Warren Kett - Academia.edu (original) (raw)

Papers by Warren Kett

Journal of Biological Chemistry, 2015

Interactions between chemokines such as CCL5 and glycosaminoglycans (GAGs) are essential for crea... more Interactions between chemokines such as CCL5 and glycosaminoglycans (GAGs) are essential for creating haptotactic gradients to guide the migration of leukocytes into inflammatory sites, and the GAGs that interact with CCL5 with the highest affinity are heparan sulfates/heparin. The interaction between CCL5 and its receptor on monocytes, CCR1, is mediated through residues Arg-17 and -47 in CCL5, which overlap with the GAG-binding (44)RKNR(47) "BBXB" motifs. Here we report that heparin and tetrasaccharide fragments of heparin are able to inhibit CCL5-CCR1 binding, with IC50 values showing strong dependence on the pattern and extent of sulfation. Modeling of the CCL5-tetrasaccharide complexes suggested that interactions between specific sulfate and carboxylate groups of heparin and residues Arg-17 and -47 of the protein are essential for strong inhibition; tetrasaccharides lacking the specific sulfation pattern were found to preferentially bind CCL5 in positions less favorable for inhibition of the interaction with CCR1. Simulations of a 12-mer heparin fragment bound to CCL5 indicated that the oligosaccharide preferred to interact simultaneously with both (44)RKNR(47) motifs in the CCL5 homodimer and engaged residues Arg-47 and -17 from both chains. Direct engagement of these residues by the longer heparin oligosaccharide provides a rationalization for its effectiveness as an inhibitor of CCL5-CCR1 interaction. In this mode, histidine (His-23) may contribute to CCL5-GAG interactions when the pH drops just below neutral, as occurs during inflammation. Additionally, an examination of the contribution of pH to modulating CCL5-heparin interactions suggested a need for careful interpretation of experimental results when experiments are performed under non-physiological conditions.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was e... more ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Carbohydrate Research, 1997

Carbohydrate Research, 2003

Glycosylpyrazoles are efficiently formed by reaction of saccharide hydrazones with pentan-2,4-dio... more Glycosylpyrazoles are efficiently formed by reaction of saccharide hydrazones with pentan-2,4-dione (acetylacetone), but in aqueous buffer, pyrazole derivatives of amino sugars couple with a further equivalent of acetylacetone affording high yields of ketoenamines. These ketoenamines were considerably more stable than the ketoenamines formed from 2-amino-2-deoxy aldoses that have been described as intermediates in the classical Elson-Morgan reaction. Moreover, high yields of perketoenamine derivatives were achieved with oligosaccharides derived from hydrolysis of chitosan. The removal of the ketoenamine moieties to regenerate the free amine was readily accomplished with aqueous hydrazine.

Carbohydrate Research, 2000

Conditions to effect the conversion of monosaccharide and disaccharide hydrazones to 1-glycosyl-3... more Conditions to effect the conversion of monosaccharide and disaccharide hydrazones to 1-glycosyl-3-methylpyrazol-5-ones were examined. The sugar pyrazolone derivatives were sensitive to oxidation, but high yields were achieved with 2,2,2-trifluoroethyl acetoacetate in mildly acidic solution. Azo coupling of the pyrazolones produced highly coloured azopyrazolone derivatives that prevented further degradation, and these may prove useful labels for chromatographic analysis of carbohydrates.

Handbook of Experimental Pharmacology, 2011

Explorations of the therapeutic potential of heparin mimetics, anionic compounds that are analogu... more Explorations of the therapeutic potential of heparin mimetics, anionic compounds that are analogues of glycosaminoglycans (GAGs), have gone hand-in-hand with the emergence of understanding as to the role of GAGs in many essential biological processes. A myriad of structurally different heparin mimetics have been prepared and examined in many diverse applications. They range in complexity from heterogeneous polysaccharides that have been chemically sulphated to well-defined compounds, designed in part to mimic the natural ligand, but with binding specificity and potency increased by conjugation to non-carbohydrate pharmacophores. The maturity of the field is illustrated by the seven heparin mimetics that have achieved marketing approval and there are several more in late-stage clinical development. An overview of the structural determinants of heparin mimetics is presented together with an indication of their activities. The challenges in developing heparin mimetics as drugs, specificity and potential toxicity issues, are highlighted. Finally, the development path of three structurally very different mimetics, PI-88(®), GMI-1070 and RGTAs, each of which is in clinical trials, is described.

