1.Construction of pSG5/TRIF and its expression in Huh7 cells.
Xue-ping WANG ; Shi-wen WANG ; Jie YANG ; Tong YIN ; Hong-juan WANG ; Bin XU ; Qing-lei ZHU ; Yang LI
Journal of Southern Medical University 2010;30(11):2498-2501
OBJECTIVETo construct the plasmid pSG5/TRIF and investigate its expression in Huh7 cells.
METHODSThe plasmid pCX4pur/Myc-TRIF was digested with Not I and the digestion product was blunted followed by further digestion with EcoR I to obtain the insert Myc-TRIF. pSG5 was digested sequentially with Sma I and EcoR I. All the digested products were analyzed with agarose gel electrophoresis. The products with the expected size were extracted and ligated, and the positive clones were screened by ampicillin and amplified. The recombinant pSG5/TRIF was extracted, purified, and identified by restriction endonuclease BamH I and agarose gel electrophoresis. The recombinant plasmids were transfected into Huh7 cells with FuGene 6 reagents and into Huh7 cells previously infected with recombinant vaccinia virus (rVV) via Lipofectin. Immunofluorescence and Western blotting were performed to detect the expression of the recombinant plasmids, and the transfection efficiency with different transfection reagents was compared.
RESULTSBamH I digestion resulted in a fragment with the expected size. Immunofluorescence staining showed successful expression of Myc-TRIF protein in Huh7 cells, and the transfection efficiency was enhanced in Huh7 cells previously infected with rVV. SDS-PAGE analysis showed that the relative molecular mass of the expressed product by pSG5/Myc-TRIF was about 100 ku, and prior infection of the cells with rVV obviously increased transfection efficiency, as was consistent with the results of immunofluorescence.
CONCLUSIONpSG5/Myc-TRIF is successfully constructed and expressed in Huh7 cells. The expression efficiency can be increased by prior infection of the cells with rVV.
Adaptor Proteins, Vesicular Transport ; genetics ; Cell Line, Tumor ; Gene Expression ; Genetic Vectors ; Hepacivirus ; genetics ; Humans ; Plasmids ; Recombinant Fusion Proteins ; genetics ; Transfection
2.Association of gene polymorphisms of MyD88 and TICAM1 and their interactions with community-acquired pneumonia in children.
Yong YANG ; Sui-Yu YANG ; Zong-Bo CHEN ; Li LIU
Chinese Journal of Contemporary Pediatrics 2023;25(8):791-799
OBJECTIVES:
To investigate the association of single nucleotide polymorphisms (SNPs) of myeloid differentiation factor 88 (MyD88) and Toll-like receptor adaptor molecule 1 (TICAM1) and their interactions with community-acquired pneumonia (CAP) in children.
METHODS:
Improved multiple ligase detection reaction assay was used for detecting the polymorphisms of nine tagging SNPs of the MyD88 and TICAM1 genes in 375 children with CAP who attended the Department of Pediatrics of the Second Affiliated Hospital of Yan'an University Medical School from August 2015 to September 2017 and 306 healthy children who underwent physical examination. A logistic regression analysis was used to evaluate the association between the distribution of genotypes and their interactions with CAP in children.
RESULTS:
The polymorphism of the TICAM1 gene at rs11466711T/C locus was closely associated with the susceptibility to CAP in children (P<0.05). The AA genotype of rs35747610G/A locus significantly reduced risk of sepsis in children with CAP (P<0.05). The AA genotype of rs6510826G/A locus was significantly associated with the increase in C-reactive protein level in children with CAP (P<0.05). The GG genotype of the MyD88 gene at rs7744A/G locus significantly increased the risk of respiratory failure and circulatory failure (P<0.05). The multiplicative interactions between MyD88 gene rs7744A/G and TICAM1 gene rs11466711T/C, rs2292151G/A, rs35299700C/T, and rs35747610G/A loci were significantly associated with the susceptibility to CAP, the severity of CAP, and the risk of sepsis in children (P<0.05).
CONCLUSIONS
The gene polymorphisms of MyD88 and TICAM1 and their interactions are closely associated with CAP in children, with a synergistic effect on the development and progression of CAP in children.
Child
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Humans
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Adaptor Proteins, Vesicular Transport/genetics*
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Community-Acquired Infections/genetics*
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Myeloid Differentiation Factor 88/genetics*
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Pneumonia/genetics*
;
Polymorphism, Single Nucleotide
;
Sepsis
3.Intersectin 1: a molecular linker in the central nervous system.
