Sci-Thur AM: Planning - 03: Extensive patient specific QA for field junction regions for craniospinal irradiation with Jagged-Junction IMRT approach without beam edge matching for field junctions.

PURPOSE: Jagged-Junction IMRT was developed for craniospinal irradiation. An extensive QA was performed for the field junction regions. METHODS AND MATERIALS: The Jagged-Junction IMRT plan employed three field sets, each with unique isocentres (Iso1,2,3). Fields from adjacent sets were overlapped and the dose was smoothly integrated inside the overlapped junction. The delivered dose in the junction regions were verified with film and ion chamber measurements on phantoms. An anthropomorphic-wax phantom was created for verifying the cranio-spinal junction. For measuring at the spinal-spinal junction, a solid water phantom was used. The influence of beam mismatching due to setup and mechanical inaccuracy was investigated by shifting all the fields from Iso1 and Iso3 superiorly and inferiorly by 3 mm and at the same time keeping all the fields from Iso2 without any shift. RESULTS: The patient-averaged difference between the measured dose with ion chamber and planned dose in the cranio-spinal junction is 0.34 % ± 0.40% and in the spinal-spinal junction this difference is 0.03% ± 0.71%. The dose profile comparison shows that measured and planned dose profiles match well to each other over a junction region. The patient-averaged dose difference discrepancy between the film measurement and the planned is 1.1% ± 1.3% at the cranio-spinal junction and -0.14% ± 1.8% at the spinal junction. CONCLUSIONS: Jagged-Junction IMRT planning provided smooth dose coverage to the target in the field junction region. The junction dose for the Jagged-Junction IMRT plan is not sensitive to the setup error during the treatment.

Poster - Thur Eve - 57: Craniospinal irradiation with jagged-junction IMRT approach without beam edge matching for field junctions.

PURPOSE: Craniospinal irradiation were traditionally treated the central nervous system using two or three adjacent field sets. A intensity-modulated radiotherapy (IMRT) plan (Jagged-Junction IMRT) which overcomes problems associated with field junctions and beam edge matching, improves planning and treatment setup efficiencies with homogenous target dose distribution was developed. METHODS AND MATERIALS: Jagged-Junction IMRT was retrospectively planned on three patients with prescription of 36 Gy in 20 fractions and compared to conventional treatment plans. Planning target volume (PTV) included the whole brain and spinal canal to the S3 vertebral level. The plan employed three field sets, each with a unique isocentre. One field set with seven fields treated the cranium. Two field sets treated the spine, each set using three fields. Fields from adjacent sets were overlapped and the optimization process smoothly integrated the dose inside the overlapped junction. RESULTS: For the Jagged-Junction IMRT plans vs conventional technique, average homogeneity index equaled 0.08±0.01 vs 0.12±0.02, and conformity number equaled 0.79±0.01 vs 0.47±0.12. The 95% isodose surface covered (99.5±0.3)% of the PTV vs (98.1±2.0)%. Both Jagged-Junction IMRT plans and the conventional plans had good sparing of the organs at risk. CONCLUSIONS: Jagged-Junction IMRT planning provided good dose homogeneity and conformity to the target while maintaining a low dose to the organs at risk. Jagged-Junction IMRT optimization smoothly distributed dose in the junction between field sets. Since there was no beam matching, this treatment technique is less likely to produce hot or cold spots at the junction in contrast to conventional techniques.

Field sports and conservation in the United Kingdom.

Many natural habitats exist on privately owned land outside protected areas, but few governments can afford to enforce or subsidize conservation of this biodiversity. Even in some developed countries, conservation subsidy schemes have only achieved limited success. Fortunately, some landowners may be willing to accept management costs in return for other benefits, although this remains controversial when it involves the killing of charismatic species. For example, participants in British field sports, such as fox hunting and game-bird shooting, may voluntarily conserve important habitats that are required by quarry species. Here we report results from a multidisciplinary study that addressed this issue by focusing on three sites across central England. We found that landowners participating in field sports maintained the most established woodland and planted more new woodland and hedgerows than those who did not, despite the equal availability of subsidies. Therefore, voluntary habitat management appears to be important for biodiversity conservation in Britain. Current debates on the future of field sports in Britain, and similar activities globally, may benefit from considering their utility as incentives to conserve additional habitat on private land.

