scholarly journals mGluR6 Transcripts in Non-neuronal Tissues

2011 ◽  
Vol 59 (12) ◽  
pp. 1076-1086 ◽  
Author(s):  
Tamar Vardi ◽  
Marie Fina ◽  
Lingli Zhang ◽  
Anuradha Dhingra ◽  
Noga Vardi
Keyword(s):  
Transgenic Mouse ◽  
Metabotropic Glutamate Receptors ◽  
Fluorescent Protein ◽  
Splice Variants ◽  
Subcommissural Organ ◽  
Wild Type Mouse ◽  
Cell Types ◽  
Protein Product ◽  
Wild Type ◽  
In The Wild

To study mGluR6 expression, the authors investigated two transgenic mouse lines that express enhanced green fluorescent protein (GFP) under control of mGluR6 promoter. In retina, GFP was expressed exclusively in all ON bipolar cell types, either uniformly across all cells of this class (line 5) or in a mosaic (patchy) fashion (line 1). In brain, GFP was found in certain cortical areas, superior colliculus, axons of the corpus callosum, accessory olfactory bulb, and cells of the subcommissural organ. Outside the nervous system, GFP was seen in the corneal endothelium, testis, the kidney’s medulla, collecting ducts and parietal layer that surround the glomeruli, and B lymphocytes. Furthermore, RT-PCR showed that most tissues that expressed GFP in the transgenic mouse also transcribed two splice variants of mGluR6 in the wild-type mouse. The alternate variant was lacking exon 8, predicting a protein product of 545 amino acids that lacks the 7-transmembrane domains of the receptor. In cornea, immunostaining for mGluR6 gave strong staining in the endothelium, and this was stronger in wild-type than in mGluR6-null mice. Furthermore, calcium imaging with Fura-2 showed that application of L-AP4, an agonist for group III metabotropic glutamate receptors including mGluR6, elevated calcium in endothelial cells.

Roles of M2 and M4 Muscarinic Receptors in Regulating Acetylcholine Release From Myenteric Neurons of Mouse Ileum

2005 ◽  
Vol 93 (5) ◽  
pp. 2841-2848 ◽  
Author(s):  
Tadayoshi Takeuchi ◽  
Kaori Fujinami ◽  
Hiroto Goto ◽  
Akikazu Fujita ◽  
Makoto M. Taketo ◽  
...  
Keyword(s):  
Muscarinic Receptors ◽  
Wild Type Mouse ◽  
Electrical Field Stimulation ◽  
Enteric Neurons ◽  
Wild Type ◽  
Ach Release ◽  
Mouse Small Intestine ◽  
Presynaptic Muscarinic Receptors ◽  
In The Wild ◽  
Mouse Ileum

We investigated the subtype of presynaptic muscarinic receptors associated with inhibition of acetylcholine (ACh) release in the mouse small intestine. We measured endogenous ACh released from longitudinal muscle with myenteric plexus (LMMP) preparations obtained from M1–M5 receptor knockout (KO) mice. Electrical field stimulation (EFS) increased ACh release in all LMMP preparations obtained from M1–M5 receptor single KO mice. The amounts of ACh released in all preparations were equal to that in the wild-type mice. Atropine further increased EFS-induced ACh release in the wild-type mice. Unexpectedly, atropine also increased, to a similar extent, EFS-induced ACh release to the wild-type mice in all M1–M5 receptor single KO mice. In M2 and M4 receptor double KO mice, the amount of EFS-induced ACh release was equivalent to an atropine-evoked level in the wild-type mouse, and further addition of atropine had no effect. M2 receptor immunoreactivity was located in both smooth muscle cells and enteric neurons. M4 receptor immunoreactivity was located in the enteric neurons, being in co-localization with M2 receptor immunoreactivity. These results indicate that both M2 and M4 receptors mediate the muscarinic autoinhibition in ACh release in the LMMP preparation of the mouse ileum, and loss of one of these subtypes can be compensated functionally by a receptor that remained. M1, M3, and M5 receptors do not seem to be involved in this mechanism.

