Biomedical subjects

M I Simon

Publications and source records attributed to M I Simon.

At least 73 records · Page 4Linked to original sources

PubMed1997

Retarding photoreceptor degeneration in Pdegtm1/Pdegtml mice by an apoptosis suppressor gene.

PURPOSE: Mice (Pdegtm1/Pdegtm1) homozygous for a mutant allele of the gamma subunit of retinal cyclic guanosine monophosphate phosphodiesterase (PDE gamma) suffer a severe photoreceptor degeneration. To determine whether the antiapoptotic BCL2 gene is effective in delaying the cell death pathway in this new strain of mutant mice, a transgene encoding the BCL2 gene product was introduced by mating into the mutant background, and the resulting mice were examined for possible rescue of the retinal degeneration. METHODS: Electroretinograms (ERGs) of the Pdegtm1/Pdegtm1 mice carrying BCL2 were taken to monitor the responses to light. Light and electron microscopy of sections were used to examine degeneration at different times after birth. RESULTS: The ERGs of the mutants with the transgene were larger than those without the transgene at 2 and 3 weeks after birth. The maximum differences occurred at 2 weeks postpartum. At 4 weeks after birth, no ERG could be detected in either strain. Histologic analysis showed a greater preservation of photoreceptor nuclei in the Pdegtm1/Pdegtm1 mice containing the BCL2 transgene, which paralleled the electroretinography. CONCLUSIONS: The introduction of an antiapoptotic transgene BCL2 can delay temporarily and partially the degeneration of photoreceptors in a new autosomal-recessive murine model of retinal degeneration.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed1996

Desensitization of inositol 1,4,5-trisphosphate/Ca2+-induced Cl- currents by prolonged activation of G proteins in Xenopus oocytes.

Expression of G protein alpha subunits of the Gq family with various G protein-coupled receptors induces activation of an inositol 1,4, 5-trisphosphate (IP3)/Ca2+-mediated Cl- conductance in Xenopus oocytes. Our present data show that two members of this family, the human Galpha16 subunit and the murine homologue Galpha15, can induce both activation and inhibition of these agonist-induced currents. Although extremely low amounts (10-50 pg) of injected Galpha16 subunit cRNA cause modest ( approximately 2-fold) enhancement of ligand-induced Cl- currents in oocytes co-injected with thyrotropin-releasing hormone (TRH) receptor cRNA 48 h postinjection, larger Galpha16 and Galpha15 cRNA injections cause >10-fold inhibition of TRH or 5HT2c receptor responses. The inhibition is analyzed in this study. The inhibited currents are recovered if various Gbetagamma subunit combinations are also expressed with the Galpha subunits. The constitutively active mutant, Galpha16Q212L, also causes a strong attenuation of the ligand-induced Cl- currents, but this inhibition is not recovered by co-expression of Gbetagamma subunits. These results indicate that the free Galpha subunit is responsible for the inhibitory signal. Although expression of TRH receptor alone produces maximum responses approximately 48 h after injection, co-expression of TRH receptor with Galpha16 results in enhanced responses 6-12 h postinjection, followed by complete attenuation at 36 h. Furthermore, injection of Galpha16 cRNA alone at comparable levels gives rise to spontaneous Cl- currents within 6-12 h postinjection, suggesting that the early spontaneous activation underlies the later suppression. Expression of other G protein alpha subunits of the Gq family, at cRNA levels considerably higher than effective for Galpha16, produces both analogous spontaneous Cl- currents and, later, inhibition of ligand-induced Cl- currents. Experiments with direct injection of IP3 and of Ca2+ suggest that this inhibition is consistent with the down-regulation of IP3 receptors. These data indicate that both enhancement and inhibition of signaling through G protein-coupled receptors can be mediated by the expression level and/or activity of an individual G protein.

Animals↗

PubMed1996

Phospholipase C beta 4 is involved in modulating the visual response in mice.

