Biomedical subjects

Ying Chen

Publications and source records attributed to Ying Chen.

At least 127 records · Page 7Linked to original sources

PubMed2005

A novel cryoprotection scheme for enhancing the diffraction of crystals of recombinant cytochrome ba3 oxidase from Thermus thermophilus.

Cytochrome ba(3) oxidase is an integral membrane protein identified in the thermophilic bacterium Thermus thermophilus. The enzyme has now been expressed recombinantly and purified with a histidine tag. As such, it crystallizes under similar conditions and in the same space group (P4(3)2(1)2) as the native protein. A novel cryoprotection scheme is described here to obtain high-resolution diffraction from these crystals, which involves soaking in a mixture of glycerol and ethylene glycol under a layer of oil. The unit-cell parameters for these crystals are larger than the native protein, apparently deriving from increased ordering of the N-terminus and an internal loop (residues 495-500) in subunit I. Hence, compared with native cytochrome ba(3) oxidase, the recombinant His-tagged protein is accommodated in an expanded but equally well ordered lattice via an alternate set of specific intermolecular contacts. The structure was refined against data to 2.3 angstroms resolution to an R factor of 21.7% and an R(free) of 23.7%.

Crystallography, X-Ray↗

PubMed2005

DNA methyltransferase 1 regulates reelin mRNA expression in mouse primary cortical cultures.

The polygenic nature of complex psychiatric disorders suggests a common pathway that may be involved in the down-regulation of multiple genes through an epigenetic mechanism. To investigate the role of methylation in down-regulating the expression of mRNAs that may be associated with the schizophrenia phenotype, we have adopted a cell-culture model amenable to this line of investigation. We have administered methionine (2 mM) to primary cultures of cortical neurons prepared from embryonic day 16 mice and show that this treatment down-regulated reelin and glutamic acid decarboxylase 67 (GAD67) mRNA expression but not that corresponding to neuron-specific enolase mRNA. Moreover, methionine increased methylation of the reelin promoter, suggesting a possible mechanism for the observed change. These cultures contain a mixed population of neurons and glia. Approximately 83% of the neurons are GABAergic based on GAD immunoreactivity, and these neurons coexpress high levels of reelin and DNA methyltransferase (Dnmt) 1 immunoreactivity. To examine whether Dnmt1 regulates reelin gene expression, we used an antisense approach to reduce (knock down) Dnmt1 expression. The reduced Dnmt1 mRNA and protein were accompanied by increased reelin mRNA expression. More importantly, the Dnmt1 knockdown blocked the methionine-induced reelin and GAD67 mRNA down-regulation. These data support the hypothesis that the reduced amounts of reelin and GAD67 mRNAs documented in postmortem schizophrenia brain may be the consequence of a Dnmt1-mediated hypermethylation of the corresponding promoters.

Animals↗

PubMed2005

Evolutionary genomics: codon volatility does not detect selection.

Plotkin et al. introduce a method to detect selection that is based on an index called codon volatility and that uses only the sequence of a single genome, claiming that this method is applicable to a large range of sequenced organisms. Volatility for a given codon is the ratio of non-synonymous codons to all sense codons accessible by one point mutation. The significance of each gene's volatility is assessed by comparison with a simulated distribution of 10(6) synonymous versions of each gene, with synonymous codons drawn randomly from average genome frequencies. Here we re-examine their method and data and find that codon volatility does not detect selection, and that, even if it did, the genomes of Mycobacterium tuberculosis and Plasmodium falciparum, as well as those of most sequenced organisms, do not meet the assumptions necessary for application of their method.

Animals↗

PubMed2005

Effects of extramitochondrial ADP on permeability transition of mouse liver mitochondria.

