Mammalian cells respond to nutrient deprivation by inhibiting energy consuming processes, such as proliferation and protein synthesis, and by stimulating catabolic processes, such as autophagy. p70 S6 kinase (S6K1) plays a central role during nutritional regulation of translation. S6K1 is activated by growth factors such as insulin, and by mammalian target of rapamycin (mTOR), which is itself regulated by amino acids. The Class IA phosphatidylinositol (PI) 3-kinase plays a well recognized role in the regulation of S6K1. We now present evidence that the Class III PI 3-kinase, hVps34, also regulates S6K1, and is a critical component of the nutrient sensing apparatus. Overexpression of hVps34 or the associated hVps15 kinase activates S6K1, and insulin stimulation of S6K1 is blocked by microinjection of inhibitory anti-hVps34 antibodies, overexpression of a FYVE domain construct that sequesters the hVps34 product PI3P, or small interfering RNA-mediated knock-down of hVps34. hVps34 is not part of the insulin input to S6K1, as it is not stimulated by insulin, and inhibition of hVps34 has no effect on phosphorylation of Akt or TSC2 in insulin-stimulated cells. However, hVps34 is inhibited by amino acid or glucose starvation, suggesting that it lies on the nutrient-regulated pathway to S6K1. Consistent with this, hVps34 is also inhibited by activation of the AMP-activated kinase, which inhibits mTOR/S6K1 in glucose-starved cells. hVps34 appears to lie upstream of mTOR, as small interfering RNA knock-down of hVps34 inhibits the phosphorylation of another mTOR substrate, eIF4E-binding protein-1 (4EBP1). Our data suggest that hVps34 is a nutrient-regulated lipid kinase that integrates amino acid and glucose inputs to mTOR and S6K1.

Aggregation of human washed platelets with collagen is accompanied by a concentration-dependent increase in cyclic GMP but not cyclic AMP. NG-Monomethyl-L-arginine (L-MeArg), a selective inhibitor of nitric oxide (NO) synthesis from L-arginine, reduces this increase and enhances aggregation. L-Arginine, which has no effect on the basal levels of cyclic GMP, augments the increase in this nucleotide induced by collagen and also inhibits aggregation. Both of these effects of L-arginine are attenuated by L-MeArg. The anti-aggregatory action of L-arginine is potentiated by prostacyclin and by M&B22948, a selective inhibitor of the cyclic GMP phosphodiesterase, but not by HL725, a selective inhibitor of the cyclic AMP phosphodiesterase. L-Arginine also inhibits platelet aggregation in whole blood in a similar manner, although the concentrations required are considerably higher. L-Arginine stimulates the soluble guanylate cyclase and increases cyclic GMP in platelet cytosol. This stimulation is dependent on NADPH and Ca2+ and is associated with the formation of NO. Both the formation of NO and the stimulation of the soluble guanylate cyclase induced by L-arginine are enantiomer specific and abolished by L-MeArg. Thus, human platelets contain an NO synthase which is activated when platelets are stimulated. The consequent generation of NO modulates platelet reactivity by increasing cyclic GMP. Changes in the activity of this pathway in platelets may have physiological, pathophysiological, and therapeutic significance.

Formation of the Escherichia coli division septum is catalyzed by a number of essential proteins (named Fts) that assemble into a ring-like structure at the future division site. Several of these Fts proteins are intrinsic transmembrane proteins whose functions are largely unknown. Although these proteins appear to be recruited to the division site in a hierarchical order, the molecular interactions underlying the assembly of the cell division machinery remain mostly unspecified. In the present study, we used a bacterial two-hybrid system based on interaction-mediated reconstitution of a cyclic AMP (cAMP) signaling cascade to unravel the molecular basis of septum assembly by analyzing the protein interaction network among E. coli cell division proteins. Our results indicate that the Fts proteins are connected to one another through multiple interactions. A deletion mapping analysis carried out with two of these proteins, FtsQ and FtsI, revealed that different regions of the polypeptides are involved in their associations with their partners. Furthermore, we showed that the association between two Fts hybrid proteins could be modulated by the coexpression of a third Fts partner. Altogether, these data suggest that the cell division machinery assembly is driven by the cooperative association among the different Fts proteins to form a dynamic multiprotein structure at the septum site. In addition, our study shows that the cAMP-based two-hybrid system is particularly appropriate for analyzing molecular interactions between membrane proteins.

