ReducedS-adenosylmethionine: Protein-lysineN-methyltransferase activity (protein methylase III) in shiverer mutant mouse brain

Mice with the dysmyelinating mutation shiverer were studied by measuring the activity of two protein methylases and myelin marker enzymes in the brain. It was observed thatS-adenosylmethionine: protein-lysineN-methyltransferase (protein methylase III, EC. 2.1.1.43) activity is significantly reduced in phenotypically affected homozygous shiverer (shi/shi) mutant mouse brain compared to the unaffected heterozygous littermate brain. This reduction in enzyme activity is manifested mainly by reduced formation of trimethyllysine during the in vitro methylation of histone. In contrast, myelin marker enzymes such as 2,3-cyclic nucleotide 3-phosphohydrolase and 5-nucleotidase as well asS-adenosyl-methionine: protein-carboxylO-methyltransferase (protein methylase II, EC. 2.1.1.24) activities were not significantly affected in these strains of mice.     

Characterization of cloned cDNA representing rat myelin basic protein: absence of expression in brain of shiverer mutant mice

A cDNA library was constructed from mRNA isolated from the brains of 18-day-old rats, the age at which myelin biosynthesis is maximal. A synthetic DNA probe synthesized based on reverse translation of the amino acid sequence of rat myelin basic protein (MBP) was used to select two cDNA clones encoding MBP. A 1.5 kb Eco RI fragment from one clone was completely sequenced. When translated, a portion of this sequence was identical at 126 of 127 positions with the reported amino acid sequence for small MBP from the rat. Brains from mice of the homozygous shiverer genotype contained neatly reduced amounts of MBP mRNA relative to wild type. A deletion of MBP sequences in the genome of shiverer mice was also demonstrated. cDNAs for MBP will allow molecular investigation of the role this gene plays in both dysmyelinating and demyelinating diseases, as well as questions of MBP biosynthesis.     

Altered topological properties of functional network connectivity in schizophrenia during resting state: a small-world brain network study

Aberrant topological properties of small-world human brain networks in patients with schizophrenia (SZ) have been documented in previous neuroimaging studies. Aberrant functional network connectivity (FNC, temporal relationships among independent component time courses) has also been found in SZ by a previous resting state functional magnetic resonance imaging (fMRI) study. However, no study has yet determined if topological properties of FNC are also altered in SZ. In this study, small-world network metrics of FNC during the resting state were examined in both healthy controls (HCs) and SZ subjects. FMRI data were obtained from 19 HCs and 19 SZ. Brain images were decomposed into independent components (ICs) by group independent component analysis (ICA). FNC maps were constructed via a partial correlation analysis of ICA time courses. A set of undirected graphs were built by thresholding the FNC maps and the small-world network metrics of these maps were evaluated. Our results demonstrated significantly altered topological properties of FNC in SZ relative to controls. In addition, topological measures of many ICs involving frontal, parietal, occipital and cerebellar areas were altered in SZ relative to controls. Specifically, topological measures of whole network and specific components in SZ were correlated with scores on the negative symptom scale of the Positive and Negative Symptom Scale (PANSS). These findings suggest that aberrant architecture of small-world brain topology in SZ consists of ICA temporally coherent brain networks.     

Automated pipeline for atlas-based annotation of gene expression patterns: Application to postnatal day 7 mouse brain

Massive amounts of image data have been collected and continue to be generated for representing cellular gene expression throughout the mouse brain. Critical to exploiting this key effort of the post-genomic era is the ability to place these data into a common spatial reference that enables rapid interactive queries, analysis, data sharing, and visualization. In this paper, we present a set of automated protocols for generating and annotating gene expression patterns suitable for the establishment of a database. The steps include imaging tissue slices, detecting cellular gene expression levels, spatial registration with an atlas, and textual annotation. Using high-throughput in situ hybridization to generate serial sets of tissues displaying gene expression, this process was applied toward the establishment of a database representing over 200 genes in the postnatal day 7 mouse brain. These data using this protocol are now well-suited for interactive comparisons, analysis, queries, and visualization.     

