Generation of membrane-bound catechol-O-methyl transferase deficient mice with disctinct sex dependent behavioral phenotype.

Catechol-O-methyltransferase (COMT) has two isoforms: soluble (S-COMT), which resides in the cytoplasm, and membrane-bound (MB-MT), anchored to intracellular membranes. COMT is involved in the O-methylation of L-DOPA, dopamine and other catechols. The exact role of MB-COMT is still mostly unclear. We wanted to create a novel genetically modified mouse model that specifically lacks MB-COMT activity and to study their behavioral phenotype. MB-COMT knock-in mutant mice were generated by introducing two point mutations in exon 2 of the Comt gene (ATGCTG->GAGCTC disabling the function of the P2 promoter and allowing only the P1-regulated S-COMT transcription. The first mutation changes methionine to glutamic acid whereas the second one does not affect coding. The expression of the two COMT isoforms, total COMT activity in several areas of the brain and peripheral tissues and extracellular dopamine concentrations after L-DOPA (10 mg/kg) and carbidopa (30 mg/kg) subcutaneous administration were assessed. A battery of behavioral tests was performed to compare MB-COMT deficient mice and their wild type littermates of both sexes. MB-COMT deficient mice were seemingly normal, bred usually and had unaltered COMT activity in the brain and periphery despite a complete lack of the MB-COMT protein. MB-COMT deficient male mice showed higher extracellular dopamine levels than their wild-type littermates in the striatum, but not in the mPFC. In addition, the MB-COMT deficient male mice exhibited a distinct endophenotype characterized by schizophrenia-related behaviors like aggressive behavior and reduced prepulse inhibition. They also had prolonged immobility in the tail suspension test. Both sexes were sensitized to acute pain and had normal motor activity but disturbed short-term memory. Hence the behavioral phenotype was not limited to schizophrenia-related endophenotype and some behavioural findings were not sex-dependent. Our findings indicate that MB-COMT is critical for behavior, and its function in COMT-dependent brain areas cannot be entirely substituted by the remaining S-COMT.

Purification, crystallization and preliminary crystallographic studies of an integral membrane protein, cytochrome bo3 ubiquinol oxidase from Escherichia coli.

Cytochrome bo(3) ubiquinol oxidase has been successfully purified for crystallization. Single crystals of this integral membrane protein diffract X-rays to 3.5 A resolution and belong to the orthorhombic space group C222(1). From the diffraction data, the unit-cell parameters were determined to be a = 91.3, b = 370.3, c = 232.4 A. The crystals have a solvent content of 59% and contain two molecules per asymmetric unit. A search model generated from the structures of cytochrome c oxidase from Paracoccus denitrificans and the extrinsic domain of cytochrome bo(3) ubiquinol oxidase from Escherichia coli was used for molecular-replacement studies, resulting in a solution with sensible molecular packing.

Peripheral enhancement and spatial contrast uptake heterogeneity of primary breast tumours: quantitative assessment with dynamic MRI.

PURPOSE: The purpose of our study was to determine if quantitative analysis of peripheral enhancement and spatial contrast uptake heterogeneity is useful in the characterisation of abnormalities seen at breast MRI. METHOD: Ninety-one women underwent dynamic gadolinium-enhanced breast MRI. Regions of interest were processed by a parallel thinning algorithm to define central and peripheral subregions of lesions. Peripheral enhancement was quantified at every time point of the dynamic sequence as a signal difference-to-noise ratio. Moreover, a radiologist assessed the images for the presence of peripheral enhancement and classified the regional and subregional time-intensity profiles of each lesion. RESULTS: Sixty-four invasive carcinomas and 30 benign lesions were analysed. Significant differences were found between benign and malignant lesions in peripheral enhancement as determined from the dynamic images (p = 0.0002; sensitivity, 0.34; specificity, 1.00) in time-intensity profiles (p < 0.000005; sensitivity, 0.67; specificity, 0.93) and in peripheral percentage signal changes at 1 min postcontrast (p = 0.001). There was a much higher relative signal increase centrally than peripherally (p < 0.0005), but peripheral signal changes had greater diagnostic value than central ones (Az = 0.72 vs. 0.63; p = 0.02). Carcinomas showed higher peripheral enhancement than benign lesions (p = 0.001). Peripheral enhancement reached maximum diagnostic value at 4 min postcontrast (Az = 0.80) and performed best as a highly sensitive but moderately specific diagnostic index. CONCLUSION: Quantification of peripheral enhancement is diagnostically useful and offers insight into the enhancement mechanisms encountered in breast MRI. Primary breast tumours show substantial spatial contrast uptake heterogeneity. Lesion differentiation based on percentage signal changes is improved by restricting sampling to the periphery of tumours.

