Isolation and characterization of a new non-toxic two-chain ribosome-inactivating protein from fruits of elder (Sambucus nigra L.)

Sambucus (Caprifoliaceae) species contain nigrin b and ebulinI, which are two-chain ribosomeinactivating proteins (RIPs)belonging to a new type of RIPS which are non-toxic to miceand cultured human cells. In this work the presence in fruitsof elder (S. nigra L.) of a new non-toxic type 2 RIP (nigrinf) that co-exists with a lectin known as SNA IV is described.Nigrin f strongly inhibited protein synthesis in mammalian,but not in plant, ribosomes, promoting the depurination of sensitiveribosomes and thus allowing the release of the RIP diagnosticRNA fragment. Nigrin f is composed of two dissimilar subunitslinked by disulphide bridges with apparent M_r values of 31 600and 26 300. The N-terminal amino acid sequence revealed closehomology of the catalytic A chain with type 1 RIPs, especiallythose from Cucurbitaceae, and the B chain with several lectinspreviously isolated from Sambucus species. Nigrin f was nottoxic to mice when injected intraperitoneally up to 2 mg kg^–1.In addition, NHC human cells were also insensitive to nigrinf up to 60 µg ml^–1. Anti-nigrin b rabbit polyclonalantibodies reacted with nigrin f, indicating that nigrin b andnigrin f are proteins with similar structures. Key words: Sambucus nigra, elder fruits, nigrin f, ribosomeinactivating protein, characterization     

Synthesis and induction of apoptosis signaling pathway of ent-kaurane derivatives

Thirty one ent-kaurane derivatives were prepared from kaurenoic acid (1), grandiflorenic acid (16), 15α-acetoxy-kaurenoic acid (26) and 16α-hydroxy-kaurenoic acid (31). They were tested for their ability to inhibit cell viability in the mouse leukemic macrophagic RAW 264.7 cell line. The most effective compounds were 12, 20, 21, and 23. These were selected for further evaluation in other human cancer cell lines such as Hela, HepG2, and HT-29. Similar effects were obtained although RAW 264.7 cells were more sensitive. In addition, these compounds were significantly less cytotoxic in non-transformed cells. The apoptotic potential of the most active compounds was investigated and they were able to induce apoptosis with compound 12 being the best inducer. The caspase-3, -8 and -9 activities were measured. The results obtained showed that compounds 12, 21, and 23 induce apoptosis via the activation of caspase-8, whereas compound 20 induces apoptosis via caspase-9. Immunoblot analysis of the expression of p53, Bax, Bcl-2, Bcl-xl, and IAPs in RAW 264.7 cells was also carried out. When cells were exposed to 5 μM of the different compounds, expression levels of p53 and Bax increased whereas levels of antiapoptotic proteins such as Bc1-2, Bc1-x1, and IAPs decreased. In conclusion, kaurane derivatives (12, 20, 21, and 23) induce apoptosis via both the mitochondrial and membrane death receptor pathways, involving the Bcl-2 family proteins. Taken together these results provide a role of kaurane derivatives as apoptotic inducers in tumor cells.     

Tolerance to herbivory in lupin genotypes with different alkaloid concentration: Interspecific differences between Lupinus albus L. and L. angustifolius L.

Lupin genotypes accumulate alkaloids that act as feeding deterrents for several kinds of herbivores. Breeding sweet (low alkaloid) genotypes resulted in a greater dependence on pesticides. Besides the concentration of defensive chemicals, plants possess another way to deal with herbivory, to allocate post-damage resources to growth in order to reach compensation in biomass (tolerance).

These two ways to deal with herbivores were postulated as alternative strategies, as scarce resources allocated to one function (growth or secondary metabolism) would not be available for the other function. Genotypes could differ in the way they respond to herbivory; identifying those genotypes with greater ability to overcome the damage would be useful to decrease the use of pesticides.

The aim of this work was to compare tolerance to herbivory in Lupinus albus and Lupinus angustifolius genotypes with contrasting alkaloid concentration. Tolerance was evaluated by comparing growth and grain yield of field-grown cut and uncut plants. Cutting treatments were performed at flowering by cutting 50% of the upper shoot biomass (including the main apex, stems, flowers and leaves). Differences between species were found in their tolerance to herbivory. While L. angustifolius showed full compensation in growth or grain yield that allowed cut plants to equal controls biomass or yield after damage, simulated herbivory reduced growth and grain yield in L. albus.     

