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Elevating cyclic AMP with a combination of forskolin and IBMX (Fskn/IBMX) was found as the cause of G1 growth arrest in Jurkat T-cells, concomitant with an induction of the cyclin-dependent kinase inhibitor, p27Kip1. The protein kinase inhibitor H-89, which can discriminate between EPAC and PKA pathways, blocked the inhibition in cell growth and induction of p27Kip1, indicating an involvement of PKA, but not EPAC. The EPAC-specific cyclic AMP analogue, 8-CPT-2Me-cAMP was able to activate Rap1, but failed to induce growth arrest or induction p27Kip1. These results demonstrate that PKA, and not EPAC, mediates cyclic AMP-dependent growth arrest in Jurkat T-cells. To further investigate a role for EPAC in these cells, we carried out cDNA microarray analysis of cells stimulated with 8-CPT-2Me-cAMP. We identified separate groups of genes whose expression was either induced or repressed in response to 8-CPT-2Me-cAMP. This provides the first demonstration that EPAC can regulate gene expression, although it may not be involved in cell cycle control. Finally, we identify c-Jun as a transcription factor whose activity is specifically down-regulated following EPAC activation, but not PKA. The control of gene expression by cyclic AMP in Jurkat T-cells therefore requires input from the EPAC signalling cascade.

Genetic variation in the human angiotensin I-converting enzyme (ACE) gene has been associated with many heritable traits, including obesity. Herein, we report the results of a study of obesity-related phenotypes and lifestyle in 1016 teen-aged Greeks. We show that there is a strong association (p = 0.001) between subcutaneous fat and the ACE insertion/deletion (I/D) polymorphism in females, possession of genotypes containing the D allele being associated with increased fat thickness. This association is strongest in females who participate in no extra exercise and accounts for 6.5% of the phenotypic variance in fat thickness by ANOVA. The association is additive, with the mean phenotypic values in heterozygotes intermediate between the means of the two homozygotes, and the association acts at both extremes of the fat thickness distribution in a classical polygenic manner. Other ACE polymorphisms (rs4424958, rs4311) that define major haplotypes in European populations fail to provide stronger associations with the subcutaneous fat phenotype. Because ACE I/D is the polymorphism most strongly associated with circulating ACE levels in European populations, we propose that the functional allelic differences that influence circulating ACE levels also mediate the associations with the obesity-related phenotypes studied here.

Huntington disease (HD) is an inherited, late onset, progressive neurodegenerative disorder. Primary degeneration appears to selectively occur in striatal medium spiny neurones but this is most likely preceded by a period of neuronal dysfunction. Altered levels of neurotransmitter receptors may disrupt neuronal function and contribute to a toxic environment within the brain. In the present study, a knock-in HD mouse modelling early stages of the disease was used to determine whether alterations in neurotransmitter receptor densities occurred before overt neuronal loss. Receptor autoradiography demonstrated reduced dopamine D2 and increased benzodiazepine receptor binding in the striatum of HD animals compared to wild-type littermates. The density of benzodiazepine receptor binding was also increased in the cerebral cortex of the HD mice. Changes in opioid and dopamine D1 receptor densities were more subtle and influenced by the genetic background of the mice. Our findings are consistent with the hypothesis that alterations in neurotransmitter receptor density precede cell loss and may be an active cellular response to the initial stages of HD pathogenesis.

Insulin stimulates glucose transport in adipocytes and muscle by inducing the redistribution of Glut4 from intracellular locations to the plasma membrane. The fusion of Glut4-containing vesicles at the plasma membrane is known to involve the target SNAREs syntaxin 4 and SNAP-23 and the vesicle SNARE VAMP2. Little is known about the initial docking of Glut4 vesicles with the plasma membrane. A recent report has implicated Exo70, a component of the mammalian exocyst complex, in the initial interaction of Glut4 vesicles with the adipocyte plasma membrane. Here, we have examined the role of two other exocyst components, rsec6 and rsec8. We show that insulin promotes a redistribution of rsec6 and rsec8 to the plasma membrane and to cytoskeletal fractions within 3T3-L1 adipocytes but does not modulate levels of these proteins co-localized with Glut4. We further show that adenoviral-mediated overexpression of either rsec6 or rsec8 increases the magnitude of insulin-stimulated glucose transport in 3T3-L1 adipocytes. By contrast, overexpression of rsec6 or rsec8 did not increase the extent of the secretion of adipsin or ACRP30 from adipocytes and had no discernible effect on transferrin receptor traffic. Collectively, our data support a role for the exocyst in insulin-stimulated glucose transport and suggest a model by which insulin-dependent relocation of the exocyst to the plasma membrane may contribute to the specificity of Glut4 vesicle docking and fusion with the adipocyte plasma membrane.

