Search

Purpose: To accurately reconstruct, and interactively reshape 3D anatomy structures' surfaces using small numbers of 2D contours drawn in the most visually informative views of 3D imagery. The innovation of this program is that the number of 2D contours can be very much smaller than the number of transverse sections, even for anatomy structures spanning many sections. This program can edit 3D structures from prior segmentations, including those from autosegmentation programs. The reconstruction and surface editing works with any image modality., Methods: Structures are represented by variational implicit surfaces defined by weighted sums of radial basis functions (RBFs). Such surfaces are smooth, continuous, and closed and can be reconstructed with RBFs optimally located to efficiently capture shape in any combination of transverse (T), sagittal (S), and coronal (C) views. The accuracy of implicit surface reconstructions was measured by comparisons with the corresponding expert-contoured surfaces in 103 prostate cancer radiotherapy plans. Editing a pre-existing surface is done by overdrawing its profiles in image views spanning the affected part of the structure, deleting an appropriate set of prior RBFs, and merging the remainder with the new edit contour RBFs. Two methods were devised to identify RBFs to be deleted based only on the geometry of the initial surface and the locations of the new RBFs., Results: Expert-contoured surfaces were compared with implicit surfaces reconstructed from them over varying numbers and combinations of T/S/C planes. Studies revealed that surface-surface agreement increases monotonically with increasing RBF-sample density, and that the rate of increase declines over the same range. These trends were observed for all surface agreement metrics and for all the organs studied-prostate, bladder, and rectum. In addition, S and C contours may convey more shape information than T views for CT studies in which the axial slice thickness is greater than the pixel size. Surface editing accuracy likewise improves with larger sampling densities, and the rate of improvement similarly declines over the same conditions., Conclusions: Implicit surfaces based on RBFs are accurate representations of anatomic structures and can be interactively generated or modified to correct segmentation errors. The number of input contours is typically smaller than the number of T contours spanned by the structure.

Purpose: To validate and clinically evaluate autocontouring using atlas-based autosegmentation (ABAS) of computed tomography images., Methods and Materials: The data from 10 head-and-neck patients were selected as input for ABAS, and neck levels I-V and 20 organs at risk were manually contoured according to published guidelines. The total contouring times were recorded. Two different ABAS strategies, multiple and single subject, were evaluated, and the similarity of the autocontours with the atlas contours was assessed using Dice coefficients and the mean distances, using the leave-one-out method. For 12 clinically treated patients, 5 experienced observers edited the autosegmented contours. The editing times were recorded. The Dice coefficients and mean distances were calculated among the clinically used contours, autocontours, and edited autocontours. Finally, an expert panel scored all autocontours and the edited autocontours regarding their adequacy relative to the published atlas., Results: The time to autosegment all the structures using ABAS was 7 min/patient. No significant differences were observed in the autosegmentation accuracy for stage N0 and N+ patients. The multisubject atlas performed best, with a Dice coefficient and mean distance of 0.74 and 2 mm, 0.67 and 3 mm, 0.71 and 2 mm, 0.50 and 2 mm, and 0.78 and 2 mm for the salivary glands, neck levels, chewing muscles, swallowing muscles, and spinal cord-brainstem, respectively. The mean Dice coefficient and mean distance of the autocontours vs. the clinical contours was 0.8 and 2.4 mm for the neck levels and salivary glands, respectively. For the autocontours vs. the edited autocontours, the mean Dice coefficient and mean distance was 0.9 and 1.6 mm, respectively. The expert panel scored 100% of the autocontours as a "minor deviation, editable" or better. The expert panel scored 88% of the edited contours as good compared with 83% of the clinical contours. The total editing time was 66 min., Conclusion: Multiple-subject ABAS of computed tomography images proved to be a useful novel tool in the rapid delineation of target and normal tissues. Although editing of the autocontours is inevitable, a substantial time reduction was achieved using editing, instead of manual contouring (180 vs. 66 min)., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Treatment planning for high precision radiotherapy of head and neck (H&N) cancer patients requires accurate delineation of many structures and lymph node regions. Manual contouring is tedious and suffers from large inter- and intra-rater variability. To reduce manual labor, we have developed a fully automated, atlas-based method for H&N CT image segmentation that employs a novel hierarchical atlas registration approach. This registration strategy makes use of object shape information in the atlas to help improve the registration efficiency and robustness while still being able to account for large inter-subject shape differences. Validation results showed that our method provides accurate segmentation for many structures despite difficulties presented by real clinical data. Comparison of two different atlas selection strategies is also reported.

