Biomedical subjects

Yi Gong

Publications and source records attributed to Yi Gong.

At least 19 recordsLinked to original sources

PubMed2024

Integrated ubiquitomics characterization of hepatocellular carcinomas.

BACKGROUND AND AIMS: Patients with aggressive HCC have limited therapeutic options. Therefore, a better understanding of HCC pathogenesis is needed to improve treatment. Genomic studies of HCC have improved our understanding of cancer biology. However, the ubiquitomic characteristics of HCC remain poorly understood. We aimed to reveal the ubiquitomic characteristics of HCC and provide clinical feature biomarkers of the aggressive HCC that may be used for diagnosis or therapy in the clinic. APPROACH AND RESULTS: The comprehensive proteomic, phosphoproteomic, and ubiquitomic analyses were performed on tumors and adjacent normal liver tissues from 85 patients with HCC. HCCs displayed overexpression of drugable targets CBR1-S151 and CPNE1-S55. COL4A1, LAMC1, and LAMA4 were highly expressed in the disease free survival-poor patients. Phosphoproteomic and ubiquitomic features of HCC revealed cross talk in metabolism and metastasis. Ubiquitomics predicted diverse prognosis and clarified HCC subtype-specific proteomic signatures. Expression of biomarkers TUBA1A, BHMT2, BHMT, and ACY1 exhibited differential ubiquitination levels and displayed high prognostic risk scores, suggesting that targeting these proteins or their modified forms may be beneficial for future clinical treatment. We validated that TUBA1A K370 deubiquitination drove severe HCC and labeled an aggressive subtype of HCCs. TUBA1A K370 deubiquitination was at least partly attributed to protein kinase B-mediated USP14 activation in HCC. Notably, targeting AKT-USP14-TUBA1A complex promoted TUBA1A degradation and blocked liver tumorigenesis in vivo. CONCLUSIONS: This study expands our knowledge of ubiquitomic signatures, biomarkers, and potential therapeutic targets in HCC.

Humans↗

PubMed2007

[Preparation and preliminary application of monoclonal antibody against alpha-zearalanol].

AIM: To prepare monoclonal antibody (mAb) against alpha-zearalanol (alpha-ZER) and develop an immunoassay for the detection of alpha-ZER and its analogues residues in food derived from animal tissues. METHODS: alpha-ZER was conjugated to BSA as immunogen to immunize BALB/c mice and mAb were prepared by hybridoma technique. mAb's characteristics (titer, Ig subclass, specificity and relative affinity) were identified by ELISA. Standard inhibitive cure was made and sensitivity of the mAb was identified. 37 samples derived from animal liver were detected for alpha-ZER residues by competitive ELISA established in the study. RESULTS: 8 hybridoma cell lines stably secreting anti-alpha-ZER mAb were obtained. The titer of one of them (4E5) was 5.142x10(7). The Ig subclass was IgG1. The mAb was specific for alpha-ZER and its analogues and had no cross-reactivity with other compounds.8 positive results were found from the 37 samples derived from animal liver which were negative detected by HPLC. CONCLUSION: Anti-alpha-ZER mAb has been prepared successfully. A rapid method using the mAb for detecting alpha-ZER and its analogues residues in animal tissues has been established.

Animals↗

PubMed2006

Angiopoietin-related growth factor (AGF) supports adhesion, spreading, and migration of keratinocytes, fibroblasts, and endothelial cells through interaction with RGD-binding integrins.

Angiopoietin-related growth factor (AGF) is a newly identified member of angiopoietin-related proteins (ARPs)/angiopoietin-like proteins (Angptls). AGF has been considered as a novel growth factor in accelerating cutaneous wound healing, as it is capable of stimulating keratinocytes proliferation as well as angiogenesis. But in our paper, we demonstrate that AGF stimulates keratinocytes proliferation only at high protein concentration, however, it can potently promote adhesion, spreading, and migration of keratinocytes, fibroblasts, and endothelial cells. Furthermore, we confirm that the adhesion and migration cellular events are mediated by RGD-binding integrins, most possibly the alpha(v)-containing integrins, by in vitro inhibition assays using synthetic competitive peptides. Our results strongly suggest that AGF is an integrin ligand as well as a mitogenic growth factor and theoretically participates in cutaneous wound healing in a more complex mechanism.

