PL Prostacyclin and thromboxane synthases: Structural and functional characteristics

Kenneth K. Wu, M.D., Ph.D.

National Health Research Institutes

 

Prostacyclin synthase (PGIS) and thromboxane synthase (TXAS) occupy a pivotal position in arachidonic acid metabolism. They catalyze the synthesis of two active molecules, PGI2 and TXA2, respectively, which play a critical role in vascular homeostasis and cardiovascular diseases. These two enzymes belong to cytochrome p450 (CYP) superfamily but are distinct from classic CYP in that they do not possess oxygenase activity. They function as isomerases that convert a common substrate PHG2 to PGI2 and TXA2, respectively. PGI2 and TXA2 have distinct chemical structures. It is of considerable interests to understand the catalytic mechanisms of these two enzymes. Our laboratory was the first to clone these two membrane proteins and characterize their structure-function relationship. We have also explored their roles in arachidonic acid metabolism and disease state by biochemical and molecular genetic approaches. In this talk, the structure-function, topology and function of these enzymes will be discussed.

 

 

PL1

Phyllotaxis and Leopard¡¦s spots
(¸­§Ç»P°\¯¾)

Sy-Sang Liaw

Department of Physics, National Chung-Hsing University
250 Guo-Kuang Road, Taichung, Taiwan

 

Nature is full of patterns. Their beauty is sometimes beyond human's imagination. One would agree that the shape of a rock, the pattern of cloud, and the orbit of a planet are governed by physical laws. However, it is to many people's surprise that shape and form of living creatures are also determined by physical laws, not by "natural selection". In this talk, I will discuss two examples, phyllotaxis of plants and coat pattern of the leopard, and show the plausibility of the physical models for determining biological forms.

 

 

PL2 Mechanistic Studies of An Unusual C-O Bond Formation in Fosfomycin Biosynthesis

Hung-wen Liu

Division of Medicinal Chemistry, College of Pharmacy,
and Department of Chemistry and Biochemistry, University of Texas, Austin, Texas, 78712, USA

 

The last step of the biosynthesis of fosfomycin is the conversion of (S)-2-hydroxypropylphosphonic acid (HPP) to fosfomycin. Since the ring oxygen in fosfomycin is derived from the hydroxyl group of HPP, this oxirane formation reaction is effectively a dehydrogenation process. To study this unusual C-O bond formation step, we have overexpressed and purified the desired HPP epoxidase. Our recent experiments showed that HPP epoxidase is an iron-dependent enzyme, and both NAD(P)H and a reductase are required for its activity. Although the identity of the reductase remains elusive, an iron-sulfur flavoprotein (E3) that mediates electron transfer in a deoxygenation reaction is capable of enhancing fosfomycin production. Interestingly, the flavin coenzyme (FMN/FAD) was also found to be an effective substitute for the putative reductase. While ether ring formation as a result of dehydrogenation of a secondary alcohol has precedence in the literature, these catalyses require £\-ketoglutarate for activity. In contrast, HPP epoxidase is £\-ketoglutarate independent. Thus, the cyclization of HPP to fosfomycin clearly represents an intriguing conversion beyond the scope entailed by common biological epoxidation and C-O bond formation. The X-ray structures of this enzyme, both alone and in complex with substrates, were recently determined. These structural data suggest how this enzyme is able to recognize its substrate and respond with a conformational change that protects the radical-based intermediates formed during catalysis. Further characterization of the iron core and elucidation of the mechanisms of the oxirane formation will be presented.

 

 

PL3 NEUTRON DIFFRACTION NOW AND IN THE FUTURE: HOW IT WILL AFFECT BIOMOLECULAR RESEARCH

Robert Bau

Chemistry Department, University of Southern California, Los Angeles, CA 90089, U.S.A.

 

Single-crystal neutron diffraction data up to a resolution of 1.5 Å have been collected at room temperature on two forms of rubredoxin using the BIX-3 diffractometer at the JRR-3 reactor of JAERI. Rubredoxin is a small iron-sulfur redox protein with 53 amino acid residues, and the source of this particular protein is the hyperthermophile Pyrococcus furiosus, a microorganism that normally lives at temperatures near that of boiling water. Data were collected on crystals of the wild-type protein and on a mutant in which three of the residues have been replaced. We were originally interested to see if the results of the neutron analysis would provide some reasons to explain the remarkable thermal stability of the wild-type protein from this intriguing microorganism.

