Electrophysiological and behavioural studies of the biting midge, Culicoides impunctatus Goetghebuer (Diptera, Ceratopogonidae): interactions between some plant-derived repellent compounds and a host-odour attractant, 1-octen-3-ol

Abstract. Electroantennogram (EAG) and y-tube bioassays have been used to demonstrate the repellent properties of five plant compounds with host-seeking parous female Culicoides impunctatus Goetghebuer. The compounds were methyl salicylate and allyl-, butyl-, phenyl- and 2-phenylethyl isothiocyanate. EAG thresholds were 1 times 10^-3 to 1 μg. In the bioassays, maximal repellencies occurred with 1 times 10˜² to lOug. When each compound was combined with 1-octen-3-ol, a confirmed host-odour attractant for C.impunctatus females, additive effects were recorded in EAG assays and in bioassays, all of the compounds either reduced or reversed the attractancy of l-octen-3-ol. Of the isothiocyanates, allyl isothiocyanate was the most potent and when combined with 1-octen-3-ol in field trials, the attractant effect of l-octen-3-ol was reduced.     

Effects of glucobrassicin, epiprogoitrin and related breakdown products on locusts feeding: Schouwia purpurea and desert locust relationships

The glucosinolates of a Saharan crucifer Schouwia purpurea (Forskål) (Brassicaceae) were determined by liquid chromatography. Two of these glucosinolates and sinigrin were tested for their deterrent effect on Schistocerca gregaria (Forskål) (Orthoptera: Acrididae). Glucobrassicin, three indolyls and epigoitrin were synthesized for this purpose. Epiprogoitrin was extracted from Crambe seeds. Choice tests on artificial substrate compared feeding responses to glucosinolates and to related breakdown products released when the plant is eaten. Breakdown products were more efficient in deterring the generalist locust than were glucosinolates. Two patterns of dose responses were recorded: glucosidic compounds deterred or stimulated feeding, depending on the concentration tested; aglycones did not stimulate feeding at any concentration. Allyl isothiocyanate, a volatile compound, was a 100-fold higher deterrent than its substrate (sinigrin).     

MicroRNA-567 inhibits cell proliferation and induces cell apoptosis in A549 NSCLC cells by regulating cyclin-dependent kinase 8

MicroRNA-567 (miR-567) plays a decisive role in cancers whereas its role in non-small cell lung cancer (NSCLC) is still unexplored. This study was therefore planned to explore the regulatory function of miR-567 in A549 NSCLC cells and investigate its possible molecular mechanism that may help in NSCLC treatment. In the current study, miR-567 expression was examined by quantitative real time-polymerase chain reaction (qRT-PCR) in different NSCLC cell lines in addition to normal cell line. A549 NSCLC cells were transfected by miR-567 mimic, miR-567 inhibitor, and negative control siRNA. Cell proliferation was evaluated by MTT and 5-bromo-2′deoxyuridine assays. Cell cycle distribution and apoptosis were studied by flow cytometry. Bioinformatics analysis programs were used to expect the putative target of miR-567. The expression of cyclin-dependent kinase 8 (CDK8) gene at mRNA and protein levels were evaluated by using qRT-PCR and western blotting. Our results found that miR-567 expressions decreased in all the studied NSCLC cells as compared to the normal cell line. A549 cell proliferation was suppressed by miR-567 upregulation while cell apoptosis was promoted. Also, miR-567 upregulation induced cell cycle arrest at sub-G1 and S phases. CDK8 was expected as a target gene of miR-567. MiR-567 upregulation decreased CDK8 mRNA and protein expression while the downregulation of miR-567 increased CDK8 gene expression. These findings revealed that miR-567 may be a tumor suppressor in A549 NSCLC cells through regulating CDK8 gene expression and may serve as a novel therapeutic target for NSCLC treatment.     

A novel antibody which precipitates 7.5S RNA is isolated from a patient with autoimmune disease

We have isolated a nobel antibody from a patient with autoimmune disease which reacts with the ribonucleoprotein complex containing 7.5S RNA. The 7.5S RNA consists of two species having slightly different electrophoretic mobilities. Fingerprinting analysis of these two species demonstrates that nucleotide sequences of the RNAs are very similar to each other. Nucleotide composition of the 7.5S RNA is found to be; A/U/G/C=20/18/32/30, indicating that the ratio of GC content of the RNA(62%) is relatively high. The RNA contains a pseudouridylic acid residue as a modified nucleotide. Immunofluorescence pattern stained with the antibody suggests that the ribonucleoprotein complex containing 7.5S RNA is located both in the nucleolus and the cytoplasm.     

