Characterization of the composition of the postsynaptic proteome (PSP) provides a framework for understanding the overall organization and function of the synapse in normal and pathological conditions. We have identified 698 proteins from the postsynaptic terminal of mouse CNS synapses using a series of purification strategies and analysis by liquid chromatography tandem mass spectrometry and large-scale immunoblotting. Some 620 proteins were found in purified postsynaptic densities (PSDs), nine in AMPA-receptor immuno-purifications, 100 in isolates using an antibody against the NMDA receptor subunit NR1, and 170 by peptide-affinity purification of complexes with the C-terminus of NR2B. Together, the NR1 and NR2B complexes contain 186 proteins, collectively referred to as membrane-associated guanylate kinase-associated signalling complexes. We extracted data from six other synapse proteome experiments and combined these with our data to provide a consensus on the composition of the PSP. In total, 1124 proteins are present in the PSP, of which 466 were validated by their detection in two or more studies, forming what we have designated the Consensus PSD. These synapse proteome data sets offer a basis for future research in synaptic biology and will provide useful information in brain disease and mental disorder studies.

OBJECTIVE

The efficacy and safety of the dipeptidyl peptidase-4 inhibitor, sitagliptin, added to ongoing metformin therapy, were assessed in patients with type 2 diabetes who had inadequate glycemic control (HbA(1c) [A1<em>C</em>]>>or=7 and <or=10%) with metformin alone.

METHODS

After a screening diet/exercise run-in period, a metformin dose titration/stabilization period, and a 2-week, single-blind, placebo run-in period, 701 patients, aged 19-78 years, with mild to moderate hyperglycemia (mean A1C 8.0%) receiving ongoing metformin >>or=1,500 mg/day) were randomly assigned to receive the addition of placebo or sitagliptin 100 mg once-daily in a 1:2 ratio for 24 weeks. Patients exceeding specific glycemic limits were provided rescue therapy (pioglitazone) until the end of the study. The efficacy analyses were based on an all-patients-treated population using an ANCOVA and excluded data obtained after glycemic rescue.

RESULTS

At week 24, sitagliptin treatment led to significant reductions compared with placebo in A1C (-0.65%), fasting plasma glucose, and 2-h postmeal glucose. Fasting insulin, fasting C-peptide, fasting proinsulin-to-insulin ratio, postmeal insulin and C-peptide areas under the curve (AUCs), postmeal insulin AUC-to-glucose AUC ratio, homeostasis model assessment of beta-cell function, and quantitative insulin sensitivity check index were significantly improved with sitagliptin relative to placebo. A significantly greater proportion of patients achieved an A1C <7% with sitagliptin (47.0%) than with placebo (18.3%). There was no increased risk of hypoglycemia or gastrointestinal adverse experiences with sitagliptin compared with placebo. Body weight decreased similarly with sitagliptin and placebo.

CONCLUSIONS

Sitagliptin 100 mg once-daily added to ongoing metformin therapy was efficacious and well tolerated in patients with type 2 diabetes who had inadequate glycemic control with metformin alone.

We have isolated a cDNA clone for one of the HLA-B locus alloantigens by hybridization with a 30-nucleotide-long DNA probe. The probe was isolated from a reverse transcriptase (RNA-dependent DNA nucleotidyltransferase)-catalyzed cDNA synthesis reaction on poly(A)-mRNA in which an oligonucleotide (5'-32P)dC-T-T-C-T-C-C-A-C-A-TOH served as a primer and in which dideoxynucleoside triphosphates were used to reduce the size and heterogeneity of the cDNA products. The desired cDNA clone was isolated from a library of recombinant cDNA clones in the plasmid pBR322. The partial nucleotide sequence of the cDNA clone corresponds to the amino acid sequence of HLA-B7 antigen. The approach described in this paper is extremely sensitive and may be useful in cloning other genes for which the corresponding mRNA is present at low levels. This cDNA clone is nearly full length and can be used to isolate and to study the genes within the HLA region and to obtain expression of HLA-B peptides in cells.

