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Focused ultrasound (FUS) is a rapidly developing stimulus technology with the potential to uncove... more Focused ultrasound (FUS) is a rapidly developing stimulus technology with the potential to uncover novel mechanosensory dependent cellular processes. Since it is noninvasive, it holds great promise for future therapeutic applications in patients used either alone or as a complement to boost existing treatments. For example, FUS stimulation causes invasive but not noninvasive cancer cell lines to exhibit marked activation of calcium signaling pathways. Here, we identify the membrane channel PANNEXIN1 (PANX1) as a mediator for activation of calcium signaling in invasive cancer cells. Knockdown of PANX1 decreases calcium signaling in invasive cells, while PANX1 overexpression enhances calcium elevations in non-invasive cancer cells. We demonstrate that FUS may directly stimulate mechanosensory PANX1 localized in endoplasmic reticulum to evoke calcium release from internal stores. This process does not depend on mechanosensory stimulus transduction through an intact cytoskeleton and does not depend on plasma membrane localized PANX1. Plasma membrane localized PANX1 however plays a different role in mediating the spread of intercellular calcium waves via ATP release. Additionally, we show that FUS stimulation evokes cytokine/chemokine release from invasive cancer cells, suggesting that FUS could be an important new adjuvant treatment to improve cancer immunotherapy.
Frontiers in Neuroscience
We describe the construction and phenotypic analysis of a human embryonic stem cell model of prog... more We describe the construction and phenotypic analysis of a human embryonic stem cell model of progressive Aβ-dependent neurodegeneration (ND) with potential relevance to Alzheimer's disease (AD). We modified one allele of the normal APP locus to directly express a secretory form of Aβ40 or Aβ42, enabling expression from this edited allele to bypass the normal amyloidogenic APP processing pathway. Following neuronal differentiation, edited cell lines specifically accumulate intracellular aggregated/oligomeric Aβ, exhibit a synaptic deficit, and have an abnormal accumulation of endolysosomal vesicles. Edited cultures progress to a stage of overt ND. All phenotypes appear at earlier culture times for Aβ42 relative to Aβ40. Whole transcriptome RNA-Seq analysis identified 23 up and 70 down regulated genes (differentially expressed genes) with similar directional fold change but larger absolute values in the Aβ42 samples suggesting common underlying pathogenic mechanisms. Pathway/annotation analysis suggested that down regulation of extracellular matrix and cilia functions is significantly overrepresented. This cellular model could be useful for uncovering mechanisms directly linking Aβ to neuronal death and as a tool to screen for new therapeutic agents that slow or prevent human ND.
The Journal of Neuroscience
ABSTRACT
Journal of Neurochemistry, 2017
Cholinergic Mechanisms, 2004
Journal of Comparative Neurology, Feb 8, 1989
Using a monoclonal antibody to choline acetyltransferase (ChAT), we have identified immunoreactiv... more Using a monoclonal antibody to choline acetyltransferase (ChAT), we have identified immunoreactive synaptic terminals in the neuropil regions of the cephalic ganglion of Drosophila melanogaster. This study demonstrates the distribution of antibody-labeled structures within the optic lobe, and then investigates the immunoreactivity altered by mutation in two temperature-sensitive ChAT alleles, chats-1 and chats-2. The general structure of the optic lobe was first observed by means of the silver impregnation technique. Then the presence of ChAT immunoreactivity was determined by the application of antibody [1G4] conjugated with HRP to frozen sections, followed by the 3,3'-diamino-benzidine tetratinct layers, which correspond to the three synaptic layers of the laminarneurons, in the medulla. Also, staining appeared in four distinct layers in the lobula. In addition, weaker staining was observed in the lamina, which corresponds to the retinula cell terminals. Somal layers were not stained. In Canton-S (wild-type), the three medullar layers stain distinctly at both 19 degrees C and 30 degrees C. In chats-1 at 19 degrees C, the stain appeared in the same layers as that of Canton-S, but with somewhat lower density. In chats-2 at 19 degrees C, the density of the stain was even lower. The densities of the stain in these mutants were further decreased after exposing the flies to 30 degrees C. The decreases were dependent on the length of exposure to the higher temperature. The decrease in stain of the specimens obtained after 24 hours exposure to 30 degrees C was clearly recognizable in both chats-1 and chats-2.(ABSTRACT TRUNCATED AT 250 WORDS)
Journal of Neurochemistry, 1995
J Neurochem, 2002
Antibodies to the plant glycoprotein horseradish peroxidase (HRP) are used extensively to identif... more Antibodies to the plant glycoprotein horseradish peroxidase (HRP) are used extensively to identify neurons in Drosophila and other insects. We are interested in characterizing the gene product(s) recognized by anti-HRP antibodies because it may be important for nervous system function and/or development. Here we identify and purify from adult Drosophila heads an anti-HRP-reactive Mr 42K glycoprotein that is likely to be the major contributor to neuronal specific anti-HRP staining. Several different monoclonal antibodies to the purified 42K glycoprotein recognize up to three proteins with distinct mobilities between Mr 38K and 42K that vary as a function of developmental age. We have collectively named these components Nervana (nerve antigen), because the monoclonal antibodies also specifically stain cultured neurons and embryonic nervous system with a pattern indistinguishable from anti-HRP staining. Western blots indicate the presence of immunologically similar proteins in a wide variety of insect species and in nac (neurally altered carbohydrate) mutant Drosophila flies that lack anti-HRP staining in adult nervous system. It should now be possible to undertake a full biochemical and functional characterization of Nervana in Drosophila.
