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Eukaryotic initiation factor 2B (eIF2B) is a guanine nucleotide exchange factor (GEF) whose activity is both tightly regulated and rate-controlling with regard to global rates of protein synthesis. Skeletal muscle eIF2B activity and expression of its catalytic [epsilon]-subunit (eIF2B[epsilon]) have been implicated as potential contributors to the altered rates of protein synthesis in a number of physiological conditions and experimental models. The objective of this study was to directly examine the effects of exogenously expressed eIF2B[epsilon] in vivo on GEF activity and protein synthetic rates in rat skeletal muscle. A plasmid encoding FLAGeIF2B[epsilon] was transfected into the tibialis anterior (TA) of one leg, while the contralateral TA received a control plasmid. Ectopic expression of eIF2B[epsilon] resulted in increased GEF activity in TA homogenates of healthy rats, demonstrating that the expressed protein was catalytically active. In an effort to restore a deficit in eIF2B activity, we utilized an established model of chronic sepsis in which skeletal muscle eIF2B activity is known to be impaired. Ectopic expression of eIF2B[epsilon] in the TA rescued the sepsis-induced deficit in GEF activity and muscle protein synthesis. The results demonstrate that modulation of eIF2B[epsilon] expression may be sufficient to correct deficits in skeletal muscle protein synthesis associated with sepsis and other muscle-wasting conditions. mRNA translation; in vivo electroporation; muscle wasting doi: 10.1152/ajpendo.00151.2010.

Macroautophagy (hereafter autophagy) is a ubiquitous process in eukaryotic cells that is integrally involved in various aspects of cellular and organismal physiology. The morphological hallmark of autophagy is the formation of double-membrane cytosolic vesicles, autophagosomes, which sequester cytoplasmic cargo and deliver it to the lysosome or vacuole. Thus, autophagy involves dynamic membrane mobilization, yet the source of the lipid that forms the autophagosomes and the mechanism of membrane delivery are poorly characterized. The TRAPP complexes are multimeric guanine nucleotide exchange factors (GEFs) that activate the Rab GTPase Ypt1, which is required for secretion. Here we describe another form of this complex (TRAPPIII) that acts as an autophagy-specific GEF for Ypt1. The Trs85 subunit of the TRAPPIII complex directs this Ypt1 GEF to the phagophore assembly site (PAS) that is involved in autophagosome formation. Consistent with the observation that a Ypt1 GEF is directed to the PAS, we find that Ypt1 is essential for autophagy. This is an example of a Rab GEF that is specifically targeted for canonical autophagosome formation. Rab | stress | trafficking | vacuole | yeast doi/ 10.1073/pnas.1000063107

In Bacillus subtilis cells, the GTP level decreases and the ATP level increases upon a stringent response. This reciprocal change in the concentrations of the substrates of RNA polymerase affects the rate of transcription initiation of certain stringent genes depending on the purine species at their transcription initiation sites. DNA microarray analysis suggested that not only the rrn and ilv-leu genes encoding rRNAs and the enzymes for synthesis of branched-chain amino acids, respectively, but also many genes, including genes involved in glucose and pyruvate metabolism, might be subject to this kind of stringent transcription control. Actually, the ptsGHI and pdhABCD operons encoding the glucose-specific phosphoenolpyruvate:sugar phosphotransferase system and the pyruvate dehydrogenase complex were found to be negatively regulated, like rrn, whereas the pycA gene encoding pyruvate carboxylase and the alsSD operon for synthesis of acetoin from pyruvate were positively regulated, like ilv-leu. Replacement of the guanine at position 1 and/or position 2 of ptsGHI and at position 1 of pdhABCD (transcription initiation base at position 1) by adenine changed the negative stringent control of these operons in the positive direction. The initiation bases for transcription of pdhABCD and pycA were newly determined. Then the promoter sequences of these stringent operons were aligned, and the results suggested that the presence of a guanine(s) and the presence of an adenine(s) at position 1 and/or position 2 might be indispensable for negative and positive stringent control, respectively. Such stringent transcription control that affects the transcription initiation rate through reciprocal changes in the GTP and ATP levels likely occurs for numerous genes of B. subtilis. doi: 10.1128/JB.01394-09

