Jianhua Huo

Xi'an Jiaotong University, Ch’ang-an, Shaanxi, China

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Publications (6)18.48 Total impact

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    ABSTRACT: Objective To construct a human ether-a-go-go-related gene (HERG) nonsense mutant L539fs/47-*558W into the autonomously fluorescent, eukaryotic expression vector pEGFP-C2, and to verify expression of the reconstruct in human embryonic kidney-293 (HEK293) cells. Methods The mutational fragment was subcloned into pEGFP-C2-HERG by double digestion of Sbf I, Eco91 I and rejoining of T4 ligase. After verification, the recombinant pEGFP-C2-L539fs/47-*558W and pEGFP-C2-HERG were respectively transfected into HEK293 cells for 48 h by the Lipofect method to observe the expression location of the fusion protein by laser confocal imaging scanning in vivo. pcDNA3 -L539fs/47-*558W and pcDNA3-HERG were transfected to observe the expression location of the HERG protein by immunofluoresceoce. The mutant protein size was determined by Western blotting. Results The about 1 kb-sized mutation region cDNA fragment from pcDNA3-L539fs/47-*558W and the about 7.2 kb-sized target vector fragment from pcDNA3-HERG were ligated after purification and gel recovery. pEGFP-C2-L539fs/47*-558W, approximately 8.2 kb, was demonstrated successfully been constructed under agarose gel electrophoresis and further sequencing. Laser confocal imaging showed that pEGFP-C2-HERG was mainly expressed in the membrane, whereas truncated mutant-type HERG in the pEGFP-C2 vector was partially located in the cytoplasm, the others were transported to the cell membrane in living HEK293 cells. The same as the immunofluoresceoce results after transfection of pcDNA3-HERG and pcDNA3-L539fs/47-558W. Wild-type HERG-GFP fusion protein expressed 160 and 180 kDa bands. The mutant and mutant-GFP fusion proteins were 70 and 100 kDa, respectively. Conclusion pEGFP-C2-L539fs/47-*558W was successfully constructed by double digestion method GFP had no effect on its protein expression and trafficking in HEK293 cells, which laid a foundation for the further study on L539fs/47-*558W.
    No preview · Article · Aug 2013 · Journal of Medical Colleges of PLA
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    Wenting Chen · Jianhua Huo · Aiqun Ma · Ling Bai · Ping Liu
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    ABSTRACT: The LMNA gene, which encodes the nuclear envelope protein lamin A/C, is considered to be the most common autosomal disease gene associated with familial dilated cardiomyopathy. To date, each mutation of the LMNA gene has been associated with a specific disease phenotype. Clinical data, family histories, and blood samples were collected from 27 biological members of a family with dilated cardiomyopathy, prominently occurring as heart failure and conduction system disease with a high incidence of sudden cardiac death in young females. Twelve exons of the LMNA gene were screened for nucleotide alterations. A novel insertion mutation (nucleotide 1526insA, amino acid T510Y) in exon nine of the LMNA gene was identified in seven subjects (7/27, 25.9 %). This reveals that the LMNA gene insertion mutation (T510Y frameshift mutation) can cause dilated cardiomyopathy, conduction system disease, and sudden cardiac death without skeletal myopathy, clinically manifested with early onset, severe symptoms, and poor prognosis.
    Preview · Article · Jun 2013 · Molecular and Cellular Biochemistry
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    ABSTRACT: The congenital long QT syndrome is a heterogeneous genetic disease associated with delayed cardiac repolarization, prolonged QT intervals, the development of ventricular arrhythmias and sudden death. Type 2 congenital long QT syndrome (LQT2) results from KCNH2 or hERG gene mutations. hERG encodes the K(v)11.1 alpha subunit of the rapidly activating delayed rectifier K(+) current in the heart. Studies of mutant hERG channels indicate that most LQT2 missense mutations generate trafficking-deficient K(v)11.1 channels. To identify the mechanism underlying G572R-hERG by using molecular and electrophysiological analyses. To elucidate the electrophysiological properties of the G572R-hERG mutant channels, mutant hERG subunits were heterologously expressed in HEK293 cells alone or in combination with wild-type (WT)-hERG subunits. Patch-clamp techniques were used to record currents, and double immunofluorescence protein tagging and Western blotting were performed to examine the cellular trafficking of mutant subunits. When expressed alone, G572R-hERG subunits were not present in the cell membrane and did not produce detectable currents. When coexpressed with WT-hERG subunits, G572R-hERG decreased current density and altered gating properties of the WT-hERG channel. The hERG-associated missense mutation G572R, like most LQT2 missense mutations, generates a trafficking-deficient phenotype. Furthermore, G572R-hERG causes a loss of function in hERG by a strong dominant negative effect on the WT-hERG channel.
    Full-text · Article · Oct 2010 · The Canadian journal of cardiology
