The mitochondrial targeting function of randomly generated peptide sequences correlates with predicted helical amphiphilicity.

A pool of oligonucleotides encoding a start methionine and nine random amino acids was inserted at the 5'-end of the gene for the yeast cytochrome oxidase subunit IV lacking its own mitochondrial targeting sequence. Approximately one-quarter of the randomly generated sequences targeted subunit IV to its correct intramitochondrial location in vivo. Sequence analysis of 89 randomly generated sequences showed that their efficiencies as mitochondrial targeting signals correlated with the potential to fold into an amphiphilic alpha-helix. Functional targeting sequences were enriched in arginine and isoleucine residues but contained few aspartate, glutamate, and proline residues. Nonfunctional sequences predicted to have significant helical amphiphilicity often had at least one acidic or multiple helix-breaking residues that would be expected to interfere with targeting functioning. These results support the hypothesis that the signal for targeting a protein into the mitochondrial matrix is usually a positively charged amphiphilic helix.

Photoaffinity labeling of the T cell receptor on cloned cytotoxic T lymphocytes by covalent photoreactive ligand.

The interaction of the T cell antigen receptor with a photoreactive antigenic peptide derivative bound covalently to the H-2Kd (Kd) molecule was studied by photoaffinity labeling on cloned, CD8 positive cytotoxic T lymphocytes. The Kd-restricted Plasmodium berghei circumsporozoite peptide 253-260 (YIPS-AEKI) was conjugated with iodo-4-azidosalicylic acid at the N terminus and with 4-azidobenzoic acid at the T cell receptor residue Lys-259. Cell-associated or soluble Kd molecules were photoaffinity-labeled with the peptide derivative by selective photoactivation of the N-terminal photoreactive group. Incubation of cell-associated or soluble covalent Kd-peptide derivative complexes (ligands) with cytotoxic T lymphocytes that recognized this peptide derivative and activation of the orthogonal photoreactive group resulted in specific photoaffinity labeling of the T cell receptor. The labeling was inhibitable by an anti-Kd antibody and was absent on Kd-restricted cytotoxic T lymphocytes of different specificity. The binding of the soluble ligand reached a maximum after 2-4 min at 37 degrees C, after 30 min at 18 degrees C, and after 3 h at 4 degrees C. In contrast, binding of the cell-associated ligand reached a transient maxima after 50 and 110 min at 37 and 18 degrees C, respectively. The degree of binding at 37 degrees C was approximately 30% lower than that at 18 degrees C. No binding took place at 4 degrees C. Inhibition studies with antibodies and drugs indicated that the binding of the cell-associated, but not the soluble ligand, was highly dependent on T cell-target cell conjugate formation, whereas the binding of the soluble ligand was greatly dependent on CD8.

The role of antibody concentration and avidity in antiviral protection.

Neutralizing antibodies are necessary and sufficient for protection against infection with vesicular stomatitis virus (VSV). The in vitro neutralization capacities and in vivo protective capacities of a panel of immunoglobulin G monoclonal antibodies to the glycoprotein of VSV were evaluated. In vitro, neutralizing activity correlated with avidity and with neutralization rate constant, a measure of on-rate. However, in vivo, protection was independent of immunoglobulin subclass, avidity, neutralization rate constant, and in vitro neutralizing activity; above a minimal avidity threshold, protection depended simply on a minimum serum concentration. These two biologically defined thresholds of antibody specificity offer hope for the development of adoptive therapy with neutralizing antibodies.

Eliminating heterophilic antibody interference for ferritin detection using Olympus F(ab')2 based reagent.

Interferences with the Olympus immunoturbidimetric assay for ferritin have been reported because the antibodies used in the immunoassay are derived from rabbits. Rabbits are familiar pets known to be a risk factor for developing heterophilic (or interfering) antibodies. This report shows how the current Olympus Ferritin assay has been improved to eliminate the interference from heterophilic antibodies.

