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1.  Wilson disease: Identification of two novel mutations and clinical correlation in Eastern Chinese patients 
AIM: To study mutations in the P-type ATPase (ATP7B) gene responsible for Wilson disease (WD) in the Eastern Chinese population, and the possible correlation of specific mutations with clinical characteristics.
METHODS: Mutations of the ATP7B gene were sought by means of direct sequencing in 50 Eastern Chinese WD patients of Han ethnic origin.
RESULTS: Two novel mutations, Asp96Gly and Asp196Glu, were first identified. We also compared the characterization of mutations in ATP7B with the clinical findings, and a significant correlation with hepatic manifestations between patients carrying the Arg778Leu mutation and those without was found.
CONCLUSION: Gene sequencing analysis was shown to have a high detection rate and accuracy. It may become the first priority in screening of WD patients.
PMCID: PMC4434647  PMID: 17876883
Wilson disease; ATP7B gene; Mutations; Polymorphisms
2.  In vitro and in vivo suppression of hepatocellular carcinoma growth by midkine-antisense oligonucleotide-loaded nanoparticles 
AIM: To synthesize antisense oligonucleotides (ASODNs) of midkine (MK), package the ASODNs with nanoparticles, and to inhibit hepatocellular carcinoma (HCC) growth using these nanoparticles.
METHODS: HepG2 cell proliferation was analyzed in vitro using the 3-(4,5-dimethythiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium, inner salt assay. The in vivo activity of nanoparticles delivering the MK-ASODNs was analyzed by histopathological and immunohistochemical staining and quantitative real time polymerase chain reaction (PCR).
RESULTS: The in vitro proliferation of HepG2 cells was significantly inhibited by the nanoparticles packaged with MK-ASODNs (NANO-ASODNs). Furthermore, the NANO-ASODNs significantly inhibited the growth of HCC in the mouse model.
CONCLUSION: NANO-ASODNs can significantly suppress the growth of HCC in vitro and in vivo.
PMCID: PMC2675086  PMID: 19399928
Midkine; Nanoparticles; Hepatocellular carcinoma; Inhibition; Drug delivery
3.  Effective inhibition of expression of hepatitis B virus genes by DNAzymes 
AIM: To evaluate the inhibitory effects of DNAzymes on the expressions of hepatitis B virus (HBV) s (HBsAg) and e (HBeAg) in 2.2.15 cells, and to explore the potential therapeutic effects of DNAzymes on replication of HBV genome.
METHODS: DNAzymes DrzBS and DrzBC specific to HBV (ayw subtype) s gene ORF A157UG and e gene ORF A1816UG, were designed and synthesized. Inhibitory effects of DrzBS or DrzBC on the expressions of HBV s and e genes as well as HBV DNA levels in culture supernatants were observed in 2.2.15 cells.
RESULTS: After being treated with DrzBS or DrzBC, the expression of HBV s or e genes in 2.2.15 cells was depressed dramatically. The maximum inhibition rate was 94.2% and 91.8% for DrzBS and DrzBC, respectively. The concentration for effective inhibition of both DrzBS and DrzBC was within 0.1-2.5 µmol/L, showing a dose-dependence. The efficiency of inhibiting HBsAg, HBeAg in 2.2.15 cells by DrzBS or DrzBC was higher than that of the same target genes by antisense oligonucleotides (ASON). The concentration for effective inhibition of DNAzymes was at least 10-fold lower compared with ASON controls. Neither inhibition on the replication of HBV DNA nor toxicity to 2.2.15 cells was observed.
CONCLUSION: DrzBS and DrzBC can block the expression of HBV s- and e-genes in 2.2.15 cells and provide a specific and effective anti-HBV gene therapeutic means.
PMCID: PMC4315949  PMID: 15962363
DNAzyme; HBV; Gene expression; Inhibition
4.  Development and Characterization of a Novel Fusion Protein of a Mutated Granulocyte Colony-Stimulating Factor and Human Serum Albumin in Pichia pastoris 
PLoS ONE  2014;9(12):e115840.
