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Biomedical subjects

Y Fang

Publications and source records attributed to Y Fang.

At least 19 recordsLinked to original sources
PubMed2001

Activated Notch2 signaling inhibits differentiation of cerebellar granule neuron precursors by maintaining proliferation.

In the developing cerebellar cortex, granule neuron precursors (GNPs) proliferate and commence differentiation in a superficial zone, the external granule layer (EGL). The molecular basis of the transition from proliferating precursors to immature differentiating neurons remains unknown. Notch signaling is an evolutionarily conserved pathway regulating the differentiation of precursor cells of many lineages. Notch2 is specifically expressed in proliferating GNPs in the EGL. Treatment of GNPs with soluble Notch ligand Jagged1, or overexpression of activated Notch2 or its downstream target HES1, maintains precursor proliferation. The addition of GNP mitogens Jagged1 or Sonic Hedgehog (Shh) upregulates the expression of HES1, suggesting a role for HES1 in maintaining precursor proliferation.

Animals↗
PubMed2001

A 10-kDa structural protein of porcine reproductive and respiratory syndrome virus encoded by ORF2b.

The major structural proteins of porcine reproductive and respiratory syndrome virus (PRRSV) are derived from ORFs 5, 6, and 7. Western blots of sucrose gradient-purified virions and PRRSV-infected MARC-145 cells, probed with immune pig serum, showed the presence of an additional 10-kDa protein. Nucleotide sequence analysis of North American PRRSV isolate SDSU-23983 revealed a small ORF within ORF2, named ORF2b, which, when translated, produced a 73-amino-acid nonglycosylated protein. Recombinant 2b protein expressed by a baculovirus clone, AcVR2, comigrated with the 10-kDa virus-associated protein. The loss of 10-kDa protein immunoreactivity after absorption of immune sera with lysates from AcVR2-infected insect cells demonstrated that the 2b and 10-kDa proteins are immunologically similar. Immunoblots were also used for the detection of anti-2b activity in serum samples from experimentally infected adult pigs. Antibodies against PRRSV were apparent by 14 days postinfection, followed by anti-2b activity and serum neutralizing activity. The putative ORF2b start codon is only 6 nucleotides downstream of the adenine of the ORF2a start codon. The expression of ORF2a and 2b as enhanced green fluorescent fusion proteins showed that both proteins were translated; however, the ORF2b was preferentially expressed. These results suggest that the 2b protein is virion associated and the principal product of ORF2.

Amino Acid Sequence↗
PubMed2001

ATR/ATM-mediated phosphorylation of human Rad17 is required for genotoxic stress responses.

Genotoxic stress triggers the activation of checkpoints that delay cell-cycle progression to allow for DNA repair. Studies in fission yeast implicate members of the Rad family of checkpoint proteins, which includes Rad17, Rad1, Rad9 and Hus1, as key early-response elements during the activation of both the DNA damage and replication checkpoints. Here we demonstrate a direct regulatory linkage between the human Rad17 homologue (hRad17) and the checkpoint kinases, ATM and ATR. Treatment of human cells with genotoxic agents induced ATM/ATR-dependent phosphorylation of hRad17 at Ser 635 and Ser 645. Overexpression of a hRad17 mutant (hRad17AA) bearing Ala substitutions at both phosphorylation sites abrogated the DNA-damage-induced G2 checkpoint, and sensitized human fibroblasts to genotoxic stress. In contrast to wild-type hRad17, the hRad17AA mutant showed no ionizing-radiation-inducible association with hRad1, a component of the hRad1-hRad9-hHus1 checkpoint complex. These findings demonstrate that ATR/ATM-dependent phosphorylation of hRad17 is a critical early event during checkpoint signalling in DNA-damaged cells.

Animals↗
PubMed2001

Isolation of a novel candidate oncogene within a frequently amplified region at 3q26 in ovarian cancer.

Amplification of 3q25-q26 was one of the most frequent chromosomal alterations in human ovarian carcinoma. A chromosome microdissection-hybrid selection method was applied to isolate transcribed sequences from a primary ovarian cancer containing high-copy-number amplification of 3q26 using 3q26 band-specific DNAs generated by chromosome microdissection. Using this method, we have isolated a novel candidate oncogene eIF-5A2 (eukaryotic initiation factor 5A2). eIF-5A2 shares 82% identity of amino acid sequence with eIF-5A including the minimum domain needed for eIF-5A maturation by hypusine modification at lysine-50 residue. Amplification and overexpression of eIF-5A2 was frequently detected in primary ovarian cancers and ovarian cancer cell lines. The proliferation-related function of eIF-5A supports that eIF-5A2 is a candidate oncogene related to the development of ovarian cancer.

