Studying Local Stable Segments to Reconstruct the Three-Dimensional Structures of Proteins Hueih Min CHEN (PI), Tian Yow TSONG
RGC Project no. HKUST 519/94M ($540,000)
Conversion of Trichosanthin, An Anti-Viral and Abortifacient, to Human Acceptable Protein Hueih Min CHEN
UGC Project no. CPDG 94/95.SC21 ($11,000)
Trichosanthin (TCS) is one of the useful proteins found in the root tuber of Trichosanthes kirilowii Maximowicz, a Chinese medicinal herb used as an abortifacient agent in China. This plant protein consisting of 247 amino acids (27 kD) is identified as a ribosome-inactivating protein since it can inhibit the synthesis of proteins in the cells both in vivo and in vitro. Besides the above mentioned biological function, TCS possesses other pharmacological properties including: (i) inhibition of human immunodeficiency virus (HIV), (ii) inhibition of tumor cells, (iii) immunomodulation, and (iv) termination of gestation. These properties may give TCS great potential to be a versatile drug. In clinical trial, However, patients experience serious side-effects upon taking TCS. For therapeutic purposes, therefore, the mutants with minimal side effects have to be generated. The basic aim of this proposal is to convert plant TCS to a form acceptable to humans. We will preliminarily study the structure/function relationships, especially on alpha helices. Assays for side effects caused by the modified proteins will then be conducted
Construction of a Plant Gene Overexpression System King-Chuen CHOW
RGC Project no. DAG95/96/SC01 ($80,000)
A plasmid carrying the left and right borders of the T-DNA, the kanamycin resistance conferring gene, a heat shock protein promoter derived from Glycine max, the gus gene and Nos terminator has been constructed.
When this plasmid was used to transform the alga Chlamydomonas reinhardtti through electroporation, all the kanamycin resistant transformants showed the gus activity. When five of the transformants were subjected to a high temperature treatment (35 degree celsius) for two hours, the gus activity could be increased from three to five fold. Southern blot analyses of these five tested transformants showed that the gus gene had integrated into the host chromosome at different sites. The difference in expression rate possibly is a reflection of the position effect of the integration sites. This study shows that the Agrobacterium derived T-DNA borders are likely to be functional in an algal system other than dicot or monocot plants. Besides, the heat inducible nature of the heat shock protein promoter of the Glycine max is also preserved in the alga.
The Development of an Expression System, Based upon Hybrid Salmonellajlagellins, for the Synthesis of Protective Epitopes of Staphylococcus aureus
James A. HACKETT
BRI Project no. BRI92-VI-1 ($313,000)
The development of antibodies which can react with particular regions of proteins is an area of interest and importance in several spheres of biotechnological activity. First, vaccination against pathogens may be effected, in the future, by injection of humans or animals with a recombinant protein which expresses, in an appropriately
"folded" form, a piece of a protein characteristic of the pathogen; immunity to this area of the protein would be protective. In the present work, it is proposed to construct recombinant genes, in which regions of vaccinally important proteins are included in the primary sequence of the genes for bacterial flagellins; these hybrid proteins are produced in large amounts by the cells, and may be readily purified by simple shearing of cell suspensions. It is known that Salmonellajlage!lin genes may be manipulated in this way.
The Development of a New Plasmid Partition System to Effect High-Efficiency Stabilization of Recombinant Plasmids in Various Strains Used as Hosts in Biological Fermentations
James HACKETT
RGC Project no. HKUST 518/94M ($467,000)
Fermentations of yeasts and bacteria which carry foreign genes yield high-value products (such as insulin or enzymes) in quantity. Foreign genes may not always be stable in the host strains, as they are usually expressed from plasmids (autonomously replicating DNA fragments), which can be lost from the host cell. The project addresses this problem. Genes which confer very high stability on a plasmid naturally present in Salmonella have been isolated and sequenced; the gene functions are now being explored. These genes, incorporated into recombinant plasmids, will help increase product yield from commercial fermentations.
Controls on the Expression and Function of the rfc Gene, which Encodes the Polymerase Assembling the 0-Antigen Coat of Salmonella Bacteria
James HACKETT (PI), Christina MORRIS RGC Project no. HKUST 556/95M
Many bacteria which cause infections possess an outer coat composed of polymerized sugar residues. The rfc gene of the Salmonella bacteria encodes an enzyme important in the polymerization process. The operation of the gene is not well understood. The objective of this work is to examine the gene in detail: it may be that the gene controls both the polymerization process and the length of polymer formed. The gene, and nearby DNA, is cloned; specific rfc functions will be sought in various genetic regions. When completed, the work will explain the mechanics of the polymerization process, and the work will perhaps permit the design of drugs to interrupt the normal assembly of the surface sugar residues. Such drugs would be of value in human and veterinary medicine.
