Browsing by Author "Wang, Yu-Kuo"

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AC electrokinetics assisted impedance biosensors for rapid bacteria detection
(2012)
Ho, Yi-Tse
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Pan, Chun-Liang
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Sheu, Jeng-Tzong
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Wang, Yu-Kuo
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Wu, Tung-Kung
Early detection of living bacteria attracts a lot of attention in infection diseases applications, especially at low concentration. Nowadays, the detection of specific bacteria faces challenges including enhancement of trapping efficiency, and increase of sensor sensitivity and specificity. In this study, we improved the trapping efficiency and increased sensor sensitivity and specificity by using AC electrokinetics and selective modification of antibody on gold electrode in a new structure of gold electrode design. COMSOL simulations of AC electrokinetics include dielectrophoresis (DEP), and AC electroosmosis (ACEO) were performed on different electrode designs and bias configurations. The simulation results were consistent with the fluorescence observations showing that the bacteria were trapping selectively on gold electrodes. The impedance measurements for Vibrio parahaemolyticus (Vp) detection at 10(5) cfu/ml with AC elctrokinetics showed a nearly 90% change in normalized impedance change (NIC).
AMP-Activated Protein Kinase Directly Phosphorylates and Destabilizes Hedgehog Pathway Transcription Factor GLI1 in Medulloblastoma
(2015-07-28)
Li, Yen-Hsing
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Luo, Jia
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Mosley, Yung-Yi C.
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Hedrick, Victoria E.
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Paul, Lake N.
The Hedgehog (Hh) pathway regulates cell differentiation and proliferation during development by controlling the Gli transcription factors. Cell fate decisions and progression toward organ and tissue maturity must be coordinated, and how an energy sensor regulates the Hh pathway is not clear. AMP-activated protein kinase (AMPK) is an important sensor of energy stores and controls protein synthesis and other energy-intensive processes. AMPK is directly responsive to intracellular AMP levels, inhibiting a wide range of cell activities if ATP is low and AMP is high. Thus, AMPK can affect development by influencing protein synthesis and other processes needed for growth and differentiation. Activation of AMPK reduces GLI1 protein levels and stability, thus blocking Sonic-hedgehog-induced transcriptional activity. AMPK phosphorylates GLI1 at serines 102 and 408 and threonine 1074. Mutation of these three sites into alanine prevents phosphorylation by AMPK. This leads to increased GLI1 protein stability, transcriptional activity, and oncogenic potency.
Angiotensin I-Converting enzyme insertion-related genotypes and allele are associated with higher susceptibility of endometriosis and leiomyoma
(2007-07-01)
Hsieh, Yao-Yuan
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Lee, Cheng-Chun
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Chang, Chi-Chen
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Wang, Yu-Kuo
;
Yeh, Lian-Shun
Endometriosis and leiomyoma display features similar to malignancy, requiring neovascularization to proliferation and growth. Altered vascular-related genes might be related to the development of endometriosis and leiomyoma. Polymorphisms of the angiotensin-converting enzyme (ACE) insertion/deletion (I/D) genes have been linked with some vascular diseases. This study investigates whether ACE I/D gene polymorphisms could be used as markers of susceptibility in endometriosis and leiomyoma. Women were divided into three groups: (4) endometriosis (n = 125); (2) leiomyoma (n = 120); () normal controls (n = 128). Genomic DNA was obtained from peripheral leukocyte. ACE I/D gene polymorphisms in intron 16 were amplified by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) Genotypes and allelic frequencies in both groups were compared. We observed the genotype distribution and allele frequency of ACE I/ D gene polymorphisms in both groups were significantly different. Proportions of ACE(star)I homozygote/ heterozygote/D homozygote in both groups were: (1) 50.4/24/25.6%; (2) 25/23.33/51.67%; (3) 10.2/ 29.7/60.1%. Proportions of I/D alleles in each group Were: (1) 62.4/37.6%; (2) 36.7/63.3%; (3) 25/75%. We concluded that ACE(star)I/D gene polymorphisms are associated with endometriosis and leiomyoma suspeptibilities. ACE(star)I-related genotypes and allele are strongly related to the occurrence of endometriosis and moderately related to the occurrence of leiomyoma.
