Michael J Schell
Johns Hopkins University, Cell Biology, Faculty Member
- Has worked at Indiana University Medical Center, Johns Hopkins, University of Cambridge, and Uniformed Services Universityedit
The cellular machinery responsible for copper-stimulated delivery of the Wilson Disease protein ATP7B to the apical domain of hepatocytes is poorly understood. We demonstrate that myosin Vb regulates the copper-stimulated delivery of... more
The cellular machinery responsible for copper-stimulated delivery of the Wilson Disease protein ATP7B to the apical domain of hepatocytes is poorly understood. We demonstrate that myosin Vb regulates the copper-stimulated delivery of ATP7B to the apical domain of polarized hepatic cells, and that disruption of the ATP7B-myosin Vb interaction reduces ATP7B apical surface expression. Myosin Vb tail overexpression, which competes for binding of subapical cargoes to myosin Vb bound to subapical actin, disrupted the surface expression of ATP7B, leading to reduced cellular copper export. The myosin Vb-dependent targeting step arose in parallel with hepatocyte-like polarity. If the myosin Vb tail was expressed acutely in cells just prior to the establishment of polarity, it appeared as part of an intracellular apical compartment, centered on gamma tubulin. ATP7B became arrested in this compartment selectively in high copper in the presence of myosin Vb tail, suggesting that these sites are...
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Three inositol 1,4,5-trisphosphate receptor (IP3R) cDNAs, designated IP3R-II, -III, and -IV, were cloned from a mouse placenta cDNA library. All three display strong homology in membrane-spanning domains M7 and M8 to the originally cloned... more
Three inositol 1,4,5-trisphosphate receptor (IP3R) cDNAs, designated IP3R-II, -III, and -IV, were cloned from a mouse placenta cDNA library. All three display strong homology in membrane-spanning domains M7 and M8 to the originally cloned cerebellar IP3R-I, with divergences predominantly in cytoplasmic domains. Levels of mRNA for the three additional IP3Rs in general are substantially lower than for IP3R-I, though in the gastrointestinal tract the levels of IP3R-III may be comparable to IP3R-I. Cerebellar Purkinje cells express at least two and possibly three distinct IP3Rs, suggesting heterogeneity of IP3 action within a single cell.
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gamma-Hexachlorocyclohexane was found to exert profound effects on the phosphatidylinositol cycle, cytosolic calcium level, and the respiratory burst of human neutrophils. Exposure of neutrophils prelabelled with 32P to 4 X 10(-4) M... more
gamma-Hexachlorocyclohexane was found to exert profound effects on the phosphatidylinositol cycle, cytosolic calcium level, and the respiratory burst of human neutrophils. Exposure of neutrophils prelabelled with 32P to 4 X 10(-4) M gamma-hexachlorocyclohexane almost tripled radioactivity in phosphatidic acid and correspondingly decreased radioactivity in phosphatidylinositol 4,5 bisphosphate. Under similar conditions, gamma-hexachlorocyclohexane evoked the generation of superoxide at a rate of over 11 nmol/min/10(6) cells and more than doubled cytosolic-free calcium concentration as monitored by Quin-2 fluorescence. Because intermediates of the phosphatidylinositol cycle, via increases in available calcium levels or activated protein kinase C, are considered potential second messengers for activation of the NADPH-dependent O-2-generating system, we compared neutrophil responses to gamma-hexachlorocyclohexane with responses to phorbol myristate acetate, an activator of protein kinase C with well known effects on neutrophils. Like phorbol myristate acetate, gamma-hexachlorocyclohexane induced neutrophil degranulation but was not an effective chemotactic stimulus. The ability of gamma-hexachlorocyclohexane to induce a pattern of oxidative activation in neutrophil cytoplasts similar to that in intact cells indicated that concurrent degranulation was not required for sustained O-2 generation in response to this agent. When neutrophils or neutrophil cytoplasts exposed to gamma-hexachlorocyclohexane were centrifuged and resuspended in stimulus-free medium, O-2 generation ceased entirely but could be reinitiated by addition of the same stimulus. This finding was in contrast to the continued O-2 production by phorbol myristate acetate-stimulated neutrophils similarly washed and resuspended in stimulus-free medium. Unlike subcellular fractions of phorbol myristate acetate-stimulated neutrophils, corresponding fractions prepared from gamma-hexachlorocyclohexane-stimulated neutrophils contained almost no detectable NADPH-dependent O-2-generating activity. Subcellular oxidase activity was not recovered when cells and membrane fractions were continuously exposed to gamma-hexachlorocyclohexane during disruption and fractionation after cell stimulation, nor could it be induced by the addition of the stimulus to the subcellular fractions. Thus, the stimulus dependence of continuous neutrophil superoxide release evoked by gamma-hexachlorocyclohexane does not merely reflect a physical interaction of the agonist with the enzyme system involved.(ABSTRACT TRUNCATED AT 400 WORDS)
