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1.
Nat Commun ; 14(1): 23, 2023 01 12.
Article in English | MEDLINE | ID: mdl-36635268

ABSTRACT

Sickle cell disease (SCD) is a heritable disorder caused by ß-globin gene mutations. Induction of fetal γ-globin is an established therapeutic strategy. Recently, epigenetic modulators, including G9a inhibitors, have been proposed as therapeutic agents. However, the molecular mechanisms whereby these small molecules reactivate γ-globin remain unclear. Here we report the development of a highly selective and non-genotoxic G9a inhibitor, RK-701. RK-701 treatment induces fetal globin expression both in human erythroid cells and in mice. Using RK-701, we find that BGLT3 long non-coding RNA plays an essential role in γ-globin induction. RK-701 selectively upregulates BGLT3 by inhibiting the recruitment of two major γ-globin repressors in complex with G9a onto the BGLT3 gene locus through CHD4, a component of the NuRD complex. Remarkably, BGLT3 is indispensable for γ-globin induction by not only RK-701 but also hydroxyurea and other inducers. The universal role of BGLT3 in γ-globin induction suggests its importance in SCD treatment.


Subject(s)
Anemia, Sickle Cell , RNA, Long Noncoding , Mice , Humans , Animals , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , gamma-Globins/genetics , Erythroid Cells/metabolism , Anemia, Sickle Cell/drug therapy , Anemia, Sickle Cell/genetics , Anemia, Sickle Cell/metabolism , Gene Expression , Fetal Hemoglobin/genetics , Fetal Hemoglobin/metabolism
2.
Sci Rep ; 10(1): 14187, 2020 08 25.
Article in English | MEDLINE | ID: mdl-32843708

ABSTRACT

Down syndrome is a complex genetic disorder caused by the presence of three copies of the chromosome 21 in humans. The most common models, carrying extra-copies of overlapping fragments of mouse chromosome 16 that is syntenic to human chromosome 21, are Ts2Cje, Ts1Cje and Ts1Rhr mice. In electrophysiological analyses using hippocampal slices, we found that the later phase of the depolarization during tetanic stimulation, which was regulated by GABAB receptors, was significantly smaller in Ts1Cje and Ts2Cje mice than that in WT controls but not in Ts1Rhr mice. Furthermore, isolated GABAB receptor-mediated inhibitory synaptic responses were larger in Ts1Cje mice. To our knowledge, this is the first report that directly shows the enhancement of GABAB receptor-mediated synaptic currents in Ts1Cje mice. These results suggest that GABAB receptor-mediated synaptic inhibition was enhanced in Ts1Cje and Ts2Cje mice but not in Ts1Rhr mice. The Cbr1 gene, which is present in three copies in Ts1Cje and Ts2Cje but not in Ts1Rhr, encodes carbonyl reductase that may facilitate GABAB-receptor activity through a reduction of prostaglandin E2 (PGE2). Interestingly, we found that a reduction of PGE2 and an memory impairment in Ts1Cje mice were alleviated when only Cbr1 was set back to two copies (Ts1Cje;Cbr1+/+/-). However, the GABAB receptor-dependent enhancement of synaptic inhibition in Ts1Cje was unaltered in Ts1Cje;Cbr1+/+/- mice. These results indicate that Cbr1 is one of the genes responsible for DS cognitive impairments and the gene(s) other than Cbr1, which is included in Ts1Cje but not in Ts1Rhr, is responsible for the GABAB receptor-dependent over-inhibition.


Subject(s)
Alcohol Oxidoreductases/genetics , Down Syndrome/genetics , Down Syndrome/metabolism , Receptors, GABA-B/genetics , Spatial Memory/physiology , Alcohol Oxidoreductases/metabolism , Animals , Brain/metabolism , Brain/pathology , DNA Copy Number Variations , Disease Models, Animal , Down Syndrome/pathology , Down Syndrome/psychology , Female , Hippocampus/metabolism , Hippocampus/pathology , Inhibition, Psychological , Male , Mice , Mice, Inbred C57BL , Receptors, GABA-B/metabolism , Synapses/genetics , Synapses/metabolism , gamma-Aminobutyric Acid/genetics , gamma-Aminobutyric Acid/metabolism
3.
Biosci Rep ; 39(7)2019 07 31.
Article in English | MEDLINE | ID: mdl-31243120