Increased plasma levels of N-terminal brain natriuretic peptide (NT-proBNP) in type 2 diabetic patients with vascular complications

Résumé : Les concentrations plasmatiques du peptide natriurétique de type B sont augmentées chez les diabétiques de type 2 atteints de complications vasculaires. But : Les concentrations plasmatiques du peptide natriurétique de type B (NT-proBNP) sont augmentées chez les diabétiques de type 2 atteints de complications vasculaires. Les concentrations plasmatiques du peptide natriurétique de type B (BNP), ou de sa pro-hormone (NT-proBNP), sont reconnues depuis peu comme marqueur de choix de la dysfonction cardiaque. Les diabétiques de type 2 sont à haut risque de développer des complications cardiovasculaires. L'objectif de cette étude a été de déterminer si les concentrations plasmatiques de NT-proBNP étaient comparables chez des diabétiques de type 2 avec ou sans complications vasculaires. Méthodes : Nous avons mesuré le NT-proBNP plasmatique chez 54 diabétiques de type 2, 27 sans complications micro- ou macrovasculaires et 27 présentant des complications soit micro- soit macrovasculaires, soit les deux. Le même dosage a été effectué chez 38 témoins sains, appariés pour l'âge et le sexe avec les diabétiques. Résultat : Le NT-proBNP plasmatique était plus élevé chez les diabétiques avec complications (médiane 121 pg/ml, intervalle interquartile 50-240 pg/ml) que chez ceux sans complications (37 pg/ml, 21-54 pg/ml, P < 0,01). Comparés au groupe témoin (55 pg/ml, 40-79 pg/ml), seuls les diabétiques avec complications vasculaires avaient un NT-proBNP plasmatique significativement augmenté (P < 0,001). Chez les diabétiques la maladie coronarienne et la néphropathie (définie selon l'excrétion urinaire d'albumine) étaient chacune associée de façon indépendante avec une augmentation des concentrations plasmatiques de NT-proBNP. Conclusion : Chez les diabétiques de type 2 souffrant de complications micro- ou macrovasculaires, les concentrations plasmatiques de NT-proBNP sont augmentées par rapport à celles des malades indemnes de complications vasculaires. L'augmentation de sécrétion de ce peptide est associée de façon indépendante avec la maladie coronarienne et la néphropathie. La mesure du NT-proBNP plasmatique pourrait donc être utile pour dépister la présence de complications micro- ou macrovasculaires.

Immunoscintigraphic localization of inflammatory lesions: concept, radiolabelling and in vitro testing of a granulocyte specific antibody.

Current nuclear medicine techniques for the localization of inflammatory processes are based on injection of 111In labelled autologous granulocytes which need to be isolated and radiolabelled in vitro before reinjection. A new technique is presented here that obviates the need for cell isolation by the direct intravenous injection of a granulocyte specific 123I labelled monoclonal antibody. In this publication the basic parameters of the antibody granulocyte interaction are described. Antibody binding does not inhibit vital functions of the granulocytes, such as chemotaxis and superoxide generation. Scatchard analysis of binding data reveals an apparent affinity of the antibody for granulocytes of 6.8 X 10(9) l/mol and approximately 7.1 X 10(4) binding sites per cell. Due to the high specificity of the antibody, the only expected interference is from CEA producing tumors.

Early neutralizing antibody response against mouse mammary tumor virus: critical role of viral infection and superantigen-reactive T cells.

Infectious mouse mammary tumor virus (MMTV) is a retrovirus that expresses a superantigen shortly after infection of B cells. The superantigen first drives the polyclonal activation and proliferation of superantigen-reactive CD4+ T cells, which then induce the infected B cells to proliferate and differentiate. Part of the MMTV-induced B cell response leads to the production of Abs that are specific for the viral envelope protein gp52. Here we show that this Ab response has virus-neutralizing activity and confers protection against superinfection by other MMTV strains in vivo as soon as 4 to 7 days after infection. A protective Ab titer is maintained lifelong. Viral infection as well as the superantigen-induced T-B collaboration are required to generate this rapid and long lasting neutralizing Ab response. Polyclonal or superantigen-independent B cell activation, on the contrary, does not lead to detectable virus neutralization. The early onset of this superantigen-dependent neutralizing response suggests that viral envelope-specific B cells are selectively recruited to form part of the extrafollicular B cell response and are subsequently amplified and maintained by superantigen-reactive Th cells.