The purpose of the present work was to develop a novel, long-acting and potent human serum albumin/granulocyte colony stimulating factor (HSA/G-CSF) therapeutic fusion protein. The novel fusion protein, called HMG, was constructed by genetically fusing mutated human derived G-CSF (mG-CSF) to the C-terminal of HSA and then prepared in Pichia pastoris. The molecular mass of HMG was about 85 kDa and the isoelectric point was 5.3. Circular dichroism spectroscopy suggested that mG-CSF retained nearly all of its native secondary structure, regardless of fusion. The binding capabilities of mG-CSF moiety to G-CSF receptor and HSA moiety to warfarin showed very little change after fusing. The bioactivity of HMG (11.0×106 IU/mg) was more than twice that of rHSA/G-CSF (4.6×106 IU/mg). A mutation was made at the 718th amino acid of HMG, substituting Ala for Thr, to investigate the glycosylation of HMG expressed in P. pastoris. Data indicated that HMG was modified at Thr718, speculatively with the addition of a mannose chain. In conclusion, a novel HSA/G-CSF fusion protein was successfully constructed based on a mutated G-CSF. This protein showed more potent bioactivity than rHSA/G-CSF and thus may be a suitable long-acting G-CSF.
PMCID: PMC4275271  PMID: 25535738
5.  Polymorphisms in the sialic acid-binding immunoglobulin-like lectin-8 (Siglec-8) gene are associated with susceptibility to asthma 
Sialic acid-binding immunoglobulin-like lectin-8 (Siglec-8) promotes the apoptosis of eosinophils and inhibits FcɛRI-dependent mediator release from mast cells. We investigated the genetic association between sequence variants in Siglec-8 and diagnosis of asthma, total levels of serum IgE (tIgE), and diagnosis of eosinophilic esophagitis (EE) in diverse populations. The effect of sequence variants on Siglec-8 glycan ligand-binding activity was also examined. Significant association with asthma was observed for SNP rs36498 (odds ratios (OR), 0.69, P=8.8 × 10−5) among African Americans and for SNP rs10409962 (Ser/Pro) in the Japanese population (OR, 0.69, P=0.019). Supporting this finding, we observed association between SNP rs36498 and current asthma among Brazilian families (P=0.013). Significant association with tIgE was observed for SNP rs6509541 among African Americans (P=0.016), and replicated among the Brazilian families (P=0.02). In contrast, no association was observed with EE in Caucasians. By using a synthetic polymer decorated with 6′-sulfo-sLex, a known Siglec-8 glycan ligand, we did not find any differences between the ligand-binding activity of HEK293 cells stably transfected with the rs10409962 risk allele or the WT allele. However, our association results suggest that the Siglec8 gene may be a susceptibility locus for asthma.
PMCID: PMC2987348  PMID: 20087405
sialic acid-binding immunoglobulin-like lectin-8 (Siglec8); polymorphisms; asthma; eosinophilic esophagitis
6.  IQGAP1 is overexpressed in hepatocellular carcinoma and promotes cell proliferation by Akt activation 
Experimental & Molecular Medicine  2010;42(7):477-483.
The scaffold protein IQGAP1 shows elevated levels in several cancer types, but its expression in hepatocellular carcinoma is unknown. We found that 58% of human hepatocellular carcinoma tissue samples had increased IQGAP1 expression compared to adjacent normal tissue. Overexpressing IQGAP1 raised the in vivo tumorigenicity of hepatocellular carcinoma cells, and forced overexpression of IQGAP1 in vitro stimulated cell proliferation. Cell growth was reduced by knockdown or mutation of IQGAP1, or by treatment of cells with a phosphotidylinositol 3-kinase inhibitor. To determine the mechanism by which IQGAP1 overexpression affected hepatocellular carcinoma cells, we confirmed its interaction in these cells with mammalian target of rapamycin (mTOR), a serine/threonine kinase that integrates signals about nutrient and energy status with downstream effectors that influence cell division. In addition, we discovered a new interaction involving IQGAP1, mTOR and Akt, which is a downstream target of mTOR. Akt phosphorylation on Ser-473, which is catalyzed by mTOR and required for Akt activation, increased with increasing amounts of IQGAP1, and decreased with IQGAP1 mutation. We hypothesize that IQGAP1 is a scaffold that facilitates mTOR and Akt interaction.
