17ß-Estradiol/N-acetylcysteine interaction enhances the neuroprotective effect on dopaminergic neurons in the weaver model of dopamine deficiency.

The weaver mouse, is a phenocopy of Parkinson's disease (PD) in which dopaminergic neurons degenerate gradually during development, reaching at P21 a neurodegeneration of 55%. Thus, the weaver mouse constitutes an appropriate in vivo PD model for investigating the effect of neuroprotective agents. In the present study, long-term treatment (from P1 to P21) with 17ß-estradiol (17ß-estradiol) significantly protected the dopaminergic neurons in the substantia nigra (SN) of weaver mouse by 54%, as was detected by immunohistochemical experiments, using the specific antibody against tyrosine hydroxylase (TH). This dopaminergic neuroprotection is in line with our biochemical results showing that 17ß-estradiol treatment significantly decreased the high lipid peroxidation levels seen in the SN of weaver mouse, indicating high oxidative stress. Interestingly, co-administration of 17ß-estradiol with N-acetylcysteine (NAC, precursor molecule of glutathione (GSH)) further significantly increased the survival of dopaminergic neurons in the SN (by 85%), with a parallel further decrease of lipid peroxidation to normal levels. Our results show the in vivo neuroprotective effect of 17ß-estradiol, which is strongly enhanced by co administration of NAC, indicating a strong synergistic effect of the two drugs. Furthermore, the main mechanism underlying this neuroprotective action seems to be the reversal of the oxidative stress shown by the high peroxidation levels. These results could be of clinical relevance since both drugs are already used separately in the clinic, 17ß-estradiol for treatment of PD and NAC as a mucolytic agent and for the treatment of several disorders.

Cadmium versus copper toxicity: insights from an integrated dissection of protein synthesis pathway in the digestive glands of mussel Mytilus galloprovincialis.

The main purpose of this study was to investigate the impact of metal-mediated stress on the protein-synthesis pathway in mussels. To this end, mussels (Mytilus galloprovincialis) underwent a 15 days exposure to 100 µg/L Cu(2+) or Cd(2+). Both metals, in particular Cd(2+), accumulated in mussel digestive glands and generated a specific status of oxidative-stress. Exposure of mussels to each metal resulted in 40% decrease of the tRNA-aminoacylation efficiency, at the end of exposure. Cu(2+) also caused a progressive loss in the capability of 40S-ribosomal subunits to form 48S pre-initiation complex, which reached 34% of the control at the end of exposure. Other steps of translation underwent less pronounced, but measurable damages. Mussels exposed to Cd(2+) for 5 days presented a similar pattern of translational dysfunctions in digestive glands, but during the following days of exposure the ribosomal efficiency was gradually restored. Meanwhile, metallothionein levels significantly increased, suggesting that upon Cd(2+)-mediated stress the protein-synthesizing activity was reorganized both quantitatively and qualitatively. Conclusively, Cd(2+) and Cu(2+) affect translation at several levels. However, the pattern of translational responses differs, largely depending on the capability of each metal to affect cytotoxic pathways in the tissues, such as induction of antioxidant defense and specific repair mechanisms.

Hydrogen peroxide is involved in the sclerotial differentiation of filamentous phytopathogenic fungi.