The antimicrobial peptide database (APD, http://aps.unmc.edu/AP/) is an original database initially online in 2003. The APD2 (2009 version) has been regularly updated and further expanded into the APD3. This database currently focuses on natural antimicrobial peptides (AMPs) with defined sequence and activity. It includes a total of 2619 AMPs with 261 bacteriocins from bacteria, 4 AMPs from archaea, 7 from protists, 13 from fungi, 321 from plants and 1972 animal host defense peptides. The APD3 contains 2169 antibacterial, 172 antiviral, 105 anti-HIV, 959 antifungal, 80 antiparasitic and 185 anticancer peptides. Newly annotated are AMPs with antibiofilm, antimalarial, anti-protist, insecticidal, spermicidal, chemotactic, wound healing, antioxidant and protease inhibiting properties. We also describe other searchable annotations, including target pathogens, molecule-binding partners, post-translational modifications and animal models. Amino acid profiles or signatures of natural AMPs are important for peptide classification, prediction and design. Finally, we summarize various database applications in research and education.

Identification of the ATPase involved in fast axonal transport of membranous organelles has proven difficult. Myosin and dynein, other ATPases known to be involved in cell motility, have properties that are inconsistent with the established properties of fast axonal transport, an essential component of which is readily solubilized in physiological buffer conditions rather than being stably associated with either membranous organelles or cytoskeletal elements. Adenylyl imidodiphosphate (AMP-PNP), a nonhydrolysable analogue of ATP, is a potent inhibitor of fast axonal transport that results in a stable interaction of membranous organelles with microtubules. Here we report the identification and partial characterization of an ATPase activity from brain whose binding to microtubules is stabilized by AMP-PNP. This ATPase activity seems to be associated with a polypeptide of relative molecular mass (Mr) 130,000 that is highly enriched in microtubule pellets after incubation with AMP-PNP and a soluble fraction from chick brain. This novel ATPase fraction has the predicted characteristics of the motor involved in fast axonal transport. Common features between the ATPase and fast axonal transport include interaction with the cytoskeleton in the presence of AMP-PNP, ready extractability, no Ca2+ dependence and inhibition by EDTA.

The intracellular signaling proteins that lead to exercise-stimulated glucose transport in skeletal muscle have not been identified, although it is clear that there are separate signaling mechanisms for exercise- and insulin-stimulated glucose transport. We have hypothesized that the 5'AMP-activated protein kinase (AMPK) functions as a signaling intermediary in exercise-stimulated glucose uptake. This hypothesis was based on recent studies showing the following: 1) muscle contraction increases AMPK activity and 2) perfusion of rat hindlimb skeletal muscles with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a compound that results in increased AMPK activity, increased insulin-stimulated glucose uptake. In the current study, isolated rat epitrochlearis muscles were treated to contract in vitro (via electrical stimulation for 10 min) and/or incubated in the absence or presence of AICAR (2 mmol/l), insulin (1 micromol/l), or wortmannin (100 nmol/l). Both contraction and AICAR significantly increased AMPK activity, while the enzyme was not activated by insulin. AICAR, contraction, and insulin all increased 3-O-methylglucose (3MG) transport by threefold to fivefold above basal. The phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin completely blocked insulin-stimulated transport, but did not inhibit AICAR- or contraction-stimulated transport. The increase in glucose transport with the combination of maximal AICAR plus maximal insulin treatments was partially additive, suggesting that these stimuli increase glucose transport by different mechanisms. In contrast, there was no additive effect on glucose transport with the combination of AICAR plus contraction. These data suggest that AICAR and contraction stimulate glucose transport by a similar insulin-independent signaling mechanism and are consistent with the hypothesis that AMPK is involved in exercise-stimulated glucose uptake.

