José Saldanha - Academia.edu (original) (raw)

Papers by José Saldanha

The area of Beirã, belonging to the municipality of Marvão, hosted during the last two weeks of J... more The area of Beirã, belonging to the municipality of Marvão, hosted during the last two weeks of July 2018, the 5th Traditional Architecture and Urbanism Summer School. There, teachers and students coming from all over the world studied the traditional construction, architecture and urbanism of the region and took them as the basis to develop different design proposals for the future development of this place. The Summer School was once again organized by INTBAU and by the Rafael Manzano Prize for New Traditional Architecture. It was possible thanks to the support provided by the Richard H. Driehaus Charitable Lead Trust (through a contribution to the Chicago Community Foundation for the Richard H. Driehaus Charitable Fund), the Fundação Serra Henriques and Kalam. Equally important was the collaboration of the Câmara Municipal de Marvão, the Junta de Freguesia de Beirã, the A Anta Association, also from Beirã, and the Infrastructures of Portugal, all of which provided constant advice and commitment. A large number of Portuguese and foreign universities also took part, providing teachers, lectures and students: the Escola Superior Gallaecia, the Instituto Universitário de Lisboa (ISCTE-IUL), the Universidade do Algarve and the Universidade de Évora (Portugal), the Pontifical and Royal University of Santo Tomas (Philippines), the Schools of Architecture of the Judson University, the University of Miami and the University of Notre Dame (USA), the Universidad Alfonso X el Sabio, the Universidad de Castilla-La Mancha and the Universidad Politécnica de Madrid (Spain) and the Centro de Investigación de Arquitectura Tradicional (CIAT-UPM).

Nucleic acids research, Jan 8, 2017

Mycobacterium tuberculosis (MTb) is the causative agent of pulmonary tuberculosis (TB). MTb colon... more Mycobacterium tuberculosis (MTb) is the causative agent of pulmonary tuberculosis (TB). MTb colonizes the human lung, often entering a non-replicating state before progressing to life-threatening active infections. Transcriptional reprogramming is essential for TB pathogenesis. In vitro, Cmr (a member of the CRP/FNR super-family of transcription regulators) bound at a single DNA site to act as a dual regulator of cmr transcription and an activator of the divergent rv1676 gene. Transcriptional profiling and DNA-binding assays suggested that Cmr directly represses dosR expression. The DosR regulon is thought to be involved in establishing latent tuberculosis infections in response to hypoxia and nitric oxide. Accordingly, DNA-binding by Cmr was severely impaired by nitrosation. A cmr mutant was better able to survive a nitrosative stress challenge but was attenuated in a mouse aerosol infection model. The complemented mutant exhibited a ∼2-fold increase in cmr expression, which led to...

Orphanet journal of rare diseases, Jul 2, 2016

TRNT1 (CCA-adding transfer RNA nucleotidyl transferase) enzyme deficiency is a new metabolic dise... more TRNT1 (CCA-adding transfer RNA nucleotidyl transferase) enzyme deficiency is a new metabolic disease caused by defective post-transcriptional modification of mitochondrial and cytosolic transfer RNAs (tRNAs). We investigated four patients from two families with infantile-onset cyclical, aseptic febrile episodes with vomiting and diarrhoea, global electrolyte imbalance during these episodes, sideroblastic anaemia, B lymphocyte immunodeficiency, retinitis pigmentosa, hepatosplenomegaly, exocrine pancreatic insufficiency and renal tubulopathy. Other clinical features found in children include sensorineural deafness, cerebellar atrophy, brittle hair, partial villous atrophy and nephrocalcinosis. Whole exome sequencing and bioinformatic filtering were utilised to identify recessive compound heterozygous TRNT1 mutations (missense mutation c.668T>C, p.Ile223Thr and a novel splice mutation c.342+5G>T) segregating with disease in the first family. The second family was found to have a ...

