1.Expression of ATP synthase CF1 alpha subunit gene (CTL-spn) as screened by the cDNA-SRAP approach is correlated with spininess in Carthamus tinctorius L.
Dan-dan GUO ; Qing-hua GUO ; Yue GAO ; Mei-li GUO
Acta Pharmaceutica Sinica 2015;50(8):1052-1059
The safflower floret is a traditional Chinese medicine used to promote blood circulation and remove obstruction in the channels. The spines on its bracts are considered a handicap when manual harvest is involved. In this study, cDNA-SRAP was used to systematically investigate which genes are associated with the spines. Sixty pairs of possible primer combinations were used on two cDNA pools representing spininess and spinelessness. Six transcript-derived fragments were identified, of which two with low recombination were sequenced successfully and named as GPY-1 and GPY-2. By using the RACE method, the full-length cDNA of GPY-2 is cloned and named as CTL-spn. The full-length cDNA of CTL-spn was 1 679 bp long with a 1 524 bp ORF encoding a 508 aminoacid protein. The deduced amino acid sequence of the CTL-spn gene shared a high homology (97%) with other known ATP synthase CF1 alpha subunits. Semiquantitative RT-PCR analysis revealed that the mRNA of GPY-1 and GPY-2 accumulated in only spiny lines. Considering the important role of ATP synthase CF1 alpha subunit in plants, it may directly take part in the formation process of spininess and enhancing resistance reaction of spiny safflower. Also, our results provide the important insights for breeding spineless cultivars of safflower.
Adenosine Triphosphate
;
Amino Acid Sequence
;
Carthamus tinctorius
;
enzymology
;
genetics
;
Chloroplast Proton-Translocating ATPases
;
genetics
;
DNA Primers
;
DNA, Complementary
;
Plant Proteins
;
genetics
2.Mitochondrial Permeability Transition Pore and Cardioprotection Against Ischemia-reperfusion Injury.
Journal of the Korean Medical Association 2009;52(10):1007-1019
Opening of mitochondrial permeability transition pore (mPTP) was found to have a critical role in cell death from ischemia/reperfusion (I/R) injury experimentally in the late 1980's. Thereafter, tremendous efforts have been made to define the molecular composition of mPTP and underlying mechanisms of its opening. mPTP opening, so far, has been demonstrated with the conformational changes of the mitochondrial protein components including cyclophilin-D, adenine nucleotide translocase, and voltage-dependent anion channel, which were induced by the modification of the levels of Ca2+, phosphate, mitochondrial membrane potential, intracellular pH and adenine nucleotide. At present, genetic modulation of the expression of protein components are being used in the investigation of its properties, presenting novel mechanisms of mPTP opening, including phosphate carrier. For therapeutic intervention, cyclosporin A and its analogues were first to be demonstrated to inhibit the opening of mPTP, affecting cyclophilin-D. There are numerous pharmacological substances that have direct or indirect effects on mPTP opening, including bongkrekic acid, reactive oxygen species scavengers, calcium channel blockers, and Na+/H+ exchanger-1 inhibitors, but only cyclosporin A was clinically tried to limit the myocardial infarction. Conditioning interventions, ischemic or anesthetic, have also been shown to be effective in limiting the detrimental effects of I/R injury. These interventions are commonly related to specific receptors on cell membrane and then signal transduction pathway consisting of many protein kinases, which eventually lead to mitochondria. And being presented are experimental evidences that inhibition of mPTP opening is a primary mechanism of these conditioning interventions. In conclusion, mPTP opening is now presented as primary mechanism and therapeutic target of I/R injury, but precise mechanism and standardized treatment method are needed to be clarified.
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine
;
Adenine
;
Bongkrekic Acid
;
Calcium Channel Blockers
;
Cell Death
;
Cell Membrane
;
Cyclosporine
;
Hydrogen-Ion Concentration
;
Membrane Potential, Mitochondrial
;
Mitochondria
;
Mitochondrial ADP, ATP Translocases
;
Mitochondrial Membrane Transport Proteins
;
Mitochondrial Proteins
;
Myocardial Infarction
;
Myocardium
;
Permeability
;
Protein Kinases
;
Reactive Oxygen Species
;
Reperfusion Injury
;
Signal Transduction
3.Association of Norepinephrine Transporter Gene and Side Effects of Osmotic-Release Oral System Methylphenidate in Attention-Deficit Hyperactivity Disorder.