Image segmentations based on maximum likelihood or maximum a posteriori analyses of object textures usually assume parametric models (e.g., Gaussian) for distributions of these features. For real images, parameter accuracy and model stationarity may be elusive, so that model-free inference methods ought to have an advantage over those that are model-dependent. Functions of the relative entropy (RE) from information theory can produce minimum error, model-free inferences, and can detect the boundary of an image object by maximizing the RE between the pixel distributions inside and outside a flexible curve contour. A generalization of the RE -- the Jensen-Rényi divergence (JRD) -- computes optimal n-way decisions and can contour multiple objects in an image simultaneously. Seed regions expand naturally and multiple contours tend not to overlap. An edge detector based on the JRD, combined with multivariate pixel segmentation, generally improved the error of the segmentation. We apply these functions to contour patient anatomy in X-ray computed tomography for radiotherapy treatment planning.

Electrophysiological data from the rodent whisker/barrel cortex indicate that GABAergic, presumed inhibitory, neurons respond more vigorously to stimulation than glutamatergic, presumed excitatory, cells. However, these data represent very small neuronal samples in restrained, anesthetized, or narcotized animals or in cortical slices. Histochemical data from primate visual cortex, stained for the mitochondrial enzyme cytochrome oxidase (CO) and for GABA, show that GABAergic neurons are more highly reactive for CO than glutamatergic cells, indicating that inhibitory neurons are chronically more active than excitatory neurons but leaving doubt about the short-term stimulus dependence of this activation. Taken together, these results suggest that highly active inhibitory neurons powerfully influence relatively inactive excitatory cells but do not demonstrate directly the relative activities of excitatory and inhibitory neurons in the cortex during normal behavior. We used a novel double-labeling technique to approach the issue of excitatory and inhibitory neuronal activation during behavior. Our technique combines high-resolution 2-deoxyglucose (2DG), immunohistochemical staining for neurotransmitter-specific antibodies, and automated image analysis to collect the data. We find that putative inhibitory neurons in barrel cortex of behaving animals are, on average, much more heavily 2DG-labeled than presumed excitatory cells, a pattern not seen in animals anesthetized at the time of 2DG injection. This metabolic activation is dependent specifically on sensory inputs from the whiskers, because acute trimming of most whiskers greatly reduces 2DG labeling in both cell classes in columns corresponding to trimmed whiskers. Our results provide confirmation of the active GABAergic cell hypothesis suggested by CO and single-unit data. We conclude that strong activation of inhibitory cortical neurons must confer selective advantages that compensate for its inherent energy inefficiency.

