Peptide transporter DtpA has two alternate conformations, one of which is promoted by inhibitor binding

Peptide transporters (PTRs) of the large PTR family facilitate the uptake of di- and tripeptides to provide cells with amino acids for protein synthesis and for metabolic intermediates. Although several PTRs have been structurally and functionally characterized, how drugs modulate peptide transport remains unclear. To obtain insight into this mechanism, we characterize inhibitor binding to the Escherichia coli PTR dipeptide and tripeptide permease A (DtpA), which shows substrate specificities similar to its human homolog hPEPT1. After demonstrating that Lys[Z-NO2]-Val, the strongest inhibitor of hPEPT1, also acts as a high-affinity inhibitor for DtpA, we used single-molecule force spectroscopy to localize the structural segments stabilizing the peptide transporter and investigated which of these structural segments change stability upon inhibitor binding. This characterization was done with DtpA embedded in the lipid membrane and exposed to physiologically relevant conditions. In the unbound state, DtpA adopts two main alternate conformations in which transmembrane α-helix (TMH) 2 is either stabilized (in ∼43% of DtpA molecules) or not (in ∼57% of DtpA molecules). The two conformations are understood to represent the inward- and outward-facing conformational states of the transporter. With increasing inhibitor concentration, the conformation characterized by a stabilized TMH 2 becomes increasingly prevalent, reaching ∼92% at saturation. Our measurements further suggest that Lys[Z-NO2]-Val interacts with discrete residues in TMH 2 that are important for ligand binding and substrate affinity. These interactions in turn stabilize TMH 2, thereby promoting the inhibited conformation of DtpA.

Guineensine is a novel inhibitor of endocannabinoid uptake showing cannabimimetic behavioral effects in BALB/c mice

High-content screening led to the identification of the N-isobutylamide guineensine from Piper nigrum as novel nanomolar inhibitor (EC50 = 290 nM) of cellular uptake of the endocannabinoid anandamide (AEA). Noteworthy, guineensine did not inhibit endocannabinoid degrading enzymes fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL) nor interact with cannabinoid receptors or fatty acid binding protein 5 (FABP5), a major cytoplasmic AEA carrier. Activity-based protein profiling showed no inhibition of serine hydrolases. Guineensine also inhibited the cellular uptake of 2-arachidonoylglycerol (2-AG). Preliminary structure–activity relationships between natural guineensine analogs indicate the importance of the alkyl chain length interconnecting the pharmacophoric isobutylamide and benzodioxol moieties for AEA cellular uptake inhibition. Guineensine dose-dependently induced cannabimimetic effects in BALB/c mice shown by strong catalepsy, hypothermia, reduced locomotion and analgesia. The catalepsy and analgesia were blocked by the CB1 receptor antagonist rimonabant (SR141716A). Guineensine is a novel plant natural product which specifically inhibits endocannabinoid uptake in different cell lines independent of FAAH. Its scaffold may be useful to identify yet unknown targets involved in endocannabinoid transport.

Proteasome inhibitor (MG132) rescues Nav1.5 protein content and the cardiac sodium current in dystrophin-deficient mdx (5cv) mice

The cardiac voltage-gated sodium channel, Nav1.5, plays a central role in cardiac excitability and impulse propagation and associates with the dystrophin multiprotein complex at the lateral membrane of cardiomyocytes. It was previously shown that Nav1.5 protein content and the sodium current (l Na) were both decreased in cardiomyocytes of dystrophin-deficient mdx (5cv) mice. In this study, wild-type and mdx (5cv) mice were treated for 7 days with the proteasome inhibitor MG132 (10 μg/Kg/24 h) using implanted osmotic mini pumps. MG132 rescued both the total amount of Nav1.5 protein and l Na but, unlike in previous studies, de novo expression of dystrophin was not observed in skeletal or cardiac muscle. This study suggests that the reduced expression of Nav1.5 in dystrophin-deficient cells is dependent on proteasomal degradation.

