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1.  UbcH10 overexpression increases carcinogenesis and blocks ALLN susceptibility in colorectal cancer 
Scientific Reports  2014;4:6910.
Cyclins are essential for cell proliferation, the cell cycle and tumorigenesis in all eukaryotes. UbcH10 regulates the degradation of cyclins in a ubiquitin-dependent manner. Here, we report that UbcH10 is likely involved in tumorigenesis. We found that cancer cells exposed to n-acetyl-leu-leu-norleucinal (ALLN) treatment and UbcH10 depletion exhibit a synergistic therapeutic effect. Abundant expression of UbcH10 drives resistance to ALLN-induced cell death, while cells deficient in UbcH10 were susceptible to ALLN-induced cell death. The depletion of UbcH10 hindered tumorigenesis both in vitro and in vivo, as assessed by colony formation, growth curve, soft agar and xenograft assays. These phenotypes were efficiently rescued through the introduction of recombinant UbcH10. In the UbcH10-deficient cells, alterations in the expression of cyclins led to cell cycle changes and subsequently decreases in tumorigenesis. The tumorigenesis of xenograft tumors from UbcH10-deficient cells treated with ALLN was decreased relative to wild-type cells treated with ALLN in nude mice. On the molecular level, we observed that UbcH10 deficiency enhances the activation of caspase 8 and caspase 3 but not caspase 9 to impair cell viability upon ALLN treatment. Collectively, our results suggest that, as an oncogene, UbcH10 is a potential drug target for the treatment of colorectal cancer.
PMCID: PMC4223683  PMID: 25376843
2.  Glycation of Apoprotein A-I Is Associated With Coronary Artery Plaque Progression in Type 2 Diabetic Patients 
Diabetes Care  2013;36(5):1312-1320.
To investigate whether glycation level of apoprotein (apo)A-I is associated with coronary artery disease (CAD) and plaque progression in patients with type 2 diabetes.
Among 375 consecutive type 2 diabetic patients undergoing quantitative coronary angiography (QCA) and intravascular ultrasound (IVUS), 82 patients with nonsignificant stenosis (luminal diameter narrowing <30% [group I]) and 190 patients with significant CAD (luminal diameter stenosis ≥70% [group II]) were included for analysis of apoA-I glycation level and serum activity of lecithin: cholesterol acyltransferase (LCAT). The control group had 136 healthy subjects. At the 1-year follow-up, angiography and IVUS were repeated mainly in group II patients for plaque progression assessment.
Relative intensity of apoA-I glycation by densitometry was increased, and serum LCAT activity was decreased stepwise across groups control, I, and II. These two measurements were associated with the number of diseased coronary arteries and extent index in group II. During 1-year follow-up, QCA detected 45 patients with plaque progression in 159 subjects, and IVUS found 38 patients with plaque progression in 127 subjects. Baseline relative intensity of apoA-I glycation was significantly increased in patients with plaque progression compared with those without, with values associated with changes in QCA and IVUS measurements. Multivariable regression analysis revealed that baseline relative intensity of apoA-I glycation was an independent determinant of CAD and plaque progression in type 2 diabetic patients.
ApoA-I glycation level is associated with the severity of CAD and coronary artery plaque progression in type 2 diabetic patients.
PMCID: PMC3631856  PMID: 23230102
3.  Increase of ADAM10 Level in Coronary Artery In-Stent Restenosis Segments in Diabetic Minipigs: High ADAM10 Expression Promoting Growth and Migration in Human Vascular Smooth Muscle Cells via Notch 1 and 3 
PLoS ONE  2013;8(12):e83853.
This study aimed to identify major proteins in the pathogenesis of coronary artery in-stent restenosis (ISR) in diabetic minipigs with sirolimus-eluting stenting, and to investigate the roles of key candidate molecules, particularly ADAM10, in human arterial smooth muscle cells (HASMCs).
Methods and Results
The stents were implanted in the coronary arteries of 15 diabetic and 26 non-diabetic minipigs, and angiography was repeated at six months. The intima of one vascular segment with significant ISR and one with non-ISR in diabetic minipigs were isolated and cultured in conditioned medium (CM). The CM was analyzed by LC-MS/MS to uncover proteins whose levels were significantly increased (≥1.5-fold) in ISR than in non-ISR tissues. After literature searching, we focused on the identified proteins, whose biological functions were most potentially related to ISR pathophysiology. Among them, ADAM10 was significantly increased in diabetic and non-diabetic ISR tissues as compared with non-ISR controls. In cell experiments, retrovirus-mediated overexpression of ADAM10 promoted growth and migration of HASMCs. The effects of ADAM10 were more remarkable in high-glucose culture than in low-glucose culture. Using shRNA and an inhibitor of γ-secretase (GSI), we found that the influences of ADAM10 were in part mediated by Notch1 and notch 3 pathway, which up-regulated Notch downstream genes and enhanced nuclear translocation of the small intracellular component of Notch1 and Notch3.