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D-Serine is localized in mammalian brain to a discrete population of glial cells near NMDA receptors, suggesting that D-serine is an endogenous agonist of the receptor-associated glycine site. To explore this possibility, we have compared... more
D-Serine is localized in mammalian brain to a discrete population of glial cells near NMDA receptors, suggesting that D-serine is an endogenous agonist of the receptor-associated glycine site. To explore this possibility, we have compared the immunohistochemical localizations of D-serine, glycine, and NMDA receptors in rat brain. In the telencephalon, D-serine is concentrated in protoplasmic astrocytes, which are abundant in neuropil in close vicinity to NMDA receptor 2A/B subunits. Ultrastructural examination of the CA1 region of hippocampus reveals D-serine in the cytosolic matrix of astrocytes that ensheath neurons and blood vessels, whereas NR2A/B is concentrated in dendritic spines. By contrast, glycine immunoreactivity in telencephalon is the lowest in brain. During postnatal week 2, D-serine levels in cerebellum are comparable to those in adult cerebral cortex but fall to undetectable levels by day 26. During week 2, we observe parallel ontogeny of D-serine in Bergmann glia a...
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Research Interests: Pharmacology, Biochemistry, Bioinformatics, Evolutionary Biology, Genetics, and 43 moreMarine Biology, Neuroscience, Environmental Science, Geophysics, Physics, Materials Science, Quantum Physics, Developmental Biology, Immunology, Climate Change, Molecular Biology, Structural Biology, Genomics, RNA, Computational Biology, Transcriptomics, Biotechnology, Systems Biology, Cancer, Biology, Metabolomics, Cell Cycle, Proteomics, Ecology, Drug Discovery, Evolution, Cell Biology, Nanotechnology, Astrophysics, Neurobiology, Medicine, Palaeobiology, Functional Genomics, Nature, Signal Transduction, Astronomy, Biological Sciences, DNA, Cell Signalling, Medical Research, Water soluble polymers, Second Messengers, and Earth Science
Research Interests: Macromolecular X-Ray Crystallography, Biological Sciences, Humans, Animals, Phosphorylation, and 10 moreMolecular Cell Biology, Enzyme, Substrate Specificity, Isoenzymes, Amino Acid Sequence, Protein Binding, Second Messengers, Molecular Structure, Adenosine Triphosphate, and Molecular Sequence Data
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Diverse patterns of Ca(2+)(i) release differentially regulate Ca(2+)-sensitive enzymes and gene transcription, and generally the extent of agonist activation of phospholipase C-linked G protein-coupled receptors determines the type of... more
Diverse patterns of Ca(2+)(i) release differentially regulate Ca(2+)-sensitive enzymes and gene transcription, and generally the extent of agonist activation of phospholipase C-linked G protein-coupled receptors determines the type of Ca(2+) signal. We have studied global Ca(2+) oscillations arising through activation of the metabotropic glutamate receptor mGluR5a expressed in Chinese hamster ovary cells and find that these oscillations are largely insensitive to agonist concentration. Using an inducible receptor expression system and a non-competitive antagonist, in conjunction with the translocation of eGFP-PH(PLCdelta) to monitor inositol 1,4,5-trisphosphate (InsP(3)) oscillations in single cells, we show that mGluR5a density determines the frequency of these oscillations. The predominant underlying mechanism resulted from a negative feedback loop whereby protein kinase C (PKC) inhibited InsP(3) generation. Down-regulation of PKC by prolonged exposure to phorbol ester revealed a second form of Ca(2+)(i) oscillation at low agonist concentrations. These Ca(2+)(i) signals showed features typical of classic repetitive Ca(2+)-induced Ca(2+) release and were sensitive to agonist concentration. Therefore, a single receptor can stimulate two types of InsP(3)-mediated Ca(2+) signal dependent upon feedback inhibition, producing two distinct means of controlling the final pattern of Ca(2+)(i) release. Our results have physiological implications for Ca(2+) signaling in general and emphasize the importance of mGluR5 surface expression for modulating synaptic plasticity.