Antibody Drug Discovery, 2011

Dübel/Handbook of Therapeutic Antibodies, 2014

Protein Science, 2008

The Aeromonas proteolytica aminopeptidase~AMP!, Pseudomonas sp.~RS-16! carboxypeptidase G2~CPG2!,... more The Aeromonas proteolytica aminopeptidase~AMP!, Pseudomonas sp.~RS-16! carboxypeptidase G2~CPG2!, and Streptomyces griseus aminopeptidase~SGAP! are zinc dependent proteolytic enzymes with cocatalytic zinc ion centers and a conserved aminopeptidase fold. A BLAST search with the sequence of the solved AMP structure indicated that a similar domain could be found in prostate-specific membrane antigen~PSMA! and the transferrin receptor~TfR!. When the PSMA or TfR sequence was input into the THREADER program, the top structural matches were SGAP and AMP confirming that these are structurally conserved domains. Optimal sequence alignment of PSMA and TfR using the known three-dimensional structures of AMP, CPG2, and SGAP shows that the critical amino acids involved in forming the catalytic pocket are conserved in PSMA but absent in the TfR. The specificity pocket in AMP is formed from four aromatic side chains and the equivalent region in CPG20PSMA has a changed sequence pattern. Since CPG2 and PSMA are folate hydrolases, the changed specificity pocket leaves space to accommodate the large pteroate moiety of folic acid. In contrast, no enzyme function has been ascribed to the TfR.

"Protein Engineering, Design and Selection", 1993

Mouse mAb TES-C21(C21) recognizes an epitope on human IgE and, therefore, has potential as a ther... more Mouse mAb TES-C21(C21) recognizes an epitope on human IgE and, therefore, has potential as a therapeutic agent in patients with IgE-mediated allergies such as hay fever, food and drug allergies and extrinsic asthma. The clinical usefulness of mouse antibodies is limited, however, due to their immunogenicity in humans. Mouse C21 antibody was humanized by complementarity determining region (CDR) grafting with the aim of developing an effective and safe therapeutic for the treatment of IgE-mediated allergies. The CDR-grafted, or reshaped human, C21 variable regions were carefully designed using a specially constructed molecular model of the mouse C21 variable regions. A key step in the design of reshaped human variable regions is the selection of the human framework regions (FRs) to serve as the backbones of the reshaped human variable regions. Two approaches to the selection of human FRs were tested: (i) selection from human consensus sequences and (ii) selection from individual human antibodies. The reshaped human and mouse C21 antibodies were tested and compared using a biosensor to measure the kinetics of binding to human IgE. Surprisingly, a few of the reshaped human C21 antibodies exhibited patterns of binding and affinities that were essentially identical to those of mouse C21 antibody.

"Protein Engineering, Design and Selection", 1991

A knowledge-based approach to the modelling of enzyme-peptide inhibitor complexes is described. G... more A knowledge-based approach to the modelling of enzyme-peptide inhibitor complexes is described. Given the structure of an enzyme, and knowledge of its binding site, the method seeks to predict the binding geometry of a peptide ligand. This novel method involves using examples of side-chain packing derived from proteins of known three-dimensional structure to define possible packing arrangements of a peptide inhibitor group to its binding site. A suite of programs, GEMINI, was written and used to predict the packing of pairs of amino acid groups from three inhibitors complexed to their enzymes for which the X-ray structures were available. These included the Phe group of the inhibitor H142 bound to endothiapepsin, the Leu group of CLT complexed to thermolysin and the C-terminus of Gly-L-Tyr bound to carboxypeptidase A. A detailed comparison of the modelled and observed inhibitor coordinates was made. This approach may be extended to modelling other types of protein interactions.