Molecular basis of Bacterial Pathogenesis: Species-Specificity in Pathogenic Salmonella Serovars James A. HACKETT (PM), Gordan DOUGAN (Imperial College, UK)
British Council/RGC Project no. JRS93/49 (£5,536). Project completed.
This project aims to identify genes on the Salmonella chromosome which are involved in the determination of host specificity. The specific scientific aims are (a) to defme DNA sequences that are present in Salmonella typhi but not in other serovars of Salmonella which are not invasive for humans when given orally, and (b) to define DNA sequences that are present in Salmonella typhimurium but not in other serovars of Salmonella which are not invasive for the mouse when given orally. As S. typhi is completely specific for humans, the inference is that unique sequences will play a role in this host-specificity. It further follows that proteins encoded by such sequences may be useful antigens in vaccination programmes and in studying the epidemiology of Salmonella in the clinical environment. For example, if one such protein mediated attachment of S. typhi to human gastrointestinal cells, then the development of gut immunity against this protein might be an effective means of vaccination against the disease. The gene could also provide a highly specific probe for identifying Salmonella species in clinical situations.
Comparison of Memory Enhancing Effects of Huperzine A and Tacrine in Rats with and without Central Cholinergic Deficiencies
YifanHAN
BRI Project no. BRI 94-I-3 ($100,000)
Comparative Studies of a New Memory Enhancer Huperzine A with Tacrine YifanHAN
RGC Project no. DAG94/95.SC11 ($60,000) Project completed
Development of Chinese Medicinal Products with Antioxidant Properties
Robert K.M. KO (PI), Chun-Tao CHE (CHEM), Yun Cheung KONG (Biochemistry, CUHK) BRI Project no. BRI91-II-2 ($200,000)
growing body of scientific evidence now indicates that the accumulation of free radical (oxidant)- induced tissue damage is an important determinant in the aging process, resulting in a cluster of degenerative diseases in the terminal part of the life-span. This points to the possibility of preventing the onset and/or progression of age-related diseases by promoting the ability of the body to combat free radicals, possibly through nutritional/therapeutic manipulation. In view of the enormous potential of developing naturally occurring anti-aging agents/regimens from traditional Chinese medicine, we have embarked on the present project to provide a biotechnological basis for the design and manufacture of optimal herbal preparations with demonstrable effectiveness in retarding age-related debilitation.
Development of a New Class of Antioxidants for Therapeutic Applications
Robert K.M. KO (PI), Chun-Tao CHE (CHEM), Yun Cheung KONG (Biochemistry, CUHK) BRI Project no. BRI94-II-1 ($150,000)
A growing body of scientific evidence now indicates that the accumulation of free radical-induced tissue damage is an important determinant in the aging process, resulting in a cluster of degenerative diseases in the terminal part of human life-span. An antioxidant compound isolated from a tonic Chinese herb may produce a widespread beneficial effect on age-related diseases. The structure-activity-relationship and the biological activity profile of the compound are being investigated in order to develop a novel class of antioxidants, prefenably in collaboration with pharmaceutical firms, for the treatment of free radical diseases.
Brain Angiotensin II, Ventrolateral Medulla and Hypertension
Peter H. Y. LAM (CI), T.M. WONG (PI, Physiology, HKU), Y.S. CHAN (Physiology, HKU), RGC Project no. HKU 331/93M ($236,000; $118,000 for HKUST) University of Hong Kong project.
Both the brain angiotensin II (Ang II) and sympathetic nervous system are essential in the genesis of hypertension. The sympathetic nervous system has been shown to mediate the effect of brain Ang II leading to alterations in arterial blood pressure (BP). However, the site(s) of action of Ang II in the brain leading to the activation of sympathetic nervous system has not been determined. One of the possible sites of action is the ventrolateral medulla (VLM), which has recently been shown to: (1) play a crucial role in the control of circulation, (2) be an important site of action of brain Ang II, and (3) be involved in the genesis of hypertentsion. The primary aim of this study is to evaluate the importance of the interaction of brain Ang II and VLM in the genesis of hypertension by: (1) correlating BP with the presence of brain Ang II, the spontaneous activities of cardiovascular neurons in VLM and the development of high BP in rats, and (2) studying the changes in BP and properties of VLM neurons after artificial suppression of the biosynthesis of brain Ang II.
These experiments will provide useful information on the cause-effect relationship among brain Ang II, VLM cardiovascular neurons and BP. Another aim of this project is to determine electrophysiologically the origin of brain Ang II that exerts a tonic influence on VLM. The results of the study will be of clinical importance in the prevention and treatment of essential hypertension.
