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Green tea polyphenols (GTP) have been shown to ameliorate lipid metabolic disorders by regulating intestinal bacteria. Given the significant role of intestinal bacteriophages in shaping the gut microbiota, this study investigates GTP's influence on gut bacteriophage-bacteria interactions and lipid metabolism using metagenomics and metabonomics. The research results indicated that GTP significantly reduced body weight, serum triglycerides, leptin, insulin resistance, interleukin-6, and TNF-α levels while increasing adiponectin in ob/ob mice fed high-fat diet, aiding intestinal repair. GTP improved gut health by decreasing Enterobacter , Siphoviridae and Enterobacteria _phage_sfv, increasing Bifidobacterium and intestinal metabolites SCFA and hippuric acid. Correlation analysis showed negative correlations between Enterobacter sp. 50,588,862 and Enterobacteria _phages, Shigella _phages with 4-hydroxyphenylpyruvate and hippuric acid. Bifidobacterium choerinum and Bifidobacterium sp. AGR2158 were positively correlated with fatty acids and bile acids. In conclusion, GTP reduced fat accumulation and inflammation, enhanced gut barrier function in obese mice, closely associated with changes in the gut bacteriophage community. [Display omitted] • GTP's impact on gut bacteria and phages, altering lipid metabolism in ob/ob mice. • Used bioinformatics to link gut health biomarkers with differential microorganism. • Found potential targets for treating metabolic issues in obesity. [ABSTRACT FROM AUTHOR]

Genetic or pharmacological inhibition of enzymes involved in GTP biosynthesis has substantial biological effects, underlining the need to better understand the function of GTP levels in regulation of cellular processes and the significance of targeting GTP biosynthesis enzymes for therapeutic intervention. Our current understanding of spatiotemporal regulation of GTP metabolism and its role in physiological and pathological cellular processes is far from complete. Novel methodologies such as genetically encoded sensors of free GTP offered insights into intracellular distribution and function of GTP molecules. In the current Review, we provide analysis of recent discoveries in the field of GTP metabolism and evaluate the key enzymes as molecular targets. GTP is essential for nuclear acid biosynthesis. In addition, through its effects on G proteins, GTP regulates a large array of cellular processes including protein synthesis, cytoskeleton biogenesis and function, and intracellular signaling. The development of genetically encoded GTP sensors has led to the realization that GTP molecules are not homogenously distributed in the cell but rather form gradients which regulate cellular processes. De novo biosynthesis acts as the primary source of GTP in most cell types. It is regulated via both transcriptional and post-translational mechanisms that are far from being completely elucidated. Inhibitors of the de novo GTP biosynthesis are used in organ transplantation as immunosuppressive drugs and in antiviral therapy. Although targeting GTP biosynthesis is an attractive anticancer strategy, no GTP-depleting agents are approved for cancer treatment. [ABSTRACT FROM AUTHOR]

Over the last decade, cancer immunotherapy has progressed from an academically interesting field to one of the most promising forms of new treatments in which not the cancer but the immune system is treated. In particular, genetic modification for purposeful redirection of autologous T cells is providing hope to many treatment-resistant patients. This personalized form of medicine is radically different from more traditional oncologic drugs. With these evolving medical advancements and more cellular therapies becoming available, some regulatory agencies have created new regulatory requirements to manage the production of these types of products. The regulations are specifically suited for the manufacture of gene and cell therapy products, as they use a risk-based approach towards product development and manufacturing, when there is limited characterization available. The correct interpretation of how and when requirements apply is crucial, since theoretical approaches to implementing GMP can easily lead to disproportionate and unwarranted restrictions that may not address the specific risks that regulators were intending to control. This is especially relevant for cell collection and biopreservation preceding the manufacturing process for products manufactured from autologous T cells. Both the fresh and cryopreserved apheresis materials can be filed as minimally manipulated starting materials to the authorities. The preservation of such cellular material can then routinely be managed using the available regulations for tissues and cells, allowing for a more fit-for-purpose approach to the control measures implemented. [ABSTRACT FROM AUTHOR]

The translation elongation factor 1α (EF1α) protein is a highly conserved G protein that is crucial for protein translation in all eukaryotic organisms. EF1α quickly became insoluble at temperatures 42 °C treatment for 2h in vitro , but generally remained soluble in vivo even after being exposed to temperatures as high as 45 °C for an extended period, which suggests that protective mechanisms exist for keeping EF1α soluble in plant cells under heat stress. EF1α had fast in vivo insolubilization when exposed to 45 °C, resulting in about 40% of the protein aggregating after 9 h. Given its established role in protein translation, heat-induced aggregation is most likely to impact the function of the elongation factor. Overexpression of constitutive mutants in both GTP-bound and GDP-bound forms of EF1α resulted in significantly decreased heat tolerance. These findings provide evidence to support the critical role of EF1α, a thermosensitive protein, in the heat tolerance of plants. • EF1a is highly prone to aggregation under heat stress in vivo and in vitro. • Heat tolerance was significantly increased by overexpression of EF1a. • Plants overexpressing mutant versions of EF1α compromised heat tolerance. • Soluble EF1a is critical to plant heat response. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (M. tuberculosis H 37 Rv) utilizes the signal recognition particle pathway (SRP pathway) system for secretion of various proteins from ribosomes to the extracellular surface which plays an important role in the machinery running inside the bacterium. This system comprises of three major components FtsY, FfH and 4.5S rRNA. This manuscript highlights essential factors responsible for the optimized enzymatic activity of FtsY. Kinetic parameters include V max and K m for the hydrolysis of GTP by ftsY which were 20.25±5.16 μM/min/mg and 39.95±7.7 μM respectively. k cat and catalytic efficiency of the reaction were 0.012±0.003 s−1 and 0.00047±0.0001 μM/s−1 respectively. These values were affected upon changing the standard conditions. Cations (Mg2+ and Mn2+) play important role in FtsY enzymatic activity as increasing Mg2+ decrease the activity. Mn2+on the other hand is required at higher concentration around 60 mM for carrying optimum GTPase activity. FtsY is hydrolyzing ATP and GDP as well and GDP acts as an inhibitor of the reaction. MD simulation shows effective binding and stabilization of the FtsY complexed structure with GTP, GDP and ATP. Mutational analysis was done at two important residues of GTP binding motif of FtsY, namely, GXXXXGK (K236) and DXXG (D367) and showed that these mutations significantly decrease FtsY GTPase activity. FtsY is comprised of α helices, but this structural pattern was shown to change with increasing concentrations of GTP and ATP which symbolize that these ligands cause significant conformational change by variating the secondary structure to transduce signals required by downstream effectors. This binding favors the functional stabilization of FtsY by destabilization of α-helix integrity. Revealing the hidden aspects of the functioning of FtsY might be an essential part for the understanding of the SRP pathway which is one of the important contributors of M. tuberculosis virulence. • SRP pathway involve ftsY as an essential pathway for translocation of co-translational proteins. • FtsY efficiently interacts with GTP and this interaction is necessary for further downstream signaling. • FtsY can also hydrolyze other nucleotides but less efficiently as compare to GTP hydrolysis. • Lysine (K236) and aspartate (D367) residues are important for GTPase activity of ftsY. • GDP acts as a inhibitor of ftsY GTPase activity as increasing GDP concentration decreases ftsY activity. [ABSTRACT FROM AUTHOR]

