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PubMed2006

Cross-interval histograms for analysis of brain electrical activity.

A method for constructing cross-interval histograms for time-localized EEG fragments of particular types is described. The basic principles of the method are presented. Examples of cross-interval histograms constructed for EEG extrema and their derivatives are presented. Cross-interval histograms were shown to include peaks, troughs, and other features. Cross-interval histogram and cross-correlation histograms are compared. The method yields qualitatively new data on interactions between changes in biopotentials in different areas of the brain and allows rapid processes to be studied.

Brain↗
PubMed1998

The death inhibitory molecules CED-9 and CED-4L use a common mechanism to inhibit the CED-3 death protease.

The apoptotic machinery of Caenorhabditis elegans includes three core interacting components: CED-3, CED-4, and CED-9. CED-3 is a death protease composed of a prodomain and a protease domain. CED-4 is a P-loop-containing, nucleotide-binding molecule that complexes with the single polypeptide zymogen form of CED-3, promoting its activation by autoprocessing. CED-9 blocks death by complexing with CED-4 and suppressing its ability to promote CED-3 activation. A naturally occurring alternatively spliced form of CED-4 that contains an insertion within the nucleotide-binding region (CED-4L) functions as a dominant negative inhibitor of CED-3 processing and attenuates cell death. Domain mapping studies revealed that distinct regions within CED-4 bind to the CED-3 prodomain and protease domain. Importantly, the CED-4 P-loop was involved in prodomain binding. Disruption of P-loop geometry because of mutation of a critical lysine (K165R) or insertional inactivation (CED-4L) abolished prodomain binding. Regardless, K165R and CED-4L still retained CED-3 binding through the protease domain but were unable to initiate CED-3 processing. Therefore, the P-loop-prodomain interaction is critical for triggering CED-4-mediated CED-3 processing. Underscoring the importance of this interaction was the finding that CED-9 contacted the P-loop and selectively inhibited its interaction with the CED-3 prodomain. These results provide a simple mechanism for how CED-9 functions to block CED-4-mediated CED-3 processing and cell death.

Apoptosis↗
PubMed2005

Relearning sound localization with a new ear.

Human sound localization results primarily from the processing of binaural differences in sound level and arrival time for locations in the horizontal plane (azimuth) and of spectral shape cues generated by the head and pinnae for positions in the vertical plane (elevation). The latter mechanism incorporates two processing stages: a spectral-to-spatial mapping stage and a binaural weighting stage that determines the contribution of each ear to perceived elevation as function of sound azimuth. We demonstrated recently that binaural pinna molds virtually abolish the ability to localize sound-source elevation, but, after several weeks, subjects regained normal localization performance. It is not clear which processing stage underlies this remarkable plasticity, because the auditory system could have learned the new spectral cues separately for each ear (spatial-mapping adaptation) or for one ear only, while extending its contribution into the contralateral hemifield (binaural-weighting adaptation). To dissociate these possibilities, we applied a long-term monaural spectral perturbation in 13 subjects. Our results show that, in eight experiments, listeners learned to localize accurately with new spectral cues that differed substantially from those provided by their own ears. Interestingly, five subjects, whose spectral cues were not sufficiently perturbed, never yielded stable localization performance. Our findings indicate that the analysis of spectral cues may involve a correlation process between the sensory input and a stored spectral representation of the subject's ears and that learning acts predominantly at a spectral-to-spatial mapping level rather than at the level of binaural weighting.

Acoustic Stimulation↗
PubMed2001

Characterization of differently processed forms of enolase 2 from Saccharomyces cerevisiae by two-dimensional gel electrophoresis and mass spectrometry.

Two-dimensional gel electrophoresis, bioinformatics, and mass spectrometry are key analysis tools in proteome analysis. The further characterization of post-translational modifications in gel-separated proteins relies fully on data obtained by mass spectrometric analysis. In this study, stress-induced changes in protein expression in Saccharomyces serevisiae were investigated. A total of eleven spots on a silver-stained two-dimensional (2-D) gel were identified by matrix-assisted laser desorption/ionization (MALDI) peptide mass mapping to represent C and/or N-terminal processed forms of enolase 2. The processing sites were determined by MALDI peptide mass mapping using a variety of proteolytic enzymes, by optimizing the sample preparation procedure and by specific labeling of all C-termini derived from in-gel digestion using a buffer containing 16O:18O (1:1). Out of eleven processed forms of enolase 2, six were fully characterized and the approximate processing sites identified for the remaining five.