Melamine toxicity in mammals has been attributed to the blockage of kidney tubules by insoluble complexes of melamine with cyanuric acid or uric acid. Bacteria metabolize melamine via three consecutive deamination reactions to generate cyanuric acid. The second deamination reaction, in which ammeline is the substrate, is common to many bacteria, but the genes and enzymes responsible have not been previously identified. Here, we combined bioinformatics and experimental data to identify guanine deaminase as the enzyme responsible for this biotransformation. The ammeline degradation phenotype was demonstrated in wild-type Escherichia coli and Pseudomonas strains, including E. coli K12 and Pseudomonas putida KT2440. Bioinformatics analysis of these and other genomes led to the hypothesis that the ammeline deaminating enzyme was guanine deaminase. An E. coli guanine deaminase deletion mutant was deficient in ammeline deaminase activity, supporting the role of guanine deaminase in this reaction. Two guanine deaminases from disparate sources (Bradyrhizobium japonicum USDA 110 and Homo sapiens) that had available X-ray structures were purified to homogeneity and shown to catalyze ammeline deamination at rates sufficient to support bacterial growth on ammeline as a sole nitrogen source. In silico models of guanine deaminase active sites showed that ammeline could bind to guanine deaminase in a similar orientation to guanine, with a favorable docking score. Other members of the amidohydrolase superfamily that are not guanine deaminases were assayed in vitro, and none had substantial ammeline deaminase activity. The present study indicated that widespread guanine deaminases have a promiscuous activity allowing them to catalyze a key reaction in the bacterial transformation of melamine to cyanuric acid and potentially contribute to the toxicity of melamine. doi:10.1128/JB.01243-09

In enteric bacteria, the cellular response to oxidative stress is activated by oxidation of the iron-sulfur clusters in SoxR, which then induces transcription of soxS, turning on a battery of defense genes. Here we demonstrate both in vitro and in cells that activation of SoxR can occur in a DNA-mediated reaction with guanine radicals, an early genomic signal of oxidative stress, serving as the oxidant. SoxR in its reduced form is found to inhibit guanine damage by repairing guanine radicals. Moreover, cells treated with a DNA-binding photooxidant, which generates guanine radicals, promotes the expression of soxS. In vitro, this photooxidant, tethered to DNA 80 bp from the soxS promoter, induces transcription by activating SoxR upon irradiation. Thus, transcription can be activated from a distance through DNA-mediated charge transport. This chemistry offers a general strategy for DNA-mediated signaling of oxidative stress. DNA charge transport | guanine radicals | oxidative stress

A new chip-based method to identify protein--protein interactions was developed using the guanine nucleotide exchange factor GRF2 and two interacting proteins, Ras and calmodulin, as model proteins. A generic immobilization strategy for FLAG-tagged bait proteins on a protein-repellent streptavidin chip surface was implemented by presentation of an oriented anti-FLAG antibody. A flow cell device, integrating different chip surfaces, was developed, and the interaction of immobilized GRF2 with the two analytes was verified by fluorescence assays. On-chip tryptic digest assays were then performed on the capture surface and analyzed by [micro]LC-MS/MS. The interaction of GRF2 with calmodulin and Ras was demonstrated, and the lower limit of detection was determined. We also implemented an on-chip immunoprecipitation assay to identify GRF2-binding partners from complex protein mixtures. Cells overexpressing FLAG-GRF2 were lysed and then incubated with the anti-FLAG chip. In addition to detecting GRF2, we also identified calmodulin, demonstrating that this technique can successfully identify endogenous levels of proteins, bound to recombinant bait proteins. This chip-based method has the advantage that no subsequent gel separations of protein complexes prior to LC-MS analysis are required and is therefore amenable to miniaturized high-throughput determination of protein--protein interactions.