A good deal is now known about the neural circuitry involved in how conditioned fear can augment a simple reflex (fear-potentiated startle). This involves visual or auditory as well as shock pathways that project via the thalamus and perirhinal or insular cortex to the basolateral amygdala (BLA). The BLA projects to the central (CeA) and medial (MeA) nuclei of the amygdala, which project indirectly to a particular part of the acoustic startle pathway in the brainstem. N-methyl-D-aspartate (NMDA) receptors, as well as various intracellular cascades in the amygdala, are critical for fear learning, which is then mediated by glutamate acting in the CeA. Less predictable stimuli, such as a long-duration bright light or a fearful context, activate the BLA, which projects to the bed nucleus of the stria terminalis (BNST), which projects to the startle pathway much as the CeA does. The anxiogenic peptide corticotropin-releasing hormone increases startle by acting directly in the BNST. CeA-mediated behaviors may represent stimulus-specific fear, whereas BNST-mediated behaviors are more akin to anxiety. NMDA receptors are also involved in extinction of conditioned fear, and both extinction in rats and exposure-based psychotherapy in humans are facilitated by an NMDA-partial agonist called D-cycloserine. ((c) 2006 APA, all rights reserved).

Hepatitis C virus (HCV) is the major causative pathogen associated with liver cirrhosis and hepatocellular carcinoma. The virus has a positive-sense RNA genome encoding a single polyprotein with the virion components located in the N-terminal portion. During biosynthesis of the polyprotein, an internal signal sequence between the core protein and the envelope protein E1 targets the nascent polypeptide to the endoplasmic reticulum (ER) membrane for translocation of E1 into the ER. Following membrane insertion, the signal sequence is cleaved from E1 by signal peptidase. Here we provide evidence that after cleavage by signal peptidase, the signal peptide is further processed by the intramembrane-cleaving protease SPP that promotes the release of core protein from the ER membrane. Core protein is then free for subsequent trafficking to lipid droplets. This study represents an example of a potential role for intramembrane proteolysis in the maturation of a viral protein.

Glucagon-like peptide 1 (GLP-1) has been shown to inhibit gastric emptying of liquid meals in type 2 diabetic patients. It was the aim of the present study to compare the action of physiological and pharmacological doses of intravenous GLP-1-(7-36) amide and GLP-1-(7-37) on gastric emptying in normal volunteers. Nine healthy subjects participated (26 +/- 3 yr; body mass index 22.9 +/- 1.6 kg/m2; hemoglobin A1C 5.0 +/- 0.2%) in five experiments on separate occasions after an overnight fast. A nasogastric tube was positioned for the determination of gastric volume by use of a dye-dilution technique (phenol red). GLP-1-(7-36) amide (0.4, 0.8, or 1.2 pmol.kg-1.min-1), GLP-1-(7-37) (1.2 pmol.kg-1.min-1), or placebo was infused intravenously from -30 to 240 min. A liquid meal (50 g sucrose, 8% amino acids, 440 ml, 327 kcal) was administered at 0 min. Glucose, insulin, and C-peptide were measured over 240 min. Gastric emptying was dose dependently slowed by GLP-1-(7-36) amide (P < 0.0001). Effects of GLP-1-(7-37) at 1.2 pmol.kg-1.min-1 were virtually identical. GLP.1 dose dependently stimulated fasting insulin secretion (-30 to 0 min) and slightly reduced glucose concentrations. After the meal (0-240 min), integrated incremental glucose (P < 0.0001) and insulin responses (P = 0.01) were reduced (dose dependently) rather than enhanced. In conclusion, 1) GLP-1-(7-36) amide or -(7-37) inhibits gastric emptying also in normal subjects, 2) physiological doses (0.4 pmol.kg-1.min-1) still have a significant effect, 3) despite the known insulinotropic actions of GLP-1-(7-36) amide and -(7-37), the net effect of administering GLP-1 with a meal is no change or a reduction in meal-related insulin responses. These findings suggest a primarily inhibitory function for GLP-1 (ileal brake mechanisms).