Effect of temporal resolution on the diagnostic efficacy of contrast-enhanced MRI in the conservatively treated breast.

PURPOSE: Our goal was to assess the effect of image acquisition rate on the diagnostic efficacy of contrast-enhanced MRI in the conservatively treated breast. METHOD: Sixty-seven women, treated with breast-conserving surgery and radiotherapy for early-stage breast cancer, were imaged at 1.5 T using a dynamic contrast-enhanced sequence with a temporal resolution of 12 s. Enhancement was recorded over time for the dominant lesion in each patient. Data were subsequently removed to simulate various acquisition rates and the enhancement indexes of benign and malignant lesions were compared. RESULTS: Seventeen patients had confirmed local recurrence and 50 remained disease-free. There were significant differences in the enhancement indexes of benign and malignant lesions 24-264 s after contrast agent administration. Acquisition rate had a negligible effect upon diagnostic efficacy. CONCLUSION: Two image data sets collected before contrast agent administration and between 1.5 and 3.5 min afterward may be sufficient to differentiate recurrent and benign disease.

Primary breast abnormalities: selective pixel sampling on dynamic gadolinium-enhanced MR images.

PURPOSE: To evaluate a region-of-interest (ROI) analytic method that involves selective sampling of pixels within predetermined ranges of contrast material enhancement values ("thresholding") on magnetic resonance (MR) images of primary breast abnormalities. MATERIALS AND METHODS: Dynamic gadolinium-enhanced MR images were obtained in 105 women. ROIs were drawn to outline the full extent of lesions. Relative signal intensity increase was determined on a pixel-by-pixel basis on 1- and 2-minute postcontrast images, as was the maximum relative signal intensity increase. Thresholding was used to analyze each ROI, with the upper boundary defined by the highest pixel value and lower boundaries of 0%-100%. RESULTS: Seventy-one invasive carcinomas and 37 benign lesions were analyzed. Narrower thresholding (i.e., larger percentage) resulted in an increase in all enhancement ratios (P < .0005). The enhancement ratio on 1-minute postcontrast images differed between benign lesions and carcinomas (P < .0005), but there were no significant differences in 2-minute and maximum ratios. Mean enhancement differences between benign and malignant lesions increased with narrower thresholding, but variability also increased linearly. Results of receiver operating characteristic analysis showed that thresholding did not affect the diagnostic usefulness of enhancement ratios. CONCLUSION: Contrary to current opinion, selective sampling of the most enhancing areas of breast abnormalities may not provide any diagnostic advantage over the use of easily drawn, lesion-encompassing ROIs.

Glutamic acid 286 in subunit I of cytochrome bo3 is involved in proton translocation.

Glutamic acid 286 (E286; Escherichia coli cytochrome bo3 numbering) in subunit I of the respiratory heme-copper oxidases is highly conserved and has been suggested to be involved in proton translocation. We report a technique of enzyme reconstitution that yields essentially unidirectionally oriented cytochrome bo3 vesicles in which proton translocation can be measured. Such experiments are not feasible in the E286Q mutant due to strong inhibition of respiration, but this is not the case for the mutants E286D and E286C. The reconstituted E286D mutant enzyme readily translocates protons whereas E286C does not. Loss of proton translocation in the D135N mutant, but not in D135E or D407N, also is verified using proteoliposomes. Stopped-flow experiments show that the peroxy intermediate accumulates in the reaction of the E286Q and E286C mutant enzymes with O2. We conclude that an acidic function of the 286 locus is essential for the mechanism of proton translocation.