C1 esterase inhibitor reduces lower extremity ischemia/reperfusion injury and associated lung damage

BACKGROUND Ischemia/reperfusion injury of lower extremities and associated lung damage may result from thrombotic occlusion, embolism, trauma, or surgical intervention with prolonged ischemia and subsequent restoration of blood flow. This clinical entity is characterized by high morbidity and mortality. Deprivation of blood supply leads to molecular and structural changes in the affected tissue. Upon reperfusion inflammatory cascades are activated causing tissue injury. We therefore tested preoperative treatment for prevention of reperfusion injury by using C1 esterase inhibitor (C1 INH). METHODS AND FINDINGS Wistar rats systemically pretreated with C1 INH (n = 6), APT070 (a membrane-targeted myristoylated peptidyl construct derived from human complement receptor 1, n = 4), vehicle (n = 7), or NaCl (n = 8) were subjected to 3h hind limb ischemia and 24h reperfusion. The femoral artery was clamped and a tourniquet placed under maintenance of a venous return. C1 INH treated rats showed significantly less edema in muscle (P<0.001) and lung and improved muscle viability (P<0.001) compared to controls and APT070. C1 INH prevented up-regulation of bradykinin receptor b1 (P<0.05) and VE-cadherin (P<0.01), reduced apoptosis (P<0.001) and fibrin deposition (P<0.01) and decreased plasma levels of pro-inflammatory cytokines, whereas deposition of complement components was not significantly reduced in the reperfused muscle. CONCLUSIONS C1 INH reduced edema formation locally in reperfused muscle as well as in lung, and improved muscle viability. C1 INH did not primarily act via inhibition of the complement system, but via the kinin and coagulation cascade. APT070 did not show beneficial effects in this model, despite potent inhibition of complement activation. Taken together, C1 INH might be a promising therapy to reduce peripheral ischemia/reperfusion injury and distant lung damage in complex and prolonged surgical interventions requiring tourniquet application.

Loss of Raf-1 kinase inhibitor protein (RKIP) is strongly associated with high-grade tumor budding and correlates with an aggressive phenotype in pancreatic ductal adenocarcinoma (PDAC)

BACKGROUND Raf-1 kinase inhibitor protein (RKIP) has emerged as a significant metastatic suppressor in a variety of human cancers and is known to inhibit Ras/Raf/MEK/ERK signaling. By suppressing the activation of the NFkB/SNAIL circuit, RKIP can regulate the induction of epithelial-mesenchymal transition (EMT). The aim of this study was to evaluate RKIP expression and to determine its association with clinicopathological features, including EMT in form of tumor budding in pancreatic ductal adenocarcinoma (PDAC). METHODS Staining for RKIP was performed on a multipunch Tissue Microarray (TMA) of 114 well-characterized PDACs with clinico-pathological, follow-up and adjuvant therapy information. RKIP-expression was assessed separately in the main tumor body and in the tumor buds. Another 3 TMAs containing normal pancreatic tissue, precursor lesions (Pancreatic Intraepithelial Neoplasia, PanINs) and matched lymph node metastases were stained in parallel. Cut-off values were calculated by receiver operating characteristic (ROC) curve analysis. RESULTS We found a significant progressive loss of RKIP expression between normal pancreatic ductal epithelia (average: 74%), precursor lesions (PanINs; average: 37%), PDAC (average 20%) and lymph node metastases (average 8%, p<0.0001). RKIP expression was significantly lower in tumor buds (average: 6%) compared to the main tumor body (average 20%; p<0.005). RKIP loss in the tumor body was marginally associated with advanced T-stage (p=0.0599) as well as high-grade peritumoral (p=0.0048) and intratumoral budding (p=0.0373). RKIP loss in the buds showed a clear association with advanced T stage (p=0.0089). CONCLUSIONS The progressive loss of RKIP seems to play a major role in the neoplastic transformation of pancreas, correlates with aggressive features in PDAC and is associated with the presence of EMT in form of tumor budding.

