Oomycete plant pathogens deliver effector proteins inside host cells to modulate plant defense circuitry and to enable parasitic colonization. These effectors are defined by a conserved motif, termed RXLR (for Arg, any amino acid, Leu, Arg), that is located downstream of the signal peptide and that has been implicated in host translocation. Because the phenotypes of RXLR effectors extend to plant cells, their genes are expected to be the direct target of the evolutionary forces that drive the antagonistic interplay between pathogen and host. We used the draft genome sequences of three oomycete plant pathogens, Phytophthora sojae, Phytophthora ramorum, and Hyaloperonospora parasitica, to generate genome-wide catalogs of RXLR effector genes and determine the extent to which these genes are under positive selection. These analyses revealed that the RXLR sequence is overrepresented and positionally constrained in the secretome of Phytophthora relative to other eukaryotes. The three examined plant pathogenic oomycetes carry complex and diverse sets of RXLR effector genes that have undergone relatively rapid birth and death evolution. We obtained robust evidence of positive selection in more than two-thirds of the examined paralog families of RXLR effectors. Positive selection has acted for the most part on the C-terminal region, consistent with the view that RXLR effectors are modular, with the N terminus involved in secretion and host translocation and the C-terminal domain dedicated to modulating host defenses inside plant cells.

Atrial natriuretic peptide (ANP) receptors have been described on rodent adipocytes and expression of their mRNA is found in human adipose tissue. However, no biological effects associated with the stimulation of these receptors have been reported in this tissue. A putative lipolytic effect of natriuretic peptides was investigated in human adipose tissue. On isolated fat cells, ANP and brain natriuretic peptide (BNP) stimulated lipolysis as much as isoproterenol, a nonselective beta-adrenergic receptor agonist, whereas C-type natriuretic peptide (CNP) had the lowest lipolytic effect. In situ microdialysis experiments confirmed the potent lipolytic effect of ANP in abdominal s.c. adipose tissue of healthy subjects. A high level of ANP binding sites was identified in human adipocytes. The potency order defined in lipolysis (ANP>> BNP>> CNP) and the ANP-induced cGMP production sustained the presence of type A natriuretic peptide receptor in human fat cells. Activation or inhibition of cGMP-inhibited phosphodiesterase (PDE-3B) (using insulin and OPC 3911, respectively) did not modify ANP-induced lipolysis whereas the isoproterenol effect was decreased or increased. Moreover, inhibition of adenylyl cyclase activity (using a mixture of alpha(2)-adrenergic and adenosine A1 agonists receptors) did not change ANP- but suppressed isoproterenol-induced lipolysis. The noninvolvement of the PDE-3B was finally confirmed by measuring its activity under ANP stimulation. Thus, we demonstrate that natriuretic peptides are a new pathway controlling human adipose tissue lipolysis operating via a cGMP-dependent pathway that does not involve PDE-3B inhibition and cAMP production.

The binding of the human immunodeficiency virus (HIV) envelope glycoprotein gp120 to the cell surface receptor CD4 has been considered a primary determinant of viral tropism. A number of cell types, however, can be infected by the virus, or bind gp120, in the absence of CD4 expression. Human placenta was identified as a tissue that binds gp120 in a CD4-independent manner. A placental cDNA library was screened by expression cloning and a cDNA (clone 11) encoding a gp120-binding protein unrelated to CD4 was isolated. The 1.3-kilobase cDNA predicts a protein of 404 amino acids with a calculated M(r) of 45,775 and organized into three domains: an N-terminal cytoplasmic and hydrophobic region, a set of seven complete and one incomplete tandem repeat, and a C-terminal domain with homology to C-type (calcium-dependent) lectins. A type II membrane orientation (N-terminal cytoplasmic) is predicted both by the cDNA sequence and by the reactivity of C-terminal peptide-specific antiserum with the surface of clone 11 transfected cells. Native and recombinant gp120 and whole virus bind transfected cells. gp120 binding is high affinity (kd, 1.3-1.6 nM) and inhibited by mannan, D-mannose, and L-fucose; once bound, gp120 is internalized rapidly. Collectively, these data demonstrate that the gp120-binding protein is a membrane-associated mannose-binding lectin. Proteins of this type may play an important role in the CD4-independent association of HIV with cells.