We report the characterization of BopE, a type III secreted protein that is encoded adjacent to the Burkholderia pseudomallei bsa locus and is homologous to Salmonella enterica SopE/SopE2. Inactivation of bopE impaired bacterial entry into HeLa cells, indicating that BopE facilitates invasion. Consistent with this notion, BopE expressed in eukaryotic cells induced rearrangements in the subcortical actin cytoskeleton, and purified BopE exhibited guanine nucleotide exchange factor activity for Cdc42 and Rac1 in vitro.

The phase-variable phage growth limitation (Pgl) system of Streptomyces coelicolor A3(2) is an unusual bacteriophage resistance mechanism that confers protection against the temperate phage [phi]C31 and homo-immune relatives. Pgl is subject to phase variation, and data presented here show that this is at least partially due to expansion and contraction of a polyguanine tract present within the putative adenine-specific DNA methyltransferase gene, pglX. Furthermore, the pglX paralogue SC6G9.02, here renamed pglS, was shown to be able to interfere with the Pgl phenotype, suggesting that PglS could provide an alternative activity to that conferred by PglX.

Rho-family GTPases regulate many cellular functions. To visualize the activity of Rho-family GTPases in living cells, we developed fluorescence resonance energy transfer (FRET)-based probes for Rac1 and Cdc42 previously (Itoh, R.E., K. Kurokawa, Y. Ohba, H. Yoshizaki, N. Mochizuki, and M. Matsuda. 2002. Mol. Cell. Biol. 22:6582-6591). Here, we added two types of probes for RhoA. One is to monitor the activity balance between guanine nucleotide exchange factors and GTPase-activating proteins, and another is to monitor the level of GTP-RhoA. Using these FRET probes, we imaged the activities of Rho-family GTPases during the cell division of HeLa cells. The activities of RhoA, Rac1, and Cdc42 were high at the plasma membrane in interphase, and decreased rapidly on entry into M phase. From after anaphase, the RhoA activity increased at the plasma membrane including cleavage furrow. Rac1 activity was suppressed at the spindle midzone and increased at the plasma membrane of polar sides after telophase. Cdc42 activity was suppressed at the plasma membrane and was high at the intracellular membrane compartments during cytokinesis. In conclusion, we could use the FRET-based probes to visualize the complex spatio-temporal regulation of Rho-family GTPases during cell division.

Certain C8-substituted and N7, C8-disubstituted guanine ribonucleosides comprise a class of small molecules with immunostimulatory activity. In a variety of animal models, these agents stimulate both humoral and cellular immune responses. The antiviral actions of these guanosine analogs have been attributed to their ability to induce type I IFNs. However, the molecular mechanisms by which the guanosine analogs potentiate immune responses are not known. Here, we report that several guanosine analogs activate Toll-like receptor 7 (TLR7). 7-Thia-8-oxoguanosine, 7-deazaguanosine, and related guanosine analogs activated mouse immune cells in a manner analogous to known TLR ligands, inducing cytokine production in mouse splenocytes (IL-6 and IL-12, type I and II IFNs), bone marrow-derived macrophages (IL-6 and IL-12), and in human peripheral blood leukocytes (type I IFNs, tumor necrosis factor [alpha] and IL-12). The guanosine congeners also up-regulated costimulatory molecules and MHC I/II in dendritic cells. Genetic complementation studies in human embryonic kidney 293 cells confirmed that the guanosine analogs activate cells exclusively via TLR7. The stimulation of TLR7 by the guanosine analogs in human cells appears to require endosomal maturation because inhibition of this process with chloroquine significantly reduced the downstream activation of NF-[kappa]B. However, TLR8 activation by R-848 and TLR2 activation by {S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitol-R-Cys-S-Ser-Lys4-OH, trihydrochloride)} were not inhibited by chloroquine, whereas TLR9 activation by CpG oligodeoxynucleotides was abolished. In summary, we present evidence that guanosine analogs activate immune cells via TLR7 by a pathway that requires endosomal maturation. Thus, the B cell-stimulating and antiviral activities of the guanosine analogs may be explained by their TLR7-activating capacity.

