Progress 01/01/01 to 12/31/05
Outputs
Purified respiratory mucous glycoproteins have been isolated from respiratory secretions from bovine cattle suffering from bovine respiratory disease complex (BRDC), or 'shipping fever', and has been the ongoing primary focus for structural characterization of these molecules. As reported in pass Progress Reports, our investigations have shown that these very large molecular weight glycoproteins possess structures and blood-group activities that are unique to bovine cattle. The carbohydrate side-chains of BRCD mucins are complex in structure and possess 22-28 degrees of polymerization. For 2005, our laboratories have continued to make progress in determining the exact structure of the isolated carbohydrate side-chains. As in previous years, our attention has been focused on the charged, ionic oligosaccharide species. Because many molecules possess the exact same molecular weight but possess very different structures, we continue in developing methods that can
distinguish these differences. Many variations employing high performance liquid chromatography and affinity chromatography have been tested. As these ionic structures are purified they will be subjected to mass spectrometry or nuclear magnetic resonance analysis. As noted in our previous Progress Reports, our difficulty in binding these glycoprotein oligosaccharide structures to plastic wells to be used in binding studies had been a major drawback in our progress. We designed several hydrophobic linkers and are in the process of isolating and purifying them. As this is the last Progress Report it is important to note that we intend to continue this research line of developing a glycomics array and have now written two complete book chapters reporting on many of our structural findings. Interestingly, many of our bovine respiratory results have had parallel importance in human respiratory health and disease.
Impacts
The expected result from our research is to provide insight into the molecular basis of 'shipping fever',which should, in turn, yield a basis for manipulative or pharmacological intervention. The process in which the biological defense mechanism works within the bovine animal (cattle) should provide ways of interfering with viral and bacterial infections that are encountered during the stress of shipping.
Publications
- Mawhinney ,T.P., and Chance, D.L. 2005 Characterization of fucosylated-sulfated oligosaccharides isolated from human respiratory mucous glycoproteins. Current Advances in Carbohydrate Research, S.G.Pandalai, Ed., TRN, 2:67-92.
- Krishnan, H.B., Bennett, J.O., Kim, W.S., Krishnan, A.H., Mawhinney ,T.P. 2005. Nitrogen Lowers the sulfur amino acid content of soybean (Glycine max [L.] Merr.) by regulating the accumulation of Bowman-Birk protease inhibitor. J. Agric. and Food Chem. 53:6347-6354.
- Chance, D., Jensen, C.A., Reilly, T.J. and Mawhinney, T.P. 2005 TEM Analysis of bacterial colony biology using microwave-assisted processing and LR White Resin. Microscopy and Microanalysis, 11(Suppl 2):968-969.
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Progress 01/01/04 to 12/31/04
Outputs
Purified respiratory mucous glycoproteins, isolated from respiratory secretions from bovine cattle suffering from bovine respiratory disease complex (BRDC), or 'shipping fever', is the ongoing primary focus for structural characterization of these molecules. As previously reported, our investigations have shown that these very large molecular weight glycoproteins possess structures and blood-group activities that are unique to bovine cattle. The carbohydrate side-chains of BRCD mucins are complex in structure and possess 22-28 degrees of polymerization. For 2004, our laboratories have continued to make progress in determining the exact structure of the isolated carbohydrate side-chains. As in previous years, our attention has been focused on the charged, ionic oligosaccharide species. Because many molecules possess the exact same molecular weight but possess very different structures, we continue in developing methods that can distinguish these differences. Many
variations employing high performance liquid chromatography and affinity chromatography have been tested. As these ionic structures are purified they will be subjected to mass spectrometry or nuclear magnetic resonance analysis. As noted in our preious report, our difficulty in binding these glycoprotein oligosaccharide structures to plastic wells to be used in binding studies was a major drawback in our progress. We have designed several hydrophobic linkers and are in the process of isolating and purifying them. It is our intention to choose the best compound and synthetic pathway to produce linkers for the bovine oligosaccharide library. The current goal of our research is to develop this binding method which will enable us to quickly screen libraries of bacterial receptor proteins and glycolipids that are common in respiratory infections in the bovine cattle.
Impacts
The expected result from our research is to provide insight into the molecular basis of 'shipping fever',which should, in turn, yield a basis for manipulative or pharmacological intervention. The process in which the defense mechanism works within the bovine animal should provide ways of interfering with viral and bacterial infections that are encountered during the stress of shipping.
Publications
- Chance, D.L. and Mawhinney, T.P. 2004. SEM Examination of Host-Pathogen Interactions in the Respiratory Mucosa with drying by HMDS and by Critical Point Method. I.M. Anderson et al. Eds. Microscopy Society of America, Proceedings, Cambridge University Press, England/New York, v. 10, Suppl. 2, pp. 238-239.