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    ABSTRACT: Our previous works revealed that human ribosomal protein S13 (RPS13) was up-regulated in multidrug-resistant gastric cancer cells and overexpression of RPS13 could protect gastric cancer cells from drug-induced apoptosis. The present study was designed to explore the role of RPS13 in tumorigenesis and development of gastric cancer. The expression of RPS13 in gastric cancer tissues and normal gastric mucosa was evaluated by immunohistochemical staining and Western blot analysis. It was found RPS13 was expressed at a higher level in gastric cancer tissues than that in normal gastric mucosa. RPS13 was then genetically overexpressed in gastric cancer cells or knocked down by RNA interference. It was demonstrated that up-regulation of RPS13 accelerated the growth, enhanced in vitro colony forming and soft agar cologenic ability and promoted in vivo tumour formation potential of gastric cancer cells. Meanwhile, down-regulation of RPS13 in gastric cancer cells resulted in complete opposite effects. Moreover, overexpression of RPS13 could promote G1 to S phase transition whereas knocking down of RPS13 led to G1 arrest of gastric cancer cells. It was further demonstrated that RPS13 down-regulated p27(kip1) expression and CDK2 kinase activity but did not change the expression of cyclin D, cyclin E, CDK2, CDK4 and p16(INK4A). Taken together, these data indicate that RPS13 could promote the growth and cell cycle progression of gastric cancer cells at least through inhibiting p27(kip1) expression.
    No preview · Article · Nov 2009 · Journal of Cellular and Molecular Medicine
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    ABSTRACT: To investigate common downstream mechanism of PGE2 and O2-sensitive voltage-dependent potassium (Kv) channels in preterm and term DA tone regulations, for suggesting respective prescriptions for preterm and term PDA. The expressions of Kv1.2, 1.5 and 2.1 were compared between preterm and term in rabbit and human DAs at mRNA and protein levels; DA contracting responses caused by O2, Kv channels blocker 4-AP, EP4 antagonist GW627368X, and PGE2 reduce using vessels rings and Whole-Cell Patch-Clamp were explored. Kv 1.2 and 2.1 expressions were developed with pregnant age in preterm DA and decreased after birth with oxygen stimulation in term DA. GW627368X led significant DA constriction and DASMC IK current decrease in preterm, which was slimier to 4-AP effects, but just slightly influenced on DA tension and DASMC IK current at term. In addition, PGE2 led great DA dilation and IK current increase of DASMC in preterm but not in term. These DA tension and IK current changes were in line with Kv channel expressions. Higher levels of PGE2 binds with GPCR EP4, which activates G-protein to couple with O2-sensitive Kv channels and to open them, leading to DA vasorelaxation in the fetus. It indicates that EP4 inhibitors, instead of PGE2 or its analogue PGE1, may be a selectable strategy for preterm PDA.
    No preview · Article · Oct 2009 · International journal of cardiology
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    ABSTRACT: We have recently identified a missense mutation, G604S, in the human ether-a-go-go related gene (hERG) that results in a malignant phenotype in a full pedigree of a Chinese congenital long QT syndrome (LQTS) family. The present study characterized the pathophysiological consequences of the mutation at the cellular level. Mutant G604S-hERG channels were expressed in HEK293 cells using a lipofectamine method. hERG currents were recorded using the voltage clamp technique. The expression of hERG protein was detected by Western blotting, and the subcellular location of hERG channels in cell was analyzed by confocal microscopy. We found that the G604S mutation did not lead to any expression of detectable currents, which was consistent with Western blotting analysis that the G604S-hERG mutation only expressed a band at 135 kDa. When coexpressed with wild-type (WT)-hERG, G604S-hERG exhibited strong dominant-negative current suppression resulting in decreased current density and altered gating properties of the WT-hERG channel, as well as interference with the trafficking of WT-hERG channel protein. In addition, confocal microscopy demonstrated that G604S-hERG subunits could be inserted into the cell membrane when forming heteromultimeric channels with WT-hERG channel subunits. Our results suggest that G604S mutation causes a loss of function in hERG through a strong dominant-negative effect on WT-hERG channel function that caused by impaired trafficking of WT-hERG channels, and further accentuates this suppression by forming heteromultimeric functional channels with WT-hERG subunits.
    No preview · Article · Sep 2008 · Pflügers Archiv - European Journal of Physiology

Publication Stats

39 Citations
18.48 Total Impact Points


  • 2008-2013
    • Xi'an Jiaotong University
      • Key Laboratory of Environment and Genes Related to Diseases
      Ch’ang-an, Shaanxi, China
  • 2009-2010
    • Shanghai Jiao Tong University
      Shanghai, Shanghai Shi, China