17th Philippine Chemistry Congress
May 23-25, 2001
Cagayan de Oro CityABSTRACTS: BIOCHEMISTRY
TS3 B1
LIFE IN THE POST-GENOME ERA: OPPORTUNITIES FOR CHEMISTS
Nina Rosario L. Rojas, Niña Socorro dJ. Cortina, Lieza Marie A. Danan,
Abigail T. Go and Bernadette M. Henares
Department of Chemistry, Ateneo de Manila University, Loyola Heights, Quezon City 1108 Philippines
In the last month, the world witnessed the publication of the sequence of the human genome, as investigated by a publicly funded international consortium (The Human Genome Project Consortium) and by a private corporation (Celera Genomics, a division of Perkin-Elmer). Along with the sequence were articles in both scientific journals and the popular press on the implications of this milestone in the life sciences. Yet as much as this project represents a major breakthrough in our understanding of life, many questions are yet to be answered.
This paper presents some of the post-genome questions, and the current strategies scientists use to answer them. Already, new buzzwords such as "proteomics" and "transcriptomics" and "functional genomics" and "functional proteomics" are floating from the lab into the popular consciousness. These words represent the need to bridge the gap between the genetic information, which is like the master list of potential properties of an organism, and the actual, living, dynamic reality of the organism interacting with a concrete and changing environment.
It is in this more complex reality that the much-hyped gifts of the genome project will bear fruit. Many of the questions here will require understanding what chemists understand best: what molecules are, how they interact with each other, and how they are transformed. In particular, proteins are a central piece to the puzzle of life, and strategies for profiling and characterizing proteins offer an exciting follow-up to the human genome discoveries.
TS3 B2
GENERATION AND CHARACTERIZATION OF A SINGLE-CHAIN HUMANIZED ANTITUMOR ANTIBODY WITH IGE EFFECTOR FUNCTION
Ameurfina D. Santos1, Noreen R. Gonzales2, Jonathan Housden3, Birgit A. Helm3,
Syed V. Kashimiri4 and Eduardo A. Padlan5
1National Institute of Molecular Biology and Biotechnology, College of Science, University of the Philippines, Diliman, Quezon City
2Department of Chemistry, Ateneo de Manila University, Loyola Heights, Quezon City
3Krebs Institute for Biomolecular Research, University of Sheffield, UK
4Laboratory of Tumor Immunology and Biology, National Cancer Institute
5Laboratory of Molecular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA
Recombinant DNA technology has made possible the generation of antibodies with desired properties. For example, reduced immunogenicity and preferred effector function may be realized by humanizing the antibody and by using the fitting isotype respectively. We wanted to generate an antitumor antibody with an IgE effector function because the reaction of the antibody with the antigen on a tumor, and with the Fce receptor on the effector cell results in the release of mediators which cause inflammation in the tumor site, and may result in tumor regression. We made a single-gene construct that encodes a single-chain variable fragment scFv of an antibody that has been humanized by CDR grafting. The antibody targets the tumor associated glycoprotein TAG72, an antigen found in a variety of carcinomas including cancer of the colon, breast and lung. The scFv was joined to the DNA of a small part of Ce 2, and the whole of Ce 3 and Ce 4 of the human IgE Fc to impart the desired effector function. The gene was expressed in insect cells and the antibody generated was characterized. In vitro assays show that the antibody is functional; it is reactive with the antigen and with the Fce receptors causing the activation of the effector cells. This immunoglobulin molecule is a very promising reagent for the therapy of human carcinomas.
TS3 B3
BIODESULFURIZATION OF GAS OIL BY Rhodococcus erythropolis Q1A-22
James A. Villanueva1, Ma. Auxilia T. Siringan1 and Michelle A. Demata1
1Natural Sciences Research Insitute, Univesity of the Philipines, Diliman
2Institute of Chemistry, University of the Philippines, Diliman
Elimination of sulfur in fuels is essential due to the release of sulfoxides upon combustion. Sulfur oxides are environmental pollutants and lead to the formation of acid rain. Existing chemical desulfurization techniques are becoming inadequate in lowering sulfur levels. Biodesulfurization is offered as a potential alternative process since it is both inexpensive and metabolically specific. We present in this study desulfurization of gas oil by Rhodococcus erythropolis Q1a-22. R. erythropolis Q1a-22 was found to have highly efficient desulfurization activity on dibenzothiophene (DBT), a recalcitrant organic sulfur compound found in fuels. Desulfurization of petroleum by the strain was determined by growing the bacteria in mineral salts sulfur-free (MSSF) medium with gas oil. Liquid-liquid extraction was performed using methylene chloride as extracting solvent (ratio = 1:1). Elemental analysis, HPLC, ion-exchange chromatography and turbidimetric analyses were done to follow the progress of desulfurization.
