized functions. Most endocrine studies to date have relied on primary cell culture or on the use of tumor cell lines. This report describes the characterization of three endocrine cell lines immortalized by transfecting endocrine cells with a temperature-sensitive mutant SV40 virus. Rabbit endometrium (HRE-H9), human placenta (SPA209-10) and rat pituitary (RP) cells were immortalized by SV40 virus, a temperature-sensitive (ts) mutant in the A gene, which encodes the large tumor antigen that is required for the maintenance of transformation. The transformed phenotype of the SV40 tsA mutant-immortalized cell line can be reversed simply by a shift in temperature. At the permissive temperature (34 degrees C), all three types of cells exhibited a transformed phenotype, which is characterized by high cell density growth and by the overgrowth of nontransformed cell layers. However, at the non-permissive temperature (40 degrees C) these cells reverted to a non-transformed phenotype as demonstrated by a marked decrease in the overgrowth of nontransformed layers and by the expression of differentiated functions. At the non-permissive temperature (40 degrees C), the endometrial cell line was capable of synthesizing beta-endorphin, and it exhibited hormonally regulated expression of the transfected hybrid uteroferrin gene construct. The human placenta cell line was capable of secreting GnRH upon stimulation by cAMP, forskolin, theophyllin, PGE, catecholamine and Ca++ channel stimulators. Moreover, the rat pituitary cell line was capable of synthesizing and secreting growth hormone (GH) which was stimulated by GHRH and cAMP. The advantage of the temperature-sensitive cell lines is that a single cell line is the source of both the normal and transformed states; thus, studies are internally controlled. These results demonstrate that tsA mutants of SV40 virus are the best available agents for immortalizing mammalian endocrine cells that retain differentiated functions.

Animals↗
PubMed1990

Induction of phosphoenolpyruvate carboxykinase gene expression by retinoic acid in an adult rat hepatocyte line.

Regulation of expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene was examined in an adult rat hepatocyte line, RALA255-10G, that was immortalized with an SV40 temperature-sensitive (ts) A mutant. These hepatocytes express a transformed phenotype at the permissive temperature (33 degrees C) but a differentiated liver phenotype at the nonpermissive temperature (40 degrees C). We have shown previously that RALA255-10G cells express only low levels of liver-specific genes such as albumin and tyrosine aminotransferase at 33 degrees C. In the present study, we demonstrated that at 33 degrees C, PEPCK synthesis and mRNA expression could be detected only in the simultaneous presence of dexamethasone (DEX), retinoic acid, and dibutyryl-cAMP (Bt2cAMP). At 40 degrees C, PEPCK synthesis and mRNA expression were demonstrated in the presence of Bt2cAMP alone, but not in the presence of either DEX or retinoic acid. However, at 40 degrees C, PEPCK gene expression was stimulated by the combination of DEX plus retinoic acid; additionally, DEX and retinoic acid potentiated the Bt2cAMP-mediated PEPCK induction. In RALA255-10G cells, optimal PEPCK gene expression required the simultaneous presence of DEX, retinoic acid, and Bt2cAMP; DEX had to be present at all times. Triiodothyronine (T3) also potentiated the Bt2cAMP-mediated PEPCK gene expression but failed to increase further the induction by DEX/retinoic acid/Bt2cAMP. By performing nuclear runoff assays, we demonstrated that the PEPCK gene transcription rate in the absence or presence of inducing agents was closely related to the levels of the corresponding mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Effects of sodium butyrate on the synthesis of human pregnancy-specific beta 1-glycoprotein.