Crystal structure of a soluble form of the intracellular chloride ion channel CLIC1 (NCC27) at 1.4-A resolution.

CLIC1 (NCC27) is a member of the highly conserved class of chloride ion channels that exists in both soluble and integral membrane forms. Purified CLIC1 can integrate into synthetic lipid bilayers forming a chloride channel with similar properties to those observed in vivo. The structure of the soluble form of CLIC1 has been determined at 1.4-A resolution. The protein is monomeric and structurally homologous to the glutathione S-transferase superfamily, and it has a redox-active site resembling glutaredoxin. The structure of the complex of CLIC1 with glutathione shows that glutathione occupies the redox-active site, which is adjacent to an open, elongated slot lined by basic residues. Integration of CLIC1 into the membrane is likely to require a major structural rearrangement, probably of the N-domain (residues 1-90), with the putative transmembrane helix arising from residues in the vicinity of the redox-active site. The structure indicates that CLIC1 is likely to be controlled by redox-dependent processes.

Interaction between the P14 residue and strand 2 of beta-sheet B is critical for reactive center loop insertion in plasminogen activator inhibitor-2.

The molecular interactions driving reactive center loop (RCL) insertion are of considerable interest in gaining a better understanding of the serpin inhibitory mechanism. Previous studies have suggested that interactions in the proximal hinge/breach region may be critical determinants of RCL insertion in serpins. In this study, conformational and functional changes in plasminogen activator inhibitor-2 (PAI-2) following incubation with a panel of synthetic RCL peptides indicated that the P14 residue is critical for RCL insertion, and hence inhibitory activity, in PAI-2. Only RCL peptides with a P14 threonine were able to induce the stressed to relaxed transition and abolish inhibitory activity in PAI-2, indicating that RCL insertion into beta-sheet A of PAI-2 is dependent upon this residue. The recently solved crystal structure of relaxed PAI-2 (PAI-2.RCL peptide complex) allowed detailed analysis of molecular interactions involving P14 related to RCL insertion. Of most interest is the rearrangement of hydrogen bonding around the breach region that accompanies the stressed to relaxed transition, in particular the formation of a side chain hydrogen bond between the threonine at P14 and an adjacent tyrosine on strand 2 of beta-sheet B in relaxed PAI-2. Structural alignment of known serpin sequences showed that this pairing (or the equivalent serine/threonine pairing) is highly conserved ( approximately 87%) in inhibitory serpins and may represent a general structural basis for serpin inhibitory activity.

Crystal structure of the complex of plasminogen activator inhibitor 2 with a peptide mimicking the reactive center loop.

The structure of the serpin, plasminogen activator inhibitor type-2 (PAI-2), in a complex with a peptide mimicking its reactive center loop (RCL) has been determined at 1.6-A resolution. The structure shows the relaxed state serpin structure with a prominent six-stranded beta-sheet. Clear electron density is seen for all residues in the peptide. The P1 residue of the peptide binds to a well defined pocket at the base of PAI-2 that may be important in determining the specificity of protease inhibition. The stressed-to-relaxed state (S --> R) transition in PAI-2 can be modeled as the relative motion between a quasirigid core domain and a smaller segment comprising helix hF and beta-strands s1A, s2A, and s3A. A comparison of the Ramachandran plots of the stressed and relaxed state PAI-2 structures reveals the location of several hinge regions connecting these two domains. The hinge regions cluster in three locations on the structure, ensuring a cooperative S --> R transition. We hypothesize that the hinge formed by the conserved Gly(206) on beta-strand s3A in the breach region of PAI-2 effects the S --> R transition by altering its backbone torsion angles. This torsional change is due to the binding of the P14 threonine of the RCL to the open breach region of PAI-2.

