Biomedical subjects

K S Wilson

Publications and source records attributed to K S Wilson.

At least 55 records · Page 3Linked to original sources

PubMed2000

Primary chemo-radiotherapy and selective oesophagectomy for oesophageal cancer: goal of cure with organ preservation.

PURPOSE: The role of definitive chemo-radiotherapy in squamous cell oesophageal carcinoma has been established by the Radiation Therapy Oncology Group (RTOG). We have studied a modification of the RTOG chemo-radiotherapy protocol in patients with any histologic type of oesophageal carcinoma. We planned oesophagectomy for patients with post-treatment positive endoscopic biopsy or <75% regression on CAT scan, or with resectable local recurrence. Study end-points were histologic response, toxicity, oesophagectomy and survival rates after primary chemo-radiotherapy. METHODS: Consecutive patients with any T or N status, M0, disease encompassable in radical radiotherapy ports, no prior surgical excision, and fit for chemo-radiotherapy, were eligible. Treatment plan was three cycles of cisplatin/5-fluorouracil chemotherapy and radical external radiation therapy (50 Gy in 25 fractions) starting with cycle 2. Selective oesophagectomy was performed in patients with post-treatment positive biopsy or <75% regression on CT scan, or with localized recurrence. RESULTS: From 1993-1996, 32 patients were treated. Post-treatment complete histologic response rate was 77% (95% confidence limits 58-90%). Grade 3 or 4 toxicities occurred in 31 and 3 patients, respectively. Minimum follow-up time was 12 months. Median disease-specific survival time was 16.1 months for all patients, and was not significantly different according to histologic type (17 squamous, 12 adenocarcinoma). Oesophagectomy was performed in six of 15 surviving and five of 17 deceased patients. CONCLUSION: It is possible to cure oesophageal cancer with chemo-radiotherapy and selective oesophagectomy, and achieve organ preservation in the majority of long term survivors.

Adenocarcinoma↗

PubMed2000

Functional sites of interaction between release factor RF1 and the ribosome.

Translational release factors decipher stop codons in mRNA and activate hydrolysis of peptidyl-tRNA in the ribosome during translation termination. The mechanisms of these fundamental processes are unknown. Here we have mapped the interaction of bacterial release factor RF1 with the ribosome by directed hydroxyl radical probing. These experiments identified conserved domains of RF1 that interact with the decoding site of the 30S ribosomal subunit and the peptidyl transferase site of the 50S ribosomal subunit. RF1 interacts with a binding pocket formed between the ribosomal subunits that is also the interaction surface of elongation factor EF-G and aminoacyl-tRNA bound to the A site. These results provide a basis for understanding the mechanism of stop codon recognition coupled to hydrolysis of peptidyl-tRNA, mediated by a protein release factor.

Amino Acid Sequence↗

PubMed2000

Digestion of single crystals of mannan I by an endo-mannanase from Trichoderma reesei.

The enzymatic degradation of single crystals of mannan I with the catalytic core domain of a beta-mannanase (EC 3.2.1.78 or Man5A) from Trichoderma reesei was investigated by transmission electron microscopy and electron diffraction. The enzyme attack took place at the edge of the crystals and progressed towards their centres. Quite remarkably the crystalline integrity of the crystals was preserved almost to the end of the digestion process. This behaviour is consistent with an endo-mechanism, where the enzyme interacts with the accessible mannan chains located at the crystal periphery and cleaves one mannan molecule at a time. The endo mode of digestion of the crystals was confirmed by an analysis of the soluble degradation products.

Chromatography, Gel↗

PubMed2000

A flexible and efficient procedure for the solution and phase refinement of protein structures.

An ab initio method is described for solving protein structures for which atomic resolution (better than 1.2 A) data are available. The problem is divided into two stages. Firstly, a substructure composed of a small percentage ( approximately 5%) of the scattering matter of the unit cell is positioned. This is used to generate a starting set of phases that are slightly better than random. Secondly, the full structure is developed from this phase set. The substructure can be a constellation of atoms that scatter anomalously, such as metal or S atoms. Alternatively, a structural fragment such as an idealized alpha-helix or a motif from some distantly related protein can be orientated and sometimes positioned by an extensive molecular-replacement search, checking the correlation coefficient between observed and calculated structure factors for the highest normalized structure-factor amplitudes |E|. The top solutions are further ranked on the correlation coefficient for all E values. The phases generated from such fragments are improved using Patterson superposition maps and Sayre-equation refinement carried out with fast Fourier transforms. Phase refinement is completed using a novel density-modification process referred to as dynamic density modification (DDM). The method is illustrated by the solution of a number of known proteins. It has proved fast and very effective, able in these tests to solve proteins of up to 5000 atoms. The resulting electron-density maps show the major part of the structures at atomic resolution and can readily be interpreted by automated procedures.

