Biomedical subjects

M Wu

Publications and source records attributed to M Wu.

At least 73 records · Page 4Linked to original sources

PubMed2001

Formation of a GNRA tetraloop in P5abc can disrupt an interdomain interaction in the Tetrahymena group I ribozyme.

The secondary structure of a truncated P5abc subdomain (tP5abc, a 56-nucleotide RNA) of the Tetrahymena thermophila group I intron ribozyme changes when its tertiary structure forms. We have now used heteronuclear NMR spectroscopy to determine its conformation in solution. The tP5abc RNA that contains only secondary structure is extended compared with the tertiary folded form; both forms coexist in slow chemical exchange (the interconversion rate constant is slower than 1 s(-1)) in the presence of magnesium. Kinetic experiments have shown that tertiary folding of the P5abc subdomain is one of the earliest folding transitions in the group I intron ribozyme, and that it leads to a metastable misfolded intermediate. Previous mutagenesis studies suggest that formation of the extended P5abc structure described here destabilize a misfolded intermediate. This study shows that the P5abc RNA subdomain containing a GNRA tetraloop in P5c (in contrast to the five-nucleotide loop P5c in the tertiary folded ribozyme) can disrupt the base-paired interdomain (P14) interaction between P5c and P2.

Animals↗

PubMed2001

[Genotyping of multiple genetic alterations of intrahepatic cholangiocarcinoma].

OBJECTIVE: To provide genetic evidence for understanding the mechanism of oncogenesis of intrahepatic cholangiocarcinoma (ICC) by detecting the multiple genetic spectrums. METHODS: Twenty-two cases of paraffin-embedded ICC tissue sections were microdissected and genotyped by PCR-based DNA sequencing to detect the frequencies of loss of heterozygosity (LOH) of tumor suppressor genes APC, MCC, DCC, OGG1, p53, and RB1 and of point mutation of Ki-ras-2 oncogene. RESULTS: The genetic alteration rate was 68.8% for APC, 46.2% for DCC, 41.7% for OGG1, 37.5% for p53, 27.3% for Ki-ras-2, 22.2% for RB1, and 14.3% for MCC. CONCLUSION: ICC-related genetic spectrum is basically composed of APC, DCC, OGG1, p53, and Ki-ras-2. Multiple genetic alteration plays an important role in the carcinogenesis of ICC.

Adenomatous Polyposis Coli Protein↗

PubMed2001

Cadmium response of the hairy root culture of the endangered species Adenophora lobophylla.

We generated hairy root cultures from two closely related species, Adenophora lobophylla and A. potaninii (Campanulaeae) and carried out a comparative study on their cadmium (Cd) response. A. lobophylla is an endangered species while A. potaninii is widely distributed in the same habitat. Upon exposure to Cd concentrations higher than 50 µM, more extensive growth inhibition and higher Cd accumulation were detected in the hairy root of A. lobophylla. Cd treatment affected the protein content in both the species. Phytochelatins (PCs) have been isolated and characterized from the hairy roots for both species. They shared structure similarities but showed different accumulation kinetics. The content of reduced glutathione (GSH) and cysteine (Cys) differs in both the species and they show different changes upon Cd challenge. The results suggested that these two species might employ different strategy for Cd detoxification. A. lobophylla is capable of synthesizing high level of PCs while a Cd exclusion system and a tighter homeostasis mechanism(s) to maintain the cellular GSH level could have been evolved in A. potanini in additon to its capability of synthesizing PCs.

Journal Article↗

PubMed2001

Gene expression profile changes in initiation and progression of squamous cell carcinoma of esophagus.

Tumorigenesis is a complex process involving multiple genes. As a step toward understanding the complicated changes between normal and malignant cells, this report focused on gene expression profile variations among normal and abnormal esophageal epithelium tissues. The cDNA microarray approach was used to investigate gene expression profiles of 5 different stages during initiation and progression of esophageal cancer. According to pathological characteristics, these 5 stages were normal, dysplasia I (mild dysplasia), dysplasia II (moderate dysplasia), carcinoma in situ (CIS) and squamous cell carcinoma of esophagus (SCC). Comparing and analyzing those gene expression profiles, we observed that the expression levels of many genes changed in dysplasia I and some known tumor-related genes were over-expressed or under-expressed in all 4 abnormal stages. Using principle component analysis we identified a set of genes that may play an important role in tumor development. Hybridization data were confirmed by semi-quantitative reverse transcription-polymerase chain reaction and immunohistochemistry. These results suggest that cDNA microarray technology is a useful tool to discover genes frequently involved in esophageal neoplasia and provides novel clues to diagnosis, early detection and intervention of SCC.

