The applicability of radiophotoluminescence dosemeter (RPLD) for measuring medical radiation (MR) doses.

The applicability of radiophotoluminescence dosimetry was determined by assessing various radiophotoluminescence dosemeter (RPLD) properties for measuring medical radiation doses from radiation sources of a continuous spectrum. The RPLD was found to be accurate for measuring doses in diagnostics (50-125 keV) and radiation therapy (6, 10 and 18 MV photons, 6 and 15 MeV electrons). The RPLD shows excellent dose linearity (R(2) > 0.99), reproducibility and batch uniformity, and minimal fading and accurate accumulated dose measurement. The dosemeter material is independent of photon energy in the diagnostic range; however, the dosemeter requires additional calibration in the mammography energy range and also for accurate dose measurement with photon or electron energies in radiation therapy. RPLD measurements with a tin filter show considerable angular dependence at angles exceeding 50° between the photon beam and the normal to the long axis of the dosemeter. The RPLD measurement accuracy at high doses can be improved with optimised pre-heating schemes.

Structure-function studies of a Melanocarpus albomyces laccase suggest a pathway for oxidation of phenolic compounds.

Melanocarpus albomyces laccase crystals were soaked with 2,6-dimethoxyphenol, a common laccase substrate. Three complex structures from different soaking times were solved. Crystal structures revealed the binding of the original substrate and adducts formed by enzymatic oxidation of the substrate. The dimeric oxidation products were identified by mass spectrometry. In the crystals, a 2,6-dimethoxy-p-benzoquinone and a C-O dimer were observed, whereas a C-C dimer was the main product identified by mass spectrometry. Crystal structures demonstrated that the substrate and/or its oxidation products were bound in the pocket formed by residues Ala191, Pro192, Glu235, Leu363, Phe371, Trp373, Phe427, Leu429, Trp507 and His508. Substrate and adducts were hydrogen-bonded to His508, one of the ligands of type 1 copper. Therefore, this surface-exposed histidine most likely has a role in electron transfer by laccases. Based on our mutagenesis studies, the carboxylic acid residue Glu235 at the bottom of the binding site pocket is also crucial in the oxidation of phenolics. Glu235 may be responsible for the abstraction of a proton from the OH group of the substrate and His508 may extract an electron. In addition, crystal structures revealed a secondary binding site formed through weak dimerization in M. albomyces laccase molecules. This binding site most likely exists only in crystals, when the Phe427 residues are packed against each other.

A near atomic resolution structure of a Melanocarpus albomyces laccase.

We have solved a crystal structure from Melanocarpus albomyces laccase expressed in the filamentous fungus Trichoderma reesei (rMaL) at 1.3A resolution by using synchrotron radiation at 100K. At the moment, this is the highest resolution that has been attained for any multicopper oxidase. The present structure confirmed our earlier proposal regarding the dynamic behaviour of the copper cluster. Thermal ellipsoids of copper atoms indicated movements of trinuclear site coppers. The direction of the type-3 copper motion was perpendicular to the type-2 copper. In addition, the structure at 1.3A resolution allowed us to describe important solvent cavities of the enzyme and the structure is also compared with other known multicopper oxidases. T2 and T3 solvent cavities, and a putative SDS-gate, formed by Ser142, Ser510 and the C-terminal Asp556 of rMaL, are described. We also observed a 2-oxohistidine, an oxidized histidine, possibly caused by a metal-catalysed oxidation by the trinuclear site coppers. To our knowledge, this is the first time that 2-oxohistidine has been observed in a protein crystal structure.

The stress on the anterior lens surface during human in vivo accommodation.

PURPOSE: To determine the qualitative change in stress on the lens capsule during in vivo human accommodation. METHODS: Nine subjects (mean age: 30 years; range: 25-38 years) were studied, each of whom had undergone a phakic refractive intraocular lens (PRL) surgical procedure. The change, during accommodation, of stress on the surface of the anterior lens capsule (ALS) was determined by employing high-resolution anterior segment optical coherence tomography (OCT). This was done by comparing the ratio of the intensity of the image from the anterior surface of the natural lens (ALS) to the images of the anterior corneal surface (ACS), posterior corneal surface (PCS) and the posterior surface of the phakic refractive intraocular lens (PPRLS) before and during accommodation. RESULTS: The intensities of the OCT images of the ACS and PPRLS did not significantly change during accommodation when compared with their respective baselines, while the intensity ratios: ALS/ACS, ALS/PCS and ALS/PPRLS all significantly increased during accommodation (p<0.01). CONCLUSIONS: The stress on the anterior lens capsule is increased during in vivo human accommodation. This observation is consistent with a mechanism of accommodation in which zonular tension increases with accommodation, which is opposite to the predictions of the Helmholtz theory.

