1.Calculated parameters for the diagnosis of Wilson disease.
Nada Syazana ZULKUFLI ; Pavai STHANESHWAR ; Wah-Kheong CHAN
Singapore medical journal 2023;64(3):188-195
INTRODUCTION:
The diagnosis of Wilson disease (WD) is plagued by biochemical and clinical uncertainties. Thus, calculated parameters have been proposed. This study aimed to: (a) compare the diagnostic values of non-caeruloplasmin copper (NCC), NCC percentage (NCC%), copper-caeruloplasmin ratio (CCR) and adjusted copper in WD; and (b) derive and evaluate a discriminant function in diagnosing WD.
METHODS:
A total of 213 subjects across all ages who were investigated for WD were recruited. WD was confirmed in 55 patients, and the rest were WD free. Based on serum copper and caeruloplasmin values, NCC, NCC%, CCR and adjusted copper were calculated for each subject. A function was derived using discriminant analysis, and the cut-off value was determined through receiver operating characteristic analysis. Classification accuracy was found by cross-tabulation.
RESULTS:
Caeruloplasmin, total copper, NCC, NCC%, CCR, adjusted copper and discriminant function were significantly lower in WD compared to non-WD. Discriminant function showed the best diagnostic specificity (99.4%), sensitivity (98.2%) and classification accuracy (99.1%). Caeruloplasmin levels <0.14 g/L showed higher accuracy than the recommended 0.20 g/L cut-off value (97.7% vs. 87.8%). Similarly, molar NCC below the European cut-off of 1.6 umol/L showed higher accuracy than the American cut-off of 3.9 umol/L (80.3% vs. 59.6%) (P < 0.001). NCC%, mass NCC, CCR and adjusted copper showed poorer performances.
CONCLUSION
Discriminant function differentiates WD from non-WD with excellent specificity, sensitivity and accuracy. Performance of serum caeruloplasmin <0.14 g/L was better than that of <0.20 g/L. NCC, NCC%, CCR and adjusted copper are not helpful in diagnosing WD.
Humans
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Hepatolenticular Degeneration/diagnosis*
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Copper/analysis*
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Ceruloplasmin/metabolism*
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Repressor Proteins
2.Dissecting the molecular mechanism of nuclear receptor action: transcription coactivators and corepressors.
Jae Woon LEE ; Jae Hun CHEONG ; Young Chul LEE ; Soon Young NA ; Soo Kyung LEE
Experimental & Molecular Medicine 2000;32(2):53-60
No abstract available.
Acetyltransferases/metabolism
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Animal
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Carrier Proteins/metabolism
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DNA-Binding Proteins/metabolism
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Human
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Nuclear Proteins/metabolism
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Receptors, Cytoplasmic and Nuclear/metabolism*
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Repressor Proteins/metabolism*n
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Trans-Activators/metabolism*
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Transcription Factors/metabolism
4.Wnt pathway antagonists and angiogenesis.
Protein & Cell 2010;1(10):898-906
Dysregulation of the Wnt pathway has been extensively studied in multiple diseases, including some angiogenic disorders. Wnt signaling activation is a major stimulator in pathological angiogenesis and thus, Wnt antagonists are believed to have therapeutic potential for neovascular disorders. Actually, some Wnt antagonists have been identified directly from the anti-angiogenic factor family. This review summarizes the recent progress toward understanding of the roles of Wnt pathway antagonists in angiogenic regulation and their mechanism of action, and exploring their therapeutic potential.
Adaptor Proteins, Signal Transducing
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metabolism
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Animals
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Humans
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Neovascularization, Pathologic
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physiopathology
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Repressor Proteins
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metabolism
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Signal Transduction
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physiology
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Wnt Proteins
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antagonists & inhibitors
5.Expression and characterization of ArgR, an arginine regulatory protein in Corynebacterium crenatum.
