PubMed Health⌕ Search

Biomedical subjects

C J Pan

Publications and source records attributed to C J Pan.

33 records · Page 2Linked to original sources
PubMed1993

Mutations in the glucose-6-phosphatase gene that cause glycogen storage disease type 1a.

Glycogen storage disease (GSD) type 1a is caused by the deficiency of D-glucose-6-phosphatase (G6Pase), the key enzyme in glucose homeostasis. Despite both a high incidence and morbidity, the molecular mechanisms underlying this deficiency have eluded characterization. In the present study, the molecular and biochemical characterization of the human G6Pase complementary DNA, its gene, and the expressed protein, which is indistinguishable from human microsomal G6Pase, are reported. Several mutations in the G6Pase gene of affected individuals that completely inactivate the enzyme have been identified. These results establish the molecular basis of this disease and open the way for future gene therapy.

Amino Acid Sequence↗
PubMed1993

Isolation of the gene for murine glucose-6-phosphatase, the enzyme deficient in glycogen storage disease type 1A.

Glycogen storage disease (GSD) type 1a (von Gierke disease) is caused by a deficiency in glucose-6-phosphatase, the key enzyme in glucose homeostasis catalyzing the terminal step in gluconeogenesis and glycogenolysis. Despite its clinical importance, this membrane-bound enzyme has eluded molecular characterization. Here we report the cloning and characterization of a murine glucose-6-phosphatase cDNA by screening a mouse liver cDNA library differentially with mRNA populations representing the normal and the albino deletion mouse known to express markedly reduced glucose-6-phosphatase activity. Additionally, we identified the gene that consists of 5 exons. Biochemical analyses indicate that the in vitro expressed enzyme is indistinguishable from mouse liver microsomal glucose-6-phosphatase exhibiting essentially identical kinetic constants, latency, thermal lability, and vanadate sensitivity. The characterization of the murine glucose-6-phosphatase gene opens the way for studying the molecular basis of GSD type 1a in humans and its etiology in an animal model.

Amino Acid Sequence↗
PubMed1993

Characterization of two allelic variants of a human pregnancy-specific glycoprotein gene.

The pregnancy-specific glycoproteins (PSGs) of the human placenta are a group of proteins that together with the carcinoembryonic antigens comprise a subfamily within the immunoglobulin superfamily. To study the control of PSG expression, we isolated and characterized PSG genes and identified cis-acting DNA elements in the 5'-flanking gene regions essential for PSG expression. Two overlapping PSG cosmid clones, which contain two allelic variants of a PSG gene (PSG12 and PSG12 psi), were isolated from an unamplified library made from a single individual. Cosmid 1 contains exons 1 (5'/L) and 2 (L/N) of the PSG12 gene located downstream of a previously identified PSG1-I gene. Cosmid 6 contains a portion of the PSG1-I gene lacking exons 1 and 2 upstream of a complete PSG12 psi transcription unit. Sequence comparison indicates that exons 5'/L and L/N in PSG12 and PSG12 psi are 99% identical, except that the L/N exon in the PSG12 psi gene contains a stop codon. Both PSG12 and PSG12 psi transcripts were detected in the human placenta, indicating that both genes are actively transcribed. However, the PSG12 psi gene may represent an allelic pseudogene variant of the PSG12 gene, because all identified PSGs contain a functional N-domain. Primer extension analysis showed that the PSG12 gene starts at a cluster of sites located at -106 to -104 base pairs with respect to the translation start site. In transient transfection assays using a chloramphenicol acetyltransferase reporter gene, we demonstrated that the -835 to -34 DNA region upstream of the translation start site of PSG12 or PSG12 psi contained both positive and negative elements that control PSG expression. Deletion analysis showed that nucleotides -172 to -34 in the PSG12 gene could function as a promoter. Gel retardation analysis showed that protein factors in human placental cell extract formed four complexes (I, II, IIa, and III) with the PSG12(-172/-34) DNA. Site-directed mutagenesis that prevents protein factor binding to the PSG12 promoter resulted in a marked reduction in transcription activation, locating the core enhancers at nucleotides -148 to -141 and -60 to -55. Mutagenesis studies also showed that the ACAGC repeats at nucleotides -84 to -68 in the PSG12 5'-flanking are essential for expression of the PSG12 gene in human placental cells.

Alleles↗
PubMed1992

Cloning and expression of genes encoding human pregnancy-specific glycoproteins.