USP7/HAUSP is a key regulator of p53 and Mdm2 and is targeted by the Epstein-Barr nuclear antigen 1 (EBNA1) protein of Epstein-Barr virus (EBV). We have determined the crystal structure of the p53 binding domain of USP7 alone and bound to an EBNA1 peptide. This domain is an eight-stranded beta sandwich similar to the TRAF-C domains of TNF-receptor associated factors, although the mode of peptide binding differs significantly from previously observed TRAF-peptide interactions in the sequence (DPGEGPS) and the conformation of the bound peptide. NMR chemical shift analyses of USP7 bound by EBNA1 and p53 indicated that p53 binds the same pocket as EBNA1 but makes less extensive contacts with USP7. Functional studies indicated that EBNA1 binding to USP7 can protect cells from apoptotic challenge by lowering p53 levels. The data provide a structural and conceptual framework for understanding how EBNA1 might contribute to the survival of Epstein-Barr virus-infected cells.

The improper activation of the Abl tyrosine kinase results in chronic myeloid leukemia (CML). The recognition of an inactive conformation of Abl, in which a catalytically important Asp-Phe-Gly (DFG) motif is flipped by approximately 180 degrees with respect to the active conformation, underlies the specificity of the cancer drug imatinib, which is used to treat CML. The DFG motif is not flipped in crystal structures of inactive forms of the closely related Src kinases, and imatinib does not inhibit c-Src. We present a structure of the kinase domain of Abl, determined in complex with an ATP-peptide conjugate, in which the protein adopts an inactive conformation that resembles closely that of the Src kinases. An interesting aspect of the Src-like inactive structure, suggested by molecular dynamics simulations and additional crystal structures, is the presence of features that might facilitate the flip of the DFG motif by providing room for the phenylalanine to move and by coordinating the aspartate side chain as it leaves the active site. One class of mutations in BCR-Abl that confers resistance to imatinib appears more likely to destabilize the inactive Src-like conformation than the active or imatinib-bound conformations. Our results suggest that interconversion between distinctly different inactive conformations is a characteristic feature of the Abl kinase domain.

The persistent activation of signaling cascades results in dramatic consequences that include loss of cellular growth control and neoplastic transformation. We show here that phosphoinositide 3-kinase (PI 3-kinase) and its mediator Akt were constitutively activated in pancreatic cancer and that this might be due to the aberrant expression of their natural antagonist MMAC/PTEN. Indeed, our results show that MMAC/PTEN expression was either lost or significantly reduced in five of eight cell lines and in twelve of seventeen tumor specimens examined. That the poor expression of MMAC/PTEN in pancreatic cancer cells could be due to promoter methylation was indicated by methylation-specific PCR analysis. Our studies also indicated that PI 3-kinase targeted two important transcription factors in pancreatic cancer cells. The ability of constitutively activated NF-kappaB to induce gene expression and the stabilization of c-MYC protein by decreased phosphorylation of Thr58 were both dependent on PI 3-kinase activity. When pancreatic cancer cells were treated with a peptide antagonist of NF-kappaB nuclear translocation, or stably transfected with a dominant-negative mutant of MYC, their proliferation was markedly inhibited. Taken together, these data indicate that the aberrant expression of MMAC/PTEN contributes to the activation of the PI 3-kinase/Akt pathway and its transcription factor mediators in pancreatic cancer.

BACKGROUND

Clinical studies have demonstrated that 50% of individuals with chronic renal disease (CRD) die of cardiovascular causes, including advanced calcific arterial and valvular disease; however, the mechanisms of accelerated calcification in CRD remain obscure, and no therapies can prevent disease progression. We recently demonstrated in vivo that inflammation triggers cardiovascular calcification. In vitro evidence also indicates that elastin degradation products may promote osteogenesis. Here, we used genetically modified mice and molecular imaging to test the hypothesis in vivo that cathepsin S (catS), a potent elastolytic proteinase, accelerates calcification in atherosclerotic mice with CRD induced by 5/6 nephrectomy.