This study has identified significantly increased expression of ADAM10 in the ISR versus non-ISR segment in diabetic minipigs and implicates ADAM10 in the enhanced neointimal formation observed in diabetes after vascular injury.
PMCID: PMC3873985  PMID: 24386293
4.  The effects of low-dose Nepsilon-(carboxymethyl)lysine (CML) and Nepsilon-(carboxyethyl)lysine (CEL), two main glycation free adducts considered as potential uremic toxins, on endothelial progenitor cell function 
Patients with chronic kidney disease (CKD) are at high risk of cardiovascular disease (CVD). Endothelial progenitor cell (EPCs) dysfunction plays a key role in this pathogenesis. Uremic retention toxins have been reported to be in associated with EPC dysfunction. Advanced glycation end-products (AGEs) free adducts, including Nepsilon-(carboxymethyl)lysine (CML) and Nepsilon-(carboxyethyl)lysine (CEL), are formed by physiological proteolysis of AGEs and released into plasma for urinary excretion. They are retained in CKD patients and are considered to be potential uremic toxins. Though AGEs have been demonstrated to impair EPC function in various ways, the effect of AGE free adducts on EPC function has not been studied. Thus, we examined the role of CML and CEL in the regulation of growth-factor-dependent function in cultured human EPCs and the mechanisms by which they may affect EPC function.
Late outgrowth EPCs were incubated with different concentrations of CML or CEL for up to 72 hours. Cell proliferation was determined using WST-1 and BrdU assays. Cell apoptosis was tested with annexin V staining. Migration and tube formation assays were used to evaluate EPC function.
Though CML and CEL were determined to have anti-proliferative effects on EPCs, cells treated with concentrations of CML and CEL in the range found in CKD patients had no observable impairment on migration or tube formation. CML and CEL did not induce EPC apoptosis. The reduced growth response was accompanied by significantly less phosphorylation of mitogen-activated protein kinases (MAPKs).
Our study revealed that CML and CEL at uremic concentrations have low biological toxicity when separately tested. The biologic effects of AGE free adducts on the cardiovascular system merit further study.
PMCID: PMC3471041  PMID: 22853433
Endothelial progenitor cells; Mitogen-activated protein kinases; Nϵ-(carboxyethyl)lysine; Nϵ-(carboxymethyl)lysine; Uremic toxins
5.  Insulin resistance predicts progression of de novo atherosclerotic plaques in patients with coronary heart disease: a one-year follow-up study 
The aim of our study was to explore and evaluate the relationship between insulin resistance and progression of coronary atherosclerotic plaques. With the great burden coronary heart disease is imposing on individuals, healthcare professionals have already embarked on determining its potential modifiable risk factors in the light of preventive medicine. Insulin resistance has been generally recognized as a novel risk factor based on epidemiological studies; however, few researches have focused on its effect on coronary atherosclerotic plaque progression.
From June 7, 2007 to December 30, 2011, 366 patients received their index coronary angiogram and were subsequently found to have coronary atherosclerotic plaques or normal angiograms were consecutively enrolled in the study by the department of cardiology at the Ruijin Hospital, which is affiliated to the Shanghai Jiaotong University School of Medicine. All patients had follow-up angiograms after the 1-year period for evaluating the progression of the coronary lesions. The modified Gensini score was adopted for assessing coronary lesions while the HOMA-IR method was utilized for determining the state of their insulin resistance. Baseline characteristics and laboratory test results were described and the binomial regression analysis was conducted to investigate the relationship between insulin resistance and coronary atherosclerotic plaque progression.
Index and follow-up Gensini scores were similar between the higher insulin lower insulin resistant groups (9.09 ± 14.33 vs 9.44 ± 12.88, p = 0.813 and 17.21 ± 18.46 vs 14.09 ± 14.18, p =0.358). However the Gensini score assessing coronary lesion progression between both visits was significantly elevated in the higher insulin resistant group (8.13 ± 11.83 versus 4.65 ± 7.58, p = 0.019). Multivariate logistic binomial regression analysis revealed that insulin resistance (HOMA-IR > 3.4583) was an independent predictor for coronary arterial plaque progression (OR = 4.969, p = 0.011). We also divided all the participants into a diabetic (n = 136) and a non-diabetic group (n = 230), and HOMA-IR remained an independent predictor for atherosclerosis plaque progression.
Insulin resistance is an independent predictor of atherosclerosis plaque progression in patients with coronary heart disease in both the diabetic and non-diabetic population.
