Sachiko Ohmori

Oxidoreductive modification of two cysteine residues in paired domain by Ref-1 regulates DNA-binding activity of Pax-8

We have reported reversible oxidoreductive regulation of DNA-binding activity of Pax-8: oxidation inhibits its binding and subsequent reduction restores the binding. Here, we show that Cys-45 and Cys-57 in the paired domain of rat Pax-8, which are conserved in all Pax members, are responsible for the redox regulation of its binding. Electrophoretic mobility shift assay using deletion mutants and mutants with substitution of cysteine with serine revealed that oxidation by diamide of either Cys-45 or Cys-57 loses the DNA binding of Pax-8. An intracellular oxidoreductive enzyme redox factor-1 (Ref-1) could reduce the oxidized Cys-45 or Cys-57 and restored the binding. Furthermore, reporter gene assay showed that transcriptional activity of wild-type Pax-8 was enhanced by co-expression of Ref-1. When the mutant with double substitutions of Cys-45 and Cys-57, which was insensitive to oxidation, was transfected, the basal transactivation level was much higher than that of wild-type Pax-8, while it was not enhanced by Ref-1. These results demonstrated that oxidoreductive modification of Cys-45 and Cys-57 via Ref-1 plays a role in redox regulation of Pax-8 in living cells.

Expression of Adrenocorticotropin Receptor Gene in Adrenocortical Adenomas From Patients With Cushing Syndrome: Possible Contribution for the Autonomous Production of Cortisol

ObjectiveTo examine whether inhibition of endogenous adrenocorticotropin (ACTH) secretion in patients with Cushing syndrome affects the expression of the ACTH receptor (ACTH-R) gene in adrenocortical adenoma and attached atrophic normal gland. Summary Background DataACTH increases adrenal cell growth and steroidogenesis by means of ACTH-R. In vivo and in vitro studies have shown that expression of ACTH-R is upregulated by its own ligand ACTH in several species. In patients with Cushing syndrome resulting from adrenocortical adenoma, there is autonomous production of cortisol from the adenoma. This strongly inhibits endogenous ACTH secretion, giving rise to the speculation that the expression of the ACTH-R gene in these patients is also suppressed. However, previous studies have shown that administration of exogenous ACTH to these patients leads to a further increase in the production of cortisol, suggesting the expression of functional ACTH-R in the adenoma. The authors, therefore, examined the expression of the ACTH-R gene in these patients. MethodsFourteen patients with Cushing syndrome were studied. Glucocorticoid excess resulting from autonomous production from the adenomas was ascertained, and unilateral adrenalectomy was performed. The levels of ACTH-R and cytochrome P450 side chain cleavage enzyme (P450scc) mRNAs were determined by Northern blot analysis. The entire coding region of the ACTH-R gene in these patients was sequenced. ResultsACTH-R mRNA abundance in the attached atrophic normal adrenals was suppressed and invariably less than that in the normal gland obtained from a patient with renal cancer. However, the expression of ACTH-R mRNA was not suppressed in any of the adenomas. Expression of ACTH-R mRNA in the adenomas was four- to sixfold greater than that in the attached atrophic gland. No mutation in the coding sequence of the ACTH-R gene in the adenoma was detected in any of the patients. The mRNA in the adenomas appeared to be translated into functionally active receptor because intramuscular administration of ACTH resulted in significant increases in plasma cortisol before surgery but not 3 months after surgery. In addition, there was a positive linear correlation between the expressions of ACTH-R and P450scc mRNAs in the adenoma tissue. ConclusionsSuppressed ACTH secretion in patients with Cushing syndrome results in reduction of the ACTH-R mRNA expression in nonneoplastic adrenocortical cells. However, the regulatory mechanism of ACTH-R expression might be different in adenoma. Persistent expression in the adenoma of ACTH-R alone, even in the absence of ACTH, might result in increased basal adenyl cyclase activity, as observed in the case of thyroid-stimulating hormone receptor, and thereby might play a role in the autonomous production of cortisol.

