Narrow Results

This communication is devoted to nature determination and quantification by PIXE of metals contained in proteins after their separation by PolyAcrylamide Gel Electrophoresis (PAGE). After the electrophoresis, the gel is dried and each track is scanned with a 2.5 MeV proton beam which triggers metal X-ray fluorescence and then, allows to determine the type of metals contained in an electrophoretic band. For quantitative determination of the amount of the metal contained inside the band, the characteristic X-ray peak area is compared with those obtained with polyacrylamide gels doped with the same metal. The normalization has been achieved by using RBS measurements on the gel itself.

The procedure presented seems to be a very useful multielementary method for the metal content analysis and for the determination of the metal amounts inside proteins after their separation by electrophoresis. Furthermore it allows to check if metals remain bound to proteins.

Astragalosides were the main active components from a native Chinese herb Astragalus membranaceus. Recent studies have shown that Astragalosides have a protective effect on myocardial injury in rats. The present study was designed to investigate the effect of Astragalosides on intracellular calcium overload and sarcoplasmic reticulum calcium load (SR Ca²⁺ load) in cultured cardiac myocytes from neonatal rats. Astragalosides (100 μg/ml) were incubated in the presence of isoproterenol (ISO) (10^-5 M) for 72 hours in cardiomyocytes. Metoprorol (10^-6 M), a β₁-selective antagonist, was cultured in the same condition as Astragalosides. The result showed that intracellular calcium concentration ([Ca²⁺]_i) and SR Ca²⁺ load increased in ISO-treated cardiac myocytes as compared to control (P<0.01). Astragalosides prevented ISO-induced increase in [Ca²⁺]_i and SR Ca²⁺ load. Metoprolol also inhibited those increase. The mRNA expression and activity of sarcoplasmic reticulum Ca²⁺ ATPase (SERCA) were enhanced following ISO treatment in cardiac myocytes, and these increases were inhibited by Astragalosides or metoprolol (P<0.05). The decrease of superoxide dismutase (SOD) activity and the elevation of intracellular maleic dialdehyde (MDA) were observed after ISO treatment in cardiac myocytes. Both Astragalosides and metoprolol restored the SOD activity and reduced the level of MDA. We conclude that Astragalosides have the effects on reducing [Ca²⁺]_i and SR Ca²⁺ load, enhancing free radical removal and decreasing lipid peroxidation in ISO-treated cardiomyocytes, which might account for their protective effect on myocardial injury.

The efficacy of methotrexate (MTX), a widely used cytotoxic chemotherapeutic agent, is often limited by its severe hepatotoxicity. Regarding the mechanisms of these adverse effects, several hypotheses have been put forward, among which oxidative stress is noticeable. The present study was undertaken to determine whether grape seed extract (GSE), a new natural free radical scavenger, could ameliorate the MTX-induced oxidative injury in the rat liver. The animals were divided into 3 groups. Each group consisted of 12 animals. MTX-GSE group: rats were given GSE (100mg/kg body weight) orally for 15 days, and a single dose of MTX (20mg/kg, intraperitoneally) was added on the 10th day. MTX group: these received placebo distilled water (orally) instead of GSE for 15 days and the same MTX protocol applied to this group on the 10th day. Control group: rats were given distilled water (orally) through 15 days and physiological saline (intraperitoneally) instead of MTX was administered on the 10th day in a similar manner. On the 16th day, liver tissue samples were obtained under deep anaesthesia. The level of malondialdehyde (MDA), an end product of lipid peroxidation, and the activities of süperoxide dismutase (SOD) and catalase (CAT), two important endogenous antioxidants, were evaluated in the tissue homogenates. MTX administration increased the MDA level and decreased the SOD and CAT activities in the liver homogenates (p < 0.001), while these alterations were significantly reversed by GSE treatment (p < 0.001). MTX led to significantly reduced whole blood count parameters (p < 0.05). When GSE was supplemented, no significant changes in blood count parameters were noted. It appears that GSE protects the rat liver and inhibits methotrexate-induced oxidative stress. These data indicate that GSE may be of therapeutic benefit when used with MTX.