Biochem Biophys Res Commun, 1973
J Neurochem, 2002
We have previously purified and characterized a nervous system-specific glycoprotein antigen from... more We have previously purified and characterized a nervous system-specific glycoprotein antigen from adult Drosophila heads, designated Nervana [nerve antigen (NRV)] and identified two separate genes coding for three different proteins. All three proteins share homology with the beta subunits of Na+,K+-ATPase from various other species. In this study we have isolated a new Drosophila Na+,K+-ATPase alpha subunit cDNA clone (PSalpha; GenBank accession no. AF044974) and demonstrate expression of functional Na+,K+-ATPase activity when PSalpha mRNA is coinjected into Xenopus oocytes along with any of the three different Nrv mRNAs. Western blotting, RNase protection assays, and immunocytochemical staining of adult fly sections indicate that NRV2 is expressed primarily in the nervous system. Staining is most intense in the brain and thoracic ganglia and is most likely associated with neuronal elements. NRV1 is more broadly expressed in muscle and excretory tissue and also shows diffuse distribution in the nervous system. Similar to other species, Drosophila expresses multiple isoforms of Na+,K+-ATPase subunits in a tissue- and cell type-specific pattern. It will now be possible to use the advantages of Drosophila molecular and classical genetics to investigate the phenotypic consequences of altering Na+,K+-ATPase expression in various cell and tissue types.
Acta Pharmacologica Et Toxicologica, 2009
The effects on mouse behaviour in the open field situation of controlled single doses of methyl m... more The effects on mouse behaviour in the open field situation of controlled single doses of methyl mercury (MeHg) (1, 5, 10 mg Hg/kg) were investigated at varying times after intraperitoneal injection (1, 3, 72 hours). In an effort to correlate behavioural and biochemical data, the effects of dose and time after dose on the levels of selected glycolytic pathway intermediates, a-glycerophosphate, adenine nucleotides and phosphocreatine were monitored. A very good correlation between brain biochemistry and behavioural effects of MeHg was observed. That is, the dose response relationship for the open field task correlated with alterations in levels of metabolic intermediates. At 1 and 3 hours after administration of MeHg, when the levels of the metabolic intermediates were significantly different from those of controls, altered behaviour was observed. At 72 hours post administration, when the biological parameters were approaching control values, a return to normal behaviour was observed.
Neuroscience Research, 1996
In vertebrate photoreceptors, guanylate cyclase (GC) plays an important role in recovering the da... more In vertebrate photoreceptors, guanylate cyclase (GC) plays an important role in recovering the dark current following light-mediated cGMP decrease. Although it has been speculated that this enzyme binds cytoskeletal proteins, it is not clear that what sorts of proteins bind this enzyme to make Triton X-100 insoluble complex. We used GC-overlay method against cytoskeletal proteins in rod outer segments and found several protein bands that can bind the enzyme.
International Review of Neurobiology, Feb 1, 1989
ABSTRACT
Neuroimage, 2007
Neural stem cells (NSCs) hold great promise for glioma therapy due to their inherent tumor-tropic... more Neural stem cells (NSCs) hold great promise for glioma therapy due to their inherent tumor-tropic properties, enabling them to deliver therapeutic agents directly to invasive tumor sites. In the present study, we visualized and quantitatively analyzed the spatial distribution of tumor-tropic NSCs in a mouse model of orthotopic glioma in order to predict the therapeutic efficacy of a representative NSC-based glioma therapy. U251.eGFP human glioma was established in the brain of athymic mice, followed by stereotactic injection of CM-DiI-labeled human NSCs posterior-lateral to the tumor site. Confocal microscopy, three-dimensional modeling and mathematical algorithms were used to visualize and characterize the spatial distribution of NSCs throughout the tumor. The pattern of NSC distribution showed a gradient with higher densities toward the centroid of the tumor mass. We estimate that NSC-mediated therapy would eradicate 70-90% of the primary tumor mass and the majority of invasive tumor foci. Our method may serve as a model for optimizing the efficacy of NSC-based glioma therapy.