OBJECTIVE

To examine associations of neonatal adiposity with maternal glucose levels and cord serum C-peptide in a multicenter multinational study, the Hyperglycemia and Adverse Pregnancy Outcome (HAPO) Study, thereby assessing the Pederson hypothesis linking maternal glycemia and fetal hyperinsulinemia to neonatal adiposity.

METHODS

Eligible pregnant women underwent a standard 75-g oral glucose tolerance test between 24 and 32 weeks gestation (as close to 28 weeks as possible). Neonatal anthropometrics and cord serum C-peptide were measured. Associations of maternal glucose and cord serum C-peptide with neonatal adiposity (sum of skin folds >90th percentile or percent body fat >90th percentile) were assessed using multiple logistic regression analyses, with adjustment for potential confounders, including maternal age, parity, BMI, mean arterial pressure, height, gestational age at delivery, and the baby's sex.

RESULTS

Among 23,316 HAPO Study participants with glucose levels blinded to caregivers, cord serum C-peptide results were available for 19,885 babies and skin fold measurements for 19,389. For measures of neonatal adiposity, there were strong statistically significant gradients across increasing levels of maternal glucose and cord serum C-peptide, which persisted after adjustment for potential confounders. In fully adjusted continuous variable models, odds ratios ranged from 1.35 to 1.44 for the two measures of adiposity for fasting, 1-h, and 2-h plasma glucose higher by 1 SD.

CONCLUSIONS

These findings confirm the link between maternal glucose and neonatal adiposity and suggest that the relationship is mediated by fetal insulin production and that the Pedersen hypothesis describes a basic biological relationship influencing fetal growth.

The role of a cell death-associated gene, fas, in T lymphocyte development and responses to antigen has been analyzed by breeding a transgenic T cell receptor specific for the 81-104 peptide of pigeon cytochrome c into fas-defective MRL-lpr/lpr and control MRL+/+ mice. Transgene-expressing T cells mature normally in both strains and populate peripheral lymphoid tissues in normal numbers. Mature CD4+ T cells from the lpr/lpr mice are resistant to suppression by high doses of antigen and to apoptotic cell death. In vivo administration of peptide antigen causes deletion of thymic T cells in both MRL-lpr/lpr and MRL+/+ strains. By contrast, antigen-induced deletion of peripheral T cells occurs in the MRL+/+ but not in the MRL-lpr/lpr strain. Therefore, the fas gene plays an essential role in activation-induced cell death in mature T lymphocytes, but not in the negative selection of immature cells in the thymus.

Soluble oligomers of the amyloid-beta (Abeta) peptide are thought to play a key role in the pathophysiology of Alzheimer's disease (AD). Recently, we reported that synthetic Abeta oligomers bind to cellular prion protein (PrP(C)) and that this interaction is required for suppression of synaptic plasticity in hippocampal slices by oligomeric Abeta peptide. We hypothesized that PrP(C) is essential for the ability of brain-derived Abeta to suppress cognitive function. Here, we crossed familial AD transgenes encoding APPswe and PSen1DeltaE9 into Prnp-/- mice to examine the necessity of PrP(C) for AD-related phenotypes. Neither APP expression nor Abeta level is altered by PrP(C) absence in this transgenic AD model, and astrogliosis is unchanged. However, deletion of PrP(C) expression rescues 5-HT axonal degeneration, loss of synaptic markers, and early death in APPswe/PSen1DeltaE9 transgenic mice. The AD transgenic mice with intact PrP(C) expression exhibit deficits in spatial learning and memory. Mice lacking PrP(C), but containing Abeta plaque derived from APPswe/PSen1DeltaE9 transgenes, show no detectable impairment of spatial learning and memory. Thus, deletion of PrP(C) expression dissociates Abeta accumulation from behavioral impairment in these AD mice, with the cognitive deficits selectively requiring PrP(C).