Functional comparison of the role of dynamin 2 splice variants on GLUT-4 endocytosis in 3T3L1 adipocytes

2000 ◽  
Vol 278 (5) ◽  
pp. E825-E831 ◽  
Author(s):  
Aimee W. Kao ◽  
Chunmei Yang ◽  
Jeffrey E. Pessin
Keyword(s):  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Splice Variants ◽  
Wild Type ◽  
Cell Cytoplasm ◽  
Functional Specificity ◽  
Glut 4 ◽  
Green Fluorescent ◽  
Dynamin 2

Previously, we reported that expression of a dominant-interfering neuronal-specific dynamin 1 (K44A/dynamin 1) inhibited the plasma membrane internalization of GLUT-4 in 3T3L1 adipocytes (15). To investigate the role of the ubiquitously expressed isoform of dynamin, dynamin 2, on adipocyte GLUT-4 internalization, and to determine whether dynamin splice variants have functional specificity, we expressed each of the four dynamin 2 isoforms (aa, ab, ba, and bb) as either wild-type proteins or GTPase-defective mutants. When expressed as enhanced green fluorescent protein (EGFP) fusions, these isoforms were found to have overlapping subcellular distributions being localized throughout the cell cytoplasm, on punctate vesicles and in a perinuclear compartment. This distribution was qualitatively similar to that of endogenous dynamin 2 and overlapped with GLUT-4 in the basal state. Expression of wild-type dynamin 2 isoforms had no effect on the basal or insulin-stimulated distribution of GLUT-4; however, expression of the dominant-interfering dynamin 2 mutants inhibited GLUT-4 endocytosis. These data demonstrate that dynamin 2 is required for GLUT-4 endocytosis in 3T3L1 adipocytes and suggest that, relative to GLUT-4 trafficking, the dynamin 2 splice variants have overlapping functions and are probably not responsible for mediating distinct GLUT-4 budding events.

scholarly journals Spermine analogue-regulated expression of spermidine/spermine N1-acetyltransferase and its effects on depletion of intracellular polyamine pools in mouse fetal fibroblasts

2009 ◽  
Vol 422 (1) ◽  
pp. 101-109 ◽  
Author(s):  
Anne Uimari ◽  
Tuomo A. Keinänen ◽  
Anne Karppinen ◽  
Patrick Woster ◽  
Pekka Uimari ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fusion Gene ◽  
Wild Type Mouse ◽  
Egfp Expression ◽  
Wild Type ◽  
Protein Fusion ◽  
Reverse Transcription Pcr ◽  
Fetal Fibroblasts ◽  
Spermine Analogues ◽  
Ssat Activity

SSAT (Spermidine/spermine N1-acetyltransferase, also known as SAT1), the key enzyme in the catabolism of polyamines, is turned over rapidly and there is only a low amount present in the cell. In the present study, the regulation of SSAT by spermine analogues, the inducers of the enzyme, was studied in wild-type mouse fetal fibroblasts, expressing endogenous SSAT, and in the SSAT-deficient mouse fetal fibroblasts transiently expressing an SSAT–EGFP (enhanced green fluorescent protein) fusion gene. In both cell lines treatments with DENSpm (N1,N11-diethylnorspermine), CPENSpm (N1-ethyl-N11-[(cyclopropyl)-methy]-4,8-diazaundecane) and CHENSpm (N1-ethyl-N11-[(cycloheptyl)methy]-4,8-diazaundecane) led to high, moderate or low induction of SSAT activity respectively. The level of activity detected correlated with the presence of SSAT and SSAT–EGFP proteins, the latter localizing both in the cytoplasm and nucleus. RT–PCR (reverse transcription–PCR) results suggested that the analogue-affected regulation of SSAT–EGFP expression occurred, mainly, after transcription. In wild-type cells, DENSpm increased the amount of SSAT mRNA, and both DENSpm and CHENSpm affected splicing of the SSAT pre-mRNA. Depleted intracellular spermidine and spermine levels inversely correlated with detected SSAT activity. Interestingly, the analogues also reduced polyamine levels in the SSAT-deficient cells expressing the EGFP control. The results from the present study show that the distinct SSAT regulation by different analogues involves regulatory actions at multiple levels, and that the spermine analogues, in addition to inducing SSAT, lower intracellular polyamine pools by SSAT-independent mechanisms.

scholarly journals The hetF Gene Product Is Essential to Heterocyst Differentiation and Affects HetR Function in the Cyanobacterium Nostoc punctiforme

