Harmful dinoflagellate Ostreopsis cf. ovata Fukuyo: detection of ovatoxins in field samples and cell immunolocalization using antipalytoxin antibodies

Ostreopsis cf. ovata, a benthic dinoflagellate often blooming along the Mediterranean coasts, has been associated with toxic events ranging from dyspnea to mild dermatitis. In late September 2009, an Ostreopsis cf. ovata bloom occurred in the Gulf of Trieste (Northern Adriatic Sea; Italy), causing pruritus and mild dermatitis in beachgoers. An integrated study was initiated to characterize Ostreopsis cells by light and confocal microscopy, PCR techniques, immunocytochemistry, and high resolution liquid chromatography-mass spectrometry (HR LC-MS). The presence of Ostreopsis cf. ovata of the Atlantic/Mediterranean clade was unambiguously established by morphological and genetic analyses in field samples. Several palytoxin-like compounds (ovatoxin-a,-b,-c,-d,-e) were identified by HR LC-MS, ovatoxin-a being the most abundant (45-64 pg/cell). Surprisingly, no palytoxin was detected. For the first time, monoclonal and polyclonal antipalytoxin antibodies revealed the intracellular cytoplasmic localization of ovatoxins, suggesting their cross-reactivity with these antibodies. Since harmful dinoflagellates do not always produce toxins, the immunocytochemical localization of ovatoxins, although qualitative, can provide an early warning for toxic Ostreopsis cells before their massive diffusion and/or concentration in seafood.     

Immunocytochemical localization of the NMDA-R2A receptor subunit in the cat retina

Immunocytochemical studies were performed to determine the distribution and cellular localization of the NMDA-R2A receptor subunit (R2A) in the cat retina. R2A-immunoreactivity (R2A-IR) was noted in all layers of the retina, with specific localizations in the outer segments of red/green and blue cone photoreceptors, B-type horizontal cells, several types of amacrine cells, Müller cells and the majority of cells in the ganglion cell layer. In the inner nuclear layer, 48% of all cells residing in the amacrine cell layer were R2A-IR including a cell resembling the GABAergic A17 amacrine cell. Interestingly, the AII rod amacrine cell was devoid of R2A-IR. Although the localization of the R2A subunit was anticipated in ganglion cells, amacrines and Müller cells, the presence of this receptor subunit to the cells in the outer retina was not expected. Here, both the R2A and the R2B subunits were found to be present in the outer segments of cone photoreceptors and to the tips of rod outer segments. Although the function of these receptor subunits in rod and cone photoreceptors remains to be determined, the fact that both R2A and R2B receptor subunits are localized to cone outer segments suggests a possible alternative pathway for calcium entry into a region where this cation plays such a crucial role in the process of phototransduction. To further classify the cells that display NR2A-IR, we performed dual labeling experiments showing the relationship between R2A-labeled cells with GABA. Results showed that all GABAergic-amacrines and displaced amacrines express the R2A-subunit protein. In addition, approximately 11% of the NR2A-labeled amacrines, did not stain for GABA. These findings support pharmacological data showing that NMDA directly facilitates GABA release in retina and retinal cultures [I.L. Ferreira, C.B. Duarte, P.F. Santos, C.M. Carvalho, A.P. Carvalho, Release of [3H]GABA evoked by glutamate receptor agonist in cultured chick retinal cells: effect of Ca2+, Brain Res. 664 (1994) 252-256; G.D. Zeevalk, W.J. Nicklas, Action of the anti-ischemic agent ifenprodil on N-methyl-d-aspartate and kainate-mediated excitotoxicity, Brain Res. 522 (1990) 135-139; R. Huba, H.D. Hofmann, Transmitter-gated currents of GABAergic amacrine-like cells in chick retinal cultures, Vis. Neurosci. 6 (1991) 303-314; M. Yamashita, R. Huba, H.D. Hofmann, Early in vitro development of voltage- and transmitter-gated currents in GABAergic amacrine cells, Dev. Brain Res. 82 (1994) 95-102; R. Ientile, S. Pedale, V. Picciurro, V. Macaione, C. Fabiano, S. Macaione, Nitric oxide mediates NMDA-evoked [3H]GABA release from chick retina cells, FEBS Lett. 417 (1997) 345-348; R.C. Kubrusly, M.C. deMello, F.G. deMello, Aspartate as a selective NMDA agonist in cultured cells from the avian retina, Neurochem. Intl. 32 (1998) 47-52] or reduction of GABA in vivo [N.N. Osborn, A.J. Herrera, The effect of experimental ischaemia and excitatory amino acid agonist on the GABA and serotonin immunoreactivities in the rabbit retina, Neurosci. 59 (1994) 1071-1081]. Since the majority of GABAergic synapses in the inner retina are onto both rod and cone bipolar axon terminals [R.G. Pourcho, M.T. Owzcarzak, Distribution of GABA immunoreactivity in the cat retina: A light and electron-microscopic study, Vis. Neurosci. 2 (1989) 425-435], we hypothesize that the NMDA-receptor plays a crucial role in providing feedback inhibition onto rod and cone bipolar cells.     