[Display omitted] • A bipolar hemicyanine cationic probe enables simultaneous sensing of ATP and GTP in a single-step process. • The probe demonstrates a strong affinity to ATP and GTP, facilitated by electrostatic and π-π stacking interactions. • The binding constants for ATP were determined to be 6.15 × 107 M−1 and 1.57 × 106 M−1, while those for GTP were determined to be 3.19 × 107 M−1 and 3.81 × 106 M−1, respectively. • The probe exhibits high sensitivity and specificity towards ATP or GTP, distinguishing them from other biological analogues. • Due to its rapid response rate (within 10 s), the probe is well-suited for monitoring enzymatic reactions related to ATP or GTP. Adenosine 5′-triphosphate (ATP) and guanosine 5′-triphosphate (GTP) are the most essential energy source in enormous biological processes. Various probes for ATP or GTP sensing, have been widely established, but the probe that could simultaneously monitor ATP and GTP is still rarely reported. Herein, we report a bipolar hemicyanine cationic probe for simultaneous sensing of ATP and GTP via a one-step monitoring process. This probe exhibited strong affinity to ATP and GTP through intramolecular electrostatic and π-π stacking interactions, which the binding constant on each step were determined as 6.15 × 107 M−1 and 1.57 × 106 M−1 for ATP, 3.19 × 107 M−1 and 3.81 × 106 M−1 for GTP. The sensitivity and specificity of this probe toward ATP or GTP over other twelve biological analogues (adenosine 5′-diphosphate (ADP), adenosine 5′-monophosphate (AMP), guanosine 5′-diphosphate (GDP), guanosine 5′-monophosphate (GMP), Etc.) have also been successfully demonstrated. Furthermore, due to the rapid response rate (within 10 s), we also proved that this probe could be employed as a monitor tool during the ATP or GTP-related enzymatic reaction process. [ABSTRACT FROM AUTHOR]

Mitochondrial ATP-sensitive K+ channels (mitoKATP) have been recently characterized structurally, and possess a protein through which K+ enters mitochondria (MitoKIR), and a regulatory subunit (mitoSUR). The mitoSUR regulatory subunit is an ATP-binding cassette (ABC) protein isoform 8 (ABCB8). Opening these channels is known to be cardioprotective, but the molecular and physiological mechanisms that activate them are not fully known. Here, to better understand the molecular and physiological mechanisms of activators (GTP) and inhibitors (ATP) on the activity of mitoKATP, we exposed isolated mitochondria to both nucleotides. We also used molecular docking directed to the nucleotide-binding domain of human ABCB8/mitoSUR to test a comparative model of ATP and GTP effects. As expected, we find that ATP dose-dependently inhibits mitoKATP activity (IC 50 = 21.24 ± 1.4 μM). However, simultaneous exposure of mitochondria to GTP dose-dependently (EC 50 = 13.19 ± 1.33 μM) reversed ATP inhibition. Pharmacological and computational studies suggest that GTP reverses ATP activity competitively. Docking directed to the site of crystallized ADP reveals that both nucleotides bind to mitoSUR with high affinity, with their phosphates directed to the Mg2+ ion and the walker A motif of the protein (SGGGKTT). These effects, when combined, result in GTP binding, ATP displacement, mitochondrial ATP-sensitive K+ transport, and lower formation of reactive oxygen species. Overall, our findings demonstrate the basis for ATP and GTP binding in mitoSUR using a combination of biochemical, pharmacological, and computational experiments. Future studies may reveal the extent to which the balance between ATP and GTP actions contributes toward cardioprotection against ischemic events. [Display omitted] • Molecular docking reveals how GTP and ATP interact with the mitoSUR/ABCB8 protein. • ATP dose-dependently inhibits the mitochondrial ATP-sensitive K+ channel. • GTP competes with ATP to stimulate the mitochondrial K+ current through mitoKATP. • GTP activates MitoKATP, resulting in lower mitochondrial H 2 O 2 production. [ABSTRACT FROM AUTHOR]

Summary Mechanistic (or mammalian) target of rapamycin complex 1 (mTORC1) integrates signals from growth factors and nutrients to control biosynthetic processes, including protein, lipid, and nucleic acid synthesis. We find that the mTORC1 pathway is responsive to changes in purine nucleotides in a manner analogous to its sensing of amino acids. Depletion of cellular purines, but not pyrimidines, inhibits mTORC1, and restoration of intracellular adenine nucleotides via addition of exogenous purine nucleobases or nucleosides acutely reactivates mTORC1. Adenylate sensing by mTORC1 is dependent on the tuberous sclerosis complex (TSC) protein complex and its regulation of Rheb upstream of mTORC1, but independent of energy stress and AMP-activated protein kinase (AMPK). Even though mTORC1 signaling is not acutely sensitive to changes in intracellular guanylates, long-term depletion of guanylates decreases Rheb protein levels. Our findings suggest that nucleotide sensing, like amino acid sensing, enables mTORC1 to tightly coordinate nutrient availability with the synthesis of macromolecules, such as protein and nucleic acids, produced from those nutrients. [ABSTRACT FROM AUTHOR]

NAD + catabolism and mitochondrial dynamics are important parts of normal mitochondrial function and are both reported to be disrupted in aging, neurodegenerative diseases, and acute brain injury. While both processes have been extensively studied there has been little reported on how the mechanisms of these two processes are linked. This review focuses on how downstream NAD + catabolism via NUDIX hydrolases affects mitochondrial dynamics under pathologic conditions. Additionally, several potential targets in mitochondrial dysfunction and fragmentation are discussed, including the roles of mitochondrial poly(ADP-ribose) polymerase 1(mtPARP1), AMPK, AMP, and intra-mitochondrial GTP metabolism. Mitochondrial and cytosolic NUDIX hydrolases (NUDT9α and NUDT9β) can affect mitochondrial and cellular AMP levels by hydrolyzing ADP- ribose (ADPr) and subsequently altering the levels of GTP and ATP. Poly (ADP-ribose) polymerase 1 (PARP1) is activated after DNA damage, which depletes NAD + pools and results in the PARylation of nuclear and mitochondrial proteins. In the mitochondria, ADP-ribosyl hydrolase-3 (ARH3) hydrolyzes PAR to ADPr, while NUDT9α metabolizes ADPr to AMP. Elevated AMP levels have been reported to reduce mitochondrial ATP production by inhibiting the adenine nucleotide translocase (ANT), allosterically activating AMPK by altering the cellular AMP: ATP ratio, and by depleting mitochondrial GTP pools by being phosphorylated by adenylate kinase 3 (AK3), which uses GTP as a phosphate donor. Recently, activated AMPK was reported to phosphorylate mitochondria fission factor (MFF), which increases Drp1 localization to the mitochondria and promotes mitochondrial fission. Moreover, the increased AK3 activity could deplete mitochondrial GTP pools and possibly inhibit normal activity of GTP-dependent fusion enzymes, thus altering mitochondrial dynamics. [ABSTRACT FROM AUTHOR]

Summary Pharmacologic agents that interfere with nucleotide metabolism constitute an important class of anticancer agents. Recent studies have demonstrated that mTOR complex 1 (mTORC1) inhibitors suppress de novo biosynthesis of pyrimidine and purine nucleotides. Here, we demonstrate that mTORC1 itself is suppressed by drugs that reduce intracellular purine nucleotide pools. Cellular treatment with AG2037, an inhibitor of the purine biosynthetic enzyme GARFT, profoundly inhibits mTORC1 activity via a reduction in the level of GTP-bound Rheb, an obligate upstream activator of mTORC1, because of a reduction in intracellular guanine nucleotides. AG2037 treatment provokes both mTORC1 inhibition and robust tumor growth suppression in mice bearing non-small-cell lung cancer (NSCLC) xenografts. These results indicate that alterations in purine nucleotide availability affect mTORC1 activity and suggest that inhibition of mTORC1 contributes to the therapeutic effects of purine biosynthesis inhibitors. [ABSTRACT FROM AUTHOR]

