bluuebunny/crossref_metadata_2025 huggingface dataset:
Poster Sessions BP08: Cytoskeleton and Molecular Motors. Sticking out its neck: myosin‐1c binding to hair‐cell receptors requires its IQ domains and is regulated by calmodulin
Authors: Cyr J. L.
Published: July 2010

As the sensory receptor cells of the inner ear, hair cells transduce mechanical stimuli into electrical signals. While the electrophysiological properties of hair cells are well understood, little is known about the molecular basis of mechanoelectrical transduction. In particular, the identities of the transduction channel and other components of the transduction machinery are unknown. Myosin‐1c, an unconventional myosin formerly called myosin‐1 beta, is one identified component of the hair‐cell transduction apparatus where it functions in the adaptation process. Because of its role in adaptation, myosin‐1c must interact, either directly or indirectly, with other components of the transduction machinery, including the channel itself. As a result, we sought to characterize myosin‐1c receptors in hair cells. Using recombinant fragments of myosin‐1c with its associated calmodulin light chains, we examined binding of these complexes to myosin‐1c receptors in the hair bundle, the mechanosensitive organelle housing the transduction apparatus. Astonishingly, binding to hair‐cell receptors was not dependent upon the myosin's tail sequences, the domain of the protein long proposed to bind to intracellular cargo. Instead, binding to intracellular receptors depends upon the neck region of myosin‐1c which contains four calmodulin‐binding IQ domains. Calmodulin blocks myosin‐1c interaction with its receptors by binding to the myosin and obscuring the myosin‐1c/receptor interaction domain. The calcium‐sensitive binding of calmodulin to myosin‐1c may therefore regulate myosin‐1c interactions with other components of the transduction apparatus.
Endocrinological function of brain‐derived neurotrophic factor in diabetic mice
Authors: Taiji M. , Tsuchida A. , Nakagawa T. , Ogawa W. , Kasuga M. , Noguchi H.
Published: July 2010

We previously found that chronic administration of Brain‐derived neurotrophic factor (BDNF) ameliorates glucose metabolism in diabetic animals. This demonstrated a new concept that a neurotrophic factor plays endocrinological function as well as neurological one. However, the mechanism underlying this new function is still poorly understood. In this study, we investigated the acute effects of BDNF on glucose metabolism and energy expenditure in diabetic mice. First, a single subcutaneous (s.c.) (70 mg/kg) or intracerebroventricular (i.c.v.)(0.3 mg/kg) injection of BDNF immediately enhanced insulin‐dependent hypoglycemic action in streptozotocin‐induced diabetic mice. In liver of this model, s.c. BDNF injection rapidly increased insulin‐stimulated tyrosine phosphorylation of insulin receptors (3.8 fold) and PI 3‐kinase activity (1.8 fold). Since i.c.v. injection reproduced the similar actions, BDNF may act on the central nervous system and rapidly modulate insulin signal transduction in peripheral tissues. Next, we examined the acute effects of BDNF on energy expenditure. The body temperatures of hyperphagic and obese diabetic C57BL/KsJ‐db/db mice dropped remarkably in 24 h after food removal. A single s.c. (20 mg/kg) or i.c.v. (0.125 mg/kg) injection of BDNF to db/db mice prevented the reduction of body temperature. BDNF increased the norepinephrine turnover and uncoupling protein‐1 mRNA levels in brown adipose tissue of db/db mice. Taken together, these suggest that BDNF has the direct action on the central nervous system and then regulates glucose and energy metabolisms possibly through modulating the autonomic nervous system in diabetic animals.
Expression of retrograde transport proteins during oligodendrocyte
Authors: Gould R. M. , Bukher T. , Barbarese E.
Published: July 2010

Using a combination of subcellular fractionation and suppression subtractive hybridization, we found that a high molecular weight dynein light intermediate chain‐2 mRNA copurifies with myelin basic protein mRNA in rat brain myelin. We used a combination of subtraction efficiency and Northern blot analysis to show that other dynein (dynein heavy chain, dynein intermediate chains 1 and 2 and dynein light intermediate chain‐1) and dynactin (arp‐1, p150glued, dynamitin, CLIP‐170 and capB1) mRNAs do not copurify with these mRNAs in rat brain myelin. We used immunofluorescence and Western blotting to show that all of these proteins are expressed by cultured oligodendrocytes. These results suggest that of all the dynein/dynactin proteins synthesized in oligodendrocytes, only one, dynein light intermediate chain‐2, is synthesized in the cell's processes (near sties of myelin sheath assembly). Consequently, the addition of dynein light intermediate chain‐2 molecules to other dynein/dynactin proteins at sites of myelin sheath assembly may influence retrograde transport initiated at these distal sites.

Acknowledgements:  Supported by grant RG2944A5/1 from the National Multiple Sclerosis Society.
Studies on targeting of vesicle transport using virally medicated expression system
Authors: Lin M. , Brady S. T.
Published: July 2010

