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In JoVE (1)
Other Publications (10)
- Experimental & Molecular Medicine
- Cellular & Molecular Biology Letters
- Journal of Bacteriology
- BMC Molecular Biology
- Journal of Bacteriology
- Molecular Biology Reports
- Hepatology (Baltimore, Md.)
- Journal of Cardiovascular Pharmacology
- Biochemical and Biophysical Research Communications
- Journal of Bacteriology
Articles by Chunxiao Yu in JoVE
JoVE Bioengineering
Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)
1Department of Electrical and Computer Engineering, Boston University, 2Department of Biomedical Engineering, Boston University, 3Center for Advanced Genomics Technology, Boston University, 4Department of Medicine, Section of Infectious Diseases, Boston University School of Medicine, 5Department of Microbiology, Boston University School of Medicine, 6CNR (National Research Council), Istituto di Chimica del Riconoscimento Molecolare
Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.
Other articles by Chunxiao Yu on PubMed
P53 Overexpression Represses Androgen-mediated Induction of NKX3.1 in a Prostate Cancer Cell Line
Prostate cancer is a disease involving complicated multiple-gene alterations. Both NKX3.1 and p53 are related to prostate cancer and play crucial roles in prostate cancer progression. However, little is known about the relationships and interactions between p53 and NKX3.1 in prostate cancer. We found that NKX3.1 expression is down-regulated by over-expression of wild type (wt) p53 in prostate cancer LNCaP cells. NKX3.1 is down-regulated at both the mRNA and protein levels by p53 over- expression due to either transient transfection of exogenous p53 or induction of endogenous p53. p53 over-expression represses androgen-induced transactivation of NKX3.1 by inhibiting the promoter of the androgen acceptor (AR) gene and by blocking AR-DNA binding activity. In addition, transfection with the p21 expression vector (pPSA-p21) showed that p21 does not reduce NKX3.1 expression, indicating that NKX3.1 expression is not the result of nonspecific effects of cell growth arrest. Our results provide biochemical and cellular biologic evidence that NKX3.1 is down-regulated by p53 over-expression in prostate cancer cells.
Molecular Cloning and Analysis of the Human PCAN1 (GDEP) Promoter
Human PCAN1 (prostate cancer gene 1) is a prostate-specific gene that is highly expressed in prostate epithelial tissue, and frequently mutated in prostate tumors. To better understand the regulation of the PCAN1 gene, a 2.6-kb fragment of its 5' flanking region was obtained by PCR. Its promoter activity was examined via the dual-luciferase reporter assay after it had been cloned into a pGL(3)-basic vector generating pGL(3)-p2.6 kb and transfected into LNCaP cells. pGL(3)-basic and pGL(3)-control were respectively used as the negative and positive controls. Sequence analysis with the MatInspector database showed that some possible binding sites for the transcriptional factors, NKX3.1, P53, SP1, cEBP and the PPAR/RXR heterodimers may locate on a 2.6-kb region upstream of the PCAN1 gene. To examine the relevant regulation of PCAN1, pGL(3)-p2.6 kb was transfected into the prostate cancer cell line LNCaP, which was treated with R1881 (10(-7) approximately 10(-9) mol/l), 17beta-estradiol (17beta-E(2), 10(-7) approximately 10(-9) mol/l), all-trans-retinoic acid (all-trans-RA, 10(-5) approximately 10(-7) mol/l) or 9-cis-retinoic acid (9-cis-RA, 10(-5) approximately 10(-7) mol/l), and eukaryotic expression plasmids of NKX3.1, p53, Sp1, Pten, PPARgamma or cEBPalpha were cotransfected with pGL(3)-p2.6 kb into LNCaP cells. pRL-TK, a Renilla luciferase reporter vector, was cotransfected into all the transfection lines as an internal control. The activities of pGL(3)-p2.6 kb (PCAN1 promoter) were analyzed via the dual-luciferase reporter assay 48 h after transfection. The results showed that 9-cis-RA enhanced the PCAN1 promoter activity in a dose-dependent manner, while R1881, 17beta-E(2) and all-trans-RA had no significant effect on PCAN1 promoter activities. Cotransfection with pGL(3)-p2.6kb and the expression plasmids of NKX3.1, p53, Sp1 or Pten respectively resulted in 1.66-, 2.48-, 2.00-and 1.72-fold 2.6 kb PCAN1 promoter activity increases relative to the controls, which were cotransfected with pcDNA3.1(+), while cotransfection of PPARgamma and cEBPalpha yielded no significant effect on PCAN1 promoter activities. These results could be applied for further study of the function and transcription regulation of the PCAN1 gene in prostate development and carcinogenesis.