Ning MA ; Rui-Fang NIU ; Yong-Jie MA
Neuroscience Bulletin 2008;24(6):401-405
Down syndrome (DS) is the most common cause of cognitive impairment associated with a congenital chromosomal abnormality, trisomy of chromosome 21. Mental retardation and congenital heart defects are key features of DS. All DS individuals develop early-onset Alzheimer's disease-like neuropathology. Intersectin 1 gene is localized on human chromosome 21, the critical region of DS, and it has higher expression in the brain of DS patients than in normal individuals. So fully understanding functions of intersectin 1 is critical for revealing the pathogenesis of DS. Intersectin 1 protein has two isoforms: intersectin 1-L and intersectin 1-S. This review will focus on the distribution, expression characters and functions of intersectin 1 in the central nervous system.
Adaptor Proteins, Vesicular Transport
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genetics
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metabolism
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Animals
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Central Nervous System
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cytology
;
metabolism
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Chromosomes, Human, Pair 21
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Humans
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Mental Disorders
;
genetics
;
metabolism
;
Neurons
;
metabolism
4.Genetic testing and prenatal diagnosis for two families affected with Joubert syndrome.
Zhouxian BAI ; Shuang HU ; Ning LIU ; Qinghua WU ; Xiangdong KONG
Chinese Journal of Medical Genetics 2020;37(5):509-513
OBJECTIVE:
To identify pathogenic variants in two families with patients suspected for Joubert syndrome(UBST) by cerebellar vermis hypoplasia.
METHODS:
Clinical data and peripheral venous blood and skin tissue samples were collected for the extraction of genomic DNA. Potential variants were screened by using targeted capture and next generation sequencing. Suspected variants were validated by PCR and Sanger sequencing. The frequency of the variants in the population was calculated. Pathogenicity of the variants was predicted by following the guidelines of the American College of Medical Genetics and Genomics (ACMG). Prenatal diagnosis was provided to these families upon subsequent pregnancy.
RESULTS:
The proband of family 1 was found to harbor homozygous c.2072delT (p.F691S*fs19) frameshift variant of the AHI1 gene, which may cause premature termination of translation of the Abelson helper integration site 1 after the 691st amino acid. The proband of family 2 was found to harbor compound heterozygous variants of the CPLANE1 gene, namely c.7243dupA (p.T2415Nfs*7) and c.8001delG (p.K2667Nfs*31), which can respectively lead to premature termination of translation of ciliogenesis and planar polarity effector 1 after the 2145th and 2667th amino acids. All of the three variants were previously unreported, and were predicted to be pathogenic by bioinformatic analysis.
CONCLUSION
The AHI1 c.2072delT and CPLANE1 c.7243dupA and c.8001delG variants probably underlay JBTS3 in family 1 and JBTS17 in family 2, respectively. Based on above results, prenatal diagnosis may be offered to the affected families upon their subsequent pregnancies.
Abnormalities, Multiple
;
diagnosis
;
genetics
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Adaptor Proteins, Vesicular Transport
;
genetics
;
Cerebellum
;
abnormalities
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Eye Abnormalities
;
diagnosis
;
genetics
;
Female
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Genetic Testing
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Genetic Variation
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Humans
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Kidney Diseases, Cystic
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diagnosis
;
genetics
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Membrane Proteins
;
genetics
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Mutation
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Pregnancy
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Prenatal Diagnosis
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Retina
;
abnormalities
5.Effects of neonatal isolation stress on hyperlocomotion of complexin II knockout mouse.
Huan YU ; Yong ZHAO ; Wei HE ; Xing-Nuan LI ; Yi-Chun ZHOU ; Li-Ping LIU
Acta Physiologica Sinica 2011;63(4):300-304
The aim of this study was to investigate the influence of neonatal isolation stress on hyperlocomotion in complexin II knockout mouse (Cplx2(-/-)). The mice were randomly divided into 4 groups: Cplx2(-/-) with stress, Cplx2(+/+) with stress, Cplx2(-/-) without stress and Cplx2(+/+) without stress. Isolation stress was employed on the pups of stress groups from the 2nd day after the postnatal to the 21st day. The PCR was used to determine the gene type and the hyperlocomotion test was employed to detect the change of animal behavior after methamphetamine or saline injection (i.p.). The results showed that the animals of all groups increased their movement after injection of 0.2 mg/kg methamphetamine in different levels (P < 0.01), compared with those injected with saline. The Cplx2(-/-) mouse with stress revealed a significant increase in the distance of free movement after injection of 0.2 mg/kg methamphetamine compared with the knockout mouse without stress (P < 0.001). When Cplx2(-/-) mouse with stress was compared with wild type with stress, Cplx2(-/-) mouse with stress had more movement (P < 0.001), indicating that Cplx2 has effect on the hyperlocomotion as well. These results suggest an involvement of stress and Cplx2 in the movement behavior of mice.