The fruits of Cornus officinalis have been used in traditional oriental medicine for treatment of inner ear diseases, such as tinnitus and hearing loss. In the present study, we investigated the protective effect of C. officinalis on hydrogen peroxide-induced cytotoxicity in HEI-OC1 auditory cells. The results from bioassay-guided fractionation of methanol extract of C. officinalis fruits showed that ursolic acid is a major active component. Ursolic acid (0.05–2 μg/ml) had protective effect against the HEI-OC1 cell damage and reduced lipid peroxidation in a dose-dependent manner. In addition, pre-treatment with ursolic acid significantly attenuated the decrease of activities of catalase (CAT) and glutathione peroxidase (GPX), but superoxide dismutase (SOD) activity was not significantly affected by ursolic acid. These results indicate that ursolic acid protects hydrogen peroxide-induced HEI-OC1 cell damage through inhibition of lipid peroxidation and induction of antioxidant enzymes, CAT and GPX, and may be one of the active components responsible for these effects of C. officinalis fruits.

To provide experimental and theoretical basis for the clinical application of Puerarin in acute alcohol poisoning, 30 Wistar rats were randomized into 3 groups as follows: (1) Group A (control) underwent normal sodium (N.S.) peritoneal injection (i.p.) and intragastric administration (i.g.); (2) Group B (alcohol) underwent an equivalent dosage of N.S. i.p. and 40% ethanol (8000 mg/kg. d).ig for 5 days; (3) Group C (Puerarin) underwent Puerarin 200 mg/kg. d. ip, and an equivalent dosage of ethanol for 5 days. The left lobes of livers were sampled, and the levels of MDA, SOD and GPX in plasma and liver homogenate were detected. The level of MDA in plasma and liver homogenate in the alcohol group was obviously higher than that in the control group (p < 0.05, respectively), while that in the Puerarin group was significantly lower than in the alcohol group (p < 0.05, respectively). The levels of SOD and GPX were opposite to that of MDA. Under a light microscope, the livers of the rats in the alcohol group showed unclear structure of hepatic lobules, stiffness of hepatic sinusoids, diffused lipid degeneration of hepatic cells, cellular swelling, and focal necrosis, while the structure remained clear in the Puerarin group. Under the electron microscope, lipid degeneration, cell organ decrease, enlargement of endoplasmic reticulum, reduced quantity of hepatins and swelling of mitochondria were observed in cells of the model group. However, the pathologistic changes were slight in the Puerarin group. In conclusion, Puerarin may have the function of inhibiting the oxidative stress induced by acute alcoholism.

Targeting the stemness of triple-negative breast cancer (TNBC) is a potential therapeutic approach for treating TNBC. Tetrandrine, a natural plant alkaloid, has several anticancer effects. Here, we aimed to evaluate the efficacy of tetrandrine in cancer stemness and epithelial to mesenchymal transition (EMT) in TNBC, and to explore the underlying mechanisms. The effects of tetrandrine on cell growth, cell viability, cell stemness capacity, cell migration, and cell invasion, as well as the molecules involved in these processes, were investigated in a cell culture system. An in vivo xenograft tumor and lung metastasis study was performed using nude mice to verify the effects and mechanisms of tetrandrine. Tetrandrine exhibited antiproliferative and cell cycle arrest activities in TNBC cell lines, significantly reduced aldehyde dehydrogenase and CD44${}^{+}$CD24${}^{-∕\mathrm{\text{low}}}$ characteristic subpopulation, and successfully prevented mammosphere formation. It suppressed migration and invasion, enhanced anoikis, and regulated the expression of proteins involved in the EMT, including E-cadherin, Vimentin, and Occludin, in both TNBC cells and MDA-MB-231 spheroid cells. Further studies revealed that tetrandrine downregulated the expression of superoxide dismutase 1 (SOD1) and catalase and induced reactive oxygen species (ROS) production, which subsequently contributed to the inhibition of cell EMT and stemness. The in vivo studies also showed that tetrandrine inhibited tumor growth and metastasis of both adherent normal cells, and flow cytometry sorted specific CD44${}^{+}$CD24${}^{-∕\mathrm{\text{low}}}$ breast cancer stem cells, which could be rescued by SOD1 overexpression. The results of this study suggest that tetrandrine could effectively inhibit breast cancer stem cell characteristics and the EMT process via the SOD1/ROS signaling pathway. Therefore, tetrandrine can be considered a promising anti-TNBC agent.