Crystal structure of a heptameric Sm-like protein complex from archaea: implications for the structure and evolution of snRNPs.

The Sm/Lsm proteins associate with small nuclear RNA to form the core of small nuclear ribonucleoproteins, required for processes as diverse as pre-mRNA splicing, mRNA degradation and telomere formation. The Lsm proteins from archaea are likely to represent the ancestral Sm/Lsm domain. Here, we present the crystal structure of the Lsm alpha protein from the thermophilic archaeon Methanobacterium thermoautotrophicum at 2.0 A resolution. The Lsm alpha protein crystallizes as a heptameric ring comprised of seven identical subunits interacting via beta-strand pairing and hydrophobic interactions. The heptamer can be viewed as a propeller-like structure in which each blade consists of a seven-stranded antiparallel beta-sheet formed from neighbouring subunits. There are seven slots on the inner surface of the heptamer ring, each of which is lined by Asp, Asn and Arg residues that are highly conserved in the Sm/Lsm sequences. These conserved slots are likely to form the RNA-binding site. In archaea, the gene encoding Lsm alpha is located next to the L37e ribosomal protein gene in a putative operon, suggesting a role for the Lsm alpha complex in ribosome function or biogenesis.

Host specificity in blood feeding parasites: a defining contribution by haemoglobin-degrading enzymes?

A hypothesis is presented that proposes that the compatibility between species-specific variants of haemoglobin-degrading proteases of blood-feeding parasites (e.g. hookworms, schistosomes, malarial parasites, etc.), and their natural substrates, i.e. haemoglobins from diverse species of mammals, has influenced to evolution of the host range of these parasites. Support for the hypothesis was drawn from molecular modelling studies of the three dimensional structure of an aspartic protease, Acasp, from the canine hookworm Ancylostoma caninum, and models of canine and human haemoglobins docked with the active site of Acasp. The molecular modelling suggested that Acasp, from a canine-specific hookworm, would have a higher substrate affinity for canine haemoglobin than for human haemoglobin.

Evolution of a light-harvesting protein by addition of new subunits and rearrangement of conserved elements: crystal structure of a cryptophyte phycoerythrin at 1.63-A resolution.

Cryptophytes are unicellular photosynthetic algae that use a lumenally located light-harvesting system, which is distinct from the phycobilisome structure found in cyanobacteria and red algae. One of the key components of this system is water-soluble phycoerythrin (PE) 545 whose expression is enhanced by low light levels. The crystal structure of the heterodimeric alpha(1)alpha(2)betabeta PE 545 from the marine cryptophyte Rhodomonas CS24 has been determined at 1.63-A resolution. Although the beta-chain structure is similar to the alpha and beta chains of other known phycobiliproteins, the overall structure of PE 545 is novel with the alpha chains forming a simple extended fold with an antiparallel beta-ribbon followed by an alpha-helix. The two doubly linked beta50/beta61 chromophores (one on each beta subunit) are in van der Waals contact, suggesting that exciton-coupling mechanisms may alter their spectral properties. Each alpha subunit carries a covalently linked 15,16-dihydrobiliverdin chromophore that is likely to be the final energy acceptor. The architecture of the heterodimer suggests that PE 545 may dock to an acceptor protein via a deep cleft and that energy may be transferred via this intermediary protein to the reaction center.

The crystal structure of plasminogen activator inhibitor 2 at 2.0 A resolution: implications for serpin function.

