bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of machined/turned, TiO2‐blasted and sandblasted/acid‐etched titanium oral implant surfaces on nerve conduction: A study on isolated rat sciatic nerves
Authors: Onur Mehmet A. , Çehreli Murat Cavit , Taş Zeynep , Şahin Saime
Published: October 2003

The purpose of this study was to explore the clinical relevance of the effects of machined/turned, TiO2‐blasted and sandblasted/acid‐etched titanium oral implant surfaces on nerve conduction. Isolated rat sciatic nerves were placed between two suction electrodes in a pyrex bath containing a tyrode solution. Evoked compound action potentials (cAPs) of the nerves were recorded before and after contact with the implants. The mandibular incisors of randomly selected animals were extracted and changes in cAP amplitudes were used as controls. The differences in final cAP values of Astra Tech® implants and rat natural teeth were insignificant (P < 0.05), whereas the differences between other groups were significant (P < 0.05). Machined/turned‐surface implants did not cause any change in cAPs. A slight decrease in cAPs was observed for TiO2‐blasted and sandblasted/acid‐etched implants, and the natural teeth. The reductions of cAPs in latter groups were not 50% after an application time of 300 min. The cAP changes of nerves contacting TiO2‐blasted and sandblasted/acid‐etched oral implants fall within physiologic limits in vitro. Machined/turned, TiO2‐blasted, and sandblasted/acid‐etched titanium implant surfaces do not lead to irreversible neurotoxic effects. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 67B: 772–778, 2003
Premixed calcium–phosphate cement pastes
Authors: Takagi Shozo , Chow Laurence C. , Hirayama Satoshi , Sugawara Akiyoshi
Published: October 2003

A self‐hardening calcium–phosphate cement (CPC) containing Ca4(PO4)2O and CaHPO4 has been shown in clinical studies to be efficacious for repairing bone defects. This and several other similar CPCs harden in 10 min with the use of a phosphate solution as the liquid and form hydroxyapatite (HA) as the product. The present study investigated the properties of water‐free, glycerol‐containing CPC pastes that are stable in the package and would harden only after being delivered to a defect site where glycerol–tissue fluids exchange occurs. Premixed CPC pastes were prepared by combining cement liquids containing glycerol and various amounts of hydroxypropyl methylcellulose/Na2HPO4, with CPC powders that contained (1) Ca4(PO4)2O and CaHPO4, (2) α‐Ca3(PO4)2 and CaCO3, or (3) CaHPO4 and Ca(OH)2. The hardening times and 1‐d and 7‐d diametral tensile strengths were measured on samples that hardened in an in vitro model that allowed exchange of glycerol and physiologic‐like solution (PLS) through fritted glass slides at 37 °C. All pastes had excellent washout resistance; they remained intact and hardened while immersed in PLS and formed HA as products. Newman–Keuls multiple comparison tests indicated that the Na2HPO4 amount, not the hydroxypropyl methylcellulose (HMC) amount, significantly (p < 0.05) affected the strength and hardening time. Although the premixed CPCs generally have longer hardening times and lower strengths, these pastes have excellent washout resistance before hardening and can be prepared in advance under well‐controlled conditions. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 67B: 689–696, 2003
Preparation of interconnected highly porous polymeric structures by a replication and freeze‐drying process
Authors: Hou Qingpu , Grijpma Dirk W. , Feijen Jan
Published: October 2003

Three‐dimensional degradable porous polymeric structures with high porosities (93–98%) and well‐interconnected pore networks have been prepared by freeze‐drying polymer solutions in the presence of a leachable template followed by leaching of the template. Templates of the pore network were prepared by fusing sugar or salt particles to form a well‐connected structure. The interstices of the template were then filled with a polymer solution (5–15% w/v) in 1,4‐dioxane, followed by freeze‐drying of the solvent. Subsequent leaching of the sugar template ensures the connectivity of the pore network. The scaffold architecture consists of relatively large interconnected pores modeled after the template and smaller pores resulting from the freeze‐drying process. The total porosity of the resultant porous structures is determined by the interstitial space of the leachable template and by the polymer concentration in the freeze‐drying solution. The freezing temperature also has an effect on the final morphology of the porous structures. Compared with freeze‐drying and combination of freeze‐drying /particulate leaching techniques, this method facilitates higher interconnectivity of the scaffolds. Porous structures have been prepared from several relevant polymers in the biomedical and tissue‐engineering field: poly(D,L‐lactide) (PDLLA), 1000PEOT70PBT30, a segmented poly(ether ester) based on polyethylene oxide and polybutylene terephthalate, and poly(ϵ‐caprolactone) (PCL). The mechanical properties of the porous structures prepared by this technique depend on the nature of the polymer, porosity, and the freezing temperature. With porosities in the range of 95–97%, the compression moduli of scaffolds prepared from the different polymers could be varied between 13.0 and 301.5 kPa. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 67B: 732–740, 2003
Biological response to wear debris generated in carbon based composites as potential bearing surfaces for artificial hip joints
Authors: Howling G. I. , Sakoda H. , Antonarulrajah A. , Marrs H. , Stewart T. D. , Appleyard S. , Rand B. , Fisher J. , Ingham E.
Published: October 2003