2013 International Workshop on Magnetic Particle Imaging (IWMPI), 2013

ABSTRACT The reaction has been explored in vitro in solution and the results for a DNA target are... more ABSTRACT The reaction has been explored in vitro in solution and the results for a DNA target are provided in Fig. 2. The 24 base pair DNA target can be found with 100 pM sensitivity. Shorter 20 base pair DNA target molecules bound the NPs together with less affinity so the sensitivity is only 2 nM. DNA molecules with incorrect sequencing produced no change in the MSB signal. Thrombin can also be measured with 4 nM sensitivity and streptavidin with 150 pM sensitivity. These proof of concept experiments demonstrate that the method is feasible for hormone concentrations.

PLoS ONE, 2013

The methylotrophic yeast, Pichia pastoris, is an important organism used for the production of th... more The methylotrophic yeast, Pichia pastoris, is an important organism used for the production of therapeutic proteins. However, the presence of fungal-like glycans, either N-linked or O-linked, can elicit an immune response or enable the expressed protein to bind to mannose receptors, thus reducing their efficacy. Previously we have reported the elimination of β-linked glycans in this organism. In the current report we have focused on reducing the O-linked mannose content of proteins produced in P. pastoris, thereby reducing the potential to bind to mannose receptors. The initial step in the synthesis of O-linked glycans in P. pastoris is the transfer of mannose from dolicholphosphomannose to a target protein in the yeast secretory pathway by members of the protein-Omannosyltransferase (PMT) family. In this report we identify and characterize the members of the P. pastoris PMT family. Like Candida albicans, P. pastoris has five PMT genes. Based on sequence homology, these PMTs can be grouped into three sub-families, with both PMT1 and PMT2 sub-families possessing two members each (PMT1 and PMT5, and PMT2 and PMT6, respectively). The remaining sub-family, PMT4, has only one member (PMT4). Through gene knockouts we show that PMT1 and PMT2 each play a significant role in O-glycosylation. Both, by gene knockouts and the use of Pmt inhibitors we were able to significantly reduce not only the degree of Omannosylation, but also the chain-length of these glycans. Taken together, this reduction of O-glycosylation represents an important step forward in developing the P. pastoris platform as a suitable system for the production of therapeutic glycoproteins. Citation: Nett JH, Cook WJ, Chen M , Davidson RC, Bobrowicz P, et al. (2013) Characterization of the Pichia pastoris Protein-O-mannosyltransferase Gene Family. PLoS ONE 8(7): e68325.

Nature Protocols, 2008

O-glycosylation is a post-translational protein modification that occurs in all eukaryotes. Yeast... more O-glycosylation is a post-translational protein modification that occurs in all eukaryotes. Yeasts have received increasing attention as a host for therapeutic protein production because of their ability to secrete high levels of recombinant protein. Because yeasts such as Pichia pastoris have been shown to O-glycosylate some proteins with varying effects on protein function, it is important to elucidate the nature of this modification. Methods that characterize O-glycosylation on a qualitative and quantitative basis are thus important when considering yeast as a host for therapeutic protein production. This protocol describes the release of O-glycans from a protein sample by b-elimination under alkaline conditions using sodium borohydride and sodium hydroxide. The released O-linked oligosaccharides are subsequently processed and then separated by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). An estimation of O-glycan molar occupancy and average O-mannose chain length is ultimately derived. This protocol requires B3 d for completion. This method provides an assessment of O-glycosylation and allows one to correlate the effect of O-glycosylation on protein properties.