Recently, a wide variety of bionanocomposites and biocomposites are being developed to be used as extracellular matrix for chronic wound healing. Majority of the chronic wound situations arise due to infections caused by drug-resistant microbes like Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. In particular S. aureus has become refractory to the current armamentarium of antimicrobial drugs. Therefore, it is imperative while designing nanobiocomposites for use as extracellular matrices to profile their antimicrobial activity. MWNT (multi-wall carbon nanotube) has been exploited previously in designing biocompatible nanocomposite for medical applications. Keeping in view, we studied the antimicrobial effect of MWNT on human clinical burn/wound pathogens, which were Methicillin-resistant Staphylococci and one Vancomycin intermediate Staphylococcus isolate. We also studied E. coli NCTC 10418 and Pseudomonas aeruginosa NCTC 10662, two surrogate gram negative microbes to understand their behavior in the presence of MWNT. Apart from reduction in viable counts of the test panel, organism's extracellular expression of the enzyme Superoxide dismutase (SOD) was also taken into account as this is the probable mechanism adopted by bacteria in general to survive and sustain under nutritional and other stress situation including pathogenesis. The present study indicated that all Staphylococcal isolates were susceptible to MWNT which reduced the bacterial count between 3–9 logs barring Sau G19 which only exhibited 1 log reduction. Sau G17, Sau G18, and Sau G19 expressed a higher SOD activity, Sau G9 exhibited a lower SOD expression and in Sau G16, the SOD expression was nearly the same as compared to control. Thus, from this study, it could be inferred that MWNT, apart from being antimicrobial, induces oxidative stress on S. aureus.

In this study, we have analyzed the effect a newly synthesized water-soluble alpha tetra-substituted zinc phthalocyanine (Pc) compound on superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR) activities and biomass accumulation in the Arthrospira platensis-M2 strain to test whether this compound could be used as an algaecide or not. We found that lower concentrations (3 μg mL${}^{\mathrm{\text{-1}}}$ and 6 μg mL${}^{\mathrm{\text{-1}}})$ of Pc compound were not toxic to algae cells, as indicated by enduring biomass accumulation during the study (7 days). Higher Pc concentrations, however, were toxic and inhibited biomass accumulation. This inhibition appeared on the fourth day and persisted during the study. At higher Pc concentrations, SOD activity decreased significantly, but APX and GR activity were not affected. These results may show that Pc applications did not cause the accumulation of reactive oxygen species in Arthrospira platensis-M2 cells. Our result suggests that higher Pc concentrations did not cause oxidative stress but biomass accumulation inhibited, possibly due to some different toxicity mechanism(s), which should be carried out in the future studies. As a result, we may offer use of this compound as a means to keep under control algal populations in natural environments.

Background and Aims: Endometriosis is a gynecological disorder defined as the presence of endometrial tissue outside the uterine cavity and affecting 10-15% of females of reproductive age. Oxidative stress has been reported as one of the main promoting factors of this disease. Green tea and its anti-angiogenic and antioxidant properties have promising potential to prevent the progression of endometriosis. This study aims to investigate the effect of green tea extract on endometriotic implant area and serum superoxide dismutase (SOD) levels in endometriosis rat models.

Method: Twenty-six Balb/c female rats were randomized into two groups of equal size: experimental group (EG) and control group (CG). All subjects in EG were given green tea extract 3 mg daily for 14 days. Endometriosis was induced on the fourteenth day in all subjects in both groups. The data of the endometriotic implant area and serum SOD were obtained and analyzed fourteen days after the induction of endometriosis.

Results: The endometriotic implant area of the group treated with green tea extract was significantly lower (p=0,022) RR=0,455, 95%CI=0.065-0,96) compared to the control group. The serum level of SOD in EG and CG were 5.33±0.52 ng/ml and 5.20±0.91 ng/ml, respectively (p=0.507).

Conclusion: Green tea extract inhibits endometriotic implant progression and increases the level of serum SOD in endometriosis-induced rat models.