The Immune Inhibitor A1 Protease of Bacillus anthracis

Bacillus anthracis, an organism ubiquitous in the soil and the causative agent of anthrax, utilizes multiple mechanisms to regulate secreted factors; one example is the activity of secreted proteases. One of the most abundant proteins in the culture supernates of B. anthracis is the Immune Inhibitor A1 (InhA1) protease. Here, I demonstrate that InhA1 modulates the abundance of approximately half of the proteins secreted into the culture supernates, including substrates that are known to contribute to the ability of the organism to cause virulence. For example, InhA1 cleaves the anthrax toxin proteins, PA, LF, and EF. InhA1 also targets a number of additional proteases, including Npr599, contributing to a complex proteolytic regulatory cascade with far-reaching affects on the secretome. Using an intra-tracheal mouse model of infection, I found that an inhA-null strain is attenuated in relation to the parent strain. The data indicate that reduced virulence of the inhA mutant strain may be the result of toxin protein deregulation, decreased association with macrophages, and/or the inability to degrade host antimicrobial peptides. Given the significant modulation of the secretome by InhA1, it is likely that expression of the protease is tightly regulated. To test this I examined inhA1 transcript and protein levels in the parent and various isogenic mutant strains and found that InhA1 expression is regulated by several mechanisms. First, the steady state levels of inhA1 transcript are controlled by the regulatory protein SinR, which inhibits inhA1 expression. Second, InhA1 abundance is inversely proportional to the SinR-regulated protease camelysin, indicating the post-transcriptional regulation of InhA1 by camelysin. Third, InhA1 activity is dependent on a conserved zinc binding motif, suggesting that zinc availability regulates InhA1 activity. The convergence of these regulatory mechanisms signifies the importance of tight regulation of InhA1 activity, activity that substantially affects how B. anthracis interacts with its environment.

COCAINE DEPENDENCE: THE ROLE OF SEROTONIN GENES IN

Animal studies have shown that behavioral responses to cocaine-related cues are altered by serotonergic medications. The effects of pharmacological agents on serotonin receptors 2a (5-HT2A) and 2c (5-HT2C), have yielded results suggesting that selective 5-HT2A antagonists and 5-HT2C agonists promote the disruption of cocaine-associated memories. One measure of cocaine related cues in humans is attentional bias, in which cocaine dependent individuals show greater response latency for cocaine related words than neutral words. Data from our laboratory shows that cocaine dependent subjects have altered attentional bias compared to controls. The purpose of this thesis was to investigate the role of the serotonin system in attentional bias and impulsivity in cocaine dependent individuals. We focused on the serotonin transporter, serotonin receptors 2A and 2C and tryptophan hydroxylase 1 and 2 (TPH1 and TPH2). We predicted that attentional bias and impulsivity would be higher in cocaine dependent individuals who had lower serotonin function. In the current study, we found a significant association between TPH2 genotype and attentional bias for the second block of the cocaine Stroop task. There was also a significant association between average attentional bias and HTTLPR genotype in the cocaine dependent individuals. The HT2C receptor genotype and attentional bias in our study sample also showed a significant difference. We did not find a significant difference between the serotonin 2A receptor variants or the TPH1 variants and attentional bias in the cocaine dependent group. In conclusion, the current study suggests that serotonergic medications should be utilized as pharmacotherapeutic treatment for cocaine addiction. Our results indicate that TPH2, the serotonin transporter and 2C receptor should be targeted in such a way as to modulate both, leading to increased synaptic serotonin function.

LY2109761, a novel transforming growth factor beta receptor type I and type II dual inhibitor, as a therapeutic approach to suppressing pancreatic cancer metastasis.

Most pancreatic cancer patients present with inoperable disease or develop metastases after surgery. Conventional therapies are usually ineffective in treating metastatic disease. It is evident that novel therapies remain to be developed. Transforming growth factor beta (TGF-beta) plays a key role in cancer metastasis, signaling through the TGF-beta type I/II receptors (TbetaRI/II). We hypothesized that targeting TbetaRI/II kinase activity with the novel inhibitor LY2109761 would suppress pancreatic cancer metastatic processes. The effect of LY2109761 has been evaluated on soft agar growth, migration, invasion using a fibroblast coculture model, and detachment-induced apoptosis (anoikis) by Annexin V flow cytometric analysis. The efficacy of LY2109761 on tumor growth, survival, and reduction of spontaneous metastasis have been evaluated in an orthotopic murine model of metastatic pancreatic cancer expressing both luciferase and green fluorescence proteins (L3.6pl/GLT). To determine whether pancreatic cancer cells or the cells in the liver microenvironment were involved in LY2109761-mediated reduction of liver metastasis, we used a model of experimental liver metastasis. LY2109761 significantly inhibited the L3.6pl/GLT soft agar growth, suppressed both basal and TGF-beta1-induced cell migration and invasion, and induced anoikis. In vivo, LY2109761, in combination with gemcitabine, significantly reduced the tumor burden, prolonged survival, and reduced spontaneous abdominal metastases. Results from the experimental liver metastasis models indicate an important role for targeting TbetaRI/II kinase activity on tumor and liver microenvironment cells in suppressing liver metastasis. Targeting TbetaRI/II kinase activity on pancreatic cancer cells or the cells of the liver microenvironment represents a novel therapeutic approach to prevent pancreatic cancer metastasis.

