The serotyping of Azospirillum spp. by cell-gold immunoblotting

Ten Azospirillum strains were serotyped using the method of cell-gold immunoblotting (dot-blot immune-overlay assay). Colloidal gold-protein A conjugate was used. Antibodies raised against the whole cells showed strain specificity and interacted mainly with carbohydrate antigens on the cell surface. Immunological identity for A. brasilense Sp 245 and Sp 107 strains was found. Cell-gold immunoblotting can be recommended for serotyping of a wide variety of bacterial strains.     

Sporadic naturally occurring melanoma in dogs as a preclinical model for human melanoma

Melanoma represents a significant malignancy in humans and dogs. Different from genetically engineered models, sporadic canine melanocytic neoplasms share several characteristics with human disease that could make dogs a more relevant preclinical model. Canine melanomas rarely arise in sun‐exposed sites. Most occur in the oral cavity, with a subset having intra‐epithelial malignant melanocytes mimicking the in situ component of human mucosal melanoma. The spectrum of canine melanocytic neoplasia includes benign lesions with some analogy to nevi, as well as invasive primary melanoma, and widespread metastasis. Growing evidence of distinct subtypes in humans, differing in somatic and predisposing germ‐line genetic alterations, cell of origin, epidemiology, relationship to ultraviolet radiation and progression from benign to malignant tumors, may also exist in dogs. Canine and human mucosal melanomas appear to harbor BRAF, NRAS, and c‐kit mutations uncommonly, compared with human cutaneous melanomas, although both species share AKT and MAPK signaling activation. We conclude that there is significant overlap in the clinical and histopathological features of canine and human mucosal melanomas. This represents opportunity to explore canine oral cavity melanoma as a preclinical model.     

Response of sunflower (Helianthus annuus L) genotypes to PEG-mediated water stress

Drought tolerance of two sunflower (Helianthus annuus L.) genotypes, cultivated cultivar 1114 and interspecific line H. annuus × H. mollis, was studied under laboratory conditions using PEG-6000. Four levels of osmotic stress (?0.4, ?0.6, ?0.8 and ?1.0 MPa) were created and performances were monitored against a control. Physiological and biochemical stress determining parameters such as malondialdechyde (MDA), proline content, and hydrogen peroxide (H₂O₂) were compared between seedlings of both genotypes. The results indicated that both genotypes have similar responses at four osmotic potentials for all traits studied. All seedling growth parameters such as germination percentage, root length, shoot length, root and shoot dry weight decreased with increasing osmotic stress. MDA, proline, and H₂O₂ were found to be increased at different osmotic gradients in comparison to control. Cultivar 1114 was less affected than the interspecific line under these stress conditions. The data observed in the experiments revealed that perennial wild H. mollis can hardly be considered to be an excellent candidate of drought tolerance genes.     

Evaluating Strategies to Normalise Biological Replicates of Western Blot Data

Western blot data are widely used in quantitative applications such as statistical testing and mathematical modelling. To ensure accurate quantitation and comparability between experiments, Western blot replicates must be normalised, but it is unclear how the available methods affect statistical properties of the data. Here we evaluate three commonly used normalisation strategies: (i) by fixed normalisation point or control; (ii) by sum of all data points in a replicate; and (iii) by optimal alignment of the replicates. We consider how these different strategies affect the coefficient of variation (CV) and the results of hypothesis testing with the normalised data. Normalisation by fixed point tends to increase the mean CV of normalised data in a manner that naturally depends on the choice of the normalisation point. Thus, in the context of hypothesis testing, normalisation by fixed point reduces false positives and increases false negatives. Analysis of published experimental data shows that choosing normalisation points with low quantified intensities results in a high normalised data CV and should thus be avoided. Normalisation by sum or by optimal alignment redistributes the raw data uncertainty in a mean-dependent manner, reducing the CV of high intensity points and increasing the CV of low intensity points. This causes the effect of normalisations by sum or optimal alignment on hypothesis testing to depend on the mean of the data tested; for high intensity points, false positives are increased and false negatives are decreased, while for low intensity points, false positives are decreased and false negatives are increased. These results will aid users of Western blotting to choose a suitable normalisation strategy and also understand the implications of this normalisation for subsequent hypothesis testing.     

Derivatives of the cationic plant alkaloids berberine and palmatine amplify protonophorous activity of fatty acids in model membranes and mitochondria

Previously it has been shown by our group that berberine and palmatine, penetrating cations of plant origin, when conjugated with plastoquinone (SkQBerb and SkQPalm), can accumulate in isolated mitochondria or in mitochondria of living cells and effectively protect them from oxidative damage. In the present work, we demonstrate that SkQBerb, SkQPalm, and their analogs lacking the plastoquinone moiety (C₁₀Berb and C₁₀Palm) operate as mitochondria-targeted compounds facilitating protonophorous effect of free fatty acids. These compounds induce proton transport mediated by small concentrations of added fatty acids both in planar and liposomal model lipid membranes. In mitochondria, such an effect can be carried out by endogenous fatty acids and the adenine nucleotide translocase.