I01

Structure of the multidrug resistance efflux transporter EmrE from Escherichia coli

Che Ma* and Geoffrey Chang

Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

10550 N. Torrey Pines Rd. CB-105, CA 92037, USA.

 

Multidrug resistance efflux transporters threaten to reverse the progress treating infectious disease by extruding a wide range of drug and other cytotoxic compounds. One such drug transporter, EmrE, from the Small Multidrug Resistance family, utilizes proton gradients as an energy source to drive substrate translocation. In an effort to understand the molecular structural basis of this transport mechanism, we have determined the structure of EmrE from Escherichia coli to 3.8 angstroms. EmrE is a tetramer comprised of two conformational hetero-dimers related by a pseudo two-fold symmetry axis perpendicular to the cell membrane. Based on the structure and biochemical evidence, we propose a mechanism by which EmrE accomplishes multidrug efflux by coupling conformational changes between two hetero-dimers with proton gradient. Because of its simplicity and compact size, the structure of EmrE can serve as an ideal model for understanding the general structural basis of proton:drug antiport for other drug efflux systems.

 

 

I02

Solution structure of porcine β-microseminoprotein

Iren Wang1,2, Yuan-Chao Lou3, Wen-Chang Chang1, Shih-Hsiung Wu1,2, Chinpan Chen3*

1Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan;

2Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan;

3Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan

 

A sperm motility inhibitor originally isolated from porcine seminal plasma has been confirmed to be identical to porcine β-microseminoprotein (MSP) based on peptide sequence, amino acid composition and mass spectral analysis. Porcine MSP contains 91 amino acid residues linked with five disulfide bridges. This protein competitively inhibits the activity of Na+, K+-ATPase purified from porcine cerebral cortex in a concentration-dependent manner and the half-maximal inhibition was achieved at an inhibitor concentration of 90 μM. To date, there is no known structure related to porcine MSP in Protein Data Bank, and hence knowing the structure/function relationship on MSP is an interesting and important task. For structural study, we have cloned, expressed, and purified the recombinant porcine MSP which possesses 17 extra amino acids at N-terminus than the native protein. It was confirmed that the recombinant MSP exhibits an inhibitory effect identical to the native protein. Also, CD and NMR data showed that the secondary as well as tertiary structures are very similar between them. Based on the specific NOEs obtained from 3D 13C-NOESY-HSQC in D2O, the five disulfide-bond pairings were unambiguously assigned. Surprisingly, our derived pairings are different from those recently reported for ostrich β-microseminoprotein based on MALDI-TOF analysis. On the basis of 1018 restraints, 3D solution structure of the recombinant porcine MSP has been determined. The 3D structure is clearly composed of two domains, one of which with one double-stranded and one four-stranded antiparallel β-sheets, and the other one with two double-stranded antiparallel β-sheets. We suggested the interaction between the two domains is mainly due to electrostatic force. In addition to discussing 3D structure, backbone atom dynamics and structure further refinement using RDC (residual dipolar coupling) data will also be discussed.

 

 

I03

Detection of quadruplex DNA structures in human telomeres by a

fluorescent carbazole derivative

Cheng-Chung Chang,1 I-Chun Kuo,1 I-Fang, Ling,2 Chin-Tin Chen,2

Huei-Chin Chen,3 Pei-Jen Lou,3 Jing-Jer Lin,4 Ta-Chau Chang1,*

1Institute of Atomic and Molecular Sciences, Academia Sinica, P.O. Box 23-166, Taipei

2 Centers for Optoelectronic Biomedicine, National Taiwan University, Taipei

3 Department of Otolaryngology, National Taiwan University Hospital, Taipei

4 Institute of Biopharmaceutical Science, National Yang-Ming University, Taipei

  

Single-stranded telomeric DNA tends to form a four base-paired planer structure termed G-quadruplex. This structure was easily formed in vitro in the presence of monovalent cations. However, the existence of this structure in native human telomeres is unclear. Here we address this important question through the distinctive properties of 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC) upon binding to various DNA structures.1 Although the fluorescence of BMVC increases significantly in the presence of DNA, BMVC has high sensitivity and binding preference to quadruplex d(T2AG3)4 over duplex DNA. In addition, the fluorescent emissions were characterized around 575 nm for quadruplex d(T2AG3)4 and 545 nm for most of duplex DNA. The 575 nm fluorescence emissions were detected in the mixtures of 2 nM BMVC with the chromosomal DNA that were extracted from human cells, suggesting the presence of quadruplex structure in human nucleus. Further analyzing the BMVC fluorescence at the ends of metaphase chromosomes and other regions of chromosomes, we detected the quadruplex-binding BMVC fluorescence at telomere-proximal regions. Together these results provide the first evidence for the presence of quadruplex structures in human telomeres. 