Medical Physics, 2013

ABSTRACT Purpose: To measure local biomarker concentrations longitudinally over time. Biomarker c... more ABSTRACT Purpose: To measure local biomarker concentrations longitudinally over time. Biomarker concentrations can be used to diagnose disease but the most promising application is in monitoring therapy. For example, the VEGF level provides a very direct evaluation of anti‐angiogenic therapies or heat shock proteins provide a direct evaluation of thermal therapies. In this effort, the potential of the proposed technology was evaluated for sensitivity and specificity. The potential sensitivity was also estimated. Methods: Magnetic nanoparticles (NPs) can be detected remotely by measuring their magnetization induced by an alternating magnetic field. Magnetic spectroscopy of Brownian motion (MSB) characterizes the rotational Brownian motion from the shape of the magnetization. Rotational Brownian motion characterizes the local microenvironment surrounding the NPs: temperature, viscosity and chemical binding. We are reporting on a novel application of MSB that will allow the local concentration of biomarkers or drugs to be measured in vivo longitudinally over time. Magnetic NPs in ∼200 micron porous walled containers can be deposited in the vascular bed or into the extracellular space via injection in a lesions' blood supply or directly into the lesion. The NPs are decorated so that the target molecule (either biomarker or drug) binds the NPs together into an aggregate. The aggregate has a much longer relaxation time than the free NPs so the MSB signal changes dramatically. The MSB signal can be used to estimate the quantitative concentration of the biomarker. Results: The method has been explored in vitro for the following analytes (minimum sensitivities): 24 mer DNA (100 pM sensitivity); 20 mer DNA (2 nM); thrombin (4 nM); streptavidin (150 pM); VEGF (4 nM). Conclusion: These proof of concept experiments demonstrate that MSB methods are capable of measuring hormone concentrations of biomarkers and would require only reasonable increases in sensitivity to measure cytokine concentrations. NIH‐NCI 1U54CA151662‐01; Hopeman Fund Grant: Norris Cotton Cancer Center

Matrix Biology, 2008

Abstracts S15

Journal of Medicinal Chemistry, 2003

The binding interactions of the phosphosulfomannan anticancer agent PI-88 (1) with the angiogenic... more The binding interactions of the phosphosulfomannan anticancer agent PI-88 (1) with the angiogenic growth factors FGF-1, FGF-2, and VEGF were studied by surface plasmon resonance (SPR) on a BIAcore 3000 biosensor. Compared with heparin, PI-88 has at least 11-fold higher affinity for FGF-1 and at least 3-fold higher affinity for VEGF, but at least 13-fold lower affinity for FGF-2. To define the structural features of PI-88 that are important for growth factor binding, several analogues, such as dephosphorylated PI-88 and a sulfated pentasaccharide, were prepared. The binding interactions of these analogues with FGF-1, FGF-2, and VEGF were similarly studied by SPR, and structure-activity relationships were determined.

The Journal of Immunology, 2010

Journal of Chromatography A, 1998

A simple technique is introduced to identify and quantitate cysteine (Cys) after acid hydrolysis ... more A simple technique is introduced to identify and quantitate cysteine (Cys) after acid hydrolysis of protein. The technique involves using 9-fluorenylmethyl chloroformate (Fmoc)-based amino acid analysis that recovers all of the amino acids (asparagine and glutamine are recovered in their acidic forms) except tryptophan. Cys adducts with acrylamide and iodoacetamide have been observed in hydrolysates of gel-separated proteins. To enable quantitation of Cys by amino acid analysis, different conditions of reduction [dithiothreitol (DTT) and tributylphosphine] and alkylation [vinylpyridine, acrylamide and iodoacetamide] were compared. Optimal conditions for on-blot reduction (125 mM of DTT, pH 8.5, at 808C) and alkylation (0.25 M iodoacetamide, pH 8.5, at 378C) of proteins which have been separated by gel electrophoresis and blotted onto polyvinylidenedifluoride (PVDF) membrane were established to achieve complete recovery of alkylated Cys. Even with the optimal on-blot iodoacetamide alkylation, there may still be some acrylamide adducts present and these were able to be separated by HPLC along with the other 16 amino acids. The Cys content has been successfully determined by Fmoc-amino acid analysis of PVDF-blotted proteins separated by 1D or 2D gel electrophoresis. Lysine alkylation with iodoacetamide and acrylamide has also been characterised. Protein identification using amino acid composition including Cys has been introduced.