Although there is good evidence that immunity to the blood stages of malaria parasites can be mediated by different effector components of the adaptive immune system, target antigens for a principal component, effector CD[4.sup.+] T cells, have never been defined. We generated CD[4.sup.+] T cell lines to fractions of native antigens from the blood stages of the rodent parasite, Plasmodium yoelii, and identified fraction-specific T cells that had a Th1 phenotype (producing IL-2, IFN-[gamma], and tumor necrosis factor-[alpha], but not IL-4, after antigenic stimulation). These T cells could inhibit parasite growth in recipient severe combined immunodeficient mice. N-terminal sequencing of the fraction showed identity with hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT). Recombinant HGXPRT from the human malaria parasite, Plasmodium falciparum, activated the T cells in vitro, and immunization of normal mice with recombinant HGXPRT reduced parasite growth rates in all mice after challenge.

BIG1 and BIG2 are brefeldin A-inhibited guanine nucleotide-exchange proteins that activate ADP-ribosylation factors (ARFs), critical components of vesicular trafficking pathways. These proteins can exist in macromolecular complexes and move between Golgi membranes and cytosol. In the BIG1 molecule, a centrally located Sec7 domain is responsible for ARF activation, but functions of other regions are largely unknown. Yeast two-hybrid screens of a human placenta cDNA library with BIG1 cDNA constructs revealed specific interaction of the N-terminal region (amino acids 1-331) with FKS06-binding protein 13 (FKBP13). The association was confirmed by immunoprecipitation of both endogenous BIG1 and FKBP13 from Jurkat T cells with antibodies against either one. Binding of BIG1, BIG2, and ARF to cell membranes in vitro was increased by guanosine 5'-[[gamma]-thio]triphosphate, and further increases were induced by FK506. Incubation of Jurkat T cells with FK506 increased binding of BIG1, BIG2, and ARF to Golgi and other membranes in a time- and concentration-dependent manner, without effects on clathrin or [gamma]-adaptin binding. Binding of BIG1, BIG2, and ARF to membranes was also increased by L-732,531, an agonist structurally related to FK506, but was not increased by a related antagonist, L-685,818, nor by cyclosporin A or rapamycin. These findings are consistent with a role for FKBP13 and FK506 in vesicular trafficking, influencing ARF activity through their guanine nucleotide-exchange proteins.

Rho family GTPases are important regulators of epithelial tight junctions (TJs); however, little is known about how the GTPases themselves are controlled during TJ assembly and function. We have identified and cloned a canine guanine nucleotide exchange factor (GEF) of the Dbl family of proto-oncogenes that activates Rho and associates with TJs. Based on sequence similarity searches and immunological and functional data, this protein is the canine homologue of human GEF-H1 and mouse Lfc, two previously identified Rho-specific exchange factors known to associate with microtubules in nonpolarized cells. In agreement with these observations, immunofluorescence of proliferating MDCK cells revealed that the endogenous canine GEF-H1/Lfc associates with mitotic spindles. Functional analysis based on overexpression and RNA interference in polarized MDCK cells revealed that this exchange factor for Rho regulates paracellular permeability of small hydrophilic tracers. Although overexpression resulted in increased size-selective paracellular permeability, such cell lines exhibited a normal overall morphology and formed fully assembled TJs as determined by measuring transepithelial resistance and by immunofluorescence and freeze-fracture analysis. These data indicate that GEF-H1/Lfc is a component of TJs and functions in the regulation of epithelial permeability.

CAMP controls many cellular processes mainly through the activation of protein kinase A (PKA). However, more recently PKA-independent pathways have been established through the exchange protein directly activated by cAMP (Epac), a guanine nucleotide exchange factor for the small GTPases Rap1 and Rap2. In this report, we show that cAMP can induce integrin-mediated cell adhesion through Epac and Rap1. Indeed, when Ovcar3 cells were treated with cAMP, cells adhered more rapidly to fibronectin. This cAMP effect was insensitive to the PKA inhibitor H-89. A similar increase was observed when the cells were transfected with Epac. Both the cAMP effect and the Epac effect on cell adhesion were abolished by the expression of Rap1--GTPase-activating protein, indicating the involvement of Rap1 in the signaling pathway. Importantly, a recently characterized cAMP analogue, 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate, which specifically activates Epac but not PKA, induced Rap-dependent cell adhesion. Finally, we demonstrate that external stimuli of cAMP signaling, i.e., isoproterenol, which activates the G[[alpha].sub.s]-coupled [[beta].sub.2]-adrenergic receptor can induce integrin-mediated cell adhesion through the Epac-Rap1 pathway. From these results we conclude that cAMP mediates receptor-induced integrin-mediated cell adhesion to fibronectin through the Epac-Rap1 signaling pathway.