Objective: Senile plaques (SP) are one of the characteristic neuropathologic lesions of Alzheimer's Disease (AD), and studies of SP cortical distribution, density and morphology may lead to new information about the mechanism and pathogenesis of AD. We used an automated, digital image analysis program to detect and measure SP size, shape and total fractional area in digital images of silver-stained tissue sections., Study Design: The program observed 94,000 SP in 2,800 digitized microscope fields from tissue sections from 42 postmortem cases ranging from healthy aged to severely demented subjects, studied prospectively before death., Results: Automated pattern recognition can measure SP densities in excellent agreement with an expert and can generate morphometric information not obtainable by conventional microscopy. SP densities (number of SPs/mm2) strongly correlate with tissue load (fraction of tissue area occupied by lesions). SP densities strongly correlate between cortical regions within the same subjects. SP densities, while correlating with the occurrence of AD, do not display a significant trend with respect to dementia severity; likewise, mean SP area and shape properties do not vary significantly with dementia severity. Finally, all the computed SP densities would have produced the same diagnoses of AD in these subjects as the manual SP densities according to the consensus criteria., Conclusion: This is the first fully automated program to identify SPs and measure SP morphometry; it uses well-established digital image analysis and statistical pattern recognition methods. The computed SP densities correlate highly with expert results, and the systematic differences are smaller than the interrater differences reported in several multicenter Alzheimer's disease neuropathology studies. The program measures morphometric properties that would be impractical to measure by manual means and, with program-controlled, scanning stage microscopy, can measure lesion densities exhaustively across large cortical areas without stereologic sampling. SP densities rise from near zero to significant values at the mildest diagnosed stage of AD, but beyond this point, there is no demonstrable correlation of density, or any other SP property, with dementia severity. Computed SP densities for even the mildest dementia satisfy the consensus diagnostic criteria.

The one-to-one correspondence of whiskers to barrels in layer IV of rodent somatosensory cortex can be demonstrated by a precise match between columns of heavy 2-deoxyglucose (2DG) label in layer IV barrels and other layers which correspond to stimulated whiskers. While there is specificity of peripheral-to-central mapping, the extent to which integration and/or modulation are generated by circuitry within or interactions between the barrel-defined whisker columns is not clear. Following stimulation of selected whiskers, large cells at the layer IV-V boundary throughout the barrel field are heavily labeled by 2-deoxyglucose (2DG) at high resolution. Many of these cells are outside the barrel columns of the stimulated whiskers. Further, the number of cells labeled is not directly related to the number of activated barrel columns. These neurons are not labeled in animals anesthetized before 2DG injection and are not as heavily labeled in barrel fields of somnolent animals. Most of the heavily labeled neurons immunolabel for glutamate decarboxylase (GAD) and are presumed to be inhibitory, while a smaller number of labeled neurons, presumed to be excitatory, immunolabel for glutamate (Glu). Similar populations of large, heavily 2DG-labeled neurons are found in other cortical areas. These relatively few neurons are exceptionally active and may modulate integrative functions of cerebral cortex.

The cerebral cortex is distinguished by layers of neurons of different morphologies and densities. The layers are formed by the migration of newly generated neurons from the ventricular zone to the cortical plate near the outer (pial) boundary of the cortex, along radial paths approximately perpendicular to the cortical surface. Immunochemical labelling makes these cells' patterns visible in brightfield microscopy so that layer formation can be studied. We developed a suite of programs that automatically digitize the entire cortex, identify the labelled cells and compute cell densities along local radial paths. Cell identification used supervised classification on all the significantly stained objects corresponding to maxima in lowpass filtered versions of the digital microgrphs. Classification of all the stained objects as cells or noncell objects was made by a decision rule based on morphometric and grey-level texture features, including features based on Gabor functions. Detection sensitivity and classification accuracy were jointly maximized on training data consisting of about 3000 expert-identified neurons in micrographs. Total program performance was tested on a separate (test) set of labelled neurons the same size as the training data set. The program detected 85% of the cells in the test set with a total error of 0.19. The identified cells' locations were used to compute population densities along normals to the cortical layers, and these densities served as a measure of neuronal migration. Transcortical density profiles obtained by computation and by manual cell counting were very similar. The cell identification program was built on well-established methods in statistical pattern recognition and image analysis and should generalize readily to other histological preparations.

