The use of iodine seed (I-125) as a marker for the localisation of lung nodules in minimal invasive pulmonary surgery.

AIM: Video assisted thoracic surgery (VATS) is an important tool in the field of thoracic pathology both for therapeutic and diagnostic purposes. The standard technique for localisation of non-visible or non-palpable lung lesions is the use of image guided insertion of a guide-wire. However, this method is associated with complications such as pneumothorax, bleeding and wire-dislocation. The aim of this study was to investigate the feasibility of using of iodine seeds (I-125) as a marker of lung lesions during VATS. METHODS: 28 consecutive patients with parenchymal lung lesions had I-125 seed localisation performed prior to VATS. After seed placement all patients underwent VATS with wedge resection. RESULTS: During surgery all lesions could be identified and radically resected. In six (21.4%) patients the seed was not placed optimally but none of these cases were associated with seed dislocation after placement. In four and in 5 patients the placement of the I-125 seed was complicated by a haematoma and pneumothorax respectively. However, in all of these patients a wait-and-see policy would have been justified. In one patient a conversion to a thoracotomy was necessary due to seed displacement. CONCLUSION: In patients with parenchymal lung lesions undergoing VATS and wedge resection I-125 seed localisation is a feasible technique. Complication rates are comparable to standard guide-wire localisation. Although I-125 seeds can be positioned under CT-guidance an optimal placement is of utmost importance for VATS wedge resection. Further research is needed to investigate the possible advantages of this technique.

Lipoma arborescens: An unusual cause of swelling of the knee.

We report a patient with a lipoma arborescens in the knee, a chance finding discovered on MRI. This is an unusual cause of swelling of the knee joint; if this condition is present, it is almost always located in the suprapatellar pouch. In this case, the lipoma arborescens was found in the popliteal space.

High-throughput sample handling and data collection at synchrotrons: embedding the ESRF into the high-throughput gene-to-structure pipeline.

An automatic data-collection system has been implemented and installed on seven insertion-device beamlines and a bending-magnet beamline at the ESRF (European Synchrotron Radiation Facility) as part of the SPINE (Structural Proteomics In Europe) development of an automated structure-determination pipeline. The system allows remote interaction with beamline-control systems and automatic sample mounting, alignment, characterization, data collection and processing. Reports of all actions taken are available for inspection via database modules and web services.

Characterization of a novel pectate lyase, Pel10A, from Pseudomonas cellulosa.

Biological recycling of plant material is essential for biosphere maintenance. This perpetual task involves a complex array of enzymes, including extracellular polysaccharide hydrolases and lyases. Whilst much is known about the structure and function of the hydrolases, relatively little is known about the structures and mechanisms of the corresponding lyases. To this end, crystals of the catalytic module of a novel family 10 pectate lyase, Pel10A from Pseudomonas cellulosa, were obtained using polyethylene glycol 2000 monomethylether as a precipitant. They belong to space group P2(1), with unit-cell parameters a = 47.7, b = 106.1, c = 55.4 A, beta = 92.0 degrees, and have two molecules in the asymmetric unit. The crystals diffract beyond 1.5 A using synchrotron radiation.

Crystal structure of GerE, the ultimate transcriptional regulator of spore formation in Bacillus subtilis.

The small, DNA-binding protein GerE regulates gene transcription in the terminally differentiated mother-cell compartment during late stages of sporulation in Bacillus subtilis. This versatile transcription factor shares sequence homology with the LuxR/FixJ/UhpA family of activators and modulates the expression of a number of genes, in particular those encoding the components of the coat that surrounds the mature spore. GerE orchestrates the final stages of coat deposition and maturation that lead to a spore with remarkable resistance properties but that must be responsive to low levels of germination signals. As this germination process is largely passive and can occur in the absence of de novo protein synthesis, the correct assembly of germination machinery, including germinant receptors and energy storage compounds, is crucial to the survival of the cell. The crystal structure of GerE has been solved at 2.05 A resolution using multi-wavelength anomalous dispersion techniques and reveals the nature of the GerE dimer. Each monomer comprises four alpha-helices, of which the central pair forms a helix-turn-helix DNA-binding motif. Implications for DNA-binding and the structural organisation of the LuxR/FixJ/UhpA family of transcription activator domains are discussed.