Bacteria which can grow in different environments have developed regulatory systems which allow them to exploit specific habitats to their best advantage. In the facultative anaerobe Escherichia coli two transcriptional regulators controlling independent networks of oxygen-regulated gene expression have been identified. One is a two-component sensor-regulator system (ArcB-A), which represses a wide variety of aerobic enzymes under anaerobic conditions. The other is FNR, the transcriptional regulator which is essential for expressing anaerobic respiratory processes. The purpose of this review is to summarize what is known about FNR. The fnr gene was initially defined by the isolation of some pleiotropic mutants which characteristically lacked the ability to use fumarate and nitrate as reducible substrates for supporting anaerobic growth and several other anaerobic respiratory functions. Its role as a transcriptional regulator emerged from genetic and molecular studies in which its homology with CRP (the cyclic AMP receptor protein which mediates catabolite repression) was established and has since been particularly important in identifying the structural basis of its regulatory specificities. FNR is a member of a growing family of CRP-related regulatory proteins which have a DNA-binding domain based on the helix-turn-helix structural motif, and a characteristic beta-roll that is involved in nucleotide-binding in CRP. The FNR protein has been isolated in a monomeric form (Mr 30,000) which exhibits a high but as yet non-specific affinity for DNA. Nevertheless, the DNA-recognition site and important residues conferring the functional specificity of FNR have been defined by site-directed mutagenesis. A consensus for the sequences that are recognized by FNR in the promoter regions of FNR-regulated genes, has likewise been identified. The basic features of the genes and operons regulated by FNR are reviewed, and examples in which FNR functions negatively as an anaerobic repressor as well as positively as an anaerobic activator, are included. Less is known about the way in which FNR senses anoxia and is thereby transformed into its 'active' form, but it seems likely that cysteine residues and possibly a metal ion are involved. Four of the five cysteine residues of FNR are clustered in an essential N-terminal 'domain' which is conserved in FNR and the HlyX protein of Actinobacillus pleuropneumoniae, but not in CRP or the FixK protein of Rhizobium meliloti. The relationships between FNR and other oxygen-related systems in E. coli are discussed, as well as parallel systems in other organisms.(ABSTRACT TRUNCATED AT 400 WORDS)

It is well established that catecholamine-stimulated thermogenesis in brown fat requires beta-adrenergic elevations in cyclic AMP (cAMP) to increase expression of the uncoupling protein 1 (UCP1) gene. However, little is known about the downstream components of the signaling cascade or the relevant transcription factor targets thereof. Here we demonstrate that cAMP- and protein kinase A-dependent activation of p38 mitogen-activated protein kinase (MAPK) in brown adipocytes is an indispensable step in the transcription of the UCP1 gene in mice. By phosphorylating activating transcription factor 2 (ATF-2) and peroxisome proliferator-activated receptor gamma (PPARgamma) coativator 1alpha (PGC-1alpha), members of two distinct nuclear factor families, p38 MAPK controls the expression of the UCP1 gene through their respective interactions with a cAMP response element and a PPAR response element that both reside within a critical enhancer motif of the UCP1 gene. Activation of ATF-2 by p38 MAPK additionally serves as the cAMP sensor that increases expression of the PGC-1alpha gene itself in brown adipose tissue. In conclusion, our findings illustrate that by orchestrating the activity of multiple transcription factors, p38 MAPK is a central mediator of the cAMP signaling mechanism of brown fat that promotes thermogenesis.

