In Vivo Micronucleus Assay in Mouse Bone Marrow.

The presence of genotoxic agents in the environment may cause chromosomal mutations through different mechanisms, which are associated with serious health effects. Genotoxicity is commonly evaluated for the chemical safety assessment, in which the in vivo micronucleus test is paid more attention in the field of genotoxicity as compared to other toxicological endpoints. This assay is an in vivo cytogenetic test which uses erythrocytes in the bone marrow of rodents to detect chemical damage to the chromosomes or mitotic apparatus of mammalian cells. At the time of erythroblast development into a polychromatic erythrocyte (PCEs) in bone marrow, the main nucleus is extruded, so any micronucleus (MN) that has been formed may remain behind in the otherwise anucleated cytoplasm. The damage in the chromosome appears as a small additional nucleus and is readily identifiable by light microscope. An increase in the frequency of micronucleated polychromatic erythrocytes (MN PCEs) in treated animals is an indication of genotoxicity.

Diagnosticando orinas con tinción de Giemsa / Diagnosing urines with Giemsa tintorial techniques

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Use of brillant blue dye on canned cherries

Introduction: The toxicity of erythrosine as well as other photochemical and biochemical degradation products thereof has been addressed in several studies. However, it is often employed in the preparation of canned cherries, since its use is allowed by regulatory agencies such as the FDA. Therefore, it would be important to find less risky replacement dyes for their use in food. Methodology: canned cherries were produced by a slow confit process, reaching at least 55° Brix, and were then subjected to commercial pasteurization. Results: Brilliant Blue dyed cherries met the required standard and had a suitable degree of acceptance in the tested population, with the expected parameters being attained in all trials. In addition, the stability test proved that blue dyed cherries remained unchanged, while Erb dyed product suffered an important discoloration. Conclusion: cherries colored by blue brilliant can be elaborated without problem

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In Vitro Differentiation of Embryonic Stem Cells into Hematopoietic Lineage: Towards Erythroid Progenitor's Production.

Embryonic stem cells (ESCs) differentiation via embryoid body (EB) formation is an established method that generates the three germ layers. However, EB differentiation poses several problems including formation of heterogeneous cell populations. Herein, we described a differentiation protocol on enhancing mesoderm derivation from murine ESCs (mESCs) using conditioned medium (CM) from HepG2 cells. We used this technique to direct hematopoiesis by generating "embryoid-like" colonies (ELCs) from murine (m) ESCs without following standard formation of EBs. Our CM-mESCs group yielded an almost fivefold increase in ELC formation (p ≤ 0.05) and higher expression of mesoderm genes;-Brachyury-T, Goosecoid, and Flk-1 compared with control mESCs group. Hematopoietic colony formation from CM-mESCs was also enhanced by twofold at days 7 and 14 with earlier colony commitment compared to control mESCs (p ≤ 0.05). This early clonogenic capacity was confirmed morphologically by the presence of nucleated erythrocytes and macrophages as early as day 7 in culture using standard 14-day colony-forming assay. Early expression of hematopoietic primitive (ζ-globin) and definitive (ß-globin) erythroid genes and proteins was also observed by day 7 in the CM-treated culture. These data indicate that hematopoietic cells more quickly differentiate from CM-treated, compared with those using standard EB approaches, and provide an efficient bioprocess platform for erythroid-specific differentiation of ESCs.

Identifying different types of chromatin using Giemsa staining.

Mixtures of polychrome methylene blue-eosin Y (i.e., Giemsa stain) are widely used in biological staining. They induce a striking purple coloration of chromatin DNA (the Romanowsky-Giemsa effect), which contrasts with the blue-stained RNA-containing cytoplasm and nucleoli. After specific prestaining treatments that induce chromatin disorganization (giving banded or harlequin chromosomes), Giemsa staining produces a differential coloration, with C- and G-bands appearing in purple whereas remaining chromosome regions are blue. Unsubstituted (TT) and bromo-substituted (BT) DNAs also appear purple and blue, respectively. The same occurs in the case of BT and BB chromatids.In addition to discussing the use of Giemsa stain as a suitable method to reveal specific features of chromosome structure, some molecular processes and models are also described to explain Giemsa staining mechanisms of chromatin.