"Protein Engineering, Design and Selection", 1991

Amylin is the major component of the amyloid found in the pancreases of noninsulin-dependent diab... more Amylin is the major component of the amyloid found in the pancreases of noninsulin-dependent diabetics (type 2 diabetes). It is a 37 amino acid polypeptide and has been shown to have 46% sequence identity with the neuropeptide alpha-calcitonin gene-related peptide (alpha-CGRP). Both amylin and alpha-CGRP are known to be potent inhibitors of glycogen synthesis in stripped rat soleus muscle. Secondary structure prediction and tertiary structure model-building show the two polypeptides to have an alpha-helix/beta-strand motif similar to that observed in the insulin B-chain. The results have been supported by CD spectroscopy, although there is no sequence similarity between insulin and amylin/alpha-CGRP. Aggregation states have been predicted based on the dimeric and hexameric arrangements seen in porcine insulin. Rat and hamster amylin have a changed sequence motif in the beta-strand which results in lack of amyloid formation and type 2 diabetes. This, we propose, is caused by disruption of hydrogen bonding which prevents the formation of the dimer.

Molecular Pharmacology, 2011

We have used alanine-scanning mutagenesis followed by functional expression and molecular modelin... more We have used alanine-scanning mutagenesis followed by functional expression and molecular modeling to analyze the roles of the 14 residues, Asn422 to Cys435, C-terminal to transmembrane (TM) helix 7 of the M 1 muscarinic acetylcholine receptor. The results suggest that they form an eighth (H8) helix, associated with the cytoplasmic surface of the cell membrane in the active state of the receptor. We suggest that the amide side chain of Asn422 may act as a cap to the C terminus of TM7, stabilizing its junction with H8, whereas the side chain of Phe429 may restrict the relative movements of H8 and the C terminus of TM7 in the inactive ground state of the receptor. We have identified four residues, Phe425, Arg426, Thr428, and Leu432, which are important for G protein binding and signaling. These may form a docking site for the C-terminal helix of the G protein ␣ subunit, and collaborate with G protein recognition residues elsewhere in the cytoplasmic domain of the receptor to form a coherent surface for G protein binding in the activated state of the receptor.

Molecular Immunology, 1999

Humanization of rodent mAbs by CDR-grafting (also called``reshaping'') is now a standard procedur... more Humanization of rodent mAbs by CDR-grafting (also called``reshaping'') is now a standard procedure for reducing immunogenicity and recruiting human eector functions. However, the design of the humanized mAb can sometimes prove circuitous. Attempts were made to humanize L-25, a mouse antibody against the human alpha-4 integrin subunit using the usual protocols. Despite reaching eight backmutations in the light chain, it was not possible to recover the binding activity to the level of the chimeric. In an eort to restore the binding activity, an analysis of the human kappa IV acceptor frameworks was undertaken. This analysis highlighted the Asp at position 9 in framework 1, which although a common amino acid in human kappa IV frameworks, was an unusual residue in mouse kappa frameworks. Backmutating this position to the mouse amino acid completely restored the binding of the humanized antibody and as a by-product also increased the secretion levels in cos cells. Mutating position 9 to the consensus residue for human kappa I also restored the binding and secretion levels although not to the same extent. The resulting humanized antibody had a light chain with only a single backmutation to the mouse sequence.

Computer Methods and Programs in Biomedicine, 1989

With the increasing interest in using knowledge-based approaches for protein structure prediction... more With the increasing interest in using knowledge-based approaches for protein structure prediction and modelling, there is a requirement for general techniques to convert molecular biological data into structures that can be interpreted by artificial intelligence programming languages (e.g. Prolog). We describe here an interactive program that generates files in Prolog clausal form from the most commonly distributed protein structural data collections. The program is flexible and enables a variety of clause structures to be defined bv the user through a general schema definition syslem. Ou~ method can be extended to include other types of molecular biological database or those containing non-structural information, thus providing a uniform framework for handling the increasing volume of data available to knowledgebased systems in biomedicine.