Neurons are composed of two morphologically and molecularly distinct domains, axons and dendrites. The accurate localization of proteins to these domains is critical for the neuronal functions. The proteins destined to localize into different cellular domains are probably packaged into different populations of carrier vesicles in the trans‐Golgi network. The objectives of this study are to examine the mechanisms to regulate targeting of transferrin receptor (TfR), a dendritic protein, neuron‐glia cell adhesion molecule (NgCAM), an axonal protein, and amyloid precursor protein (APP) and synaptophysin (p38), synaptic proteins, using live cell imaging system. Previous studies were complicated by overexpression artifacts. Our approach to this study was to apply an adenovirus mediated regulatable expression system for TfR, NgCAM, APP and p38 in NT‐2 cells, a neuronal precursor cell line, and BHK cells. Live cell imaging was performed with confocal microscopy. There are two specific features of our adenovirus systems: (1) proteins are tagged with green fluorescent protein (GFP) or its variants (YFP and CFP) for live cell imaging; (2) protein expression is under the control of tetracycline responsive element (Tet‐On), for tight control of both timing and levels of expression. Our results showed that APP, p38 and TfR vesicle movements in both BHK and NT‐2 cells were bi‐directional. NgCAM in BHK cells showed plasma membrane localization and minimal vesicle movement whereas expression of NgCAM in NT‐2 cells showed both plasma membrane and vesicle localization with bidirectional vesicle movements. In conclusion, our adenovirus system appears well suited for live cell imaging and could be adapted with other proteins of interest for studies of protein transport and sorting during neuronal development.
Poster Sessions CP06: Cell Surface, Cell Membrane. Glucuronyl glycoconjugates replace sulfoglucuronyl glycoconjugates in HNK‐1 knockout mice
Authors: Chou D. K. H. , Schachner M. , Jungalwala F. B.
Published: July 2010

Sulfoglucuronyl carbohydrate (SGC), reactive with HNK‐1 antibody, is expressed in several glycolipids, glycoproteins and proteoglycans of the nervous system. The interaction of SGC with SGC‐binding protein, SBP‐1 has been implicated in cell‐cell recognition, neurite outgrowth and neuronal migration during development. In sulfotransferase (ST) null mutant mice, which lack SGC, synaptic transmission in pyramidal cells of the hippocampus was increased and long‐term potentiation was reduced. However, ST null mice are viable, fertile and have wild type anatomy of all major brain areas and many non‐neural organs. Failure to observe severe phenotype in the ST null mice prompted us to determine the compensatory molecular replacement of SGC by analyzing the carbohydrate of glycolipids and glycoprotefins of the mutant nervous system. In the ST null mice, SGC containing molecules were absent and they were replaced by the precursor glucuronyl carbohydrate (GC) containing molecules. Other relevant glycolipids and proteins were not affected. The GC molecules in the mutant were localized at the same anatomical sites as the SGC molecules in the wild type. In vitro binding studies showed that similar to sulfoglucuronyl glycolipids, glucuronyl glycolipids interacted with SBP‐1, but with a lower binding capacity. In vitro studies with explant cultures of cerebellum indicated that neurite outgrowth and cell migration were not significantly affected, possibly due to interaction of SBP‐1 with the GC molecules. The results indicated that in vivo SBP‐1–GC interaction was sufficient enough for normal neurite outgrowth and cell migration in the mutant and thus having a minimal abnormal phenotype.
Activation of PKC isoforms by ATP/UTP and PMA in murine astrocytes
Authors: Sun G. Y. , Xu J. , Liao Z. , Lo S‐C. , Weisman G. A.
Published: July 2010

Protein kinase C (PKC) isoforms are known to play an important role in regulating activities of a number of receptor signalling pathways. Our earlier studies have demonstrated that in murine astrocytes, PKC and mitogen‐activated protein kinases (MAPK) are involved in the stimulation of cytosolic phospholipase A2 and arachidonic acid (AA) release by extracellular nucleotides (ATP or UTP) and phorbol myristate acetate (PMA). These studies showed that PKC inhibitors or PKC down‐regulation induced by prolonged exposure to PMA had variable effects on AA release induced by ATP/UTP or PMA. PKC down‐regulation reduced PMA‐ but not ATP/UTP‐mediated AA release. In this study, we examined responses of PKC isoforms to stimulation by ATP/UTP and PMA in primary astrocytes isolated from mouse brain. These astrocytes showed 4 major PKC isoforms, namely, PKCα, ε, ι, and λ. Prolonged treatment of astrocytes with PMA resulted in down‐regulation of PKCα and ε, but not PKCι and λ. Separation of membrane and cytosol fractions revealed that PKCα was largely localized to the cytosol, PKCε in the membrane, whereas PKCι and λ were present in both membrane and cytosol fractions. Rapid translocation of PKCα from the cytosol to cell membrane could be observed in astrocytes treated with PMA, but not ATP or UTP. Neither PMA nor ATP altered cytosol and membrane distribution of PKCε, ι and λ. The lack of an effect of ATP and UTP on PKCα translocation suggests that nucleotide‐mediated AA release occurs by a PKCα‐independent mechanism.

Acknowledgements:  Supported by P01 AG 18357 from NIA.
Effect of gangliosides on adenylyl cyclase activity in the cortex and striatum of rats
Authors: Nalivaeva N. N. , Plesneva S. A. , Dubrovskaya N. M. , Zhuravin I. A.
Published: July 2010