Structural origins of the interfacial activation in Thermomyces (Humicola) lanuginosa lipase.

The already known X-ray structures of lipases provide little evidence about initial, discrete structural steps occurring in the first phases of their activation in the presence of lipids (process referred to as interfacial activation). To address this problem, five new Thermomyces (formerly Humicola) lanuginosa lipase (TlL) crystal structures have been solved and compared with four previously reported structures of this enzyme. The bias coming from different crystallization media has been minimized by the growth of all crystals under the same crystallization conditions, in the presence of detergent/lipid analogues, with low or high ionic strength as the only main variable. Resulting structures and their characteristic features allowed the identification of three structurally distinct species of this enzyme: low activity form (LA), activated form (A), and fully Active (FA) form. The isomerization of the Cys268-Cys22 disulfide, synchronized with the formation of a new, short alpha(0) helix and flipping of the Arg84 (Arginine switch) located in the lid's proximal hinge, have been postulated as the key, structural factors of the initial transitions between LA and A forms. The experimental results were supplemented by theoretical calculations. The magnitude of the activation barrier between LA (ground state) and A (end state) forms of TlL (10.6 kcal/mol) is comparable to the enthalpic barriers typical for ring flips and disulfide isomerizations at ambient temperatures. This suggests that the sequence of the structural changes, as exemplified in various TlL crystal structures, mirror those that may occur during interfacial activation.

The trans-activation domain of the sporulation response regulator Spo0A revealed by X-ray crystallography.

Sporulation in Bacillus involves the induction of scores of genes in a temporally and spatially co-ordinated programme of cell development. Its initiation is under the control of an expanded two-component signal transduction system termed a phosphorelay. The master control element in the decision to sporulate is the response regulator, Spo0A, which comprises a receiver or phosphoacceptor domain and an effector or transcription activation domain. The receiver domain of Spo0A shares sequence similarity with numerous response regulators, and its structure has been determined in phosphorylated and unphosphorylated forms. However, the effector domain (C-Spo0A) has no detectable sequence similarity to any other protein, and this lack of structural information is an obstacle to understanding how DNA binding and transcription activation are controlled by phosphorylation in Spo0A. Here, we report the crystal structure of C-Spo0A from Bacillus stearothermophilus revealing a single alpha-helical domain comprising six alpha-helices in an unprecedented fold. The structure contains a helix-turn-helix as part of a three alpha-helical bundle reminiscent of the catabolite gene activator protein (CAP), suggesting a mechanism for DNA binding. The residues implicated in forming the sigmaA-activating region clearly cluster in a flexible segment of the polypeptide on the opposite side of the structure from that predicted to interact with DNA. The structural results are discussed in the context of the rich array of existing mutational data.

Structural analysis of a chimeric bacterial alpha-amylase. High-resolution analysis of native and ligand complexes.

