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The genetic and functional basis of isolated 17,20-lyase deficiency.
Human male sexual differentiation requires production of fetal testicular testosterone, whose biosynthesis requires steroid 17,20-lyase activity. Patients with putative isolated 17,20-lyase deficiency have been reported. The existence of true isolated 17,20-lyase deficiency, however, has been questioned because 17 alpha-hydroxylase and 17,20-lyase activities are catalyzed by a single enzyme, microsomal cytochrome P450c17, and because the index case of apparent isolated 17,20-lyase deficiency had combined deficiencies of both activities. We studied two patients with clinical and hormonal findings suggestive of isolated 17,20-lyase deficiency. We found two patients homozygous for substitution mutations in CYP17, the gene encoding P450c17. When expressed in COS-1 cells, the mutants retained 17 alpha-hydroxylase activity but had minimal 17,20-lyase activity. Substrate competition experiments suggested that the mutations did not alter the enzyme's substrate-binding capacity, but co-transfection of cells with P450 oxidoreductase, the electron donor used by P450c17, indicated that the mutants had a diminished ability to interact with redox partners. Computer-graphic modelling of P450c17 suggests that both mutations lie in or near the redox-partner binding site, on the opposite side of the haem from the substrate-binding pocket. These mutations alter electrostatic charge distribution in the redox-partner binding site, so that electron transfer for the 17,20-lyase reaction is selectively lost or diverted to uncoupling reactions. These are the first proven cases of isolated 17,20-lyase deficiency, and they demonstrate a novel mechanism for loss of enzymatic activity.
Picture this. Buoyed by a range of new technologies.
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An enzyme-substrate complex involved in bacterial cell wall biosynthesis.
The crystal structure of UDP-N-acetylenolpyruvylglucosamine reductase in the presence of its substrate, enolpyruvyl-UDP-N-acetylglucosamine, has been solved to 2.7 A resolution. This enzyme is responsible for the synthesis of UDP-N-acetylmuramic acid in bacterial cell wall biosynthesis and consequently provides an attractive target for the design of antibacterial agents. The structure reveals a novel flavin binding motif, shows a striking alignment of the flavin with the substrate, and suggests a catalytic mechanism for the reduction of this unusual enol ether.
Synthetic vaccines.
Synthetic vaccines are designed with the help of computer-graphics programs. These displays generated by Arthur J. Olson of the Research Institute of Scripps Clinic show a method whereby parts of a viral protein that are on the surface of a virus, and therefore accessible to antibodies, can be identified. The backbone of the surface domain of the protein on the outer shell of the tomato bushy-stunt virus is displayed (1) on the basis of coordinates determined by Stephen C. Harrison of Harvard University and his colleagues. A single peptide of the protein is picked out in yellow, with the side chains of its component amino acids indicated in atomic detail (2). The peptide is enlarged and a sphere representing a water molecule is displayed (3). The sphere is rolled around the peptide to generate a map of the surface accessible to water (4); it does so, following an algorithm developed by Michael L. Connolly, by placing a dot at each point of its closest contact with the peptide, taking account of the sphere's own van der Waals radius (zone of influence, in effect) and that of each atom of the peptide and the rest of the protein. A similar-dot-surface map is generated to show what parts of the peptide are still accessible to water when three copies of the protein are associated in an array on the surface of the virus (5) and when four such arrays (out of 60) are in position on the outer surface of the virus (6).
Seeing is understanding. Improvements in computer software and hardware are revolutionizing three-dimensional imaging in biology.
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Modified virtual reality technology for treatment of amblyopia.
PURPOSE: The conventional patching/occlusion treatment for amblyopia sometimes gives disappointing results for a number of reasons: it is unpopular, prolonged, frequently resulting in poor or noncompliance, and also disrupts fusion. The aim of this research was to develop a novel virtual-reality (VR)-based display system that facilitates the treatment of amblyopia with both eyes stimulated simultaneously. METHODS: We have adopted a multidisciplinary approach, combining VR expertise with a team of ophthalmologists and orthoptists to develop the Interactive Binocular Treatment (I-BiT) system. This system incorporates adapted VR technology and specially written software providing interactive 2D and 3D games and videos to the patient via a stereo (binocular) display, and a control screen for the clinician. RESULTS: We developed a prototype research system designed for treatment of amblyopia in children. CONCLUSIONS: The result is a novel way to treat amblyopia, which allows binocular treatment. It is interactive, and as it is partially software based, can be adapted to suit the age/ability, and needs of the patient. This means that the treatment can be made captivating and enjoyable. Further research is on-going to determine the efficacy of this new modality in the treatment of amblyopia.