RESULTS

Apolipoprotein-deficient (apoE(-/-))/catS(+/+) (n=24) and apoE(-/-)/catS(-/-) (n=24) mice were assigned to CRD and control groups. CRD mice had significantly higher serum phosphate, creatinine, and cystatin C levels than those without CRD. To visualize catS activity and osteogenesis in vivo, we coadministered catS-activatable and calcification-targeted molecular imaging agents 10 weeks after nephrectomy. Imaging coregistered increased catS and osteogenic activities in the CRD apoE(-/-)/catS(+/+) cohort, whereas CRD apoE(-/-)/catS(-/-) mice exhibited less calcification. Quantitative histology demonstrated greater catS-associated elastin fragmentation and calcification in CRD apoE(-/-)/catS(+/+) than CRD apoE(-/-)/catS(-/-) aortas and aortic valves. Notably, catS deletion did not cause compensatory increases in RNA levels of other elastolytic cathepsins or matrix metalloproteinases. Elastin peptide and recombinant catS significantly increased calcification in smooth muscle cells in vitro, a process further amplified in phosphate-enriched culture medium.

CONCLUSIONS

The present study provides direct in vivo evidence that catS-induced elastolysis accelerates arterial and aortic valve calcification in CRD, providing new insight into the pathophysiology of cardiovascular calcification.

In the present study, the 26-residue peptide sequence Ac-KWKSFLKTFKSAVKTVLHTALKAISS-amide (V681) was utilized as the framework to study the effects of peptide hydrophobicity/hydrophilicity, amphipathicity, and helicity (induced by single amino acid substitutions in the center of the polar and nonpolar faces of the amphipathic helix) on biological activities. The peptide analogs were also studied by temperature profiling in reversed-phase high performance liquid chromatography, from 5 to 80 degrees C, to evaluate the self-associating ability of the molecules in solution, another important parameter in understanding peptide antimicrobial and hemolytic activities. A higher ability to self-associate in solution was correlated with weaker antimicrobial activity and stronger hemolytic activity of the peptides. Biological studies showed that strong hemolytic activity of the peptides generally correlated with high hydrophobicity, high amphipathicity, and high helicity. In most cases, the D-amino acid substituted peptides possessed an enhanced average antimicrobial activity compared with L-diastereomers. The therapeutic index of V681 was improved 90- and 23-fold against Gram-negative and Gram-positive bacteria, respectively. By simply replacing the central hydrophobic or hydrophilic amino acid residue on the nonpolar or the polar face of these amphipathic derivatives of V681 with a series of selected D-/L-amino acids, we demonstrated that this method has excellent potential for the rational design of antimicrobial peptides with enhanced activities.

Superoxide is among the most abundant reactive oxygen species (ROS) produced by the mitochondria, and is involved in cellular signaling pathways. Superoxide and other ROS can damage cellular macromolecules and levels of oxidative damage products are positively correlated with aging. Superoxide dismutase (SOD) enzymes catalyze the breakdown of superoxide into hydrogen peroxide and water and are therefore central regulators of ROS levels. Genetic and transgenic manipulation of SOD activities in model systems such as S. cereviseae, mouse and Drosophila are consistent with a central role for SOD enzymes in regulating oxidative stress resistance. Over-expression of SOD in S. cereviseae and Drosophila can reduce oxidative damage and extend life span, but the mechanism(s) are not yet clear. A phylogenetic analysis of publicly available SOD protein sequences suggests several additional conserved gene families. For example, in addition to the well-characterized soluble Cu/Zn enzyme (Sod) and mitochondrial manganese-containing form (Sod2), Drosophila melanogaster is found to contain a putative copper chaperone (CCS), an extracellular Cu/Zn enzyme (Sod3), and an extracellular protein distantly related to the Cu/Zn forms (Sodq). C. elegans and blue crab are unusual in having two Mn-containing SODs, and A. gambiae contains an unusual internally repeated SOD. The most parsimonius conclusion from the analysis of the extracellular SODs is that they evolved independently multiple times by addition of a signal peptide to cytoplasmic SOD.

BACKGROUND

Local delivery of chemotactic factors represents a novel approach to tissue regeneration. However, successful chemokine protein delivery is challenged by barriers including the rapid diffusion of chemokines and cleavage of chemokines by proteases that are activated in injured tissues. Stromal cell-derived factor-1 (SDF-1) is a well-characterized chemokine for attracting stem cells and thus a strong candidate for promoting regeneration. However, SDF-1 is cleaved by exopeptidases and matrix metalloproteinase-2, generating a neurotoxin implicated in some forms of dementia.

