Dimeric structure of the coxsackievirus and adenovirus receptor D1 domain at 1.7 A resolution.

BACKGROUND: The coxsackievirus and adenovirus receptor (CAR) comprises two extracellular immunoglobulin domains, a transmembrane helix and a C-terminal intracellular domain. The amino-terminal immunoglobulin domain (D1) of CAR is necessary and sufficient for adenovirus binding, whereas the site of coxsackievirus attachment has not yet been localized. The normal cellular role of CAR is currently unknown, although CAR was recently proposed to function as a homophilic cell adhesion molecule. RESULTS: The human CAR D1 domain was bacterially expressed and crystallized. The structure was solved by molecular replacement using the structure of CAR D1 bound to the adenovirus type 12 fiber head and refined to 1.7 A resolution, including individual anisotropic temperature factors. The two CAR D1 structures are virtually identical, apart from the BC, C"D, and FG loops that are involved both in fiber head binding and homodimerization in the crystal. Analytical equilibrium ultracentrifugation shows that a dimer also exists in solution, with a dissociation constant of 16 microM. CONCLUSIONS: The CAR D1 domain forms homodimers in the crystal using the same GFCC'C" surface that interacts with the adenovirus fiber head. The homodimer is very similar to the CD2 D1-CD58 D1 heterodimer. CAR D1 also forms dimers in solution with a dissociation constant typical of other cell adhesion complexes. These results are consistent with reports that CAR may function physiologically as a homophilic cell adhesion molecule in the developing mouse brain. Adenovirus may thus have recruited an existing and conserved interaction surface of CAR to use for its own cell attachment.

The glutamine-rich domain of the Drosophila GAGA factor is necessary for amyloid fibre formation in vitro, but not for chromatin remodelling.

The Drosophila GAGA factor binds specifically to the sequence GAGAG, and synergises with nucleosome remodelling factor to remodel chromatin in vitro. It consists of an N-terminal domain (POZ/BTB) which mediates protein-protein interactions, a central region which contains the DNA-binding domain, and a C-terminal glutamine-rich region. It is shown that the glutamine-rich region is responsible for the formation of fibres in vitro which, on the basis of their tinctorial properties and CD spectra, may be classified as amyloid fibres. A large structural change, probably resulting in beta-sheet structure, is observed upon fibre formation. Mutants containing the central region, either alone or together with the glutamine-rich region, are largely lacking in secondary structure but they bind specifically to the cognate DNA and are able to remodel chromatin in vitro. Consequently, neither the N-terminal domain nor the C-terminal glutamine-rich regions of the GAGA factor are necessary for chromatin remodelling in vitro.

Automatic correction for phase shifts, frequency shifts, and lineshape distortions across a series of single resonance lines in large spectral data sets.

A new model-free method is presented that automatically corrects for phase shifts, frequency shifts, and additional lineshape distortions of one single resonance peak across a series of in vivo NMR spectra. All separate phase and frequency variations are quickly and directly derived from the common lineshape in the data set using principal component analysis and linear regression. First, the new approach is evaluated on simulated data in order to quantitatively assess the phase and frequency shifts which can be removed by the proposed correction procedure. Subsequently, the value of the method is demonstrated on in vivo (31)P NMR spectra from skeletal muscle of the hind leg of the mouse focusing on the phosphocreatine resonance which is distorted by the experimental procedure. Phase shifts, frequency shifts, and lineshape distortions with respect to the common lineshape in the spectral data set could successfully be removed.

In situ light dosimetry during whole bladder wall photodynamic therapy: clinical results and experimental verification.