Several chimeric alpha-amylases genes were constructed by an in vivo recombination technique from the Bacillus amyloliquefaciens and Bacillus licheniformis genes. One of the fusion amylases (hereafter BA2), consisting of residues 1-300 from B. amyloliquefaciens and 301-483 from B. licheniformis, has been extensively studied by X-ray crystallography at resolutions between 2.2 and 1.7 A. The 3-dimensional structure of the native enzyme was solved by multiple isomorphous replacement, and refined at a resolution of 1.7 A. It consists of 483 amino acids, organized similarly to the known B. lichiniformis alpha-amylase structure [Machius et al. (1995) J. Mol. Biol. 246, 545-559], but features 4 bound calcium ions. Two of these form part of a linear cluster of three ions, the central ion being attributed to sodium. This cluster lies at the junction of the A and B domains with one calcium of the cluster structurally equivalent to the major Ca(2+) binding site of fungal alpha-amylases. The third calcium ion is found at the interface of the A and C domains. BA2 contains a fourth calcium site, not observed in the B. licheniformis alpha-amylase structure. It is found on the C domain where it bridges the two beta-sheets. Three acid residues (Glu261, Asp328, and Asp231) form an active site similar to that seen in other amylases. In the presence of TRIS buffer, a single molecule of TRIS occupies the -1 subsite of the enzyme where it is coordinated by the three active-center carboxylates. Kinetic data reveal that BA2 displays properties intermediate to those of its parents. Data for crystals soaked in maltooligosaccharides reveal the presence of a maltotriose binding site on the N-terminal face of the (beta/alpha)(8) barrel of the molecule, not previously described for any alpha-amylase structure, the biological function of which is unclear. Data for a complex soaked with the tetrasaccharide inhibitor acarbose, at 1.9 A, reveal a decasaccharide moiety, spanning the -7 to +3 subsites of the enzyme. The unambiguous presence of three unsaturated rings in the (2)H(3) half-chair/(2)E envelope conformation, adjacent to three 6-deoxypyranose units, clearly demonstrates synthesis of this acarbose-derived decasaccharide by a two-step transglycosylation mechanism.

The X6 "thermostabilizing" domains of xylanases are carbohydrate-binding modules: structure and biochemistry of the Clostridium thermocellum X6b domain.

Many polysaccharide-degrading enzymes display a modular structure in which a catalytic module is attached to one or more noncatalytic modules. Several xylanases contain a module of previously unknown function (termed "X6" modules) that had been implicated in thermostability. We have investigated the properties of two such "thermostabilizing" modules, X6a and X6b from the Clostridium thermocellumxylanase Xyn10B. These modules, expressed either as discrete entities or as their natural fusions with the catalytic module, were assayed, and their capacity to bind various carbohydrates and potentiate hydrolytic activity was determined. The data showed that X6b, but not X6a, increased the activity of the enzyme against insoluble xylan and bound specifically to xylooligosaccharides and various xylans. In contrast, X6a exhibited no affinity for soluble or insoluble forms of xylan. Isothermal titration calorimetry revealed that the ligand-binding site of X6b accommodates approximately four xylose residues. The protein exhibited K(d) values in the low micromolar range for xylotetraose, xylopentaose, and xylohexaose; 24 microM for xylotriose; and 50 microM for xylobiose. Negative DeltaH and DeltaS values indicate that the interaction of X6b with xylooligosaccharides and xylan is driven by enthalpic forces. The three-dimensional structure of X6b has been solved by X-ray crystallography to a resolution of 2.1 A. The protein is a beta-sandwich that presents a tryptophan and two tyrosine residues on the walls of a shallow cleft that is likely to be the xylan-binding site. In view of the structural and carbohydrate-binding properties of X6b, it is proposed that this and related modules be re-assigned as family 22 carbohydrate-binding modules.

B-DNA at atomic resolution reveals extended hydration patterns.

Despite the importance of hydration around DNA in the understanding of its conformation and interactions with other molecules in many biological processes, only limited atomic resolution information is available. Crystal-engineering techniques, which were originally developed to mimic DNA base triplets in a crystal lattice, also eliminate the rotational disorder of oligonucleotides around their helical axis and thereby enhance the resolution of the structure analysis. We have determined the low-temperature crystal structure of the synthetic DNA decamer d(GGCCAATTGG) at atomic resolution (1. 15 A) using 17700 reflections and have characterized the highly organized hydration patterns in both grooves. The narrow d(AATT) minor groove is occupied by an 'extended hydration spine' alternately bridging base pairs and phosphate O1P atoms of opposite strands, while a distinctive pattern of parallel water ribbons is observed in the major groove. This analysis provides structural insight into the correlation found between narrow minor-groove width and occurrence of the B(I) conformation and can be used to design new minor-groove binders. By their location between adjacent helices, two fully hydrated magnesium ions further stabilize the crystal packing. The structure also provides details of the hydration and conformation of G.GC triple helices.

