Distribution and essential oils of Salvia pomifera subsp. pomifera (Labiatae) on the island of Crete (S Greece)

The distribution of Salvia pomifera subsp. pomifera (Cretan sage) on the island of Crete is presented. The essential oils of six populations scattered on the island are studied. The essential oil content varies from 2.1–4.2%, whereas the main oil components were in all cases α- and/or β-thujone (27.4–72.3% and 7.1–40.8%, respectively). The comparison of our results to literature data, suggest that S. pomifera can be distinguished from S. fruticosa (Greek sage), on the basis of its essential oil composition.     

Epicuticular Phenolics Over Guard Cells: Exploitation for in situ Stomatal Counting by Fluorescence Microscopy and Combined Image Analysis

Guard cells emit an alkali-induced, blue fluorescence upon excitationby ultraviolet radiation (emission maximum energy at 365 nm).Fluorescence emission of guard cells was brighter than thatof the neighbouring epidermal cells in a number of wild andcultivated plants including conifers, but the relative fluorescenceintensity and quality was species-dependent. Three representativeplants possessing stomatal complexes which differed morphologicallywere studied: Olea europaea, Vicia faba and Triticum aestivum.Immersing leaves of these plants in chloroform for 30 s (therebyremoving epicuticular waxes) significantly reduced the intensityof the fluorescence emitted by guard cells. This indicates thatguard cell fluorescence could be due to either an increasedconcentration of fluorescing compounds (probably wax-bound phenolics),or a thicker cuticular layer covering the guard cells. Giventhat the alkali-induced blue fluorescence of the guard cellsis a common characteristic of all plants examined, it couldbe used as a rapid and convenient method for in situ measurementsof the number, distribution and size of stomatal complexes.The proposed experimental procedure includes a single coatingof the leaf surface by, or immersion of the whole leaf in, a10% solution of KOH for 2 min, washing with distilled water,and direct observation of the leaf surface under the fluorescencemicroscope. Fluorescence images were suitable for digital imageanalysis and methodology was developed for stomatal countingusing Olea europaea as a model species. Copyright 2001 Annalsof Botany Company Cuticle, epicuticular waxes, fluorescence microscopy, image analysis, phenolics, stomata     

Analytical Performance of ELISA Assays in Urine: One More Bottleneck towards Biomarker Validation and Clinical Implementation

ELISA is the main approach for the sensitive quantification of protein biomarkers in body fluids and is currently employed in clinical laboratories for the measurement of clinical markers. As such, it also constitutes the main methodological approach for biomarker validation and further qualification. For the latter, specific assay performance requirements have to be met, as described in respective guidelines of regulatory agencies. Even though many clinical ELISA assays in serum are regularly used, ELISA clinical applications in urine are significantly less. The scope of our study was to evaluate ELISA assay analytical performance in urine for a series of potential biomarkers for bladder cancer, as a first step towards their large scale clinical validation. Seven biomarkers (Secreted protein acidic and rich in cysteine, Survivin, Slit homolog 2 protein, NRC-Interacting Factor 1, Histone 2B, Proteinase-3 and Profilin-1) previously described in the literature as having differential expression in bladder cancer were included in the study. A total of 11 commercially available ELISA tests for these markers were tested by standard curve analysis, assay reproducibility, linearity and spiking experiments. The results show disappointing performance with coefficients of variation>20% for the vast majority of the tests performed. Only 3 assays (for Secreted protein acidic and rich in cysteine, Survivin and Slit homolog 2 protein) passed the accuracy thresholds and were found suitable for further application in marker quantification. These results collectively reflect the difficulties in developing urine-based ELISA assays of sufficient analytical performance for clinical application, presumably attributed to the urine matrix itself and/or presence of markers in various isoforms.     

Genotypic assignment of infection by Dirofilaria repens

Dirofilariasis is a parasitic disease, which if treated inappropriately due to misdiagnosis, can cause unwanted complications particularly when the infection is located in the breast. The numerous obstacles that can cause misdiagnosis of dirofilariases by standard morphological procedures prompted the development of a Dirofilaria repens-specific direct polymerase chain reaction (PCR)-based diagnostic approach using freshly infected dog blood. Reliable amplification of nematode DNA from formalin-fixed infected human specimens by this method is only possible from relatively fresh biological material, preserved in the fixative for up to 20 days. We report here our first case of dirofilariasis since the development of PCR genotyping, where the pathogen was morphologically unrecognizable and the diagnosis was based exclusively on DNA amplification. We complete our methodological contribution to the clinical laboratory diagnosis of dirofilariasis by presenting two more cases, where the primary genotypic assignment of infection by D. repens was further confirmed by conventional morphological means.     