Characterization and epitope mapping of Dengue virus type 1 specific monoclonal antibodies
(2017-10-02)
Chen, Wen-Hung
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Chou, Feng-Pai
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Wang, Yu-Kuo
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Huang, Sheng-Cih
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Cheng, Chuan-Hung
Background: Dengue virus (DV) infection causes a spectrum of clinical diseases ranging from dengue fever to a life-threatening dengue hemorrhagic fever. Four distinct serotypes (DV1-4), which have similar genome sequences and envelope protein (E protein) antigenic properties, were divided. Among these 4 serotypes, DV1 usually causes predominant infections and fast diagnosis and effective treatments are urgently required to prevent further hospitalization and casualties. Methods: To develop antibodies specifically targeting and neutralizing DV1, we immunized mice with UV-inactivated DV1 viral particles and recombinant DV1 E protein from residue 1 to 395 (E395), and then generated 12 anti-E monoclonal antibodies (mAbs) as the candidates for a series of characterized assays such as ELISA, dot blot, immunofluorescence assay, western blot, and foci forming analyses. Results: Among the mAbs, 10 out of 12 showed cross-reactivity to four DV serotypes as well as Japanese encephalitis virus (JEV) in different cross-reactivity patterns. Two particular mAbs, DV1-E1 and DV1-E2, exhibited strong binding specificity and neutralizing activity against DV1 and showed no cross-reactivity to DV2, DV3, DV4 or JEV-infected cells as characterized by ELISA, dot blot, immunofluorescence assay, western blot, and foci forming analyses. Using peptide coated indirect ELISA, we localized the neutralizing determinants of the strongly inhibitory mAbs to a sequence-unique epitope on the later-ridge of domain III of the DV1 E protein, centered near residues T346 and D360 ((346)TQNGRLITANPIVTD(360)). Interestingly, the amino acid sequence of the epitope region is highly conserved among different genotypes of DV1 but diverse from DV2, DV3, DV4 serotypes and other flaviviruses. Conclusions: Our results showed two selected mAbs DV1-E1 and DV1-E2 can specifically target and significantly neutralize DV1. With further research these two mAbs might be applied in the development of DV1 specific serologic diagnosis and used as a feasible treatment option for DV1 infection. The identification of DV1 mAbs epitope with key residues can also provide vital information for vaccine design.
Chromosome 15q21-22-Related Polymorphisms and Haplotypes Are Associated with Susceptibility to Type-2 Diabetic Nonproliferative Retinopathy
(2012-05-01)
Hsieh, Yao-Yuan
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Huang, Yu-Chuen
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Chang, Chi-Chen
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Wang, Yu-Kuo
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Lin, Wen-Hsin
Objective: Diabetic retinopathy (DR) is a microvascular complication of diabetes with a complex multifactorial pathogenesis. We aimed to investigate whether chromosome 15q21-22-related gene polymorphisms could be used as markers of DR susceptibility in type 2 diabetic (T2D) individuals. Methods: Individuals were divided into three groups: (1) T2D with nonproliferative DR (NPDR; n = 102); (2) T2D with proliferative DR (PDR; n = 72); (3) T2D without DR (n = 573). Six single-nucleotide polymorphisms (SNPs) (rs7174997, rs3751624, rs8025011, rs17818837, rs2922220, and rs2414520) lying within chromosome 15q21-22 region were genotyped by using Illumina HumanHap550-Duo BeadChips. Genotypes/allelic frequencies and haplotypes for these polymorphisms in each group were compared. Results: The MYO5C related SNP (rs3751624)*A related genotype and allele are associated with higher susceptibilities to DR, including PDR and NPDR. The rs3751624*GG/AA + AG percentages in each group are (1) 75.5%/24.5%, (2) 73.6%/26.4%, and (3) 82.5%/17.5%. In contrast, the other five SNPs in each group were not significantly different. One haplotype (G-A-G-G-T-G) appears significantly different between T2D individuals with and without DR. Other haplotype distributions were not significantly different between each group. Conclusion: The MYO5C related SNP (rs3751624)*A related genotype/allele and haplotype (G-A-G-G-T-G) might be associated with susceptibility for retinopathy in T2D individuals. Some chromosome 15q21-22* related genetic variations might contribute to the pathogenesis of DR.
