The mechanism of acid secretion by gastric mucosa

The mechanism of acid secretion by gastric mucosa has been the subject of much experimentation in recent years. Examinations in this field have been prompted by an idea which is generally known as the redox theory of acid secretion. This theory is discussed with particular reference to the value of the ratio between acid secretion and the associated oxygen uptake by gastric mucosa. The redox theory has two interesting consequences. Firstly, it implies a spatial separation of the hydrogen and hydroxyl ions produced in the respiratory chain. Secondly, it leads to the view that the secretion of hydrogen ions, unlike other active transport processes, does not necessarily require phosphate-bond energy. In summary, it would seem that separation of hydrogen and hydroxyl ions produced in the respiratory metabolism of the oxyntic cell is a basic feature of the mechanism of acid secretion by gastric mucosa. However, the exact source and site of origin of the hydrogen ions in the oxyntic cell remain to be found.peer-reviewe

Superoxide and superoxide dismutase in red blood cells

In 1969 a previously obscure copper protein of red blood cells, erythrocuprein, was shown to catalyse the dismutation of superoxide radicals. Erythrocuprein thus became superoxide dismutase and the object of intensive study. Superoxide dismutase is typically an enzyme of aerobic organisms which utilize oxygen as the major electron acceptor. The presence of superoxide dismutase in microorganisms has been found to parallel their tolerance for oxygen. We recently described the isolation and properties of two forms of superoxide dismutase from human erythrocytes, SOD I and SOD II (Bannister et al., 1976). These can be obtained from a haemolysate of red blood cells after precipitation of the hemoglobin with a mixture of ethanol and chloroform.peer-reviewe

Aspirin-induced apoptosis of yeast cells is associated with mitochondrial superoxide radical accumulation and NAD(P)H oxidation

In previous studies, we observed that aspirin, a promising cancer-preventive agent, induces apoptosis in mitochondrial manganese superoxide dismutase(MnSOD)-deficient Saccharomyces cerevisiae cells grown aerobically in ethanol medium. In this study, we show that aspirin-induced apoptosis is associated with a significant increase in mitochondrial and cytosolic O2 and oxidation of mitochondrial NAD(P)H. A concomitant rise in the level of cytosolic CuZn-SOD activity failed to compensate for mitochondrial MnSOD deficiency. However, an observed increase in activity of Escherichia coli FeSOD targeted to the mitochondrial matrix of the MnSOD-deficient yeast cells, markedly decreased aspirin-induced accumulation of mitochondrial O 2 , significantly increased the mitochondrial NAD(P)H level and rescued the apoptotic phenotype. Indeed, recombinant yeast cells expressing E. coli FeSOD behaved in a similar manner to the parent wild-type yeast cells with native mitochondrial MnSOD activity. Wild-type cells consistently showed a decrease in mitochondrial O 2 and an increase in mitochondrial NAD(P)H levels in the presence of aspirin in ethanol medium. In fact, in wild-type cells, our studies supported an antioxidant action of aspirin. Taken together, our results indicate that a pro-oxidant effect of aspirin occurring predominantly in cells with compromised mitochondrial redox balance may be enough to overcome antioxidant defences resulting in apoptosis, as observed in MnSOD-deficient yeast cells.peer-reviewe

Abnormal hemoglobins in Malta : the significance of two fetal and an adult variant

The study of human hemoglobin variants has a special place in hemoglobin research. The discovery of hemoglobin F (Malta) by Cauchi et al. (1969) and the high incidence of this hemoglobin in Maltese newborns gave us the opportunity to initiate a systematic study of a fetal hemoglobin variant. During the course of our screening of cord blood samples we have found a new fetal variant, hemoglobin F-Malta-II. We have also found that some families with Hb-F-Malta-II have a new adult hemoglobin variant, which we have called "Hemoglobin St. Luke's". We have redesignated Hb F(Malta) as Hb-F-Malta-I. The purpose of this article is to give briefly the relevance of Hb-F-Malta-I and Hb-F-Malta-II to current concepts of the genetics of the γ-chain of fetal hemoglobin (Huisman et aI., 1972), and to give a brief description of Hb St. Luke's (Bannister et al., 1972).peer-reviewe

Prokaryotic iron superoxide dismutase replaces cytosolic copper, zinc superoxide dismutase in protecting yeast cells against oxidative stress

The iron superoxide dismutase (FeSOD) gene of Escherichia coli was cloned in Saccharomyces cerevisiae cells deficient in copper,zinc superoxide dismutase (Cu,ZnSOD). FeSOD replaced Cu,ZnSOD in protecting the yeast cells against oxidative stress. In the recombinant strains the FeSOD gene, which was under the transcriptional control of the yeast phosphoglycerate kinase gene promoter, was functionally expressed at two different levels on episomal and centromeric plasmids. Despite suppression of methionine and lysine auxotrophy, the higher level of FeSOD activity was more beneficial to growth of the mutant yeast cells only when these were exposed to higher levels of oxidative stress induced by paraquat or 100% oxygen. In the presence of paraquat, there was a novel stimulation of FeSOD activity. This was associated with a marked increase in catalase activity, and a decrease in glutathione reductase activity.peer-reviewe

Prediction of protein secondary structure from circular dichroism spectra using artificial neural network techniques