By studying the effects of gangliosides (G) on learning and memory we have found that i.p. administration of G led to a decrease in AC activity in the cortex (Cx) and striatum (Str) as well as in the threshold of the sensitivity of striatal neurons to the effect of cholinergic agonists. G also modified the sensitivity of AC from the Cx and Str to such agents as Gpp[NH]p and forskolin. The aim of this work was to analyze the correlation between the changes in the activity of AC in the Cx and Str, concentration of G in these brain structures as well as formation of motor reactions of newborn rats during the first month of postnatal ontogenesis. It was found that new born rats develop normal body rotation by sixth day, locomotion by fifteenth day and stabilization of locomotor activity and supporting body balance by 20–22nd day. As shown previously, the concentration of gangliosides in the Cx and Str is gradually increasing during the first month of animal development. The activity of AC (pmol cAMP/min/mg of protein) in the Cx was found to decrease from 34.75 to 4.09 and in the Str from 46.00 to 11.67 during the first week after birth. However in the periods of formation of general behavioural reactions we observed a statistically significant increase in AC activity: in the Str on 10th and 26th days (p < 0.01) and in the Cx from 10th to 19th days (p < 0.05) compared to the AC activity on 5th and 30th days. Thus, formation of locomotor activity and posture‐tonic reactions during development of rats in early postnatal ontogenesis correlates with increasing concentration of G and basal activity of AC. Supported by RFBR (99‐04‐49751) and RAS (99‐06‐287).
Analysis of glycosphingolipid composition of a neural progenitor cell line
Authors: Suetake K. , Liour S. S. , Tencomnao T. , Kraemer Stacy , Yu Robert K.
Published: July 2010

Continuous neurogenesis from neural stem cells in adult brain suggests the possibility that these newly generated neural tissue may be capable of repair of the neurodegeneration associated with genetic disorders, trauma, and aging. Glycosphingolipids (GSLs) play important roles in cellular growth, differentiation and cell signalling; however, little is known about the GSL composition in neural progenitor and/or stem cells. In order to characterize the GSL composition in neural stem cells, we studied the multipotential C17.2 cell line that was generated after myc‐transformation of isolated from neonatal mouse cerebellar neural progenitor cells. The developmental potential of C17.2 cells is similar to the endogenous neural stem cells, i.e. they are capable of self‐renewal and differentiation into neurons and glia (Snyder et al. Cell68, 33–51, 1992). HPTLC analysis of the total lipid fractions isolated undifferentiated C17.2 cells revealed that the major GSL expressed in these cells were glucosylceramide, GM3, GM2, GM1, and GD1a. Histochemical staining for GM1 using cholera toxin B subunit revealed punctate staining on the plasma membrane, suggesting GM1 at the plasma membrane, maybe clustered into microdomain‐like structures. Our results indicate that the undifferentiated C17.2 neural stem cell line is unique in expression of a‐series gangliosides. These gangliosides may prove to be useful markers for these cells.

Acknowledgements: Supported by an NIH grant NS11853 and a grant from the Children's Medical Research Foundation.
Transcription factors NF‐κB and Sp1 are major determinants of the basal promoter activity of the rat GD3‐synthase gene
Authors: Zeng G. , Gao L. , Xia T. , Tencomao T. , Yu R. K.
Published: July 2010

GD3‐synthase is one of the key sialyltransferases responsible for synthesis of ganglioside GD3, the substrate for initiation of the ‘b’ and ‘c’ series ganglioside synthesis. We have previously cloned the rat GD3‐synthase gene promoter, and preliminary characterization has identified a minimal 0.5‐kb region that has a strong basal promoter activity, and is GC‐rich and has no CAAT or TATA boxes. In this study, we showed that the Sp1 and NF‐κB sites in this region significantly contributed to basal GD3‐synthase promoter activity. When either the Sp1 or NF‐κB sites were deleted, a 50% decrease in promoter activity was observed. The same results were obtained by a decoy strategy using oligonucleotides containing the Sp1 or NF‐κB sites. The binding to the Sp1 and NF‐κB sites was confirmed by electrophoretic mobility shift assay (EMSA), competition and supershift EMSA. In addition, cell‐type specific activation of the promoter was also determined. The promoter was highly activated in the GD3‐expressing F‐11 cells while repressed in NG‐108 cells in which GD3 is almost undetectable. An additional band of NF‐κB family was identified only in the F‐11 nuclear extract using the NF‐κB consensus probe by EMSA. DNA pull‐down assays were further carried out to screen proteins that bound to the promoter including the basal region and the potential negative‐regulatory region between −526 and −769. More than 10 major binding proteins were pulled down, some of which were present only in the F‐11 or NG‐108 nuclear extracts. Our data demonstrate that NF‐κB and Sp1 are the major determinants for the basal promoter activity and some factors such as NF‐κB may be involved in cell type‐specific expression of the gene.
c‐Fos activates phospholipid metabolism in events associated to growth and differentiation
Authors: Caputto B. L. , Gil G. A. , Portal M. M. , Borioli G. A. , Renner M. L. , Guido M. E.
Published: July 2010

32P phospholipid labelling increases in quiescent fibroblasts stimulated with serum to re‐enter growth. This stimulation of phospholipid synthesis shows two waves that depend on the transcription and translation of the immediate early gene c‐fos. The time‐course pattern of c‐Fos protein expression resembles the activation pattern of phospholipid synthesis (The FASEB J, January 52001, 10.1096/fj.00‐0446fje). NGF‐treatment of PC12 cells promotes a strong increase in the expression of c‐Fos and enhances phospholipid synthesis in a c‐Fos‐concentration‐dependent way. Specifically blocking Fos expression with a c‐Fos mRNA antisense oligonucleotide impairs activation of phospholipid synthesis and also greatly inhibits cell differentiation. By contrast, inhibition of Fos degradation in NGF‐treated cells significantly increases c‐Fos content, phospholipid synthesis and neurite elongation. Blocking nuclear import of c‐Fos (AP‐1) at the initiation of NGF treatment completely blocks neurite formation. However, if c‐Fos nuclear import is blocked 16 h after addition of NGF to the cell cultures, neurite elongation continues almost normally. These results indicate that c‐Fos has a dual function in developing cells: it first releases the nuclear program for differentiation and then sustains growth by activating key components required for membrane genesis, in the cytoplasm. The mechanisms underlying this novel regulatory activity of c‐Fos on phospholipid synthesis, that is independent of its transcription factor activity, are being studied.