Electrophoresis, 1997

Preparative two-dimensional polyacrylamide electrophoresis (2-D PAGE) is a method of separation w... more Preparative two-dimensional polyacrylamide electrophoresis (2-D PAGE) is a method of separation which for the first time allows protein isoforms to be readily purified for subsequent analysis. The profile of the 2-D separation of the protein complement (proteome) of eukaryotic cells and tissues typically contains obvious 'trains' of spots which differ in p l and/or apparent molecular mass. These are usually isoforms of the same protein and result from posttranslational modifications. There is growing evidence that alterations to the glycosylation and/or phosphorylation of a protein can be correlated with developmental and pathological changes; these changes can be visualised on the 2-D separation. It is not clear, however, how these modifications alter the structural properties of the protein and affect their migration in this mode of separation. Strategies need to be developed to obtain a more detailed understanding of the reason for the appearance of isoforms as discrete spots on 2-D PAGE. Standard proteins, fetuin and ovalbumin, were used to monitor the effect of the removal of glycans and phosphates on the migration of the glycoproteins in the 2-D system. The isoforms were not simply explained by the presence or absence of a single modification. To further investigate the reasons for the different migration of the isoforms it is necessary to characterise the modifications in more detail. Unlike protein analysis, until recently the available methodology for the analysis of the glycans attached to proteins has not been sensitive enough to allow analysis of single spots in gels or blots resulting from 2-D electrophoresis. In this paper we review current and future strategies for characterisation of protein modifications using single spots from 2-D gels.

CMLS Cellular and Molecular Life Sciences, 2005

Heparin and the related glycosaminoglycan, heparan sulfate, bind a myriad of proteins. The struct... more Heparin and the related glycosaminoglycan, heparan sulfate, bind a myriad of proteins. The structural diversity of heparin and heparan sulfates is enormous, but differences in the conformational flexibility of the monosaccharide constituents add extra complexity and may influence protein binding. Silencing genes for heparin/ heparan sulfate biosynthetic enzymes profoundly affects mammalian development. Thus, altering the structure of heparan sulfate chains can alter protein binding and embryo development. Different heparan sulfate structures are located in particular tissue sites, and these CMLS, Cell. Mol. Life Sci. 62 (2005) 410-424

Biosensors and Bioelectronics, 2013

Functionalized magnetic nanoparticles (mNPs) have shown promise in biosensing and other biomedica... more Functionalized magnetic nanoparticles (mNPs) have shown promise in biosensing and other biomedical applications. Here we use functionalized mNPs to develop a highly sensitive, versatile sensing strategy required in practical biological assays and potentially in vivo analysis. We demonstrate a new sensing scheme based on magnetic spectroscopy of nanoparticle Brownian motion (MSB) to quantitatively detect molecular targets. MSB uses the harmonics of oscillating mNPs as a metric for the freedom of rotational motion, thus reflecting the bound state of the mNP. The harmonics can be detected in vivo from nanogram quantities of iron within 5 s. Using a streptavidin-biotin binding system, we show that the detection limit of the current MSB technique is lower than 150 pM (0.075 pmole), which is much more sensitive than previously reported techniques based on mNP detection. Using mNPs conjugated with two anti-thrombin DNA aptamers, we show that thrombin can be detected with high sensitivity (4 nM or 2 pmole). A DNA-DNA interaction was also investigated. The results demonstrated that sequence selective DNA detection can be achieved with 100 pM (0.05 pmole) sensitivity. The results of using MSB to sense these interactions, show that the MSB based sensing technique can achieve rapid measurement (within 10 s), and is suitable for detecting and quantifying a wide range of biomarkers or analytes. It has the potential to be applied in variety of biomedical applications or diagnostic analyses.

Biochimica et Biophysica Acta (BBA) - General Subjects, 2003

The specificity, affinity and stoichiometry of the interaction between avidin and glycosaminoglyc... more The specificity, affinity and stoichiometry of the interaction between avidin and glycosaminoglycans (GAGs) have been investigated using heparin-coated microtiter-plate assays, a filter binding assay and surface plasmon resonance (SPR) analysis using a BIAcore 2000 biosensor. Avidin binds heparin and heparan sulfate, and chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate or hyaluronan were unable to compete for binding. Highest-affinity binding was observed with heparin, and weaker binding was seen when using heparan sulfate or low molecular weight heparin preparations. This indicated that only specific polysaccharide structures tightly interact with avidin. Approximately two avidin molecules bind to each heparin molecule with an overall affinity of 160 nM. The interaction is pH dependent, increasing five-fold upon decreasing the pH from 7.5 to 5.5, while binding was negligible at pH 9. We demonstrate the potential of fluorescent avidin derivatives as a tool for the detection of heparin and heparan sulfates on surfaces by application to both heparin immobilized on polystyrene plates and heparan sulfate on cell surfaces.