Signal peptide peptidase (SPP) catalyzes intramembrane proteolysis of some signal peptides after they have been cleaved from a preprotein. In humans, SPP activity is required to generate signal sequence-derived human lymphocyte antigen-E epitopes that are recognized by the immune system, and to process hepatitis C virus core protein. We have identified human SPP as a polytopic membrane protein with sequence motifs characteristic of the presenilin-type aspartic proteases. SPP and potential eukaryotic homologs may represent another family of aspartic proteases that promote intramembrane proteolysis to release biologically important peptides.

Two distinct receptors for tumor necrosis factor (TNF) of 55 and 75 kd are expressed at low levels by various cells. The 55 kd TNF receptor was purified from HL60 cells, and partial amino acid sequences were determined. Short degenerate sense and antisense oligonucleotide primers encoding the N- and C-terminal ends of a peptide of 22 amino acid residues were used to amplify a 66 bp cDNA fragment from HL60 RNA by reverse transcriptase-polymerase chain reaction. The cDNA fragment as a probe identified several overlapping clones in a human placenta cDNA library. The open reading frame of the cDNA predicts a 455 amino acid TNF receptor protein with leader, extracellular, transmembrane, and intracellular domains. When expressed in COS-1 cells or in a baculovirus system, the cDNA conferred TNF binding properties comparable to the native receptor. Surprisingly, the 55 kd TNF receptor shows a high degree of sequence homology to the NGF receptor extracellular domain.

Type 1 diabetes (T1D) is an autoimmune disease that occurs in genetically susceptible individuals. Regulatory T cells (Tregs) have been shown to be defective in the autoimmune disease setting. Thus, efforts to repair or replace Tregs in T1D may reverse autoimmunity and protect the remaining insulin-producing β cells. On the basis of this premise, a robust technique has been developed to isolate and expand Tregs from patients with T1D. The expanded Tregs retained their T cell receptor diversity and demonstrated enhanced functional activity. We report on a phase 1 trial to assess safety of Treg adoptive immunotherapy in T1D. Fourteen adult subjects with T1D, in four dosing cohorts, received ex vivo-expanded autologous CD4(+)CD127(lo/-)CD25(+) polyclonal Tregs (0.05 × 10(8) to 26 × 10(8) cells). A subset of the adoptively transferred Tregs was long-lived, with up to 25% of the peak level remaining in the circulation at 1 year after transfer. Immune studies showed transient increases in Tregs in recipients and retained a broad Treg FOXP3(+)CD4(+)CD25(hi)CD127(lo) phenotype long-term. There were no infusion reactions or cell therapy-related high-grade adverse events. C-peptide levels persisted out to 2+ years after transfer in several individuals. These results support the development of a phase 2 trial to test efficacy of the Treg therapy.

Amyloid beta-protein (Abeta) assembly into toxic oligomeric and fibrillar structures is a seminal event in Alzheimer's disease, therefore blocking this process could have significant therapeutic benefit. A rigorous mechanistic understanding of Abeta assembly would facilitate the targeting and design of fibrillogenesis inhibitors. Prior studies have shown that Abeta fibrillogenesis involves conformational changes leading to the formation of extended beta-sheets and that an alpha-helix-containing intermediate may be involved. However, the significance of this intermediate has been a matter of debate. We report here that the formation of an oligomeric, alpha-helix-containing assembly is a key step in Abeta fibrillogenesis. The generality of this phenomenon was supported by conformational studies of 18 different Abeta peptides, including wild-type Abeta(1-40) and Abeta(1-42), biologically relevant truncated and chemically modified Abeta peptides, and Abeta peptides causing familial forms of cerebral amyloid angiopathy. Without exception, fibrillogenesis of these peptides involved an oligomeric alpha-helix-containing intermediate and the kinetics of formation of the intermediate and of fibrils was temporally correlated. The kinetics varied depending on amino acid sequence and the extent of peptide N- and C-terminal truncation. The pH dependence of helix formation suggested that Asp and His exerted significant control over this process and over fibrillogenesis in general. Consistent with this idea, Abeta peptides containing Asp->>Asn or His->>Gln substitutions showed altered fibrillogenesis kinetics. These data emphasize the importance of the dynamic interplay between Abeta monomer conformation and oligomerization state in controlling fibrillogenesis kinetics.