Computational Biology and Chemistry, 2004

A series of agonists to the rat muscarinic receptor have been docked computationally to the activ... more A series of agonists to the rat muscarinic receptor have been docked computationally to the active site of a homology model of rat M1 muscarinic receptor. The agonists were modelled on the X-ray crystal structure of atropine, which is reported here and the docking studies are shown to reproduce correctly the order of experimental binding affinities for the agonists as well as indicate where there appear to be inconsistencies in the experimental data. The crystal and molecular structure of atropine (tropine tropate; ␣-[hydroxymethyl]benzeneacetic acid 8-methyl[3.2.1]oct-3-yl ester C 17 H 23 NO 3) has been determined by X-ray crystallography using an automated Patterson search method, and refined by full-matrix least-squares to a final R of 0.0452 for 2701 independent observed reflections and 192 parameters using Mo K␣ radiation, λ = 0.71073Å at 150 K. The compound crystallises in space group Fdd2 with Z = 16 molecules per unit cell.

Bioinformatics, 1991

A logical design that describes the overall structure of proteins, together with a more detailed ... more A logical design that describes the overall structure of proteins, together with a more detailed design describing secondary and some supersecondary structures, has been constructed using the computer-aided software engineering (CASE) tool, Auto-mate. Auto-mate embodies the philosophy of the Structured Systems Analysis and Design Method (SSADM) which enables the logical design of computer systems. Our design will facilitate the building of large information systems, such as databases and knowledgebases in the field of protein structure, by the derivation of system requirements from our logical model prior to producing the final physical system. In addition, the study has highlighted the ease of employing SSADM as a formalism in which to conduct the transferral of concepts from an expert into a design for a knowledge-based system that can be implemented on a computer (the knowledge-engineering exercise). It has been demonstrated how SSADM techniques may be extended for the purpose of modelling the constituent Prolog rules. This facilitates the integration of the logical system design model with the derived knowledge-based system.

Molecular cancer therapeutics, 2003

The aurora kinases are a novel oncogenic family of mitotic serine/threonine kinases (S/T kinases)... more The aurora kinases are a novel oncogenic family of mitotic serine/threonine kinases (S/T kinases) that are overexpressed in a number of solid tumors, including pancreas and colorectal cancer. A PSI-BLAST search [National Center for Biotechnology Information (NCBI)] with the sequence of the S/T kinase domain of human aurora1 kinase [also known as AUR1, ARK2, AIk2, AIM-1, and STK12] and human aurora2 kinase (also known as AUR2, ARK1, AIK, BTAK, and STK15) showed a high sequence similarity to the three-dimensional structures of bovine cAMP-dependent kinase [Brookhaven Protein Data Bank code 1CDK], murine cAMP-dependent kinase (1APM), and Caenorhabditis elegans twitchin kinase (1KOA). When the aurora1 or aurora2 sequence was input into the tertiary structure prediction programs THREADER and 3D-PSSM (three-dimensional position-sensitive scoring matrix), the top structural matches were 1CDK, 1APM, and 1KOA, confirming that these domains are structurally conserved. The structural models of...

… Biology and Chemistry, 2009

Recently published detailed X-ray structures of the three common forms, A, B and C, of Oligomycin... more Recently published detailed X-ray structures of the three common forms, A, B and C, of Oligomycin, including absolute configurations, have been employed in order to investigate their binding to ATP synthase. The X-ray studies revealed regions with differences in threedimensional structure and hydrogen bonding propensity between the Oligomycins, which may in turn be associated with their potential to bind to sites on the ATP synthase molecule. In the x-ray structure of Oligomycin A most of the potential H-bond donors and acceptors are employed in the formation of four intramolecular interactions. The three groups OH(24), OH(28) and O(36) participate in intermolecular H-bonds. In the x-ray structure of Oligomycin B and C only one intramolecular H-bond is formed. The six groups OH(2), O(7), OH(24), OH(25), OH(28) and O(36 participate in intermolecular H-bonds in Oligomycin B and four OH(2), OH(24), OH(28) and O(36) in Oligomycin C. Therefore the x-ray data suggest that Form A is thus possibly the most stable, and form B the most versatile with respect to receptor binding. Computational docking studies carried out in MOE using the x-ray structures above and an homology model of the F 0 domain of the ATP synthase from E. Coli (also built using MOE), were used to derive an induced fit pocket.