Crystal Structure and Inhibition Studies of Transglutaminase from Streptomyces mobaraense
(2011-03-04)
Yang, Ming-Te
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Chang, Cheng-Hsiang
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Wang, Jiou Ming
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Wu, Tung Kung
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Wang, Yu-Kuo
The crystal structure of the microbial transglutaminase (MTGase) zymogen from Streptomyces mobaraense has been determined at 1.9-angstrom resolution using the molecular replacement method based on the crystal structure of the mature MTGase. The overall structure of this zymogen is similar to that of the mature form, consisting of a single disk-like domain with a deep active cleft at the edge of the molecule. A major portion of the prosequence (45 additional amino acid residues at the N terminus of the mature transglutaminase) folds into an L-shaped structure, consisting of an extended N-terminal segment linked with a one-turn short helix and a long alpha-helix. Two key residues in the short helix of the prosequence, Tyr-12 and Tyr-16, are located on top of the catalytic triad (Cys-110, Asp-301, and His-320) to block access of the substrate acyl donors and acceptors. Biochemical characterization of the mature MTGase, using N-alpha-benzyloxycarbonyl-L-glutaminylglycine as a substrate, revealed apparent K(m) and k(cat)/K(m) values of 52.66 mM and 40.42 mM(-1) min(-1), respectively. Inhibition studies using the partial prosequence SYAETYR and homologous sequence SQAETYR showed a noncompetitive inhibition mechanism with IC(50) values of 0.75 and 0.65 mM, respectively, but no cross-linking product formation. Nevertheless, the prosequence homologous oligopeptide SQAETQR, with Tyr-12 and Tyr-16 each replaced with Gln, exhibited inhibitory activity with the formation of the SQAETQR-monodansylcadaverine fluorophore cross-linking product (SQAETQR-C-DNS). MALDI-TOF tandem MS analysis of SQAETQR-C-DNS revealed molecular masses corresponding to those of (N)SQAETQ(C)-C-DNS and C-DNS-(N)QR(C) sequences, suggesting the incorporation of C-DNS onto the C-terminal Gln residue of the prosequence homologous oligopeptide. These results support the putative functional roles of both Tyr residues in substrate binding and inhibition.
Crystal Structure and Mutational Analysis of Aminoacylhistidine Dipeptidase from Vibrio alginolyticus Reveal a New Architecture of M20 Metallopeptidases
(2010-12-01)
Chang, Chin-Yuan
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Hsieh, Yin-Cheng
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Wang, Ting-Yi
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Chen, Yi-Chin
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Wang, Yu-Kuo
Aminoacylhistidine dipeptidases (PepD, EC 3.4.13.3) belong to the family of M20 metallopeptidases from the metallopeptidase H clan that catalyze a broad range of dipeptide and tripeptide substrates, including L-carnosine and L-homocarnosine. Homocarnosine has been suggested as a precursor for the neurotransmitter gamma-aminobutyric acid (GABA) and may mediate the antiseizure effects of GABAergic therapies. Here, we report the crystal structure of PepD from Vibrio alginolyticus and the results of mutational analysis of substrate-binding residues in the C-terminal as well as substrate specificity of the PepD catalytic domain-alone truncated protein PepD(CAT). The structure of PepD was found to exist as a homodimer, in which each monomer comprises a catalytic domain containing two zinc ions at the active site center for its hydrolytic function and a lid domain utilizing hydrogen bonds between helices to form the dimer interface. Although the PepD is structurally similar to PepV, which exists as a monomer, putative substrate-binding residues reside in different topological regions of the polypeptide chain. In addition, the lid domain of the PepD contains an "extra" domain not observed in related M20 family metallopeptidases with a dimeric structure. Mutational assays confirmed both the putative di-zinc allocations and the architecture of substrate recognition. In addition, the catalytic domain-alone truncated PepD(CAT) exhibited substrate specificity to L-homocarnosine compared with that of the wild-type PepD, indicating a potential value in applications of PepD(CAT) for GABAergic therapies or neuroprotection.