This scientifically describes a simple, cost-effective, and sensitive colorimetric method for the quantitative detection of biothiols and Hg2+ based on the peroxidase-like activity of GTP. [Display omitted] • GTP as a peroxidase-like mimic. • There is avoiding the synthesis of nanomaterials. • A facile colorimetric strategy for biothiols and Hg2+ detection has been established. Guanosine-5′-triphosphate (GTP) not only plays a key role in a majority of cellular processes but also be proposed as a peroxidase-like mimic. Compared with nanozymes, GTP shows good tolerance under harsh conditions, which can be used to construct an easy colorimetric analysis for the detection of biomolecules. Here, on the basis of the peroxidase-like activity of GTP which can catalyze the oxidation of 3,3′,5,5′-tetramethyl benzidine dihydrochloride (TMB), colorimetric sensing was established for biothiols and Hg2+. Biothiols reduced the oxTMB back to colorless TMB, and Hg2+ restored the formation of oxTMB, leading to the recovery of color. This method not only provides a platform for the detection of metal ions and biothiols, but also shows that GTP has great potential for analytical detection. [ABSTRACT FROM AUTHOR]

[Display omitted] • SEC-eluted cold tubulin dimers assembled into 1D oligomers and rings but did not hydrolyze GTP. • Tubulin 1D oligomers remained curved also at 36 °C. • Dimers with exposed E-sites exchanged nucleotide 10 times more than dimers with buried E-sites. We investigated how the self-association of isolated tubulin dimers affects the rate of GTP hydrolysis and the equilibrium of nucleotide exchange. Both reactions are relevant for microtubule (MT) dynamics. We used HPLC to determine the concentrations of GDP and GTP and thereby the GTPase activity of SEC-eluted tubulin dimers in assembly buffer solution, free of glycerol and tubulin aggregates. When GTP hydrolysis was negligible, the nucleotide exchange mechanism was studied by determining the concentrations of tubulin-free and tubulin-bound GTP and GDP. We observed no GTP hydrolysis below the critical conditions for MT assembly (either below the critical tubulin concentration and/or at low temperature), despite the assembly of tubulin 1D curved oligomers and single-rings, showing that their assembly did not involve GTP hydrolysis. Under conditions enabling spontaneous slow MT assembly, a slow pseudo-first-order GTP hydrolysis kinetics was detected, limited by the rate of MT assembly. Cryo-TEM images showed that GTP-tubulin 1D oligomers were curved also at 36 °C. Nucleotide exchange depended on the total tubulin concentration and the molar ratio between tubulin-free GDP and GTP. We used a thermodynamic model of isodesmic tubulin self-association, terminated by the formation of tubulin single-rings to determine the molar fractions of dimers with exposed and buried nucleotide exchangeable sites (E-sites). Our analysis shows that the GDP to GTP exchange reaction equilibrium constant was an order-of-magnitude larger for tubulin dimers with exposed E-sites than for assembled dimers with buried E-sites. This conclusion may have implications on the dynamics at the tip of the MT plus end. [ABSTRACT FROM AUTHOR]

Prochilodus nigricans , locally known as curimatã, is an Amazonian commercial fish that endures adverse environmental conditions, in particular low dissolved oxygen, during its migration. Poorer environmental conditions are expected in the near future. Prochilodus nigricans overcomes current seasonal and diurnal changes in dissolved oxygen by adjusting erythrocytic levels of ATP and GTP, modulators of Hb-O 2 affinity. Will this fish species be endangered under more extreme environmental conditions as hypoxia and acidification tend to occur in a shorter period of time? As P. nigricans does not exhibit any apparent morphological alterations to exploit the air–water interface, it must rely on fast adjustments of blood properties. To investigate this aspect, basic hematology indices, pHe, pHi, plasma lactate, erythrocytic levels of ATP and GTP and functional properties of the hemolysate of P. nigricans were analyzed over a period of 6 h in hypoxia and subsequent recovery in normoxia. The levels of erythrocytic GTP were four times higher than ATP and were reduced to ¼ of the original level after 3 h under hypoxia. Erythrocytic levels of ATP were unaffected over the experimental period. All other analyzed blood parameters exhibited a time-course change in animals under hypoxia and returned to normoxic levels. Considering the hemolysate functional properties and the ability to regulate the above mentioned blood characteristics, P. nigricans is able to endure short-term changes in dissolved oxygen. [ABSTRACT FROM AUTHOR]

Unlike most kinases, phosphatidylinositol 5-phosphate 4-kinase β (PI5P4Kβ) utilizes GTP as a physiological phosphate donor and regulates cell growth under stress (i.e., GTP-dependent stress resilience). However, the genesis and evolution of its GTP responsiveness remain unknown. Here, we reveal that PI5P4Kβ has acquired GTP preference by generating a short dual-nucleotide-recognizing motif called the guanine efficient association (GEA) motif. Comparison of nucleobase recognition with 660 kinases and 128 G proteins has uncovered that most kinases and PI5P4Kβ use their main-chain atoms for adenine recognition, while the side-chain atoms are required for guanine recognition. Mutational analysis of the GEA motif revealed that the acquisition of GTP reactivity is accompanied by an extended activity toward inosine triphosphate (ITP) and xanthosine triphosphate (XTP). Along with the evolutionary analysis data that point to strong negative selection of the GEA motif, these results suggest that the GTP responsiveness of PI5P4Kβ has evolved from a compromised trade-off between activity and specificity, underpinning the development of the GTP-dependent stress resilience. [Display omitted] • GTP sensing of PI5P4Kβ is enabled by the dual ATP/GTP-recognizing GEA motif • As in kinases, the main-chain atoms of the GEA motif contribute to adenine binding • Similar to G proteins, the side-chain atoms are required for guanine recognition • We propose an evolutionary path for how PI5P4Kβ became a GTP-sensing kinase PI5P4Kβ is a unique GTP-utilizing kinase that functions as an intracellular GTP sensor. Takeuchi et al. show that GTP binding requires the GTP/ATP dual recognition motif, named GEA, which emerged in a vertebrate. Further analyses reveal that limited substitutions in the GEA motif turned PI5P4Kβ into an unusual GTP-utilizing kinase. [ABSTRACT FROM AUTHOR]

Imidazole-based metal-organic frameworks (MOFs) are easy to prepare as well-dispersed nanoparticles, which have attracted a lot of interest in sensing. Metal substitution is an effective way to regulate the composition and performance of MOFs. Herein, by tuning the contents of Co and Zn, a series of homobimetallic Co x Zn 100-x -ZIF (x = 0–100) were synthesized. Using a fluorescently-labeled DNA oligonucleotide probe, guanosine triphosphate (GTP) can readily displace the adsorbed DNA from Co 50 Zn 50 -ZIF, resulting in over 30-fold fluorescence enhancement with 1 mM GTP. Co 80 Zn 20 -ZIF could specifically recognize adenosine triphosphate (ATP), whereas Co 65 Zn 35 -ZIF and Co 20 Zn 80 -ZIF responded to both ATP and GTP. For comparison, Co 50 Ni 50 -ZIF and Co 50 Cu 50 -ZIF were also prepared, but none of them were selective for any of the molecules, indicating a synergetic effect of cobalt and zinc in Co 50 Zn 50 -ZIF for the selective recognition of GTP. This system can sensitively detect GTP with a detection limit of 0.13 μM. Moreover, based on the varying binding affinities of these CoZn-ZIFs towards different nucleoside triphosphates (NTPs), a ZIF fluorescent sensor array was also designed for the discrimination of the four types of NTPs. [Display omitted] • A series of homobimetallic Co x Zn 100-x -ZIF (x = 0–100) were synthesized, and investigated the interaction between them and different phosphate-containing molecules. • Co 50 Zn 50 -ZIF exhibited excellent recognition ability of GTP. • CoZn-ZIF sensor array was designed for the discrimination of the four types of NTPs. [ABSTRACT FROM AUTHOR]