In Huntington's Disease and related expanded CAG repeat diseases, a polyglutamine [poly(Gln)] sequence containing 36 repeats in the corresponding disease protein is benign, whereas a sequence with only 2-3 additional glutamines is associated with disease risk. Above this threshold range, longer repeat lengths are associated with earlier ages-of-onset. To investigate the biophysical basis of these effects, we studied the in vitro aggregation kinetics of a series of poly(Gln) peptides. We find that poly(Gln) peptides in solution at 37 degrees C undergo a random coil to beta-sheet transition with kinetics superimposable on their aggregation kinetics, suggesting the absence of soluble, beta-sheet-rich intermediates in the aggregation process. Details of the time course of aggregate growth confirm that poly(Gln) aggregation occurs by nucleated growth polymerization. Surprisingly, however, and in contrast to conventional models of nucleated growth polymerization of proteins, we find that the aggregation nucleus is a monomer. That is, nucleation of poly(Gln) aggregation corresponds to an unfavorable protein folding reaction. Using parameters derived from the kinetic analysis, we estimate the difference in the free energy of nucleus formation between benign and pathological length poly(Gln)s to be less than 1 kcal/mol. We also use the kinetic parameters to calculate predicted aggregation curves for very low concentrations of poly(Gln) that might obtain in the cell. The repeat-length-dependent differences in predicted aggregation lag times are in the same range as the length-dependent age-of-onset differences in Huntington's disease, suggesting that the biophysics of poly(Gln) aggregation nucleation may play a major role in determining disease onset.

Integrated insulin secretion rates calculated from peripheral venous C-peptide measurements by two-compartment kinetic analysis were measured in six young normal subjects after increasing oral glucose loads of 25, 50, and 100 g and respective isoglycemic glucose infusions. The differences in B-cell secretory responses between oral and iv glucose challenges were attributed to factors other than glycemia itself (incretin effect). Both insulin and C-peptide concentrations as well as calculated integrated insulin secretion rates increased with increasing oral glucose loads. Due to the similarity in the glucose profiles after all oral loads, almost identical amounts of iv glucose (approximately 20 g) were infused in all "isoglycemic" infusion experiments, with resulting similar hormone profiles and insulin secretion rates. The percent contribution of incretin factors to total immunoreactive insulin responses after 25, 50, and 100 g glucose (85.6%, 74.9%, and 93.0%; response to oral load, 100%) was significantly higher than their contribution to integrated C-peptide responses (27.6-62.9%) or calculated integrated insulin secretion rates (19.2-61.0%). These findings indicate that the degree of incretin stimulation of insulin secretion depends on the amount of glucose ingested. A discrepancy between the estimates of the incretin effect derived from peripheral venous insulin responses, on the one hand, and C-peptide responses or calculated insulin secretion rates, on the other hand, exists. Inasmuch as peripheral insulin values reflect both insulin secretion and hepatic insulin removal, this discrepancy suggests that elimination kinetics of insulin differ between oral and iv glucose administration. This difference can be related to a significantly reduced fractional hepatic insulin extraction after oral (46.9-54.6%) compared to iv (63.4-76.5%) glucose administration when calculated by a three-compartment kinetic model. This reduction in fractional hepatic insulin extraction could be caused by gastrointestinal factors (hormones or nerves) stimulated in the course of glucose ingestion.