Angiopoietin-Like Protein 6↗

PubMed2006

Nonylphenol-induced oxidative stress and cytotoxicity in testicular Sertoli cells.

Nonylphenol (NP) as an environment contaminant has been demonstrated to adversely affect male reproduction. The main objective of this study was to evaluate NP-induced oxidative stress and toxicity in testicular Sertoli cells. Sertoli cells were exposed to 10-40 microM NP for 24 h. Cell death and growth inhibition were observed by flow cytometric analysis and 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT) assay, while fluorescein diacetate (FDA) and propidium iodide (PI) staining was used to examine the morphological changes following NP exposure. Subsequently, we found that short-term treatment (2 h) of NP caused intracellular accumulation of reactive oxygen species (ROS), which was evaluated by loading of 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) without visible morphological changes. Loss of mitochondrial membrane potential (MMP) was detected following 12 and 24 h treatment of NP and assessment by Rhodamine 123 (Rh123) staining. In addition, incubation with NP for 12 h also increased lipid peroxidation of Sertoli cells. These results indicated that low micromolar concentrations of NP induce an adverse oxidative stress in rat Sertoli cells.

Animals↗

PubMed2006

Juxtamembranous aspartic acid in Insig-1 and Insig-2 is required for cholesterol homeostasis.

Insig-1 and Insig-2 are closely related proteins of the endoplasmic reticulum (ER) that mediate feedback control of cholesterol synthesis by sterol-dependent binding to the following two membrane proteins: the escort protein Scap, thus preventing proteolytic processing of sterol regulatory element-binding proteins; and the cholesterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl CoA reductase, thus inducing the ubiquitination and ER-associated degradation of the enzyme. Here, we report that the conserved Asp-205 in Insig-1, which abuts the fourth transmembrane helix at the cytosolic side of the ER membrane, is essential for its dual function. When Asp-205 was mutated to alanine, the mutant Insig-1 lost the ability to bind to Scap and, thus, was unable to suppress the cleavage of sterol regulatory element-binding proteins. The mutant Insig-1 was ineffective also in accelerating sterol-stimulated degradation of 3-hydroxy-3-methylglutaryl CoA reductase. Alanine substitution of the corresponding aspartic acid in Insig-2 produced the same dual defects. These studies identify a single amino acid residue that is crucial for the function of Insig proteins in regulating cholesterol homeostasis in mammalian cells.

Amino Acid Sequence↗

PubMed2006

Proteasomal degradation of ubiquitinated Insig proteins is determined by serine residues flanking ubiquitinated lysines.

Insig-1 and Insig-2 are closely related proteins of the endoplasmic reticulum that play crucial roles in cholesterol homeostasis by inhibiting excessive cholesterol synthesis and uptake. In sterol-depleted cells Insig-1 is degraded at least 15 times more rapidly than Insig-2, owing to ubiquitination of Lys-156 and Lys-158 in Insig-1. In this study, we use domain-swapping methods to localize amino acid residues responsible for this differential degradation. In the case of Insig-2, Glu-214 stabilizes the protein by preventing ubiquitination. When Glu-214 is changed to alanine, Insig-2 becomes ubiquitinated, but it is still not degraded as rapidly as ubiquitinated Insig-1. The difference in the degradation rates is traced to two amino acids: Ser-149 in Insig-1 and Ser-106 in Insig-2. Ser-149, which lies NH(2)-terminal to the ubiquitination sites, accelerates the degradation of ubiquitinated Insig-1. Ser-106, which is COOH-terminal to the ubiquitination sites, retards the degradation of ubiquitinated Insig-2. The current studies indicate that the degradation of ubiquitinated Insigs is controlled by serine residues flanking the sites of ubiquitination.

Amino Acid Sequence↗

PubMed2006

Changes in coordination of sterically demanding hybrid imidazolylphosphine ligands on Pd(0) and Pd(II).