Fate of rubrospinal neurons after unilateral section of the cervical spinal cord in adult macaque monkeys: effects of an antibody treatment neutralizing Nogo-A.

The present study describes in primates the effects of a spinal cord injury on the number and size of the neurons in the magnocellular part of the red nucleus (RNm), the origin of the rubrospinal tract, and evaluates whether a neutralization of Nogo-A reduces the lesioned-induced degenerative processes observed in RNm. Two groups of monkeys were subjected to unilateral section of the spinal cord affecting the rubrospinal tract; one group was subsequently treated with an antibody neutralizing Nogo-A; the second group received a control antibody. Intact animals were also included in the study. Counting neurons stained with a monoclonal antibody recognizing non-phosphorylated epitopes on neurofilaments (SMI-32) indicated that their number in the contralesional RNm was consistently inferior to that in the ipsilesional RNm, in a proportion amounting up to 35%. The lesion also induced shrinkage of the soma of the neurons detected in the contralesional RNm. Infusing an anti-Nogo-A antibody at the site of the lesion did not increase the proportion of SMI-32 positive rubrospinal neurons in the contralesional RNm nor prevent shrinkage.

A peptide inhibitor of c-Jun N-terminal kinase for the treatment of endotoxin-induced uveitis.

PURPOSE: To evaluate the effect of XG-102 (formerly D-JNKI1), a TAT-coupled dextrogyre peptide that selectively inhibits the c-Jun N-terminal kinase, in the treatment of endotoxin-induced uveitis (EIU). METHODS: EIU was induced in Lewis rats by LPS injection. XG-102 was administered at the time of LPS challenge. The ocular biodistribution of XG-102 was evaluated using immunodetection at 24 hours after either 20 microg/kg IV (IV) or 0.2 microg/injection intravitreous (IVT) administrations in healthy or uveitic eyes. The effect of XG-102 on EIU was evaluated using clinical scoring, infiltration cell quantification, inducible nitric oxide synthase (iNOS) expression and immunohistochemistry, and cytokines and chemokines kinetics at 6, 24, and 48 hours using multiplex analysis on ocular media. Control EIU eyes received vehicle injection IV or IVT. The effect of XG-102 on c-Jun phosphorylation in EIU was evaluated by Western blot in eye tissues. RESULTS: After IVT injection, XG-102 was internalized in epithelial cells from iris/ciliary body and retina and in glial and microglial cells in both healthy and uveitic eyes. After IV injection, XG-102 was concentrated primarily in inflammatory cells of uveitic eyes. Using both routes of administration, XG-102 significantly inhibited clinical signs of EIU, intraocular cell infiltration, and iNOS expression together with reduced phosphorylation of c-Jun. The anti-inflammatory effect of XG-102 was mediated by iNOS, IFN-gamma, IL-2, and IL-13. CONCLUSIONS: This is the first evidence that interfering with the JNK pathway can reduce intraocular inflammation. Local administration of XG-102, a clinically evaluated peptide, may have potential for treating uveitis.

Glucagon-like peptide-1 protects beta-cells against apoptosis by increasing the activity of an IGF-2/IGF-1 receptor autocrine loop.