BACKGROUND: Plasminogen activator inhibitor 2 (PAI-2) is a member of the serpin family of protease inhibitors that function via a dramatic structural change from a native, stressed state to a relaxed form. This transition is mediated by a segment of the serpin termed the reactive centre loop (RCL); the RCL is cleaved on interaction with the protease and becomes inserted into betasheet A of the serpin. Major questions remain as to what factors facilitate this transition and how they relate to protease inhibition. RESULTS: The crystal structure of a mutant form of human PAI-2 in the stressed state has been determined at 2.0 A resolution. The RCL is completely disordered in the structure. An examination of polar residues that are highly conserved across all serpins identifies functionally important regions. A buried polar cluster beneath betasheet A (the so-called 'shutter' region) is found to stabilise both the stressed and relaxed forms via a rearrangement of hydrogen bonds. CONCLUSIONS: A statistical analysis of interstrand interactions indicated that the shutter region can be used to discriminate between inhibitory and non-inhibitory serpins. This analysis implied that insertion of the RCL into betasheet A up to residue P8 is important for protease inhibition and hence the structure of the complex formed between the serpin and the target protease.

Determination of the structure of seleno-methionine-labelled hydroxymethylbilane synthase in its active form by multi-wavelength anomalous dispersion.

The enzyme hydroxymethylbilane synthase (HMBS, E.C. 4.3.1.8) catalyzes the conversion of porphobilinogen into hydroxymethylbilane, a key intermediate for the biosynthesis of heme, chlorophylls, vitamin B12 and related macrocycles. The enzyme is found in all organisms, except viruses. The crystal structure of the selenomethionine-labelled enzyme ([SeMet]HMBS) from Escherichia coli has been solved by the multi-wavelength anomalous dispersion (MAD) experimental method using the Daresbury SRS station 9.5. In addition, [SeMet]HMBS has been studied by MAD at the Grenoble ESRF MAD beamline BM14 (BL19) and this work is described especially with respect to the use of the ESRF CCD detector. The structure at ambient temperature has been refined, the R factor being 16.8% at 2. 4 A resolution. The dipyrromethane cofactor of the enzyme is preserved in its reduced form in the crystal and its geometrical shape is in full agreement with the crystal structures of authentic dipyrromethanes. Proximal to the reactive C atom of the reduced cofactor, spherical density is seen consistent with there being a water molecule ideally placed to take part in the final step of the enzyme reaction cycle. Intriguingly, the loop with residues 47-58 is not ordered in the structure of this form of the enzyme, which carries no substrate. Direct experimental study of the active enzyme is now feasible using time-resolved Laue diffraction and freeze-trapping, building on the structural work described here as the foundation.

Schistosomes express two forms of cathepsin D.

We report here a cDNA and its deduced amino acid sequence encoding a cathepsin D-like, aspartic protease expressed by adult stages of the human blood fluke Schistosoma mansoni. The cDNA encodes a short signal peptide, a pro-enzyme peptide of 37 amino acid residues, and a mature enzyme of 377 residues which has strong homology with mammalian cathepsins D. This aspartic protease, although 84% identical in amino acids of the mature enzyme region to the previously reported cathepsin D from the Asian schistosome S. japonicum, differs remarkably from the S. japonicum enzyme in having a carboxyl terminal extension of 43 amino acid residues. These cathepsins D of schistosomes may play pivotal roles in the degradation of hemoglobin obtained by the parasites from ingested host erythrocytes.

Structure of a human lysosomal sulfatase.

