Biomedical subjects

Yan Wu

Publications and source records attributed to Yan Wu.

At least 55 records · Page 3Linked to original sources

PubMed2006

The Rad51/RadA N-terminal domain activates nucleoprotein filament ATPase activity.

Proteins in the RecA/RadA/Rad51 family form helical filaments on DNA that function in homologous recombination. While these proteins all have the same highly conserved ATP binding core, the RadA/Rad51 proteins have an N-terminal domain that shows no homology with the C-terminal domain found in RecA. Both the Rad51 N-terminal and RecA C-terminal domains have been shown to bind DNA, but no role for these domains has been established. We show that RadA filaments can be trapped in either an inactive or active conformation with respect to the ATPase and that activation involves a large rotation of the subunit aided by the N-terminal domain. The G103E mutation within the yeast Rad51 N-terminal domain inactivates the filament by failing to make proper contacts between the N-terminal domain and the core. These results show that the N-terminal domains play a regulatory role in filament activation and highlight the modular architecture of the recombination proteins.

Adenosine Triphosphatases↗

PubMed2006

Terpenoid indole alkaloids from Winchia calophylla.

Three new indole alkaloids, N(4)-demethyl-12-methoxyalstogustine (1), 17-carboxyl-N(4)-methylechitamidine chloride (2), and 17-carboxyl-12-methoxy-N(4)-methylechitamidine chloride (3), along with 15 known alkaloids, were isolated from the ethanolic extract of the stem bark of Winchia calophylla. The structures of 1-3 were elucidated on the basis of spectroscopic means and chemical methods. The determination of relative configurations at C-19 and C-20 for 1-3 was aided by 13C NMR spectroscopic data. The absolute configurations of alkaloids 1-3 were determined by direct comparison of their CD spectra with those of known alkaloids. All the alkaloids were tested in cytotoxic assays against P-388 and A-549 tumor cell lines, and only two of them showed weak activity against the A-549 cell line.

Animals↗

PubMed2006

Challenges in the analytical method development and validation for an unstable active pharmaceutical ingredient.

A sensitive high-performance liquid chromatography (HPLC) impurity profile method for the antibiotic ertapenem is developed and subsequently validated. The method utilizes an Inertsil phenyl column at ambient temperature, gradient elution with aqueous sodium phosphate buffer at pH 8, and acetonitrile as the mobile phase. The linearity, method precision, method ruggedness, limit of quantitation, and limit of detection of the impurity profile HPLC method are found to be satisfactory. The method is determined to be specific, as judged by resolving ertapenem from in-process impurities in crude samples and degradation products that arise from solid state thermal and light stress, acid, base, and oxidative stressed solutions. In addition, evidence is obtained by photodiode array detection studies that no degradate or impurity having a different UV spectrum coeluted with the major component in stressed or unstressed samples. The challenges during the development and validation of the method are discussed. The difficulties of analyzing an unstable active pharmaceutical ingredient (API) are addressed. Several major impurities/degradates of the API have very different UV response factors from the API. These impurities/degradates are synthesized or prepared by controlled degradation and the relative response factors are determined.

Chromatography, High Pressure Liquid↗

PubMed2006

Improving IMRT dose accuracy via deliverable Monte Carlo optimization for the treatment of head and neck cancer patients.

