A comparative study of human placental and fetal liver catalase during development.

Cultured Renal Epithelial Cells From Birds and Mice: Enhanced Resistance of Avian Cells to Oxidative Stress and DNA Damage

Abstract: Current mechanistic theories of aging would predict that many species of birds, given their unusually high metabolic rates, body temperatures, and blood sugar levels, should age more rapidly than mammals of comparable size. On the contrary, many avian species display unusually long life spans. This finding suggests that cells and tissues from some avian species may enjoy unusually robust and/or unique protective mechanisms against fundamental aging processes, including a relatively high resistance to oxidative stress. We therefore compared the sensitivities of presumptively homologous epithelial somatic cells derived from bird and mouse kidneys to various forms of oxidative stress. When compared to murine cells, we found enhanced resistance of avian cells from three species (budgerigars, starlings, canaries) to 95% oxygen, hydrogen peroxide, paraquat, and gamma-radiation. Differential resistance to 95% oxygen was demonstrated with both replicating and quiescent cultures. Hydrogen peroxide was shown to induce DNA single-strand breaks. There were fewer breaks in avian cells than in mouse cells when similarly challenged.

Changes in concentration, localization and activity of catalase within the human placenta during early gestation

Abstract: Using villous tissue from accurately dated gestational age placentae, this study identified significant changes in the protein concentration, enzyme activity and localization of catalase, an enzyme responsible for the intracellular metabolism of hydrogen peroxide, during the first and early second trimester of pregnancy. Enzyme activity was found to increase approximately threefold between weeks 6 and 17, with the greatest increase between 12 and 17 weeks. Immunostaining of tissue sections was supportive of these findings, demonstrating a progressively stronger signal between weeks 6 and 17. Immunostaining also demonstrated that the main cell types expressing catalase were the cytotrophoblast cells as well as a subset of the stromal cells. Between 13-17 weeks gestation, however, it was possible to detect catalase within the syncytiotrophoblast also, although with a much reduced intensity of staining. At the ultrastructural level, immunogold labelling of catalase clearly showed that staining was predominately compartmentalized within peroxisomes, although non-peroxisomal staining was also seen. Immunoreactivity also demonstrated, via morphological identification, that the stromal cells containing detectable levels of catalase were placental macrophages (Hofbauer cells). These results are in agreement with the proposal that the placenta exists in a physiologically low oxygen environment during the early part of gestation. In this environment oxidative activity of the sort resulting in the generation of hydrogen peroxide would presumably be suppressed, thereby limiting the requirement for catalase until oxygen tension begins to rise.

Differences in the proteome profile in placenta from normal term and preeclamptic preterm pregnancies.

Abstract: The aim of this study was to use proteomic approaches to examine differences in protein expression in placentae from normal term and preterm preeclamptic pregnancies and to validate the data thus obtained by other independent methods. Using 2-DE we found that 80% of the proteins were present in both normal and preeclamptic placentae. However, 26 proteins in the normal term placentae were not matched in the preterm preeclamptic group. Six proteins showed increased intensity and one protein was down-regulated in preeclampsia. Four of the seven proteins that were altered in preeclampsia were further analyzed by Western blot and immunohistochemistry. Identification by MS techniques revealed these proteins to be involved in regulatory pathways activated by stress. This is significant because preeclampsia is a multisystem disorder in human pregnancies that results in considerable oxidative and nitrative stress. Three proteins identified by MS to be Hsp27, catalase, and glucose-regulated protein were confirmed by Western blot analysis to be significantly up-regulated in preeclampsia. Endothelial monocyte-activating polypeptide was shown to be down-regulated in preeclampsia by 2-DE and MS.

Protein Measurement with the Folin Phenol Reagent

Abstract: Since 1922 when Wu proposed the use of the Folin phenol reagent for the measurement of proteins, a number of modified analytical procedures utilizing this reagent have been reported for the determination of proteins in serum, in antigen-antibody precipitates, and in insulin. Although the reagent would seem to be recommended by its great sensitivity and the simplicity of procedure possible with its use, it has not found great favor for general biochemical purposes. In the belief that this reagent, nevertheless, has considerable merit for certain application, but that its peculiarities and limitations need to be understood for its fullest exploitation, it has been studied with regard to effects of variations in pH, time of reaction, and concentration of reactants, permissible levels of reagents commonly used in handling proteins, and interfering substances. Procedures are described for measuring protein in solution or after precipitation with acids or other agents, and for the determination of as little as 0.2 gamma of protein.

A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase.

Abstract: Several methods have been developed for following the breakdown of hydrogen peroxide catalyzed by catalase, but these either have not been sufficiently quantitative or have not proved rapid enough to yield reliable data during the critical 1st or 2nd minute of the reaction. Chemical procedures in which residual peroxide is titrated with permanganate (l-3) or an excess of permanganate is measured calorimetrically (4) are accurate except for reaction times of less than a minute, although Lemberg and Foulkes (5) developed a micromethod for obtaining data every 10 seconds (see also Ogura et al. (6)). Considerable variability is unavoidable, however, when samples must-be taken at such short intervals. The manometric method for measuring oxygen evolved from the system proved in detailed studies to be unsuited for following the rapid breakdown of peroxide in which a diffusion process across the liquid-air interface becomes limiting. This is manifested by changes in both the order of the reaction and the rate of evolution of oxygen with variations in the rate of agitation of the reaction mixture (7). Direct measurement of hydrogen peroxide by polarography provides good quantitative data during the 1st minute of the reaction which fit first order kinetics (8). However, an elaborate, special, electronic circuit is needed for such measurements. Furthermore, as pointed out by Bonmschen, Chance, and Theorell (8), this method appears to give lower values for catalase activity than do titration techniques. Preliminary experiments for following the breakdown of hydrogen peroxide by observing the decrease in light absorption of peroxide solutions in the ultraviolet were reported by Chance (9) and Chance and Herbert (10). The potentialities of this method have been investigated and a quantitative, spectrophotometric technique for following the breakdown of hydrogen peroxide has been developed for routine studies of catalase kinetics.