AIMS: The purpose of this study was to investigate the role of H(2) O(2) and the related oxidative stress markers catalase (CAT) and lipid peroxidation in the sclerotial differentiation of the phytopathogenic filamentous fungi Sclerotium rolfsii, Sclerotinia minor, Sclerotinia sclerotiorum and Rhizoctonia solani. METHODS AND RESULTS: Using the H(2) O(2) -specific scopoletin fluorometric assay and the CAT-dependent H(2) O(2) consumption assays, it was found that the production rate of intra/extracellular H(2) O(2) and CAT levels in the sclerotiogenic fungi were significantly higher and lower, respectively, than those of their nondifferentiating counterpart strains. They peaked in the transition between the undifferentiated and the differentiated state of the sclerotiogenic strains, suggesting both a cell proliferative and differentiative role. In addition, the indirect indicator of oxidative stress, lipid peroxidation, was substantially decreased in the nondifferentiating strains. CONCLUSIONS: These findings suggest that the differentiative role of H(2) O(2) is expressed via induction of higher oxidative stress in the sclerotiogenic filamentous phytopathogenic fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that the direct marker of oxidative stress H(2) O(2) is involved in the sclerotial differentiation of the phytopathogenic filamentous fungi S. rolfsii, S. minor, S. sclerotiorum and R. solani, which could have potential biotechnological implications in terms of developing antifungal strategies by regulating intracellular H(2) O(2) levels.

Thiol redox state and oxidative stress affect sclerotial differentiation of the phytopathogenic fungi Sclerotium rolfsii and Sclerotinia sclerotiorum.

AIMS: To investigate the involvement of oxidative stress and thiol redox state (TRS) in sclerotial differentiation of Sclerotium rolfsii and Sclerotinia sclerotiorum. METHODS AND RESULTS: Oxidative stress in these fungi was assessed by lipid peroxidation, which was higher in comparison with their nonsclerotiogenic counterpart strains. TRS [measured as glutathione (GSH) and cysteine] was associated with oxidative stress and differentiation using the TRS modulator and antioxidant Nu-acetylcysteine (AcCSH) and the GSH biosynthesis inducer and inhibitor l-2-oxo-thiazolidine-4-carboxylate and L-buthionine-S,R-sulphoximine (BSO) respectively. Differentiation and oxidative stress was decreased by AcCSH in both fungi. The decrease of differentiation by BSO was not associated with oxidative stress in these fungi. CONCLUSIONS: Differentiation and oxidative stress in both fungi depends on the availability of antioxidant noncytotoxic -SH groups and is not depended on any direct antioxidant role of GSH and its precursor cysteine. SIGNIFICANCE AND IMPACT OF THE STUDY: This study helps to understand the mechanism(s) of sclerotial differentiation in these agriculturally important phytopathogenic fungi and proposes that AcCSH can be used as potent fungicide by (i) acting as growth inhibiting cytotoxic oxidant and (ii) sustaining these fungi in their undifferentiated hyphal stage where they are vulnerable to degradation by soil micro-organisms.

Indoor radon levels in primary schools of Patras, Greece.

Radon activity concentrations have been measured in 53 from a total of 66 public primary schools throughout of Patras, Greece, during December 1999 to May 2000 using solid-state nuclear track detectors (LR-115 II). The indoor radon levels in the classrooms were generally low, ranging from 10 to 89 Bqm(-3). The mean (arithmetic mean) indoor concentration was 35 +/- 17 Bq m(-3) and an estimated annual effective dose of 0.1 +/- 0.1 mSv y(-1) was calculated for students and 0.2 +/- 0.1 mSv y(-1) for teachers, assuming an equilibrium factor of 0.4 and occupancy factor of 12 and 14%, respectively. The research was also focused on parameters affecting radon concentration levels such as floor number of the classrooms and the age of the buildings in relation to building materials.

Evidence for oxidative stress in lens epithelial cells in pseudoexfoliation syndrome.

PURPOSE: To investigate the oxidative status in lens epithelial cells of patients with pseudoexfoliation (PEX) syndrome. METHODS: Lens capsule samples obtained during cataract surgery of patients with PEX syndrome and normal age-matched control subjects were examined for changes in the levels of glutathione (GSH), glutathione disulphide (GSSG), protein concentration, and lipid peroxidation. Concentrations of GSH, GSSG, lipid peroxidation, and protein concentration were determined by specific fluorescent assays. RESULTS: This study shows a 2.2- and 2.0-fold decrease in GSH and GSSG levels, respectively, in PEX lens epithelial lens compared with non-PEX lens epithelial cells, as well as a 2.5-fold increase in lipid peroxidation product malondialdehyde (MDA) levels. CONCLUSION: The increased MDA and decreased GSH levels indicate high oxidative stress. On the other hand, GSSG usually increases in cases of high-oxidative stress, but this is not always the case, as it may not always accumulate in cells. Our findings suggest a role for oxidative stress in the pathogenesis and the progression of PEX syndrome.

