Biomedical subjects

P A Bates

Publications and source records attributed to P A Bates.

At least 37 records · Page 2Linked to original sources

PubMed1999

Model building by comparison at CASP3: using expert knowledge and computer automation.

Ten models were constructed for the comparative modeling section of the Critical Assessment of Techniques for Protein Structure Prediction-3 (CASP3). Sequence identity between each target and the best possible parent(s) ranged between 12% and 64%. The modeling protocol is a mixture of automated computer algorithms with human intervention at certain critical stages. In particular, intervention is required to check sequence alignments and the selection of parameters for various computer programs. Seven of the targets were constructed from single-parent templates, and three were constructed from multiple parents. The reasons for such a high ratio of modeling from single parents only are discussed. Models constructed from multiple parents were found to be more accurate than models constructed from single parents only. A novel loop-modeling algorithm is presented that consists of fragment database searches, several fragment libraries, and mean-field calculations on representative fragment candidates.

Algorithms↗

PubMed1999

Humanisation and characterisation of PR1A3, a monoclonal antibody specific for cell-bound carcinoembryonic antigen.

Carcinoembryonic antigen (CEA) is highly expressed by most tumours of gastrointestinal origin, but its use as a target for tumour therapy is complicated by the high levels of soluble CEA that are found circulating in the blood of cancer patients. A monoclonal antibody PR1A3 has been prepared, which binds preferentially to cell-surface rather than soluble CEA, this cell selectivity should make PR1A3 an ideal candidate for antibody-targeted tumour therapy. PR1A3 has been humanised and shown to retain its cell-surface specificity and affinity. Stable expression of the humanised antibody from chinese hamster ovary (CHO) cells has been achieved after transfection and amplification. Since PR1A3 binds preferentially to cell-associated CEA, a cell-free enzyme-linked immunosorbent assay (ELISA) has been developed to allow characterisation and routine assay of the antibody. This assay was developed using a recombinant chimeric protein constructed by cloning the domain of CEA that is bound by PR1A3 (the B3 domain) into a hybrid gene containing the Fc portion of IgG and three domains of biliary glycoprotein. Stable expression of this hybrid protein has been achieved from CHO cells. In ELISA both humanised and murine PR1A3 bound strongly to this antigen but only at a minimal level to soluble CEA. Two binding sites for the antibody were found on the gastric carcinoma cell line MKN45, one of higher affinity (1 nM) and the other at lower affinity (60 nM). Similar affinities were found for both murine and humanised antibodies. The data presented make it unlikely that the differential binding to cell-surface as distinct from soluble CEA can be accounted for by low affinity of PR1A3 for CEA, and provides further support for the hypothesis that some conformational change takes place on CEA release from cells and that it is this change that blocks PR1A3 binding to its epitope.

Amino Acid Sequence↗

PubMed1999

Filamentous proteophosphoglycan secreted by Leishmania promastigotes forms gel-like three-dimensional networks that obstruct the digestive tract of infected sandfly vectors.

Development of Leishmania parasites in the digestive tract of their sandfly vectors involves several morphological transformations from the intracellular mammalian amastigote via a succession of free and gut wall-attached promastigote stages to the infective metacyclic promastigotes. At the foregut midgut transition of Leishmania-infected sandflies a gel-like plug of unknown origin and composition is formed, which contains high numbers of parasites, that occludes the gut lumen and which may be responsible for the often observed inability of infected sandflies to draw blood. This "blocked fly" phenotype has been linked to efficient transmission of infectious metacyclic promastigotes from the vector to the mammalian host. We show by immunofluorescence and immunoelectron microscopy on two Leishmania/sandfly vector combinations (Leishmania mexicana/Lutzomyia longipalpis and L. major/Phlebotomus papatasi) that the gel-like mass is formed mainly by a parasite-derived mucin-like filamentous proteophosphoglycan (fPPG) whereas the Leishmania polymeric secreted acid phosphatase (SAP) is not a major component of this plug. fPPG forms a dense three-dimensional network of filaments which engulf the promastigote cell bodies in a gel-like mass. We propose that the continuous secretion of fPPG by promastigotes in the sandfly gut, that causes plug formation, is an important factor for the efficient transmission to the mammalian host.

Acid Phosphatase↗

PubMed1999

Progress in protein structure prediction: assessment of CASP3.

The third comparative assessment of techniques of protein structure prediction (CASP3) was held during 1998. This is a blind trial in which structures are predicted prior to having knowledge of the coordinates, which are then revealed to enable the assessment. Three sections at the meeting evaluated different methodologies - comparative modelling, fold recognition and ab initio methods. For some, but not all of the target coordinates, high quality models were submitted in each of these sections. There have been improvements in prediction techniques since CASP2 in 1996, most notably for ab initio methods.