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We have characterized the effects of the antimitotic drug paclitaxel (Taxol(TM)) on the Ca(2+) signaling cascade of terminally differentiated mouse pancreatic acinar cells. Using single cell fluorescence techniques and whole-cell patch... more
We have characterized the effects of the antimitotic drug paclitaxel (Taxol(TM)) on the Ca(2+) signaling cascade of terminally differentiated mouse pancreatic acinar cells. Using single cell fluorescence techniques and whole-cell patch clamping to record cytosolic Ca(2+) and plasma membrane Ca(2+)-dependent Cl(-) currents, we find that paclitaxel abolishes cytosolic Ca(2+) oscillations and in more than half of the cells it also induces a rapid, transient cytosolic Ca(2+) response. This response is not affected by removal of extracellular Ca(2+) indicating that paclitaxel releases Ca(2+) from an intracellular Ca(2+) store. Using saponin-permeabilized cells, we show that paclitaxel does not affect Ca(2+) release from an inositol trisphosphate-sensitive store. Furthermore, up to 15 min after paclitaxel application, there is no significant effect on either microtubule organization or on endoplasmic reticulum organization. The data suggest a non-endoplasmic reticulum source for the intracellular Ca(2+) response. Using the mitochondrial fluorescent dyes, JC-1 and Rhod-2, we show that paclitaxel evoked a rapid decline in the mitochondrial membrane potential and a loss of mitochondrial Ca(2+). Cyclosporin A, a blocker of the mitochondrial permeability transition pore, blocked both the paclitaxel-induced loss of mitochondrial Ca(2+) and the effect on Ca(2+) spikes. We conclude that paclitaxel exerts rapid effects on the cytosolic Ca(2+) signal via the opening of the mitochondrial permeability transition pore. This work indicates that some of the more rapidly developing side effects of chemotherapy might be due to an action of antimitotic drugs on mitochondrial function and an interference with the Ca(2+) signal cascade.
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There has been much controversy over the possibility that inositol 1,3,4,5-tetrakisphosphate (InsP4) may have a second messenger function. A possible resolution to this controversy may stem from the recent cloning of two putative... more
There has been much controversy over the possibility that inositol 1,3,4,5-tetrakisphosphate (InsP4) may have a second messenger function. A possible resolution to this controversy may stem from the recent cloning of two putative receptors for InsP4, GAP1IP4BP and GAP1m. Both these proteins are expressed at high levels in neurones, as is inositol 1,4,5-trisphosphate 3-kinase, the enzyme that makes InsP4. In this review we discuss the possible relevance of these high expression levels to the complex way in which neurones control Ca2+ and use it as a second messenger.