Acknowledgements:  Financed by Beca Carrillo‐Oñativia, FONCyT, Agencia Córdoba. Ciencia (CONICOR), SeCyT‐UNC, and CONICET.
Aluminum triggers NFκB signalling, inflammatory and apoptotic gene expression in human neural cells
Authors: Lukiw W. J. , Bazan N. G.
Published: July 2010

Aluminum, the most abundant neurotoxic metal in the biosphere, has been implicated in the etiology of neurodegenerative disorders including Alzheimers disease (AD). To further understand aluminum's influence on brain gene expression we examined 2360 RNA message levels in normal human neural progenitor (NHNP) cells using DNA microarrays. Of the 53 gene expression levels that were altered by a factor of >3‐fold (p < 0.05) over control, 44 were down‐regulated. This group included transcripts encoding 17 transcription factors (TFs) or TF kinases, 12 membrane receptors, genes encoding 10 growth factors, neurotransmitters, DNA ligase, DNA repair enzymes and antiapoptotic factors such as bcl‐2. The only TFs found to be consistently induced by aluminum on these panels were members of the pro‐inflammatory (PI) NFκB gene family. As verified independently by RT–PCR, eight of the nine genes found to be induced by aluminum encoded PI or proapoptotic signalling elements, including NFκB p52/p65, IL‐1 precursor, COX‐2 and DAXX, a FAS binding protein known to induce apoptosis and repress transcription. These PI gene promoters contain multiple NFκB binding sites; electrophoretic mobility shift assay and [kB]4 vector transfection analysis indicated that aluminum strongly induced NFκB‐DNA binding, suggesting a role for aluminum and NFκB in driving PI gene expression in neural cells. Interestingly, many aluminum‐induced PI genes are also elevated in AD. These data show that aluminum triggered NHNP cells emulate many of the gene expression patterns observed in AD including the rampant up‐regulation of NFκB‐sensitive and PI signalling genes.

Acknowledgements:  Supported in part by NIH AG18031, NS23002 and the EENT Foundation.
Sodium thiosulfate as an effective antioxidant substance at experimental mycotoxin zearalenon poisoning
Authors: Karagyozyan M. K.
Published: July 2010

Mycotoxin zearalenon (MZ) in concentration 2.5–15.0 μg/mL of alcohol solution activates the reactions of monoamines biosynthesis, while 20.0–25.0 μg/mL of MZ has a contrarary effect. The molecular mechanism of changes noticed is conditioned by action of MZ on numerous proteins functioning in chromaffin granules mainly in their membranes, such as the cytochrome B561, an acidic cooper‐containing protein and the membrane‐bound form of dopamine‐β‐monooxigenase, which catalyses the reaction of transformation of dopamine into noradrenaline. It has been established also that in the brain mitochondrial and microsomal membranes the MZ induces pronounced abnormalities in the ratio of phospholipid‐phospholipid interrelations. These changes are conditioned by significant intensification of the phospholipase A2 and phosphatidylserine (PS) decarboxylase activites, with formation of high concentrations of lysophosphatidylcholines, free polyenic fatty acids, lipid peroxides and by increasing of PS quantity in the systems studied. Using on this background single intramuscular administration of 1.0 mL of 10.0% aquaus solution of sodium thiosulfate (ST) normalizes and establishes the initial level of phospholipid (PL) metabolism intensity. The content of PL in the investigated membranes remains unchanged if ST was administrated before the MZ poisoning modulation. Antioxidant properties of ST are conditioned in particular by elevation of PS quantity, which are of great importance in stimulation of cell respiratory function, hence the cell activity in general.
Mechanisms of inflammation in spinal cord injury
Authors: Rafati D. S. , Perez‐Polo J. R.
Published: July 2010

The inflammatory response initiated after spinal cord injury (SCI) is characterized by an increase in cytokines, notably IL‐1β. Among its effects are the transcriptional control of cyclooxygenase 2 (COX‐2) and the inducible form of nitric oxide synthase (iNOS). While they may ameliorate inflammation, they may also cause cellular damage via production of reactive oxygen species (O2 − ., OH·, NO·). The transcription factor NF‐κB is a key intermediary in the signalling pathways leading from IL‐1β expression to COX‐2 and iNOS stimulation. Binding of NF‐κB can be abrogated by the use of oligonucleotide ‘decoys’ that compete for the cognate endogenous NF‐κB proteins. Using injections of fluorescent ‘decoy’ oligonucleotides into two‐month‐old‐male rats after SCI into the site of injury, we found prompt, robust and transient uptake of labelled ‘decoys’ into both cytoplasm and nuclei of resident cells. Injection of ‘decoys’ containing the NF‐κB binding sequences present in the COX‐2 promoter region, together with a ‘nonsense’ sequence, showed selective effects on iNOS expression at the site of injury. These results are consistent with the hypothesis that NF‐κB transcriptional regulation of COX‐2 and iNOS are linked and may be an element in the pathophysiology and recovery of mammalian spinal cord after contusion injury.