Simultaneous quantification of methylene blue and its major metabolite, azure B, in plasma by LC-MS/MS and its application for a pharmacokinetic study.

A simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of methylene blue (MB) and its major metabolite, azure B (AZB), in rat plasma. A simple protein precipitation using acetonitrile was followed by injection of the supernatant on to a Zorbax HILIC Plus column (3.5 µm, 2.1 × 100 mm) with isocratic mobile phase consisting of 5 mM ammonium acetate in 10:90 (v/v) water:methanol at a flow rate of 0.3 mL/min and detection in positive ionization mode. The standard curve was linear over the concentration range from 1 to 1000 ng/mL for MB and AZB with coefficient of determination above 0.9930. The lower limit of quantification was 1 ng/mL using 20 µL of rat plasma sample. The intra- and inter-assay precision and accuracy were <12%. The developed analytical method was successfully applied to the pharmacokinetic study of MB and AZB in rats.

A robust procedure for distinctively visualizing zebrafish retinal cell nuclei under bright field light microscopy.

To simultaneously visualize individual cell nuclei and tissue morphologies of the zebrafish retina under bright field light microscopy, it is necessary to establish a procedure that specifically and sensitively stains the cell nuclei in thin tissue sections. This necessity arises from the high nuclear density of the retina and the highly decondensed chromatin of the cone photoreceptors, which significantly reduces their nuclear signals and makes nuclei difficult to distinguish from possible high cytoplasmic background staining. Here we optimized a procedure that integrates JB4 plastic embedding and Feulgen reaction for visualizing zebrafish retinal cell nuclei under bright field light microscopy. This method produced highly specific nuclear staining with minimal cytoplasmic background, allowing us to distinguish individual retinal nuclei despite their tight packaging. The nuclear staining is also sensitive enough to distinguish the euchromatin from heterochromatin in the zebrafish cone nuclei. In addition, this method could be combined with in situ hybridization to simultaneously visualize the cell nuclei and mRNA expression patterns. With its superb specificity and sensitivity, this method may be extended to quantify cell density and analyze global chromatin organization throughout the retina or other tissues.

Simple method for culture of peripheral blood lymphocytes of Testudinidae.

We developed and optimized a simple, efficient and inexpensive method for in vitro culture of peripheral blood lymphocytes from the Brazilian tortoise Chelonoidis carbonaria (Testudinidae), testing various parameters, including culture medium, mitogen concentration, mitotic index, culture volume, incubation time, and mitotic arrest. Peripheral blood samples were obtained from the costal vein of four couples. The conditions that gave a good mitotic index were lymphocytes cultured at 37°C in minimum essential medium (7.5 mL), with phytohemagglutinin as a mitogen (0.375 mL), plus streptomycin/penicillin (0.1 mL), and an incubation period of 72 h. Mitotic arrest was induced by 2-h exposure to colchicine (0.1 mL), 70 h after establishing the culture. After mitotic arrest, the cells were hypotonized with 0.075 M KCl for 2 h and fixed with methanol/acetic acid (3:1). The non-banded mitotic chromosomes were visualized by Giemsa staining. The diploid chromosome number of C. carbonaria was found to be 52 in females and males, and sex chromosomes were not observed. We were able to culture peripheral blood lymphocytes of a Brazilian tortoise in vitro, for the preparation of mitotic chromosomes.