Jungeun SONG ; Hyun Ju HONG ; Byung Ook LEE ; Ki Hwan YOOK
Journal of the Korean Academy of Child and Adolescent Psychiatry 2014;25(2):82-88
OBJECTIVES: The aim of our study was to investigate association of norepinephrine transporter gene (SLC6A2) polymorphism and side effects of osmotic-release oral system methylphenidate (OROS MPH) in children with attention-deficit hyperactivity disorder (ADHD). METHODS: We recruited drug naive children with ADHD (N=97). We administered OROS MPH by tolerable dosage. At week 8 of treatment, parents completed the Barkley's side effect rating scale. We analyzed two SLC6A2 single nucleotide polymorphisms (SNPs), rs192303 and rs3785143, with blood of subjects. We compared the frequency and severity of each side effect among SLC6A2 genotypes of 2 SNPs. RESULTS: In the analysis of frequency of each side effect, irritability differed according to rs192303 and rs3785143 genotype. In comparisons of severity, talking less and disinterest differed according to rs192303 genotype. In the case of rs3785143, severities of disinterest and irritability were involved with genotype. CONCLUSION: Side effects of OROS MPH showed an association with SLC6A2 genotype.
Child
;
Genotype
;
Humans
;
Methylphenidate*
;
Norepinephrine Plasma Membrane Transport Proteins*
;
Parents
;
Polymorphism, Single Nucleotide
4.Application of next generation sequencing for the diagnosis of congenital hearing loss.
Shumin REN ; Xiangdong KONG ; Huirong SHI ; Qinghua WU ; Ning LIU
Chinese Journal of Medical Genetics 2019;36(4):301-305
OBJECTIVE:
To identify genetic mutations among patients with hearing loss but without common GJB2, SLC26A4, 12 SrRNA mutations.
METHODS:
Thirty-three patients were subjected to next-generation sequencing (NGS). Suspected mutations were verified by Sanger sequencing.
RESULTS:
Four patients were found to harbor previously known pathogenic variations, and four were found to carry suspicious pathogenic variations, which yielded a detection rate of 24.2%.
CONCLUSION
NGS can improve the detection rate for mutations underlying congenital hearing loss and improve the efficiency and accuracy of the diagnosis.
Connexins
;
Deafness
;
Hearing Loss, Sensorineural
;
High-Throughput Nucleotide Sequencing
;
Humans
;
Membrane Transport Proteins
;
Mutation
;
Sulfate Transporters
5.Proteomic Evaluation of Cellular Responses of Saccharomyces cerevisiae to Formic Acid Stress.
Sung Eun LEE ; Byeoung Soo PARK ; Jeong Jun YOON
Mycobiology 2010;38(4):302-309
Formic acid is a representative carboxylic acid that inhibits bacterial cell growth, and thus it is generally considered to constitute an obstacle to the reuse of renewable biomass. In this study, Saccharomyces cerevisiae was used to elucidate changes in protein levels in response to formic acid. Fifty-seven differentially expressed proteins in response to formic acid toxicity in S. cerevisiae were identified by 1D-PAGE and nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) analyses. Among the 28 proteins increased in expression, four were involved in the MAP kinase signal transduction pathway and one in the oxidative stress-induced pathway. A dramatic increase was observed in the number of ion transporters related to maintenance of acid-base balance. Regarding the 29 proteins decreased in expression, they were found to participate in transcription during cell division. Heat shock protein 70, glutathione reductase, and cytochrome c oxidase were measured by LC-MS/MS analysis. Taken together, the inhibitory action of formic acid on S. cerevisiae cells might disrupt the acid-base balance across the cell membrane and generate oxidative stress, leading to repressed cell division and death. S. cerevisiae also induced expression of ion transporters, which may be required to maintain the acid-base balance when yeast cells are exposed to high concentrations of formic acid in growth medium.