Human pregnancy-specific beta 1-glycoprotein (PS beta G) is a polymorphic placental protein which shows strong sequence similarity with the oncofetal protein, carcinoembryonic antigen. To better understand the role of PS beta G in pregnancy, we examined its synthesis and regulation in placental fibroblasts, which had been shown to express the PS beta G gene. The major placental PS beta G is a 72-kDa glycoprotein, while the major fibroblast PS beta G is a 62-kDa species. Administration of sodium butyrate to these fibroblasts slightly stimulated the synthesis of the 62-kDa species but markedly increased the production of two additional PS beta Gs of 72 and 48 kDa. The similarity between the PS beta Gs synthesized by butyrate-treated fibroblasts and human placenta was confirmed by cell-free protein synthesis. Poly(A)+ RNA from butyrate-treated fibroblasts and placenta directed the synthesis of two polypeptides of 48 and 36 kDa, which form the polypeptide backbone of the 72- and 48-kDa glycoproteins. Moreover, the predicted molecular weights of PS beta Gs encoded by the two types of PS beta G cDNA clones were 48,000 and 36,000. Most PS beta G cDNAs identified to date, including the three cDNAs (PSG16, PSG93, and PSG95) isolated in this laboratory, share strong sequence similarity at the 5' region (designated PSG-5') but differ in sequences at their 3' regions. The PSG-5', PSG93-specific, PSG16/PSG93-3', and PSG95-3' probes, which identify the majority of PS beta G mRNAs, hybridized with three PS beta G mRNAs of 2.3, 2.2, and 1.7 kilobases from placental fibroblasts. Butyrate increased the steady-state levels of all three mRNAs. Ribonuclease protection analysis showed that butyrate increased the PS beta G mRNAs containing the PSG-5' or PSG93-specific sequence to approximately 20% of human placental levels. However, unlike human term placenta, which predominantly expressed PS beta G mRNAs with 3'-sequences similar to PSG16/PSG93, the butyrate-treated fibroblasts expressed roughly equal levels of PS beta G mRNAs with the PSG16/PSG93-3' and PSG95-3' ends. All PS beta G cDNAs identified encode proteins with distinct carboxyl termini, suggesting that the composition of the 72-kDa species in placenta and butyrate-treated fibroblasts is likely to be different. Placental fibroblasts provide a unique model for the study of the mechanisms responsible for the differential expression of the PS beta G gene.

Amino Acid Sequence↗
PubMed1990

Linkage of two human pregnancy-specific beta 1-glycoprotein genes: one is associated with hydatidiform mole.

A genomic clone containing two linked human pregnancy-specific beta 1-glycoprotein (PS beta G) genes has been isolated and characterized. The two genes are arranged in the same 5'----3' orientation; the 3' region (including the A2 and B-C exons) of the upstream gene, PSGGA, is linked to the 5' region (including the 5'/L and L/N exons) of PSGGB, the downstream gene. Depending upon the domains compared, PSGGA and PSGGB share 92-98% nucleotide and 86-95% amino acid sequence identity with PSG93, the most abundant PS beta G transcript. The 3' exon (B-C) of PSGGA contains four alternative splice sites and three polyadenylylation sites, which account for the 3' heterogeneity previously reported in the PS beta G family. Each of the predicted PSGGA-encoded proteins would have a different carboxyl terminus. PSGGB corresponds to the previously identified cDNA PSG6, which encodes proteins containing a 34-amino acid leader peptide and a 108-amino acid N domain, which is one amino acid shorter than the majority of PS beta G N domains. Additionally, the PSGGB-encoded proteins contain the cell-surface recognition tripeptide Arg-Gly-Asp, shared by several previously reported PS beta Gs as deduced from cDNA sequences. Northern blot hybridization performed with a PSGGB-specific oligonucleotide probe to the N domain revealed that PSGGB or a PSGGB-like gene encodes a major 1.7-kilobase mRNA in hydatidiform mole tissues and a major 2.0-kilobase mRNA in term placenta tissues. Moreover, the PSGGB-specific probe hybridized most strongly with mRNA from molar trophoblastic tissue, suggesting that the PSGGB-like species may be the gene preferentially expressed in gestational trophoblastic disease. Additionally, the sequence of a 2315-base-pair PS beta G cDNA (PSG95) that contains an N-A1-A2-B2-C domain arrangement is reported. The coding region of PSG95 is identical to the previously reported cDNA clones PSG1d and FL-NCA, but PSG95 contains an additional 518 and 523 base pairs in the 3' end as compared with PSG1d and FL-NCA, respectively.