The Construction and Assessment of Insecticidal Activity of Recombinant Baculoviruses Expressing Insect Oostatic Hormone and Diuretic Hormone Genes: Potential Biological Insect Pest Control Agents Peter H. Y. LAM
RGC Project no. DAG94/95.SC17 ($96,320)
Vegetable growers commonly use chemicals to control insect pests, especially foliage feeders. Improper use of these toxic chemical insecticides has resulted in cases of consumer poisoning in Hong Kong and China. Other drawbacks of chemical insecticides include decreasing effectiveness as target insects develop resistance to the chemicals and the eradication of beneficial insects. Alternative means of controlling crop pests are urgently needed.
Baculovirus, an insect-specific virus, is one of several promising biological control candidates being evaluated.
Advantages of using baculovirus include: (1) host specificity; (2) no harmful effects on mammals and other vertebrates; (3) environmentally friendly; and (4) self-spreading, in that infected insects act as virus-generating factories such that more target insects are killed. One disadvantage of this system, however, is the slow action of the virus. This project aims to construct baculoviruses with more efficient insect-killing capabilities by incorporating insect hormone genes into the virus genome to form recombinant viruses. The outcome of this project promises to have an important impact on regional plant protection practices and benefit local consumers by lowering their exposure to chemical pesticides.
Regulation of Inositol Containing Phospholipids on Protein Kinase C Substrates (MARCKS, Neuromodulin, and Neurogranin) Phosphorylation
Fwu-Shan SHEU
RGC Project no. DAG94/95.SC10 ($60,000) Project completed.
Protein kinase C (PKC) gene family regulate several neural functions including transmitter release, ion channel activity, neural growth and plasticity. Since acidic phospholipids (in particular the inositol phospholipids) are generated in specific cell surface receptor-mediated membrane phospholipids turnover pathways to transduce cellular functions, it is likely that the inositol phospholipids could regulate cellular functions not only through supporting the activity of PKC but also regulating the phosphorylation ·of PKC substrates. To test this hypothesis and gain further understanding of functional roles played by PKC substrates in the signal transduction pathways, we will explore the possibility of interaction of inositol containing phospholipids with three purified PKC substrates, MARCKS, neuromodulin and neurogranin in this project.
This project will involve purification of PKC and its three major substrates from rat brains to homogeneity.
With these purified enzyme and substrates as materials, in vitro phosphorylation assay will be used to assess whether inositol phospholipids affect the phosphorylation of PKC substrates. For comparison, the effect of some acidic phospholipids without inositol, such as phosphatidylserine, phosphatidic acid, and phosphatidylglycerol, on PKC substrates phosphorylation will be tested. To distinguish the effect of these lipids on enzyme or on substrates, the enzyme activity against histone substrate will be compared with thost of the three physiological PKC substrates. The results will be useful for determining a linkage between agonist-induced phosphoinositol turnover and protein kinase C/substrates phosphorylation system. The elucidation of these signal transduction parthways may help us developing a microinjection system for neuronal culture to directly monitor their cellular effect
Structure Study of Neuron Specific Protein Kinase C Substrate, Neurogranin, by Multidimentional Nuclear Magnetic Resonance
Fwu-Shan SHEU (PI), Guang Zhu
RGC Project no. DAG95/96.SC02 ($80,000)
Several physiological target substrates of PKC have been identified. Among them, neurogranin (Ng) is of particular interest. The structure change and significance of oxidation of Ng on its function will be further explored in this project by multidimensional nuclear magnetic resonance (NMR) spectroscopy. We have recently found that using either hydrogen peroxide or some nitric oxide (NO) generating agents, Ng can be oxidized in a time- and dose-dependent manner. This type of oxidation reversibly drives Ng predominantly to form intra- rather than inter-molecular disulfide bond(s) which implies a thermodynamic favor state for Ng to form intramolecular disulfide bond(s) upon oxidation. With respect to the function ofNg, oxidation reduces the binding affinity ofNg to calmodulin (CaM) which binds toNg in low [Ca2+] condition. The released CaM will then be able to trigger many biological effects through Ca2+/CaM dependent signal transduction pathways. To understand how the structural change ofNg leads to its biological function, we propose to purify~ 10 mg Ng from rat brain and use pure halo-protein to study the three-dimensional structure ofNg in its reduced state and oxidized state by multidimensional NMR.
Recent development in NMR spectroscopic techniques and hardware technologies enables scientists to study structures and functions of the biomolecules, especially proteins in aqueous solution which possibly mimics their physiological conditions. Multidimensional (2-D, 3-D and 4-D) NMR techniques have been used to study proteins with their molecular weight up to 30 kDa. The NMR spectroscopic methods that we will apply in this initial project consist of the following two dimensional (2-D) NMR experiments for structural study ofNg:(1) COSY and TOCSY experiments for proton assignments. (2) NOESY and ROESY experiments for proton assignments and structure calculation.