ABSTRACT

Although wasting marmoset syndrome (WMS) is one of the biggest problems facing captive marmoset colonies, the mechanisms underlying its pathogenesis remain unclear. In our clinical experience, it is difficult to cure WMS-affected marmosets with severe hypoalbuminemia. Thus, the mechanisms underlying hypoalbuminemia in WMS must be understood. In the present study, we investigated whether intestinal protein loss, a known reason for hypoalbuminemia, occurs in this disease. Fecal α1-proteinase inhibitor (α1-PI, also known as α1-antitrypsin) has been used to diagnose intestinal protein loss in other species. To develop an assay system for this protein, marmoset α1-PI was purified from plasma and antibodies against it were developed using the purified protein. Using the antibodies, a sandwich enzyme-linked immunosorbent assay (ELISA) to measure marmoset α1-PI was developed, and its detection sensitivity for fecal samples was ∼20-fold higher than that of a commercial kit for human α1-PI. From this ELISA, the reference intervals for serum and feces of healthy marmosets were 0.87-1.85 mg/ml and 0.53-395.58 µg/g, respectively. The average concentrations of α1-PI in serum and feces of seven WMS-affected marmosets were 1.17 mg/ml and 1357.58 µg/g, respectively. Although there were no significant differences in the serum concentrations between healthy and WMS-affected marmosets, the fecal concentrations were significantly higher in WMS-affected marmosets than in healthy individuals, suggesting that intestinal protein loss occurs in WMS. Intestinal protein loss of WMS-affected marmosets was significantly attenuated with treatment, suggesting that it is one of the mechanisms involved in the hypoalbuminemia observed in WMS.


Subject(s)
Callithrix/blood , Hypoalbuminemia/blood , Wasting Syndrome/blood , alpha 1-Antitrypsin/blood , Animals , Antibodies/pharmacology , Enzyme-Linked Immunosorbent Assay , Feces/chemistry , Humans , Hypoalbuminemia/pathology , Intestines/pathology , Wasting Syndrome/drug therapy , Wasting Syndrome/pathology , Wasting Syndrome/veterinary , alpha 1-Antitrypsin/genetics , alpha 1-Antitrypsin/immunology
4.
Mol Cell ; 58(6): 1015-27, 2015 Jun 18.
Article in English | MEDLINE | ID: mdl-25959394

ABSTRACT

Blood pressure maintenance is vital for systemic homeostasis, and angiotensin II is a critical regulator. The upstream mechanisms that regulate angiotensin II are not completely understood. Here, we show that angiotensin II is regulated by ERp44, a factor involved in disulfide bond formation in the ER. In mice, genetic loss of ERp44 destabilizes angiotensin II and causes hypotension. We show that ERp44 forms a mixed disulfide bond with ERAP1, an aminopeptidase that cleaves angiotensin II. ERp44 controls the release of ERAP1 in a redox-dependent manner to control blood pressure. Additionally, we found that systemic inflammation triggers ERAP1 retention in the ER to inhibit hypotension. These findings suggest that the ER redox state calibrates serum angiotensin II levels via regulation of the ERp44-ERAP1 complex. Our results reveal a link between ER function and normotension and implicate the ER redox state as a potential risk factor in the development of cardiovascular disease.


Subject(s)
Aminopeptidases/metabolism , Blood Pressure , Endoplasmic Reticulum/metabolism , Membrane Proteins/metabolism , Molecular Chaperones/metabolism , Amino Acid Sequence , Aminopeptidases/genetics , Angiotensin II/blood , Angiotensin II/metabolism , Animals , Blotting, Western , Cells, Cultured , HeLa Cells , Humans , Membrane Proteins/genetics , Mice, Inbred C57BL , Mice, Knockout , Minor Histocompatibility Antigens , Molecular Chaperones/genetics , Molecular Sequence Data , Oxidation-Reduction , Protein Binding , RNA Interference , Sequence Homology, Amino Acid
5.
PLoS One ; 8(2): e53855, 2013.
Article in English | MEDLINE | ID: mdl-23408933

