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J Y Chou

Publications and source records attributed to J Y Chou.

At least 55 records · Page 3Linked to original sources
PubMed1997

Characterization and cellular localization of PSG in rat testis.

In order to establish the rat testis as a model system for studying the human pregnancy-specific beta1-glycoprotein (PSG), expression and cellular distribution of PSG in rat testis were examined. Three partial PSG cDNAs, namely, rnCGM6, rnGCM7, and rnCGM8 were obtained when rat testis cDNA libraries were screened with a human placental PSG cDNA probe. Unlike the human PSGs, the rat PSGs show less nucleotide and amino acid sequence homology among family members. The rat PSGs also have multiple truncated leader sequences followed by immunoglobulin variable-like N domains while human PSGs have a single N domain. Examination of the testis, intestine, kidney, liver, lung, and muscle of male rats by reverse transcription-polymerase chain reaction (RT-PCR) with nested gene-specific primers showed that rnCGM6 was present only in the testis, while rnCGM8 was present in the testis, intestine and lung. On the other hand rnCMG7 was found in all tissues examined. Furthermore, rnCGM7 transcript was present in all somatic cells examined whereas rnCGM6 was predominantly in myoid cells and rnCMG8 in Leydig cells. These results suggest that there is cell-specificity in the expression of PSGs in the rat testis and that the rat testis is a good model for studying the biological activities of the PSGs.

Amino Acid Sequence↗
PubMed1997

Dominant inheritance of isolated hypermethioninemia is associated with a mutation in the human methionine adenosyltransferase 1A gene.

Methionine adenosyltransferase (MAT) I/III deficiency, characterized by isolated persistent hypermethioninemia, is caused by mutations in the MAT1A gene encoding MAT(alpha)1, the subunit of major hepatic enzymes MAT I ([alpha1]4) and III([alpha1]2). We have characterized 10 MAT1A mutations in MAT I/III-deficient individuals and shown that the associated hypermethioninemic phenotype was inherited as an autosomal recessive trait. However, dominant inheritance of hypermethioninemia, also hypothesized to be caused by MAT I/III deficiency, has been reported in two families. Here we show that the only mutation uncovered in one of these families, G, is a G-->A transition at nt 791 in exon VII of one MAT1A allele that converts an arginine at position 264 to a histidine (R264H). This single allelic R264H mutation was subsequently identified in two hypermethioninemic individuals in an additional family, C. Family C members were also found to inherit hypermethioninemia in a dominant fashion, and the available affected members analyzed carried the single allelic R264H mutation. Substitution of R-264 with histidine (R264H, the naturally occurring mutant), leucine (R264L), aspartic acid (R264D), or glutamic acid (R264E) greatly reduced MAT activity and severely impaired the ability of the MAT(alpha)1 subunits to form homodimers essential for optimal catalytic activity. On the other hand, when lysine was substituted for R-264 (R264K), the mutant alpha1 subunit was able to form dimers that retain significant MAT activity, suggesting that amino acid 264 is involved in intersubunit salt-bridge formation. Cotransfection studies show that R264/R264H MAT(alpha)1 heterodimers are enzymatically inactive, thus providing an explanation for the R264H-mediated dominant inheritance of hypermethioninemia.

Adolescent↗
PubMed1997

Gene transfer into hepatocytes mediated by helper virus-free HSV/AAV hybrid vectors.