Functional analysis of the amine substrate specificity domain of pepper tyramine and serotonin N-hydroxycinnamoyltransferases.

Pepper (Capsicum annuum) serotonin N-hydroxycinnamoyltransferase (SHT) catalyzes the synthesis of N-hydroxycinnamic acid amides of serotonin, including feruloylserotonin and p-coumaroylserotonin. To elucidate the domain or the key amino acid that determines the amine substrate specificity, we isolated a tyramine N-hydroxycinnamoyltransferase (THT) gene from pepper. Purified recombinant THT protein catalyzed the synthesis of N-hydroxycinnamic acid amides of tyramine, including feruloyltyramine and p-coumaroyltyramine, but did not accept serotonin as a substrate. Both the SHT and THT mRNAs were found to be expressed constitutively in all pepper organs. Pepper SHT and THT, which have primary sequences that are 78% identical, were used as models to investigate the structural determinants responsible for their distinct substrate specificities and other enzymatic properties. A series of chimeric genes was constructed by reciprocal exchange of DNA segments between the SHT and THT cDNAs. Functional characterization of the recombinant chimeric proteins revealed that the amino acid residues 129 to 165 of SHT and the corresponding residues 125 to 160 in THT are critical structural determinants for amine substrate specificity. Several amino acids are strongly implicated in the determination of amine substrate specificity, in which glycine-158 is involved in catalysis and amine substrate binding and tyrosine-149 plays a pivotal role in controlling amine substrate specificity between serotonin and tyramine in SHT. Furthermore, the indisputable role of tyrosine is corroborated by the THT-F145Y mutant that uses serotonin as the acyl acceptor. The results from the chimeras and the kinetic measurements will direct the creation of additional novel N-hydroxycinnamoyltransferases from the various N-hydroxycinnamoyltransferases found in nature.

THE HISTONE DEACETYLASE INHIBITOR, MS-275, SENSITIZES METASTATIC OSTEOSARCOMA TO FASL-INDUCED CELL DEATH: A ROLE FOR C-FLIP

The purpose of this study was to determine the effects of the histone deacetylase inhibitor, MS-275, on the Fas signaling pathway and susceptibility of osteosarcoma (OS) to Fas ligand (FasL)-induced cell death. OS metastasizes almost exclusively to the lungs. We have shown that Fas expression in OS cells is inversely correlated with their metastatic potential. Fas+ cells are rapidly eliminated when they enter the lungs via interaction with FasL, which is constitutively expressed in the lungs. Fas- OS cells escape this FasL-induced apoptosis and survive in the lung microenvironment. Moreover, upregulation of Fas in established OS lung metastases results in tumor regression. Therefore, agents that upregulate Fas expression or activate the Fas signaling pathway may have therapeutic potential. Treatment of Fas- metastatic OS cell lines with 2 μM MS-275 sensitized cells to FasL-induced cell death in vitro. We found that MS-275 did not alter the expression of Fas on the cell surface; rather it resulted in increased levels of Fas within the membrane lipid rafts, as demonstrated by an increase in Fas expression in detergent insoluble lipid raft fractions. We further demonstrated that following MS-275 treatment, Fas colocalized with GM1+ lipid rafts and that there was a decrease in c-FLIP (cellular FLICE-inhibitory protein) mRNA and protein. Downregulation of c-FLIP correlated with caspase activation and apoptosis induction. Transfection of cells with shRNA to c-FLIP also resulted in the localization of Fas to lipid rafts. These studies indicate that MS-275 sensitizes OS cells to FasL by upregulating the expression of Fas in membrane lipid rafts, which correlated with the downregulation of c-FLIP. Treatment of nu/nu-mice with established OS lung metastases with oral MS-275 resulted in increased apoptosis, a significant inhibition of c-FLIP expression in tumors and tumor regression. Histopathological examination of mice showed no significant organ toxicity. Overall, these results suggest that the mechanism by which MS-275 sensitizes OS cells and lung metastases to FasL-induced cell death may be by a reduction in the expression of c-FLIP.