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Progress 01/01/03 to 12/31/03
Outputs
Respiratory mucous glycoproteins, purified from respiratory secretions from bovine cattle suffering from bovine respiratory disease complex (BRDC), or 'shipping fever', are the primary focus for structural characterization. In our ongoing research, these exceptionally large molecular weight glycoproteins have been determined to possess structures and blood-group activities that are unique to the bovine animal. Side-chain oligosaccharides of BRCD mucins are also more complex in structure, compared to human samples; now determined to possess an average of 22-28 sugars per chain. For this 2003 year report, these laboratories have continued with the determination of the isolated carbohydrate structures. Our attention is still fixed on our first purified panel of ionic carbohydrate structures. Because of the complexity of these structures, many of these purified oligosaccharides are still isomeric mixtures and alternate purification methods of these oligosaccharides via
high performance liquid chromatography and affinity chromatography are being tested. As these ionic structures are purified they will be subjected to mass spectrometry or nuclear magnetic resonance analysis. Our difficulty in binding these glycoprotein oligosaccharide structures to plastic wells to be used in binding studies still persists. Attempts to bind hydrophobic linkers to these oligosaccharides to aid in their binding are being designed. Some of the linkers, so far tested, do not bind the oligosaccharides in a useful manner. As stated in my previous report, the development of this binding method will enable us to quickly screen libraries of bacterial receptor proteins and glycolipids that are common in respiratory infections in the bovine cattle.
Impacts
The expected result from our research is to provide insight into the molecular basis of 'shipping fever',which should, in turn, yield a basis for manipulative or pharmacological intervention. The process in which the defense mechanism works within the bovine animal should provide ways of interfering with viral and bacterial infections that are encountered during the stress of shipping.
Publications
- Mawhinney,T.P., Chance, D.L., Waters, J.K., Mossine, V.V., He, S. and Cassity, N.A. 2003. Characterization of sulfated blood group antigen-containing oligosaccharides isolated from human respiratory mucous glycoproteins. Current Advances in Carbohydrate Research, S.G.Pandalai, Ed., Transworld Research Network Publishers.
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Progress 01/01/02 to 12/31/02
Outputs
Purified respiratory mucous glycoproteins from respiratory secretions from bovine cattle suffering from bovine respiratory disease complex (BRDC), or 'shipping fever', are still continuing to undergo structural characterization. As a follow up to our previous and earlier findings, these large molecular weight glycoproteins have been determined to possess structures and blood-group activities that are unique to the bovine animal. Side-chain oligosaccharides of BRCD mucins are also more complex in structure, compared to human samples, containing an average of 20-24 sugars per chain. For the 2002 year, these laboratories have continued with the carbohydrate structural analyses. The first, in a series of ionic carbohydrate structures, have been isolated. Final purification and analysis of these ionic structures for mass spectrometry or nuclear magnetic resonance are in progress. Lastly, our initial attempts to develop methods of binding these glycoprotein oligosaccharide
structures to plastic wells for future binding analysis of pulmonary bacteria have not met with great success. I am currently working on some synthetic linkers to assist in the binding. It is hoped, that in the the development of this latter method, it will be possible to quickly screen libraries of bacterial receptor proteins and glycolipids.
Impacts
The expected result from our research is to provide insight into the molecular basis of 'shipping fever',which should, in turn, yield a basis for manipulative or pharmacological intervention. The process in which the defense mechanism works within the bovine animal should provide ways of interfering with viral and bacterial infections that are encountered during the stress of shipping.
Publications
- Mossine, V.V., Barnes, C.W., Feather, M.S. and Mawhinney, T.P. 2002. Acyclic tautomers in crystylline carbohydrates: the keto forms of 1-deoxy-1-carboxymethylamino-D-2-pentuloses (Pentulose-glycines). J. Amer. Chem. Soc. 124:15178-15179.
- Yang, W.M., Boles, R.L. and Mawhinney, T.P. 2002. Determination of phosphorus in fertilizers by inductively coupled plasma atomic emission spectrometry. J. of AOAC International 85:1241-1246.