Abstract: The unstable bonds of ATP (adenosine triphosphate) are investigated in relation to those for the GTP, CTP and UTP. Two existing efficacies Eff A: GTP > UTP > CTP > ATP and Eff B: ATP > UTP > CTP > GTP can be substantiated, respectively, by two least variance inequalities Var A: ΔRGTP >ΔRUTP >ΔRCTP >ΔRATP and Var B: ΔRATP >ΔRUTP >ΔRCTP >ΔRGTP . The verification involves computing the R-integrals and their variances ΔR from the nanometer displacements of the molecules of the S49 wild-type lymphoma cells. They are relevant to the coupling of the β2-adrenoreceptor (β2AR) to Gs protein, expressed as β2AR-Gαs. Application of the least variance principle, Eff A and B is identified with the NTP (nucleoside 5′-triphosphates). They can enhance or inhibit the agonist-stimulated AC (adenylyl cyclase) activity. Eff A is found to be more stable with a longer sustaining time than Eff B. These findings are consistent with the behavior of potentially reactive molecules. Corrective measures of ATP production can be used as an adjustment at the molecular scale to compensate for the unstable character of the ATP molecules. Quantitative assessment of the reactive process of ATP can also provide information on the breakdown of RNA and DNA. A possible approach is to couple the use of energy-release and non-equilibrium thermodynamics with the principle of least variance. [Copyright &y& Elsevier]

Abstract: Evidence is emerging that Foxo family proteins serve as biochemical signal integrators in complex signaling networks mediating and modulating diverse cellular functions. Herein, we report that besides the well-established function of Foxo3a as a transcriptional regulator of multiple target genes in nucleus, a substantial fraction of Foxo3a translocates to mitochondria leading to disruption of mitochondrial membrane potential, release of cytochrome c and caspase activation during erythroid differentiation mediated by guanosine 5′-triphosphate (GTP). In fact, non transcriptional role of Foxo3a in mitochondria was achieved through the protein–protein interaction with pro-apoptotic protein Bax and its translocation to mitochondrial membrane. Furthermore, our results revealed that mitochondrial localization of Foxo3a was modulated by intracellular GTP content which is sensed by PKC signaling element. Collectively, our findings provided insight into a novel Foxo3a mechanism in leukemia cells which led to engagement of cells in the maturation pathway. [Copyright &y& Elsevier]

Abstract: ATP was previously reported to bind to the chaperone α-crystallin resulting in a significant effect on the protein''s ability to suppress the aggregation of a thermally denatured protein. Here, we have investigated the binding of GTP to α-crystallin. Unlike ATP, binding of GTP to α-crystallin did not affect its ability to suppress the aggregation of thermally denatured rhodanese. GTP binding induced a conformational change on α-crystallin, however the degree of exposed hydrophobic surfaces, which are believed to be involved in the binding of the chaperone to denaturing proteins did not change. Here, we report that GTP binds to α-crystallin and this results in a decreased stability of the chaperone as indicated by urea denaturation. [Copyright &y& Elsevier]

Abstract: Time-resolved Fourier transform infrared (FTIR) difference spectroscopy is a valuable tool for monitoring the dynamics of protein reactions and interactions. Absorbance changes can be monitored with time resolutions down to nanoseconds and followed for time periods that range over nine orders of magnitude. Membrane proteins bound to solid supported lipid bilayers can be investigated in near physiological conditions with the attenuated total reflection (ATR) technique. Here, we review the basics of time-resolved FTIR with a focus on Ras, a GTPase that is mutated in 25% of human tumors. We show the first time-resolved measurements of membrane anchored Ras and observed the switching between its activated and its inactivated state. We compared those measurements with measurements of the truncated Ras in solution. We found that both the kinetics and the functional groups involved were very similar. This suggested that the membrane did not have a major influence on the hydrolysis reaction. [Copyright &y& Elsevier]

Abstract: Transglutaminase 2 (TG2) is the most widely distributed member of the transglutaminase family with almost all cell types in the body expressing TG2 to varying extents. In addition to being widely expressed, TG2 is an extremely versatile protein exhibiting transamidating, protein disulphide isomerase and guanine and adenine nucleotide binding and hydrolyzing activities. TG2 can also act as a protein scaffold or linker. This unique protein also undergoes extreme conformational changes and exhibits localization diversity. Being mainly a cytosolic protein; it is also found in the nucleus, associated with the cell membrane (inner and outer side) and with the mitochondria, and also in the extracellular matrix. These different activities, conformations and localization need to be carefully considered while assessing the role of TG2 in physiological and pathological processes. For example, it is becoming evident that the role of TG2 in cell death processes is dependent upon the cell type, stimuli, subcellular localization and conformational state of the protein. In this review we discuss in depth the conformational and functional diversity of TG2 in the context of its role in numerous cellular processes. In particular, we have highlighted how differential localization, conformation and activities of TG2 may distinctly mediate cell death processes. [Copyright &y& Elsevier]

Abstract: The year 2000 radiative forcing (RF) due to changes in O3 and CH4 (and the CH4-induced stratospheric water vapour) as a result of emissions of short-lived gases (oxides of nitrogen (NOx), carbon monoxide and non-methane hydrocarbons) from three transport sectors (ROAD, maritime SHIPping and AIRcraft) are calculated using results from five global atmospheric chemistry models. Using results from these models plus other published data, we quantify the uncertainties. The RF due to short-term O3 changes (i.e. as an immediate response to the emissions without allowing for the long-term CH4 changes) is positive and highest for ROAD transport (31 mW m−2) compared to SHIP (24 mW m−2) and AIR (17 mW m−2) sectors in four of the models. All five models calculate negative RF from the CH4 perturbations, with a larger impact from the SHIP sector than for ROAD and AIR. The net RF of O3 and CH4 combined (i.e. including the impact of CH4 on ozone and stratospheric water vapour) is positive for ROAD (+16(±13) (one standard deviation) mW m−2) and AIR (+6(±5) mW m−2) traffic sectors and is negative for SHIP (−18(±10) mW m−2) sector in all five models. Global Warming Potentials (GWP) and Global Temperature change Potentials (GTP) are presented for AIR NOx emissions; there is a wide spread in the results from the 5 chemistry models, and it is shown that differences in the methane response relative to the O3 response drive much of the spread. [ABSTRACT FROM AUTHOR]

Abstract: The functional isolation of proteome subsets based on small molecule–protein interactions is an increasingly popular and promising field in functional proteomics. Entire protein families may be profiled on the basis of their common interaction with a metabolite or small molecule inhibitor. This is enabled by novel multifunctional small molecule probes. One platform approach in this field are Capture Compounds that contain a small molecule of interest to bind target proteins, a photo-activatable reactivity function to covalently trap bound proteins, and a sorting function to isolate Capture Compound–protein conjugates from complex biological samples for direct trypsinisation and protein identification by liquid chromatography/mass spectrometry (CCMS). We here present the synthesis and application of a novel GDP-Capture Compound for the functional enrichment of GTPases, a pivotal protein family that exerts key functions in signal transduction. We present data from CCMS experiments on two biological lysates from Escherichia coli and from human-derived Hek293 cells. The GDP-Capture Compound robustly captures a wide range of different GTPases from both systems and will be a valuable tool for the proteomic profiling of this important protein family. [Copyright &y& Elsevier]

Abstract: Introduction: The purpose of this study was to evaluate the antimicrobial efficacy of Triphala, green tea polyphenols (GTP), MTAD, and 5% sodium hypochlorite against E. faecalis biofilm formed on tooth substrate. Methods: Extracted human teeth were biomechanically prepared, vertically sectioned, placed in the tissue culture wells exposing the root canal surface to E. faecalis to form a biofilm. At the end of the 3rd and 6th weeks all groups were treated for 10 minutes with the test solutions and control and were analyzed qualitatively and quantitatively. Results: Qualitative assay with 3-week biofilm showed complete inhibition of bacterial growth with Triphala, MTAD and NaOCl, except GTP and saline, which showed presence of bacterial growth. In quantitative analysis, GTP- and saline-treated tooth samples have shown 1516 ± 17.2 CFU/mL, 156.4 × 109 ± 3.1 × 109 CFU/mL respectively. Qualitative assay with 6-week biofilm showed growth when treated with Triphala, GTP and MTAD whereas NaOCl has shown complete inhibition. All groups except NaOCl showed eight log reduction when compared to control when analyzed quantitatively. Conclusions: 5% sodium hypochlorite showed maximum antibacterial activity against E. Faecalis biofilm formed on tooth substrate. Triphala, green tea polyphenols and MTAD showed statistically significant antibacterial activity. The use of herbal alternatives as a root canal irrigant might prove to be advantageous considering the several undesirable characteristics of NaOCl. [Copyright &y& Elsevier]