The purpose of this work is to investigate the effect of dose-calculation accuracy on head and neck (H&N) intensity modulated radiation therapy (IMRT) plans by determining the systematic dose-prediction and optimization-convergence errors (DPEs and OCEs), using a superposition/convolution (SC) algorithm. Ten patients with locally advanced H&N squamous cell carcinoma who were treated with simultaneous integrated boost IMRT were selected for this study. The targets consisted of gross target volume (GTV), clinical target volume (CTV), and nodal target volumes (CTV nodes). The critical structures included spinal cord, parotid glands, and brainstem. For all patients, three IMRT plans were created: A: an SC optimized plan (SCopt), B: an SCopt plan recalculated with Monte Carlo [MC(SCopt)], and C: an MC optimized plan (MCopt). For each structure, DPEs and OCEs were estimated as DPE(SC)=D(B)-D(A) and OCE(SC)=D(C)-D(B) where A, B, and C stand for the three different optimized plans as defined above. Deliverable optimization was used for all plans, that is, a leaf-sequencing step was incorporated into the optimization loop at each iteration. The range of DPE(SC) in the GTV D98 varied from -1.9% to -4.9%, while the OCE(SC) ranged from 0.9% to 7.0%. The DPE(SC) in the contralateral parotid D50 reached 8.2%, while the OCE(SC) in the contralateral parotid D50 varied from 0.91% to 6.99%. The DPE(SC) in cord D2 reached -3.0%, while the OCE(SC) reached to -7.0%. The magnitude of the DPE(SC) and OCE(SC) differences demonstrate the importance of using the most accurate available algorithm in the deliverable IMRT optimization process, especially for the estimation of normal structure doses.

Algorithms↗

PubMed2006

Depigmentation of melanocytes by the treatment of extracts from traditional Chinese herbs: a cell culture assay.

OBJECTIVE: To obtain potential skin whitening agents from traditional Chinese herbs, we tested changes of melanin content in melanocyte cell lines after treatment with extracts of 90 traditional Chinese herbs. METHODS: Mouse melanocyte cell lines were used. Depigmentation activity of the herb extracts were first screened in Mel-Ab cells, and then re-evaluated in melan-a cells and co-culture of melan-a and SP-1 cells. Melanin content and cell viability were the two indications for evaluation. Tyrosinase activity and the expression of melanin synthesis related enzymes in cells treated with the herb extracts were also tested. RESULTS: Nine herb extracts were proved to have depigmentation activity similar to or better than that of arbutin and low cytotoxicity to melanocytes. Two of them were more effective in co-cultured melan-a cells. Most of the effective herb extracts inhibited tyrosinase activity and the expression of tyrosinase. Some of them also inhibited tyrosinase related protein-1 and/or tyrosinase related protein-2 in cultured cells. CONCLUSIONS: We have found 9 herb extracts to be promising skin whitening agents. Among them, water extract of Galla Chinensis and ethanol extract of Radix Clematidis exhibited higher depigmentation activity and caused lower tyrosinase activity in cell culture assays and are worthy to be further studied.

Animals↗

PubMed2006

[Responses of grafted eggplant seedling roots to low temperature stress].

The study on the changes of physiological and biochemical properties of grafted eggplant seedling roots under low temperature stress and the relationships between these changes and cold tolerance showed that at the initial stage of treating with 10 degrees C (daytime) /3 degrees C (night), eggplant seedling roots were most impressible to low temperature, with the MDA content, chilling injury index, POD activity, proline content and soluble protein content increased significantly, and root respiration rate, SOD activity and CAT activity decreased rapidly. With the extending of low temperature stress, root respiration rate and osmotic adjustment reduced continually, while chilling injury index increased. After 3 days recovery, the respiration rate, osmotic adjustment and SOD activity were increased. Among the test materials, T2 (with Hiranasu as rootstock) had the best recovery capability, followed by T1 (with Taibyo as rootstock) and CK (Jinong 2000). Grafting with stronger cold tolerance rootstock could improve eggplant root activity, and thus, its cold resistance markedly.

Catalase↗

PubMed2006

[Removal of SO2 from flue gas by water vapor DC corona discharge].

The influence of several factors on removal rate of SO2 from flue gas in unsaturated water vapor DC corona discharge was researched. Furthermore, the experiments of the removal rate of SO2 in pulsed discharge increased by water vapor DC corona discharge plasma were conducted. The experiment system is supplied with multi-nozzle-plate electrodes and the flow of simulated flue gas is under 70 m3/h. The results show that removal rate of SO2 can be improved by increasing the concentration of water vapor, intensity of electric field or decreasing flow of simulated flue gas. In unsaturated water vapor DC corona discharge, removal rate of SO2 can be improved by 10%, when NH3 is added as NH3 and SO2 is in a mole ratio of two to one, it can reach 60%. The removal rate of SO2 can be increased by 5% in pulsed corona discharge and reach above 90%.