RESULTS

We designed a new chemokine called S-SDF-1(S4V) that is resistant to matrix metalloproteinase-2 and exopeptidase cleavage but retains chemotactic bioactivity, reducing the neurotoxic potential of native SDF-1. To deliver S-SDF-1(S4V), we expressed and purified fusion proteins to tether the chemokine to self-assembling peptides, which form nanofibers and allow local delivery. Intramyocardial delivery of S-SDF-1(S4V) after myocardial infarction recruited CXCR4+/c-Kit+ stem cells (46+/-7 to 119+/-18 cells per section) and increased capillary density (from 169+/-42 to 283+/-27 per 1 mm2). Furthermore, in a randomized, blinded study of 176 rats with myocardial infarction, nanofiber delivery of the protease-resistant S-SDF-1(S4V) improved cardiac function (ejection fraction increased from 34.0+/-2.5% to 50.7+/-3.1%), whereas native SDF-1 had no beneficial effects.

CONCLUSIONS

The combined advances of a new, protease-resistant SDF-1 and nanofiber-mediated delivery promoted recruitment of stem cells and improved cardiac function after myocardial infarction. These data demonstrate that driving chemotaxis of stem cells by local chemokine delivery is a promising new strategy for tissue regeneration.

The preferential in vitro methylation of histone H3 by coactivator-associated arginine methyltransferase 1 (CARM1) has been proposed as a basis for its ability to enhance gene transcription [Chen, D., et al. (1999) Science 284, 2174-2177]. To further evaluate the significance of H3 methylation, we studied the kinetics and site specificity of its modification by CARM1. Affinity-purified CARM1 methylated recombinant chick H3, which is free of posttranslational modifications, and calf thymus H3, which is heterogeneous with regard to preexisting modifications, equally well, exhibiting a V(max) of 4500 pmol min(-1) (mg of enzyme)(-1) and an apparent K(m) for H3 of < or = 0.2 microM. The catalytic efficiency (k(cat)/K(m)) of CARM1 toward H3 was at least 1000 times that toward R1 (GGFGGRGGFGG-amide), a highly effective substrate for protein arginine methyltransferase 1. Peptide mapping of 3H-methyl-labeled H3 indicated methylation at Arg-2, Arg-17, and Arg-26 in the N-terminal region and at one or more of four arginines (128/129/131/134) at the C-terminus. Two of the N-terminal sites, Arg-17 and Arg-26, occur in the sequence KAXRK and appear to be more efficiently methylated than Arg-2. CARM1 catalyzed formation of N(G),N(G)-dimethylarginine (asymmetric) but little or no N(G),N'(G)-dimethylarginine (symmetric) and no form of methyllysine. Amino acid analysis of untreated calf thymus H3 revealed that 3.7% of the molecules naturally contain asymmetric dimethylarginine and/or monomethylarginine. Our findings support the hypothesis that methylation of H3 may be involved in the mechanism of transcriptional coactivation by CARM1 of genes whose expression is under the control of nuclear receptors.

OBJECTIVE

The aim of the study was to examine the 48 month outcome of treating recent-onset type 1 diabetic patients for 6 days with humanised CD3-antibody, ChAglyCD3.

METHODS

Eighty patients, aged 12-39 years, were recruited for a phase 2 multicentre trial and randomised to placebo (n=40) or ChAglyCD3 (n=40) treatment by a third party member; participants and care-givers were blinded. The change in insulin dose (U kg(-1)day(-1)) over 48 months was chosen as primary endpoint and compared in 31 placebo-and 33 ChAglyCD3-treated patients. Adverse events were followed in 35 and 38 patients, respectively.

RESULTS

Treatment with ChAglyCD3 delayed the rise in insulin requirements of patients with recent-onset diabetes and reduced its amplitude over 48 months (+0.09 vs +0.32 U kg(-1)day(-1) in the placebo group). Using multivariate analysis this effect was correlated with higher baseline residual beta cell function and a younger age. It was associated with better outcome variables in subgroups selected according to these variables. In the ChAglyCD3 subgroup with higher initial beta cell function, 0/11 patients became C-peptide-negative over 48 months vs 4/9 in the corresponding placebo subgroup. In the subgroup aged <27 years old, antibody treatment preserved initial beta cell function for 36 months (vs >80% decline within 24 months in the placebo subgroup <27 years old), resulted in lower HbA1c concentrations and tended to reduce glycaemic variability (p=0.08). No longterm adverse events were observed.