Gen Pharmacol Vasc Syst, 1976
Focused ultrasound (FUS) is a rapidly developing stimulus technology with the potential to uncove... more Focused ultrasound (FUS) is a rapidly developing stimulus technology with the potential to uncover novel mechanosensory dependent cellular processes. Since it is noninvasive, it holds great promise for future therapeutic applications in patients used either alone or as a complement to boost existing treatments. For example, FUS stimulation causes invasive but not noninvasive cancer cell lines to exhibit marked activation of calcium signaling pathways. Here, we identify the membrane channel PANNEXIN1 (PANX1) as a mediator for activation of calcium signaling in invasive cancer cells. Knockdown of PANX1 decreases calcium signaling in invasive cells, while PANX1 overexpression enhances calcium elevations in non-invasive cancer cells. We demonstrate that FUS may directly stimulate mechanosensory PANX1 localized in endoplasmic reticulum to evoke calcium release from internal stores. This process does not depend on mechanosensory stimulus transduction through an intact cytoskeleton and does not depend on plasma membrane localized PANX1. Plasma membrane localized PANX1 however plays a different role in mediating the spread of intercellular calcium waves via ATP release. Additionally, we show that FUS stimulation evokes cytokine/chemokine release from invasive cancer cells, suggesting that FUS could be an important new adjuvant treatment to improve cancer immunotherapy.
Frontiers in Neuroscience
We describe the construction and phenotypic analysis of a human embryonic stem cell model of prog... more We describe the construction and phenotypic analysis of a human embryonic stem cell model of progressive Aβ-dependent neurodegeneration (ND) with potential relevance to Alzheimer's disease (AD). We modified one allele of the normal APP locus to directly express a secretory form of Aβ40 or Aβ42, enabling expression from this edited allele to bypass the normal amyloidogenic APP processing pathway. Following neuronal differentiation, edited cell lines specifically accumulate intracellular aggregated/oligomeric Aβ, exhibit a synaptic deficit, and have an abnormal accumulation of endolysosomal vesicles. Edited cultures progress to a stage of overt ND. All phenotypes appear at earlier culture times for Aβ42 relative to Aβ40. Whole transcriptome RNA-Seq analysis identified 23 up and 70 down regulated genes (differentially expressed genes) with similar directional fold change but larger absolute values in the Aβ42 samples suggesting common underlying pathogenic mechanisms. Pathway/annotation analysis suggested that down regulation of extracellular matrix and cilia functions is significantly overrepresented. This cellular model could be useful for uncovering mechanisms directly linking Aβ to neuronal death and as a tool to screen for new therapeutic agents that slow or prevent human ND.
The Journal of Neuroscience
ABSTRACT
Journal of Neurochemistry, 2017
Cholinergic Mechanisms, 2004
Journal of Comparative Neurology, Feb 8, 1989
Using a monoclonal antibody to choline acetyltransferase (ChAT), we have identified immunoreactiv... more Using a monoclonal antibody to choline acetyltransferase (ChAT), we have identified immunoreactive synaptic terminals in the neuropil regions of the cephalic ganglion of Drosophila melanogaster. This study demonstrates the distribution of antibody-labeled structures within the optic lobe, and then investigates the immunoreactivity altered by mutation in two temperature-sensitive ChAT alleles, chats-1 and chats-2. The general structure of the optic lobe was first observed by means of the silver impregnation technique. Then the presence of ChAT immunoreactivity was determined by the application of antibody [1G4] conjugated with HRP to frozen sections, followed by the 3,3'-diamino-benzidine tetratinct layers, which correspond to the three synaptic layers of the laminarneurons, in the medulla. Also, staining appeared in four distinct layers in the lobula. In addition, weaker staining was observed in the lamina, which corresponds to the retinula cell terminals. Somal layers were not stained. In Canton-S (wild-type), the three medullar layers stain distinctly at both 19 degrees C and 30 degrees C. In chats-1 at 19 degrees C, the stain appeared in the same layers as that of Canton-S, but with somewhat lower density. In chats-2 at 19 degrees C, the density of the stain was even lower. The densities of the stain in these mutants were further decreased after exposing the flies to 30 degrees C. The decreases were dependent on the length of exposure to the higher temperature. The decrease in stain of the specimens obtained after 24 hours exposure to 30 degrees C was clearly recognizable in both chats-1 and chats-2.(ABSTRACT TRUNCATED AT 250 WORDS)
Journal of Neurochemistry, 1995
J Neurochem, 2002
Antibodies to the plant glycoprotein horseradish peroxidase (HRP) are used extensively to identif... more Antibodies to the plant glycoprotein horseradish peroxidase (HRP) are used extensively to identify neurons in Drosophila and other insects. We are interested in characterizing the gene product(s) recognized by anti-HRP antibodies because it may be important for nervous system function and/or development. Here we identify and purify from adult Drosophila heads an anti-HRP-reactive Mr 42K glycoprotein that is likely to be the major contributor to neuronal specific anti-HRP staining. Several different monoclonal antibodies to the purified 42K glycoprotein recognize up to three proteins with distinct mobilities between Mr 38K and 42K that vary as a function of developmental age. We have collectively named these components Nervana (nerve antigen), because the monoclonal antibodies also specifically stain cultured neurons and embryonic nervous system with a pattern indistinguishable from anti-HRP staining. Western blots indicate the presence of immunologically similar proteins in a wide variety of insect species and in nac (neurally altered carbohydrate) mutant Drosophila flies that lack anti-HRP staining in adult nervous system. It should now be possible to undertake a full biochemical and functional characterization of Nervana in Drosophila.