AMP-activated protein kinase (AMPK) regulates cellular metabolism in response to the availability of energy and is therefore a target for type II diabetes treatment. It senses changes in the ratio of AMP/ATP by binding both species in a competitive manner. Thus, increases in the concentration of AMP activate AMPK resulting in the phosphorylation and differential regulation of a series of downstream targets that control anabolic and catabolic pathways. We report here the crystal structure of the regulatory fragment of mammalian AMPK in complexes with AMP and ATP. The phosphate groups of AMP/ATP lie in a groove on the surface of the gamma domain, which is lined with basic residues, many of which are associated with disease-causing mutations. Structural and solution studies reveal that two sites on the gamma domain bind either AMP or Mg.ATP, whereas a third site contains a tightly bound AMP that does not exchange. Our binding studies indicate that under physiological conditions AMPK mainly exists in its inactive form in complex with Mg.ATP, which is much more abundant than AMP. Our modelling studies suggest how changes in the concentration of AMP ([AMP]) enhance AMPK activity levels. The structure also suggests a mechanism for propagating AMP/ATP signalling whereby a phosphorylated residue from the alpha and/or beta subunits binds to the gamma subunit in the presence of AMP but not when ATP is bound.

The cellular protein p300 is a target of the adenoviral E1A oncoprotein and is thought to participate in preventing the G0/G1 transition in the cell cycle, activating certain enhancers and stimulating differentiation pathways. CBP is a protein that is associated with and coactivates the transcription factor CREB, mediating the induction by cyclic AMP of certain responsive promoters. The sequences of p300 and CBP are highly related. We show here that p300, like CBP2, can stimulate transcription. This activity is directly and specifically inhibited by E1A. We also find that CBP exists in a DNA-bound complex containing a member of the CREB family and that E1A and CBP interact with one another in vivo. In keeping with the idea that E1A functionally targets CBP, cAMP-dependent transcription is repressed by E1A. Thus, p300 and CBP define a family of transcriptional adaptor proteins that are specifically targeted by the E1A oncoprotein.

In premenopausal women, the ovaries are the principle source of estradiol, which functions as a circulating hormone to act on distal target tissues. However, in postmenopausal women when the ovaries cease to produce estrogen, and in men, this is no longer the case, because estradiol is no longer solely an endocrine factor. Instead, it is produced in a number of extragonadal sites and acts locally at these sites as a paracrine or even intracrine factor. These sites include the mesenchymal cells of adipose tissue including that of the breast, osteoblasts and chondrocytes of bone, the vascular endothelium and aortic smooth muscle cells, and numerous sites in the brain. Thus, circulating levels of estrogens in postmenopausal women and in men are not the drivers of estrogen action, they are reactive rather than proactive. This is because in these cases circulating estrogen originates in the extragonadal sites where it acts locally, and if it escapes local metabolism then it enters the circulation. Therefore, circulating levels reflect rather than direct estrogen action in postmenopausal women and in men. Tissue-specific regulation of CYP19 expression is achieved through the use of distinct promoters, each of which is regulated by different hormonal factors and second messenger signaling pathways. Thus, in the ovary, CYP19 expression is regulated by FSH which acts through cyclic AMP via the proximal promoter II, whereas in placenta the distal promoter I.1 regulates CYP19 expression in response to retinoids. In adipose tissue and bone by contrast, another distal promoter--promoter I.4--drives CYP19 expression under the control of glucocorticoids, class 1 cytokines and TNFalpha. The importance of this unique aspect of the tissue-specific regulation of aromatase expression lies in the fact that the low circulating levels of estrogens which are observed in postmenopausal women have little bearing on the concentrations of estrogen in, for example, a breast tumor, which can reach levels at least one order of magnitude greater than those present in the circulation, due to local synthesis within the breast. Thus, the estrogen which is responsible for breast cancer development, for the maintenance of bone mineralization and for the maintenance of cognitive function is not circulating estrogen but rather that which is produced locally at these specific sites within the breast, bone and brain. In breast adipose of breast cancer patients, aromatase activity and CYP19 expression are elevated. This occurs in response to tumor-derived factors such as prostaglandin E2 produced by breast tumor fibroblasts and epithelium as well as infiltrating macrophages. This increased CYP19 expression is associated with a switch in promoter usage from the normal adipose-specific promoter I.4 to the cyclic AMP responsive promoter, promoter II. Since these two promoters are regulated by different cohorts of transcription factors and coactivators, it follows that the differential regulation of CYP19 expression via alternative promoters in disease-free and cancerous breast adipose tissue may permit the development of selective aromatase modulators (SAMs) that target the aberrant overexpression of aromatase in cancerous breast, whilst sparing estrogen synthesis in other sites such as normal adipose tissue, bone and brain.

