Search results for "rase"

RPGR ORF15 isoform co-localizes with RPGRIP1 at centrioles and basal bodies and interacts with nucleophosmin

2005

The ORF15 isoform of RPGR (RPGR(ORF15)) and RPGR interacting protein 1 (RPGRIP1) are mutated in a variety of retinal dystrophies but their functions are poorly understood. Here, we show that in cultured mammalian cells both RPGR(ORF15) and RPGRIP1 localize to centrioles. These localizations are resistant to the microtubule destabilizing drug nocodazole and persist throughout the cell cycle. RPGR and RPGRIP1 also co-localize at basal bodies in cells with primary cilia. The C-terminal (C2) domain of RPGR(ORF15) (ORF15(C2)) is highly conserved across 13 mammalian species, suggesting that it is a functionally important domain. Using matrix-assisted laser desorption ionization time-of-flight mas…

Production of ceramides causes apoptosis during early neural differentiation in vitro.

2000

To investigate signal transduction pathways leading to apoptosis during the early phase of neurogenesis, we employed PCC7-Mz1 cells, which cease to proliferate and begin to differentiate into a stable pattern of neurons, astroglial cells, and fibroblasts upon incubation with retinoic acid (RA). As part of lineage determination, a sizable fraction of RA-treated cultures die by apoptosis. Applying natural long-chain C(16)-ceramides as well as membrane-permeable C(2)/C(6)-ceramide analogs caused apoptosis, whereas the biologically nonactive C(2)-dihydroceramide did not. Treating PCC7-Mz1 stem cells with a neutral sphingomyelinase or with the ceramidase inhibitor N-oleoylethanolamine elevated t…

Ceramide Mediates Acute Oxygen Sensing in Vascular Tissues

2014

AbstractAims: A variety of vessels, such as resistance pulmonary arteries (PA) and fetoplacental arteries and the ductus arteriosus (DA) are specialized in sensing and responding to changes in oxygen tension. Despite opposite stimuli, normoxic DA contraction and hypoxic fetoplacental and PA vasoconstriction share some mechanistic features. Activation of neutral sphingomyelinase (nSMase) and subsequent ceramide production has been involved in hypoxic pulmonary vasoconstriction (HPV). Herein we aimed to study the possible role of nSMase-derived ceramide as a common factor in the acute oxygen-sensing function of specialized vascular tissues. Results: The nSMase inhibitor GW4869 and an anticera…

Streptolysin O-permeabilized granulocytes shed L-selectin concomitantly with ceramide generation via neutral sphingomyelinase

2000

Abstract Cleavage of membrane-associated L-selectin regulates leukocyte rolling on vascular endothelium at sites of inflammation. We report that rapid and massive shedding of L-selectin occurs from granulocytes attacked by the pore-forming bacterial toxin streptolysin O (SLO). Shedding was not induced by an SLO mutant that retained binding capacity but lacked pore-forming activity. Cells permeabilized with SLO exhibited a 1.5-fold increase in the activity of neutral sphingomyelinase, which was accompanied by increased ceramide formation. L-selectin cleavage was inducible by treatment of cells with bacterial sphingomyelinase, and also through exogenous application of a cell-permeable ceramid…

The angiopoietin-Tie2 pathway regulates Purkinje cell dendritic morphogenesis in a cell-autonomous manner.

2021

Neuro-vascular communication is essential to synchronize central nervous system development. Here, we identify angiopoietin/Tie2 as a neuro-vascular signaling axis involved in regulating dendritic morphogenesis of Purkinje cells (PCs). We show that in the developing cerebellum Tie2 expression is not restricted to blood vessels, but it is also present in PCs. Its ligands angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) are expressed in neural cells and endothelial cells (ECs), respectively. PC-specific deletion of Tie2 results in reduced dendritic arborization, which is recapitulated in neural-specific Ang1-knockout and Ang2 full-knockout mice. Mechanistically, RNA sequencing reveals that Tie…

Aspartate aminotransferase in brain tissue cultures

1969

Quantification of GABA(A) receptor subunit mRNAs by non-radioisotopic competitive RT-PCR utilizing plate-based EIA methodology.

2000

We developed a non-radioisotopic quantitative competitive RT-PCR method for the measurement of gamma-aminobutyric acid (GABA) type A receptor subunit mRNA levels. The specificity of the method was optimized by the use of four subunit-specific oligonucleotides in the sequential steps: reverse transcription, polymerase chain reaction (PCR), and detection. The biotinylated PCR products were bound on streptavidin-coated microtiter plates allowing detection of the products using dinitrophenyl (DNP)-labeled probes and anti-DNP alkaline phosphatase conjugate. The method was set up for the six major cerebellar GABA(A) receptor subunits: alpha1; alpha6; beta2; beta3; gamma2 and delta. The method is …

Comparative computational analysis of different active site conformations and substrates in a chalcone isomerase catalyzed reaction.

2006

Chalcone isomerase catalyzes the transformation of chalcones to flavanones. We present a computational study of the rate-limiting chemical step, an intramolecular Michael addition of a 2'-oxyanion to the alpha,beta-double bound. By using quantum mechanical/molecular mechanical hybrid methods we traced the free-energy profiles associated with the reaction of two different substrates (chalcone and 6'-deoxychalcone) in two different conformations of the active site that are described in the different crystallographic structures available. We have obtained significant differences (about 4 kcal/mol) in the free-energy barriers calculated for the two active sites. According to our results, the ac…

Enzymatic effects on reactant and transition states. The case of chalcone isomerase.

2007

Chalcone isomerase catalyzes the transformation of chalcone to naringerin as a part of flavonoid biosynthetic pathways. The global reaction takes place through a conformational change of the substrate followed by chemical reaction, being thus an excellent example to analyze current theories about enzyme catalysis. We here present a detailed theoretical study of the enzymatic action on the conformational pre-equilibria and on the chemical steps for two different substrates of this enzyme. Free-energy profiles are obtained in terms of potentials of mean force using hybrid quantum mechanics/molecular mechanics potentials. The role of the enzyme becomes clear when compared to the counterpart eq…

A Novel Strategy to Study Electrostatic Effects in Chemical Reactions: Differences between the Role of Solvent and the Active Site of Chalcone Isomer…

2015

The electrostatic behavior of active site residues in enzyme catalysis is quite different from that of water molecules in solution. To highlight the electrostatic differences between both environments, we propose a QM/MM strategy to study the role of the environment in chemical reactions. The novelty of the present communication is that free energy surfaces are generated by means of two distinguished reaction coordinates: a solute coordinate and the electrostatic potential created by the environment. This is applied to analyze the origin of catalysis in the transformation of a chalcone into a flavanone, a Michael addition that requires the desolvation of the nucleophile.