 

1. Chang, C. C.; Wu, J. Y.; Chien, C. W.; Wu, W. S.; Liu, H.; Kang, C. C.; Yu, L. J.; Chang, T.-C. (2003) Anal. Chem. 75, 6177-6183.

 

 

I04

Issues and prospects of diffusion magnetic resonance imaging

Ching-nien Chen, Ph. D.

National Institutes of Health

9000 Rockville Pike, Bethesda, MD 20892, USA

 

Magnetic resonance imaging (MRI) has been a focus of attention in the medical and bioengineering world for many years. It culminated last winter in the 2003 Nobel Prize of Medicine or Physiology award ceremony to two scientists for their pioneering work in this field. For the past three decades, MRI has gone from its infancy to the present status of youth, and ever going on strong and vibrant. The combination of an old NMR technique for measuring diffusion effects and MRI created the so-called “diffusion MRI” (DMRI). DMRI is a typical product of interdisciplinary stimulation and cross- fertilization. Because DMRI measures the diffusion characteristics of water molecules in the tissues, it in turn explores the state of their environment (i.e. the tissues). DMRI and especially the diffusion tensor MRI developed in recent years played important roles to probe the human brains, e.g. stroke, brain microstructures and brain functions. Since DMRI is inherently a method of low sensitivity, the obstacles of creating a successful diffusion-weighted image are many. But with the development of faster and more sensitive imagers, powerful computing processors, and image processing software the prospect of DMRI is quite encouraging. The leading application of DMRI has been in the area of acute brain ischaemia.  The diffusion tensor imaging also tells the anisotropy behavior of the water molecules in the brain white matter, and thus its structure and orientation. Thus it is possible to follow the growth, development, and disease states of the brain by DMRI. We will touch upon some of the basic principles, fundamental requirements, current status, and some clinical applications of DMRI.

 

 

I05

Issues structure and function of Malic enzymes

Gu-Gang Chang

Faculty of Life Sciences, Institute of Biochemistry, Structural Biology Program,

and Proteome Research Center, National Yang-Ming University

Taipei 112, Taiwan

 

Malic enzyme catalyzes a reversible oxidative decarboxylation of l-malate to give carbon dioxide and pyruvate in the concomitant reduction of NAD(P)+ to NAD(P)H. The reaction also needs an essential divalent metal ion (Mn2+ or Mg2+) for the catalysis. In C4 plants, ME is involved in the anaplerotic replenish of tricarboxylic acid-cycle intermediate. Human m-NAD-ME has received much attention because of its involvement in the energy metabolism in neuron or neoplasia tissues. Malic enzyme is a tetrameric protein with double dimer structure in which the dimer interface is more intimate contacted than the tetramer interface. For human mitochondrial malic enzyme, there are an exo nucleotide-binding site for the inhibitor ATP and an allosteric site for the activator fumarate, located at the tetramer and dimer interfaces, respectively. Crystal structures of the enzyme in various complexed forms indicate that the enzyme may exist in equilibrium among two open and two closed forms. Interconversion among these forms involves rigid-body movements of the four structural domains. Substrate binding at the active site shifts the open form to the closed form that represents an active site closure. Fumarate binding at the allosteric site induces the interconversion between forms I and II and is related to the allosteric regulation of the enzyme activity. Structures of malic enzyme from different sources are compared with an emphasis on the differences and their implications to structure-function relationships. The binding modes of the substrate, product, cofactors, and transition-state analog at the active site, as well as ATP and fumarate at the exo site and allosteric site, respectively, provide a clear account for the catalytic mechanism, nucleotide specificities, allosteric regulation, and functional roles of the quaternary structure.