Air Pollutants↗

PubMed2006

Effect of hydrodynamic environment on tablet dissolution using flow-through dissolution apparatus.

The main objective of this research is to investigate the principles underlying the dissolution process, study the phenomena of drug release in laminar flow, and better understand the effect of hydrodynamic condition on drug dissolution, in order to predict drug dissolution from a solid dosage form. Two drug models were selected, theophylline (Class I) and naproxen (Class II), and were formulated into conventional tablets containing 105 mg theophylline or 300 mg naproxen using wet granulation method. Additionally theophylline (105 mg) and naproxen (300 mg) matrices containing 30% hydroxypropylmethylcellulose (HPMC) polymer were prepared by direct compression and tested for dissolution using both USP II and IV dissolution apparatus. Tablets were tested for dissolution (USP IV) using different cell diameter, flow rate, and different position of the tablet inside the cell. In general, the drug dissolution at a given time is a direct function of the flow rate, increasing the flow rate increases drug release. The use of a small cell resulted in faster drug dissolution and higher Reynold's Number than using a large cell. Tablet position in the cell, also has an effect on drug dissolution, inserting the tablet in a horizontal position inside the cell gave faster dissolution than a vertical position. The hydrodynamic conditions did not affect the drug dissolution from HPMC controlled release tablets indicating that the drug dissolution is controlled by the matrix. An equation to predict drug dissolution from conventional tablets was established: Sh=-21.36+10.58Re(1/2) where R2=0.98. This study demonstrated that hydrodynamic conditions, and type of dissolution testing apparatus used have an effect on dissolution rate, mass transfer rate, and film thickness underlying dissolution process.

Solubility↗

PubMed2005

VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche.

The cellular and molecular mechanisms by which a tumour cell undergoes metastasis to a predetermined location are largely unknown. Here we demonstrate that bone marrow-derived haematopoietic progenitor cells that express vascular endothelial growth factor receptor 1 (VEGFR1; also known as Flt1) home to tumour-specific pre-metastatic sites and form cellular clusters before the arrival of tumour cells. Preventing VEGFR1 function using antibodies or by the removal of VEGFR1(+) cells from the bone marrow of wild-type mice abrogates the formation of these pre-metastatic clusters and prevents tumour metastasis, whereas reconstitution with selected Id3 (inhibitor of differentiation 3)-competent VEGFR1+ cells establishes cluster formation and tumour metastasis in Id3 knockout mice. We also show that VEGFR1+ cells express VLA-4 (also known as integrin alpha4beta1), and that tumour-specific growth factors upregulate fibronectin--a VLA-4 ligand--in resident fibroblasts, providing a permissive niche for incoming tumour cells. Conditioned media obtained from distinct tumour types with unique patterns of metastatic spread redirected fibronectin expression and cluster formation, thereby transforming the metastatic profile. These findings demonstrate a requirement for VEGFR1+ haematopoietic progenitors in the regulation of metastasis, and suggest that expression patterns of fibronectin and VEGFR1+VLA-4+ clusters dictate organ-specific tumour spread.

Animals↗

PubMed2005

L-canavanine is a time-controlled mechanism-based inhibitor of Pseudomonas aeruginosa arginine deiminase.

The mechanism for inhibition of the Pseudomonas aeruginosa arginine deiminase (PaADI) by the arginine analogue l-canavanine was investigated. Inhibition by this substance (kinact = 0.31 +/- 0.03 min-1 and Ki = 1.7 +/- 0.5 mM) is associated with the formation of a modestly stable S-alkylthiouronium intermediate, detected by using kinetic techniques and identified by using electrospray ionization mass spectrometry. The electronic and/or orientation effects, caused by oxygen-for-methylene substitution in l-canavanine, on the rate of enzyme regeneration from the S-alkylthiouronium intermediate could serve as the basis for a strategy for the rational design of new slow substrate inhibitors of ADI.

Arginine↗

PubMed2005

Crystal structure of Methanococcus voltae RadA in complex with ADP: hydrolysis-induced conformational change.