Mycoremediation of Congo red dye by filamentous fungi

Azo, anthroquinone and triphenylmethane dyes are the major classes of synthetic colourants, which are difficult to degrade and have received considerable attention. Congo red, a diazo dye, is considered as a xenobiotic compound, and is recalcitrant to biodegradative processes. Nevertheless, during the last few years it has been demonstrated that several fungi, under certain environmental conditions, are able to transfer azo dyes to non toxic products using laccases. The aim of this work was to study the factors influencing mycoremediation of Congo red. Several basidiomycetes and deuteromycetes species were tested for the decolourisation of Congo red (0.05 g/l) in a semi synthetic broth at static and shaking conditions. Poor decolourisation was observed when the dye acted as the sole source of nitrogen, whereas semi synthetic broth supplemented with fertilizer resulted in better decolourisation. Decolourisation of Congo red was checked in the presence of salts of heavy metals such as mercuric chloride, lead acetate and zinc sulphate. Decolourisation parameters such as temperature, pH, and rpm were optimized and the decolourisation obtained at optimized conditions varied between 29.25- 97.28 percent at static condition and 82.1- 100 percent at shaking condition. Sodium dodecyl sulphate polyacrylamide gel electrophoretic analysis revealed bands with molecular weights ranging between 66.5 to 71 kDa, a characteristic of the fungal laccases. High efficiency decolourisation of Congo red makes these fungal forms a promising choice in biological treatment of waste water containing Congo red.

Photocatalytic degradation of Azure and Sudan dyes using nano TiO2.

The present study investigates the dependence of photocatalytic rate on molecular structure of the substrate that is degraded. The photocatalytic degradation of Azure (A and B) and Sudan (III and IV) dyes, having similar structure, but different functional groups, were investigated with two catalysts. The photocatalytic activity of solution combustion synthesized TiO(2) (CS TiO(2)) was compared with that of Degussa P-25 for degrading these dyes. The effect of solvents and mixed-solvent system on photodegradation of Sudan III was investigated. The photodegradation rate was found to be higher in solvents with higher polarity. The effect of pH and the presence of metal ions in the form of chloride and nitrate salt, on degradation rate of Azure A was also investigated. The metal ions significantly reduced the photocatalysis rates. A detailed Langmuir-Hinshelwood kinetic model has been developed to explain the effect of metal ions on degradation rate of the substrate. This model elucidates the contribution of holes and electrons towards degradation of the dye.

[Study on the interaction between alginic sodium diester and azur A and the application].

The interaction of alginic sodium diester (ASD) and azur A (AA) was studied by absorption spectra. The influence of pH and the molar ratio of AA/ASD on the spectra was investigated. The maximum binding number (N = 168) and the binding equilibrium constant (K = 3.25 x 10(6)) of the alginic sodium diester and azur A were obtained by the theoretical model. The new method for the determination of ASD was developed by the formation of ASD-AA ion-association complex that caused the fading of the AA. The detection limit is 0.009 microg x mL(-1). The method has the advantages of simplicity, stable system, good accuracy and selectivity, and was applied to the determination of alginic sodium diester in tablets with satisfactory results.

The effects of autoclaving on the physical properties and biological activity of parenteral heparin preparations.

Heparin ampoules have been autoclaved at 115 degrees C, 121 degrees C, 126 degrees C and 130 degrees C for time intervals up to 50 min and the biological potency and physicochemical integrity of the preparations assessed. The anticoagulant activity, determined with the APTT assay, did not change significantly for the autoclaved samples but a decrease was observed using the BP assay. Autoclaving was also associated with a depolymerization process, as confirmed by high performance liquid chromatography, and the formation of 'species' with an absorption in the UV spectrum. However, autoclaving had no detrimental effect on the integrity of the anionic sites on heparin. It is proposed that Maillard-type reactions are responsible for these observations and the results indicate that autoclaving could be used to sterilize parenteral heparin solutions.

Quality control of azure B preparations by liquid chromatography and standardization with azure B tetrafluoroborate.

A liquid chromatographic procedure for the quantitative determination of the thiazine dye azure B, the principal constituent of Romanowsky stains, is presented. Unlike previous methods relying on peak area normalization, the present approach involves real quantitation through calibration with the reference standard azure B tetrafluoroborate. The method has been used for the quality control of commercial azure B preparations and to study their stability in stock and staining solutions, either or not in the presence of eosin Y. Results suggest that highly pure azure B perchlorate meets the requirements of a reference material, useful for standardization of Romanowsky-Giemsa staining in haematology.