CONCLUSIONS

A 6 day ChAglyCD3 treatment can suppress the rise in insulin requirements of recent-onset type 1 diabetic patients over 48 months, depending on their age and initial residual beta cell function. In younger patients this effect is associated with reduced deterioration of metabolic variables. These observations help to define inclusion criteria for prevention trials.

BACKGROUND

ClinicalTrials.gov NCT00627146

BACKGROUND

Center grants from the Juvenile Diabetes Research Foundation (4-2001-434, 4-2005-1327) and grants from the Belgian Fund for Scientific Research-Flanders and from Brussels Free University-VUB.

The deficits characteristic of Alzheimer's disease (AD) are believed to result, at least in part, from the neurotoxic effects of beta-amyloid peptides, a set of 39-43 amino acid fragments derived proteolytically from beta-amyloid precursor protein (APP). APP also is cleaved intracytoplasmically at Asp-664 to generate a second cytotoxic peptide, APP-CC-terminal processing of APP plays a role in the pathogenesis of AD is unknown. Therefore, we compared elements of the Alzheimer's phenotype in transgenic mice modeling AD with vs. without a functional Asp-664 caspase cleavage site. Surprisingly, whereas beta-amyloid production and plaque formation were unaltered, synaptic loss, astrogliosis, dentate gyral atrophy, increased neuronal precursor proliferation, and behavioral abnormalities were completely prevented by a mutation at Asp-664. These results suggest that Asp-664 plays a critical role in the generation of Alzheimer-related pathophysiological and behavioral changes in human APP transgenic mice, possibly as a cleavage site or via protein-protein interactions.

BACKGROUND

Cirrhosis is a diffuse process of hepatic fibrosis and regenerative nodule formation of unknown pathogenesis. Transforming growth factor (TGF) beta 1 induces the production of extracellular matrix proteins by liver cells and has been implicated in the pathogenesis of hepatic fibrosis in laboratory animals. TGF alpha is a hepatocyte mitogen that participates in liver regeneration.

METHODS

Using Northern blot analysis, we studied the expression of TGF beta 1 messenger RNA (mRNA) in liver specimens from 42 patients with chronic hepatitis and cirrhosis and 12 subjects with either normal or fatty livers. The results were correlated with measurements of procollagen Type I mRNA in liver tissue, procollagen Type III peptide in serum, and the degree of histologic injury. We also investigated whether TGF alpha mRNA would be detectable in biopsy specimens of livers with proliferative activity.

RESULTS

TGF beta 1 mRNA expression correlated closely with the expression of procollagen Type I mRNA (r = 0.94) and serum procollagen Type III peptide (r = 0.89) and with the histologic activity index (r = 0.73). All patients with increased fibrogenic activity (serum procollagen Type III peptide level, greater than 11.9 micrograms per liter) had increased levels of TGF beta 1 mRNA (2 to 14 times the levels in the control group or in patients with normal fibrogenic activity), and both TGF alpha and H3 histone (a marker of DNA synthesis) mRNAs were detectable in patients with regenerative nodules. Six of eight patients with hepatitis C treated with interferon alfa for one year had sustained clinical responses with normalization of serum procollagen Type III peptide and aminotransferase activity. All these patients had normal levels of TGF beta 1 mRNA in liver specimens obtained at the end of the year.

CONCLUSIONS

TGF beta 1 may have an important role in the pathogenesis of fibrosis in patients with chronic liver disease, and TGF alpha expression may be associated with liver regeneration in these patients.