Influence of the digital image display monitor on observer performance.
OBJECTIVE: To assess the influence of the display monitor on observer performance. MATERIAL AND METHODS: Artificial enamel lesions were created in 40 extracted teeth at random using 1/4 and 1/2 round burs. Teeth were mounted in dental stone blocks to simulate a hemidentition. Approximate exposures were recorded at 70 kVp using a Digota (Soredex, Orion Corp, Helsinki, Finland) digital imaging system, calibrated to achieve optimum density. Six dentists rated each image on a five-point scale for the presence or absence of a lesion. Radiographic images were viewed on the following monitors: (1) AlphaScan 711 (Sampo Corp.); (2) Multiscan 17 Se II (Sony Electronics Inc.); (3) DS 2000 (Clinton Electronics Corp.) and (4) Latitude CP Laptop (Dell Computer Corp.). Raters were allowed to magnify and to adjust density and contrast of each image at will. Receiver Operating Characteristic (ROC) analysis was performed and curves were plotted for each image. Data was subjected to repeated measures analysis of variance and ordinal logistic regression to test for significance between variables and to determine odds ratios. RESULTS: Mean ROC curve areas ranged from 0.8728 for the Sampo monitor to 0.8395 for the Sony. Repeated measures analysis of variance showed significant differences between observers (P < 0.0001), lesion size (P < 0.0001), examiner/monitor interaction (P < 0.033) and examiner/block interaction (P < 0.013). However, no significant difference was found between monitors. CONCLUSION: This study suggests that observer performance is independent of the visual characteristics of the display monitor.
Coypu insulin. Primary structure, conformation and biological properties of a hystricomorph rodent insulin.
Insulin from a hystricomorph rodent, coypu (Myocaster coypus), was isolated and purified to near homogeneity. Like the other insulins that have been characterized in this Suborder of Rodentia, coypu insulin also exhibits a very low (3%) biological potency, relative to pig insulin, on lipogenesis in isolated rat fat-cells. The receptor-binding affinity is significantly higher (5-8%) in rat fat-cells, in rat liver plasma membranes and in pig liver cells, indicating that the efficacy of coypu insulin on receptors is about 2-fold lower than that of pig insulin. The primary structures of the oxidized A- and B-chains were determined, and our sequence analysis confirms a previous report [Smith (1972) Diabetes 21, Suppl. 2, 457-460] that the C-terminus of the A-chain is extended by a single residue (i.e. aspartate-A22), in contrast with most other insulin sequences, which terminate at residue A21. In spite of a large number of amino acid substitutions (relative to mammalian insulins), computer-graphics model-building studies suggest a similar spatial arrangement for coypu insulin to that for pig insulin. The substitution of the zinc-co-ordinating site (B10-His----Gln) along with various substitutions on the intermolecular surfaces involved in the formation of higher aggregates are consistent with the observation that this insulin is predominantly 'monomeric' in nature. The c.d. spectrum of coypu insulin is relatively similar to those of casiragua insulin and of bovine insulin at low concentration.
Engineering N-terminal domain of tissue inhibitor of metalloproteinase (TIMP)-3 to be a better inhibitor against tumour necrosis factor-alpha-converting enzyme.
We previously reported that full-length tissue inhibitor of metalloproteinase-3 (TIMP-3) and its N-terminal domain form (N-TIMP-3) displayed equal binding affinity for tissue necrosis factor-alpha (TNF-alpha)-converting enzyme (TACE). Based on the computer graphic of TACE docked with a TIMP-3 model, we created a number of N-TIMP-3 mutants that showed significant improvement in TACE inhibition. Our strategy was to select those N-TIMP-3 residues that were believed to be in actual contact with the active-site pockets of TACE and mutate them to amino acids of a better-fitting nature. The activities of these mutants were examined by measuring their binding affinities (K(app)(i)) and association rates (k(on)) against TACE. Nearly all mutants at position Thr-2 exhibited slightly impaired affinity as well as association rate constants. On the other hand, some Ser-4 mutants displayed a remarkable increase in their binding tightness with TACE. In fact, the binding affinities of several mutants were less than 60 pM, beyond the sensitivity limits of fluorimetric assays. Further studies on cell-based processing of pro-TNF-alpha demonstrated that wild-type N-TIMP-3 and one of its tight-binding mutants, Ser-4Met, were capable of inhibiting the proteolytic shedding of TNF-alpha. Furthermore, the Ser-4Met mutant was also significantly more active (P<0.05) than the wild-type N-TIMP-3 in its cellular inhibition. Comparison of N-TIMP-3 and full-length TIMP-3 revealed that, despite their identical TACE-interaction kinetics, the latter was nearly 10 times more efficient in the inhibition of TNF-alpha shedding, with concomitant implications for the importance of the TIMP-3 C-terminal domain in vivo.