UHMWPE wear particles have been implicated in osteolysis, implant loosening, and long‐term failure of total hip arthroplasties in vivo. This study examined four carbon‐based composite materials as alternatives for UHMWPE in joint bearings. These materials were HMU‐CVD, SMS‐CVD, P25‐CVD, and CFR‐PEEK. New bearing materials should satisfy certain criteria: they should have good wear properties that at least match UHMWPE, and produce wear particles with low levels of biological activity. Of the four materials tested in multidirectional pin‐on‐plate tribological tests, SMS‐CVD, P25‐CVD, and CFR‐PEEK showed lower volumetric wear factors than UHMWPE. P25‐CVD had the lowest wear factor of 0.54 ± 0.34 × 10−7 mm3/Nm. Analysis of P25‐CVD wear particles by transmission electron microscopy showed that the debris was very small, with the vast majority of particles being under 100 nm in size, which was similar in size to metal wear particles. The P25‐CVD particles were isolated and cultured with L929 fibroblasts and U937 monocytic cells to assess their effect on cell viability. P25‐CVD particles were significantly less cytotoxic (p < 0.01, ANOVA) to both cell types than CoCr metal wear particles. This work suggests that carbon–carbon composite materials may have potential for use in total hip replacement bearings. Of the materials tested P25‐CVD had the lowest wear factor, and produced very small wear debris that had minimal cytotoxic effect on L929 and U937 cells in vitro. Therefore carbon–carbon composites, such as P25‐CVD, may be important in the development of next‐generation implants with lower wear rates and reduced cytotoxic potential. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 67B: 758–764, 2003
In vitro cytotoxicity testing of AB‐polymer networks based on oligo(ϵ‐caprolactone) segments after different sterilization techniques
Authors: Rickert Dorothee , Lendlein A. , Schmidt A. M. , Kelch S. , Roehlke W. , Fuhrmann R. , Franke R. P.
Published: October 2003

Recently developed versatile biodegradable polymeric biomaterial offer new therapeutic options in numerous medical fields. Biocompatibility is a crucial requirement for the biomedical application of biomaterials, including the sterilization of these materials with the use of accepted protocols. Ethylene‐oxide (EO) and low‐temperature plasma (LTP) sterilization are frequently used low‐temperature sterilization technologies for heat‐sensitive materials. The agarose diffusion assay is a recommended cell‐screening test to assess the cytotoxicity of biomaterials in vitro. The sensitivity of the agarose assay can be increased by using a modified computer‐based image‐analysis system. The influence of EO and LTP sterilization on the cytotoxicity of a versatile polymer system of shape‐memory polymer networks based on oligo (ϵ‐caprolactone) dimethacrylate and n‐butyl acrylate was investigated. Statistically significant differences in the rate of cell lysis after EO and LTP sterilization of the polymer samples were detected by using this modified quantification system. The influence of the different sterilization techniques on the cytotoxicity of the polymeric material, as well as the clinical relevance of the described differences, are discussed. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 67B: 722–731, 2003
The fall and rise of active chloride transport: Implications for regulation of intraocular pressure
Authors: Civan Mortimer M.
Published: October 2003

Early study of transepithelial salt transfer focused on Cl− and not Na+, partly because Cl− was readily measureable. The advent of flame photometry and tracer techniques brought Na+ to the fore, especially since short‐circuited frog skin (Rana temporaria) produces baseline net movement of Na+ and not of Cl−. Zadunaisky was among the first to describe what is currently termed secondary active Cl− transport, helping stimulate interest in Cl− handling by other tissues, notably the thick ascending limb of the loop of Henle important in renal counter‐current multiplication. More recently, molecules responsible for electroneutral and electrogenic Cl− transfer have been cloned, and specific diseases resulting from their faulty expression have been identified. The clinical importance of transepithelial Cl− transfer is illustrated by studies of aqueous humor formation by the eye's bilayered ciliary epithelium. NaCl is taken up from the stroma by the pigmented ciliary epithelial (PE) layer, diffuses through gap junctions into the nonpigmented ciliary epithelial (NPE) layer, and is released into the aqueous humor largely through Na+ pumps and Cl− channels. ATP released by NPE cells can be ecto‐enzymatically metabolized to adenosine. Adenosine can mediate paracrine/autocrine stimulation of Cl− channels and aqueous humor secretion by occupying A3 adenosine receptors (ARs). A3AR agonists indeed elevate, and A3AR antagonists lower, intraocular pressure (IOP) in wild‐type mice. A3AR knockout mice have low IOP and their responses to A3AR agonists and antagonists are blunted; this suggests that reducing Cl−‐channel activity with A3AR antagonists may provide a novel approach for treating glaucoma. J. Exp. Zool. 300A:5–13, 2003. © 2003 Wiley‐Liss, Inc.
Purinergic signaling in the rabbit ciliary body epithelium
Authors: Farahbakhsh Nasser A.
Published: October 2003

In the past forty years, a wealth of information has accumulated that points to the presence of adenosine and adenine nucleotides in the anterior segment of the eye and a number of hypotheses have been introduced to describe the possible role of these agents in the regulation of aqueous humor flow. However, in the absence of a generally accepted model for the cellular and molecular mechanisms of aqueous humor formation by the ciliary body epithelium, efforts to identify the signal transduction pathway(s) responsible for regulation of the ion and water transport have not been successful. This article briefly reviews the evidence for (i), the presence in aqueous humor of adenine nucleotides, cyclic adenosine monophosphate and adenosine, their metabolic product, (ii), the possible role of these agents in the regulation of aqueous humor dynamics, and (iii), the expression of ecto‐nucleotidases, receptors, and second messengers that may mediate such regulation. Finally, a model for the regulation of aqueous humor formation by adenosine and ATP is proposed. J. Exp. Zool. 300A:14–24, 2003. © 2003 Wiley‐Liss, Inc.
Cl− absorption in European eel intestine and its regulation
Authors: Schettino T. , Lionetto M.G.
Published: October 2003