Acknowledgements:  Supported in part by NINDS Grant NS‐39161 and Shriners Hospital Grant 8710.
Poster Sessions CP13: Hypoxia, Ischemia and Oxidative Stress. Changes of brain nitric oxide production in experimental cerebral ischemia
Authors: Zhang H. , Hu Y. Y. , Murong S. X.
Published: July 2010

In order to study the role of nitric oxide (NO) in ischemic brain injury. Global cerebral ischemia was established in SD rats by modified Pulsinelli's method. The activities of constitutive nitric oxide synthase (cNOS), inducible NOS (iNOS), neuronal NOS (nNOS), nitrite (NO2) and cyclic GMP in cerebral cortex, hippocampus, striatum and cerebellum at different time intervals were measured by radioimmunoassy, NADPH‐d histochemistry and fluorometry methods. The results showed that the activities of cNOS increased at 5 min in four regions and decreased in cortex, hippocampus and striatum at 60 min, in cerebellum at 15 min iNOS increased in cortex and striatum at 15 min, in hippocampus and cerebellum at 10 min, and persisted to 60 min. The expression of nNOS increased after 5 min ischemia in cortex, striatum and hippocampus, and return to normal at 30–60 min. The NO2 and cGMP also increased after 5–15 min ischemia and returned to normal after 30–60 min ischemia. These results indicated that the NO participated in the pathogenesis of cerebral ischemia injury and different types of NOS play different role in the cerebral ischemia injuries. Selected specific NOS inhibitors to decreased the excessive production of NO at early stage may help to decrease the ischemic injury.
Sphingolipids in rat model of transient focal cerebral ischemia: implication for stroke injury
Authors: Khan M. , Sekhon B. K. , Sekhon C. S. , Singh I. , Singh A. K.
Published: July 2010

Lipids are essential for signal transduction in response to trauma leading to neurodegeneration. Ceramide is an important mediator of apoptosis and cell proliferation. We studied the involvement of ceramide/sphingomyelin pathway in rat brain (stroke model) after 45 min ischemia followed by 24‐h reperfusion. Ischemia was performed through occlusion of right middle cerebral artery (MCA). The level of ceramide was found increased (70–100% in ischemic side of brain v/s contralateral side of brain). Sphingomyelin levels were also decreased by 20–25% in ischemic brain v/s contralateral side of brain. Increase in ceramide and decrease in sphingomyelin were in good agreement with observed apoptotic cell loss (TUNEL assay) and decrease in the level of cardiolipin (a mitochondrian specific phospholipids) in affected ischemic brain. N‐acetyl cysteine (NAC), a therapeutic agent recognized as potent antioxidant provided protective effect. Pretreatment with NAC before ischemia reduced the infarct volume size, suppressed apoptosis, restored cardiolipin level and decreased the levels of free fatty acids. However, NAC did not normalize the ceramide level. These interesting observations raise a question about the role of ceramide and its relationship with apoptosis and oxidative stress in rat brain ischemia.

Acknowledgements:  Supported by NIH grants NS‐40144, NS‐40810, NS‐22576, NS‐34741 and NS‐37766.
Diazepam prevents changes in intracellular Cl− and Ca2+ and restores neuronal activity after ischemia in vitro
Authors: Galeffi F. , Sah R. , Schwartz‐Bloom R. D.
Published: July 2010

We have shown that diazepam (DZ) protects hippocampal neurons when administered early after transient cerebral ischemia. To examine mechanisms of DZ action, in the absence of systemic effects, we used an in vitro model of ischemia [oxygen‐glucose deprivation (OGD)] in the adult hippocampal slice. We are interested in determining if DZ can prevent upstream events such as loss of ionic homeostasis and its consequences on neuronal transmission. We measured the effect of OGD on changes in intracellular Cl− and Ca2+. Application of DZ after OGD prevented the increase in intracellular Cl− in area CA1 pyramidal neurons, measured by optical imaging with the Cl− sensitive fluorescent probe, MEQ. The increase in intracellular Cl− after OGD led to a reduction in GABA responses 2 h later, measured in the presence of the GABA agonist, muscimol. When DZ was added for 1 h after OGD, muscimol responses were restored completely. To measure changes in intracellular Ca2+ after OGD, we bath‐loaded adult hippocampal slices with calcium green‐1 AM. OGD increased intracellular Ca2+ in CA1 pyramidal neurons. The addition of DZ prior to OGD reduced the increase in intracellular Ca2+ by approximately 50%. To determine the effect of DZ on neuronal transmission within the hippocampus, we measured field responses in area CA1 pyramidal neurons. Area CA1 population spikes were suppressed by OGD and they did not recover 1 h later. When DZ was included in the reoxygenation buffer for only 30 min, it restored the population spike amplitude to within 85% of that in normal slices. Thus, after OGD, DZ restores ionic homeostasis, preserves GABA responses and restores neuronal activity within the hippocampus.

Acknowledgements:  Supported by NIH grant #NS28791.
Oxidative modification of mitochondrial proteins and cell death in Parkinson's disease
Authors: Maruyama W. , Naoi M.
Published: July 2010

Oxidative stress is one of the cell death mechanisms in neurodegenerative disorders, such as Parkinson's disease (PD) and Alzheimer's disease. Most of reactive oxygen species (ROS) generate in mitochondria through oxidative phosphorylation, and a part of them are not scavenged by antioxidative system and react with bioactive molecules. Recently, alpha‐synuclein containing nitrotyrosine, a marker for oxidative modification by peroxynitrite, was identified in Lewy body. In addition, inhibitors of mitochondrial respiratory chain were reported to induce formation of Lewy body‐like inclusion in vivo and in vitro. In this paper it was examined whether ROS and reactive nitrogen species (RNS) generated in mitochondria oxidize mitochondrial respiratory enzymes and induce the formation of inclusion body and cell death in PD. Human neuroblastoma SH‐SY5Y cells were treated with a peroxynitrite donor, SIN‐1, or an inhibitor of complex I, rotenone. After the treatment, proteins modified with toxic aldehydes, 4‐hydroxynonenal and acrolein, and containing nitrotyrosine were analyzed by immunoblotting. Particularly in mitochondrial fraction, the oxidized protein was characterized by two‐dimensional immunoblotting. Most of the oxidized proteins were detected in subunits proteins of complex I. These results indicate that mitochondrial complex I is a main target of oxidative stress in dopamine neurons and its dysfunction may be involved in the death mechanism in neurodegenerative disorders.
Poster Sessions CP14: Transplantation and Neurological Repair. GDNF and VMC cotransplantation helps in restoration of neurobehavioral function in Parkinson's disease
Authors: Chaturvedi R. , Agrawal A. K. , Aziz M. H. , Seth K. , Khanna V. K. , Seth P. K.
Published: July 2010