Brain ischemia and reperfusion engage multiple independently-fatal terminal pathways involving loss of membrane integrity in partitioning ions, progressive proteolysis, and inability to check these processes because of loss of general translation competence and reduced survival signal-transduction. Ischemia results in rapid loss of high-energy phosphate compounds and generalized depolarization, which induces release of glutamate and, in selectively vulnerable neurons (SVNs), opening of both voltage-dependent and glutamate-regulated calcium channels. This allows a large increase in cytosolic Ca(2+) associated with activation of mu-calpain, calcineurin, and phospholipases with consequent proteolysis of calpain substrates (including spectrin and eIF4G), activation of NOS and potentially of Bad, and accumulation of free arachidonic acid, which can induce depletion of Ca(2+) from the ER lumen. A kinase that shuts off translation initiation by phosphorylating the alpha-subunit of eukaryotic initiation factor-2 (eIF2alpha) is activated either by adenosine degradation products or depletion of ER lumenal Ca(2+). Early during reperfusion, oxidative metabolism of arachidonate causes a burst of excess oxygen radicals, iron is released from storage proteins by superoxide-mediated reduction, and NO is generated. These events result in peroxynitrite generation, inappropriate protein nitrosylation, and lipid peroxidation, which ultrastructurally appears to principally damage the plasmalemma of SVNs. The initial recovery of ATP supports very rapid eIF2alpha phosphorylation that in SVNs is prolonged and associated with a major reduction in protein synthesis. High catecholamine levels induced by the ischemic episode itself and/or drug administration down-regulate insulin secretion and induce inhibition of growth-factor receptor tyrosine kinase activity, effects associated with down-regulation of survival signal-transduction through the Ras pathway. Caspase activation occurs during the early hours of reperfusion following mitochondrial release of caspase 9 and cytochrome c. The SVNs find themselves with substantial membrane damage, calpain-mediated proteolytic degradation of eIF4G and cytoskeletal proteins, altered translation initiation mechanisms that substantially reduce total protein synthesis and impose major alterations in message selection, down-regulated survival signal-transduction, and caspase activation. This picture argues powerfully that, for therapy of brain ischemia and reperfusion, the concept of single drug intervention (which has characterized the approaches of basic research, the pharmaceutical industry, and clinical trials) cannot be effective. Although rigorous study of multi-drug protocols is very demanding, effective therapy is likely to require (1) peptide growth factors for early activation of survival-signaling pathways and recovery of translation competence, (2) inhibition of lipid peroxidation, (3) inhibition of calpain, and (4) caspase inhibition. Examination of such protocols will require not only characterization of functional and histopathologic outcome, but also study of biochemical markers of the injury processes to establish the role of each drug.

Currently, no vaccine exists for hepatitis C virus (HCV), a major pathogen thought to infect 170 million people globally. Many studies suggest that host T cell responses are critical for spontaneous resolution of disease, and preclinical studies have indicated a requirement for T cells in protection against challenge. We aimed to elicit HCV-specific T cells with the potential for protection using a recombinant adenoviral vector strategy in a phase 1 study of healthy human volunteers. Two adenoviral vectors expressing NS proteins from HCV genotype 1B were constructed based on rare serotypes [human adenovirus 6 (Ad6) and chimpanzee adenovirus 3 (ChAd3)]. Both vectors primed T cell responses against HCV proteins; these T cell responses targeted multiple proteins and were capable of recognizing heterologous strains (genotypes 1A and 3A). HCV-specific T cells consisted of both CD4+ and CD8+ T cell subsets; secreted interleukin-2, interferon-γ, and tumor necrosis factor-α; and could be sustained for at least a year after boosting with the heterologous adenoviral vector. Studies using major histocompatibility complex peptide tetramers revealed long-lived central and effector memory pools that retained polyfunctionality and proliferative capacity. These data indicate that an adenoviral vector strategy can induce sustained T cell responses of a magnitude and quality associated with protective immunity and open the way for studies of prophylactic and therapeutic vaccines for HCV.