Carcinoma in Situ↗

PubMed2001

Cloning and characterization of full-length human ribosomal protein L15 cDNA which was overexpressed in esophageal cancer.

The aim of this investigation was trying to identify the genes differentially expressed in esophageal cancer. By combining suppression subtractive hybridization (SSH) with reverse Northern high density blots, a gene named EC45 was obtained, which dramatically overexpressed in 70% esophageal cancer (18/26). EC45 was mapped to 3p12-3p11.2 by radiation hybrid mapping (RH mapping). The putative full length EC45 cDNA (1987 bp) was identified by cDNA libraries screening of esophageal cancer. EC45 encoded 204 amino acids, and it shared a 100% similarity with ribosomal protein L15 (635 bp, mRNA) in ORF, but no similarity in 5' UTR or 3' UTR. Northern blot panel of multiple adult human normal tissues showed EC45 distributed in almost normal tissues tested. All these data suggested that EC45, encoding ribosomal protein L15 and overexpressing in esophageal cancer might play a possible role in carcinogenesis of esophagus.

Amino Acid Sequence↗

PubMed2001

The role of rigidity in DNA looping-unlooping by AraC.

We applied two experiments useful in the study of ligand-regulated DNA binding proteins to AraC, the dimeric regulator of the Escherichia coli l-arabinose operon. In the absence of arabinose, AraC prefers to loop DNA by binding to two half-sites that are separated by 210 base pairs, and in the presence of arabinose it prefers to bind to adjacently located half-sites. The basis for this ligand-regulated shift in binding appears to result from a shift in the rigidity of the system, where rigidity both in AraC protein in the absence of arabinose, and in the DNA are required to generate the free energy differences that produce the binding preferences. Eliminating the dimerization domains and connecting the two DNA binding domains of AraC by a flexible peptide linker should provide a protein whose behavior mimics that of AraC when there is no interaction between its dimerization and DNA binding domains. The resulting protein bound to adjacent half-sites on the DNA, like AraC protein in the presence of arabinose. When the two double-stranded DNA half-sites were connected by 24 bases of single-stranded, flexible DNA, wild-type AraC protein bound to the DNA in the presence and absence of arabinose with equal affinity, showing that AraC modulates its DNA binding affinity in response to arabinose by shifting the relative positions of its DNA binding domains. These results are consistent with the light switch mechanism for the action of AraC, refine the model, and extend the range of experimental tests to which it has been subjected.

Amino Acid Sequence↗

PubMed2001

Factors affecting quantitation of DNA bands in gels using a charge-coupled device imaging system.

Quantitation of DNA bands separated in polyacrylamide or agarose gels was tested under a variety of conditions to examine key factors contributing to the ability to obtain quantitative data. Variations tested included comparison of different fluorescent stains (ethidium bromide and GelStar stain), and variations of other parameters relating to the electrophoretic separation, e.g. gel and sample geometry, mode of staining, and mode of excitation of stains. The results showed that linear results were seen for a similar 20-30-fold range of DNA concentrations with both stains tested and that it is critical to standardize separations to obtain consistent results. Variations in separation and detection could lead to relatively large changes in fluorescent signals seen for similar amounts of DNA.

DNA↗

PubMed2001

Fatty acid composition of lysophosphatidic acid and lysophosphatidylinositol in plasma from patients with ovarian cancer and other gynecological diseases.