TS3 B4
A BIOINFORMATICS TOOLKIT FOR PROTEOME ANALYSIS
Abigail T. Go and Nina Rosario L. Rojas
Department of Chemistry, Ateneo de Manila University, Loyola Heights, Quezon City 1108, Philippines
Protein identification and characterization has been refined greatly in the recent years to a highly systematized method that begins in the wet laboratory and ends in front of a workstation or a personal computer. The final step, which involves biological informatics or bioinformatics, is particularly vital to this streamlined process. As such, there is an increasing need to not only create protein databases and the corresponding tools to search these databases but also to evaluate and review them. This need is further highlighted by the contrast of the strong Philippine focus on the study of plants, both medicinal and agricultural interest and the comparatively sparse amount of information available on plant proteins.
As such, a protein bioinformatics toolkit and tutorial has been assembled to aid the protein researcher. The toolkit assembles, classifies, and annotates a select group of bioinformatics tools available (at no cost to non-profit institutions) on the World Wide Web. These tools and the scientific concepts behind them are discussed and introduced at every level. A tutorial is included to illustrate the concepts and uses of the toolkit. It is interactive and walks the user through the actual use of these tools. Both the toolkit and tutorial were developed with an eye towards the needs and resources of the plant protein chemist and biochemist. An advanced version for users who are familiar with the tools and their complexities is also provided.
TS4 B5
Chromatographic evidence of the interaction of Paralytic Shellfish Poisoning (PSP) toxins with Ca-Alginate and k -Carrageenan gels
Marco Nemesio E. Montaño, Socrates Jose P. Cañete, Cecilia B. Conaco and Lourdes J. Cruz
Marine Science Institute, University of the Philippines, Diliman
The positively charged, highly potent paralytic shellfish poison (PSP) has caused major menace in the Philippine community with a casualty toll of over 700 individuals from 1998 to 1998 and, by far, a fool-proof antidote for these toxins has not yet been reported. Alginates and carrageenans are two groups of polysaccharides that are derived from seaweeds containing negatively charged moieties. Both polysaccharides have been proven to possess cation exchange properties. It is highly possible that these compounds could interact with the cationic paralytic shellfish toxins and can be used as PSP-sequestering agents. This paper presents evidence of the interaction between PSP and the gel forms of alginic acid and k -carrageenan. Chromatograms of interacted PSP extracts show that there is a 57.37%-diminution in the amount of saxitoxin for the carrageenan-interacted extract and 42.66%-diminution for the alginate interacted extract. Differences in chromatographic profiles show that the concentration reduction is not brought about by sample dilution. It further indicates that the action of carrageenan gel on PSP is likely sequestration or retention while the action of alginate is likely conversion to another compound that is not toxic to mammals. These data were corroborated by toxicity studies where the carrageenan interacted extract showed a toxicity reduction of 65.12% and a 61.49%-toxicity reduction for that of the alginate-treated extract. These in vitro results strengthen the promise of developing these polysaccharides as antidotes for paralytic shellfish poisoning.