Amino Acid Sequence↗
PubMed2001

Two-dimensional direct-reading fluorescence spectrograph for DNA sequencing by capillary array electrophoresis.

We report a compact, two-dimensional direct-reading fluorescence spectrograph and demonstrate its application to DNA sequencing by capillary array electrophoresis. The detection cuvette is based on sheath flow, wherein the capillaries terminate in a two-dimensional array in a fluid-filled chamber that is pressurized with buffer. A thin metal plate is located downstream from the capillaries. This barrier plate has an array of holes that precisely matches the location of the capillaries. Buffer flows through the holes, drawing analyte from the capillaries in a well-defined array of thin filaments. Fluorescence is excited in the upper chamber with an elliptically shaped laser beam. The bottom chamber is sealed with a glass window and drained from the side. Fluorescence is detected by imaging the illuminated sample streams through the holes in the barrier plate. A prism is used to disperse fluorescence from each sample across a CCD camera so that the emission spectrum is monitored simultaneously from each capillary. The instrument is demonstrated in a 32-capillary configuration but can be scaled to several thousand capillaries.

DNA↗
PubMed2001

Characterization of single-stranded DNA on chitosan-modified electrode and its application to the sequence-specific DNA detection.

Single-strand DNA could bind with chitosan on a platinum electrode via forming a tight DNA-chitosan complex. The salt concentration of the ssDNA solution had an obvious effect on the surface coverage, the immobilization was remarkably reduced at high salt concentration. The sample ssDNA immobilized on the chitosan-modified electrode can hybridize efficiently with the complementary sequences and be successfully used for the sequence-specific DNA detection. The same results could be obtained using a gold or graphite electrode modified with chitosan. The stability of this electrode has been also discussed.

Biopolymers↗
PubMed2001

Analysis of genetic alterations in primary nasopharyngeal carcinoma by comparative genomic hybridization.

To identify genetic alterations associated with the development and progression of human nasopharyngeal carcinoma (NPC), 57 tumors were analyzed by comparative genomic hybridization (CGH). In 47 cases, chromosomal imbalances were found. Several recurrent chromosomal abnormalities were identified in the present study. The most frequently detected chromosomal gains involved chromosome arms 12q (24 cases, 51%), 4q (17 cases, 36%), 3q (16 cases, 34%), 1q (15 cases, 32%), and 18q (15 cases, 32%). Common regions of gain involved 12q13--q15, 4q12--q21, and 3q21--q26. High-copy-number increases of chromosomal materials were detected in four chromosomal regions, 3q21--q26.2, 4p12--q21, 8p, and 12q14--q15. The most frequently detected loss of chromosomal materials involved chromosome arms 16q (26 cases, 55%), 14q (21 cases, 45%), 1p (20 cases, 43%), 3p (20 cases, 43%), 16p (19 cases, 40%), 11q (17 cases, 36%), and 19p (16 cases, 34%). The most common regions of loss involved 14q24--qter, 1pter--p36.1, 3p22--p21.3, 11q21--qter, and the distal region of 19p. Genomic alterations detected by CGH were compared and found to be largely consistent with those identified in banding analysis and loss of heterozygosity studies. However, several previously unrecognized recurrent alterations were also identified in the present study, including gain of 4q and 18q, and loss of 16q, 14q, and 19p. In addition, gain of 1q, 8q, 18q, and loss of 9q showed a statistically significant association with advanced clinical stages (P < 0.05). Identification of recurrent sites of chromosomal gain and loss identify regions of the genome that may contain oncogenes or tumor suppressor genes, respectively, which may be involved in the tumorigenesis of NPC. Published 2000 Wiley-Liss, Inc.

Adult↗
PubMed2001

Studies on the effect of surface properties on the biocompatibility of polyurethane membranes.

To study the effect of surface properties on the biocompatibility of biomaterials based on the same material, polyurethane membranes with different surface properties were prepared. Myoblast culture and interleukin-1 (IL-1) generation in an air pouch model and in vitro monocyte culture were used to examine biocompatibility of different polyurethane membranes. Polyurethane membranes were found to exhibit significant differences depending on their surface properties prepared by different fabrication processes. When myoblasts were cultured on polyurethane surfaces, the smooth and hydrophobic membrane (F1), prepared by the solvent evaporation process, showed the greatest inhibition of myoblast adhesion compared with other porous and hydrophilic membranes (F2, F3 and F4), prepared by immersing the polymer solution into a precipitation bath. In contrast, IL-1 generation by monocytes/macrophages on the membrane F1 was more severe than those on the porous and hydrophilic membranes. Based on our results, the interaction of biomaterials with various cells is discussed.