Magnetic resonance imaging of the uterus after endometrial resection.

OBJECTIVE: Despite the increasing popularity of endometrial resection for the treatment of menstrual problems, the long term sequelae of this procedure are poorly recognised. As diagnostic hysteroscopy following endometrial resection is frequently unrewarding and transvaginal ultrasound is incapable of detecting subtle changes in endometrial morphology, magnetic resonance imaging was employed to evaluate the uterus. DESIGN: Retrospective study of unselected post-operative women. METHODS: Fifty-nine women, of whom 22 were amenorrhoeic, were studied a mean number of 34 months after endometrial resection, using a 1.5 Tesla magnetic resonance imaging system with a pelvic phased array coil for signal reception. T2-weighted FSE images were acquired through the long and short uterine axis and volumetric assessment of each uterine layer performed using an ISG Allegro workstation. MAIN OUTCOME MEASURES: The location and volume of residual endometrium, the volume of junctional zone and myometrial tissue, and the presence of additional intrauterine and pelvic pathology were recorded. RESULTS: Residual endometrium was demonstrated in all except three amenorrhoeic women, with a similar mean volume present in menstruating and amenorrhoeic groups. Additional findings included adenomyosis, haematometra, fallopian tube dilatation and free intraperitoneal fluid. CONCLUSIONS: The majority of amenorrhoeic and all menstruating women have residual endometrium after endometrial resection. The lack of communication of islands of residual endometrium with the uterine cavity results in haematometra formation, fallopian tube dilatation and possibly free intraperitoneal fluid.

Dynamic MR imaging of the breast combined with analysis of contrast agent kinetics in the differentiation of primary breast tumours.

OBJECTIVE: To assess dynamic Gd-DTPA-enhanced magnetic resonance (MR) imaging in the diagnosis of primary breast pathology, and to test the hypothesis that analysis of contrast agent kinetics increases specificity. METHODS: Forty-seven women underwent breast MR imaging using three-dimensional and dynamic spoiled gradient-recalled sequences. Image interpretation was based on the evaluation of lesion conspicuity, signal intensity, contour and enhancement pattern from the static acquisitions. Assessment of contrast kinetics was based on pixel-by-pixel analysis of the dynamic data. A two-compartment model described by three parameters (amplitude of uptake, exchange rate and washout rate), and a three-compartment model described by two parameters (permeability and exchange rate) were used. Regions of interest were drawn for all lesions found in the dynamic sections. Mean regional pixel values were calculated for each parameter and tested for diagnostic efficacy. RESULTS: Twenty-two malignant and 36 benign lesions were examined. Fibroadenomas accounted for 86% of the benign tumours. Image interpretation had a sensitivity of 0.95 and specificity of 0.86. The fat-suppressed post-contrast images permitted good visualization of the contour and matrix characteristics of fibroadenomas, but all non-fibroadenomatous benign lesions were classified as indeterminate or suspicious. Significant differences were found between benign and malignant lesions in the amplitude of uptake (P = 0.0008) and exchange rate (P < 0.00005) of the two-compartment model, and permeability (P=0.0001) and exchange rate (P < 0.00005) of the three-compartment model. However, image interpretation was superior to the isolated use of quantitative indices (P=0.02). The most discriminating parameters were the exchange rates of both models, with no significant difference between them. CONCLUSION: Assessment of lesion morphology is essential and probably sufficient for the differentiation of fibroadenomas from malignant tumours. However, specificity of conventional MR imaging may be much lower for other types of primary benign breast pathology. Analysis of Gd-DTPA kinetics improves the specificity obtained using simple enhancement measurements and can be used to produce parametric images that provide information about lesion heterogeneity, permeability and vascularity.