Computer-aided lead optimization derives a unique, orally bioavailable inhibitor of the signal transducer and activator of transcription (Stat)3 Src homology 2 domain. BP-1-102 binds Stat3 with an affinity (K(D)) of 504 nM, blocks Stat3-phospho-tyrosine (pTyr) peptide interactions and Stat3 activation at 4-6.8 μM, and selectively inhibits growth, survival, migration, and invasion of Stat3-dependent tumor cells. BP-1-102-mediated inhibition of aberrantly active Stat3 in tumor cells suppresses the expression of c-Myc, Cyclin D1, Bcl-xL, Survivin, VEGF, and Krüppel-like factor 8, which is identified as a Stat3 target gene that promotes Stat3-mediated breast tumor cell migration and invasion. Treatment of breast cancer cells with BP-1-102 further blocks Stat3-NF-κB cross-talk, the release of granulocyte colony-stimulating factor, soluble intercellular adhesion molecule 1, macrophage migration-inhibitory factor/glycosylation-inhibiting factor, interleukin 1 receptor antagonist, and serine protease inhibitor protein 1, and the phosphorylation of focal adhesion kinase and paxillin, while enhancing E-cadherin expression. Intravenous or oral gavage delivery of BP-1-102 furnishes micromolar or microgram levels in tumor tissues and inhibits growth of human breast and lung tumor xenografts.

The mechanism of antitumor effect of monoclonal antibodies (mAbs) is not fully understood. Here we show that coating myeloma cells with anti-syndecan-1 antibody promotes cross-presentation of cellular antigens by dendritic cells (DCs) to autologous T cells from healthy donors. The tumor cells treated with anti-syndecan-1 or isotype-matched control antibody were fed to HLA-mismatched monocyte-derived immature DCs. Tumor cell-loaded mature DCs induced a strong CD8(+) T cell response that was specific for the cancer-testis (C-T) antigens expressed in the tumor. The CD8(+) T cells killed peptide-pulsed targets, as well as myeloma tumor cells. Importantly, mAbs-coated tumor-loaded DCs were consistently superior to DCs loaded with peptides or dying cells for eliciting tumor-specific killer T cells. This enhanced cross-presentation was not due to enhanced tumor cell uptake or to DC maturation. When mixtures of NY-Eso-1-positive and -negative myeloma cells were captured by DCs, the anti-syndecan-1 antibody had to be on the NY-Eso-1-positive cells to elicit NY-Eso-1-specific response. Cross-presentation was inhibited by pretreatment of DCs with Fc gamma receptor blocking antibodies. Targeting of mAb-coated tumors to DCs may contribute to the efficacy of tumor-reactive mAb and offers a new strategy for immunotherapy.

The map position of the coding sequence of glycoprotein H of herpes simplex virus type 1 was determined by marker transfer studies in which DNA fragments cloned from a virus resistant to neutralisation by an anti-gH monoclonal antibody were used to transfer antibody resistance to wild type virus DNA following cotransfection. The gH coding sequence was mapped to the BglII "m" fragment of HSV-1 DNA (map coordinates 0.27-0.312), confirming the map position previously determined by intertypic recombinant analysis (Buckmaster et al., 1984). The complete nucleotide sequence of the BglII "m" fragment revealed two large open reading frames in addition to the thymidine kinase gene. The open reading frame lying immediately 3' of the thymidine kinase gene has a predicted translation product with the features of a large glycoprotein. This open reading frame translates to an amino acid sequence of 90,323 mol wt with a signal peptide, a membrane anchor sequence, a large external domain containing potential N-glycosylation sites, and a charged C- terminal cytoplasmic domain. We suppose that this amino acid sequence corresponds to gH of HSV-1, and A. Davison (personal communication) has noted the existence of homologous glycoproteins predicted from the nucleotide sequences of Varicella-zoster virus and Epstein-Barr virus. The properties of monoclonal antibody LP11, directed against gH show remarkable similarities to the properties for gD antibodies. LP11 efficiently neutralizes virus infectivity, blocks cell fusion by syncytial virus strains, and inhibits the formation of plaques when added to cell monolayers after infection. These similarities in antibody activity imply functional relatedness between gH and gD of herpes simplex virus.

OBJECTIVE

Glucagon-like peptide-1 (GLP-1), a polypeptide hormone secreted by the l-cells in the gastrointestinal tract, has shown promising effects as a new treatment modality for patients with Type II (non-insulin-dependent) diabetes mellitus. However, the pharmacokinetic profile of native GLP-1 with a rapid elimination has limited its therapeutic potential. NN2211 is a fatty acid derivative of GLP-1, which pre-clinically has shown a protracted pharmacokinetic profile, while maintaining its biological activity. This study aimed to investigate the safety, tolerability, pharmacokinetics and pharmacodynamics of NN2211 in healthy male subjects following seven days treatment.