The Influence of Genetic Variability and Proinflammatory Status on the Development of Bone Disease in Patients with Gaucher Disease

Gaucher disease, the most common lysosomal storage disorder, is caused by β-glucocerebrosidase deficiency. Bone complications are the major cause of morbidity in patients with type 1 Gaucher disease (GD1). Genetic components strongly influence bone remodelling. In addition, chronic inflammation produced by Gaucher cells induces the production of several cytokines, which leads to direct changes in the bone remodelling process and can also affect the process indirectly through other immune cells. In this study, we analysed the association between bone mineral density (BMD), bone marrow burden score, and relevant genetic polymorphisms related to bone metabolism, as well as profiles of proinflammatory cytokines in a GD1 cohort. This study included 83 patients distributed according to bone status. BMD was measured with DXA and broadband ultrasound attenuation; bone marrow involvement was evaluated using MRI. We also analysed 26 SNPs located in 14 genes related to bone metabolism. To assess proinflammatory status, we analysed IL-4, IL-6, IL-7, IL-10, IL-13, MIP-1α, MIP-1β, and TNFα in plasma samples from 71 control participants and GD1 patients. SNP genotype proportions and BMD differed significantly between ESRI c.453-397T>C and VDR c.1024+283G>A variants. We also observed significant associations between GD1 genotypes and bone affectation. When patients were stratified by spleen status, we observed significant correlations between non-/splenectomized groups and Spanish MRI (S-MRI) score. Across genotype proportions of non-/splenectomized patients and S-MRI, we observed significant differences in ESRI c.453-397T>C, VDR c.-83-25988G>A, and TNFRSF11B c.9C>G polymorphisms. We observed different significant proinflammatory profiles between control participants, treatment-naïve patients, and patients on enzyme replacement therapy (ERT); between non-/splenectomized patients (between untreated and ERT-treated patients) and among those with differing GBA genotypes. The data suggest that patients with GD1 have increased susceptibility to developing bone disease owing to the coexistence of genetic variants, and that genetic background in GD1 is fundamental to regulate the impact of proinflammatory status on the development of bone disease.     

Tolerance Associated Gene Expression following Allogeneic Hematopoietic Cell Transplantation

Biologic markers of immune tolerance may facilitate tailoring of immune suppression duration after allogeneic hematopoietic cell transplantation (HCT). In a cross-sectional study, peripheral blood samples were obtained from tolerant (n = 15, median 38.5 months post-HCT) and non-tolerant (n = 17, median 39.5 post-HCT) HCT recipients and healthy control subjects (n = 10) for analysis of immune cell subsets and differential gene expression. There were no significant differences in immune subsets across groups. We identified 281 probe sets unique to the tolerant (TOL) group and 122 for non-tolerant (non-TOL). These were enriched for process networks including NK cell cytotoxicity, antigen presentation, lymphocyte proliferation, and cell cycle and apoptosis. Differential gene expression was enriched for CD56, CD66, and CD14 human lineage-specific gene expression. Differential expression of 20 probe sets between groups was sufficient to develop a classifier with > 90% accuracy, correctly classifying 14/15 TOL cases and 15/17 non-TOL cases. These data suggest that differential gene expression can be utilized to accurately classify tolerant patients following HCT. Prospective investigation of immune tolerance biologic markers is warranted.     