BACKGROUND: Vectors based on herpes simplex virus type 1 (HSV-1) can efficiently transduce hepatocytes in the mouse liver, and vector genomes can persist for at least 2 months. However, 24 hr after gene transfer, the number of cells that express the transgene decreases rapidly and no transduced cells are detectable after 7 days. In this study, we examined the capability of a helper virus-free HSV/AAV hybrid amplicon vector to extend transgene expression in hepatocytes in vivo. MATERIALS AND METHODS: HSV-1 amplicon or HSV/AAV hybrid amplicon vectors that express reporter genes from different transcriptional regulatory sequences were packaged into HSV-1 virions using a helper virus-free packaging system. To determine relative transduction efficiencies, vector stocks were titered on four different cell lines, including hamster kidney (BHK21) and human lung (Hs913T) fibroblasts, and mouse (G6Pase-/-) and human (NPLC) hepatocytes. After in vivo injection of vector stocks into mouse liver, tissue sections were examined for reporter gene expression and cellular inflammatory response. Blood samples were collected to measure serum transaminase levels as a biochemical index of liver toxicity. RESULTS: Expression of a reporter gene from liver-specific promoter sequences was consistently more effective in hepatic cells compared with fibroblasts, whereas the opposite was true when using an HSV-1 immediate-early promoter. Expression in hepatocytes in vivo was markedly longer from HSV/AAV hybrid vector compared with traditional HSV-1 amplicon vector: the number of transduced cells (approximately 2% of all hepatocytes) remained stable over 7 days after injection of HSV/AAV hybrid vector, whereas no transduced cells were detected 7 days after gene transfer with standard HSV-1 amplicon vector. The rapid decline in reporter gene expression from standard amplicons was not solely caused by a B or T lymphocyte-mediated immune response, as it also occurred in RAG2-/- mice. Hepatocyte toxicity and cellular inflammatory effects associated with HSV/AAV hybrid vector-mediated gene transfer were minimal, and readministration of vector stock proved equally effective in naive mice and in animals that received a first vector dose 4 weeks earlier. CONCLUSIONS: HSV/AAV hybrid amplicon vectors support gene expression in vivo for considerably longer than do traditional HSV-1 amplicon vectors. Moreover, expression from these vectors does not provoke an overt inflammatory or immune response, allowing efficacious expression following repeated in vivo dosing. These characteristics suggest that such vectors may hold future promise for hepatic gene replacement therapy.

Animals↗
PubMed1996

Demyelination of the brain is associated with methionine adenosyltransferase I/III deficiency.

Individuals deficient in hepatic methionine adenosyltransferase (MAT) activity (MAT I/III deficiency) have been demonstrated to contain mutations in the gene (MATA1) that encodes the major hepatic forms, MAT I and III. MAT I/III deficiency is characterized by isolated persistent hypermethioninemia and, in some cases, unusual breath odor. Most individuals with isolated hypermethioninemia have been free of major clinical difficulties. Therefore a definitive diagnosis of MAT I/III deficiency, which requires hepatic biopsy, is not routinely made. However, two individuals with isolated hypermethioninemia have developed abnormal neurological problems, including brain demyelination, suggesting that MAT I/III deficiency can be deleterious. In the present study we have examined the MATA1 gene of eight hypermethioninemic individuals, including the two with demyelination of the brain. Mutations that abolish or reduce the MAT activity were detected in the MATA1 gene of all eight individuals. Both patients with demyelination are homozygous for mutations that alter the reading frame of the encoded protein such that the predicted MATalpha1 subunits are truncated and enzymatically inactive. The product of MAT, S-adenosylmethionine (AdoMet), is the major methyl donor for a large number of biologically important compounds including the two major myelin phospholipids, phosphatidylcholine and sphingomyelin. Both are synthesized primarily in the liver. Our findings demonstrate that isolated persistent hypermethioninemia is a marker of MAT I/III deficiency, and that complete lack of MAT I/III activity can lead to neurological abnormalities. Therefore, a DNA-based diagnosis should be performed for individuals with isolated hypermethioninemia to assess if therapy aimed at the prevention of neurological manifestations is warranted.

Adolescent↗
PubMed1996

Differential expression and butyrate response of human alkaline phosphatase genes are mediated by upstream DNA elements.

Human placentas express high levels of the placental alkaline phosphatase (PLAP) gene and low levels of a highly related gene, germ cell AP (GCAP). Malignant transformation of the placenta is accompanied by a reversal of this pattern of expression. Three Sp1-binding GC-rich DNA elements (sites I-III) located within the first 156 base pairs upstream of the GCAP gene have been shown to direct optimal GCAP gene expression in choriocarcinoma cells. Here we show that the first 100 base pairs upstream of the GCAP gene, which contains sites I and II, constitutes a minimal GCAP promoter. The simultaneous presence of both sites I and II is necessary for GCAP expression and its induction by sodium butyrate. The PLAP promoter directs only a very low level of gene expression in choriocarcinoma cells; the expression does not respond to butyrate. The -100/-1 DNA regions between the GCAP and PLAP promoters differ by only eight base pairs. However, the GC-rich stretches in sites I and II of the GCAP promoter are disrupted in the corresponding PLAP promoter. This disruption blocks or markedly reduces the binding of choriocarcinoma nuclear factors to the PLAP promoter, leading to a reduction in expression and a loss of butyrate response. We further demonstrate that nucleotides -75 to -58 in both AP promoters, which bind a human Y-box binding protein, appear to down-regulate GCAP expression.