More importantly, much of the talk will be focused on present and future single-crystal neutron diffraction facilities: what are the problems that can be solved with current instruments, and what improvements lie in the future. As neutron sources become more and more intense, it will be possible to collect data on smaller and smaller crystals, and more information about the location of critical hydrogen atoms near the active sites of an enzyme, and the orientation of key water molecules, will become possible. It is anticipated that, in the coming years, the field of neutron diffraction on biological macromolecules will undergo a significant renaissance, as more users become attracted to this hitherto-underutilized but powerful technique.

 

 

PL4 Experiences of Molecular Images in Chang Gung Memorial University Hospital

Tzu-Chen Yen, MD, PhD

Molecular Imaging Center and Department of Nuclear Medicine, Chang Gung Memorial Hospital and University

 

The Molecular Imaging Center (MIC) at Chang Gung Memorial Hospital at Linko was established from June, 2006. The mission of this MIC was to establish a ¡§Translational Research Center¡¨. Under this mission, the center has two parts. One is for early phase clinical trial and the other is for pre-clinical trial. For early phase clinical trial, a 3T-MRI, a PET/CT, a cyclotron, a CGMP and more than 20 PIs are comprised. For pre-clinical trial, a microPET, a microSPECT/microCT, an optic image, whole set instrumentations for autoradiography (two microtones, a beta-imager and a phosphoimager) and qualified satellite animal room have been setup.
For early phase clinical trial, we have cooperated with domestic and international industry in pipeline drug test and instrumentations. So far, most of the drugs are for oncology or neuropsychiatry but will include cardiology in the future. As for instrumentation, we are now cooperating with SIEMENS in our 3T MRI (as a research site) and are focusing PET/MRI in the future. For pre-clinical trial, though the first microPET and microSPECT/microCT were established by the end of the Feb (2007), several projects have already had some preliminary data. Parts of the results have also been accepted in some leading journals.
Herein, we report the experiences of molecular imaging center at this hospital. We especially focus on what we have done in animal images in the past three months.
In human 3T-MRI at animal researches, there are two directions. One is using MR-guided blood-barrier barrier (BBB) disruption induced by High Intensity Focused ultrasound (HIFU) and the second is in vivo animal imaging using a clinical MR scanner. For the use of BBB disruption, the basic mechanism of ultrasound induced BBB disruption was explored by using in vivo MR Imaging. The energy window was calibrated and designed for the safe introduction of ultrasound. The real-time monitoring of the occurrence of hemorrhage during the procedure has been made possible. The changes in diffusion and perfusion properties by the increase of BBB permeability were under further investigation, which could be essential to studies of MR guided, locally targeted drug delivery. For in vivo animal imaging, the clinical scanner has been used to explore various disease models on small animals, including graft versus host disease, ischemia and several tumor models. For this specific purpose, the scanner configuration has been revised with dedicated home made animal coils and specifically written MR sequences. The relativity and bio-distribution of MR contrast agents were measured and calibrated for the scanner, which is essential for projects related to drug development. Recently, we applied optic imaging to identified Grp78 overexpressed head neck cancer (HNC) model from our group. In order to understand whether Grp78 can serve as a molecular therapeutic target, we employ siRNA technique to knockdown Grp78 expression of xenograft HNC in BALB/C nude mice. IVIS technique was used to monitor the status of tumor growth. Results showed that administration of Grp78-siRNA plasmid significantly inhibited both lines (Fadu, Detroit) of xenograft tumor growth in situ (~70% inhibition at day 34), as well as inhibition of liver metastatic potential. For microPET studies, we have applied it in our cholangiocarcinoma mice model. Results of microPET versus our cholangiocarcinoma mice model have also been verified with autoradiography. We also used microPET to evaluate the therapeutic effect of with and without radiation therapy in prostate cancer mice model. For microSPECT/microCT, we have successfully used it to detect the BBB disruption under HIFU in SD rats. Moreover, microSPECT/microCT has also bee applied in rodent serotonin transporter imaging and the correlation between imaging results and quantitative autoradiography was satisfied.

