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Immunolocalization techniques are standard in biomedical research. Tissue fixation with aldehydes and cell membrane permeabilization with detergents can distort the specific binding of antibodies to their high affinity epitopes. In immunofluorescence protocols, it is desirable to quench the sample’s autofluorescence without reduction of the antibody-dependent signal. Here we show that adding glycine to the blocking buffer and diluting the antibodies in a phosphate saline solution containing glycine, Triton X-100, Tween20 and hydrogen peroxide increase the specific antibody signal in tissue immunofluorescence and immunogold electron microscopy. This defined antibody signal enhancer (ASE) solution gives similar results to the commercially available Pierce Immunostain Enhancer (PIE). Furthermore, prolonged tissue incubation in resin and fixative and application of ASE or PIE are described in an improved protocol for triple immunogold electron microscopy that is used to show co-localization of GABA-A ρ2 and dopamine D2 receptors in GFAP-positive astrocytes in the mouse striatum. The addition of glycine, Triton X-100, Tween20 and hydrogen peroxide during antibody incubation steps is recommended in immunohistochemistry methods.

Conserved polypeptides of the chitin synthase genes UmCHS3 and UmCHS6 from the phytopathogenic fungus Ustilago maydis were utilized as immunogens to obtain polyclonal antibodies that were purified by affinity procedures. Because of their similarities at the regions encoded by either polypeptide, it was concluded that anti-Chs3 antibodies recognized both Chs3 and Chs4 chitin synthases, whereas anti-Chs6 antibodies recognized Chs6 and Chs8 polypeptides. These antibodies were used to analyze the localization of the corresponding chitin synthases in U. maydis cells, using both indirect immunofluorescence microscopy and immunoelectron microscopy with colloidal-gold-labeled secondary antibodies. It was observed that chitin synthase proteins were accumulated both in the surface and in the cytoplasm of the fungal cells. Electron microscopy images revealed the accumulation of clusters of gold particles in vesicles, providing evidence for the possible origin and destination of chitin synthases in the fungal cells.

The Casiopeínas ® are mixed chelate copper (II) complexes and promising antineoplastics agents against cancer cells and tumors in vitro and in vivo. However, the action mode of these compounds is poorly characterized. In this work the effect of the antineoplastic Casiopeína IIIEa on the metabolism and ultrastructure of the yeast Saccharomyces cerevisiae was investigated. Exposure of cells growing in rich or in low-iron medium to 5 μM of the compound decreased duplication time and reduced oxygen consumption. Those cells formed smaller colonies when growing in a non-fermentable carbon source and low-iron medium, and under the light microscope, multiple folds were observed along the plasma membrane accompanied with a reduction in the diameter of the yeast. These observations were confirmed under the electron microscope, which also revealed a slight reduction of the mitochondrial size. A correlation was found with smaller colonies exhibiting lower rates of oxygen consumption, and yeast labelled with fluorescent MitoTracker TM consistently exhibited reduced mitochondrial activity. It appears that Casiopeína IIIEa gives rise to smaller yeast and petite -like colonies by reducing the mitochondrial respiratory activity without significantly affecting the mitochondrial structure.

Expression of GEF1 in Xenopus laevis oocytes and HEK-293 cells gave rise to a Cl⁻ channel that remained permanently open and was blocked by nitro-2-(3-phenylpropylamino) benzoic acid and niflumic acid. NPPB induced petite-like colonies, resembling the GEF1 knock-out. The fluorescent halide indicator SPQ was quenched in a wild-type strain, in contrast to both a GEF1 knock-out strain and yeast grown in the presence of NPPB. Immunogold and electron microscopy located Gef1p in the plasma membrane, vacuole, endoplasmic reticulum and Golgi apparatus. Eleven substitutions in five residues forming the ion channel of GEF1 were introduced; some of them (S186A, I188N, Y459D, Y459F, Y459V, I467A, I467N and F468N) did not rescue the pet phenotype, whereas F468A, A558F and A558Y formed normal colonies. All the pet mutants showed reduced O₂ consumption, small mitochondria and mostly disrupted organelles. Finally, electron microscopy revealed that the plasma membrane of the mutants develop multiple foldings and highly ordered cylindrical protein-membrane complexes. All the experiments above suggest that Gef1p transports Cl⁻ through the plasma membrane and reveal the importance of critical amino acids for the proper function of the protein as suggested by structural models. However, the mechanism of activation of the channel has yet to be defined.

Cyst walls of Entamoeba invadens were isolated and purified. Both whole cysts and purified walls appeared intensely fluorescent when stained with Calcofluor white M2R. Examination of positive replicas of purified cyst walls with the electron microscope revealed the presence of a microfibrillar structure. The main sugars detected in acid hydrolysates from the walls were hexosamines. X-ray diffraction analysis of purified cyst walls demonstrated that the crystalline polymer present was chitin.

Chitin was located in the cyst wall of Entamoeba invadens with colloidal gold-linked wheat germ agglutinin. Cysts stained differentially from trophozoites when encysting cultures were treated with the gold tracer; cysts acquired a wine-red coloration while, in general trophozoites remained unstained. Observation of cells with the electron microscope revealed that the tracer particles were bound specifically to the walls of the surface of the cyst when cells were exposed in suspension, and to the cyst wall cross-section, when cells were exposed to the tracer in thin section, indicating that chitin fibers were distributed on the surface as well as throughout the matrix of the cyst wall.

Thinners are chemical mixtures used as industrial solvents. Humans can come into contact with thinner by occupational exposure or by intentional inhalation abuse. Thinner sniffing causes damage to the brain, kidney, liver, lung, and reproductive system. We discuss some proposed mechanism by which thinner induces damage. Recently, the induction of oxidative stress has been suggested as a possible mechanism of damage. This paper reviews the current evidence for oxidative stress effects induced by thinner inhalation. Early ideas about the effects of thinner on lipids are discussed in one section. We discuss several studies that have shown the oxidative effects of thinner inhalation on: lipid peroxidation, levels of antioxidant enzymes, glutathione depletion, and oxidation of proteins and DNA. We have also included studies about oxidative stress effects induced by toluene, the principal component (60-70%) of thinner. Finally, work describing the effects of oxidative stress induced by thinner inhalation on different organs is discussed.

Nitrogen-fixing activity in the phyllosphere of 12 species of Tillandsia from different Mexican habitats was evaluated by the acetylene reduction assay, and nitrogen-fixing microorganisms were isolated and characterized. The leaves from eight of the 12 Tillandsia species examined exhibited nitrogenase activity in enrichment cultures. Among the microorganisms implicated--Agrobacterium, Bacillus, Erwinia, Pseudomonas, Rahnella, Vibrio, and Xanthomonas--only Bacillus megatherium reduced acetylene in pure culture. Our findings suggest that nitrogen fixation in the phyllosphere of the sampled epiphytes occurs under suitable conditions and that most of the bacteria involved are primarily soil and water inhabitants. The results also suggest a relationship between the composition of the nitrogen-fixing microbial communities grown on the leaf and the different development of the leaf area in Tillandsia due to the aerial components (wings) of the trichomes