Nucleic acid-containing amyloid fibrils potently induce type I interferon and stimulate systemic autoimmunity.

The immunopathophysiologic development of systemic autoimmunity involves numerous factors through complex mechanisms that are not fully understood. In systemic lupus erythematosus, type I IFN (IFN-I) produced by plasmacytoid dendritic cells (pDCs) critically promotes the autoimmunity through its pleiotropic effects on immune cells. However, the host-derived factors that enable abnormal IFN-I production and initial immune tolerance breakdown are largely unknown. Previously, we found that amyloid precursor proteins form amyloid fibrils in the presence of nucleic acids. Here we report that nucleic acid-containing amyloid fibrils can potently activate pDCs and enable IFN-I production in response to self-DNA, self-RNA, and dead cell debris. pDCs can take up DNA-containing amyloid fibrils, which are retained in the early endosomes to activate TLR9, leading to high IFNα/β production. In mice treated with DNA-containing amyloid fibrils, a rapid IFN response correlated with pDC infiltration and activation. Immunization of nonautoimmune mice with DNA-containing amyloid fibrils induced antinuclear serology against a panel of self-antigens. The mice exhibited positive proteinuria and deposited antibodies in their kidneys. Intriguingly, pDC depletion obstructed IFN-I response and selectively abolished autoantibody generation. Our study reveals an innate immune function of nucleic acid-containing amyloid fibrils and provides a potential link between compromised protein homeostasis and autoimmunity via a pDC-IFN axis.

The region of mouse mammary tumor virus DNA containing the long terminal repeat includes a long coding sequence and signals for hormonally regulated transcription.

Starting from a biologically active recombinant DNA clone of exogenous unintegrated GR mouse mammary tumor virus, we have generated three subclones of PstI fragments of 1.45, 1.1, and 2.0 kb in the plasmid vector PBR322. The nucleotide sequence has been determined for the clone of 1.45 kb which includes almost the complete region of the long terminal repeat (LTR) plus an adjacent stretch of unique sequence DNA. A short region of the 2.0 kb clone, containing the beginning of the LTR, has also been sequenced. Starting with the A of an initiation codon outside the LTR, we detected an open reading frame of 960 nucleotides, potentially coding for a protein of 320 amino acids (36K). Two hundred nucleotides downstream from the termination codon, and approximately 25 nucleotides upstream from the presumptive initiation site of viral RNA synthesis, we found a promoter-like sequence. The sequence AGTAAA was detected approximately 15-20 nucleotides upstream from the 3' end of virion RNA and probably serves as a polyadenylation signal. The 1.45 kb PstI fragment has been transfected into Ltk- cells together with a plasmid containing the thymidine kinase gene of herpes simplex virus. The virus-specific RNA synthesis detected in a Tk+ cell clone was strongly stimulated by the addition of dexamethasone.

Protein kinase A-dependent phosphorylation of GLUT2 in pancreatic beta cells.

In pancreatic beta cells, cyclic AMP-dependent protein kinase regulates many cellular processes including the potentiation of insulin secretion. The substrates for this kinase, however, have not been biochemically characterized. Here we demonstrate that the glucose transporter GLUT2 is rapidly phosphorylated by protein kinase A following activation of adenylyl cyclase by forskolin or the incretin hormone glucagon-like peptide-1. We show that serines 489 and 501/503 and threonine 510 in the carboxyl-terminal tail of the transporter are the in vitro and in vivo sites of phosphorylation. Stimulation of GLUT2 phosphorylation in beta cells reduces the initial rate of 3-O-methyl glucose uptake by approximately 48% but does not change the Michaelis constant. Similar differences in transport kinetics are observed when comparing the transport activity of GLUT2 mutants stably expressed in insulinoma cell lines and containing glutamates or alanines at the phosphorylation sites. These data indicate that phosphorylation of GLUT2 carboxyl-terminal tail modifies the rate of transport. This lends further support for an important role of the transporter cytoplasmic tail in the modulation of catalytic activity. Finally, because activation of protein kinase A stimulates glucose-induced insulin secretion, we discuss the possible involvement of GLUT2 phosphorylation in the amplification of the glucose signaling process.

