Identification of molecular targets for selective elimination of TRAIL‐resistant leukemia cells. From spots to in vitro assays using TOP15 charts

The resistance of malignant cells to chemotherapy calls for the development of novel anti‐cancer drugs. TNF‐related apoptosis‐inducing ligand (TRAIL) is a pro‐apoptotic cytokine, which selectively induces apoptosis in malignant cells. We derived two TRAIL‐resistant HL‐60 subclones, HL‐60/P1 and HL‐60/P2, from a TRAIL‐sensitive HL‐60 acute promyelocytic leukemia cell line. To identify therapeutically exploitable “weaknesses” of the TRAIL‐resistant leukemia cells that could be used as molecular targets for their elimination, we performed proteomic (2‐DE) analysis and compared both TRAIL‐resistant subclones with the original TRAIL‐sensitive HL‐60 cells. We identified over 40 differentially expressed proteins. To significantly narrow the lists of candidate proteins, we excluded proteins that are known to be often differentially expressed, regardless of experiment type and tissue (the so‐called “TOP15” proteins). Decreased expression of DNA replication and maintenance proteins MCM7 and RPA32 in HL‐60/P1 cells, and the marked down‐regulation of enzyme adenosine deaminase in HL‐60/P2 cells, suggests increased sensitivity of these cells to DNA‐interfering drugs, and adenosine and its homologues, respectively. In a series of in vitro assays, we confirmed the increased toxicity of etoposide and cisplatin to TRAIL resistant HL‐60/P1 cells, and adenosine and vidarabine to HL‐60/P2, compared with TRAIL‐sensitive HL‐60 cells.     

Backbone dynamics of the antifungal Psd1 pea defensin and its correlation with membrane interaction by NMR spectroscopy

Plant defensins are cysteine-rich cationic peptides, components of the innate immune system. The antifungal sensitivity of certain exemplars was correlated to the level of complex glycosphingolipids in the membrane of fungi strains. Psd1 is a 46 amino acid residue defensin isolated from pea seeds which exhibit antifungal activity. Its structure is characterized by the so-called cysteine-stabilized α/β motif linked by three loops as determined by two-dimensional NMR. In the present work we explored the measurement of heteronuclear Nuclear Overhauser Effects, R1 and R2 ¹⁵N relaxation ratios, and chemical shift to probe the backbone dynamics of Psd1 and its interaction with membrane mimetic systems with phosphatidylcholine (PC) or dodecylphosphocholine (DPC) with glucosylceramide (CMH) isolated from Fusarium solani. The calculated R2 values predicted a slow motion around the highly conserved among Gly12 residue and also in the region of the Turn3 His36-Trp38. The results showed that Psd1 interacts with vesicles of PC or PC:CMH in slightly different forms. The interaction was monitored by chemical shift perturbation and relaxation properties. Using this approach we could map the loops as the binding site of Psd1 with the membrane. The major binding epitope showed conformation exchange properties in the μs-ms timescale supporting the conformation selection as the binding mechanism. Moreover, the peptide corresponding to part of Loop1 (pepLoop1: Gly12 to Ser19) is also able to interact with DPC micelles acquiring a stable structure and in the presence of DPC:CMH the peptide changes to an extended conformation, exhibiting NOE mainly with the carbohydrate and ceramide parts of CMH.     

The tick saliva immunosuppressor, Salp15, contributes to Th17-induced pathology during Experimental Autoimmune Encephalomyelitis

Salp15 is a tick saliva protein that inhibits CD4⁺ T cell differentiation through its interaction with CD4. The protein inhibits early signaling events during T cell activation and IL-2 production. Because murine Experimental Autoimmune Encephalomyelitis development is mediated by central nervous system-infiltrating CD4⁺ T cells that are specific for myelin-associated proteins, we sought to determine whether the treatment of mice with Salp15 during EAE induction would prevent the generation of proinflammatory T cell responses and the development of the disease. Surprisingly, Salp15-treated mice developed more severe EAE than control animals. The treatment of EAE-induced mice with the tick saliva protein did not result in increased infiltration of T cells to the central nervous system, indicating that Salp15 had not affected the permeability of the blood-brain barrier. Salp15 treatment did not affect the development of antibody responses against the eliciting peptide or the presence of IFNγ in the sera. The treatment with Salp15 resulted, however, in the increased differentiation of Th17 cells in vivo, as evidenced by higher IL-17 production from PLP_139-151-specific CD4⁺ T cells isolated from the central nervous system and the periphery. In vitro, Salp15 was able to induce the differentiation of Th17 cells in the presence of IL-6 and the absence of TGFβ These results suggest that a conductive milieu for the differentiation of Th17 cells can be achieved by restriction of the production of IL-2 during T cell differentiation, a role that may be performed by TGFβ and other immunosuppressive agents.     

Phylogenetic distances for neighbour dependent substitution processes

We consider models of nucleotidic substitution processes where the rate of substitution at a given site depends on the state of the neighbours of the site. We first estimate the time elapsed between an ancestral sequence at stationarity and a present sequence. Second, assuming that two sequences are issued from a common ancestral sequence at stationarity, we estimate the time since divergence. In the simplest non-trivial case of a Jukes-Cantor model with CpG influence, we provide and justify mathematically consistent estimators in these two settings. We also provide asymptotic confidence intervals, valid for nucleotidic sequences of finite length, and we compute explicit formulas for the estimators and for their confidence intervals. In the general case of an RN model with YpR influence, we extend these results under a proviso, namely that the equation defining the estimator has a unique solution.     