OBJECTIVE

This review evaluates recent findings on the mechanisms by which lipogenic enzymes are upregulated or activated in cancer cells, the implications of increased lipogenesis for cancer cell biology and the feasibility of exploiting this pathway and its regulators as targets for antineoplastic intervention.

RESULTS

The list of cancer types showing increased lipogenic enzyme expression keeps growing and further evidence is accumulating that growth factor signaling and particularly activation of the phosphatidylinositol 3'-kinase/protein kinase B pathway plays a role in this process. This signaling pathway stimulates lipogenic gene transcription through activation of the lipogenic transcription factor sterol regulatory element-binding protein-1 and directly activates lipogenic enzymes such as ATP-citrate lyase, linking the upregulation of lipogenesis in cancer cells to the well known tumor-associated increase in glycolysis. Steroid hormones, overexpression of the ubiquitin-specific protease-2a and mutations in breast cancer susceptibility gene 1 may further enhance lipid synthesis. While fatty acid synthase is further established as a target for antineoplastic intervention, recent findings show that interference with acetyl-CoA carboxylase-alpha, ATP citrate lyase or the AMP-activated protein kinase limits cancer cell proliferation and survival.

CONCLUSIONS

The same disturbances in signaling pathways responsible for oncogenic transformation may also contribute to the increased lipogenesis observed in tumor cells. Increased lipogenesis involves modulation of multiple lipogenic enzymes at both transcriptional and posttranscriptional level and is linked to other cancer-associated metabolic changes. Not only fatty acid synthase, but in fact all key enzymes involved in fatty acid synthesis as well as key metabolic regulators are potential targets for antineoplastic intervention.

Bioluminescent marine bacteria of the species Vibrio fischeri are the specific light organ symbionts of the sepiolid squid Euprymna scolopes. Although they share morphological and physiological characteristics with other strains of V. fischeri, when cultured away from the light organ association the E. scolopes symbionts depress their maximal luminescence over 1,000-fold. The primary cause of this reduced luminescence is the underproduction by these bacteria of luciferase autoinducer, a molecule involved in the positive transcriptional regulation of the V. fischeri lux operon. Such an absence of visible light production outside of the symbiotic association has not been previously reported among light organ symbionts of this or any other species of luminous bacteria. Levels of luminescence approaching those of the E. scolopes bacteria in the intact association can be restored by the addition of exogenous autoinducer to bacteria in laboratory culture and are affected by the presence of cyclic AMP. We conclude that some condition(s) specific to the internal environment of the light organ is necessary for maximal autoinduction of luminescence in the symbionts of this squid-bacterial association.

An X-linked recessive disease is reported in a large pedigree. The disease is characterised by a triad of dilated cardiomyopathy, neutropenia and skeletal myopathy. The untreated patients, all boys, died in infancy or early childhood from septicemia or cardiac decompensation. Ultrastructural abnormalities were observed in mitochondria in cardiac muscle cells, neutrophil bone marrow cells and to a lesser extent (0-9%) in skeletal muscle cells. Membrane-bound vacuoles were seen in neutrophil bone marrow cells. Intramuscular fat droplets were increased in type I skeletal muscle fibres. An affected patient had intermittent lactic acidemia, borderline low plasma carnitine, the latter decreasing during periods of illness, and low muscle carnitine (27% pretreatment; 35-40% posttreatment). While on treatment with oral carnitine he had less weakness and no cardiac complaints, but his neutropenia was not affected. Respiratory chain abnormalities were observed in this patient's isolated skeletal muscle mitochondria. These were: (1) diminished concentrations of cytochromes c1 + c, b and aa3 to 29, 47 and 64% of the averaged controls, and (2) a lowered P:0 ratio for oxidation of ascorbate + TMPD, with diminished uncoupler stimulated Mg2+-ATPase activity. Muscle AMP deaminase was deficient (5 resp. 17%). Only one previous report (Neustein et al. 1979) on X-linked mitochondrial cardiomyopathy exists, which probably refers to the same entity. Biochemical studies and haematological abnormalities (neutropenia) are reported for the first time.

