Electron microscopic and microprobe analysis of calciuminduced differentiation of the white mutant (LU887 x LU897) strain of Physarum polycephalum

Differentiation of the white mutant (LU887 x LU897) strain of Physarum polycephalum leading to spherule formation can be induced by CaCl(2) if the concentration in the nutrient medium is increased by 5mM prior to the transfer to a non-nutrient salts medium. All stages previously reported for the typical (M(3)cVII) strain of Physarum polycephalum from microplasmodia to spherules are seen but the mutant lacks the synchrony that the replacement technique induces in the typical strain. X-ray microanalyses locate calcium and phosphorus in granules in mitochondria and in the cytoplasm of specimens fixed without osmium. Mitochondria accumulate calcium-containing granules during early differentiation and appear to be essentially without granules in mature spherules. Mobilization of mitochondrial calcium is implicated in the initiation of differentiation. A longitudinally striated cytoplasmic inclusion is abundant in microplasmodia grown in media that have not been supplemented with additional calcium and is seen more rarely during calcium-induced spherulation. Whether or not this inclusion represents cytoplasmic contractile elements is unknown. The calcium-treated mutant strain, previously considered non-differentiating, may prove to be a good alternate model for the study of factors influencing differentiation. It was employed earlier as a control in studies of strains that readily spherulate in response to routine procedures.     

Effects of the free radical generator paraquat on differentiation, superoxide dismutase, glutathione and inorganic peroxides in microplasmodia of Physarum polycephalum

The herbicide paraquat was used to investigate the effects of oxidative stress on the spherulation of Physarum polycephalum microplasmodia. The responses of a white non-differentiating strain of Physarum were compared with those of a common yellow strain that readily spherulates in salts-only starvation medium. The addition of paraquat to the salts medium increased the specific activity of superoxide dismutase in both strains; it also induced an increase in the intracellular inorganic peroxide concentration in both strains. Glutathione concentration was higher in the paraquat-treated yellow strain than in the controls. Paraquat had no effect on glutathione concentration in white microplasmodia. Paraquat accelerated spherulation in yellow microplasmodia. The white microplasmodia responded to the herbicide by cleaving into structures similar to immature spherules; however, these structures were not viable. The results of this study support the hypothesis that free radicals are involved in cell state transitions.     

Effects of the Free Radical Generator Paraquat On Differentiation, Superoxide Dismutase, Glutathione and Inorganic Peroxides In Microplasmodia of Physarum Polycephalum

Abstract. The herbicide paraquat was used to investigate the effects of oxidative stress on the spherulation of Physarum polycephalum microplasmodia. the responses of a white non-differentiating strain of Physarum were compared with those of a common yellow strain that readily spherulates in salts-only starvation medium. the addition of paraquat to the salts medium increased the specific activity of superoxide dismutase in both strains; it also induced an increase in the intracellular inorganic peroxide concentration in both strains. Glutathione concentration was higher in the paraquat-treated yellow strain than in the controls. Paraquat had no effect on glutathione concentration in white microplasmodia. Paraquat accelerated spherulation in yellow microplasmodia. the white microplasmodia responded to the herbicide by cleaving into structures similar to immature spherules; however, these structures were not viable. the results of this study support the hypothesis that free radicals are involved in cell state transitions.     

Involvement of Glutathione in the Differentiation of the Slime Mold Physarum polycephalum

The effects of experimentally-altered glutathione concentration on differentiation of the slime mold, Physarum polycephalum were examined. Spherulation was induced by transfer of Physarum from growth medium to a salts-only starvation medium. As differentiation proceeded, superoxide dismutase (SOD) activity in control cultures increased by as much as 21-fold. This increase in SOD activity paralleled the rate of differentiation. Glutathione (GSH) concentration decreased during differentiation by more than 80% in all cultures, regardless of the initial concentration. The rate of differentiation was inversely related to the initial GSH concentration and directly proportional to the SOD activity. These observations suggest that a free radical mechanism may be involved in the differentiation of Physarum microplasmodia into spherules.     