Role of cilia in structural birth defects: Insights from ciliopathy mutant mouse models

Structural birth defect (SBD) is a major cause of morbidity and mortality in the newborn period. Although the etiology of SBD is diverse, a wide spectrum of SBD associated with ciliopathies points to the cilium as having a central role in the pathogenesis of SBDs. Ciliopathies are human diseases arising from disruption of cilia structure and/or function. They are associated with developmental anomalies in one or more organ systems and can involve defects in motile cilia, such as those in the airway epithelia or from defects in nonmotile (primary cilia) that have sensory and cell signaling function. Availability of low cost next generation sequencing has allowed for explosion of new knowledge in genetic etiology of ciliopathies. This has led to the appreciation that many genes are shared in common between otherwise clinically distinct ciliopathies. Further insights into the relevance of the cilium in SBD has come from recovery of pathogenic mutations in cilia‐related genes from many large‐scale mouse forward genetic screens with differing developmental phenotyping focus. Our mouse mutagenesis screen for congenital heart disease (CHD) using noninvasive fetal echocardiography has yielded a marked enrichment for pathogenic mutations in genes required for motile or primary cilia function. These novel mutant mouse models will be invaluable for modeling human ciliopathies and further interrogating the role of the cilium in the pathogenesis of SBD and CHD. Overall, these findings suggest a central role for the cilium in the pathogenesis of a wide spectrum of developmental anomalies associated with CHD and SBDs. Birth Defects Research (Part C) 102:115–125, 2014. © 2014 Wiley Periodicals, Inc.     

Meeting report of the European mouse complex genetics network SYSGENET

The second scientific meeting of the European systems genetics network for the study of complex genetic human disease using genetic reference populations (SYSGENET) took place at the Center for Cooperative Research in Biosciences in Bilbao, Spain, December 10–12, 2012. SYSGENET is funded by the European Cooperation in the Field of Scientific and Technological Research (COST) and represents a network of scientists in Europe that use mouse genetic reference populations (GRPs) to identify complex genetic factors influencing disease phenotypes (Schughart, Mamm Genome 21:331–336, 2010). About 50 researchers working in the field of systems genetics attended the meeting, which consisted of 27 oral presentations, a poster session, and a management committee meeting. Participants exchanged results, set up future collaborations, and shared phenotyping and data analysis methodologies. This meeting was particularly instrumental for conveying the current status of the US, Israeli, and Australian Collaborative Cross (CC) mouse GRP. The CC is an open source project initiated nearly a decade ago by members of the Complex Trait Consortium to aid the mapping of multigenetic traits (Threadgill, Mamm Genome 13:175–178, 2002). In addition, representatives of the International Mouse Phenotyping Consortium were invited to exchange ongoing activities between the knockout and complex genetics communities and to discuss and explore potential fields for future interactions.     

Kinetic analyses of activation-induced changes in the hydrodynamic and surface properties of the glucocorticoid-receptor complex in mouse brain

Unactivated, molybdate-stabilized, [³H]triamcinolone acetonide-labeled, glucocorticoid receptors from mouse whole brain were activated by removal of the molybdate and incubation at 22°C for 1.5 to 24 min and then rapidly quenched at 0°C with molybdate. The loss of the 9.2 S (unactivated) form of the [³H]TA-receptor complex and the concomitant formation of the 3.8 S (activated) form displayed first-order kinetics with a half-time of less than two min. The increase in the 3.8 S form correlated nearly perfectly with an increased binding to DNA-cellulose, and with a decreased and increased adsorption to DEAE-cellulose and glass fiber filters, respectively. The changes in adsorption to these filters, which occurred at a faster rate than did the changes in binding to DNA-C, are thought to reflect an increase in the relative number of positive charges and hydrophobic groups on the surface of the activated complex.     