Xue Lan CHEN ; Bin ZHANG ; Li TANG ; Hai Tao JIAO ; Heng Yi XU ; Feng XU ; Hong XU ; Hua WEI ; Yong Hua XIONG
Biomedical and Environmental Sciences 2014;27(6):436-443
OBJECTIVECorynebacterium crenatum MT, a mutant from C. crenatum AS 1.542 with a lethal argR gene, exhibits high arginine production. To confirm the effect of ArgR on arginine biosynthesis in C. crenatum, an intact argR gene from wild-type AS 1.542 was introduced into C. crenatum MT, resulting in C. crenatum MT. sp, and the changes of transcriptional levels of the arginine biosynthetic genes and arginine production were compared between the mutant strain and the recombinant strain.
METHODSQuantitative real-time polymerase chain reaction was employed to analyze the changes of the related genes at the transcriptional level, electrophoretic mobility shift assays were used to determine ArgR binding with the argCJBDF, argGH, and carAB promoter regions, and arginine production was determined with an automated amino acid analyzer.
RESULTSArginine production assays showed a 69.9% reduction in arginine from 9.01 ± 0.22 mg/mL in C. crenatum MT to 2.71 ± 0.13 mg/mL (P<0.05) in C. crenatum MT. sp. The argC, argB, argD, argF, argJ, argG, and carA genes were down-regulated significantly in C. crenatum MT. sp compared with those in its parental C. crenatum MT strain. The electrophoretic mobility shift assays showed that the promoter regions were directly bound to the ArgR protein.
CONCLUSIONThe arginine biosynthetic genes in C. crenatum are clearly controlled by the negative regulator ArgR, and intact ArgR in C. crenatum MT results in a significant descrease in arginine production.
Arginine ; biosynthesis ; Bacterial Proteins ; chemistry ; genetics ; metabolism ; Corynebacterium ; genetics ; metabolism ; Gene Expression Regulation, Bacterial ; Repressor Proteins ; chemistry ; genetics ; metabolism
6.Expression of neural salient serine/arginine-rich protein 1 (NSSR1) in the development of mouse brain.
Wei ZHANG ; Li-mei FAN ; Lin-lin LI ; Zheng-yu PENG
Journal of Zhejiang University. Medical sciences 2014;43(1):51-57
OBJECTIVETo investigate the expression of neural salient serine/arginine-rich protein 1 (NSSR1) in the development of mouse brain.
METHODSBrain samples were collected from mice with different developmental stages: 9, 12, 14 d before birth (E9, E12, E14) and 1 d, 3 weeks and 3 months after birth. The expression of NSSR1 in mouse brain at different developmental stages was detected by Western blot and the distribution of NSSR1 was analyzed by immunohistochemical staining. The expression and distribution of NSSR1 in mouse brain were compared among embryos, neonatal and adult animals.
RESULTSDuring embryogenesis, the expression of NSSR1 proteins increases significantly from 0.186(E9) to 0.445(E14) and reached a high level after birth. Immunohistochemical analysis showed that in E12 embryos, NSSR1 was specifically distributed in the marginal and mantle layers. The expression of NSSR1 in hippocampus was very low in neonatal animals but stronger in adults. In cerebellar cortex, NSSR1 was widely expressed in purkinje and granule cells of adult animals, but mainly expressed in Purkinje cells in neonates.
CONCLUSIONThe expression of NSSR1 is regulated by the development of mouse brain and presents dynamic changes.
Animals ; Brain ; embryology ; growth & development ; metabolism ; Cell Cycle Proteins ; metabolism ; Mice ; Neoplasm Proteins ; metabolism ; RNA-Binding Proteins ; metabolism ; Repressor Proteins ; metabolism
7.Purification and functional identification of the recombinant human CREG/myc-His glycoprotein.
Ming-Yu SUN ; Ya-Ling HAN ; Peng GUO ; Jian KANG ; Cheng-Hui YAN
Chinese Journal of Applied Physiology 2010;26(3):297-301
OBJECTIVETo purify the recombinant human cellular repressor of EIA stimulated gene (hCREG)/myc-His glycoprotein and confirm the biological function of hCREG/myc-His which could inhibit the proliferation of human internal thoracic artery smooth muscle cells (HITASY) cultured in vitro.