Superoxide radical is involved in the sclerotial differentiation of filamentous phytopathogenic fungi: identification of a fungal xanthine oxidase

This study shows that the direct indicator of oxidative stress superoxide radical (O·??) is involved in the sclerotial differentiation of the phytopathogenic filamentous fungi Rhizoctonia solani, Sclerotinia sclerotiorum, Sclerotium rolfsii, and Sclerotinia minor. The production rate of O·?? and the antioxidant enzyme superoxide dismutase (SOD) levels in the sclerotiogenic fungi were significantly higher and lower, respectively, than those of their non-differentiating counterpart strains, which strongly suggests that the oxidative stress of the sclerotium differentiating fungi is higher than that of the non-differentiating ones. Xanthine oxidase (XO), which was detected for the first time in fungi in general, was localized in the cytoplasmic membrane. The contribution of XO in the overall O·??production was very significant, reaching 30-70% among the strains, especially in the transition developmental stage between the undifferentiated and the differentiated state, suggesting a sclerotium triggering and a phytopathogenic role of XO during plant infection. The additional finding that these fungi secrete extracellular SOD can be related to their protection from the response of plants to produce O·?? at infection sites.     

Electron microscopic observations of an unusual cytoplasmic inclusion in the calcium-starved white mutant (LU887 × LU897) of Physarum polycephalum

Electron micrographs of Physarum polycephalum microplasmodia (LU887 × LU897) reveal cytoplasmic inclusions that appear “striated” at low magnifications; at higher magnifications these exhibit a structure that we have interpreted as microtubule bundles. The light and dark regions in the inclusions are due to the affinity of some microtubules for osmic acid; these appear to have dense regions while other microtubules remain electron lucent. The diameters of the microtubules are about 32–33nm; the subunits forming the tubule walls measure about 8–9nm in diameter. The diameter measurements are slightly larger than the dimensions assigned to vertebrate microtubules (28nm); however, the diameter of the subunits in the microtubule wall measures about 8–9nm which is essentially the same measurement reported for vertebrate tubulin dimers.     

The use of [125I]iodocyanopindolol as a specific probe for beta-adrenergic receptors in differentiating cultured rat skeletal muscle

In order to examine more precisely the role of beta-adrenergic receptors in the process of differentiation we used the new radioligand iodocyanopindolol ([125I]ICYP), which we found to be a very useful probe to identify beta receptors. Binding characteristics conformed to those expected for a physiologically relevant beta receptor. L6E9 cells grown in horse serum, which allows differentiation, exhibit increased beta receptor density in intact cells as a function of age. In contrast, cells grown in fetal calf serum, which does not allow differentiation, exhibit constant beta receptor density. In broken cells, however, both differentiating and non-differentiating cells show an increase in beta receptors. These results suggest that the process of differentiation is associated with an unmasking of beta receptors which are increasing but cryptic in undifferentiated cells.     

Effect of l-valine and l-isoleucine on fatty acid composition of Streptomyces hygroscopicus and S. griseus

The effects of L-valine and L-isoleucine on the composition of mycelial fatty acids were investigated during growth of differentiating parent strains of Streptomyces hygroscopicus and Streptomyces griseus as well as their non-differentiating derivatives (Amy-strains) on a synthetic medium. Both in the Amy+ and Amy- strains, in the presence of L-valine, the portion of the isopalmitic acid (iC16:0) increased, but the addition of L-isoleucine led to an elevated level of the 12-methyltetradecanoic acid (aC15:0). The results suggest that the genetically determined alterations in the ratio of both fatty acids in the non-differentiating derivatives may be due to specific changes in the biosynthetic pathways of both amino acid precursors rather than due to changes of their catabolism.     

A Comparative Study on Protein Metabolism During Subculture and Differentiation of Tobacco Callus

Comparative study on the subcultured callus of tobacco (Nicotiana tabacum L. cv. Willow leaf) has revealed that protein contents and pr otease activities slowly decreased in the callus during differentiation and bud formation. The synthetic rates of fraction Ⅱ protein (water soluble protein and enzymes) and ribosomal hist one, the levels of total ribosomes, especially the levels of polyribosomes which function the protein synthesis, were higher in the differentiating callus than those in the subcultured callus. This indicates that protein synthesis in differentiating callus is greater than that in non-differentiating callus, and that the protein pattern synthesized in differentiating callus may differ from that in non-differentiating callus. During the late period of culture, the protease activities in subcultured callus rapidly increased, and the levels of polyribosomes, protein synthetic rates and protein contents apparently declined, which may be the result of metabolic changes in callus senescence. Meanwhile in the differentiating callus the protein contents, protein synthesis rates and polyribosome levels although somewhat declined accempanying the growth of formed bud, were still much higher than those in the subcultured callus.     