Model-based mutagenesis to improve the enantioselective fractionation properties of an antibody.

The binding affinity and specificity of recombinant antibodies can be modified by site-directed mutagenesis. Here we have used molecular modelling of the variable domains of an enantiospecific antibody fragment to fine-tune its affinity so it is more suitable for the fractionation of the drug enantiomers. We have shown earlier that the Fab fragment of this antibody specifically recognizes one enantiomer from the racemic mixture of a medical drug and that it can be used for the fractionation of these enantiomers by affinity chromatography. However, the affinity was unnecessarily high, requiring harsh elution conditions to release the bound enantiomer. Thus, the continuous use of the antibody affinity columns was impossible. We made a homology model of the antibody and designed mutations to the antigen-binding site to decrease the affinity. Four out of five point mutations showed decreased affinity for the hapten. Two of the mutations were also combined to construct a double mutant. The affinity columns made using one of the single mutants with lowered affinity and the double mutant were capable of multiple rounds of enantioseparation.

Apparent diffusion coefficients and T2 relaxation time measurements to evaluate disc degeneration. A quantitative MR study of young patients with previous vertebral fracture.

PURPOSE: To assess the suitability of apparent diffusion coefficient (ADC) measurements to evaluate degeneration processes of the vertebral disc and to compare the results with T2 relaxation time measurements in both degenerated and normal intervertebral discs. MATERIAL AND METHODS: Fourteen young patients (8.8-20.8 years old) who had had a vertebral compression fracture at least 1 year earlier, underwent MR studies with diffusion imaging in three orthogonal directions and T2 relaxation time measurements. ADC values and T2 relaxation times of both degenerated and normal intervertebral discs were compared to the values of 20 healthy young asymptomatic control subjects. RESULTS: In the degenerated discs of patients, the ADCx and ADCy values were decreased compared to earlier determined values of healthy controls. ADC values in the z-direction in degenerated discs did not differ significantly from the values of controls. T2 relaxation times were shorter in the degenerated discs of patients compared to the values of controls. The greatest changes in both these values were observed in degenerated discs followed by discs with normal signal intensity adjacent to primary trauma area and secondary trauma area. CONCLUSION: We suggest that decreased ADC values reflect the lost integrity of the intervertebral disc. ADC measurements at MR may prove sensitive depicting of early degenerative changes in vertebral discs.

Engineering of a glycosidase Family 7 cellobiohydrolase to more alkaline pH optimum: the pH behaviour of Trichoderma reesei Cel7A and its E223S/ A224H/L225V/T226A/D262G mutant.

The crystal structures of Family 7 glycohydrolases suggest that a histidine residue near the acid/base catalyst could account for the higher pH optimum of the Humicola insolens endoglucanase Cel7B, than the corresponding Trichoderma reesei enzymes. Modelling studies indicated that introduction of histidine at the homologous position in T. reesei Cel7A (Ala(224)) required additional changes to accommodate the bulkier histidine side chain. X-ray crystallography of the catalytic domain of the E223S/A224H/L225V/T226A/D262G mutant reveals that major differences from the wild-type are confined to the mutations themselves. The introduced histidine residue is in plane with its counterpart in H. insolens Cel7B, but is 1.0 A (=0.1 nm) closer to the acid/base Glu(217) residue, with a 3.1 A contact between N(epsilon2) and O(epsilon1). The pH variation of k(cat)/K(m) for 3,4-dinitrophenyl lactoside hydrolysis was accurately bell-shaped for both wild-type and mutant, with pK(1) shifting from 2.22+/-0.03 in the wild-type to 3.19+/-0.03 in the mutant, and pK(2) shifting from 5.99+/-0.02 to 6.78+/-0.02. With this poor substrate, the ionizations probably represent those of the free enzyme. The relative k(cat) for 2-chloro-4-nitrophenyl lactoside showed similar behaviour. The shift in the mutant pH optimum was associated with lower k(cat)/K(m) values for both lactosides and cellobiosides, and a marginally lower stability. However, k(cat) values for cellobiosides are higher for the mutant. This we attribute to reduced non-productive binding in the +1 and +2 subsites; inhibition by cellobiose is certainly relieved in the mutant. The weaker binding of cellobiose is due to the loss of two water-mediated hydrogen bonds.

Cellulose-binding domains promote hydrolysis of different sites on crystalline cellulose.