A new device to measure the structural properties of the femur-anterior cruciate ligament-tibia complex

Previous studies of biomechanical properties of femur-anterior cruciate ligament-tibia complex (FATC) utilized a wide variety of testing methodologies, particularly with respect to ligament orientation relative to loading direction. A new device was designed and built to test the anterior-posterior displacement of the intact porcine knee at 30 and 90 deg of flexion, as well as the tensile properties of the FATC at any loading direction and flexion angle. Tensile tests were performed with the knees at 30 and 90 deg of flexion with the loading direction along either the axis of the tibia (tibial axis) or the axis of the anterior cruciate ligament (ligament axis). The results showed that the stiffness, ultimate load and energy absorbed were all significantly increased when the FATC was tested along the ligament axis. This study demonstrates the importance of alignment in the evaluation of the biomechanical characteristics of the femur-ACL-tibia complex.     

Kinetic and structural analysis of mutant Escherichia coli dihydroorotases: a flexible loop stabilizes the transition state

Dihydroorotase (DHOase) catalyzes the reversible cyclization of N-carbamyl-l-aspartate (CA-asp) to l-dihydroorotate (DHO) in the de novo biosynthesis of pyrimidine nucleotides. Two different conformations of the surface loop (residues 105-115) were found in the dimeric Escherichia coli DHOase crystallized in the presence of DHO (PDB code 1XGE). The loop asymmetry reflected that of the active site contents of the two subunits: the product, DHO, was bound in the active site of one subunit and the substrate, CA-asp, in the active site of the other. In the substrate- (CA-asp-) bound subunit, the surface loop reaches in toward the active site and makes hydrogen bonds with the bound CA-asp via two threonine residues (Thr109 and Thr110), whereas the loop forms part of the surface of the protein in the product- (DHO-) bound subunit. To investigate the relationship between the structural states of this loop and the catalytic mechanism of the enzyme, a series of mutant DHOases including deletion of the flexible loop were generated and characterized kinetically and structurally. Disruption of the hydrogen bonds between the surface loop and the substrate results in significant loss of catalytic activity. Furthermore, structures of these mutants with low catalytic activity have no interpretable electron density for parts of the flexible loop. The structure of the mutant (Delta107-116), in which the flexible loop is deleted, shows only small differences in positions of other substrate binding residues and in the binuclear zinc center compared with the native structure, yet the enzyme has negligible activity. The kinetic and structural analyses suggest that Thr109 and Thr110 in the flexible loop provide productive binding of substrate and stabilize the transition-state intermediate, thereby increasing catalytic activity.     

Monoclonal antibody to galactosylceramide: discrimination of structural difference in the ceramide moiety

A mouse monoclonal antibody (mAb) was developed against monohexaosylceramide. This mAb differentially reacted on thin-layer chromatograms with 3 types of galactosylceramide (GalCer) obtained from bovine brain. Structural analysis of the 3 glycolipids revealed that they consisted of the same galactose and sphingosine but of apparently different fatty acids. Among the GalCers, the mAb reacted with teh two GalCers which contained alpha-hydroxy fatty acids, but not with GalCer composed of nonhydroxy fatty acids. These findings suggest that not only that the mAb discriminated the fatty acid composition in the ceramide moiety of GalCer, but also that the ceramide structure defines the immunological epitope as it is known to do for the carbohydrate moiety of glycosphingolipid.     

QSAR and complex network study of the chiral HMGR inhibitor structural diversity

Efficient drugs such as statins or mevinic acids are inhibitors of the rate-limiting enzyme of cholesterol biosynthesis, 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGR), an enzyme responsible for the double reduction of 3-hydroxy-3-methyl-glutaryl coenzyme A into mevalonic acid. These compounds promoted the synthesis and evaluation of new inhibitors for HMGR, named HMGRIs. The high number of possible candidates creates the necessity of Quantitative Structure–Activity Relationship models in order to guide the HMGRI synthesis. There are two main problems of the reported QSAR models: the homogeneous series of the compounds and the chirality of many candidates. In this work, we propose for the first time a QSAR model for a very large and heterogeneous series of HMGRIs. The model is based on the Topological Indices (TIs) of molecular structures. Using the predictions of this model as input, we construct the first complex network that describes the drug–drug similarity relationships for more than 1600 experimentally non-explored chiral HMGRIs isomers. We also presented a reduced version of this network (Giant Component) that contains the most representative set of chiral HMGRI candidates. The work suggests a new mixed application in the QSAR study of relevant aspects of structural diversity by using chiral/non-chiral TIs, combined with complex networks.     