Carboxyatractylate (CAT) and atractylate inhibit the mitochondrial adenine nucleotide translocator (ANT) and stimulate the opening of permeability transition pore (PTP). Following pretreatment of mouse liver mitochondria with 5 microM CAT and 75 microM Ca2+, the activity of PTP increased, but addition of 2 mM ADP inhibited the swelling of mitochondria. Extramitochondrial Ca2+ concentration measured with Calcium-Green 5N evidenced that 2 mM ADP did not remarkably decrease the free Ca2+ but the release of Ca2+ from loaded mitochondria was stopped effectively after addition of 2 mM ADP. CAT caused a remarkable decrease of the maximum amount of calcium ions, which can be accumulated by mitochondria. Addition of 2 mM ADP after 5 microM CAT did not change the respiration, but increased the mitochondrial capacity for Ca2+ at more than five times. Bongkrekic acid (BA) had a biphasic effect on PT. In the first minutes 5 microM BA increased the stability of mitochondrial membrane followed by a pronounced opening of PTP too. BA abolished the action about of 1 mM ADP, but was not able to induce swelling of mitochondria in the presence of 2 mM ADP. We conclude that the outer side of inner mitochondrial membrane has a low affinity sensor for ADP, modifying the activity of PTP. The pathophysiological importance of this process could be an endogenous prevention of PT at conditions of energetic depression.

Adenine Nucleotide Translocator 3↗

PubMed2005

Phosphoinositide binding by the disabled-1 PTB domain is necessary for membrane localization and Reelin signal transduction.

Disabled-1 (Dab1) is an essential adaptor protein that functions in the Reelin signaling pathway and is required for the regulation of neuronal migration during embryonic development. Dab1 interacts with NPXY motifs in the cytoplasmic tails of the lipoprotein receptors ApoER2 and very low density lipoprotein receptor through an amino-terminal phosphotyrosine binding (PTB) domain. Binding of Reelin to these receptors leads to tyrosine phosphorylation of Dab1 and the initiation of a signaling cascade that results in remodeling of the cytoskeleton. Structural and biochemical studies of the Dab1 PTB domain have demonstrated that this domain binds to both the NPXY peptide motif in the lipoprotein receptor tails as well as to the head group of phosphoinositide 4,5-P2 through energetically independent mechanisms. Here we have investigated how phosphoinositide binding by the Dab1 PTB domain influences Reelin signal transduction. Our findings in cultured primary neurons that have been transduced with lentiviral constructs expressing mutant Dab1 forms reveal that phosphoinositide binding by the Dab1 PTB domain is necessary for proper membrane localization of Dab1 and for effective transduction of a Reelin signal.

Amino Acid Sequence↗

PubMed2005

Gene therapy targeting the Tie2 function ameliorates collagen-induced arthritis and protects against bone destruction.

OBJECTIVE: In a previous study, we demonstrated that Tie2 regulates angiogenesis in arthritis. The current study was performed to determine whether systemic delivery of a soluble Tie2 receptor (ExTek) using an adenoviral vector (AdExTek) as a Tie2 inhibitor affects arthritis development and progression in an animal model. METHODS: We used a collagen-induced arthritis (CIA) mouse model to study the outcome of treatment with either AdExTek or a control vector. The onset, incidence, and severity of arthritis were quantified. Immunohistologic analysis of endothelium obtained from the paws was performed. Bone destruction in paws was analyzed using phase-contrast radiography. RESULTS: The data showed that systemic delivery of ExTek before disease development significantly inhibited the onset, incidence, and severity of arthritis. When AdExTek was given after disease onset, the severity of disease in mice treated with AdExTek was significantly lower than that in the control group at 35 days postimmunization, which correlated with significantly diminished angiogenesis in mouse paws. Strikingly, AdExTek treatment protected bone from erosion in the CIA model and reduced levels of RANKL. No differences in collagen-specific antibodies were detected between these 2 groups. CONCLUSION: We demonstrated that blocking Tie2 receptor activation inhibits angiogenesis and arthritis development and protects against bone destruction in a CIA mouse model. These findings identify Tie2 as a therapeutic target for arthritis treatment and imply that interventions designed to target the Tie2 pathway could be clinically beneficial.

Adenoviridae↗

PubMed2005

On the exact null distribution of the generalised likelihood ratio test for analysing unreplicated factorial designs.

Al-Shiha and Yang (1999) proposed a multistage procedure for analysing unreplicated factorial experiments, which is based on the statistic that is derived from the generalised likelihood ratio test statistic under the assumption of normality. It was shown by their simulation study that the method is quite competitive with Lenth's (1989) method. In their paper, because of the difficulty of determining the null distribution analytically, the quantiles of the null distribution were empirically simulated. In this paper, we give the exact null distribution of their test statistic, which makes it possible to calculate the critical values of the test.