Using EPAC1 (exchange protein directly activated by cAMP 1) as bait in two-hybrid screens of foetal and adult human brain libraries, we identified the LC2 (light chain 2) of MAP1A (microtubule-associated protein 1A) as a protein capable of interaction with EPAC1. We applied an immunoprecipitation assay to demonstrate protein interaction between EPAC1 and LC2 in co-transfected human embryonic kidney 293 cells. EPAC2 also co-immunoprecipitated with LC2 from extracts of rat cerebellum. Immunolocalization in co-transfected human embryonic kidney 293 cells revealed that EPAC1 co-localizes with LC2 throughout the cell body. We found that endogenous EPAC2 is also immunolocalized with LC2 in PC12 cells. Immunolocalization of EPAC1 in transfected COS1 cells showed that EPAC1 is associated with the perinuclear region surrounding the nucleus and filamentous structures throughout the cell. Removal of the cAMP-binding domain of EPAC1 (DeltacAMP-EPAC1) appeared to disrupt targeting of EPAC1 in cells resulting in a more dispersed staining pattern. Using two-hybrid assay, we tested the ability of LC2 to interact with DeltacAMP-EPAC1 and DeltaDEP-EPAC1, which lacks a DEP domain (dishevelled, Egl-10 and pleckstrin homology domain). We found that deletion of the cAMP-binding domain inhibited interaction between EPAC1 and LC2 in a two-hybrid assay, but removal of the DEP domain had little effect. LC2 was found to interact with a glutathione-S-transferase-fusion protein of the cAMP-binding domain of EPAC1 in a pull-down assay, but not the DEP, REM (Ras exchange motif) or CAT (catalytic) domains. Together with our two-hybrid results, this suggests that the cAMP-binding domain of EPAC1 mediates interaction with LC2.

The cells of organisms as diverse as bacteria and humans can enter stable, nonproliferating quiescent states. Quiescent cells of eukaryotic and prokaryotic microorganisms can survive for long periods without nutrients. This alternative state of cells is still poorly understood, yet much benefit is to be gained by understanding it both scientifically and with reference to human health. Here, we review our knowledge of one "model" quiescent cell population, in cultures of yeast grown to stationary phase in rich media. We outline the importance of understanding quiescence, summarize the properties of quiescent yeast cells, and clarify some definitions of the state. We propose that the processes by which a cell enters into, maintains viability in, and exits from quiescence are best viewed as an environmentally triggered cycle: the cell quiescence cycle. We synthesize what is known about the mechanisms by which yeast cells enter into quiescence, including the possible roles of the protein kinase A, TOR, protein kinase C, and Snf1p pathways. We also discuss selected mechanisms by which quiescent cells maintain viability, including metabolism, protein modification, and redox homeostasis. Finally, we outline what is known about the process by which cells exit from quiescence when nutrients again become available.

Immunoblotting with a monoclonal Rap1 antibody, we found that elevation of cyclic AMP, with forskolin and IBMX or CPT-cAMP, led to a rapid reduction in the levels of Rap1 protein associated with particulate, nuclear/perinuclear fractions from PC12 and COS1 cells. In contrast, cytoplasmic levels of Rap1 remained constant following cyclic AMP stimulation. To gain independent confirmation that cyclic AMP promoted loss of Rap1 in nuclear/perinuclear fractions we used a polyclonal Rap1 antibody, which gave similar results to the monoclonal antibody. This demonstrated that the loss in Rap1 immunoreactivity was not due to phosphorylation-dependent changes that alter immunorecognition. The reduction in Rap1 levels was blocked by PKA inhibitors and by a Rap1 serine to alanine PKA-phosphorylation site mutant (S180A). Peptide inhibitors of the proteasome, cathespin, and calpain II also inhibited the decrease in Rap1 levels, indicating that proteolytic degradation may contribute to maintaining Rap1 levels in the nuclear/perinuclear fraction of cells.