The protein responsible for malignant transformation by avian sarcoma viruses (ASVs) has been identified as a phosphoprotein of molecular weight 60,000 designated pp60src (refs 1--4). It has been suggested that this protein has a functional role in cellular transformation involving the phosphorylation of cellular proteins, for it was discovered that specific immunoprecipitates from ASV-transformed cells that contain pp60src catalysed the transfer of phosphate from [gamma-32P]ATP to the heavy chain of rabbit immunoglobulin. Additional studies involving the cell-free synthesis of the ASV src protein further demonstrated that the presence of the src polypeptide correlated with that presence of a phosphotransferase activity. Our studies, involving the biochemical purification of this protein, have demonstrated that the ASV-transforming gene product, pp60src, is itself a protein kinase. We have purified the pp60src protein approximately 5,000-fold using either conventional ion-exchange chromatography or immunoaffinity chromatography. The resultant partially purified preparations contain a cyclic AMP-independent protein kinase activity. We report here that the soluble phosphotransferase activity of partially purified pp60src results in the phosphorylation of exclusively tyrosine residues in a variety of proteins that serve as substrates.

Aggregation of human platelets induced by a variety of agonists was inhibited by 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl] amino]hexyl]-1H-pyrrole-2,5-dionel (U-73122) (IC50 values 1-5 microM), but not by the close analog 1-[6-[[17 beta-3-methoxyestra- 1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidine-dione (U-73343) in which pyrrolidinedione was substituted for pyrroledione. Inhibition by U-73122 was not mediated by an increase in intracellular cyclic AMP. In contrast, the production of inositol 1,4,5-trisphosphate (IP3) and the subsequent rapid increase in cytosolic Ca++ induced by either thrombin or the thromboxane-mimetic, (5Z,9 alpha, 11 alpha, 13E, 15S) 15-hydroxy-11,9-(epoxymethano)prosta- 5,13,-dien-1-oic acid (U-46619), was inhibited by U-73122 but not by U-73343. Reduction of IP3 levels appeared to reflect an inhibition of IP3 production because the hydrolysis of phosphatidyl[3H]inositol and phosphatidyl[3H]inositol 4,5-bisphosphate catalyzed by a soluble fraction from platelets was inhibited by U-73122 (Ki = 9 and 40 microM, respectively). In addition, U-73122 inhibited thromboxane B2 production induced by collagen but not that supported by exogenously added arachidonic acid, suggesting that U-73122 also inhibited receptor-coupled mobilization of arachidonic acid. After preincubation of platelets with [3H]arachidonic acid, the loss of [3H]phosphatidylinositol and accumulation of [3H]phosphatidic acid induced by thrombin was attenuated by U-73122. U-73122 did not inhibit the activities of phospholipases A2 purified either from porcine pancreas or from the venoms of Crotalus adamanteus and Naja naja. Although U-73122 inhibited neither the conversion of exogenous arachidonic acid to thromboxane B2 nor the binding of the thromboxane receptor antagonist [1S-[1 alpha, 2 beta (5Z), 3 beta, 4 alpha]]-7-[3-[[2- [2-[(phenylamino)-carbonyl]- hydrazino]methyl]-7-oxabicyclo [2.2.1]-hept-2-yl-5-heptenoic acid to platelet membranes, it was an effective inhibitor of arachidonic acid-induced aggregation of platelets. These data are consistent with the observed inhibition by U-73122 of platelet activation by the thromboxane receptor agonist, U-46619, via a mechanism that involves inhibition of a phospholipase C-dependent component(s) of signal transduction. U-73122, but not U-73343, inhibited also N-formyl-methionyl-leucyl-phenylalanine-induced aggregation of human polymorphonuclear neutrophils (PMN) and the associated production of IP3 and diacyglycerol. Diradylglycerol produced in PMN stimulated with N-formyl- methionyl-leucyl-phenylalanine was 74 +/- 7% saponifiable and inhibited by U-73122 (Ki = 2 microM).(ABSTRACT TRUNCATED AT 400 WORDS)