TS4 B6
EFFECT OF DIFFERENT CULTURE SUBSTRATES AND EXTRACELLULAR MATRIX ON THE SURVIVAL AND DIFFERENTIATION OF, NEURAL PRECURSOR CELLS
Ann Marie G. Moran and Cynthia P. Palmes-Saloma
Laboratory of Molecular and Cell Biology, National Institute of Molecular Biology and Biotechnology, University of the Philippines, Diliman
Neurons in culture require specialized conditions, such as a matrix coating on the growth vessel, in addition to the regular requirements for culture of cells. In this paper, we studied the effect of varying substrate as well as serum concentration. Initial survival of neural precursor cells is largely affected by their attachment to the substrate. Different types of substrates have been described such as poly-lysine, poly-ornithine and collagen. We cultured neural precursor cells from mouse embryos using various matrices. An analysis of the substrates mentioned was done by comparison of growth properties and morphology of differentiated cells. Cells cultured on all three substrates survived and exhibited normal neural morphology in vitro. Neurons cultured in clumps extended neurites towards other neurons. Collagen coated dishes provided inadequate anchorage for neurons since cells were easily washed off during medium change. Among the substrates tested, poly-ornithine provides the best results at the most reasonable expense.
TS4 B7
THE EFFECT OF MOLECULAR WEIGHT AND THE PRESENCE OF OTHER COUNTER-CATIONS ON THE BINDING CAPACITY OF i -CARRAGEENAN
TO LEAD, CADMIUM AND ZINC
Dahlia C. Apodaca and Clovia Isabel Z. Holdsworth
Chemistry Department, De La Salle University, Taft Avenue, Manila
Carrageenan is an anionic biopolymer extracted from numerous species of red seaweeds and extensively used as gelling, thickening and stabilizing agents in the food, pharmaceutical and cosmetics industries. k - and i -carrageenan are produced in large volumes in the Philippines. Recent studies aimed at expanding the applications of carrageenan explored the possibility of using the polysaccharide as a remediation agent and metal sorbent. The results of these studies showed that both k - and i -carrageenan are capable of binding with heavy metal ions, particularly lead, cadmium and zinc, and that the iota form shows a higher binding capacity.
To date, extensively studied remediation agents include ethylenediaminetetraacetic acid (EDTA) and nitriloacetic acid (NTA). They are both known to bind metals by chelation. On the other hand, the mechanism of metal binding to carrageenan has not yet been established. There is no doubt that ion-exchange is predominant but other mechanisms are also possible. Investigation into the heavy metal binding capacity of carrageenan has been pursued without regard to the mechanism of metal-carrageenan interaction.
In order to understand wholly the efficiency of carrageenan as metal sorbent, it is essential to get a clear perception of its metal-binding mechanism by investigating the effect of Group I and II counter-cations and molecular size (weight). Results of this study show that the ability of i - carrageenan to bind to lead, cadmium and zinc ions is affected by the presence of Group I and II counter-cations. Ion-exchange with lead proved to be more efficient. The amount of lead, cadmium and zinc bound to i -carrageenan has also been observed to be lower in hydrolysed (lower molecular weight) samples suggesting that metal-binding mechanisms other than ion-exchange operate. Except for studies done locally, no other studies on the metal-binding ability of carrageenan has yet been reported.
BP1
METHOD DEVELOPMENT FOR MOLECULAR WEIGHT DETERMINATION OF PROTEINS USING HIGH RESOLUTION ELECTROSPRAY IONIZATION MASS
SPECTROMETRY (ESI-MS)
Lieza Marie A. Danan1, Milady R. Niñonuevo2 and Nina Rosario L. Rojas1
1Department of Chemistry, Ateneo de Manila University, Loyola Heights, Quezon City
2National Chemistry Instrumentation Center, Ateneo de Manila University,
Loyola Heights, Quezon City
One of the key attributes used to identify proteins is its mass. However, ordinary mass spectrometry cannot handle proteins due to their large mass and the difficulty of obtaining them in the gas phase. Electrospray ionization (ESI) is one of the ionization methods, which have been developed for non-volatile or heat-sensitive molecules, such as proteins.
ESI-MS has generally been performed on proteins using quadrupole mass spectrometers. On the other hand, the National Center for Chemistry Instrumentation is equipped with a B/Z double-sector high resolution Finnigan MAT95T mass spectrometer with electrospray ionization capability. In this study, parameters were developed to adapt protein molecular weight determination methods from the literature to the MAT95T.
The parameters were initially tested using a readily available protein, egg white lysozyme from chicken. This protein is basic and its mass spectrum can be obtained at low pH using positive-mode detection. Results indicate that the amount of acid in the sample solution is a key factor, because it determines the extent of protonation of the protein. Tests using lysozyme have shown that the instrument can analyze as low as 3 x 10-9 M samples of this protein, although better results are obtained at higher concentrations (e.g., 3 x 10-5 M).