Triplet state properties of the methylmercury(II)-tyrosine complex

CH₃Hg(II)OH forms complexes at pH 8 with tyrosine and with tyrosine ethyl ester (TEE) that are detected by ultraviolet difference absorption spectra. With K_f defined by CH₃HgOH + HB

CH₃HgB + H₂O, we find log K_f = 3.61 (tyrosine) and 3.36 (TEE). A heavy-atom effect is observed in frozen glasses of the complexes; this indicates a close interaction between Hg and the chromophore. No UV difference spectrum or heavy-atom effect is observed with N-acetyl tyrosine ethyl ester, indicating that complexing at the phenol O does not occur, and suggesting that binding occurs at the amine N. Zero field optically detected magnetic resonance (ODMR) measurements of the CH₃Hg(II)-tyrosine triplet state give (D, E) = (0.129, 0.047) or (0.134, 0.041) cm^?1 depending upon assignment of transitions. D of tyrosine is relatively unaffected, but E is reduced by CH₃Hg(II) complexing. Low-temperature kinetic measurements show that the shortest lived sublevel of the complex is T_z, where z lies along the phenol long axis in tyrosine. A dominant 11.6-msec component in the 77 K decay of the phosphorescence is consistent with the individual sublevel lifetimes obtained by ODMR.     

Fine structural analysis of calcitonin gene-related peptide in the mouse inferior olivary complex

Climbing fiber afferents to the cerebellum, from the inferior olivary complex, have a powerful excitatory effect on Purkinje cells. Changes in the responsiveness of olivary neurons to their afferent inputs, leading to changes in the firing rate or pattern of activation in climbing fibers, have a significant effect on the activation of cerebellar neurons and ultimately on cerebellar function. Several neuropeptides have been localized in both varicosities and cell bodies of the mouse inferior olivary complex, one of which, calcitonin gene related peptide (CGRP), has been shown to modulate the activity of olivary neurons. The purpose of the present study was to investigate the synaptic relationships of CGRP-containing components of the caudal medial accessory olive and the principal olive of adult mice, using immunohistochemistry and electron microscopy. The vast majority of immunoreactive profiles were dendrites and dendritic spines within and outside the glial boundaries of synaptic glomeruli (clusters). Both received synaptic inputs from non-CGRP labeled axon terminals. CGRP was also present within the somata of olivary neurons as well as in profiles that had cytological characteristics of axons, some of which were filled with synaptic vesicles. These swellings infrequently formed synaptic contacts. At the LM level, few, if any, CGRP-immunoreactive climbing fibers, were seen, suggesting that CGRP is compartmentalized within the somata and dendrites of olivary neurons and is not transported to their axon terminals. Thus, in addition to previously identified extrinsic sources of CGRP, the widespread distribution of CGRP within olivary somata and dendrites identifies an intrinsic source of the peptide suggesting the possibility of dendritic release and a subsequent autocrine or paracrine function for this peptide within olivary circuits.     

Brain water and electrolytes in response to respiratory acidosis and brain edema in the mutant mouse,Jimpy

Compared to littermate controls, unstressed Jimpy mice have higher brain water, sodium, potassium and chloride contents and lower carbonic anhydrase activity. When stressed by CO₂ to produce a respiratory acidosis or by injection of distilled water to produce brain edema, the Jimpy mouse brain has water and ionic responses essentially like those in controls.