Alkaline Phosphatase↗
PubMed1996

Glucose-6-phosphatase dependent substrate transport in the glycogen storage disease type-1a mouse.

Glycogen storage disease type 1a (GSD-1a) is caused by a deficiency in microsomal glucose-6-phosphatase (G6Pase), the key enzyme in glucose homeostasis. A G6Pase knockout mouse which mimics the pathophysiology of human GSD-1a patients was created to understand the pathogenesis of this disorder, to delineate the mechanisms of G6Pase catalysis, and to develop future therapeutic approaches. By examining G6Pase in the liver and kidney, the primary gluconeogenic tissues, we demonstrate that glucose-6-P transport and hydrolysis are performed by separate proteins which are tightly coupled. We propose a modified translocase catalytic unit model for G6Pase catalysis.

Animals↗
PubMed1995

Structure-function analysis of human glucose-6-phosphatase, the enzyme deficient in glycogen storage disease type 1a.

Glucose-6-phosphatase (G6Pase) is the enzyme deficient in glycogen storage disease type 1a, an autosomal recessive disorder. We have previously identified six mutations in the G6Pase gene of glycogen storage disease type 1a patients and demonstrated that these mutations abolished or greatly reduced enzymatic activity of G6Pase, a hydrophobic protein of 357 amino acids. Of these, four mutations (R83C, R295C, G222R, and Q347X) are missense and one (Q347X) generates a truncated G6Pase of 346 residues. To further understand the roles and structural requirements of amino acids 83, 222, 295, and those at the carboxyl terminus in G6Pase catalysis, we characterized mutant G6Pases generated by near-saturation mutagenesis of the aforementioned amino acids. Substitution of Arg-83 with amino acids of diverse structures including Lys, a conservative change, yielded mutant G6Pase with no enzymatic activity. On the other hand, substitution of Arg-295 with Lys (R295K) retained high activity, and R295N, R295S, and R295Q exhibited moderate activity. All other substitutions of Arg-295 had no G6Pase activity, suggesting that the role of Arg-295 is to stabilize the protein either by salt bridge or hydrogen-bond formation. Substitution of Gly-222, however, remained functional unless a basic (Arg or Lys), acidic (Asp), or large polar (Gln) residue was introduced, consistent with the hydrophobic requirement of codon 222, which is predicted to be in the fourth membrane-spanning domain. It is possible that Arg-83 is involved in stabilizing the enzyme (His)-phosphate intermediate formed during G6Pase catalysis. There exist 9 conserved His residues in human G6Pase. His-9, His-119, His-252, and His-353 reside on the same side of the endoplasmic reticulum membrane as Arg-83. Whereas H119A mutant G6Pase had no enzymatic activity, H9A, H252A, and H353A mutant G6Pases retained significant activity. Substitution of His-119 with amino acids of diverse structures also yielded mutant G6Pase with no activity, suggesting that His-119 is the phosphate acceptor in G6Pase catalysis. We also present data demonstrating that the carboxyl-terminal 8 residues in human G6Pase are not essential for G6Pase catalysis.

Amino Acid Sequence↗
PubMed1995

Role of the transcription factor C/EBP beta in expression of a rat pregnancy-specific glycoprotein gene.