Evidence for intestinal oxidative stress in patients with obstructive jaundice.

BACKGROUND: Obstructive jaundice results in failure of the intestinal barrier with consequent systemic endotoxemia associated with septic complications. We have recently shown that gut barrier failure in experimental obstructive jaundice is associated with high intestinal oxidative stress. This study was undertaken to investigate whether oxidative alterations occur in the intestinal mucosa of patients with obstructive jaundice. PATIENTS AND METHODS: Fifteen patients with malignant biliary obstruction and no signs of cholangitis and 15 control patients were subjected to duodenal biopsy to assess intestinal oxidative stress, estimated by lipid peroxidation (malondialdehyde - MDA) and glutathione redox state [reduced glutathione (GSH), glutathione disulphide (GSSG) and GSH/GSSG ratio]. In addition, mucosal biopsies were examined histologically and intestinal mucosal protein content was determined biochemically as an index of intestinal trophic state. RESULTS: Patients with obstructive jaundice presented high levels of intestinal oxidative stress, with significantly increased lipid peroxidation (P < 0.001). Glutathione redox state was also suggestive of high intestinal oxidative stress in jaundiced patients, indicated by significantly decreased GSH (P = 0.001) and GSH/GSSG ratio (P = 0.006) and increased GSSG (P = 0.026). Histological examination showed a mild infiltration of the lamina propria by chronic inflammatory cells in obstructive jaundice, whereas duodenal architecture remained intact and epithelial continuity was retained. Duodenal mucosa was atrophic in jaundiced patients as indicated by a significant reduction of mucosal protein content compared with controls (P = 0.001). Among oxidative stress parameters, intestinal GSH exhibited a significant positive correlation with mucosal protein content (r = 0.588, P = 0.021). CONCLUSIONS: Obstructive jaundice in humans induces intestinal oxidative stress, which may be a key factor contributing to intestinal barrier failure and the development of septic complications in this patient population.

What is metamorphosis?

Metamorphosis (Gr. meta- "change" + morphe "form") as a biological process is generally attributed to a subset of animals: most famously insects and amphibians, but some fish and many marine invertebrates as well. We held a symposium at the 2006 Society for Integrative and Comparative Biology (SICB) annual meeting in Orlando, FL (USA) to discuss metamorphosis in a comparative context. Specifically, we considered the possibility that the term "metamorphosis" could be rightly applied to non-animals as well, including fungi, flowering plants, and some marine algae. Clearly, the answer depends upon how metamorphosis is defined. As we participants differed (sometimes quite substantially) in how we defined the term, we decided to present each of our conceptions of metamorphosis in 1 place, rather than attempting to agree on a single consensus definition. Herein we have gathered together our various definitions of metamorphosis, and offer an analysis that highlights some of the main similarities and differences among them. We present this article not only as an introduction to this symposium volume, but also as a reference tool that can be used by others interested in metamorphosis. Ultimately, we hope that this article-and the volume as a whole-will represent a springboard for further investigations into the surprisingly deep mechanistic similarities among independently evolved life cycle transitions across kingdoms.

Glutathione and lipid peroxide changes in pseudoexfoliation syndrome.