Abstract: Proteasome degradation of endoplasmic reticulum (ER)-misfolded proteins requires retrograde transport from ER to the cytosol. To date, it is not clear whether this event constitutes the exclusive ER degradation process for non-native membrane proteins. Here we describe the role of GTP in the degradation of ΔF508-CFTR and the α subunit of the T-cell receptor (TCRα), representative misfolded ER membrane proteins. Selective intracellular GTP depletion extended the ΔF508-CFTR half-life sixfold, whereas ATP depletion accelerated its turnover and inhibited only 80% of the proteasome activity that was not affected by GTP depletion. AlF4 −, a well-known inhibitor of heterotrimeric G proteins, but not of AlF3, delayed the mutant CFTR turnover in vivo, in semi-intact cells and in ER-enriched microsomes, without affecting ER to Golgi cargo transport. ΔF508-CFTR degradation was also inhibited by alkaline stripping of ER-associated membrane proteins. We propose that at the ER, GTP may participate in the disposal of misfolded membrane proteins through activation of heterotrimeric G proteins. [Copyright &y& Elsevier]

Abstract: Voltage dependant calcium channels (VDCC) play a critical role in coupling electrical excitability to important physiological events such as secretion by neuronal and endocrine cells. Rem2, a GTPase restricted to neuroendocrine cell types, regulates VDCC activity by a mechanism that involves interaction with the VDCC β subunit (CaVβ). Mapping studies reveal that Rem2 binds to the guanylate kinase domain (GK) of the CaVβ subunit that also contains the high affinity binding site for the pore forming and voltage sensing VDCC α subunit (CaVα) interaction domain (AID). Moreover, fine mapping indicates that Rem2 binds to the GK domain in a region distinct from the AID interaction site, and competitive inhibition studies reveal that Rem2 does not disrupt CaVα - CaVβ binding. Instead, the CaVβ subunit appears to serve a scaffolding function, simultaneously binding both Rem2 and AID. Previous studies have found that in addition to CaVβ binding, Rem2 must be localized to the plasma membrane to inhibit VDCC function. Plasma membrane localization requires the C-terminus of Rem2 and binding studies indicate that this domain directs phosphorylated phosphatidylinositide (PIP) lipids association. Plasma membrane localization may provide a unique point of regulation since the ability of Rem2 to bind PIP lipids is inhibited by the phosphoserine dependant binding of 14-3-3 proteins. Thus, in addition to CaVβ binding, VDCC blockade by Rem2 is likely to be controlled by both the localized concentration of membrane PIP lipids and direct 14-3-3 binding to the Rem2 C-terminus. [Copyright &y& Elsevier]

Abstract: c-Kit encodes for the receptor tyrosine kinase (RTK) and belongs to type III receptor family. This includes platelet derived growth factor (PDGF) α and β and macrophage colony stimulating factor (mCSF) apart from others. Their characteristic features are the presence of five immunologlobulin like domains in the extracellular region and 70–100 residues long kinase insert domain in the cytoplasmic region. The RTKs activate several signaling pathways within the cells leading to cell proliferation, differentiation, migration or metabolic changes. The Kit ligand-stem cell factor (SCF) induces a rapid and complete receptor dimerization resulting in activation by autophosphorylation of the catalytic tyrosine kinase and generation of signal transduction leading to regulation of cell growth. Various mutations in c-kit such as insertions and deletions (without affecting reading frame) and point mutations in the inhibitory juxtamembrane (JM) domain encoded by exon 11 have been reported in gastrointestinal stromal tumors (GISTs). Thus, c-kit signaling is believed to play a role in tumorigenesis. Efforts are being made to control and treat these tumors by blocking kit signaling using Imatinib with varying degrees of success. This review deals with the features of c-kit, its ligand and roles in gastrointestinal stromal tumors. [Copyright &y& Elsevier]

Abstract: Oxidation of the guanosine moiety in DNA has become a hallmark biomarker in assessing oxidative stress. The oxidation of guanosine in the nucleotide triphosphate pool has been overlooked due to the lack of a reliable methodology. This method describes a sample processing and high performance liquid chromatography with electrochemical detection protocol for the analysis of the cellular pool of guanosine triphosphates and oxidized guanosine triphosphates. Validation of this method is demonstrated along with evaluation of these analytes in control and oxidizing conditions in vitro and in HEK 293T cells. Oxidation of this triphosphate pool occurred independently of oxidation to DNA. [Copyright &y& Elsevier]

Abstract: Since differentiation therapy is one of the promising strategies for treatment of leukemia, universal efforts have been focused on finding new differentiating agents. In that respect, it was recently shown that guanosine 5′-triphosphate (GTP) induced the differentiation of K562 cells, suggesting its possible efficiency in treatment of chronic myelogenous leukemia (CML). However, further investigations are required to verify this possibility. Here, the effects of GTP on activation of mitogen-activated protein kinases (MAPKs) and caspases in K562 cells were examined. Exposure of K562 cells to 100μM GTP markedly inhibited growth (4–70%) and increased percent glycophorin A positive cells after 1–6 days. GTP-induced terminal erythroid differentiation of K562 cells was accompanied with activation of three key caspases, i.e., caspase-3, -6 and -9. More detailed studies revealed that mitochondrial pathway is activated along with down-regulation of Bcl-xL and releasing of cytochrome c into cytosol. Among MAPKs, ERK1/2and p38 were modulated after GTP treatment. Western blot analyses showed that sustained phosphorylation of p38 MAPK was accompanied by a decrease in ERK1/2 activation. These modulatory effects of GTP were observed at early exposure times before the onset of differentiation (3h), and followed for 24–96h. Interestingly, inhibition of p38 MAPK pathway by SB202190 impeded GTP-mediated caspases activation and differentiation in K562 cells, suggesting that p38 MAPK may act upstream of caspases in our system. These results point to a pivotal role for p38 MAPK pathway during GTP-mediated erythroid differentiation of K562 cells and will hopefully have important impact on pharmaceutical evaluation of GTP for CML treatment in differentiation therapy approaches. [Copyright &y& Elsevier]

Abstract: The brain neuropeptide Neuropeptide Y (NPY) is an important modulator of a number of centrally mediated processes including feeding, anxiety-like behaviors, blood pressure and others. NPY produces its effects through at least four functional G-protein coupled receptors termed Y1, Y2, Y4 and Y5. In the brain, the Y1 and Y2 receptor subtypes are the predominant receptor population. To better understand the roles of NPY, genetically modified mice lacking NPY were produced but lacked the expected phenotypes. These mice have previously been reported to have a marked increase in Y2 receptor binding. In the present study, we found an upregulation of both Y1 and Y2 receptor binding and extended these findings to the female. These increases were as large as 10-fold or greater in many brain regions. To assess functional coupling of the receptors, we performed agonist-induced [35S]GTPγS autoradiography. In the mouse brain, the Y1/Y4/Y5 agonist Leu31,Pro34-NPY increased [35S]GTPγS binding with a regional distribution consistent with that produced when labeling adjacent sections with [125I]-Leu31,Pro34-PYY. In a few brain regions, minor increases were noted in the agonist-induced binding when comparing knock out mice to wild type. The Y2 agonist C2-NPY stimulated [35S]GTPγS binding in numerous brain areas with a regional distribution similar to the binding observed with [125I]-PYY3-36. Again, no major increases were noted in the functional activation of Y2 receptors between knock out and wild type mice. Therefore, the increased Y1 and Y2 binding observed in the NPY knock out mice does not represent an increase in NPY receptor mediated signaling and is likely due to an increase in spare (uncoupled) receptors. [Copyright &y& Elsevier]