Pregnancy-specific glycoproteins (PSGs), which are the major placental proteins, and the carcinoembryonic antigens comprise a subfamily within the immunoglobulin superfamily. To understand the molecular mechanisms underlying the control of PSG expression, we characterized the promoter elements of a rodent PSG gene, rnCGM3, and showed that DNA elements at nucleotides -326 to -185 (PI) relative to the translation start site of rnCGM3 function as a promoter. The rnCGM3 PI promoter contains two placental factor binding sites, PISI and PISII. Both are transcription activation elements. In the present report, we screened a placental expression cDNA library with a rnCGM3-PISII probe (nucleotides -263 to -233) encompassing two overlapping palindromes (TGTTGCTCAACATGTTG) and demonstrated that the PISII-binding factor is C/EBP beta, a leucine zipper family of transcription factor. Gel mobility-shift and transient expression analyses showed that C/EBP beta and C/EBP isoforms, C/EBP alpha and C/EBP delta, bind to the PISII element and trans-activate rnCGM3 gene expression. Deletion of PISII from the rnCGM3 PI promoter greatly reduced the basal as well as the C/EBP-activated rnCGM3 expression. Gel supershift assays demonstrated that C/EBP beta is the placental isoform that binds to the PISII site rnCGM3. Moreover, C/EBP beta is expressed in high levels in the placenta, ovary, liver, lung, heart, and spleen, in contrast to C/EBP alpha, which is expressed primarily in the liver and only low levels in the placenta. Our results demonstrate that C/EBP beta is one of the transcription factors that positively regulate rnCGM3 expression during pregnancy.

Animals↗
PubMed1995

Mutations in the glucose-6-phosphatase gene are associated with glycogen storage disease types 1a and 1aSP but not 1b and 1c.

Glycogen storage disease (GSD) type 1, which is caused by the deficiency of glucose-6-phosphatase (G6Pase), is an autosomal recessive disease with heterogenous symptoms. Two models of G6Pase catalysis have been proposed to explain the observed heterogeneities. The translocase-catalytic unit model proposes that five GSD type 1 subgroups exist which correspond to defects in the G6Pase catalytic unit (1a), a stabilizing protein (1aSP), the glucose-6-P (1b), phosphate/pyrophosphate (1c), and glucose (1d) translocases. Conversely, the conformation-substrate-transport model suggests that G6Pase is a single multifunctional membrane channel protein possessing both catalytic and substrate (or product) transport activities. We have recently demonstrated that mutations in the G6Pase catalytic unit cause GSD type 1a. To elucidate whether mutations in the G6Pase gene are responsible for other GSD type 1 subgroups, we characterized the G6Pase gene of GSD type 1b, 1c, and 1aSP patients. Our results show that the G6Pase gene of GSD type 1b and 1c patients is normal, consistent with the translocase-catalytic unit model of G6Pase catalysis. However, a mutation in exon 2 that converts an Arg at codon 83 to a Cys (R83C) was identified in both G6Pase alleles of the type 1aSP patient. The R83C mutation was also demonstrated in one homozygous and five heterogenous GSD type 1a patients, indicating that type 1aSP is a misclassification of GSD type 1a. We have also analyzed the G6Pase gene of seven additional type 1a patients and uncovered two new mutations that cause GSD type 1a.

Alleles↗
PubMed1995

Molecular mechanisms of an inborn error of methionine pathway. Methionine adenosyltransferase deficiency.

Methionine adenosyltransferase (MAT) is a key enzyme in transmethylation, transsulfuration, and the biosynthesis of polyamines. Genetic deficiency of alpha/beta-MAT causes isolated persistent hypermethioninemia and, in some cases, unusual breath odor or neural demyelination. However, the molecular mechanism(s) underlying this deficiency has not been clearly defined. In this study, we characterized the human alpha/beta-MAT transcription unit and identified several mutations in the gene of patients with enzymatically confirmed diagnosis of MAT deficiency. Site-directed mutagenesis and transient expression assays demonstrated that these mutations partially inactivate MAT activity. These results establish the molecular basis of this disorder and allow for the development of DNA-based methodologies to investigate and diagnose hypermethioninemic individuals suspected of having abnormalities at this locus.

Amino Acid Metabolism, Inborn Errors↗
PubMed1995

Development and characterization of a human marrow stromal cell line that enhances osteoclast-like cell formation.

