Biomedical subjects

Min-Jen Tseng

Publications and source records attributed to Min-Jen Tseng.

9 recordsLinked to original sources

PubMed2006

Using surface plasmon resonance to directly measure slow binding of low-molecular mass inhibitors to a VanX chip.

VanX, a d,d-dipeptidase, is one of five gene products responsible for vancomycin resistance in pathogenic bacteria and is an attractive drug target in circumventing clinical drug resistance. Our previous combinatorial search of VanX substrates in a dipeptide library of d-X(1)-d-X(2) (19(2)=361) forms has led to the discovery of three new compounds (d-Ala-d-Phe, d-Ala-d-Tyr, and d-Ala-d-Trp) having higher k(cat)/K(M) values than those of its natural substrate, d-Ala-d-Ala. Based on structures of newly identified substrates, two representative transition state analogs of substrates, d-Ala(P,O)d-Phe (6a) and d-Ala(P,O)d-Ala (6b) dipeptide phosphonates, used as VanX inhibitor were rationally designed and chemically synthesized. In the synthesis, eight synthetic steps in total were employed for preparing each VanX inhibitor, and their overall isolated yields were 21 and 11% for 6a and 6b, respectively. Binding interactions of d-Ala(P,O)d-Phe (6a) and d-Ala(P,O)d-Ala (6b) with VanX were confirmed unambiguously and measured quantitatively by surface plasmon resonance. The result reveals that both dipeptide phosphonates are slow-binding inhibitors of VanX (for 6a, k(on)=1.18 x 10(3)M(-1)s(-1), k(off)=2.31 x 10(-3) s(-1), K(D)=1.96 microM, chi(2)=0.0737; for 6b, k(on)=1.09 x 10(3)M(-1)s(-1), k(off)=1.80 x 10(-2)s(-1), K(D)=16.5 microM, chi(2)=0.0599). This suggests that only a fraction of the conformers of the inhibitors in solution adopts a conformation best suited for binding interaction with VanX and that the VanX-inhibitor complex may concomitantly undergo a conformational isomerization from an initial but fast weak-binding adduct to slowly convert to a tight-binding complex with a more stable bound geometry. Moreover, in comparison with 6b, an additional aromatic interaction of 6a with the Phe79 residue in the active site of the enzyme, through an energetically favorable face-to-face offset stacked orientation, may account for its higher affinity than 6b to VanX.

Bacterial Proteins↗

PubMed2006

A novel follicle-stimulating hormone-induced G alpha h/phospholipase C-delta1 signaling pathway mediating rat sertoli cell Ca2+-influx.

FSH is known to activate Gs/cAMP signaling pathway in Sertoli cells (SCs) to support spermatogenesis. However, the molecular mechanism of FSH-induced Gs/cAMP-independent Ca2+-influx in SCs is not clear. In this study, FSH indeed induced an immediate and dose-dependent intracellular Ca2+-elevation in rat SCs. In the presence of EDTA (2.5 mm) or in the absence of extracellular Ca2+, the FSH-induced intracellular Ca2+-elevation was abolished. The confocal microscopic observation of Ca2+ image revealed that the SC cellular Ca2+ level was gradually increased after 50 sec of FSH treatment. Dantrolene, a blocker of intracellular Ca2+ release, did not affect this FSH-induced intracellular Ca2+ elevation. The pretreatment of rat SCs with phosphatidylinositol-phospholipase C (PLC)-specific inhibitor, U73122 (3 and 10 microm), inhibited the FSH-induced Ca2+-influx in a dose-dependent manner, but treatment with Gs-specific inhibitor, NF449 (0.1 and 0.3 microm), did not. On the other hand, the activation of G alpha h was immediately induced by FSH in the rat SCs within 5 sec of treatment. The translocation of PLC-delta1 from cytosol to cell membrane and the formation of G alpha h /PLC-delta1 complexes occurred within 5 and 10 sec, respectively, of FSH exposure. The intracellular inositol 1,4,5-triphosphate (IP3) production was also detected after 30 sec of FSH treatment. The synthetic peptide of PLC-delta1 (TIPWNSLKQGYRHVHLL), not Gs inhibitor, predominantly inhibited the FSH-induced PLC-delta1 translocation, formation of G alpha h /PLC-delta1 complex, intracellular IP3 production, and Ca2+ influx. In contrast, the peptide did not interfere with FSH-induced intracellular cAMP accumulation. In conclusion, the FSH-induced immediate Ca2+ influx is unambiguously mediated by an alternative G alpha h /PLC-delta1/IP3 pathway that is distinct from the Gs/cAMP pathway in rat SCs.

