Biomedical subjects

Wei Lian

Publications and source records attributed to Wei Lian.

17 recordsLinked to original sources

PubMed2006

Preparation and optical properties of ThO(2) and Eu-doped ThO(2) nanotubes by the sol-gel method combined with porous anodic aluminum oxide template.

In this paper, we report the synthesis of thorium oxide and Eu-doped thorium oxide nanotubes for the first time using the sol-gel method in porous anodic aluminum oxide template. Transmission electron microscopy, X-ray diffraction, and X-ray photoelectron spectroscopy were applied to characterize the morphology and structure of the as-prepared nanotubes. It has been demonstrated that Eu(3+) ions were homogeneously doped into the ThO(2) crystal lattice. The optical properties resulting from Eu-doped products were investigated by means of photoluminescence spectroscopy. Strong visible light emissions were observed at low doping concentration, and the luminescent intensity decreased at high doping concentration. The luminescent centers were concluded to be the Eu(3+) ions in the cubic (O(h)) sites rather than the C(3v) sites, which accounted well for the decrease of luminescent intensity at high doping concentration.

Journal Article↗

PubMed2006

Ricin detection by biological signal amplification in a well-in-a-well device.

This paper presents a ricin detection method based on ricin's inhibitory effects on protein synthesis. Biological synthesis (expression) of a protein includes the steps of gene transcription (DNA --> RNA) and protein translation (RNA --> proteins); these reactions can be coupled into a one-step operation and carried out in a cell-free medium. Ricin is known to inhibit protein synthesis by interacting with 28S ribosome RNA; the inhibitory effect is exploited as the sensing mechanism in this work. For each copy of DNA, thousands of copies of proteins can be produced. As a result, the inhibitory effects of ricin are amplified, leading to a significantly enhanced detection signal (the difference between the positive control and samples). An array of protein expression units is developed to accommodate positive/negative controls and multiple samples. The array device contains a solution without any reagent captured on a solid surface, offering flexibility without comprising the activities of biomolecules. The miniaturized well-in-a-well design possesses a mechanism to supply nutrients continuously and remove byproducts, leading to higher protein expression yields and thus larger detection signals (lower detection limit) when ricin is present. We demonstrate the production of green fluorescent protein and luciferase in the device. A calibration curve has been obtained between the luciferase expression yield and the ricin concentration, showing a detection limit of 0.01 nM (0.3 ng/mL) ricin. The nested-well device is also used for measuring the toxicity level of ricin after physical or chemical treatment.

Calibration↗

PubMed2006

ExoS of Pseudomonas aeruginosa induces apoptosis through a Fas receptor/caspase 8-independent pathway in HeLa cells.

Pseudomonas aeruginosa infection is a serious complication in immunocompromised individuals and in patients with cystic fibrosis. We have previously shown that the type III secreted effector ExoS triggers apoptosis in various cultured cell lines via its ADP-ribosyltransferase (ADPRT) activity. The apoptosis process was further shown to involve intrinsic signalling pathway requiring c-Jun N-terminal kinase (JNK)-initiated mitochondrial pathway. In the present study, we investigated the role of Fas pathway activation in P. aeruginosa-induced apoptosis. P. aeruginosa infection resulted in caspase 8 cleavage in HeLa cells, which was inhibited by overexpression of a dominant negative version of Fas-associated death domain (FADD), suggesting that Fas pathway was activated. In fact, confocal laser scanning microscopy showed that P. aeruginosa induced clustering of FasR. In addition, the ADPRT activity of the ExoS was required for the induction of FasR clustering and caspase 8 cleavage. However, blocking the FasR-FasL interaction by antagonistic antibodies to FasR or to FasL had no effect on P. aeruginosa-induced caspase 8 and caspase 3 activation, neither did the silencing of FasR by small interfering RNA (siRNA), suggesting that caspase 8 activation through the FADD bypasses FasR/FasL-mediated signalling. Thus, FADD-mediated caspase 8 activation involves intracellular ExoS in an ADPRT-dependent manner. Furthermore, silencing of caspase 8 by siRNA did not interfere with P. aeruginosa-induced apoptosis, whereas it rendered HeLa cells markedly increased resistance towards FasL-induced apoptosis. In conclusion, our findings indicate that ExoS of P. aeruginosa induces apoptosis through a mechanism that is independent of Fas receptor/caspase 8 pathway.