Journal of Biological Chemistry, 2015

Interactions between chemokines such as CCL5 and glycosaminoglycans (GAGs) are essential for crea... more Interactions between chemokines such as CCL5 and glycosaminoglycans (GAGs) are essential for creating haptotactic gradients to guide the migration of leukocytes into inflammatory sites, and the GAGs that interact with CCL5 with the highest affinity are heparan sulfates/heparin. The interaction between CCL5 and its receptor on monocytes, CCR1, is mediated through residues Arg-17 and -47 in CCL5, which overlap with the GAG-binding (44)RKNR(47) "BBXB" motifs. Here we report that heparin and tetrasaccharide fragments of heparin are able to inhibit CCL5-CCR1 binding, with IC50 values showing strong dependence on the pattern and extent of sulfation. Modeling of the CCL5-tetrasaccharide complexes suggested that interactions between specific sulfate and carboxylate groups of heparin and residues Arg-17 and -47 of the protein are essential for strong inhibition; tetrasaccharides lacking the specific sulfation pattern were found to preferentially bind CCL5 in positions less favorable for inhibition of the interaction with CCR1. Simulations of a 12-mer heparin fragment bound to CCL5 indicated that the oligosaccharide preferred to interact simultaneously with both (44)RKNR(47) motifs in the CCL5 homodimer and engaged residues Arg-47 and -17 from both chains. Direct engagement of these residues by the longer heparin oligosaccharide provides a rationalization for its effectiveness as an inhibitor of CCL5-CCR1 interaction. In this mode, histidine (His-23) may contribute to CCL5-GAG interactions when the pH drops just below neutral, as occurs during inflammation. Additionally, an examination of the contribution of pH to modulating CCL5-heparin interactions suggested a need for careful interpretation of experimental results when experiments are performed under non-physiological conditions.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was e... more ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Carbohydrate Research, 1997

Carbohydrate Research, 2003

Glycosylpyrazoles are efficiently formed by reaction of saccharide hydrazones with pentan-2,4-dio... more Glycosylpyrazoles are efficiently formed by reaction of saccharide hydrazones with pentan-2,4-dione (acetylacetone), but in aqueous buffer, pyrazole derivatives of amino sugars couple with a further equivalent of acetylacetone affording high yields of ketoenamines. These ketoenamines were considerably more stable than the ketoenamines formed from 2-amino-2-deoxy aldoses that have been described as intermediates in the classical Elson-Morgan reaction. Moreover, high yields of perketoenamine derivatives were achieved with oligosaccharides derived from hydrolysis of chitosan. The removal of the ketoenamine moieties to regenerate the free amine was readily accomplished with aqueous hydrazine.

Carbohydrate Research, 2000

Conditions to effect the conversion of monosaccharide and disaccharide hydrazones to 1-glycosyl-3... more Conditions to effect the conversion of monosaccharide and disaccharide hydrazones to 1-glycosyl-3-methylpyrazol-5-ones were examined. The sugar pyrazolone derivatives were sensitive to oxidation, but high yields were achieved with 2,2,2-trifluoroethyl acetoacetate in mildly acidic solution. Azo coupling of the pyrazolones produced highly coloured azopyrazolone derivatives that prevented further degradation, and these may prove useful labels for chromatographic analysis of carbohydrates.

Handbook of Experimental Pharmacology, 2011

Explorations of the therapeutic potential of heparin mimetics, anionic compounds that are analogu... more Explorations of the therapeutic potential of heparin mimetics, anionic compounds that are analogues of glycosaminoglycans (GAGs), have gone hand-in-hand with the emergence of understanding as to the role of GAGs in many essential biological processes. A myriad of structurally different heparin mimetics have been prepared and examined in many diverse applications. They range in complexity from heterogeneous polysaccharides that have been chemically sulphated to well-defined compounds, designed in part to mimic the natural ligand, but with binding specificity and potency increased by conjugation to non-carbohydrate pharmacophores. The maturity of the field is illustrated by the seven heparin mimetics that have achieved marketing approval and there are several more in late-stage clinical development. An overview of the structural determinants of heparin mimetics is presented together with an indication of their activities. The challenges in developing heparin mimetics as drugs, specificity and potential toxicity issues, are highlighted. Finally, the development path of three structurally very different mimetics, PI-88(®), GMI-1070 and RGTAs, each of which is in clinical trials, is described.