 

 

I06

Phase contrast microradiography

Yeukuang Hwu

Institute of Physics, Academia Sinica

 

One hundred years after the discovery of X-ray, radiography has reached a new level of sophistication and power.  With higher coherence of the X-ray source produced by synchrotron, phase difference is added to the absorption as anther, yet much improved, contrast mechanism.  Together with the improvement in the detecting system, we can now looking deep inside into matters with unprecedented precision and speed.  Sub-μm resolution radiograph can now be obtained with ease and fast phenomenon can be observed in a real time fashion.  The application of this new tool has already helped researches in materials science, biology and medicine.  Ongoing developments in the instrumentation and reconstruction algorithms have generated even higher excitement with the recent demonstration of nanometer scale resolution in 3D. It is anticipated that with the new installation of X-ray free electron laser, X-rays can be used to “image” a single protein molecule with atomic resolution in the near future and to continue the legacy started by Röntgen.  Review of this advent of this technology, its application to various domains in science, the relevance to the medicine, nano-science and -technology, and the future potential will be presented.

 

 

I07

Seeing macromolecular complexes through glasses

 

Wei-hau Chang

Institute of Chemistry, Academia Sinica

128 Sec. 2 Academia Rd., Taipei, Taiwan 115

 

Recent years have witnessed cryo electron microscopy emerging as a powerful tool in macromolecular structural determination for systems not amenable to X-ray crystallography or NMR spectroscopy. Such advances are made mainly because of introduction of new generation of microscopy instruments, fast computers and sophisticated image analysis programs. Near atomic resolution information can be retrieved from cryo microscope images if the samples are with symmetry, for example, viruses and 2-D crystals. While 1.5 nm structure can be routinely obtained from samples with homogenious single molecules. Here, two successful examples and relevant issues of determining structures of macromolecular complexes by electron microscopy are offered, one by 2-D crystallography and the other by single molecule reconstruction. In the former case, a core complex of a basal transcription and DNA repair factor, TFIIH, was solved by 2-D crystallography. In the latter case, in the light of solved X-ray structures of RNA polymerases, suprise came when structures of RNA polymerase II were re-determined as they were preserved as single molecules in frozen-hydrated state. 

 

 

I08

Computational approaches in protein sequence and structure analysis

An-Suei Yang
Dept of Parmacology and Columbia Genome Center
Columbia University

 

Information on the function and interacting network for proteins encoded in a genome is critical for the success in the drive to construct a computational model at the cellular level. We have devised computational methodologies aimed at extracting and applying knowledge towards understanding the data-rich disciplines in protein sequence, structure, and function. In the first part of the presentation, statistical and energetic evidence, which suggested that a protein’s native fold is determined largely by forming local structures with specific conformations, will be demonstrated. The body of evidence also indicated that some segments of the protein could be encoded with strong propensities biasing toward specific substructures that are prevalent among proteins with grossly different global fold. The conclusion further suggested that protein local structure prediction based solely on local sequences can be achieved with useful accuracy. Along this line of argument, the second part of the presentation will describe several local structure prediction strategies and the benchmark results indicating the expected prediction accuracy and the comparison with other available methods. Reasonable prediction accuracy from these methodologies indeed reflected well on the protein folding mechanism centered on the local structure formation theme, and thus provided the basis for several applications in protein sequence/structure/functional analysis and in protein structure modeling. These applications will be elaborated in the third part of the presentation. In conclusion, computational methodologies based on protein sequence and structure segments provide useful tools toward understanding protein function on the basis of protein three-dimensional structure. The methodologies are useful not only as bioinformatic tools for computational modeling but also as designing tools for polypeptides geared towards novel structure and function.

 

 

I09

Binding of actinomycin D to single-stranded DNA, old drug with new tricks

Fu-Ming Chen

Department of Chemistry, Tennessee State University

3500 John Merritt Blvd., Nashville, Tennessee, 37209-1561, USA

 

Actinomycin D (ACTD) is an anticancer antibiotic best known for inhibiting transcription by binding to double-stranded DNA. It is a chromopeptide consisting of a phenoxazinone planar chromophore with two pentapeptide rings attached. Its mode of DNA binding has been shown to be intercalative, in which the chromophore inserts in-between the DNA base pairs while the two chains of pentapeptide rest in the minor groove. The GpC sequence specificity of this drug has been well characterized and originates from its ability to form two hydrogen bonds between each guanine and a pentapeptide ring. However, a report appeared in 1989 to indicate that ACTD can also bind strongly and cooperatively to d(CGTCGACG) with 2:1 drug to duplex binding stoichiometry. This talk will attempt to tell a story as to how our efforts on delineating the ACTD binding mode to this non-GpC-containing oligomer have led us to the finding that this drug is capable of binding to several sequence-specific single-stranded DNA oligonucleotides with high affinity. Binding models will be proposed to account for the observed strong binding.