Further studies are being performed to develop methods to determine protein masses in mixtures.
BP2
TWO DIMENSIONAL GEL ELECTROPHORESIS FOR PROTEIN PROFILING: A STUDY OF VITEX NEGUNDO
Niña Socorro dJ. Cortina, Bernadette M. Henares and Nina Rosario L. Rojas
Department of Chemistry, Ateneo de Manila University, Loyola Heights, Quezon City
Proteomics is the large-scale study of the proteins in a cell, organism, or biological fluid. Unlike the genome, the proteome is dynamic, readily responding to changes in metabolic activity and environmental conditions. One of the most important tools for separating and visualizing the protein repertoire is two dimensional gel electrophoresis (2-D PAGE). Proteins are separated according to their isoelectric point by isoelectric focusing (IEF) in the first dimension. On the second dimension, the proteins are resolved by their molecular weights using sodium dodecyl sulfate electrophoresis (SDS-PAGE). This technique allows the resolution of a large fraction of the proteome in a single experiment.
Two-dimensional PAGE was initially tested on a simple mixture of proteins, i.e., hen egg white. The reproducibility of the protein separation was tested to permit comparison between two or more gels within the same pI range and gel concentration. The method was then used to obtain a protein profile from the leaves of the plant Vitex negundo (lagundi). The plant extract contained a complex mixture of proteins, many of which have high molecular weights. The method developed and the protein profile obtained in this study can be used in subsequent work to track the plant's responses to various environmental conditions.
BP3
A Comparative Study of the Cholesterol Level in the Serum
and Liver of Mice Fed with Orlistat and Chitosan
Nancy Lazaro-Llanos, Sol Teresa B. Bautista and Jennifer O. Sia
Chemistry Department, De La Salle University, Taft Ave., Manila
The effects of Orlistat and Chitosan on liver and serum cholesterol levels in mice were compared. Orlistat is a synthetic prescription drug, which is a derivative of lipstatin. Its action is to inhibit the enzyme pancreatic lipase in the lumen of the intestine. Chitosan is a polysaccharide in which the repeating unit is b -D-glucosamine. It is used as a source of dietary fiber. Groups of mice were weighed and fed with the specified diet, weighed and sacrificed after three weeks, blood collected individually and the liver excised. Cholesterol was extracted from the serum and liver and determined spectrophotometrically using the Lieberman-Burchard Reaction. Results show that mice that had Xenical added to the diet had a lower level of cholesterol in both the serum and the liver compared to the group of mice that took the chitosan and the normal diet.
BP4
THE Y-CHROMOSOME STR SYSTEM AND FORENSIC DNA ANALYSIS
IN THE PHILIPPINES
Frederick C. Delfin, Gayvelline C. Calacal, Saturnina C. Halos and Ma. Corazon A. de Ungria
DNA Analysis Laboratory, Natural Sciences Research Institute,
University of the Philippines, Diliman
Empirical examination of records of reported crimes in the National Capital Region (physical injury, theft, robbery, murder, homicide and sexual assault), reveals a high probability of isolating biological materials from crime scenes and/or victims. Samples may include blood, various body secretions (e.g., semen, saliva, perspiration), muscle tissue and other isolated body parts.
We initially reported procedures for forensic DNA analysis using autosomal DNA short tandem repeat (STR) system and Amelogenin sex-determining marker for effective profiling. These systems were previously validated by the laboratory and were found to be effective in typing human DNA. However in certain instances such as rape and father-son paternity cases, identification of male specific DNA is necessary.
To facilitate the detection and profiling of male DNA from biological evidence, a Y- chromosome database consisting of short tandem repeat markers, namely DYS19, DYS390, DYS393, DYS385 was constructed. This system was validated to analyze different types of biological samples namely blood, buccal cells, muscles and bone tissues. Our results show that the Y-STR system was successful in first, determining the presence of male DNA and second, in identifying the male source of the DNA with relatively high probability in all samples tested. Overall, we present here a new system that can compliment the already existing autosomal database of the Philippine population for forensic and paternity applications.