OBJECTIVE: We previously reported that plasma levels of total lysophosphatidic acid (LPA) represented a potential biomarker for ovarian cancer and other gynecological cancers [1]. However, total LPA is composed of different LPA species with distinct fatty acid chains. The major objective of the current study, therefore, was to determine whether one or more specific fatty acid LPA species was associated with disease or disease staging. If this was determined, these species could be useful in further improving the sensitivity and/or specificity of this biomarker for the diagnosis and/or prognosis of the disease. Because lysophosphatidylinositol (LPI) co-migrates with LPA, this study represents the analysis of combined molecular species from both lysolipid classes. METHODS: The patient population, sample collection, and analyses have been reported previously [1]. Lipids were hydrolyzed from the LPA band on thin-layer chromatography plates. The following individual fatty acid species were analyzed by gas chromatography: palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), arachidonic acid (20:4), and docosahexaenoic acid (22:6). The LPA/LPI fatty acid composition levels were analyzed and compared with disease status. RESULTS: Distinct plasma LPA/LPI fatty acid chain species were not associated with ovarian or other gynecological cancers, compared to patients with benign gynecological disease or healthy controls. However, an increased presence of unsaturated fatty acids in plasma LPA/LPI was found in patients with late-stage or recurrent ovarian cancer and possibly with other gynecological cancers. CONCLUSIONS: Analysis of individual fatty acid species present in plasma LPA/LPI do not appear to enhance the sensitivity or specificity of total LPA/LPI as a marker for gynecological cancer detection. However, our results suggest that increased LPA/LPI species with unsaturated fatty acid chains may be associated with late-stage or recurrent ovarian cancer.

Adult↗

PubMed2001

Studies on metal phthalocyanine as a dual functional mimic enzyme.

Four phthalocyanines (iron tetracarboxylphthalocyanine, copper tetracarboxylphthalocyanine, manganese tetracarboxylphthalocyanine, cobalt tetracarboxylphthalocyanine) were used as dual functional mimic enzymes of superoxide dismutase (SOD) and catalase (CAT). The first function, eliminating O2-, was proved by using riboflavine-methionine photoreduction method in the concentration range of 10(-5) to 10(-6) mol/L. The second function, clearing out H2O2, was demonstrated by means of spectrophotometry with the decomposing percentage being increased with the increase of the concentration of the imitating compounds. Measurements of metal phthalocyanines, SOD and CAT by the liver homogenate technique of mice showed that they had obvious action of decreasing the lipid peroxidation.

Animals↗

PubMed2001

Analysis of nuclear apoptotic process in a cell-free system.

We report an analysis of the apoptotic process of mouse liver nuclei induced in a cell-free carrot cytosol system by cytochrome c. Typical characteristics of apoptosis were observed, such as chromatin condensation, margination, apoptotic bodies and DNA ladders. Furthermore, transmission and scanning electron microscope analysis of the apoptotic nuclei detected chromatin-free nuclear vesicles before apoptotic bodies appeared at a comparatively late phase. When AC-YVAD-CHO, an inhibitor of caspase 6, was introduced into the system, these vesicles and apoptotic bodies disappeared completely within our study sections. We confirmed the results using whole-mount electron microscopy, and found that although the nuclear lamina was destroyed early, the nuclear matrix largely remained intact during the course of apoptosis. The nuclear matrix played an important role in maintaining the integrity of apoptotic cells and connecting the apoptotic bodies and apoptotic nucleus.

Animals↗

PubMed2001

Kaposi's sarcoma-associated herpesvirus/human herpesvirus-8 ORF50 gene product contains a potent C-terminal activation domain which activates gene expression via a specific target sequence.

The ART (Activator of Replication and Transcription) protein of Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus-8 (HHV-8), is encoded by the ORF50 gene. It is expressed as an immediate-early gene and plays a crucial role in the transition between latency and productive infection. HHV-8 ART is a transcriptional transactivator which can up-regulate viral gene expression. Transient expression assays showed that ART strongly activated ORF57 and K8 promoter-directed gene expression in both CV-1 and BJAB cells. The ART target site was mapped to a 40-bp region compassing nt 81904 to 81943 on the ORF57 promoter. When linked upstream to a heterologous SV40 promoter, this region by itself was able to confer ART responsiveness. This 40-bp segment contains a 16-bp consensus sequence which is also found in the K8 promoter region located between nt 74769 to 74784. Deletion of the fragment including this 16-bp consensus abrogated the ART responsiveness of the K8 promoter. The role of this 16-bp consensus in ART transactivation was further supported by site-directed mutagenesis. Mutations of the conserved nucleotides within the 16-bp consensus in the ORF57 promoter dramatically impaired its responsiveness to ART. Fusion protein analysis with chimeric proteins containing the DNA binding domain of yeast transactivator Gal4 (residues 1 to 147) and different ART segments defined an acidic C-terminal region (amino acids [aa] 527 to 634) as a potent activator. Deletions of this activation domain in the ART protein resulted in a decrease or loss of its ability to activate ORF57 and K8 promoters containing the ART responsive element in transfected cells. How the ART activation domain activates ORF57 and K8 gene expression through the 16-bp consensus sequence remains to be determined.