During transplantation of VMC in Parkinson's disease their degeneration is very high due to lack of trophic support and mismatch conditions. To overcome this problem, Glial cell line‐derived Neurotrophic Factor (GDNF) known to increase the functional viability and regeneration of dopaminergic cells. In the present study an attempt has been made to validate the role of GDNF cotransplanted with fetal VMC in functional restoration in rat model of Parkinson's disease. A significant restoration was observed in apomorphine induced rotation in rats co transplanted with GDNF and VMC (66%) as compare to VMC alone (42%). Apomorphine induced locomotor activity was restored by 67, 38% in cotransplanted and VMC alone transplanted rats, respectively. Level of dopamine and 3,4 dihydroxy‐phenyl acetic acid (DOPAC) in the striatum were significantly restored by 67 and 62, 42 and 33% in cotransplanted and VMC alone transplanted rats, respectively. A significant restoration was observed in striatum dopamine receptors by 69% in rats cotransplanted with VMC & GDNF, and 45% in those transplanted with VMC alone. GDNF alone transplantation did not show significant restoration in either of the parameters. Functional viabilty of dopaminergic neurons was further confirmed by Tyrosine Hydroxylase (TH) immunopositivity in striatal region where a significantly high expression was observed in cotransplanted animals when compared with VMC alone.Results of the present study suggests that cotransplantation of GDNF and VMC may help in better functional restoration in 6‐OHDA lesioned rat model of Parkinson's disease studied at 4 weeks post transplantation.
Oral Presentations OP01: Neurodegenerative Diseases. BCL‐W Plays a key neuroprotective role in Alzheimer's disease
Authors: Zhu X. , Wang Y. , Ogawa O. , Lee H. G. , Raina A. K. , Fujioka H. , Shimohama S. , Atwood C. S. , Petersen R. B. , Perry G. , Smith M. A.
Published: July 2010

The mechanisms underlying neuronal degeneration in Alzheimer's disease (AD) are very controversial and none more so than whether apoptosis plays a role. Although neurons in AD face a wide assortment of apoptogenic stimuli, the temporal dichotomy between the acuteness of apoptosis vs. the chronicity of AD suggests that apoptosis should be extremely rare in AD. In this regard, survival factor(s) must be involved. In this study, we investigated Bcl‐w, a pro‐survival member of the Bcl‐2 family. Although expressed at low levels in brains of control cases, Bcl‐w is significantly up‐regulated in AD as shown by both immunocytochemistry and immunoblot analysis. Astonishingly, increased Bcl‐w was found to be associated with neurofibrillary pathologies in AD, which was further demonstrated by an EM study. Since neuronal death in AD is thought to be triggered by increased production of amyloid‐β (Aβ), it was interesting to find that exposure of human M17 neuroblastoma cells to Aβ1–42 (1 nm−10 μm) dramatically up‐regulates Bcl‐w protein levels. Such increases may be a protective response that attenuates apoptotic processes. Consistent with this, transfected M17 cells overexpressing Bcl‐w were protected from both STS‐induced and Aβ‐induced apoptosis compared to vector‐transfected controls. Notably, both tau phosphorylation and p38 is inhibited in Bcl‐w transfected cells which may contribute to the neuroprotective role of Bcl‐w. Taken together, these set of in vitro and in vivo results suggest that Bcl‐w plays an important protective role in neurons in the AD brain.
Neuroprotection with caspase‐9 inhbition against in vitro and in vivo trauma
Authors: Wallis R. A. , Panizzon K. L. , Shin D.
Published: July 2010

Objective:  To evaluate the neuroprotective efficacy of the cell‐permeable caspase‐9 inhibitor, LEHD‐CHO, against in vitro and in vivo traumatic neuronal injury.

Methods:  The neuroprotective potential of LEHD‐CHO was assessed in vitro using rat hippocampal slices. CA1 orthodromic and antidromic population spike (PS) amplitude was monitored before and after fluid percussion injury in slices treated with or without LEHD‐CHO. Final recovery of PS amplitude was assessed 95 min after trauma. Studies of in vivo neuroprotection with LEHD‐CHO utilized a model of controlled cortical impact (CCI). Rats were given either LEHD‐CHO (10 nmol icv) or an equal volume of vehicle at 5 min following CCI. Rats were perfused 24 h after CCI and brains were processed for histological examination.

Results:  LEHD‐CHO provided significant protection against loss of CA1 evoked response after fluid percussion. The EC50 for LEHD‐CHO protection of CA1 orthodromic and antidromic PS amplitude against trauma was 2.1 μm and 2.3 μm. Protection extended to preservation of LTP after trauma. In vivo treatment with LEHD‐CHO significantly decreased the appearance of eosinophilic cells in the CA1 region after CCI from 131 ± 23 cells in vehicle‐treated animals to 24 ± 5 in LEHD‐CHO treated animals. Extensive labelling with TUNEL staining was seen in vehicle‐treated animals, whereas sections from LEHD‐CHO treated animals demonstrated little staining.