Algorithms↗

PubMed2000

The three-dimensional structure of a Trichoderma reesei beta-mannanase from glycoside hydrolase family 5.

The crystal structure of the catalytic core domain of beta-mannanase from the fungus Trichoderma reesei has been determined at a resolution of 1.5 A. The structure was solved using the anomalous scattering from a single non-isomorphous platinum complex with two heavy-metal sites in space group P2(1). The map computed with the experimental phases was enhanced by the application of an automated model building and refinement procedure using the amplitudes and experimental phases as observations. This approach is expected to be of more general application. The structure of the native enzyme and complexes with Tris-HCl and mannobiose are also reported: the mannobiose binds in subsites +1 and +2. The structure is briefly compared with that of the homologous beta-mannanase from the bacterium Thermomonospora fusca.

Actinomycetales↗

PubMed2000

Crystallization and preliminary X-ray diffraction analysis of human calcium-binding protein S100A12.

S100A12, a member of the calgranulin family, isolated from human blood, has been crystallized by vapour diffusion in the presence of Ca(2+). Crystals belong to the space group R3 with unit-cell dimensions a = b = 99.6 c = 64.2 A. There are two monomers per asymmetric unit, with a solvent content of 57.9%. The crystals diffract to at least 2.2 A resolution and complete X-ray data have been collected to 2.5 A on a conventional laboratory source.

Calcium-Binding Proteins↗

PubMed2000

Neoadjuvant hormone therapy and radical radiotherapy for localized prostate cancer: poorer biochemical outcome using flutamide alone.

Since a recent meta-analysis of non-steroidal anti-androgen therapy in metastatic prostate cancer concluded that survival was worse compared with medical or surgical androgen withdrawal, we analyzed our experience with flutamide monotherapy and other forms of neoadjuvant hormone therapy (NHT) prior to radiation therapy in clinically localized prostate cancer. A total of 45 patients received flutamide and 328 patients received other NHT. Flutamide patients had higher PSA levels at diagnosis and shorter duration of treatment, which could bias the results against flutamide monotherapy. Kaplan Meier analysis of PSA -- disease free survival showed significantly poorer outcome with flutamide monotherapy. Multivariate analysis supported this conclusion. Until equivalence to other forms of NHT is shown, we do not recommend flutamide monotherapy prior to radical radiation. A prospective randomized trial would be necessary to confirm this conclusion.

Adenocarcinoma↗

PubMed2000

Neoadjuvant hormone therapy and external beam radiation for localized prostate cancer: Vancouver Island Cancer Centre experience.

OBJECTIVES: To evaluate the effects of neo-adjuvant hormone therapy (NAHT) given prior to radiation in patients with clinically localized adenocarcinoma of the prostate. METHODS: Six hundred nine patients were treated between 1992 and 1997 with NAHT prior to radiation therapy. Clinical stage, presenting PSA and Gleason score were examined for influence on outcome. Time to post radiotherapy failure was defined from the first assessed PSA value over 4 microg/L at follow-up. Radiation therapy was confined to the prostate and seminal vesicles. Median follow-up was 2.6 years. RESULTS: PSA disease free survival (PDFS) decreased with increasing cancer risk factors (p <.0.0001). The overall duration of NAHT was significant with patients receiving >8 months having a lower failure rate than those on therapy for <3 months (p <0.0001). The PSA prior to starting radiation correlated with outcome, a PSA <=0.1 microg\L having a better PDFS than those with a PSA >=4 microg\L (P <0.0001). NAHT for >8 months gave improved PDFS in intermediate grade Gleason score 5-7, (n = 256, p <0.0001), high grade Gleason score 8-10 (n = 80, p =0.005), but not in low grade, Gleason score <=4. CONCLUSION: Neo-adjuvant hormone therapy for >8 months offers prolonged PSA disease free survival in patients with less well differentiated tumors, Gleason score >4. Clinical trials are required to confirm this.

Adenocarcinoma↗

PubMed1999

Protein titration in the crystal state.