Brain-derived neurotrophic factor (BDNF) is a prototypic neurotrophin that regulates diverse developmental events from the selection of neural progenitors to the terminal dendritic differentiation and connectivity of neurons. We focus here on activity-dependent synaptic regulation by BDNF and its receptor, full length TrkB. BDNF-TrkB signaling is involved in transcription, translation, and trafficking of proteins during various phases of synaptic development and has been implicated in several forms of synaptic plasticity. These functions are carried out by a combination of the three signaling cascades triggered when BDNF binds TrkB: The mitogen-activated protein kinase (MAPK), the phospholipase Cgamma (PLC PLCgamma), and the phosphatidylinositol 3-kinase (PI3K) pathways. MAPK and PI3K play crucial roles in both translation and/or trafficking of proteins induced by synaptic activity, whereas PLCgamma regulates intracellular Ca(2+) that can drive transcription via cyclic AMP and a protein kinase C. Conversely, the abnormal regulation of BDNF is implicated in various developmental and neurodegenerative diseases that perturb neural development and function. We will discuss the current state of understanding BDNF signaling in the context of synaptic development and plasticity with a focus on the postsynaptic cell and close with the evidence that basic mechanisms of BDNF function still need to be understood to effectively treat genetic disruptions of these pathways that cause devastating neurodevelopmental diseases.

Cell-to-cell communication is a major process that allows bacteria to sense and coordinately react to the fluctuating conditions of the surrounding environment. In several pathogens, this process triggers the production of virulence factors and/or a switch in bacterial lifestyle that is a major determining factor in the outcome and severity of the infection. Understanding how bacteria control these signaling systems is crucial to the development of novel antimicrobial agents capable of reducing virulence while allowing the immune system of the host to clear bacterial infection, an approach likely to reduce the selective pressures for development of resistance. We provide here an up-to-date overview of the molecular basis and physiological implications of cell-to-cell signaling systems in Gram-negative bacteria, focusing on the well-studied bacterium Pseudomonas aeruginosa. All of the known cell-to-cell signaling systems in this bacterium are described, from the most-studied systems, i.e., N-acyl homoserine lactones (AHLs), the 4-quinolones, the global activator of antibiotic and cyanide synthesis (GAC), the cyclic di-GMP (c-di-GMP) and cyclic AMP (cAMP) systems, and the alarmones guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), to less-well-studied signaling molecules, including diketopiperazines, fatty acids (diffusible signal factor [DSF]-like factors), pyoverdine, and pyocyanin. This overview clearly illustrates that bacterial communication is far more complex than initially thought and delivers a clear distinction between signals that are quorum sensing dependent and those relying on alternative factors for their production.

The 2.9 A resolution crystal structure of Escherichia coli catabolite gene activator protein (CAP) complexed with cyclic AMP reveals two distinct structural domains separated by a cleft. The smaller carboxy-terminal domain is presumed to bind DNA while the amino-terminal domain is seen to bind cyclic AMP. Model building studies suggest that CAP binds to left-handed B-type DNA, contracting its major groove via two alpha-helices. It is possible that the CAP conversion of right- to left-handed DNA in a closed supercoil, is what activates transcription by RNA polymerase.