Identification of a novel myosin-Va mutation in an ataxic mutant rat, dilute-opisthotonus

Abstract. Mutations of the myosin-Va gene (Myo5a) cause diluted coat color in mice and are occasionally associated with severe neurological disorders. Dilute-opisthotonus (dop) is a spontaneous gene mutation in the rat, and phenotypes of the homozygote (dop/dop) are similar to those of the Myo5a-deficient mouse, suggesting that the mutation resides in the rat Myo5a gene. To elucidate the molecular basis of the dop mutation, we cloned the rat Myo5a cDNA from the wild type and the dop/dop. The wild-type rat Myo5a cDNA contained a 5487-bp ORF and showed higher homology with Myo5a of the other species than Myr6 (Myo5b) in the rat. A 141-bp in-frame deletion was detected in the head region in the dop cDNA. An intragenic rearrangement consisting of a 306-bp inversion associated with 17-bp and 217-bp deletions were identified in the Myo5a gene of the dop genome. This rearrangement involved a 141-bp exon, which was skipped in the dop transcript. The MyoVA protein expression was severely impaired in the dop/dop brain. This is the first report to define the dop mutation as the Myo5a gene abnormality in the rat.

Molecular cloning of prostaglandin EP3 receptors from canine sensory ganglia and their facilitatory action on bradykinin-induced mobilization of intracellular calcium

We previously demonstrated that the activation of prostaglandin E‐prostanoid‐3 (EP3) receptor sensitized the canine nociceptor response to bradykinin (BK). To elucidate the molecular mechanism for this sensitization, we cloned two cDNAs encoding EP3s with different C‐terminals, from canine dorsal root ganglia, and established the transformed cell lines stably expressing them. In both transformants, EP3 agonist did not increase intracellular cAMP levels, but it attenuated forskolin‐dependent cAMP accumulation in a pertussis toxin (PTX)‐sensitive manner and increased intracellular calcium levels in a PTX‐resistant manner, indicating that both EP3s can couple with Gi and Gq, but not with Gs proteins. As the nociceptor response to BK is mediated by BK B2 receptor, it was transfected into the transformants and the effects of EP3 agonist on BK‐dependent calcium mobilization were investigated. When BK was applied twice with a 6‐min interval, the second response was markedly attenuated. Pre‐treatment with EP3 agonist had no effect on the initial response, but restored the second response in a PTX‐sensitive manner. A protein kinase A inhibitor mimicked the effect of EP3 agonist. These results demonstrate that the activation of EP3 restores the response to BK by attenuating the desensitization of BK B2 receptor activity via Gi protein.

Expression of E16/CD98LC/hLAT1 is responsive to 2,3,7,8-tetrachlorodibenzo-p-dioxin

We employed cDNA representational difference analysis to identify new genes that are upregulated by 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) in a human hepatoblastoma cell line HepG2. We isolated several TCDD‐responsive cDNAs. Sequence analysis revealed that one of them encodes E16/CD98LC/hLAT1, an integral membrane protein involved in multiple cellular functions including cellular transport of L‐type amino acids. Northern blot analysis confirmed the TCDD‐dependent upregulation of the mRNA. Induction of E16/CD98LC/hLAT1 mRNA by TCDD did not require de novo protein synthesis as revealed by the experiment using cycloheximide. Consistent with the changes at mRNA level, the transport of 3H‐leucine into HepG2 cells was significantly increased by TCDD treatment. These findings provide a novel aspect of biological effects of TCDD on human hepatocytes.

