PURSE 1999

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Browsing PURSE 1999 by Author "Athauda, S. B. P."

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    Bovine Beta-Lactoglobulin: The Role in Cow Milk Allergy in Infants
    (1999-11-20) Horadagoda, A.; Hemamala, H. G. I. K.; Athauda, S. B. P.; Wijekoon, A.
    Hypersensitivity to cow milk proteins is frequently observed during infancy and is believed to be an antigenic response to milk proteins that are absorbed through the immature intestinal mucosa. This hypothesis has been verified by identifying the proteins following in vitro digestibility of milk and determining the presence of such proteins in the serum of infants with cow milk allergy. In Sri Lanka, over 80 per cent of milk is consumed in the form of powdered milk in contrast to other countries where fresh milk is popular. The high keeping quality, availability, and the popular notion that powdered milk is less of a health hazard compared to fresh cow milk has led to increased powdered milk consumption. Fresh cow's milk, human milk and six cow milk formulae were subjected to in vitro proteolytic digestion in pepsin (1%) at pH 2 and trypsin (1%) at pH 8. Following the initiation of the in vitro digestive process, aliquots were collected at 0,0.5, 1,2,3,4, 6 and 24 hour and subjected to SDS-PAGE. Protein analysis by SDS-PAGE indicated that most cow milk proteins were completely digested in 4 hours but beta-lactoglobulin remained undigested even after 24 hours. Human milk is devoid of beta-lactoglobulin. All human milk proteins were completely digested by the proteolytic enzymes. Immunoblotting studies using rabbit anti-bovine beta-lactoglobulin indicated the presence of bovine beta-lactoglobulin only in the sera of infants with cow milk allergy and not those individuals without an allergic reaction despite the consumption of cow milk. Beta-lactoglobulin was not present in the serum of infants on human milk. The results indicate that the cow milk whey protein, beta-lactoglobulin is poorly digested and may play a role in the hypersensitive reaction in infants following its entry into circulation.
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    Characterization of aspartic proteinase inhibitor from Spondias Pinnata
    (Unviersity of Peradeniya, Peradeniya, Sri Lanka, 1999-11-20) Kumari, H. M. P. S.; Athauda, S. B. P.; Perera, P. A. J.; Wickramasinghe, A.; Takaashashi, Kenji
    Study of natural inhibitors of aspartic proteinases have become very important and essential. They were identified as very important therapeutic target points in the control of AIDS, Malaria and Hypertension. In this study potential inhibitorls of aspartic proteinases were isolated and partially characterized from Spondias pinnata. Fresh stem bark of Spondias pinnata was ground at room temperature and at -70 °C by using liquid Nitrogen. Crude extract of powder was prepared in H20. Assay procedure to determine inhibitory activity of aspartic proteinase was developed by using porcine pepsin as the enzyme and denatured hemoglobin as the substrate. Percentage inhibitory activity of crude extract prepared by grinding at different temperature did not differ significantly. Inhibitory activity of crude extract was not changed significantly during incubation at 37°C and at cold temperatures. This suggests the relative stability of inhibitory constituents in crude extracts at room temperature and subsequent studies were done at room temperature. But extract prepared from oven drying lowered activity compared to the fresh sample. This suggests the less stability of constituents in crude extract at higher temperatures. 60% of the inhibitory activity of crude extract was lost during dialysis against a membrane with molecular cut off point of 12,000 Da. But 100% of inhibitory activity was recovered by using a dialysis tube with cut off point of 3000. This result suggests that the inhibitory molecules have molecular weights between 3000-12.000. The inhibitor was partially purified using Diethyl amino ethyl cellulose-52 chromatography, Ammonium sulfate precipitation and Q sepharose chromatography. In DEAE-52 chromatography, inhibitor molecules were eluted with linear gradient of 0.1 M NaCI in 0.02 M phosphate buffer, pH 7.0. Inhibitory activity was detected in 2 peaks eluted at 0.2 M, 0.4 M NaCI suggesting the presence of two inhibitor molecules in the crude extract with different charges. pH stability of inhibitor was analyzed by incubating the crude extract at different 'pH's ( pH 2.0, 3.5, 4.0 and 5.0) and determining remaining inhibitory activity. Relatively higher stability of inhibitor at acidic pH was observed. Further highest pepsin inhibitory activity was observed at pH 2.0. Purification and further characterization of the two inhibitory molecules are in progress to elucidate the structure of the inhibitors.