Amino Acid Sequence↗
PubMed1998

First-pass versus second-pass parsing processes in a Wernicke's and a Broca's aphasic: electrophysiological evidence for a double dissociation.

The present paper is a first attempt to integrate the classical brain lesion behavioral impairment approach of functional neuroanatomy and the electrophysiological brain mapping approach in the domain of syntactic processing. In a group of normal age-matched controls we identified three electrophysiological components previously observed in correlation with language comprehension processes: an early left anterior negativity normally seen in correlation with syntactic first-pass parsing processes (ELAN), a centroparietal negativity seen in correlation with processes of lexical-semantic integration (N400), and a late centroparietal positivity observed in correlation with secondary syntactic processes of reanalysis and repair (P600). The early left anterior negativity was absent in a patient with an extended lesion in the anterior part of the left hemisphere sparing the temporal lobe, although the late centroparietal positivity and the centroparietal N400 were present. In a patient with a left temporal-parietal lesion the early left anterior negativity was found to be present, whereas the N400 component was absent. These findings suggest that first-pass parsing and secondary processes are subserved by distinct brain systems.

Adult↗
PubMed2006

Gene expression and genetic mapping analyses of a perennial ryegrass glycine-rich RNA-binding protein gene suggest a role in cold adaptation.

A perennial ryegrass cDNA clone encoding a putative glycine-rich RNA binding protein (LpGRP1) was isolated from a cDNA library constructed from crown tissues of cold-treated plants. The deduced polypeptide sequence consists of 107 amino acids with a single N-terminal RNA recognition motif (RRM) and a single C-terminal glycine-rich domain. The sequence showed extensive homology to glycine-rich RNA binding proteins previously identified in other plant species. LpGRP1-specific genomic DNA sequence was isolated by an inverse PCR amplification. A single intron which shows conserved locations in plant genes was detected between the sequence motifs encoding RNP-1 and RNP-2 consensus protein domains. A significant increase in the mRNA level of LpGRP1 was detected in root, crown and leaf tissues during the treatment of plants at 4 degrees C, through which freezing tolerance is attained. The increase in the mRNA level was prominent at least 2 h after the commencement of the cold treatment, and persisted for at least 1 week. Changes in mRNA level induced by cold treatment were more obvious than those due to treatments with abscisic acid (ABA) and drought. The LpGRP1 protein was found to localise in the nucleus in onion epidermal cells, suggesting that it may be involved in pre-mRNA processing. The LpGRP1 gene locus was mapped to linkage group 2. Possible roles for the LpGRP1 protein in adaptation to cold environments are discussed.

Abscisic Acid↗
PubMed1998

Characterization of proteins utilized in the desulfurization of petroleum products by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF/MS) with delayed extraction is utilized in linear, reflected-ion and post-source decay (PSD) modes to directly characterize enzymes being developed for use in a petroleum desulfurization process. The DNA sequence for the genes isolated from Rhodococcus sp. strain IGTS8 that produce three of the four enzymes under study had been previously reported with a discrepancy in residue assignments for one of the enzymes, dsz-C. The use of proteolytic digests followed by MALDI/TOF/MS with delayed extraction in the reflected-ion mode provided sequence-specific information with mass accuracies exceeding 40 ppm over a range of masses and signal-to-noise values. Peptide mapping of >80% of the residues was accomplished for all four proteins. The use of PSD established the true sequence for dsz-C, resolving the discrepancy in the literature. A posttranslational loss of N-terminal methionine was observed for each of the four proteins in linear MALDI/MS and was reconfirmed by peptide mapping for three of the proteins.

Amino Acid Sequence↗
PubMed1997

Regional gene mapping using mixed radiation hybrids and reverse chromosome painting.