Basic-Leucine Zipper Transcription Factors↗

PubMed2001

A fast and efficient method for isolation of the BAC end.

As a new developmental vector system, the bacterial artificial chromosome (BAC) has been used widely in constructing genomic libraries and in generating transgenic animals. Isolation of the BAC insert end is useful to analyze the BAC clone. Here, we describe a fast and efficient method to obtain the BAC end by ligating the BAC fragments digested with Not I and another selected restriction enzyme into universal cloning vector, followed by determining the correct clones with HindIII digestion. Further DNA sequencing analysis verified the results mentioned above.

Chromosomes, Artificial, Bacterial↗

PubMed2001

Application of laser measuring, numerical simulation and rapid prototyping to titanium dental castings.

OBJECTIVES: This paper describes a method of making titanium dental crowns by means of integrating laser measuring, numerical simulation and rapid prototype (RP) manufacture of wax patterns for the investment casting process. METHODS: Four real tooth crowns (FDI No. 24, 25, 26, 27) were measured by means of 3D laser scanning. The laser digitized geometry of the crowns was processed and converted into standard CAD models in STL format, which is used by RP systems and numerical simulation software. Commercial software (MAGMASOFT) was used to simulate the casting process and optimize the runner and gating system (sprue) design. RP crowns were 'printed' directly on a ModelMaker II 3D Plotting System. A silicone negative mold (soft tool) was made from the RP crowns, then more than hundreds wax crowns were duplicated. The duplicated crowns were joined to the optimized runner and gating system. By using the investment casting process 20-25 replicas of each crown were made on a centrifugal casting machine. All castings were examined for porosity by X-ray radiographs. RESULTS: By using the integrated scanning, simulation, RP pattern and casting procedure, cast crowns, free of porosity, with excellent functional contour and a smooth surface finish, were obtained from the first casting trial. SIGNIFICANCE: The coupling of laser digitizing and RP indicates a potential to replace the traditional 'impression taking and waxing' procedure in dental laboratory, with the quality of the cast titanium prostheses also being improved by using the numerically optimized runner and gating system design.

Calcium Sulfate↗

PubMed2001

Characterization of the pharmacology, signal transduction and internalization of the fluorescent PACAP ligand, fluor-PACAP, on NIH/3T3 cells expressing PAC1.

Fluor-PACAP, a fluorescent derivative of PACAP-27, has been confirmed to share a high affinity for PAC1 receptors transfected into NIH/3T3 cells and to have comparable pharmacological characteristics to the unconjugated, native form. Through competitive binding with 125I-PACAP-27, the two ligands exhibited similar dose- dependent inhibition. Additional examination of the efficacy of activating adenylyl cyclase revealed that both ligands analogously stimulated the production of cyclic AMP. Furthermore, PAC1 internalization visualized by our Fluor-PACAP, is compareable to that performed with the radioligand, 125I-PACAP-27, with maximal internalization achieved within thirty minutes. Thus, Fluor-PACAP exhibits intracellular signaling abilities homologous to the native ligand.

3T3 Cells↗

PubMed2001

Furosemide alters nonlinear capacitance in isolated outer hair cells.

The outer hair cell (OHC) from the organ of Corti plays a crucial role in hearing through its unique voltage-dependent mechanical responses. Furosemide, one of the loop diuretics, disrupts normal cochlear function. Here we report on direct effects of furosemide on OHC motility-related, voltage-dependent capacitance using the whole-cell patch-clamp technique. Extracellularly applied furosemide reversibly shifted the voltage at peak capacitance (V(pkC(m))) to positive levels. The shift, whose maximum approached 90 mV, evidenced a Hill coefficient of 1.5 and K(1/2) of 10 mM. Changes in the magnitude of nonlinear capacitance were not fully reversible. While it is clear that the overwhelming effect of furosemide on hearing results via its effects on the endolymphatic potential, the present results indicate that furosemide directly alters OHC motility and may, in part, contribute to sensory dysfunction.