2001 ◽  
Vol 183 (8) ◽  
pp. 2654-2661 ◽  
Author(s):  
Francis C. Y. Wong ◽  
John C. Meeks
Keyword(s):  
Fluorescent Protein ◽  
Filamentous Cyanobacterium ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
Nostoc Punctiforme ◽  
Combined Nitrogen ◽  
In The Wild ◽  
Step Down ◽  
Green Fluorescent ◽  
Heterocyst Development

ABSTRACT A novel gene, hetF, was identified as essential for heterocyst development in the filamentous cyanobacterium Nostoc punctiforme strain ATCC 29133. In the absence of combined nitrogen, hetF mutants were unable to differentiate heterocysts, whereas extra copies of hetF intrans induced the formation of clusters of heterocysts. Sequences hybridizing to a hetF probe were detected only in heterocyst-forming cyanobacteria. The inactivation and multicopy effects of hetF were similar to those of hetR, which encodes a self-degrading serine protease thought to be a central regulator of heterocyst development. Increased transcription ofhetR begins in developing cells 3 to 6 h after deprivation for combined nitrogen (N step-down), and the HetR protein specifically accumulates in heterocysts. In the hetFmutant, this increase in hetR transcription was delayed, and a hetR promoter::green fluorescent protein (GFP) transcriptional reporter indicated that increased transcription of hetR occurred in all cells rather than only in developing heterocysts. When a fully functional HetR-GFP fusion protein was expressed in the hetF mutant from a multicopy plasmid, HetR-GFP accumulated nonspecifically in all cells under nitrogen-replete conditions; when expressed in the wild type, HetR-GFP was observed only in heterocysts after N step-down. HetF therefore appears to cooperate with HetR in a positive regulatory pathway and may be required for the increased transcription of hetR and localization of the HetR protein in differentiating heterocysts.

Enhanced sodium-dependent extrusion of magnesium in mutant cells established from a mouse renal tubular cell line

2005 ◽  
Vol 289 (4) ◽  
pp. F742-F748 ◽  
Author(s):  
Masaru Watanabe ◽  
Masato Konishi ◽  
Ichiro Ohkido ◽  
Senya Matsufuji
Keyword(s):  
Culture Media ◽  
Cell Types ◽  
Renal Tubular Cell ◽  
Wild Type ◽  
Tubular Cells ◽  
Renal Tubular Cells ◽  
In The Wild ◽  
Renal Tubular ◽  
Mutant Cells ◽  
Very High

To study the regulatory mechanisms of intracellular Mg2+ concentration ([Mg2+]i) in renal tubular cells as well as in other cell types, we established a mutant strain of mouse renal cortical tubular cells that can grow in culture media with very high extracellular Mg2+ concentrations ([Mg2+]o > 100 mM: 101Mg-tolerant cells). [Mg2+]i was measured with a fluorescent indicator furaptra (mag-fura 2) in wild-type and 101Mg-tolerant cells. The average level of [Mg2+]i in the 101Mg-tolerant cells was kept lower than that in the wild-type cells either at 51 mM or 1 mM [Mg2+]o. When [Mg2+]o was lowered from 51 to 1 mM, the decrease in [Mg2+]i was significantly faster in the 101Mg-tolerant cells than in the wild-type cells. These differences between the 101Mg-tolerant cells and the wild-type cells were abolished in the absence of extracellular Na+ or in the presence of imipramine, a known inhibitor of Na+/Mg2+ exchange. We conclude that Na+-dependent Mg2+ transport activity is enhanced in the 101Mg-tolerant cells. The enhanced Mg2+ extrusion may prevent [Mg2+]i increase to higher levels and may be responsible for the Mg2+ tolerance.

scholarly journals Reciprocal Regulation between SigK and Differentiation Programs in Streptomyces coelicolor

2009 ◽  
Vol 191 (21) ◽  
pp. 6473-6481 ◽  
Author(s):  
Xu-Ming Mao ◽  
Zhan Zhou ◽  
Xiao-Ping Hou ◽  
Wen-Jun Guan ◽  
Yong-Quan Li
Keyword(s):  
Fluorescent Protein ◽  
Streptomyces Coelicolor ◽  
Wild Type ◽  
Inhibitory Effects ◽  
Reciprocal Regulation ◽  
Enhanced Production ◽  
In The Wild ◽  
Negative Role ◽  
Green Fluorescent ◽  
Similar Dynamic