Members of a superfamily of RecA-like recombinases facilitate a central strand exchange reaction in the DNA repair process. Archaeal RadA and Rad51 and eukaryal Rad51 and meiosis-specific DMC1 form a closely related group of recombinases distinct from bacterial RecA. Nevertheless, all such recombinases share a conserved core domain which carries the ATPase site and putative DNA-binding sites. Here we present the crystal structure of an archaeal RadA from Methanococcus voltae (MvRadA) in complex with ADP and Mg2+ at 2.1 A resolution. The crystallized RadA-ADP filament has an extended helical pitch similar to those of previously determined structures in the presence of nonhydrolyzable ATP analogue AMP-PNP. Structural comparison reveals two recurrent conformations with an extensive allosteric effect spanning the ATPase site and the putative DNA-binding L2 region. Varied conformations of the L2 region also imply a dynamic nature of recombinase-bound DNA.

Adenosine Diphosphate↗

PubMed2005

VEGFR-1 (FLT-1) activation modulates acute lymphoblastic leukemia localization and survival within the bone marrow, determining the onset of extramedullary disease.

The presence of persistent circulating leukemia cells, or engrafted into extramedullary tissues, is a bad prognostic factor for patients with acute leukemia. However, little is known about the mechanisms that regulate the exit of leukemia cells from the bone marrow (BM) microenvironment. We reveal that vascular endothelial growth factor receptor 1 (FLT-1) modulates acute leukemia distribution within the BM, along VEGF and PlGF gradients, regulating leukemia survival and exit into the peripheral circulation. FLT-1 activation on acute lymphoblastic leukemia (ALL) cells results in cell migration and proliferation in vitro, whereas in vivo FLT-1-overexpressing cells accumulate in the BM epiphysis of nonobese diabetic-severe combined immunodeficient (NOD-SCID) recipients and are detected in circulation 2 weeks after inoculation. In turn, FLT-1 neutralization affects leukemia localization (now in the BM diaphysis), increases leukemia apoptosis, and impedes the exit of ALL cells, prolonging the survival of inoculated mice. We demonstrate further that FLT-1-induced cell migration involves actin polymerization and lipid raft formation. Taken together, we show that FLT-1 regulates the BM localization of ALL cells, determining their survival and exit into the circulation and ultimately the survival of inoculated recipients. FLT-1 targeting on subsets of acute leukemias may delay the onset of extramedullary disease, which may be advantageous in combinatorial therapeutic settings.

Apoptosis↗

PubMed2005

Transcriptional characterizations of differences between eutopic and ectopic endometrium.

Endometriosis, defined as the presence of endometrial glandular and stromal cells outside the uterine cavity, is a common gynecological disease with poorly understood pathogenesis. Using laser capture microdissection and a cDNA microarray with 9600 genes/expressed sequence tags (ESTs), we have conducted a comprehensive profiling of gene expression differences between the ectopic and eutopic endometrium taken from 12 women with endometriosis adjusted for menstrual phase and the location of the lesions. With dye-swapping and replicated arrays, we found 904 genes/ESTs that are differentially expressed. We validated the gene expression using real-time RT-PCR. We found that the expression patterns of these genes/ESTs correctly classified the 12 patients into ovarian and nonovarian endometriosis. We identified gene clusters that are location-specific. In addition, we identified several biological themes using Expression Analysis Systematic Explorer. Finally, we identified 79 pathways with over 100 genes with known functions, which include oxidative stress, focal adhesion, Wnt signaling, and MAPK signaling. The identification of these genes and their associated pathways provides new insight. Our findings will stimulate future investigations on molecular genetic mechanisms underlying the pathogenesis of endometriosis.

Base Sequence↗

PubMed2005

Changes of apoptosis-related proteins in hippocampus of SAM mouse in development and aging.