Proteins are complex structures whose overall stability critically depends on a delicate balance of numerous interactions of similar strength, which are markedly influenced by their environment. Here, we present an analysis of the effect of pH on a protein structure in the crystalline state using RNase A as a model system. By altering only one physico-chemical parameter in a controlled manner, we are able to quantify the structural changes induced in the protein. Atomic resolution X-ray diffraction data were collected for crystals at six pH* values ranging from 5.2 to 8.8, and the six independently refined structures reveal subtle, albeit well-defined variations directly related to the pH titration of the protein. The deprotonation of the catalytic His12 residue is clearly evident in the electron density maps, confirming the reaction mechanism proposed by earlier enzymatic and structural studies. The concerted structural changes observed in the regions remote from the active-site point to an adaptation of the protein structure to the changes in the physico-chemical environment. Analysis of the stereochemistry of the six structures provided accurate estimates of p Kavalues of most of the histidine residues. This study gives further evidence for the advantage of atomic resolution X-ray crystallographic analyses for revealing small but significant structural changes which provide clues to the function of a biological macromolecule.

Binding Sites↗

PubMed1999

Common chelatase design in the branched tetrapyrrole pathways of heme and anaerobic cobalamin synthesis.

Prosthetic groups such as heme, chlorophyll, and cobalamin (vitamin B(12)) are characterized by their branched biosynthetic pathway and unique metal insertion steps. The metal ion chelatases can be broadly classed either as single-subunit ATP-independent enzymes, such as the anaerobic cobalt chelatase and the protoporphyrin IX (PPIX) ferrochelatase, or as heterotrimeric, ATP-dependent enzymes, such as the Mg chelatase involved in chlorophyll biosynthesis. The X-ray structure of the anaerobic cobalt chelatase from Salmonella typhimurium, CbiK, has been solved to 2.4 A resolution. Despite a lack of significant amino acid sequence similarity, the protein structure is homologous to that of Bacillus subtilis PPIX ferrochelatase. Both enzymes contain a histidine residue previously identified as the metal ion ligand, but CbiK contains a second histidine in place of the glutamic acid residue identified as a general base in PPIX ferrochelatase. Site-directed mutagenesis has confirmed a role for this histidine and a nearby glutamic acid in cobalt binding, modulating metal ion specificity as well as catalytic efficiency. Contrary to the predicted protoporphyrin binding site in PPIX ferrochelatase, the precorrin-2 binding site in CbiK is clearly defined within a large horizontal cleft between the N- and C-terminal domains. The structural similarity has implications for the understanding of the evolution of this branched biosynthetic pathway.

Amino Acid Sequence↗

PubMed1999

Experimental observation of bonding electrons in proteins.

We demonstrate with two examples the success and potential of recent developments in x-ray protein crystallography at ultra high resolution. Our preliminary structural analyses using diffraction data collected for the two proteins crambin and savinase show meaningful deviations from the conventional independent spherical atom approximation. A noise-reduction averaging technique enables bonding details of electron distributions in proteins to be revealed experimentally for the first time. We move one step closer to imaging directly the fine details of the electronic structure on which the biological function of a protein is based.

Animals↗

PubMed1999

X-ray structure of Novamyl, the five-domain "maltogenic" alpha-amylase from Bacillus stearothermophilus: maltose and acarbose complexes at 1.7A resolution.

The three-dimensional structure of the Bacillus stearothermophilus "maltogenic" alpha-amylase, Novamyl, has been determined by X-ray crystallography at a resolution of 1.7 A. Unlike conventional alpha-amylases from glycoside hydrolase family 13, Novamyl exhibits the five-domain structure more usually associated with cyclodextrin glycosyltransferase. Complexes of the enzyme with both maltose and the inhibitor acarbose have been characterized. In the maltose complex, two molecules of maltose are found in the -1 to -2 and +2 to +3 subsites of the active site, with two more on the C and E domains. The C-domain maltose occupies a position identical to one previously observed in the Bacillus circulans CGTase structure [Lawson, C. L., et al. (1994) J. Mol. Biol. 236, 590-600], suggesting that the C-domain plays a genuine biological role in saccharide binding. In the acarbose-maltose complex, the tetrasaccharide inhibitor acarbose is found as an extended hexasaccharide species, bound in the -3 to +3 subsites. The transition state mimicking pseudosaccharide is bound in the -1 subsite of the enzyme in a 2H3 half-chair conformation, as expected. The active site of Novamyl lies in an open gully, fully consistent with its ability to perform internal cleavage via an endo as opposed to an exo activity.

Acarbose↗

PubMed1999

Determinants of the stability of transcription elongation complexes: interactions of the nascent RNA with the DNA template and the RNA polymerase.