Algorithms↗

PubMed1998

Crystal structure at 1.95 A resolution of the breast tumour-specific antibody SM3 complexed with its peptide epitope reveals novel hypervariable loop recognition.

The anti-breast tumour antibody SM3 has a high selectivity in reacting specifically with carcinoma-associated mucin. SM3 recognises the core repeating motif (Pro-Asp-Thr-Arg-Pro) of aberrantly glycosylated epithelial mucin MUC1, and has potential as a therapeutic and diagnostic tool. Here we report the crystal structure of the Fab fragment of SM3 in complex with a 13-residue MUC1 peptide antigen (Thr1P-Ser2P-Ala3P-Pro4P-Asp5P-Thr6P -Arg7P-Pro8P-Ala9P-Pro10P-Gly11P- Ser12P-Thr13P). The SM3-MUC1 peptide structure was solved by molecular replacement, and the current model is refined at 1.95 A resolution with an R-factor of 21.3% and R-free 28.3%. The MUC1 peptide is bound both by non-polar interactions and hydrogen bonds in an elongated groove in the antibody-combining site through interactions with Complimentarity Determining Regions (CDRs), three of the light chain (L1, L2, L3) and two of the heavy chain (H1 and H3). The conformation of the peptide is mainly extended with no discernable standard secondary structure. There is a single non-proline cis-peptide bond in H3 (Val95H-Gly96H-Gln97H-Phe98H-Ala101H-Ty r102H) between Gly96H and Gln97H, which appears to play a role in SM3-peptide antigen interactions, and represents the first such example within an antibody hypervariable loop. The SM3-MUC1 peptide structure has implications for rational therapeutic and diagnostic antibody engineering.

Amino Acid Sequence↗

PubMed1998

Conformational analysis of the first observed non-proline cis-peptide bond occurring within the complementarity determining region (CDR) of an antibody.

An analysis has been performed on the first example of a non-proline cis- peptide bond found within a complementarity determining region (CDR) of an antibody. The bond is located in CDR 3 of the heavy chain (H3) and makes substantial interactions to a peptide from a breast tumour-associated antigen. The antibody-peptide complex is compared, both in H3 length (six residues) and peptide conformation, to a number of other such complexes in the Brookhaven Data Bank (PDB). There is only one other H3 loop of the same length. Analysis of loop searches of the PDB, taken over the H3 framework of SM3, suggest that there is a limited repertoire of conformations for loops of length 6 compared to loops of length 5 and 7. It is argued that the cis-peptide bond is present because of the limited number of loop conformations of length 6, plus, the requirement of the H3 loop to contact the bound peptide. Modelling suggests that an all-trans-peptide loop conformation can replace the H3 loop and this raises the question of whether there is a trans- to cis-peptide bond isomerization upon peptide binding.

Antibodies↗

PubMed1998

Structure of an XRCC1 BRCT domain: a new protein-protein interaction module.

The BRCT domain (BRCA1 C-terminus), first identified in the breast cancer suppressor protein BRCA1, is an evolutionarily conserved protein-protein interaction region of approximately 95 amino acids found in a large number of proteins involved in DNA repair, recombination and cell cycle control. Here we describe the first three-dimensional structure and fold of a BRCT domain determined by X-ray crystallography at 3.2 A resolution. The structure has been obtained from the C-terminal region of the human DNA repair protein XRCC1, and comprises a four-stranded parallel beta-sheet surrounded by three alpha-helices, which form an autonomously folded domain. The compact XRCC1 structure explains the observed sequence homology between different BRCT motifs and provides a framework for modelling other BRCT domains. Furthermore, the established structure of an XRCC1 BRCT homodimer suggests potential protein-protein interaction sites for the complementary BRCT domain in DNA ligase III, since these two domains form a stable heterodimeric complex. Based on the XRCC1 BRCT structure, we have constructed a model for the C-terminal BRCT domain of BRCA1, which frequently is mutated in familial breast and ovarian cancer. The model allows insights into the effects of such mutations on the fold of the BRCT domain.

Amino Acid Sequence↗

PubMed1998

The I domain of integrin leukocyte function-associated antigen-1 is involved in a conformational change leading to high affinity binding to ligand intercellular adhesion molecule 1 (ICAM-1).