PMCID: PMC2912475  PMID: 20530982
carcinoma, hepatocellular; IQ motif containing GTPase activating protein; mTOR protein; oncogenes; proto-oncogene proteins c-akt
7.  Genes related to the very early stage of ConA-induced fulminant hepatitis: a gene-chip-based study in a mouse model 
BMC Genomics  2010;11:240.
Due to the high morbidity and mortality of fulminant hepatitis, early diagnosis followed by early effective treatment is the key for prognosis improvement. So far, little is known about the gene expression changes in the early stage of this serious illness. Identification of the genes related to the very early stage of fulminant hepatitis development may provide precise clues for early diagnosis.
Balb/C mice were used for ConA injection to induce fulminant hepatitis that was confirmed by pathological and biochemical examination. After a gene chip-based screening, the data of gene expression in the liver, was further dissected by ANOVA analysis, gene expression profiles, gene network construction and real-time RT-PCR.
At the very early stage of ConA-triggered fulminant hepatitis, totally 1,473 genes with different expression variations were identified. Among these, 26 genes were finally selected for further investigation. The data from gene network analysis demonstrate that two genes, MPDZ and Acsl1, localized in the core of the network.
At the early stages of fulminant hepatitis, expression of twenty-six genes involved in protein transport, transcription regulation and cell metabolism altered significantly. These genes form a network and have shown strong correlation with fulminant hepatitis development. Our study provides several potential targets for the early diagnosis of fulminant hepatitis.
PMCID: PMC2867829  PMID: 20398290
8.  Arsenic trioxide, a potent inhibitor of NF-κB, abrogates allergen-induced airway hyperresponsiveness and inflammation 
Respiratory Research  2006;7(1):146.
Overactivation of nuclear factor κB (NF-κB) orchestrates airway eosinophilia, but does not dampen airway hyperresponsiveness in asthma. NF-κB repression by arsenic trioxide (As2O3) contributes to apoptosis of eosinophils (EOS) in airways. Here we provide evidence that As2O3 abrogates allergen (OVA)-induced airway eosinophilia by modulating the expression of IκBα, an NF-κB inhibitory protein, and decreases the airway hyperresponsiveness.
Using a murine model of asthma, the airway hyperresponsiveness was conducted by barometric whole-body plethysmography. Airway eosinophilia, OVA-specific IgE in serum, and chemokine eotaxin and RANTES (regulated upon activation, normal T cell expressed and secreted) in bronchoalveolar lavage fluid were measured by lung histology, Diff-Quick staining, and ELISA. Chemokine-induced EOS chemotactic activity was evaluated using EOS chemotaxis assay. Electrophoretic mobility shift assay and Western blot analysis were performed to assess pulmonary NF-κB activation and IκBα expression, respectively.
As2O3 attenuated the allergen-induced serum IgE, chemokine expression of eotaxin and RANTES, and the EOS recruitment in bronchoalveolar lavage fluid, which is associated with an increased IκBα expression as well as a decreased NF-κB activation. Also, As2O3 suppressed the chemotaxis of EOS dose-dependently in vitro. Additionally, As2O3 significantly ameliorated the allergen-driven airway hyperresponsiveness, the cardinal feature underlying asthma.
These findings demonstrate an essential role of NF-κB in airway eosinophilia, and illustrate a potential dissociation between airway inflammation and hyperresponsiveness. As2O3 likely exerts its broad anti-inflammatory effects by suppression of NF-κB activation through augmentation of IκBα expression in asthma.
