The diversity of endothelial cells is becoming more apparent and more important in defining vesse... more The diversity of endothelial cells is becoming more apparent and more important in defining vessel systems that supply blood to normal organs and to tumors. Reagents that identify expression of cell surface determinants on these cells are crucial for differentiating among different vessel types. As a first step in this process we have selected a panel of 25 scFvs from a phage display library that bind to the endothelial cell line LEII. The scFvs are of high affinity and bind to some tumor cells as well as to the target endothelial cell. The scFvs can be divided into 8 epitope groups by use of competition binding studies. DNA sequencing of the members of these groups generally support the classification. This work shows that phage display is a rapid and efficient method for identification of reagents for cell surface molecules.
Chemical crosslinking of proteins combined with mass spectrometric analysis of the tryptic digest... more Chemical crosslinking of proteins combined with mass spectrometric analysis of the tryptic digest of the products shows considerable promise as a tool for interrogating structure and geometry of proteins and protein complexes. An impediment to the use of this tool has been the difficulty of distinguishing crosslinked peptide pairs from non-crosslinked peptides, and from the products of side reactions. We describe the use of a commercially available biotinylated crosslinking reagent, sulfo-SBED, that allows affinity-based enrichment of crosslinked species. An intramolecular crosslink is prepared using the peptide neurotensin as a model system. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra show the predicted crosslinking product, as well as several side products. Finally, we describe the optimized enrichment of biotinylated species, and reduction of non-specific binding, for a batch-mode affinity separation based on immobilized monomeric avidin.
... Thrombomodulin (TM) {formerly designated Pl12 (Ford et al. ... The data show that in contrast... more ... Thrombomodulin (TM) {formerly designated Pl12 (Ford et al. ... The data show that in contrast to expression in the adult, TM is found in high concentrations in the leptomeninges of the brain andspinal cord, and the de-veloping reproductive system, suggesting that TM may have a ...
Amyloidosis is a protein-misfolding disorder characterized by the extracellular deposition of amy... more Amyloidosis is a protein-misfolding disorder characterized by the extracellular deposition of amyloid, a complex matrix composed of protein fibrils, hyper-sulphated glycosaminoglycans and serum amyloid P component (SAP). Accumulation of amyloid in visceral organs results in the destruction of tissue architecture leading to organ dysfunction and failure. Early differential diagnosis and disease monitoring are critical for improving patient outcomes; thus, whole body amyloid imaging would be beneficial in this regard. Non-invasive molecular imaging of systemic amyloid is performed in Europe by using iodine-123-labelled SAP; however, this tracer is not available in the US. Therefore, we evaluated synthetic, poly-basic peptides, designated p5 and p5+14, as alternative radiotracers for detecting systemic amyloidosis. Herein, we perform a comparative effectiveness evaluation of radiolabelled peptide p5+14 with p5 and SAP, in amyloid-laden mice, using dual-energy SPECT imaging and tissue b...
Chemical crosslinking of proteins combined with mass spectrometric analysis of the tryptic digest... more Chemical crosslinking of proteins combined with mass spectrometric analysis of the tryptic digest of the products shows considerable promise as a tool for interrogating structure and geometry of proteins and protein complexes. An impediment to the use of this tool has been the difficulty of distinguishing crosslinked peptide pairs from non-crosslinked peptides, and from the products of side reactions. We describe the use of a commercially available biotinylated crosslinking reagent, sulfo-SBED, that allows affinity-based enrichment of crosslinked species. An intramolecular crosslink is prepared using the peptide neurotensin as a model system. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra show the predicted crosslinking product, as well as several side products. Finally, we describe the optimized enrichment of biotinylated species, and reduction of non-specific binding, for a batch-mode affinity separation based on immobilized monomeric avidin.
An acousto-optic tunable filter (AOTF)-based system for dual-modality hyperspectral imaging (DMHS... more An acousto-optic tunable filter (AOTF)-based system for dual-modality hyperspectral imaging (DMHSI) has been developed for use in characterization of normal and malignant mouse tissue. The system consists of a laser, endoscope, AOTF, and two cameras coupled with optics and electronics. Initial results show that the system can delineate normal and malignant mouse tissues real-time. The analysis shows that malignant tissues consistently exhibit less fluorescent intensity in the wavelength band from 440 to 540 nm with a peak intensity of around 490 nm. The analysis also shows key spectroscopic differences between normal and malignant tissues. Further, these results are compared to real-time spectroscopic data and show good correlation.
Sequence analysis of the 5' end of the thrombomodulin (TM) gene has identified a differen... more Sequence analysis of the 5' end of the thrombomodulin (TM) gene has identified a difference in two strains of laboratory mice, BALB/cBD (maintained at the Oak Ridge National Laboratory) and the strain used for the sequence listed in the GenBank data base. An intracisternal A-particle (IAP) provirus was present in the BALB/cBD sequence but absent from the GenBank sequence. Thus, there are two different 5' regions for the TM gene in mice. Southern blot and polymerase chain reaction analyses on other mouse strains demonstrated the presence of this proviral sequence in five out of ten strains tested. Northern blot analyses demonstrated that there were differences (up to three-fold) in the steady-state levels of TM mRNA among the strains. However, TM mRNA levels did not correlate directly with the presence of the IAP DNA upstream from the TM gene. This is in contrast to other loci (the interleukin-3 gene, c-mos, and the immunoglobulin Ck gene), in which the IAP element is known to affect levels of RNA transcription. While the IAP provirus does not appear to have an effect on TM transcription under normal conditions, it is possible that transcription levels altered by factors like thrombin and tumor necrosis factor are affected by the IAP provirus.