Senile plaques (SP) are one of the characteristic neuropathologic lesions of Alzheimer's Disease (AD), and studies of SP cortical distribution, density (number of SP/mm2), and morphology are expected to lead to new information about the mechanism and pathogenesis of AD. We describe a digital image analysis procedure to detect SP, and to measure SP size, shape, and total fractional area in digital micrographs of silver-stained tissue sections. This histology is nonspecific so the program detects all the significant stained objects and a classifier sorts the SP from other tissue elements. SP vary greatly in size and form, and detection is based on multiscale template correlation. Three independent comparisons of computed versus expert-determined SP densities produced correlation coefficients greater than 0.8. The program found 94,000 SP in 2800 digital images of tissue sections from 42 postmortem cases including healthy aged controls and severely demented subjects.

Senile plaques (SP) are the most characteristic neuropathologic lesions of Alzheimer's disease (AD) and studies of plaque cortical distribution, density, and morphology may lead to new information about the origin and pathogenesis of this disease. We have developed an automated computer image analysis program to detect SP (including diffuse and mature forms) and to measure SP size, shape, and fractional area or load in digital micrographs of silver-stained tissue sections. The plaques are detected with adaptive thresholding, requiring no user interaction. Measures of SP size, morphology, and load are readily calculated from the pixel values in the detected SP features. These measurements are achieved accurately and exhaustively, and this method offers an alternative to manual SP counting. We demonstrate its application to 4 cases spanning the full range of the severity of the disease.

The primary motor cortex (M1) is thought to control movements of different body parts from somatotopically organized cortical territories. Electrical stimulation suggests, however, that territories controlling different fingers overlap. Such overlap might be artifactual or else might indicate that activation of M1 to produce a finger movement occurs over a more widespread cortical area than usually assumed. These possibilities were distinguished in monkeys moving different fingers. Recordings showed that single M1 neurons were active with movements of different fingers. Neuronal populations active with movements of different fingers overlapped extensively. Control of any finger movement thus appears to utilize a population of neurons distributed throughout the M1 hand area rather than a somatotopically segregated population.

Image alignment is an absolute requirement for three-dimensional (3-D) reconstruction from serial sections, and Fourier correlation is the most powerful way to compute alignments. The rotational and translational components of misalignment can be corrected by an iterative correlation procedure, but for images having significant differences, alignment can fail with a likelihood proportional to the extent of the differences. We found that translational correction was determined much more reliably when low-pass filters were applied to the product transforms from which the correlations were calculated. Rotational corrections based on polar analyses of the auto-correlations of the images instead of on the images directly contributed to more accurate alignments. These methods were used to generate 3-D reconstructions of brain capillary modules from serial-section mosaics of digitized transmission electron micrographs.

Extracellular action potentials of single neurons in motor cortex and rectified and integrated electromyographic activity (EMG) of gastrocnemius and anterior tibialis were recorded while a monkey performed isometric ankle plantar and dorsal flexion tasks. This study determined the consistency of neuronal behaviors across different tasks. Methods characterized neuronal behaviors by determining which behavioral event within a single task, such as the appearance of the 'go' signal, force onset, or agonist and antagonist EMG onset, was best related to changes in neuronal activity. Another method compared the temporal profiles of discharge modulation across different tasks. Of 220 neurons recorded, 44 were selected because they were consistently active in the tasks. Of these, 37 were in the precentral cortex and the remaining seven were in the postcentral cortex. Only 14 of the 33 in motor cortex were consistent in their behavioral correlations. Several had multiple changes in activity within a single task that were related to different behavioral events. Half were consistent for direction of force and a third were consistent for magnitude of force. Furthermore, there was little consistency in the temporal profiles of discharge activity for all 44 neurons across tasks. Similar modulations of discharge activity among neurons in one task were different in another task. Such inconsistencies are evidence against the cardinal cell hypothesis of physiological representation. We offer a new hypothesis analogous to connectionism in parallel distributed processing.