PMCID: PMC3441242  PMID: 22709409
Coronary heart disease; Insulin resistance; Atherosclerosis plaque progression; In-stent restenosis; one-year follow-up; HOMA-IR
6.  Protection against Respiratory Syncytial Virus Infection by DNA Immunization  
Respiratory syncytial virus (RSV) remains a major cause of morbidity and mortality in infants and the elderly and is a continuing challenge for vaccine development. A murine T helper cell (Th) type 2 response associates with enhanced lung pathology, which has been observed in past infant trials using formalin-inactivated RSV vaccine. In this study, we have engineered an optimized plasmid DNA vector expressing the RSV fusion (F) protein (DNA-F). DNA-F was as effective as live RSV in mice at inducing neutralizing antibody and cytotoxic T lymphocyte responses, protection against infection, and high mRNA expression of lung interferon γ after viral challenge. Furthermore, a DNA-F boost could switch a preestablished anti-RSV Th2 response towards a Th1 response. Critical elements for the optimization of the plasmid constructs included expression of a secretory form of the F protein and the presence of the rabbit β-globin intron II sequence upstream of the F-encoding sequence. In addition, anti-F systemic immune response profile could be modulated by the route of DNA-F delivery: intramuscular immunization resulted in balanced responses, whereas intradermal immunization resulted in a Th2 type of response. Thus, DNA-F immunization may provide a novel and promising RSV vaccination strategy.
PMCID: PMC2213364  PMID: 9705950
respiratory syncytial virus; DNA vaccine; vector design; F protein; immune modulation
7.  The Transferrin Binding Protein B of Moraxella catarrhalis Elicits Bactericidal Antibodies and Is a Potential Vaccine Antigen 
Infection and Immunity  1998;66(9):4183-4192.
The transferrin binding protein genes (tbpA and tbpB) from two strains of Moraxella catarrhalis have been cloned and sequenced. The genomic organization of the M. catarrhalis transferrin binding protein genes is unique among known bacteria in that tbpA precedes tbpB and there is a third gene located between them. The deduced sequences of the M. catarrhalis TbpA proteins from two strains were 98% identical, while those of the TbpB proteins from the same strains were 63% identical and 70% similar. The third gene, tentatively called orf3, encodes a protein of approximately 58 kDa that is 98% identical between the two strains. The tbpB genes from four additional strains of M. catarrhalis were cloned and sequenced, and two potential families of TbpB proteins were identified based on sequence similarities. Recombinant TbpA (rTbpA), rTbpB, and rORF3 proteins were expressed in Escherichia coli and purified. rTbpB was shown to retain its ability to bind human transferrin after transfer to a membrane, but neither rTbpA nor rORF3 did. Monospecific anti-rTbpA and anti-rTbpB antibodies were generated and used for immunoblot analysis, which demonstrated that epitopes of M. catarrhalis TbpA and TbpB were antigenically conserved and that there was constitutive expression of the tbp genes. In the absence of an appropriate animal model, anti-rTbpA and anti-rTbpB antibodies were tested for their bactericidal activities. The anti-rTbpA antiserum was not bactericidal, but anti-rTbpB antisera were found to kill heterologous strains within the same family. Thus, if bactericidal ability is clinically relevant, a vaccine comprising multiple rTbpB antigens may protect against M. catarrhalis disease.
PMCID: PMC108504  PMID: 9712766
8.  Cloning and Expression of the Moraxella catarrhalis Lactoferrin Receptor Genes 
Infection and Immunity  1998;66(8):3656-3665.
The lactoferrin receptor genes from two strains of Moraxella catarrhalis have been cloned and sequenced. The lfr genes are arranged as lbpB followed by lbpA, a gene arrangement found in lactoferrin and transferrin receptor operons from several bacterial species. In addition, a third open reading frame, orf3, is located one nucleotide downstream of lbpA. The deduced lactoferrin binding protein A (LbpA) sequences from the two strains were found to be 99% identical, the LbpB sequences were 92% identical, and the ORF3 proteins were 98% identical. The lbpB gene was PCR amplified and sequenced from a third strain of M. catarrhalis, and the encoded protein was found to be 77% identical and 84% similar to the other LbpB proteins. Recombinant LbpA and LbpB proteins were expressed from Escherichia coli, and antisera raised to the purified proteins were used to assess antigenic conservation in a panel of M. catarrhalis strains. The recombinant proteins were tested for the ability to bind human lactoferrin following gel electrophoresis and electroblotting, and rLbpB, but not rLbpA, was found to bind lactoferrin. Bactericidal antibody activity was measured, and while the anti-rLbpA antiserum was not bactericidal, the anti-rLbpB antisera were found to be weakly bactericidal. Thus, LbpB may have potential as a vaccine candidate.
PMCID: PMC108399  PMID: 9673246

Results 1-8 (8)