Abstract: The Rap family of small GTP-binding proteins is composed by four different members: Rap1A, Rap1B, Rap2A and Rap2B. In this work we report the identification and characterization of a fifth member of this family of small GTPases. This new protein is highly homologous to Rap2A and Rap2B, binds labeled GTP on nitrocellulose, and is recognized by a specific anti-Rap2 antibody, but not by an anti-Rap1 antibody. The protein has thus been named Rap2C. Binding of GTP to recombinant purified Rap2C was Mg2+-dependent. However, accurate comparison of the kinetics of nucleotide binding and release revealed that Rap2C bound GTP less efficiently and possessed slower rate of GDP release compared to the highly homologous Rap2B. Moreover, in the presence of Mg2+, the relative affinity of Rap2C for GTP was only about twofold higher than that for GDP, while, under the same conditions, Rap2B was able to bind GTP with about sevenfold higher affinity than GDP. When expressed in eukaryotic cells, Rap2C localized at the plasma membrane, as dictated by the presence of a CAAX motif at the C-terminus. We found that Rap2C represented the predominant Rap2 protein expressed in circulating mononuclear leukocytes, but was not present in platelets. Importantly, Rap2C was found to be expressed in human megakaryocytes, suggesting that the protein may be down-regulated during platelets generation. This work demonstrates that Rap2C is a new member of the Rap2 subfamily of proteins, able to bind guanine nucleotides with peculiar properties, and differently expressed by various hematopoietic subsets. This new protein may therefore contribute to the still poorly clarified cellular events regulated by this subfamily of GTP-binding proteins. [Copyright &y& Elsevier]

Abstract: Background: The mechanism of action of mycophenolic acid (MPA) has been described as a blockade of inosine 5′-monophosphate dehydrogenase (IMPDH) and is thought to selectively influence T- and B-lymphocytes due to their strong dependency on guanine nucleotides synthesized via the de novo purine synthesis pathway. Recent evidence suggests MPA to affect antigen-presenting cells. Methods: Using CD14+ derived human dendritic cells (DC) we have investigated the effects of MPA on differentiation, maturation and function and studied intracellular nucleotide content and IMPDH activity. Results: GTP content and IMPDH activities of DC were strongly and dose-dependently decreased when MPA was present during the entire culture period or was added after the fifth (immature DC) or the seventh (mature DC) day of culture. Concurrent to low GTP levels, a dose-dependent reduction in the expression of CD80, CD86, CD40, CD54 and CD83 was seen which was accompanied by a decreased capacity of DC to stimulate T-cells. Our data for the first time shows a direct effect of MPA on the maturation and function of human CD14+ derived DC, indicates a role of IMPDH and a dependency on the de novo purine synthesis pathway. [Copyright &y& Elsevier]

Abstract: Uptake of neurotransmitters into synaptic vesicles is driven by an electrochemical gradient generated by a vacuolar-type proton pump ATPase. This uptake implies a key role for synaptic vesicles in the regulation of neurotransmitter systems. Guanine nucleoside and nucleotides are involved in the inhibition of glutamate-induced cellular responses via an extracellular action and diverse trophic, proliferative, and modulatory effects of guanine nucleotides on neural cells have been shown. Here, we characterized the uptake of GTP into synaptic vesicles isolated from whole rat brain, by using a tritiated poorly-hydrolyzable GTP analog, 5′-guanylylimidodiphosphate ([3H]GppNHp). Uptake of GTP into synaptic vesicles is saturable, time- and temperature-dependent, and relies on a proton-eletrochemical gradient. However, [3H]GMP and [3H]GDP radioactive labeling in synaptic vesicles is not dependent on temperature and vesicular ATPase activity, which indicates that these nucleotides only bind to and are not taken up into synaptic vesicles. GTP is taken up by the same eletrochemical gradient-dependent transport system, as are neurotransmitters storage, which indicates that this guanine nucleotide may also function as a neurotransmitter. [Copyright &y& Elsevier]

Abstract: Cathodic haemoglobins of four species of anguilliform fish were characterized from a functional point of view, with special regard to the interaction with their physiological effectors. A series of oxygen-binding experiments at increasing GTP concentrations was carried out in order to compare GTP-binding activities in the absence and presence of saturating amounts of chloride. The results indicated that the cathodic haemoglobin of three species (Anguilla anguilla, Conger conger and Muraena helena) do have two sites for GTP-binding. In the absence of chloride, the two sites cannot be discriminated, whereas in the presence of chloride, a competition between the two anions occurred for the second GTP-binding site. The cathodic haemoglobin of Gymnothorax unicolor, which showed lower GTP sensitivity than the other haemoglobins examined, displayed only one GTP-binding site. The presence of an additional phosphate-binding site is not exceptional, although the way haemoglobin interacts with the two organic phosphate molecules may differ among species. This property may provide an auxiliary means of haemoglobin modulation for species that inhabit environments where oxygen availability is highly variable and haemoglobin-oxygen affinity needs to be modulated to different extents in order to satisfy physiological oxygen requirements. [Copyright &y& Elsevier]

Abstract: Lesch–Nyhan disease (LND), caused by complete deficiency of hypoxanthine guanine phosphoribosyltransferase (HPRT), is characterized by a neurological deficit, the etiology of which is unknown. Evidence has accumulated indicating that it might be related to dysfunction of the basal ganglia with a prominent loss of striatal dopamine fibers. Guanine nucleotide depletion has been shown to occur in cells from Lesch–Nyhan patients. In this study we demonstrate that chronic guanine nucleotide depletion induced by inhibition of inosine monophosphate dehydrogenase with low levels (50nM) of mycophenolic acid (MPA) lead human neuroblastoma cell lines to differentiate toward the neuronal phenotype. The MPA-induced morphological changes were more evident in the dopaminergic line LAN5, than in the cholinergic line IMR32. MPA-induced differentiation, unlike that induced by retinoic acid, caused a less extensive neurite outgrowth and branching (similar to that observed in cultured HPRT-deficient dopaminergic neurons) and involved up-regulation of p53, p21 and bax, and bcl-2 down-regulation without p27 protein accumulation. These results suggest that guanine nucleotide depletion following HPRT deficiency, might lead to earlier and abnormal brain development mainly affecting the basal ganglia, displaying the highest HPRT activity, and could be responsible for the specific neurobehavioral features of LND. [Copyright &y& Elsevier]

Abstract: In freshly isolated neurons of the ventral tegmental area of young rats, we first examined the role of G proteins in the functional modulation of the glycine receptor (GlyR). GTP-γ-S [guanosine-5′-0-(2-thiotriphosphate)] (2 mM) or GDP-β-S [guanosine 5′-0-(2-thiodiphosphate)] (2 mM) was added to the pipette solution of whole-cell recordings to regulate G protein activities. GTP-γ-S enhanced the amplitude of glycine-induced current (IGly), suggesting modulation of GlyRs via a G protein-coupled pathway. GDP-β-S suppressed IGly, suggesting that basal G protein activity positively modulates the GlyRs. We next examined effects of G proteins in ethanol potentiation of GlyR function. Activation of G proteins with 2 mM GTP-γ-S attenuated, but did not eliminate, ethanol-induced potentiation of IGly. These results suggest that GTP-γ-S and ethanol share the same pathway of activating GlyRs. When G proteins are maximally activated by GTP-γ-S, the action of ethanol was partially occluded. When 2 mM GDP-β-S was added in pipette solution, ethanol-induced potentiation of IGly was significantly attenuated, suggesting that GDP-β-S partially blocked the action of ethanol. However, the inability of GTP-γ-S (or GDP-β-S) to eliminate completely the potentiating effect of ethanol indicates that some other factors, in addition to G proteins, may also contribute to the action of ethanol on GlyRs. [Copyright &y& Elsevier]