ADP Ribose Transferases↗

PubMed2005

A framework to analyze multiple time series data: a case study with Streptomyces coelicolor.

Transcriptional regulation in differentiating microorganisms is highly dynamic involving multiple and interwinding circuits consisted of many regulatory genes. Elucidation of these networks may provide the key to harness the full capacity of many organisms that produce natural products. A powerful tool evolved in the past decade is global transcriptional study of mutants in which one or more key regulatory genes of interest have been deleted. To study regulatory mutants of Streptomyces coelicolor, we developed a framework of systematic analysis of gene expression dynamics. Instead of pair-wise comparison of samples in different combinations, genomic DNA was used as a common reference for all samples in microarray assays, thus, enabling direct comparison of gene transcription dynamics across different isogenic mutants. As growth and various differentiation events may unfold at different rates in different mutants, the global transcription profiles of each mutant were first aligned computationally to those of the wild type, with respect to the corresponding growth and differentiation stages, prior to identification of kinetically differentially expressed genes. The genome scale transcriptome data from wild type and a DeltaabsA1 mutant of Streptomyces coelicolor were analyzed within this framework, and the regulatory elements affected by the gene knockout were identified. This methodology should find general applications in the analysis of other mutants in our repertoire and in other biological systems.

Anthraquinones↗

PubMed2005

An analysis of the use of genomic DNA as a universal reference in two channel DNA microarrays.

BACKGROUND: DNA microarray is an invaluable tool for gene expression explorations. In the two-dye microarray, fluorescence intensities of two samples, each labeled with a different dye, are compared after hybridization. To compare a large number of samples, the 'reference design' is widely used, in which all RNA samples are hybridized to a common reference. Genomic DNA is an attractive candidate for use as a universal reference, especially for bacterial systems with a low percentage of non-coding sequences. However, genomic DNA, comprising of both the sense and anti-sense strands, is unlike the single stranded cDNA usually used in microarray hybridizations. The presence of the antisense strand in the 'reference' leads to reactions between complementary labeled strands in solution and may cause the assay result to deviate from true values. RESULTS: We have developed a mathematical model to predict the validity of using genomic DNA as a reference in the microarray assay. The model predicts that the assay can accurately estimate relative concentrations for a wide range of initial cDNA concentrations. Experimental results of DNA microarray assay using genomic DNA as a reference correlated well to those obtained by a direct hybridization between two cDNA samples. The model predicts that the initial concentrations of labeled genomic DNA strands and immobilized strands, and the hybridization time do not significantly affect the assay performance. At low values of the rate constant for hybridization between immobilized and mobile strands, the assay performance varies with the hybridization time and initial cDNA concentrations. For the case where a microarray with immobilized single strands is used, results from hybridizations using genomic DNA as a reference will correspond to true ratios under all conditions. CONCLUSION: Simulation using the mathematical model, and the experimental study presented here show the potential utility of microarray assays using genomic DNA as a reference. We conclude that the use of genomic DNA as reference DNA should greatly facilitate comparative transcriptome analysis.

DNA↗

PubMed2004

Ultrasensitive detection of biomolecules with fluorescent dye-doped nanoparticles.

Fluorescent-labeled molecules have been used extensively for a wide range of applications in biological detection and diagnosis. A new form of highly luminescent and photostable nanoparticles was generated by doping the fluorescent dye tris(2'2-bipyridyl)dichlororuthenium(II)hexahydrate (Rubpy) inside silica material. Because thousands of fluorescent dye molecules are encapsulated in the silica matrix that also serves to protect Rubpy dye from photodamaging oxidation, the Rubpy-dye-doped nanoparticles are extremely bright and photostable. We have used these nanoparticles successfully in various fluorescence labeling techniques, including fluorescent-linked immunosorbent assay, immunocytochemistry, immunohistochemistry, DNA microarray, and protein microarray. By combining the high-intensity luminescent nanoparticles with the specificity of antibody-mediated recognition, ultrasensitive target detection has been achieved. In all cases, assay results clearly demonstrated the superiority of the nanoparticles over organic fluorescent dye molecules and quantum dots in probe labeling for sensitive target detection. These results demonstrate the potential to apply these newly developed fluorescent nanoparticles in various biodetection systems.

Fluorescent Dyes↗

PubMed2004

Crystallization and preliminary X-ray crystallographic studies on recombinant rat choline acetyltransferase.