On T cells the leukocyte integrin leukocyte function-associated antigen-1 (LFA-1) (CD11a/CD18) can be induced to bind its ligand intercellular adhesion molecule 1 (ICAM-1) (CD54) either by increasing the affinity of the receptor with Mg2+ and EGTA or by receptor clustering following activation with phorbol ester. The existence of these two adhesion-inducing pathways implies that alternative mechanisms might exist by which LFA-1 engages ICAM-1. The LFA-1 alpha subunit I domain contains a major binding site for ICAM-1. In this study we show that soluble LFA-1 I domain blocks ICAM-1 binding of the high affinity Mg2+-induced form of LFA-1 but not the phorbol ester-induced form. Under conditions of Mg2+-activation, the soluble I domain also prevents expression of an activation dependent epitope on LFA-1, implying that it inhibits a conformational change necessary for conversion to the high affinity form of this integrin. In addition, the binding of Mg2+-activated LFA-1 to ICAM-1 is blocked by peptides covering the alpha4-beta3 loop, the beta3-alpha5 loop, and the alpha5 helix of the I domain, whereas none of the peptides tested blocks phorbol ester-mediated adhesion. The blocking peptides localize to the same face of the crystal structure of the LFA-1 I domain and define an area that, during activation, may be involved in association of the I domain with another region of LFA-1, potentially the beta-propeller domain. This is the first evidence linking a structural domain of an integrin, in this case the I domain, with a particular activation mechanism.

Amino Acid Sequence↗

PubMed1998

Automated classification of antibody complementarity determining region 3 of the heavy chain (H3) loops into canonical forms and its application to protein structure prediction.

A computer-based algorithm was used to cluster the loops forming the complementarity determining region (CDR) 3 of the heavy chain (H3) into canonical classes. Previous analyses of the three-dimensional structures of CDR loops (also known as the hypervariable regions) within antibody immunoglobulin variable domains have shown that for five of the six CDRs there are only a few main-chain conformations (known as canonical forms) that show clear relationships between sequence and structure. However, the larger variation in length and conformation of loops within H3 has limited the classification of these loops into canonical forms. The clustering procedure presented here is based on aligning the Ramachandran-coded main-chain conformation of the residues using a dynamic algorithm that allows the insertion of gaps to obtain an optimum alignment. A total of 41 H3 loops out of 62 non-identical loops, extracted from the Brookhaven Protein Data Bank, have been automatically grouped into 22 clusters. Inspection of the clusters for consensus sequences or intra-loop interactions or invariant conformation led to the proposal of 13 canonical forms representing 31 loops. These canonical forms include a consideration of the geometry of both the take-off region adjacent to the bracing beta-strands and the remaining loop apex. Subsequently a new set of 15 H3 loops not included in the initial analysis was considered. The clustering procedure was repeated and nine of these 15 loops could be assigned to original clusters, including seven to canonical forms. A sequence profile was generated for each canonical form from the original set of loops and matched against the sequences of the new H3 loops. For five out of the seven new H3 loops that were in a canonical form, the correct form was identified at first rank by this predictive scheme.

Amino Acid Sequence↗

PubMed1998

In vitro stimulation of metacyclogenesis in Leishmania braziliensis, L. donovani, L. major and L. mexicana.

Promastigotes of Leishmania braziliensis, L. donovani, L. major and L. mexicana recently derived from tissue amastigotes were cultured in Schneider's Drosophila medium supplemented with 20% (v/v) heat-inactivated foetal calf serum and 25 micrograms gentamicin sulfate/ml at pH 5.5. These cultures produced more metacyclic promastigotes in their stationary-phase populations than others cultured at pH 7.0. Metacyclic promastigotes possessed a short (< or = 8 microns) and narrow (< or = 1.5 microns) cell body with a flagellum twice or more the length of the cell body. Promastigotes from acidic cultures were more resistant to complement-mediated lysis and more infective in vivo than those grown at neutral pH. These results demonstrate that induction of metacyclogenesis by acidic pH is a response conserved across a variety of species of Leishmania.

Animals↗

PubMed1998

Recognition of analogous and homologous protein folds--assessment of prediction success and associated alignment accuracy using empirical substitution matrices.