Bias↗

PubMed2005

Prevalence and partial sequence analysis of human T cell lymphotropic virus type I in China.

A total of 145,293 serum samples were collected from volunteer blood donors in 13 provinces of China and tested for anti-HTLV antibody using an ELISA licensed in China. Thirty were positive for anti-HTLV by ELISA and 19 of those 30 samples were confirmed positive using a supplemental immunoblot assay. Anti-HTLV-I was detected only in samples from Fujian province (a Southern province) where the positivity rate was approximately 0.05%. The region encoding gp46 from 5 of the 19 antibody positive samples was amplified and sequenced. Sequence analysis indicated that these isolates belong to HTLV-I genotype A. These data suggest that HTLV-I is not endemic throughout China and the virus may be restricted to a particular region.

Antibodies, Viral↗

PubMed2005

p53 protein activates the transcription of human proliferating cell nuclear antigen in response to 4-nitroquinoline N-oxide treatment.

4-nitroquinoline N-oxide (4-NQO) is a potent mutagen and carcinogen. To elucidate the cellular response to 4-NQO, we studied the transcriptional regulation of human proliferating cell nuclear antigen (hPCNA), an essential protein in DNA replication and repair, after 4-NQO treatment. We found that hPCNA promoter was dose-dependently transactivated by 4-NQO under the concentration of 2 microM via a previously reported p53-binding element located from -236 to -217 upstream of the transcription start site. Based on our western blot analysis, the phosphorylation of serine at the 15th residue (Ser15) of p53 was activated by 4-NQO, whereas the level of p53 in the cells did not change much. It was observed that Staurosporine, a Ser/Thr kinase inhibitor, blocked the Ser15 phosphorylation of p53 and the hPCNA promoter response to 4-NQO simultaneously, suggesting that Ser15 phosphorylated p53 was the 4-NQO-responsive hPCNA regulator. The [3H]-thymidine deoxyribose (TdR) incorporation assay and the comet assay showed that DNA repair was triggered when DNA replication was inhibited after the treatment of 4-NQO, and the hPCNA transactivation seemed to contribute to DNA repair. Taken together, our data indicate that after 4-NQO treatment hPCNA is transactivated by Ser15 phosphorylated p53, and participate in DNA repair.

4-Nitroquinoline-1-oxide↗

PubMed2005

Left ventricular SERCA2a gene down-regulation does not parallel ANP gene up-regulation during post-MI remodelling in rats.

BACKGROUND: In most animal models of chronic hemodynamic overload of the left ventricle (LV) as well as in human end stage heart failure, the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a) mRNA levels are decreased in parallel with increased atrial natriuretic peptide (ANP) mRNA levels. The situation in the remote myocardium following myocardial infarction (MI) is unclear. AIMS: (1) To examine SERCA2a mRNA levels in the non-infarcted LV myocardium of rats at the chronic stage of experimental MI and (2) To examine whether a negative linear correlation exists between SERCA2a and ANP mRNA levels in this model. METHODS: Anesthetized adult male Wistar rats underwent left coronary artery ligation or sham operation. Three months later, the rats were divided into three groups: sham-operated rats (sham, n=21), HF-free rats with MI (non-failing (NF)-MI, n=29) and rats with both MI and HF (congestive heart failure (CHF)-MI, n=14). LV remodelling and function were assessed by echocardiography and hemodynamic measurements. SERCA2a and ANP mRNA levels were determined by Northern and dot blot analysis with specific cDNA probes. RESULTS: LV SERCA2a mRNA levels varied markedly in sham-operated rats (0.9-1.8). Mean ANP mRNA level increased markedly and mean SERCA2a mRNA level decreased moderately in the remote myocardium. In some NF-MI rats, SERCA2a mRNA levels were higher than those in some sham controls. Whereas ANP mRNA levels correlated well with MI severity (r2=0.79, p<0.001), this was not the case for SERCA2a mRNA levels (r2=0.42, p<0.01). We found no negative correlation between ANP and SERCA2a mRNA levels. CONCLUSION: SERCA2a gene down-regulation in the non-infarcted myocardium of rats with MI does not correlate with ANP gene up-regulation, suggesting that the two genes are not antithetically regulated.