Availability of key nutrients, such as sugars, amino acids, and nitrogen compounds, dictates the developmental programs and the growth rates of yeast cells. A number of overlapping signaling networks--those centered on Ras/protein kinase A, AMP-activated kinase, and target of rapamycin complex I, for instance--inform cells on nutrient availability and influence the cells' transcriptional, translational, posttranslational, and metabolic profiles as well as their developmental decisions. Here I review our current understanding of the structures of the networks responsible for assessing the quantity and quality of carbon and nitrogen sources. I review how these signaling pathways impinge on transcriptional, metabolic, and developmental programs to optimize survival of cells under different environmental conditions. I highlight the profound knowledge we have gained on the structure of these signaling networks but also emphasize the limits of our current understanding of the dynamics of these signaling networks. Moreover, the conservation of these pathways has allowed us to extrapolate our finding with yeast to address issues of lifespan, cancer metabolism, and growth control in more complex organisms.

We previously proposed that the production of hyperglycemia-induced mitochondrial reactive oxygen species (mtROS) is a key event in the development of diabetes complications. The association between the pathogenesis of diabetes and its complications and mitochondrial biogenesis has been recently reported. Because metformin has been reported to exert a possible additional benefit in preventing diabetes complications, we investigated the effect of metformin and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) on mtROS production and mitochondrial biogenesis in cultured human umbilical vein endothelial cells. Treatment with metformin and AICAR inhibited hyperglycemia-induced intracellular and mtROS production, stimulated AMP-activated protein kinase (AMPK) activity, and increased the expression of peroxisome proliferator-activated response-gamma coactivator-1alpha (PGC-1alpha) and manganese superoxide dismutase (MnSOD) mRNAs. The dominant negative form of AMPKalpha1 diminished the effects of metformin and AICAR on these events, and an overexpression of PGC-1alpha completely blocked the hyperglycemia-induced mtROS production. In addition, metformin and AICAR increased the mRNA expression of nuclear respiratory factor-1 and mitochondrial DNA transcription factor A (mtTFA) and stimulated the mitochondrial proliferation. Dominant negative-AMPK also reduced the effects of metformin and AICAR on these observations. These results suggest that metformin normalizes hyperglycemia-induced mtROS production by induction of MnSOD and promotion of mitochondrial biogenesis through the activation of AMPK-PGC-1alpha pathway.

OBJECTIVE

Glucagon-like peptide-1 receptor (GLP-1R) agonists are used to treat type 2 diabetes, and transient GLP-1 administration improved cardiac function in humans after acute myocardial infarction (MI) and percutaneous revascularization. However, the consequences of GLP-1R activation before ischemic myocardial injury remain unclear.

METHODS

We assessed the pathophysiology and outcome of coronary artery occlusion in normal and diabetic mice pretreated with the GLP-1R agonist liraglutide.

RESULTS

Male C57BL/6 mice were treated twice daily for 7 days with liraglutide or saline followed by induction of MI. Survival was significantly higher in liraglutide-treated mice. Liraglutide reduced cardiac rupture (12 of 60 versus 46 of 60; P = 0.0001) and infarct size (21 +/- 2% versus 29 +/- 3%, P = 0.02) and improved cardiac output (12.4 +/- 0.6 versus 9.7 +/- 0.6 ml/min; P = 0.002). Liraglutide also modulated the expression and activity of cardioprotective genes in the mouse heart, including Akt, GSK3beta, PPARbeta-delta, Nrf-2, and HO-1. The effects of liraglutide on survival were independent of weight loss. Moreover, liraglutide conferred cardioprotection and survival advantages over metformin, despite equivalent glycemic control, in diabetic mice with experimental MI. The cardioprotective effects of liraglutide remained detectable 4 days after cessation of therapy and may be partly direct, because liraglutide increased cyclic AMP formation and reduced the extent of caspase-3 activation in cardiomyocytes in a GLP-1R-dependent manner in vitro.

CONCLUSIONS

These findings demonstrate that GLP-1R activation engages prosurvival pathways in the normal and diabetic mouse heart, leading to improved outcomes and enhanced survival after MI in vivo.