The area of Beirã, belonging to the municipality of Marvão, hosted during the last two weeks of J... more The area of Beirã, belonging to the municipality of Marvão, hosted during the last two weeks of July 2018, the 5th Traditional Architecture and Urbanism Summer School. There, teachers and students coming from all over the world studied the traditional construction, architecture and urbanism of the region and took them as the basis to develop different design proposals for the future development of this place. The Summer School was once again organized by INTBAU and by the Rafael Manzano Prize for New Traditional Architecture. It was possible thanks to the support provided by the Richard H. Driehaus Charitable Lead Trust (through a contribution to the Chicago Community Foundation for the Richard H. Driehaus Charitable Fund), the Fundação Serra Henriques and Kalam. Equally important was the collaboration of the Câmara Municipal de Marvão, the Junta de Freguesia de Beirã, the A Anta Association, also from Beirã, and the Infrastructures of Portugal, all of which provided constant advice and commitment. A large number of Portuguese and foreign universities also took part, providing teachers, lectures and students: the Escola Superior Gallaecia, the Instituto Universitário de Lisboa (ISCTE-IUL), the Universidade do Algarve and the Universidade de Évora (Portugal), the Pontifical and Royal University of Santo Tomas (Philippines), the Schools of Architecture of the Judson University, the University of Miami and the University of Notre Dame (USA), the Universidad Alfonso X el Sabio, the Universidad de Castilla-La Mancha and the Universidad Politécnica de Madrid (Spain) and the Centro de Investigación de Arquitectura Tradicional (CIAT-UPM).

Nucleic acids research, Jan 8, 2017

Mycobacterium tuberculosis (MTb) is the causative agent of pulmonary tuberculosis (TB). MTb colon... more Mycobacterium tuberculosis (MTb) is the causative agent of pulmonary tuberculosis (TB). MTb colonizes the human lung, often entering a non-replicating state before progressing to life-threatening active infections. Transcriptional reprogramming is essential for TB pathogenesis. In vitro, Cmr (a member of the CRP/FNR super-family of transcription regulators) bound at a single DNA site to act as a dual regulator of cmr transcription and an activator of the divergent rv1676 gene. Transcriptional profiling and DNA-binding assays suggested that Cmr directly represses dosR expression. The DosR regulon is thought to be involved in establishing latent tuberculosis infections in response to hypoxia and nitric oxide. Accordingly, DNA-binding by Cmr was severely impaired by nitrosation. A cmr mutant was better able to survive a nitrosative stress challenge but was attenuated in a mouse aerosol infection model. The complemented mutant exhibited a ∼2-fold increase in cmr expression, which led to...

Orphanet journal of rare diseases, Jul 2, 2016

TRNT1 (CCA-adding transfer RNA nucleotidyl transferase) enzyme deficiency is a new metabolic dise... more TRNT1 (CCA-adding transfer RNA nucleotidyl transferase) enzyme deficiency is a new metabolic disease caused by defective post-transcriptional modification of mitochondrial and cytosolic transfer RNAs (tRNAs). We investigated four patients from two families with infantile-onset cyclical, aseptic febrile episodes with vomiting and diarrhoea, global electrolyte imbalance during these episodes, sideroblastic anaemia, B lymphocyte immunodeficiency, retinitis pigmentosa, hepatosplenomegaly, exocrine pancreatic insufficiency and renal tubulopathy. Other clinical features found in children include sensorineural deafness, cerebellar atrophy, brittle hair, partial villous atrophy and nephrocalcinosis. Whole exome sequencing and bioinformatic filtering were utilised to identify recessive compound heterozygous TRNT1 mutations (missense mutation c.668T>C, p.Ile223Thr and a novel splice mutation c.342+5G>T) segregating with disease in the first family. The second family was found to have a ...