Unchanged basal ganglia N-acetylaspartate and glutamate in idiopathic Parkinson's disease measured by proton magnetic resonance spectroscopy.

The lentiform nucleus of five patients with idiopathic Parkinson's disease (IPD) was studied by quantitative magnetic resonance spectroscopy (MRS), both before and after administration of apomorphine, and the spectra were compared with those from a group of age-matched normal subjects. The concentrations of the three major metabolites, choline, creatine, and N-acetylaspartate (NAA), were quantified using tissue water content as an internal concentration reference. Glutamate concentration was assessed as the (glutamate + glutamine; GLX)/creatine peak area ratio. In normal subjects, the mean +/- SD concentrations of the the three metabolites were 2.4 +/- 0.4 mumol/g wet wt for choline, 11.5 +/- 0.8 mumol/g for creatine, and 14.7 +/- 2.8 mumol/g for NAA. The Glx/creatine ratio was 1.26 +/- 0.12. There was no significant difference in these parameters in the lentiform nucleus of patients with IPD either before or after apomorphine. The absence of detectable differences in IPD in this study implies that the changes in glutamate metabolism in the basal ganglia predicted by animal work are more subtle than those currently observable by MRS.

Microvessel density of invasive breast cancer assessed by dynamic Gd-DTPA enhanced MRI.

It has been postulated that the rapid enhancement demonstrated by breast carcinomas after administration of contrast media is a direct result of tumor angiogenesis. However, to date, little quantitative data have been published to support this view. A retrospective study has been undertaken to compare dynamic contrast-enhanced data obtained from 40 patients with microvessel density (MVD) evaluated in specimens immunohistochemically stained with a factor VIII related antigen. The dynamic data were analyzed quantitatively using both simple indices of enhancement and a two-compartment kinetic model. A moderate but significant correlation was demonstrated between initial enhancement and MVD, and this correlation strengthened when node-positive tumors were considered in isolation (r = .77, P < .0005). However, the data showed considerable variability. The enhancement characteristics of the tumors could not be explained solely by their MVD; therefore, MRI cannot be used to predict MVD in vivo. Further work is required to address the exact relationship between contrast-enhanced MRI and tumor angiogenesis.

Dynamic MR imaging of invasive breast cancer: correlation with tumour grade and other histological factors.

The purpose of this study was to explore the association between dynamic MR enhancement characteristics and histopathological prognostic factors of invasive breast cancer. 53 women with primary invasive breast cancer underwent dynamic contrast enhanced breast MRI. Region of interest (ROI) analysis was performed on synthetic images obtained by kinetic modelling of the dynamic data. Operator-defined, large ROIs and computer-defined, 9-pixel ROIs were selected for each tumour. The relative increase in mean ROI pixel intensity was expressed in the form of enhancement ratios. Univariate and multivariate analyses were performed to explore the association of these ratios with standard histological factors, including tumour size, histopathological classification, histological grade, the presence of extensive in situ component and lymphovascular invasion, multifocal disease, and axillary lymph node status. All enhancement ratios showed significant differences between node-positive and node-negative tumours (max. p = 0.002). However, automated ROI ratios showed less overlap between node-positive and node-negative carcinomas than did large ROI ratios. A strongly significant association was observed between all automated ROI enhancement ratios and histological tumour grade (max. p = 0.001). Based on stepwise multiple regression analysis, node status and histological grade were the only histopathological factors with a significant independent effect on the enhancement characteristics. In summary, there is a strong association between dynamic MR characteristics and two important prognostic markers of invasive breast cancer, namely axillary node status and histological grade. This may allow MRI to be used in pre-operative predictions of tumour behaviour and biological activity.

Dynamic MRI of invasive breast cancer: assessment of three region-of-interest analysis methods.

