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Biomedical subjects

M I Simon

Publications and source records attributed to M I Simon.

At least 37 records · Page 2Linked to original sources
PubMed2000

Slowed recovery of rod photoresponse in mice lacking the GTPase accelerating protein RGS9-1.

Timely deactivation of the alpha-subunit of the rod G-protein transducin (Galphat) is essential for the temporal resolution of rod vision. Regulators of G-protein signalling (RGS) proteins accelerate hydrolysis of GTP by the alpha-subunits of heterotrimeric G proteins in vitro. Several retinal RGS proteins can act in vitro as GTPase accelerating proteins (GAP) for Galphat. Recent reconstitution experiments indicate that one of these, RGS9-1, may account for much of the Galphat GAP activity in rod outer segments (ROS). Here we report that ROS membranes from mice lacking RGS9-1 hydrolyse GTP more slowly than ROS membranes from control mice. The Gbeta5-L protein that forms a complex with RGS9-1 was absent from RGS9-/- retinas, although Gbeta5-L messenger RNA was still present. The flash responses of RGS9-/- rods rose normally, but recovered much more slowly than normal. We conclude that RGS9-1, probably in a complex with Gbeta5-L, is essential for acceleration of hydrolysis of GTP by Galphat and for normal recovery of the photoresponse.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2000

Inhibition of subsets of G protein-coupled receptors by empty mutants of G protein alpha subunits in g(o), G(11), and G(16).

We previously reported that the xanthine nucleotide binding G(o)alpha mutant, G(o)alphaX, inhibited the activation of G(i)-coupled receptors. We constructed similar mutations in G(11)alpha and G(16)alpha and characterized their nucleotide binding and receptor interaction. First, we found that G(11)alphaX and G(16)alphaX expressed in COS-7 cells bound xanthine 5'-O-(thiotriphosphate) instead of guanosine 5'-O-(thiotriphosphate). Second, we found that G(11)alphaX and G(16)alphaX interacted with betagamma subunits in the presence of xanthine diphosphate. These experiments demonstrated that G(11)alphaX and G(16)alphaX were xanthine nucleotide-binding proteins, similar to G(o)alphaX. Third, in COS-7 cells, both G(11)alphaX and G(16)alphaX inhibited the activation of G(q)-coupled receptors, whereas only G(16)alphaX inhibited the activation of G(i)-coupled receptors. Therefore, when in the nucleotide-free state, empty G(11)alphaX and G(16)alphaX appeared to retain the same receptor binding specificity as their wild-type counterparts. Finally, we found that G(o)alphaX, G(11)alphaX, and G(16)alphaX all inhibited the endogenous thrombin receptors and lysophosphatidic acid receptors in NIH3T3 cells, whereas G(11)alphaX and G(16)alphaX, but not G(o)alphaX, inhibited the activation of transfected m1 muscarinic receptor in these cells. We conclude that these empty G protein mutants of G(o)alpha, G(11)alpha, and G(16)alpha can act as dominant negative inhibitors against specific subsets of G protein-coupled receptors.

3T3 Cells↗
PubMed2000

Rapid and reproducible deactivation of rhodopsin requires multiple phosphorylation sites.

Efficient single-photon detection by retinal rod photoreceptors requires timely and reproducible deactivation of rhodopsin. Like other G protein-coupled receptors, rhodopsin contains multiple sites for phosphorylation at its COOH-terminal domain. Transgenic and electrophysiological methods were used to functionally dissect the role of the multiple phosphorylation sites during deactivation of rhodopsin in intact mouse rods. Mutant rhodopsins bearing zero, one (S338), or two (S334/S338) phosphorylation sites generated single-photon responses with greatly prolonged, exponentially distributed durations. Responses from rods expressing mutant rhodopsins bearing more than two phosphorylation sites declined along smooth, reproducible time courses; the rate of recovery increased with increasing numbers of phosphorylation sites. We conclude that multiple phosphorylation of rhodopsin is necessary for rapid and reproducible deactivation.

