A simple method to measure cell viability in proliferation and cytotoxicity assays

Resazurin dye has been broadly used as indicator of cell viability in several types of assays for evaluation of the biocompatibility of medical and dental materials. Mitochondrial enzymes, as carriers of diaphorase activities, are probably responsible for the transference of electrons from NADPH + H+ to resazurin, which is reduced to resorufin. The level of reduction can be quantified by spectrophotometers since resazurin exhibits an absorption peak at 600 çm and resorufin at 570 çm wavelengths. However, the requirement of a spectrophotometer and specific filters for the quantification could be a barrier to many laboratories. Digital cameras containing red, green and blue filters, which allow the capture of red (600 to 700 çm) and green (500 to 600 çm) light wavelengths in ranges bordering on resazurin and resorufin absorption bands, could be used as an alternative method for the assessment of resazurin and resorufin concentrations. Thus, our aim was to develop a simple, cheap and precise method based on a digital CCD camera to measure the reduction of resazurin. We compared the capability of the CCD-based method to distinguish different concentrations of L929 and normal Human buccal fibroblast cell lines with that of a conventional microplate reader. The correlation was analyzed through the Pearson coefficient. The results showed a strong association between the measurements of the method developed here and those made with the microplate reader (r² = 0.996; p < 0.01) and with the cellular concentrations (r² = 0.965; p < 0.01). We concluded that the developed Colorimetric Quantification System based on CCD Images allowed rapid assessment of the cultured cell concentrations with simple equipment at a reduced cost.

A biolistic process for in vitro gene transfer into chicken embryos

Chicken embryos kept in culture medium were bombarded using a high helium gas pressure biolistic device. To optimize the factors that affect transformation efficiency, the lacZ gene under control of the human cytomegalovirus immediate early enhancer/promoter was used as a reporter gene. There was an inverse relationship between survival rate and transformation efficiency. The best conditions obtained for high embryo survival and high transformation efficiency were achieved with 800 psi helium gas pressure, 500 mmHg vacuum, gold particles, an 8 cm DNA-coated microparticle flying distance to the embryo and embryo placement 0.5 cm from the center of the particle dispersion cone. Under these conditions, transformation efficiency was 100 percent, survival rate 25 percent and the number of expression units in the embryo body cells ranged from 100 to 1,000. Expression of green fluorescent protein was also detected in embryos bombarded under optimal conditions. Based on the results obtained, the biolistic process can be considered an efficient method for the transformation of chicken embryos and therefore can be used as a model system to study transient gene expression and tissue-specific promoters

Studies on electron donation to photosystem I sites by exogenous donors in Spirulina thylakoids.

The kinetic parameters of different sites of electron donation to photosystem I (PS I) were evaluated in Spirulina platensis thylakoids. Reduced 2,6-dichlorophenolindophenol (DCIPH2) exhibited two sites of electron donation, with apparent K(m) values of 8 and 40 microM each. The corresponding value for reduced N-tetramethyl-p-phenylenediamine (TMPDH2) and diaminodurene (DADH2) which donate electrons at a single site to PS I were 103 and 48 microM, respectively. The electron donation by these three exogenous donors were differentially inhibited by KCN (70 mM) affecting the apparent K(m) and Rmax values to varying extent. This cyanide inhibition of PS I catalyzed electron transport suggests the presence of plastocyanin in the photosynthetic electron transport chain of Spirulina platensis.

Metabolitos Reactivos del oxígeno / Reaccional metabolites of oxigen

Un gran número de factores contribuyen a la generación de radicales y metabolitos derivados del oxígeno bajo circunstancias patológicas. El oxígeno esta propenso a la formación de radicales debido a su peculiar estructura electrónica: el hecho de que estos radicales contribuyan con los mecanismos de defensa del organismo contra diferentes microorganismos al mismo tiempo podrían ocasionar "lesión tisular del huésped". Los sistemas de defensa antioxidante son esenciales para evitar este tipo de lesión y por lo tanto mantiene un delicado balance con los oxidantes. En condiciones patológicas el balance puede inclinarse hacia el lado de los oxidantes y en determinadas circunstancias ser potencialmente letal. Los antioxidantes requieren de nutrientes. Por ej: el Se es esencial para la GPO. La vit E reduce la frecuencia en la mortalidad por enfermedad cardíaca. Por lo tanto, el estado nutricional afecta el nivel y características de las defensas antioxidantes.

The reaction between ABTS radical cation and antioxidants and its use to evaluate the antioxidant status of serum samples

The 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical cation can be generated by incubation of ABTS and 2,2'-azo-bis(2-amidinopropane) at 45 degrees Celsius. The ABTS radical cation is stable for several minutes at room temperature and reacts quantitatively and instantaneously with several antioxidants, such as Trolox, ascorbic acid, uric acid, cysteine, glutathione and bilirubin. In contrast, the ABTS radical cation reacts slowly with albumin. When serum is added to a solution of the ABTS radical cation, the bleaching of the radical follows biphasic kinetics, with a fast decay followed by a slow decay that takes place within several minutes. The fast decay is primarily due to uric acid, while the slow decay is related to the protein content of the sample. We propose that this procedure can provide an independent and simultaneous evaluation of the low molecular weight and protein antioxidants present in biological samples such as serum.

Production of hydroquinol from p-benzoquinone using yeast.

Reduction of p-benzoquinone to hydroquinol has been studied using Saccharomyces cerevisae and S.uvarum. Maximum conversion of p-Benzoquinone (p-BQ) to Hydroquinol (HQ) at a substrate concentration of 6%. S. cerevisae was found to be better than S. uvarum. No further conversion did take place after 60 hr. Optical innoculam density was found to be 5-6% (v/v). The pH optima was observed at pH value 5.50. No significant improvement could be observed by doping acetone, which solubilizes p-BQ, to the medium.