Determination of bacteriocin activity with bioassays carried out on solid and liquid substrates: assessing the factor "indicator microorganism"

Background  

Successful application of growth inhibition techniques for quantitative determination of bacteriocins relies on the sensitivity of the applied indicator microorganism to the bacteriocin to which is exposed. However, information on indicator microorganisms' performance and comparisons in bacteriocin determination with bioassays is almost non-existing in the literature. The aim of the present work was to evaluate the parameter "indicator microorganism" in bioassays carried out on solid -agar diffusion assay- and liquid -turbidometric assay- substrates, applied in the quantification of the most studied bacteriocin nisin.     

Melanin protects choroidal blood vessels against light toxicity

Low ocular pigmentation and high long-term exposure to bright light are believed to increase the risk of developing age-related macular degeneration (ARMD). To investigate the role of pigmentation during bright light exposure, cell damage in retinae and choroids of pigmented and non-pigmented rats were compared. Pigmented Long Evans (LE) rats and non-pigmented (albino) Wistar rats were exposed to high intensity visible light from a cold light source with 140,000 lux for 30 min. Control animals of both strains were not irradiated. The animals had their pupils dilated to prevent light absorbance by iris pigmentation. 22 h after irradiation, the rats were sacrificed and their eyes enucleated. Posterior segments, containing retina and choroid, were prepared for light and electron microscopy. Twenty different sections of specified and equal areas were examined in every eye. In albino rats severe retinal damage was observed after light exposure, rod outer segments (ROS) were shortened and the thickness of the outer nuclear layer (ONL) was significantly diminished. Choriocapillaris blood vessels were obstructed. In wide areas the retinal pigment epithelium (RPE) was absent in albino rats after irradiation. In contrast, LE rats presented much less cell damage in the RPE and retina after bright light exposure, although intra-individual differences were observed. The thickness of the ONL was almost unchanged compared to controls. ROS were shortened in LE rats, but the effect was considerably less than that seen in the albinos. Only minimal changes were found in choroidal blood vessels of pigmented rats. The RPE showed certain toxic damage, but cells were not destroyed as in the non-pigmented animals. The number of melanin granules in the RPE of LE rats was reduced after irradiation. Ocular melanin protects the retina and choroid of pigmented eyes against light-induced cell toxicity. Physical protection of iris melanin, as possible in eyes with non-dilated pupils, does not seem to play a major role in our setup. Biochemical mechanisms, like reducing oxidative intracellular stress, are more likely to be responsible for melanin-related light protection in eyes with dilated lens aperture.     

In vivo Wnt signaling tracing through a transgenic biosensor fish reveals novel activity domains

The creation of molecular tools able to unravel in vivo spatiotemporal activation of specific cell signaling events during cell migration, differentiation and morphogenesis is of great relevance to developmental cell biology. Here, we describe the generation, validation and applications of two transgenic reporter lines for Wnt/β-catenin signaling, named TCFsiam, and show that they are reliable and sensitive Wnt biosensors for in vivo studies. We demonstrate that these lines sensitively detect Wnt/β-catenin pathway activity in several cellular contexts, from sensory organs to cardiac valve patterning. We provide evidence that Wnt/β-catenin activity is involved in the formation and maintenance of the zebrafish CNS blood vessel network, on which sox10 neural crest-derived cells migrate and proliferate. We finally show that these transgenic lines allow for screening of Wnt signaling modifying compounds, tissue regeneration assessment as well as evaluation of potential Wnt/β-catenin genetic modulators.     

Structural elucidation of Leuprolide and its analogues in solution: insight into their bioactive conformation

Leuprolide [dLeu⁶, NHEt¹⁰]GnRH, a potent gonadotropin-releasing hormone (GnRH) agonist, is used in a wide variety of hormone-related diseases like cancer and endometriosis. In this report, the conformational behaviour of Leuprolide and its linear synthetic analogues, namely [Tyr⁵(OMe), dLeu⁶, Aze⁹, NHEt¹⁰]GnRH (1) and [Tyr⁵(OMe), dLeu⁶, NHEt¹⁰]GnRH (2) have been studied in DMSO and H₂O solutions by means of 2D nuclear magnetic resonance (NMR) experiments and detailed molecular dynamics (MD) simulations. The aim was to identify the conformational requirements of GnRH analogues for agonistic activity. This approach is of value as no crystallographic data are available for the GnRH receptor (G protein-coupled receptor, GPCR). The NOE data indicate the existence of a β-turn type I in the 2–5 segments of Leuprolide and its linear analogues in the case of using DMSO-d₆ as solvent, whereas a β-turn type II in the 3–6 segments is indicated using D₂O as solvent. The final structures fulfil the conformational requirements that are known, in the literature, to play a significant role in receptor recognition and activation. Finally, the linear analogues (1) and (2) are biologically active when tested against the human breast cancer cell line, MCF-7.