 

 

I10

Charge conductivity in peptides: dynamic simulations of a bifunctional

model supporting experimental data

Sheh-Yi Sheu#, Dah-Yen Yang§, H. L. Selzle* and E. W. Schlag*

# Department of Life Science, National Yang-Ming University, Taipei, Taiwan; §Institute of Atomic and Molecular Science, Academia Sinica, Taipei, Taiwan; and *Institut für Physikalische und Theoretische Chemie, Technische Universität München, D-85747 Garching, Lichtenbergstrasse 4, Germany

 

In our bifunctional model, the charge is transported along a polypeptide chain. At each Cα-atom, the torsional angles ψ and φ are constrained in the Ramachandran plot (see Fig. 1). The charge is transported from the C-side of the Cα-atom to the N-side. Before the charge is transported, it waits in the Cside until the OO atoms between two connected amino acids collide. The rotational motion of the ψ and φ angles is similar to a Brownian particle moving inside a 2D box with a static gate where the OO-atom collide with each other and charge starts to transfer.

In our MD simulation, we provide charge energy E to the atoms attached to the ψ axis, i.e., N-, O-, and H-atoms. We define the efficiency as efficiency = successful configurations/total configuration.

The interesting new sidelight on these MD calculations is that we find that not all initial starting configurations of the calculation lead to a firing state, but in fact a fraction of initial states are dissipated, leading to an efficiency in the process of less than unity. Such an inefficiency in charge transport is experimentally well-known and leads to -values in the efficiency Y of charge transfer with distance

  with typical values in condensed media of near 1.0 .  Here A is the prefactor.              

We estimate the successful run with efficiency equal to 0.008, i.e. the -value =1.3 , a value in astonishing agreement with experiment. 

        The charge transport along each individual chain inside the -sheet is seen to have the same efficiency as it has in the -helix. But the sum of the efficiency of the -sheet is the geometric sum of each individual chain. Hence, for the example Azurin, the -sheet contains about three chains. Its efficiency is expected to be about three times higher than that of each individual -helix chain. We thus have an efficiency of the -sheet ca. 0.0244; i.e. -valuet=1.0 . Therefore, for the theoretical predicted collision distance for firing, the calculated efficiency here predicts a -value that corresponds closely to that of known experiments.

     

 Fig. 1.   Charge transport in a polypeptide.

 

References:         

1.   E.W. Schlag, Sheh-Yi Sheu, Dah-Yen Yang, H.L. Selzle and S.H. Lin, Proc. Natl. Acad. Sci. USA, 97(2000)1068.

 

 

I11

Structure of the C-terminal domain of prokaryotic topoisomerase IIA:

a novel beta-Propeller implies how gyrase wraps DNA

Tung-Ju Hsieh and Nei-Li Chan

Institute of Biochemistry, College of Life Sciences, National Chung Hsing University

250 Kuo-Kuang Rd. Taichung City, 402, Taiwan

 

The highly conserved type IIA topoisomerases alter the topological state of DNA by catalyzing ATP-dependent passage of DNA duplexes through one another.  This group of enzymes alters the topological state of DNA by catalyzing ATP-dependent passage of DNA duplexes through one another.  By regulating DNA topology, type IIA enzymes are involved in almost all aspects of DNA metabolism such as replication, transcription, recombination, sister chromatid segregation, and chromosome condensation.  In bacteria, two homologous type IIA enzymes, DNA gyrase and topoisomerase IV (TopIV), have been identified.  Although these two bacterial enzymes share a high degree of similarity, yet they appear to have distinct cellular functions.  Gyrase is responsible for introducing negative supercoil into bacterial genome, while TopIV is the decatenating enzyme required for the segregation of linked daughter chromosomes.  It has been shown that the supercoiling capability of gyrase can be attributed to the DNA binding activity of its C-terminal domain (CTD).  Interestingly, despite clear sequence homology, no such activity was found for the TopIV-CTD.  To characterize the differences between these domains, we have cloned, purified, and crystallized the 36 kDa TopIV-CTD from Bacillus stearo-thermophilus.  And the 2.0 Å crystal structure of TopIV-CTD has been determined by Se-MAD approach.  Preliminary analysis indicated that the TopIV-CTD belongs to a novel type of beta-propeller fold.  Details on structural determination and analysis will be presented.