Adaptor Proteins, Vesicular Transport
;
genetics
;
Animals
;
Animals, Newborn
;
Behavior, Animal
;
physiology
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Locomotion
;
physiology
;
Methamphetamine
;
pharmacology
;
Mice
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Mice, Inbred C57BL
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Mice, Knockout
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Mice, Mutant Strains
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Nerve Tissue Proteins
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genetics
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Social Isolation
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Stress, Psychological
;
psychology
6.Cardamine komarovii flower extract reduces lipopolysaccharide-induced acute lung injury by inhibiting MyD88/TRIF signaling pathways.
Qi CHEN ; Ke-Xin ZHANG ; Tai-Yuan LI ; Xuan-Mei PIAO ; Mei-Lan LIAN ; Ren-Bo AN ; Jun JIANG
Chinese Journal of Natural Medicines (English Ed.) 2019;17(6):461-468
In the present study, we investigated anti-inflammatory effect of Cardamine komarovii flower (CKF) on lipopolysaccharide (LPS)-induced acute lung injury (ALI). We determined the effect of CKF methanolic extracts on LPS-induced pro-inflammatory mediators NO and prostaglandin E2 (PGE2), production of pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6), and related protein expression levels of MyD88/TRIF signaling pathways in peritoneal macrophages (PMs). Nuclear translocation of NF-κB-p65 was analyzed by immunofluorescence. For the in vivo experiments, an ALI model was established to detect the number of inflammatory cells and inflammatory factors (IL-1β, TNF-α, and IL-6) in bronchoalveolar lavage fluid (BALF) of mice. The pathological damage in lung tissues was evaluated through H&E staining. Our results showed that CKF can decrease the production of inflammatory mediators, such as NO and PGE2, by inhibiting their synthesis-related enzymes iNOS and COX-2 in LPS-induced PMs. In addition, CKF can downregulate the mRNA levels of IL-1β, TNF-α, and IL-6 to inhibit the production of inflammatory factors. Mechanism studies indicated that CKF possesses a fine anti-inflammatory effect by regulating MyD88/TRIF dependent signaling pathways. Immunocytochemistry staining showed that the CKF extract attenuates the LPS-induced translocation of NF-kB p65 subunit in the nucleus from the cytoplasm. In vivo experiments revealed that the number of inflammatory cells and IL-1β in BALF of mice decrease after CKF treatment. Histopathological observation of lung tissues showed that CKF can remarkably improve alveolar clearance and infiltration of interstitial and alveolar cells after LPS stimulation. In conclusion, our results suggest that CKF inhibits LPS-induced inflammatory response by inhibiting the MyD88/TRIF signaling pathways, thereby protecting mice from LPS-induced ALI.
Acute Lung Injury
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chemically induced
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drug therapy
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genetics
;
metabolism
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Adaptor Proteins, Vesicular Transport
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genetics
;
metabolism
;
Animals
;
Anti-Inflammatory Agents
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administration & dosage
;
chemistry
;
Cardamine
;
chemistry
;
Cyclooxygenase 2
;
genetics
;
metabolism
;
Female
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Flowers
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chemistry
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Humans
;
Lipopolysaccharides
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adverse effects
;
Male
;
Mice
;
Myeloid Differentiation Factor 88
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genetics
;
metabolism
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NF-kappa B
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genetics
;
metabolism
;
Nitric Oxide Synthase Type II
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genetics
;
metabolism
;
Plant Extracts
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administration & dosage
;
chemistry
;
Signal Transduction
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drug effects
;
Tumor Necrosis Factor-alpha
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genetics
;
metabolism
7.Effect of Shensu Yin on the expression of toll-like receptors and the downstream signaling components on RAW 264.7 cells.
Bao-Sheng ZHAO ; Lan-Fang LI ; Yue-Ying MA ; Shu-Ying GUO ; Cang-Hai LI ; Hai-Ru HUO ; Ting-Liang JIANG
China Journal of Chinese Materia Medica 2007;32(4):327-332
OBJECTIVETo investigate the influences of Shensu Yin to RAW 264.7 on the expression of TLR3, TLR4 and the factors of the downstream in RAW 264. 7 cells.