Crystal Structure and Mutational Analysis of Aminoacylhistidine Dipeptidase from Vibrio alginolyticus Reveal a New Architecture of M20 Metallopeptidases
(2010-12-01)
Chang, Chin-Yuan
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Hsieh, Yin-Cheng
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Wang, Ting-Yi
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Chen, Yi-Chin
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Wang, Yu-Kuo
Aminoacylhistidine dipeptidases (PepD, EC 3.4.13.3) belong to the family of M20 metallopeptidases from the metallopeptidase H clan that catalyze a broad range of dipeptide and tripeptide substrates, including L-carnosine and L-homocarnosine. Homocarnosine has been suggested as a precursor for the neurotransmitter gamma-aminobutyric acid (GABA) and may mediate the antiseizure effects of GABAergic therapies. Here, we report the crystal structure of PepD from Vibrio alginolyticus and the results of mutational analysis of substrate-binding residues in the C-terminal as well as substrate specificity of the PepD catalytic domain-alone truncated protein PepD(CAT). The structure of PepD was found to exist as a homodimer, in which each monomer comprises a catalytic domain containing two zinc ions at the active site center for its hydrolytic function and a lid domain utilizing hydrogen bonds between helices to form the dimer interface. Although the PepD is structurally similar to PepV, which exists as a monomer, putative substrate-binding residues reside in different topological regions of the polypeptide chain. In addition, the lid domain of the PepD contains an "extra" domain not observed in related M20 family metallopeptidases with a dimeric structure. Mutational assays confirmed both the putative di-zinc allocations and the architecture of substrate recognition. In addition, the catalytic domain-alone truncated PepD(CAT) exhibited substrate specificity to L-homocarnosine compared with that of the wild-type PepD, indicating a potential value in applications of PepD(CAT) for GABAergic therapies or neuroprotection.
Expression and characterization of the biofilm-related and carnosine-hydrolyzing aminoacylhistidine dipeptidase from Vibrio alginolyticus
(2008-10-01)
Wang, Ting-Yi
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Chen, Yi-Chin
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Kao, Liang-Wei
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Chang, Chin-Yuan
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Wang, Yu-Kuo
The biofilm-related and carnosine-hydrolyzing aminoacylhistidine dipeptidase (pepD) gene from Vibrio alginolyticus was cloned and sequenced. The recombinant PepD protein was produced and biochemically characterized and the putative active-site residues responsible for metal binding and catalysis were identified. The recombinant enzyme, which was identified as a homodimeric dipeptidase in solution, exhibited broad substrate specificity for Xaa-His and His-Xaa dipeptides, with the highest activity for the His-His dipeptide. Sequence and structural homologies suggest that the enzyme is a member of the metal-dependent metallopeptidase family. Indeed, the purified enzyme contains two zinc ions per monomer. Reconstitution of His.Tag-cleaved native apo-PepD with various metal ions indicated that enzymatic activity could be optimally restored when Zn(2+) was replaced with other divalent metal ions, including Mn(2+), Co(2+), Ni(2+), Cu(2+) and Cd(2+), and partially restored when Zn(2+) was replaced with Mg(2+). Structural homology modeling of PepD also revealed a 'catalytic domain' and a 'lid domain' similar to those of the Lactobacillus delbrueckii PepV protein. Mutational analysis of the putative active-site residues supported the involvement of His80, Asp119, Glu150, Asp173 and His461 in metal binding and Asp82 and Glu149 in catalysis. In addition, individual substitution of Glu149 and Glu150 with aspartic acid resulted in the partial retention of enzymatic activity, indicating a functional role for these residues on the catalysis and zinc ions, respectively. These effects may be necessary either for the activation of the catalytic water molecule or for the stabilization of the substrate-enzyme tetrahedral intermediate. Taken together, these results may facilitate the design of PepD inhibitors for application in antimicrobial treatment and antibody-directed enzyme prodrug therapy.