We report a novel method to simultaneously extract superoxide dismutase (SOD), catalase (CAT) and carbonic anhydrase (CA) from the same sample of red blood cells (RBCs). This avoids the need to use expensive commercial enzymes, thus allowing this to be cost effective for large-scale production of a nanobiotechnological polyHb-SOD-CAT-CA with enhancements to all three RBC functions. The best concentration of phosphate buffer for ethanol-chloroform treatment results in good recovery of CAT, SOD and CA after extraction. Different concentrations of the enzymes can be used to enhance the activity of polyHb-SOD-CAT-CA to 2, 4 or 6 times that of RBCs.

Polyhemoglobin-superoxide dismutase-catalase-carbonic anhydrase (poly-[Hb-SOD-CAT-CA]) contains all three major functions of red blood cells (RBCs) at an enhanced level. It transports oxygen, removes oxygen radicals and transports carbon dioxide. Our previous studies in a 90-minute 30 mm Hg MAP-sustained hemorrhagic shock rat model showed that it was more effective than blood in the lowering of elevated intracellular PCO₂, recovery of ST-elevation and histology of the heart and intestine. This paper analyzes the storage and temperature stability. The allowable storage time for RBCs is about 1 day at room temperature and 42 days at 4°C. Also, RBCs cannot be pasteurized to remove infective agents like HIV and Ebola. PolyHb can be heat sterilized and can be stored for a year even at room temperature. However, poly-[Hb-SOD-CAT-CA] contains both Hb and enzymes, and enzymes are particularly sensitive to storage and heat. We thus carried out studies to analyze its storage stability at different temperatures and heat pasteurization stability. The results of the storage stability show that lyophillization extends the storage time to a year at 4°C and 40 days at room temperature (compared to 42 days and 1 day, respectively, for RBCs). After the freeze-dry process, the enzyme activities of poly-[SFHb-SOD-CAT-CA] was 100 ± 2% for CA, 100 ± 2% for SOD, and 93 ± 3.5% for CAT. After heat pasteurization at 70°C for 2 hours, lyophilized poly-[Hb-SOD-CAT-CA] retained good enzyme activities of CA (97 ± 4%), SOD (100 ± 2.5%) and CAT (63.8 ± 4%). More CAT can be added during the crosslinking process to maintain the same enzyme ratio after heat pasteurization. Heat pasteurization is possible only for the lyophilized form of poly-[Hb-SOD-CAT-CA] and not for the solution. It can be easily reconstituted by dissolving it in suitable solutions that continue to have good storage stability (though less than that for the lyophilized form). According to the P50 value, poly-[SFHb-SOD-CAT-CA] retains its oxygen-carrying ability before and after long-term storage.

The effects of superoxide dismutase on aging were tested using two differt experimental approaches. In the first, replicated populations with postponed aging were compared with their controls for frequencies of electrophoretic alleles at the SOD locus. Populations with postponed aging had consistently greater frequencies of the allele coding for more active SOD protein. This allele was not part of a segregating inversion polymorphism. The second experimental approach was the extraction of SOD alleles from different natural populations followed by the construction of different SOD genotypes on hybrid genetic backgrounds. This procedure did not uncover any statistical effect of SOD genotype on longevity or fecundity. There were large effects on longevity and fecundity due to the family from which a particular SOD genotype was derived. To detect the effects of SOD genotypes on longevity with high probability would require a ten-fold increase in the number of families used.

Drosophila melanogaster populations that exhibit constrasting life histories as a result of laboratory selection were compared at several potentially relevant enzyme loci. Selection regimes included postponed reproduction, accelerated development, and intermediate generation time. Each selection regime was represented by fivefold replicated populations maintained for between 50 and 500 generations. For each population, allele frequencies were calculated from frequencies of electrophoretically distinguishable allozymes of alcohol dehydrogenase, α-glycerol-3-phosphate dehydrogenase, phosphoglucomutase, and CuZn-superoxide dismutase. Based on allozyme frequency changes consistent across replicate populations, two of the studied loci responded to both selection for postponed reproduction and selection for accelerated development. The responses to contrasting selection regimes were in opposing directions, suggesting antagonistic pleiotropy.

