Humanization of murine monoclonal antibodies for human therapy has commonly been achieved by comp... more Humanization of murine monoclonal antibodies for human therapy has commonly been achieved by complementarity-determining region (CDR) grafting, in which murine CDR loops are grafted onto human framework regions. Difficulties with that method have revealed the importance of certain framework residues in determining both the 3-D structure of CDR loops and the overall affinity of the molecule for its specific ligand. In the general model of structure-function relationships presented here, each amino acid position in the variable region is classified according to the benefit of achieving a more human-like antibody versus the risk of decreasing or abolishing specific binding affinity. Substitutions of human residues at low-risk positions (exposed to solvent but not contributing to antigen binding or antibody structure) are likely to decrease immunogenicity with little or no effect on binding affinity. Changes at high-risk positions (directly involved in antigen binding, CDR stabilization or internal packing) are avoided to preserve the biological activity of the antibody. Moderate-risk changes are made with caution. This model has been tested experimentally using H65, an anti-CD5 murine monoclonal antibody, whose binding activity had been greatly reduced by two previous attempts at humanization by conventional CDR grafting. The new 'human-engineered' H65 antibody containing 20 low-risk human consensus substitutions (expressed as either IgG or Fab) retains the full binding avidity of parental murine and chimeric H65 antibodies. A human-engineered antibody with an additional 14 moderate-risk substitutions has unexpectedly enhanced avidity (3- to 7-fold). This method is generally applicable to the design of other human-engineered antibodies with therapeutic potential.
Biochemical and Biophysical Research Communications, 2006
The SIRT 1 enzyme is a NAD dependent deacetylase implicated in ageing, cell protection, and energ... more The SIRT 1 enzyme is a NAD dependent deacetylase implicated in ageing, cell protection, and energy metabolism in mammalian cells. How the endogenous activity of SIRT 1 is modulated is not known. The enzyme CD38 is a multifunctional enzyme capable of synthesis of the second messenger, cADPR, NAADP, and ADPR. However, the major enzymatic activity of CD38 is the hydrolysis of NAD. Of particular interest is the fact that CD38 is present on the inner nuclear membrane. Here, we investigate the modulation of the SIRT 1 activity by CD38. We propose that by modulating availability of NAD to the SIRT1 enzyme, CD38 may regulate SIRT1 enzymatic activity. We observed that in CD38 knockout mice, tissue levels of NAD are significantly increased. We also observed that incubation of purified recombinant SIRT1 enzyme with CD38 or nuclear extracts of wild-type mice led to a significant inhibition of its activity. In contrast, incubation of SIRT1 with cellular extract from CD38 knockout mice was without effect. Furthermore, the endogenous activity of SIRT1 was several time higher in nuclear extracts from CD38 knockout mice when compared to wild-type nuclear extracts. Finally, the in vivo deacetylation of the SIRT1 substrate P53 is increased in CD38 knockout mice tissue. Our data support the novel concept that nuclear CD38 is a major regulator of cellular/nuclear NAD level, and SIRT1 activity. These findings have strong implications for understanding the basic mechanisms that modulate intracellular NAD levels, energy homeostasis, as well as ageing and cellular protection modulated by the SIRT enzymes.
Biochemical and Biophysical Research Communications, 2006
The SIRT 1 enzyme is a NAD dependent deacetylase implicated in ageing, cell protection, and energ... more The SIRT 1 enzyme is a NAD dependent deacetylase implicated in ageing, cell protection, and energy metabolism in mammalian cells. How the endogenous activity of SIRT 1 is modulated is not known. The enzyme CD38 is a multifunctional enzyme capable of synthesis of the second messenger, cADPR, NAADP, and ADPR. However, the major enzymatic activity of CD38 is the hydrolysis of NAD. Of particular interest is the fact that CD38 is present on the inner nuclear membrane. Here, we investigate the modulation of the SIRT 1 activity by CD38. We propose that by modulating availability of NAD to the SIRT1 enzyme, CD38 may regulate SIRT1 enzymatic activity. We observed that in CD38 knockout mice, tissue levels of NAD are significantly increased. We also observed that incubation of purified recombinant SIRT1 enzyme with CD38 or nuclear extracts of wild-type mice led to a significant inhibition of its activity. In contrast, incubation of SIRT1 with cellular extract from CD38 knockout mice was without effect. Furthermore, the endogenous activity of SIRT1 was several time higher in nuclear extracts from CD38 knockout mice when compared to wild-type nuclear extracts. Finally, the in vivo deacetylation of the SIRT1 substrate P53 is increased in CD38 knockout mice tissue. Our data support the novel concept that nuclear CD38 is a major regulator of cellular/nuclear NAD level, and SIRT1 activity. These findings have strong implications for understanding the basic mechanisms that modulate intracellular NAD levels, energy homeostasis, as well as ageing and cellular protection modulated by the SIRT enzymes.