 

 

PL5 Optical manometrology

Yia-Chung Chang

Research Center for Applied Sciences, Academia Sinica
128, Sec. 2, Academia Road, Taipei 115, Taiwan

 

Ellipsometric measurements coupled with efficient theoretical modeling are used to determine the size, density, and distribution of various nanostructures on a multilayer film. Examples considered include metallic nanospheres on a substrate and semiconductor nanorods. These samples were investigated by variable-angle spectroscopic ellipsometry (VASE) in the ultraviolet to near infrared region (with wavelengths ranging from 140nm to 1100nm). The spectroscopic ellipsometry measurements were carried out at several angles of incidence. It is found that the results at 60o angle of incidence, which is close to the Brewster angle of the sample, show more distinct responses for different samples. To understand the distribution of nanoparticles, rigorous coupled-wave analysis (RCWA)1 and finite-element Green¡¦s function method2 were used to model the polarization-dependent reflectivity. Our model calculations are in reasonable agreement with the ellipsometric measurements assuming the size distribution of nanoparticels is uniform. Via the inverse scattering technique, we are also able to determine the average geometry of semiconductor nanorods with resolution much smaller than the wavelength. This demonstrates that the spectroscopic ellipsometry could be a useful tool to provide information about the size, shape and distribution of nanostructures on a substrate or buried inside a multilayer film.

1. M.G. Moharam, E.B. Grann, and D.A. Pommet, J. Opt. Soc. A12, 1068 (1995).
2. Y.-C. Chang, G. Li, H. Chu, J. Opsal, J. Opt. Soc. A 23, 638 (2006).

 

 

PL6 Translational bioinformatics: a bioinformatics approach to discover the disease genes and mechanisms

Ueng-Cheng Yang

Bioinformatics Research Center, National Yang-Ming University

 

Disease is derived from a perturbation of normal regulation, which may lead to observable phenotypic changes. Since more and more diseases were found to be predisposed by genetic factors, the interaction of genes may play an important role in disease formation. By integrating the phenome with protein interaction network, the functional links of known risk factors can be revealed. We have taken advantage of this correlation to predict the candidate factors that may associate with diseases. On the other hand, studying the disease mechanism is more complicated, because different patients may have their unique way to develop the disease as a result of a specific gene-environment interaction. From another point of view, this complication may open a window for us to compare different disease states. Therefore, the process of disease formation and the regulatory circuit can be studied by comparing these states. By using the microarray data of each individual, it is possible to discover disease subtypes. An algorithm has been developed to discover the state-specific pathways. The disease-related pathways were first retrieved by using the Microarray Annotation and Profile (MAP) tool. The state-specific gene expression patterns were then mapped to known pathways. The function of pathways were annotated by gene ontology (GO). Therefore, the pathways can be classified by their functions in the cell. In the case of breast cancers, the IGFR and EGF pathways appear to be the predominant pathway for proliferation in estrogen receptor positive and negative subtypes, respectively. This observation was made by a pure informatics approach, but it was supported by wet laboratory experiments published previously. Taken together, these methods have successfully predicted phenomena that have been observed experimentally. Therefore, this strategy has the potential to analyze unknown phenomena in the future.

 

 

PL7 Non-classical Pathway of Fibroblast Growth Factor Releasing

Chin Yu

Chemistry Department, National Tsing Hua University

 

Fibroblast growth factors (FGF) are key regulators of cell proliferation, differentiation and migration. They are crucial for early embryonic development, organ formation and angiogenesis. They also play important roles in tumor formation, inflammation, wound healing and restenosis. The biological functions of FGFs are mediated by a family of transmembrane tyrosine kinase receptors, FGFR. Thus to exert their activities, FGFs have to be released into the extracellular compartment. However, acidic FGF (FGF-1) and basic FGF (FGF-2), two prototypic members of the FGF family, which are represented in practically all tissues, lack the conventional amino-terminal signal sequence needed for the secretion of proteins through the classical endoplasmic reticulum (ER)-Golgi apparatus secretory pathway. Maciag et al. demonstrated that under cellular stress, FGF-1 is exported through a non-classical release pathway involving the formation of a specific copper (Cu2+)-dependent multiprotein complex. The protein constituents of this complex include the FGF-1 homodimer, the small calcium (Ca2+) binding protein, S100A13, and the p40 form of synaptotagmin 1 (Syt1), displaying two lipid binding domains, C2A and C2B, is a membrane docking protein which participates in exocytosis. The exact mechanism(s) by which the protein constituents specifically interact with each other and the molecular events which occur during the release of the multiprotein complex is still not clear. We propose a hypothetical model for the sequence of molecular events leading to the formation of the multiprotein release complex. The main aim of this proposal is to elucidate the molecular mechanism(s) underlying the non-classical export of FGF-1 through understanding the structure of the FGF-1 release complex.