Functional Mapping of an Oligomeric Autotransporter Adhesin of Aggregatibacter Actinomycetemcomitans
Extracellular matrix protein adhesin A (EmaA) is a 202-kDa nonfimbrial adhesin, which mediates the adhesion of the oral pathogen Aggregatibacter actinomycetemcomitans to collagen. EmaA oligomers form surface antenna-like protrusions consisting of a long helical rod with an ellipsoidal ending. The functional analysis of in-frame emaA deletion mutants has located the collagen binding activity to the amino terminus of the protein corresponding to amino acids 70 to 386. The level of collagen binding of this deletion mutant was comparable to the emaA mutant strain. Transmission electron microscopy studies indicate that the first 330 amino acids of the mature protein form the ellipsoidal ending of the EmaA protrusions, where the activity resides. Amino acid substitution analysis within this sequence has identified a critical amino acid, which is essential for the formation of the ellipsoidal ending and for collagen binding activity.
Characterization of Two Functional NKX3.1 Binding Sites Upstream of the PCAN1 Gene That Are Involved in the Positive Regulation of PCAN1 Gene Transcription
NKX3.1 and PCAN1 are both prostate-specific genes related to prostate development and prostate cancer. So far, little is known about the regulatory mechanisms of the expression of these two genes. In the present study, we found that NKX3.1 upregulated PCAN1 gene transcription in LNCaP prostate cancer cells. To understand the regulatory mechanisms, our work focused on identifying the functional NKX3.1 binding sites upstream of the PCAN1 gene, which might be involved in the positive regulation of PCAN1 expression by NKX3.1.
Investigation of the Three-dimensional Architecture of the Collagen Adhesin EmaA of Aggregatibacter Actinomycetemcomitans by Electron Tomography
The periodontal pathogen Aggregatibacter actinomycetemcomitans displays on the bacterial surface a nonfimbrial adhesin, EmaA, which is required for collagen binding. In this study, electron tomography was used to characterize the three-dimensional (3D) architecture of this adhesin. The antenna-like surface appendages, corresponding to EmaA, were found to be composed of an ellipsoidal domain capping a rod-like domain that adopts either a straight or a bent conformation at various positions along the length. The most common flexible point along the length of the EmaA appendage was localized 29.4 nm away from the distal end. One-fifth of the appendages were straight and the remaining showed angles distributed between 140 degrees and 170 degrees at this location. Deletion analysis mapped this bend to amino acids 611 to 640 of the protein sequence. The 3D structure of the collagen binding domain of EmaA was generated by alignment and averaging of 9 subvolumes of the adhesin extracted from tomograms. The structure contains three subdomains: a globular structure with a diameter of approximately 5 nm and a cylindrical domain ( approximately 4.4 nm by 5.8 nm) separated by a linker region with a diameter of approximately 3 nm, followed by a cylindrical domain ( approximately 4.6 nm by 6.6 nm). This is the first 3D structure of a trimeric autotransporter protein of A. actinomycetemcomitans.
Molecular Cloning and Preliminary Analysis of the Human Alpha-methylacyl-CoA Racemase Promoter
Alpha-methylacyl-CoA racemase (AMACR) is an enzyme involved in beta-oxidation of branched-chain fatty acids and bile acid intermediates. Recent works have revealed that AMACR is overexpressed in prostate cancer and functionally important for the growth of prostate cancer cells. Despite the recent interest in AMACR as a diagnostic marker for prostate cancer, little is known about the transcriptional regulation of AMACR in prostate cancer. To elucidate the regulation of the AMACR gene, a 2.3-kb fragment of its 5' flanking region was cloned into pGL3-Basic, then using tansfection and Dual-luciferase reporter assay, a preliminary analysis on promoter activity and function of this 2.3-kb sequence was carried out. This 2.3-kb fragment represented promoter activity that consistent with the expression level in LNCaP and PC-3 cells respectively. Transfection experiments of 5'-deletion mutants into LNCaP cells revealed a positive-regulatory region located between nucleotides -423 and -93 that may be responsible for AMACR transactivation in LNCaP cells. Cotransfection experiments revealed that promoter activity of this 2.3-kb sequence was down-regulated by C/EBPalpha, p53, NF-kappaB p50. And data from luciferase-based reporter assays suggest that the promoter function of AMACR is independent of androgen receptor-mediated signaling.