PMCID: PMC1769498  PMID: 17178007
9.  Surveillance of viral contamination of invasive medical instruments in dentistry*  
Objective: To investigate the viral contamination of invasive medical instruments in dentistry and to provide health administrative institutions with surveillance data. Methods: Sterilized samples were randomly collected from the department of dentistry to detect HBV-DNA, HCV-RNA, HIV-RNA and HBsAg. Results: Of the invasive medical instruments that were sterilized with 2% glutaraldehyde, one of the samples was positive for HBV-DNA, and another sample was positive for HBsAg. Conclusion: Though massive virus contamination of invasive medical instruments in dentistry has been reduced to a low level, the occurrence of contamination still remains.
PMCID: PMC1559799  PMID: 16909477
Glutaraldehyde; Viral contamination; Invasive
10.  Immunomodulatory function of orally administered thymosin α1*  
Objective: To investigate the immunological function of a yeast expression system for thymosin α1 (Tα1). Methods: A constructed Tα1 yeast expression system was used to investigate the immunological function of orally administered Tα1. Dried yeast containing three different concentration of Tα1 was fed to normal Balb/c mice and other Balb/c mice whose immunities were inhibited in advance by cyclophosphamide. Synthesized Tα1 peptide was used as positive control and dried yeast with empty plasmid was used as negative control. CD4+ and CD8+ levels were detected by flow cytometry assay. TNF-α, IFN-γ, IL-2, IL-6 and IL-10 levels were detected by liquid chip. Results: In normal Balb/c mice or immune inhibition Balb/c mice, CD8+ levels were significantly increased. Especially in immune inhibition Balb/c mice, CD8+ levels in synthesized Tα1 group (18.77%±4.72%), small dose group (13.48%±6.17%) and large dose group (22.74%±1.09%) were significantly higher than that in empty yeast control group (7.49%±2.14%). Conclusion: Orally administered Tα1 has its certain immunomodulatory function.
PMCID: PMC1389904  PMID: 16130188
Thymosin α1; Yeast expression system; Biological activity; Orally administered
11.  Proapoptotic and pronecrosis effect of different truncated hepatitis C virus core proteins*  
Objective: To study the roles of different truncated hepatitis C virus (HCV) core proteins (CORE) in the pathogenesis of HCV persistent infection and hepatocellular carcinoma (HCC) and to assess intracellular localization in transiently transfected cells. Methods: Seven truncated CORE-GFP (green fluorescent protein) fusion protein expression plasmids were constructed, which contained HCV CORE sequences derived from tumor tissues (BT) and non-tumor tissues (BNT) from one patient infected with HCV. Amino acid (aa) lengths were BT: 1–172 aa, 1–126 aa, 1–58 aa, 59–126 aa, 127–172 aa; BNT: 1–172 aa and C191: 1–172 aa respectively. Subcellular localization of CORE-GFP was analyzed by con-focal laser scanning microscope. Apoptosis and necrosis were quantified by flow cytometry. Results: Different truncated CORE-GFP localized mainly in the cytoplasm, but nuclear staining was also observed. HCV CORE could induce apoptosis and necrosis, and different truncated COREs could induce cell apoptosis and necrosis at different levels. Among the same length 1–172 aa of BT, BNT and C191, the cell apoptosis and necrosis percentage of BT is highest, and C191 is the lowest (BT>BNT>C191). To the different fragment COREs of BT, N-terminal of CORE induced apoptosis and necrosis higher, compared with that of C-terminal (1–172 aa>1–126 aa>1–58 aa>127–172 aa>59–126 aa). Conclusion: These results suggest HCV CORE could induce apoptosis and necrosis of cells, which might play an important role in the pathogenesis of HCV persistent infection and HCC and the different CORE domains of different HCV quasi-species might have some difference in their pathogenesis.
PMCID: PMC1389739  PMID: 15754428
Hepatitis C virus; Core protein; Apoptosis; Necrosis

Results 1-11 (11)