Adult↗
PubMed2001

Kinetics of tumorigenic vascular endothelial growth factor signalling and its significance in human hepatocellular carcinoma cells.

Ras-VEGF-concerned angiogenesis is correlated with oncogene maintenance, tumorigenesis, metastasis and resistance to anti-cancer therapies; however, this association is not clearly elucidated by serum VEGF, due to VEGF signalling in blood cells themselves. The present study aimed to elucidate tumorigenic VEGF signalling in eight human HCC cell types and reveal the kinetics of tumorigenic VEGF signalling in three time intervals, thereby discovering the relationships of VEGF-concerned angiogenesis signalling with the extent of the human HCC cell growth, metastasis and resistance to anti-cancer drugs, by using the poorly metastatic SMMC7721, 7402/D+ (doxorubicin-resistance) and 7402/D- (doxorubicin-withdrawal), the highly metastatic MHCC1 non-transfected human HCC cell lines, and the highly metastatic A3-1, F8, F11 and E3 human HCC cell lines transfected with expressing green fluorescence protein into the phenotype of MHCC1 cells, and quantitative 'sandwich' ELISA analyses. The unique results indicated attributes and objective laws as follows. Human HCC cell growth requires time-dependent tumorigenic VEGF signalling; levels of VEGF signalling are positively correlated with each cell phenotype itself; and levels of VEGF signalling are inversely correlated with the possibility of metastasis and drug resistance. The contrast data first reveal important clues for exploring dual metastatic mechanisms via tumor cell-generated non-endothelium vasculogenesis and VEGF-endothelium-attached angiogenesis that may be essential for developing novel strategies aimed at VEGF-concerned signal networks in ischemic/metastatic diseases and transgenic models.

Carcinoma, Hepatocellular↗
PubMed2001

Electrochemical detection of sequence-specific DNA using a DNA probe labeled with aminoferrocene and chitosan modified electrode immobilized with ssDNA.

The electrochemical detection of sequence-specific DNA using a DNA probe labeled with aminoferrocene (AFC) is reported. Sample ssDNA was immobilized on a chitosan modified glassy carbon electrode. A sequence-known DNA with 256 bp [obtained by polymerase chain reaction (PCR)] was successfully labeled with the electro-active reagent AFC by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide for the first time. This DNA probe labeled with AFC was applied to hybridize with a sequence-unknown DNA sample. Only the complementary sequence (cDNA) could form a double-stranded DNA (dsDNA) with the DNA probe labeled with AFC. The anodic peak currents (ipa) of the AFC bound to the dsDNA by differential pulse voltammetry were used for the determination of cDNA. The ipa of AFC was linearly related to the concentration of cDNA sequence between 1.0 x 10(-8) and 6.0 x 10(-6) mol L-1. The detection limit was 2.0 x 10(-9) mol L-1 using 3 sigma (where sigma is the standard deviation of blank solution, n = 11). The probe showed high sensitivity and selectivity.

Animals↗
PubMed2001

Study of the separation and determination of monosaccharides in soluble coffee by capillary zone electrophoresis with electrochemical detection.

A simple, fast and reliable method, based on capillary electrophoresis with electrochemical detection, for the separation and determination of six monosaccharides, namely glucose, galactose, arabinose, fructose, xylose and ribose, in soluble coffees was developed. A copper disk electrode was used as the working electrode. The optimum conditions for separation and detection were 50 mmol L-1 sodium hydroxide buffer (pH 12.7), separation voltage 5 kV and detection potential 0.65 V (vs. Ag/AgCl). The linear ranges were from 5.0 x 10(-3) to 0.5 mmol L-1 for all six sugars. All regression coefficients were > 0.99. The detection limits for all the sugars were 1.0 x 10(-3) mmol L-1. The RSD of the peak current was < 4.2% (n = 5). The proposed method was applied directly to the separation and determination of the six sugars without prior derivatization, and the assay results were satisfactory.

Journal Article↗
PubMed2001

Determination of nucleic acids based on shifting the association equilibrium between tetrasulfonated aluminium phthalocyanine and acridine orange.

Based on the ability of nucleic acids to shift the association equilibrium of the ion-association complex of Acridine Orange and tetrasulfonated aluminium phthalocyanine, thus leading to an increase in the phthalocyanine fluorescence, a method is suggested for the fluorimetric determination of nucleic acids. Investigations were carried out on the spectral characteristics, order of addition of reagents, selection of the buffer system, effect of pH, influence of reaction time, effect of salt, the usage of reagents, interference of foreign substances and the effect of different acridine derivatives. Under the optimum conditions, the calibration graphs for the determination of calf thymus DNA (CT DNA), salmon DNA (SM DNA) and yeast RNA were linear over the ranges 0.04-1.2, 0.04-1.2 and 0.1-1.2 micrograms cm-1, respectively. The detection limits for CT DNA, SM DNA and RNA were 17, 24 and 98 micrograms cm-3, respectively. The relative standard deviation (n = 6) was within 4.6% for the detection of samples. The method was applied to the determination of Staphylococcus aureus DNA and the result was in agreement with that achieved by a UV method.