OBJECTIVE: The gluco-incretin hormones glucagon-like peptide (GLP)-1 and gastric inhibitory peptide (GIP) protect beta-cells against cytokine-induced apoptosis. Their action is initiated by binding to specific receptors that activate the cAMP signaling pathway, but the downstream events are not fully elucidated. Here we searched for mechanisms that may underlie this protective effect. RESEARCH DESIGN AND METHODS: We performed comparative transcriptomic analysis of islets from control and GipR(-/-);Glp-1-R(-/-) mice, which have increased sensitivity to cytokine-induced apoptosis. We found that IGF-1 receptor expression was markedly reduced in the mutant islets. Because the IGF-1 receptor signaling pathway is known for its antiapoptotic effect, we explored the relationship between gluco-incretin action, IGF-1 receptor expression and signaling, and apoptosis. RESULTS: We found that GLP-1 robustly stimulated IGF-1 receptor expression and Akt phosphorylation and that increased Akt phosphorylation was dependent on IGF-1 but not insulin receptor expression. We demonstrated that GLP-1-induced Akt phosphorylation required active secretion, indicating the presence of an autocrine activation mechanism; we showed that activation of IGF-1 receptor signaling was dependent on the secretion of IGF-2. We demonstrated, both in MIN6 cell line and primary beta-cells, that reducing IGF-1 receptor or IGF-2 expression or neutralizing secreted IGF-2 suppressed GLP-1-induced protection against apoptosis. CONCLUSIONS: An IGF-2/IGF-1 receptor autocrine loop operates in beta-cells. GLP-1 increases its activity by augmenting IGF-1 receptor expression and by stimulating secretion; this mechanism is required for GLP-1-induced protection against apoptosis. These findings may lead to novel ways of preventing beta-cell loss in the pathogenesis of diabetes.

Desensitization and phosphorylation of the glucagon-like peptide-1 (GLP-1) receptor by GLP-1 and 4-phorbol 12-myristate 13-acetate.

Glucagon-like peptide-1 (GLP-1) stimulates glucose-induced insulin secretion by binding to a specific G protein-coupled receptor linked to activation of the adenylyl cyclase pathway. Here, using insulinoma cell lines, we studied homologous and heterologous desensitization of GLP-1-induced cAMP production. Preexposure of the cells to GLP-1 induced a decrease in GLP-1-mediated cAMP production, as assessed by a 3- to 5-fold rightward shift of the dose-response curve and an approximately 20 percent decrease in the maximal production of cAMP. Activation of protein kinase C by the phorbol ester phorbol 12-myristate 13-acetate (PMA) also induced desensitization of the GLP-1-mediated response, leading to a 6- to 9-fold shift in the EC50 and a 30% decrease in the maximal production of cAMP. Both forms of desensitization were additive, and the protein kinase C inhibitor RO-318220 inhibited PMA-induced desensitization, but not agonist-induced desensitization. GLP-1- and PMA-dependent desensitization correlated with receptor phosphorylation, and the levels of phosphorylation induced by the two agents were additive. Furthermore, PMA-induced, but not GLP-1-induced, phosphorylation was totally inhibited by RO-318220. Internalization of the GLP-1 receptor did not participate in the desensitization induced by PMA, as a mutant GLP-1 receptor lacking the last 20 amino acids of the cytoplasmic tail was found to be totally resistant to the internalization process, but was still desensitized after PMA preexposure. PMA and GLP-1 were not able to induce the phosphorylation of a receptor deletion mutant lacking the last 33 amino acids of the cytoplasmic tail, indicating that the phosphorylation sites were located within the deleted region. The cAMP production mediated by this deletion mutant was not desensitized by PMA and was only poorly desensitized by GLP-1. Together, our results indicate that the production of cAMP and, hence, the stimulation of insulin secretion induced by GLP-1 can be negatively modulated by homologous and heterologous desensitization, mechanisms that involve receptor phosphorylation.

A novel interferon-gamma regulated human melanoma-associated antigen, gp33-38, defined by monoclonal antibody Me14-D12. II. Molecular cloning of a genomic probe.

The human Me14-D12 antigen is a cell surface glycoprotein regulated by interferon-gamma (IFN-gamma) on tumor cell lines of neuroectodermal origin. It consists of two non-convalently linked subunits with apparent mol. wt sizes of 33,000 and 38,000. Here we describe the molecular cloning of a genomic probe for the Me14-D12 gene using the gene transfer approach. Mouse Ltk- cells were stably cotransfected with human genomic DNA and the Herpes Simplex virus thymidine kinase (TK) gene. Primary and secondary transfectants expressing the Me14-D12 antigen were isolated after selection in HAT medium by repeated sorting on a fluorescence activated cell sorter (FACS). A recombinant phage harboring a 14.3 kb insert of human DNA was isolated from a genomic library made from a positive secondary transfectant cell line. A specific probe derived from the phage DNA insert allowed the identification of two mRNAs of 3.5 kb and 2.2 kb in primary and secondary L cell transfectants, as well as in human melanoma cell lines expressing the Me14-D12 antigen. The regulation of Me14-D12 antigen by INF-gamma was retained in the L cell transfectants and could be detected both at the level of protein and mRNA expression.