A putative consensus site for GTP/ATP binding has been recently identified within human annexin A6 (AnxA6) by using molecular modeling, infrared (IR), and fluorescence spectroscopies. In this report, we describe the structural changes of human recombinant AnxA6 caused by binding of a non-hydrolyzable GTP analog, GTP-γ-S photoreleased from GTP-γ-S[1-(4,5-dimethoxy-2-nitrophenyl)-ethyl] (caged GTP-γ-S). Recording the infrared spectra of AnxA6 before and after photorelease of GTP-γ-S and measuring directly their differences revealed time-dependent structural changes caused by the photorelease of GTP-γ-S and its binding to AnxA6. The infrared difference spectra consisted of a decrease in the intensity of the 1660–1655-cm-1 band and a concomitant increase of the 1622–1617-cm-1 component band. This suggests that carbonyl groups of peptide backbone are affected due to a small distortion of secondary structure of AnxA6 upon nucleotide binding. The infrared spectra measured as function of time, served to generate 2D IR correlation spectra. Our findings indicated that both bands were correlated, suggesting that they belong to the same molecular species. [Copyright &y& Elsevier]

Increased galanin (GAL) may be associated with the cognitive deficits characteristic of Alzheimer’s disease (AD). However, both increased and decreased GAL receptor density has been reported in AD brain. Previous studies indicate pre-treatment with guanine nucleotides displaces endogenous GAL from GAL receptors (GALR), providing an indirect measurement of GALR occupancy. In addition, pre-treatment with guanine nucleotides may provide a more accurate measurement of GALR density since it would avoid the masking of GALRs by residual binding of endogenous GAL. Thus, in the present study, we examined the influence of pre-treatment with guanine nucleotides on 125I-GAL binding in multiple regions of normal and AD brain. Our results indicate that GTP pre-treatment enhances GAL binding in specific regions in normal and AD brain. In addition, our results suggest an increase in the number of GALRs occupied by endogenous GAL in the deep layers of the frontal cortex and the lateral hypothalamus of AD subjects compared to normal subjects. The regional differences in GALR density and receptor occupancy between normal and AD subjects may play a role in the cognitive disturbances associated with the disease. [Copyright &y& Elsevier]

In addition to its use for heroin addiction pharmacotherapy in general, buprenorphine has advantages in treating maternal heroin abuse. To examine the gestational effects of buprenorphine on opioid receptor signaling, the [35S]-GTPγS in situ binding induced by the μ agonist [D-Ala2,MePhe4,Gly5-ol] enkephalin (DAMGO) or the nociceptin/orphanin FQ (N/OFQ) agonist was measured in mesolimbic structures of pup brains from pregnant rats administered with buprenorphine±naloxone, naloxone, or methadone by osmotic minipump. Drug- and gender-based changes in DAMGO- and N/OFQ-induced GTPγS binding were discovered in mesolimbic regions of dam, P2, and P7 brains. Buprenorphine and/or methadone gestational treatment attenuated DAMGO-induced GTPγS binding in some dam and male P2 mesolimbic regions. Methadone diminished DAMGO-induced GTPγS binding in almost all monitored brain regions of the dam but had few effects on their N/OFQ-induced GTPγS binding. Naloxone used in combination with buprenorphine blocked the inhibition by buprenorphine alone on DAMGO-induced GTPγS binding. In contrast to its inhibitory effects on DAMGO-induced GTPγS binding, buprenorphine stimulated N/OFQ-induced GTPγS binding in male P2 nucleus accumbens and lateral septum. Brain region-dependent gender differences in DAMGO-induced GTPγS binding were seen in P2 pups, and males showed greater sensitivity to buprenorphine and methadone than females. Our findings on μ opioid receptor (MOR) GTP-binding regulatory protein (G protein) coupling and its gender dependency are consistent with our earlier studies on μ receptor binding adaptation induced by buprenorphine in dams and neonatal rats after in utero treatment regimens, and they extend the gestational effects of this opiate to μ and N/OFQ receptor functionality. [Copyright &y& Elsevier]

Cytidine 5′-triphosphate (CTP) synthase catalyzes the ATP-dependent formation of CTP from UTP using either ammonia or l-glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as a positive allosteric effector to promote catalysis of glutamine hydrolysis. We show that at concentrations exceeding ∼0.15 mM, GTP actually behaves as a negative allosteric effector of E. coli CTP synthase, inhibiting glutamine-dependent CTP formation. In addition, GTP inhibits NH3-dependent CTP formation in a concentration-dependent manner. However, GTP does not inhibit the enzyme''s intrinsic glutaminase activity. Although the activation of CTP synthase by GTP does not display cooperative behavior, inhibition of both CTP synthase-catalyzed ammonia- and glutamine-dependent CTP synthesis by GTP do exhibit positive cooperativity. These results suggest that GTP binding affects CTP synthase catalysis in two ways: it activates enzyme-catalyzed glutamine hydrolysis and it inhibits the utilization of NH3 as a substrate by the synthase domain. [Copyright &y& Elsevier]

Voltage-gated Na+ currents were recorded from anatomically identified postganglionic muscular sympathetic neurons without and with ATP and GTP in the intracellular solution. The main findings of the study were that cells without ATP and GTP in the intracellular solution express a higher amplitude and greater density of voltage-gated Na+ current, and their Na+ current activates faster and also inactivates faster time dependently. The current is also steady-state inactivated to a lesser degree and recovers from inactivation more slowly in cells without added ATP and GTP. These findings suggest that the presence of ATP and GTP, substrates for channel phosphorylation, changes the kinetic properties of Na+ currents. [Copyright &y& Elsevier]

Neuropeptide Y, one of the most abundant brain peptides, has been found to modulate several important biological functions via a family of G-protein coupled receptors. To investigate the localization of functional NPY receptor subtypes in the rat brain, we performed agonist-induced [35S]GTPγS autoradiography. The Y1/Y4/Y5 agonist Leu31, Pro34-NPY increased [35S]GTPγS binding in several brain areas with a regional distribution consistent with that produced when labeling adjacent sections with [125I]-Leu31, Pro34-PYY. The Y1 selective antagonist BIBP3226 antagonized the Leu31, Pro34-NPY stimulated increase in [35S]GTPγS binding in all areas examined. The Y2 agonist C2-NPY stimulated [35S]GTPγ binding in numerous brain areas with a regional distribution similar to the binding observed with [125I]-PYY 3-36. No increase in [35S]GTPγS binding above basal was observed in any brain area evaluated using Y4 and Y5 selective agonists. This study demonstrates abundant Y1 and Y2 receptor activation in the rat brain, while evidence for functional Y4 and Y5 receptors was not observed. [Copyright &y& Elsevier]

Calexcitin (CE) is a calcium-binding protein, closely related to sarcoplasmic calcium-binding proteins, that is involved in invertebrate learning and memory. Early reports indicated that both Hermissenda and squid CE also could bind GTP; however, the biochemical significance of GTP-binding and its relationship to calcium binding have remained unclear. Here, we report that the GTPase activity of CE is strongly regulated by calcium. CE possessed a P-loop-like structure near the C-terminal similar to the phosphate-binding regions in other GTP-binding proteins. Site-directed mutagenesis of this region showed that Gly182, Phe186 and Gly187 are required for maximum affinity, suggesting that the GTP-binding motif is G-N-x-x-[FM]-G. CE cloned from Drosophila CNS possessed a similar C-terminal sequence and also bound and hydrolyzed GTP. GTPase activity in Drosophila CE was also strongly regulated by Ca2+, exhibiting over 23-fold higher activity in the presence of 0.3 μM calcium. Analysis of the conserved protein motifs defines a new family of Ca2+-binding proteins representing the first example of proteins endowed with both EF-hand calcium binding domains and a C-terminal, P-loop-like GTP-binding motif. These results establish that, in the absence of calcium, both squid and Drosophila CE bind GTP at near-physiological concentrations and hydrolyze GTP at rates comparable to unactivated ras. Calcium functions to increase GTP-binding and GTPase activity in CE, similar to the effect of GTPase activating proteins in other low-MW GTP-binding proteins. CE may, therefore, act as a molecular interface between Ca2+ cytosolic oscillations and the G protein-coupled signal transduction. [Copyright &y& Elsevier]