MRI of liver metastases: limitation of spleen-liver model in optimizing pulse sequences.

The spleen-liver model, as a predictor for contrast-to-noise ratio (C/N) in liver metastases, was verified for seven sequences in 22 patients with 70 colorectal metastases. Optimization of conventional spin-echo, T1-magnetization-prepared gradient-echo and fat frequency-selective presaturation inversion-recovery fast spin echo can be done using the spleen-liver model. C/N of liver-spleen and liver-metastases, however, differed significantly on our T1 gradient-echo and T2-weighted fast spin-echo images, with and without fat-selective saturation.

Role of carotid sonography as a first examination in the evaluation of patients with transient ischemic attacks and strokes: benefit in relation to age.

PURPOSE: We studied the influence of age on the utility of carotid sonography in patients with transient ischemic attacks and strokes. METHODS: The results of Doppler ultrasound examinations of the carotid arteries in 613 consecutive patients with transient ischemic attacks (n = 450) or strokes (n = 163) were analyzed for different age groups. For each patient, the grade of stenosis was scored for the ipsilateral internal carotid artery. The results of the ultrasound examinations were correlated with angiographic findings and findings at endarterectomy. The extent of atherosclerosis for each age group was expressed as the ratio between the number of grade II-IV stenoses (> or = 50%) in the carotid arteries and the number of patients in that group ("atherosclerosis ratio"). RESULTS: Under the age of 40 years, high-grade atherosclerotic stenoses were not found. However, 3 relatively young patients had dissections of the internal carotid arteries. The atherosclerosis ratio exceeded 0.5 for age groups 65-69 years through 80+ years. Among the patients with high-grade stenoses, ischemic heart disease prevented endarterectomy in 63% of patients in age group 80+ years, 44% in age group 75-79 years, and 26% in age group 70-74 years. CONCLUSIONS: Carotid sonography did not detect any significant atherosclerotic changes in young patients but was useful for diagnosing other etiologies of ischemic cerebral disease, eg, carotid dissection. At the other end of the spectrum, the impact of carotid sonography on patient management appears to be limited in patients over the age of 70 years. Carotid sonography seems to be most useful for patients 40-69 years old.

Modern developments in molecular replacement.

Molecular replacement is a possible route to obtaining initial phasing for an unknown structure from a known, structurally related molecule. Recent years have seen an explosive growth in the number of protein structures solved using this technique. Automated packages can make the application quite straightforward. Progress has been made in the placing of fragments of complexes, and in the use of imprecise models from NMR or homology modelling. Such models have necessitated the development of new approaches to rebuilding and refinement.

DNA-drug refinement: a comparison of the programs NUCLSQ, PROLSQ, SHELXL93 and X-PLOR, using the low-temperature d(TGATCA)-nogalamycin structure.