PURPOSE: In this study, three region-of-interest (ROI) analysis methods based on operator-defined and semiautomated sampling of pharmacokinetic breast maps of contrast uptake are described. The observer variability and impact of the methods on the estimated enhancement characteristics of invasive cancer are also presented. METHOD: Fifty-four women with invasive breast cancer underwent dynamic Gd-DTPA-enhanced MRI. ROIs were drawn by two observers on parametric images obtained from compartmental modeling of the dynamic data. Three methods were used: (a) An irregular ROI was drawn to include as much of the enhancing part of the tumor as possible (large ROI); (b) a 12 pixel circular ROI was placed at the most rapidly enhancing part of the large region (small ROI); and (c) a computer algorithm interrogated the large region pixel by pixel using a 9 pixel square mask and selected the region with the highest mean parameter value (semiautomated ROI). RESULTS: Significant observer variability and bias were found in the enhancement measurements using the large ROI method. There was no observer bias associated with the other methods, but the variability of the small ROI method was substantial. An almost perfect observer agreement was achieved using the semiautomated method. The small and semiautomated ROI methods produced significantly higher enhancement ratios than the large ROI method, especially in grade III carcinomas. CONCLUSION: Variability is inherent in subjective ROI analysis, but the semiautomated method of ROI selection and sampling of parameter images of the breast is an efficient and reliable alternative that may allow better standardization of the MR technique.

Prediction of axillary lymph node status in invasive breast cancer with dynamic contrast-enhanced MR imaging.

PURPOSE: To determine if magnetic resonance (MR) imaging can be used to predict axillary lymph node status in patients with breast cancer. MATERIALS AND METHODS: Fifty-one women with primary invasive breast cancer underwent dynamic contrast material-enhanced MR imaging of the breast Region-of-interest (ROI) analysis was performed on parametric images obtained with kinetic modeling of the data. Large and automated ROIs were selected. Typical enhancement ratios that represented the relative increase in mean pixel signal intensity were calculated for each ROI. Stepwise logistic regression analysis was applied to identify prognostic factors of axillary node status. Receiver operating characteristic analysis was performed and a Brier score and calibration curve were calculated to assess the diagnostic efficacy and predictive capability of the logistic regression model. RESULTS: The maximum enhancement ratio of the automated ROI was found to be the strongest predictor of node status (P < .001). Patient age (P = .007) and ROI size (P = .045) were also significant predictor variables. The model showed good accuracy (area beneath the fitted binormal receiver operating characteristic curve [Az] = 0.90; Brier score, 0.133). In 12 (24%) of the patients, a less than 5% or greater than 95% probability of positive-node status was correctly identified. CONCLUSION: The suggested predictive model may decrease the need for surgical staging of the axilla in patients with breast cancer.

In vivo quantification of citrate concentration and water T2 relaxation time of the pathologic prostate gland using 1H MRS and MRI.

We have previously reported a striking correlation between water T2 relaxation time and citrate concentration in the normal prostate (Liney G.P.; Lowry M.; Turnbull L.W.; Manton D.J.; Knowles A.J.; Blackband S.J.; Horsman A. Proton MR T2 maps correlate with the citrate concentration in the prostate. NMR Biomed. 9:59-64; 1996). In this study we present data from similar studies of the pathologic gland. The findings support the hypothesis that measurement of both citrate concentration and water T2 relaxation time in vivo may aid the differentiation of prostatic carcinoma from benign disease and normal tissue.

Tumour volume determination from MR images by morphological segmentation.

Accurate tumour volume measurement from MR images requires some form of objective image segmentation, and therefore a certain degree of automation. Manual methods of separating data according to the various tissue types which they are thought to represent are inherently prone to operator subjectivity and can be very time consuming. A segmentation procedure based on morphological edge detection and region growing has been implemented and tested on a phantom of known adjustable volume. Comparisons have been made with a traditional data thresholding procedure for the determination of tumour volumes on a set of patients with intracerebral glioma. The two methods are shown to give similar results, with the morphological segmentation procedure having the advantages of being automated and faster.