Choline acetyltransferase (ChAT) catalyzes the biosynthesis of the neurotransmitter acetylcholine from acetyl-CoA and choline in cholinergic neurons. Rat ChAT (rChAT) was overexpressed in Escherichia coli, purified by affinity chromatography and crystallized. Diffraction data were collected from a single crystal under cryoconditions at the F1 beamline at the Cornell High Energy Synchrotron Source, with a maximal useful diffraction pattern to 1.55 A resolution. The crystals were shown to belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 138.97, b = 77.67, c = 59.67 A and a scaling R(sym) of 0.054 for 72 446 unique reflections. Packing considerations indicate there to be one molecule per asymmetric unit. It is expected that in the near future the structure of rChAT will be obtained using molecular-replacement methods. Elucidation of the structure of rChAT will aid in the development of therapeutic agents for Alzheimer's disease.

Animals↗

PubMed2004

Structural and mutational characterization of L-carnitine binding to human carnitine acetyltransferase.

We report the crystal structure of a binary complex of human peroxisomal carnitine acetyltransferase and the substrate l-carnitine, refined to a resolution of 1.8 Angstrom with an R(factor) value of 18.9% (R(free)=22.3%). L-carnitine binds to a preformed pocket in the active site tunnel of carnitine acetyltransferase aligned with His(322). The quaternary nitrogen of carnitine forms a pi-cation interaction with Phe(545), while Arg(497) forms an electrostatic interaction with the negatively charged carboxylate group. An extensive hydrogen bond network also occurs between the carboxylate group and Tyr(431), Thr(444), and a bound water molecule. Site-directed mutagenesis and kinetic characterization reveals that Tyr(431), Thr(444), Arg(497), and Phe(545) are essential for high affinity binding of L-carnitine.

Binding Sites↗

PubMed2004

Structural insights and functional implications of choline acetyltransferase.

The biosynthetic enzyme for the neurotransmitter acetylcholine, choline acetyltransferase (ChAT) (E.C. 2.3.1.6), is essential for the development and neuronal activities of cholinergic systems involved in many fundamental brain functions. ChAT catalyzes the transfer of an acetyl group from acetyl-coenzyme A to choline to form the neurotransmitter acetylcholine. Since its discovery more than 60 years ago much research has been devoted to the kinetic studies of this enzyme. For the first time we report the crystal structure of rat ChAT (rChAT) to 1.55 A resolution. The structure of rChAT is a monomer and consists of two domains with an interfacial active site tunnel. This structure, with the modeled substrate binding, provides critical insights into the molecular basis for the production of acetylcholine and may further our understanding of disease causing mutations.

Acetylcholine↗

PubMed2004

Diagnosis and treatment of the pituitary metastases.

Three cases of pituitary metastases were reported. They all had operations and the pathological examination confirming the diagnosis. The clinical features of diabetes insipidus and extraocular nerve palsy were presented. In two cases, the original tumors were bronchioloalveolar carcinoma; in the other one, the original tumor was unknown. All three cases had poor outcome. These cases illustrate the fact that a pituitary metastasis can closely mimic a pituitary benign tumor, such as pituitary adenoma. Especially in the presence of suggestive symptoms such as diabetes insipidus and/or cranical nerve paralysis, the possibility of metastatic disease in the differential diagnosis of a pituitary mass should always be considered.

Adenocarcinoma↗

PubMed2004

[Diaphyseal defect repair with nacre/polylactic acid composite artificial bone in rabbits].

OBJECTIVE: To evaluate the biocompatibility, degradation and bone formation activity of a new bone substitute for bone grafting, nacre/polylactic acid composite artificial bone (NPCB). METHODS: Radial bone defects 1.5 cm in length were induced in 32 New Zealand rabbits and immediately filled with NPCB or nothing. The animals' local and whole body responses to the implants were observed after the operation, and the serum calcium levels were detected 1 week and 4 weeks postoperatively. Tissue response, new bone formation in the defects and degradation of the implants were evaluated by X-ray, and examination of the bone mineral content (BMC) in the defects and histomorphological analysis were performed after the rabbits were sacrificed. RESULTS: All the rabbits survived the operation and the incisions healed smoothly. No significant difference was noted in the serum calcium level between 1 day before operation, 1 week and 4 weeks postoperation. BMC in the defects of the rabbits with NPCB grafts increased more rapidly than that in the control group 6-12 weeks after the operation and began to decrease after 12 weeks. Gross, X-ray and histological observations revealed that NPCB possessed osteoconductive activity and new bone ingrowth in the implants was found during the whole experiment. At 16 weeks, the defects grafts were almost completely repaired with NPCB while the defects in the control group remained nonunion. Biodegradation of the implants was observed early at 6 weeks, with numerous macrophage and multinuclear giant cells containing phagocytosed NPCB particles around the implants at 12 weeks, but NPCB remnants were still visible at 16 weeks. CONCLUSION: As a bone substitute, NPCB possesses good biocompatibility, biodegradability and osteoconductive activity.