We established a human bone marrow stromal cell line (Saka) by infecting marrow adherent cells from semisolid marrow cultures with a recombinant simian virus-40 (SV40) virus. The cells expressed SV40 large tumor antigen, had a fibroblast-like shape, and expressed fibronectin and vimentin. They did not contain detectable alkaline phosphatase activity; express myeloid, lymphoid, or factor VIII-associated antigens; or develop adipocyte-like characteristics with dexamethasone treatment. Polymerase chain reaction analysis of Saka cell RNA detected expression of messenger RNAs for interleukin-6 (IL-6), IL-1 beta, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, stem cell factor, and the 1,25-dihydroxyvitamin D3 receptor. Coculture of Saka cells with human marrow mononuclear cells enhanced formation of osteoclast-like multinucleated cells (MNC) in long term human bone marrow cultures. These MNC expressed calcitonin receptors and formed resorption lacunae on dentine. In contrast, coculture of marrow mononuclear cells with other SV40-transformed human marrow stromal cell lines did not increase MNC formation. Conditioned medium from Saka cells or coculture of bone marrow and Saka cells separated by a Millipore membrane did not enhance MNC formation. Addition of a neutralizing antibody to IL-6 or IL-1 beta blocked the effects of Saka cells on MNC formation. These results suggest that marrow stromal cells enhance osteoclast formation in part through direct cell to cell contact and production of IL-6 and/or IL-1 beta.

Base Sequence↗
PubMed1995

Development and characterization of a simian virus 40-transformed, temperature-sensitive rat antimesometrial decidual cell line.

The rat decidual tissue is formed by two cell populations, which express different genes and play diverse roles in pregnancy. Cells that decidualize in the antimesometrial region secrete several hormones and serve as a true endocrine gland. Isolation and maintenance of these decidual cells in primary culture is difficult. The goal of these experiments was to develop a cell line to serve as a model to study the expression and regulation of various genes specific to the antimesometrial decidual cells. Decidualization was induced in pseudopregnant rats. The antimesometrial decidua was dissected out, and cells were enzymatically dissociated and were cultured for 18 h at 37 C in RPMI-1640 medium containing 10% fetal bovine serum. Cells were washed repeatedly and then infected with a temperature-sensitive mutant of the simian virus. Transformed cells were maintained at the permissive temperature (33 C) until colonies were identified and harvested. Whereas primary cells in culture did not divide, the cloned decidual cell lines demonstrated transformed features; they multiplied at 33 C and formed multilayers. At the nonpermissive temperature (39 C), cell replication decreased, and after 4 days of culture the cells lost their transformed phenotype and continued to grow as a monolayer similar to primary cells. Cells under these conditions also assumed morphological characteristics similar to antimesometrial cells: polynucleated, large, and having cytoplasm filled with lipid droplets. Interestingly, cells cultured at 39 C that were shifted back to 33 C resumed rapid growth. To determine whether these cells also express messenger RNAs (mRNAs) found in normal antimesometrial decidual cells, the presence of activin beta A mRNA was investigated by Northern analysis and reverse transcription polymerase chain reaction. A single 6.8-kilobase activin beta A transcript was expressed abundantly at both 33 and 39 C, indicating that even when cells are rapidly dividing they express activin beta A. Activin beta B mRNA was also expressed in these cells, although in lower abundance, as were two binding proteins for activin, activin receptor II and follistatin. The activin beta A and beta B genes were responsive to cAMP stimulation in these cells. Since the hallmark of the antimesometrial decidual cells is the secretion of PRL-related hormones, the expression of decidual PRL-related protein and PRL-like protein B was examined. Northern analysis revealed a major 1.2-kilobase transcript of PRL-like protein B expressed equally at both temperatures.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1995

Estrogen receptors are present in human granulosa cells.