Human lymphocyte antigen B-associated transcript 2, 3, and 5 polymorphisms and haplotypes are associated with susceptibility of Kawasaki disease and coronary artery aneurysm (vol 24, pg 262, 2010)
(2011)
Hsieh, Yao-Yuan
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Lin, Ying-Ju
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Chang, Chi-Chen
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Chen, Da-Yuan
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Hsu, Chin-Mu
Identification of a Vibtio furnissii oligopeptide permease and characterization of its in vitro hemolytic activity
(2007-11-01)
Wu, Tung-Kung
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Wang, Yu-Kuo
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Chen, Yi-Chin
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Feng, Jen-Min
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Liu, Yen-Hsi
We describe purification and characterization of an oligopeptide permease protein (Hly-OppA) from Vibrio furnissii that has multifaceted functions in solute binding, in in vitro hemolysis, in antibiotic resistance, and as a virulence factor in bacterial pathogenesis. The solute-binding function was revealed by N-terminal and internal peptide sequences of the purified protein and was confirmed by discernible effects on oligopeptide binding, by accumulation of fluorescent substrates, and by fluorescent substrate-antibiotic competition assay experiments. The purified protein exhibited host-specific in vitro hemolytic activity against various mammalian erythrocytes and apparent cytotoxicity in CHO-K1 cells. Recombinant Hly-OppA protein and an anti-Hly-OppA monoclonal antibody exhibited and neutralized the in vitro hemolytic activity, respectively, which further confirmed the hemolytic activity of the gene product. In addition, a V. furnissii hly-oppA knockout mutant caused less mortality than the wild-type strain when it was inoculated into BALB/c mice, indicating the virulence function of this protein. Finally, the in vitro hemolytic activity was also confirmed with homologous ATP-binding cassette-type transporter proteins from other Vibrio species.
Identification of novel peptides specifically binding to endometriosis by screening phage-displaying peptide libraries
(2009-12-01)
Chang, Chi-Chen
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Hsieh, Yao-Yuan
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Wang, Yu-Kuo
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Hsu, Kung-Hao
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Tsai, Horng-Der
Objective: To search for novel peptides and common binding motif that specifically bind to endometriosis. Design: Prospective study. Setting: Department of Biological Science and Technology in national university. Patient(s): Specimens were divided into [1] ectopic endometrium (n = 10); [2] eutopic endometrium (n = 10). Intervention(s): Peptides specifically binding to endometriosis are screened from a phage-displaying peptide library (Ph.D.-12) by using whole-cell screening technique after an adsorption elution amplification procedure. Main Outcome Measure(s): Combinatorial peptide libraries were used to identify small molecules that bind with high affinity to receptor molecules and mimic the interaction with natural ligands. Few pans of positive phage clones with significantly positive signals were identified by ELISA and analyzed by DNA sequencing. Result(s): During the biopanning processes, the recovered phage number (10(6) pfu/mL) in parts 1, 2, 3, 4, and 5 of the study were 9, 33, 82, 142, and 169. Nine phages consistently had residue Arg, whereas six clones had a consensus motif of Arg-X-Arg-X-X-X-X-Arg. The biotin-labeled peptide bound to endometriosis cells in a dose-dependent manner, yet the control peptide revealed lesser binding activity. Conclusion(s): The novel motif is associated with higher affinity of endometriosis, which might be useful in endometriosis targeting and as potential antiendometriosis therapies. We provide one potential approach for novel therapies toward endometriosis. (Fertil Steril (R) 2009;92:1850-5. (C)2009 by American Society for Reproductive Medicine.)
Immunocapture couples with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for rapid detection of type 1 dengue virus
(2013-05-03)
Chen, Wen-Hung
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Hsu, I-Hsiang
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Sun, Yuh-Chang
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Wang, Yu-Kuo
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Wu, Tung-Kung
A facile method for accurate detection of type I dengue virus (DV1) infection from complex biological mixtures, using type specific immunocapture coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), was developed. The biological mixtures were treated with magnetic particles coated with a monoclonal antibody directly against type 1 dengue virus. After immunocapture purification, the DV1 was eluted with 30% acetic acid, directly spotted with seed-layer method, and analyzed by MALDI-TOF MS for DV1 capsid protein. The detection limit of the assay was similar to 10(5) pfu/mL by MALDI-TOF MS. The immunocapture could unambiguously differentiate the DV1 from other serotypes of the dengue viruses and Japanese encephalitis virus, and could be used as a specific probe to detect DV1 from complex biological mixtures. (C) 2013 Elsevier B.V. All rights reserved.