Role of protein kinase C in short-term sensitization of {\it Aplysia}

Plasticity at the connections between sensory neurons and their follower cells in Aplysia has been used extensively as a model system to examine mechanisms of simple forms of learning, such as sensitization. Sensitization is induced, at least in part, by the transmitter serotonin (5-HT) and expressed in several forms, including facilitation of sensorimotor connections. Spike broadening has been believed to be a key mechanism underlying facilitation of nondepressed synapses. Previously, this broadening was believed to be dependent primarily on cAMP/protein kinase A (PKA)-mediated reduction of a noninactivating, relatively voltage-independent K$\sp{+}$ current termed the S-K$\sp+$ current (I$\sb{\rm K{,}S}$). Recent evidence, however, suggests that 5-HT-induced somatic spike broadening is composed of at least two components: a cAMP-dependent, rapidly developing component and a cAMP-independent, slowly developing component.^ Phorbol esters, activators of protein kinase C (PKC), mimicked the cAMP-independent component of 5-HT-induced broadening. Staurosporine, which inhibits PKC, had little effect on the rapidly developing component of 5-HT-induced broadening, but inhibited significantly the slowly developing component. These results suggest that PKC is involved in the cAMP-independent component of 5-HT-induced broadening. The membrane currents responsible for the slowly developing component of broadening were examined. Activation of PKC mimicked, and partially occluded, 5-HT-induced modulation of membrane currents above 0 mV, where a voltage-dependent K$\sp+$ current (I$\sb{\rm K{,}V}$) is significantly activated. This modulation was complex because it was associated with a reduction in the magnitude of I$\sb{\rm K{,}V}$, as well as a slowing of both activation and inactivation kinetics of I$\sb{\rm K{,}V}$. These results support the hypothesis that PKC modulates I$\sb{\rm K{,}V}$ and that this modulation contributes to the slowly developing component of 5-HT-induced broadening. Based on these results and others, a new scheme for 5-HT-induced spike broadening is proposed in which the modulatory effects are mediated via two second messenger/protein kinase systems converging and diverging on multiple ionic conductances.^ The relationship between spike broadening and synaptic facilitation was also examined. Pharmacological reduction of I$\sb{\rm K{,}V}$ by low concentrations of 4-aminopyridine (4-AP) led to spike broadening and facilitation of the nondepressed sensorimotor connections, indicating that spike broadening via the reduction of I$\sc{K,V}$ can facilitate the synaptic connection. Further analyses, however, revealed that 4-AP-induced facilitation has qualitative differences from 5-HT- and PKC-induced facilitation. These results suggest that 5-HT- and PKC-induced facilitation of nondepressed synapses is mediated, at least in part, by spike-duration independent (SDI) processes. Under certain conditions, the PKC inhibitor, staurosporine, significantly inhibited the 5-HT-induced facilitation of sensorimotor connections.^ Finally, it was found that activation of PKC increased a basal level of cAMP and that PKC caused desensitization of the 5-HT receptor, which may be a possible negative feedback mechanism through which an extracellular ligand, 5-HT, is regulated. These results suggest that these two second messenger/protein kinase pathways can interact in the sensory neuron. Thus, neuronal plasticity that may contribute to learning and memory appears to involve several complex and interactive processes. ^

