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Modeling the haemodynamic response in functional magnetic resonance (fMRI) experiments is an important aspect of the analysis of functional neuroimages. This has been done in the past using parametric response function, from a limited family. In this contribution, we adopt a semi-parametric approach based on finite impulse response (FIR) filters. In order to cope with the increase in the number of degrees of freedom, we introduce a Gaussian process prior on the filter parameters. We show how to carry on the analysis by incorporating prior knowledge on the filters, optimizing hyper-parameters using the evidence framework, or sampling using a Markov Chain Monte Carlo (MCMC) approach. We present a comparison of our model with standard haemodynamic response kernels on simulated data, and perform a full analysis of data acquired during an experiment involving visual stimulation.

Zellweger syndrome is a fatal recessively inherited disease with disturbed function of many organs. The disease is caused by a defect of peroxisomes, subcellular organelles, which are absent in these patients. Several genes are necessary for the formation and function of the peroxisomes. The clinical picture of Zellweger syndrome can be caused by defects in a number of genes. On the other hand, clinically different diseases such as neonatal adrenoleucodystrophy and infantile Refsum disease have been shown to be allelic to Zellweger syndrome. We describe a typical Zellweger patient belonging to complementation group 1, which is by far the largest group containing more than half of the Zellweger patients.

Several bioartificial liver devices have been developed as temporary therapy for patients suffering from fulminant hepatic failure. Some of these devices contain porcine hepatocytes entrapped in collagen matrices. In order to improve the function of these BAL devices, there exists a need to optimize metabolic function of cultured hepatocytes. The goal of these investigations was to evaluate the effect of altering culture conditions on rifampin-mediated induction of CYP3A isoforms in cultured porcine hepatocytes. Midazolam metabolism was compared in porcine hepatocytes cultured in a monolayer configuration on collagen gels, in a sandwich configuration between collagen gels and a Matrigel overlay, and in spheroidal cultures. The effect of culture conditions was evaluated, by measuring CYP3A-mediated metabolism of midazolam and by immunoblotting to detect CYP3A proteins, in control cultures and in rifampin-treated cultures. Results obtained by normalizing the metabolism rate data to cell numbers (based on DNA content) present at the end of the culture experiment, showed that there was no difference between the different culture conditions tested. Our results suggest that culturing porcine hepatocytes as spheroids or in a sandwich configuration between collagen and Matrigel, offers no advantage in terms of CYP3A-mediated metabolic function on a per cell basis compared to culturing on collagen gels.

Diagnosis of acute osteomyelitis in childhood is conventionally established by a combination of clinical symptoms and results of inflammation parameters in blood and blood culture, plain radiography, bone scintigraphy and ultrasound. High sensitivity of bone scintigraphy enables it to be positive in the majority of cases, especially when clinical symptoms and blood tests are inconclusive. We present a case of a nine month old girl with an established diagnosis of vertebral osteomyelitis. Her two bone scintigraphies were negative despite obvious destruction of vertebra L5 and S1, documented by plain radiography and magnetic resonance imaging (MRI). MRI should be used when conventional examinations are inconclusive.

Unlabelled: Abnormal glucose metabolic patterns in the brain have been reported for HIV-1 seropositive (HIV+) patients when compared with seronegative healthy individuals. The metabolic covariance pattern obtained from voxel- or volume-of-interest (VOI)-based multivariate data analysis techniques can be used to characterize disease and potentially to detect and monitor disease progression in the early stage of AIDS dementia complex. However, the arbitrary smoothing typically applied to PET data during reconstruction and processing to reduce noise has an unknown effect on the data, especially for the voxel-based analysis., Methods: To investigate the impact of smoothing on a discrimination task, we applied principal component analysis with scaled subprofile-model preprocessing (SSM/PCA) followed by Fisher discriminant analysis to FDG PET data that were reconstructed and processed with different degrees of smoothing. Receiver operating characteristic curves were used to compare the ability of derived metabolic covariance patterns to discriminate HIV+ patients from healthy volunteers., Results: For the voxel-based analysis, we found that the spatial distribution of voxel weights from the SSM/PCA analysis suggested edge effects along major tissue and cerebrospinal fluid boundaries, indicative of a disease-specific pattern of cerebral atrophy for the HIV+ patients. In terms of its discrimination performance, this metabolic covariance pattern is stable and insensitive to a wide range of smoothing kernels, except for ramp reconstruction and Hanning reconstruction with 7 x 7 x 7 block smoothing. In these reconstructions, the discrimination performance decreased as a result of high image noise and excessive smoothing, respectively. Our results also indicated that if sufficient variance from the VOI measurements is included, the overall performance of a conventional VOI-based analysis can be similar to that of the voxel-based analysis for the same discrimination task. However, the VOI-based analysis performed poorly at low false-positive fraction and is less tolerant to noise in the metabolic covariance pattern than the voxel-based analysis., Conclusion: We have obtained a unique covariance pattern of brain glucose metabolism for HIV+ patients compared with healthy volunteers. Discrimination based on this covariance pattern was found to be insensitive to a wide range of image smoothness.