Antibody Drug Discovery, 2011

Dübel/Handbook of Therapeutic Antibodies, 2014

Protein Science, 2008

The Aeromonas proteolytica aminopeptidase~AMP!, Pseudomonas sp.~RS-16! carboxypeptidase G2~CPG2!,... more The Aeromonas proteolytica aminopeptidase~AMP!, Pseudomonas sp.~RS-16! carboxypeptidase G2~CPG2!, and Streptomyces griseus aminopeptidase~SGAP! are zinc dependent proteolytic enzymes with cocatalytic zinc ion centers and a conserved aminopeptidase fold. A BLAST search with the sequence of the solved AMP structure indicated that a similar domain could be found in prostate-specific membrane antigen~PSMA! and the transferrin receptor~TfR!. When the PSMA or TfR sequence was input into the THREADER program, the top structural matches were SGAP and AMP confirming that these are structurally conserved domains. Optimal sequence alignment of PSMA and TfR using the known three-dimensional structures of AMP, CPG2, and SGAP shows that the critical amino acids involved in forming the catalytic pocket are conserved in PSMA but absent in the TfR. The specificity pocket in AMP is formed from four aromatic side chains and the equivalent region in CPG20PSMA has a changed sequence pattern. Since CPG2 and PSMA are folate hydrolases, the changed specificity pocket leaves space to accommodate the large pteroate moiety of folic acid. In contrast, no enzyme function has been ascribed to the TfR.

"Protein Engineering, Design and Selection", 1993

Mouse mAb TES-C21(C21) recognizes an epitope on human IgE and, therefore, has potential as a ther... more Mouse mAb TES-C21(C21) recognizes an epitope on human IgE and, therefore, has potential as a therapeutic agent in patients with IgE-mediated allergies such as hay fever, food and drug allergies and extrinsic asthma. The clinical usefulness of mouse antibodies is limited, however, due to their immunogenicity in humans. Mouse C21 antibody was humanized by complementarity determining region (CDR) grafting with the aim of developing an effective and safe therapeutic for the treatment of IgE-mediated allergies. The CDR-grafted, or reshaped human, C21 variable regions were carefully designed using a specially constructed molecular model of the mouse C21 variable regions. A key step in the design of reshaped human variable regions is the selection of the human framework regions (FRs) to serve as the backbones of the reshaped human variable regions. Two approaches to the selection of human FRs were tested: (i) selection from human consensus sequences and (ii) selection from individual human antibodies. The reshaped human and mouse C21 antibodies were tested and compared using a biosensor to measure the kinetics of binding to human IgE. Surprisingly, a few of the reshaped human C21 antibodies exhibited patterns of binding and affinities that were essentially identical to those of mouse C21 antibody.

"Protein Engineering, Design and Selection", 1991

A knowledge-based approach to the modelling of enzyme-peptide inhibitor complexes is described. G... more A knowledge-based approach to the modelling of enzyme-peptide inhibitor complexes is described. Given the structure of an enzyme, and knowledge of its binding site, the method seeks to predict the binding geometry of a peptide ligand. This novel method involves using examples of side-chain packing derived from proteins of known three-dimensional structure to define possible packing arrangements of a peptide inhibitor group to its binding site. A suite of programs, GEMINI, was written and used to predict the packing of pairs of amino acid groups from three inhibitors complexed to their enzymes for which the X-ray structures were available. These included the Phe group of the inhibitor H142 bound to endothiapepsin, the Leu group of CLT complexed to thermolysin and the C-terminus of Gly-L-Tyr bound to carboxypeptidase A. A detailed comparison of the modelled and observed inhibitor coordinates was made. This approach may be extended to modelling other types of protein interactions.

