Evidence of the importance of pH and salt concentration on column chromatography with Sepharose gel filtration in separation of proteins.

Gel filtration chromatography with Sepharose agarose gel has been widely applied in the purification of enzymes because of its capability to separate macromolecules according to molecular size. Although a wide range of pH and salt concentrations have been suggested for its use, we have found that the selectivity, or efficiency, of separation is strongly affected by the pH and salt concentrations actually used. Separation is best at neutral pH with low salt concentrations. Increasing the molarity of the buffer or salt content (such as ammonium sulfate) in the protein sample will either broaden protein peaks resulting in poor separation or displace the peaks to a position of much lower apparent hydrodynamic volume. Rabbit plasma monoamine oxidase (MAO), a protein of 150,000 MW, when combined with 1.3 m (NH₄)₂SO₄ at pH 5.4, was found to be retained in Sepharose 6B column until the very end and elute with ammonium sulfate molecules. This behavior was attributed to severe morphological changes on the gel surface at acidic pH leading to a loss of selectivity. Evidence for this interpretation is provided by parallel experiments with Sephadex columns under identical conditions which excludes the possibility of dissociation of MAO into subunits and by scanning electron microscopy which demonstrates the change of surface morphology of the gel. The necesslty of a careful selection of optimum conditions for Sepharose gel chromatographic separation is therefore suggested.     

A Quantitative Real-Time PCR Method Using an X-Linked Gene for Sex Typing in Pigs

At present, a wide range of molecular sex-typing protocols in wild and domestic animals are available. In pigs, most of these methods are based on PCR amplification of X–Y homologous genes followed by gel electrophoresis which is time-consuming and in some cases expensive. In this paper, we describe, for the first time, a SYBR green-based quantitative real-time PCR (qPCR) assay using an X-linked gene, the glycoprotein M6B, for genetic sexing of pigs. Taking into account the differences in the glycoprotein M6B gene copy number between genders, we determine the correct sex of 54 pig samples from either diaphragm or hair follicle from different breeds using the 2^?ΔΔCT method for relative quantification. Our qPCR assay represents a quick, inexpensive, and reliable tool for sex determination in pigs. This new protocol could be easily adapted to other species in which the sex determination was required.     

The modifications produced in allergic alveolitis and in Goodpasture's syndrome due to exposure to cigarette smoke.

Two groups of rats with experimental alveolitis were exposed to cigarette smoke. After comparing the results, the possible muffling effect of the cigarette smoke related to interstitial lung disease was discussed. 180 rats were divided into 6 groups of 30 animals each: Group 1: untreated controls; Group 2: exposed to cigarette smoke for 2 months; Group 3: sensitized with bovine albumin (BA) and exposed to an atmosphere with this antigen for two months, to reproduce a type of extrinsic allergic alveolitis (EAA); Group 4: treated with a single daily dose of anti-lung serum for three days followed by two days without treatment, to reproduce a type of Goodpasture's syndrome; Group 5: exposed to cigarette smoke and to BA; Group 6: exposed to cigarette smoke and treated with anti-lung serum. The animals were sacrificed and their lungs were treated for: Bronchoalveolar lavage (BAL), percentage lymphocyte count, polymorphonuclear (PMN) and alveolar macrophages (AM); semiquantitative and morphometric histological study. The semiquantitative study determined the area of the studied lung incision, affected by granulomae, increased alveolar aerial spaces, thickened alveolar walls and haemosiderine lung area. The morphometric study, based on the linear integration method, evaluated: the distance between two alveolar walls, the amount of interstice per field; and the number of AM with haemosiderine per field was counted. From the results we point out that the treated animals had significantly higher lymphocyte and BAL PMN counts than the untreated ones; no significant differences were found between the singly and doubly treated animals. The animals exposed to cigarette smoke and treated with anti-lung serum were those that showed the least number of lymphocytes and PMN of all the treated animals. The semiquantitative variables studied were all increased in comparison to the control group, most of the increases being significant. The morphometric variables revealed significant differences with respect to the untreated group, except for the animals which were treated with anti-lung serum and cigarette smoke, which showed a minimum decrease in the alveolar size and a slight increase of the interstitial tissue. Only one morphometric variable showed a significant difference between the group treated with anti-lung serum and the one treated with anti-lung serum and cigarette smoke: the number of AM with haemosiderine in the lung.(ABSTRACT TRUNCATED AT 400 WORDS)     

The singular properties of photosynthetic cytochrome c550 from the diatom Phaeodactylum tricornutum suggest new alternative functions

Cytochrome c₅₅₀ is an extrinsic component in the luminal side of photosystem II (PSII) in cyanobacteria, as well as in eukaryotic algae from the red photosynthetic lineage including, among others, diatoms. We have established that cytochrome c₅₅₀ from the diatom Phaeodactylum tricornutum can be obtained as a complete protein from the membrane fraction of the alga, although a C‐terminal truncated form is purified from the soluble fractions of this diatom as well as from other eukaryotic algae. Eukaryotic cytochromes c₅₅₀ show distinctive electrostatic features as compared with cyanobacterial cytochrome c₅₅₀. In addition, co‐immunoseparation and mass spectrometry experiments, as well as immunoelectron microscopy analyses, indicate that although cytochrome c₅₅₀ from P. tricornutum is mainly located in the thylakoid domain of the chloroplast – where it interacts with PSII – , it can also be found in the chloroplast pyrenoid, related with proteins linked to the CO₂ concentrating mechanism and assimilation. These results thus suggest new alternative functions of this heme protein in eukaryotes.     

P-Aminodimethylaniline Monohydrochloride as an Indicator of Microbial Action on Fats

Flooding p-aminodimethylaniline monohydrochloride on fat emulsion agar inoculated with certain types of microorganisms frequently results in marked color changes in the fat globules. It is shown in this paper that the colors result from the increased solubility in fat and fatty acids of this dye as it becomes oxidized. Some of the acids oxidize the dye on contact and therefore color very quickly; fats become colored only when some other agent oxidizes the dye. The characteristic color reactions with certain fats and fatty acids are described for various degrees of oxidation of the dye; this suggests the explanation for the colors observed in the inoculated globules flooded with this dye. A table is included showing the colors in globules of oil that were inoculated with 39 pure cultures of bacteria.     

A cytochemical stain for glutathione in rat hepatocytes cultured on plastic

Thiol groups of glutathione react with the organomercurial azo dye mercury orange at a faster rate than with -SH groups of proteins. This property makes possible visualization of glutathione in cells without appreciable interference from other -SH groups. To render this method useful for cytochemical localization of glutathione in plastic cultured cells, it was necessary to adapt this reaction to the specific characteristics of the biological samples to be assayed. First, the choice of a solvent that would allow a convenient solubility of the dye and at the same time be compatible with the plastic culture plate was crucial. Second, to avoid diffusion of glutathione out of the cell the procedure for staining cells was also important. Satisfactory results were obtained after 30-40 sec reaction with 50 microM mercury orange in acetone/water 9:1, v/v, at room temperature. Glutathione-mercury orange complexes exhibited orange fluorescence on excitation with blue light. No diffusion of glutathione out of the cells was observed, and the hepatocytes stained with the dye showed orange fluorescence which paralleled their glutathione content.