Green tea polyphenol-reduced graphene oxide: derivatisation, reduction efficiency, reduction mechanism and cytotoxicity

This paper reports on the derivatisation, reduction efficiency, reduction mechanism and cytotoxicity of green tea polyphenol-reduced graphene oxide (GTP-RGO). The reduction of graphene oxide (GO) at 90°C using a weight ratio (WR) of GTP/GO=1 resulted in the production of a stable GTP-RGO dispersion in aqueous media, as indicated by the results of ultravioletvisible (UV-Vis) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, thermogravimetric analysis (TGA) and the measurement of zeta potential and electrophoretic mobility. In addition, the results from UV-Vis spectroscopy and X-ray photoelectron spectroscopy (XPS) analysis indicated the comparable reduction ability of GTP relative to the standard reducing agent, hydrazine (N2H4). The removal mechanism of epoxy group from GO via reduction reaction with GTP was investigated by implementing hybrid functional method of Becke-3-parameters-Lee-Yang-Parr (B3LYP)using Gaussian 09 software. The energy and frequency calculations showed that the GO reduction using GTP was more spontaneous and relatively took place faster than the reduction using N2H4, as evidenced by higher entropy change (ΔS) (0.039 kcal/mol·K) and lower Gibbs free energy (ΔG) barrier (58.880 kcal/mol).The cytotoxicities of GO and GTP-RGO samples were evaluated against human colonic fibroblasts cells (CCD-18Co). The GO sample was determined to be toxic even at low concentration (6.25 μg/mL), while the GTP-RGO sample possesses notably low toxicity at the same concentration. The cell culture experiments revealed that the incorporation of GTP led to a decrease in the toxicity of GTP-RGO samples

Persistent activation of transducin by bleached rhodopsin in salamander rods.

The hydrolysis-resistant GTP analogue GTP-gamma-S was introduced into rods isolated from the retina of the salamander Ambystoma tigrinum to study the origin of the persistent excitation induced by intense bleaching illumination. Dialysis of a dark-adapted rod with a whole-cell patch pipette containing 2 mM GTP-gamma-S resulted in a gradual decrease in circulating current. If the rod was first bleached and its sensitivity allowed to stabilize for at least 30 min, then dialysis with GTP-gamma-S produced a much faster current decay. The circulating current could be restored by superfusion with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, suggesting that the decay in current originated from persistent excitation of the phosphodiesterase by transducin bound to GTP-gamma-S. We conclude that the persistent excitation which follows bleaching is likely to involve the GTP-binding protein transducin, which mediates the normal photoresponse. This observation suggests that a form of rhodopsin which persists long after bleaching can activate transducin much as does photoisomerized rhodopsin, although with considerably lower gain

A guanosine 5′-triphosphate-dependent protein kinase is localized in the outer envelope membrane of pea chloroplasts

A guanosine 5-triphosphate (GTP)-dependent protein kinase was detected in preparations of outer chloroplast envelope membranes of pea (Pisum sativum L.) chloroplasts. The protein-kinase activity was capable of phosphorylating several envelope-membrane proteins. The major phosphorylated products were 23- and 32.5-kilo-dalton proteins of the outer envelope membrane. Several other envelope proteins were labeled to a lesser extent. Following acid hydrolysis of the labeled proteins, most of the label was detected as phosphoserine with only minor amounts detected as phosphothreonine. Several criteria were used to distinguish the GTP-dependent protein kinase from an ATP-dependent kinase also present in the outer envelope membrane. The ATP-dependent kinase phosphorylated a very different set of envelope-membrane proteins. Heparin inhibited the GTP-dependent kinase but had little effect upon the ATP-dependent enzyme. The GTP-dependent enzyme accepted phosvitin as an external protein substrate whereas the ATP-dependent enzyme did not. The outer membrane of the chloroplast envelope also contained a phosphotransferase capable of transferring labeled phosphate from [-32P]GTP to ADP to yield (-32P]ATP. Consequently, addition of ADP to a GTP-dependent protein-kinase assay resulted in a switch in the pattern of labeled products from that seen with GTP to that typically seen with ATP

Effect of GTP and Ca2+ on inositol 1,4,5-trisphosphate induced Ca2+ release from permeabilized rat exocrine pancreatic acinar cells

The effects of Ca2+ and GTP on the release of Ca2+ from the inositol 1,4,5-trisphosphate (IP3) sensitive Ca2+ compartment were investigated with digitonin permeabilized rat pancreatic acinar cells. The amount of Ca2+ released due to IP3 directly correlated with the amount of stored Ca2+ and was found to be inversely proportional to the medium free Ca2+ concentration. Ca2+ release induced by 0.18 μM IP3 was half maximally inhibited at 0.5 μM free Ca2+, i.e. at concentrations observed in the cytosol of pancreatic acinar cells. GTP did not cause Ca2+ release on its own, but a single addition of GTP (20 μM) abolished the apparent desensitization of the Ca2+ release which was observed during repeated IP3 applications. This effect of GTP was reversible. GTPγS could not replace GTP. Desensitization still occurred when GTPγS was added prior to GTP. The reported data indicate that GTP, stored Ca2+ and cytosolic free Ca2+ modulate the IP3 induced Ca2+ release. EGTA, Ethylene-glycol-bis (2-aminoethylether)-N,N,N′,N′- tetra acetic acid; GTPγS, Guanosine 5′-O-[3-thio]triphosphate; GDPβS, Guanosine 5′-O-[2-thio]diphosphate; IP3, Inositol 1,4,5-trisphosphate; IP2, Inositol 1,4-bisphosphate; IP4, Inositol 1,3,4,5-tetrakisphosphate; MOPS, Morpholinopropane sulfonic acid; HEPES, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid; pHMB, Parahydroxymercuribenzoat