Animals↗

PubMed2005

Residues and source identification of persistent organic pollutants in farmland soils irrigated by effluents from biological treatment plants.

Sewage and industrial effluents from biological treatment plant have been widely used for agricultural irrigation in north part of China. However, effluents after biological treatment still contain heavy metals and persistent organic contaminants. The persistent organic contaminants accumulated in soil may transfer through the food chains and cause adverse health effects on human or biological effects on soil fauna and flora after long-term application. In present study, field surveys were carried out in the farmlands irrigated by effluents from biological treatment plants that receive sewage wastewater and industrial discharges. Residues of polycyclic aromatic hydrocarbons (PAHs) and organochlorine pesticides (OCPs) in the soils irrigated using both ground water and effluents were compared. The origins of PAHs in the soils were discussed. The results showed that wastewater irrigation could cause accumulation of PAHs in soils close to the pollution discharge. Significantly higher concentrations of PAHs were observed in the sampling sites close to the entrance of main channel in contrast to those along branches and the reference sites. There was no significant relationship between the accumulation of persistent organic pollutants and organic matter content in soil (TOC). Soil contamination of these persistent organic pollutants as affected by effluent irrigation was characterized by the dominant accumulation of high-molecular-weight PAHs (HMW-PAHs). In the case study, concentration of benzo[a]pyrane (BaP, 45.6 ng/g), indeno[1,2,3-cd]pyrene (IcP, 86.3 ng/g), benzo[g,h,i]perlene (BgP, 66.9 ng/g) could exceed the limits of the soil quality standard for biodegraded soils. In identification of the sources, the IcP/BgP values of PAHs in soils were more close to that in air particulates from coal/coke source (1.09+/-0.03 ng/g) [Dickhut RM, Canuel EA, Gustafson KE, Liu K, Arzayus KM, Walkers E, et al. Automotive sources of carcinogenic polycyclic aromatic hydrocarbons associated with particulate matter in the chesapeake bay region. Environ Sci Technol 2000;34:4635-40]. Therefore, both of the PAHs residues in effluents and emission from a nearby coal/coke plant were responsible. Also in this case study, low levels of the OCPs were observed and were not of significant concern in this wastewater irrigation area. Among the different OCPs analyzed, DDTs (mean 8.41 ng/g) and HCHs (mean 2.91 ng/g) were the major components. From the ratios of DDT/DDTs and beta-HCH/HCHs, it indicated that OCPs residues should be from historical usage.

Agriculture↗

PubMed2005

A homologous expression system for obtaining engineered cytochrome ba3 from Thermus thermophilus HB8.

Cytochrome ba3 is an integral membrane protein that serves as a terminal oxidase of the respiratory chain in some prokaryotes. We have cloned the complete cba operon of Thermus thermophilus HB8 in an Escherichia coli/T. thermophilus shuttle vector. The ba3-encoding operon, cba, was eliminated from the chromosome of T. thermophilus strain MT111 using the pyrE system of Yamagishi and co-workers. Expression of functional cytochrome ba3 occurred in cells grown at reduced dioxygen levels. A hepta-histidine tag was placed at the N-terminus of subunit I, and a purification method for this form of the enzyme was developed. Growth conditions were investigated for moderate sized cultures (2L) with typical yields of approximately 2 mg of highly pure enzyme per liter of culture medium. The physical properties and enzymatic activities of these recombinant enzymes were compared with those of native enzyme. Recombinant enzyme lacking the histidine tag is spectrally identical to wild-type enzyme. Histidine-tagged cytochrome ba3 shows minor differences from wild-type, and it appears be somewhat less active as a cytochrome c552 oxidase. Exemplary mutants were also produced and compared to native protein. Tyrosine I-237, previously found to be covalently bonded to I-His-233, was changed to phenylalanine (I-Y237F) and to histidine (I-Y237H) in the hepta-histidine tagged cytochrome ba3. The Y to F mutant is devoid of enzyme activity whereas the Y to H mutant possesses approximately 5% wild-type oxidase activity; their properties are compared with those of wild-type enzyme. The above versions of the histidine-tagged enzyme have been crystallized, and our analysis of a 2.3 angstrom resolution electron-density map will be discussed elsewhere.