Rat prostate transglutaminase is characterized by a high degree of complexity. In fact, as previously demonstrated, it is highly glycosilated and possesses a lipid anchor which is retained during enzyme apocrine secretion. In order to assess the importance of such modifications upon enzyme functionality, full length rat prostate transglutaminase cDNA has been synthesized by RT-PCR and stably expressed in MDCK cells. The recombinant form has been partially purified by GTP-affinity chromatography, a technique which has been used to purify the enzyme produced from rat prostate secretion. The recombinant protein is endowed with enzymatic activity even though, as we have demonstrated by immunological studies, it lacks post-translational modifications which occur in the prostate enzyme. Moreover, we have demonstrated that a deletion mutant, which gives rise to a protein lacking 103 amino acid residues at the N-terminus end, loses enzymatic activity and the capability of binding GTP. This study shows that, while post-translational modifications are not essential for enzymatic activity, the N-terminus end is responsible for both transglutaminase functionality and GTP-binding. [Copyright &y& Elsevier]

The pseudopeptide [Nphe1]N/OFQ(1–13)NH2 (Nphe) has been shown to act as a pure, selective and competitive antagonist of nociceptin/orphanin FQ (N/OFQ) receptors in different tissues. However, Nphe displayed a highly variable potency, with pA2 values ranging from 5.96 to 8.45. In the present study, we show that sodium ions and GTP markedly affect the potency of Nphe in blocking N/OFQ receptors coupled to cyclic AMP inhibition in different cellular systems. In intact N1E-115 neuroblastoma cells, the pA2 value of Nphe increased from 7.13 to 8.02 when the extracellular sodium concentration was reduced from 138 to 2.5 mM. When N/OFQ inhibition of adenylyl cyclase activity was assayed in cell membranes, 100 mM NaCl decreased the pKi value of Nphe from 8.38 to 7.32, but increased that of the nonpeptide N/OFQ receptor antagonist CompB from 8.61 to 8.92. Similar effects of sodium ions on the potencies of Nphe and CompB were observed when the compounds were used to antagonize the N/OFQ inhibition of adenylyl cyclase activity in membranes of the external plexiform layer of the rat olfactory bulb. In the same assay, the increase of GTP concentration from 0.1 to 200 μM decreased Nphe potency by 8-fold. These data demonstrate that sodium ions and GTP affect the potency of Nphe in a manner similar to that of agonists but not of pure antagonists and suggest that these factors may contribute to the reported variability of Nphe affinity constant. [Copyright &y& Elsevier]

The greenhouse gas emissions associated with bioenergy are often temporally dispersed and can be a mixture of long-term forcers (such as carbon dioxide) and short-term forcers (such as methane). These factors affect the timing and magnitude of climate-change impacts associated with bioenergy in ways that cannot be clearly communicated with a single metric. This is critical as key comparisons that determine incentives and policy for bioenergy are based upon climate-change impacts expressed as carbon dioxide equivalent calculated with GWP100. This paper explores these issues further and presents a spreadsheet tool to facilitate quick assessment of these temporal effects. The potential effect of (i) a mix of GHGs and (ii) emissions that change with time are illustrated through two case studies. In case study 1, variations in the mix of greenhouse gases mean that apparently similar impacts after 100-years, mask radically different impacts before then. In case study 2, variations in the timing of emissions cause their climate-change impacts (integrated radiative-forcing and temperature change) to differ from the impacts that an emissions-balance would suggest. The effect of taking alternative approaches to considering "CO 2 -equivalence" are also assessed. In both cases, a single metric for climate-change effects was found to be wanting. A simple tool has been produced to help practitioners evaluate whether this is the case for any given system. If complex dynamics are apparent, it is recommended that additional metrics, more detailed inventory, or full time-series impact results are used in order to accurately communicate these climate-change effects. • Climate-change effects of two case studies compared to results from carbon-balance. • Single metrics do not always fully convey climate-change impacts. • Spreadsheet tool made available to facilitate assessment of temporal variation in impacts. • Inclusion of additional results recommended to provide complete picture. [ABSTRACT FROM AUTHOR]

Allosteric modulation of the CB1Rs could represent an alternative strategy for the treatment of diseases in which these receptors are involved, without the undesirable effects associated with their orthosteric stimulation. PSNCBAM-1 is a reference diaryl urea derivative that positively affects the binding affinity of orthosteric ligands (PAM) and negatively affects the functional activity of orthosteric ligands (NAM) at CB1Rs. In this work we reported the design, synthesis and biological evaluation of three different series of compounds, derived from structural modifications of PSNCBAM-1 and its analogs reported in the recent literature. Almost all the new compounds increased the percentage of binding affinity of CP55940 at CB1Rs, showing a PAM profile. When tested alone in the [35S]GTPγS functional assay, only a few derivatives lacked detectable activity, so were tested in the same functional assay in the presence of CP55940. Among these, compounds 11 and 18 proved to be functional NAMs at CB1Rs, dampening the orthosteric agonist-induced receptor functionality by approximately 30%. The structural features presented in this work provide new CB1R-allosteric modulators (with a profile similar to the reference compound PSNCBAM-1) and an extension of the structure-activity relationships for this type of molecule at CB1Rs. Image 1 • We synthesized and evaluated novel PSNCBAM-1 analogs as CB1R allosteric modulators. • Some compounds acted as PAMs for the binding of orthosteric agonist CP55940 at CB1Rs. • Compounds 11 and 18 proved to be functional NAMs at CB1Rs. • New insights for SAR of diaryl urea derivatives at CB1Rs were provided. [ABSTRACT FROM AUTHOR]

Bacteria are sophisticated systems with high capacity and flexibility to adapt to various environmental conditions. Each prokaryote however possesses a defined metabolic network, which sets its overall metabolic capacity, and therefore the maximal growth rate that can be reached. To achieve optimal growth, bacteria adopt various molecular strategies to optimally adjust gene expression and optimize resource allocation according to the nutrient availability. The resulting physiological changes are often accompanied by changes in the growth rate, and by global regulation of gene expression. The growth-rate-dependent variation of the abundances in the cellular machineries, together with condition-specific regulatory mechanisms, affect RNA metabolism and fate and pose a challenge for rational gene expression reengineering of synthetic circuits. This article is part of a Special Issue entitled: RNA and gene control in bacteria, edited by Dr. M. Guillier and F. Repoila. • Regulation of RNA synthesis, turnover and fate in bacteria depend on growth rate. • Global regulation results from the growth-rate dependency of molecular machines. • (p)ppGpp coordinates global regulation and cell adaptation to changing environments. • Gene expression from synthetic sequences reveals complex regulatory interactions. [ABSTRACT FROM AUTHOR]

GTP cyclohydrolase (GCYH-I) is an enzyme in the folate biosynthesis pathway that has not been previously exploited as an antibiotic target, although several pathogens including N. gonorrhoeae use a form of the enzyme GCYH-IB that is structurally distinct from the human homologue GCYH-IA. A comparison of the crystal structures of GCYH-IA and -IB with the nM inhibitor 8-oxo-GTP bound shows that the active site of GCYH-IB is larger and differently shaped. Based on this structural information, we designed and synthesized a small set of 8-oxo-G derivatives with ether linkages at O 6 and O 8 expected to displace water molecules from the expanded active site of GCYH-IB. The most potent of these compounds, G3 , is selective for GCYH-IB, supporting the premise that potent and selective inhibitors of GCYH-IB could constitute a new class of small molecule antibiotics. [ABSTRACT FROM AUTHOR]