bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sequential exoproteolysis as a structural probe: a cautionary note
Authors: Beynon Robert J. 
Published: February 2004

AbstractIn a recent paper, Villaneuvaet al.(J. Mass Spectrom.2002;37: 974) described the use of exoproteases as probes of higher order structure in proteins. Their model assumes that the proteins are attacked sequentially from either the N‐terminus or the C‐terminus, depending on the type of exoprotease (aminopeptidase or carboxypeptidase) used. The products of this presumed exoproteolysis were then analysed by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. The pattern of fragments obtained was mapped on to the primary sequence of the protein, and the exoproteolysis was interpreted as comprising a series of fast and slow phases, the rates of the different phases being directly related to the higher order structure of particular segments of the protein. Here, it is shown that this explanation is unlikely, that both kinetic and practical considerations suggest that alternative explanations for the data should be sought, and that exoproteolysis will perhaps not be as valuable as a conformational probe as the authors suggest. Copyright © 2004 John Wiley & Sons, Ltd.
Bromazepam determination in human plasma by high‐performance liquid chromatography coupled to tandem mass spectrometry: a highly sensitive and specific tool for bioequivalence studies
Authors: Laurito Tiago L. , Mendes Gustavo D. , Santagada Vincenzo , Caliendo Giuseppe , de Moraes Maria Elisabete A. , De Nucci Gilberto 
Published: February 2004

AbstractA rapid, sensitive and specific method to quantify bromazepam in human plasma using diazepam as the internal standard (IS) is described. The analyte and the IS were extracted from plasma by liquid–liquid extraction using diethyl ether–hexane (80 : 20, v/v). The extracts were analyzed by high‐performance liquid chromatography (HPLC) coupled to electrospray tandem mass spectrometry (MS/MS). Chromatography was performed isocratically on a Genesis C18 analytical column (100 × 2.1 mm i.d., film thickness 4 µ m). The method had a chromatographic run time of 5.0 min and a linear calibration curve over the range 5.0–150 ng ml−1 (r2 > 0.9952). The limit of quantification was 5 ng ml−1. This HPLC/MS/MS procedure was used to assess the bioequivalence of two bromazepam 6 mg tablet formulations (bromazepam from Medley SA Indústria Farmacêutica as the test formulation and Lexotan from Produtos Roche Químico e Farmacêutico SA as the reference formulation). A single 6 mg dose of each formulation was administered to 24 healthy volunteers (12 males and 12 females). The study was conducted using an open, randomized, two‐period crossover design with a 3 week washout interval. Since the 90% CI for Cmax, AUClast, AUC0–240 h (linear) and AUC(0–∞) ratios were all inside the 80–125% interval proposed by the US Food and Drug Administration, it was concluded that the bromazepam formulation from Medley is bioequivalent to the Lexotan formulation for both the rate and the extent of absorption. Copyright © 2004 John Wiley & Sons, Ltd.
Improved detection of multi‐phosphorylated peptides in the presence of phosphoric acid in liquid chromatography/mass spectrometry
Authors: Kim Jeongkwon , Camp David G. , Smith Richard D. 
Published: February 2004

AbstractIn contrast to lower phosphorylation states (e.g. the tryptic monophosphopeptide FQpSEEQQQTEDELQDK from bovine β‐casein), the specific detection of multi‐phosphorylated peptides (e.g. the tetraphosphopeptide RELEELNVPGEIVEpSLpSpSpSEESITR from tryptic digestion of bovine β‐casein) has often been problematic for liquid chromatographic mass spectrometric (LC/MS) analysis owing to their high affinity for adsorption to exposed surfaces. We observed an enhancement in the overall detection of phosphopeptides on addition of phosphoric acid (0.1–1.0%) to the sample solution; a 10‐fold increase in sensitivity was determined for the detection of two tryptic phosphopeptides and also a significant improvement in the detection of the tetraphosphopeptide. Using capillary LC with ion trap tandem MS for detection and identification, the achievable detection limits were 50 fmol and 50 pmol for the monophosphopeptide and the tetraphosphopeptide, respectively. Phosphoric acid is believed to act as a blocking agent to available silanol groups on both the silica capillary surface and the C18‐bonded stationary phase silica surface. Copyright © 2004 John Wiley & Sons, Ltd.
Full hydrodynamic simulation of GaAs MESFETs
Authors: Aste Andreas , Vahldieck Rüdiger , Rohner Marcel 
Published: February 2004