The intestinal epithelium of the euryhaline teleost fish, Anguilla anguilla, absorbs Cl− transepithelially. This gives rise to a negative transepithelial potential at the basolateral side of the epithelium and to a measured short circuit current. Cl− absorption occurs via bumetanide‐sensitive Na+‐K+‐2Cl− cotransport, localized on the luminal membrane. The cotransport operates in parallel with a luminal K+ conductance that recycles the ion into the lumen. Cl− leaves the cell across the basolateral membrane by way of Cl− conductance and presumably via a KCl cotransport. The driving force for this process is provided by the electrochemical sodium gradient across the plasma membrane, generated and maintained by the basolateral Na+‐K+‐ATPase. The resulting NaCl absorption process is active and enables marine fish to take up water, thereby compensating for water that was lost passively from the body. Fresh water acclimatized eel also absorb Cl− actively, although in smaller quantities, utilizing the same ion transport mechanisms as marine eels. This mechanism is basically the same as the model proposed for the thick ascending limb (cTAL). Cl− absorption is regulated by a number of cellular factors, such as HCO3−, pH, Ca2+, cyclic nucleotides, and cytoskeletal elements. It is sensitive to osmotic stress, and therefore is a good physiological model to study ion transport mechanisms that are activated when osmotic stress induces cell volume regulation. The activation of these various ion transport pathways is dependent on cellular transduction mechanisms in which phosphorylation events (mainly by PKC and MLCK for the hypertonic response) and cytoskeletal elements, either microfilaments or microtubules, seem to play key roles. J. Exp. Zool. 300A:63–68, 2003 © 2003 Wiley‐Liss, Inc.
The xenobiotic transporter, MRP2, in epithelia from insects, sharks, and the human breast: Implications for health and disease
Authors: Karnaky Karl John , Hazen‐Martin Debra , Miller David S.
Published: October 2003

A large number of mechanisms, including special excretory transporters, have evolved to help organisms excrete deleterious xenobiotics and endogenous molecules. We have examined the xenobiotic transport function of a putative multidrug resistance associated protein, MRP2, in three different epithelia: the insect renal (Malpighian) tubules, the secretory tubule of the shark rectal gland, and in ductules of the human breast. In the case of the insect and shark, transporter activity occurs in epithelia capable of great fluid transport. In the case of the insect Malpighian tubule, understanding the underlying mechanisms of this transporter may help with efforts to control populations of disease‐carrying agriculturally important insects. In striking contrast, ductule architecture in nonlactating human breast ductules is that of an epithelium with a closed lumen. Immunocytochemical studies show that MRP2 is localized in the apical region of the ductule epithelial cells. In this unique case, MRP2 substrates transported into the lumen could possibly be concentrated. Transport substrates of MRP2 include carcinogens as well as antioxidants and other salutary molecules. Thus, in the breast ductule, MRP2 may play a significant role in breast epithelial cell health and cancer carcinogenesis. J. Exp. Zool. 300A:91–97, 2003. © 2003 Wiley‐Liss, Inc.
Proteasome‐mediated degradation of the C‐terminus of the Alzheimer's disease β‐amyloid protein precursor: Effect of C‐terminal truncation on production of β‐amyloid protein
Authors: Nunan Janelle , Williamson Nicholas A. , Hill Andrew F. , Sernee M. Fleur , Masters Colin L. , Small David H.
Published: October 2003

The β‐amyloid protein (Aβ) is derived by proteolytic processing of the amyloid protein precursor (APP). Cleavage of APP by β‐secretase generates a C‐terminal fragment (APP‐CTFβ), which is subsequently cleaved by γ‐secretase to produce Aβ. Our previous studies have shown that the proteasome can cleave the C‐terminal cytoplasmic domain of APP. To identify proteasome cleavage sites in APP, two peptides homologous to the C‐terminus of APP were incubated with recombinant 20S proteasome. Cleavage of the peptides was monitored by reversed phase high‐performance liquid chromatography and mass spectrometry. Proteasome cleaved the APP C‐terminal peptides at several sites, including a region around the sequence YENPTY that interacts with several APP‐binding proteins. To examine the effect of this cleavage on Aβ production, APP‐CTFβ and mutant forms of APP‐CTFβ terminating on either side of the YENPTY sequence were expressed in CHO cells. Truncation of APP‐CTFβ on the N‐terminal side of the YENPTY sequence at residue 677 significantly decreased the amount of Aβ produced, whereas truncation on the C‐terminal side of residue 690 had little effect. The results suggest that proteasomal cleavage of the cytosolic domain of APP at the YENPTY sequence decreases γ‐secretase processing, and consequently inhibits Aβ production. Therefore, the proteasome‐dependent trafficking pathway of APP may be a valid therapeutic target for altering Aβ production in the Alzheimer's disease brain. © 2003 Wiley‐Liss, Inc.
Regulators of the neuropeptide‐degrading enzyme, EC 3.4.24.15 (thimet oligopeptidase), in cerebrospinal fluid
Authors: Shrimpton Corie N. , Wolfson Adele J. , Smith A. Ian , Lew Rebecca A.
Published: October 2003