2013 International Workshop on Magnetic Particle Imaging (IWMPI), 2013

ABSTRACT The reaction has been explored in vitro in solution and the results for a DNA target are... more ABSTRACT The reaction has been explored in vitro in solution and the results for a DNA target are provided in Fig. 2. The 24 base pair DNA target can be found with 100 pM sensitivity. Shorter 20 base pair DNA target molecules bound the NPs together with less affinity so the sensitivity is only 2 nM. DNA molecules with incorrect sequencing produced no change in the MSB signal. Thrombin can also be measured with 4 nM sensitivity and streptavidin with 150 pM sensitivity. These proof of concept experiments demonstrate that the method is feasible for hormone concentrations.

PLoS ONE, 2013

The methylotrophic yeast, Pichia pastoris, is an important organism used for the production of th... more The methylotrophic yeast, Pichia pastoris, is an important organism used for the production of therapeutic proteins. However, the presence of fungal-like glycans, either N-linked or O-linked, can elicit an immune response or enable the expressed protein to bind to mannose receptors, thus reducing their efficacy. Previously we have reported the elimination of β-linked glycans in this organism. In the current report we have focused on reducing the O-linked mannose content of proteins produced in P. pastoris, thereby reducing the potential to bind to mannose receptors. The initial step in the synthesis of O-linked glycans in P. pastoris is the transfer of mannose from dolicholphosphomannose to a target protein in the yeast secretory pathway by members of the protein-Omannosyltransferase (PMT) family. In this report we identify and characterize the members of the P. pastoris PMT family. Like Candida albicans, P. pastoris has five PMT genes. Based on sequence homology, these PMTs can be grouped into three sub-families, with both PMT1 and PMT2 sub-families possessing two members each (PMT1 and PMT5, and PMT2 and PMT6, respectively). The remaining sub-family, PMT4, has only one member (PMT4). Through gene knockouts we show that PMT1 and PMT2 each play a significant role in O-glycosylation. Both, by gene knockouts and the use of Pmt inhibitors we were able to significantly reduce not only the degree of Omannosylation, but also the chain-length of these glycans. Taken together, this reduction of O-glycosylation represents an important step forward in developing the P. pastoris platform as a suitable system for the production of therapeutic glycoproteins. Citation: Nett JH, Cook WJ, Chen M , Davidson RC, Bobrowicz P, et al. (2013) Characterization of the Pichia pastoris Protein-O-mannosyltransferase Gene Family. PLoS ONE 8(7): e68325.

Nature Protocols, 2008

O-glycosylation is a post-translational protein modification that occurs in all eukaryotes. Yeast... more O-glycosylation is a post-translational protein modification that occurs in all eukaryotes. Yeasts have received increasing attention as a host for therapeutic protein production because of their ability to secrete high levels of recombinant protein. Because yeasts such as Pichia pastoris have been shown to O-glycosylate some proteins with varying effects on protein function, it is important to elucidate the nature of this modification. Methods that characterize O-glycosylation on a qualitative and quantitative basis are thus important when considering yeast as a host for therapeutic protein production. This protocol describes the release of O-glycans from a protein sample by b-elimination under alkaline conditions using sodium borohydride and sodium hydroxide. The released O-linked oligosaccharides are subsequently processed and then separated by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). An estimation of O-glycan molar occupancy and average O-mannose chain length is ultimately derived. This protocol requires B3 d for completion. This method provides an assessment of O-glycosylation and allows one to correlate the effect of O-glycosylation on protein properties.