Absorbable Implants↗

PubMed2004

Elevated soluble epidermal growth factor receptor level in pituitary adenoma and carcinoma.

OBJECTIVE: To investigate effect of the soluble epidermal growth factor receptor (sEGFR/sErbB1) level in the peripheral blood in development, invasiveness, apoplexy of each type of pituitary tumor. METHODS: The sEGFR level was determined in peripheral serum from 190 patients with pituitary diseases by enzyme linked immunosorbent assay. The sEGFR levels were measured in 10 pituitary Rathke's pouch, 18 pituitary hyperplasia, 161 pituitary adenomas including 30 microadenomas, 83 large adenomas, 48 giant adenomas, 1 pituitary carcinoma, and 28 healthy controls. RESULTS: In the patients with pituitary hyperplasia, microadenoma, large adenoma, giant adenoma, and pituitary carcinoma, the sEGFR level was 188.92 +/- 32.62, 209.83 +/- 19.01, 333.20 +/- 69.33, 405.85 +/- 37.38, and 617.45 fmol/mL independently. They were all significantly higher than patients with pituitary Rathke's pouch (156.78 +/- 18.24 fmol/mL, P < 0.001) and healthy control group (159.11 +/- 40.50 fmol/mL, P < 0.05). The sEGFR level in pituitary carcinoma was higher than pituitary adenoma. In patients with pituitary adenoma, the sEGFR level was positive correlated to the size of pituitary adenomas (r=0.998), the significant difference was observed for the sEGFR level in each group of the patients with pituitary adenomas (P < 0.001). Furthermore, in patients with pituitary ACTH-secreting microadenomas, the serum sEGFR levels in invasiveness (295.00 +/- 77.80 fmol/mL) was higher than that in non-invasiveness (210.60 +/- 16.4 fmol/mL, P < 0.05). In patients with pituitary ACTH-secreting, PRL-secreting, GH-secreting, and non-functioning large adenomas, the serum sEGFR levels in invasiveness (407.86 +/- 28.50, 399.25 +/- 30.10, 386.00 +/- 13.08, and 369.25 +/- 36.70 fmol/mL) was higher than that in non-invasiveness (335.25 +/- 63.49, 300.64 +/- 47.57, 297.00 +/- 61.93, and 269.30 +/- 25.68 fmol/mL) respectively (P < 0.05). In patients with invasive pituitary PRL-secreting, GH-secreting, and non-functioning giant adenomas, the serum sEGFR levels not significantly different in between invasiveness (417.50 +/- 35.94, 409.50 +/- 69.14, and 417.50 +/- 44.13 fmol/mL) and non-invasiveness (386.00 +/- 49.64, 417.50 +/- 44.03, and 409.51 +/- 35.17 fmol/mL) (P > 0.05). In patients with pituitary large adenomas, the sEGFR levels in pituitary apoplexy (377.48 +/- 39.18 fmol/mL) was higher than that in non-pituitary apoplexy (343.18 +/- 68.17 fmol/mL, P > 0.05). CONCLUSIONS: The increased level of peripheral serum sEGFR is concomitant with development, proliferous size of the adenomas in patients with pituitary adenomas. In addition, the elevated levels of serum sEGFR occur in pituitary apoplexy as clinical active tumors, and the non-invasive ACTH secreting adenomas. The sEGFR levels could be differentiated helpfully between pituitary adenomas and non-pituitary adenomas. These data suggest that serum sEGFR could be as a referable marker of the size and activation of proliferation in pituitary adenoma.

Adenoma↗

PubMed2003

Redox stress regulates cell proliferation and apoptosis of human hepatoma through Akt protein phosphorylation.