Low-coordinate organometallic complexes are important in structure and catalysis, and hemilability or secondary interactions such as hydrogen bonding enabled by hybrid ligands are receiving increasing attention. To study the factors controlling these phenomena, three new imidazol-2-ylphosphine ligands, L, were made. In these ligands, the bulk around P and the hindrance at the basic and potentially coordinating imidazole N-3 were varied. Remarkably, L(2)Pd(0) complexes 3a-c were shown to be two-coordinate, 12-electron species, despite the availability of imidazole N-3 to enter into eta(2)-P,N chelation. In oxidative additions of C-X bonds to the Pd(0) complexes, reaction rates and products could be controlled by the nature of the C and X groups and the R groups on the phosphine. Most significantly, whereas 4c-PhI and 4c-MeOTf from 3c are normal trans-bis(phosphine)Pd(R)(X) species, 5a-PhI, 5a-PhBr, and 5b-PhI from 3a and 3b were shown by X-ray diffraction to be a monomeric species with a single eta(2)-P,N-chelating phosphine. From 3a and methyl triflate, an ionic complex [6a-Me](+)[OTf](-) with one chelating and one nonchelating phosphine was formed, with temperature-dependent windshield-wiper exchange of the two, showing hemilability. Thus, large phosphine substituents (R = tert-butyl rather than isopropyl) favor chelation. The chelate Pd-imidazole N-3 bond is longer when the heterocyclic nitrogen is hindered by an adjacent tert-butyl group at C-4 (comparing 5a-PhI and 5b-PhI). Finally, whereas in [8b-Ph](+)[OTf](-) from 5b-PhI and isopropylamine, the amine coordinates without chelate opening or hydrogen bonding, in [10c-Me](+)[OTf](-) made from 4c-MeOTf and isopropylamine, the amine is not only coordinated at N but also donates a hydrogen bond to each phosphine imidazol-2-yl substituent.

Journal Article↗

PubMed2006

Sterol-regulated ubiquitination and degradation of Insig-1 creates a convergent mechanism for feedback control of cholesterol synthesis and uptake.

This paper describes a convergent mechanism for the feedback control of cholesterol synthesis and uptake mediated by SREBPs, membrane bound transcription factors. Endoplasmic reticulum (ER) bound SREBPs form complexes with Scap, a polytopic ER protein. In sterol-overloaded cells, Scap/SREBP binds to Insig-1, which retains the complex in the ER. Upon sterol deprivation, the Scap/SREBP complex dissociates from Insig-1, which is then ubiquitinated on lysines 156 and 158 and degraded in proteasomes. Scap/SREBP moves to the Golgi, where SREBP is processed to liberate a nuclear fragment that activates genes for cholesterol synthesis and uptake and the gene for Insig-1. Ubiquitination is not necessary for release of Scap/SREBP from Insig-1, but it establishes a requirement for synthesis of new Insig-1 for feedback inhibition. When the new Insig-1 and cholesterol converge on Scap, Scap/SREBP binds to Insig-1, preventing ubiquitination. The Insig-1/Scap/SREBP complex accumulates in the ER, ready for liberation when the cell is again sterol deprived.

Animals↗

PubMed2006

Effect of nonylphenol on steroidogenesis of rat Leydig cells.

Nonylphenol is the primary final biodegradation product of nonylphenol polyethoxylate (NPE), a non-ionic surfactant that is frequently incorporated into pesticide and detergent formulation. Recent researchers have hypothesized that environmental/ occupational exposure to nonylphenol poses adverse effects on reproductive system of humans and wildlife species. During our study, in vivo and in vitro experiments were performed to examine the effect of nonylphenol on testosterone biosynthesis of rat Leydig cells. In experiment in vivo, serum testosterone (T) as well as luteinizing hormone (LH) levels were detected after animals had been treated with different doses (0 mg/kg/day, 125 mg/kg/day, and 250 mg/kg/day) of nonylphenol for 50 days by gavage, and the final result revealed that testosterone level dramatically declined at the dose of 250 mg/kg/day, while LH level ascended at the dose of 125 mg/kg/day and 250 mg/kg/day. In experiment in vitro, primary cultured Leydig cells were exposed to nonylphenol for 48 h, including low concentrations (0 mg/L, 0.0011 mg/L, 0.0033 mg/L, 0.0055 mg/L, 0.011 mg/L, 0.022 mg/L) and higher concentrations (0.11 mg/L, 0.55 mg/L, 1.1 mg/L, 1.65 mg/L, 2.2 mg/L, 2.75 mg/L, 3.3 mg/L, 5.5 mg/L). Increase of testosterone levels was observed at low concentrations of nonylphenol while reduction was detected at higher concentrations.

Animals↗

PubMed2006

Two observed regions in B lymphocyte stimulator important for its biological activity.