The cohesin-dockerin interaction in Clostridium thermocellum cellulosome mediates the tight binding of cellulolytic enzymes to the cellulosome-integrating protein CipA. Here, this interaction was used to study the effect of different cellulose-binding domains (CBDs) on the enzymatic activity of C. thermocellum endoglucanase CelD (1,4-beta-d endoglucanase, EC) toward various cellulosic substrates. The seventh cohesin domain of CipA was fused to CBDs originating from the Trichoderma reesei cellobiohydrolases I and II (CBD(CBH1) and CBD(CBH2)) (1,4-beta-d glucan-cellobiohydrolase, EC), from the Cellulomonas fimi xylanase/exoglucanase Cex (CBD(Cex)) (beta-1,4-d glucanase, EC), and from C. thermocellum CipA (CBD(CipA)). The CBD-cohesin hybrids interacted with the dockerin domain of CelD, leading to the formation of CelD-CBD complexes. Each of the CBDs increased the fraction of cellulose accessible to hydrolysis by CelD in the order CBD(CBH1) < CBD(CBH2) approximately CBD(Cex) < CBD(CipA). In all cases, the extent of hydrolysis was limited by the disappearance of sites accessible to CelD. Addition of a batch of fresh cellulose after completion of the reaction resulted in a new burst of activity, proving the reversible binding of the intact complexes despite the apparent binding irreversibility of some CBDs. Furthermore, burst of activity also was observed upon adding new batches of CelD-CBD complexes that contained a CBD differing from the first one. This complementation between different CBDs suggests that the sites made available for hydrolysis by each of the CBDs are at least partially nonoverlapping. The only exception was CBD(CipA), whose sites appeared to overlap all of the other sites.

Apparent diffusion coefficient in thoracolumbar intervertebral discs of healthy young volunteers.

Apparent diffusion coefficient values (ADC) of healthy intervertebral discs of young volunteers in the thoracolumbar spine were determined using a single-shot EPI sequence. ADC(z) was in the lumbar spine slightly higher than ADC(x) or ADC(y). In vivo diffusion measurements of intervertebral discs may offer a novel diagnostic tool to evaluate disc diseases in early phases.

Characterization of Trichoderma reesei cellobiohydrolase Cel7A secreted from Pichia pastoris using two different promoters.

Heterologous expression of T. reesei cellobiohydrolase Cel7A in a methylotrophic yeast Pichia pastoris was tested both under the P. pastoris alcohol oxidase (AOX1) promoter and the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter in a fermentor. Production of Cel7A with the AOX1 promoter gave a better yield, although part of the enzyme expressed was apparently not correctly folded. Cel7A expressed in P. pastoris is overglycosylated at its N-glycosylation sites as compared to the native T. reesei protein, but less extensive than Cel7A expressed in Saccharomyces cerevisiae. The k(cat) and K(m) values for the purified protein on soluble substrates are similar to the values found for the native Trichoderma Cel7A, whereas the degradation rate on crystalline substrate (BMCC) is somewhat reduced. The measured pH optimum also closely resembles that of purified T. reesei Cel7A. Furthermore, the hyperglycosylation does not affect the thermostability of the enzyme monitored with tryptophane fluorescence and activity measurements. On the other hand, CD measurements indicate that the formation of disulfide bridges is an important step in the correct folding of Cel7A and might explain the difficulties encountered in heterologous expression of T. reesei Cel7A. The constitutive GAP promoter expression system of P. pastoris is nevertheless well suited for activity screening of cellulase activities in microtiter plates. With this type of screening method a faster selection of site-directed and random mutants with, for instance, an altered optimum pH is possible, in contrast to the homologous T. reesei expression system.

Initial experience with a wireless personal digital assistant as a teleradiology terminal for reporting emergency computerized tomography scans.

A new type of terminal device, a wireless personal digital assistant (PDA) based on a GSM digital cellular phone, was used to transmit computerized tomography scans of 21 patients to a neuroradiologist. All transmitted images were suitable for a preliminary consultation and in one case a final report could be made. In 18 cases the findings were compatible with the reference film reading performed later and in three cases there were minor differences of no clinical importance. Transmission of a single image lasted 1 min 30 s and the transmission of a complete brain scan (14 images) took on average 21 min. The total process of transmission and interpretation of a brain examination series took on average 40 min. In this pilot study the neuroradiologist gained essential information in 24% of the cases and beneficial information in 62%. The neuroradiologist considered that the image consultation saved a hospital visit in 15 cases (71%). Although PDA technology is at an early stage of development and has numerous limitations, it is likely that future technical improvements will allow easier clinical consultations for neurosurgeons and neurologists.

Hydrolyses of alpha- and beta-cellobiosyl fluorides by Cel6A (cellobiohydrolase II) of Trichoderma reesei and Humicola insolens.