Medical Physics, 2013

ABSTRACT Purpose: To measure local biomarker concentrations longitudinally over time. Biomarker c... more ABSTRACT Purpose: To measure local biomarker concentrations longitudinally over time. Biomarker concentrations can be used to diagnose disease but the most promising application is in monitoring therapy. For example, the VEGF level provides a very direct evaluation of anti‐angiogenic therapies or heat shock proteins provide a direct evaluation of thermal therapies. In this effort, the potential of the proposed technology was evaluated for sensitivity and specificity. The potential sensitivity was also estimated. Methods: Magnetic nanoparticles (NPs) can be detected remotely by measuring their magnetization induced by an alternating magnetic field. Magnetic spectroscopy of Brownian motion (MSB) characterizes the rotational Brownian motion from the shape of the magnetization. Rotational Brownian motion characterizes the local microenvironment surrounding the NPs: temperature, viscosity and chemical binding. We are reporting on a novel application of MSB that will allow the local concentration of biomarkers or drugs to be measured in vivo longitudinally over time. Magnetic NPs in ∼200 micron porous walled containers can be deposited in the vascular bed or into the extracellular space via injection in a lesions' blood supply or directly into the lesion. The NPs are decorated so that the target molecule (either biomarker or drug) binds the NPs together into an aggregate. The aggregate has a much longer relaxation time than the free NPs so the MSB signal changes dramatically. The MSB signal can be used to estimate the quantitative concentration of the biomarker. Results: The method has been explored in vitro for the following analytes (minimum sensitivities): 24 mer DNA (100 pM sensitivity); 20 mer DNA (2 nM); thrombin (4 nM); streptavidin (150 pM); VEGF (4 nM). Conclusion: These proof of concept experiments demonstrate that MSB methods are capable of measuring hormone concentrations of biomarkers and would require only reasonable increases in sensitivity to measure cytokine concentrations. NIH‐NCI 1U54CA151662‐01; Hopeman Fund Grant: Norris Cotton Cancer Center

Matrix Biology, 2008

Abstracts S15

Journal of Medicinal Chemistry, 2003

The binding interactions of the phosphosulfomannan anticancer agent PI-88 (1) with the angiogenic... more The binding interactions of the phosphosulfomannan anticancer agent PI-88 (1) with the angiogenic growth factors FGF-1, FGF-2, and VEGF were studied by surface plasmon resonance (SPR) on a BIAcore 3000 biosensor. Compared with heparin, PI-88 has at least 11-fold higher affinity for FGF-1 and at least 3-fold higher affinity for VEGF, but at least 13-fold lower affinity for FGF-2. To define the structural features of PI-88 that are important for growth factor binding, several analogues, such as dephosphorylated PI-88 and a sulfated pentasaccharide, were prepared. The binding interactions of these analogues with FGF-1, FGF-2, and VEGF were similarly studied by SPR, and structure-activity relationships were determined.

The Journal of Immunology, 2010

Journal of Chromatography A, 1998

A simple technique is introduced to identify and quantitate cysteine (Cys) after acid hydrolysis ... more A simple technique is introduced to identify and quantitate cysteine (Cys) after acid hydrolysis of protein. The technique involves using 9-fluorenylmethyl chloroformate (Fmoc)-based amino acid analysis that recovers all of the amino acids (asparagine and glutamine are recovered in their acidic forms) except tryptophan. Cys adducts with acrylamide and iodoacetamide have been observed in hydrolysates of gel-separated proteins. To enable quantitation of Cys by amino acid analysis, different conditions of reduction [dithiothreitol (DTT) and tributylphosphine] and alkylation [vinylpyridine, acrylamide and iodoacetamide] were compared. Optimal conditions for on-blot reduction (125 mM of DTT, pH 8.5, at 808C) and alkylation (0.25 M iodoacetamide, pH 8.5, at 378C) of proteins which have been separated by gel electrophoresis and blotted onto polyvinylidenedifluoride (PVDF) membrane were established to achieve complete recovery of alkylated Cys. Even with the optimal on-blot iodoacetamide alkylation, there may still be some acrylamide adducts present and these were able to be separated by HPLC along with the other 16 amino acids. The Cys content has been successfully determined by Fmoc-amino acid analysis of PVDF-blotted proteins separated by 1D or 2D gel electrophoresis. Lysine alkylation with iodoacetamide and acrylamide has also been characterised. Protein identification using amino acid composition including Cys has been introduced.