There is currently much interest in designing synthetic substrates incorporating the cell binding motif RGD for tissue engineering. In this paper, hepatocyte function was examined on two synthetic RGD substrates and compared to that on fibronectin (Fn). One is a 2.3 kD RGD peptide (P-2) containing a single RGD, a short spacer in the middle and an end basic sequence to enhance adsorption. On bacteriological plastic, P-2 induced a rounded cell shape, enhanced differentiated function, and inhibited DNA synthesis. The other, a 73 kD synthetic RGD protein Pronectin F (PnF), contains repeating RGD units interspersed with a structural peptide. PnF induced cell spreading, dedifferentiation, and enhanced DNA synthesis, similar to Fn. In addition, only P-2 showed distinct differences in cell shape and DNA synthesis when coated on bacteriological plastic, or on Immulon II plastic, both intrinsically non-adhesive to cells. On bacteriologic plates coated with P-2, cells were round and showed diminished DNA synthesis while on Immulon II plates, they were spread and showed enhanced DNA synthesis. These results demonstrate that synthetic RGD peptides can induce very different hepatocyte function depending on the context in which they are presented to cells. It is likely that the RGD peptide conformation determines the specificity of cellular response.

The induction of putative CYP3A isoforms in cultured porcine hepatocytes was evaluated by measurement of midazolam metabolism, a model substrate of the CYP3A family. The induction was also studied at the molecular level by quantitation of mRNA and protein levels, by Northern blotting and Western blotting, respectively. Pretreatment with rifampin (50 microM) resulted in a 5.5- to 9-fold higher rate of midazolam metabolism when compared with control cultures. No induction was observed when the cultures were pretreated with 50 microM dexamethasone. A 12-fold increase in the CYP3A mRNA signal (approximately 2.4 kB) was observed in induced cultures over control cultures. Microsomal proteins were separated by SDS-polyacrylamide gel electrophoresis and detected by immunoblotting with a polyclonal antibody raised against human CYP3A4. The immunoblots showed the presence of four bands in microsomes prepared from pig livers, with two bands (51.5 and 52 kD) that showed intense staining. Microsomes prepared from a pig pretreated with rifampin showed marked induction of these two bands. Immunoblotting of microsomes from rifampin-induced cultures also showed significantly greater intensity than in control cultures. Our results indicate that rifampin, but not dexamethasone, is an inducer of midazolam hydroxylase in pig hepatocytes. This induction may be regulated at the transcriptional level as detected by an increase in mRNA with a CYP3A oligonucleotide probe. Finally, there appears to be a multiplicity of the CYP3A isoforms in pig hepatocytes, similar to that observed in humans and rats.

We apply nine analytic methods employed currently in imaging neuroscience to simulated and actual BOLD fMRI signals and compare their performances under each signal type. Starting with baseline time series generated by a resting subject during a null hypothesis study, we compare method performance with embedded focal activity in these series of three different types whose magnitudes and time courses are simple, convolved with spatially varying hemodynamic responses, and highly spatially interactive. We then apply these same nine methods to BOLD fMRI time series from contralateral primary motor cortex and ipsilateral cerebellum collected during a sequential finger opposition study. Paired comparisons of results across methods include a voxel-specific concordance correlation coefficient for reproducibility and a resemblance measure that accommodates spatial autocorrelation of differences in activity surfaces. Receiver-operating characteristic curves show considerable model differences in ranges less than 10% significance level (false positives) and greater than 80% power (true positives). Concordance and resemblance measures reveal significant differences between activity surfaces in both data sets. These measures can assist researchers by identifying groups of models producing similar and dissimilar results, and thereby help to validate, consolidate, and simplify reports of statistical findings. A pluralistic strategy for fMRI data analysis can uncover invariant and highly interactive relationships between local activity foci and serve as a basis for further discovery of organizational principles of the brain. Results also suggest that a pluralistic empirical strategy coupled formally with substantive prior knowledge can help to uncover new brain-behavior relationships that may remain hidden if only a single method is employed., (Copyright 1999 Academic Press.)

Rat hepatocytes adherent to a rigid film of type I collagen will spread and enter S phase, while those attached to collagen gel or a dried collagen substrate remain round and quiescent. The current studies were initiated to determine the mechanism by which these different substrates differentially influence cell cycle progression. Cyclin D1 mRNA and protein expression and associated kinase activity was low on dried collagen relative to collagen film. In contrast, cyclin E and cdk2 protein levels were similar on the two substrates. Although cyclin E and cdk2 were present, cells on dried collagen lacked cdk2 kinase activity. p27 protein levels did not differ between dried collagen and film, but more p27 was associated with cdk2 in cells on dried collagen than those on collagen film. Cyclin D1 expression on collagen film was inhibited by cytochalasin D and exoenzyme C3, suggesting a role for the GTP-binding protein, Rho, in regulating cyclin D1 expression. Cyclin D1 over-expression induced hepatocytes into S phase in the absence of cell shape change on dried collagen or collagen gel. These results demonstrate a novel, substrate-dependent mechanism for cyclin D1 expression in hepatocytes, and also demonstrate that cyclin D1 over-expression allows shape-independent S phase entry.