We describe a new approach for low-resolution physical mapping using pooled DNA probe from mixed (non-clonal) populations of human-CHO cell hybrids and reverse chromosome painting. This mapping method is based on a process in which the human chromosome fragments bearing a complementing gene were selectively retained in a large non-clonal population of CHO-human hybrid cells during a series of 12- to 15-Gy gamma irradiations each followed by continuous growth selection. The location of the gene could then be identified by reverse chromosome painting on normal human metaphase spreads using biotinylated DNA from this population of "enriched" hybrid cells. We tested the validity of this method by correctly mapping the complementing human HPRT gene, whose location is well established. We then demonstrated the method's usefulness by mapping the chromosome location of a human gene which complemented the defect responsible for the hypersensitivity to ionizing radiation in CHO irs-20 cells. This method represents an efficient alternative to conventional concordance analysis in somatic cell hybrids where detailed chromosome analysis of numerous hybrid clones is necessary. Using this approach, it is possible to localize a gene for which there is no prior sequence or linkage information to a subchromosomal region, thus facilitating association with known mapping landmarks (e.g. RFLP, YAC or STS contigs) for higher-resolution mapping.

Animals↗
PubMed2001

Proteomic analysis of proteins in PC12 cells before and after treatment with nerve growth factor: increased levels of a 43-kDa chromogranin B-derived fragment during neuronal differentiation.

Proteomic analysis is an important approach to characterizing the proteome and studying protein function in the post-genomic era. It is also a powerful screening method for detecting unexpected alterations in protein expression that may be missed by conventional biochemical techniques. The aim of this study was to perform a preliminary proteomic analysis of PC12 cells in order to investigate the effect of nerve growth factor (NGF) on protein expression in PC12 cells during neurite outgrowth. PC12 cell proteins were separated by two-dimensional electrophoresis (2DE) and visualized by silver staining, then certain proteins were identified by N-terminal amino acid microsequencing and a homology search of a protein sequence database. Over 400 proteins were detected, 10% of which showed a significant (greater than 30%) increase or decrease in expression during NGF-induced neuronal differentiation. Seven proteins in the 2DE map were identified; the levels of five of these were unaffected by NGF treatment, whereas the levels of the other two, beta-tubulin and a novel 43-kDa chromogranin B-derived fragment, were significantly increased by more than 30 and 200%, respectively. Our results suggest that chromogranin B processing is enhanced in PC12 cells during NGF-induced neuronal differentiation. In addition, since this increase in the levels of the chromogranin B-derived fragment was specifically blocked by PD98059, we suggest that the increased processing can be ascribed to activation of the MAP kinase pathway, and that the 43-kDa chromogranin B-derived fragment can serve as a new marker of neuronal differentiation for proteomic studies.

Animals↗
PubMed1996

Olfactory information processing in the brain: encoding chemical and temporal features of odors.

A fundamental problem in studying the neural mechanisms of odor recognition and discrimination in the olfactory system lies in determining the features or "primitives" of an odor stimulus that are analyzed by glomerular circuits at the first level of processing in the brain. Several recent studies support the idea that it is not simply the molecular features of odors that contain important information, but also the intermittent pattern of their presentation to the olfactory epithelium that helps determine the behavioral response to odor.

Animals↗
PubMed1999

Synergistic role of the phosphatidylinositol 3-kinase and mitogen-activated protein kinase cascade in the regulation of insulin receptor trafficking.

To examine the molecular mechanism of insulin receptor trafficking, we investigated the intracellular signaling molecules that regulate this process in Rat1 fibroblasts overexpressing insulin receptors. Cellular localization of insulin receptors was assessed by confocal laser microscopy with indirect immunofluorescence staining. Insulin receptors were visualized diffusely in the basal state. Insulin treatment induced the change of insulin receptor localization to perinuclear compartment. This insulin-induced insulin receptor trafficking was not affected by treatment of the cells with PI3-kinase inhibitor (wortmannin), whereas treatment with MEK [mitogen-activated protein (MAP) kinase-Erk kinase] inhibitor (PD98059) partly inhibited the process in a dose-dependent manner. Interestingly, treatment with both wortmannin and PD98059 almost completely inhibited insulin receptor trafficking. The functional importance of PI3-kinase and MAP kinase in the trafficking process was directly assessed by using single cell microinjection analysis. Microinjection of p85-SH2 and/or catalytically inactive MAP kinase ([K71A]Erk1) GST fusion protein gave the same results as treatment with wortmannin and PD98059. Furthermore, to determine the crucial step for the requirement of PI3-kinase and MAP kinase pathways, the effect of wortmannin and PD98059 on insulin receptor endocytosis was studied. Insulin internalization from the plasma membrane and subsequent insulin degradation were not affected by treatment with wortmannin and PD98059. In contrast, insulin receptor down-regulation from the cell surface and insulin receptor degradation, after prolonged incubation with insulin, were markedly impaired by the treatment. These results suggest that PI3-kinase and MAP kinase pathways synergistically regulate insulin receptor trafficking at a step subsequent to the receptor internalization.