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Progress 01/01/01 to 12/31/01
Outputs
Isolated and purified respiratory mucous glycoproteins from pulmonary secretions from bovine cattle suffering from bovine respiratory disease complex (BRDC), or 'shipping fever', are still continuing to undergo structural characterization. In conjunction with our previous and earlier findings, these large molecular weight glycoproteins have been determined to possess structures and blood-group activities that are unique to the bovine animal. Side-chain oligosaccharides of BRCD mucins are also more complex in structure, compared to human samples, containing an average of 20-24 sugars per chain. For the 2001 year, these laboratories have continued with the carbohydrate structural analyses. The first 24 carbohydrate structures, as indicated as a goal for this year, have been confirmed by gel permeation size exclusion. Final purification and analysis of these structures for mass spectrometry and nuclear magnetic resonance are in progress. Also, development of binding
these structures to plastic wells for future binding analysis of bacteria to them is also underway. Also, it is hoped, that in the the development of this latter method, it will be possible to quickly screen libraries of bacterial receptor proteins and glycolipids
Impacts
The expected result from our research is to provide insight into the molecular basis of 'shipping fever' which should, in turn, provide the basis for manipulative or pharmaceutical intervention. The process in which the defense mechanism works within the bovine animal should yield ways of interfering with viral and bacterial infections that are encountered during the stress of shipping.
Publications
- No publications reported this period
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Progress 01/02/00 to 12/31/00
Outputs
Purified, isolated respiratory mucous glycoproteins from respiratory secretions from cattle suffering from bovine respiratory disease complex (BRDC), or 'shipping fever', are continuing to undergo structural characterization. In conjunction with our previous and earlier findings, these large molecular weight glycoproteins have been determined to possess blood-group activities that are unique to the bovine animal. These blood group structures are more complex and varied than that observed in human mucins isolated from similar tissue. The protein core of the bovine glycoprotein, though, is remarkedly similar to to human tracheobronchial glycoproteins, indicating the reason for previously observed cross-reactivity. Side-chain oligosaccharides of BRCD mucins are also more complex in structure, containing an average of 20-24 sugars per chain. This past year, continuing structural analyses, including chemical makeup and complete carbohydrate sequences are ongoing and
include specific exoglycosidase digestion, mass spectrometry and 500 mHz nuclear magnetic resonance. Structures appear more branched, but, I am awaiting the gel permeation size exclusion studies before proceeding with the final analyses of the first structures.
Impacts
Research into the 'shipping fever' of bovine cattle is undertaken to provide research in an area where more than 38 million dollars are spent annually to treat these animals, prior to market.
Publications
- No publications reported this period
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Progress 01/01/99 to 12/31/99
Outputs
Purified, isolated respiratory mucous glycoproteins from respiratory secretions from cattle suffering from bovine respiratory disease complex (BRDC), or 'shipping fever', have undergone futher structural characterization. In conjunction with our earlier findings, these large molecular weight glycoproteins have been determined to possess blood-group activities that are unique to the bovine animal. These blood group structure are more complex and varied than that observed in human mucins isolated from similar tissue. The protein core of the bovine glycoprotein, though, is remarkedly similar to to human tracheobronchial glycoproteins, indicating the reason for previously observed cross-reactivity. Side-chain oligosaccharides of BRCD mucins are also more complex in structure, containing an average of 20-24 sugars per chain. Continuing structural analyses, including chemical makeup and complete carbohydrate sequences are ongoing and include specific exoglycosidase
digestion, mass spectrometry and 500 mHz nuclear magnetic resonance.
Impacts
Research into the 'shipping fever' of bovine cattle is undertaken to provide basic research in an area where more than 38 million dollars are spent annually to treat these animals, prior to market.
Publications
- Chance, D.L. and Mawhinney, T.P. 1999. Carbohydrate sulfation effects on growth of Pseudomonas aeruginosa in nutrient limited conditions. Microbiology, in press.
- Sun, Da-Hai, Waters, J.K. and Mawhinney, T.P. 1999. Determination of 13 common elements in food samples by inductively coupled plasma atomic emission spectrometry: comparison of five digestion methods. J. Assoc. Official Analyt. Chemist Assoc. Internat. in press.
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Progress 01/01/98 to 12/31/98
Outputs
Since the main focus of this research project is to purify isolated respiratory mucous glycoproteins from respiratory secretions from cattle suffering from bovine respiratory disease complex (BRDC, better known as shipping fever), we have used our earlier findings that these large molecular weight glycoproteins cross-reacted with antisera against human tracheobronchial glycoproteins and human colonic mucins to assist in mucin purification by utilizing laboratory generated, highly specific, affinity columns. During the last research period (1998), we have developed these affinity columns and, because of the high purity of the glycoprotein product, have isolated more than 200 oligosaccharide side-chains from these mucins for characterization. Currently, these oligosaccharides fall into a variety of types, i.e., neutral, sulfated, and those possessing N-acetylneuraminic acid. The N-acetylneuraminic acid containing oligosaccharides appear to be the predominant form found
on these glycoproteins, to date. Final structural analyses, including chemical makeup and complete carbohydrate sequences are ongoing and include specific exoglycosidase digestion, mass spectrometry and 500 mHz nuclear magnetic resonance.