A prototype computer system for de novo protein design.
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The role of computer imaging in facial plastic surgery consultation: a clinical study.
The influence of using computerized visual communication on preoperative communication between the surgeon and the patient was analysed. This was a retrospective study based on a questionnaire completed by 50 patients who had undergone various facial plastic surgical procedures. Prediction tracings and postoperative slides were compared by the surgeon. The role of computer imaging in communication between doctor and patient, as well as the patient-doctor relationship and trust in the judgement of the doctor was considered to be positive by most of the patients. The vast majority of patients thought computer imaging should be a routine part of preoperative evaluation. Both the surgeon and the patients agreed that the representative value of prediction tracing was about 80%. In view of the possible positive influences on communication and relationship in the preoperative phase, computer imaging may help to provide a clear and realistic preoperative informed consent.
The effectiveness of a new algorithm on a three-dimensional finite element model construction of bone trabeculae in implant biomechanics.
More validity of finite element analysis (FEA) in implant biomechanics requires element downsizing. However, excess downsizing needs computer memory and calculation time. To evaluate the effectiveness of a new algorithm established for more valid FEA model construction without downsizing, three-dimensional FEA bone trabeculae models with different element sizes (300, 150 and 75 micron) were constructed. Four algorithms of stepwise (1 to 4 ranks) assignment of Young's modulus accorded with bone volume in the individual cubic element was used and then stress distribution against vertical loading was analysed. The model with 300 micron element size, with 4 ranks of Young's moduli accorded with bone volume in each element presented similar stress distribution to the model with the 75 micron element size. These results show that the new algorithm was effective, and the use of the 300 micron element for bone trabeculae representation was proposed, without critical changes in stress values and for possible savings on computer memory and calculation time in the laboratory.
APPENDIX. A computer model of the Land Mondrian retinex experiment.
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Hierarchy of metabolic compounds based on their synthesising capacity.
The concept of scopes is applied to analyse large metabolic networks. Scopes are defined as sets of metabolites that can be synthesised by a metabolic network when it is provided with given seeds (Sets of initial metabolic compounds). Thus, scopes represent synthesising capacities of the seeds in the network. A hierarchy is discussed in the sense that compounds, which are part of the scope of another compound, possess scopes themselves that are subsets of the former scope. This hierarchy is analysed by means of a directed acyclic graph. Using a simple chemical model, it is found that this hierarchy contains specific structures that can, to a large extent, be explained by the chemical composition of the participating compounds. In this way, it represents a new kind of map of metabolic networks, arranging the metabolic compounds according to their chemical capacity.
[Assessment of the optical image quality of the eye using raytracing technique of corneal topography data].