Pleurotus ostreatus is an edible white-rot basidiomycete with medicinal and bioremediation properties. In this research, the activity of key intracellular anti-oxidative stress enzymes, superoxide dismutase (SOD), catalase (CAT) and peroxidase (POX), was investigated and characterized in the mushroom. A Pleurotus ostreatus homogenate was centrifuged at 3,000 g for 10 min; the supernatant was centrifuged at 35,000 g for 30 min, and the new supernatant obtained, called "crude extract" was used for our studies. Results showed that up to 7.4 units SOD, 11.7 units CAT and 0.037 units POX were detectable per mg protein in Pleurotus ostreatus extract. All three enzymes were sensitive to KCN. Non-denaturing polyacrylamide gel electrophoresis of the extract, followed by activity staining, revealed one SOD band (estimated M.W. 44 kD), one CAT band (estimated M.W. 280 kD) and three POX bands (estimated M.W. 77, 65, and 60 kD, respectively).

Doxorubicin (DOXO), an anthracycline antibiotic, is a potent anticancer agent with severe toxic side-effects that have been attributed to its ability to generate free radicals (ROS), particularly in mitochondria. In this study, a prokaryotic organism, Salmonella typhimurium, was used to evaluate the effect of the drug on ROS-scavenging enzymes, catalase (CAT) and superoxide dismutase (SOD) activity, and on cell growth and viability. Increasing DOXO concentrations in the culture medium led to increasingly prolonged lag period, reduced growth rate and cell viability, and inhibition of SOD and CAT activity. SOD activity dropped to 66% of the control value in cells grown with 1 µg/ml DOXO and to 34% and 28% in cells grown with 150 and 300 µg/ml DOXO. CAT activity decreased progressively from 93% in 1 µg/ml DOXO to 30% in 150 µg/ml DOXO and was undetectable in 300 µg/ml. Growth in the presence of DOXO led to alterations in cytokinesis, as seen upon examination of cells under fluorescence microscope.

The golden chanterelle mushroom (Cantharellus cibarius), also known as girolle, is an edible mushroom, renowned as a delicacy especially prized for its unique flavor. As an ectomycorrhizal fungus, C. cibarius grows in symbiosis with the root tips of forest trees, giving rise to an inextricable relationship between tree vitality and mushroom thriving. In this research, the activity of key intracellular anti-oxidative stress enzymes, superoxide dismutase (SOD), catalase (CAT) and peroxidase (POX), was investigated and characterized in C. cibarius mushroom. A C. cibarius homogenate was centrifuged at 3,000 g for 10 min, then at 35,000 g for 30 min; the supernatant (“crude extract”) was used for our studies. Data showed that up to 18 units SOD, 10 units CAT, and 0.11 units POX, were detectable per mg protein in the extract. Non-denaturing polyacrylamide gel electrophoresis of the extract, followed by activity staining, revealed one SOD band (estimated M.W. 30.2 kD), at least two CAT bands (estimated M.W. 280 and 350 kD), and at least one POX band (estimated M.W. 45 kD).

The aim of this work was to characterize the cellular stress, the production of reactive oxygen species (ROS), the liberation of nitric oxide (NO) and the enzymatic activity of superoxide dismutase (SOD), in the two bacterial species Staphylococcus aureus and Escherichia coli, which have both been associated with the formation of biofilms. We studied the influence of a different range of culture conditions (sugar, osmotic stress and reduction conditions). The extracellular production of ROS was evaluated by the reduction of nitro blue tetrazolium (NBT), the NO production was detected as nitrite using the Griess reagent and SOD activity being assayed using the inhibition of NBT. We observed that ROS, RNI and their downstream derivatives played important roles in biofilm development. This suggests that cellular stress affected the biofilms grown under different conditions. Our results suggest that biofilm formation was influenced by the different conditions, with a close relation seen between cellular stress (ROS and NO) and biofilm formation. Under favourable culture conditions for biofilm formation, the polysaccharide matrix was increased because the sessile cells were present at lower cellular stresses. The decrease in the extracellular matrix observed under unfavorable conditions for increasing the cellular stress, caused radical oxidizers to accummulate in an extracellular medium and thereby affects the matrix. In conclusion, we suggest that biofilm formations in a variety of environmental conditions are influenced by cellular stress. An improved knowledge of ROS, RNI and enzymatic pathway regulation may help to clarify the relevance of biofilm formation, and new advances in this aspect could also be of great value in the development of better preventive and therapeutic measures.