Cyclin D1 is widely believed to regulate progression through G1 phase of the cell cycle, and previous studies have shown that this protein is induced during hepatocyte proliferation in culture and in vivo. In this study, the role of cyclin D1 in the cell cycle of primary rat hepatocytes was further examined. Following epidermal growth factor stimulation, cyclin D1 was upregulated at time points corresponding to the mitogen restriction point, and this was associated with enhanced cyclin D1-associated kinase activity. To test whether cyclin D1 expression was sufficient to promote mitogen-independent progression through the G1-S transition, we constructed a replication-defective adenovirus that overexpressed human cyclin D1. Transfection with the cyclin D1 vector but not a control vector resulted in hepatocyte DNA synthesis in the absence of growth factor that was similar to that seen in mitogen-treated cells. Furthermore, cyclin D1 transfection led to activation of downstream biochemical events, including cyclin A and proliferating cell nuclear antigen expression and cyclin E- and cyclin A-associated kinase activation. These results suggest that cyclin D1 expression is sufficient to promote progression of hepatocytes through the G1 restriction point.

Generalization can be defined quantitatively and can be used to assess the performance of principal component analysis (PCA). The generalizability of PCA depends on the number of principal components retained in the analysis. We provide analytic and test set estimates of generalization. We show how the generalization error can be used to select the number of principal components in two analyses of functional magnetic resonance imaging activation sets., (Copyright 1999 Academic Press.)

This paper addresses the problem of neuro-anatomical registration across individuals for functional [15O] water PET activation studies. A new algorithm for three-dimensional (3-D) nonlinear structural registration (warping) of MR scans is presented. The method performs a hierarchically scaled search for a displacement field, maximizing one of several voxel similarity measures derived from the two-dimensional (2-D) histogram of matched image intensities, subject to a regularizer that ensures smoothness of the displacement field. The effect of the nonlinear structural registration is studied when it is computed on anatomical MR scans and applied to coregistered [15O] water PET scans from the same subjects: in this experiment, a study of visually guided saccadic eye movements. The performance of the nonlinear warp is evaluated using multivariate functional signal and noise measures. These measures prove to be useful for comparing different intersubject registration approaches, e.g., affine versus nonlinear. A comparison of 12-parameter affine registration versus non-linear registration demonstrates that the proposed nonlinear method increases the number of voxels retained in the cross-subject mask. We demonstrate that improved structural registration may result in an improved multivariate functional signal-to-noise ratio (SNR). Furthermore, registration of PET scans using the 12-parameter affine transformations that align the coregistered MR images does not improve registration, compared to 12-parameter affine alignment of the PET images directly.

Analysis of fMRI time series is often performed by extracting one or more parameters for the individual voxels. Methods based, e.g., on various statistical tests are then used to yield parameters corresponding to probability of activation or activation strength. However, these methods do not indicate whether sets of voxels are activated in a similar way or in different ways. Typically, delays between two activated signals are not identified. In this article, we use clustering methods to detect similarities in activation between voxels. We employ a novel metric that measures the similarity between the activation stimulus and the fMRI signal. We present two different clustering algorithms and use them to identify regions of similar activations in an fMRI experiment involving a visual stimulus., (Copyright 1999 Academic Press.)

Objectives: To evaluate the reproducibility and the accuracy of pediatric radiologists' assessments of chest radiographs with respect to the presence or absence of heart defects in children with an asymptomatic heart murmur., Design: Ninety-eight children, ages 1 month to 15 years (median, 30.1 months), referred for evaluation of a heart murmur were consecutively included. They all had a standard chest radiograph and a color Doppler echocardiograph (CDE) performed. Six specialists in pediatric radiology evaluated the chest radiographs independently on two occasions 6 months apart. The radiologists were asked to classify each set of films into one of two categories: heart disease or no heart disease. The outcome of the CDE was considered the definite diagnosis. kappa statistics were used to analyze the reproducibility of the radiologic assessments. Sensitivity, specificity, and the predictive value of a positive and a negative test were used for evaluation of the accuracy of the radiologic assessments., Results: Mean intra- and interobserver kappa values were all <0.6, and the majority were <0.4. Mean sensitivity was 0.3 (range: 0.17-0.52), mean predictive value of a positive test was 0.4, implying that 60% of the positive assessments were falsely positive. Mean specificity was 0.86 (range: 0.75-0.93) and the mean predictive value of a negative test was 0.80 implying that 20% of the negative assessments were falsely negative., Conclusion: We found a low reproducibility, as well as a low accuracy, of the radiologic assessments of the chest radiographs of children with an asymptomatic heart murmur with respect to the presence or absence of heart disease. A false-positive radiologic assessment of the chest radiograph with respect to heart defects causes unnecessary anxiety and further examinations, whereas a false-negative assessment might result in omission of relevant investigations and proper identification of the heart defect. We cannot recommend the use of chest radiographs in the initial evaluation of the asymptomatic child with a heart murmur. If a heart defect cannot be excluded by clinical examination a CDE must be performed.