Photodynamic therapy (PDT) using Photofrin IIR (PII) as a photosensitizer is currently being evaluated as a new treatment modality for superficial bladder cancer. An optimum therapeutic ratio requires uniform illumination of the whole bladder wall and accurate light dosimetry. The first clinical light dosimetry results (16 patients) are reported, obtained using a system which allows in situ measurement and control of the light fluence at the bladder wall. The true light fluence at the bladder wall (i.e. non-scattered incident light plus scattered light) appeared to be on the average a factor beta = 4.8 +/- 1.2 (mean +/- SD) larger than the non-scattered incident light fluence. The latter is often, but incorrectly, used in reporting light fluence. The factor beta varied between patients with extremes of 2.5 and 7.1. Because such large variations were unexpected, but may have significant clinical consequences, experiments in plastic bladder models were performed to study separately the various factors (e.g. bladder shape, air bubble) affecting the dosimetry in clinical treatments. The results imply that the clinical variations are most likely to be the result of variations in optical properties of the bladder wall mucosa, probably due to the disease and prior treatments. If light dosage is based on non-scattered light only (without in situ light dosimetry, according to a current clinical protocol) the present results indicate variations in the true (total) light fluence between patients by a factor of at least 2. At the least this may cause unnecessary discomfort to a number of patients.

A comparison of three sampling techniques to estimate the population size of caterpillars in trees.

Three sampling techniques commonly used to estimate the population size of caterpillars and sawfly larvae in trees (branch samples, frass production, water basins), were compared with respect to sampling error and economic costs. At the level of tree populations (e.g. forests), on an arbitrary date, the mean caterpillar intensity per tree (expressed in numbers of larvae or their biomass per 100 shoots) was predicted from the mean frass production per tree (expressed in mg frass per m2 forest floor per day). At the level of the single tree, the frass production on an arbitrary date was related to the population intensity, but, due to the large sampling error, did not provide an accurate prediction. Summing the frass produced over the whole season reduced this error and predicted the seasonal abundance of larvae in single trees, estimated as their maximum intensity or their density (numbers of larvae or their biomass per m2 forest floor). The maximum population intensity was not related to the population density. The sampling techniques suffer from large errors unrelated to larval abundance. The main sources of error (i.e. weather or predation of the larvae) usually cause an underestimation of population size. Labour, the main cause of high costs, was low in the basin technique and high in the frass production technique. Possible ways of reducing errors and applications of the three techniques are discussed.

Leaf quality and insect herbivory in model tropical plant communities after long-term exposure to elevated atmospheric CO2.

Results from laboratory feeding experiments have shown that elevated atmospheric carbon dioxide can affect interactions between plants and insect herbivores, primarily through changes in leaf nutritional quality occurring at elevated CO2. Very few data are available on insect herbivory in plant communities where insects can choose among species and positions in the canopy in which to feed. Our objectives were to determine the extent to which CO2-induced changes in plant communities and leaf nutritional quality may affect herbivory at the level of the entire canopy. We introduced equivalent populations of fourth instar Spodoptera eridania, a lepidopteran generalist, to complex model ecosystems containing seven species of moist tropical plants maintained under low mineral nutrient supply. Larvae were allowed to feed freely for 14 days, by which time they had reached the seventh instar. Prior to larval introductions, plant communities had been continuously exposed to either 340 µl CO2 l-1 or to 610 µl CO2 l-1 for 1.5 years. No major shifts in leaf nutritional quality [concentrations of N, total non-structural carbohydrates (TNC), sugar, and starch; ratios of: C/N, TNC/N, sugar/N, starch/N; leaf toughness] were observed between CO2 treatments for any of the species. Furthermore, no correlations were observed between these measures of leaf quality and leaf biomass consumption. Total leaf area and biomass of all plant communities were similar when caterpillars were introduced. However, leaf biomass of some species was slightly greater-and for other species slightly less (e.g. Cecropia peltata)-in communities exposed to elevated CO2. Larvae showed the strongest preference for C. peltata leaves, the plant species that was least abundant in all communites, and fed relatively little on plants species which were more abundant. Thus, our results indicate that leaf tissue quality, as described by these parameters, is not necessarily affected by elevated CO2 under relatively low nutrient conditions. Hence, the potential importance of CO2-induced shifts in leaf nutritional quality, as determinants of herbivory, may be overestimated for many plant communities growing on nutrient-poor sites if estimates are based on traditional laboratory feeding studies. Finally, slight shifts in the abundance of leaf tissue of various species occurring under elevated CO2 will probably not significantly affect herbivory by generalist insects. However, generalist insect herbivores appear to become more dependent on less-preferred plant species in cases where elevated CO2 results in reduced availability of leaves of a favoured plant species, and this greater dependency may eventually affect insect populations adversely.

