Growth and swainsonine production of Swainsona galegifolia (Andr.) R. Br. untransformed and transformed root cultures

Untransformed and transformed root cultures of Swainsona galegifollawere established for swainsonine production. Transformed rootsgrew faster and produced higher swainsonine levels (62.3 µgg^–1 DW) than untransformed roots (23.6 ,µg g^–1DW) or roots of intact plants (8.7 µg g^–1 DW). Transformationof a number of plant genotypes using A. rhizogenes strain LBA9402 showed that plant genotype Influences swainsonine levelin transformed roots but that a wide range of swainsonine levelscan be induced by separate transformation events in the samegenotype. Enhancement of swainsonine production was attemptedby treatment with sugars and induction of polyploid roots. Key words: Agrobacterium rhizogenes, root cultures, Swainsona galegifolia, swainsonine     

Bi-specific TCR-anti CD3 redirected T-cell targeting of NY-ESO-1- and LAGE-1-positive tumors

NY-ESO-1 and LAGE-1 are cancer testis antigens with an ideal profile for tumor immunotherapy, combining up-regulation in many cancer types with highly restricted expression in normal tissues and sharing a common HLA-A*0201 epitope, 157–165. Here, we present data to describe the specificity and anti-tumor activity of a bifunctional ImmTAC, comprising a soluble, high-affinity T-cell receptor (TCR) specific for NY-ESO-1_157–165 fused to an anti-CD3 scFv. This reagent, ImmTAC-NYE, is shown to kill HLA-A2, antigen-positive tumor cell lines, and freshly isolated HLA-A2- and LAGE-1-positive NSCLC cells. Employing time-domain optical imaging, we demonstrate in vivo targeting of fluorescently labelled high-affinity NYESO-specific TCRs to HLA-A2-, NY-ESO-1_157–165-positive tumors in xenografted mice. In vivo ImmTAC-NYE efficacy was tested in a tumor model in which human lymphocytes were stably co-engrafted into NSG mice harboring tumor xenografts; efficacy was observed in both tumor prevention and established tumor models using a GFP fluorescence readout. Quantitative RT-PCR was used to analyze the expression of both NY-ESO-1 and LAGE-1 antigens in 15 normal tissues, 5 cancer cell lines, 10 NSCLC, and 10 ovarian cancer samples. Overall, LAGE-1 RNA was expressed at a greater frequency and at higher levels than NY-ESO-1 in the tumor samples. These data support the clinical utility of ImmTAC-NYE as an immunotherapeutic agent for a variety of cancers.     

Neutralizing Nanobodies Targeting Diverse Chemokines Effectively Inhibit Chemokine Function

Chemokine receptors and their ligands play a prominent role in immune regulation but many have also been implicated in inflammatory diseases such as multiple sclerosis, rheumatoid arthritis, allograft rejection after transplantation, and also in cancer metastasis. Most approaches to therapeutically target the chemokine system involve targeting of chemokine receptors with low molecular weight antagonists. Here we describe the selection and characterization of an unprecedented large and diverse panel of neutralizing Nanobodies (single domain camelid antibodies fragment) directed against several chemokines. We show that the Nanobodies directed against CCL2 (MCP-1), CCL5 (RANTES), CXCL11 (I-TAC), and CXCL12 (SDF-1α) bind the chemokines with high affinity (at nanomolar concentration), thereby blocking receptor binding, inhibiting chemokine-induced receptor activation as well as chemotaxis. Together, we show that neutralizing Nanobodies can be selected efficiently for effective and specific therapeutic treatment against a wide range of immune and inflammatory diseases.     

Hair testing for drugs of abuse

Hair testing for drugs of abuse is a developing technology, which offers the possibility of longer detection times than is commonly obtained with urine analysis. It is the main method for evaluation of an individual's drugs of abuse history. In many countries hair analysis is routinely used to detect drug abuse in forensic cases, occupational and traffic medicine and clinical toxicology. Hair analysis in pregnant women, neonates and infants is a useful tool for the detection of drug exposure in utero. In Croatia hair testing for drugs of abuse is performed at the Institute for Medical Research and Occupational Health. Three-year experience in drugs of abuse analysis in hair is described. In 331 hair samples (270 from adolescents and 61 from adults) opiates and metabolites, cocaine, methadone, and amphetamines were analyzed by gas chromatography/mass spectrometry. Most prevalent drugs of abuse in adolescents were amphetamines, and in adults heroin. From the examples cited and samples analyzed it is evident that hair testing is emerging as a reliable biological marker for cumulative account of individual exposure to drugs of abuse.     

Antioxidant Properties of Selected <Emphasis Type="Italic">Boletus</Emphasis> Mushrooms