PURPOSE: To investigate the oxidative status of the aqueous humor of patients with pseudoexfoliation (PEX) syndrome. METHODS: Aqueous humor samples obtained during cataract surgery of patients with PEX syndrome and normal age-matched control subjects were examined for changes in the levels of glutathione (GSH), glutathione disulfide (GSSG), and TBA reactive species (TBARS), products of lipid peroxidation. GSH, GSSG, and TBARS were determined by specific fluorescent assays. RESULTS: Compared to normal controls, PEX syndrome aqueous humor samples showed a decrease of up to 28% of GSH concentration, and GSSG was increased up to 23%. The ratio of GSH/GSSG was 1.7-fold decreased in PEX syndrome samples. TBARS levels were increased by 100% in the PEX aqueous humor samples as compared to the controls. CONCLUSIONS: High levels of GSSG and TBARS indicate high oxidative stress, as well as the decrease in the ratio of GSH/GSSG. Our findings suggest a role for oxidation stress in the pathogenesis and the progression of PEX syndrome.

beta-Carotene production and its role in sclerotial differentiation of Sclerotium rolfsii.

The fungus Sclerotium rolfsii produces beta-carotene, the main detected carotenoid, in levels dependent upon oxidative growth conditions and upon differentiation. beta-Carotene accumulation is 5-, 6.5-, and 6.7-fold higher in undifferentiated mycelia, sclerotia, and differentiated mycelia, respectively, at high than at low oxidative stress. It accumulates more in older than in younger mycelia and is 2-fold higher in differentiated than in undifferentiated mycelia. We propose that beta-carotene is formed possibly to help the fungus reduce oxidative stress that develops during growth. This is supported by the finding that exogenous beta-carotene at non-growth-inhibiting concentrations causes a concentration-dependent reduction of oxidative stress (lipid peroxidation) of undifferentiated mycelia, which results in an equally proportional reduction of sclerotial differentiation. The data of this study support our hypothesis that sclerotial differentiation is induced by oxidative stress.

An apparatus (Georgiou-Petri dish) for growing fungi and other microorganisms on liquid media in a Petri dish.

This work describes a new apparatus for growing fungi and other microorganisms on liquid nutrient media in a Petri dish. The apparatus is composed of a net supporting a cellophane membrane stretched between an outer and an inner ring that is placed inside a Petri dish. This modification of the standard Petri dish offers many advantages for studying growth, metabolism, differentiation, and other aspects of fungi in liquid cultures with minimal waste of expensive chemicals. Monitoring of excreted or absorbed substances by the fungi, the aseptic transfer of undisturbed fungal colonies from dish to dish, and harvesting are made easier, using this apparatus.

Modified, large-scale purification of the cytochrome o complex (bo-type oxidase) of Escherichia coli yields a two heme/one copper terminal oxidase with high specific activity.

The cytochrome o complex is a bo-type ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli. This complex has a close structural and functional relationship with the eukaryotic and prokaryotic aa3-type cytochrome c oxidases. The specific activity, subunit composition, and metal content of the purified cytochrome o complex are not consistent for different preparative protocols reported in the literature. This paper presents a relatively simple preparation of the enzyme starting with a strain of Escherichia coli which overproduces the oxidase. The pure enzyme contains four subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Partial amino acid sequence data confirm the identities of subunit I, II, and III from the SDS-PAGE analysis as the cyoB, cyoA, and cyoC gene products, respectively. A slight modification of the purification protocol yields an oxidase preparation that contains a possible fifth subunit which may be the cyoE gene product. The pure four-subunit enzyme contains 2 equivs of iron but only 1 equiv of copper. There is no electron paramagnetic resonance detectable copper in the purified enzyme. Hence, the equivalent of CuA of the aa3-type cytochrome c oxidases is absent in this quinol oxidase. There is also no zinc in the purified quinol oxidase. Finally, monoclonal antibodies are reported that interact with subunit II. One of these monoclonals inhibits the quinol oxidase activity of the detergent-solubilized, purified oxidase. Hence, although subunit II does not contain CuA and does not interact with cytochrome c, it still must have an important function in the bo-type ubiquinol oxidase.

Beta-galactosidase gene fusions as probes for the cytoplasmic regions of subunits I and II of the membrane-bound cytochrome d terminal oxidase from Escherichia coli.