Endopeptidase EC 3.4.24.15 (EP 24.15; thimet oligopeptidase) is a soluble metalloendopeptidase implicated in the metabolism of a number of neuropeptides, including neurotensin, gonadotropin‐releasing hormone, and opioid peptides. We have shown previously that thiol reducing agents, such as dithiothreitol, activate EP 24.15 by mediating the conversion of inactive multimeric forms to active monomers and that this conversion involves the disruption of intermolecular disulfide bonds involving cysteine residues 246, 248, and 253. We have identified two components of cerebrospinal fluid that activate recombinant EP 24.15, but have no effect on a thiol‐independent cysteine mutant form of the enzyme. The low molecular weight (<10 kDa) component co‐elutes with glutathione by reversed‐phase HPLC, whereas the high molecular weight component (>50 kDa) is sensitive to digestion with trypsin, suggesting it is proteinaceous in nature. These results suggest that EP 24.15 activity in the brain may be modulated by factors released into cerebrospinal fluid. © 2003 Wiley‐Liss, Inc.
ADAMs family members as amyloid precursor protein α‐secretases
Authors: Allinson Tobias M.J. , Parkin Edward T. , Turner Anthony J. , Hooper Nigel M.
Published: October 2003

In the non‐amyloidogenic pathway, the Alzheimer's amyloid precursor protein (APP) is cleaved within the amyloid‐β domain by α‐secretase precluding deposition of intact amyloid‐β peptide. The large ectodomain released from the cell surface by the action of α‐secretase has several neuroprotective properties. Studies with protease inhibitors have shown that α‐secretase is a zinc metalloproteinase, and several members of the adamalysin family of proteins, tumour necrosis factor‐α convertase (TACE, ADAM17), ADAM10, and ADAM9, all fulfil some of the criteria required of α‐secretase. We review the evidence for each of these ADAMs acting as the α‐secretase. What seems to be emerging from numerous studies, including those with mice in which each of the ADAMs has been knocked out, is that there is a team of zinc metalloproteinases able to cleave APP at the α‐secretase site. We also discuss how upregulation of α‐secretase activity by muscarinic agonists, cholesterol‐lowering drugs, steroid hormones, non‐steroidal anti‐inflammatory drugs, and metal ions may explain some of the therapeutic actions of these agents in Alzheimer's disease. © 2003 Wiley‐Liss, Inc.
Caspase regulation of genotoxin‐induced neural precursor cell death
Authors: D'Sa Cleta , Klocke Barbara J. , Cecconi Francesco , Lindsten Tulia , Thompson Craig B. , Korsmeyer Stanley J. , Flavell Richard A. , Roth Kevin A.
Published: October 2003

Neural precursor cells (NPCs) critically regulate brain morphogenesis and recent studies have revealed an unexpectedly high frequency of NPC chromosomal abnormalities and apoptosis in the developing brain. We have shown previously that the apoptotic response of NPCs to genotoxic agents is dependent on p53 and caspase‐9, but not Bax or caspase‐3 expression. In this study, we found that NPCs deficient in Apaf‐1, or both the pro‐apoptotic multidomain Bcl‐2 family members Bax and Bak, were resistant to cytosine arabinoside and γ‐irradiation‐induced apoptosis. Inhibitors of gene transcription, protein translation, and caspase activity also blocked genotoxin‐induced NPC apoptosis. Although caspase‐3 and caspase‐6 were both cleaved in response to DNA damage, neither of these effector caspases was critical for apoptosis. Genotoxin‐induced NPC death was accompanied by the generation of reactive oxygen species and could be inhibited by several known antioxidants. Conversely, DNA damage‐induced reactive oxygen species generation was inhibited significantly by gene disruption of p53, Apaf‐1, or caspase‐9, and combined deficiency of Bax and Bak, but not by caspase‐3 or caspase‐6 deficiency. These studies suggest that caspase‐9 activation is both necessary and sufficient for genotoxin‐induced neural precursor cell reactive oxygen species generation and death. © 2003 Wiley‐Liss, Inc.
Role of proteolysis in polyglutamine disorders
Authors: Tarlac V. , Storey E.
Published: October 2003

To date, nine polyglutamine disorders have been characterised, including Huntington's disease (HD), spinobulbar muscular atrophy (SBMA), dentatorubral‐pallidoluysian atrophy (DRPLA), and spinocerebellar ataxias 1, 2, 3, 6, 7 and 17 (SCAs). Although knockout and transgenic mouse experiments suggest that a toxic gain of function is central to neuronal death in these diseases (with the probable exception of SCA6), the exact mechanisms of neurotoxicity remain contentious. A further conundrum is the characteristic distribution of neuronal damage in each disease, despite ubiquitous expression of the abnormal proteins. One mechanism that could possibly underlie the specific distribution of neuronal toxicity is proteolytic cleavage of the full‐length expanded polyglutamine tract‐containing proteins. There is evidence found in vitro or in vivo (or both) of proteolytic cleavage in HD, SBMA, DRPLA, and SCAs 2, 3, and 7. In HD, cleavage has been demonstrated to be regionally specific, occurring as a result of caspase activation. These diseases are also characterised by development of intraneuronal aggregates of the abnormal protein that co‐localise with components of the ubiquitin‐proteasome pathway. It remains unclear whether these aggregates are pathogenic or merely disease markers; however, at least in the case of ataxin‐3, cleavage promotes aggregation. Inhibition of specific proteases constitutes a potential therapeutic approach in these diseases. © 2003 Wiley‐Liss, Inc.
JLK isocoumarin inhibitors: Selective γ‐secretase inhibitors that do not interfere with notch pathway in vitro or in vivo
Authors: Petit A. , Pasini A. , Alves da Costa C. , Ayral E. , Hernandez J.F. , Dumanchin‐Njock C. , Phiel C.J. , Marambaud P. , Wilk S. , Farzan M. , Fulcrand P. , Martinez J. , Andrau D. , Checler F.
Published: October 2003