METHODS

In a double-blind, randomized, dose escalation, placebo controlled study, healthy male subjects were enrolled at five consecutive dose levels of NN2211 (1.25, 5.0, 7.5, 10.0, 12.5 microg/kg). Six subjects were allocated at random at each dose level to active or placebo treatment with a ratio of 2:1. Dosing with NN2211 was performed on day 1, and days 5-11. The 84-h pharmacokinetics and 24-h glucose and insulin profiles were assessed on day 1 and day 11.

RESULTS

Following s. c. administration the half-life of NN2211 was found to be 12.6 +/- 1.1 h, with a subsequent accumulation index after a daily dose for seven days of 1.4-1.5. There were dose-proportional increases in exposure (AUC and C(max)) with increasing doses. Overall, there were no statistically significant differences from placebo in the 24-h glucose and insulin profiles. In subjects treated with NN2211 rather than placebo, there was a higher incidence of adverse events, most notably dizziness and adverse events related to the gastrointestinal system. There were no serious adverse events but three subjects were nonetheless withdrawn because of dizziness, fever and nausea. There were no clinically relevant changes in vital signs, ECG parameters, physical examination or safety laboratory parameters. A significantly lower diuresis was observed in the actively treated subjects, without a clinically relevant change in packed cell volume.

CONCLUSIONS

This study shows NN2211 has a pharmacokinetic profile supporting a daily dose in human beings, but also that subjects treated with NN2211 rather than placebo, had a higher incidence of adverse events, most notably dizziness and adverse events related to the gastrointestinal system.

Despite the prevalence of obesity and its related diseases, the signaling pathways for appetite control and satiety are not clearly understood. Here we report C. elegans quiescence behavior, a cessation of food intake and movement that is possibly a result of satiety. C. elegans quiescence shares several characteristics of satiety in mammals. It is induced by high-quality food, it requires nutritional signals from the intestine, and it depends on prior feeding history: fasting enhances quiescence after refeeding. During refeeding after fasting, quiescence is evoked, causing gradual inhibition of food intake and movement, mimicking the behavioral sequence of satiety in mammals. Based on these similarities, we propose that quiescence results from satiety. This hypothesized satiety-induced quiescence is regulated by peptide signals such as insulin and TGF-beta. The EGL-4 cGMP-dependent protein kinase functions downstream of insulin and TGF-beta in sensory neurons including ASI to control quiescence in response to food intake.

Mitochondrial cytochrome c oxidase plays an essential role in aerobic cellular respiration, reducing dioxygen to water in a process coupled with the pumping of protons across the mitochondrial inner membrane. An aspartate residue, Asp-51, located near the enzyme surface, undergoes a redox-coupled x-ray structural change, which is suggestive of a role for this residue in redox-driven proton pumping. However, functional or mechanistic evidence for the involvement of this residue in proton pumping has not yet been obtained. We report that the Asp-51 ->> Asn mutation of the bovine enzyme abolishes its proton-pumping function without impairment of the dioxygen reduction activity. Improved x-ray structures (at 1.8/1.9-A resolution in the fully oxidized/reduced states) show that the net positive charge created upon oxidation of the low-spin heme of the enzyme drives the active proton transport from the interior of the mitochondria to Asp-51 across the enzyme via a water channel and a hydrogen-bond network, located in tandem, and that the enzyme reduction induces proton ejection from the aspartate to the mitochondrial exterior. A peptide bond in the hydrogen-bond network critically inhibits reverse proton transfer through the network. A redox-coupled change in the capacity of the water channel, induced by the hydroxyfarnesylethyl group of the low-spin heme, suggests that the channel functions as an effective proton-collecting region. Infrared results indicate that the conformation of Asp-51 is controlled only by the oxidation state of the low-spin heme. These results indicate that the low-spin heme drives the proton-pumping process.