Liver disease continues to be a challenge clinically, with 30,000 patients dying each year from liver failure (1). Although liver transplantation can successfully treat many patients undergoing liver failure, the scarcity of donor organs severely limits this treatment's application. For this reason, many investigators are pursuing alternatives to total organ transplantation, from living donors to cell transplantation. One additional approach is the development of a hybrid, bioartificial liver as an extracorporeal device for the temporary treatment of acute liver failure. This approach has demonstrated early success, and may provide an important clinical treatment in the near future. In addition, a bioartificial liver reactor is useful for prolonged in vitro studies of hepatocyte function. This chapter will provide information on the design and use of such a reactor for in vitro applications.

Several investigators have demonstrated that freshly harvested hepatocytes self-assemble into three-dimensional, compacted, freely suspended aggregates known as spheroids (1-3). These aggregates have smooth, undulating surfaces and average approx 120 µm in diameter. Hepatocyte spheroids exhibit enhanced liver-specific activities and prolonged viability, compared to cells maintained as a monolayer (4,5). Extensive cell-cell contacts, tight junctions, and microvilli-lined channels that resemble bile canaliculi have been observed between hepatocytes in spheroids (6,7). Thus, these cells appear to mimic the morphology and ultrastructure of an in vivo liver lobule. Reorganization of hepatocytes into these three-dimensional structures is hypothesized to contribute to their enhanced liver-specific functions. Because of their enhanced function and tissue-like ultrastructure, hepatocyte spheroids show great promise for use in tissue-engineering applications and drug metabolism studies.

This paper presents a quantitative comparison of three types of information available for 52 patients scheduled for carotid endarterectomy: subjective classification of the ultrasound images obtained during scanning before operation, first-and second-order statistical features extracted from regions of the plaque in still ultrasound images from three orthogonal scan planes and finally a histological analysis of the surgically removed plaque. The quantitative comparison was made with the linear model and with separation of the available data into training and test sets. The comparison of subjective classification with features from still ultrasound images revealed an overall agreement of 60% for classification of echogenicity and 70% for classification of structure. Comparison of the histologically determined relative volume of soft materials with features from the still images revealed a correlation coefficient of r = -0.42(p = 0.002). for mean echogenicity of the plaque region. The best performing feature was of second order and denoted Contrast (r = -0.5). Though significant, the latter correlation is probably not strong enough to be useful for clinical prediction of relative volume of soft materials for individual patients. Reasons for this is discussed in the paper, together with suggestions for improvements.

Familial neurohypophyseal diabetes insipidus (FNDI) is an autosomal dominant (adFNDI) or X-linked recessive (xrFNDI) disorder characterized by the development in early childhood of an irreversible deficiency of arginine vasopressin (AVP) secretion. Autopsy data in adFNDI reveal selective destruction of the posterior pituitary magnocellular neurons that normally produce the hormone. These abnormalities are due to a variety of mutations in the gene that encodes the AVP-neurophysin II precursor. Each one predicts a change in the primary structure of the preprohormone, and all but one are of a type known or reasonably presumed to impair the folding and cellular trafficking of the preprohormone. This pattern and the uniform clinical characteristics of adFNDI suggest that the disease is due to the production of a mutant precursor that is toxic for magnocellular neurons, because it cannot be folded, processed, or otherwise disposed of efficiently. Although the gene responsible for xrFNDI has not yet been cloned, the striking clinical similarities between adFNDI and xrFNDI suggest that similar pathophysiologic mechanisms may be involved.

A better understanding of pruning methods based on a ranking of weights according to their saliency in a trained network requires further information on the statistical properties of such saliencies. We focus on two-layer networks with either a linear or nonlinear output unit, and obtain analytic expressions for the distribution of saliencies and their logarithms. Our results reveal unexpected universal properties of the log-saliency distribution and suggest a novel algorithm for saliency-based weight ranking that avoids the numerical cost of second derivative evaluations.

We investigate the use of a regularization prior and its pruning properties. We illustrate the behavior of this prior by conducting analyses both using a Bayesian framework and with the generalization method, on a simple toy problem. Results are thoroughly compared with those obtained with a traditional weight decay. Copyright 1997 Elsevier Science Ltd.

The objective of this study was to analyze retrospectively the results of color Doppler echocardiographic (CDE) investigations performed for the first time on a child under circumstances where there was unrestricted access to CDE. The records were obtained from a tertiary referral center at a university hospital serving a population of about one million. The patients were children (n = 284) ages 0 days to 15 years 9 months referred for CDE over a 1-year period. Heart abnormalities were found in 119 children (42%). Among the children <3 months old, a heart abnormality was detected by CDE in 71%. The most prevalent diagnosis was atrial septal defect, particularly during the first 3 months of life, where the intraatrial communications accounted for 78% of the heart abnormalities. It is concluded that there is high prevalence of atrial communications during the first 3 months of life. The spectrum of diagnoses among newborns differs from the previously reported frequencies of congenital heart diseases. Better equipment (CDE) seems to diagnose abnormalities that might not be diagnosed otherwise.

The reproducibility of patterns from brain activation experiments has been examined only for suprathreshold spatially localized foci. Scatter plots comparing signal levels across all pairs of Talairach voxels for pairs of functional activation images provide an alternative approach for assessing reproducibility. Image-wide, signal-level reproducibility may be quantitatively summarized using pattern similarity measures such as the Pearson product-moment correlation, rho. Empirical population distributions of rho for many pair-wise image comparisons, generated using statistical resampling techniques, may be used to examine the impact of a wide range of experimental variables. We demonstrate the use of such empirical rho-histograms to measure changes in reproducibility for [(15)O]-water PET scans of a simple motor task as a function of group size and data analysis model., (Copyright (c) 1997 Wiley-Liss, Inc.)