Recent studies failed to detect estrogen receptors in primate follicles. This study was initiated to determine whether estrogen receptor (ER) messenger ribonucleic acid (mRNA) is present in human granulosa cells and, further, if functional ER proteins are present. To evaluate the presence of ER, RNA from human granulosa cells obtained at the time of oocyte retrieval for assisted reproduction was extracted, and complementary DNA synthesis was performed by the reverse transcriptase-polymerase chain reaction. Oligonucleotide primers were used to amplify basepairs 570-852 in the B- and C- domains of the ER mRNA. Southern blotting was performed and confirmed that the amplified DNA fragment identified in granulosa cells represented ER. By reverse transcriptase-polymerase chain reaction, mRNA for the ER is clearly identified in primary human granulosa cells obtained at the time of oocyte retrieval. To expand these studies and determine whether functional ER were present in human granulosa cells in culture, a simian virus-40-transformed human granulosa cell line was studied. Cells were transfected with a plasmid containing as estrogen response element up-stream from the bacterial reporter gene chloramphenicol acetyltransferase (CAT). In transfected cells, CAT activity is inducible by estradiol if endogenous functional ER are present. In these studies, the transfection analysis confirmed that functional, transcriptionally competent ER are present in a human granulosa cell line, with a 4- to 5-fold enhancement of CAT activity demonstrated after the addition of estradiol compared to that in nonhormone-treated cells. In conclusion, ER mRNA is present in human granulosa cells. Functional ER are also demonstrated in a transformed human granulosa cell line. We hypothesize that low, but biologically significant, amounts of ER protein are present in human granulosa cells, which are not routinely detectable by standard assays.

Blotting, Southern↗
PubMed1995

A motif in PSG11s mediates binding to a receptor on the surface of the promonocyte cell line THP-1.

The pregnancy-specific glycoproteins (PSG) form a large family of essential pregnancy proteins, but their biological function is unknown. We have investigated whether one function of the PSG is to interact with cells of the maternal immune system. Normal human peripheral blood mononuclear cells, activated with phorbol ester, are shown to bind to purified placental PSG. This binding activity can be mimicked using a chemically synthesized peptide ligand containing the Arg-Gly-Asp (RGD) motif present in the N-terminal domain of PSG11s. The PSG11s receptors are present on cells of the myeloid cell lineage but not of the T cell or B cell lineages. The binding is mediated in part by the RGD motif and can be competed against by appropriate RGD-containing, but not Arg-Ala-Asp (RAD)-containing, ligands. Ligand binding requires a functional cytoskeleton. By examining the U937 and THP-1 promonocyte cell lines, the presence of receptors with two different binding characteristics are demonstrated. The THP-1 receptor is identified by chemical cross-linking as a protein of 46 kilodaltons (kDa), and affinity chromatography demonstrates the presence of three protein species of 32 kDa, 16.8 kDa, and 15.9 kDa, suggesting the receptor has multiple subunits.

Amino Acid Sequence↗
PubMed1995

Characterization of a prolactin-synthesizing rat pituitary cell line immortalized by the temperature-sensitive mutant SV40.

A prolactin (PRL) synthesizing rat anterior pituitary (AP) cell line named tAP-CLC-2 was established by transformation with mutant temperature-sensitive A (tsA) simian virus 40 (SV40). The transformed cells were temperature-sensitive for morphology, cell propagation, and expression of phenotypic genes. Monolayer primary rat AP cells were transfected with a mutant SV40 (tsA 205) at 10 times the multiplicity of infection (MOI) at 34 degrees C. After cell clones formed, each clone was isolated and transferred to separate flask until sufficient cells were grown for freezing and characterization. At the permissive temperature (34 degrees C), these cells were spindle-shaped and grew rapidly like tumor cells. However, at the nonpermissive temperature (40 degrees C), the cells exhibited a rounded shape and ceased to propagate because the gene for maintenance of transformation was not expressed. Cell extracts from the cell line tAP-CLC-2 showed an inhibition curve parallel to that of normal rat pituitary cell extracts in a PRL radioimmunoassay (RIA). The gel filtration profile of immunoassayable PRL obtained from fast performance liquid chromatography (FPLC) showed that tAP-CLC-2 cell extract exhibited a PRL peak coincidental with primary rat pituitary cell extracts. Western blot showed that cell extracts from the tAP-CLC-2 cell line and from normal rat pituitary glands shared a similar, major immunoreactive PRL band. The tAP-CLC-2 cell line was responsive to estradiol (E 2; 10(-7) M), progesterone (10(-8) M), and gonadotropin-releasing hormone (GnRH; 10(-9) M) treatments. These hormonal treatments increased (p < 0.05) cell PRL content. Interestingly, treatment with a high dose (10(-7) M) of ovine luteinizing hormone (oLH) also increased (p < 0.05) PRL content in the cell line; a low dose (10(-9) M) of oLH did not. The cell line appeared to synthesize growth hormone (GH) as well. These results indicate this cell line has properties shared by primary AP cells and can provide a unique model for the study of the synthesis and gene regulation of PRL in vitro.