Conclusions:  These findings indicate that the caspase 9 inhibitor LHED‐CHO provides concentration‐dependent protection against in vitro CA1 neuronal injury, which extends to protection against in vivo CA1 injury from CCI. They further suggest that inhibition of caspase 9 may be a useful treatment strategy for traumatic brain injury.

Acknowledgement:  Supported by VA Research and UCLA BIRC.
‘Stop and go’ movement of cytoskeletal polymers in axons
Authors: Brown A.
Published: July 2010

Axonal cytoskeletal and cytosolic proteins are synthesized in the cell body of nerve cells and transported along axons by slow axonal transport, at average rates on the order of millimeters per day. My laboratory has recently succeeded in observing the movement of two cytoskeletal proteins, neurofilament protein and tubulin, in cultured nerve cells using live‐cell fluorescence imaging. Both proteins move in the form of filamentous structures, which appear to represent single cytoskeletal polymers. Contrary to expectations, these polymers move very rapidly, at rates on the order of micrometers per second, but their movements are also infrequent and highly asynchronous. Our data suggest that rate of movement in slow axonal transport is actually fast, but that the overall rate is slow because the rapid movements are interrupted by lengthy pauses.
Neurotrophic factor gene delivery supports axonal regeneration after injury
Authors: Shine H. D. , Zhou L. , Grider M. H. , McGowen L. R. , Menesses‐Diaz D. A. , Zheng X. , Le W‐D. , Mamounas L.
Published: July 2010

A successful treatment for spinal cord injury (SCI) must include means to induce axonal regeneration and synaptogenesis. Though much research has demonstrated the effectiveness of neurotrophic factors (NFs) in supporting axonal regeneration, systemic delivery of doses sufficient to reach therapeutic concentrations and overcome their short half‐lives has caused adverse effects. Local expression of NFs would overcome these limitations. We tested whether local expression of NFs would induce axonal regeneration without adverse effects in two models of neural injury. In a chemical injury model the rat serotonergic system was lesioned with p‐chloroamphetamine. When an adenoviral vector carrying the gene for brain‐derived neurotrophic factor (BDNF) was injected into the denervated cortex BDNF expressed by the transfected cells induced serotonergic axon reinnervation only in area around the injection site. In a mechanical injury model the cortical spinal tract (CST) in rats was lesioned unilaterally at the level of the hindbrain. Neurotorphin‐3 (NT‐3) was expressed locally in the spinal cord either by direct injection of an adenoviral vector carrying the gene for NT‐3 or by retrograde delivery of the vector from the sciatic nerve. Axons were observed growing from the unlesioned CST across the midline to the denervated side. These data demonstrate that local expression of NFs will induce and support axonal regeneration in a circumscribed area after injury without adverse effects and suggest that a therapy for SCI based upon this strategy may include NF gene delivery.

Acknowledgements:  Supported by NIH grant NS35280 and Mission Connect of the TIRR Foundation.
Optimally designed nanolayered metal-dielectric particles as probes for massively multiplexed and ultrasensitive molecular assays
Authors: Kodali Anil K. , Llora Xavier , Bhargava Rohit
Published: July 2010

An outstanding challenge in biomedical sciences is to devise a palette of molecular probes that can enable simultaneous and quantitative imaging of tens to hundreds of species down to ultralow concentrations. Addressing this need using surface-enhanced Raman scattering-based probes is potentially possible. Here, we theorize a rational design and optimization strategy to obtain reproducible probes using nanospheres with alternating metal and reporter-filled dielectric layers. The isolation of reporter molecules from metal surfaces suppresses chemical enhancement, and consequently signal enhancements are determined by electromagnetic effects alone. This strategy synergistically couples interstitial surface plasmons and permits the use of almost any molecule as a reporter by eliminating the need for surface attachment. Genetic algorithms are employed to optimize the layer dimensions to provide controllable enhancements exceeding 11 orders of magnitude and of single molecule sensitivity for nonresonant and resonant reporters, respectively. The strategy also provides several other opportunities, including a facile route to tuning the response of these structures to be spectrally flat and localization of the enhancement within a specific volume inside or outside the probe. The spectrally uniform enhancement for multiple excitation wavelengths and for different shifts enables generalized probes, wheras enhancement tuning permits a large dynamic range by suppression of enhancements from outside the probe. Combined, these theoretical calculations open the door for a set of reproducible and robust probes with controlled sensitivity for molecular sensing over a concentration range of over 20 orders of magnitude.
Towards a conceptual and methodological framework for determining robot believability
Authors: Rose Robert , Scheutz Matthias , Schermerhorn Paul
Published: July 2010

Making interactions between humans and artificial agents successful is a major goal of interaction design. The aim of this paper is to provide researchers conducting interaction studies a new framework for the evaluation of robot believability. By critically examining the ordinary sense of believability, we first argue that currently available notions of it are underspecified for rigorous application in an experimental setting. We then define four concepts that capture different senses of believability, each of which connects directly to an empirical methodology. Finally, we show how this framework has been and can be used in the construction of interaction studies by applying it to our own work in human–robot interaction.
Room temperature ferromagnetism in Ni-doped HfO2 thin films
Authors: Sharma M K , Kanjilal Aloke , Voelskow Matthias , Kanjilal D , Chatterjee Ratnamala
Published: July 2010