Magnetic and in vitro heating properties of implants formed in situ from injectable formulations and containing superparamagnetic iron oxide nanoparticles (SPIONs) embedded in silica microparticles for magnetically induced local hyperthermia

The biological and therapeutic responses to hyperthermia, when it is envisaged as an anti-tumor treatment modality, are complex and variable. Heat delivery plays a critical role and is counteracted by more or less efficient body cooling, which is largely mediated by blood flow. In the case of magnetically mediated modality, the delivery of the magnetic particles, most often superparamagnetic iron oxide nanoparticles (SPIONs), is also critically involved. We focus here on the magnetic characterization of two injectable formulations able to gel in situ and entrap silica microparticles embedding SPIONs. These formulations have previously shown suitable syringeability and intratumoral distribution in vivo. The first formulation is based on alginate, and the second on a poly(ethylene-co-vinyl alcohol) (EVAL). Here we investigated the magnetic properties and heating capacities in an alternating magnetic field (141 kHz, 12 mT) for implants with increasing concentrations of magnetic microparticles. We found that the magnetic properties of the magnetic microparticles were preserved using the formulation and in the wet implant at 37 degrees C, as in vivo. Using two orthogonal methods, a common SLP (20 Wg(-1)) was found after weighting by magnetic microparticle fraction, suggesting that both formulations are able to properly carry the magnetic microparticles in situ while preserving their magnetic properties and heating capacities. (C) 2010 Elsevier B.V. All rights reserved.

A peptide encoded by the human MAGE3 gene and presented by HLA-B44 induces cytolytic T lymphocytes that recognize tumor cells expressing MAGE3.

The human MAGE3 gene is expressed in a significant proportion of tumors of various histological types, but is silent in normal adult tissues other than testis and placenta. Antigens encoded by MAGE3 may therefore be useful targets for specific antitumor immunization. Two antigenic peptides encoded by the MAGE3 gene have been reported previously. One is presented to cytolytic T lymphocytes (CTL) by HLA-A1, the other by HLA-A2 molecules. Here we show that MAGE3 also codes for a peptide that is presented to CTL by HLA-B44. MAGE3 peptides containing the HLA-B44 peptide binding motif were synthesized. Peptide MEVDPIGHLY, which showed the strongest binding to HLA-B44, was used to stimulate blood T lymphocytes from normal HLA-B44 donors. CTL clones were obtained that recognized not only HLA-B44 cells sensitized with the peptide, but also HLA-B44 tumor cell lines expressing MAGE3. The proportion of metastatic melanomas expressing the MAGE3/HLA-B44 antigen should amount to approximately 17% in the Caucasian population, since 24% of individuals carry the HLA-B44 allele and 76% of these tumors express MAGE3.

Fas-associated death domain protein (FADD) and caspase-8 mediate up-regulation of c-Fos by Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) via a FLICE inhibitory protein (FLIP)-regulated pathway.

Fas, a death domain-containing member of the tumor necrosis factor receptor family and its ligand FasL have been predominantly studied with respect to their capability to induce cell death. However, a few studies indicate a proliferation-inducing signaling activity of these molecules too. We describe here a novel signaling pathway of FasL and the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) that triggers transcriptional activation of the proto-oncogene c-fos, a typical target gene of mitogenic pathways. FasL- and TRAIL-mediated up-regulation of c-Fos was completely dependent on the presence of Fas-associated death domain protein (FADD) and caspase-8, but caspase activity seemed to be dispensable as a pan inhibitor of caspases had no inhibitory effect. Upon overexpression of the long splice form of cellular FADD-like interleukin-1-converting enzyme (FLICE) inhibitory protein (cFLIP) in Jurkat cells, FasL- and TRAIL-induced up-regulation of c-Fos was almost completely blocked. The short splice form of FLIP, however, showed a rather stimulatory effect on c-Fos induction. Together these data demonstrate the existence of a death receptor-induced, FADD- and caspase-8-dependent pathway leading to c-Fos induction that is inhibited by the long splice form FLIP-L.

Interaction of antigenic peptides with MHC class I molecules on living cells studied by photoaffinity labeling.