Impacts
(N/A)
Publications
- Sun, Da-Hai, Waters, J.K., and Mawhinney, T.P. 1998, Determination of Total Boron in Soils by Microwave-assisted Digestion-Inductively Coupled Plasma Atomic Emission Spectrometry. Commun. in Soil Sci. and Plant Analysis, 29(15&16), 2493-2503.
- Waters, J.K, Hughes, B.L. II, Purcell, L.C., Gerhardt, K.O., Mawhinney, T.P. and Emerich, D.W. 1998. Alanine, not ammonia, is excreted from N2-fixing soybean nodule. Proc. Nat. Acad. Sci, 95(20)12038-12042.
- Chance, D.L, Gerhardt, K.O. and Mawhinney, T.P. 1998. Gas-liquid hromatography/mass spectrometry of hydroxy fatty acids as their methyl esters tert.-butyldimethylsilyl ethers. J. Chromatogr. A, 793(1)91-98.
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Progress 01/01/97 to 12/31/97
Outputs
The general objective for this research project is to isolate and purify respiratory mucous glycoproteins from respriatory secretions from cattle suffering from bovine respiratory disease complex (BRDC); also known as shipping fever. Research has determined that these large molecular weight glycoproteins cross reacted with antisera against human tracheobronchial glycoproteins and human colonic mucins clearly demonstrating that bovine respiratory mucins have comparable antigenic sites to human secreted mucins and that other compositional analyses showed that they were very similar to the human glycoprotein values. During the last research period (1997), continuing research has focused primarily on completing the isolation and preparation of highly purified glycoproteins batches from which individual side-chain oligosaccharides can be isolated. As in the last report, following Bio-Gel, A-5m chromatography side chain oligosaccharides were removed from the glyproteins by
alkaline cleavage and reduction of treatment with 0.1 N KOH containing O.4 M NaBH(4) for 16 h at 45 degrees C in a sealed container under nitrogen. After reaction termination and desalting, several small structures have been determined by both mass spectrometry and by nuclear magnetic resonance. Over 120 different oligosaccharides have been isolated, to date, and their structures will be determined in subsequent research periods.
Impacts
(N/A)
Publications
- Sun, D.H. and Mawhinney, T.P. 1997. Determination of boron in plants and plant derived foods by UN-ICP with mannitol. J. Assoc. Official Analyt. Chemist Assoc. Internat. 80(1):20-24.
- Sun, D. H. and Mawhinney, T.P. 1997. Determination of Aluminum, Boron and 13 Other Elements in Plants by ICP. J. Assoc. Official Analyt. Chyemist Assoc. Internat. 80(3):647-650.
- Chance, D.L., Gerhardt, K. and Mawhinney, T.P. 1997. GLC-MS of primary and secondary fatty alcohols and diols as their tert.-BDMS derivatives. J. Chromatogr. A771, 191-201.
- Sun, D. H., Watters, J.K. and Mawhinney, T.P. 1997. Microwave Digestion for Determination of Boron in Animal Tissues by ICP. J. Analyst. Atomic Spectrometry 12, 675-679.
- Sun, D.H., Waters, J. K., and Mawhinney, T.P. 1997. Microwave Digestion with HNO(3)-H(2)0(2)-HF for Total Aluminum in Seafood and Meat by ICP. J. Agricultural and Food Chem. 45(6):2115-2119.
- Chance, D. L., Gerhardt, K. and Mawhinney, T.P. 1997. GLC of hydroxy fatty acids as their methyl esters tert.-butyldimethylsilyl ethers. J. Chromatogr. A793(1)91-98.
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Progress 01/01/96 to 12/30/96
Outputs
Our original goal for this report period was to collect, isolate and purify respiratory mucous glycoproteins from crude preparations that were secreted by cattle suffering from bovine respiratory disease complex (BRDC); also known as shipping fever'. Within this past year we continued our effort to collect samples from the field via collaborative arrangements with veterinarians in constant contact with feedlots and cattle farms. To date, over 135 samples have been collected of which 25 are now being subjected to separation techniques to isolate purified mucins and associated proteins. As in the past, these methods primarily include size exclusion chromatography and polyacrylamide gel electrophoresis. Normal bovine tracheal mucins are also being collected and will be employed in our comparative studies. Results have confirmed that bovine exocrine gland mucins possess large molecular weights in the range of 0.5 to 1.5 x 10(6) and appear to be primarily sialo- and sulfo-
mucins. Antigenic similarities to human lung and colonic mucins have also been confirmed.