 

 

I12

Molecular interactions with DNAs studied by surface enhanced Raman scattering

W.-Y. Lo and T.-S. Yang*

Department of Chemistry and Biochemistry, National Chung Cheng University,

160 Sna-Hsing, Min-Hsiung, Chia Yi, 621, Taiwan

 

  The interactions between spermine and the potential anti-cancer drug molecule 3,6-Bis(1-methyl-4-vinylpyridium iodine) carbazole (BMVC) with various duplex and quadruplex DNAs were studied by surface enhanced Raman scattering using Ag nanoparticles as metal substrates. The spermine molecule had four positive charges which could neutralize the negatively charged DNA backbone so that DNAs could adsorb on Ag nanoparticles which are also negatively charged. As the result, the SERS signals of DNAs became reproducible upon adding spermine. The concentrations of DNAs used in SERS experiments were as low as 10-7M, about four order magnitude less than those used by the conventional Raman spectroscopy. The SERS signals of DNA/spermine indicated that spermine molecules cross link the DNA backbones with two end positive charges clamping the phosphate oxygens on the backbone chains. The SERS signals of BMVC with DNAs depicted that BMVC favored the interactions with the adenine base. The central nitrogen atom of BMVC interacted with the amine group on C6 of adenine and the two end positive charged nitrogens of BMVC grasped the phosphate oxygens on the backbone.

 

  

I13

A minimal off-lattice model for structural prediction of small proteins

C. I. Chou a*, R.S Han b stext, H. S. Huang b, Y. W. Wu b and etextT. K. Lee b

a Department of Physics, Chinese Culture University, Taipei, Taiwan and b Institute of Physics,

Academia Sinica, Nankang, Taipei, Taiwan.

 

We develop an off-lattice minimal model to predict the structures of small proteins. The model is composed of a polypeptide chain with Ramachandran angles as its degrees of freedom. The force field of this model is based on hydrogen bonds and the anisotropic hydrophobic forces. The energy form of the anisotropic hydrophobic forces is designed by analyzing the protein data banks. By using this model, we obtain several small proteins with secondary structures including the a-helix, b-sheet and the a/b structures. Our results are compared with the real protein structures.

 

 

I14

Information, universality and self-similarity in whole genomes

T.Y. Chen1, L.C. Hsieh1,2, C.H. Chang1, and H.C. Lee1-3*

1Department of Physics, 2Center for Complex Systems and 3Department of Life Sciences,

National Central University, Chungli, Taiwan  *hclee@phy.ncu.edu.tw 

 

Genomes are books of life and necessarily carry a huge amount of information. Here we measured the Shannon information associated with word frequencies in allprokaryotes and eukaryotes - 155 microbial and 127 eukaryotic sequences whose complete genomes have been sequenced and are available in publicly accessible database. We find that not only do all complete genomes have a very high content of Shannon information, the amount of Shannon information in complete genome sequences follow an extremely simple pattern. With the exception of one eukaryote the Shannon information in all complete genomes is a genome-independent universal constant that depends only on word length.  This defines a universality class given by a simple formula.  Furthermore, genomes exhibit a high degree of self-similarity.  We find that with few exceptions any randomly selected segment in a gneome as short as a few thousand bases has the same Shannon information as the whole genome. We show that this body of data imposes strict constraints on the modes by which genomes grew and evolved. Through computer simulation of sequence growth, evolution and divergence, we have found a simple and robust model for genome growth that generates sequences whose statistical properties agree with observed data. Our results suggest that the ancestor or ancestors of present day genomes grew, evolved and diverged mainly by maximally stochastic and selectively neutral duplications and by random point mutation, and this process most likely began when they were not more than 300 bases long. The two most important inferences of this work are: (a) genomes are very close to being self-organized critical systems and (b) the notion of selective neutralism implied by the growth model independently corroborates Kimura’s neutral theory of evolution.