METHODRAW 264.7 cell line was stimulated with Lipopolysaccharide and POLY I: C, respectively, and treated with the drug serum of Shensuyin simultaneously. 24 hours later, collected the supernatant and measured the inflammatory factors TNF-alpha and IFN-beta, extracted mRNA and measured the expression of TLR3, TLR4 and other correlated indexes of the downstream, analyzed and evaluated Shensu Yin's substance basis of pharmacodynamic actions.
RESULTShensu Yin drug serum depressed the expression of TLR4, MyD88, TRAF-6, TRAM and TRIF mRNA, as a result, it decreased the amount of TNF-alpha and IFN-beta.
CONCLUSIONDepressing the expression of TLR3, MyD88, TRAM and TRIF mRNA may be the elementary basis of Shensu Yin to play heat-clearing and detoxicating effect.
Adaptor Proteins, Vesicular Transport ; genetics ; Animals ; Cell Line ; Drug Combinations ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Interferon-beta ; secretion ; Lipopolysaccharides ; pharmacology ; Macrophages ; cytology ; drug effects ; metabolism ; Male ; Mice ; Myeloid Differentiation Factor 88 ; genetics ; Plants, Medicinal ; chemistry ; Poly I-C ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, Interleukin ; genetics ; Signal Transduction ; drug effects ; Toll-Like Receptor 3 ; genetics ; Toll-Like Receptor 4 ; genetics ; Tumor Necrosis Factor-alpha ; secretion
8.Myeloid differentiation primary response protein 88 blockade upregulates indoleamine 2,3-dioxygenase expression in rheumatoid synovial fibroblasts.
Mi Kyung PARK ; Hye Jwa OH ; Yang Mi HEO ; Eun Mi PARK ; Mi La CHO ; Ho Youn KIM ; Sung Hwan PARK
Experimental & Molecular Medicine 2011;43(8):446-454
Indoleamine 2,3-dioxygenase (IDO) is a key negative regulator of immune responses and has been implicated in tumor tolerance, autoimmune disease and asthma. IDO was detected in the joint synovial tissue in the inflammatory microenvironment of rheumatoid arthritis (RA), but IDO expression in joint synovial tissue is not sufficient to overcome the inflamed synovial environment. This study aimed to unravel the mechanisms involving the failure to activate tolerogenic IDO in the inflamed joint. We demonstrate that both poly (I:C) and lipopolysaccharide (LPS) induce expression of IDO in synovial fibroblasts. However, inflammatory cytokines such as IL-17, TNF-alpha, IL-12, IL-23 and IL-16 did not induce IDO expression. Poly (I:C) appeared to induce higher IDO expression than did LPS. Surprisingly, toll-like receptor (TLR)4-mediated IDO expression was upregulated after depletion of myeloid differentiation primary response protein 88 (MyD88) in synovial fibroblasts using small interfering RNA (siRNA). IDO, TLR3 and TLR4 were highly expressed in synovial tissue of RA patients compared with that of osteoarthritis patients. In addition, RA patients with severe disease activity had higher levels of expression of IDO, TLR3 and TLR4 in the synovium than patients with mild disease activity. These data suggest that upregulation of IDO expression in synovial fibroblasts involves TLR3 and TLR4 activation by microbial constituents. We showed that the mechanisms responsible for IDO regulation primarily involve MyD88 signaling in synovial fibroblasts, as demonstrated by siRNA-mediated knockdown of MyD88.
Adaptor Proteins, Vesicular Transport/genetics/metabolism
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Arthritis, Rheumatoid/*metabolism
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Blotting, Western
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Cells, Cultured
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Fibroblasts/drug effects/*metabolism
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Humans
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Immunohistochemistry
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Indoleamine-Pyrrole 2,3,-Dioxygenase/genetics/*metabolism
;
Interleukin-12/pharmacology
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Interleukin-16/pharmacology
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Interleukin-17/pharmacology
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Interleukin-23/pharmacology
;
Lipopolysaccharides/pharmacology
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Myeloid Differentiation Factor 88/genetics/*metabolism
;
Poly I-C/pharmacology
;
Polymerase Chain Reaction
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RNA, Small Interfering/genetics/physiology
;
Synovial Membrane/*cytology
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Toll-Like Receptor 4/genetics/metabolism
;
Tumor Necrosis Factor-alpha/pharmacology