Research has been conducted on recombinant human O (super)6 -alkylguanine-DNA alkyltransferase (hAGT). The role of zinc in hAGT and its effects on hAGT properties have been investigated and discussed.

Research has been conducted on MutT enzyme from Escherischia coli. The hypothesis that 8-oxo-guanine nucleotide binds more tightly to MutT than the corresponding guanine nucleotide has been tested via calorimetric, NMR and kinetic methods, and the details are reported.

Research has been conducted on the 8-oxoguanine-containing RNA. Results indicate that mammalian Y box-binding protein 1 can bind to this RNA.

The ADP ribosylation factor (ARF) GTP binding proteins are believed to mediate cytoskeletal remodeling and vesicular trafficking along the secretory pathway. Here we show that ARF6 is specifically associated with dense-core secretory granules in neuroendocrine PC12 cells. Stimulation with a secretagogue triggers the recruitment of secretory granules to the cell periphery and the concomitant activation of ARF6 by the plasma membrane-associated guanine nucleotide exchange factor, ARF nucleotide binding site opener (ARNO). Expression of the constitutively inactive ARF6(T27N) mutant inhibits secretagogue-dependent exocytosis from PC12 cells. Using a mutant of ARF6 specifically impaired for PLD1 stimulation, we find that ARF6 is functionally linked to phospholipase D (PLD)1 in the exocytotic machinery. Finally, we show that ARNO, ARF6, and PLD1 colocalize at sites of exocytosis, and we demonstrate direct interaction between ARF6 and PLD1 in stimulated cells. Together, these results provide the first direct evidence that ARF6 plays a role in calcium-regulated exocytosis in neuroendocrine cells, and suggest that ARF6-stimulated PLD1 activation at the plasma membrane and consequent changes in membrane phospholipid composition are critical for formation of the exocytotic fusion pore.

Research has been conducted on the N,N'-dimethyl-2,3-diaminobutane retro model adduct. The properties and conformer distribution of these adducts have been investigated and the results are reported.

Byline: Sylvie Cornelie (1), Eric Wiel (2), Niels Lund (3), Gilles Lebuffe (2), Catherine Vendeville (1), Gilles Riveau (1), Benoit Vallet (2), Elisabeth Ban (1) Keywords: Cytosine-phosphate-guanine (CpG) oligodeoxynucleotide Lipopolysaccharide (LPS) D-galactosamine Tumor necrosis factor [alpha] Septic shock Abstract: Abstract Objective. Unmethylated cytosine-phosphate-guanine (CpG) oligodeoxynucleotides are highly frequent motifs in bacterial DNA and rare in the mammalian genome. They are potent inducers of inflammatory cytokines and act synergistically with lipopolysaccharide (LPS) for the induction of tumor necrosis factor [alpha] (TNF-[alpha]) production in vivo. It has therefore been suggested that innate immune reaction to bacterial unmethylated CpG motifs might contribute to the development of septic shock. We designed this study to assess the sensitization role of CpG motifs in LPS-induced shock using the D-galactosamine (D-GalN)-sensitized mouse model. Design. A prospective, randomized in vivo animal laboratory study. Setting. Experimental research laboratory. Intervention. We performed experiments in which CpG, LPS and D-GalN were administrated sequentially in various orders or simultaneously in 8week-old BALB/c mice. Measurements and results. Cytosine-phosphate-guanine treatment potentiated LPS action only if injected prior to LPS. A combination of predefined sublethal doses of CpG (1 nmol/mouse) and LPS (1 ng/mouse) not only had a synergetic effect on TNF-[alpha] production (20.3+-9.2 IU/ml versus 2.5+-1.4 IU/ml and 5.6+-3.4 IU/ml for CpG and LPS groups, respectively, p Conclusion. Our findings indicate that CpG motifs act synergistically with LPS by initializing the synthesis of TNF-[alpha] and/or TNF-[alpha] regulating factors, thereby acting as a sensitizing agent. Author Affiliation: (1) Unite INSERM U547, Institut Pasteur de Lille, 59019 Lille Cedex,France (2) Departement d'Anesthesie-Reanimation 2, Hopital Claude Huriez, CHRU de Lille, 59037 Lille Cedex, France (3) Department of Anesthesiology, University of Rochester Medical Center, 14642 Rochester, NY, USA Article History: Received Date: 27/12/2001 Accepted Date: 12/06/2002 Article note: Electronic Publication