The diversity of endothelial cells is becoming more apparent and more important in defining vesse... more The diversity of endothelial cells is becoming more apparent and more important in defining vessel systems that supply blood to normal organs and to tumors. Reagents that identify expression of cell surface determinants on these cells are crucial for differentiating among different vessel types. As a first step in this process we have selected a panel of 25 scFvs from a phage display library that bind to the endothelial cell line LEII. The scFvs are of high affinity and bind to some tumor cells as well as to the target endothelial cell. The scFvs can be divided into 8 epitope groups by use of competition binding studies. DNA sequencing of the members of these groups generally support the classification. This work shows that phage display is a rapid and efficient method for identification of reagents for cell surface molecules.
Chemical crosslinking of proteins combined with mass spectrometric analysis of the tryptic digest... more Chemical crosslinking of proteins combined with mass spectrometric analysis of the tryptic digest of the products shows considerable promise as a tool for interrogating structure and geometry of proteins and protein complexes. An impediment to the use of this tool has been the difficulty of distinguishing crosslinked peptide pairs from non-crosslinked peptides, and from the products of side reactions. We describe the use of a commercially available biotinylated crosslinking reagent, sulfo-SBED, that allows affinity-based enrichment of crosslinked species. An intramolecular crosslink is prepared using the peptide neurotensin as a model system. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra show the predicted crosslinking product, as well as several side products. Finally, we describe the optimized enrichment of biotinylated species, and reduction of non-specific binding, for a batch-mode affinity separation based on immobilized monomeric avidin.
... Thrombomodulin (TM) {formerly designated Pl12 (Ford et al. ... The data show that in contrast... more ... Thrombomodulin (TM) {formerly designated Pl12 (Ford et al. ... The data show that in contrast to expression in the adult, TM is found in high concentrations in the leptomeninges of the brain andspinal cord, and the de-veloping reproductive system, suggesting that TM may have a ...
Amyloidosis is a protein-misfolding disorder characterized by the extracellular deposition of amy... more Amyloidosis is a protein-misfolding disorder characterized by the extracellular deposition of amyloid, a complex matrix composed of protein fibrils, hyper-sulphated glycosaminoglycans and serum amyloid P component (SAP). Accumulation of amyloid in visceral organs results in the destruction of tissue architecture leading to organ dysfunction and failure. Early differential diagnosis and disease monitoring are critical for improving patient outcomes; thus, whole body amyloid imaging would be beneficial in this regard. Non-invasive molecular imaging of systemic amyloid is performed in Europe by using iodine-123-labelled SAP; however, this tracer is not available in the US. Therefore, we evaluated synthetic, poly-basic peptides, designated p5 and p5+14, as alternative radiotracers for detecting systemic amyloidosis. Herein, we perform a comparative effectiveness evaluation of radiolabelled peptide p5+14 with p5 and SAP, in amyloid-laden mice, using dual-energy SPECT imaging and tissue b...
Chemical crosslinking of proteins combined with mass spectrometric analysis of the tryptic digest... more Chemical crosslinking of proteins combined with mass spectrometric analysis of the tryptic digest of the products shows considerable promise as a tool for interrogating structure and geometry of proteins and protein complexes. An impediment to the use of this tool has been the difficulty of distinguishing crosslinked peptide pairs from non-crosslinked peptides, and from the products of side reactions. We describe the use of a commercially available biotinylated crosslinking reagent, sulfo-SBED, that allows affinity-based enrichment of crosslinked species. An intramolecular crosslink is prepared using the peptide neurotensin as a model system. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra show the predicted crosslinking product, as well as several side products. Finally, we describe the optimized enrichment of biotinylated species, and reduction of non-specific binding, for a batch-mode affinity separation based on immobilized monomeric avidin.
An acousto-optic tunable filter (AOTF)-based system for dual-modality hyperspectral imaging (DMHS... more An acousto-optic tunable filter (AOTF)-based system for dual-modality hyperspectral imaging (DMHSI) has been developed for use in characterization of normal and malignant mouse tissue. The system consists of a laser, endoscope, AOTF, and two cameras coupled with optics and electronics. Initial results show that the system can delineate normal and malignant mouse tissues real-time. The analysis shows that malignant tissues consistently exhibit less fluorescent intensity in the wavelength band from 440 to 540 nm with a peak intensity of around 490 nm. The analysis also shows key spectroscopic differences between normal and malignant tissues. Further, these results are compared to real-time spectroscopic data and show good correlation.
Sequence analysis of the 5' end of the thrombomodulin (TM) gene has identified a differen... more Sequence analysis of the 5' end of the thrombomodulin (TM) gene has identified a difference in two strains of laboratory mice, BALB/cBD (maintained at the Oak Ridge National Laboratory) and the strain used for the sequence listed in the GenBank data base. An intracisternal A-particle (IAP) provirus was present in the BALB/cBD sequence but absent from the GenBank sequence. Thus, there are two different 5' regions for the TM gene in mice. Southern blot and polymerase chain reaction analyses on other mouse strains demonstrated the presence of this proviral sequence in five out of ten strains tested. Northern blot analyses demonstrated that there were differences (up to three-fold) in the steady-state levels of TM mRNA among the strains. However, TM mRNA levels did not correlate directly with the presence of the IAP DNA upstream from the TM gene. This is in contrast to other loci (the interleukin-3 gene, c-mos, and the immunoglobulin Ck gene), in which the IAP element is known to affect levels of RNA transcription. While the IAP provirus does not appear to have an effect on TM transcription under normal conditions, it is possible that transcription levels altered by factors like thrombin and tumor necrosis factor are affected by the IAP provirus.
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Papers by Stephen Kennel