We use a synthetic "primed bubble-duplex" model elongation complex developed previously to examine certain structural and thermodynamic features of the transcription elongation complex of Escherichia coli. The nucleic acid framework of this model complex consists of a linear base-paired DNA molecule with a central "bubble" of non-complementary nucleotide residues, together with a single-stranded RNA molecule that is complementary (at its 3'-end) to three to 12 nucleotide residues of one of the DNA strands within the bubble. RNA polymerase is added to this framework in trans, and on addition of rNTPs the resulting complex can elongate the 3'-end of the RNA primer in a template-dependent manner with functional properties that are indistinguishable from those of a "natural" promoter-initiated transcription elongation complex operating under the same conditions. In this study we use this model system to show that the formation of a stable elongation complex at any particular template position can be treated as an equilibrium process, and that semi-quantitative dissociation constants can be estimated for the complex by using a gel band-shift assay to monitor the binding of the RNA oligomer to the complex. We then show that the formation of a stable complex depends on the presence of a complementary RNA-DNA hybrid that is at least 9 bp in length, and in addition that several nucleotide residues of non-complementary RNA located upstream of the RNA-DNA hybrid bind strongly to the putative single-stranded RNA binding site of the polymerase and significantly enhance the stability of the resulting elongation complex. Finally, we demonstrate that the measured stabilities of the model constructs in which the length of the RNA-DNA hybrid is varied correlate well with the transcriptional processivity of the functioning complex that results when rNTPs are added. These findings are discussed in the context of related studies of both model systems and natural elongation complexes. The general concepts that emerge are used to define some central structural and functional features of the transcription complex.

DNA, Bacterial↗

PubMed1999

The crystal structure of the monomeric human SOD mutant F50E/G51E/E133Q at atomic resolution. The enzyme mechanism revisited.

The crystal structure of the engineered monomeric human Cu,ZnSOD triple mutant F50E/G51E/E133Q (Q133M2SOD) is reported at atomic resolution (1.02 A). This derivative has about 20 % of the wild-type activity. Crystals of Q133M2SOD have been obtained in the presence of CdCl2. The metal binding site is disordered, with both cadmium and copper ions simultaneously binding to the copper site. The cadmium (II) ions occupy about 45 % of the copper sites by binding the four histidine residues which ligate copper in the native enzyme, and two further water molecules to complete octahedral coordination. The copper ion is tri-coordinate, and the fourth histidine (His63) is detached from copper and bridges cadmium and zinc. X-ray absorption spectroscopy performed on the crystals suggests that the copper ion has undergone partial photoreduction upon exposure to the synchrotron light. The structure is also disordered in the disulfide bridge region of loop IV that is located at the subunit/subunit interface in the native SOD dimer. As a consequence, the catalytically relevant Arg143 residue is disordered. The present structure has been compared to other X-ray structures on various isoenzymes and to the solution structure of the same monomeric form. The structural results suggest that the low activity of monomeric SOD is due to the disorder in the conformation of the side-chain of Arg143 as well as of loop IV. It is proposed that the subunit-subunit interactions in the multimeric forms of the enzyme are needed to stabilize the correct geometry of the cavity and the optimal orientation of the charged residues in the active channel. Furthermore, the different coordination of cadmium and copper ions, contemporaneously present in the same site, are taken as models for the oxidized and reduced copper species, respectively. These properties of the structure have allowed us to revisit the enzymatic mechanism.

Binding Sites↗

PubMed1999

Molecular, crystal and solution structure of a beta-cyclodextrin complex with the bromide salt of 2-(3-dimethylaminopropyl)tricyclo[3.3.1.1(3,7)]decan-2-ol, a potent antimicrobial drug.

The pharmacological properties of a cyclomaltoheptaose (beta-cyclodextrin) series of adamantane-group-bearing compounds that exhibit potent antibacterial activity have been studied, both isolated and in complex with beta-cyclodextrins (betaCDs). In this work, the structure of the bromide salt of 2-(3-dimethylaminopropyl)-tricyclo[3.3.1.1(3,7)]decan-2-ol(A DM-10) complexed with betaCD and ten water molecules was studied in the solid state by X-ray crystallography and in solution by NMR spectroscopy. X-ray crystallographic studies of the complex were performed both at room and cryogenic temperatures. The long aliphatic chain of ADM-10 adopts a single conformation at low temperature in contrast to what is observed at room temperature, where two side chain conformations are seen. Both NMR and X-ray crystallography studies indicate that the adamantane moiety of ADM-10 is buried in the betaCD cavity. Chemical shifts in NMR experiments can be explained on the basis of the crystal structure of the complex.

Anti-Bacterial Agents↗

PubMed1999

The crystal structure of a 2-fluorocellotriosyl complex of the Streptomyces lividans endoglucanase CelB2 at 1.2 A resolution.