Solid-state NMR spectroscopy and Fourier transform IR (FTIR) spectroscopy have been used to study... more Solid-state NMR spectroscopy and Fourier transform IR (FTIR) spectroscopy have been used to study the degradation of cellulose in the form of transformer insulating paper and Whatman ®lter paper in air at temperatures from 200°C to 550°C for 1 h with and without 0.01 wt.% NaCl, ZnCl 2 and CuCl 2 . The NMR studies included 1 H wide line measurements of the spin-lattice relaxation time (T 1 ) and spin-lattice relaxation time in the rotating frame (T 1q ), and 13 C magic-angle spinning experiments. The 1 H spectra and relaxation times indicated the presence of absorbed water and oxygen in the papers. On degradation, the 1 H intensity and line width decreased, indicating the loss of hydrogen nuclei, and the 1 H relaxation times decreased due to the formation of paramagnetic species. The 13 C-NMR spectra and FTIR spectra indicated the formation of aromatic structures on degradation above 250°C, the conversion being essentially complete at 450°C. The presence of the salts had little or no eect on the rate or mechanism of degradation. Ó
Solid-state NMR spectroscopy and Fourier transform IR (FTIR) spectroscopy have been used to study... more Solid-state NMR spectroscopy and Fourier transform IR (FTIR) spectroscopy have been used to study the degradation of cellulose in the form of transformer insulating paper and Whatman ®lter paper in air at temperatures from 200°C to 550°C for 1 h with and without 0.01 wt.% NaCl, ZnCl 2 and CuCl 2 . The NMR studies included 1 H wide line measurements of the spin-lattice relaxation time (T 1 ) and spin-lattice relaxation time in the rotating frame (T 1q ), and 13 C magic-angle spinning experiments. The 1 H spectra and relaxation times indicated the presence of absorbed water and oxygen in the papers. On degradation, the 1 H intensity and line width decreased, indicating the loss of hydrogen nuclei, and the 1 H relaxation times decreased due to the formation of paramagnetic species. The 13 C-NMR spectra and FTIR spectra indicated the formation of aromatic structures on degradation above 250°C, the conversion being essentially complete at 450°C. The presence of the salts had little or no eect on the rate or mechanism of degradation. Ó
Humanization of murine monoclonal antibodies for human therapy has commonly been achieved by comp... more Humanization of murine monoclonal antibodies for human therapy has commonly been achieved by complementarity-determining region (CDR) grafting, in which murine CDR loops are grafted onto human framework regions. Difficulties with that method have revealed the importance of certain framework residues in determining both the 3-D structure of CDR loops and the overall affinity of the molecule for its specific ligand. In the general model of structure-function relationships presented here, each amino acid position in the variable region is classified according to the benefit of achieving a more human-like antibody versus the risk of decreasing or abolishing specific binding affinity. Substitutions of human residues at low-risk positions (exposed to solvent but not contributing to antigen binding or antibody structure) are likely to decrease immunogenicity with little or no effect on binding affinity. Changes at high-risk positions (directly involved in antigen binding, CDR stabilization or internal packing) are avoided to preserve the biological activity of the antibody. Moderate-risk changes are made with caution. This model has been tested experimentally using H65, an anti-CD5 murine monoclonal antibody, whose binding activity had been greatly reduced by two previous attempts at humanization by conventional CDR grafting. The new 'human-engineered' H65 antibody containing 20 low-risk human consensus substitutions (expressed as either IgG or Fab) retains the full binding avidity of parental murine and chimeric H65 antibodies. A human-engineered antibody with an additional 14 moderate-risk substitutions has unexpectedly enhanced avidity (3- to 7-fold). This method is generally applicable to the design of other human-engineered antibodies with therapeutic potential.
Biochemical and Biophysical Research Communications, 2006
The SIRT 1 enzyme is a NAD dependent deacetylase implicated in ageing, cell protection, and energ... more The SIRT 1 enzyme is a NAD dependent deacetylase implicated in ageing, cell protection, and energy metabolism in mammalian cells. How the endogenous activity of SIRT 1 is modulated is not known. The enzyme CD38 is a multifunctional enzyme capable of synthesis of the second messenger, cADPR, NAADP, and ADPR. However, the major enzymatic activity of CD38 is the hydrolysis of NAD. Of particular interest is the fact that CD38 is present on the inner nuclear membrane. Here, we investigate the modulation of the SIRT 1 activity by CD38. We propose that by modulating availability of NAD to the SIRT1 enzyme, CD38 may regulate SIRT1 enzymatic activity. We observed that in CD38 knockout mice, tissue levels of NAD are significantly increased. We also observed that incubation of purified recombinant SIRT1 enzyme with CD38 or nuclear extracts of wild-type mice led to a significant inhibition of its activity. In contrast, incubation of SIRT1 with cellular extract from CD38 knockout mice was without effect. Furthermore, the endogenous activity of SIRT1 was several time higher in nuclear extracts from CD38 knockout mice when compared to wild-type nuclear extracts. Finally, the in vivo deacetylation of the SIRT1 substrate P53 is increased in CD38 knockout mice tissue. Our data support the novel concept that nuclear CD38 is a major regulator of cellular/nuclear NAD level, and SIRT1 activity. These findings have strong implications for understanding the basic mechanisms that modulate intracellular NAD levels, energy homeostasis, as well as ageing and cellular protection modulated by the SIRT enzymes.