Animals↗
PubMed2000

Gq protein alpha subunits Galphaq and Galpha11 are localized at postsynaptic extra-junctional membrane of cerebellar Purkinje cells and hippocampal pyramidal cells.

Following cell surface receptor activation, the alpha subunit of the Gq subclass of GTP-binding proteins activates the phosphoinositide signalling pathway. Here we examined the expression and localization of Gq protein alpha subunits in the adult mouse brain by in situ hybridization and immunohistochemistry. Of the four members of the Gq protein alpha subunits, Galphaq and Galpha11 were transcribed predominantly in the brain. The highest transcriptional level of Galphaq was observed in cerebellar Purkinje cells (PCs) and hippocampal pyramidal cells, while that of Galpha11 was noted in hippocampal pyramidal cells. Antibody against the C-terminal peptide common to Galphaq and Galpha11 strongly labelled the cerebellar molecular layer and hippocampal neuropil layers. In these regions, immunogold preferentially labelled the cytoplasmic face of postsynaptic cell membrane of PCs and pyramidal cells. Immunoparticles were distributed along the extra-junctional cell membrane of spines, dendrites and somata, but were almost excluded from the junctional membrane. By double immunofluorescence, Galphaq/Galpha11 was extensively colocalized with metabotropic glutamate receptor mGluR1alpha in dendritic spines of PCs and with mGluR5 in those of hippocampal pyramidal cells. Together with concentrated localization of mGluR1alpha and mGluR5 in a peri-junctional annulus on PC and pyramidal cell synapses (Baude et al. 1993, Neuron, 11, 771-787; Luján et al. 1996, Eur. J. Neurosci., 8, 1488-1500), the present molecular-anatomical findings suggest that peri-junctional stimulation of the group I metabotropic glutamate receptors is mediated by Galphaq and/or Galpha11, leading to the activation of the intracellular effector, phospholipase Cbeta.

Amino Acid Sequence↗
PubMed2000

Synergistic effect of Bcl-2 and BAG-1 on the prevention of photoreceptor cell death.

PURPOSE: Ectopic expression of Bcl-2 in photoreceptors of mice with retinal degenerative disease slows progression of the disease. BAG-1 has previously been shown to augment the inhibitory effect of Bcl-2 on programmed cell death in cultured cell systems. This study was designed to determine whether the coexpression of BAG-1 and Bcl-2 in the photoreceptors of mice with an autosomal dominant form of retinitis pigmentosa (RP) would enhance the protective effect provided by Bcl-2 alone. METHODS: An expression vector using the 5' regulatory region of the murine opsin gene was used to target the expression of BAG-1 specifically to photoreceptor cells of mice. The BAG-1 transgenic mice were crossed to Bcl-2 transgenics to obtain animals that coexpress the two transgenes in photoreceptor cells. BAG-1/Bcl-2 animals were then crossed to an RP mouse model (a transgenic line overexpressing the S334ter rhodopsin mutant) to assess the effect of coexpression of BAG-1 and Bcl-2 on retinal degeneration. Morphologic analysis was performed on retinas isolated at various times after birth to monitor disease progression. RESULTS: High levels of BAG-1 expression resulted in retinal degeneration that was not prevented by Bcl-2 expression. However, coexpression of appropriate levels of BAG-1 and Bcl-2 was found to have a profound inhibitory effect on retinal degeneration caused by overexpression of a mutant rhodopsin transgene. Whereas expression of Bcl-2 alone was previously found to delay degeneration of the retina from 2 weeks to approximately 4 weeks of age, coexpression of BAG-1 and Bcl-2 inhibited photoreceptor cell death for as long as 7 to 9 weeks. CONCLUSIONS: The synergistic effect against photoreceptor cell death produced by the coexpression of Bcl-2 and BAG-1 indicates that these proteins can function in concert to prevent cell death. At the correct dosage, coexpression of Bcl-2 and BAG-1 may serve as a potential means to treat retinal degenerative diseases.