B lymphocyte stimulator (BLyS), a member of the tumor necrosis factor superfamily of ligands, is a crucial survival factor for B cells. We successfully constructed seven mutants of the functional soluble fragment of human BLyS (named cBLyS, amino acid 134-285), including three deletion mutants and four site-directed mutants. All the mutant proteins were expressed in Escherichia coli and purified by Ni-NTA affinity chromatography. The biological activities of these mutants were assessed by the ligand-receptor binding assay, B cell proliferation assay and immune effect response in vivo. Our results indicated that four residues, H218, F220, T228 and L229, are indispensable for the biological activity of cBLyS, whereas two regions, amino acid 134-148 and amino acid 271-285, are related to the biological activity of BLyS. The protein of deletion of amino acid 134-148 leads to a complete defection in raising the antigen-specific IgM titer. The deletion of amino acid 271-285 reduces the effectiveness compared with the native cBLyS. This indicates that the region of amino acid 134-148 is indispensable for cBLyS to function normally.

Amino Acid Sequence↗

PubMed2006

[Isolation of a novel peptide that binds to BLyS from a 12-mer phage display peptide library].

A 12-mer phage display peptide library was screened for specific binders against B lymphocyte stimulator (BLyS). After 3 rounds of panning, positive phage clones were enriched. ELISAs were used to identify positive phages, and the DNA encoding the positive peptide (RHKIQLRQNIIT) was cloned and expressed as a GST fusion protein in E. coli. After purification, the identity of the fusion protein was confirmed through its specific binding to BLyS by ELISA. We have obtained a peptide that can bind to BLyS and probably act as an antagonistic peptide against the natural BLyS receptor.

B-Cell Activating Factor↗

PubMed2006

[Effect of nonylphenol on testosterone secretion of rat leydig cells in vitro].

OBJECTIVE: To investigate the effect of nonylphenol (NP) on testosterone secretion of Leydig cells. METHODS: A primary culture system of Leydig cells was set up, followed by identification of Leydig cells with 3beta-HSD staining. After treatment with different concentrations of NP, testosterone secretion was detected and morphological examination was performed. RESULTS: Following treatment with NP, morphologic changes of Leydig cells were detected, with decreased cell density at high doses of NP. Testosterone increased at lower concentrations of NP while decreased at high concentrations of NP. CONCLUSION: Lower doses of NP can stimulate the secretion of testosterone, but increased exposure to NP will inhibit testosterone secretion of Leydig cells. Besides, high concentrations of NP can cause death of Leydig cells in vitro.

Animals↗

PubMed2005

Deleterious genomic mutation rate for viability in Drosophila melanogaster using concomitant sibling controls.

New deleterious mutations may reduce health and fitness and are involved in the evolution and maintenance of numerous biological processes. Hence, it is important to estimate the deleterious genomic mutation rate (U) in representative higher organisms. However, these estimated rates vary widely, mainly because of inadequate experimental controls. Here we describe an experimental design (the Binscy assay) with concomitant sibling controls and estimate U for viability in Drosophila melanogaster to be 0.31. This estimate, like most published studies, focuses on viability mutations and the overall deleterious genomic mutation rate would therefore be higher.

Animals↗

PubMed2005

Establishment of a yeast system that stably expresses human cytochrome P450 reductase: application for the study of drug metabolism of cytochrome P450s in vitro.

Cytochrome P450s (CYPs) hold a balance in studying pharmacokinetics, toxico-kinetics, drug metabolism, and drug-drug interactions, which require association with cytochrome P450 reductase (CPR) to achieve optimal activity. A novel system of Saccharomyces cerevisiae useful for expression studies of mammalian microsomal CYPs was established. Human CPR (hCPR) was co-expressed with human CYP3A4 (hCYP3A4) in this system, and two expression plasmids pTpLC and pYeplac195-3A4 containing the cDNA of hCPR and hCYP3A4 were constructed, respectively. The two plasmids were applied first and controlled by phosphoglycerate kinase (PGK) promoter. S. cerevisiae BWG1-7alpha transformed with the expression plasmids produced the respective proteins in the expected molecular sizes reactive with both anti-hCYP3A4 immunoglobulin (Ig) and anti-hCPR Ig. The activity of hCPR in yeast BWG-CPR was 443.2 nmol reduced cytochrome c/min/mg, which was about three times the CPR activity of the microsome prepared from the parental yeast. The protein amount of hCYP3A4 in BWG-CPR/3A4 was 35.53 pmol/mg, and the 6beta-hydroxylation testosterone formation activity of hCYP3A4 expressed was 7.5 nmol/min/nmol CYP, 30 times higher than the activity of hCYP3A4 expressed in the parental yeast, and almost two times the activity of hCYP3A4 from homologous human liver microsome. Meanwhile, BWG-CPR/3A4 retained 100 generations under nonselective culture conditions, indicating this yeast was a mitotically stable transformant. BWG-CPR was further tested daily by the PCR amplification of hCPR of yeast genome, Western blot analysis, and the activity assay of hCPR of yeast microsome. This special expression host for CYPs was validated to be stable and efficient for the expression of CYPs, applying as an effective selection model for the drug metabolism in vitro.