Observer variability in the interpretation of contrast enhanced MRI of the breast.

The purpose of this study was to determine observer variability in the interpretation of contrast enhanced breast MRI and to evaluate its effect on the detection and differentiation of breast cancer. 57 women underwent breast MRI using spin echo and dynamic spoiled gradient-recalled sequences. Images were independently reviewed by three radiologists, two experienced and one newly trained in breast MRI interpretation. One of the experienced readers reviewed all examinations twice. Interpretation was based on lesion conspicuity, signal intensity, contour and enhancement pattern. Contrast uptake was assessed using region of interest (ROI) analysis of the dynamic images and calculation of a maximum enhancement index. Sensitivity and specificity in the diagnosis of malignancy irrespective of disease extent, and in the diagnosis of multifocal malignancy were estimated. 113 lesions were reported. Kappa coefficient estimations showed only a moderate agreement between the two experienced readers in rating morphological characteristics; the agreement between the newly trained reader and the experienced readers was even worse. Moreover, there was significant interobserver and intraobserver variation in the enhancement index measurements. Weighted kappa values indicated good agreement between the experienced readers in lesion and overall interpretation, excellent intraobserver agreement, but substantial disagreement between the newly trained reader and both experienced readers. All readers showed good sensitivity in cancer detection, but specificity was substantially lower. There is significant observer variability and a substantial learning curve in the interpretation of breast MRI, and variability in the ROI analysis of dynamic data. Further efforts to improve the reliability of ROI analysis and image interpretation are needed to help MRI realise its full potential in the clinical management of breast cancer.

Maturation effects on the NMR microimaging characteristics of single neurons.

We have used an isolated neuronal preparation of single Aplysia californica L7 cells to study the effects of development on the nuclear magnetic resonance (NMR) characteristics, T2 spin-spin relaxation rate (RT2) and apparent diffusion coefficient (D), of intracellular water within the nuclear and cytoplasmic compartments. These studies demonstrate a significant correlation of animal weight, but not age, with RT2. On the other hand, D was not significantly different as a function of either age or weight in single L7 neurons. Demonstration of maturation dependence in single cells is important in understanding the cellular origins of developmental effects on NMR characteristics in cell assemblies such as brain.

Quantification of citrate concentration in the prostate by proton magnetic resonance spectroscopy: zonal and age-related differences.

A commercial phased-array multicoil was used to acquire water-suppressed localized proton spectra of the two major anatomical regions of the prostate. The signal-to-noise ratio and spectral resolution allowed identification of peaks from choline and creatine, as well as a major peak from citrate. Quantification of the citrate peak using experimentally determined relaxation parameters with tissue water as an internal concentration reference revealed a marked variability between different volunteers. Nevertheless, in each case, the citrate concentration was up to fourfold greater in the peripheral zone than in the central gland. Furthermore, the difference in citrate concentration between these two regions was positively correlated with the subjects age. The results indicate a consistent difference in cellular function between the major anatomical regions within the prostate and may have important consequences for the application of magnetic resonance spectroscopy to the diagnosis of prostatic pathology.

Comparison of conventional single echo and multi-echo sequences with a fast spin-echo sequence for quantitative T2 mapping: application to the prostate.

The accuracy of water T2 maps generated from a fast spin-echo (FSE) sequence was compared with data obtained by conventional single and multi-echo spin-echo pulse sequences using a commercial gel phantom. Spatially localized stimulated echo acquisition mode (STEAM) proton spectroscopy was also used to confirm the reported water T2 values of the gels contained in the phantom. The FSE sequence was shown to be superior in accuracy to both the single and multi-echo spin echo sequences and comparable to STEAM, producing results that were within 10% of known values. The effectiveness of the FSE sequence was further demonstrated by generating T2 maps of the normal and diseased prostate in clinically acceptable imaging times, resulting in comparable T2 values to those obtained using STEAM. Accurate quantitative T2 maps can be produced with the FSE sequence.