BACKGROUND: Optical aberrations in the optical system may downgrade image quality and cannot be fully compensated by spherocylindrical glasses. The subjectively evaluated visual acuity may be significantly reduced. The purpose of this study was to calculate the image forming properties of the eye using a spotlight source or alternatively extended objects. METHODS: A convex and first derivative continuous (C1) surface from the rough height data of the anterior corneal surface (TMS-1, Tomey, Erlangen) or the anterior and posterior corneal surface (Orbscan, Orbtec, USA) was calculated by means of an interpolating subdivision scheme (modified Butterfly algorithm). The characteristics of the residual refractive surfaces were used according to Navarro's eye model. The focal distance was calculated from the exact raytracing calculation (Snellius' law) of the point-spread function by minimising the variance of the point-spread function. The diffraction property of the aperture stop was implemented with a transmission characteristic according to a radially symmetrical Bessel function within the entrance pupil. The algorithm was realised with a C code on the LINUX platform and applied to a normal eye (example 1, TMS-1), an eye with severe keratoconus (example 2, TMS-1) and an eye with corneal scars (example 3, Orbscan). RESULTS: The focal distance in example 1 (22.5 mm, 22.6 mm, and 22.8 mm) increased with the pupil diameter (2 mm, 3 mm, and 5 mm). The variance of the approximately radially symmetrical point-spread function in the focal plane attained a minimum value with a pupil size of 3 mm (0.164, 0.104, and 0.230). In example 2, the focal distance changed inconclusively (21.1 mm, 21.0 mm, and 21.3 mm) with the pupil size (2 mm, 3 mm, and 5 mm). The variance of the markedly asymmetrical point-spread function in the focal plane was systematically higher compared to the values of example 1 and reached a minimum value with a pupil size of 3 mm (0.255, 0.224, and 0.371). The imaging of the sinus-modulated pattern is anisotropic due to the asymmetry of the point-spread function. In example 3, the focal distance (22.3 mm, 22.3 mm, and 22.5 mm) did not change systematically with the pupil size (2 mm, 3 mm, and 5 mm). The variance of the nearly radially symmetrical point-spread function changed only marginally between pupil sizes of 2 mm and 3 mm (0.231, 0.239, and 0.338). CONCLUSIONS: Raytracing of corneal topography height data based on refined eye models with the option of auto-focussing has the potential to trace the optical resolution of the eye for arbitrary objects. Further studies on contrast sensitivity and the conversion of the real image to a perceived image by the retina and brain are required for complete modeling of subjective visual acuity.
Virtual simulation of neuroendoscopic procedures: early clinical experience with ventricular lesions.
BACKGROUND: Virtual endoscopy (VE) is a new and promising imaging technology. Applied to neuroendoscopy it allows preoperative simulation of a procedure and evaluation of the individual intraventricular anatomy in selected cases. Along with neuronavigation and real time intraoperative imaging, VE is expected to improve the safety and efficacy of neuroendoscopic procedures. PATIENTS AND METHODS: Between April 2003 and February 2004 VE simulation was performed in 13 randomly selected patients subjected to endoscopic procedures. Pathological entities included 4 cases with aqueduct stenosis, 4 with suprasellar arachnoid cysts, 2 tumors of the posterior third ventricle, 1 colloid cyst, 1 hyperplasia of the choroid plexus and 1 case with multiloculated hydrocephalus due to intraventricular septations. In 8 patients VE was accomplished preoperatively, in another 5 it was done after the operation, using data sets from neuronavigation imaging planning in 4 patients, and in one case using postoperative imaging studies. T (1)-weighted 3D image sets were acquired on a 1.5 T GE Genesis SIGNA MR scanner and VE reconstruction was performed using the General Electric Navigator software. The VE images were compared with the real images obtained during the endoscopic procedures and evaluated for their impact on the planning of the operative approach. RESULTS: VE implementation succeeded in all 13 patients. Major neuroanatomic reference structures were easily recognizable in all cases. Membranous structures such as the thinned floor of the third ventricle or cyst walls were identifiable in only 46 % of the cases. In 6 cases (46 %) VE showed anatomical variants and details relevant for the endoscopic procedure that were not identified on conventional MR images. CONCLUSIONS: VE has proved to be an important adjunct to the preoperative planning of neuroendoscopic procedures and its routine application is suggested.
[Possibilities of 3-dimensional ultrasound imaging].
This is a report on the clinical use of sonographic imaging. Experimental studies have shown that parallel sonographic sections are available only in vitro, which means no in-vivo three-dimensional reconstruction is possible as it is with magnetic resonance and computed tomography. The idea that a coordinated sequence of sections can be obtained by spatial rotation of the plane of sound so that the individual sections differ from one another by defined angular distances, enables three-dimensional reconstruction of sonographic in-vivo sections. A transducer was constructed that enables the production of sonographic sections rotating around a fixed center of rotation. Its clinical usefulness was tried and confirmed in the imaging of early pregnancies, benign and malignant carcinomas of the breast, and in imaging a gallbladder with a solitary gallstone. Three-dimensional imaging can be achieved either as a ring-shaped structure or with a continuous surface. This points to the possibility of diagnosing malformations early in pregnancy if the number of sections is sufficiently high. In tumour imaging the malignant tumour seems to be clearly distinguishable from the benign one; three-dimensional diagnosis is likely to furnish important additional criteria in diagnosis. Further clinical studies will have to verify this.