Impacts
(N/A)
Publications
- CHOUDHURY, T. K., GERHARDT, K.O and MAWHINNEY, T.P. 1996. Solid-phase microextraction of phosphorus-containing pesticides in drinking water. Environmental. Science and Technology 30(11): 3259-3265.
- CHANCE, D.L and MAWHINNEY, T.P. 1996. Disulfated oligosaccharides derived from tracheo-bronchial mucous glycoproteins of a patient suffering from cystic fibrosis, Journal of Carbohydrate Chemistry 295: 157-177.
- KRISHNAN, H. B., WHITE, J.A. and MAWHINNEY, T.P. 1996 Purification, characterization, and localization of a 29 kilodalton glycoprotein from the edible tubers of Apios americana Medikus. Journal of Plant Physiology 149: 322-328.
- SUN, DA-HAI, et al. 1997. in press. Determination of boron in plants and plant derived foods by ultrasonic nebulization-ICP AES with addition of mannitol. Journal of the Assoc. Official Analytical Chemist Association, International.
- CHANCE, D.L, GERHARDT. K. and MAWHINNEY, T.P. 1997. in press. Gas-liquid chromatography/mass spectrometry of primary and secondary fatty alcohols and diols as their tert.-butyldimethylsilyl derivatives. J. of Chromatography.
- SUN, DA-HAI, et al. 1997. in press. Microwave Digestion for the Determination of15 Elements in Plants by ICP Atomic Emission Spectrometry. Journal of the Association Official Analytical Chemist Association, International.
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Progress 01/01/95 to 12/30/95
Outputs
The overall objective for this research project was to isolate and purify respiratory mucous glycoproteins from respiratory samples that are secreted by cattle suffering from bovine respiratory disease complex (BRDC); also known as shipping fever'. It was determined that these large molecular weight glycoproteins cross reacted with antisera against human tracheobronchial glycoproteins and against human colonic mucins clearly indicating that bovine respiratory mucins possess similar antigenic sites to human secreted mucins and that other compositional analyses showed that they were very similar to the human glycoprotein values. During the last research period, ongoing research continued primarily involved in the isolation and preparation of highly purified glycoproteins from which individual side-chain oligosaccharides could be isolated. Following Bio-Gel A-5m chromatography side chain oligosaccharides were removed from the glycoproteins by alkaline cleavage and
reduction by treatment with 0.1 N KOH containing 0.4 M NaBH4 for 16 h at 45deg.C in a sealed container under nitrogen. The reaction was terminated by bringing the reaction mixture to pH 5 with acetic acid at 4deg.C and then each sample was desalted on a Bio-Gel P-2 column. Structural analysis of the HPLC separated oligosaccharides that were separated into their respective degrees of polymerization will continue.
Impacts
(N/A)
Publications
- PHILLIPS, R., T. MAWHINNEY, M. HARMATA, AND D. SMITH. 1995. Characterization of Gallus domesticus alpha-N-acetylgalatosaminidase blood group A2 activity. Artificial Cells, Blood Substitutes and Immobilization Biotechnology 23:63-79.
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Progress 01/01/94 to 12/30/94
Outputs
For this research period I have focused on the isolation of the side-chain oligosaccharides from purified mucins from bovine tracheal lung secretions in an effort to elucidate their secondary structures. It was previously determined that guanidinium hydrochloride solubilization of crude mucins followed by removal of bacterial products, large proteins, cell debris, deoxyribonucleic acids and ribonucleic acids produced a product from which nasopharyngeal and tracheal mucins of high purity could be accomplished by Bio-Gel A-0.5m size exclusion chromatography. Upon @er examinations of these mucins by polyacrylamide gel electrophoresis it was confirmed that the preparations were free of any contaminating proteins. Side-chain oligosaccharides from these purified mucins were removed from their covalent linkage to the central protein core of the glycoproteins by alkaline cleavage and reduction in 0.1 N KOH containing 0.4 M NaBH(subscript 4). Upon chromatographic separation on
Bio-Gel P2 columns, twelve neutral oligosaccharides were attained in sufficient purity that enabled them to be immediately subjectable to the determination of their primary structures. Presently, as an ongoing process, these oligosaccharides are being sequentially subjected to classical carbohydrate chemistry employing total carbohydrate analysis, specific glycosidase degradation, methylation analysis and periodate oxidation and analysis.
Impacts
(N/A)
Publications
- No publications reported this period.
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Progress 01/01/93 to 12/30/93
Outputs
See MO-BCHB0237.
Impacts
(N/A)
Publications
- No publications reported this period.
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