Alkylation of Hydroquinone with tert-butanol over Silica-immobilized Triflate Derivatives

Friedel-Crafts alkylations of α-methylnaphthalene with various alkylating agents were first carried out in the presence of methanesulfonic acid (MeSA). The Brønsted acid catalyst MeSA exhibited outstanding catalytic performance, and was found to be excellent catalyst and solvent for alkylation reaction of aromatic hydrocarbon. It’s found that alkenes can be used as excellent alkylating agent for alkylation of α-methylnaphthalene. The effects of various reaction parameters like type of alkylating agent, dosages of catalysts, reaction temperature and reaction time were investigated in detail. Moreover, the performance of reuse for catalysts was also studied. It’s found that, under the optimal reaction conditions, more than 90% of conversion for olefins and 100% of selectivity for the desired products were obtained. Compared with traditional catalysis technology, the reaction, catalyzed by MeSA catalyst, no volatile solvents needed, good selectivity for desired products could be obtained. The catalyst can be isolated easily from the reaction mixtures by decantation, and was successfully reused. The methanesulfonic acid could be considered as environmentally friendly novel catalyst and solvent for long-chain alkylation of α-methylnaphthalene with alkenes. The catalytic reaction mechanism for alkylation in the presence of MeSA was proposed as well.     

The micromilling process for high aspect ratio microstructures

High aspect ratio microstructures are currently created by several processes which include lithography (X-ray, deep ultraviolet, etc.) and mechanical machining (diamond machining, microdrilling, etc.) The lithographic processes require more extensive processing equipment such as an energy source, mask/mask holder/mask aligner, photoresist and substrate, and chemical development capacity. In addition, these processes are serial in nature and each adds to the tolerances of the finished structure. The current mechanical processes provide for the direct removal of the substrate material in a single step but are more limited in the geometric patterns which can be created. In conventional machining, the process which provides the most versatility in geometric patterns is milling. The micromilling process has two basic components. The first is the fabrication of small milling cutters with very sharp cutting edges. The second is the actual removal of the workpiece material with a very precise and repeatable machine tool. Several basic cutter designs have been fabricated using focused ion beam micromachining and are undergoing testing. The cutter diameters are nominally 100 micrometers and 22 micrometers. Results have been obtained which show that this process can be very effective for the rapid fabrication of molds and mask structures.     

Micrometer-scale machining: tool fabrication and initial results

Conventional milling techniques scaled to ultrasmall dimensions have been used to machine polymethyl methacrylate (PMMA) with micrometer-sized milling tools. The object of this work is to achieve machining of a common material over dimensions exceeding 1 mm while holding submicrometer tolerances and micrometer size features. Fabricating the milling tools themselves was also an object of the study. A tool geometry for nominal 25 micrometer diameter cutting tools was found that cuts PMMA with submicrometer tolerances over trench lengths of 2 mm. The tool shape is a simple planar facet cut by focused ion beam milling on ground and polished 25 micrometer diameter steel tool blanks. Pairs of trenches 24 micrometers wide, 26 micrometers deep, 2.3 mm long, with a 14 micrometer separation were milled under various machining conditions. The results indicate that the limits of the machining process in terms of speed, pattern complexity, and tolerances have not been approached. This is the first demonstration of a generic method for microtool making by focused ion beam machining combined with ultraprecision numerically controlled milling. The method is shown to be capable of producing structures and geometries that are considered inaccessible by conventional materials removal techniques, and generally regarded as applications for deep X-ray lithography.     

Total cross-section of chromium at low neutron energies

The total neutron cross-section of chromium has been measured in the range 1.3 × 10^?4 to 30 eV by the transmission method with pulsed neutron time of flight techniques. A theoretical fit was performed giving a value of (3.25 ± 0.15) b for σ_abs at 0.0253 eV.     

Modification of (CH)x chains during thermal cis-trans isomerization

It is shown that the isomerization of cis polyacetylene at 195°C for long periods is accompanied by structural modifications. Infra-red and pyrolysis gas-chromatography data suggest that the aromatization process occurs even at this temperature.     

Effect of water content on thermal and dynamic mechanical properties of xanthan powder: A comparison between standard and novel techniques

In this work, the sorption isotherms of xanthan powder obtained under static and dynamic conditions and their fitting with mathematical models were discussed. The transitions within xanthan-water systems have been investigated using a standard material characterization technique - Differential Scanning Calorimetry (DSC) - and two novel techniques - Dynamic Mechanical Thermal Analysis (DMTA) material pocket and Phase Transition Analysis (PTA). It was found that within water-xanthan systems, the onset temperature for the melting of freezable water obtained from DSC method was in agreement with freezable water melting peaks obtained via DMTA material pocket at levels of hydration when the freezable water was present. Also, low-temperature transitions, related to polysaccharide-water interactions, were observed by both techniques within xanthan systems which contained no freezable water. However, the use of the PTA technique was limited to xanthan systems with low water content.     

The compatibility of the polystyrene/acrylonitrile-butadiene-styrene grafted copolymer system

Summary The study of polystyrene (PS)/acrylonitrile-butadiene-styrene (ABS) grafted copolymer/cyclohexanone or methylene chloride system compatibility both in concentrated solutions and solid state has been carried out by phase separation, viscosimetrical measurements and thermal behaviour.Having in view the obtained data it has been ascertained that the systems present different degrees of compatibility depending on the mixtures composition. At higher temperatures, the compatibility of the components is changed.