Expression of G protein-regulated phospholipase C (PLC) beta 4 in the retina, lateral geniculate nucleus, and superior colliculus implies that PLC beta 4 may play a role in the mammalian visual process. A mouse line that lacks PLC beta 4 was generated and the physiological significance of PLC beta 4 in murine visual function was investigated. Behavioral tests using a shuttle box demonstrated that the mice lacking PLC beta 4 were impaired in their visual processing abilities, whereas they showed no deficit in their auditory abilities. In addition, the PLC beta 4-null mice showed 4-fold reduction in the maximal amplitude of the rod a- and b-wave components of their electroretinograms relative to their littermate controls. However, recording from single rod photoreceptors did not reveal any significant differences between the PLC beta 4-null and wild-type littermates, nor were there any apparent differences in retinas examined with light microscopy. While the behavioral and electroretinographic results indicate that PLC beta 4 plays a significant role in mammalian visual signal processing, isolated rod recording shows little or no apparent deficit, suggesting that the effect of PLC beta 4 deficiency on the rod signaling pathway occurs at some stage after the initial phototransduction cascade and may require cell-cell interactions between rods and other retinal cells.

Animals↗

PubMed1996

RGS-r, a retinal specific RGS protein, binds an intermediate conformation of transducin and enhances recycling.

G proteins regulate intracellular signaling by coupling a cycle of guanine nucleotide binding and hydrolysis to transient changes of cellular functions. The mechanisms that control the recycling of transducin, the "pacesetting" G protein that regulates mammalian phototransduction, are unclear. We show that a novel retinal specific RGS-motif protein specifically binds to an intermediate conformation involved in GTP hydrolysis by transducin and accelerates phosphate release and the recycling of transducin. This specific interaction further rationalizes the kinetics of the phototransduction cascade and provides a general hypothesis to explain the mechanism of interaction of RGS proteins with other G proteins.

Amino Acid Sequence↗

PubMed1996

A novel form of the G protein beta subunit Gbeta5 is specifically expressed in the vertebrate retina.

The G protein beta subunit, Gbeta5, is predominantly expressed in the central nervous system. In rodent brain, Gbeta5 is expressed as a protein with an apparent molecular mass of 39,000 daltons (39 kDa). We have identified an additional Gbeta5 immunoreactive protein of apparent size 44 kDa in the vertebrate retina. Molecular cloning and sequencing of polymerase chain reaction products revealed that the cDNA encoding the larger species of Gbeta5 (Gbeta5L) was identical to the shorter form with the addition of 126 base pairs of 5' DNA sequence potentially encoding an in-frame 42-amino acid extension. Sequencing of mouse Gbeta5 genomic clones demonstrated that the 126-base pair of retinal-specific coding material is derived from a hitherto undetected 5' exon. During sucrose density gradient fractionation of bovine retinas, the 44-kDa Gbeta5L protein co-purified with rod outer segment membranes. Incubation of rod outer segment membranes with the nonhydrolyzable guanine nucleotide, GTPgammaS (guanosine 5'-3-O-(thio)triphosphate), which released the Gbeta subunit of transducin (Gbeta1), failed to remove Gbeta5L. The 39-kDa Gbeta5 protein displayed differential association with retinal and brain membranes. In the retina, Gbeta5 was present as a soluble protein and was undetectable in the membrane fraction, whereas in the brain approximately 70% of Gbeta5 was associated with cellular membranes. In transient COS-7 cell expression experiments, Gbeta5L formed functional Gbetagamma dimers and Galphabetagamma heterotrimers, and activated phosphoinositide-specific phospholipase Cbeta2 in a manner indistinguishable from the 39-kDa Gbeta5 protein. The cloning of the retinal-specific Gbeta5L cDNA suggests the existence of potentially novel G protein-mediated signaling cascades in photoreception.

Amino Acid Sequence↗

PubMed1996

Galpha12 and galpha13 are phosphorylated during platelet activation.

The ubiquitously expressed G-proteins G12 and G13 whose function is currently not clear have been shown to be activated in platelet membranes through receptors that stimulate platelet aggregation. We used intact human platelets to determine whether alpha subunits of both G-proteins can be phosphorylated under physiological conditions. Activation of human platelets by thrombin and the thromboxane A2 receptor agonist U46619 lead to phosphorylation of Galpha12 and Galpha13. Phosphorylation occurred rapidly after addition of thrombin and was not mediated by glycoprotein IIb/IIIa (integrin alphaIIbbeta3) activation. Phosphorylation of Galpha12 and Galpha13 could be mimicked by phorbol 12-myristate 13-acetate, and thrombin-induced phosphorylation was inhibited by the protein kinase C inhibitor calphostin C indicating an involvement of protein kinase C in Galpha12/13 phosphorylation induced by thrombin in human platelets. The phosphorylation of both G protein alpha subunits was reconstituted in COS-7 cells cotransfected with Galpha12 or Galpha13 and different protein kinase C isoforms. Among the protein knase C isoforms tested, protein kinase C beta, delta, and epsilon were most effective in promoting phosphorylation of Galpha12 and Galpha13 in a phorbol 12-myristate 13-acetate-dependent manner. These data demonstrate that Galpha12 and Galpha13 are phosphorylated under in vivo conditions and that this phosphorylation involves protein kinase C.