Caspases orchestrate the controlled demise of a cell after an apoptotic signal through specific protease activity and cleavage of many substrates altering protein function and ensuring apoptosis proceeds efficiently. Comparing a variety of substrates of each apoptotic caspase (2, 3, 6, 7, 8, 9 and 10) showed that the cleavage sites had a general motif, sometimes specific for one caspase, but other times specific for several caspases. Using commercially available short peptide-based substrates and inhibitors the promiscuity for different cleavage motifs was indicated, with caspase-3 able to cleave most substrates more efficiently than those caspases to which the substrates are reportedly specific. In a cell-free system, immunodepletion of caspases before or after cytochrome c-dependent activation of the apoptosome indicated that the majority of activity on synthetic substrates was dependent on caspase-3, with minor roles played by caspases-6 and -7. Putative inhibitors of individual caspases were able to abolish all cytochrome c-induced caspase activity in a cell-free system and inhibit apoptosis in whole cells through the extrinsic and intrinsic pathways, raising issues regarding the use of such inhibitors to define relevant caspases and pathways. Finally, caspase activity in cells lacking caspase-9 displayed substrate cleavage activity of a putative caspase-9-specific substrate underlining the lack of selectivity of peptide-based substrates and inhibitors of caspases.

Thermal unfolding curves have been measured for a series of short alanine-based peptides that contain repeating sequences and varying chain lengths. Standard helix-coil theory successfully fits the observed transition curves, even for these short peptides. The results provide values for sigma, the helix nucleation constant, delta H0, the enthalpy change on helix formation, and for s (0 degree C), the average helix propagation parameter at 0 degree C. The enthalpy change agrees with the value determined calorimetrically. The success of helix-coil theory in describing the unfolding transitions of short peptides in water indicates that helical propensities, or s values, can be determined from substitution experiments in short alanine-based peptides.

Carbohydrate-responsive element binding protein (ChREBP) is a transcription factor that activates lipogenic genes in liver in response to excess carbohydrate in the diet. ChREBP is regulated in a reciprocal manner by glucose and cAMP. cAMP-dependent protein kinase (protein kinase A) phosphorylates two physiologically important sites in ChREBP, Ser-196, which is located near nuclear localization signal sequence (NLS), and Thr-666, within the basic helix-loop-helix (bHLH) site, resulting in inactivation of nuclear translocation of ChREBP and of the DNA-binding activity, respectively. We demonstrate here that crude cytosolic extracts from livers of rats fed a high carbohydrate diet contained protein phosphatase (PPase) activity that dephosphorylated a peptide containing Ser-196, whereas a PPase in the nuclear extract catalyzed dephosphorylation of Ser-568 and Thr-666. All these PPases are activated specifically by xylulose 5-phosphate (Xu5P), but not by other sugar phosphates. Furthermore, addition of Xu5P elevated PPase activity to the level observed in extracts of fed liver cells. These partially purified PPases were characterized as PP2A-AB delta C by immunoblotting with specific antibodies. These results suggest that (ia) Xu5P-dependent PPase is responsible for activation of transcription of the L-type pyruvate kinase gene and lipogenic enzyme genes, and (ii) Xu5P is the glucose signaling compound. Thus, we propose that the same Xu5P-activated PPase controls both acute and long-term regulation of glucose metabolism and fat synthesis.

We introduced previously an on-line resource, RANKPEP that uses position specific scoring matrices (PSSMs) or profiles for the prediction of peptide-MHC class I (MHCI) binding as a basis for CD8 T-cell epitope identification. Here, using PSSMs that are structurally consistent with the binding mode of MHC class II (MHCII) ligands, we have extended RANKPEP to prediction of peptide-MHCII binding and anticipation of CD4 T-cell epitopes. Currently, 88 and 50 different MHCI and MHCII molecules, respectively, can be targeted for peptide binding predictions in RANKPEP. Because appropriate processing of antigenic peptides must occur prior to major histocompatibility complex (MHC) binding, cleavage site prediction methods are important adjuncts for T-cell epitope discovery. Given that the C-terminus of most MHCI-restricted epitopes results from proteasomal cleavage, we have modeled the cleavage site from known MHCI-restricted epitopes using statistical language models. The RANKPEP server now determines whether the C-terminus of any predicted MHCI ligand may result from such proteasomal cleavage. Also implemented is a variability masking function. This feature focuses prediction on conserved rather than highly variable protein segments encoded by infectious genomes, thereby offering identification of invariant T-cell epitopes to thwart mutation as an immune evasion mechanism.