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IP3K (inositol 1,4,5-trisphosphate 3-kinase) catalyses the Ca2+-regulated phosphorylation of the second messenger Ins(1,4,5)P3, thereby inactivating the signal to release Ca2+ and generating Ins(1,3,4,5)P4. Here we have investigated the... more
IP3K (inositol 1,4,5-trisphosphate 3-kinase) catalyses the Ca2+-regulated phosphorylation of the second messenger Ins(1,4,5)P3, thereby inactivating the signal to release Ca2+ and generating Ins(1,3,4,5)P4. Here we have investigated the localization and activity of IP3KB and its modulation by proteolysis. We found that the N- and C-termini (either side of residue 262) of IP3KB localized predominantly to the actin cytoskeleton and ER (endoplasmic reticulum) respectively, both in COS-7 cells and in primary astrocytes. The functional relevance of this was demonstrated by showing that full-length (actin-localized) IP3KB abolished the histamine-induced Ca2+ response in HeLa cells more effectively than truncated constructs localized to the ER or cytosol. The superior efficacy of full-length IP3KB was also attenuated by disruption of the actin cytoskeleton. By transfecting COS-7 cells with double-tagged IP3KB, we show that the translocation from actin to ER may be a physiologically regulated process caused by Ca2+-modulated constitutive proteolysis in intact cells.
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A highly specific and sensitive mass assay for inositol hexakisphosphate (InsP6) was characterized. This centres around phosphorylating InsP6 with [32P]ATP using a recombinant InsP6 kinase from Giardia lambia, followed by HPLC of the... more
A highly specific and sensitive mass assay for inositol hexakisphosphate (InsP6) was characterized. This centres around phosphorylating InsP6 with [32P]ATP using a recombinant InsP6 kinase from Giardia lambia, followed by HPLC of the 32P-labelled products with an internal [3H]InsP7 standard. This assay was used to quantify InsP6 levels in a variety of biological samples.Concentrations of InsP6 in rat tissues varied from 10-20 microM (assuming 64% of wet weight of tissue is cytosol water), whereas using the same assumption axenic Dictyostelium discoideum cells contained 352 +/- 11 microM InsP6. HeLa cells were seeded at low density and grown to confluence, at which point they contained InsP6 levels per mg of protein similar to rat tissues. This amounted to 1.952 +/- 0.117 nmol InsP6 per culture dish, despite the cells being grown in serum shown to contain no detectable(less than 20 pmol per dish) InsP6. These results demonstrate that mammalian cells synthesize all their own InsP6. Human blood was analysed, and although the white cell fraction contained InsP6 at a concentration comparable with other tissues, in serum and platelet-free plasma no InsP6 was detected (<1 nM InsP6). Human urine was also examined, and also contained no detectable (<5 nM) InsP6. These results suggest that dietary studies purporting to measure InsP6 at micromolar concentrations in human plasma or urine may not have been quantifying this inositol phosphate. Therefore claims that administrating InsP6 in the diet or applying it topically can produce health benefits by increasing extracellular InsP6 levels may require reassessment.
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Ca(2+) and cAMP are widely used in concert by neurons to relay signals from the synapse to the nucleus, where synaptic activity modulates gene expression required for synaptic plasticity. Neurons utilize different transcriptional... more
Ca(2+) and cAMP are widely used in concert by neurons to relay signals from the synapse to the nucleus, where synaptic activity modulates gene expression required for synaptic plasticity. Neurons utilize different transcriptional regulators to integrate information encoded in the spatiotemporal dynamics and magnitude of Ca(2+) and cAMP signals, including some that are Ca(2+)-responsive, some that are cAMP-responsive and some that detect coincident Ca(2+) and cAMP signals. Because Ca(2+) and cAMP can influence each other's amplitude and spatiotemporal characteristics, we investigated how cAMP acts to regulate gene expression when increases in intracellular Ca(2+) are buffered. We show here that cAMP-mobilizing stimuli are unable to induce expression of the immediate early gene c-fos in hippocampal neurons in the presence of the intracellular Ca(2+) buffer BAPTA-AM. Expression of enzymes that attenuate intracellular IP(3) levels also inhibited cAMP-dependent c-fos induction. Synaptic activity induces c-fos transcription through two cis regulatory DNA elements - the CRE and the SRE. We show here that in response to cAMP both CRE-mediated and SRE-mediated induction of a luciferase reporter gene is attenuated by IP(3) metabolizing enzymes. Furthermore, cAMP-induced nuclear translocation of the CREB coactivator TORC1 was inhibited by depletion of intracellular Ca(2+) stores. Our data indicate that Ca(2+) release from IP(3)-sensitive pools is required for cAMP-induced transcription in hippocampal neurons.