Fold recognition methods aim to use the information in the known protein structures (the targets) to identify that the sequence of a protein of unknown structure (the probe) will adopt a known fold. This paper highlights that the structural similarities sought by these methods can be divided into two types: remote homologues and analogues. Homologues are the result of divergent evolution and often share a common function. We define remote homologues as those that are not easily detectable by sequence comparison methods alone. Analogues do not have a common ancestor and generally do not have a common function. Several sets of empirical matrices for residue substitution, secondary structure conservation and residue accessibility conservation have previously been derived from aligned pairs of remote homologues and analogues (Russell et al., J. Mol. Biol., 1997, 269, 423-439). Here a method for fold recognition, FOLDFIT, is introduced that uses these matrices to match the sequences, secondary structures and residue accessibilities of the probe and target. The approach is evaluated on distinct datasets of analogous and remotely homologous folds. The accuracy of FOLDFIT with the different matrices on the two datasets is contrasted to results from another fold recognition method (THREADER) and to searches using mutation matrices in the absence of any structural information. FOLDFIT identifies at top rank 12 out of 18 remotely homologous folds and five out of nine analogous folds. The average alignment accuracies for residue and secondary structure equivalencing are much higher for homologous folds (residue approximately 42%, secondary structure approximately 78%) than for analogues folds (approximately 12%, approximately 47%). Sequence searches alone can be successful for several homologues in the testing sets but nearly always fail for the analogues. These results suggest that the recognition of analogous and remotely homologous folds should be assessed separately. This study has implications for the development and comparative evaluation of fold recognition algorithms.

Evolution, Molecular↗

PubMed1998

The polyserine tract of herpes simplex virus ICP4 is required for normal viral gene expression and growth in murine trigeminal ganglia.

ICP4 of herpes simplex virus (HSV) is essential for productive infection due to its central role in the regulation of HSV transcription. This study identified a region of ICP4 that is not required for viral growth in culture or at the periphery of experimentally inoculated mice but is critical for productive growth in the trigeminal ganglia. This region of ICP4 encompasses amino acids 184 to 198 and contains 13 nearly contiguous serine residues that are highly conserved among the alphaherpesviruses. A mutant in which this region is deleted (DeltaSER) was able to grow on the corneas of mice and be transported back to the trigeminal ganglia. DeltaSER did not grow in the trigeminal ganglia but did express low levels of several immediate-early (ICP4 and ICP27) and early (thymidine kinase [tk] and UL42) genes. It expressed very low levels of the late gC gene and did not appear to replicate DNA. This pattern of gene expression was similar to that observed for a tk mutant, dlsptk. Both DeltaSER and dlsptk expressed higher levels of the latency-associated transcript (LAT) per genome earlier in infected ganglia than did the wild-type virus, KOS. However, infected ganglia from all three viruses accumulated the same level of LAT per genome at 30 days postinfection (during latency). The data suggest that the polyserine tract of ICP4 provides an activity that is required for lytic infection in ganglia to progress to viral DNA synthesis and full lytic gene expression. In the absence of this activity, higher levels of LAT per genome accumulate earlier in infection than with wild-type virus.

Amino Acid Sequence↗

PubMed1998

Transformation, development, and transmission of axenically cultured amastigotes of Leishmania mexicana in vitro and in Lutzomyia longipalpis.

Axenic cultures of Leishmania mexicana amastigotes were transformed to promastigotes in vitro and in vivo in Lutzomyia longipalpis. In vitro, both exponential phase and stationary phase amastigotes were capable of transforming and growing as promastigotes, but exponential phase amastigotes completed this transition more quickly. In vivo, both populations were capable of establishing infections in sand flies by membrane feeding and could be transmitted to BALB/c mice via bite. A variety of morphologic forms could be observed in vivo, including putative metacyclic promastigotes. Infection rates in sandflies with axenic amastigotes were comparable with those achieved with lesion-derived amastigotes, supporting the use of these cultured forms in studies of parasite biology.

Animals↗

PubMed1998

Phenol red method for measuring the pH of the gut contents in Lutzomyia longipalpis (Psychodidae:Diptera).

AIM: To develop a method to estimate the pH of the gut contents of female sandflies using a microcapillary feeding technique. METHODS: Female Lutzomyia longipalpis were fed with a small quantity of phenol red solution (indicator range pH 6.8-8.4) before and after a bloodmeal. The colour patterns of the gut contents were recorded by video microscopy immediately after the alimentary canal was dissected out of the sandfly body, and used to determine the pH level. RESULTS: In unfed flies the thoracic mid-gut (TMG) is normally neutral, with the pH ranging between 7.0 to 7.3; and the abdominal mid-gut(AMG) is mildly alkaline from pH 7.1 to 8.4 with the maximum pH observed at the junction with the hind-gut. The presence of sugar in the crop reduced the pH of the TMG to 6.8, and the presence of a recently ingested bloodmeal raised the pH of the TMG to 7.4. However, as bloodmeal digestion proceeded the pH of the TMG was reduced to acidic levels, pH 6.8 or below. CONCLUSION: The new method could be integrated with the investigation of metacyclogenesis of Leishmania parasites in vivo.