A Novel Role for Thyroid-stimulating Hormone: Up-regulation of Hepatic 3-hydroxy-3-methyl-glutaryl-coenzyme A Reductase Expression Through the Cyclic Adenosine Monophosphate/protein Kinase A/cyclic Adenosine Monophosphate-responsive Element Binding Protein Pathway
Elevated thyroid-stimulating hormone (TSH) and hypercholesterolemia commonly coexist, as typically seen in hypothyroidism, but there is no known mechanism directly linking the two. Here, we demonstrated that in liver cells, TSH promoted the expression of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR), a rate-limiting enzyme in cholesterol synthesis, by acting on the TSH receptor in hepatocyte membranes and stimulating the cyclic adenosine monophosphate / protein kinase A / cyclic adenosine monophosphate-responsive element binding protein (cAMP/PKA/CREB) signaling system. In thyroidectomized rats, the production of endogenous thyroid hormone was eliminated and endogenous TSH was suppressed through pituitary suppression with constant administration of exogenous thyroid hormone, and hepatic HMGCR expression was increased by administration of exogenous TSH. These results suggested that TSH could up-regulate hepatic HMGCR expression, which indicated a potential mechanism for hypercholesterolemia involving direct action of TSH on the liver.
Hydrogen Sulfide Protects Against High-glucose-induced Apoptosis in Endothelial Cells
Hydrogen sulfide (H2S) is the third endogenous gaseous mediator identified after nitric oxide and carbon monoxide. It has been demonstrated that H2S has protective effects on myocardial ischemia/reperfusion-induced cell apoptosis. To date, little is known about the role of H2S in the pathophysiology of diabetic vascular complications. In this study, we investigated the effects of sodium hydrosulfide on high-glucose-induced apoptosis of primary human umbilical vein endothelium cells. Exposure to high glucose (25 mmole/L) for 48 hours resulted in the induction of apoptosis by 41.6% ± 1.01%, which was attenuated by pretreatment with sodium hydrosulfide (50 μmole/L) for 30 minutes. Further investigation of the apoptotic mechanisms in the cells demonstrated that high glucose upregulated the ratio of Bax/Bcl-2 and activated caspase-3 and also increased the levels of reactive oxygen species and malondialdehyde while reducing superoxide dismutase activity. All the above responses could be prevented by pretreatment with 50 μmole/L of sodium hydrosulfide. These results indicated that the protective effects of H2S on endothelial cells in the condition of high glucose might involve an antioxidative stress mechanism.
Diosgenin Induces Apoptosis in IGF-1-stimulated Human Thyrocytes Through Two Caspase-dependent Pathways
Insulin-like growth factor-1 (IGF-1) is a growth factor of the thyroid that has been shown in our previous study to possess proliferative and antiapoptotic effects in FRTL-5 cell lines through the upregulation of cyclin D and Fas-associated death domain-like interleukin-1-converting enzyme (FLICE)-inhibitory protein (FLIP). Diosgenin, a natural steroid sapogenin from plants, has been shown to induce apoptosis in many cell lines, with the exception of thyroid cells. In this report, we investigated the apoptotic effect and mechanism of diosgenin in IGF-1-stimulated primary human thyrocytes. Primary human thyrocytes were preincubated with or without IGF-1 for 24h and subsequently exposed to varying concentrations of diosgenin for different times. We found that diosgenin induced apoptosis in human thyrocytes pretreated with IGF-1 in a dose-dependent manner through the activation of caspase cascades. Moreover, diosgenin inhibited FLIP and activated caspase-8 in the FAS-related apoptotic pathway. Diosgenin increased the production of ROS, regulated the balance of Bax and Bcl-2 and cleaved caspase-9 in the mitochondrial apoptotic pathway. These results indicate that diosgenin induces apoptosis in IGF-1-stimulated primary human thyrocytes through two caspase-dependent pathways.
Fur-Mediated Activation of Gene Transcription in the Human Pathogen Neisseria Gonorrhoeae
It is well established that the Ferric uptake regulatory protein (Fur) functions as a transcriptional repressor in diverse microorganisms. Recent studies demonstrated that Fur also functions as a transcriptional activator. In this study we defined Fur-mediated activation of gene transcription in the sexually transmitted disease pathogen Neisseria gonorrhoeae. Analysis of 37 genes previously determined to be iron-induced and which contained putative Fur boxes revealed that only 30 of these genes exhibited reduced transcription in a gonococcal fur mutant strain. Fur-mediated activation was established by examining binding of Fur to the putative promoter regions of 16 Fur-activated genes with variable binding affinities observed. Only ∼50% of the newly identified Fur-regulated genes bound Fur in vitro, suggesting that additional regulatory circuits exist which may function through a Fur-mediated indirect mechanism. The gonococcal Fur-activated genes displayed variable transcription patterns in a fur mutant strain which correlated with the position of the Fur box in each (promoter) region. These results suggest that Fur-mediated direct transcriptional activation is fulfilled by multiple mechanisms involving either competing with a repressor or recruiting RNA polymerase. Collectively, our studies have established that gonococcal Fur functions as an activator of gene transcription through both direct and indirect mechanisms.