 

 

I15

Optical biopsy using multiphoton microscopy

J Chen-Yuan Dong1*, Sung-Jan Lin2, Shiou-Hwa Jee2, Hsin-Yuan Tan4, Hsuan-Shu Lee5, Wen Lo1,

Yen Sun1, Shu-Wen Teng1, Yuan Liu1, Peter T. Fwu1,

1Department of Physics, National Taiwan University, Taipei 106, Taiwan

2Department of Dermatology, National Taiwan University Hospital, Taipei 100, Taiwan

3Department of Dermatology, National Taiwan University College of Medicine, Taipei 100, Taiwan

4Department of Internal Medicine, National Taiwan University Hospital, Taipei 100, Taiwan

5Department of Ophthalmology, Chang Gung Memorial Hospital, Linko, Taiwan

 

In recent years, multiphoton microscopy has developed into a powerful bioimaging technology. High image contrast, reduced photodamage, and enhanced penetration depths are some of the major advantages of this technique. A particularly promising application of multiphoton excited fluorescence and second harmonic generation (SHG) imaging is the diagnosis of the physical and chemical states of biological tissues. This unique feature enables multiphoton microscopy to be developed into an in vivo optical biopsy tool, accompanying conventional histological procedures. In this presentation, I will present imaging results of some of the tissue types accessible with multiphoton microscopy and discuss their relevance to genomic research in Taiwan.

 

 

I16

Cancer cell’s secreted proteome as a basis for searching potential tumor markers - nasopharyngeal carcinoma as a model

Chih-Ching Wu1,5, Kuan-Yi Chien2,6, Ngan-Ming Tsang3, Kai-Ping Chang4, Sheng-Po Hao4,

Chuang-Hui Tsao1,6, Wen-Chuan Huang1, Yu-Sun Chang5, and Jau-Song Yu1,5*

1Department of Cell and Molecular Biology, 2Department of Biochemistry, 5Graduate Institute of Basic Medical Sciences, and 6Chang Gung Proteomic Core Laboratory, Medical College of Chang Gung University, Tao-Yuan, Taiwan; 3Department of Radiation Oncology and 4Department of Otolaryngology-Head Neck Surgery, Chang Gung Memorial Hospital, Lin-Kou, Taiwan

259, Wen-Hwa 1st Road, Kwei-Shan, Tao-Yuan 333, Taiwan, ROC

 

Advances in proteomic technologies have provided researchers new tools for identifying tumor markers systematically. To search potential biomarkers for nasopharyngeal carcinoma (NPC), the profile of proteins secreted from two NPC cell lines NPC-TW02 and NPC-TW04 were examined. Proteins in the conditioned medium from both cell lines cultured in serum-free medium were resolved by 8-14% SDS-PAGE and silver-stained. All the bands were excised, digested with trypsin, and analyzed by MALDI-TOF mass spectrometry. By this approach, 33 (36) proteins with significant score and matched molecular weight were identified in 42 (35) out of 61 (46) bands derived from NPC-TW02 (NPC-TW04) cells, and 23 proteins were identified from both cell lines. Among them, 15 proteins have been reported previously to be highly expressed or dysregulated in certain types of tumor. In addition, 14 of them could be detected in serum in previous reports, and 7 of the 14 proteins were considered as potential serum tumor markers. Most of these proteins have not been described previously to be secreted by NPC cell lines. The presence of four of these identified proteins (fibronectin, Mac-2 BP, fascin and PAI-1) in the conditioned medium from the two NPC cell lines were confirmed by Western blotting. Some of the four proteins could also be detected in the conditioned medium of other 11 cancer cell lines from different origins. Immunohistochemical analysis further revealed the high expression of fibronetin, Mac-2 BP and PAI-1 in tumor biopsies from animal model and NPC patients. Fluorimetric sandwich ELISA for fibronectin, Mac-2 BP and PAI-1 were then developed to measure their serum levels in nude mice bearing with or without NPC tumor and in NPC patients and healthy controls. The serum levels of Mac-2 BP and PAI-1 were significantly elevated in nude mice bearing NPC tumor and in NPC patients when compared with tumor-free mice and healthy controls, respectively. The serum fibronectin level in NPC patients was also significantly higher than that of healthy controls, although its level in NPC-bearing mice was lower than that of tumor-free mice. Collectively, our results show that elevated serum levels of Mac-2 BP, fibronectin and PAI-1 are associated with NPC. In addition, the results also point out that systematic identification of secreted proteins from cancer cell lines in conjunction with animal model can be a feasible strategy for searching potential tumor markers.