Animals↗

PubMed1997

Crystallization of an antitumour antibody SM3 complexed with a peptide epitope.

SM3 antibody binds to a tumour-associated epitope on polymorphic epithelial mucin (PEM). Crystals of the Fab fragment of SM3 in complex with a peptide antigen were obtained by vapour diffusion against mother liquor containing acetate buffer, pH 6.5, cadmium chloride and polyethylene glycol (PEG) 4000 as precipitating agent. Crystals belong to the monoclinic space group P2(1) with cell dimensions a = 42.2, b = 83.9, c = 64.5 A and beta = 93.4 degrees. One Fab-antigen complex is present in the asymmetric unit. Diffracted intensities up to 1.95 A resolution have been measured from a frozen crystal using synchrotron radiation.

Journal Article↗

PubMed1997

Detection of lectin activity in Leishmania promastigotes and amastigotes.

Cell lysates from 16 strains of eight Leishmania species were used to test haemagglutination activity (HA) against a variety of RBC. HA was detected using native or neuraminidase-treated rabbit RBC; it was found in promastigotes of all the Leishmania strains tested and in axenic amastigotes of L. mexicana. The HA was trypsin-sensitive, heat-resistant and partially dependent on divalent cations. The HA was inhibited by amino-sugars, LPS from E. coli K 235, fetuin and heparin. The HA is probably located on the surface of promastigotes, as shown by the same sugar-binding specificity when live cells were used in inhibition tests. Leishmania promastigotes were agglutinated with neoglycoproteins NAc-glc-BSA and NAc-gal-BSA. This agglutination was blocked by galactosamine, glucosamine and sialic acid, but not by glcNAc or galNAc. The level of HA is increased in axenic amastigotes when compared to promastigotes. In general, HA was found at a higher titre in infective compared to uninfective strains of Leishmania. These results suggest that the haemagglutinin could play a role in the vertebrate phase of the parasite life cycle, possibly in macrophage attachment or invasion.

Agglutination↗

PubMed1997

Crystallization of the Fab fragment of the tumour-specific antibody PR1A3.

PR1A3 antibody binds specifically to the tumour-associated cell-surface antigen, carcinoembryonic antigen. Crystals of the Fab fragment of the PR1A3 antibody were obtained by vapour diffusion against mother liquor containing Tris-HC1 buffer, pH 8.6, magnesium chloride and polyethylene glycol 4000 as precipitating agent. Crystals belong to the monoclinic space group P2(1) with cell dimensions a = 42.2, b = 216.7, c = 45.9 A and beta = 95.6 degrees. Two Fab fragments are proesent in the asymmetric unit. Diffracted intensities up to 2.9 A resolution have been measured from frozen crystals.

Journal Article↗

PubMed1997

Recognition of analogous and homologous protein folds: analysis of sequence and structure conservation.

An analysis was performed on 335 pairs of structurally aligned proteins derived from the structural classification of proteins (SCOP http://scop.mrc-lmb.cam.ac.uk/scop/) database. These similarities were divided into analogues, defined as proteins with similar three-dimensional structures (same SCOP fold classification) but generally with different functions and little evidence of a common ancestor (different SCOP superfamily classification). Homologues were defined as pairs of similar structures likely to be the result of evolutionary divergence (same superfamily) and were divided into remote, medium and close sub-divisions based on the percentage sequence identity. Particular attention was paid to the differences between analogues and remote homologues, since both types of similarities are generally undetectable by sequence comparison and their detection is the aim of fold recognition methods. Distributions of sequence identities and substitution matrices suggest a higher degree of sequence similarity in remote homologues than in analogues. Matrices for remote homologues show similarity to existing mutation matrices, providing some validity for their use in previously described fold recognition methods. In contrast, matrices derived from analogous proteins show little conservation of amino acid properties beyond broad conservation of hydrophobic or polar character. Secondary structure and accessibility were more conserved on average in remote homologues than in analogues, though there was no apparent difference in the root-mean-square deviation between these two types of similarities. Alignments of remote homologues and analogues show a similar number of gaps, openings (one or more sequential gaps) and inserted/deleted secondary structure elements, and both generally contain more gaps/openings/deleted secondary structure elements than medium and close homologues. These results suggest that gap parameters for fold recognition should be more lenient than those used in sequence comparison. Parameters were derived from the analogue and remote homologue datasets for potential used in fold recognition methods. Implications for protein fold recognition and evolution are discussed.

Computer Simulation↗