Recently, a novel class of murine type C virus (MCF), some strains of which are highly oncogenic in the AKR acceleration test, has been isolated from premalignant and malignant thymuses of AKR mice. The biology of these viruses suggested that MCFs are the product of recombination between endogenous ecotropic and xenotropic viruses and, further, that the recombination has taken place within the envelope (env) gene which encodes the surface glycoprotein (gp70) of the virion. We have compared by tryptic peptide analysis, the gp70s of four MCF isolates with the gp70s of various possible parental viruses. In addition, we have compared the tryptic peptides of the gag gene products p30 and p15 from several of these viruses. The results allow the following conclusions: (i) the gp70s of the MCF viruses are not identical to one another and are different from the gp70s of the possible parental viruses tested; (ii) the MCF virus gp70s have tryptic peptides in common with xenotropic virus gp70s as well as with ecotropic virus gp70s; and (iii) the gap region protein, p30, of the MCFs tested is identical to p30 of AKR ecotropic virus (Akv-1 or Akv-2) and distinct from p30 of xenotropic viruses, suggesting that the 5' end of the recombinant viruses is of Akv origin. The findings are discussed with respect to the possible role a recombinant virus might play in leukemogenesis in AKR mice.

The cytoplasmic protein Dishevelled (Dvl) and the associated membrane-bound receptor Frizzled (Fz) are essential in canonical and noncanonical Wnt signaling pathways. However, the molecular mechanisms underlying this signaling are not well understood. By using NMR spectroscopy, we determined that an internal sequence of Fz binds to the conventional peptide binding site in the PDZ domain of Dvl; this type of site typically binds to C-terminal binding motifs. The C-terminal region of the Dvl inhibitor Dapper (Dpr) and Frodo bound to the same site. In Xenopus, Dvl binding peptides of Fz and Dpr/Frodo inhibited canonical Wnt signaling and blocked Wnt-induced secondary axis formation in a dose-dependent manner, but did not block noncanonical Wnt signaling mediated by the DEP domain. Together, our results identify a missing molecular connection within the Wnt pathway. Differences in the binding affinity of the Dvl PDZ domain and its binding partners may be important in regulating signal transduction by Dvl.

The receptor for platelet-derived growth factor (PDGF) binds two proteins containing SH2 domains, GTPase activating protein (GAP) and phosphatidylinositol 3-kinase (PI3-kinase). The sites on the receptor that mediate this interaction were identified by using phosphotyrosine-containing peptides representing receptor sequences to block specifically binding of either PI3-kinase or GAP. These results suggested that PI3-kinase binds two phosphotyrosine residues, each located in a 5 aa motif with an essential methionine at the fourth position C-terminal to the tyrosine. Point mutations at these sites caused a selective elimination of PI3-kinase binding and loss of PDGF-stimulated DNA synthesis. Mutation of the binding site for GAP prevented the receptor from associating with or phosphorylating GAP, but had no effect on PI3-kinase binding and little effect on DNA synthesis. Therefore, GAP and PI3-kinase interact with the receptor by binding to different phosphotyrosine-containing sequence motifs.

The seven-membrane-spanning angiotensin II type 1A receptor activates the mitogen-activated protein kinases extracellular signal-regulated kinases 1 and 2 (ERK1/2) by distinct pathways dependent on either G protein (likely G(q)/G(11)) or beta-arrestin2. Here we sought to distinguish the kinetic and spatial patterns that characterize ERK1/2 activated by these two mechanisms. We utilized beta-arrestin RNA interference, the protein kinase C inhibitor Ro-31-8425, a mutant angiotensin II receptor (DRY/AAY), and a mutant angiotensin II peptide (SII-angiotensin), which are incapable of activating G proteins, to isolate the two pathways in HEK-293 cells. G protein-dependent activation was rapid (peak <2 min), quite transient (t((1/2)) approximately 2 min), and led to nuclear translocation of the activated ERK1/2 as assessed by confocal microscopy. In contrast, beta-arrestin2-dependent activation was slower (peak 5-10 min), quite persistent with little decrement noted out to 90 min, and entirely confined to the cytoplasm. Moreover, ERK1/2 activated via beta-arrestin2 accumulated in a pool of cytoplasmic endosomal vesicles that also contained the internalized receptors and beta-arrestin. Such differential regulation of the temporal and spatial patterns of ERK1/2 activation via these two pathways strongly implies the existence of distinct physiological endpoints.