Employing a spin trapping agent combined with electron spin resonance spectroscopy, we were able to capture reactive oxygen species (ROS) in living hepatoma cells and first found that the trapped ROS was superoxide anion (O(2)(z.rad;-)). O(2)(z.rad;-) suppressed by treatment with diphenylene iodonium, a flavoprotein inhibitor, was generated by the flavoprotein-containing NADPH-oxidase complex. Applying endogenous/exogenous pro-oxidant or antioxidant causes different redox states in hepatoma cells. Akt activity and cell growth were significantly stimulated by treating hepatoma cells with low concentration of ROS, which could be abolished by adding antioxidants. The phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin (0.15 microM) inhibited Akt phosphorylation induced by ROS. Our results indicate that hepatoma cell growth is ROS-dependent, and fluctuation of the intracellular redox state may regulate hepatoma cell growth through Akt phosphorylation and the PI3K/Akt pathway, resulting in a broad array of responses from cellular proliferation to apoptosis.

Antioxidants↗

PubMed2003

Structure of human carnitine acetyltransferase. Molecular basis for fatty acyl transfer.

Carnitine acyltransferases are a family of ubiquitous enzymes that play a pivotal role in cellular energy metabolism. We report here the x-ray structure of human carnitine acetyltransferase to a 1.6-A resolution. This structure reveals a monomeric protein of two equally sized alpha/beta domains. Each domain is shown to have a partially similar fold to other known but oligomeric enzymes that are also involved in group-transfer reactions. The unique monomeric arrangement of the two domains constitutes a central narrow active site tunnel, indicating a likely universal feature for all members of the carnitine acyltransferase family. Superimposition of the substrate complex of a related protein, dihydrolipoyl trans-acetylase, reveals that both substrates localize to the active site tunnel of human carnitine acetyltransferase, suggesting the location of the ligand binding sites for carnitine and coenzyme A. Most significantly, this structure provides critical insights into the molecular basis for fatty acyl chain transfer and a possible common mechanism among a wide range of acyltransferases utilizing a catalytic dyad.

Amino Acid Sequence↗

PubMed2003

Production of laccase by Coriolus versicolor and its application in decolorization of dyestuffs: (II). Decolorization of dyes by laccase containing fermentation broth with or without self-immobilized mycelia.

The capability of decolorization for commercial dyes by Coriolus versicolor fermentation broth containing laccase with or without immobilized mycelium was evaluated. With cell-free fermentation broth containing laccase, high decolorization ratio was achieved foracid orange 7, but not for the other dyes concerned. The immobilized mycelium was proved to be more efficient than the cell-free system. All the four dyestuffs studied were found being decolourized with certain extent by immobilized mycelium. The repeated-batch decolorization was carried out with satisfactory results. The experimental data showed that the continuous decolorization of wastewater from a printing and dyeing industry was possible by using the self-immobilized C. versicolor.

Basidiomycota↗

PubMed2002

Protein microarrays to detect protein-protein interactions using red and green fluorescent proteins.

Proteomics, the study of protein function on a global scale, will play an important role in furthering our understanding of gene functions, complex biological pathways, and discovery of novel drug targets. A number of techniques have been developed for proteomic studies to identify and analyze proteins, compare protein expression levels, and study protein-protein interactions. Recent developments have applied a DNA array-type approach to immobilize proteins on a surface for high-throughput analysis. Here we report the development and construction of protein chips using derivatized glass and nitrocellulose-coated slides and the employment of recombinant proteins fused with green and red fluorescent proteins for detection. Fluorescent signals were found to be proportional to the amount of arrayed proteins and could be readily detected with a conventional fluorescence slide scanner. This technique allows the investigation of protein-protein interactions without the need for additional labeling steps of probe proteins.

Antibodies↗

PubMed2002

Crystallization and preliminary X-ray crystallographic studies on recombinant human carnitine acetyltransferase.

In this paper, the purification, crystallization and preliminary X-ray crystallographic studies of human carnitine acetyltransferase are reported. Recombinant human carnitine acetyltransferase crystals were grown by the hanging-drop vapor-diffusion method and belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 137.65, b = 84.76, c = 57.65 A and one molecule per asymmetric unit. The intensity data were collected from a cryocooled crystal to 1.6 A resolution using a conventional X-ray source.

Carnitine O-Acetyltransferase↗