METHODSThe recombinant hCREG/myc-His protein was purified with Ni-NTA column according to 6 x His affinity chromatographic theory. The recombinant hCREG/myc-His protein was desalted by HiTrap Desalting Column. The effect of recombinant hCREG/myc-His glycoprotein of different concentration (0.5 microg/ml, 1 microg/ml and 2 microg/ml) on proliferation of HITASY cells was studied by flow cytometric analysis and the effect of recombinant protein on proliferation of HITASY cells was confirmed by BrdU incorporation method.
RESULTSThe recombinant hCREG protein was purified with Ni-NTA column according to 6 x His affinity chromatographic theory. The concentration of recombinant hCREG protein which has been concentrated and desalted was determined to be 1.6 mg/ml and the purity of recombinant protein reached 92%. The protein was identified to be glycosylated. The recombinant hCREG protein was identified to inhibit the proliferation of HITASY cells cultured in vitro and the inhibition effect was stronger in low-dosage group than that in high-dosage group by flow cytometric analysis. The proliferation of HITASY cells cultured in vitro with 2 microg/ml recombinant hCREG protein was inhibited significantly compared with that in control group according to the BrdU incorporation result. There was statistical difference among the groups (P < 0.05).
CONCLUSIONThe purification of recombinant hCREG/myc-His glycoprotein with biological activity provides an experiment platform for function study and engineering production of hCREG protein.
Adenoviridae ; Cell Division ; Cells, Cultured ; Glycoproteins ; isolation & purification ; Glycosylation ; Humans ; Recombinant Proteins ; isolation & purification ; metabolism ; Repressor Proteins ; isolation & purification ; metabolism
8.Silencing DNMT1 Attenuates the Effect of WIF-1 Gene Promoter Methylation on the Biological Behavior of Chronic Myeloid Leukemia K562 Cells.
Kun WU ; Zhen JIN ; Yi-Xun LI ; Xin LI ; Shen-Ju CHENG ; Yan-Hong LI ; Chong GUO
Journal of Experimental Hematology 2021;29(6):1768-1774
OBJECTIVE:
To investigate the effect of silencing DNA methyltransferase 1(DNMT1) to the methylation of the promoter of the tumor suppressor gene wnt-1 (WIF-1) in human chronic myeloid leukemia (CML) cells.
METHODS:
DNMT1 siRNAi plasmid was constructed and DNMT1 siRNAi was transfected into CML K562 cells. RT-PCR and Western blot were used to detect the expression of DNMT1 gene and related protein, and methylation PCR was used to detect WIF-1 gene promoter methylation level. The trypan blue exclusion and MTT assay were used to detect the cell proliferation, flow cytometry were used to detect the cell apoptosis rate, colony formation assay was used to detect cell colony formation ability. Expression of Wnt/β- catenin and its downstream signaling pathway proteins were detected by Western blot after DNMT1 gene was silenced.
RESULTS:
The expression level of DNMT1 mRNA and its related protein in the experimental group were significantly lower than those in the control group and negative control group (P<0.05). After 72 hours of successful transfection, the WIF-1 gene in the control group and negative control group were completely methylated, while in the experimental group, the methylation level significantly decreased. The results of MSP showed that the PCR product amplified by the unmethylated WIF-1 primer in the experimental group increased significantly,while by the methylated WIF-1 primer decreased significantly. After 48 h of transfection, the OD value, viable cell number and colony formation of the cells in experimental group were significantly lower than those in the negative control group and the control group (P<0.05). The apoptosis rate of the cells in experimental group was significantly higher than those in the negative control group and control group (P<0.05). The expression levels of β- actin, myc, cyclin D1 and TCF-1 in K562 cells in the experimental group were significantly lower than those in the negative control group and control group (P<0.05).