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We examined a variety of tissues for the presence of cytosolic cofactor activity that would support arachidonate-dependent cell-free activation of NADPH-oxidase in isolated human neutrophil membranes. Cofactor activity was not found in... more
We examined a variety of tissues for the presence of cytosolic cofactor activity that would support arachidonate-dependent cell-free activation of NADPH-oxidase in isolated human neutrophil membranes. Cofactor activity was not found in cytosol isolated from erythrocytes, lymphocytes, placenta, brain, liver, or the human promyelocytic leukemic cell line HL-60. Induction of differentiation in HL-60 cells led to expression of cytosolic cofactor activity. In dimethylsulphoxide-induced HL-60 cells the level of cytosolic cofactor activity was closely correlated with phorbol myristate acetate-stimulated whole cell superoxide production. These results strongly suggest that the cytosolic cofactor is a phagocyte-specific regulatory protein of physiologic importance in NADPH-oxidase activation.
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Though l-amino acids predominate in living organisms, substantial levels of free d-serine and d-aspartate occur in mammals, especially in nervous and endocrine tissues. Using an antibody specific for glutaraldehyde-fixed d-aspartate, we... more
Though l-amino acids predominate in living organisms, substantial levels of free d-serine and d-aspartate occur in mammals, especially in nervous and endocrine tissues. Using an antibody specific for glutaraldehyde-fixed d-aspartate, we have localized d-aspartate in rat tissues. In the brain we observe discrete neuronal localizations of d-aspartate, especially in the external plexiform layer of the olfactory bulb, hypothalamic supraoptic and paraventricular nuclei, the medial habenula, and certain brainstem nuclei. In rats 3-4 weeks old, we observe d-aspartate in septal nuclei and in a subset of stellate and basket cells of the cerebellum. d-aspartate is also concentrated in glands, including the epinephrine cells of the adrenal medulla, the posterior pituitary, and the pineal gland. Levels in the pineal gland are the highest of any mammalian tissue. d-aspartate oxidase, visualized by enzyme histochemistry, is concentrated in neurons of the hippocampus, cerebral cortex, and olfactory epithelium, as well as choroid plexus and ependyma. Localizations of d-aspartate oxidase are reciprocal to d-aspartate, suggesting that the enzyme depletes endogenous stores of the amino acid and might inactivate synaptically released d-aspartate.
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Brain injury is a major cause of long-term disability. The possibility exists for exogenously derived neural progenitor cells to repair damage resulting from brain injury, although more information is needed to successfully implement this... more
Brain injury is a major cause of long-term disability. The possibility exists for exogenously derived neural progenitor cells to repair damage resulting from brain injury, although more information is needed to successfully implement this promising therapy. To test the ability of neural progenitor cells (NPCs) obtained from rats to repair damaged neocortex, we transplanted neural progenitor cell suspensions into normal and injured slice cultures of the neocortex acquired from rats on postnatal day 0-3. Donor cells from E16 embryos were obtained from either the neocortex, including the ventricular zone (VZ) for excitatory cells, ganglionic eminence (GE) for inhibitory cells or a mixed population of the two. Cells were injected into the ventricular/subventricular zone (VZ/SVZ) or directly into the wounded region. Transplanted cells migrated throughout the cortical plate with GE and mixed population donor cells predominately targeting the upper cortical layers, while neocortically deri...