Considering the growing interest for mushrooms and the demand search of natural antioxidants sources, the aim of this study was to investigate the antioxidant properties of two edible widely used Boletus species, Boletus edulis, and Boletus auranticus, collected from Istra region in Croatia in late summer 2007. To evaluate the antioxidant properties and content of antioxidant compounds, scavenging capacity on DPPH˙, OH˙, and O₂˙⁻ radicals, reducing power and capacity to inhibit lipid peroxidation has been investigated. It is determined that content of total phenols (41.82 ± 0.08 mg gallic acid equivalent per gram of dry extract) was higher for B. edulis. Using high performance liquid chromatography/diode array detector analysis, the main antioxidant compound, variegatic acid, has been detected and quantified. 1,1-Diphenyl-2-picryl-hydrazyl-hydrate assay was used as a preliminary free radical–scavenging evaluation. By this assay, it has been found that B. edulis dry mushroom extract exhibits 50% of inhibition value at the extract concentration of 0.016 ± 0.0003 mg/ml. The extracts were capable of reducing iron(III) and, thus, are capable of donating electrons. Using electron paramagnetic resonance spin-trapping and spin-probing techniques, activity against relevant reactive species, ˙OH and O₂˙⁻ radical, was analyzed for both mushroom extracts. Both investigated extracts are determined as good inhibitors for ˙OH radical reduction, and both exhibited significant capacity for scavenging O₂˙⁻ radical and for that could help to prevent or meliorate oxidative damage. Only B. edulis extract prevents lipid peroxidation. Investigated mushroom extracts could represent easily accessible natural antioxidant resource.     

The antioxidant properties of polypore mushroom <Emphasis Type="Italic">Daedaleopsis confragosa</Emphasis>

Daedaleopsis confragosa belong to a large and remarkable group of mushrooms called polypores. This type of mushroom could be easily said to be quite unexplored and unused when it comes to its antioxidant properties. Thus, in order to evaluate its antioxidant activity, the investigation had to include the total phenolics and flavonoide content, the content of Selenium, the content of Zinc, the scavenging capacity on DPPH^· and OH^· radicals, reducing power and capacity to remove lipid peroxidation. The investigated mushroom extract contained 54.17 mg GAE/g of total phenols and 48.46 mg CE/g of total flavonoides. Zinc and Selenium were detected and quantified by using inductively coupled plasma mass spectroscopy. The scavenging activity of the radicals was found to exhibit 50% of the inhibition value (IC₅₀ value) at the extract concentration of 0.015±0.007 mg/ml for the investigated D. confragosa extract. By using electron paramagnetic resonant spectroscopy it was found that the investigated extract does not have a significant role in the prevention of lipid peroxidation. It was effective in scavenging on ^·OH radical, RI was 56±2%.     

RETRACTED ARTICLE: An analysis of population structure and linkage disequilibrium using multilocus data in 187 maize inbred lines

This study analyzes population structure and linkage disequilibrium (LD) among 187 commonly used Chinese maize inbred lines, representing the genetic diversity among public, commercial and historically important lines for corn breeding. Seventy SSR loci, evenly distributed over 10 chromosomes, were assayed for polymorphism. The identified 290 alleles served to estimate population structure and analyze the genome-wide LD. The population of lines was highly structured, showing 6 subpopulations: BSSS (American BSSS including Reid), PA (group A germplasm derived from modern U.S. hybrids in China), PB (group B germplasm derived from modern U.S. hybrid in China), Lan (Lancaster Surecrop), LRC (derivative lines from Lvda Reb Cob, a Chinese landrace) and SPT (derivative lines from Si-ping-tou, a Chinese landrace). Forty lines, which formerly had an unknown and/or miscellaneous origin and pedigree record, were assigned to the appropriate group. Relationship estimates based on SSR marker data were quantified in a Q matrix, and this information will inform breeder’s decisions regarding crosses. Extensive inter- and intra-chromosomal LD was detected between 70 microsatellite loci for the investigated maize lines (2109 loci pairs in LD with D′ > 0.1 and 93 out of them at P < 0.01).This suggests that rapidly evolving microsatellites may track recent population structure. Interlocus LD decay among the diverse maize germplasm indicated that association studies in QTLs and/or candidate genes might avoid nonfunctional and spurious associations since most of the LD blocks were broken between diverse germplasm. The defined population structure and the LD analysis present the basis for future association mapping. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Llama-derived Single Variable Domains (Nanobodies) Directed against Chemokine Receptor CXCR7 Reduce Head and Neck Cancer Cell Growth in Vivo

The chemokine receptor CXCR7, belonging to the membrane-bound G protein-coupled receptor superfamily, is expressed in several tumor types. Inhibition of CXCR7 with either small molecules or small interference (si)RNA has shown promising therapeutic benefits in several tumor models. With the increased interest and effectiveness of biologicals inhibiting membrane-bound receptors we made use of the “Nanobody platform” to target CXCR7. Previously we showed that Nanobodies, i.e. immunoglobulin single variable domains derived from naturally occurring heavy chain-only camelids antibodies, represent new biological tools to efficiently tackle difficult drug targets such as G protein-coupled receptors. In this study we developed and characterized highly selective and potent Nanobodies against CXCR7. Interestingly, the CXCR7-targeting Nanobodies displayed antagonistic properties in contrast with previously reported CXCR7-targeting agents. Several high affinity CXCR7-specific Nanobodies potently inhibited CXCL12-induced β-arrestin2 recruitment in vitro. A wide variety of tumor biopsies was profiled, showing for the first time high expression of CXCR7 in head and neck cancer. Using a patient-derived CXCR7-expressing head and neck cancer xenograft model in nude mice, tumor growth was inhibited by CXCR7-targeting Nanobody therapy. Mechanistically, CXCR7-targeting Nanobodies did not inhibit cell cycle progression but instead reduced secretion of the angiogenic chemokine CXCL1 from head and neck cancer cells in vitro, thus acting here as inverse agonists, and subsequent angiogenesis in vivo. Hence, with this novel class of CXCR7 inhibitors, we further substantiate the therapeutic relevance of targeting CXCR7 in head and neck cancer.