"Protein Engineering, Design and Selection", 1991

Amylin is the major component of the amyloid found in the pancreases of noninsulin-dependent diab... more Amylin is the major component of the amyloid found in the pancreases of noninsulin-dependent diabetics (type 2 diabetes). It is a 37 amino acid polypeptide and has been shown to have 46% sequence identity with the neuropeptide alpha-calcitonin gene-related peptide (alpha-CGRP). Both amylin and alpha-CGRP are known to be potent inhibitors of glycogen synthesis in stripped rat soleus muscle. Secondary structure prediction and tertiary structure model-building show the two polypeptides to have an alpha-helix/beta-strand motif similar to that observed in the insulin B-chain. The results have been supported by CD spectroscopy, although there is no sequence similarity between insulin and amylin/alpha-CGRP. Aggregation states have been predicted based on the dimeric and hexameric arrangements seen in porcine insulin. Rat and hamster amylin have a changed sequence motif in the beta-strand which results in lack of amyloid formation and type 2 diabetes. This, we propose, is caused by disruption of hydrogen bonding which prevents the formation of the dimer.

Molecular Pharmacology, 2011

We have used alanine-scanning mutagenesis followed by functional expression and molecular modelin... more We have used alanine-scanning mutagenesis followed by functional expression and molecular modeling to analyze the roles of the 14 residues, Asn422 to Cys435, C-terminal to transmembrane (TM) helix 7 of the M 1 muscarinic acetylcholine receptor. The results suggest that they form an eighth (H8) helix, associated with the cytoplasmic surface of the cell membrane in the active state of the receptor. We suggest that the amide side chain of Asn422 may act as a cap to the C terminus of TM7, stabilizing its junction with H8, whereas the side chain of Phe429 may restrict the relative movements of H8 and the C terminus of TM7 in the inactive ground state of the receptor. We have identified four residues, Phe425, Arg426, Thr428, and Leu432, which are important for G protein binding and signaling. These may form a docking site for the C-terminal helix of the G protein ␣ subunit, and collaborate with G protein recognition residues elsewhere in the cytoplasmic domain of the receptor to form a coherent surface for G protein binding in the activated state of the receptor.

Molecular Immunology, 1999

Humanization of rodent mAbs by CDR-grafting (also called``reshaping'') is now a standard procedur... more Humanization of rodent mAbs by CDR-grafting (also called``reshaping'') is now a standard procedure for reducing immunogenicity and recruiting human eector functions. However, the design of the humanized mAb can sometimes prove circuitous. Attempts were made to humanize L-25, a mouse antibody against the human alpha-4 integrin subunit using the usual protocols. Despite reaching eight backmutations in the light chain, it was not possible to recover the binding activity to the level of the chimeric. In an eort to restore the binding activity, an analysis of the human kappa IV acceptor frameworks was undertaken. This analysis highlighted the Asp at position 9 in framework 1, which although a common amino acid in human kappa IV frameworks, was an unusual residue in mouse kappa frameworks. Backmutating this position to the mouse amino acid completely restored the binding of the humanized antibody and as a by-product also increased the secretion levels in cos cells. Mutating position 9 to the consensus residue for human kappa I also restored the binding and secretion levels although not to the same extent. The resulting humanized antibody had a light chain with only a single backmutation to the mouse sequence.

Computer Methods and Programs in Biomedicine, 1989

With the increasing interest in using knowledge-based approaches for protein structure prediction... more With the increasing interest in using knowledge-based approaches for protein structure prediction and modelling, there is a requirement for general techniques to convert molecular biological data into structures that can be interpreted by artificial intelligence programming languages (e.g. Prolog). We describe here an interactive program that generates files in Prolog clausal form from the most commonly distributed protein structural data collections. The program is flexible and enables a variety of clause structures to be defined bv the user through a general schema definition syslem. Ou~ method can be extended to include other types of molecular biological database or those containing non-structural information, thus providing a uniform framework for handling the increasing volume of data available to knowledgebased systems in biomedicine.

