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Mass spectrometric identification of serine hydrolase OVCA2 in the medulloblastoma cell line DAOY.

OVCA2 is a putative serine-hydrolase. Performing protein profiling in human tumour cell lines, OVCA2 was detected in DAOY medulloblastoma cells as a high abundance protein. The protein was unambiguously identified by 2D gel-electrophoresis and MALDI-MS and MS/MS, its presence was confirmed by western blotting. Immunohistochemistry revealed expression in medulloblastoma and predominantly in oligodendrocytes. Computational approaches predicted functional motifs and domains, interaction with apoptosis-related protein BAG and 3D structure. In addition to the presence of OVCA2 in medulloblastoma, it was furthermore detectable in three out of 10 human tumour cell-lines as a high abundance protein probably suggesting a role in the tumour biology.

Amino Acid Sequence↗

Characterization of histones and their post-translational modifications by mass spectrometry.

Histone proteins and their accompanying post-translational modifications have received much attention for their ability to affect chromatin structure and, hence, regulate gene expression. Recently, mass spectrometry has become an important complementary tool for the analysis of histone variants and modification sites, for determining the degree of occupancy of these modifications and for quantifying differential expression of these modifications from various samples. Additionally, as advancements in mass spectrometry technologies continue, the ability to read entire 'histone codes' across large regions of histone polypeptides or intact protein is possible. As chromatin biology demands, mass spectrometry has adapted and continues as a key technology for the analysis of gene regulation networks involving histone modifications.

Amino Acid Sequence↗

Proprioceptive event related potentials: gating and task effects.

OBJECTIVE: The integration of proprioception with vision, touch or audition is considered basic to the developmental formation of perceptions, conceptual objects and the creation of cognitive schemes. Thus, mapping of proprioceptive information processing is important in cognitive research. A stimulus of a brisk change of weight on a hand held load elicit a proprioceptive evoked potential (PEP). Here this is used to examine early and late information processing related to weight discrimination by event related potentials (ERP). METHODS: A gating paradigm having 1s between the proprioceptive stimuli of 100 g weight increase was recorded in 12 runs of 40 pairs and an 1:4 oddball task of discriminating between 40 and 100 g weight increase was both recorded in 24 healthy men. The subjects were stratified in 3 groups according to their discrimination errors. RESULTS: The proprioceptive event related potential (PERP) consisted of a contralateral parietal P60, frontal N70, midline P100, initial contralateral later widespread N160, vertex P200, parietal N290 and target related widespread P360 and posterior N500. The target related components were augmented in the best performers, while the bad performers had delayed P60 and attenuated N70. The amplitudes of N160, P200 and N290 were unrelated to performance. Gating was seen as attenuation of P100, N160 and P200 amplitude. CONCLUSIONS: The proprioceptive stimulus feature processing seem to be accomplished in the first 100 ms, while later components are modified by context as expected from previous findings in the somatosensory modality. SIGNIFICANCE: The PERP could be a useful research tool in the investigation of bodily information processing in neuropsychiatric disorders.

Adult↗

Optical imaging of breast tumor through temporal log-slope difference mappings.

A novel optical temporal log-slope difference mapping approach is proposed for cancerous breast tumor detection. In this method, target tissues are illuminated by near-infrared (700-1000 nm) ultrashort laser pulses from various surface source points, and backscattered time-resolved light signals are collected at the same surface points. By analyzing the log-slopes of decaying signals over all points on the source-detection grid, a log-slope distribution on the surface is obtained. After administration of absorption contrast agents, the presence of cancerous tumors increases the decaying steepness of the transient signals. The mapping of log-slope difference between native tissue and absorption-enhanced cancerous tissue indicates the location and projection of tumors on the detection surface. In this paper, we examine this method in the detection of breast tumors in two model tissue phantoms through computer simulation. The first model has a spherical tumor of 6mm in diameter embedded at the tissue center. The second model is a large tissue phantom embedded with a non-centered spherical tumor 8mm in diameter. Monte Carlo methods were employed to simulate the light transport and signal measurement. It is shown that the tumor in both the tissue models will be accurately projected on the detection surface by the proposed log-slope difference mapping method. The image processing is very fast and does not require any inverse optimization in image reconstruction.