Three-dimensional reconstructions from serial section images require the accurate registration of those images. Image correlation is the most powerful computed alignment method and its performance on identical images, or parts thereof, has been thoroughly studied. Correlation alignments of complex, dissimilar images can fail, however, with a likelihood proportional to the magnitude of the differences. We report that alignments can be computed more reliably and more accurately (higher-valued correlation coefficients) by the combined use of lowpass-filtered product transforms (from which the correlation functions are formed), autocorrelation correction of rotational misalignment, and covariance correction of translation misalignment. A simple rule is proposed for the lowpass filter cutoff radius depending on measures of the images' differences. These methods are demonstrated with a reconstruction of a capillary loop in the median eminence of the hypothalamus.

The effect of Althesin, an anesthetic comprising two steroids, on regional cerebral function was determined by measurement of regional cerebral glucose utilization. Rats were anesthetized with an intravenous dose of 4, 8, or 20 mg total steroid/kg. These doses produced anesthesia for 12, 18, and 37 min, respectively. There were no physiologically significant effects of Althesin (20 mg/kg) on body temperature, blood pH, or blood gases. Blood pressure and heart rate decreased slightly after administration of Althesin. Althesin had a profound effect on glucose consumption in many, but not all, cerebral structures. The forebrain (especially cerebral cortex) was affected most, while the hindbrain was much less so or not at all. This pattern of functional depression is in accord with the minimal effects observed on physiologic variables. The effects of Althesin differ from those of other known anesthetics and suggest a unique mechanism. The possibility of action through naturally occurring steroid receptors is considered.

Quantitative autoradiography of brain glucose metabolism has been combined with digital image processing to represent the brain as a three-dimensional (3-D) reconstruction of brain energy use. Autoradiographs contain enormous amounts of potentially useful data, but conventional analyses, based on tedious manual methods, can sample and analyze only a small portion of this information. Computer 3-D reconstruction provides a mechanism for observing and analyzing all the data; therefore, a system of computer programs was developed for this purpose. The programs use digital imaging methods for image registration, superimpose whole brain data sets, and allow resampling of the 3-D data in arbitrary planes for pixel-by-pixel comparisons among multiple 3-D sets. These programs operate on the mathematical properties of the images alone, obviating the need for manual image alignment. Various statistical analyses can be applied to the data directly to study the patterns of metabolic changes in different experiments. The system is applied to data from experiments on the influence of injectable anesthetics on cerebral glucose metabolism.

Regional cerebral glucose influx was measured using quantitative autoradiography after the intravenous infusion of [2-14C]glucose for a period of 10 or 20 s. Glucose influx varied considerably among structures over an almost threefold range. When compared with rates of regional glucose utilization, a significant correlation by region was found between glucose influx and utilization, demonstrating that the glucose supply to individual cerebral structures is closely matched to their metabolic needs.

The efficacy of [14C]glucose molecules labeled in various positions as tracers of regional cerebral glucose utilization (rCMRGlc) was examined in rats. Arteriovenous differences of different [14C]-glucose species and 14CO2 were measured across brain to determine the relative rates of 14CO2 loss. As anticipated, 14CO2 evolution decreased in the order: [U-14C]glucose greater than [2-14C]glucose greater than [1-14C]glucose greater than [6-14C]glucose. Release of 14CO2 from [6-14C]glucose was undetectable at 5 min and barely detectable at 10 min, and release from [1-14C]glucose, which includes the pentose phosphate pathway, was only slightly greater. rCMRGlc was measured with [1-14C]-,[2-14C]-, or [6-14C]glucose in 5-min experiments. The results of [1-14C]- and [6-14C]glucose were indistinguishable; no difference due to the activity of the pentose phosphate pathway was found. Both [1-14C]- and [6-14C]-glucose gave values similar to, but on the whole slightly higher than, [2-14C]glucose. It was concluded that when knowledge of total rCMRGlc is required, [6-14C]glucose is the labeled substrate of choice. When the experimental objective is measurement of energy metabolism, use of [1-14C]glucose avoids inclusion of the nonenergy-yielding pentose phosphate pathway.