The pregnancy-specific glycoproteins (PSGs) of the human placenta and the carcinoembryonic antigens comprise a subfamily within the immunoglobulin superfamily. There may be as many as 20 different PSG genes which are predominantly expressed in the placenta. As an initial step toward understanding the control of PSG expression, we isolated and characterized two nearly identical PSG genes, PSG1 and PSG1-I. PSG1, which lacks exon 1 (5'/L), but contains exons 2 (L/N), 3 (A1), 4 (A2), and 5 (B2-C), encodes five previously identified type I transcripts, PSG1a, 1b, 1c, 1d, and 1e in a L/N-A1-A2-B2-C domain arrangement. PSG1-I, which contains a complete transcriptional unit consisting of exons 5'/L, L/N, A1, and B2-C, encodes type II PSG transcripts in a L/N-A1-B2-C domain arrangement. The predicted PSG1-I-encoded proteins share nearly complete sequence identity with the PSG1-encoded members, except the latter contain extra A domains. Amplification by polymerase chain reaction of placental or hydatidiform mole cDNA demonstrates that PSG1-I is a functional type II PSG gene. Using transient expression assays, we demonstrated that the -834/-34 region upstream of the translational start site of the PSG1-I gene contained the PSG promoter elements and the -834 to -456 region contained negative control elements. Sodium butyrate, an inducer of PSG synthesis, greatly stimulated expression of all PSG1-I-chloramphenicol acetyltransferase (CAT) fusion gene constructs. However, butyrate was at least 2-fold more effective in stimulating CAT activity of fusion genes containing upstream sequences (-834 to -576) than those containing proximal sequences (-456 to -172), suggesting two regions in the PSG1-I gene that mediate the butyrate response.

Amino Acid Sequence↗
PubMed1992

Inhibition of tyrosine aminotransferase gene expression by retinoic acid.

Regulation of tyrosine aminotransferase (TAT) gene expression was examined in RALA255-10G, a simian virus-40 tsA mutant-immortalized adult rat hepatocyte line. At the nonpermissive temperature (40 C), these hepatocytes exhibited a differentiated phenotype and actively expressed the TAT gene, but only in the presence of dexamethasone (DEX). The glucocorticoid-mediated TAT expression was inhibited by cycloheximide, a protein synthesis inhibitor, and by RU486, a glucocorticoid antagonist, suggesting that glucocorticoid induction requires protein synthesis and may be mediated through hormone receptors. (Bu)2cAMP (Bt2cAMP) or retinoic acid, individually or in combination, failed to increase TAT mRNA levels. However, Bt2cAMP greatly potentiated the induction by DEX, whereas retinoic acid inhibited the induction by DEX or DEX/Bt2cAMP. Nuclear run-on assays demonstrated that the induction of TAT expression by DEX or DEX/Bt2cAMP in RALA255-10G cells is regulated primarily at the transcriptional level. In contrast, retinoic acid antagonized the DEX- or DEX/Bt2cAMP-mediated induction without affecting the rate of TAT gene transcription. Instead, retinoic acid destabilized TAT mRNA. The half-life values of TAT mRNA in DEX/Bt2cAMP- and DEX/Bt2cAMP/retinoic acid-treated cells were approximately 235-270 min and 90-100 min, respectively. Our results indicate that inhibition of TAT expression by retinoic acid was regulated primarily at the posttranscriptional level.

Animals↗
PubMed1992

Immortalization of virus-free human placental cells that express tissue-specific functions.

Human pregnancy-specific glycoproteins (PSGs) are a family of closely related placental proteins that, together with the carcinoembryonic antigen members, comprise a subfamily within the immunoglobulin superfamily. To facilitate study of the control of PSG expression, we immortalized human placental cell lines with adenovirus-origin-minus (ori-)-simian virus-40 (SV40) recombinant viruses containing either wild-type or temperature-sensitive (ts) A mutants of SV40. Cells transformed with the SV40 tsA chimera (HP-A1 and HP-A2), but not the SV40 wild-type chimera (HP-W1), were temperature sensitive for transformation. All three cell lines expressed trophoblast-specific genes, including PSG and the alpha- and beta-subunits of hCG. Human CG alpha expression was greatly stimulated by (Bu)2cAMP in all three cell lines; shifting HP-A1 and HP-A2 cells to the nonpermissive temperature (39.5 C) further increased hCG alpha expression. At both 33 C (permissive temperature) and 39.5 C, the transformed placental cells expressed PSG mRNAs of 2.2 and 1.7 kilobases; expression was greatly stimulated by sodium butyrate. In the absence of an inducer, the three placental lines synthesized a PSG of 64 kilodaltons (kDa). In the presence of butyrate, they synthesized PSGs of 72, 64, and 54 kDa, similar to the placental PSGs. However, in placenta the predominant species is the 72-kDa product. At 39.5 C, butyrate selectively increased synthesis of the 72-kDa PSG in HP-A1 and HP-A2 cells. To characterize PSG promoter activity, we constructed chloramphenicol acetyltransferase (CAT) fusion genes containing -809 to -44 basepairs up-stream of the translational start site of the PSG6 gene. Using transient expression assays, we demonstrated that the -809/-44 region of the PSG6 gene contained cis-acting sequences that can direct CAT expression in human placental cells. Sodium butyrate, which stimulates PSG expression, greatly increased CAT activity, indicating that butyrate-induced PSG expression is regulated primarily at the level of gene transcription.

Base Sequence↗
PubMed1992

[The ultrastructural and immunohistochemical observations of anaplastic meningioma].