Biochem Biophys Res Commun, 1973
J Neurochem, 2002
We have previously purified and characterized a nervous system-specific glycoprotein antigen from... more We have previously purified and characterized a nervous system-specific glycoprotein antigen from adult Drosophila heads, designated Nervana [nerve antigen (NRV)] and identified two separate genes coding for three different proteins. All three proteins share homology with the beta subunits of Na+,K+-ATPase from various other species. In this study we have isolated a new Drosophila Na+,K+-ATPase alpha subunit cDNA clone (PSalpha; GenBank accession no. AF044974) and demonstrate expression of functional Na+,K+-ATPase activity when PSalpha mRNA is coinjected into Xenopus oocytes along with any of the three different Nrv mRNAs. Western blotting, RNase protection assays, and immunocytochemical staining of adult fly sections indicate that NRV2 is expressed primarily in the nervous system. Staining is most intense in the brain and thoracic ganglia and is most likely associated with neuronal elements. NRV1 is more broadly expressed in muscle and excretory tissue and also shows diffuse distribution in the nervous system. Similar to other species, Drosophila expresses multiple isoforms of Na+,K+-ATPase subunits in a tissue- and cell type-specific pattern. It will now be possible to use the advantages of Drosophila molecular and classical genetics to investigate the phenotypic consequences of altering Na+,K+-ATPase expression in various cell and tissue types.
Acta Pharmacologica Et Toxicologica, 2009
The effects on mouse behaviour in the open field situation of controlled single doses of methyl m... more The effects on mouse behaviour in the open field situation of controlled single doses of methyl mercury (MeHg) (1, 5, 10 mg Hg/kg) were investigated at varying times after intraperitoneal injection (1, 3, 72 hours). In an effort to correlate behavioural and biochemical data, the effects of dose and time after dose on the levels of selected glycolytic pathway intermediates, a-glycerophosphate, adenine nucleotides and phosphocreatine were monitored. A very good correlation between brain biochemistry and behavioural effects of MeHg was observed. That is, the dose response relationship for the open field task correlated with alterations in levels of metabolic intermediates. At 1 and 3 hours after administration of MeHg, when the levels of the metabolic intermediates were significantly different from those of controls, altered behaviour was observed. At 72 hours post administration, when the biological parameters were approaching control values, a return to normal behaviour was observed.
Neuroscience Research, 1996
In vertebrate photoreceptors, guanylate cyclase (GC) plays an important role in recovering the da... more In vertebrate photoreceptors, guanylate cyclase (GC) plays an important role in recovering the dark current following light-mediated cGMP decrease. Although it has been speculated that this enzyme binds cytoskeletal proteins, it is not clear that what sorts of proteins bind this enzyme to make Triton X-100 insoluble complex. We used GC-overlay method against cytoskeletal proteins in rod outer segments and found several protein bands that can bind the enzyme.
International Review of Neurobiology, Feb 1, 1989
ABSTRACT
Neuroimage, 2007
Neural stem cells (NSCs) hold great promise for glioma therapy due to their inherent tumor-tropic... more Neural stem cells (NSCs) hold great promise for glioma therapy due to their inherent tumor-tropic properties, enabling them to deliver therapeutic agents directly to invasive tumor sites. In the present study, we visualized and quantitatively analyzed the spatial distribution of tumor-tropic NSCs in a mouse model of orthotopic glioma in order to predict the therapeutic efficacy of a representative NSC-based glioma therapy. U251.eGFP human glioma was established in the brain of athymic mice, followed by stereotactic injection of CM-DiI-labeled human NSCs posterior-lateral to the tumor site. Confocal microscopy, three-dimensional modeling and mathematical algorithms were used to visualize and characterize the spatial distribution of NSCs throughout the tumor. The pattern of NSC distribution showed a gradient with higher densities toward the centroid of the tumor mass. We estimate that NSC-mediated therapy would eradicate 70-90% of the primary tumor mass and the majority of invasive tumor foci. Our method may serve as a model for optimizing the efficacy of NSC-based glioma therapy.
Gen Pharmacol Vasc Syst, 1976