The cytochrome d terminal oxidase complex is a component of the aerobic respiratory chain of Escherichia coli. This enzyme catalyzes the oxidation of ubiquinol-8 within the cytoplasmic membrane and the reduction of molecular oxygen to water along with the concomitant generation of a proton-motive force across the membrane. Previous studies have established that the oxidase is composed of one copy of each of two subunits (I and II), and contains four heme prosthetic groups. The hydropathy profiles of the amino acid sequences suggest that each subunit has multiple transmembrane-spanning helical segments. The goal of the current work is to obtain experimental information about which portions of the two polypeptide chains are facing the cytoplasm. This is part of an effort to determine the topological folding of the two subunits across the membrane. A number of random gene fusions were generated in vitro which encode hybrid proteins in which the amino-terminal portion is provided by one of the two subunits of the oxidase, and the carboxyl-terminal portion is beta-galactosidase. Studies from other systems have indicated that the only hybrid proteins which will manifest high beta-galactosidase specific activity and be membrane-bound will be those where the fusion junction is in a region of the cytochrome polypeptides facing the cytoplasm. Fusions were obtained in eight positions within subunit I and 11 positions within subunit II. These identified four cytoplasmic-facing regions within subunit II, consistent with its hydropathy profile showing eight transmembrane helices. The data with subunit I are less conclusive.

The nucleotide sequence of the cyd locus encoding the two subunits of the cytochrome d terminal oxidase complex of Escherichia coli.

The cytochrome d terminal oxidase complex is one of two terminal oxidases in the aerobic respiratory chain of Escherichia coli. The enzyme is located in the cytoplasmic membrane where it oxidizes ubiquinol-8 in the bilayer and reduces oxygen to water. Enzyme turnover is coupled to the generation of a proton-motive force, resulting in electrogenic translocation across the membrane of one proton per electron passing through the system. The enzyme is an alpha beta heterodimer containing four hemes. The cyd locus, encoding both subunits, has previously been genetically mapped and cloned. This work describes an insertion and deletion analysis of cyd which indicates the direction of transcription, defines the coding regions, and suggests that cyd is an operon. In addition, the complete DNA sequence of the cyd gene is reported. Two open reading frames, separated by 18 base pairs, encode the two subunits of the oxidase complex. Hydropathy profiles of the deduced protein sequence indicate that subunits I and II are each likely to have multiple transmembrane elements. There are only 10 histidines in both subunits, several of which are likely to serve as heme axial ligands.

Relationships between membrane-bound cytochrome o from Vitreoscilla and that of Escherichia coli.

The cytochrome o terminal oxidases from the bacteria Vitreoscilla and Escherichia coli are structurally and functionally related. They have similar optical spectra, both exhibit ubiquinol-1 oxidase activity and are inhibited similarly. Both enzymes contain four subunits by SDS-polyacrylamide gel electrophoresis analysis and contain protoheme IX and Cu2+ prosthetic groups. Antibodies raised against the oxidase purified from E. coli crossreact with the Vitreoscilla oxidase.

Regulation of expression of the cytochrome d terminal oxidase in Escherichia coli is transcriptional.

The cytochrome d complex is one of the two terminal oxidases in the aerobic respiratory system of Escherichia coli. This enzyme is not present in cells grown with high levels of dissolved oxygen in the culture medium but accumulates after mid-exponential growth, reaching high levels in stationary-phase cells. In this study, the transcriptional activity of the cyd operon, encoding the two subunits of the enzyme, was examined under a variety of growth conditions. This was accomplished by the use of a chromosomal operon fusion, cyd-lacZ, generated in vivo by a lambda plac-Mu hopper bacteriophage and also by the use of a cyd-lacZ protein fusion created in vitro on a plasmid, transferred onto a lambda transducing phage, and examined as a single-copy lysogen. Transcription of the gene fusions was monitored by determination of beta-galactosidase activity. The data clearly show that cyd is transcriptionally regulated and that induction is observed when the culture reaches a sufficient cell density so as to substantially reduce the steady-state levels of dissolved oxygen. The transcriptional activity is also regulated by other growth conditions, including the carbon source. The turn-on of cyd under semianaerobic conditions does not require the fnr gene product, cyclic AMP, or the cyclic AMP-binding protein.