Electrophoresis, 1997

Preparative two-dimensional polyacrylamide electrophoresis (2-D PAGE) is a method of separation w... more Preparative two-dimensional polyacrylamide electrophoresis (2-D PAGE) is a method of separation which for the first time allows protein isoforms to be readily purified for subsequent analysis. The profile of the 2-D separation of the protein complement (proteome) of eukaryotic cells and tissues typically contains obvious 'trains' of spots which differ in p l and/or apparent molecular mass. These are usually isoforms of the same protein and result from posttranslational modifications. There is growing evidence that alterations to the glycosylation and/or phosphorylation of a protein can be correlated with developmental and pathological changes; these changes can be visualised on the 2-D separation. It is not clear, however, how these modifications alter the structural properties of the protein and affect their migration in this mode of separation. Strategies need to be developed to obtain a more detailed understanding of the reason for the appearance of isoforms as discrete spots on 2-D PAGE. Standard proteins, fetuin and ovalbumin, were used to monitor the effect of the removal of glycans and phosphates on the migration of the glycoproteins in the 2-D system. The isoforms were not simply explained by the presence or absence of a single modification. To further investigate the reasons for the different migration of the isoforms it is necessary to characterise the modifications in more detail. Unlike protein analysis, until recently the available methodology for the analysis of the glycans attached to proteins has not been sensitive enough to allow analysis of single spots in gels or blots resulting from 2-D electrophoresis. In this paper we review current and future strategies for characterisation of protein modifications using single spots from 2-D gels.

CMLS Cellular and Molecular Life Sciences, 2005

Heparin and the related glycosaminoglycan, heparan sulfate, bind a myriad of proteins. The struct... more Heparin and the related glycosaminoglycan, heparan sulfate, bind a myriad of proteins. The structural diversity of heparin and heparan sulfates is enormous, but differences in the conformational flexibility of the monosaccharide constituents add extra complexity and may influence protein binding. Silencing genes for heparin/ heparan sulfate biosynthetic enzymes profoundly affects mammalian development. Thus, altering the structure of heparan sulfate chains can alter protein binding and embryo development. Different heparan sulfate structures are located in particular tissue sites, and these CMLS, Cell. Mol. Life Sci. 62 (2005) 410-424

Biosensors and Bioelectronics, 2013

Functionalized magnetic nanoparticles (mNPs) have shown promise in biosensing and other biomedica... more Functionalized magnetic nanoparticles (mNPs) have shown promise in biosensing and other biomedical applications. Here we use functionalized mNPs to develop a highly sensitive, versatile sensing strategy required in practical biological assays and potentially in vivo analysis. We demonstrate a new sensing scheme based on magnetic spectroscopy of nanoparticle Brownian motion (MSB) to quantitatively detect molecular targets. MSB uses the harmonics of oscillating mNPs as a metric for the freedom of rotational motion, thus reflecting the bound state of the mNP. The harmonics can be detected in vivo from nanogram quantities of iron within 5 s. Using a streptavidin-biotin binding system, we show that the detection limit of the current MSB technique is lower than 150 pM (0.075 pmole), which is much more sensitive than previously reported techniques based on mNP detection. Using mNPs conjugated with two anti-thrombin DNA aptamers, we show that thrombin can be detected with high sensitivity (4 nM or 2 pmole). A DNA-DNA interaction was also investigated. The results demonstrated that sequence selective DNA detection can be achieved with 100 pM (0.05 pmole) sensitivity. The results of using MSB to sense these interactions, show that the MSB based sensing technique can achieve rapid measurement (within 10 s), and is suitable for detecting and quantifying a wide range of biomarkers or analytes. It has the potential to be applied in variety of biomedical applications or diagnostic analyses.

Biochimica et Biophysica Acta (BBA) - General Subjects, 2003

The specificity, affinity and stoichiometry of the interaction between avidin and glycosaminoglyc... more The specificity, affinity and stoichiometry of the interaction between avidin and glycosaminoglycans (GAGs) have been investigated using heparin-coated microtiter-plate assays, a filter binding assay and surface plasmon resonance (SPR) analysis using a BIAcore 2000 biosensor. Avidin binds heparin and heparan sulfate, and chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate or hyaluronan were unable to compete for binding. Highest-affinity binding was observed with heparin, and weaker binding was seen when using heparan sulfate or low molecular weight heparin preparations. This indicated that only specific polysaccharide structures tightly interact with avidin. Approximately two avidin molecules bind to each heparin molecule with an overall affinity of 160 nM. The interaction is pH dependent, increasing five-fold upon decreasing the pH from 7.5 to 5.5, while binding was negligible at pH 9. We demonstrate the potential of fluorescent avidin derivatives as a tool for the detection of heparin and heparan sulfates on surfaces by application to both heparin immobilized on polystyrene plates and heparan sulfate on cell surfaces.