Objective: This paper describes an exposure assessment of an American Indian population using blood and hair samples as indicators of mercury and polychlorinated biphenyl exposure from the consumption of fish taken from the Great Lakes region., Methods: Questionnaires regarding fish consumption were completed by 89 Ojibwa tribal members. Mercury concentrations were determined in human hair and blood samples, and polychlorinated biphenyl concentrations were determined in serum., Results: Fish were consumed at the highest rates in April, May, June, and July. Lake trout, whitefish, and walleye were the preferred fish consumed by 91.4% of the respondents. Concentrations of blood mercury were all below 55 micrograms/L (ppb), while concentrations of mercury in hair were all less than 3 mg/L (ppm). Hair mercury concentrations were correlated with the previous year's fish consumption (p = .05). Dental amalgams and blood mercury concentrations were also significantly correlated (p < .002). Serum polychlorinated biphenyl concentrations, determined as the sum of 89 congeners, were all below 9.6 ppb total polychlorinated biphenyls. Subject age and total serum polychlorinated biphenyls were correlated (p < .001)., Conclusions: The concentrations of mercury and polychlorinated biphenyls in this Ojibwa population were relatively low, but several individuals were identified as having elevated concentrations and additional testing may be warranted. Since the accumulation of contaminants was related to fish consumption and age, a long-term monitoring program that assesses chronic exposure to fish diets would be beneficial.

Sixteen segments of Arabidopsis thaliana DNA that function as enhancers in transgenic tobacco plants were isolated using the pROA97 enhancer cloning vehicle and library transformation of Nicotiana tabacum. The sequences were compared for AT content, homology, repeated motifs, and expression pattern in transgenic N. tabacum. The sequences were average with respect to the AT content of A. thaliana DNA. They could be placed into seven homology groups. Five of the sequences are single-copy sequences. The remaining eleven sequences represent two homology groups. Homology Group I contains seven sequences with minor differences. Homology Group II contains four sequences with minor differences. Two repeated motifs were identified (5'-CCTCT-3' and 5'-AAGGAT-3'). Both repeated motifs are found in other plant enhancers, and in the promoter region of the cauliflower mosaic virus 35S gene. In the 35S gene TATA region, the motifs can form two alternative stem-loop structures. The TATATAA sequence is located in the loop region of both stem-loop structures.

The Ojibwa Health Study is a descriptive epidemiology study of six Ojibwa reservations in the Upper Great Lakes. Fish consumption habits, contaminant (mercury [Hg], polychlorinated biphenyls [PCBs], and other organochlorines [OCs]) residues in dietary fish and humans, and chronic disease outcomes currently are being documented. Four hundred and fifty questionnaires and approximately 200 biological samples had been collected as of December 1994. Fish collections from reservations included lake trout, walleye, lake whitefish, and lake herring from Lakes Superior (three sites), Michigan, and Huron. Hg and OC residue analyses have been completed for the fish composite samples. A preliminary examination of the data has revealed regional differences in the contaminant burdens of the fish. Lake Michigan and Lake Huron whitefish composite samples had approximately twice the OC concentrations of Lake Superior whitefish samples. In general, the whitefish composite samples were far below the U.S. Food and Drug Administration or any state advisory limits for OC or Hg residues. Lake trout samples contained higher amounts of all residues than did the whitefish samples.

Myxoedema, later known as hypothyroidism, was first described in England in 1873. During the following years it was thought that the disease was caused by a lesion in the nervous system. However, towards the end of the century it was established that the thyroid gland produced an essential substance later known as a hormone. An efficient treatment was introduced, consisting of different forms of tissue extracts. The thyroid gland and myxoedema became a model for the development of the concept of internal secretion. Norwegian physicians were well informed about the scientific advances and quickly accepted the new principles of treatment. This article describes the first Norwegian reference to the disease and its treatment.

We investigated the diagnostic value of the chest radiograph and ECG in the evaluation of whether asymptomatic children with a cardiac murmur had a heart disease as defined by echocardiography. One hundred children aged 1 month to 15 years (median 30.1 months) were included. After physical examination, a tentative diagnosis was made: 53 children had no heart disease, 24 had a probable heart disease and 23 children were thought to have heart disease on the basis of clinical assessment alone. After information from chest radiography and electrocardiography was obtained, the diagnoses were re-evaluated. This resulted in a change of the tentative diagnosis in three children. However, the diagnosis in relation to the definite diagnosis by colour Doppler echocardiography was not changed to the correct diagnosis in any of these cases. In 24 cases, radiography suggested the presence of heart disease; however, only 10 of these had heart abnormalities on the colour Doppler echocardiogram (CDE). Three children had an abnormal ECG; all of these had abnormalities on the CDE, but they were already diagnosed as having heart disease by physical examination. We conclude that chest radiography and electrocardiography is of no help in the discrimination between heart disease and no heart disease in asymptomatic children referred for a cardiac murmur.