We have measured the hydrolyses of alpha- and beta-cellobiosyl fluorides by the Cel6A [cellobiohydrolase II (CBHII)] enzymes of Humicola insolens and Trichoderma reesei, which have essentially identical crystal structures [Varrot, Hastrup, Schülein and Davies (1999) Biochem. J. 337, 297-304]. The beta-fluoride is hydrolysed according to Michaelis-Menten kinetics by both enzymes. When the approximately 2.0% of beta-fluoride which is an inevitable contaminant in all preparations of the alpha-fluoride is hydrolysed by Cel7A (CBHI) of T. reesei before initial-rate measurements are made, both Cel6A enzymes show a sigmoidal dependence of rate on substrate concentration, as well as activation by cellobiose. These kinetics are consistent with the classic Hehre resynthesis-hydrolysis mechanism for glycosidase-catalysed hydrolysis of the 'wrong' glycosyl fluoride for both enzymes. The Michaelis-Menten kinetics of alpha-cellobiosyl fluoride hydrolysis by the T. reesei enzyme, and its inhibition by cellobiose, previously reported [Konstantinidis, Marsden and Sinnott (1993) Biochem. J. 291, 883-888] are withdrawn. (1)H NMR monitoring of the hydrolysis of alpha-cellobiosyl fluoride by both enzymes reveals that in neither case is alpha-cellobiosyl fluoride released into solution in detectable quantities, but instead it appears to be hydrolysed in the enzyme active site as soon as it is formed.

Crystallographic evidence for substrate ring distortion and protein conformational changes during catalysis in cellobiohydrolase Ce16A from trichoderma reesei.

BACKGROUND: Cel6A is one of the two cellobiohydrolases produced by Trichoderma reesei. The catalytic core has a structure that is a variation of the classic TIM barrel. The active site is located inside a tunnel, the roof of which is formed mainly by a pair of loops. RESULTS: We describe three new ligand complexes. One is the structure of the wild-type enzyme in complex with a nonhydrolysable cello-oligosaccharide, methyl 4-S-beta-cellobiosyl-4-thio-beta-cellobioside (Glc)(2)-S-(Glc)(2), which differs from a cellotetraose in the nature of the central glycosidic linkage where a sulphur atom replaces an oxygen atom. The second structure is a mutant, Y169F, in complex with the same ligand, and the third is the wild-type enzyme in complex with m-iodobenzyl beta-D-glucopyranosyl-beta(1,4)-D-xylopyranoside (IBXG). CONCLUSIONS: The (Glc)(2)-S-(Glc)(2) ligand binds in the -2 to +2 sites in both the wild-type and mutant enzymes. The glucosyl unit in the -1 site is distorted from the usual chair conformation in both structures. The IBXG ligand binds in the -2 to +1 sites, with the xylosyl unit in the -1 site where it adopts the energetically favourable chair conformation. The -1 site glucosyl of the (Glc)(2)-S-(Glc)(2) ligand is unable to take on this conformation because of steric clashes with the protein. The crystallographic results show that one of the tunnel-forming loops in Cel6A is sensitive to modifications at the active site, and is able to take on a number of different conformations. One of the conformational changes disrupts a set of interactions at the active site that we propose is an integral part of the reaction mechanism.

A comparative study of two retaining enzymes of Trichoderma reesei: transglycosylation of oligosaccharides catalysed by the cellobiohydrolase I, Cel7A, and the beta-mannanase, Man5A.

HPLC, MALDI-TOF MS and NMR spectroscopy were used to investigate the hydrolysis of cello- and mannooligosaccharides by Cel7A and Man5A from Trichoderma reesei. The experimental progress curves were analysed by fitting the numerically integrated kinetic equations, which provided cleavage patterns for oligosaccharides. This data evaluation procedure accounts for product inhibition and avoids the initial slope approximation. In addition, a transglycosylation step had to be included in the model to reproduce the experimental progress curves. For the hydrolysis of manno-oligosaccharides, Man4-6, by Man5A no mannose was detected at the beginning of the reaction showing that only the internal linkages are hydrolysed. For cellotriose and cellotetraose hydrolysis by Cel7A, the main product is cellobiose and glucose is released from the non-reducing end of the substrate. Intermediary products longer than the substrates were detected by MALDI-TOF MS when oligosaccharides (Glc4-6 or Man4-6) were hydrolysed by either Cel7A or Man5A. Interestingly, two distinct transglycosylation pathways could be observed. Cel7A produced intermediates that are one unit longer than the substrate, whereas Man5A produced intermediates that are two units longer than the substrate.