AbstractA finite difference upwind discretization scheme in two dimensions is presented in detail for the transient simulation of the highly coupled non‐linear partial differential equations of the full hydrodynamic model, providing thereby a practical engineering tool for improved charge carrier transport simulations at high electric fields and frequencies. The discretization scheme preserves the conservation and transportive properties of the equations. The hydrodynamic model is able to describe inertia effects which play an increasing role in different fields of micro‐ and optoelectronics, where simplified charge transport models like the drift–diffusion model and the energy balance model are no longer applicable. Results of extensive numerical simulations are shown for a two‐dimensional MESFET device. A comparison of the hydrodynamic model to the commonly used energy balance model is given and the accuracy of the results is discussed. Copyright © 2004 John Wiley & Sons, Ltd.
Partial element equivalent circuit (PEEC) models for on‐chip passives and interconnects
Authors: Verbeek Menno E. 
Published: February 2004

AbstractThe accurate simulation of the electromagnetic behaviour of interconnects and on‐chip inductors is becoming more and more important for chip designs. For this purpose, we investigate the use of the electric field integral equation, which can be used to construct a full‐wave electromagnetic model. To facilitate the combined analysis of the chip's circuit and the electromagnetic behaviour, the electromagnetic model can be formulated as an equivalent circuit. We have conducted some simple experiments to see the method at work and to obtain an idea of the frequencies for which retardation effects are important. Copyright © 2004 John Wiley & Sons, Ltd.
Calcium channel blockers in the treatment of disease
Authors: Elmslie Keith S. 
Published: February 2004

AbstractOver the last 20 years the combination of patch clamp and molecular biology techniques have resulted in an explosion in our knowledge of the different calcium channel types and their roles in physiology. A crucial component to this advance has been the discovery of specific blockers for different calcium channel types. These blockers have not only permitted researchers to assign specific functions to different channel types, but their specificity allowed them to be used to treat diseases that resulted from enhanced calcium channel function. In some cases, the enhanced calcium channel function is secondary to other problems leading to increased cellular excitability. The specificity of calcium channel blockers, however, has provided a means to treat symptoms of the pathophysiology until more effective treatments become available to address the underlying problems. The goal of this review is to introduce the various types of calcium channels, their primary physiologic roles, drugs that block these channels, and diseases that are currently being treated with these drugs. © 2004 Wiley‐Liss, Inc.
Asymmetric localization of LGN but not AGS3, two homologs of Drosophila pins, in dividing human neural progenitor cells
Authors: Fuja Tannin J. , Schwartz Philip H. , Darcy Dan , Bryant Peter J. 
Published: February 2004

AbstractHuman neural progenitor cells (hNPCs) can be recovered from postmortem human brains and used to study the molecular basis of neurogenesis. Human NPCs are being used to investigate the molecular basis of cell fate determination during stem cell divisions, based on comparison with the Drosophila model system. Drosophila neuroblasts and sensory organ precursors undergo well‐defined asymmetric cell divisions (ACD), under the control of a genetically defined set of apical and basal determinants that are localized tightly and dynamically during division. We show by indirect immunofluorescence, confocal microscopy, and time‐lapse video‐microscopy that LGN and AGS3, two human homologs of the Drosophila ACD determinant Pins, have distinct patterns of localization in hNPCs. When cells are grown under conditions favoring proliferation, LGN is distributed asymmetrically in a cell cycle‐dependent manner; it localizes to one side of the dividing cell and segregates into one of the daughter cells. When the cells are grown under conditions favoring differentiation, LGN accumulates in double foci similar to those containing the mitotic apparatus protein NuMA, and in a pattern shown previously for LGN and NuMA in differentiated cells. AGS3, a slightly more distant Pins homolog than LGN, does not show asymmetric localization in these cells. The progenitor cell marker nestin also localizes asymmetrically in colcemid‐treated hNPCs and colocalizes with LGN. The results suggest that hNPCs undergo ACD and that similar molecular pathways may underlie these divisions in Drosophila and human cells. © 2004 Wiley‐Liss, Inc.
Somatodendritic localization and mRNA association of the splicing regulatory protein Sam68 in the hippocampus and cortex
Authors: Grange Julien , Boyer Véronique , Fabian‐Fine Ruth , Fredj Naïla Ben , Sadoul Rémy , Goldberg Yves 
Published: February 2004