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

PubMed1996

The hybrid histidine kinase DokA is part of the osmotic response system of Dictyostelium.

We have used PCR to identify a Dictyostelium homolog of the bacterial two-component system. The gene dokA codes for a member of the hybrid histidine kinase family which is defined by the presence of conserved amino acid sequence motifs corresponding to an N-terminal receptor domain, a central kinase and a C-terminal response regulator moiety. Potential function of the regulator domain was demonstrated by phosphorylation in vitro. dokA mutants are deficient in the osmoregulatory pathway, resulting in premature cell death under high osmotic stress. Under less stringent osmotic conditions, cells grow at a normal rate, but development at the multicellular stage is altered. dokA is a member of a family of histidine kinase-like genes that play regulatory roles in eukaryotic cell function.

Amino Acid Sequence↗

PubMed1996

bcl-2 overexpression reduces apoptotic photoreceptor cell death in three different retinal degenerations.

Apoptosis of photoreceptors occurs infrequently in adult retina but can be triggered in inherited and environmentally induced retinal degenerations. The protooncogene bcl-2 is known to be a potent regulator of cell survival in neurons. We created lines of transgenic mice overexpressing bcl-2 to test for its ability to increase photoreceptor survival. Bcl-2 increased photoreceptor survival in mice with retinal degeneration caused by a defective opsin or cGMP phosphodiesterase. Overexpression of Bcl-2 in normal photoreceptors also decreased the damaging effects of constant light exposure. Apoptosis was induced in normal photoreceptors by very high levels of bcl-2. We conclude that bcl-2 is an important regulator of photoreceptor cell death in retinal degenerations.

Aging↗

PubMed1996

Regulation by cAMP-dependent protein kinease of a G-protein-mediated phospholipase C.

The heterotrimeric G proteins mediate a variety of cellular processes by coupling transmembrane receptors to different effector molecules, including adenylyl cyclases and inositol-phospholipid-specific phospholipase C (PLC)1-3. Activation of adenylyl cyclases results in the production of cyclic AMP and activation of cAMP-dependent protein kinase (PKA). Phospholipase C catalyses the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PtdInsP2) to generate diacylglycerol and inositol-1,4,5-triphosphate (InsP2), leading to the activation of protein kinase C (PKC) and the mobilization of intracellular calcium. The various PLC isoforms appear to be activated by different receptors, and in some cases by different G-protein components. There are four well-characterized forms of PLC-beta and all of them are activated to various extents by the G alpha q family of G proteins. Specific activation of PLC isoforms beta 2 and beta 3 by G-protein beta gamma subunits has also been reported. Although it has been suggested that PLC activity might be modulated by the adenylyl cyclase pathway, no clear link has been established between the two pathways. Here we report that cAMP-dependent protein kinase specifically inhibits G beta gamma-activated PLC-beta 2 activity but not that of the G alpha-activated PLC isoforms, and that the effect of PKA is not mimicked by PKC isozymes. Furthermore, we show that PKA directly phosphorylates serine residues of the PLC-beta 2 protein both in vivo and in vitro. Our results provide an insight into the specificity and nature of the crosstalk between the two G-protein-coupled signal transduction pathways.

Amino Acid Sequence↗

PubMed1996

Archaeal ubiquity.

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Adaptation, Physiological↗

PubMed1996

A bacterial artificial chromosome-based framework contig map of human chromosome 22q.

We have constructed a physical map of human chromosome 22q using bacterial artificial chromosome (BAC) clones. The map consists of 613 chromosome 22-specific BAC clones that have been localized and assembled into contigs using 452 landmarks, 346 of which were previously ordered and mapped to specific regions of the q arm of the chromosome by means of chromosome 22-specific yeast artificial chromosome clones. The BAC-based map provides immediate access to clones that are stable and convenient for direct genome analysis. The approach to rapidly developing marker-specific BAC contigs is relatively straightforward and can be extended to generate scaffold BAC contig maps of the rest of the chromosomes. These contigs will provide substrates for sequencing the entire human genome. We discuss how to efficiently close contig gaps using the end sequences of BAC clone inserts.