Insulin-like Growth Factors II exon 9 and E-cadherin-Pml I but not Myeloperoxidase Promoter-463, Urokinase-ApaL I nor Xeroderma Pigmentosum Polymorphisms Are Associated with Higher Susceptibility to Leiomyoma
(2010-06-01)
Hsieh, Yao-Yuan
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Chang, Chi-Chen
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Wang, Yu-Kuo
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Hsu, Kung-Hao
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Chen, Chih-Ping
Objectives: To investigate the roles of insulin-like growth factor II (IGF2), myeloperoxidase (MPO), E-cadherin (CDH1), urokinase and xeroderma pigmentosum group A and D (XPA, XPD) polymorphisms upon leiomyoma susceptibility. Study Design: Women were divided into: group I, leiomyoma (n=158); group 2, non-leiomyoma (n=156). Polymorphisms (IGF2 exon 9*A/G, MPO-463*A/G, CDH1-Pml I, urokinase-ApaL, XPA*A-23G, XPD*Lys751Gln) were amplified by polymerase chain reaction and detected by electrophoresis after restriction enzyme digestion. Genotype and allelic frequencies were compared between both groups. Results: Associations between leiomyoma with IGF2 and CDH1 polymorphism exist. Proportions of IGF2 exon 9*AA/AG/GG in and CDH1* CC/CT/TT in the groups were: group I, 38/39.2/22.8% and 27.8/66.5/5.7%; group 2, 22.4/53.9/23.7% and 21.2/64.1/14.7. MPO, urokinase, XPA and XPD in both groups were non-significantly different. Proportions of MPO*AA/AG/GG, urokinase*CC/CT/TT, XPA*AA/AG/GG and XPD*AA/AC/CC were: group 1: 1.9/23.4/74.7%, 0.6/7/92.4%, 20.9/55.1/24%, 85.4/14.6/0%; group 2: 3.8/24.4/71.8%, 1.3/4.5/94.2%, 22.4/53.9/23.7%, 84.6/15.4/0%. Conclusion: IGF2*A allele and CDH1*C allele were correlated with leiomyoma susceptibility, which may be associated with leiomyoma development. MPO, urokinase, XPA and XPD polymorphisms are not related to leiomyoma susceptibilities.
Ligand-Functionalization of BPEI-Coated YVO4:Bi3+,Eu3+ Nanophosphors for Tumor-Cell-Targeted Imaging Applications
(2013-11-01)
Chen, Yi-Chin
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Huang, Sheng-Cih
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Wang, Yu-Kuo
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Liu, Yuan-Ting
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Wu, Tung-Kung
In this study, surface-functionalized, branched polyethylenimine (BPEI)-modified YVO4:Bi3+,Eu3+ nanocrystals (NCs) were successfully synthesized by a simple, rapid, solvent-free hydrothermal method. The BPEI-coated YVO4:Bi3+,Eu3+ NCs with high crystallinity show broad-band excitation in the =250 to 400nm near-ultraviolet (NUV) region and exhibit a sharp-line emission band centered at =619nm under excitation at =350nm. The surface amino groups contributed by the capping agent, BPEI, not only improve the dispersibility and water/buffer stability of the BPEI-coated YVO4:Bi3+,Eu3+ NCs, but also provide a capability for specifically targeted biomolecule conjugation. Folic acid (FA) and epidermal growth factor (EGF) were further attached to the BPEI-coated YVO4:Bi3+,Eu3+ NCs and exhibited effective positioning of fluorescent NCs toward the targeted folate receptor overexpressed in HeLa cells or EGFR overexpressed in A431 cells with low cytotoxicity. These results demonstrate that the ligand-functionalized, BPEI-coated YVO4:Bi3+, Eu3+ NCs show great potential as a new-generation biological luminescent bioprobe for bioimaging applications. Moreover, the unique luminescence properties of BPEI-coated YVO4:Bi3+,Eu3+ NCs show potential to combine with a UVA photosensitizing drug to produce both detective and therapeutic effects for human skin cancer therapy.
Multiple Pleomorphic Tetramers of Thermostable Direct Hemolysin from Grimontia hollisae in Exerting Hemolysis and Membrane Binding
(2019-07-08)
Wang, Yu-Kuo
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Huang, Sheng-Cih
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Chang, Chin-Yuan
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Huang, Wan-Ting
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Liao, Man-Jun
Oligomerization of protein into specific quaternary structures plays important biological functions, including regulation of gene expression, enzymes activity, and cell-cell interactions. Here, we report the determination of two crystal structures of the Grimontia hollisae (formally described as Vibrio hollisae) thermostable direct hemolysin (Gh-TDH), a pore-forming toxin. The toxin crystalized in the same space group of P2(1)2(1)2, but with two different crystal packing patterns, each revealing three consistent tetrameric oligomerization forms called Oligomer-I, -II, and -III. A central pore with comparable depth of similar to 50 angstrom but differing in shape and size was observed in all determined toxin tetrameric oligomers. A common motif of a toxin dimer was found in all determined structures, suggesting a plausible minimum functional unit within the tetrameric structure in cell membrane binding and possible hemolytic activity. Our results show that bacterial toxins may form a single or highly symmetric oligomerization state when exerting their biological functions. The dynamic nature of multiple symmetric oligomers formed upon release of the toxin may open a niche for bacteria survival in harsh living environments.