Animals↗

PubMed2006

Identification of a new chromophoric substrate in the library of amino acid p-nitroanilides for continuous assay of VanX, a D,D-dipeptidase essential for vancomycin resistance.

As one of key bacterial proteins involved in vancomycin resistance, VanX is a D,D-dipeptidase that impedes bacterial cell wall biosynthesis by hydrolyzing the essential D-Ala-D-Ala dipeptide. Based on a report by Crowder and co-workers that L-alanine-p-nitroanilide (L-Ala-pNA) was a useful substrate for continuous assay of VanX, we constructed a library of 35 L- and D-amino acid p-nitroanilides to provide the needed diversity to discover new substrates that are more specific than L-Ala-pNA. We report here that, among all compounds tested, D-leucine-p-nitroanilide (D-Leu-pNA) was found to be the best substrate for VanX enzyme (KM=8.9+/-1.2 mM, kcat=0.0102+/-0.0016 s(-1), kcat/KM=0.0012 mM(-1)s(-1)). Although it is catalytically inefficient, this new VanX substrate needs essentially no sophisticated synthetic chemistry for preparation and therefore offers a convenient means for routine analysis of enzyme catalysis and the screening of potential inhibitors. Moreover, because it is the uncommon leucine in its D form in D-Leu-pNA, enzymatic activities due to other contaminated species in Escherichia coli used for VanX overproduction should be greatly reduced.

Anilides↗

PubMed2005

Protein microarray using alpha-amino acids as metal tags on chips.

Procedures for synthesizing alpha-amino acids on a chip for coordination with transitional metal ions and a His-Tagged protein have been successfully developed as a stable protein microarray. Using the recombinant His-Tagged 3CL-protease (3CLpro) as a model for attachment to chips containing D-/L-Glu, Asp, Orn, Ser via different transitional metal ions, it was found that the Orn chip was the best of affinity binding and stability by which Zn2+ was the best metal ion for affinity while Co2+ was the best metal ion for stability. Thus, this protein microarray can be alternatively used as a high throughput screening method for rapid detection against SARS CoV 3CLpro and/or efficient purification of other Tagged proteins.

Amino Acids↗

PubMed2004

Nuclear betaII-tubulin associates with the activated notch receptor to modulate notch signaling.

The Notch signal pathway plays important roles in proliferation, apoptosis, and differentiation. Abnormalities in Notch signaling are linked to many human diseases. After ligand binding, Notch signaling is activated through the cleavage of Notch receptors to release and translocate the Notch intracellular domain into the nucleus. The Notch1 receptor intracellular domain (N1IC), the activated form of the Notch1 receptor, can modulate downstream target genes via C promoter-binding factor 1-dependent and -independent pathways. To further dissect the Notch1 signaling pathway, we screened the N1IC-associated proteins using a yeast two-hybrid system and identified nuclear beta(II)-tubulin as a candidate for the N1IC-associated proteins. It was suggested that the presence of beta(II)-tubulin in nuclei might be correlated with the cancerous state of cells. However, the function of beta(II)-tubulin locating in the nucleus still is unknown. Herein, we show that the complex of alpha- and beta(II)-tubulin is associated with N1IC in cancer cells by a coimmunoprecipitation analysis. The ankyrin domain of the Notch1 receptor alone was sufficient to associate with beta(II)-tubulin. Furthermore, alpha- and beta(II)-tubulin were localized in the nucleus and formed a complex with N1IC. Treatment with Taxol increased the amounts of nuclear alpha- and beta(II)-tubulin in K562 and HeLa cells and promoted the C promoter-binding factor 1-dependent transactivation activity of N1IC. We also show that nuclear beta(II)-tubulin was bound on the C promoter-binding factor 1 response elements via the association with N1IC. These results suggest that nuclear beta(II)-tubulin can modulate Notch signaling through interaction with N1IC in cancer cells.

Cell Line, Tumor↗

PubMed2004

Cloning and characterization of two thermostable xylanases from an alkaliphilic Bacillus firmus.