Animals↗
PubMed1999

Abnormal photoresponses and light-induced apoptosis in rods lacking rhodopsin kinase.

Phosphorylation is thought to be an essential first step in the prompt deactivation of photoexcited rhodopsin. In vitro, the phosphorylation can be catalyzed either by rhodopsin kinase (RK) or by protein kinase C (PKC). To investigate the specific role of RK, we inactivated both alleles of the RK gene in mice. This eliminated the light-dependent phosphorylation of rhodopsin and caused the single-photon response to become larger and longer lasting than normal. These results demonstrate that RK is required for normal rhodopsin deactivation. When the photon responses of RK-/- rods did finally turn off, they did so abruptly and stochastically, revealing a first-order backup mechanism for rhodopsin deactivation. The rod outer segments of RK-/- mice raised in 12-hr cyclic illumination were 50% shorter than those of normal (RK+/+) rods or rods from RK-/- mice raised in constant darkness. One day of constant light caused the rods in the RK-/- mouse retina to undergo apoptotic degeneration. Mice lacking RK provide a valuable model for the study of Oguchi disease, a human RK deficiency that causes congenital stationary night blindness.

Animals↗
PubMed1999

Activation of G12/G13 results in shape change and Rho/Rho-kinase-mediated myosin light chain phosphorylation in mouse platelets.

Platelets respond to various stimuli with rapid changes in shape followed by aggregation and secretion of their granule contents. Platelets lacking the alpha-subunit of the heterotrimeric G protein Gq do not aggregate and degranulate but still undergo shape change after activation through thromboxane-A2 (TXA2) or thrombin receptors. In contrast to thrombin, the TXA2 mimetic U46619 led to the selective activation of G12 and G13 in Galphaq-deficient platelets indicating that these G proteins mediate TXA2 receptor-induced shape change. TXA2 receptor-mediated activation of G12/G13 resulted in tyrosine phosphorylation of pp72(syk) and stimulation of pp60(c-src) as well as in phosphorylation of myosin light chain (MLC) in Galphaq-deficient platelets. Both MLC phosphorylation and shape change induced through G12/G13 in the absence of Galphaq were inhibited by the C3 exoenzyme from Clostridium botulinum, by the Rho-kinase inhibitor Y-27632 and by cAMP-analogue Sp-5,6-DCl-cBIMPS. These data indicate that G12/G13 couple receptors to tyrosine kinases as well as to the Rho/Rho-kinase-mediated regulation of MLC phosphorylation. We provide evidence that G12/G13-mediated Rho/Rho-kinase-dependent regulation of MLC phosphorylation participates in receptor-induced platelet shape change.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1999

Structure of CheA, a signal-transducing histidine kinase.

Histidine kinases allow bacteria, plants, and fungi to sense and respond to their environment. The 2.6 A resolution crystal structure of Thermotoga maritima CheA (290-671) histidine kinase reveals a dimer where the functions of dimerization, ATP binding, and regulation are segregated into domains. The kinase domain is unlike Ser/Thr/Tyr kinases but resembles two ATPases, Gyrase B and Hsp90. Structural analogies within this superfamily suggest that the P1 domain of CheA provides the nucleophilic histidine and activating glutamate for phosphotransfer. The regulatory domain, which binds the homologous receptor-coupling protein CheW, topologically resembles two SH3 domains and provides different protein recognition surfaces at each end. The dimerization domain forms a central four-helix bundle about which the kinase and regulatory domains pivot on conserved hinges to modulate transphosphorylation. Different subunit conformations suggest that relative domain motions link receptor response to kinase activity.

Adenosine Triphosphatases↗
PubMed1999

Human genome anatomy: BACs integrating the genetic and cytogenetic maps for bridging genome and biomedicine.