Using a direct binding assay based on photoaffinity labeling, we have studied the interaction of antigenic peptides with murine MHC class I molecules on living cells. Photoreactive derivatives were prepared by N-terminal amidation with iodo, 4-azido salicylic acid of the Kd restricted Plasmodium berghei circumsporozoite (P.b. CS) peptide 253-260 (YIPSAEKI) and the Db-restricted Adenovirus 5 early region 1A (Ad5 E1A) peptide 234-243 (SGPSNTPPEI). As assessed in functional competition experiments, both peptide derivatives retained the specific binding activity of the parental peptides for Kd or Dd, respectively. The P.b. CS photoprobe specifically labeled Kd molecules on P815 (H-2d) cells, but failed to label RMA (H-2b) cells. Conversely, the Ad5 E1A photoprobe specifically labeled Db molecules on RMA cells, but failed to label P815 cells. When the two photoprobes were tested on a panel of Con A-activated spleen cells expressing 10 different H-2 haplotypes, significant photoaffinity labeling was observed only on H-2d cells with the P.b. CS photoprobe and on H-2b cells with the Ad5 E1A photoprobe. Labeling of cell-associated Kd or Db molecules with the photoprobes was specifically inhibited by antigenic peptides known to be presented by the same class I molecule. Photoaffinity labeling of Kd with the P.b. CS photoprobe was used to study the dynamics of peptide binding on living P815 cells. Binding increased steadily with the incubation period (up to 8 h) at 37 degrees C and at ambient temperature, but was greatly reduced (greater than 95%) at 0 to 4 degrees C or in the presence of ATP synthesis inhibitors. The magnitude of the labeling was twofold higher at room temperature than at 37 degrees C. In contrast, binding to isolated Kd molecules in solution rapidly reached maximal binding, particularly at 37 degrees C. Dissociation of the photoprobe from either cell-associated or soluble Kd molecules was similar, with a half time of approximately 1 h at 37 degrees C, whereas the complexes were long-lived at 4 degrees C in both instances.

Malaria vaccine candidate: design of a multivalent subunit α-helical coiled coil poly-epitope.

A new strategy for the rapid identification of new malaria antigens based on protein structural motifs was previously described. We identified and evaluated the malaria vaccine potential of fragments of several malaria antigens containing α-helical coiled coil protein motifs. By taking advantage of the relatively short size of these structural fragments, we constructed different poly-epitopes in which 3 or 4 of these segments were joined together via a non-immunogenic linker. Only peptides that are targets of human antibodies with anti-parasite in vitro biological activities were incorporated. One of the constructs, P181, was well recognized by sera and peripheral blood mononuclear cells (PBMC) of adults living in malaria-endemic areas. Affinity purified antigen-specific human antibodies and sera from P181-immunized mice recognised native proteins on malaria-infected erythrocytes in both immunofluorescence and western blot assays. In addition, specific antibodies inhibited parasite development in an antibody dependent cellular inhibition (ADCI) assay. Naturally induced antigen-specific human antibodies were at high titers and associated with clinical protection from malaria in longitudinal follow-up studies in Senegal.

Esfingolipids i senyal•lització cel.lular: l'(E)-2-hexadecenal com a possible lípid bioactiu

L’esfingosina-1-fosfat (S1P) és un lípid bioactiu amb funcions crucials en la biologia cel•lular. Entre aquestes, la seva activitat mitogènica i citoprotectora són les més estudiades. L’S1P és catabolitzada intracel•lularment mitjançant l’esfingosina-1-fosfat liasa (SGPL1) per generar (E)-2-hexadecenal i fosforiletanolamina. L’objectiu d’aquest projecte és explorar si l’(E)-2-hexadecenal és realment un catabòlit innocu o bé si, pel seu caràcter acceptor de Michael, és capaç de reaccionar amb pèptids o proteïnes específics. Aquesta interacció podria traduïr-se en funcions biològiques determinades, algunes de les quals són possiblement atribuïdes a l’esfingosina-1-fosfat com a tal. Per poder explorar el potencials adductes proteïcs amb l’aldehid, s’han emprat, sobre cèl•lules HeLa que sobreexpressen SGPL1, sondes anàlegs a esfingosina i esfinganina (i els seus derivats fosforil•lats) que presenten una funció azida en la posició omega de la cadena esfingoide. Aquestes, mitjançant química click sense coure, s’han fet reaccionar amb una molècula que presenta un dibenzociclooctí unit a biotina DBCObiotina). Després d’aïllar les proteïnes així biotinilades amb una reïna d’estreptavidina, aquestes es van separar per electroforesi. Les bandes proteïques observades es van extreure del gel i es van digerir amb tripsina, per posteriorment analitzar els pèptids per MALDI-TOF, el que permetria l’identificació de proteïnes a partir de “peptide mass fingerprinting”. Lamentablement, a la fi d’aquest contracte, encara no s’ha pogut identificar cap proteïna que s’uneixi a l’aldehid alliberat per la reacció de l’esfingosina-1- fosfat liasa. No obstant, durant aquest temps s’ha millorat el mètode per detectar aquests adductes proteïcs. Per això, si la recerca continua en aquesta línia, properament es podria saber amb certesa si existeixen o no aquestes interaccions covalents entre determinades proteïnes i l’(E)-2-hexadecenal.