Expression of Caspase and Bcl-2 proteins was examined in the hippocampus of senescence-accelerated mice (SAM, P8 and R1 strain) from E19 to 16 months of age. Immunoblotting analysis showed no upregulation of pro-apoptotic proteins (caspase-2L, -3, -6, -8, -9, and Bax) with age while all the anti-apoptotic proteins (caspase-2S, Bcl-2, and Bcl-XL) remained unchanged during aging. Terminal dUTP nick end labeling (TUNEL) and electron microscopy on the hippocampus of 3- and 16-month-old SAM revealed very few TUNEL positive cells in both groups. Morphometric study further showed neuronal loss in the hippocampus was not age-related. Our results suggest apoptosis and cell loss are minor events in the hippocampus of SAM mice and are unlikely to be the cause of functional decline during aging in SAM.

Aging↗

PubMed2005

Function of the vascular endothelial growth factor receptors Flt-1 and Flk-1/KDR in the alloimmune response in vivo.

BACKGROUND: We have recently reported that vascular endothelial growth factor (VEGF) functions as a proinflammatory cytokine to regulate the trafficking of leukocytes into allografts in the early posttransplant period. VEGF binds two major VEGF receptors: VEGFR-1 (flt-1) and VEGFR-2 (flk-1/KDR). Here, we wished to investigate the expression and function of VEGF receptors in the process of acute allograft rejection in vivo. METHODS: We performed fully MHC-mismatched C57BL/6 (H-2b) into BALB/c (H-2d) vascularized heterotopic murine cardiac transplants and we examined the expression of VEGF and VEGF receptors by immunohistochemistry during acute allograft rejection. Next, we treated mice with specific neutralizing monoclonal antibodies against murine VEGFR-1 and VEGFR-2 and examined their effect on the development of acute allograft rejection by histology and by analysis of graft survival. The intragraft expression of cytokines and chemokines were also evaluated by quantitative real-time PCR analysis. RESULTS: The expression of VEGF, VEGFR-1 and VEGFR-2 were significantly up-regulated during allograft rejection as compared to isografts. Administration of either anti-VEGFR-1 or anti-VEGFR-2 alone failed to inhibit allograft rejection. However, coadministration of both antibodies together inhibited leukocyte infiltration of allografts and prolonged allograft survival. Furthermore, the effect of VEGFR blockade was associated with the downregulation of intragraft cytokine and chemokine expression. CONCLUSIONS: Our data suggest that VEGF-VEGFR interactions function in the alloimmune response in vivo. Targeting VEGFRs may represent a novel therapy to protect allografts following clinical transplantation.

Acute Disease↗

PubMed2005

Vascular endothelial growth factor receptor-1 promotes migration and invasion in pancreatic carcinoma cell lines.

BACKGROUND: Vascular endothelial growth factor receptor-1 (VEGFR-1) is one of three receptor tyrosine kinases for VEGF, a key regulator of angiogenesis in cancer. Although VEGFRs initially were believed to be expressed exclusively on endothelial cells (ECs), recent studies have demonstrated the presence of VEGFR-1 on non-EC types. The authors hypothesized that VEGFR-1 is present and functional in pancreatic carcinoma cells, contributing to the malignant phenotype. METHODS: The authors assessed the expression of VEGFR-1 and its ligands in 11 pancreatic carcinoma cell lines by reverse-transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and/or Western blot analysis. The function of VEGFR-1 was evaluated by treating two representative cell lines with VEGF-B, a selective ligand for VEGFR-1, and/or a specific anti-VEGFR-1 antibody and assessing the effects on signaling, migration, invasion, and proliferation. RESULTS: All 11 pancreatic carcinoma cell lines expressed VEGFR-1 mRNA and protein, as well as the VEGFR-1 ligands VEGF-A and VEGF-B. Two representative cell lines (L3.6 and Panc-1) exhibited VEGF-B-induced mitogen-activated protein kinase signaling. A VEGFR-1 neutralizing antibody abrogated signaling, confirming that the ligand effect was mediated through VEGFR-1. VEGFR-1 stimulation by VEGF-A or VEGF-B was found to promote migration in both cell lines. Panc-1 cells also demonstrated enhanced Matrigel invasion after VEGFR-1 stimulation. VEGFR-1-dependent migration and invasion were blocked by the VEGFR-1 neutralizing antibody. VEGFR-1 activation did not appear to enhance cell proliferation. CONCLUSIONS: VEGFR-1 appears to be expressed ubiquitously in pancreatic carcinoma cell lines, in which it induces signaling and promotes migration and invasion. Overexpression of VEGF in tumors may activate tumor cells bearing VEGFR-1 via an autocrine pathway. Agents targeting VEGF or its receptors may have a dual inhibitory effect on tumor growth by suppressing both angiogenesis and tumor cell function.