The studies reported here were undertaken to determine whether the thrombin precursors prothrombin, prethrombin 1, prethrombin 2, and Meizo thrombin interact with the fluorescent, reversible thrombin inhibitor dansylarginine N,N-(3-ethyl-1,5-pentanediyl)amide (DAPA) [Nesheim, M. E., Prendergast, F. G., & Mann, K. G. (1979) Biochemistry 18, 996--1003]. The results indicate that prothrombin and prethrombin 1, in which the cleavage sites at Arg274-Thr275 and Arg323-Ile324 both remain intact, do not bind DAPA, while prethrombin 2 or Meizo thrombin, which results respectively from a single cleavage of prothrombin at Arg274-Thr275 or Arg323-Ile324, do bind the inhibitor. Since prethrombin 2 is a precursor of thrombin without measurable enzymatic activity, a thorough characterization of its interaction with DAPA was undertaken. The interaction of DAPA with bovine thrombin similarly was studied for comparative purposes. The binding of DAPA to either protein is accompanied by changes in the fluorescence properties of the dansyl moiety including increases in emission intensity, excited-state lifetime, polarization, and a slight blue shift in the wavelength of maximum emission intensity. Corrected excitation spectra indicate energy transfer to DAPA from one or more aromatic side chains of both proteins. Values of P0 for both complexes were extrapolated from Perrin plots of polarization vs. temperature and suggest that the dansyl moiety is held more rigidly in thrombin than in prethrombin 2. With excitation at either 280 or 335 nm the emission intensity of DAPA-prethrombin 2 is substantially less than that of the DAPA-thrombin complex. In contrast, the intensity of the Meizo thrombin-DAPA complex is greater than that of the DAPA-thrombin complex. From measurements of intensity changes the dissociation constants and stoichiometry of DAPA binding to thrombin and prethrombin 2 were measured. Prethrombin 2 binds to DAPA with a Kd = 5.9 x 10(-7) M (n = 1) while thrombin binds about 30 times more tightly with a Kd = 2.0 x 10(-8) M (n = 1). The active site directed irreversible thrombin inhibitors diisopropyl phosphorofluoridate and D-phenylalanylprolylarginyl chloromethyl ketone displace DAPA from thrombin but not from prethrombin 2. The results of these studies indicate the binding of a presumed substrate analogue (DAPA) to an inactive zymogen, prethrombin 2. In addition, the lack of DAPA binding to prothrombin and prethrombin 1, under conditions in which it binds to prethrombin 2, implicates events that accompany cleavage at Arg274-Arg275 in the "progressive" formation of an active site, even though further cleavage at Arg323-Ile324 is required for expression of enzymatic activity.

Autoradiography can generate large quantities of information related to brain metabolism, blood flow, transport across the blood-brain barrier, neurotransmitter-receptor binding and other aspects of brain function. Three-dimensional (3D) reconstruction of digitized autoradiograms provides a mechanism for efficient analysis of function, in detail, over the entire brain. 3D reconstructions of the mean and variance can be obtained by superimposing data from similar experiments, leading ultimately to 3D reconstructions of differences with statistical tests of significance. Image registration is essential for reconstruction, and this article reports two independent algorithms for coronal image alignment that have been successfully implemented in computer programs. The first algorithm superimposes the centroids and principal axes of serial images; the extent and direction of the translation and rotation required for each image is obtained from an analysis of the inertia matrix of that image. The second algorithm matches the edges of structure features in serial-adjacent images, from analyses of the cross-correlation function of each pair of adjacent images. The cross-correlation method requires a great deal more computation than the principal axes method, but it can align damaged sections not reliably treated by the principal axes method. The methods are described in detail, and a quantitative assessment of the registration of non-identical images is considered.