In an earlier study [Smith, Davies, Dodson & Moore (1995). Biochemistry, 34, 415-425] the crystal structure of the d(TGATCA)-nogalamycin complex was determined to 1.8 A and refined with PROLSQ to R = 19.5% against 4767 reflections with F> 1sigma(F). A low-temperature crystallographic study on this complex has now been performed. Native data collection at liquid-nitrogen temperature (120 K) improved the resolution to 1.4 A. The structure has now been refined against these new diffraction data in the resolution range 8-1.4 A using NUCLSQ, PROLSQ, SHELXL93 and X-PLOR, in order to determine to what extent the resulting DNA conformation and associated solvent structure would differ and to examine the suitability of these programs for the refinement of oligonucleotide structures. With the advent of more DNA-protein structure determinations, it is of interest to see how well the protein-refinement packages, PROLSQ and X-PLOR, and the small-molecule program, SHELXL93, are able to accommodate DNA. Comparisons are made between the dictionaries, weights and restraints used and the final models obtained from each program. Although the final R values, using all data in the resolution range 8.0-1.4 A, from PROLSQ (22.8%), SHELXL93 (R1 =21.7% after isotropic refinement) and X-PLOR (24.4%) are higher than the R value from the NUCLSQ refinement (21.2%), the root-mean-square deviations between the four final models are very small. Using this high-quality 8.0-1.4 A data set neither the dictionary nor the refinement program leave an imprint on the final fully refined complex. Likewise, the helical parameters and backbone conformation including sugar-puckering modes are not influenced by the refinement procedure used. Although a different number of water molecules is found in each refinement, varying from 62 (X-PLOR) to 86 (NUCLSQ), the first hydration sphere is well conserved in all four models.

Case report: aneurysm of an aberrant right subclavian artery diagnosed with MR imaging.

An aberrant right subclavian artery (ARSCA), also known as arteria lusoria, is the most common congenital anomaly of the aortic arch, with a reported prevalence ranging from 0.4 to 2%. The ARSCA arises as the last branch of the aortic arch and crosses the mediastinum from left to right indenting the oesophagus posteriorly. Aneurysms of this aberrant vessel, whether or not arising from a Kommerell's diverticulum, are rare. A 79-year-old woman is presented, in whom a partly thrombosed aneurysm of an ARSCA was diagnosed with magnetic resonance (MR) imaging.

Crystal structure of an extracellular fragment of the rat CD4 receptor containing domains 3 and 4.

BACKGROUND: CD4 is a transmembrane protein on the surface of T lymphocytes that interacts with MHC class II proteins at the surface of accessory cells, and is involved in the triggering of the lymphocytes by foreign antigens. It is also the major receptor for the human immunodeficiency virus. The extracellular portion of CD4 was predicted to contain four immunoglobulin superfamily domains and this has been confirmed by X-ray crystallography, but no detailed structure of domains 3 and 4 has been available. RESULTS: We now report the expression of a form of rat CD4 containing only domains 3 and 4, its crystallization, and the refinement and analysis of its structure by X-ray crystallography with 2.6 A spacing data. Both domains show variations in core residues when compared with immunoglobulin domains. Features of the structure are discussed with respect to the structure of the complete extracellular part of CD4 and its function. CONCLUSIONS: Domains 3 and 4 of CD4 show considerable similarity to domains 1 and 2, although there is a 25 degrees rotation in the relative positions of the domains with respect to one another. The absence of the disulphide bond in domain 3 is associated with an alteration in the packing of the beta-sheets, which may be important for interactions with domain 2 in the overall receptor structure. The location of N-linked glycosylation on one face of domain 3 appears to preclude the dimerization that is observed in antibodies.

Structure of a ternary complex of an allosteric lactate dehydrogenase from Bacillus stearothermophilus at 2.5 A resolution.

We report the refined structure of a ternary complex of an allosterically activated lactate dehydrogenase, including the important active site loop. Eightfold non-crystallographic symmetry averaging was utilized to improve the density maps. Interactions between the protein and bound coenzyme and oxamate are described in relation to other studies using site-specific mutagenesis. Fructose 1,6-bisphosphate (FruP2) is bound to the enzyme across one of the 2-fold axes of the tetramer, with the two phosphate moieties interacting with two anion binding sites, one on each of two subunits, across this interface. However, because FruP2 binds at this special site, yet does not possess an internal 2-fold symmetry axis, the ligand is statistically disordered and binds to each site in two different orientations. Binding of FruP2 to the tetramer is signalled to the active site principally through two interactions with His188 and Arg173. His188 is connected to His195 (which binds the carbonyl group of the substrate) and Arg173 is connected to Arg171 (the residue that binds the carboxylate group of the substrate).