The 60-residue C-terminal region of the single-stranded DNA binding protein of herpes simplex virus type 1 is required for cooperative DNA binding.

ICP8 is the major single-stranded DNA (ssDNA) binding protein of the herpes simplex virus type 1 and is required for the onset and maintenance of viral genomic replication. To identify regions responsible for the cooperative binding to ssDNA, several mutants of ICP8 have been characterized. Total reflection X-ray fluorescence experiments on the constructs confirmed the presence of one zinc atom per molecule. Comparative analysis of the mutants by electrophoretic mobility shift assays was done with oligonucleotides for which the number of bases is approximately that occluded by one protein molecule. The analysis indicated that neither removal of the 60-amino-acid C-terminal region nor Cys254Ser and Cys455Ser mutations qualitatively affect the intrinsic DNA binding ability of ICP8. The C-terminal deletion mutants, however, exhibit a total loss of cooperativity on longer ssDNA stretches. This behavior is only slightly modulated by the two-cysteine substitution. Circular dichroism experiments suggest a role for this C-terminal tail in protein stabilization as well as in intermolecular interactions. The results show that the cooperative nature of the ssDNA binding of ICP8 is localized in the 60-residue C-terminal region. Since the anchoring of a C- or N-terminal arm of one protein onto the adjacent one on the DNA strand has been reported for other ssDNA binding proteins, this appears to be the general structural mechanism responsible for the cooperative ssDNA binding by this class of protein.

A novel quaternary structure of the dimeric alpha-crystallin domain with chaperone-like activity.

alphaB-crystallin, a member of the small heat-shock protein family and a major eye lens protein, is a high molecular mass assembly and can act as a molecular chaperone. We report a synchrotron radiation x-ray solution scattering study of a truncation mutant from the human alphaB-crystallin (alphaB57-157), a dimeric protein that comprises the alpha-crystallin domain of the alphaB-crystallin and retains a significant chaperone-like activity. According to the sequence analysis (more than 23% identity), the monomeric fold of the alpha-crystallin domain should be close to that of the small heat-shock protein from Methanococcus jannaschii (MjHSP16.5). The theoretical scattering pattern computed from the crystallographic model of the dimeric MjHSP16.5 deviates significantly from the experimental scattering by the alpha-crystallin domain, pointing to different quaternary structures of the two proteins. A rigid body modeling against the solution scattering data yields a model of the alpha-crystallin domain revealing a new dimerization interface. The latter consists of a strand-turn-strand motif contributed by each of the monomers, which form a four-stranded, antiparallel, intersubunit composite beta-sheet. This model agrees with the recent spin labeling results and suggests that the alphaB-crystallin is composed by flexible building units with an extended surface area. This flexibility may be important for biological activity and for the formation of alphaB-crystallin complexes of variable sizes and compositions.

Common processing of in vivo MR spectra.

This introductory article addresses approaches currently in use to process in vivo spectra. First, a brief overview is given of the information content represented by the parameters of MR signals. Subsequently, common steps in the processing of MR spectra such as pre-processing, normalisation and quantification and the use of prior knowledge are described. Finally, some prospects for more advanced processing are given.

Lead extraction in young patients with and without congenital heart disease using the subclavian approach.