Acridine Orange↗
PubMed2001

Focal adhesion kinase affects the sensitivity of human hepatocellular carcinoma cell line SMMC-7721 to tumor necrosis factor-alpha/cycloheximide-induced apoptosis by regulating protein kinase B levels.

Most cell lines are resistant to tumor necrosis factor-alpha (TNF-alpha) cytotoxicity and require cotreatment of TNF-alpha with cycloheximide (Chx) to undergo apoptosis. Recently, the serine/threonine protein kinase, protein kinase B has been demonstrated to protect cells from apoptosis induced by TNF-alpha. In this study, we have shown that the human hepatocellular carcinoma cell line, SMMC-7721, was insensitive to TNF-alpha cytotoxicity and underwent apoptosis quickly in the presence of TNF-alpha and Chx. PKB levels decreased during TNF-alpha/Chx-induced apoptosis. No significant change in PKB levels was found in the presence of TNF-alpha or Chx alone. It seemed that the level of PKB closely correlated with apoptosis. The protein level of focal adhesion kinase (FAK) was reduced by 66% by transfecting FAK antisense cDNA recombinant vector into SMMC-7721 cells. We determined the apoptosis-induced effect of TNF-alpha/Chx on the FAK antisense cDNA transfectant cells. The results indicated that the percentage of apoptotic cells was enhanced at lower doses of TNF-alpha (10, 20 or 50 U.mL(-1)) and decreased at a higher dose of TNF-alpha (1000 U.mL(-1)) in the transfected cells as compared to the control. Correspondingly, in the FAK antisense cDNA transfectant cells treated with lower doses of TNF-alpha in presence of 10 microg.mL(-1) Chx, the PKB level was lower, but in the FAK antisense cDNA transfectants treated with higher doses of TNF-alpha in presence of 10 microg.mL(-1) Chx, the PKB level was higher. In response to TNF-alpha alone, FAK antisense cDNA transfectants showed a decrease in the level of PKB. However, in the case of TNF-alpha cotreated with wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PtdIns3K), the FAK antisense cDNA transfectants produced significantly less amounts of PKB than the control. It seemed that FAK could stimulate PKB levels through a pathway not involving PtdIns3K. These results suggest that FAK can affect the sensitivity of SMMC-7721 cells to TNF-alpha/Chx-induced apoptosis in a biphasic manner by regulating PKB levels.

Androstadienes↗
PubMed2001

Interaction between the vitamin D receptor gene and collagen type Ialpha1 gene in susceptibility for fracture.

Osteoporosis is a common disease with a strong genetic component. Polymorphisms in the vitamin D receptor (VDR) gene have been implicated in osteoporosis but explain only a small part of the genetic effect on bone mineral density (BMD) while their effect on fractures is still uncertain. Recently, a G to T polymorphism in an Sp1 site in the collagen type Ialpha1 (COLIA1) gene was found to be associated with reduced BMD and with increased fracture risk. To analyze the combined influence of polymorphisms in the VDR gene and the COLIA1 gene in determining the susceptibility to osteoporotic fracture, we studied 1004 postmenopausal women. The "baT" VDR haplotype, constructed from three adjacent restriction fragment length polymorphisms, was found to be overrepresented among fracture cases (p = 0.009). This corresponded to an odds ratio (OR) of 1.8 (95% CI, 1.0-3.3) for heterozygous carriers and 2.6 (95% CI, 1.4-5.0) for homozygous carriers of the risk haplotype. The effect was similar for vertebral and nonvertebral fractures and, most importantly, independent of BMD. We observed significant interaction (p = 0.03) between VDR and COLIA1 genotype effects. Fracture risk was not VDR genotype-dependent in the COLIA1 "reference" group (genotype GG) while in the COLIA1 "risk" group (genotypes GT and TT) the risk of fracture was 2.1 (95% CI, 1.0-4.4) for heterozygous and 4.4 (95% CI, 2.0-9.4) for homozygous carriers of the VDR risk haplotype. We conclude that both the VDR and the COLIA1 polymorphisms are genetic markers for osteoporotic fracture in women, independent of BMD. Our data indicate that interlocus interaction is likely to be an important component of osteoporotic fracture risk.

Cohort Studies↗