Computational Biology and Chemistry, 2004

A series of agonists to the rat muscarinic receptor have been docked computationally to the activ... more A series of agonists to the rat muscarinic receptor have been docked computationally to the active site of a homology model of rat M1 muscarinic receptor. The agonists were modelled on the X-ray crystal structure of atropine, which is reported here and the docking studies are shown to reproduce correctly the order of experimental binding affinities for the agonists as well as indicate where there appear to be inconsistencies in the experimental data. The crystal and molecular structure of atropine (tropine tropate; ␣-[hydroxymethyl]benzeneacetic acid 8-methyl[3.2.1]oct-3-yl ester C 17 H 23 NO 3) has been determined by X-ray crystallography using an automated Patterson search method, and refined by full-matrix least-squares to a final R of 0.0452 for 2701 independent observed reflections and 192 parameters using Mo K␣ radiation, λ = 0.71073Å at 150 K. The compound crystallises in space group Fdd2 with Z = 16 molecules per unit cell.

Bioinformatics, 1991

A logical design that describes the overall structure of proteins, together with a more detailed ... more A logical design that describes the overall structure of proteins, together with a more detailed design describing secondary and some supersecondary structures, has been constructed using the computer-aided software engineering (CASE) tool, Auto-mate. Auto-mate embodies the philosophy of the Structured Systems Analysis and Design Method (SSADM) which enables the logical design of computer systems. Our design will facilitate the building of large information systems, such as databases and knowledgebases in the field of protein structure, by the derivation of system requirements from our logical model prior to producing the final physical system. In addition, the study has highlighted the ease of employing SSADM as a formalism in which to conduct the transferral of concepts from an expert into a design for a knowledge-based system that can be implemented on a computer (the knowledge-engineering exercise). It has been demonstrated how SSADM techniques may be extended for the purpose of modelling the constituent Prolog rules. This facilitates the integration of the logical system design model with the derived knowledge-based system.

Molecular cancer therapeutics, 2003

The aurora kinases are a novel oncogenic family of mitotic serine/threonine kinases (S/T kinases)... more The aurora kinases are a novel oncogenic family of mitotic serine/threonine kinases (S/T kinases) that are overexpressed in a number of solid tumors, including pancreas and colorectal cancer. A PSI-BLAST search [National Center for Biotechnology Information (NCBI)] with the sequence of the S/T kinase domain of human aurora1 kinase [also known as AUR1, ARK2, AIk2, AIM-1, and STK12] and human aurora2 kinase (also known as AUR2, ARK1, AIK, BTAK, and STK15) showed a high sequence similarity to the three-dimensional structures of bovine cAMP-dependent kinase [Brookhaven Protein Data Bank code 1CDK], murine cAMP-dependent kinase (1APM), and Caenorhabditis elegans twitchin kinase (1KOA). When the aurora1 or aurora2 sequence was input into the tertiary structure prediction programs THREADER and 3D-PSSM (three-dimensional position-sensitive scoring matrix), the top structural matches were 1CDK, 1APM, and 1KOA, confirming that these domains are structurally conserved. The structural models of...

… Biology and Chemistry, 2009

Recently published detailed X-ray structures of the three common forms, A, B and C, of Oligomycin... more Recently published detailed X-ray structures of the three common forms, A, B and C, of Oligomycin, including absolute configurations, have been employed in order to investigate their binding to ATP synthase. The X-ray studies revealed regions with differences in threedimensional structure and hydrogen bonding propensity between the Oligomycins, which may in turn be associated with their potential to bind to sites on the ATP synthase molecule. In the x-ray structure of Oligomycin A most of the potential H-bond donors and acceptors are employed in the formation of four intramolecular interactions. The three groups OH(24), OH(28) and O(36) participate in intermolecular H-bonds. In the x-ray structure of Oligomycin B and C only one intramolecular H-bond is formed. The six groups OH(2), O(7), OH(24), OH(25), OH(28) and O(36 participate in intermolecular H-bonds in Oligomycin B and four OH(2), OH(24), OH(28) and O(36) in Oligomycin C. Therefore the x-ray data suggest that Form A is thus possibly the most stable, and form B the most versatile with respect to receptor binding. Computational docking studies carried out in MOE using the x-ray structures above and an homology model of the F 0 domain of the ATP synthase from E. Coli (also built using MOE), were used to derive an induced fit pocket.