Aggression, impulsivity and serotonin in drug dependence

Aggression, impulsivity, and central serotonergic function were evaluated in two groups of human volunteers; one group having a history of substance dependence (DRUG+) and another group with no drug use history (DRUG$-$). The hypothesis was that DRUG+ subjects would be more aggressive, more impulsive, and have attenuated serotonergic function. Results showed that DRUG+ subjects behaved more aggressively in a computer paradigm of aggression and also reported more aggression on questionnaires than DRUG$-$ subjects. In a computer paradigm of impulsivity, the DRUG+ group showed a lesser ability to delay gratification than the DRUG$-$ group in the last session of testing. The DRUG+ subjects also reported more venturesomeness and problems associated with low impulse control on questionnaires. Serotonergic function was measured through the neuroendocrine and hypothermic response to an orally administered serotonin (5-HT) agonist specific to the 5-HT$\rm\sb{1A}$ receptor subtype (ipsapirone). The neuroendocrine responses did not differ between DRUG$\pm$ groups, indicating no difference in the sensitivity of the presynaptic or postsynaptic 5-HT$\rm\sb{1A}$ receptors. An unexpected result was that the indicator hormone, cortisol, was at a lower baseline level in the DRUG+ group than the DRUG$-$ group. Lowered cortisol levels have been previously noted in children at high risk foul antisociality and future drug use. A principal components analysis including impulsivity, aggression, and serotonergic function measures produced three unique factors. The factors, Antisocial Tendency and Self-Control and Serotonergic Function combined to produce a significant regression equation explaining 36% of variability in the DRUG$\pm$ groups. These factors included measures of aggression, impulsivity, mood, and educational attainment. These results suggest that the current measures of aggression and impulsivity were predictive of a drug dependence disorder but that neuroendocrine function is not yet a useful indicator of drug dependence status. ^

Analysis of pp60$\sp{{\rm c}{-}src}$ and pp62$\sp{{\rm c}{-}yes}$ intracellular protein tyrosine kinase function in colon tumor cell growth regulation using herbimycin A, a tyrosine kinase specific inhibitor

Two approaches were utilized to investigate the role of pp60c-src activation in growth control of model colon tumor cell lines. The first approach involved analysis of pp60c-src activity in response to growth factor treatment to determine if transient activation of the protein was associated with ligand induced mitogenic signal transduction as occurs in non-colonic cell types. Activation of pp60c-src was detected using colon tumor cell lysates after treatment with platelet derived growth factor (PDGF). Activation of pp60c-src was also detected in response to epidermal growth factor (EGF) treatment using cellular lysates and intact cells. In contrast, down-regulation of purified pp60c-src occurred after incubation with EGF-treated EGFr immune complexes in vitro suggesting additional cellular events were potentially required for the stimulatory response observed in intact cells. The results demonstrated activation of pp60c-src in colon tumor cells in response to PDGF and EGF which is consistent with the role of the protein in mitogenic signal transduction in non-colonic cell types.^ The second approach used to study the role of pp60c-src activation in colonic cell growth control focused on analysis of the role of constitutive activation of the protein, which occurs in approximately 80% of colon tumors and cell lines, in growth control. These studies involved analysis of the effects of the tyrosine kinase specific inhibitor Herbimycin A (HA) on monolayer growth and pp60c-src enzymatic activity using model colon tumor cell lines. HA induced dose-dependent growth inhibition of all colon tumor cell lines examined possessing elevated pp60c-src activity. In HT29 cells the dose-dependent growth inhibition induced by HA correlated with dose-dependent pp60c-src inactivation. Inactivation of pp60c-src was shown to be an early event in response to treatment with HA which preceded induction of HT29 colon tumor cell growth inhibition. The growth effects of HA towards the colon tumor cells examined did not appear to be associated with induction of differentiation or a cytotoxic mechanism of action as changes in morphology were not detected in treated cells and growth inhibition (and pp60c-src inactivation) were reversible upon release from treatment with the compound. The results suggested the constitutive activation of pp60c-src functioned as a proliferative signal in colon tumor cells. Correlation between pp60c-src inactivation and growth inhibition was also observed using HA chemical derivatives confirming the role of tyrosine kinase inactivation by these compounds in inhibition of mitogenic signalling. In contrast, in AS15 cells possessing specific antisense mRNA mediated inactivation of pp60c-src, HA-induced inactivation of the related pp62c-yes tyrosine kinase, which is also activated during colon tumor progression, was not associated with induction of monolayer growth inhibition. These results suggested a function for the constitutively activated pp62c-yes protein in colon tumor cell proliferation which was different from that of activated pp60c-src. (Abstract shortened by UMI.) ^

Long-term synaptic facilitation of tail sensorimotor connections in {\it Aplysia\/}