Pacemaker lead removal using interlocking stylets and dilator sheaths has greatly reduced the need for major surgical intervention when lead extraction is required. Previous reports have shown the utility of this method in older patients, most of whom have anatomically normal hearts. The purpose of this study is to report the results of this technique in young patients with and without congenital heart disease. There were 13 patients (M:F = 7:6) aged 9-26 years (median 13). Congenital heart disease was present in 8 of 13 patients. A total of 17 leads required removal; they had been implanted for 54 +/- 24 months (range 19-94). Leads were removed from the left subclavian vein (13) or right subclavian vein (4) only. Seventeen of 18 leads were completely removed and one partially retained in the left subclavian vein. New leads were implanted from the same vein in 11 of 13 patients. Interlocking stylets and metal or flexible dilator sheaths were used in all cases except two. There was one surgical complication: a late wound dehiscence, which was easily managed. No patient required a transfusion, and there was no structural damage noted in any patient on the postoperative echocardiogram. We conclude that lead removal using interlocking stylets and dilator sheaths from the subclavian approach is an effective technique that can be used in young patients, including those with congenital heart disease.

Effects of ischemia on skeletal muscle energy metabolism in mice lacking creatine kinase monitored by in vivo 31P nuclear magnetic resonance spectroscopy.

The aim of this study was to provide in vivo experimental evidence for the proposed biological significance of the creatine kinase (CK)/phosphocreatine (PCr) system in the energy metabolism of skeletal muscle. As a test system we compared hindlimb muscle of knockout mice lacking the cytosolic M-type (M-CK(-)/(-)), the mitochondrial ScMit-type (ScCKmit(-)/(-)), or both creatine kinase isoenzymes (CK(-)/(-)), and in vivo 31P-NMR was used to monitor metabolic responses during and after an ischemic period. Although single mutants show some subtle specific abnormalities, in general their metabolic responses appear similar to wild type, in contrast to CK(-)/(-) double mutants. This implies that presence of one CK isoform is both necessary and sufficient for the system to be functional in meeting ischemic stress conditions. The global ATP buffering role of the CK/PCr system became apparent in a 30% decline of ATP in the CK(-)/(-) mice during ischemia. Both M-CK(-)/(-) and CK(-)/(-) showed increased phosphomonoester levels during ischemia, most likely reflecting adaptation to a more efficient utilization of glycogenolysis. While in M-CK(-)/(-) muscle PCr can still be hydrolyzed to provide Pi for this process, in CK(-)/(-) muscle only Pi from ATP breakdown is available and Pi levels increase much more slowly. The experiments also revealed that the system plays a role in maintaining pH levels; the CK(-)/(-) mice showed a faster and more pronounced acidification (pH = 6.6) than muscles of wild type and single knockout mutants (pH = 6.9).

Tunicamycin-inhibited carrot somatic embryogenesis can be restored by secreted cationic peroxidase isoenzymes.

Somatic embryogenesis of carrot (Daucus carota L.) is inhibited by the glycosylation inhibitor tunicamycin. This inhibition is reversible by the addition of correctly glycosylated glycoproteins which have been secreted into the culture medium. To identify the proteins responsible for complementation, glycoproteins present in the medium of embryo cultures were purified and tested for their activity in the tunicamycin inhibition/ complementation assay. A 38-kDa glycoprotein was purified that could restore embryogenesis to more than 50% of that in untreated controls. This 38-kDa glycoprotein was identified as a heme-containing peroxidase on the basis of its A405/A280 ratio (Reinheit Zahl or RZ) and enzyme activity. The 38-kDa peroxidase consisted of four different cationic isoenzymes of which only one or possibly two appeared active in the complementation assay. The cationic peroxidase isoenzymes from the carrot medium could be effectively replaced by cationic horseradish peroxidases which depended on their catalytic properties for their ability to restore tunicamycin-inhibited somatic embryogenesis.

Proton MR spectroscopy of wild-type and creatine kinase deficient mouse skeletal muscle: dipole-dipole coupling effects and post-mortem changes.