In order to analyse the non-specialized paediatrician's ability to evaluate children with a heart murmur and the consequences of this evaluation for the need of further investigations, we made a prospective and consecutive study concerning 100 children who were referred for initial evaluation of a heart murmur. Based on the clinical evaluation, the children were assigned to one of the three categories of "no heart disease" "heart disease" or "definite heart disease". Within two months each child underwent a complete colour Doppler echocardiographic study. Except for one child with an insignificant peripheral pulmonary artery stenosis, 53% of the children were correctly diagnosed as having no heart disease. We conclude that clinical evaluation without laboratory tests performed by paediatricians without any special training in cardiology could correctly acquit half of the children of heart disease, with no need for further investigation.

This study was undertaken to characterize the potential heparin affinity of an amino-acid sequence within the 70 kDa heat-shock family of proteins (HSPs) that shares homology with a heparin-binding sequence present in the carboxy-terminus of fibronectin (FN), defined by the synthetic peptide, FN-C/H-II (KNNQKSEPLIGRKKT). To first define the heparin binding sequence within FN-C/H-II, solid phase binding assays were performed using overlapping, short (7 amino acids) synthetic peptides corresponding to the amino-acid sequence within FN-C/H-II. Only the sequence LIGRKKT bound [3H] heparin, and the LIGRKKT peptide blocked heparin binding to intact fibronectin by 47% (+/- 0.4, p < 0.001). A computer-generated homology search revealed that two members of the 70 kDa HSP family, HSP70 and HSC70, contain the sequences LIGRK and LIGRR, respectively. Examination of heparin binding using affinity chromatography indicated that while native HSC70 binds heparin, native HSP70 does not. Treatment of the heparin-unbound fraction with heat or urea led to enhanced HSP70 binding to heparin affinity columns. Soluble LIGRKKT peptide or anti-FN-C/H-II IgG also significantly inhibited heparin binding to HSC70 that had been purified by heparin affinity chromatography. Finally, Western blot analysis of HSC70 purified by heparin affinity chromatography demonstrated that polyclonal anti-FN-C/H-II IgG could recognize HSC70. These data demonstrate that LIGRK or LIGRR represent a a common heparin binding motif in fibronectin, HSP70, and HSC70, and are consistent with a proposed role for heparin or similar polyanionic structures in the function of the 70 kDa heat-shock proteins.

Unlabelled: In a 9-week-old boy with Kawasaki disease an active parvovirus B19 infection was diagnosed serologically., Conclusion: This case supports the hypothesis of an aetiological relationship between parvovirus B19 infection and Kawasaki disease.

Crystals of elastase from North Atlantic salmon have been grown from 2-methyl-2,4-pentanediol by the hanging-drop vapour-diffusion method at room temperature. They grow to dimensions of 0.7 x 0.4 x 0.3 mm in three weeks. The crystals belong to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2 with cell dimensions a = b = 68.0 A and c = 84.0 A. There are eight molecules in the unit cell. The crystals diffract to at least 1.6 A resolution and are suitable for a high-resolution crystal structure determination.

Lysozymes (E.C. 3.2.1.17) are well characterized ubiquitous enzymes that have an antibacterial effect. The lysozymes from rainbow trout (RBTL) (Oncorhynchus mykiss) could be particularly interesting in aquaculture since they show higher activity than egg-white lysozyme and lysozymes from other fish species against a variety of pathogenic bacteria. Two lysozymes, I and II, differing only in a single amino acid, were purified from the kidney of rainbow trout and shown to belong to the c-type class of lysozymes. The type II form was shown to be much more potent against a variety of bacteria than the type I enzyme. We have grown crystals from a mixture containing about 80% type I and 20% type II lysozyme from rainbow trout, and solved the X-ray crystal structure. The crystals are trigonal with a = 76.68, c = 54.46 A and space group P3(1)21. The phase problem was solved by the molecular-replacement method, and the structure was refined to an R-factor of 17.4% using data to 1.8 A resolution. The crystal structure shows that the three-dimensional structure of rainbow trout lysozyme is very similar to the previously solved structures of other c-type lysozymes. The single polypeptide of 129 amino acids is folded into two domains separated by a deep cleft which contains the active site. Secondary-structure elements, four alpha-helices and a three-stranded beta-sheet, are located in the same sequential positions as in the hen, turkey and human enzymes. The beta-sheet is found to be common for structures of both c- and g-type lysozymes. We suggest that differences in antibiotic activity of the two forms of RBTL are probably due to small differences in the hydophobicity of a small surface region.

Ataxia telangiectasia is a genetically determined multi-system disorder in which senile skin changes are at an early stage. The patients have progressive neurological disability, cellular and humoral immunodeficiencies and increased cancer incidence. Heterozygous carriers of the gene, estimated to comprise about 1% of the general population, have an increased risk of cancer. We have initiated an identification of a cohort of patients and their relatives in order to evaluate their cancer risk.