Androstadienes↗
PubMed1985

Growth cone-target interactions in the frog retinotectal pathway.

The growth cones of retinal ganglion cell axons were studied in the optic tract and tectum with horseradish peroxidase (HRP) histochemistry and electron microscopy. The ganglion cell growth cones has many morphological features similar to those described in vitro and in other in vivo systems. However, we found that some processes formed highly differentiated terminal arborizations, while retaining growth cones on many of their branches. In addition, ultrastructural examination of the tectal neuropil revealed that many ganglion cell axonal processes had characteristics of both growth cones and presynaptic endings. These findings are discussed in the context of the hypothesis of shifting connections and the evidence that retinotectal map formation involves several mechanisms, including a process that depends on the action potential activity in the optic fibers.

Animals↗
PubMedDate not supplied

The inferior temporal cortex: architecture, computation, and representation.

Neurons in the inferior temporal cortex (IT), an area crucially involved in visual object recognition in monkeys, show the visual response properties and anatomical/chemical nature which are distinct from those in the cortical areas that feed visual inputs to the IT. Earlier physiological studies showed that IT neurons have large receptive fields covering the center and contralateral (often bilateral) visual fields, stimulus selectivity for images of complex objects or shapes, and translation invariance of the stimulus selectivity. Recent studies have revealed new aspects of their properties such as invariant selectivity for shapes despite drastic changes in various physical attributes of stimuli, latent excitatory inputs masked by stimulus-specific GABAergic inhibition, selectivity for binocular disparity and 3-dimensional surface structures, profound effects of learning on the stimulus selectivity, and columnar clustering of neurons with similar stimulus selectivity for shapes and other object features. Another line of research using histological techniques have revealed that pyramidal neurons in the IT are larger in the size of dendritic arbors, in the number of dendritic branches and spines, and in the size and distribution of horizontal axonal arbors than those in the earlier areas, allowing them to integrate a larger population of afferents and process more diverse inputs. The concentration of several neurochemicals including those related to synaptic transmission or plasticity changes systematically towards the IT along the occipitotemporal pathway. Many of the characteristics of IT neurons parallel or explain certain aspects of visual object perception, although the behavioral relevance has yet to be addressed experimentally.

Animals↗
PubMed1987

Human alpha 2-HS-glycoprotein: the A and B chains with a connecting sequence are encoded by a single mRNA transcript.

The alpha 2-HS-glycoprotein (AHSG) is a plasma protein reported to play roles in bone mineralization and in the immune response. It is composed of two subunits, the A and B chains. Recombinant plasmids containing human cDNA AHSG have been isolated by screening an adult human liver library with a mixed oligonucleotide probe. The cDNA clones containing AHSG inserts span approximately 1.5 kilobase pairs and include the entire AHSG coding sequence, demonstrating that the A and B chains are encoded by a single mRNA transcript. The cDNA sequence predicts an 18-amino-acid signal peptide, followed by the A-chain sequence of AHSG. A heretofore unseen connecting sequence of 40 amino acids was deduced between the A- and B-chain sequences. The connecting sequence demonstrates the unique amino acid doublets and collagen triplets found in the A and B chains; it is not homologous with other reported amino acid sequences. The connecting sequence may be cleaved in a posttranslational step by limited proteolysis before mature AHSG is released into the circulation or may vary in its presence because of alternative processing. The AHSG cDNA was utilized for mapping the AHSG gene to the 3q21----qter region of human chromosome 3. The availability of the AHSG cDNA clone will facilitate the analysis of its genetic control and gene expression during development and bone formation.

Amino Acid Sequence↗
PubMedDate not supplied

The N-CODES project: the first year.