Brain natriuretic peptide is able to stimulate cardiac progenitor cell proliferation and differentiation in murine hearts after birth.

Brain natriuretic peptide (BNP) contributes to heart formation during embryogenesis. After birth, despite a high number of studies aimed at understanding by which mechanism(s) BNP reduces myocardial ischemic injury in animal models, the actual role of this peptide in the heart remains elusive. In this study, we asked whether BNP treatment could modulate the proliferation of endogenous cardiac progenitor cells (CPCs) and/or their differentiation into cardiomyocytes. CPCs expressed the NPR-A and NPR-B receptors in neonatal and adult hearts, suggesting their ability to respond to BNP stimulation. BNP injection into neonatal and adult unmanipulated mice increased the number of newly formed cardiomyocytes (neonatal: +23 %, p = 0.009 and adult: +68 %, p = 0.0005) and the number of proliferating CPCs (neonatal: +142 %, p = 0.002 and adult: +134 %, p = 0.04). In vitro, BNP stimulated CPC proliferation via NPR-A and CPC differentiation into cardiomyocytes via NPR-B. Finally, as BNP might be used as a therapeutic agent, we injected BNP into mice undergoing myocardial infarction. In pathological conditions, BNP treatment was cardioprotective by increasing heart contractility and reducing cardiac remodelling. At the cellular level, BNP stimulates CPC proliferation in the non-infarcted area of the infarcted hearts. In the infarcted area, BNP modulates the fate of the endogenous CPCs but also of the infiltrating CD45(+) cells. These results support for the first time a key role for BNP in controlling the progenitor cell proliferation and differentiation after birth. The administration of BNP might, therefore, be a useful component of therapeutic approaches aimed at inducing heart regeneration.

In vivo localization of a bispecific antibody which targets human T lymphocytes to lyse human colon cancer cells.

A bispecific MAb was derived from the fusion of a hybridoma producing MAb CD3 with a hybridoma producing MAb L-DI (which is directed against a 41-kDa glycoprotein expressed in most gastro-intestinal and pancreatic carcinomas). Bispecific antibody molecules were isolated from parental antibody molecules by the use of hydroxylapatite-HPLC and shown to target human cytolytic T lymphocytes, irrespective of their original specificity, to specifically lyse human colon carcinoma cells. Localization studies in vivo using nude mice bearing human colon carcinoma xenografts showed significant accumulation of the HPLC-purified 125I-labelled bispecific antibodies into the tumor compared to 131I-labelled control CD3 antibody.

Soluble MHC-peptide complexes induce rapid death of CD8+ CTL.

Soluble MHC-peptide (pMHC) complexes, commonly referred to as tetramers, are widely used to enumerate and to isolate Ag-specific CD8(+) CTL. It has been noted that such complexes, as well as microsphere- or cell-associated pMHC molecules compromise the functional integrity of CTL, e.g., by inducing apoptosis of CTL, which limits their usefulness for T cell sorting or cloning. By testing well-defined soluble pMHC complexes containing linkers of different length and valence, we find that complexes comprising short linkers (i.e., short pMHC-pMHC distances), but not those containing long linkers, induce rapid death of CTL. This cell death relies on CTL activation, the coreceptor CD8 and cytoskeleton integrity, but is not dependent on death receptors (i.e., Fas, TNFR1, and TRAILR2) or caspases. Within minutes of CTL exposure to pMHC complexes, reactive oxygen species emerged and mitochondrial membrane depolarized, which is reminiscent of caspase-independent T cell death. The morphological changes induced during this rapid CTL death are characteristic of programmed necrosis and not apoptosis. Thus, soluble pMHC complexes containing long linkers are recommended to prevent T cell death, whereas those containing short linkers can be used to eliminate Ag-specific CTL.