Human genome sequencing is accelerating rapidly. Multiple genome maps link this sequence to problems in biology and clinical medicine. Because each map represents a different aspect of the structure, content, and behavior of human chromosomes, these fundamental properties must be integrated with the genome to understand disease genes, cancer instability, and human evolution. Cytogenetic maps use 400-850 visible band landmarks and are the primary means for defining prenatal defects and novel cancer breakpoints, thereby providing simultaneous examination of the entire genome. Recent genetic, physical, and transcript maps use PCR-based landmarks called sequence-tagged sites (STSs). We have integrated these genome maps by anchoring the human cytogenetic to the STS-based genetic and physical maps with 1021 STS-BAC pairs at an average spacing of approximately 1 per 3 Mb. These integration points are represented by 872 unique STSs, including 642 polymorphic markers and 957 bacterial artificial chromosomes (BACs), each of which was localized on high resolution fluorescent banded chromosomes. These BACs constitute a resource that bridges map levels and provides the tools to seamlessly translate questions raised by genomic change seen at the chromosomal level into answers based at the molecular level. We show how the BACs provide molecular links for understanding human genomic duplications, meiosis, and evolution, as well as reagents for conducting genome-wide prenatal diagnosis at the molecular level and for detecting gene candidates associated with novel cancer breakpoints.

Chromosome Mapping↗
PubMed1999

A 12-Mb complete coverage BAC contig map in human chromosome 16p13.1-p11.2.

We have constructed a complete coverage BAC contig map that spans a 12-Mb genomic segment in the human chromosome 16p13.1-p11.2 region. The map consists of 68 previously mapped STSs and 289 BAC clones, 51 of which-corresponding to a total of 7.721 Mb of genomic DNA-have been sequenced, and provides a high resolution physical map of the region. Contigs were initially built based mainly on the analysis of STS contents and restriction fingerprint patterns of the clones. To close the gaps, probes derived from BAC clone ends were used to screen deeper BAC libraries. Clone end sequence data obtained from chromosome 16-specific BACs, as well as from public databases, were used for the identification of BACs that overlap with fully sequenced BACs by means of sequence match. This approach allowed precise alignment of clone overlaps in addition to restriction fingerprint comparison. A freehand contig drawing software tool was developed and used to manage the map data graphically and generate a real scale physical map. The map we present here is approximately 3.5 x deep and provides a minimal tiling path that covers the region in an array of contigous, overlapping BACs.

Base Sequence↗
PubMed1999

Increased susceptibility to light damage in an arrestin knockout mouse model of Oguchi disease (stationary night blindness)

PURPOSE: To determine whether constitutive signal flow arising from defective rhodopsin shut-off causes photoreceptor cell death in arrestin knockout mice. METHODS: The retinas of cyclic-light-reared, pigmented arrestin knockout mice and wild-type littermate control mice were examined histologically for photoreceptor cell loss from 100 days to 1 year of age. In separate experiments, to determine whether constant light would accelerate the degeneration in arrestin knockout mice, these animals and wild-type control mice were exposed for 1, 2, or 3 weeks to fluorescent light at an intensity of 115 to 150 fc. The degree of photoreceptor cell loss was quantified histologically by obtaining a mean outer nuclear layer thickness for each animal. RESULTS: In arrestin knockout mice maintained in cyclic light, photoreceptor loss was evident at 100 days of age, and it became progressively more severe, with less than 50% of photoreceptors surviving at 1 year of age. The photoreceptor degeneration appeared to be caused by light, because when these mice were reared in the dark, the retinal structure was indistinguishable from normal. When exposed to constant light, the retinas of wild-type pigmented mice showed no light-induced damage, regardless of exposure duration. By contrast, the retinas of arrestin knockout mice showed rapid degeneration in constant light, with a loss of 30% of photoreceptors after 1 week of exposure and greater than 60% after 3 weeks of exposure. CONCLUSIONS: The results indicate that constitutive signal flow due to arrestin knockout leads to photoreceptor degeneration. Excessive light accelerates the cell death process in pigmented arrestin knockout mice. Human patients with naturally occurring mutations that lead to nonfunctional arrestin and rhodopsin kinase have Oguchi disease, a form of stationary night blindness. The present findings suggest that such patients may be at greater risk of the damaging effects of light than those with other forms of retinal degeneration, and they provide an impetus to restrict excessive light exposure as a protective measure in patients with constitutive signal flow in phototransduction.