Generic approach for the sensitive absolute quantification of large undigested peptides in plasma using a particular liquid chromatography-mass spectrometry setup.

A generic LC-MS approach for the absolute quantification of undigested peptides in plasma at mid-picomolar levels is described. Nine human peptides namely, brain natriuretic peptide (BNP), substance P (SubP), parathyroid hormone 1-34 (PTH), C-peptide, orexines A and B (Orex-A and -B), oxytocin (Oxy), gonadoliberin-1 (gonadothropin releasing-hormone or luteinizing hormone-releasing hormone, LHRH) and α-melanotropin (α-MSH) were targeted. Plasma samples were extracted via a 2-step procedure: protein precipitation using 1vol of acetonitrile followed by ultrafiltration of supernatants on membranes with a MW cut-off of 30 kDa. By applying a specific LC-MS setup, large volumes of filtrates (e.g., 2×750 μL) were injected and the peptides were trapped on a 1mm i.d.×10 mm length C8 column using a 10× on-line dilution. Then, the peptides were back-flushed and a second on-line dilution (2×) was applied during the transfer step. The refocalized peptides were resolved on a 0.3mm i.d. C18 analytical column. Extraction recovery, matrix effect and limits of detection were evaluated. Our comprehensive protocol demonstrates a simple and efficient sample preparation procedure followed by the analysis of peptides with limits of detection in the mid-picomolar range. This generic approach can be applied for the determination of most therapeutic peptides and possibly for endogenous peptides with latest state-of-the-art instruments.

Digestion of Streptococcus pneumoniae cell walls with its major peptidoglycan hydrolase releases branched stem peptides carrying proinflammatory activity.

The peptidoglycan of Gram-positive bacteria is known to trigger cytokine release from peripheral blood mononuclear cells (PBMCs). However, it requires 100-1000 times more Gram-positive peptidoglycan than Gram-negative lipopolysaccharide to release the same amounts of cytokines from target cells. Thus, either peptidoglycan is poorly active or only part of it is required for PBMC activation. To test this hypothesis, purified Streptococcus pneumoniae walls were digested with their major autolysin N-acetylmuramoyl-L-alanine amidase, and/or muramidase. Solubilized walls were separated by reverse phase high pressure chromatography. Individual fractions were tested for their PBMC-stimulating activity, and their composition was determined. Soluble components had a Mr between 600 and 1500. These primarily comprised stem peptides cross-linked to various extents. Simple stem peptides (Mr <750) were 10-fold less active than undigested peptidoglycan. In contrast, tripeptides (Mr >1000) were >/=100-fold more potent than the native material. One dipeptide (inactive) and two tripeptides (active) were confirmed by post-source decay analysis. Complex branched peptides represented </=2% of the total material, but their activity (w/w) was almost equal to that of LPS. This is the first observation suggesting that peptidoglycan stem peptides carry high tumor necrosis factor-stimulating activity. These types of structures are conserved among Gram-positive bacteria and will provide new material to help elucidate the mechanism of peptidoglycan-induced inflammation.

Advantages of extended bottom-up proteomics using sap9 for analysis of monoclonal antibodies.

Despite the recent advances in structural analysis of monoclonal antibodies with bottom-up, middle-down, and top-down mass spectrometry (MS), further improvements in analysis accuracy, depth, and speed are needed. The remaining challenges include quantitatively accurate assignment of post-translational modifications, reduction of artifacts introduced during sample preparation, increased sequence coverage per liquid chromatography (LC) MS experiment, and ability to extend the detailed characterization to simple antibody cocktails and more complex antibody mixtures. Here, we evaluate the recently introduced extended bottom-up proteomics (eBUP) approach based on proteolysis with secreted aspartic protease 9, Sap9, for analysis of monoclonal antibodies. Key findings of the Sap9-based proteomics analysis of a single antibody include: (i) extensive antibody sequence coverage with up to 100% for the light chain and up to 99-100% for the heavy chain in a single LC-MS run; (ii) connectivity of complementarity-determining regions (CDRs) via Sap9-produced large proteolytic peptides (3.4 kDa on average) containing up to two CDRs per peptide; (iii) reduced artifact introduction (e. g., deamidation) during proteolysis with Sap9 compared to conventional bottom-up proteomics workflows. The analysis of a mixture of six antibodies via Sap9-based eBUP produced comparable results. Due to the reasons specified above, Sap9-produced proteolytic peptides improve the identification confidence of antibodies from the mixtures compared to conventional bottom-up proteomics dealing with shorter proteolytic peptides.