Research has been conducted on the tobacco carcinogen benzo[a]pyrene. The quantitative analysis of the benzo[a]pyrene diol epoxide-DNA adducts has been carried out and the results demonstrate nonrandom lesion distribution in double-stranded DNA.

Research has been conducted on the human glioma cell line extracts which lack O (super)6 -methylguanine DNA-methyltransferase. The ability of these extracts to execute strand incision at various base mismatches in model DNA has been investigated and the results are discussed.

Research has been conducted on the cyclic guanine and adenine nucleotides. The role of these nucleotides in the intracellular signal transduction processes has been investigated via the study of their photoliable phosphotriester derivatives and the results are reported.

Research has been conducted on the dinuclear platinum(II) complexes. The reactions of these complexes with 9-ethylguanine have been monitored in aqueous solution via (super)1 NMR spectroscopy and the results are described.

Research has been conducted on the full-length colicin B and its isolated C-terminal domain. The conformational stabilities of the colicin B C-terminal domain have been investigated via unfolding induced by guanidine hydrochloride and the results indicate that this domain consists of two subdomains.

Research has been conducted on G.TA and T.CG triads in which TA and CG base pairs are recognized by guanine and thymine. The characterization of stabilities of these triads has been carried out and the effects of the triads on the canonical triads in the triple-helical DNA have been investigated.

Research has been conducted on Rac protein, an intracellular signaling integrator. Results indicate that the guanine-nucleotide exchange factor P-Rex1 is a coincidence detector in the purified from neutrophil cytasol PtdIns(3,4,5)P (sub)3-sensitive Rac activator.

Tissue transglutaminase (TG) is a [Ca.sup.2+]-dependent acyltransferase with roles in cellular differentiation, apoptosis, and other biological functions. In addition to being a transamidase, TG undergoes a GTP-binding/GTPase cycle even though it lacks any obvious sequence similarity with canonical GTP-binding (G) proteins. Guanine nucleotide binding and [Ca.sup.2+] concentration reciprocally regulate TG's transamidation activity, with nucleotide binding being the negative regulator. Here we report the x-ray structure determined to 2.8-[Angstrom] resolution of human TG complexed with GDP. Although the transamidation active site is similar to those of other known transglutaminases, the guanine nucleotide-binding site of TG differs markedly from other G proteins. The structure suggests a structural basis for the negative regulation of transamidation activity by bound nucleotide, and the positive regulation of transamidation by [Ca.sup.2+].

Research has been conducted on the Ru(II) complexes which photo-oxidize DNA guanine bases. The hypothesis that the luminescence of [Ru(TAP) (sub)2 POQ-Nmet] (super)2+ may be switched on after the interaction with DNA has been investigated and the results are reported.

Research has been conducted on the proteins containing G-protein regulatory (GPR) motifs, members of the guanine nucleotide dissociation inhibitors (GDI) for the G(alpha) subunits from the Gi family. The GPR-contact sites on the G(alpha) subunits and the GPR-protein GDI activity have been investigated and the results are reported.