Animals↗
PubMed1998

Phospholipase cbeta4 is specifically involved in climbing fiber synapse elimination in the developing cerebellum.

Elimination of excess climbing fiber (CF)-Purkinje cell synapses during cerebellar development involves a signaling pathway that includes type 1 metabotropic glutamate receptor, Galphaq, and the gamma isoform of protein kinase C. To identify phospholipase C (PLC) isoforms involved in this process, we generated mice deficient in PLCbeta4, one of two major isoforms expressed in Purkinje cells. PLCbeta4 mutant mice are viable but exhibit locomotor ataxia. Their cerebellar histology, parallel fiber synapse formation, and basic electrophysiology appear normal. However, developmental elimination of multiple CF innervation clearly is impaired in the rostral portion of the cerebellar vermis, in which PLCbeta4 mRNA is predominantly expressed. By contrast, CF synapse elimination is normal in the caudal cerebellum, in which low levels of PLCbeta4 mRNA but reciprocally high levels of PLCbeta3 mRNA are found. These results indicate that PLCbeta4 transduces signals that are required for CF synapse elimination in the rostral cerebellum.

Animals↗
PubMed1998

Interaction of the xanthine nucleotide binding Goalpha mutant with G protein-coupled receptors.

We constructed a double mutant version of the alpha subunit of Go that was regulated by xanthine nucleotides instead of guanine nucleotides (GoalphaX). We investigated the interaction between GoalphaX and G protein-coupled receptors in vitro. First, we found that the activated m2 muscarinic cholinergic receptor (MAChR) could facilitate the exchange of XTPgammaS for XDP in the GoalphaXbetagamma heterotrimer. Second, the GoalphaXbetagamma complex was able to induce the high affinity ligand-binding state in the N-formyl peptide receptor (NFPR). These experiments demonstrated that GoalphaX was able to interact effectively with G protein-coupled receptors. Third, we found that the empty form of GoalphaX, lacking a bound nucleotide and betagamma, formed a stable complex with the m2 muscarinic cholingeric receptor associated with the plasma membrane. Finally, we investigated the interaction of GoalphaX with receptor in COS-7 cells. The empty form of GoalphaX bound tightly to the receptor and was not activated because XTP was not available intracellularly. We tested the ability of GoalphaX to inhibit the activities of several different G protein-coupled receptors in transfected COS-7 cells and found that GoalphaX specifically inhibited Go-coupled receptors. Thus the modified G proteins may act as dominant-negative mutants to trap and inactivate specific subsets of receptors.

Animals↗
PubMed1998

Specific involvement of G proteins in regulation of serum response factor-mediated gene transcription by different receptors.