BACKGROUND: . Sulfatases catalyze the hydrolysis of sulfuric acid esters from a wide variety of substrates including glycosaminoglycans, glycolipids and steroids. There is sufficient common sequence similarity within the class of sulfatase enzymes to indicate that they have a common structure. Deficiencies of specific lysosomal sulfatases that are involved in the degradation of glycosamino-glycans lead to rare inherited clinical disorders termed mucopolysaccharidoses. In sufferers of multiple sulfatase deficiency, all sulfatases are inactive because an essential post-translational modification of a specific active-site cysteine residue to oxo-alanine does not occur. Studies of this disorder have contributed to location and characterization of the sulfatase active site. To understand the catalytic mechanism of sulfatases, and ultimately the determinants of their substrate specificities, we have determined the structure of N-acetylgalactosamine-4-sulfatase. RESULTS: . The crystal structure of the enzyme has been solved and refined at 2.5 resolution using data recorded at both 123K and 273K. The structure has two domains, the larger of which belongs to the alpha/beta class of proteins and contains the active site. The enzyme active site in the crystals contains several hitherto undescribed features. The active-site cysteine residue, Cys91, is found as the sulfate derivative of the aldehyde species, oxo-alanine. The sulfate is bound to a previously undetected metal ion, which we have identified as calcium. The structure of a vanadate-inhibited form of the enzyme has also been solved, and this structure shows that vanadate has replaced sulfate in the active site and that the vanadate is covalently linked to the protein. Preliminary data is presented for crystals soaked in the monosaccharide N-acetylgalactosamine, the structure of which forms a product complex of the enzyme. CONCLUSIONS: . The structure of N-acetylgalactosamine-4-sulfatase reveals that residues conserved amongst the sulfatase family are involved in stabilizing the calcium ion and the sulfate ester in the active site. This suggests an archetypal fold for the family of sulfatases. A catalytic role is proposed for the post-translationally modified highly conserved cysteine residue. Despite a lack of any previously detectable sequence similarity to any protein of known structure, the large sulfatase domain that contains the active site closely resembles that of alkaline phosphatase: the calcium ion in sulfatase superposes on one of the zinc ions in alkaline phosphatase and the sulfate ester of Cys91 superposes on the phosphate ion found in the active site of alkaline phosphatase.

Structural analysis of the catalytic site of AcCP-1, a cysteine proteinase secreted by the hookworm Ancylostoma caninum.

Previously, we have reported that hookworms secrete cysteine proteinase activity that is capable of cleaving the cathepsin L-specific substrate Z-Phe-Arg-AMC. We have also reported the gene sequences of novel cathepsin B-like proteinases from hookworms, but have been unable to locate cathepsin L-like genes that could account for the presence of the cathepsin L activity in these parasites. Here we present an homology model for the secreted hookworm cysteine proteinase AcCP-1 based upon the crystal structure co-ordinates of human cathepsin B. The model predicts that substrate binding and specificity differs between AcCP-1 and cathepsin B, and demonstrates that AcCP-1 would preferentially cleave Phe-Arg over Arg-Arg. This thereby provides an explanation for our previous observations that the hookworm proteinase, while structurally cathepsin B-like, displays a cathepsin L-like substrate specificity.

Acasp, a gene encoding a cathepsin D-like aspartic protease from the hookworm Ancylostoma caninum.

Intestinal infection with the zoonotic hookworm Ancylostoma caninum can provoke human eosinophilic enteritis. A cDNA was isolated from A. caninum, using an oligonucleotide primer designed to hybridize to the region encoding the consensus, catalytic site residues D32TGSSNLW of aspartic proteases. This novel cDNA encoded an aspartic protease zymogen of 422 amino acids, exhibiting 47% identity to the lysosomal aspartic protease of Aedes aegypti, 46% identity to the aspartic protease of Schistosoma japonicum, and 48.5% to human cathepsin D. Its deduced structure differed from that of cathepsin D in the loop 2 "flap," which holds the substrate at the active site, and by the presence of a COOH-terminal extension of approximately 30 residues.

Structure solution of a cubic crystal of concanavalin A complexed with methyl alpha-D-glucopyranoside.

The solution of the cubic crystal form (a = 167.8 A) of concanavalin A complexed with the monosaccharide methyl alpha-D-glucopyranoside is described. The space group has been determined as I2(1)3 rather than I23. The use of cadmium to replace cobalt at the transition metal-ion binding site and to replace calcium at its binding site proved to be crucial to the successful solution of the crystal structure. The relatively small isomorphous signals of 21 e(-) for the replacement of cobalt and 28 e(-) for the replacement of calcium, yielded interpretable difference Patterson maps. The electron-density map calculated in space group I2(1)3 at 5.4 A resolution, based on phases derived from single- and double-substituted cadmium differences, revealed a classical concanavalin A tetramer of 222 point symmetry, as seen in all the known crystal structures of concanavalin A. Rigid-body refinement at 3.6 A using the refined coordinates of saccharide-free concanavalin A converged to an R factor of 27.4%. A molecular-replacement analysis, consistent with this crystal structure, and initial experiences in the incorrect space group I23 are described as these also prove to be instructive.