Blotting, Western↗

PubMed2005

Is the critical role of loop 3 of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase in catalysis due to loop-3 residues arginine-84 and tryptophan-89? Site-directed mutagenesis, biochemical, and crystallographic studies.

Deletion mutagenesis, biochemical, and X-ray crystallographic studies have shown that loop 3 of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is required for the assembly of the active center, plays an important role in the stabilization of the ternary complex of HPPK with MgATP and 6-hydroxymethyl-7,8-dihydropterin (HP), and is essential for catalysis. Whether the critical functional importance of loop 3 is due to the interactions between residues R84 and W89 and the two substrates has been addressed by site-directed mutagenesis, biochemical, and X-ray crystallographic studies. Substitution of R84 with alanine causes little changes in the dissociation constants and kinetic constants of the HPPK-catalyzed reaction, indicating that R84 is not important for either substrate binding or catalysis. Substitution of W89 with alanine increases the K(d) for the binding of MgATP by a factor of 3, whereas the K(d) for HP increases by a factor of 6, which is due to the increase in the dissociation rate constant. The W89A mutation decreases the rate constant for the chemical step of the forward reaction by a factor of 15 and the rate constant for the chemical step of the reverse reaction by a factor of 25. The biochemical results of the W89A mutation indicate that W89 contributes somewhat to the binding of HP and more significantly to the chemical step. The crystal structures of W89A show that W89A has different conformations in loops 2 and 3, but the critical catalytic residues are positioned for catalysis. When these results are taken together, they suggest that the critical functional importance of loop 3 is not due to the interactions of the R84 guanidinium group or the W89 indole ring with the substrates.

Amino Acid Sequence↗

PubMed2005

SMAD pathway mediation of BDNF and TGF beta 2 regulation of proliferation and differentiation of hippocampal granule neurons.

Hippocampal granule cells self-renew throughout life, whereas their cerebellar counterparts become post-mitotic during early postnatal development, suggesting that locally acting, tissue-specific factors may regulate the proliferative potential of each cell type. Confirming this, we show that conditioned medium from hippocampal cells (CM(Hippocampus)) stimulates proliferation in cerebellar cultures and, vice versa, that mitosis in hippocampal cells is inhibited by CM(Cerebellum). The anti-proliferative effects of CM(Cerebellum) were accompanied by increased expression of the cyclin-dependent kinase inhibitors p21 and p27, as well as markers of neuronal maturity/differentiation. CM(Cerebellum) was found to contain peptide-like factors with distinct anti-proliferative/differentiating and neuroprotective activities with differing chromatographic properties. Preadsorption of CM(Cerebellum) with antisera against candidate cytokines showed that TGFbeta2 and BDNF could account for the major part of the anti-proliferative and pro-differentiating activities, an interpretation strengthened by studies involving treatment with purified TGFbeta2 and BDNF. Interference with signaling pathways downstream of TGFbeta and BDNF using dominant-negative forms of their respective receptors (TGFbeta2-RII and TRKB) or of dominant-negative forms of SMAD3 and co-SMAD4 negated the anti-proliferative/differentiating actions of CM(Cerebellum). Treatment with CM(Cerebellum) caused nuclear translocation of SMAD2 and SMAD4, and also transactivated a TGFbeta2-responsive gene. BDNF actions were shown to depend on activation of ERK1/2 and to converge on the SMAD signaling cascade, possibly after stimulation of TGFbeta2 synthesis/secretion. In conclusion, our results show that the regulation of hippocampal cell fate in vitro is regulated through an interplay between the actions of BDNF and TGFbeta.

Animals↗