Long-term sensitization in Aplysia is a well studied model for the examination of the cellular and molecules mechanisms of long-term memory. Several lines of evidence suggest long-term sensitization is mediated at least partially by long-term synaptic facilitation between the sensory and motor neurons. The sensitization training and one of its analogues, serotonin (5-HT), can induce long-term facilitation. In this study, another analogue to long-term sensitization training has been developed. Stimulation of peripheral nerves of pleural-pedal ganglia preparation induced long-term facilitation at both 24 hr and 48 hr. This is the first report that long-term facilitation in Aplysia persists for more than 24 hr, which is consistent with the observation that long-term sensitization lasts for more than one day. Thus, the data support the hypothesis that long-term facilitation is an important mechanism for long-term sensitization.^ One of the major differences between short-term and long-term facilitation is that long-term facilitation requires protein synthesis. Therefore, the effects of anisomycin, a protein synthesis inhibitor, on long-term facilitation was examined. Long-term facilitation induced by nerve stimulation was inhibited by 2 $\mu$M anisomycin, which inhibits $\sim$90% of protein synthesis. Nevertheless, at higher concentration (20 $\mu$M), anisomycin induced long-term facilitation by itself, which raises an interesting question about the function of anisomycin other than protein synthesis inhibition.^ Since protein synthesis is critical for long-term facilitation, a major goal is to identify and functionally characterize the molecules whose mRNA levels are altered during the formation of long-term facilitation. Behavioral training or its analogues (nerve stimulation and 5-HT) increases the level of mRNA of calmodulin (CaM). Thus, the role of Ca$\sp{2+}$-CaM-dependent protein kinase II (CaMKII), a major substrate of CaM, in long-term facilitation induced by nerve stimulation was examined. KN-62, a specific CaMKII inhibitor, did not block either the induction or the maintenance of long-term facilitation induced by nerve stimulation. These data indicate that CaMKII may not be involved in long-term facilitation. Another protein whose mRNA level of a molecule was increased by the behavioral training and the treatment of 5-HT is Aplysia tolloid/BMP-1-like protein 1 (apTBL-1). Tolloid in Drosophila and BMP-1 in human tissues are believed to be secreted as a metalloprotease to activate TGF-$\beta.$ Thus, the long-term effects of recombinant human TGF-$\beta1$ on synaptic strength were examined. Treatment of ganglia with TGF-$\beta1$ produced long-term facilitation, but not short-term or intermediate-term facilitation ($\le$4 hr). In addition, TGF-$\beta1$ and 5-HT were not additive in producing long-term facilitation, which indicates an interaction between two cascades. Moreover, 5-HT-induced facilitation (at both 24 hr and 48 hr) and nerve stimulation-induced facilitation (at 24 hr) were inhibited by TGF-$\beta$ sRII, a TGF-$\beta$ inhibitor. These results suggest that TGF-$\beta$ is part of the cascade of events underlying long-term sensitization, and also indicate that a signaling molecule used in development may also have functions in adult neuronal plasticity. ^

Plasminogen activator inhibitor-1, monocyte chemoattractant protein-1, e-selectin and C-reactive protein levels in response to 4-week very-high-fructose or -glucose diets.

BACKGROUND/OBJECTIVES High intake of added sweeteners is considered to have a causal role in the pathogenesis of cardiometabolic disorders. Especially, high-fructose intake is regarded as potentially harmful to cardiometabolic health. It may cause not only weight gain but also low-grade inflammation, which represents an independent risk factor for developing type 2 diabetes and cardiovascular disease. In particular, fructose has been suggested to induce plasminogen activator inhibitor-1 (PAI-1) expression in the liver and to increase circulating inflammatory cytokines. We therefore aimed to investigate, whether high-fructose diet has an impact on PAI-1, monocyte chemoattractant protein-1 (MCP-1), e-selectin and C-reactive protein (CRP) concentrations in healthy humans. SUBJECTS/METHODS We studied 20 participants (12 males and 8 females) of the TUebingen FRuctose Or Glucose study. This is an exploratory, parallel, prospective, randomized, single-blinded, outpatient, hypercaloric, intervention study. The participants had a mean age of 30.9 ± 2.1 years and a mean body mass index of 26.0 ± 0.5 kg/m(2) and they received 150 g of either fructose or glucose per day for 4 weeks.Results:There were neither significant changes of PAI-1, MCP-1, e-selectin and CRP after fructose (n=10) and glucose (n=10) intervention nor treatment effects (all P>0.2). Moreover, we did not observe longitudinal associations of the inflammatory parameters with triglycerides, liver fat, visceral fat and body weight in the fructose group. CONCLUSIONS Temporary high-fructose intake does not seem to cause inflammation in apparently healthy people in this secondary analysis of a small feeding trial.