Molecular Cloning of Sucrase-Isomaltase cDNA in the House Musk Shrew Suncus murinus and Identification of a Mutation Responsible for Isolated Sucrase Deficiency

Isolated sucrase deficiency has been demonstrated in a line of house musk shrew, Suncus murinus (laboratory name: suncus). This animal belongs to the order Insectivore and is phylogenetically different from ordinarily used laboratory animals. They are believed to have evolved with mainly animal food without sucrose. To study the molecular basis of the sucrase deficiency in suncus, we cloned 6.0-kilobase (kb) sucrase-isomaltase (SI, EC3.2.1.48–10) cDNA from suncus intestinal cDNA library. The cDNA clone contained a 5442-base pair (bp)-long open reading frame preceded by an in frame termination codon. The deduced 1813-amino acid sequence showed 68.6, 71.2, and 74.7% similarity with those of rat, rabbit, and human, respectively. A cleavage site between isomaltase and sucrase as well as the region surrounding the catalytic sites for sucrase and isomaltase were conserved among the species. Out of 18 potential N-linked glycosylation sites, 5 were common among all 4 species. In the connecting segment which was enriched with O-linked glycosylation sites in the other species, only two sites were present in suncus. Northern blot analysis revealed that the 6.0-kb SI mRNA was expressed in the KAT line with intact sucrase-isomaltase activity. In contrast, 3.0-kb SI mRNA was expressed in suncus of the MI line with isolated sucrase deficiency. The 3.0-kb mRNA cosegregated with sucrase deficiency phenotype as an autosomal recessive trait. Sequence analysis revealed a 2-nucleotide deletion at position 2767–2768, which results in a frameshift and an immature termination codon. The cDNA of the MI line diverged from that of the KAT line at position 2865, having an 18-bp unique sequence followed by a poly(A) tail. The mutant cDNA encodes 922 amino acid residues which preserves the region for isomaltase but lacks that for whole sucrase. While the cells transfected with the plasmids expressing SI in the KAT line showed both sucrase and isomaltase activity, the plasmids expressing MI line cDNA showed only isomaltase activity. Thus it was concluded that the mutation in the SI gene was responsible for isolated sucrase deficiency in the MI line.

A Japanese family with autosomal dominant growth hormone deficiency.

Abstract We report a 1-year-old Japanese boy and his father with isolated growth hormone deficiency II. In both cases, a G → A transition of the first base of the donor splice site of intron 3 of the growth hormone-1 gene was detected. All unaffected family members were homozygous normal. Conclusion This is the fourth reported case of autosomal isolated growth hormone deficiency II with a G → A transition. The CG dinucleotide at the exon 3-intron 3 junction of the growth hormone-1 gene appears to be a hot spot for point mutations.

Transgenic mice expressing a mutant human GH gene causing type II IGHD

We identified several mutations in the intron 3 of human growth hormone gene I (hGH-I) in patients with isolated GH deficiency (IGHD) type II characterized by an autosomal dominant trait. The mutations result in exon 3 skipping and generation of 17 Kd mutant GH. To elucidate how the mutation causes dominant trait, transgenic mice expressing a mutant hGH gene (the first guanine to adenine transversion in intron 3: GH-I; IVS3+1: G-A) were produced in C57BL/6 strain. Genotypes of mice were identified by PCR-amplified products of tail snip DNAs. Delivery of the mutant hGH transgene into 76 fertilized eggs resulted in production of two male heterozygous transgenic mice (hGH + / - , the zero filial generation, F0). Since the mating of the transgenic mice with the same strain was unsuccessful, they were outcrossed with CD-I (ICR) strain. Only one mouse gave birth, producing 4 male and 7 female (Fl) harboring the mutant hGH gene in one allele (hGH + / - ). F1 mice were mated again with the wild type ICR strain, generating 82 hGH + / - mice (F2: 51 males and 31 females). To study whether somatotrophs in F2 mice express the mutant hGH gene, RNA extracted from the pituitary was subjected to RT-PCR. It was demonstrated that the F2, hGH + / - mice express the mutant hGH gene, lacking exon 3. Thus, these heterozygous mice were sib-mated to generate homozygous mice (F3). The mating resulted in 27% hGH - / - , 64% hGH + / - and 9% hGH + / + mice, indicating that the transgene was carried stably to the descendants and did not interfere with the reproduction. These mice will be a valuable model to study how type II IGHD develops during the course of development.