Potential antitumor therapeutic application of Grimontia hollisae thermostable direct hemolysin mutants
(2015-04-01)
Huang, Sheng-Cih
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Wang, Yu-Kuo
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Huang, Wan-Ting
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Kuo, Tsam-Ming
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Yip, Bak-Sau
We report on the preparation of a new type of immunotoxin by conjugation of an epidermal growth factor receptor (EGFR)-binding peptide and an R46E mutation of thermostable direct hemolysin from Grimontia hollisae, (Gh-TDHR46E/EB). The hybrid immunotoxin was purified to homogeneity and showed a single band with slight slower mobility than that of Gh-TDHR46E. Cytotoxicity assay of Gh-TDHR46E/EB on EGFR highly, moderately, low, and non-expressed cells, A431, MDA-MB-231, HeLa, and HEK293 cells, respectively, showed apparent cytotoxicity on A431 and MDA-MB-231 cells but not on HeLa or HEK293 cells. In contrast, no cytotoxicity was observed for these cells treated with either Gh-TDHR46E or EB alone, indicating enhanced cytotoxic efficacy of Gh-TDHR46E by the EGFR binding moiety. Further antitumor activity assay of Gh-TDHR46E/EB in a xenograft model of athymic nude mice showed obvious shrinkage of tumor size and degeneration, necrosis, and lesions of tumor tissues compared to the normal tissues. Therefore, the combination of Gh-TDHR46E with target affinity agents opens new possibilities for pharmacological treatment of cancers and potentiates the anticancer drug's effect.
Preliminary structure determination and functional residues characterization of thermostable direct hemolysin from Grimontia hollisae
(2009-08-16)
Wang, Yu-Kuo
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Huang, Sheng-Cih
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Wu, Tung-Kung
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生物科技學系
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Department of Biological Science and Technology
Purification, crystallization and preliminary X-ray analysis of a thermostable direct haemolysin from Grimontia hollisae
(2011-02-01)
Wang, Yu-Kuo
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Huang, Sheng-Cih
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Wu, Yi-Fang
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Chen, Yu-Ching
;
Chen, Wen-Hung
Vibrio hollisae, a halophilic species recently reclassified as Grimontia hollisae, is a causative agent of gastroenteritis and septicaemia. One important pathogenic Vibrio factor, thermostable direct haemolysin (TDH), has been purified and crystallized in two crystal forms using the vapour-diffusion method. The crystals belonged to an orthorhombic space group, with unit-cell parameters a = 104.8, b = 112.4, c = 61.3 angstrom and a = 122.9, b = 123.3, c = 89.8 angstrom. The crystals contained either four or eight molecules per asymmetric unit, with predicted solvent contents of 49.4 and 46.3% and Matthews coefficients (V(M)) of 2.4 and 2.3 angstrom(3) Da(-1), respectively. These crystals were suitable for structure determination, which would yield structural details related to the cytotoxicity and oligomeric structure of this pore-forming toxin.
Purification, crystallization and preliminary X-ray crystallographic analysis of xylose reductase from Candida tropicalis
(2009-04-01)
Chen, Li-Chun
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Huang, Sheng-Cih
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Chuankhayan, Phimonphan
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Chen, Chung-Der
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Huang, Yen-Chieh
Xylose reductase (XR), which requires NADPH as a co-substrate, catalyzes the reduction of D-xylose to xylitol, which is the first step in the metabolism of D-xylose. The detailed three-dimensional structure of XR will provide a better understanding of the biological significance of XR in the efficient production of xylitol from biomass. XR of molecular mass 36.6 kDa from Candida tropicalis was crystallized using the hanging-drop vapour-diffusion method. According to X-ray diffraction data from C. tropicalis XR crystals at 2.91 angstrom resolution, the unit cell belongs to space group P3(1) or P3(2). Preliminary analysis indicated the presence of four XR molecules in the asymmetric unit, with 68.0% solvent content.