A cellular receptor for the haemagglutinating enteroviruses (HEV), and the protein that mediates haemagglutination, is the membrane complement regulatory protein decay accelerating factor (DAF; CD55). Although primate DAF is highly conserved, significant differences exist to enable cell lines derived from primates to be utilized for the characterization of the DAF binding phenotype of human enteroviruses. Thus, several distinct DAF-binding phenotypes of a selection of HEVs (viz. coxsackievirus A21 and echoviruses 6, 7, 11-13, 29) were identified from binding and infection assays using a panel of primate cells derived from human, orang-utan, African Green monkey and baboon tissues. These studies complement our recent determination of the crystal structure of SCR(34) of human DAF [Williams, P., Chaudhry, Y., Goodfellow, I. G., Billington, J., Powell, R., Spiller, O. B., Evans, D. J. & Lea, S. (2003). J Biol Chem 278, 10691-10696] and have enabled us to better map the regions of DAF with which enteroviruses interact and, in certain cases, predict specific virus-receptor contacts.

A typical purple bacterial photosynthetic unit consists of two types of light-harvesting complex (LH1 and LH2) together with a reaction centre. This short review presents a description of the structure of the LH2 complex from Rhodopseudomonas acidophila, which has recently been improved to a resolution of 2.0 A [Papiz et al., J. Mol. Biol. 326 (2003) 1523-1538]. We show how this structure has helped to reveal the details of the various excitation energy transfer events in which it is involved.

Fluorescent molecules bound to receptors can show their location and, if binding is reversible, can provide pharmacological information such as affinity and proximity between interacting molecules. The spatial precision offered by visualisation transcends the diverse localisation and low molecular concentration of receptor molecules. Consequently, the relationships between receptor location and function and life cycles of receptors have become better understood as a result of fluorescent labeling. Each of these aspects contributes new insights to drug action and potential new targets. The relationships between spatial distribution of receptor and function are largely unknown. This is particularly apparent for native receptors expressed in their normal host tissues where communication between heterogeneous cell types influences receptor distribution and function. In cultured cell systems, particularly for G-protein-coupled receptors (GPCR), fluorescence-based methods have enabled the visualisation of the cycle of agonist-stimulated receptor clustering, endocytic internalisation to the perinuclear region, degradation of the receptor-ligand complex, and recycling back to the surface membrane. Using variant forms of green fluorescent protein (GFP), antibodies, or fluorescent ligands, it is possible to detect or visualise the formation of oligomeric receptor complexes. Careful selection of fluorescent molecules based on their spectral properties enables resonance energy transfer and multilabel visualisation with colocalisation studies. Fluorescent agonist and antagonist ligands are now being used in parallel with GFP to study receptor cycling in live cells. This review covers how labeling and visualisation technologies have been applied to the study of major pharmacologically important receptors and illustrates this by giving examples of recent techniques that have relied on GFP, antibodies, or fluorescent ligands alone or in combination for the purpose of studying GPCR.

The lymphomagenic action of myc genes in conjunction with Epstein-Barr virus nuclear antigen-1 (EBNA-1) have been examined using transgenic mice in several separate tests. Synergy between Myc and EBNA-1 in lymphomagenesis was revealed in a cross breed study where co-expression of transgenic myc and EBNA-1 led to a tumor latency period reduced significantly in some crosses. In the resulting bitransgenic tumors, expression of the Emu-myc genes was not affected by EBNA-1 expression. MoMLV was utilized as a transposon tag to activate cellular oncogenes by infection of EmuEBNA-1 mice. Rearrangement at the c-myc locus in B cell tumors from these mice again suggests a cooperative action between myc and EBNA-1. Tumors arising in EmuEBNA-1 mice typically showed a trisomy of chromosome 15, upon which the c-myc locus resides. Bitransgenic tumors (EBNA-1/c-myc) did not show trisomy 15. This raises the possibility that amplification of c-myc is factorial in the selection of trisomy 15 in these tumors. These data indicate that myc and EBNA-1 act cooperatively and are not redundant in lymphomagenesis. Expression of EBNA-1 by EBV may provide a selection pressure in addition to translocation of the c-myc locus in the genesis of endemic Burkitt's lymphoma (BL)., (Copyright 2003 Wiley-Liss, Inc.)