Breast Neoplasms↗

Simultaneous functional magnetic resonance imaging in the rat spinal cord and brain.

Functional magnetic resonance imaging (fMRI) method was developed to investigate the pattern and temporal relationship in neuronal pathways of brain and spinal cord. Signal intensity changes correlating with stimulation patterns were observed simultaneously in the rat spinal cord and brain using fMRI at 9.4 T. Electrical stimulation of the forepaw was used to elicit activity. A quadrature volume RF coil covering both brain and the cervical spinal cord was used. Sets of fast spin echo (FSE) images were acquire simultaneously for both brain and spinal cord fMRI. Experiments were repeated in single animal and across animals. Activities within the dorsal horn of the spinal cord and within the somatosensory cortex were observed consistently within each animal as well as across animals.

Animals↗

The 3' termini of small RNAs in Trypanosoma brucei.

Post-transcriptional 3' end formation is an essential step in the maturation of most small RNAs. Knowledge of the precise 3' ends of mature RNAs is essential for defining 3'-processing activities. We have mapped the mature 3' ends of Spliced Leader RNA, the U1, U2, U4, U5, and U6 small nuclear RNAs, the U3 small nucleolar RNA, and the 5S and 5.8S ribosomal RNAs by ligation-mediated PCR in Trypanosoma brucei. With the exception of U5, two classes of 3' ends were observed: flush with the base of a stem-loop structure, and 1-2 nt extended from a stem-loop. Multiple mechanisms and structural features are likely to influence 3' end maturation of RNAs.

Animals↗

Characterization by liquid chromatography combined with mass spectrometry of monoclonal anti-IGF-1 receptor antibodies produced in CHO and NS0 cells.

7H2HM is a new humanized recombinant monoclonal antibody (MAb) directed against insulin-like growth factor-1 receptor and produced in CHO cells. Homogeneity of intact antibody, reduced light and heavy chains, Fab and Fc fragments were investigated by analytical methods based on mass (SDS-PAGE, SEC), charge (IEF, C-IEX) and hydrophobicity differences (RP-HPLC, HIC) and compared side-by-side with A2CHM, produced in NS0 cells. Primary structures and disulfide bridge pairing were analyzed by microsequencing (Edman degradation), mass spectrometry (MALDI-TOF, ES-TOF) and peptide mapping after enzymatic digestion (Trypsin, endoprotease Lys-C, papain). The light chains demonstrated the expected sequences. The heavy chains yielded post-translational modifications previously reported for other recombinant humanized or human IgG1 such as N-terminal pyroglutamic acid, C-terminal lysine clipping and N-glycosylation for asparagine 297. More surprisingly, two-thirds of the 7H2HM heavy chains were shown to contain an additional 24-amino-acid sequence, corresponding to the translation of an intron located between the variable and the constant domains. Taken together these data suggest that 7H2HM is a mixture of three families of antibodies corresponding (i) to the expected structure (17%; 14,9297 Da; 1330 amino acids), (ii) a variant with a translated intron in one heavy chains (33%; 15,2878 Da; 1354 amino acids) and (iii) a variant with translated introns in two heavy chains (50%; 15,4459 Da; 1378 amino acids), respectively. RP-HPLC is not a commonly used chromatographic method to assess purity of monoclonal antibodies but unlike to SEC and SDS-PAGE, was able to show and to quantify the family of structures present in 7H2HM, which were also identified by peptide mapping, mass spectrometry and microsequencing.

Amino Acid Sequence↗

Multi-factorial retainer design analysis of posterior resin-bonded fixed partial dentures: a finite element study.