Research has been conducted on the Escherichia coli elongation factor Tu. The interaction of this elongation factor with the elongation factor Ts and guanine nucleotides has been investigated via the stop-flow technique, and the rate constants of all the association and dissociation reactions between the elongation factor-Tu, elongation factor-Ts, GTP and GDP have been determined.

The authors reviews the publications on electron transport through DNA. The topics of interest include hole transfer and hole transport through DNA, excess electron transport through DNA and electric conduction through DNA.

Research has been conducted on the cationic adducts which combine guanine with the phenylnitrenium ion. Results of the density functional calculations predict that that these adducts are lower in energy than the separated reactants.

Research has been conducted on the 5-hydroxy-4-hydroxymethyl-2-cyclohexenylguanine. The facile synthesis of this compound has been carried out and the involvement of the Diels-Alder reaction of ethyl (2E)-3-acetyloxy-2-propenoate with Danishefsky's diene in building up the ring skeleton is reported.

Research has been conducted on the formation, repair and replication of the DNA lesion O (super)6 -methylguanine. The context-dependent O (super)6 -methylguanine processing in vivo in cells with different genetic backgrounds has been defined and the results are presented.

Batiuk, Thomas D., Carol Schnizlein-Bick, Zoya Plotkin, and Pierre C. Dagher. Guanine nucleosides and Jurkat cell death: roles of ATP depletion and accumulation of deoxyribonucleotides. Am J Physiol Cell Physiol 281: C1776-C 1784, 2001.--Guanine nucleosides are toxic to some forms of cancer. This toxicity is pronounced in cancers with upregulated guanine nucleotide synthesis, but the mechanisms are poorly understood. We investigated this toxicity by measuring the effects of guanine nucleosides on nucleotides in Jurkat cells using HPLC. We also measured proliferation and cell death with microscopy and fluorescence-activated cell sorting. Guanosine increased GTP to 600% and reduced ATP to 40% of control. This resulted in cell death with a predominance of necrosis. Deoxyguanosine caused similar increases in GTP but at earlier time points. Cell death was severe with a predominance of apoptosis. Deoxyguanosine but not guanosine increased dGTP to 800% of control. Adenosine inhibited the effects of guanosine, in part by competing for uptake. In stimulated leukocytes, guanosine and deoxyguanosine altered the nucleotide pools in a way qualitatively similar to that observed in Jurkat cells. However, proliferation was enhanced rather than impaired. In conclusion, guanosine and deoxyguanosine are toxic to Jurkat cells through two mechanisms: ATP depletion, causing necrosis, and the accumulation of dGTP, resulting in apoptosis. cancer; lymphocytes; apoptosis; necrosis; adenosine 5'-triphosphate; guanosine 5'-triphosphate

The Dbl family of guanine nucleotide exchange factors are multifunctional molecules that transduce diverse intracellular signals leading to the activation of Rho GTPases. The tandem Dbl-homology and pleckstrin-homology domains shared by all members of this family represent the structural module responsible for catalyzing the GDP-GTP exchange reaction of Rho proteins. Recent progress in genomic, genetic, structural and biochemical studies has implicated Dbl family members in diverse biological processes, including growth and development, skeletal muscle formation, neuronal axon guidance and tissue organization. The detailed pictures of their autoregulation, agonist-controlled activation and mechanism of interaction with Rho GTPase substrates, have begun to emerge.

Research has been conducted on the purified human OGG1 protein and mammalian XRS5 nuclear extracts. The relative efficiency of these protein and extracts in excising 8-oxo-guanine from clustered damages has been investigated via the use of the oligonucleotide constructs containing 8-oxo-7,8-dihydroguanine in locations opposite to the damage.

Research has been conducted on the artificial metallopeptidase with a well-defined active site. The construction of this metallopeptidase on the cross-linked polystyrene backbone has been carried out via adjoining a guanidinium moiety to the tetraaza ligand's copper(II) complex.

Research has been conducted on the 8-oxoguanine, an oxidized guanine form. Results indicate that the Escherichia coli cells contain proteins binding specifically to RNA molecules that carry this oxoguanine and that the mutants defective in the protein are hyperresistant to the drug inducing oxidative stress.