Regulation of serum response factor (SRF)-mediated gene transcription by G protein subunits and G protein-coupled receptors was investigated in transfected NIH3T3 cells and in a cell line that was derived from mice lacking Galphaq and Galpha11. We found that the constitutively active forms of the alpha subunits of the Gq and G12 class of G proteins, including Galphaq, Galpha11, Galpha14, Galpha16, Galpha12, and Galpha13, can activate SRF in NIH3T3 cells. We also found that the type 1 muscarinic receptor (m1R) and alpha1-adrenergic receptor (AR)-mediated SRF activation is exclusively dependent on Galphaq/11, while the receptors for thrombin, lysophosphatidic acid (LPA), thromboxane A2, and endothelin can activate SRF in the absence of Galphaq/11. Moreover, RGS12 but not RGS2, RGS4, or Axin was able to inhibit Galpha12 and Galpha13-mediated SRF activation. And RGS12, but not other RGS proteins, blocked thrombin- and LPA-mediated SRF activation in the Galphaq/11-deficient cells. Therefore, the thrombin, LPA, thromboxane A2, and endothelin receptors may be able to couple to Galpha12/13. On the contrary, receptors including beta2- and alpha2-ARs, m2R, the dopamine receptors type 1 and 2, angiotensin receptors types 1 and 2, and interleukin-8 receptor could not activate SRF in the presence or absence of Galphaq/11, suggesting that these receptors cannot couple to endogenous G proteins of the G12 or Gq classes.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1998

Tec/Bmx non-receptor tyrosine kinases are involved in regulation of Rho and serum response factor by Galpha12/13.

A transient transfection system was used to identify regulators and effectors for Tec and Bmx, members of the Tec non-receptor tyrosine kinase family. We found that Tec and Bmx activate serum response factor (SRF), in synergy with constitutively active alpha subunits of the G12 family of GTP-binding proteins, in transiently transfected NIH 3T3 cells. The SRF activation is sensitive to C3, suggesting the involvement of Rho. The kinase and Tec homology (TH) domains of the kinases are required for SRF activation. In addition, kinase-deficient mutants of Bmx are able to inhibit Galpha13- and Galpha12-induced SRF activation, and to suppress thrombin-induced SRF activation in cells lacking Galphaq/11, where thrombin's effect is mediated by G12/13 proteins. Moreover, expression of Galpha12 and Galpha13 stimulates autophosphorylation and transphosphorylation activities of Tec. Thus, the evidence indicates that Tec kinases are involved in Galpha12/13-induced, Rho-mediated activation of SRF. Furthermore, Src, which was previously shown to activate kinase activities of Tec kinases, activates SRF predominantly in Rho-independent pathways in 3T3 cells, as shown by the fact that C3 did not block Src-mediated SRF activation. However, the Rho-dependent pathway becomes significant when Tec is overexpressed.

3T3 Cells↗
PubMed1998

Genetic analysis of mammalian G-protein signalling.

Heterotrimeric G-proteins are important signalling proteins which function in all cells of the mammalian organism. Inactivating mutations in a variety of G-protein alpha-subunit genes in mice resulted in mostly unexpected phenotypes and have provided interesting new insight into their biological roles. Whereas the inactivation of some G alpha genes led to mild phenotypes suggesting the presence of redundant or compensatory mechanisms, other G-proteins appear to play highly specific biological or developmental roles. The purpose of this review is to summarize the current knowledge about G-protein functions based on gene-inactivation studies.

Animals↗
PubMed1998

Embryonic cardiomyocyte hypoplasia and craniofacial defects in G alpha q/G alpha 11-mutant mice.

Heterotrimeric G proteins of the Gq class have been implicated in signaling pathways regulating cardiac growth under physiological and pathological conditions. Knockout mice carrying inactivating mutations in both of the widely expressed G alpha q class genes, G alpha q and G alpha 11, demonstrate that at least two active alleles of these genes are required for extrauterine life. Mice carrying only one intact allele [G alpha q(-/+);G alpha 11(-/-) or G alpha q(-/-);G alpha 11(-/+)] died shortly after birth. These mutants showed a high incidence of cardiac malformation. In addition, G alpha q(-/-);G alpha 11(-/+) newborns suffered from craniofacial defects. Mice lacking both G alpha q and G alpha 11 [G alpha q(-/-);G alpha 11(-/-)] died at embryonic day 11 due to cardiomyocyte hypoplasia. These data demonstrate overlap in G alpha q and G alpha 11 gene functions and indicate that the Gq class of G proteins plays a crucial role in cardiac growth and development.

Alleles↗