K(+) channel expression distinguishes subpopulations of parvalbumin- and somatostatin-containing neocortical interneurons

Kv3.1 and Kv3.2 K+ channel proteins form similar voltage-gated K+ channels with unusual properties, including fast activation at voltages positive to −10 mV and very fast deactivation rates. These properties are thought to facilitate sustained high-frequency firing. Kv3.1 subunits are specifically found in fast-spiking, parvalbumin (PV)-containing cortical interneurons, and recent studies have provided support for a crucial role in the generation of the fast-spiking phenotype. Kv3.2 mRNAs are also found in a small subset of neocortical neurons, although the distribution of these neurons is different. We raised antibodies directed against Kv3.2 proteins and used dual-labeling methods to identify the neocortical neurons expressing Kv3.2 proteins and to determine their subcellular localization. Kv3.2 proteins are prominently expressed in patches in somatic and proximal dendritic membrane as well as in axons and presynaptic terminals of GABAergic interneurons. Kv3.2 subunits are found in all PV-containing neurons in deep cortical layers where they probably form heteromultimeric channels with Kv3.1 subunits. In contrast, in superficial layer PV-positive neurons Kv3.2 immunoreactivity is low, but Kv3.1 is still prominently expressed. Because Kv3.1 and Kv3.2 channels are differentially modulated by protein kinases, these results raise the possibility that the fast-spiking properties of superficial- and deep-layer PV neurons are differentially regulated by neuromodulators. Interestingly, Kv3.2 but not Kv3.1 proteins are also prominent in a subset of seemingly non-fast-spiking, somatostatin- and calbindin-containing interneurons, suggesting that the Kv3.1–Kv3.2 current type can have functions other than facilitating high-frequency firing.

Autosis is a Na+,K+-ATPase-regulated form of cell death triggered by autophagy-inducing peptides, starvation, and hypoxia-ischemia.

A long-standing controversy is whether autophagy is a bona fide cause of mammalian cell death. We used a cell-penetrating autophagy-inducing peptide, Tat-Beclin 1, derived from the autophagy protein Beclin 1, to investigate whether high levels of autophagy result in cell death by autophagy. Here we show that Tat-Beclin 1 induces dose-dependent death that is blocked by pharmacological or genetic inhibition of autophagy, but not of apoptosis or necroptosis. This death, termed "autosis," has unique morphological features, including increased autophagosomes/autolysosomes and nuclear convolution at early stages, and focal swelling of the perinuclear space at late stages. We also observed autotic death in cells during stress conditions, including in a subpopulation of nutrient-starved cells in vitro and in hippocampal neurons of neonatal rats subjected to cerebral hypoxia-ischemia in vivo. A chemical screen of ~5,000 known bioactive compounds revealed that cardiac glycosides, antagonists of Na(+),K(+)-ATPase, inhibit autotic cell death in vitro and in vivo. Furthermore, genetic knockdown of the Na(+),K(+)-ATPase α1 subunit blocks peptide and starvation-induced autosis in vitro. Thus, we have identified a unique form of autophagy-dependent cell death, a Food and Drug Administration-approved class of compounds that inhibit such death, and a crucial role for Na(+),K(+)-ATPase in its regulation. These findings have implications for understanding how cells die during certain stress conditions and how such cell death might be prevented.

Synthesis and anticancer activity of long-chain isonicotinic ester ligand-containing arene ruthenium complexes and nanoparticles

Arene ruthenium complexes containing long-chain N-ligands L1 = NC5H4-4-COO-C6H4-4-O-(CH2)9-CH3 or L2 = NC5H4-4-COO-(CH2)10-O-C6H4-4-COO-C6H4-4-C6H4-4-CN derived from isonicotinic acid, of the type [(arene)Ru(L)Cl2] (arene = C6H6, L = L1: 1; arene = p-MeC6H4Pr i , L = L1: 2; arene = C6Me6, L = L1: 3; arene = C6H6, L = L2: 4; arene = p-MeC6H4Pr i , L = L2: 5; arene = C6Me6, L = L2: 6) have been synthesized from the corresponding [(arene)RuCl2]2 precursor with the long-chain N-ligand L in dichloromethane. Ruthenium nanoparticles stabilized by L1 have been prepared by the solvent-free reduction of 1 with hydrogen or by reducing [(arene)Ru(H2O)3]SO4 in ethanol in the presence of L1 with hydrogen. These complexes and nanoparticles show a high anticancer activity towards human ovarian cell lines, the highest cytotoxicity being obtained for complex 2 (IC50 = 2 μM for A2780 and 7 μM for A2780cisR)