Nucleotides in the terminal loop of the poliovirus 2C cis-acting replication element (2C(CRE)), a 61 nt structured RNA, function as the template for the addition of two uridylate (U) residues to the viral protein VPg. This uridylylation reaction leads to the formation of VPgpUpU, which is used by the viral RNA polymerase as a nucleotide-peptide primer for genome replication. Although VPg primes both positive- and negative-strand replication, the specific requirement for 2C(CRE)-mediated uridylylation for one or both events has not been demonstrated. We have used a cell-free in vitro translation and replication reaction to demonstrate that 2C(CRE) is not required for the initiation of the negative-sense strand, which is synthesized in the absence of 2C(CRE)-mediated VPgpUpU formation. We propose that the 3' poly(A) tail could serve as the template for the formation of a VPg-poly(U) primer that functions in the initiation of negative-sense strands.

Arfophilin is an ADP ribosylation factor (Arf) binding protein of unknown function. It is identical to the Rab11 binding protein eferin/Rab11-FIP3, and we show it binds both Arf5 and Rab11. We describe a related protein, arfophilin-2, that interacts with Arf5 in a nucleotide-dependent manner, but not Arf1, 4, or 6 and also binds Rab11. Arfophilin-2 localized to a perinuclear compartment, the centrosomal area, and focal adhesions. The localization of arfophilin-2 to the perinuclear compartment was selectively blocked by overexpression of Arf5-T31N. In contrast, a green fluorescent protein-arfophilin-2 chimera or arfophilin-2 deletions were localized around the centrosome in a region that was also enriched for transferrin receptors and Rab11 but not early endosome markers, suggesting that the distribution of the endosomal recycling compartment was altered. The arfophilins belong to a conserved family that includes Drosophila melanogaster nuclear fallout, a centrosomal protein required for cellularization. Expression of green fluorescent protein-nuclear fallout in HeLa cells resulted in a similar phenotype, indicative of functional homology and thus implicating the arfophilins in mitosis/cytokinesis. We suggest that the novel dual GTPase-binding capacity of the arfophilins could serve as an interface of signals from Rab and Arf GTPases to regulate membrane traffic and integrate distinct signals in the late endosomal recycling compartment.

The unfolding of shikimate kinase (SK) from Erwinia chrysanthemi by urea and its subsequent refolding on dilution of the denaturing agent has been studied in detail [Eur. J. Biochem. 269 (2002) 2124]. Comparison of the effects of urea on the enzyme with those of guanidinium chloride (GdmCl) and NaCl indicated that chloride ions significantly weakened the binding of shikimate. This finding prompted a more detailed examination of the effects of salts on the structure, function and stability of the enzyme; the effects of NaCl and Na(2)SO(4) were investigated in detail. These salts have very small effects on the secondary structure as judged by far UV CD circular dichroism (CD), although the near UV CD spectra suggest that some limited changes in the environment of aromatic amino acids may occur. Both salts inhibit SK activity and analysis of the kinetic and substrate binding parameters point to a complex mechanism for the inhibition. Inclusion of salts leads to a marked stabilisation against unfolding of the enzyme by urea. When the enzyme is unfolded by incubation in 4 M urea, addition of NaCl or Na(2)SO(4) leads to a relatively slow refolding of the enzyme as judged by the regain of native-like CD and fluorescence. In addition, the refolded enzyme can bind shikimate, though more weakly than the native enzyme. However, the refolded enzyme does not appear to be capable of binding nucleotides, nor does it possess detectable catalytic activity. The refolding process brought about by addition of salt in the presence of 4 M urea is not associated with any change in the fluorescence of the probe 8-anilino-1-naphthalenesulfonic acid (ANS), indicating that an intermediate formed by hydrophobic collapse is unlikely to be significantly populated. The results point to both specific and general effects of salts on SK. These are discussed in the light of the structural information available on the enzyme.