ABSTRACT

Bipolar disorder is a severe mental illness characterized by recurrent manic and depressive episodes. In bipolar disorder, family and twin studies suggest contributions from genetic and environmental factors; however, the detailed molecular pathogenesis is yet unknown. Thus, identification of biomarkers may contribute to the clinical diagnosis of bipolar disorder. Monozygotic twins discordant for bipolar disorder are relatively rare but have been reported. Here we performed a comparative proteomic analysis of whole cell lysate derived from lymphoblastoid cells of monozygotic twins discordant for bipolar disorder by using two-dimensional differential in-gel electrophoresis (2D-DIGE). We found approximately 200 protein spots to be significantly differentially expressed between the patient and the co-twin (t test, p<0.05). Some of the proteins were subsequently identified by liquid chromatography tandem mass spectrometry and included proteins involved in cell death and glycolysis. To examine whether these proteins could serve as biomarkers of bipolar disorder, we performed Western blot analysis using case-control samples. Expression of phosphoglycerate mutase 1 (PGAM1), which is involved in glycolysis, was significantly up-regulated in patients with bipolar disorder (t test, p<0.05). Although PGAM1 cannot be regarded as a qualified biomarker of bipolar disorder from this preliminary finding, it could be one of the candidates for further study to identify biomarkers of bipolar disorder.


Subject(s)
Bipolar Disorder/diagnosis , Lymphocytes/metabolism , Proteomics , Twins, Monozygotic , Adult , Biomarkers/metabolism , Bipolar Disorder/genetics , Blotting, Western , Case-Control Studies , Chromatography, Liquid , Databases, Protein , Electrophoresis, Gel, Two-Dimensional , Female , Humans , Male , Middle Aged , Tandem Mass Spectrometry
6.
Cell Mol Neurobiol ; 24(1): 37-50, 2004 Feb.
Article in English | MEDLINE | ID: mdl-15049509

ABSTRACT

Mastoparan (MP), a tetradecapeptide in wasp venom, has been reported to evoke catecholamine release, but also reported to inhibit secretory response upon nicotinic stimulation in adrenal chromaffin cells. To elucidate the inhibitory mechanism of MP, we examined the effect of two MP fragments (INLK-NH2 and KKIL-NH2) on catecholamine release in bovine adrenal chromaffin cells. These MP fragments inhibited catecholamine release induced by nicotinic stimulation in a noncompetitive manner. These fragments did not affect catecholamine release evoked by high [K+] or by other secretagogues, neither caused catecholamine release by themselves. Replacement by hydrophobic and basic amino acids of the MP fragments enhanced the inhibitory effects on ACh-evoked catecholamine release. Among 23 analogs of the MP fragments, (Nle)3-R-NH2 showed the most potent inhibition with IC50 = 541 microM. These results suggested that the MP fragments selectively inhibit the secretory response to nicotinic stimulation by attacking nAChR on the site(s) made up of hydrophobic and acidic amino acids but other than ACh-binding sites. This mechanism may explain the inhibitory action of MP on nicotine-evoked catecholamine release.


Subject(s)
Adrenal Medulla/drug effects , Catecholamines/metabolism , Chromaffin Cells/drug effects , Peptide Fragments/pharmacology , Receptors, Nicotinic/drug effects , Wasp Venoms/pharmacology , Adrenal Medulla/metabolism , Amino Acids/chemistry , Amino Acids/physiology , Animals , Binding Sites/drug effects , Binding Sites/physiology , Cattle , Cells, Cultured , Chromaffin Cells/metabolism , Hydrophobic and Hydrophilic Interactions , Intercellular Signaling Peptides and Proteins , Nicotinic Antagonists/chemistry , Nicotinic Antagonists/pharmacology , Peptide Fragments/chemistry , Peptides , Potassium/metabolism , Potassium/pharmacology , Protein Structure, Tertiary/physiology , Receptors, Nicotinic/metabolism , Wasp Venoms/chemistry
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