AbstractThe RNA‐binding protein Sam68 has been implicated in the signal‐dependent processing of pre‐mRNA and in the utilization of intron‐containing retroviral mRNAs. Sam68 is predominantly nuclear but exhibits remarkable binding affinity for signalling proteins located at the membrane. We have investigated the subcellular distribution of Sam68 in adult rat cortex and hippocampus. Subcellular fractionation showed that the protein was most abundant in nuclei but also was present at a significant level in the cytosol and membrane fractions, including light and synaptic membranes derived from crude synaptosomes. Sam68 extracted from the synaptosomal fraction cosedimented with polysomes on sucrose gradients. In agreement with these findings, immunohistochemical staining indicated that Sam68 was concentrated in neuronal nuclei but was also detectable in the soma and dendrites. Sam68 immunoreactivity examined at the ultrastructural level was found to associate with dendritic microtubules, endoplasmic reticulum, and free polyribosomes, sometimes close to synapses. A combination of immunoprecipitation and RT‐PCR directly confirmed that Sam68 was bound to polyadenylated mRNA in cortical lysates. The αCaMKII mRNA was identified as one of the coprecipitated transcripts; in contrast, the gephyrin and NR1‐1 mRNAs were not coprecipitated, indicating a certain degree of sequence specificity in the association. In electrophoretic mobility shift assays, recombinant GST‐Sam68 as well as brain‐derived Sam68 bound with high affinity to the αCaMKII 3′ untranslated region. These results suggest that Sam68 may accompany and, conceivably, regulate mature mRNAs during nuclear export, somatodendritic transport, and translation. © 2004 Wiley‐Liss, Inc.
Structure and function of long‐lived olfactory organotypic cultures from postnatal mice
Authors: Josephson E.M. , Yilma S. , Vodyanoy V. , Morrison E.E. 
Published: February 2004

AbstractThe first synapse in the olfactory pathway mediates a significant transfer of information given the restricted association of specific olfactory receptor neurons with specific glomeruli in the olfactory bulb. To understand better how this connection is made and what the functional capacities of the participating cells are, we created a long‐lived culture system composed of olfactory epithelium and olfactory bulb tissues. Using the roller tube method of culturing, we grew epithelium‐bulb cocultures, explanted from 1–4‐day‐old Swiss Webster mice, on Aclar for periods ranging from 18 hr to 68 days. The explants flattened so that in some areas the culture was only a few cells thick, making individual cells distinguishable. From 107 cultures studied, we identified the following cell types by expression of specific markers (oldest culture expressing marker, days in vitro, DIV): olfactory receptor neurons (neural cell adhesion molecule, 42 DIV); mature receptor neurons (olfactory marker protein, 28 DIV); postmitotic olfactory receptor neurons and olfactory bulb neurons (β‐tubulin, 68 DIV); astrocytes (glial fibrillary acidic protein, glutamate/aspartate transporter, 68 DIV); olfactory horizontal basal cells (cytokeratin, 22 DIV). Neuronal processes formed glomeruli in 2–4‐week‐old cultures. We also recorded electro‐olfactography responses to puffs of vapor collected over an odorant mixture containing ethyl butyrate, eugenol, (+) carvone, and (−) carvone from cultures as old as 21 DIV. These features of our olfactory culture system make this model useful for studying properties of immature and mature olfactory receptor neurons, pathfinding strategies of receptor axons, and mechanisms of information transfer in the olfactory glomerulus. © 2004 Wiley‐Liss, Inc.
Glucose deprivation increases hydrogen peroxide level in immunostimulated rat primary astrocytes
Authors: Choi Ji Woong , Shin Chan Young , Yoo Byoung Kwon , Choi Min Sik , Lee Woo Jong , Han Byoung Hee , Kim Won‐Ki , Kim Hyoung‐Chun , Ko Kwang Ho 
Published: February 2004