Dysfunction of the gene encoding DMPK (myotonic dystrophy protein kinase) has been implicated in the human neuromuscular disease myotonic dystrophy (DM1). The cardiac features of the disease include progressive conduction defects and ventricular arrhythmias. These defects have been observed in hearts of mice deficient for DMPK function. We have investigated the role of DMPK in the function of ventricular cardiomyocytes using dmpk knockout (KO) mice. A deficit in DMPK caused enhanced basal contractility of single cardiomyocytes and an associated increase in intracellular Ca(2+), measured using fura-2. Biochemical measurements indicated hyperphosphorylation of phospholamban (PLB) in KO mice. This suggests increased Ca(2+) uptake into the sarcoplasmic reticulum (SR) as the underlying cause of enhanced contractility. This conclusion was supported by the larger amplitude of caffeine-induced Ca(2+) release from the SR in KO cardiomyocytes. Concurrent with hyperphosphorylated PLB, the response to isoprenaline was reduced. These observations suggest dmpk has a modulatory role in the control of intracellular Ca(2+) concentration in mouse ventricular cardiomyocytes, loss of which may contribute to cardiac dysfunction in DM1.

Cellular response to scaffold materials is of great importance in cellular and tissue engineering, and it is perhaps the initial cell contact with the scaffold that determines development of new tissue. Material surface morphology has strong effects on cell cytoskeleton and morphology, and it is thought that cells may react to the topography of collagen and surrounding cells during tissue embryology. A poorly understood area is, however, gene-level responses to topography. Thus, this paper used microarray to probe for consistent gene changes in response to lithographically produced topography (12.5 x 2-microm grooves) with time. The results showed many initial gene changes and also down-regulation of gene response with time. Cell and nucleus morphology were also considered, with nuclear deformation linked to cell signaling.

The poliovirus cis-acting replication element (CRE) templates the uridylylation of VPg, the protein primer for genome replication. The CRE is a highly conserved structural RNA element in the enteroviruses and located within the polyprotein-coding region of the genome. We have determined the native structure of the CRE, defined the regions of the structure critical for activity, and investigated the influence of genomic location on function. Our results demonstrate that a 14-nucleotide unpaired terminal loop, presented on a suitably stable stem, is all that is required for function. These conclusions complement the recent analysis of the 14-nucleotide terminal loop in the CRE of human rhinovirus type 14. The CRE can be translocated to the 5' noncoding region of the genome, at least 3.7-kb distant from the native location, without adversely influencing activity, and CRE duplications do not adversely influence replication. We do not have evidence for a specific interaction between the CRE and the RNA-binding 3CD(pro) complex, an essential component of the uridylylation reaction, and the mechanism by which the CRE is coordinated and orientated during the reaction remains unclear. These studies provide a detailed overview of the structural determinants required for CRE function, and will facilitate a better understanding of the requirements for picornavirus replication.

Insulin stimulates the fusion of intracellular vesicles containing the glucose transporter Glut4 with the plasma membrane in adipocytes and muscle cells. Glut4 vesicle fusion is thought to be catalyzed by the interaction of the vesicle soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptor VAMP2 with the target soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptors SNAP-23 and syntaxin 4. Here, we use combined membrane fractionation, detergent solubility, and sucrose gradient flotation to demonstrate that the large majority (>70%) of SNAP-23 and a significant proportion of syntaxin 4 ( approximately 35%) are associated with plasma membrane lipid rafts in 3T3-L1 adipocytes. Furthermore, VAMP2 is shown to be concentrated in lipid rafts isolated from intracellular membranes. Insulin stimulation had no effect on the plasma membrane raft association of SNAP-23 or syntaxin 4 but promoted VAMP2 insertion into plasma membrane rafts. Immunofluorescence analysis revealed that SNAP-23 was clustered at the plasma membrane and almost completely segregated from the transferrin receptor. SNAP-23 distribution seemed to be distinct from caveolin-1, and clusters of SNAP-23 were dispersed after cholesterol extraction with methyl-beta-cyclodextrin, suggesting that the majority of SNAP-23 is associated with non-caveolar, cholesterol-rich lipid rafts. The results described implicate lipid rafts as important platforms for Glut4 vesicle fusion and suggest the hypothesis that such rafts may represent a spatial integration point of insulin signaling and membrane traffic.