Amino Acid Sequence↗
PubMed1990

Expression of the pregnancy-specific beta 1-glycoprotein gene in cultured human trophoblasts.

Human pregnancy-specific beta 1-glycoprotein (PS beta G), the major placental glycoprotein, shares strong sequence similarity with carcinoembryonic antigen and is a member of the immunoglobulin superfamily. To understand the role of PS beta G in placental ontogeny during pregnancy, we examined its synthesis and regulation in primary cultures of trophoblast cells. Freshly plated (12 h) cytotrophoblasts expressed little PS beta G transcripts; however, within 24 h of culture, PS beta G mRNAs became detectable. PS beta G synthesis and mRNA expression increased with time in culture, and maximal synthesis was achieved at 4 days, indicating that primary trophoblasts continue differentiating in vitro. Molecular cloning revealed that PS beta G is an extremely polymorphic protein. Most PS beta G cDNAs identified to date, including the three cDNAs (PSG16, PSG93, and PSG95) isolated in this laboratory, share strong sequence similarity in the 5' (designated PSG-5') and coding regions, but differ in sequences at the 3' region. The PSG-5', PSG93-specific, PSG16/PSG93-3', and PSG95-3' probes, which identify the majority of PS beta G mRNAs, hybridized with three mRNAs of 2.3, 2.2, and 1.7 kilobases in primary trophoblasts and human term placental tissue. Ribonuclease protection analysis demonstrated that primary trophoblasts expressed most of the placental PS beta G transcripts. However, culturing in vitro altered PS beta G gene expression, and the level of PS beta G transcripts containing a PSG95-3' sequence was preferentially increased in primary trophoblasts. Moreover, primary trophoblasts synthesized a 64K PS beta G polypeptide in variable amounts and three PS beta Gs of 72K, 62K, and 54K in roughly equal amounts, whereas purified human term placental PS beta G consists of a major polypeptide of 72K and two minor ones of 64K and 54K. PS beta G gene expression in primary trophoblasts was slightly reduced by 8-bromo-cAMP, but was markedly inhibited by sodium butyrate.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1989

Expression of the germ cell alkaline phosphatase gene in human choriocarcinoma cells.

Alkaline phosphatase (ALP) in human choriocarcinoma cells (malignant trophoblasts) was characterized by its greater sensitivity to EDTA and L-leucine inhibition as compared with the placental isozyme. In addition, both the fully processed and the nonglycosylated forms of choriocarcinoma ALP migrated faster than the corresponding placental enzyme in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Choriocarcinoma cells express a 2.6-kilobase (kb) ALP mRNA unlike normal human placenta which expresses a 2.8-kb ALP mRNA. Administration of sodium butyrate to choriocarcinoma cells greatly increased the steady-state levels of the 2.6-kb choriocarcinoma ALP mRNA, which resulted in an increase in enzyme activity and biosynthesis. S1 nuclease analysis using probes derived from a placental ALP cDNA and ribonuclease protection assays employing probes derived from the germ cell ALP gene demonstrated that choriocarcinoma cells express the germ cell ALP gene. The germ cell ALP gene encodes the placental ALP-like isozyme that is primarily expressed in the thymus, testis, and germ cell tumors. The structures of the internal exons (II-X) of the germ cell ALP gene were determined previously based on their similarity to the placental ALP gene. However, the boundaries of exons I and XI (3' exon) of the germ cell ALP gene were not defined due to sequence divergence between the two genes at the 5' and 3' regions. Ribonuclease protection and primer extension assays demonstrated that exon I of this gene is 119 base pairs in length and that germ cell ALP mRNA contains one major transcription initiation site. The isolation and characterization of germ cell ALP cDNA clones from a butyrate-treated choriocarcinoma cDNA library showed that the germ cell ALP mRNA is 2487 bases in length and exon XI of this gene is 1135 base pairs long.