MAD Phasing Strategies Explored with a Brominated Oligonucleotide Crystal at 1.65A Resolution.

The crystal structure of a brominated oligonucleotide d(CGCG(Br)CG), chemical formula C(114)N(48)O(68)P(10)Br(2), has been analysed by multiwavelength anomalous dispersion (MAD) methods. The oligonucleotide crystallizes in space group P2(1)2(1)2(1) with a = 17.97, b = 30.98, c = 44.85 A, alpha = beta = gamma 90 degrees . Data to a resolution of 1.65 A were collected at four wavelengths about the K-absorption edge of the bromine atom (lambda(1) = 0.9323 A, a reference wavelength at the long-wavelength side of the edge; lambda(2) = 0.9192 A, at the absorption-edge inflection point; lambda(3) = 0.9185 A, at the ;white line' absorption maximum; lambda(4) = 0.8983 A, a reference wavelength at the short-wavelength side) using synchrotron radiation at Station PX9.5, SRS, Daresbury. Multiwavelength data could be collected on a single-crystal as the sample was radiation stable. Anomalous and dispersive Patterson maps were readily interpretable to give the bromine anomalous scatterer positions. Phase calculations to 1.65 A, resolution, using all four wavelengths, gave a figure of merit of 0.825 for 2454 reflections. The electron-density map was readily interpretable showing excellent connectivity for the sugar/phosphate backbone and each base was easily characterized. The two nucleotide strands paired up as expected in an antiparallel Watson-Crick-type manner. The structure was refined to 1.65 A using all the data (R-factor = 17.0% based on 3151 reflections, with a data-to-parameter ratio of 2.6). In addition to the four-wavelength analysis, a variety of other phasing strategies, and the associated quality of the resulting electron-density maps, were compared. These included use of either of the reference wavelength data sets in the two possible three-wavelength phasing combinations to assess their relative effectiveness. Moreover, the time dependence upon measuring the Bijvoet differences and its effect upon phasing was also investigated. Finally, the use of only two wavelengths, including Friedel pairs, is demonstrated (the theoretical minimum case); this is of particular interest when considering overall beam time needs and is clearly a feasible experimental strategy, as shown here.

Toddler's fracture.

"Toddler's fracture" can be difficult to diagnose but should be suspected whenever a child presents to the accident and emergency department with a limp or fails to bear weight on the leg. Irritable hip and subacute osteomyelitis must feature in the differential diagnosis. The history may or may not include an obvious traumatic episode. Rather than fracture, elastic bowing of the bone and consequent periosteal stripping may explain symptoms in some cases.

Cloning and characterization of the Schistosoma japonicum aspartic proteinase involved in hemoglobin degradation.

A cDNA encoding a Schistosoma japonicum aspartic proteinase was cloned, sequenced, and found to encode a zymogen of 380 amino acid residues, and its gene was shown to be present as a single copy in the S. japonicum genome. Identity comparisons showed that the enzyme (Sjpasp) was most closely related to the cathepsin Ds. The deduced amino acid sequence has four potential glycosylation sites, two of which are in identical positions to the two glycosylation sites of human kidney lysosomal cathepsin D. Furthermore, all four disulfide bonds found in mammalian cathepsin D sequences are present in Sjpasp, although the beta-hairpin (loop 3), which is cleaved during maturation of vertebrate cathepsin Ds to yield light and heavy chain subunits, is absent from Sjpasp. While most residues involved in substrate specificity and catalysis of aspartic proteinases are preserved in Sjpasp, several residues in these regions exhibit changes that may result in a novel substrate specificity. Aspartic proteinase activity is present in extracts of adult S. japonicum and Schistosoma mansoni and in culture media in which schistosomes were maintained and was capable of digesting hemoglobin. The schistosome aspartic proteinase may play a pivotal role in the catabolism of hemoglobin obtained from host erythrocytes.