Cloning, Molecular↗

PubMed2005

Genetic difference in susceptibility to the blood-retina barrier breakdown in diabetes and oxygen-induced retinopathy.

The breakdown of the blood-retina barrier (BRB) is a common feature of diabetic retinopathy. The purpose of the present study is to determine whether there are genetic differences in susceptibility to the breakdown of the BRB in diabetic retinopathy using two rat models. In streptozotocin (STZ)-induced diabetes, Brown Norway (BN) rats developed sustained vascular hyperpermeability in the retina during the entire experimental period (16 weeks of diabetes), while diabetic Sprague Dawley (SD) rats only showed retinal hyperpermeability from 3 to 10 days after the onset of diabetes. The strain difference in permeability was not correlated with the blood glucose levels in these two strains. In oxygen-induced retinopathy (OIR), BN rats developed retinal vascular hyperpermeability from postnatal day 12 (P12) to P22 with a peak at P16, which was 8.7-fold higher than that in the age-matched normal controls. In OIR-SD rats, however, hyperpermeability was observed from P14 to P18, with a peak only 2.2-fold higher than that in the controls. The strain difference in vascular hyperpermeability was correlated with the different overexpression of vascular endothelial growth factor (VEGF) in the retina of these two models. This finding suggests that genetic backgrounds contribute to the susceptibility to diabetic retinopathy.

Animals↗

PubMed2005

Macrophage activating effects of new alkamides from the roots of Echinacea species.

Chemical investigation of the roots of Echinacea angustifolia, E. purpurea, and E. pallida yielded two new alkamides, identified by analysis of spectroscopic data and comparison with reported alkamides. The new compounds were dodeca-2Z,4E,10Z-trien-8-ynoic acid isobutylamide (1) from E. angustifolia and dodeca-2Z,4E-diene-8,10-diynoic acid isobutylamide (2) from E. purpurea and E. pallida. These two components, as well as previously identified alkamides, exerted inhibition on LPS-mediated activation of a murine macrophage line, RAW264.7. These data suggest that these alkamides may have anti-inflammatory activity. The cytotoxicity of these alkamides using MTT assays was also investigated.

Amides↗

PubMed2005

Immortalization of pancreatic stellate cells as an in vitro model of pancreatic fibrosis: deactivation is induced by matrigel and N-acetylcysteine.

Tissue fibrosis is one of the characteristics of chronic pancreatitis and pancreatic adenocarcinoma. Activated pancreatic stellate cells (PSC) play a central role in this process. However, analysis of the molecular mechanisms leading to PSC activation is hampered by the lack of an established human PSC line. To overcome this problem, we immortalized and characterized primary human PSC. The cells were isolated by the outgrowth method and were immortalized by transfection with SV40 large T antigen and human telomerase (hTERT). Primary human PSC served as controls. An immortalized line, RLT-PSC, was analyzed for the expression of stellate cell markers. Moreover, the effects of transforming growth factor beta 1(TGFbeta1) or platelet-derived growth factor stimulation and of cultivation on basement membrane components or N-acetylcysteine (NAC) treatment on gene and protein expression and proliferation were analyzed. Immortal RLT-PSC cells retained the phenotype of activated PSC proven by the expression of alpha-smooth muscle actin (alphaSMA), vimentin, desmin and glial fibrillary acidic protein (GFAP). TGFbeta1 treatment upregulated the expression of alphaSMA, collagen type I (Col I), fibronectin and TGFbeta1. Incubation of RLT-PSC cells and primary human activated PSC on Matrigel plus NAC treatment resulted in a deactivated phenotype as evidenced by a decrease of alphaSMA, connective tissue growth factor and Col I expression and by a decreased proliferation of the cells. Moreover, this treatment restored the ability of the cells to store vitamin A in cytoplasmic vesicles. In conclusion, we have established an immortal pancreatic stellate cell line, without changing the characteristic phenotype. Importantly, we were able to demonstrate that besides soluble factors, the matrix surrounding PSC plays a pivotal role in the maintenance of the activation process of PSC. Cultivation of activated PSC on a reconstituted basement membrane plus treatment with NAC was able to deactivate the cells, thus pointing to the possibility of an antifibrosis therapy in chronic pancreatitis.