γ‐Secretase activity is involved in the generation of Aβ and therefore likely contributes to the pathology of Alzheimer's disease. Blocking this activity was seen as a major therapeutic target to slow down or arrest Aβ‐related AD progression. This strategy seemed more doubtful when it was established that γ‐secretase also targets other substrates including Notch, a particularly important transmembrane protein involved in vital functions, at both embryonic and adulthood stages. We have described previously new non‐peptidic inhibitors able to selectively inhibit Aβ cellular production in vitro without altering Notch pathway. We show here that in vivo, these inhibitors do not alter the Notch pathway responsible for somitogenesis in the zebrafish embryo. In addition, we document further the selectivity of JLK inhibitors by showing that, unlike other described γ‐secretase inhibitors, these agents do not affect E‐cadherin processing. Finally, we establish that JLKs do not inhibit β‐site APP cleaving enzymes (BACE) 1 and BACE2, α‐secretase, the proteasome, and GSK3β kinase. Altogether, JLK inhibitors are the sole agents to date that are able to prevent Aβ production without triggering unwanted cleavages of other proteins. © 2003 Wiley‐Liss, Inc.
Consolidation of memory for odour‐reward association requires transient polysialylation of the neural cell adhesion molecule in the rat hippocampal dentate gyrus
Authors: Foley Andrew G. , Hedigan Kate , Roullet Pascal , Moricard Yves , Murphy Keith J. , Sara Susan J. , Regan Ciaran M.
Published: October 2003

Cell adhesion molecule function is involved in hippocampal synaptic plasticity and is associated with memory consolidation. At the infragranular zone of the dentate gyrus, neurons expressing the polysialylated form of the neural cell adhesion molecule (NCAM PSA) transiently increase their frequency at the 12‐hr posttraining time in behaviours elicited by stressful stimuli, such as those associated with conditioned avoidance, water maze, and fear conditioning paradigms. To determine whether learning‐induced modulation of NCAM polysialylation is limited to stressful paradigms, we employed a reward‐based odour discrimination task. Animals show a rapid acquisition and recall of this task in terms of latency to identify the food‐associated odour and the number of choice errors. Immunohistochemical procedures were employed to determine the change in NCAM PSA expression following task acquisition. NCAM PSA immunoreactivity in the hippocampal formation was most intense on the granule‐like neurons in the infragranular zone of the dentate gyrus, and their frequency transiently increased in the 12‐hr posttraining period. The nature of the transient increase in NCAM PSA‐immunoreactive neurons was indistinguishable from that observed following avoidance conditioning or spatial learning, in that it occurred at the same time. The transient increase in NCAM PSA expression is suggested to facilitate dendritic elaboration in response to the acquisition of novel behavioural repertoires. © 2003 Wiley‐Liss, Inc.
Identifying loci for behavioral traits using genome‐tagged mice
Authors: Liu Dahai , Singh Ram Pyare , Khan Arshad H. , Bhavsar Kinnar , Lusis Aldons J. , Davis Richard C. , Smith Desmond J.
Published: October 2003

Identification of behavioral loci through complex trait mapping remains a widely employed approach but suffers from poor gene localization and low replicability. Genome‐tagged mice (GTMs) are overlapping sets of congenic strains spanning the whole genome and offer the possibilities of superior mapping power and reproducibility. In this study, three GTM strains each consisting of an average ∼27 cM DBA/2J genomic intervals introgressed onto a C57BL/6J background were employed for localization of behavioral traits. These GTMs were chosen because the corresponding chromosomal regions had been previously identified as containing loci for learning and memory. Analysis of the GTMs allowed confirmation of the learning and memory loci, and one on chromosome 3 was in addition fine mapped to an 8.8‐cM region of overlap between two of the GTMs. Moreover, loci for prepulse inhibition of the startle response, acoustic startle response, and spontaneous locomotor activity were also mapped. These results suggest that the GTMs should be a valuable resource for mapping and confirmation of loci contributing to complex behavioral traits in the mouse. © 2003 Wiley‐Liss, Inc.
Ethanol‐induced oxidative stress precedes mitochondrially mediated apoptotic death of cultured fetal cortical neurons
Authors: Ramachandran Vinitha , Watts Lora Talley , Maffi Shivani Kaushal , Chen Juanjuan , Schenker Steven , Henderson George
Published: October 2003

In utero ethanol exposure elicits apoptotic cell death in the fetal brain, and this may be mediated by oxidative stress. Our studies utilize cultured fetal rat cortical neurons and illustrate that ethanol elicits a rapid onset of oxidative stress, which culminates in mitochondrially mediated apoptotic cell death. Cells exposed to ethanol (2.5 mg/ml) remained attached to their polylysine matrix during a 24‐hr exposure, but they exhibited distinct signs of oxidative stress, decreased viability, and apoptosis. Confocal microscopy of live cortical neurons pretreated with dichlorodihydrofluorescein diacetate demonstrated an increase in reactive oxygen species (ROS) within 5 min of ethanol exposure. The levels of ROS further increased by 58% within 1 hr (P < .05) and by 82% within 2 hr (P < .05), accompanied by increases of mitochondrial 4‐hydroxynonenal (HNE). These early events were followed by decreased trypan blue exclusion of 10% to 32% (P < .05) at the 6‐ to 24‐hr time points, respectively. This culminates in apoptotic death, with increases of Annexin V binding of 43%, 89%, 123%, and 238%, at 2, 6, 12, and 24 hr of ethanol treatment, respectively, as well as DNA fragmentation increases of 50% and 65% by 12 and 24 hr, respectively. Release of cytochrome c by mitochondria increased by 53% at 6 hr of exposure (P < .05), concomitant with activation of caspase 3 (52% at 12 hr, P < .05). Pretreatment with N‐acetylcysteine increased cellular glutathione and prevented apoptosis. These studies provide a time line illustrating that oxidative stress and formation of a proapoptotic lipid peroxidation product, HNE, precede a cascade of mitochondrially mediated events in cultured fetal cortical neurons, culminating in apoptotic death. The prevention of apoptosis by augmentation of glutathione stores also strongly supports a role for oxidative stress in ethanol‐mediated apoptotic death of fetal cortical neurons. © 2003 Wiley‐Liss, Inc.
Isolation of neural stem cells from the forebrain of deceased early postnatal and adult rats with protracted post‐mortem intervals
Authors: Xu Yi , Kimura Kazushi , Matsumoto Naoya , Ide Chizuka
Published: October 2003