Alkaline Phosphatase↗
PubMed1989

Expression of the alpha-fetoprotein gene in adult rat liver.

The adult rat liver contains three alpha-fetoprotein (AFP) mRNAs of 2.2 (minor), 1.7, and 1.5 kb. These transcripts share a common 3' sequence, but the 1.7- and 1.5-kb AFP mRNAs lack sequences present in the first seven 5' exons of the 2.2-kb AFP mRNA. S1 nuclease analysis maps the 1.7-kb mRNA at the 5' boundary of the eighth exon of the 2.2-kb AFP mRNA and the 1.5-kb mRNA in the middle of the eight exon. In a transformed fetal rat liver cell line, we have previously identified a 1.7-kb AFP mRNA which is encoded by an AFP cDNA (ARFP5) isolated from an adult rat liver cDNA library. The 90-bp 5' sequence of ARFP5, which is located in the seventh intron of the rat AFP gene, is not present in the 2.2-kb fetal AFP mRNA, although ARFP5 does contain nucleotide sequence present in the 2.2-kb AFP mRNA extending from the beginning of its eighth exon (nucleotide 873) to the 3' end. The 1.7-kb RNA in adult liver also hybridizes with the 90-bp 5' sequence of ARFP5, suggesting that the two 1.7-kb AFP mRNAs are similar. The developmental profile of these AFP transcripts shows that fetal rat liver contains mainly the 2.2-kb mRNA which decreases to a very low level around the fifth week after birth. The 1.7- and 1.5-kb AFP mRNAs can be visualized about the third and fourth weeks after birth, respectively, and are the major AFP mRNAs in the liver of rats that are older than 4 weeks. The levels of the AFP mRNAs in adult liver are approximately 0.4% of the AFP mRNA level in 18-day-old fetal liver. Both the 1.7- and 1.5-kb AFP mRNAs are actively translated; they direct the cell-free synthesis of two polypeptides of 50K and 44K. A 50K polypeptide is also the encoded product of the 1.7-kb mRNA in the transformed fetal rat liver cell line, suggesting that the 44K polypeptide may be encoded by the 1.5-kb AFP mRNA.

Animals↗
PubMed1989

Characterization of a temperature-sensitive beta-endorphin-secreting transformed endometrial cell line.

An endometrial cell line (HRE-H9) was established and characterized to study the mechanism by which gene expression of POMC-derived peptides is controlled in uterine tissues. The HRE-H9 cell line was isolated by transforming primary rabbit endometrial cell cultures, derived from hCG-treated pseudopregnant rabbits, with a temperature-sensitive A209 mutant (tsA209) simian virus 40 at a permissive temperature (33 C). The resulting cells exhibited temperature sensitivity in growth and synthesis of immunoreactive beta-endorphin (ir-beta END). The ir-beta-END present in the cell extracts and culture media was assayed by a specific beta END RIA. Sephadex G-50 gel filtration chromatography of the transformed cell extracts showed three peaks of beta END immunoreactivity. The first peak eluted at the void volume, the second peak coeluted with the beta-lipotropin standard, and the third peak coincided with the porcine beta END standard, ir-beta-END was also detectable in endometrial culture media, suggesting that the transformed endometrial cells secreted POMC-derived peptides. Our data indicate that the tsA209 mutant virus-transformed endometrial cell line provides a suitable model for study of the synthesis and regulation of POMC-derived peptides in extrapituitary tissues.

Animals↗
PubMed1989

Characterization of pregnancy-specific beta 1-glycoprotein synthesized by human placental fibroblasts.