Localized proton MR spectra of mouse skeletal muscle obtained at 7 T show dipole-dipole coupling effects for creatine and putative taurine resonances and for the lactate methine signal. These effects are independent of the presence of creatine kinase. The intensity of the methylene (1)H resonance of creatine is not different between wild-type and creatine kinase deficient mice, which have a lower phosphocreatine content. (1)H-MR spectra acquired post-mortem from wild-type mouse skeletal muscle parallel to B(0) show a linewidth decrease for the methyl resonance of creatine and a 20% signal intensity loss for its methylene peak concurrent with the total breakdown of phosphocreatine as observed by (31)P-MR spectroscopy. However, with the muscle at the magic angle no changes in the appearance and intensity of creatine (and taurine) resonances are observed. These results indicate that the changes observed for creatine resonances are related to altered dipolar couplings and that the intensity of the methylene peak does not necessarily reflect muscular phosphocreatine content.

Crystallization and preliminary X-ray crystallographic studies on the adenovirus ssDNA binding protein in complex with ssDNA.

Crystals of the C-terminal domain of the adenovirus single-stranded DNA binding protein (DBP) in complex with the single-stranded oligonucleotide (dT)16 have been obtained by a batch method from material obtained by chymotryptic digest of full-length DBP. The colorless crystals grow as hexagonal prisms to a maximal size of approximately 0.85 x 0.55 x 0.55 mm. The spacegroup is P3(1)21 with unit cell constants a = 151.5 A and c = 124.0 A. There are either three or four molecules of DBP per asymmetrical unit. To improve the reproducibility of crystallization, recombinant protein has been produced using a baculovirus expression system and shown to crystallize under the same conditions.

Introduction to in vivo 31P magnetic resonance spectroscopy of (human) skeletal muscle.

31P magnetic resonance spectroscopy (MRS) offers a unique non-invasive window on energy metabolism in skeletal muscle, with possibilities for longitudinal studies and of obtaining important bioenergetic data continuously and with sufficient time resolution during muscle exercise. The present paper provides an introductory overview of the current status of in vivo 31P MRS of skeletal muscle, focusing on human applications, but with some illustrative examples from studies on transgenic mice. Topics which are described in the present paper are the information content of the 31P magnetic resonance spectrum of skeletal muscle, some practical issues in the performance of this MRS methodology, related muscle biochemistry and the validity of interpreting results in terms of biochemical processes, the possibility of investigating reaction kinetics in vivo and some indications for fibre-type heterogeneity as seen in spectra obtained during exercise.

Presence of (phospho)creatine in developing and adult skeletal muscle of mice without mitochondrial and cytosolic muscle creatine kinase isoforms.

We assessed the relationship between phosphocreatine (PCr) and creatine (Cr) content and creatine kinase (CK) activity in skeletal muscle of mice. The PCr and total Cr (tCr) concentrations, as well as CK activity, in hindlimb muscles of mice, with or without the cytosolic and mitochondrial isoforms of muscle creatine kinase (wild-type or CK--/-- mice), were determined by in vivo magnetic resonance (MR) spectroscopy and by biochemical means during postnatal growth and adulthood. In wild-type muscle the [tCr], PCr/ATP ratio and CK activity increased rapidly in the first 4-7 weeks. Remarkably, CK--/-- mice showed a similar increase in the PCr/ATP ratio during the first month in the presence of only minor brain-type BB-CK activity. Uptake of Cr in muscle was seemingly unrelated to CK activity as tCr increased in the same way in the muscles of both mouse types. At older ages the PCr/ATP ratio decreased in CK--/-- muscles, in contrast to wild-type where it still slowly increased, whereas [tCr] was similar for muscle of both mouse types. Using a new in vivo MR approach with application of [4-13C]Cr, a lower PCr/tCr ratio was also observed in CK--/-- muscle. From these data it follows that in vivo global ATP levels at rest are similar in the presence or absence of CK. Although Cr could still be converted to PCr in mature CK--/-- muscle, the immediate availability of PCr decreased, and PCr became partly inconvertible at older ages. Apparently, catalysis of the CK reaction by BB-CK, although significant in muscles of newborn mice, gradually declines to very low levels in adulthood. Part or all of this BB-CK may arise from satellite cells fusing with myotubes, a process that is most active during the first months of life. Finally, our observation that the MR and chemical assessment of muscle [tCr] and PCr/tCr ratio were similar for all mice does not support the existence of a significant MR-invisible or immobile pool of Cr, with a role for CK in this phenomenon.