Comparative study of chemiluminescence and resistance of serum and its components after radiation exposure

Kinetics of spontaneous chemiluminescence (CL) and electrochemiluminescence (ECL) and resistance of blood serum and its protein, lipid and carbohydrate components under the effect of X-rays (3 to 1622 Gy) and the indirect effect of radiation initiated by the addition of hydrogen peroxide (1.5 X 10(-5)-1.5%) was studied to estimate the contribution of each of the serum components to cumulative changes in the kinetics of free radical oxidation initiated by the effect of radiation. There was a parametric dependence between the absorbed dose, the rate of ECL and the resistance of blood serum and its components. As the absorbed dose or hydrogen peroxide concentration increased ECL contribution to the cumulative luminescence signal regularly decreased. Changes in CL and ECL of blood serum induced by ionizing radiation and H2O2 were qualitatively similar. The kinetics of free radical oxidation of blood serum initiated by irradiation was determined integrally (according to CL and ECL parameters) by a complex of changes in its components.     

Biochemical properties and localization of the chromosomal protein IP25.

The protein IP25, which has previously been reported to accumulate in the chromatin during erythroid differentiation of Friend-virus-transformed erythroleukemia cells (FL cells), is shown to behave like histone H1 without being structurally related to it. Like H1, IP25 is not released by digestion of FL cells nuclei with DNAse I. After micrococcal digestion IP25 and H1 are differentially distributed in the nucleosome monomers and dimers. This distribution suggests an internucleosomal location for IP25 and H1. Different rates of digestion are observed between nuclei of differentiating and non-differentiating FL cells with both DNAse I and micrococcal nuclease. These differences could be due to the presence of IP25 in the chromatin of differentiating cells.     

荒漠土壤中两株抗氧化细菌的抗氧化生理生化特征

【背景】微生物在荒漠生态系统中经常面临多重胁迫,包括干旱、高温、UV辐射,这些环境胁迫使得荒漠土壤微生物极易在体内外积累大量的超氧离子或过氧化物,抑制其生长或者直接造成死亡。【目的】荒漠土壤细菌为适应荒漠环境表现出抗氧化特性,作为荒漠生态系统重要组成部分,对其抗氧化特性的研究为荒漠地区抗氧化资源的开发提供科学依据和技术基础,也对荒漠微生物抗氧化机制的挖掘奠定了基础。【方法】利用过氧化氢氧化筛选出两株具有强抗氧化性的荒漠土壤细菌:海床动性微菌AX6(PlanomicrobiumokeanokoitesAX6)和海洋考克氏菌KD4(Kocuriamarina KD4),通过测定其在过氧化氢条件下的生长曲线、细胞受损程度、抗氧化酶活性以及自由基清除能力,探究荒漠土壤微生物的抗氧化生理生化特征。【结果】两株细菌在低浓度过氧化氢中细胞丙二醛含量显著低于阴性对照大肠杆菌,在1.5mmol/L过氧化氢中菌株AX6的谷胱甘肽过氧化物酶活性可达108.33 U/mL,同时DPPH、超氧阴离子自由基的清除能力显著升高;此外,在3 mmol/L过氧化氢中菌株KD4的过氧化氢酶活性升高至1.16 U/mL,显著高于阳性对照耐辐射球菌,羟自由基的清除能力也显著升高。【结论】不同荒漠土壤细菌的活性抗氧化酶种类、自由基清除能力存在较大差异,表明荒漠土壤微生物抗氧化过程的多样性。     

Changes in the ascorbate metabolism of apoplastic and symplastic spaces are associated with cell differentiation