AbstractActivated astrocytes produce a large amount of bioactive molecules, including reactive oxygen and nitrogen species. Astrocytes are in general resistant to those reactive species. However, we previously reported that immunostimulated astrocytes became highly vulnerable to metabolic insults, such as glucose deprivation. In this study, we investigated whether H2O2 production was associated with the increased vulnerability. Glucose deprivation for up to 8 hr did not change the intracellular level of H2O2 in astrocytes. Treatment with lipopolysaccharide plus interferon‐γ for 48 hr evoked astroglial H2O2 production; however, no apparent death or injury was observed in immunostimulated astrocytes. Glucose deprivation after 48 hr of immunostimulation markedly increased H2O2 level, depleted adenosine triphosphate (ATP), and enhanced lactate dehydrogenase (LDH) release. The ATP depletion and LDH release were in part prevented by catalase, mannitol, and N‐acetyl‐L‐cysteine. The enhanced level of H2O2 in glucose‐deprived immunostimulated astrocytes appeared to be secondary to the depletion of reduced glutathione. 4‐(2‐Aminoethyl)bebzenesulfonyl fluoride (AEBSF), an inhibitor of NADPH oxidase, reduced H2O2 level and LDH release in glucose‐deprived immunostimulated astrocytes. H2O2, either endogenously produced or exogenously added, depolarized mitochondrial transmembrane potential in glucose‐deprived astrocytes, leading to their ATP depletion and death. The present results strongly indicate that glucose deprivation causes deterioration of immunostimulated astrocytes by increasing the intracellular concentration of H2O2. © 2004 Wiley‐Liss, Inc.
JNK activation by tetrahydrobiopterin: Implication for Parkinson's disease
Authors: Choi Hyun Jin , Lee So Yeon , Cho Yuri , Hwang Onyou 
Published: February 2004

AbstractParkinson's disease (PD) is a progressive neurologic disease associated with selective degeneration of dopaminergic neurons in the substantia nigra. Despite extensive studies to understand the underlying cause of dopaminergic degeneration, the pathologic factors leading to this neuronal loss in PD remain obscure. We have observed previously that tetrahydrobiopterin (BH4) exerts selective toxicity and oxidative stress on dopaminergic cells, suggesting that BH4 might participate endogenously in dopaminergic neurodegeneration in PD. We investigated signaling events leading to BH4 toxicity in dopaminergic CATH.a cells. We show that c‐Jun N‐terminal kinase (JNK), but not extracellular signal‐regulated kinase (ERK) or p38 mitogen‐activated protein kinase (MAPK), is phosphorylated significantly by BH4 exposure. BH4 also leads to c‐Jun phosphorylation and an increase in c‐Jun protein level. The JNK inhibitor SP600125 protects cells against BH4 toxicity and inhibits cytochrome c release and apoptotic nuclear condensation induced by BH4. These data indicate that activation of the JNK pathway is important in mediating BH4‐induced dopaminergic cell death. © 2004 Wiley‐Liss, Inc.
Binding and transport of [3H](2S,4R)‐ 4‐methylglutamate, a new ligand for glutamate transporters, demonstrate labeling of EAAT1 in cultured murine astrocytes
Authors: Apricò Karina , Beart Philip M. , Crawford Duncan , O'Shea Ross D. 
Published: February 2004

AbstractTransporters for L‐glutamate (excitatory amino acid transporters; EAATs), localized to astrocytes, are involved intimately in intermediary metabolism within the brain. Because (2S,4R)‐4‐methylglutamate (4MG) has affinity for glial EAATs, we employed [3H]4MG to define the characteristics of EAATs in cultured murine astrocytes and describe new approaches to analyze EAAT function. Specific binding of [3H]4MG in astrocytic membranes at 4°C represented 90% of total binding. Binding was rapid (apparent t1/2 ∼7 min) and saturable. Saturation and Scatchard analyses indicated a single binding site (nH = 0.8) with a Kd of 6.0 ± 1.5 μM and Bmax = 9.7 ± 2.9 pmol/mg protein. Binding of [3H]4MG to astrocytic homogenates was Na+‐dependent and inhibited by K+. Compounds acting at EAATs, such as L‐glutamate (Glu), D‐aspartate (D‐Asp), L‐(2S,3S,4R)‐2‐(carboxycyclopropyl)glycine and L‐trans‐pyrrolidine‐2,4‐dicarboxylate displaced binding to nonspecific levels. L‐Serine‐O‐sulphate, an EAAT1‐preferring ligand, fully displaced binding of [3H]4MG. In contrast, inhibitors having preferential affinity for EAAT2, L‐threo‐3‐methylglutamate, dihydrokainate, and kainate, were relatively ineffective binding displacers. Agonists and antagonists for Glu receptors failed to significantly inhibit [3H]4MG binding. Studies with [3H]D‐Asp reinforced evidence that [3H]4MG was binding to EAATs. These data were consistent with Western blot analyses, which indicated abundant expression of EAAT1 but not EAAT2. [3H]4MG was also accumulated rapidly (apparent t1/2 ∼4 min) into whole astrocytes by a sodium‐ and temperature‐sensitive process (Km of 146 ± 24 μM, Vmax = 336 ± 27 nmol/mg protein/min), which possessed an EAAT1‐like pharmacologic profile. These findings confirm that 4MG is a substrate for EAAT1 and that the binding assay developed using [3H]4MG can be utilized in various preparations including cultured astrocytes. © 2004 Wiley‐Liss, Inc.
Increased expression of the MBP mRNA binding protein HnRNP A2 during oligodendrocyte differentiation
Authors: Maggipinto M. , Rabiner C. , Kidd G.J. , Hawkins A.J. , Smith R. , Barbarese E. 
Published: February 2004