Identification of b, c, and d cytochromes in the membrane of Vitreoscilla.

Cytochromes b, c, d, and o were identified by spectroscopic analysis of respiratory membrane fragments from Vitreoscilla sp., strain C1. Carbon monoxide difference spectra of the reduced membranes had absorption maxima at 416, 534, and 571 nm (ascribed to cytochrome o) and 632 nm (cytochrome d). Derivative spectra of the pyridine hemochromogen spectra of the membranes identified the presence of b- and c-type cytochromes in Vitreoscilla. The cyanide binding curve of the membranes was biphasic with dissociation constants of 2.14 mM and 10.7 which were assigned to cytochrome o and cytochrome d, respectively. Membranes bound carbon monoxide with dissociation constant 3.9 microM, which was assigned to cytochrome o. Cytochrome c556 and a NADH-p-iodonitrotetrazolium violet reductase component were partially purified from Vitreoscilla membranes.

Purification and partial characterization of the membrane-bound cytochrome o(561,564) from Vitreoscilla.

Cytochrome o(561,564) terminal oxidase was solubilized from the membrane fraction of the bacterium Vitreoscilla sp., strain C1, and purified by differential pH dialysis, gel filtration chromatography, and ion-exchange chromatography. Subunit molecular weights, determined on sodium dodecyl sulfate-polyacrylamide gels by the Ferguson plot method, were 49,500 and 23,500. There were two protohemes IX, two coppers, and 45 mol of phosphorus per mole of protomer (73,000). The molecular weight of the cytochrome o complex estimated by chromatography on Sephacryl-400 in deoxycholate was 265,000, which is consistent with the enzyme complex under these conditions being a dimer (146,000) with the remaining molecular weight contribution arising from bound phospholipid, deoxycholate, and possibly other, smaller subunits. Difference spectra of the dithionite-reduced enzyme have split alpha absorption maxima at 561 and 564 nm at room temperature and 558 and 561 nm at 77 K. The CO difference spectrum at room temperature has absorption maxima at 570, 534, and 416 nm. Dissociation constants for CO and cyanide binding to the reduced and oxidized forms of the oxidase are 5.2 microM and 3.5 mM, respectively. The hemes in the cytochrome are one electron accepting centers, both with midpoint potentials around +165 mV at pH 7.0. The enzyme is highly autoxidizable, and its menadiol oxidizing activity is stimulated by phospholipids.

Identification of the cydC locus required for expression of the functional form of the cytochrome d terminal oxidase complex in Escherichia coli.

The aerobic respiratory chain of Escherichia coli contains two terminal oxidases which are differentially regulated. The cytochrome o complex predominates under growth conditions of high aeration, whereas the cytochrome d complex predominates when the oxygen tension is low. Either terminal oxidase will support aerobic growth. The goal of the work presented in this paper was to identify genes required for the expression of the functional form of the cytochrome d complex, other than the genes encoding the polypeptide components of the oxidase complex (cyd locus). A strain lacking the cytochrome o complex (cyo mutant strain) was mutagenized by using a lambda-Mu hybrid hopper bacteriophage, lambda placMu53, which inserts randomly into the chromosome and carries a kanamycin resistance marker. Strains were isolated and examined which were unable to grow aerobically, i.e., which lacked functional cytochrome d complex, and which could not be complemented by introduction of the cyd gene on F-prime episomes. One strain was selected for characterization. The phage insert was mapped to min 18.9 on the genetic linkage map, defining a new genetic locus, cydC. Evidence described in the text suggests that the gene product is probably required for the synthesis of the unique heme d component of the cytochrome d complex.