Phenethyl isothiocyanate potentiates anti‐tumour effect of doxorubicin through Akt‐dependent pathway

The present study aims to investigate the in vivo and in vitro anti‐tumour properties of phenethyl isothiocyanate (PEITC) alone and in combination with doxorubicin (Dox). The anti‐tumour activity was evaluated in vitro by MTT assay using cultured human breast cancer cell line (MCF‐7) and human hepatoma cell line (HepG‐2) cell lines. In vivo, Ehrlich solid tumour model was used. Tumour volume, weight and antioxidant parameters were determined. Immunohistochemistry analysis for active (cleaved) caspase‐3 was also performed. We tested the effect of PEITC treatment on pAkt/Akt ratio, NF‐κB p65 DNA binding activity and caspase‐9 enzyme activity in both MCF‐7 and HepG‐2 cell lines. Effect of PEITC treatment on cell migration was assessed by wound healing assay. PEITC and/or Dox treatment significantly inhibited solid tumour volume and tumour weight when compared with control mice. PEITC treatment significantly reduced oxidative stress caused by Dox treatment as indicated by significant increase in total antioxidant capacity and decrease in malondialdehyde level. Microscopic examination of tumour tissues showed a significant increase in active (cleaved) caspase‐3 expression in PEITC and/or Dox treated groups. PEITC showed a dose‐dependent inhibition of MCF‐7 and HepG‐2 cellular viability. PEITC inhibited Akt and NF‐κB activation and increased caspase‐9 activity in a dose‐dependent manner. PEITC treatment effectively inhibited both MCF‐7 and HepG‐2 cell migration. We can conclude that PEITC acts via multiple molecular targets to elicit anti‐carcinogenic activity. PEITC/Dox combination therapy might be a potential novel strategy, which may benefit patients with breast and liver cancers. Copyright © 2015 John Wiley & Sons, Ltd.     

Bispecific antibodies with Fab-arms featuring exchanged antigen-binding constant domains

Monoclonal antibodies can acquire the property of engagement of a second antigen via fusion methods or modification of their CDR loops, but also by modification of their constant domains, such as in the mAb² format where a set of mutated amino acid residues in the C_H3 domains enables a high-affinity specific interaction with the second antigen. We tested the possibility of introducing multiple binding sites for the second antigen by replacing the Fab C_H1/C_L domain pair with a pair of antigen-binding C_H3 domains in a model scaffold with trastuzumab variable domains and VEGF-binding C_H3 domains. Such bispecific molecules were produced in a “Fab-like” format and in a full-length antibody format. Novel constructs were of expected molecular composition using mass spectrometry. They were expressed at a high level in standard laboratory conditions, purified as monomers with Protein A and gel filtration and were of high thermostability. Their high-affinity binding to both target antigens was retained. Finally, the Her2/VEGF binding domain-exchanged bispecific antibody was able to mediate a potentiated surface Her2-internalization effect on the Her2-overexpressing cell line SK-BR-3 due to improved level of cross-linking with the endogenously secreted cytokine. To conclude, bispecific antibodies with Fabs featuring exchanged antigen-binding C_H3 domains offer an alternative solution in positioning and valency of antigen binding sites.     

A yeast chemical genetics approach identifies the compound 3,4,5-trimethoxybenzyl isothiocyanate as a calcineurin inhibitor

The phosphatase enzyme calcineurin controls gene expression in a variety of biological contexts however few potent inhibitors are currently available. A screen of 360 plant extracts for inhibition of calcineurin-dependent gene expression in the model organism Saccharomyces cerevisiae identified the compound 3,4,5-trimethoxybenzyl isothiocyanate as an inhibitor. The compound was subsequently shown to inhibit human calcineurin via a mixed inhibition mechanism. To gain further mechanistic insight a yeast haploinsufficiency screen of 1152 deletion strains was carried out using a novel liquid medium screening method. The resulting haploinsufficiency profile is similar to that reported for the known calcineurin inhibitor FK506.