To test the hypothesis that the median eminence microvasculature has a direct regulatory role in the hormonal communication between the brain and the pituitary gland, it is necessary to determine whether the physical means for such control (e.g. smooth muscle sphincters strategically located in the capillary plexus) actually exists. Our approach is to search for such structures in transmission electron micrographs of thin serial sections of the median eminence. The complexity of these images and the anticipated need to include large numbers of them in the study led us to consider computer reconstruction for this problem. We report here the successful three-dimensional reconstruction of capillary modules using digital image processing techniques for capillary feature detection/extraction, for construction of montages (mosaics) of overlapping images of the same section, and for automatic image registration by two independent methods without the use of fiducial marks. These tasks have been performed manually in nearly all the published neurobiological reconstructions; here they are performed by programs using only the mathematical properties of the images. Methods like those described here provide the only practical means for executing large scale reconstructions and gaining significant new information about the regulation of blood flow in this region of the brain.

An endogenous brain ligand which competes with [3H]-flunitrazepam for the binding to benzodiazepine receptor has been isolated and purified to homogeneity. The purification procedures involve the extraction of the endogenous ligand by homogenizing the brain tissue in water containing various protease inhibitors followed by filtration through a PM 10 membrane (exclusion limit: 10,000-dalton), column chromatographies on Sephadex G-50, Bio-Rad P2 and a series of C18 reverse phase HPLC columns. The purified endogenous ligand was eluted as a single and symmetrical peak monitored at either 220 or 280 nm. Furthermore, the ligand activity coincided with the absorption peak. The purified endogenous ligand is thermostable, insensitive to various peptidases and proteolytic enzymes, resistant to DNAse, RNAse, and carbohydrate enzyme e.g. neuraminidase (EC 3.2.1.18) and acid treatment. It has a major absorption peak at 220 nm and a minor one at 313 nm. The endogenous ligand appears to be quite specific since it only inhibits the binding of ligand to the central type benzodiazepine receptor but not to other receptors, e.g. peripheral type benzodiazepine receptor, alpha 1-adrenoceptor, alpha 2-adrenoceptor, beta-adrenoceptor and muscarinic cholinergic receptor. Furthermore, the inhibition of the receptor binding by the endogenous ligand is enhanced by GABA suggesting that the endogenous ligand is a benzodiazepine receptor agonist. The structure of the endogenous ligand is unknown.

Crystals of alpha-chymotrypsin (CHT) at equilibrium in solutions of 2.0 M guanidine hydrochloride and 3.0 M urea at pH 3.6 were prepared, three-dimensional X-ray intensities were measured, and difference electron-density maps were calculated and examined. The guanidine hydrochloride derivative displayed changes occurring exclusively on the surface of the protein. The difference peaks represented mostly small changes in various protein surface groups and in the adjacent solvent regions, and some displayed convincing evidence of binding of the guanidinium ion to the protein. The urea difference map likewise showed that changes had occurred on the surface of the protein, but also that numerous changes in the structure occurred in the hydrophobic interior of the CHT molecule. Further, the urea difference map contained evidence for two kinds of interactions of urea with protein groups. There are examples of bound urea either causing or accompanying structural changes and examples of urea binding with no accompanying changes to the protein. Examples of both kinds of binding were observed in both the surface regions and in the hydrophobic interior of the molecule. From an examination of these two derivatives, it is clear that guanidine hydrochloride and urea unfold proteins by different mechanisms.

Spleen cells obtained from mice immunized with partially purified human coagulation Factor V were fused with NS-1 mouse myeloma cells, and hybrids were selected. Culture media were screened for anti-Factor V activity, and an antibody-positive clone was obtained and passaged as an ascites tumor in mice. The ascitic fluid from the hybridoma-bearing mouse could be diluted 1:10(6) before losing reactivity in an anti-Factor V radioimmunoassay. When immobilized on agarose, the monoclonal antibody quantitatively removed Factor V activity from human plasma. Factor V activity could be eluted with 1.2 M NaCl at pH 6.5. Homogeneous Factor V was isolated by chromatography of barium citrate-adsorbed, polyethylene glycol 6000 precipitated plasma on the antibody column followed by chromatography on phenyl-Sepharose. The isolated Factor V exhibited a single band upon gel electrophoresis in sodium dodecyl sulfate with an apparent Mr comparable to that of bovine Factor V (330,000). Upon exposure to thrombin, the activity of Factor V increased 53-fold when measured in Factor V-deficient plasma. This increased activity was associated with discrete proteolytic cleavages of the parent molecule.