Glycoside hydrolases have been classified into over 66 families on the basis of amino acid sequence. Recently a number of these families have been grouped into "clans" which share a common fold and catalytic mechanism [Henrissat, B., and Bairoch, A. (1996) Biochem. J. 316, 695-696]. Glycoside hydrolase Clan GH-C groups family 11 xylanases and family 12 cellulases, which share the same jellyroll topology, with two predominantly antiparallel beta-sheets forming a long substrate-binding cleft, and act with net retention of anomeric configuration. Here we present the three-dimensional structure of a family 12 endoglucanase, Streptomyces lividans CelB2, in complex with a 2-deoxy-2-fluorocellotrioside. Atomic resolution (1.2 A) data allow clear identification of two distinct species in the crystal. One is the glycosyl-enzyme intermediate, with the mechanism-based inhibitor covalently linked to the nucleophile Glu 120, and the other a complex with the reaction product, 2-deoxy-2-fluoro-beta-D-cellotriose. The active site architecture of the complex provides insight into the double-displacement mechanism of retaining glycoside hydrolases and also sheds light on the basis of the differences in specificity between family 12 cellulases and family 11 xylanases.

Cellulase↗

PubMed1999

A new proposal for urease mechanism based on the crystal structures of the native and inhibited enzyme from Bacillus pasteurii: why urea hydrolysis costs two nickels.

BACKGROUND: Urease catalyzes the hydrolysis of urea, the final step of organic nitrogen mineralization, using a bimetallic nickel centre. The role of the active site metal ions and amino acid residues has not been elucidated to date. Many pathologies are associated with the activity of ureolytic bacteria, and the efficiency of soil nitrogen fertilization with urea is severely decreased by urease activity. Therefore, the development of urease inhibitors would lead to a reduction of environmental pollution, to enhanced efficiency of nitrogen uptake by plants, and to improved therapeutic strategies for treatment of infections due to ureolytic bacteria. Structure-based design of urease inhibitors would require knowledge of the enzyme mechanism at the molecular level. RESULTS: The structures of native and inhibited urease from Bacillus pasteurii have been determined at a resolution of 2.0 A by synchrotron X-ray cryogenic crystallography. In the native enzyme, the coordination sphere of each of the two nickel ions is completed by a water molecule and a bridging hydroxide. A fourth water molecule completes a tetrahedral cluster of solvent molecules. The enzyme crystallized in the presence of phenylphosphorodiamidate contains the tetrahedral transition-state analogue diamidophosphoric acid, bound to the two nickel ions in an unprecedented mode. Comparison of the native and inhibited structures reveals two distinct conformations of the flap lining the active-site cavity. CONCLUSIONS: The mode of binding of the inhibitor, and a comparison between the native and inhibited urease structures, indicate a novel mechanism for enzymatic urea hydrolysis which reconciles the available structural and biochemical data.

Bacillus↗

PubMed1999

Crystal structure of dUTPase from equine infectious anaemia virus; active site metal binding in a substrate analogue complex.

The X-ray structures of dUTPase from equine infectious anaemia virus (EIAV) in unliganded and complexed forms have been determined to 1.9 and 2.0 A resolution, respectively. The structures were solved by molecular replacement using Escherichia coli dUTPase as search model. The exploitation of a relatively novel refinement approach for the initial model, combining maximum likelihood refinement with stereochemically unrestrained updating of the model, proved to be of crucial importance and should be of general relevance.EIAV dUTPase is a homotrimer where each subunit folds into a twisted antiparallel beta-barrel with the N and C-terminal portions interacting with adjacent subunits. The C-terminal 14 and 17 amino acid residues are disordered in the crystal structure of the unliganded and complexed enzyme, respectively. Interactions along the 3-fold axis include a water-containing volume (size 207 A3) which has no contact with bulk solvent. It has earlier been shown that a divalent metal ion is essential for catalysis. For the first time, a putative binding site for such a metal ion, in this case Sr2+, is established. The positions of the inhibitor (the non-hydrolysable substrate analogue dUDP) and the metal ion in the complex are consistent with the location of the active centre established for trimeric dUTPase structures, in which subunit interfaces form three surface clefts lined with evolutionary conserved residues. However, a detailed comparison of the active sites of the EIAV and E. coli enzymes reveals some structural differences. The viral enzyme undergoes a small conformational change in the uracil-binding beta-hairpin structure upon dUDP binding not observed in the other known dUTPase structures.

Amino Acid Sequence↗