Optimal management of neuropathic pain is a major clinical challenge. We investigated the involvement of c-Jun N-terminal kinase (JNK) in neuropathic pain produced by spinal nerve ligation (SNL) (L5). SNL induced a slow (>3 d) and persistent (>21 d) activation of JNK, in particular JNK1, in GFAP-expressing astrocytes in the spinal cord. In contrast, p38 mitogen-activated protein kinase activation was found in spinal microglia after SNL, which had fallen to near basal level by 21 d. Intrathecal infusion of a JNK peptide inhibitor, D-JNKI-1, did not affect normal pain responses but potently prevented and reversed SNL-induced mechanical allodynia, a major symptom of neuropathic pain. Intrathecal D-JNKI-1 also suppressed SNL-induced phosphorylation of the JNK substrate, c-Jun, in spinal astrocytes. However, SNL-induced upregulation of GFAP was not attenuated by spinal D-JNKI-1 infusion. Furthermore, SNL induced a rapid (<12 h) but transient activation of JNK in the L5 (injured) but not L4 (intact) DRG. JNK activation in the DRG was mainly found in small-sized C-fiber neurons. Infusion of D-JNKI-1 into the L5 DRG prevented but did not reverse SNL-induced mechanical allodynia. Finally, intrathecal administration of an astroglial toxin, l-alpha-aminoadipate, reversed mechanical allodynia. Our data suggest that JNK activation in the DRG and spinal cord play distinct roles in regulating the development and maintenance of neuropathic pain, respectively, and that spinal astrocytes contribute importantly to the persistence of mechanical allodynia. Targeting the JNK pathway in spinal astroglia may present a new and efficient way to treat neuropathic pain symptoms.

Secondary bacterial pneumonia frequently claimed the lives of victims during the devastating 1918 influenza A virus pandemic. Little is known about the viral factors contributing to the lethality of the 1918 pandemic. Here we show that expression of the viral accessory protein PB1-F2 enhances inflammation during primary viral infection of mice and increases both the frequency and severity of secondary bacterial pneumonia. The priming effect of PB1-F2 on bacterial pneumonia could be recapitulated in mice by intranasal delivery of a synthetic peptide derived from the C-terminal portion of the PB1-F2. Relative to its isogenic parent, an influenza virus engineered to express a PB1-F2 with coding changes matching the 1918 pandemic strain was more virulent in mice, induced more pulmonary immunopathology, and led to more severe secondary bacterial pneumonia. These findings help explain both the unparalleled virulence of the 1918 strain and the high incidence of fatal pneumonia during the pandemic.

Cytochrome c release and the mitochondrial permeability transition (PT), including loss of the transmembrane potential (Deltapsi), play an important role in apoptosis. Using isolated mitochondria, we found that recombinant Bax and Bak, proapoptotic members of the Bcl-2 family, induced mitochondrial Deltapsi loss, swelling, and cytochrome c release. All of these changes were dependent on Ca2+ and were prevented by cyclosporin A (CsA) and bongkrekic acid, both of which close the PT pores (megachannels), indicating that Bax- and Bak-induced mitochondrial changes were mediated through the opening of these pores. Bax-induced mitochondrial changes were inhibited by recombinant Bcl-xL and transgene-derived Bcl-2, antiapoptotic members of the Bcl-2 family, as well as by oligomycin, suggesting a possible regulatory effect of F0F1-ATPase on Bax-induced mitochondrial changes. Proapoptotic Bax- and Bak-BH3 (Bcl-2 homology) peptides, but not a mutant BH3 peptide nor a mutant Bak lacking BH3, induced the mitochondrial changes, indicating an essential role of the BH3 region. A coimmunoprecipitation study revealed that Bax and Bak interacted with the voltage-dependent anion channel, which is a component of PT pores. Taken together, these findings suggest that proapoptotic Bcl-2 family proteins, including Bax and Bak, induce the mitochondrial PT and cytochrome c release by interacting with the PT pores.