Cells may sense heat shock via the accumulation of thermally misfolded proteins. To explore this possibility, we determined the effect of protein misfolding on gene expression in the absence of temperature changes. The imino acid analog azetidine-2-carboxylic acid (AZC) is incorporated into protein competitively with proline and causes reduced thermal stability or misfolding. We found that adding AZC to yeast at sublethal concentrations sufficient to arrest proliferation selectively induced expression of heat shock factor-regulated genes to a maximum of 27-fold and that these inductions were dependent on heat shock factor. AZC treatment also selectively repressed expression of the ribosomal protein genes, another heat shock factor-dependent process, to a maximum of 20-fold. AZC treatment thus strongly and selectively activates heat shock factor. AZC treatment causes this activation by misfolding proteins. Induction of HSP42 by AZC treatment required protein synthesis; treatment with ethanol, which can also misfold proteins, activated heat shock factor, but treatment with canavanine, an arginine analog less potent than AZC at misfolding proteins, did not. However, misfolded proteins did not strongly induce the stress response element regulon. We conclude that misfolded proteins are competent to specifically trigger activation of heat shock factor in response to heat shock.

Assembly of the infectious herpes simplex virus type 1 virion is a complex, multistage process that begins with the production of a procapsid, which is formed by the condensation of capsid shell proteins around an internal scaffold fashioned from multiple copies of the scaffolding protein, pre-VP22a. The ability of pre-VP22a to interact with itself is an essential feature of this process. However, this self-interaction must subsequently be reversed to allow the scaffolding proteins to exit from the capsid to make room for the viral genome to be packaged. The nature of the process by which dissociation of the scaffold is accomplished is unknown. Therefore, to investigate this process, the properties of isolated scaffold particles were investigated. Electron microscopy and gradient sedimentation studies showed that the particles could be dissociated by low concentrations of chaotropic agents and by moderate reductions in pH (from 7.2 to 5.5). Fluorescence spectroscopy and circular dichroism analyses revealed that there was relatively little change in tertiary and secondary structures under these conditions, indicating that major structural transformations are not required for the dissociation process. We suggest the possibility that dissociation of the scaffold may be triggered by a reduction in pH brought about by the entry of the viral DNA into the capsid.

Protein kinase C, encoded by PKC1, regulates construction of the cell surface in vegetatively growing yeast cells. Pkc1 in part acts by regulating Mpk1, a MAP kinase. Mutants lacking Bck1, a component of the MAP kinase branch of the pathway, fail to respond normally to nitrogen starvation, which causes entry into quiescence. Given that the Tor1 and Tor2 proteins are key inhibitors of entry into quiescence, the Pkc1 pathway may regulate these proteins. We find that pkc1Delta and mpk1Delta mutants rapidly die by cell lysis upon carbon or nitrogen starvation. The Pkc1 pathway does not regulate the TOR proteins: transcriptional changes dependent on inhibition of the TORs occur normally in pkc1Delta and mpk1Delta mutants when starved for nitrogen; pkc1Delta and mpk1Delta mutants die rapidly upon treatment with rapamycin, an inhibitor of the TORs. We find that Mpk1 is transiently activated by rapamycin treatment via a novel mechanism. Finally, we find that rapamycin treatment or nitrogen starvation induces resistance to the cell wall-digesting enzyme zymolyase by a Pkc1-dependent mechanism. Thus, the Pkc1 pathway is not a nutrient sensor but acts downstream of TOR inhibition to maintain cell integrity in quiescence.