Clinical decision making is a complex task, and particularly challenging for the novice nurse. Little assistance is available, and decision supports such as standardized guidelines are difficult to access in the hectic flow of practice. The Nursing Computer Decision Support (N-CODES) project, directed by investigators for nursing and computer engineering, addresses this problem by developing a prototype of a point-of-care system to deliver clinical knowledge via a handheld computer. This article reports on the progress made during the first year of the project. The nursing investigators have developed a novice-nurse decision-making model, a comprehensive knowledge development process, and a series of computerized practice maps. The focus of engineering has been on designing the database architecture and the knowledge representation, extraction, and discovery algorithms used to mimic nursing knowledge and clinical decision processes in software. But the success of an interdisciplinary collaborative project depends on establishing tasks and boundaries, clarifying perspectives and language, and developing a productive process. Therefore, along with the progress of each discipline, strategies used to promote collaboration are discussed.

Acute Disease↗
PubMed1989

Physical linkage of mouse lambda genes by pulsed-field gel electrophoresis suggests that the rearrangement process favors proximate target sequences.

The first complete map of a mammalian immunoglobulin gene locus is presented. Mouse lambda genes were mapped by pulsed-field gel electrophoresis. The gene order is V2-Vx-C2-C4-V1-C3-C1. The distance between V2 or Vx and the C2-C4 cluster is 74 or 55 kilobases (kb), respectively, whereas that between V1 and C3-C1 is only 19 kb; V2 and C3-C1 are at least 190 kb apart. Thus, the distances between the lambda subloci are inversely proportional to their frequencies of rearrangement. The related gene lambda 5 is not within the 500 kb of the lambda locus mapped here.

Animals↗
PubMed1996

Gene expression in the cos region of the Streptomyces temperate actinophage phi C31.

A transcription map of a 5.12 kb region containing the cos ends of actinophage phi C31 was determined using RNA prepared from induced and uninduced cultures of the temperature-sensitive lysogen, Streptomyces coelicolor A3(2) (phi C31cts1). In induced cultures, RNA synthesis was detected only late in the lytic cycle. A late operon initiated downstream of the int gene on the right-hand end of the genome traversed the cos ends and extended at least 3.6 kb along the left-hand end. Shorter, possibly processed, mRNAs were also present. The map was superimposed on the DNA sequence of 2.8 kb of the region, part of which had been determined previously and part of which is presented here. The late-expressed transcripts contained a tRNA(Thr)-like gene and four ORFs (1, 2, 3 and 5) detected on the basis of codon bias. Analysis of the putative protein products showed that one of the ORFs could encode a lysis protein and at least one may be involved in DNA maturation. Transcription mapping of RNA from uninduced cultures demonstrated a 620 nt transcript, xRNA1, of ORF6. So far this is the only gene in phi C31 found to be expressed right to left with respect to the standard map of phi C31; its function during lysogenic growth could not be deduced from database searches.

Amino Acid Sequence↗
PubMed2005

Non-random chromosome positioning in mammalian sperm nuclei, with migration of the sex chromosomes during late spermatogenesis.

Chromosomes are highly organized and compartmentalized in cell nuclei. The analysis of their position is a powerful way to monitor genome organization in different cell types and states. Evidence suggests that the organization of the genome could be functionally important for influencing different cellular and developmental processes, particularly at early stages of development (i.e. fertilization and the consequent entry of the sperm nucleus into the egg). The position of chromosomes in the sperm nucleus might be crucial, because their location could determine the time at which particular chromatin domains are decondensed and remodelled, allowing some epigenetic level of control or influence over subsequent paternal gene expression in the embryo. Here, we analyse genome organization by chromosome position in mammalian sperm nuclei from three breeds of pig, as a model species. We have mapped the preferential position of all chromosomes (bar one) in sperm nuclei in two dimensions and have established that the sex chromosomes are the most internally localized chromosomes in mature sperm. The distribution of two autosomes and chromosomes X and Y in sperm heads was compared in primary and secondary spermatocytes and spermatids in porcine testes. The sex chromosomes were found at the nuclear edge in primary spermatocytes, which correlates with the known position of the XY body and their position in somatic cells, whereas, in spermatids, the sex chromosomes were much more centrally located, mirroring the position of these chromosomes in ejaculated spermatozoa. This study reveals the temporal repositioning of chromosome territories in spermatogenesis.

Animals↗

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