 

 

I17

Laser desorption mass spectrometry for DNA sequencing and its applications

Chung-Hsuan Chen, Lan-Yang Chang1, V. V. Golovlev, K. Tang, Y. F. Zhu, L. Smamartano,

S. L. Allman, N. R. Isola, N. I. Taranenko, H. Farquar, K. J. Matteson2, N. T. Potter2

Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN. 37831-6378.

1Institute of Biomedical Science, Academia Sinica

2University of Tennessee Medical Center

 

DNA sequence is among the most important information among biomolecular structures. Due to the critical importance of DNA sequence to biomedical science, Human Genome Project has put tremendous effort and time to finish the sequencing of entire human genome. Sequences of genome of mouse, rice and many microbes have also been revealed. Recently, discovery of new genes has been speeded up due to the extensive study of genomic and proteomic research. The sequence of DNA becomes the foundation for diagnosis of diseases and identification of human, animals and microbes. It is expected the need of re-sequencing will increase dramatically. Most routine DNA analysis nowadays still relies on gel electrophoresis. Nevertheless, the speed of gel electrophoresis for sequencing is still relatively slow. The requirement of radioactivity or dye labeling also increase the cost and producing undesirable wastes. With laser desorption mass spectrometry for DNA fragment sizing and sequencing, the speed can be much faster and no labeling is required.

The basic principle of a gel electrophoresis sequencing is to separate different sizes of DNA with electric field in gel medium. A time-of-flight mass spectrometer can be use to separate different sizes of DNA in gas phase. The time needed for the separation of different biomolecules in a mass spectrometer is typically less than 1 millisecond compared to minutes to hours for gel electrophoresis. We have developed several different approaches to use laser desorption mass spectrometry for DNA size measurements and sequencing. They include (1) sequencing DNA with Sanger=s approach with DNA ladders (2) sequencing DNA with Maxam-Gilbert=s chemical degradation method and (3) direct DNA sequencing without the need of chemical processing. In addition to DNA sequencing, we also apply laser desorption mass spectrometry for biomedical and forensic applications. They are (1) microarray hybridization detection (2) Single Nucleotide Polymorphism (SNP) detection (3) Short tandem Repeat (STR) detection for disease and forensic applications, (4) genetic disease diagnosis and (5) microbial analysis.

Experimental details will be presented in the meeting.

 

 

I18

The double-stranded RNA-binding motif of RNA helicase A (RHA) is

a versatile ligand-docking module capable of accommodating

a variety of bio-molecules.

Ming-Lung Hung , Ping Chao, Chung-Te Chang, Wen-Hau Yang, Mei-Chi Su and Kung-Yao Chang*

Institute of Biochemistry, National Chung-Hsing University,

250 Kuo-Kung Road, Taichung, 402 Taiwan.

 

The double-stranded RNA-binding motif (dsRBM) is a widely distributed motif frequently found within proteins with sequence-nonspecific RNA duplex binding activity. In addition to the binding of double-stranded RNA, some dsRBMs also participate in complex formation via protein-protein interactions. Interestingly, a lot of proteins containing multiple dsRBMs have only some of their dsRBMs with the expected RNA duplex-binding competency proven, while the functions of the other dsRBMs remaining unknown. RNA helicase A (RHA) is a multi-functional protein with ability to interact with different classes of macromolecules. Among them, the CREB-binding protein (CBP), the constitute transport element (CTE) RNA and specific dsDNA promoters were all reported to interact with the N-terminal region of RHA that containing two copies of double-stranded RNA-binding motif (dsRBM).We show here that the dsRBM1 of RNA helicase A (RHA) not only can interact with the CBP but also can cooperate with a carboxyl-terminal domain of proline-rich content to gain novel nucleic acid- binding activities. This integrated nucleic acid binding module is capable of associating with the consensus sequences of the constitute transport element (CTE) RNA of type D retrovirus against RNA duplex competitors. Remarkably, binding activity for double-stranded DNA also resides within this composite nucleic acid binder. It thus suggests that the widely distributed dsRBM fold has the potential to serve as a scaffold by itself or by cooperating with a flanking sequence to accommodate a variety of bio-molecules, and functional assessment for a newly identified protein containing dsRBM fold should be more cautious.