The chloroplast import receptor Toc34 functions as preprotein-regulated GTPase

Toc34 is a protein of the chloroplast outer envelope membrane that acts as receptor for preproteins containing a transit sequence. The recognition of preproteins by Toc34 is regulated by GTP binding and phosphorylation. The phosphorylation site of Toc34 is located at serine 113, close to the postulated triphosphate binding site. This can explain the down-regulation of Toc34 by phosphorylation, resulting in the loss of GTP binding. Vice versa, GTP but not GDP binding of Toc34 influences the phosphorylation. The nucleotide specificity of Toc34 is not only determined by the classical nucleotide binding domains but by a non-typical region at the N-terminus of the protein. As a result, the GTP binding properties are unusual, since the triphosphate moiety of GTP is bound with higher affinity than the purine base. Purified Toc34 hydrolyses GTP at a low rate, which could regulate the receptor function. The rate of hydrolysis is greatly stimulated by a precursor protein

Dual positive and negative regulation of GPCR signaling by GTP hydrolysis

G protein-coupled receptors (GPCRs) regulate a variety of intracellular pathways through their ability to promote the binding of GTP to heterotrimeric G proteins. Regulator of G protein signaling (RGS) proteins increase the intrinsic GTPase activity of G-subunits and are widely regarded as negative regulators of G protein signaling. Using yeast we demonstrate that GTP hydrolysis is not only required for desensitization, but is essential for achieving a high maximal (saturated level) response. Thus RGS-mediated GTP hydrolysis acts as both a negative (low stimulation) and positive (high stimulation) regulator of signaling. To account for this we generated a new kinetic model of the G protein cycle where GGTP enters an inactive GTP-bound state following effector activation. Furthermore, in vivo and in silico experimentation demonstrates that maximum signaling output first increases and then decreases with RGS concentration. This unimodal, non-monotone dependence on RGS concentration is novel. Analysis of the kinetic model has revealed a dynamic network motif that shows precisely how inclusion of the inactive GTP-bound state for the G produces this unimodal relationship

Reciprocal regulation of PKA and rac signaling

Activated G protein-coupled receptors (GPCRs) and receptor tyrosine kinases relay extracellular signals through spatial and temporal controlled kinase and GTPase entities. These enzymes are coordinated by multifunctional scaffolding proteins for precise intracellular signal processing. The cAMP-dependent protein kinase A (PKA) is the prime example for compartmentalized signal transmission downstream of distinct GPCRs. A-kinase anchoring proteins tether PKA to specific intracellular sites to ensure precision and directionality of PKA phosphorylation events. Here, we show that the Rho-GTPase Rac contains A-kinase anchoring protein properties and forms a dynamic cellular protein complex with PKA. The formation of this transient core complex depends on binary interactions with PKA subunits, cAMP levels and cellular GTP-loading accounting for bidirectional consequences on PKA and Rac downstream signaling. We show that GTP-Rac stabilizes the inactive PKA holoenzyme. However, β-adrenergic receptor-mediated activation of GTP-Rac–bound PKA routes signals to the Raf-Mek-Erk cascade, which is critically implicated in cell proliferation. We describe a further mechanism of how cAMP enhances nuclear Erk1/2 signaling: It emanates from transphosphorylation of p21-activated kinases in their evolutionary conserved kinase-activation loop through GTP-Rac compartmentalized PKA activities. Sole transphosphorylation of p21-activated kinases is not sufficient to activate Erk1/2. It requires complex formation of both kinases with GTP-Rac1 to unleash cAMP-PKA–boosted activation of Raf-Mek-Erk. Consequently GTP-Rac functions as a dual kinase-tuning scaffold that favors the PKA holoenzyme and contributes to potentiate Erk1/2 signaling. Our findings offer additional mechanistic insights how β-adrenergic receptor-controlled PKA activities enhance GTP-Rac–mediated activation of nuclear Erk1/2 signaling

Revisiting the microtubule based quantum models of mind: tubulin bound GTP cannot pump microtubule coherence or provide energy for alpha <-> beta computation in stable microtubules

The current paper investigates the biological models of stable brain microtubules as quantum or classical computers whose function is based on electron hopping associated with kinking of the tubulin dimer. Hameroff (1998a, 1998b, 2003a, 2003b), Tuszynski et al. (1998), Hagan et al. (2000), Mershin et al. (1999); Mershin (2003) suppose that the energy needed could be somehow delivered via guanosine diphosphate (GDP) exchange for guanosine triphosphate (GTP) or via cycles of tubulin bound GTP hydrolysis. Here is presented biological and structural data from electron diffraction studies performed by Lowe et al. (2001) and computer simulation with MDL ® Chime Version 2.6 SP4, explaining and visualizing the inconsistency of the proposed tubulin bit (qubit) GTP energized alpha <-> beta computation and/or tubulin bound GTP pumped coherence in stable microtubules