Ascorbate levels and redox state, as well as the activities of the ascorbate related enzymes, have been analysed both in the apoplastic and symplastic spaces of etiolated pea (Pisum sativum L.) shoots during cellular differentiation. The ascorbate pool and the ascorbate oxidizing enzymes, namely ascorbate oxidase and ascorbate peroxidase, were present in both pea apoplast and symplast, whereas ascorbate free radical reductase and dehydroascorbate reductase were only present in the symplastic fractions. During cell differentiation the ascorbate redox enzymes changed in different ways, since a decrease in ascorbate levels, ascorbate peroxidase and ascorbate free radical reductase occurred from meristematic to differentiated cells, whereas ascorbate oxidase and dehydroascorbate reductase increased. The activity of secretory peroxidases has also been followed in the apoplast of meristematic and differentiating cells. These peroxidases increased their activity during differentiation. This behaviour was accompanied by changes in their isoenzymatic profiles. The analysis of the kinetic characteristics of the different peroxidases present in the apoplast suggests that the presence of ascorbate and ascorbate peroxidase in the cell wall could play a critical role in regulating the wall stiffening process during cell differentiation by interfering with the activity of secretory peroxidases.     

蝉虫草菌丝体胞内和胞外多糖抗肝细胞氧化损伤比较研究

蝉虫草（蝉花）作为我国传统的中药材,是一种药食两用的虫生真菌,因含有丰富的活性物质而具有广泛的医疗保健价值。本研究以自由基清除率为指标分析蝉虫草胞内和胞外多糖的化学抗氧化活性,再以H₂O₂诱导的人肝LO2细胞氧化损伤为模型,进而分析比较二者对肝细胞氧化应激损伤的改善作用。结果表明,在化学抗氧化能力比较上,蝉虫草菌丝体胞外多糖有效清除?OH自由基、ABTS自由基和DPPH自由基的EC₅₀值分别为1.06mg/mL、0.96mg/mL和0.63mg/mL,而胞内多糖的EC₅₀值分别为3.71mg/mL、2.83mg/mL和1.70mg/mL,表明蝉虫草胞外多糖的化学抗氧化能力更强;在改善细胞氧化应激损伤比较上,与模型组对比,二者均能随着浓度递增而显著地提高细胞存活率,但胞外多糖比胞内多糖更强,当多糖浓度为5mg/mL时,胞外多糖细胞存活率达到92.36%,胞内多糖只达到82.07%;在调节细胞抗氧化酶清除ROS的机制上,与模型组对比,胞外多糖分别上调SOD酶活力2.51倍和CAT酶活力2.91倍,极显著地降低了细胞ROS水平（P<0.01）来改善细胞的氧化应激损伤作用。相应地,胞内多糖只上调了1.85倍和2.33倍,显著性地清除了ROS（P<0.05）,表明蝉虫草菌丝体胞外多糖具有更显著的抗肝细胞氧化损伤作用。本研究结果显示蝉虫草菌丝体胞外和胞内多糖均具有良好的抗肝氧化损伤活性,且胞外多糖比胞内多糖活性更好,为蝉虫草菌丝体多糖在保肝产品中的开发和应用提供了科学依据。     

Effect of the H, K-ATPase inhibitor, esomeprazole magnesium, on gut total antioxidant capacity in mice

Antioxidant depletion is believed to be a mechanism involved in the pathophysiology of several upper gastrointestinal disorders, and H, K-ATPase inhibitors can alter free radical production by neutrophils. We hypothesized that the H, K-ATPase inhibitor esomeprazole magnesium would decrease gut free radical production with a concomitant increase in gut total antioxidant capacity. A/J mice (n = 10/group) received either vehicle (control) or one of three concentrations of esomeprazole magnesium in vehicle by once-daily gavage for 10 days. Using tissue extracts from stomach and colon, total antioxidant capacity, lipid peroxide levels, and constitutive Cu/Zn-superoxide dismutase were measured using validated assays. There was a dose-related increase in total antioxidant capacity (analysis of variance, P < 0.001) in stomach, but there was no change in the colon. In the assessment of free radical production, there was a trend toward decreased lipid peroxide levels in stomach from mice receiving esomeprazole. In stomach, Cu/Zn-superoxide dismutase activity was increased (ANOVA: p=.03) in mice receiving esomeprazole. In conclusion, gastric total antioxidant capacity and Cu/Zn-superoxide dismutase activity are increased by esomeprazole, and these changes may result in part from decreased free radical production. The present results support the notion that the pharmacological effects of this agent on upper intestinal tissue are more complex than previously thought, and appear to involve both enzymatic and nonenzymatic tissue antioxidants.     