AbstractHeterogeneous nuclear ribonucleoprotein (hnRNP) A2, a trans‐acting factor that mediates intracellular trafficking of myelin basic protein (MBP) mRNA to the myelin compartment in oligodendrocytes, is most abundant in the nucleus, but shuttles between the nucleus and cytoplasm. In the cytoplasm, it is associated with granules that transport mRNA from the cell body to the processes of oligodendrocytes. We found that the overall level of hnRNP A2 increased in oligodendrocytes as they differentiated into MBP‐positive cells, and that this augmentation was reflected primarily in the cytoplasmic pool of hnRNP A2 present in the form of granules. The extranuclear distribution of hnRNP A2 was also observed in brain during the period of myelination in vivo. Methylation and phosphorylation have been implicated previously in the nuclear to cytoplasmic distribution of hnRNPs, so we used drugs that block methylation and phosphorylation of hnRNPs to assess their effect on hnRNP A2 distribution and mRNA trafficking. Cultures treated with adenosine dialdehyde (AdOx), an inhibitor of S‐adenosyl‐L‐homocysteine hydrolase, or with 5,6‐dichloro‐1‐β‐D‐ribofuranosylbenzimidazole (DRB), a drug that inhibits casein kinase 2 (CK2), maintained the preferential nuclear distribution of hnRNP A2. Treatment with either drug affected the transport of RNA trafficking granules that remained confined to the cell body. © 2004 Wiley‐Liss, Inc.
Distinctive expression of midkine in the repair period of rat brain during neurogenesis: Immunohistochemical and immunoelectron microscopic observations
Authors: Kikuchi‐Horie Kae , Kawakami Emiko , Kamata Makiko , Wada Manabu , Hu Jian‐Guo , Nakagawa Hachiro , Ohara Kazuhiko , Watabe Kazuhiko , Oyanagi Kiyomitsu 
Published: February 2004

AbstractDistinctive expression of midkine (MK) was observed during the repair period of fetal brain neuroepithelium. MK is a heparin‐binding growth factor that occurs as a product of a retinoic acid‐inducible gene, and has a molecular mass of 13 kDa. MK expression was examined immunohistochemically and by immunoelectron microscopy during a period of repair in developing rat brain at the neurogenesis stage. Injury was induced in rat fetuses by transplacental administration of ethylnitrosourea (ENU) on embryonic Day (E) 16, and histological changes were examined up to 48 hr thereafter (i.e., up to E 18). In normal rat fetuses, MK immunostaining was observed in the cytoplasm and radial and horizontal processes of all cells in the neuroepithelium (NE), subventricular zone (SV), and intermediate zone (IMZ). In ENU‐administered brains, cells in the NE, SV, and IMZ were damaged severely, especially 16–24 hr after ENU administration. The remaining neuroepithelial cells, with the exception of those in M‐phase and the tips of processes at the ventricular surface, were negative for MK immunohistochemistry 16–24 hr after the administration of ENU. Forty‐eight hours after the administration, the cytoplasm and processes of cells in the NE, SV, and IMZ were MK immunopositive. Our previous data reported that the cell cycle of most NE cells is synchronized to the S‐phase 16 hr after ENU administration and to the M‐phase at 24 hr, and many NE cells were recovered 48 hr after ENU administration. The previous results taken together with the present results indicate that: (1) MK expression does not increase during the repair period of the NE, being different from adults; (2) MK expression is likely to be suppressed at S‐phase according to the condition of the NE; and (3) MK expression is not essential for every cell cycle phase of NE cells; but (4) is necessary to maintain the M‐phase of NE cells. © 2004 Wiley‐Liss, Inc.