Tryptophan 272: an essential determinant of crystalline cellulose degradation by Trichoderma reesei cellobiohydrolase Cel6A.

Trichoderma reesei cellobiohydrolase Cel6A (formerly CBHII) has a tunnel shaped active site with four internal subsites for the glucose units. We have predicted an additional ring stacking interaction for a sixth glucose moiety with a tryptophan residue (W272) found on the domain surface. Mutagenesis of this residue selectively impairs the enzyme function on crystalline cellulose but not on soluble or amorphous substrates. Our data shows that W272 forms an additional subsite at the entrance of the active site tunnel and suggests it has a specialised role in crystalline cellulose degradation, possibly in guiding a glucan chain into the tunnel.

Digital wireless radiology consultations with a portable computer.

A wireless system for radiological subspecialist consultation based on a portable personal computer and a GSM cellular phone was tested. A link with secure access to the hospital image network was built. A total of 68 emergency computerized tomography (CT) examinations were transmitted. Transmission time via GSM for a single CT image was 1 min and for a complete head scan was 18 min. The transmitted images were acceptable for final diagnosis in 72% of the cases, the rest being acceptable for preliminary diagnosis. The diagnosis from the transmitted images did not change after a later review of the original images in 97% of cases. The wireless link saved a hospital visit by the senior radiologist in 24% of cases. The results show that a remote consultation link can be built with readily available technology and that the technique is useful in radiological subspecialist consultations for CT images.

Image feature based automatic correction of low-frequency spatial intensity variations in MR images.

An automatic method of compensating for low-frequency variations in magnetic resonance images is presented. Small variations within a tissue type are modelled and a correction function is generated. The method is based completely on image features and does not need a phantom or user interaction to generate the compensation function. This image correction simplifies digital image analysis and may enhance clinical evaluation. As a result, the correction technique reduces inhomogeneity and improves contrast. Our results show that the radiofrequency response variation of coils can be reduced. The segmentation process, even with a simple threshold method, produces more reliable results when corrected images are used. The presented method is most useful for images acquired in the sagital and coronal planes with circular local coils, or using surface coils, e.g., spine coils.

Computed tomography data based rapid prototyping model of the temporal bone before cochlear implant surgery.

Rapid prototyping (RP) technique allows automatic fabrication of 3D model parts. This method was applied to make a temporal bone model before cochlear implant surgery. A helical CT scan is used to acquire high resolution data from the middle and the inner ear of the patient. From the scanning data bone structures and soft tissues can be separated because their different grayscale pixel values. By using a guided image processing tool the desired parts of the anatomy can be extracted and 3D data created. The segmented data are processed to the form suitable for creating a high accuracy RP model. The RP model is made in the stereolithography (SLA) process by means of a computer guided HeCd laser beam inducing polymerisation of acrylic solution as it passes layer by layer over the surface of the polymer solution. In this prototype model the anatomy of the temporal bone can be clearly visualised, including, e.g., mastoid cells, tympanic cavity, bony canal of facial nerve, and round and oval windows. The inner ear spaces including vestibule, semicircular canals and cochlear turn are also shaped. The transparent acrylic material allows bonelike mechanical handling. The RP model can be dissected and used in individual surgical planning and simulation prior to cochlear implantation.

Immunoaffinity Chromatographic Purification of Cellobiohydrolase II Mutants from Recombinant Trichoderma reesei Strains Devoid of Major Endoglucanase Genes

Efficient purification of Trichoderma reesei cellobiohydrolase II (CBHII) requires the use of affinity chromatography based on a substrate analogue. Due to altered substrate binding, the purification of many active-site mutants of CBHII from the complex fungal culture media represents a considerable challenge. Here we describe a combination of two approaches to facilitate the purification: the first is based on the construction of novel engineered T. reesei strains devoid of the major contaminating endoglucanases, and the second uses immunoaffinity chromatography as the final purification step. Two different procedures for the preparation of the antibody matrix were tested. Crosslinking of the monoclonal antibody to Protein G matrix instead of the conventional immobilization via cyanogen bromide increased the binding efficiency. Three different active-site mutants of CBHII bound to the immunoaffinity column in neutral pH and were eluted in pH 2.7. The purity of the CBHII mutant preparations was tested using small chromophoric substrates and hydroxyethyl cellulose, which are hydrolyzed by many other cellulases but not by CBHII. The immunoaffinity column purified the CBHII mutants over 800-fold in a single step and resulted in homogeneous protein preparations free of proteolytically cleaved forms of CBHII. The use of the double replacement T. reesei production strains, especially the one lacking the genes coding for both the endogeneous CBHII and the endoglucanase II (EGII), helped to reduce the total endoglucanase activity in the preparations.