ABSTRACT Here we reported that deletion of SigK (SCO6520), a sigma factor in Streptomyces coelicolor, caused an earlier switch from vegetative mycelia to aerial mycelia and higher expression of chpE and chpH than that in the wild type. Loss of SigK also resulted in accelerated and enhanced production of antibiotics, actinorhodin, and undecylprodigiosin and increased expression of actII-orf4 and redD. These results suggested that SigK had a negative role in morphological transition and secondary metabolism. Furthermore, the sigK promoter (sigKp) activity gradually increased and sigK expression was partially dependent on SigK, but this dependence decreased during the developmental course of substrate mycelia. Meanwhile, two potentially nonspecific cleavages occurred between SigK and green fluorescent protein, and the SigK fusion proteins expressed under the constitutive promoter ermEp* sharply decreased and disappeared when aerial mycelia emerged. If expressed under sigKp, 3FLAG-SigK showed similar dynamic patterns but did not decrease as sharply as SigK expressed under ermEp*. These data suggested that the climbing expression of sigK might reduce the prompt degradation of SigK during vegetative hypha development for the proper timing of morphogenesis and that SigK vanished to remove the block for the emergence of aerial mycelia. Thus, we proposed that SigK had inhibitory roles on developmental events and that these inhibitory effects may be released by SigK degradation.

The effect of pressure on the excited-state proton transfer in the wild-type green fluorescent protein

2008 ◽  
Vol 455 (4-6) ◽  
pp. 303-306 ◽  
Author(s):  
Pavel Leiderman ◽  
Dan Huppert ◽  
S. James Remington ◽  
Laren M. Tolbert ◽  
Kyril M. Solntsev
Keyword(s):  
Excited State ◽  
Green Fluorescent Protein ◽  
Proton Transfer ◽  
Fluorescent Protein ◽  
Wild Type ◽  
Effect Of Pressure ◽  
Excited State Proton Transfer ◽  
In The Wild ◽  
Green Fluorescent

Arabidopsis gynoecium structure in the wild and in ettin mutants

Development ◽  
1995 ◽  
Vol 121 (5) ◽  
pp. 1519-1532 ◽  
Author(s):  
R.A. Sessions ◽  
P.C. Zambryski
Keyword(s):  
Cell Types ◽  
Flowering Plants ◽  
Wild Type ◽  
Reproductive Structure ◽  
Developmental Abnormalities ◽  
Transmitting Tract ◽  
Type Pattern ◽  
In The Wild ◽  
Gynoecium Development ◽  
Scanning Electron

The gynoecium is the female reproductive structure of flowering plants. Here we present a description of the Arabidopsis thaliana gynoecium at anthesis. The cylindrical organ can be broken down into three longitudinal regions arranged in an apical-basal order: stigma, style, and ovary. Each region can be distinguished histologically and morphologically. The transmitting (pollination) tract is axially positioned along the center of the gynoecium and spans stigma, style and ovary. Histochemistry, scanning electron microscopy and a style-specific reporter gene are used to compare the wild-type pattern of gynoecium cell types and regions, with patterns formed in gynoecia of individuals homozygous for a series of allelic mutations at the ETTIN locus. ettin gynoecia show morphological and histological alterations that appear to result from the merging of apical and basal regions and the development of abaxial into adaxial tissues. These developmental abnormalities result in a reduction of the ovary region, an expansion of the stylar and stigmatic regions, and the abaxial (outward) proliferation of transmitting tract tissue. The alterations in the mutants can be interpreted as resulting from misspecifications of position along the longitudinal and transverse axes during gynoecium development. The results suggest that early patterning events underlie wild-type gynoecium development, and that ETT functions during this early programming.

scholarly journals HE6/GPR64 adhesion receptor co-localizes with apical and subapical F-actin scaffold in male excurrent duct epithelia

Reproduction ◽  
2008 ◽  
Vol 136 (2) ◽  
pp. 235-245 ◽  
Author(s):  
Christiane Kirchhoff ◽  
Caroline Osterhoff ◽  
Annemarie Samalecos
Keyword(s):  
Wild Type Mouse ◽  
Cell Types ◽  
Receptor Protein ◽  
Wild Type ◽  
Ductuli Efferentes ◽  
Apical Compartment ◽  
Tubulin Antibodies ◽  
Subcellular Domains ◽  
Human And Mouse ◽  
Monospecific Antibodies