Eighteen cases of anaplastic meningioma were studied by LM, EM and immunohistochemistry for vimentin, EMA, keratin, GFAP and S-100. Microscopically, there were four histologic types, i.e. fibrosarcoma-like, angiosarcoma-like, polymorphic giant cell sarcoma-like and angiopapillary structure. By EM, four kinds of cells: undifferentiated cell, intermediate transitional cell, spindle-shaped cell, and giant cell, were found and variant transitions from undifferentiated or poorly, differentiated to meningioma cells were observed. Their ultrastructures and immunohistochemical features are similar to those of malignant mesothelioma. Since these two kinds of neoplasm showed both mesenchymal and epithelial cells in the features, the authors consider that their histogenesis may also be similar.

Adolescent↗
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↗
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↗
PubMed1982

Methylation of ribonucleic acid in a cell-free system from mouse myeloma cells.

Isolated nuclei incorporate few methyl groups into RNA when they are incubated with S-adenosyl[methyl-3H3]methionine and four ribotriphosphates. When the nuclei were supplemented with a soluble total cell protein extract, the incorporation of methyl groups into RNA was stimulated 3-6-fold. All classes of RNA were methylated. Methylation of the 2'-OH of ribose and the bases of ribosomal RNA occurred predominantly on endogenous ribosomal RNA precursors, with a minority (20%) occurring on the newly synthesized rRNA precursor. Methylation of the tRNA precursor occurred on both endogenous (40%) and newly synthesized (60%) molecules. The methylation of adenosine in hnRNA occurred predominantly on molecules transcribed in vitro and was sensitive to 1 microgram/mL alpha-amanitin. A final site of methylation was the 7 position of guanosine of the cap structure. About 10% of the RNA polymerase II transcripts were capped in vitro. Capping was blocked 90% by 1 microgram/mL alpha-amanitin and was independent of the presence of the cell protein extract.

Animals↗
PubMed1981

Isolation of two clusters of mouse histone genes.

Histone mRNA was partially purified from mouse myeloma cells synchronized in S phase by isoleucine starvation. A cDNA was prepared that contained sequences complementary to all five mouse histone genes. This cDNA was used to screen a library of mouse DNA in lambda phage. The positive clones were screened by hybridization with sea urchin histone gene-specific probes to identify those clones that contained histone genes. Confirmation of this identification was obtained by hybridization with Drosophila histone genes. Two independent clusters of histone genes were isolated. One, MM531, contains regions hybridizing specifically to H3, H4, and H1 and the other, MM221, contains two regions hybridizing specifically to H3 and single regions complementary to H4, H2b, and H2a. They are not part of a simple repeating structure. The nucleotide sequence of the coding region of the H3 gene in MM531 has been determined. This gene could code for a variant H3 protein that has several amino acid substitutions not reported in other H3 proteins.

Animals↗
PubMed1978

RNA synthesis in myeloma cells synchronized by isoleucine starvation.

Myeloma cells have been synchronized by isoleucine starvation. Changes in RNA synthetic rates as a result of starvation have been studied. The ability of isolated nuclei to synthesize RNA declines on starvation and increases subsequently on refeeding isoleucine. There is a coordinate drop in synthetic rate for all three polymerases both in vivo and in vitro. The chain elongation rate in vitro is the same in starved and normal cells, so the difference is in the number of active polymerases in vitro. However, the nuclei do not exactly parallel the state of the cell from which they were isolated, but the in vitro RNA synthesis increases more slowly than the in vivo RNA synthesis. There is no change in relative amounts of synthesis by the different RNA polymerases. The in vitro RNA product is similar in starved and growing cells.

Cell Division↗
PubMed1977

Studies of human histone messenger RNA. I. Methods for the isolation and partial characterization of RNA fractions containing human histone message from HeLa S3 polyribosomes.

Large quantities of nonpolyadenylated [poly(A(-))] 4 to 18 S RNA were isolated from the polyribosomes of S phase HeLa S3 cells and were fractionated into multiple discrete RNA components by continuous elution preparative electrophoresis. Previous studies have shown that treatment os S phase HeLa cells with cytosine arabinoside inhibits DNA replication and causes translatable histone messenger RNA (mRNA) species to disappear from cytoplasmic polyribosomes (Borun, T. W., Scharff, M.D., and Robbins, E. (1967) Proc. Natl. Acad. Sci. U.S.A. 58, 1977-1983; Gallwitz, D., and Mueller, G. C. (1969) J. Biol. Chem. 244, 5948-5952; Borun, T. W., Gabrielli, F., Ajiro, K., Zweidler, A., and Baglioni, C. (1975) Cell 4, 59-67; Gallwitz, D. (1975) Nature 257, 247-248). In the present study it was found that cytosine arabinoside treatment does not appreciably affect major 7.5 to 8 S RNA species but does cause the disappearance of 8.6 to 13 S RNA components from preparative electrophoresis elution profiles of S phase polyribosomal 4 to 18 S RNA. Base ratio analysis of the 8.6 to 13 S putative histone mRNA species indicates that they are GC-rich but not like the HeLa 18 or 28 S rRNA in base composition.

Chromatography, Affinity↗