The flexibility and conformational behaviour of salmon and bovine trypsins were modelled with a 300 ps molecular dynamics simulation in aqueous solution. Trajectories from both trypsins were analysed to eventually detect differences in mobility that could explain observed variations in stability and activity. The simulations were performed at 300 K with all the acidic groups deprotonated and the basic groups protonated. The radius of gyration, the overall r.m.s. deviation from the starting structure as a function of time, together with the r.m.s. deviation from the starting structures as a function of residue number, demonstrated that the simulations were stable and representative of the X-ray structures of both enzymes. Isotropic Debye-Waller factors were calculated from the fluctuations for main-chain atoms and were in good agreement with experimental values. The overall dynamic properties of the two enzymes were similar. Based on the present 300 ps molecular dynamics simulation, it cannot be concluded that either of the two enzymes is more 'flexible' than the other. However, there are clearly differences in mobility on a more detailed level and for particular regions.

Unlabelled: To analyse the ability of the non-specialized paediatrician to evaluate children with a heart murmur and the consequences of this evaluation for the need of further investigations, we undertook a prospective and consecutive study of 100 children, referred for initial evaluation of a heart murmur. Except for 1 child with a slight peripheral pulmonary artery stenosis, 53% of the children were correctly diagnosed as having no heart disease. In a project design where 47% of the children were referred for an echocardiographic study, no clinically significant heart disease was missed., Conclusion: Clinical evaluation without laboratory tests performed by paediatricians without any special training in cardiology could correctly acquit 50% of the children of heart disease, with no need for further investigation.

Cells have evolved an autoregulatory mechanism to dampen variations in the concentration of tubulin monomer that is available to polymerize into microtubules (MTs), a process that is known as tubulin autoregulation. However, thermodynamic analysis of MT polymerization predicts that the concentration of free tubulin monomer must vary if MTs are to remain stable under different mechanical loads that result from changes in cell adhesion to the extracellular matrix (ECM). To determine how these seemingly contradictory regulatory mechanisms coexist in cells, we measured changes in the masses of tubulin monomer and polymer that resulted from altering cell-ECM contacts. Primary rat hepatocytes were cultured in chemically defined medium on bacteriological petri dishes that were precoated with different densities of laminin (LM). Increasing the LM density from low to high (1-1000 ng/cm2), promoted cell spreading (average projected cell area increased from 1200 to 6000 microns2) and resulted in formation of a greatly extended MT network. Nevertheless, the steady-state mass of tubulin polymer was similar at 48 h, regardless of cell shape or ECM density. In contrast, round hepatocytes on low LM contained a threefold higher mass of tubulin monomer when compared with spread cells on high LM. Furthermore, similar results were obtained whether LM, fibronectin, or type I collagen were used for cell attachment. Tubulin autoregulation appeared to function normally in these cells because tubulin mRNA levels and protein synthetic rates were greatly depressed in round cells that contained the highest level of free tubulin monomer. However, the rate of tubulin protein degradation slowed, causing the tubulin half-life to increase from approximately 24 to 55 h as the LM density was lowered from high to low and cell rounding was promoted. These results indicate that the set-point for the tubulin monomer mass in hepatocytes can be regulated by altering the density of ECM contacts and changing cell shape. This finding is consistent with a mechanism of MT regulation in which the ECM stabilizes MTs by both accepting transfer of mechanical loads and altering tubulin degradation in cells that continue to autoregulate tubulin synthesis.

This study was undertaken to determine the importance of integrin binding and cell shape changes in the control of cell-cycle progression by extracellular matrix (ECM). Primary rat hepatocytes were cultured on ECM-coated dishes in serum-free medium with saturating amounts of growth factors (epidermal growth factor and insulin). Integrin binding and cell spreading were promoted in parallel by plating cells on dishes coated with fibronectin (FN). Integrin binding was separated from cell shape changes by culturing cells on dishes coated with a synthetic arg-gly-asp (RGD)-peptide that acts as an integrin ligand but does not support hepatocyte extension. Expression of early (junB) and late (ras) growth response genes and DNA synthesis were measured to determine whether these substrata induce G0-synchronized hepatocytes to reenter the growth cycle. Cells plated on FN exhibited transient increases in junB and ras gene expression (within 2 and 8 h after plating, respectively) and synchronous entry into S phase. Induction of junB and ras was observed over a similar time course in cells on RGD-coated dishes, however, these round cells did not enter S phase. The possibility that round cells on RGD were blocked in mid to late G1 was confirmed by the finding that when trypsinized and replated onto FN-coated dishes after 30 h of culture, they required a similar time (12-15 h) to reenter S phase as cells that had been spread and allowed to progress through G1 on FN. We have previously shown that hepatocytes remain viable and maintain high levels of liver-specific functions when cultured on these RGD-coated dishes. Thus, these results suggest that ECM acts at two different points in the cell cycle to regulate hepatocyte growth: first, by activating the G0/G1 transition via integrin binding and second, by promoting the G1/S phase transition and switching off the default differentiation program through mechanisms related to cell spreading.

Two siblings with molybdenum cofactor deficiency are presented. They showed clinical, biochemical and neuroradiological features very similar to those of the few previously described cases. Difficulties in diagnosis are emphasised.