OBJECTIVES: Biomechanical aspects related to prosthesis design of RBFPD have been proposed as the predominant contributor to unpredictable clinical retention. The aim of this study was to investigate the biomechanics of multiple posterior retainer designs and their interactions using three-dimensional finite element analysis. METHODS: To understand the interactions among the retainer design factors, three values of retainer thicknesses (1.2, 0.8 and 0.4mm), heights (100, 75 and 50% of the distance from 2mm above the CEJ to the occlusal surface) and angle of the axial surface extensions (150, 180 and 210 degrees ) were selected as the design parameters. Twenty-seven RBFPD FE models with three retainer-design parameters were created by image processing, contour stacking, and mapping mesh procedures. The maximum principal and von Mises stresses in remaining tooth and prosthesis, respectively, were recorded in 54 FE analyses (27 FE models with axial and lateral occlusal loadings). RESULTS: The simulated results showed that the averaged stress values of the remaining tooth and prosthesis decreased with greater retainer thickness and height as a result of increasing prosthesis stiffness and maximizing of the bonding area between the enamel and the retainer, respectively. However, no significant stress differences were found according to the angle of the retainer extension because stress transmission was concentrated at the connectors. CONCLUSIONS: The stress elevation rate (termed as VSR-stress elevation by decreasing one unit volume of the remaining tooth) addresses that the height was the most influential factor for the remaining tooth structure, followed by retainer thickness and extension angle.

Dental Prosthesis Retention↗

The high and middle molecular weight neurofilament subunits regulate the association of neurofilaments with kinesin: inhibition by phosphorylation of the high molecular weight subunit.

Kinesin participates in axonal transport of neurofilaments (NFs), but the mode by which they attach to kinesin is unclear. We compared the association of NFs with kinesin in mice expressing or lacking NF-H or NF-M. In normal and M-/- mice, the leading edge of metabolically labeled NF subunits was selectively co-precipitated with kinesin. By contrast, the entire wave of radiolabeled subunits co-precipitated with kinesin in H-/- mice. Similar bulk levels of NFs co-precipitated with kinesin from normal and H-/- mice, but reduced levels co-precipitated from M-/- mice. These data suggest that both NF-H and NF-M regulate the association of NFs with kinesin. They further indicate that phosphorylation of NF-H dissociates NFs from kinesin and provides a mechanism by which NF-H phosphorylation can contribute to the slowing of NF axonal transport.

Animals↗

The inhibitory leukocyte-associated Ig-like receptor-1 (LAIR-1) is expressed at high levels by human naive T cells and inhibits TCR mediated activation.

Human leukocyte-associated Ig-like receptor-1 (LAIR-1) is a transmembrane glycoprotein with a single extracellular Ig-like domain and a cytoplasmic tail containing two immunoreceptor tyrosine-based inhibition motifs (ITIMs). It is constitutively expressed on the majority of human mononuclear leukocytes and functions as an inhibitory receptor. In this study, we show that freshly isolated peripheral blood T cells are heterogeneous in their expression levels of LAIR-1. We have found that naive T cells express the highest levels of LAIR-1, even more than memory cells. The cross-linking of LAIR-1 inhibits T cell receptor (TCR) mediated signals in freshly isolated human naive T cells and whole populations of CD4+ or CD8+ T cells. TCR cross-linking increased cell surface expression of LAIR-1 in a process that requires p38 MAP kinase and ERK signaling. Altogether, these results indicate that LAIR-1 is capable of negatively regulating T cell functions, and its high level of expression by naive T cells suggests that it may function at an early stage in the development of an immune response.

Animals↗

Conserved residues of the bare lymphocyte syndrome transcription factor RFXAP determine coordinate MHC class II expression.