Cell Line↗

PubMed1996

Pertussis toxin-sensitive activation of phospholipase C by the C5a and fMet-Leu-Phe receptors.

Signal transduction pathways that mediate C5a and fMet-Leu-Phe (fMLP)-induced pertussis toxin (PTx)-sensitive activation of phospholipase C (PLC) have been investigated using a cotransfection assay system in COS-7 cells. The abilities of the receptors for C5a and fMLP to activate PLC beta2 and PLC beta3 through the Gbetagamma subunits of endogenous Gi proteins in COS-7 cells were tested because both PLC beta2 and PLC beta3 were shown to be activated by the betagamma subunits of G proteins in in vitro reconstitution assays. Neither of the receptors can activate endogenous PLC beta3 or recombinant PLC beta3 in transfected COS-7 cells. However, both receptors can clearly activate PLC beta2 in a PTx-sensitive manner, suggesting that the receptors may interact with endogenous PTx-sensitive G proteins and activate PLC beta2 probably through the Gbetagamma subunits. These findings were further corroborated by the results that PLC beta3 could only be slightly activated by Gbeta1gamma1 or Gbeta1gamma5 in the cotransfection assay, whereas the Gbetagamma subunits strongly activated PLC beta2 under the same conditions. PLC beta3 can be activated by Galphaq, Galpha11, and Galpha16 in the cotransfection assay. In addition, the Ggamma2 and Ggamma3 mutants with substitution of the C-terminal Cys residue by a Ser residue, which can inhibit wild type Gbetagamma-mediated activation of PLC beta2, were able to inhibit C5a or fMLP-mediated activation of PLC beta2. These Ggamma mutants, however, showed little effect on m1-muscarinic receptor-mediated PLC activation, which is mediated by the Gq class of G proteins. These results all confirm that the Gbetagamma subunits are involved in PLC beta2 activation by the two chemoattractant receptors and suggest that in COS-7 cells activation of PLC beta3 by Gbetagamma may not be the primary pathway for the receptors.

Amino Acid Sequence↗

PubMed1996

Construction and characterization of a human bacterial artificial chromosome library.

We have constructed an arrayed human genomic BAC library with approximately 4x coverage that is represented by 96,000 BAC clones with average insert size of nearly 140 kb. A new BAC vector that allows color-based positive screening to identify transformants with inserts has increased BAC cloning efficiency. The library was gridded onto hybridization filters at high density for efficient identification of BAC clones by colony hybridization. The library was also formulated into characteristic DNA pools to allow for PCR screening of the library for STS content. We have characterized the library mainly by screening with more than 300 different landmarks that include cDNA, STSs, and cosmid clones. We describe methods for using BAC clones and discuss the implications for genome characterization, mapping, and sequencing.

Cell Line↗

PubMed1996

A human chromosome 22 fosmid resource: mapping and analysis of 96 clones.

We have created a resource for chromosome 22 consisting of 96 unique, well-characterized Fosmids. The Fosmid vector permits efficient cloning of DNA fragments averaging 40 kb in a single-copy vector based on the F factor of Escherichia coli. We have found that Fosmid clones from human chromosome 22 show remarkable stability and are useful for a wide variety of applications in genome analysis. These 96 clones have been localized by FISH, using high-resolution fluorescent banding and multicolor mapping techniques, and their position on the chromosome was correlated with their content of a number of common repeated sequence elements. We identified a subset of clones likely to contain genes by restriction analysis using the enzymes NotI, MluI, SacII, and BssHII. This collection of cytogenetically anchored clones, representing nearly 7% of the chromosome, is of immediate value for detecting chromosomal rearrangements, for use in gene isolation, and as a framework for physical mapping.

Binding Sites↗

PubMed1996

Hyphal development in Neurospora crassa: involvement of a two-component histidine kinase.