CONCLUSION
Silencing DNMT1 gene can inhibit the proliferation and promote the apoptosis of K562 cells. The mechanism may be related to reverse the hypermethylation level of the WIF-1 gene promoter, thereby inhibit the activity of the Wnt/β- catenin signaling pathway.
Adaptor Proteins, Signal Transducing/metabolism*
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DNA Methylation
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Humans
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K562 Cells
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Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics*
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Repressor Proteins/metabolism*
9.Casein kinase 2 interacts with and phosphorylates ataxin-3.
Rui-Song TAO ; Er-Kang FEI ; Zheng YING ; Hong-Feng WANG ; Guang-Hui WANG
Neuroscience Bulletin 2008;24(5):271-277
OBJECTIVEMachado-Joseph disease (MJD)/Spinocerebellar ataxia type 3 (SCA3) is an autosomal dominant neurodegenerative disorder caused by an expansion of polyglutamine tract near the C-terminus of the MJD1 gene product, ataxin-3. The precise mechanism of the MJD/SCA3 pathogenesis remains unclear. A growing body of evidence demonstrates that phosphorylation plays an important role in the pathogenesis of many neurodegenerative diseases. However, few kinases are known to phosphorylate ataxin-3. The present study is to explore whether ataxin-3 is a substrate of casein kinase 2 (CK2).
METHODSThe interaction between ataxin-3 and CK2 was identified by glutathione S-transferase (GST) pull-down assay and co-immunoprecipition assay. The phosphorylation of ataxin-3 by CK2 was measured by in vitro phosphorylation assays. Results (1) Both wild type and expanded ataxin-3 interacted with CK2alpha and CK2beta in vitro. (2) In 293 cells, both wild type and expanded ataxin-3 interacted with CK2beta, but not CK2alpha. (3) CK2 phosphorylated wild type and expanded ataxin-3.
CONCLUSIONAtaxin-3 is a substrate of protein kinase CK2.
Ataxin-3 ; Casein Kinase II ; metabolism ; Cell Line, Transformed ; Glutathione Transferase ; metabolism ; Humans ; Immunoprecipitation ; methods ; Nerve Tissue Proteins ; metabolism ; Nuclear Proteins ; metabolism ; Phosphorylation ; Repressor Proteins ; metabolism ; Transfection ; methods
10.Optimizing expression of recombinant jasmonate ZIM-domain protein from Salvia miltiorrhiza.
Li-Hua ZHANG ; Wen-Yan WU ; Lu-Qi HUANG ; Ye SHEN
China Journal of Chinese Materia Medica 2012;37(24):3712-3716
OBJECTIVEAccumulation of tanshinton in Salvia miltiorrhiza are enhanced by exogenous application of jasmonates. The core JA signaling module COI1/JAZ/MYC2 play a central role on control of downstream gene expression in the JA pathway. To obtained the antibody of SmJAZ, SmJAZ recombinant protein was expressed in Escherichia coli and optimal expression was performed.
METHODThe full-length SmJAZ1 ORF was sub-cloned in a prokaryotic expression vector pET32a. The recombinant fusion protein had high expression level in BL21 (DE3) strain of E. coli, and SDS-PAGE analysis showed its molecular weight was about 24 kDa.
RESULTThe induction of E. coli [pET32-JAZ1] in different temperature, induction time, IPTG concentrations and IPTG adding time of E. coli were performed. The induction time and the induction temperature are positively related trends with SmJAZ1 protein expression, and IPTG concentration had no significant impact in protein expression, whereas IPTG adding time had significant impact on protein expression.
CONCLUSIONShaking the culture at 30 degrees C until the A600 is approximately 0.9 (2 h in LB), and add IPTG to a final concentration of 0.1 mmol x L(-1), and then the optimal expression of SmJAZ1 recombinant protein were accumulated after the induction time of 20 h.
Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; Gene Expression ; Plant Proteins ; genetics ; metabolism ; Plasmids ; genetics ; metabolism ; Recombinant Proteins ; metabolism ; Repressor Proteins ; genetics ; metabolism ; Salvia miltiorrhiza ; genetics ; metabolism ; Time Factors