The PEA-15 Protein Regulates Autophagy via Activation of JNK

PEA-15/PED (phosphoprotein enriched in astrocytes 15 kDa/phosphoprotein enriched in diabetes) is a death effector domain-containing protein which is known to modulate apoptotic cell death. The mechanism by which PEA-15 inhibits caspase activation and increases ERK (extracellular-regulated kinase) activity is well characterized. Here, we demonstrate that PEA-15 is not only pivotal in the activation of the ERK pathway but also modulates JNK (c-Jun N-terminal kinase) signaling. Upon overexpression of PEA-15 in malignant glioma cells, JNK is potently activated. The PEA-15-induced JNK activation depends on the phosphorylation of PEA-15 at both phosphorylation sites (serine 104 and serine 116). The activation of JNK is substantially inhibited by siRNA-mediated down-regulation of endogenous PEA-15. Moreover, we demonstrate that glioma cells overexpressing PEA-15 show increased signs of autophagy in response to classical autophagic stimuli such as ionizing irradiation, serum deprivation, or rapamycin treatment. In contrast, the non-phosphorylatable mutants of PEA-15 are not capable of promoting autophagy. The inhibition of JNK abrogates the PEA-15-mediated increase in autophagy. In conclusion, our data show that PEA-15 promotes autophagy in glioma cells in a JNK-dependent manner. This might render glioma cells more resistant to adverse stimuli such as starvation or ionizing irradiation.     

Nuclear Magnetic Resonance Studies on Huwentoxin-Ⅺ from the Chinese Bird Spider Ornithoctonus huwena:~(15)N Labeling and Sequence-specific ~1H,~(15)N Nuclear Magnetic Resonance Assignments

Huwentoxin-XI purified from the Chinese bird spider Ornithoctonus huwena is a toxin withboth antiprotease activity and potassium channel blocking activity.To determine its solution structure,huwentoxin-Ⅺ was expressed in a yeast eukaryotic expression system and studied by NMR.The ~(15)N labelingstrategy was used to facilitate the process of resonance assignments.The nearly complete sequence-specificassignments of proton and nitrogen resonances were obtained by analyzing a series of two-dimensional(2D)and three-dimensional(3D)spectra,including DQF-COSY,TOCSY,NOESY,~(15)N-~1H HSQC,~(15)N-~1H HNHA,~(15)N-~1H HNHB,~(15)N-~1H TOCSY-HSQC and ~(15)N-~1H NOESY-HSQC spectra.Secondary structure analysis ofhuwentoxin-Ⅺ showed that it mainly contains an N-terminal 3_(10)-helix from Thr3 to Arg5 and a C-terminalα-helix from Gln45 to Cys52,plus a triple-stranded antiparallel β-sheet of Glul 8-Asn23,Thr26-Ile31 andAsn40-Lys41.These studies provide a solid basis for the final structure determination of huwentoxin-Ⅺ.     

Use of isotopic signatures to assess the food web in a tropical shallow marine ecosystem of Southeastern Brazil

A dual isotope approach was used to assess the relative importance of terrestrial vegetation detritus and other primary producers in the trophic web of Flamengo Sound (Ubatuba, SP), SE Brazil, surrounded by the Atlantic Rain Forest. Primary producers showed distinct C signatures and the observed values suggest that little terrestrial or bulk sediment organic matter enter the food web of the sound. Suspended particulate organic matter (POM, supports the bulk of the consumers, with some contribution by macroalgae . Consumers C values ranged from −17.4 to . At least three trophic levels were detectable in the food web. The N value of POM was , while that of sediment and detritus was . The N values of suspension feeding benthic invertebrates were 8.2–, deposit feeders 8.3–, and carnivores 10.7–. Values for fishes were for detritivore, 11.4– for benthic feeders, 12.4– for zooplanktivores, and for piscivores/benthic invertebrate feeders. Squid mean value was . There is a reasonable agreement between feeding habits information from the literature and N values from this study. In the sound, the first and second trophic steps seem to be about 1– higher than those of similar organisms studied in temperate waters and this may reflect an input of allochtonous anthropogenic nitrogen enriched in ¹⁵N from human activities.     

Feeding ecology of the highly threatened common bottlenose dolphin of the Gulf of Ambracia,Greece, through stable isotope analysis

The Gulf of Ambracia, in northwestern Greece, hosts a highly threatened community of about 150 common bottlenose dolphins (Tursiops truncatus). Until now, information on their feeding habits was derived exclusively from fish scale samples collected during surface-feeding events targeting small schooling epipelagic fish. The aim of this study was to determine the diet of bottlenose dolphins living in the Gulf of Ambracia through the application of Bayesian isotopic mixing models. Skin biopsy samples of 16 dolphins were analyzed and no difference related to sex or age-class was found in δ¹³C and δ¹⁵N values. Results suggested that the dolphin diet was mainly based on Trachurus trachurus, species belonging to the family Sparidae: Diplodus annularis, Lithognathus mormyrus, and Sepia officinalis, which represented together about 42% ± 15% of the biomass ingested, followed by species belonging to the order Clupeiformes (Engraulis encrasicolus, Sardinella aurita, and Sardina pilchardus) and the genus Gobius (37% ± 17%). A better understanding of the feeding habits of these dolphins sheds light on the feeding ecology of this highly threatened population by, for instance, evidencing interactions with artisanal fisheries sharing the same target species, and is key for identifying adequate management measures consistent with an ecosystem-based approach.