An Innovative Technology for Manufacturing Monoclonal Antibodies Tian Yow TSONG
ITDC Project no. AF/327/94 ($950,000)
Preparation of a monoclonal antibody involves two steps: fusion of myelomas with B-lymphocytes to prepare hybridomas, and screening of hybridomas to obtain productive hybridomas. This project will, 1) improve a surface-specific-cell-selection procedure to locate correct B-lymphocytes for electrofusion with myelomas, and
suspension, to prepare hybridomas. Irrelevant B-cells will be excluded from fusion because, without decoration of the antigen/avidin complex on their surfaces, they cannot form cell doublets with myeloma.
Selective B-Lymphocyte-Myeloma Cell Fusion for Antibody Production Tian Yow TSONG
UGC Project no. CPDG94/95.SC01 ($31,500) Project completed.
The idea was to use antigen to select productive B-lymphocytes from non-productive ones and then use the electrofusion method to fuse these productive B-cells to myelomas for preparing hybridomas. An antigen was cross-linked to myeloma surface. These antigen-decorated myelomas were mixed with B-lymphocytes from immunized mice. Electrofusion was applied to cell mixtures. We are testing the technology to assess its efficiency as compared to the efficiency which use the conventional PEG fusion method.
Construction and Analysis of Peptide Libraries for Therapeutically Active Peptides Tian-Yow TSONG (PI), James A. HACKETT, Peter H.Y. LAM
RGC Project no. HKUST 166/93M ($1,035,000)
Peptide libraries are large collections of peptides with wide sequence varieties in which peptides with various biological activities can be sought. We will use currently available DNA technologies to construct peptide libraries. From these libraries we will isolate and characterize peptides of three types. 1) Peptides which bind to fibrin. Such peptides will be fused to proteins such as tissue plasminogen activator (TPA) to increase the affinity of the TPA for blood clots. 2) Peptides which bind to human hepatocytes. The purpose is to search for TPA analogs which may have improved biological effects. 3) Peptides which mimic the interaction of the hormone calcitonin with its receptor. These peptides are candidates as drugs for the treatment of various diseases with altered calcium metabolism.
Molecular Mechanisms of Cell Interactions with Electromagnetic Fields Tian-Yow TSONG
RGC Project no. HKUST 555/95M ($531 ,000)
We use oscillatory electric field to activate Na,K-Pump of human erythrocyte. It was found that there are windows of field strength, frequency, and ligand concentration for an optimal activation. This result indicates that the Na,K-Pump can recognize an electric signal. The finding was analyzed with model analysis. The next experiment was to impose a white-noise electric field on an electric signal and examine effects of noises on signal recognition of the Pump. Our result indicates that a white noise did not diminish the ability of the enzyme to recognize the signal. In fact, up to certain level of noise, improved signal transduction by the enzyme. This is considered a novel finding.
Conformational Stability and Folding Mechanism of Engineered Protein Analogs Tian Yow TSONG
BRI Project no. BRI93-I-1 ($584,000)
A major goal of biotechnology is to produce medically and agriculturally valuable proteins cheaply using modem genetic, molecular biology and chemical synthetic techniques. In the process of large scale biotech manufacturing, much of the product--when it is a protein--is often either inactivated or degraded before purification. These problems are caused by protein instability and difficulties in folding. This project will find out how these difficulties can be solved. Staphylococcal nuclease, cytochrome c, and ribonuclease A have been selected as model proteins for study using various physicochemical methods: differential scanning microcalorimetry measures protein stability; circular dichroism and fluorescence spectroscopies measure protein structure in solution; stopped-flow rapid kinetics measures time course of the folding/unfolding transition and characterizes folding intermediates; amino-acid substitutions determine how different types of molecular interactions contribute to protein stability and dictate protein folding pathways of protein analogs. Data will be obtained to understand effects of salts, pH, temperature, and amino acid substitutions on protein stability and folding mechanisms, with the goal of designing superior protein analogs with therapeutic activity.
Design of Specific Substrates of Cdc2-Like Protein Kinases Jerry H. C. WANG
APSB Second International Symposium and Workshop Jerry H.C. WANG
ITDC Project no. AF/224/95 ($391,500)
The Asia Pacific Society of Bioscientists (APSB) is a professional society with the objectives of stimulating interactions between its members and between communities of bioscientists, biotechnologists and industrialists of the region. The biennial symposium and workshop held under the auspices of the society is one of the most effective mechanisms to achieve thse objectives.
The Second International APSB Symposium and Workshop will consist of three days of intense scientific activities with formal sessions on current bioscience and biotechnology research. There will be plenary, symposium and poster sessions where the areas covered will range from basic bioscience topics to medically
The Second International APSB Symposium and Workshop will consist of three days of intense scientific activities with formal sessions on current bioscience and biotechnology research. There will be plenary, symposium and poster sessions where the areas covered will range from basic bioscience topics to medically