Androgens↗

PubMed2005

The influence of premeiotic clusters of mutation on indirect estimations of mutation rate.

Based on the hypothesis that rare alleles are in mutation and random loss equilibrium, mutation rate can be indirectly estimated by measuring the number of rare variants and the average existing time of a mutant allele. This method can be applied to estimate the mutation rate in humans. However, this estimation of mutation rate is affected by the presence of premeiotic clusters of mutation. Mutation clusters change both the number of initial mutants and the average existing time of a mutant allele. As a result, the formula indirectly estimating mutation rate should be modified. The influence of premeiotic clusters is more obvious when the population size is small or the average cluster size is big. For example, if the population size is 3,000 and average cluster size is two, instead of one, the mutation rate is increased by about 9.4%.

Alleles↗

PubMed2005

Proteomic analysis of global alteration of protein expression in squamous cell carcinoma of the esophagus.

Squamous cell carcinoma of the esophagus (ESCC), a major subtype of esophageal carcinoma, is one of the aggressive cancers with worst prognosis in the world. The dismal outcome of ESCC is attributed to multiple reasons including its aggressive nature, largely unknown molecular mechanism of its progression, and the lack of biomarkers for early detection and effective prediction of its clinical behavior. To identify proteins with prognostic and/or predictive value, we applied a proteomics strategy to quantify proteins differentially expressed in ESCC using matched samples of carcinoma and adjacent normal epithelial cells. The analysis led to identification of 28 proteins aberrantly expressed in cancer cells with changes of at least three-fold in ESCC relative to normal squamous epithelial cells. These changes represent functional alterations of essential proteins for normal cellular physiology, accounting for many cellular changes involved in development of ESCC, including cell transformation, loss of differentiation, tumor growth, apoptosis, tumor invasion, and cell metabolism. The differentially expressed proteins shed new insights on the mechanism of tumorigenesis and provide candidate biomarkers for early detection of ESCC.

Amino Acid Sequence↗

PubMed2005

Construction and expression of a new fusion protein, thymosin alpha1-cBLyS, E. coli.

A fusion thymosin alpha1-soluble B lymphocyte stimulator (TM alpha1-cBLyS) gene was generated to engineer a bifunctional lymphokine, which was then over-produced in Escherichia coli. The molecular weight of the expressed fusion protein was approximately 28 kDa. After being purified by Ni-NTA affinity column, the fusion protein had full activity of BLyS with a slightly higher immunological action than synthetic TMalpha1. Because TM alpha1 regulates the cellular immune response and cBLyS amplifies the humoral response, this bifunctional lymphokine could be useful in the treatment of various immunodeficiency syndromes and serve as an immunomodulator to enhance the host's response to vaccination.

Animals↗

PubMed2005

Generation and characterization of C305, a murine neutralizing scFv antibody that can inhibit BLyS binding to its receptor BCMA.

B-lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor (TNF) family and a key regulator of B cell response. Neutralizing single-chain fragment variable (scFv) antibody against BLyS binding to its receptor BCMA has the potential to play a prominent role in autoimmune disease therapy. A phage display scFv library constructed on pIII protein of M13 filamentous phage was screened using BLyS. After five rounds of panning, their binding activity was characterized by phage-ELISA. Nucleotide sequencing revealed that at least two different scFv gene fragments (C305 and D416) were obtained. The two different scFv gene fragments were expressed to obtain the soluble scFv antibodies, then the soluble scFv antibodies were characterized by means of competitive ELISA and in vitro neutralization assay. The results indicated that C305 is the neutralizing scFv antibody that can inhibit BLyS binding to its receptor BCMA.

Amino Acid Sequence↗