Biochemical and Biophysical Research Communications, 2006
The SIRT 1 enzyme is a NAD dependent deacetylase implicated in ageing, cell protection, and energ... more The SIRT 1 enzyme is a NAD dependent deacetylase implicated in ageing, cell protection, and energy metabolism in mammalian cells. How the endogenous activity of SIRT 1 is modulated is not known. The enzyme CD38 is a multifunctional enzyme capable of synthesis of the second messenger, cADPR, NAADP, and ADPR. However, the major enzymatic activity of CD38 is the hydrolysis of NAD. Of particular interest is the fact that CD38 is present on the inner nuclear membrane. Here, we investigate the modulation of the SIRT 1 activity by CD38. We propose that by modulating availability of NAD to the SIRT1 enzyme, CD38 may regulate SIRT1 enzymatic activity. We observed that in CD38 knockout mice, tissue levels of NAD are significantly increased. We also observed that incubation of purified recombinant SIRT1 enzyme with CD38 or nuclear extracts of wild-type mice led to a significant inhibition of its activity. In contrast, incubation of SIRT1 with cellular extract from CD38 knockout mice was without effect. Furthermore, the endogenous activity of SIRT1 was several time higher in nuclear extracts from CD38 knockout mice when compared to wild-type nuclear extracts. Finally, the in vivo deacetylation of the SIRT1 substrate P53 is increased in CD38 knockout mice tissue. Our data support the novel concept that nuclear CD38 is a major regulator of cellular/nuclear NAD level, and SIRT1 activity. These findings have strong implications for understanding the basic mechanisms that modulate intracellular NAD levels, energy homeostasis, as well as ageing and cellular protection modulated by the SIRT enzymes.
Solid-state NMR spectroscopy and Fourier transform IR (FTIR) spectroscopy have been used to study... more Solid-state NMR spectroscopy and Fourier transform IR (FTIR) spectroscopy have been used to study the degradation of cellulose in the form of transformer insulating paper and Whatman ®lter paper in air at temperatures from 200°C to 550°C for 1 h with and without 0.01 wt.% NaCl, ZnCl 2 and CuCl 2 . The NMR studies included 1 H wide line measurements of the spin-lattice relaxation time (T 1 ) and spin-lattice relaxation time in the rotating frame (T 1q ), and 13 C magic-angle spinning experiments. The 1 H spectra and relaxation times indicated the presence of absorbed water and oxygen in the papers. On degradation, the 1 H intensity and line width decreased, indicating the loss of hydrogen nuclei, and the 1 H relaxation times decreased due to the formation of paramagnetic species. The 13 C-NMR spectra and FTIR spectra indicated the formation of aromatic structures on degradation above 250°C, the conversion being essentially complete at 450°C. The presence of the salts had little or no eect on the rate or mechanism of degradation. Ó
Solid-state NMR spectroscopy and Fourier transform IR (FTIR) spectroscopy have been used to study... more Solid-state NMR spectroscopy and Fourier transform IR (FTIR) spectroscopy have been used to study the degradation of cellulose in the form of transformer insulating paper and Whatman ®lter paper in air at temperatures from 200°C to 550°C for 1 h with and without 0.01 wt.% NaCl, ZnCl 2 and CuCl 2 . The NMR studies included 1 H wide line measurements of the spin-lattice relaxation time (T 1 ) and spin-lattice relaxation time in the rotating frame (T 1q ), and 13 C magic-angle spinning experiments. The 1 H spectra and relaxation times indicated the presence of absorbed water and oxygen in the papers. On degradation, the 1 H intensity and line width decreased, indicating the loss of hydrogen nuclei, and the 1 H relaxation times decreased due to the formation of paramagnetic species. The 13 C-NMR spectra and FTIR spectra indicated the formation of aromatic structures on degradation above 250°C, the conversion being essentially complete at 450°C. The presence of the salts had little or no eect on the rate or mechanism of degradation. Ó
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Papers by Sandra Soares