Animals↗

PubMed2001

Akt-mediated phosphorylation of the G protein-coupled receptor EDG-1 is required for endothelial cell chemotaxis.

The role of the protein kinase Akt in cell migration is incompletely understood. Here we show that sphingosine-1-phosphate (S1P)-induced endothelial cell migration requires the Akt-mediated phosphorylation of the G protein-coupled receptor (GPCR) EDG-1. Activated Akt binds to EDG-1 and phosphorylates the third intracellular loop at the T(236) residue. Transactivation of EDG-1 by Akt is not required for G(i)-dependent signaling but is indispensable for Rac activation, cortical actin assembly, and chemotaxis. Indeed, T236AEDG-1 mutant sequestered Akt and acted as a dominant-negative GPCR to inhibit S1P-induced Rac activation, chemotaxis, and angiogenesis. Transactivation of GPCRs by Akt may constitute a specificity switch to integrate rapid G protein-dependent signals into long-term cellular phenomena such as cell migration.

Actins↗

PubMed2001

Cyclohexene ring and Fjord region twist inversion in stereoisomeric DNA adducts of enantiomeric benzo[c]phenanthrene diol epoxides.

The sterically hindered, nonplanar fjord region polycyclic aromatic hydrocarbons (PAHs) have been of great interest because of the exceptionally high mutagenic and tumorigenic activity of certain of their metabolically activated diol epoxides. Benzo[c]phenanthrene (B[c]Ph), a representative fjord region PAH, is metabolically activated to a pair of enantiomers, 1S,2R,3R,4S-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene, (+)-anti-B[c]PhDE, and the corresponding 1R,2S,3S,4R enantiomer, (-)-anti-B[c]PhDE. Both of these can bind covalently to the amino group of purines in DNA via trans addition. In the present work we carry out an extensive computational investigation of the 1R(+) and 1S(-)-trans-anti-B[c]Ph adducts to the base guanine, with the goal of delineating the conformational possibilities for the fjord region and the adjacent cyclohexene-type benzylic ring and their relevance to DNA duplexes. We created 10 369 starting structures for each adduct and minimized the energy using AMBER 5.0. A limited set of conformational families is computed, in which the R isomer structures are near mirror images of the S isomer. The benzylic rings are essentially all half-chair-type. Cyclohexene-type ring inversion as well as fjord region twist inversion are possible for each isomer and are correlated. DNA duplexes modified by fjord region adducts select conformers from the allowed families that optimize stacking interactions, which contributes to the stability of the carcinogen-intercalated DNA duplex structures [Cosman et al. (1993) Biochemistry 32, 12488-12497; Cosman et al. (1995) Biochemistry 34, 1295-1307; Suri et al. (1999) J. Mol. Biol. 292, 289-307; Lin et al. (2001) J. Mol. Biol. 306, 1059-1080]. In turn, this stability could contribute to the resistance to repair by the human nucleotide excision system observed in fjord region adducts [Buterin et al. (2000) Cancer Res. 60, 1849-1856].

Cyclohexanes↗

PubMed2001

Numerical simulation of the casting process of titanium tooth crowns and bridges.

The objectives of this paper were to simulate the casting process of titanium tooth crowns and bridges; to predict and control porosity defect. A casting simulation software, MAGMASOFT, was used. The geometry of the crowns with fine details of the occlusal surface were digitized by means of laser measuring technique, then converted and read in the simulation software. Both mold filling and solidification were simulated, the shrinkage porosity was predicted by a "feeding criterion", and the gas pore sensitivity was studied based on the mold filling and solidification simulations. Two types of dental prostheses (a single-crown casting and a three-unit-bridge) with various sprue designs were numerically "poured", and only one optimal design for each prosthesis was recommended for real casting trial. With the numerically optimized design, real titanium dental prostheses (five replicas for each) were made on a centrifugal casting machine. All the castings endured radiographic examination, and no porosity was detected in the cast prostheses. It indicates that the numerical simulation is an efficient tool for dental casting design and porosity control.

Journal Article↗