A role for HE6/GPR64 in male excurrent ducts in the regulation of water balance was suggested from targeted gene mutation in the mouse. Results of the present immunolocalization study strengthen this hypothesis. Employing monospecific antibodies and laser confocal microscopy, we studied the localization of the receptor protein in the human and wild-type mouse ductuli efferentes and epididymis. We show that HE6/GPR64 is specifically associated with cell types and subcellular domains involved in the process of fluid reabsorption. In the mouse, dual labelling with anti-tubulin antibodies revealed that HE6/GPR64 was absent from the (kino-) cilia of ciliated cells. Instead, the receptor protein accumulated in the non-ciliated principal cells. Specifically, strong immunofluorescence was observed in the apical compartment of these cells. Dual labelling with phalloidin and anti-ezrin antibodies revealed that in the mouse the bulk amount of HE6/GPR64 protein co-localized with the F-actin–ezrin scaffold in brush border-like microvilli of ductuli efferentes and long stereocilia of the epididymis proper. In the ductuli efferentes, HE6/GPR64 also co-localized with the subapical F-actin network immediately below the microvilli. Comparable immunostaining patterns were observed in human and mouse; however, a specific feature of the human ductuli efferentes was an intense HE6/GPR64-related labelling of crypt-like grooves or furrows of hitherto unknown function.

scholarly journals Cell-Matrix Adhesions Differentially Regulate Fascin Phosphorylation

1999 ◽  
Vol 10 (12) ◽  
pp. 4177-4190 ◽  
Author(s):  
Josephine C. Adams ◽  
James D. Clelland ◽  
Georgina D.M. Collett ◽  
Fumio Matsumura ◽  
Shigeko Yamashiro ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Fluorescent Protein ◽  
Cell Types ◽  
C2c12 Cells ◽  
Actin Binding ◽  
Wild Type ◽  
Inhibitory Antibody ◽  
Thrombospondin 1 ◽  
Laminin 1 ◽  
In Cells

Cell adhesion to individual macromolecules of the extracellular matrix has dramatic effects on the subcellular localization of the actin-bundling protein fascin and on the ability of cells to form stable fascin microspikes. The actin-binding activity of fascin is down-regulated by phosphorylation, and we used two differentiated cell types, C2C12 skeletal myoblasts and LLC-PK1 kidney epithelial cells, to examine the hypothesis that cell adhesion to the matrix components fibronectin, laminin-1, and thrombospondin-1 differentially regulates fascin phosphorylation. In both cell types, treatment with the PKC activator 12-tetradecanoyl phorbol 13-acetate (TPA) or adhesion to fibronectin led to a diffuse distribution of fascin after 1 h. C2C12 cells contain the PKC family members α, γ, and λ, and PKCα localization was altered upon cell adhesion to fibronectin. Two-dimensional isoelectric focusing/SDS-polyacrylamide gels were used to determine that fascin became phosphorylated in cells adherent to fibronectin and was inhibited by the PKC inhibitors calphostin C and chelerythrine chloride. Phosphorylation of fascin was not detected in cells adherent to thrombospondin-1 or to laminin-1. LLC-PK1 cells expressing green fluorescent protein (GFP)-fascin also displayed similar regulation of fascin phosphorylation. LLC-PK1 cells expressing GFP-fascin S39A, a nonphosphorylatable mutant, did not undergo spreading and focal contact organization on fibronectin, whereas cells expressing a GFP-fascin S39D mutant with constitutive negative charge spread more extensively than wild-type cells. In contrast, C2C12 cells coexpressing S39A fascin with endogenous fascin remained competent to form microspikes on thrombospondin-1, and cells that expressed fascin S39D attached to thrombospondin-1 but did not form microspikes. Blockade of PKCα activity by TPA-induced down-regulation led to actin association of wild-type fascin in fibronectin-adherent C2C12 and LLC-PK1 cells but did not alter the distribution of S39A or S39D fascins. The association of fascin with actin in fibronectin-adherent cells was also evident in the presence of an inhibitory antibody to integrin α5 subunit. These novel results establish matrix-initiated PKC-dependent regulation of fascin phosphorylation at serine 39 as a mechanism whereby matrix adhesion is coupled to the organization of cytoskeletal structure.

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