Neural stem cells were isolated from deceased early postnatal and adult rats with varying post‐mortem intervals. Animals were killed by deep anesthesia and stored in a refrigerator at 4°C for 1–6 days before use. Neurospheres were obtained from the forebrain tissue, including the lateral ventricle in the early postnatal rats, and from the striatal wall of lateral ventricle, including the subventricular zone (SVZ) in adult rats. The number of neurospheres obtained in the primary culture from early postnatal animals was much larger than that from the adult rats. There was no significant difference in the population of neurospheres between the living and the deceased animals at least within 2 days after death. A few neurospheres were still obtainable at 6 days after death in early postnatal animals, but almost no neurospheres were obtained at 5 days after death in the adult rats. The differentiation capacity of neural stem cells in neurospheres was similar between the deceased and the living animals. The rich vascular bed in the SVZ of the lateral ventricle suggests that the vascular architecture might be in part responsible for the survival of the neural stem cells in the deceased animals. Neurosphere cells derived from deceased adult rats survived and differentiated mainly into glial cells in the host spinal cord tissue after transplantation into the injured spinal cord. Therefore, the neural stem cells from deceased animals express the same phenotypes as those from living animals in terms of neurosphere formation, proliferation, and differentiation at least 2 days after death. The neural stem cells from cadavers have great significance in terms of their clinical use as homografts for CNS regeneration. © 2003 Wiley‐Liss, Inc.
Plasmalemmal sealing of transected mammalian neurites is a gradual process mediated by Ca2+‐regulated proteins
Authors: Yoo Soonmoon , Nguyen Michael P. , Fukuda Mitsunori , Bittner George D. , Fishman Harvey M.
Published: October 2003

Cultured mammalian PC12 or B104 cells do not instantaneously restore a plasmalemmal barrier (seal) after neurite transection, as measured using fluorescent dye probes of various sizes and saline solutions with different [Ca2+]o. Rather, transected cells gradually (from 15 to 60 min postseverance) exclude probes (dye molecules) of progressively smaller size. Furthermore, an inhibitor (calpeptin) of a Ca2+‐activated cysteine protease (calpain) and antibodies or toxins to a Ca2+‐regulated protein (synaptotagmin) and other membrane fusion proteins (syntaxin and synaptobrevin) inhibit plasmalemmal sealing. These data obtained using molecular probes on mammalian cell lines are consistent with previous data on invertebrate giant axons indicating that Ca2+ plays many roles in the formation, accumulation, and fusion/interaction of vesicles gradually forming a seal at a site of plasmalemmal damage. © 2003 Wiley‐Liss, Inc.
Specific inhibitor of FGF receptor signaling: FGF‐2‐mediated effects on proliferation, differentiation, and MAPK activation are inhibited by PD173074 in oligodendrocyte‐lineage cells
Authors: Bansal Rashmi , Magge Suma , Winkler Susan
Published: October 2003

Multiple studies have shown that migration, proliferation, and differentiation of oligodendrocyte (OL) lineage cells are influenced by fibroblast growth factor‐2 (FGF‐2) signaling through its receptors (FGFR) FGFR‐1, FGFR‐2, and FGFR‐3. We report the effectiveness and specificity of a unique inhibitor, PD173074, for inhibiting FGF receptor signaling in OL‐lineage cells. Three FGF‐mediated responses of OL progenitors and two of differentiated OLs were examined by immunofluorescence microscopy and immunoblotting. PD173074 effectively antagonized the effect of FGF‐2 on proliferation and differentiation of OL progenitors in culture. One dose of PD173074 at nanomolar concentrations was sufficient to inhibit ongoing FGF‐2 mediated proliferation for prolonged periods, in a non‐toxic, dose‐dependent manner. In contrast, platelet‐derived growth factor (PDGF)‐induced proliferation was unaffected by PD173074. Similarly, mitogen‐activated protein kinase (MAPK) activation, a downstream event after activation of either FGFR or PDGFR, was also blocked by PD173074 in OL progenitors stimulated with FGF‐2 but not PDGF. A general tyrosine kinase inhibitor (PD166285), however, antagonized both FGF‐2‐ and PDGF‐mediated responses. PD173074 also completely antagonized two phenotypic alterations of differentiated OLs, specifically downregulation of myelin proteins, and their re‐entry into the cell cycle. We conclude that PD173704 is an effective and specific inhibitor for multiple FGF‐2‐mediated responses of both OL progenitors and differentiated OLs. This inhibitor provides a direct approach for identifying the importance of FGF signaling, comparable in effect to a knockout of all FGF receptors and all FGF ligands, while leaving other pathways unaffected. Thus, PD173704 is an excellent tool for investigating the role of FGF signaling in vivo in the context of combinatorial interactions of other signals. © 2003 Wiley‐Liss, Inc.
Distinct presenilin‐dependent and presenilin‐independent γ‐secretases are responsible for total cellular Aβ production
Authors: Wilson Christina A. , Doms Robert W. , Lee Virginia M.‐Y.
Published: October 2003