Acid-Base Equilibrium
;
Biomass
;
Cell Division
;
Cell Membrane
;
Electron Transport Complex IV
;
Formates
;
Glutathione Reductase
;
HSP70 Heat-Shock Proteins
;
Ion Transport
;
Mass Spectrometry
;
Oxidative Stress
;
Phosphotransferases
;
Proteins
;
Proteomics
;
Saccharomyces
;
Saccharomyces cerevisiae
;
Signal Transduction
;
Yeasts
6.Mutation analysis of two pedigrees with suspected oculocutaneous albinism.
Haiyun YE ; Xiaoping LAN ; Tong QIAO ; Wuhen XU ; Xiaojun TANG ; Yongchen YANG ; Hong ZHANG
Chinese Journal of Medical Genetics 2019;36(3):212-216
OBJECTIVE:
To analyze the clinical presentation and gene of 2 pedigrees with suspected oculocutaneous albinism(OCA), and provide basis for clinical classification, genetic counseling and prenatal diagnosis.
METHODS:
Variants were identified using next-generation sequencing(NGS) and confirmed by Sanger sequencing in 2 pedigrees with suspected OCA. The pathogenicity of the variants was analyzed according to the American College of Medical Genetics and Genomics (ACMG) standard.
RESULTS:
Two compound heterozygous mutations of TYR and OCA2 genes were identified respectively in 2 pedigrees with suspected OCA. The mutation of c.819+3insATATGCC in TYR and the mutation of c.1870G>C in OCA2 are first reported in this study. The pathogenicity analysis shows that two novel mutations are likely pathogenic by combination of prediction of SIFT, Polyphen-2 and Human Splicing Finder.
CONCLUSION
The findings of this study expand the mutational spectrum of OCA. Compound heterozygous mutations in the TYR and OCA2 gene may be responsible for clinical manifestations of 2 pedigrees with suspected OCA.
Albinism, Oculocutaneous
;
DNA Mutational Analysis
;
Female
;
High-Throughput Nucleotide Sequencing
;
Humans
;
Membrane Transport Proteins
;
Monophenol Monooxygenase
;
Mutation
;
Pedigree
;
Pregnancy
7.Clinical characteristics and gene variants of patients with infantile intrahepatic cholestasis.
Mei-Juan WANG ; Xue-Mei ZHONG ; Xin MA ; Hui-Juan NING ; Dan ZHU ; You-Zhe GONG ; Meng JIN
Chinese Journal of Contemporary Pediatrics 2021;23(1):91-97
OBJECTIVE:
To explore the clinical characteristics and genetic findings of patients with infantile intrahepatic cholestasis.
METHODS:
The clinical data were collected in children who were admitted to the Department of Gastroenterology in Children's Hospital, Capital Institute of Pediatrics from June 2017 to June 2019 and were suspected of inherited metabolic diseases. Next generation sequencing based on target gene panel was used for gene analysis in these children. Sanger sequencing technology was used to verify the genes of the members in this family.
RESULTS:
Forty patients were enrolled. Pathogenic gene variants were identified in 13 patients (32%), including
CONCLUSIONS
The etiology of infantile intrahepatic cholestasis is complex. Next generation sequencing is helpful in the diagnosis of infantile intrahepatic cholestasis.
Alagille Syndrome/genetics*
;
Child
;
Cholestasis, Intrahepatic/genetics*
;
Citrullinemia
;
Genetic Testing
;
High-Throughput Nucleotide Sequencing
;
Humans
;
Mitochondrial Membrane Transport Proteins
;
Mutation
8.Lysosomal membrane protein Sidt2 knockout induces apoptosis of human hepatocytes in vitro independent of the autophagy-lysosomal pathway.
Jiating XU ; Mengya GENG ; Haijun LIU ; Wenjun PEI ; Jing GU ; Mengxiang QI ; Yao ZHANG ; Kun LÜ ; Yingying SONG ; Miaomiao LIU ; Xin HU ; Cui YU ; Chunling HE ; Lizhuo WANG ; Jialin GAO
Journal of Southern Medical University 2023;43(4):637-643
OBJECTIVE:
To explore the regulatory mechanism of human hepatocyte apoptosis induced by lysosomal membrane protein Sidt2 knockout.
METHODS:
The Sidt2 knockout (Sidt2-/-) cell model was constructed in human hepatocyte HL7702 cells using Crispr-Cas9 technology.The protein levels of Sidt2 and key autophagy proteins LC3-II/I and P62 in the cell model were detected using Western blotting, and the formation of autophagosomes was observed with MDC staining.EdU incorporation assay and flow cytometry were performed to observe the effect of Sidt2 knockout on cell proliferation and apoptosis.The effect of chloroquine at the saturating concentration on autophagic flux, proliferation and apoptosis of Sidt2 knockout cells were observed.