RFXAP is required for the transcriptional regulation of MHC-II genes. Mutations in RFXAP are the genetic basis for complementation group D cases of the bare lymphocyte syndrome (BLS) immunodeficiency. Comparative genomic sequence analysis was conducted and found that only the C-terminal half of the protein is conserved among vertebrates. The C-terminal third of RFXAP, which contained an extensive glutamine-rich tract, could rescue HLA-DR, but not HLA-DQ or HLA-DP expression in a BLS cell line. To understand this phenomenon, a detailed analysis of the role of specific sequences in the C-terminal third of RFXAP with respect to MHC-II regulation was undertaken. Surprisingly, mutation of the conserved glutamine residues had no effect on activity, whereas mutation of hydrophobic and other conserved residues resulted in discoordinate MHC-II isotype expression. Moreover, mutation of potential phosphorylation sites abolished RFXAP activity. The ability of RFXAP mutants to rescue one isotype, but not another was investigated by their ability to form RFX complexes, bind DNA in vivo, recruit CIITA to promoters and to activate a series of chimeric reporter genes. The results suggest that certain RFXAP mutants exaggerate isotype promoter-specific differences and form transcriptionally inefficient activation complexes with factors at the neighboring cis-acting elements. These results show a distinction in factor recognition that is associated with specific MHC-II isotypes and may explain the basis of allele-specific expression differences.

Amino Acid Sequence↗

Type II phosphatidylinositol 4-kinase beta associates with TCR-CD3 zeta chain in Jurkat cells.

Phosphatidylinositol lipid signaling cascades are integral part of TCR-CD3 signaling. The mechanisms by which phosphatidylinositol kinases are coupled to TCR-CD3 complex remain elusive. Here we report an association of type II PtdIns 4-kinase with TCR-CD3 zeta chain upon cross-linking. Mapping studies have revealed that the C-terminal ITAM is critical for docking of the enzyme on the zeta chain. The association is shown to be tyrosyl phosphorylation dependent as mutation of Y-151 and Y-142 on the C-terminal ITAM disrupts interaction of the two proteins. Identification of the associated type II PtdIns 4-kinase revealed that the beta isoform of the enzyme interacts with the zeta chain in vivo.

1-Phosphatidylinositol 4-Kinase↗

Affine registration: a comparison of several programs.

Several registration programs with an affine model for the displacement field were tested on various 2D and 3D MRI of the same modality. The following programs were considered: AIR 3.0 (Woods, J. Comp. Assist. Tomogr, 22(1): 139-152, 1998), COCGV (Ostuni, JMRI, 7(2): 410-415, 1997), FLIRT (Jenkinson, Med. Image Analysis, 5(2): 143-156, 2001), Intramodal Registration (Thevenaz, IEEE Trans. Image Proc., 7(1): 27-41, 1998), SPM (Friston, Human Brain Mapping, 2: 165-189, 1995), and Patch Algorithm (Zhilkin, MRI 18(9): 1143-1150, 2000). Although some of these programs can perform multimodal registration, none was used in such a mode. This paper attempts a fair comparison of the performance of the Patch Algorithm with other programs. However, different settings of the programs' parameters may further improve the quality of the registration and/or change execution speed. The registered images, the CPU time required to perform the registration, and the error between the registered and reference images, are presented. Most of the programs give comparable accuracies of registration, but their execution times vary considerably. In general the AIR and Patch Algorithm require the least time. The Patch Algorithm can be easily parallelizable on a multi-processor computer.

Algorithms↗

Space, time and learning in the hippocampus: how fine spatial and temporal scales are expanded into population codes for behavioral control.

The hippocampus participates in multiple functions, including spatial navigation, adaptive timing and declarative (notably, episodic) memory. How does it carry out these particular functions? The present article proposes that hippocampal spatial and temporal processing are carried out by parallel circuits within entorhinal cortex, dentate gyrus and CA3 that are variations of the same circuit design. In particular, interactions between these brain regions transform fine spatial and temporal scales into population codes that are capable of representing the much larger spatial and temporal scales that are needed to control adaptive behaviors. Previous models of adaptively timed learning propose how a spectrum of cells tuned to brief but different delays are combined and modulated by learning to create a population code for controlling goal-oriented behaviors that span hundreds of milliseconds or even seconds. Here it is proposed how projections from entorhinal grid cells can undergo a similar learning process to create hippocampal place cells that can cover a space of many meters that are needed to control navigational behaviors. The suggested homology between spatial and temporal processing may clarify how spatial and temporal information may be integrated into an episodic memory. The model proposes how a path integration process activates a spatial map of grid cells. Path integration has a limited spatial capacity, and must be reset periodically, leading to the observed grid cell periodicity. Integration-to-map transformations have been proposed to exist in other brain systems. These include cortical mechanisms for numerical representation in the parietal cortex. As in the grid-to-place cell spatial expansion, the analog representation of number is extended by additional mechanisms to represent much larger numbers. The model also suggests how visual landmarks may influence grid cell activities via feedback projections from hippocampal place cells to the entorhinal cortex.