γ‐Secretase is the second of two proteolytic enzymes involved in the liberation of the β‐amyloid peptide (Aβ) from the amyloid precursor protein (APP). γ‐Secretase cleavage occurs at several intracellular sites, including the Golgi network and the endoplasmic reticulum/intermediate compartment (ER/IC) to produce multiple forms of the Aβ peptide that can be either secreted from the cell or remain intracellular. To date, most evidence has suggested that members of the presenilin protein family are required for γ‐secretase activity. Although it seems that presenilins are indeed necessary for the production of most secreted and intracellular Aβ particularly that generated in downstream organelles, it was shown recently that a presenilin‐independent γ‐secretase is active in the ER/IC and is responsible for the production of a portion of intracellular Aβ42. We discuss the implications of this finding for the understanding of presenilin biology and speculate on the putative identity of the presenilin‐independent cleavage activity. © 2003 Wiley‐Liss, Inc.
EP24.15 is associated with lipid rafts
Authors: Jeske Nathaniel A. , Glucksman Marc J. , Roberts James L.
Published: October 2003

Metalloendopeptidase EC 3.4.24.15 (EP24.15, thimet oligopeptidase) is a neuropeptide‐metabolizing peptidase expressed throughout the body, but primarily in the brain, gonads, and pituitary. For EP24.15 to have its greatest effect upon peptides in the periphery, it must be targeted and released into the extracellular space. Western blot analysis of fractions taken from discontinuous sucrose density gradients carried out on crude plasma membrane fractions from AtT‐20 cells reveals colocalization of EP24.15 and flotillin‐1, a known lipid raft marker. Further analysis revealed that an intracellular membrane marker and non‐lipid raft, plasma membrane marker, failed to colocalize, supporting EP24.15/lipid raft association. Furthermore, EP24.15 immunoreactivity in lipid raft fractions generated from cells treated with methyl β‐cyclodextrin (MβCD) was greatly reduced. Finally, treatment with MβCD resulted in the accumulation of EP24.15 in the media of drug‐treated cells over vehicle‐treated cells, suggesting that a large percentage of EP24.15 associating with lipid rafts resides on the extracellular surface of the plasma membrane. With this exofacial localization, EP24.15 could have ample access to neuropeptides not only in the immediate microenvironment, but the ability to degrade or modify peptides bound for receptor interaction. © 2003 Wiley‐Liss, Inc.
Extracellular matrix and matrix metalloproteinases in sciatic nerve
Authors: Platt C.I. , Krekoski C.A. , Ward R.V. , Edwards D.R. , Gavrilovic J.
Published: October 2003

Although matrix metalloproteinases (MMPs) are increasingly being implicated in several pathologies of the nervous system, it is not yet clear what role they play in normal neurobiological processes. We review the expression of extracellular matrix (ECM) components as well as MMPs and tissue inhibitors of metalloproteinases (TIMPs) in the peripheral nervous system. We explore the expression of certain MMPs and the four TIMPs at the mRNA level in the postnatal mouse sciatic nerve. In addition, we have used substrate gel and in situ zymography to determine levels of MMP‐2 and ‐9 and TIMP activity in rat sciatic nerve after crush and during regeneration. A rapid and transient increase in MMP‐9 localised at and immediately distal to the site of injury was observed, whereas an increase in MMP‐2 activity was delayed, prolonged, and extended proximal and distal to the injury site. This activity coincides with periods of axonal elongation, suggesting that it could act to facilitate axonal extension along the nerve matrix. We also detected multiple species of gelatinolytic inhibitory activity, including TIMP‐1 and ‐3 in control and injured nerve. These activities probably act to prevent uncontrolled gelatinolytic activity, maintaining nerve integrity at the level essential for axonal regrowth. © 2003 Wiley‐Liss, Inc.
Cysteine proteases are the major β‐secretase in the regulated secretory pathway that provides most of the β‐amyloid in Alzheimer's disease: Role of BACE 1 in the constitutive secretory pathway
Authors: Hook Vivian Y.H. , Reisine Terry D.
Published: October 2003

This article focuses on β‐amyloid (Aβ) peptide production and secretion in the regulated secretory pathway and how this process relates to accumulation of toxic Aβ in Alzheimer's disease. New findings are presented demonstrating that most of the Aβ is produced and secreted, in an activity‐dependent manner, through the regulated secretory pathway in neurons. Only a minor portion of cellular Aβ is secreted via the basal, constitutive secretory pathway. Therefore, regulated secretory vesicles contain the primary β‐secretases that are responsible for producing the majority of secreted Aβ. Investigation of β‐secretase activity in regulated secretory vesicles of neuronal chromaffin cells demonstrated that cysteine proteases account for the majority of the β‐secretase activity. BACE 1 is present in regulated secretory vesicles but provides only a small percentage of the β‐secretase activity. Moreover, the cysteine protease activities prefer to cleave the wild‐type β‐secretase site, which is relevant to the majority of AD cases. In contrast, BACE 1 prefers to cleave the Swedish mutant β‐secretase site that is expressed in a minor percentage of the AD population. These new findings lead to a unifying hypothesis in which cysteine proteases are the major β‐secretases for the production of Aβ in the major regulated secretory pathway and BACE 1 is the β‐secretase responsible for Aβ production in the minor constitutive secretory pathway. These results indicate that inhibition of multiple proteases may be needed to decrease Aβ production as a therapeutic strategy for Alzheimer's disease. © 2003 Wiley‐Liss, Inc.
Effects of immunosuppression on regrowth of adult rat retinal ganglion cell axons into peripheral nerve allografts
Authors: Gillon Russell S. , Cui Qi , Dunlop Sarah A. , Harvey Alan R.
Published: October 2003