Two genes encoding thermostable xylanases, named xyn10A and xyn11A, from an alkaliphilic Bacillus firmus were cloned and expressed in Escherichia coli. The E. coli harboring either gene showed clear zone with Congo red clearance assay on xylan plate. The Xyn10A and Xyn11A have molecular weights of 45 and 23kDa, respectively, and both show activities on xylan-zymogram. The xyn10A encodes 396 amino acid residues and is very similar to an alkaliphilic xylanase A from alkaliphilic Bacillus halodurans. The Xyn11A contains 210 amino acid residues and only one amino acid different from an endo-beta-1,4-xylanase from B. halodurans. From alignment of the amino acid sequences with other xylanases, Xyn10A and Xyn11A belong to family 10 and 11 glycosyl hydrolases, respectively. Both show activities over the pH range of 4-11 at 37 degrees C and over 80% activities at 70 degrees C. Interestingly both still retain over 70% activities after 16h preincubation at 62 degrees C.

Amino Acid Sequence↗

PubMed2004

Capillary electrophoresis using immobilized whole cells with overexpressed endothelin receptor for specific ligand screening.

A new capillary electrophoresis method using immobilized cells as the stationary phase has been developed. The power of this method is demonstrated by the separation and identification of endothelin antagonists on a capillary column coated by the transfected Chinese hamster ovary (CHO) cells with overexpressing endothelin receptors. The screening results are validated by functional assays suppressing the increase of intracellular calcium concentration induced by endothelin-1. Instead of making efforts in isolating protein receptors, the easily prepared whole-cell capillary column provides a superior tool on the basis of ligand/receptor affinity for a rapid screening of potent drug candidates from compound libraries.

Amino Acid Sequence↗

PubMed2003

Association of transcription factor YY1 with the high molecular weight Notch complex suppresses the transactivation activity of Notch.

Notch receptors are evolutionarily conserved from Drosophila to human and play important roles in cell fate decisions. After ligand binding, Notch receptors are cleaved to release their intracellular domains. The intracellular domains, the activated form of Notch receptors, are then translocated into the nucleus where they interact with other transcriptional machinery to regulate the expression of cellular genes. To dissect the molecular mechanisms of Notch signaling, the cellular targets that interact with Notch1 receptor intracellular domain (N1IC) were screened. In this study, we found that endogenous transcription factor Ying Yang 1 (YY1) was associated with exogenous N1IC in human K562 erythroleukemic cells. The ankyrin (ANK) domain of N1IC and zinc finger domains of YY1 were essential for the association of N1IC and YY1 according to the pull-down assay of glutathione S-transferase fusion proteins. Furthermore, both YY1 and N1IC were present in a large complex of the nucleus to suppress the luciferase reporter activity transactivated by Notch signaling. The transcription factor YY1 indirectly regulated the transcriptional activity of the wild-type CBF1-response elements via the direct interaction of N1IC and CBF1. We also demonstrated the association between endogenous N1IC and intrinsic YY1 in human acute T-cell lymphoblastic leukemia cell lines. Taken together, these results indicate that transcription factor YY1 may modulate Notch signaling via association with the high molecular weight Notch complex.

Avian Proteins↗

PubMed2002

Biomic study of human myeloid leukemia cells differentiation to macrophages using DNA array, proteomic, and bioinformatic analytical methods.

A biomic approach by integrating three independent methods, DNA microarray, proteomics and bioinformatics, is used to study the differentiation of human myeloid leukemia cell line HL-60 into macrophages when induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Analysis of gene expression changes at the RNA level using cDNA against an array of 6033 human genes showed that 5950 (98.6%) of the genes were expressed in the HL-60 cells. A total of 624 genes (10.5%) were found to be regulated during HL-60 cell differentiation. Most of these genes have not been previously associated with HL-60 cells and include genes encoded for secreted proteins as well as genes involved in cell adhesion, signaling transduction, and metabolism. Protein analysis using two-dimensional gel electrophoresis showed a total of 682 distinct protein spots; 136 spots (19.9%) exhibited quantitative changes between HL-60 control and macrophages. These differentially expressed proteins were identified by mass spectrometry. We developed a bioinformatics program, the Bulk Gene Search System (BGSS, http://www.sinica.edu.tw:8900/perl/genequery.pl) to search for the functions of genes and proteins identified by cDNA microarrays and proteomics. The identified regulated proteins and genes were classified into seven groups according to subcellular locations and functions. This powerful holistic biomic approach using cDNA microarray, proteomics coupled to bioinformatics can provide in-depth information on the impact and importance of the regulated genes and proteins for HL-60 differentiation.

Cell Differentiation↗