An approach to predict protein secondary structure is presented using circular dichroism (CD) spectra as input to two types of artificial neural networks (ANNs): (i) a three-layer backpropagation network and (ii) a hybrid self-organisation to backpropagation network. The dataset comprised the CD spectra of 22 proteins in the 178-260 nm wavelength range whose secondary structures were known. A total of 22 networks were trained by each method, using the jackknife technique for testing the prediction on each protein in turn. The performance, measured in terms of root mean square residuals. and Pearson product-moment correlation coefficients, compares well with that obtained by other statistical and ANN methods, and is likely to improve with the growth of the dataset.peer-reviewe

Cloning, expression and characterisation of two manganese superoxide dismutases from Caenorhabditis elegans

Two genes encoding manganese superoxide dismutase (sod-2 and sod-3) have been identified in the nematode Caenorhabditis elegans. Each gene is composed of five exons, and intron positions are identical; however, intron sizes and sequences are not the same. The predicted protein sequences are 86.3% homologous (91.8% conservative), and the cDNAs are only 75.2% homologous. Both deduced protein sequences contain the expected N-terminal mitochondrial transit peptides. Reverse transcriptase polymerase chain reaction analysis shows that both genes are expressed under normal growth conditions and that their RNA transcripts aretrans-spliced to the SL-1 leader sequence. The latter result together with Northern blot analysis indicate that both genes have mono-cistronic transcripts. The sod-3 gene was mapped to chromosome X, and the location of sod-2 was confirmed to be chromosome I. Polymerase chain reaction was used to amplify the cDNA regions encoding the predicted mature manganese superoxide dismutase proteins and each was cloned and expressed to high levels in Escherichia coli cells deficient in cytosolic superoxide dismutases. Both proteins were shown to be active in E. coli, providing similar protection against methyl viologen-induced oxidative stress. The expressed enzymes, which were not inhibited by hydrogen peroxide or cyanide, are dimeric, show quite different electrophoretic mobilities and isoelectric points, but exhibit comparable specific activities.peer-reviewe

Cloned prokaryotic iron superoxide dismutase protects yeast cells against oxidative stress depending on mitochondrial location

Superoxide dismutase (SOD) is considered to be the first line of defense against oxygen toxicity. It exists as a family of three metalloproteins with copper,zinc (Cu,ZnSOD), manganese (MnSOD), and iron (FeSOD) forms. In this work, we have targetedEscherichia coliFeSOD to the mitochondrial intermembrane space (IMS) of yeast cells deficient in mitochondrial MnSOD. Our results show that FeSOD in the IMS increases the growth rate of the cells growing in minimal medium in air but does not protect the MnSOD-deficient yeast cells when exposed to induced oxidative stress. Cloned FeSOD must be targeted to the mitochondrial matrix to protect the cells from both physiological and induced oxidative stress. This confirms that the superoxide radical is mainly generated on the matrix side of the inner mitochondrial membrane of yeast cells, without excluding its potential appearance in the mitochondrial IMS where its elimination by SOD is beneficial to the cells.peer-reviewe

Aspirin commits yeast cells to apoptosis depending on carbon source

The effect of aspirin on the growth of a wild-type Saccharomyces cerevisiae strain (EG103), containing both copper,zinc superoxide dismutase (CuZnSOD) and manganese superoxide dismutase (MnSOD), a strain deficient in MnSOD (EG110) and a strain deficient in CuZnSOD (EG118) was measured in media containing different carbon sources. Aspirin inhibited the fermentative growth of all three strains in glucose medium. It inhibited the non-fermentative growth of the MnSOD-deficient strain very drastically in ethanol medium and had no effect on this strain in glycerol or acetate medium. The non-fermentative growth of the other two strains was not affected by aspirin. The growth inhibition of strain EG110 was associated with early necrosis in glucose medium and late apoptosis in ethanol medium. The apoptosis was preceded by a pronounced loss of cell viability. The growth inhibitory effect of aspirin was not reversed by the antioxidants N-acetylcysteine and vitamin E. Furthermore, aspirin itself appeared to act as an antioxidant until the onset of overt apoptosis, when a moderate increase in the intracellular oxidation level occurred. This suggested that reactive oxygen species probably do not play a primary role in the apoptosis of cells exposed to aspirin.peer-reviewe

Superoxide dismutases : recent advances and clinical applications

The Escherichia coli iron superoxide dismutase (FeSOD) gene was expressed, at two different levels (using episomal and centromeric plasmid systems), in Saccharomyces cerevisiae cells deficient in copper, zinc superoxide dismutase. Levels of antioxidant enzymes were studied in the recombinant strains in the presence and absence of 1 mM paraquat in minimal medium. Exposure to paraquat resulted in: (1) increase in the levels of total SOD, FeSOD, and catalase activities in both yeast strains expressing the FeSOD gene, and (2) decrease in the levels of glutathione reductase in yeast cells expressing the cloned FeSOD gene on the episomal, but not on the centromeric, plasmid The increase in FeSOD activity suggested that there is stimulation of the yeast 3-phosphoglycerate kinase gene (PGK) promoter controlling the cloned FeSOD gene in the presence of paraquat This could be either due to the oxidative stress induced by paraquat, or as a result of an inherent effect of paraquat itself. This hypothesis, that is, induction of the PGK promoter in S. cerevisiae under oxidative stress, is presently being studied in our laboratory. If confirmed, this finding would complement previous observations that transcription of S. cerevisiae PGK increases on heat-shock (Piper et al. (1986) Eur. 1. Bioc/zem., 161,525-531). Thus the PGK gene could be implicated as being one of the genes that are induced as part of the general stress response of S. cerevisiae.peer-reviewe