Analysis of the effectiveness of allografts and immunosuppression in the repair of nerve defects in the adult peripheral nervous system (PNS) has a long experimental and clinical history. There is little information, however, on the use of allografts in peripheral nerve (PN) transplantation into the injured central nervous system (CNS). We assessed the ability of PN allografts (from Dark‐Agouti rats) to support regeneration of adult rat retinal ganglion cell (RGC) axons in immunosuppressed host Lewis rats. PN allografts were sutured onto intraorbitally transected optic nerves. Three weeks after grafting, regenerating RGC axon numbers were determined using retrograde fluorescent labelling, and total axons within PN grafts were assessed using pan‐neurofilament immunohistochemistry. In the absence of immunosuppression, PN allografts contained few axons and there were very few labelled RGC. These degenerate grafts contained many T cells and macrophages. Systemic (intraperitoneal) application of the immunosuppressants cyclosporin‐A or FK506 reduced cellular infiltration into allografts and resulted in extensive axonal regrowth from surviving RGCs. The average number of RGCs regenerating axons into immunosuppressed allografts was not significantly different from that seen in PN autografts in rats sham‐injected with saline. Many pan‐neurofilament‐positive axons, a proportion of which were myelinated, were seen in immunosuppressed allografts, particularly in proximal regions of the grafts toward the optic nerve‐PN interface. This study demonstrates that PN allografts can support axonal regrowth in immunosuppressed adult hosts, and points to possible clinical use in CNS repair. © 2003 Wiley‐Liss, Inc.
Opposing effects of low and high‐dose clozapine on survival of transgenic amyotrophic lateral sclerosis mice
Authors: Turner B.J. , Rembach A. , Spark R. , Lopes E.C. , Cheema S.S.
Published: October 2003

Clozapine is a potent atypical neuroleptic or antipsychotic agent used to relieve symptoms of early‐diagnosed schizophrenia. Aside from well‐described dopamine and serotonin receptor blockade effects, clozapine may also be neuroprotective through its modulation of the p75 neurotrophin receptor (p75NTR) and superoxide dismutase 1 (SOD1) expression. The death‐signalling activities of both p75NTR and mutant SOD1 are implicated in motor neuron degeneration in humans and transgenic mice with amyotrophic lateral sclerosis (ALS). We therefore investigated the effects of clozapine in cell culture and mouse models of ALS. Clozapine dose‐dependently inhibited full‐length and cleaved p75NTR but not SOD1 protein expression in the motor neuron‐like (NSC‐34) cell line. Furthermore, low concentrations of clozapine protected NSC‐34 cells from paraquat‐mediated superoxide toxicity, nerve growth factor (NGF)‐induced death signalling, and serum deprivation, whereas high concentrations potentiated death. Systemic thrice‐weekly administration of low and high‐dose clozapine to mutant superoxide dismutase 1 (SOD1G93A) mice produced differential effects on disease onset and survival. Low‐dose treatment was associated with delayed locomotor impairment and death, compared to high‐dose clozapine, which accelerated paralysis and mortality (P < 0.05). Increased death was not attributable to toxicity, as clozapine‐induced agranulocytosis was not detected from blood analysis. High‐dose clozapine, however, produced extrapyramidal symptoms in mice manifest by hindlimb rigidity, despite reducing spinal cord p75NTR levels overall. These results suggest that although clozapine may exert p75NTR‐mediated neuroprotective activity in vitro, its profound antagonistic effects on dopaminergic and serotonergic systems in vivo at high doses may exacerbate the phenotype of transgenic ALS mice. © 2003 Wiley‐Liss, Inc.
Centrally administered norepinephrine modifies the behavior induced by corticotropin‐releasing factor in neonatal chicks
Authors: Zhang Rong , Tachibana Tetsuya , Takagi Tomo , Koutoku Tomoyuki , Denbow D. Michael , Furuse Mitsuhiro
Published: October 2003

We previously reported that glucagon‐like peptide‐1 decreased corticotropin‐releasing factor (CRF)‐induced behaviors in neonatal chicks, and such an effect is hypothesized to act through norepinephrine (NE). Experiments were designed to explore the effect of the NE on CRF‐induced behaviors. In experiment 1, the chicks were intracerebroventricularly (i.c.v.) administered saline, 0.1 μg of CRF, 50.0 μg of NE, or 0.1 μg of CRF with 50.0 μg of NE. Behavior was monitored for the 10 min immediately after i.c.v. injection, and plasma corticosterone was analyzed at the end of behavior tests. Compared with the control, chicks were excited by CRF as evidenced by increased spontaneous activity and distress vocalizations (DVs). NE decreased the spontaneous activity of chicks, and the differences diverged with time. DVs completely disappeared in the presence of NE, and sleep‐like (sitting with eyes closed) behavior was observed in the same birds. NE‐treated birds spent most of the time in a sleep‐like posture irrespective of CRF treatment. CRF‐treated chicks had increased plasma corticosterone, whereas NE injection caused a decrease in corticosterone. In experiment 2, the effect of NE was further studied using i.c.v. administration with either 0.1 μg of CRF alone or 0.1 μg of CRF plus 12.5, 25.0, or 50.0 μg of NE. NE dose dependently modified the CRF‐induced locomotor activity in the open field, and DVs disappeared when chicks were given any dose of NE with CRF. With these findings taken together, it is suggested that central NE interacts the CRF‐induced behaviors in neonatal chicks. © 2003 Wiley‐Liss, Inc.