Animals↗

Three-dimensional ultrasonography of monkey brain.

Three-dimensional (3D) ultrasonography is new technology based on automatic volume acquisition of ultrasound images, and computer-aided image reconstruction. In the present study, we used a commercially available 3D ultrasonographic instrument for electrophysiological and neuroanatomical experiments in the macaque monkey. 3D reconstruction for multiplanar analysis and surface rendering were useful to observe the microelectrode or microinjection pipette in the cerebral cortex or deep brain structure. The present results indicate that the 3D ultrasonography for monkey brain is a more powerful tool for experiments in vivo than conventional two-dimensional (2D) ultrasonography.

Animals↗

Fusion of autoradiographs with an MR volume using 2-D and 3-D linear transformations.

In the past years, the development of 3-D medical imaging has enabled the 3-D imaging of in vivo tissues, from an anatomical (MR, CT) or even functional (fMRI, PET, SPECT) point of view. However, despite immense technological progress, the resolution of these images is still short of the level of anatomical or functional details that in vitro imaging (e.g., histology, autoradiography) permits. The motivation of this work is to compare fMRI activations to activations observed in autoradiographic images from the same animals. We aim to fuse post-mortem autoradiographic data with a pre-mortem anatomical MR image. We first reconstruct a 3-D volume from the 2-D autoradiographic sections, coherent both in geometry and intensity. Then, this volume is fused with the MR image. This way, we ensure that the reconstructed 3-D volume can be superimposed onto the MR image that represents the reference anatomy. We demonstrate that this fusion can be achieved by using only simple global transformations (rigid and/or affine, 2-D and 3-D), while yielding very satisfactory results.

Animals↗

Retinotopy within rat primary visual cortex using optical imaging.

The purpose of this study was to determine the retinotopic organization of rat primary visual cortex (area 17) using optical imaging technology. Stimulating discrete regions of visual space resulted in localised changes in the remitted light during optical imaging of visual cortex in rat. From these localised changes, our results confirm previous electrophysiological studies on the location, size and organization of rat primary visual cortex. Small differences in the cortical magnification factor (CMF) were found between visual field areas with the highest CMF confined to the upper nasal region. No significant CMF differences were found within the horizontal and vertical visual field axes. No secondary visual areas were activated either anterior or medial to area 17 with the pattern stimuli used in the current study. However, there was evidence of activity to upper nasal stimulation on the posterior lateral extrastriate area. The location of area 17 from optical imaging activity was confirmed anatomically using conventional immunohistochemical techniques. This study shows the retinotopic organization of rat primary visual cortex and serves as a precursor before examining animal models of retinal degeneration and the effectiveness of potential therapies to stem retinal disease.

Animals↗

Bootstrap white matter tractography (BOOT-TRAC).

White matter tractography is a noninvasive method for estimating and visualizing the white matter connectivity patterns in the human brain using diffusion tensor imaging (DTI) data. Sources of experimental noise may induce errors in the measured fiber directions, which will reduce the accuracy of the estimated white matter trajectories. In this study, a statistical nonparametric bootstrap method is described for estimating the dispersion and other errors in white matter tractography results. Prior studies have derived models of tractography error using the signal-to-noise ratio (SNR) and diffusion anisotropy of the DTI data. Tractography errors measured using bootstrap methods were generally consistent with an analytic model of tractography error except in areas where branching was evident. White matter tractography with bootstrap resampling is also applied to estimate the probabilities of connection between brain regions. The approach was used to generate probabilistic connectivity maps between the cerebral peduncles and specific cortical regions.

Brain↗