BP5
ISOLATION OF YEAST INVERTASE USING CHITOSAN AS SOLID SUPPORT
Elizabeth C. Colat, Veronica C. Sabularse and Marivic S. Lacsamana
Institute of Chemistry, College of Arts and Sciences
University of the Philippines, Los Bas, Laguna
Yeast invertase was immobilized on glutaraldehyde-activated chitosan. Optimum conditions for activity of both free and immobilized invertase were determined by pH 4.5, a temperature of 55oC and a sucrose concentration of 0.3 M. The immobilized invertase exhibited an activity of 85.45% relative to that of free enzyme and was found to have lower energy of activation. Immobilization of invertase did not alter its stability towards storage.
BP6
"LAHAR" AS A POTENTIAL INORGANIC SOLID SUPPORT FOR YEAST
INVERTASE IMMOBILIZATION
C. G. Mendoza, V. C. Sabularse and D. C. Sabularse
Institute of Chemistry, College of Arts and Sciences
University of the Philippines, Los Bas, Laguna
The use of lahar as an inorganic solid support for immobilization of yeast invertase was evaluated. Yeast invertase was covalently immobilized on activated lahar. Activation involved ignition at 55oC for 5 hours followed by treatment with concentrated hydrochloric acid. The two components of lahar were separated (white and black lahar) and were activated by treating with g -aminopropyltriethoxysilane followed by reaction with glutaraldehyde. The activated lahar was then incubated with invertase for immobilization.
The covalent coupling of silane and glutaraldehyde onto lahar were verified by chemical tests and FT-IR analyses.
Invertase covalently coupled with lahar retained 76.5% of its activity. Storage stability of both immobilized and free enzymes were compared and result show that the stability of the former was enhanced. The performance of the immobilized enzyme towards repeated use was also studied and results tend to show that its performance is maintained even after repeated use.
BP7
EFFICACY OF VARIOUS TRANSFECTION TECHNIQUES TO TRANSIENTLY EXPRESS GFP IN PRIMARY CULTURES OF NEURAL PRECURSOR CELLS
Cynthia P. Palmes-Saloma and Ann Marie G. Moran
Laboratory of Molecular and Cell Biology, National Institute of Molecular Biology and Biotechnology University of the Philippines, Diliman
The introduction of a gene of interest into eukaryotic cells either by biochemical or physical methods is a crucial tool used to study the dynamics as well as the mechanisms of eukaryotic gene expression. However, the process of transfection of nucleic acids contained in appropriate mammalian vectors is highly dependent on the cell type to be transfected and it is nearly impossible to predict which method will yield the most reproducible result with the least cytotoxicity. Hence, it is essential to test and optimize various techniques for every cell type to be studied. We are interested in transiently transfecting primary cultures of murine neural precursor cells (NPCS) with various genes to test their effects on the cells’ differentiation repertoire, i.e. which genes would promote differentiation into neurons or glia. In our experiment, we utilized enhanced green fluorescent protein, a GFP variant, as our live reporter of gene expression in three transfection techniques: calcium phosphate co-precipitation, electroporation and lipofection using the polycationic LipofectAmineTM reagent. In this paper, we report the conditions we have tested and the desirability of each method vis a vis the efficiency of DNA delivery, reproducibility of results, convenience and the financial requirements which should be considered in deciding the technique to be employed routinely in the laboratory.
BP8
AXON PROJECTION DEFECTS IN OPEN BRAIN MUTANTS OF TOPICAL TRETINOIN (all-trans Retinoic Acid)
Ann Marie G. Moran and Cynthia P. Palmes-Saloma
Laboratory of Molecular and Cell Biology, National Institute of Molecular Biology and Biotechnology, University of the Philippines, Diliman
Tretinoin or all-trans Retinoic Acid is a vitamin A derivative that is commonly used as an anti- acne and anti-photoaging topical preparation. We have previously reported that the topical application of tretinoin to female mice at the start of pregnancy could result to embryofetal defects particularly affecting the brain and other neural crest-derived tissues. Here, we report that in addition to gross morphological defects such as exencephaly or open brain, cranial ganglia development are also affected in mutant embryos. We used monoclonal antibodies against neuron-specific proteins to visualize in wholemount the path neurons take as they project axons to their target tissues. In particular, axon projections of the mesencephalic trigeminal neurons are misdirected as they descend caudally. Moreover, in severe cases, axon arborizations are fewer or missing when compared to wild type controls. These results suggest that retinoic acid is involved in the regulation of axon projection and arborization and that tight regulation of its levels during embryonic development is essential for the correct neural network formation in the mammalian brain.