Trichothiodystrophy is a neuroectodermal syndrome with features of a specific hair dysplasia associated with disorders in organs derived from ectoderm and neuroectoderm. The key finding is brittle hair with low sulfur content. Polarizing microscopy shows alternating dark and light bands with trichoscisis and absent or defective cuticle. Patients with trichothiodystrophy have short, sparse, dry and easily broken hair. Growth retardation and mental retardation are almost obligatory findings. The skin and the nails are very often affected; other neuroectodermal disorders are associated, but none is a constant feature. The disease is transmitted as an autosomal recessive trait.

We report here the crystal structure of the complex formed between phospholipase C (PLC) from Bacillus cereus and the widely used biochemical buffer tris (hydroxymethyl)-methylamine (Tris). The structure has been determined at 1.9 A resolution and refined to R = 20.3%. Tris has metal-binding properties, especially to Zn2+, and has been reported to reduce the activity of PLC. The amine nitrogen atom in Tris is co-ordinated to one of the three Zn2+ ions in the active site of the enzyme, thus confirming its chelating properties and the involvement of the metal ions in the catalytic process. The occupancy of the Zn2+ ion in site 2 in native PLC is 0.6 which could imply the presence of Ca2+ rather than Zn2+. The fact that Tris binds to this metal ion, the nature of the site 2 co-ordination shell and comparison with several homologous Zn-metalloenzymes indicate that PLC is a 3-Zn metalloenzyme. This study is one of a series which explores the active site of PLC by complexing the enzyme with inhibitors and substrate analogues.

The optimal brain damage (OBD) scheme of Le Cun, Denker and Solla for pruning of feedforward networks has been implemented and applied to the contiguity classification problem. It is shown that OBD improves the learning curve (the test error as a function of the number of examples). By inspecting the architectures obtained through pruning, it is found that the networks with less parameters have the smallest test error in agreement with "Ockhams Razor". Based on this, we propose a heuristic which selects the smallest successful architecture among a group of pruned networks and we show that it leads to very efficient optimization of the architecture. The validity of the approximations involved in OBD are discussed and it is found that they are surprisingly accurate for the problem studied.

The crystal structure of the complex formed between phospholipase C (PLC) from Bacillus cereus and inorganic phosphate (Pi), which is an inhibitor, has been determined and refined to 2.1 A resolution. The final R-factor is 19.7%. We have also studied the binding of two other inhibitors, iodide and iodate, to PLC. X-ray data for these two complexes were collected to 2.8 A resolution during the search for heavy-atom derivatives. A series of screening experiments where PLC crystals have been treated with several reaction products and a substrate analogue were carried out to clarify the question of substrate binding. The results have so far been ambiguous but are discussed briefly. Phosphate and iodate are both found to bind to the three metal ions in the protein molecule, suggesting that these ions are involved directly in the catalytic process and thereby identifying the active site. PLC also binds nine iodide ions, eight of which are on the surface of the molecule and of lower occupancy. The ninth blocks the entrance to the active site cleft and is of higher occupancy. Altogether, these results suggest that the substrate, a phospholipid, is associated directly with the metal ions during catalysis.

Purification of hemoglobin from North Atlantic salmon (Salmo salar) gave three different types. The CO-complexes of types I and III have been crystallized by the batch method at 4 degrees C from solutions 18% (w/v) in polyethylene glycol 2000, 50 mg/ml in hemoglobin and 0.05 M in phosphate buffer (pH 8.3). Orthorhombic crystals, space group P2(1)2(1)2(1), were obtained for both, with cell dimensions a = 53.9 A, b = 80.4 A, c = 132.4 A, and a = 58.7 A, b = 95.0 A, c = 107.4 A, for types I and III, respectively.

A subset of heat shock proteins, HSP90 alpha, HSP90 beta, and a member of the HSP70 family, HSC70, shows enhanced synthesis following mitogenic activation as well as heat shock in human peripheral blood mononuclear cells. In this study, we have examined expression of mRNA for these proteins, including the major 70-kDa heat shock protein, HSP70, in mononuclear cells following either heat shock or mitogenic activation with phytohemagglutinin (PHA), ionomycin, and the phorbol ester, tetradecanoyl phorbol acetate. The results demonstrate that the kinetics of mRNA expression of these four genes generally parallel the kinetics of enhanced protein synthesis seen following either heat shock or mitogen activation and provide clear evidence that mitogen-induced synthesis of HSC70 and HSP90 is due to increased mRNA levels and not simply to enhanced translation of preexisting mRNA. Although most previous studies have focused on cell cycle regulation of HSP70 mRNA, we found that HSP70 mRNA was only slightly and transiently induced by PHA activation, while HSC70 is the predominant 70-kDa heat shock protein homologue induced by mitogens. Similarly, HSP90 alpha appears more inducible by heat shock than mitogens while the opposite is true for HSP90 beta. These results suggest that, although HSP70 and HSC70 have been shown to contain similar promoter regions, additional regulatory mechanisms which result in differential expression to a given stimulus must exist. They clearly demonstrate that human lymphocytes are an important model system for determining mechanisms for regulation of heat shock protein synthesis in unstressed cells. Finally, based on kinetics of mRNA expression, the results are consistent with the hypothesis that HSC70 and HSP90 gene expression are driven by an IL-2/IL-2 receptor-dependent pathway in human T cells.