Two-component signal transduction systems are most often found in prokaryotic organisms where they are responsible for mediating the cellular responses to many environmental stimuli. These systems are composed of an autophosphorylating histidine kinase and a response regulator. We have found evidence for the existence of two-component histidine kinases in the eukaryotic filamentous fungus Neurospora crassa based on screening with degenerate primers to conserved regions of these signaling proteins. Subsequent cloning and sequencing of one member of this newly discovered group, nik-1+, shows that the predicted protein sequence shares homology with both the kinase and response regulator modules of two-component signaling proteins. In addition, the N-terminal region of the protein has a novel repeating 90-amino acid motif. Deletion of the nik-1+ gene in N. crassa results in an organism that displays aberrant hyphal structure, which is enhanced under conditions of high osmostress. Increased osmotic pressure during growth on solid medium leads to restricted colonial growth, loss of aerial hyphae formation, and no subsequent conidiophore development. This finding may have implications for mechanisms of fungal colonization and pathogenicity.

Amino Acid Sequence↗

PubMed1996

Framework for a physical map of the human 22q13 region using bacterial artificial chromosomes (BACs).

Detailed physical maps of entire chromosomes based on combined genetic, cytogenetic, and structural information are essential components for positional cloning and genomic sequencing. Despite the wealth of genetic information of the known diseases in the chromosome 22q13, the construction of a detailed physical map of the terminal region is difficult due to the sparsity of the genetic markers. We present here a map of bacterial artificial chromosome (BAC) contigs that cover a number of genetic loci in the 22q13 region. One hundred thirty-six BACs with an average insert size of 140 kb are assembled into 35 contigs defined by 64 markers in 22q13-qter. Twenty-three anonymous markers are now linked to the previously mapped genetic anchor points.

Chromosome Mapping↗

PubMed1996

Isolation and in vitro characterization of CheZ suppressors for the Escherichia coli chemotactic response regulator mutant CheYN23D.

The phosphorylated form of the response regulator CheY promotes the tumble signal in Escherichia coli chemotaxis. Phospho-CheY is thought to interact with the switch at the base of the flagellar motor and cause reversal of flagellar rotation from counterclockwise to clockwise changing the swimming direction. Thus the level of phospho-CheY controls the direction of flagellar rotation. The decay of the tumble signal is caused by dephosphorylation of CheY. CheY has an intrinsic autophosphatase activity; however, this reaction is greatly accelerated by the presence of the CheZ protein. We have shown previously that mutations at residues Asn-23 and Lys-26 in CheY confer resistance to the dephosphorylation activity of CheZ (Sann, M.G., Swanson, R.V., Bourret, R.B., and Simon, M.I. (1995) Mol. Microbiol. 15, 1069-79). Here we show that mutant CheY(N23D) is impaired in binding to CheZ, which provides a possible explanation for its resistance to the dephosphorylation activity of CheZ. Moreover, we isolated CheZ second-site suppressors of CheY(N23D), which restore both dephosphorylation and binding activity in a CheY(N23D) background. When the CheZ suppressor mutations are mapped, they are found in two clusters at the N and C termini of the CheZ protein which could define two regions of interaction with CheY. Furthermore, these regions may generate a surface in the folded three-dimensional structure of CheZ required for interaction with CheY.

Amino Acid Sequence↗

PubMed1996

Phosphotransfer and CheY-binding domains of the histidine autokinase CheA are joined by a flexible linker.

Multidimensional heteronuclear NMR techniques were applied to study a protein fragment of the histidine autokinase CheA from Escherichia coli. This fragment (CheA1-233) contains the phosphotransfer domain and the CheY-binding domain joined by a linker region. Comparison of chemical shift and NOE cross-peak patterns indicates that the structures of the two domains in CheA1-233 remain nearly the same as in the two individual domain fragments, CheA1-134 and CheA124-257. Relaxation properties of the backbone 15N nuclei were measured to study the rotational correlations of the two domains and properties of the linker region. Dynamics data were analyzed both by an isotropic motional model and an anisotropic motional model. The experimental T1 and T2 values, the derived rotational correlation times, and motional anisotropy are significantly different for the two domains, indicating the two domains reorient independently and the linker region is highly flexible. Dynamics data of CheA1-233 were also compared with those of CheA1-134. Our studies show that flexible domain linkers and extended and flexible terminal polypeptide chains can have significant effects on the motional properties of the adjacent structured regions. These observations suggest a model for the graded regulation of CheA autophosphorylation activity. In this model, the various activity states of the receptor are generated by controlling the access of the mean position of the kinase domain to the phosphotransfer domain. This would then modulate the diffusional encounter rate of the domains and hence activity over a wide and graded range of values.

Amino Acid Sequence↗