In this paper detailed studies on modification of structural and magnetic properties of Ni-doped hafnium oxide (HfO2) thin films are reported. We used 200 keV Ni beam for doping of Ni. For homogeneous dispersion and activation of doped Ni ions, 120 MeV Ni swift heavy ions (SHI) irradiation was used. This unique combination of Ni doping by ion beam and dispersing and activating by Ni SHI irradiation of HfO2 films is reported for the first time. The origin of ferromagnetism in the Ni-doped HfO2 thin films is investigated. We demonstrate the cluster free nature of our film using cross-sectional high resolution transmission microscopy and magnetization versus temperature data. Rutherford backscattering data are used to establish that Ni ions are implanted in the HfO2 matrix at the predicted location. Dispersion of implanted Ni and lattice defects such as oxygen vacancies are attributed to be the main source of ferromagnetism. The observed magnetic moment is too large to be attributed to any secondary phase/magnetic clusters.
Lateral confinement of laser ablation plasma in magnetic field
Authors: Pagano C , Lunney J G
Published: July 2010

The expansion of copper laser-produced plasma along a magnetic field was investigated with the aim of confining the lateral expansion of the plasma. Time-resolved optical imaging, time- and space-resolved optical emission spectroscopy and time-of-flight Langmuir ion probe measurements were used to study the plasma dynamics. Thin films of copper were deposited without, and with, the magnetic field. It was observed that the magnetic field gives rise to substantial confinement of the plasma, leading to an increase by a factor of 6 in the ion yield and 2.6 in the deposition rate.
Thrust measurements in a low-magnetic field high-density mode in the helicon double layer thruster
Authors: Ling J , West M D , Lafleur T , Charles C , Boswell R W
Published: July 2010

A momentum flux measuring instrument (MFMI) has been used to take thrust measurements of the helicon double layer thruster (HDLT) prototype immersed in a vacuum chamber. The MFMI recorded a sharp increase in force when the plasma transitioned into a low-magnetic field, high-density mode. The HDLT uses a helicon antenna to generate an ion beam-containing plasma in a diverging magnetic field. The presence of a low-magnetic field mode in the HDLT prototype was demonstrated and its properties were explored at 0.3 mTorr argon at various applied rf powers, locations within the source tube and downstream vacuum chamber, and applied magnetic field strengths. The low-magnetic field mode occurs at solenoid currents that correspond to peak magnetic fields between 20 and 30 G in the source tube and Langmuir probe measurements show that the mode is associated with an increase in ion density of up to six times the density at applied magnetic fields just outside the mode. This rise in ion density correlates with a peak in plasma resistance. Furthermore, the transition into the mode is accompanied by the appearance of a higher current ion beam in the downstream region of the expanding plasma and a rise in plasma potential of approximately 30% at 250 W rf power as measured by retarding field energy analyzers. This mode is of interest for the HDLT because it generates an ion beam-containing high-density (≈1017 m−3) expanding plasma at low-magnetic fields and rf powers as low as 130 W.
Ion flux formation in asymmetric 2 MHz capacitive discharge
Authors: Schweigert I V
Published: July 2010

From two-dimensional particle-in-cell–Monte Carlo collisions simulations, the effect of chamber geometry on the ion flux is studied in a low gas pressure capacitive 2 MHz discharge. It is shown that the optimization of the reactor geometry significantly enhances the ion flux to the small electrode at a given applied voltage. The uniformity of the ion flux and the ion energy distribution at the electrode are studied for the applied voltage amplitude ranging from 2 to 4 kV. The dependence of sheath voltages from the ratio of driven and grounded electrode areas is discussed.
RPPanalyzer: Analysis of reverse-phase protein array data
Authors: Mannsperger Heiko A. , Gade Stephan , Henjes Frauke , Beissbarth Tim , Korf Ulrike
Published: July 2010

Summary: RPPanalyzer is a statistical tool developed to read reverse-phase protein array data, to perform the basic data analysis and to visualize the resulting biological information. The R-package provides different functions to compare protein expression levels of different samples and to normalize the data. Implemented plotting functions permit a quality control by monitoring data distribution and signal validity. Finally, the data can be visualized in heatmaps, boxplots, time course plots and correlation plots. RPPanalyzer is a flexible tool and tolerates a huge variety of different experimental designs.

Availability: The RPPAanalyzer is open source and freely available as an R-Package on the CRAN platform http://cran.r-project.org/

Contact:  h.mannsperger@dkfz.de

Supplementary information:  Supplementary data are available at Bioinformatics online.
LocusZoom: regional visualization of genome-wide association scan results
Authors: Pruim Randall J. , Welch Ryan P. , Sanna Serena , Teslovich Tanya M. , Chines Peter S. , Gliedt Terry P. , Boehnke Michael , Abecasis Gonçalo R. , Willer Cristen J.
Published: July 2010

Summary: Genome-wide association studies (GWAS) have revealed hundreds of loci associated with common human genetic diseases and traits. We have developed a web-based plotting tool that provides fast visual display of GWAS results in a publication-ready format. LocusZoom visually displays regional information such as the strength and extent of the association signal relative to genomic position, local linkage disequilibrium (LD) and recombination patterns and the positions of genes in the region.

Availability: LocusZoom can be accessed from a web interface at http://csg.sph.umich.edu/locuszoom. Users may generate a single plot using a web form, or many plots using batch mode. The software utilizes LD information from HapMap Phase II (CEU, YRI and JPT+CHB) or 1000 Genomes (CEU) and gene information from the UCSC browser, and will accept SNP identifiers in dbSNP or 1000 Genomes format. Single plots are generated in ∼20 s. Source code and associated databases are available for download and local installation, and full documentation is available online.

Contact:  cristen@umich.edu