Cerebral creatine kinase deficiency influences metabolite levels and morphology in the mouse brain: a quantitative in vivo 1H and 31P magnetic resonance study.

Creatine kinase (CK)-catalysed ATP-phosphocreatine (PCr) exchange is considered to play a key role in energy homeostasis of the brain. This study assessed the metabolic and anatomical consequences of partial or complete depletion of this system in transgenic mice without cytosolic B-CK (B-CK-/-), mitochondrial ubiquitous CK (UbCKmit-/-), or both isoenzymes (CK -/-), using non-invasive quantitative magnetic resonance (MR) imaging and spectroscopy. MR imaging revealed an increase in ventricle size in a subset of B-CK-/- mice, but not in animals with UbCKmit or compound CK mutations. Mice lacking single CK isoenzymes had normal levels of high-energy metabolites and tissue pH. In the brains of CK double knockouts pH and ATP and Pi levels were also normal, even though PCr had become completely undetectable. Moreover, a 20-30% decrease was observed in the level of total creatine and a similar increase in the level of neuronal N-acetyl-aspartate compounds. Although CKs themselves are not evenly distributed throughout the CNS, these alterations were uniform and concordant across different brain regions. Changes in myo-inositol and glutamate peaks did appear to be mutation type and brain area specific. Our results challenge current models for the biological significance of the PCr-CK energy system and suggest a multifaceted role for creatine in the brain.

Global HDO uptake in human glioma xenografts is related to the perfused capillary distribution.

The aim of this study is to evaluate the existence of a possible relationship between global deuterium-labeled water (HDO) uptake rates and the diffusion geometry of human glioma xenografts in nude mice. HDO diffusion times in the whole extravascular tumor volume were estimated by combining quantitative (1)H-MR diffusion imaging and morphometric analysis of intercapillary distances in two tumor lines with a different perfused vascular architecture. HDO uptake was measured independently using (2)H-magnetic resonance spectroscopy. Time constants of HDO-uptake curves (tau) were compared to estimations of maximum HDO diffusion times (t(difmax)). Tumors with a homogeneously perfused capillary distribution showed a mono-exponential HDO uptake. The t(difmax) was comparable to tau values of HDO uptake curves: t(difmax) varied between 74 and 368 sec and the range of tau values was 115-370 sec. Heterogeneously perfused tumors had a bi-exponential HDO uptake with t(difmax) in between the tau values of the fast and slow uptake phase. These findings indicate that the global HDO uptake is related to the perfused capillary distribution in human glioma xenografts. That HDO uptake rates indeed can depend on the perfused capillary distribution was substantiated in experiments with two-dimensional (2D) models. In these models with a diffusion-limited HDO uptake, HDO uptake curves could be approximated by curves derived from 2D HDO diffusion simulations. Magn Reson Med 42:479-489, 1999.

Recommendations for extraction of chronically implanted transvenous pacing and defibrillator leads: indications, facilities, training. North American Society of Pacing and Electrophysiology Lead Extraction Conference Faculty.

The procedure of lead removal has recently matured into a definable, teachable art with its own specific tools and techniques. It is now time to recognize and formalize the practice of lead removal according to the current methods of medicine and the health care industry. In addition, since at this time the only prospective scientific study of lead extraction is the PLEXES trial, we suggest that studies relating to the techniques of and indications for lead extraction be designed. Recommendations for a common set of definitions, for a framework of training and reviewing physicians in the art, for general methods of reimbursement, and for consistency among clinical trials have been made. Implementation of these recommendations will require additional effort and cooperation from practicing physicians, medical societies, hospital administrations, and industry.