RESULTS:
Sidt2-/- HL7702 cells were successfully constructed.Sidt2 knockout significantly inhibited the proliferation and increased apoptosis of the cells, causing also increased protein expressions of LC3-II/I and P62(P < 0.05) and increased number of autophagosomes.Autophagy of the cells reached a saturated state following treatment with 50 μmol/L chloroquine, and at this concentration, chloroquine significantly increased the expressions of LC3B and P62 in Sidt2-/- HL7702 cells.
CONCLUSION
Sidt2 gene knockout causes dysregulation of the autophagy pathway and induces apoptosis of HL7702 cells, and the latter effect is not mediated by inhibiting the autophagy-lysosomal pathway.
Humans
;
Lysosome-Associated Membrane Glycoproteins/metabolism*
;
Autophagy
;
Apoptosis
;
Hepatocytes
;
Lysosomes/metabolism*
;
Chloroquine/pharmacology*
;
Nucleotide Transport Proteins/metabolism*
9.Methods for the study of drug transporters.
Acta Pharmaceutica Sinica 2014;49(7):963-970
As a functional membrane protein, drug transporters play an important role in the absorption, distribution, metabolism and excretion of drugs. The functional omission or inhibition of drug transporters is believed to be involved in the drug-drug interaction and pathogenesis of certain diseases. Understanding the function of drug transporters is highly significant in terms of pharmacokinetics, pharmacodynamics and toxicity of drugs. This article summarized the methods for the study of drug transporters in vitro and in vivo.
Biological Transport
;
Drug Interactions
;
Humans
;
Membrane Transport Proteins
;
metabolism
10.Effects of chronic intermittent hypoxic exposure on myocardial mitochondria ATPase and enzyme complexes of respiratory chain in rats.
Chao-liang LONG ; Zhao-yun YIN ; Hai WANG
Chinese Journal of Applied Physiology 2004;20(3):219-222
AIMTo study the effects of acute and chronic intermittent hypoxic exposure on the activities of Na+ , K+ -ATPase, Ca2 + , Mg2 + -ATPase of myocardial mitochondria and enzyme complexes of respiratory chain in rats.
METHODSThe activities of Na , K+ -ATPase, Ca2+ , Mg2+ -ATPase of myocardial mitochondria and enzyme complexes of respiratory chain were investigated after chronic intermittent hypoxic exposure (3000 m and 5000 m, 4 h/d, 2 w respectively) and normoxic rats were exposed to hypoxia (8000 m) for 4h.
RESULTS(1) Hypoxia had no effects on the activity of Na+, K+ -ATPase in myocardial mitochondria of rats. (2) Compared with normoxic control rats, the activity of Ca2+, Mg2+ -ATPase in myocardial mitochondria of acute hypoxic rats was reduced significantly. After chronic intermittent hypoxic exposure, its activity was increased significantly compared with that of acute hypoxic rats. (3) Compared with normoxic control rats, the activities of enzyme complex I, II and IV of respiratory chain in acute hypoxic rats were reduced significantly. After chronic intermittent hypoxic exposure, their activities were increased significantly compared with those of acute hypoxic rats. Under the same experimental conditions, hypoxia had no effects on the activity of enzyme complex III.
CONCLUSIONAfter chronic intermittent hypoxic exposure, the activities of Na+, K+ -ATPase, Ca2+, Mg2+ -ATPase of myocardial mitochondria and enzyme complexes of respiratory chain were increased significantly. These suggested that chronic intermittent hypoxic exposure could improve the functions of respiratory chain in myocardial mitochondria and keep the normal energy metabolism.
Animals ; Calcium ; metabolism ; Electron Transport ; Hypoxia ; metabolism ; Male ; Mitochondria, Heart ; enzymology ; Mitochondrial Proton-Translocating ATPases ; metabolism ; Multienzyme Complexes ; metabolism ; Potassium ; metabolism ; Rats ; Rats, Wistar ; Sodium-Potassium-Exchanging ATPase ; metabolism