BP9
Optimization of Enzymatic Hydrolysis of Coconut Oil
Titos Anaeleto O. Quibuyen, Mary Ann A. Endoma and Cynthia V. Pagba
Institute of Chemistry, University of the Philippines, Diliman
Hydrolysis can be done in two ways; one is physicochemical and the other enzymatic. The former involves operation at high temperature and high pressure making it energy consuming. The latter, on the other hand, promises to conserve energy and minimize thermal degradation. It is preferred over the other technique because of its specificity, simplicity, cost-effectiveness and biodegradability of the enzymes.
Hydrolysis of coconut oil using enzymes has been studied but the process has not been optimized. One of the objectives of this study then was to determine the optimum parameters for the hydrolysis of coconut oil by three lipases from three different microorganisms namely Candida rugosa, Candida cylindracea and Rhizopus arrhizus. The activities of the enzymes against the standard triglycerides and the commercially available coconut oil were determined at different hydrolysis conditions. The parameters varied were amount of enzyme, amount of solvent (water) and reaction time. The fatty acid released for every hydrolysis was analyzed using the calorimetric assay developed by Kwon and Rhee (1986) which made use of hydrated cupric acetate as coloring agent.
The activity (19600.00 m g/g-min) of Candida cylindracea lipase was highest when the following hydrolysis conditions were employed: 0.0025 g lipase, 3 % water and five hour hydrolysis time. While, the highest activities observed for Candida rugosa and Rhizopus arrhizus lipases 4091.85 m g/g-min and 9935.80 m g/g-min, respectively were obtained using the following hydrolysis parameters: 0.0025 g lipase, 3 % water and 30 minute hydrolysis time.
The crude products were characterized by High Performance Liquid Chromatography (HPLC). The reference retention times used were those of standard fatty acids, monoglycerides, diglycerides and triglycerides found in coconut oil. The components of the products are further analyzed by Mass Spectrophotometry.
BP10
ESTABLISHMENT OF RADIO-RFCEPTOR ASSAY FOR THE MONITORING OF PARALYTIC SHELLFISH POISONING IN THE PHILIPPINES
Elvira Z. Sombritol, Azucena de Vera2, Merrian Tangonan1 Adelina Bulos1
and Ma. Celestina Honrado1
1Chemistry Research Section
2Biomedical Research Section Philippine Nuclear Research Institute Diliman, Quezon City
The assay for saxitoxins in contaminated shellfish is an important component of a red tide monitoring program. Mouse bioassay continues to be the most common basis for regulatory action on shellfish harvest ban, though it is considered as a non-specific assay with high variability (+10%), low sensitivity, and limited sample throughput. Radiometric receptor binding assay provides a sensitive assay for samples containing toxin levels near the Philippine regulatory limit of 40m g/100g. Its major drawback for its regulatory application is its time consuming and laborious nature. This drawback is addressed by the recent modification of traditional protocol to microplate format, which eliminates extensive manipulation of individual samples and reduces analysis time. The radioreceptor assay utilizing the microplate format is now in place at the Philippine Nuclear Research Institute.
The radiometric receptor binding method, like the mouse bioassay, measures the total toxic potency of seafood toxins interacting with its receptor. Competition between the tritium-labelled saxitoxin and the unlabelled toxins for the receptor site generates a competition curve which can be used to quantify the amount of saxitoxin (the red tide toxin) in the seafood sample. To address the local availability of the tritium-labelled saxitoxin, efforts were also made to label saxitoxin with tritium. A brief description of the labeling protocol is included in this report.
To standardize the radio-receptor method, comparison studies were made between mouse bioassay and receptor assay. Good intercomparison results were obtained. Initial inter-lab comparisons between PNRI and National Oceanic and Atmospheric Administration (USA), the laboratory which developed the technique, also gave acceptable results.