Quantitative autoradiography is a powerful method for studying brain function by the determination of blood flow, glucose utilization, or transport of essential nutrients. Autoradiographic images contain vast amounts of potentially useful information, but conventional analyses can practically sample the data at only a small number of points arbitrarily chosen by the experimenter to represent discrete brain structures. To use image data more fully, computer methods for its acquisition, storage, quantitative analysis, and display are required. We have developed a system of computer programs that performs these tasks and has the following features: 1) editing and analysis of single images using interactive graphics, 2) an automatic image alignment algorithm that places images in register with one another using only the mathematical properties of the images themselves, 3) the calculation of mean images from equivalent images in different experimental serial image sets, 4) the calculation of difference images (e.g., experiment-minus-control) with the option to display only differences estimated to be statistically significant, and 5) the display of serial image metabolic maps reconstructed in three dimensions using a high-speed computer graphics system.

The calcium-binding properties of the coagulation cofactor, Factor V, have been investigated using atomic absorption spectrophotometry, equilibrium dialysis, and chemical exchange experiments. Two classes of binding sites have been observed: one site containing a single Ca2+ ion bound with an unusually high affinity (Kd less than 10(-8) M), and a second site in which 2 mol of calcium are bound/mol of Factor V with an association constant Ka = 1.7 +/- 0.52 x 10(4) M-1 (Kd = 5.9 +/- 1.9 x 10(-5) M). The binding of the dissociable Ca2+ ions is apparently noncooperative. The single high affinity Ca2+ ion may be removed readily by EDTA under native conditions with an immediate loss of Factor V activity, and the activity of the Factor V can be restored by the addition of a molar excess of calcium. The high affinity Ca2+ ion will exchange with radiolabeled calcium in solution, and Factor V labeled in this way was used to provide an independent measure of the stoichiometry of this high affinity binding site, and to observe directly the interaction of calcium in this site with EDTA. In addition, elemental analyses of solid lyophilized samples of Factor V revealed that no transition metals are present and that no phosphorus (or phospholipid) is retained by the Factor V after purification.

The brain contains its own angiotensin II (AII) system. To better understand the role of central AII in cardiovascular regulation, we used 125I-[Sar1, Ile8]-AII (125I-SI-AII), radioactive AII antagonist, to autoradiographically localize putative AII receptor binding in many parts of the central nervous system of the spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. With 125I-SI-AII binding on brain membrane preparations. Scatchard analysis indicated that Kd values were from 0.10 +/- 0.04 nM to 0.13 +/- 0.05 nM, whereas Bmax values (femtomol/mg protein) were found to be from 6.95 +/- 1.60 to 15.52 +/- 4.99 among brain regions studied. Various SI-AII receptor binding activities among brain regions revealed in this study were therefore most likely due to differences in AII receptor density with high affinity binding of 125I-AII. Using 125I-SI-AII, specific binding for SI-AII was found in the nucleus tractus solitarius (NTS), paraventricular hypothalamic nucleus (PVN), subfornical organ (SFO), suprachiasmatic nucleus (SCN), area postrema, the dorsal motor nucleus of the vagus (DMX), and the nucleus of spinal tract of the trigeminal system (NSV). With quantitative receptor autoradiography in conjunction with radioactive standards, we have observed that the NTS possesses the highest SI-AII binding, followed by the PVN, SFO, NTS, DMX, and NSV. No significant differences were observed between the SHR and WKY rats in the SI-AII binding within the SFO, PVN and NTS. However, SHR at early hypertensive (7 weeks) and established hypertensive (16 weeks) stages contained significantly higher SI-AII bindings in the NSV, as compared to age-matched WKY rats.(ABSTRACT TRUNCATED AT 250 WORDS)