We have previously demonstrated that human placental fibroblasts produce a pregnancy-specific beta 1-glycoprotein (PS beta G) immunologically indistinguishable from placental PS beta G. This was confirmed by the immunocytochemical localization of PS beta G in these fibroblasts. In addition, placental fibroblasts contain all three PS beta G mRNAs of 2.3, 2.2, and 1.7 kilobases which hybridize with the three PS beta G cDNAs (PSG16, PSG93, and PSG95) identified, although at 1.4-2.5% of the levels in human term placenta. The major PS beta G species synthesized by placental fibroblasts is a 62K glycopolypeptide formed from a 58K intracellular precursor polypeptide. However, the PS beta G species found in human placenta are one major glycoprotein of 72K and two minor ones of 64K and 54K. Poly(A)+ RNA from placental fibroblasts directed the synthesis of two polypeptides of 48K and 46K (major), whereas, poly(A)+ RNA from human placenta directed the synthesis of higher levels of four polypeptides of 50 K, 48 K (major), 46 K, and 36 K. Thus, the major PS beta G species found in fibroblasts and human placenta differ. The carbohydrate side-chains are essential for the stability of fibroblast PS beta G, because PS beta G synthesis in these fibroblasts could not be detected in the presence of tunicamycin, a protein glycosylation inhibitor which did not affect PS beta G mRNA expression. Our finding that a variant PS beta G species is produced in placental fibroblasts raises the possibility that the authentic placental PS beta G species may have different functions.

Alkaloids↗
PubMed1989

Expression of insulin-like growth factor-I and its receptor by SV40-transformed rat granulosa cells.

Cellular proliferation is a dominant aspect of ovarian follicular development in the rat, and insulin-like growth factor I (IGF-I) has been proposed as a mediator of cellular growth and differentiation in the ovary. An SV40-transformed rat granulosa cell line (RGA-41S) has been established as a model for studies on dividing cells of granulosa origin. Granulosa cells from the ovaries of immature diethylstilbestrol-treated rats were infected with the tsA255 mutant of SV40, followed by cloning in serum-free medium to select transformed cell lines which were serum independent. At the permissive temperature (33 C), RGA-41S cells exhibited a transformed phenotype and rapidly formed high density multilayers of compact cells that readily overgrew nontransformed cells. At the nonpermissive temperature (40 C) cell replication declined and division ceased after 4 days. Furthermore, at 40 C the cells grew as a monolayer and assumed a tetrahedral shape with a high cytoplasm-to-nucleus ratio, and displayed reduced ability to overgrow nontransformed cells. The transformed ovarian cells did not express detectable gonadotropin receptors and steroidogenic activity but retained their epithelial phenotype as demonstrated by cytokeratin staining of the cytoskeleton, the presence of microvilli, and the formation of tight junctions between cells. In support of the proposed autocrine-paracrine actions of IGF-I in the ovary, assay of conditioned serum-free culture medium revealed secretion of IGF-I-immunoreactive material by RGA-41S cells. HPLC-purified IGF-I immunoreactivity from these cells eluted with the same retention time as recombinant human IGF-I. When hybridized with a 32P-labeled rat IGF-I cDNA probe, poly(A)+ mRNA prepared from RGA-41S cells grown at both temperatures showed the typical three size classes of IGF-I mRNA on Northern blots (7.5, 1.7, and 0.8-1.2 kilobase kb), although the levels were somewhat higher at 33 C. The presence of IGF-I receptors in transformed cells was demonstrated by specific 125I-IGF-I binding to intact cells. Scatchard analysis indicated a single class of high affinity receptors at a density of 10(5) binding sites per cell and a dissociation constant (Kd) = 0.52 x 10(-9) M. Furthermore, hybridization of a 32P-labeled IGF-I receptor probe to Northern blots of poly(A+) RNA prepared from cells grown at 33 C and 40 C revealed an 11-kilobase rat IGF-I receptor mRNA. Physiological concentrations of IGF-I increased [3H]aminoisobutyric acid uptake by RGA-41S cells grown at either temperature, attesting to the retention of responsiveness to IGF-I in these transformed granulosa cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Aminobutyrates↗