Animals↗
PubMed1994

Effect of acute diabetes on rat hepatic glucose-6-phosphatase activity and its messenger RNA level.

Glucose-6-phosphatase catalyzes the final step of glucose production by liver and kidney. Though its strategic position has sparked interest in its regulation, difficulty with isolating a pure, stable enzyme has slowed progress. Virtually all previous work examining the physiologic regulation of this enzyme has relied on estimates of glucose-6-phosphatase activity in crude microsome preparations. The recent cloning of human and murine glucose-6-phosphatase cDNAs has now allowed study of its mRNA expression. We studied the effect of acute, streptozotocin-induced diabetes on hepatic microsomal glucose-6-phosphatase activity and mRNA expression in young (89 +/- 3 g), juvenile (304 +/- 4 g) and adult (512 +/- 10 g) rats. In control rats, mRNA expression and enzyme activity was similar among the three age groups. Streptozotocin-induced diabetes significantly increased the enzyme activities in both intact and triton-treated microsomes in all groups of rats (p < 0.001). Glucose-6-phosphatase mRNA expression was increased in the diabetic rats as well (p < 0.0001). Blood glucose concentrations correlated significantly with glucose-6-phosphatase mRNA level (p < 0.005) and both intact (p < 0.002) and triton-treated (p < 0.001) microsomal glucose-6-phosphatase activity. Both intact and triton-treated microsomal glucose-6-phosphatase activity correlated with mRNA level (p < 0.001, for each). We conclude that acute streptozotocin-diabetes increase expression of glucose-6-phosphatase mRNA and this contributes to the increased glucose-6-phosphatase activity seen with diabetes mellitus.

Acute Disease↗
PubMed1994

Characterization of two promoters of a rat pregnancy-specific glycoprotein gene.

Pregnancy-specific glycoproteins (PSGs) are the major placental glycoproteins, that together with the carcinoembryonic antigens comprise a subfamily within the immunoglobulin superfamily. In order to develop an animal model for understanding the molecular mechanisms underlying the control of PSG expression, we isolated and characterized cDNA and genomic clones encoding a rodent PSG, rnCGM3. The rnCGM3 cDNA is 2761 bp in length and contains an open reading frame that encodes a 475 amino acid polypeptide with a domain arrangement of L1N1-L2N2-L3N3-A. The sequence in 5'-untranslated and L1 regions of rnCGM3 is identical to a previously identified cDNA, rnCGM6. The transcription initiation sites of both genes are located at nucleotide -197 upstream of the translation start site. In transient transfection assays using a chloramphenicol acetyltransferase (CAT) reporter gene, we demonstrated that DNA elements at nucleotides -326 to -185 (PI) and -147 and -86 (PII) relative to the translation start site of rnCGM3 could both function as promoters. The downstream promoter, PII, which is located within the first exon, shares high sequence identity with the minimal promoters of human PSG genes. Electrophoretic mobility shift assays (EMSAs) showed that protein factors in placental cell extracts formed three complexes (PIICI, PIICII, and PIICIII) with the PII promoter element. The PIICIII complex was also observed by DNase I footprinting analysis. Unlike PII, the upstream promoter, PI, contains a TATA box. DNase I footprinting analysis revealed two nuclear protein binding sites at nucleotides -311 to -290 (PISI) and -257 to -239 (PISII) in PI. EMSAs showed that protein factors in placental cell extracts bound to both sites and deletion of either site markedly reduced CAT expression. PISII contains a palindromic motif, TGTTGCTCAACA, and protein cross-linking and Southwestern hybridization analyses demonstrated that the protein factor binding to PISII had an apparent molecular mass of 40 kDa.

Amino Acid Sequence↗