Growth and quaternary alkaloid production in differentiating and non-differentiating strains of Ruta graveolens

Cultures of Ruta graveolens have been grown and maintained on Murashige and Skoog's medium supplemented with α-naphthalene acetic acid (1 mg/l.) and kinetin (0.1 mg/l.) or 2,4-dichlorophenoxyacetic acid (1 mg/l.), and glucose (30 g/l.). The production of dihydrofuroquinoline alkaloids has been investigated in these cultures. The present study showed that: (a) cultures of Ruta produced both differentiating and non-differentiating strains; (b) quaternary alkaloids (platydesminium, ribalinium and rutalinium) were present in all the strains and maximum contents were detected at 3-4 weeks growth; (c) high platydesminium and rutalinium content was associated with differentiation and callus formation, respectively; (d) changed concentrations of quaternary alkaloids were recorded in all the five strains grown on media supplemented with tryptophan and precursors (anthranilic acid, 5-methylanthranilic acid and 2,4-dihydroxyquinoline).     

Messenger RNA changes during differentiation of murine erythroleukemia cells

During differentiation of murine erythroleukemia cells, the levels of certain mRNA were observed to change. To characterize the various patterns of changes that occur during differentiation, cDNA libraries made from RNA isolated from uninduced and differentiating cells were screened with labeled cDNA or RNA labeled in vivo for different periods of time. cDNA clones that corresponded to individual mRNAs whose level remained constant, increased, or decreased during differentiation were identified. These clones were used to analyze Northern blots containing RNA from uninduced and differentiated cells. A number of characteristic changes in individual mRNAs in differentiating murine erythroleukemia cells could be identified, such as no change, increase in concentration, increase in concentration and slight change in size, decrease in concentration, decrease in concentration and change in size, appearance of new band(s) of entirely different size, and change in relative concentrations of two related mRNAs. Measurements of rates of mRNA synthesis and degradation suggest that both parameters change during differentiation and that these changes are instrumental in establishing cellular concentration of specific mRNAs. It seems that the changes in mRNA stability observed in differentiating murine erythroleukemia cells may be associated with changes in the primary structure of the transcribed portion of mRNA. The observation that specific mRNA synthesized before and after induction may have very different stabilities at the same point in differentiation supports this hypothesis.     

Possible mechanisms for antioxidant activity of glycyrrhizinic acid

Possible mechanisms of antioxidant activity of glycyrrhizinic acid (GA) were studied. GA did not exhibit antiradical properties in the experiments with stable radical 1,1-diphenyl-2-picrylhydrazyl at the concentration range of 1–100 μM. These data were confirmed by the study of GA effect on luminol chemiluminescence in a cell-free system in the presence of hydrogen peroxide. At the same time, GA decreases (in a dose-dependent manner) the generation of reactive oxygen species by neutrophils activated with both phorbol 12-myristate 13-acetate (PMA) and the chemotactic peptide, N-formyl-Met-Leu-Phe (FMLP). Using dichlorodihydrofluorescein (DCF) fluorescence it has been demonstrated that direct addition of GA to neuron culture did not decrease the level of free radical formation. However, preincubation of cells with GA resulted in the decrease in free radical production rate and increase in reduced intracellular glutathione level.     

Changes in diadenosine tetraphosphate levels in Physarum polycephalum with different oxygen concentrations.

Cellular levels of diadenosine tetraphosphate (Ap4A) were measured, by a specific high-pressure liquid chromatography method, in microplasmodia of Physarum polycephalum subjected to different degrees of hypoxia, hyperoxia, and treatment with H2O2. Ap4A levels increased three- to sevenfold under anaerobic conditions, and the microplasmodia remained viable after such treatment. Elevated levels of Ap4A returned to the basal level within 5 to 10 min upon reoxygenation of the microplasmodia. The increases in Ap4A levels were larger in stationary-phase or starved microplasmodia than in fed, log-phase microplasmodia. The maximal increase measured in log-phase microplasmodia was twofold. No significant changes in Ap4A levels occurred in microplasmodia subjected to mild hypoxia, hyperoxia, or treatment with 1 mM H2O2. These results indicate that in P. polycephalum, Ap4A may function in the metabolic response to anaerobic conditions rather than in the response to oxidative stress.