Acetylcysteine↗

PubMed2005

GABA(B) receptor modulators potentiate baclofen-induced depression of dopamine neuron activity in the rat ventral tegmental area.

2,6-Di-tert-butyl-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol (CGP7930) is a recently reported positive allosteric modulator of gamma-aminobutyric acid (GABA)(B) receptors. In this study, we assessed the ability of CGP7930 to modulate the baclofen-induced depression of dopamine (DA) neuron activity via the activation of GABA(B) receptors in the ventral tegmental area in rat midbrain slices. The selective GABA(B) receptor agonist, baclofen, depressed the spontaneous firing rate of DA neurons in a concentration-dependent manner (EC50 = 0.27 microM, n = 11). CGP7930 (30 microM) significantly (P < 0.05) shifted the baclofen concentration-response curve to the left (EC50 = 0.15 microM, n = 5). The effects of baclofen alone or baclofen coapplied with CGP7930 were fully blocked by 1 microM (2S)-3-[[(1S)-1-(3,4-dichloropheny)ethyl]amino-2-hydroxypropyl] (phenylmethyl) phosphinic acid (CGP55845), a potent and selective GABA(B) receptor antagonist. In similar experiments, N-[3,3-diphenylpropyl]-alpha-methylbenzylamine (fendiline) (30 or 50 microM), a compound shown to potentiate GABA(B) receptor-mediated cortical hyperpolarisation, also significantly enhanced the inhibitory effect of baclofen. It is therefore concluded that the recently reported GABA(B) receptor modulators, CGP7930 and fendiline, can enhance GABA(B) receptor-mediated depression of DA neuronal activity. This finding suggests a therapeutic potential for GABA(B) potentiators for the treatment of diseases associated with a hyperfunctional mesocorticolimbic system.

Animals↗

PubMed2005

Human embryonic stem cell lines derived from the Chinese population.

Six human embryonic stem cell lines were established from surplus blastocysts. The cell lines expressed alkaline phosphatase and molecules typical of primate embryonic stem cells, including Oct-4, Nanog, TDGF1, Sox2, EBAF, Thy-1, FGF4, Rex-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. Five of the six lines formed embryoid bodies that expressed markers of a variety of cell types; four of them formed teratomas with tissue types representative of all three embryonic germ layers. These human embryonic stem cells are capable of producing clones of undifferentiated morphology, and one of them was propagated to become a subline. Human embryonic stem cell lines from the Chinese population should facilitate stem cell research and may be valuable in studies of population genetics and ecology.

Animals↗

PubMed2005

Mannose-binding lectin binds to Ebola and Marburg envelope glycoproteins, resulting in blocking of virus interaction with DC-SIGN and complement-mediated virus neutralization.

Mannose-binding lectin (MBL), a serum lectin that mediates innate immune functions including activation of the lectin complement pathway, binds to carbohydrates expressed on some viral glycoproteins. In this study, the ability of MBL to bind to virus particles pseudotyped with Ebola and Marburg envelope glycoproteins was evaluated. Virus particles bearing either Ebola (Zaire strain) or Marburg (Musoke strain) envelope glycoproteins bound at significantly higher levels to immobilized MBL compared with virus particles pseudotyped with vesicular stomatitis virus glycoprotein or with no virus glycoprotein. As observed in previous studies, Ebola-pseudotyped virus bound to cells expressing the lectin DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin). However, pre-incubation of virus with MBL blocked DC-SIGN-mediated binding to cells, suggesting that the two lectins bind at the same or overlapping sites on the Ebola glycoprotein. Neutralization experiments showed that virus pseudotyped with Ebola or Marburg (Musoke) glycoprotein was neutralized by complement, while the Marburg (Ravn strain) glycoprotein-pseudotyped virus was less sensitive to neutralization. Neutralization was partially mediated through the lectin complement pathway, since a complement source deficient in MBL was significantly less effective at neutralizing viruses pseudotyped with filovirus glycoproteins and addition of purified MBL to the MBL-deficient complement increased neutralization. These experiments demonstrated that MBL binds to filovirus envelope glycoproteins resulting in important biological effects and suggest that MBL can interact with filoviruses during infection in humans.

Binding, Competitive↗