DNA barcoding and minibarcoding as a powerful tool for feather mite studies

Feather mites (Astigmata: Analgoidea and Pterolichoidea) are among the most abundant and commonly occurring bird ectosymbionts. Basic questions on the ecology and evolution of feather mites remain unanswered because feather mite species identification is often only possible for adult males, and it is laborious even for specialized taxonomists, thus precluding large‐scale identifications. Here, we tested DNA barcoding as a useful molecular tool to identify feather mites from passerine birds. Three hundred and sixty‐one specimens of 72 species of feather mites from 68 species of European passerine birds from Russia and Spain were barcoded. The accuracy of barcoding and minibarcoding was tested. Moreover, threshold choice (a controversial issue in barcoding studies) was also explored in a new way, by calculating through simulations the effect of sampling effort (in species number and species composition) on threshold calculations. We found one 200‐bp minibarcode region that showed the same accuracy as the full‐length barcode (602 bp) and was surrounded by conserved regions potentially useful for group‐specific degenerate primers. Species identification accuracy was perfect (100%) but decreased when singletons or species of the Proctophyllodes pinnatus group were included. In fact, barcoding confirmed previous taxonomic issues within the P. pinnatus group. Following an integrative taxonomy approach, we compared our barcode study with previous taxonomic knowledge on feather mites, discovering three new putative cryptic species and validating three previous morphologically different (but still undescribed) new species.     

Are SNP-Smoking Association Studies Needed in Controls? DNA Repair Gene Polymorphisms and Smoking Intensity

Variations in tobacco-related cancers, incidence and prevalence reflect differences in tobacco consumption in addition to genetic factors. Besides, genes related to lung cancer risk could be related to smoking behavior. Polymorphisms altering DNA repair capacity may lead to synergistic effects with tobacco carcinogen-induced lung cancer risk. Common problems in genetic association studies, such as presence of gene-by-environment (G x E) correlation in the population, may reduce the validity of these designs. The main purpose of this study was to evaluate the independence assumption for selected SNPs and smoking behaviour in a cohort of 320 healthy Spanish smokers. We found an association between the wild type alleles of XRCC3 Thr241Met or KLC3 Lys751Gln and greater smoking intensity (OR = 12.98, 95% CI = 2.86–58.82 and OR=16.90, 95% CI=2.09-142.8; respectively). Although preliminary, the results of our study provide evidence that genetic variations in DNA-repair genes may influence both smoking habits and the development of lung cancer. Population-specific G x E studies should be carried out when genetic and environmental factors interact to cause the disease.     

Identification of Chemical Inhibitors of β-Catenin-Driven Liver Tumorigenesis in Zebrafish

Hepatocellular carcinoma (HCC) is one of the most lethal human cancers. The search for targeted treatments has been hampered by the lack of relevant animal models for the genetically diverse subsets of HCC, including the 20-40% of HCCs that are defined by activating mutations in the gene encoding β-catenin. To address this chemotherapeutic challenge, we created and characterized transgenic zebrafish expressing hepatocyte-specific activated β-catenin. By 2 months post fertilization (mpf), 33% of transgenic zebrafish developed HCC in their livers, and 78% and 80% of transgenic zebrafish showed HCC at 6 and 12 mpf, respectively. As expected for a malignant process, transgenic zebrafish showed significantly decreased mean adult survival compared to non-transgenic control siblings. Using this novel transgenic model, we screened for druggable pathways that mediate β-catenin-induced liver growth and identified two c-Jun N-terminal kinase (JNK) inhibitors and two antidepressants (one tricyclic antidepressant, amitriptyline, and one selective serotonin reuptake inhibitor) that suppressed this phenotype. We further found that activated β-catenin was associated with JNK pathway hyperactivation in zebrafish and in human HCC. In zebrafish larvae, JNK inhibition decreased liver size specifically in the presence of activated β-catenin. The β-catenin-specific growth-inhibitory effect of targeting JNK was conserved in human liver cancer cells. Our other class of hits, antidepressants, has been used in patient treatment for decades, raising the exciting possibility that these drugs could potentially be repurposed for cancer treatment. In support of this proposal, we found that amitriptyline decreased tumor burden in a mouse HCC model. Our studies implicate JNK inhibitors and antidepressants as potential therapeutics for β-catenin-induced liver tumors.     

Detection of Peptide-Based Nanoparticles in Blood Plasma by ELISA

AimsThe aim of the current study was to develop a method to detect peptide-linked nanoparticles in blood plasma.ResultsThe ELISA based method for the detection of FITC labeled peptides had a detection limit of 1 ng/mL. We were able to accurately measure peptides bound to pentafluorophenyl methacrylate nanoparticles in blood plasma of rats, and similar results were obtained by LC/MS.ConclusionsWe detected FITC-labeled peptides on pentafluorophenyl methacrylate nanoparticles after injection in vivo. This method can be extended to detect nanoparticles with different chemical compositions.