DNA damage is an early event in doxorubicin-induced cardiac myocyte death

Anthracyclines are antitumor agents the main clinical limitation of which is cardiac toxicity. The mechanism of this cardiotoxicity is thought to be related to generation of oxidative stress, causing lethal injury to cardiac myocytes. Although protein and lipid oxidation have been documented in anthracycline-treated cardiac myocytes, DNA damage has not been directly demonstrated. This study was undertaken to determine whether anthracyclines induce cardiac myocyte DNA damage and whether this damage is linked to a signaling pathway culminating in cell death. H9c2 cardiac myocytes were treated with the anthracycline doxorubicin at clinically relevant concentrations, and DNA damage was assessed using the alkaline comet assay. Doxorubicin induced DNA damage, as shown by a significant increase in the mean tail moment above control, an effect ameliorated by inclusion of a free radical scavenger. Repair of DNA damage was incomplete after doxorubicin treatment in contrast to the complete repair observed in H2O2-treated myocytes after removal of the agent. Immunoblot analysis revealed that p53 activation occurred subsequent in time to DNA damage. By a fluorescent assay, doxorubicin induced loss of mitochondrial membrane potential after p53 activation. Chemical inhibition of p53 prevented doxorubicin-induced cell death and loss of mitochondrial membrane potential without preventing DNA damage, indicating that DNA damage was proximal in the events leading from doxorubicin treatment to cardiac myocyte death. Specific doxorubicin-induced DNA lesions included oxidized pyrimidines and 8-hydroxyguanine. DNA damage therefore appears to play an important early role in anthracycline-induced lethal cardiac myocyte injury through a pathway involving p53 and the mitochondria.     

Massive telomere loss is an early event of DNA damage-induced apoptosis

Chromosomal stability and cell viability require a proficient telomeric end-capping function. In particular, telomere dysfunction because of either critical telomere shortening or because of mutation of telomere-binding proteins results in increased apoptosis and/or cell arrest. Here, we show that, in turn, DNA damage-induced apoptosis results in a dramatic telomere loss. In particular, using flow cytometry for simultaneous detection of telomere length and apoptosis, we show that cells undergoing apoptosis upon DNA damage also exhibit a rapid and dramatic loss of telomeric sequences. This telomere loss occurs at early stages of apoptosis, because it does not require caspase-3 activation, and it is induced by loss of the mitochondrial membrane potential (Deltapsi(m)) and production of reactive oxygen species. These observations suggest a direct effect of mitochondrial dysfunction on telomeres.     

Calnexin overexpression increases manganese peroxidase production in Aspergillus niger

Heme-containing peroxidases from white rot basidiomycetes, in contrast to most proteins of fungal origin, are poorly produced in industrial filamentous fungal strains. Factors limiting peroxidase production are believed to operate at the posttranslational level. In particular, insufficient availability of the prosthetic group which is required for peroxidase biosynthesis has been proposed to be an important bottleneck. In this work, we analyzed the role of two components of the secretion pathway, the chaperones calnexin and binding protein (BiP), in the production of a fungal peroxidase. Expression of the Phanerochaete chrysosporium manganese peroxidase (MnP) in Aspergillus niger resulted in an increase in the expression level of the clxA and bipA genes. In a heme-supplemented medium, where MnP was shown to be overproduced to higher levels, induction of clxA and bipA was also higher. Overexpression of these two chaperones in an MnP-producing strain was analyzed for its effect on MnP production. Whereas bipA overexpression seriously reduced MnP production, overexpression of calnexin resulted in a four- to fivefold increase in the extracellular MnP levels. However, when additional heme was provided in the culture medium, calnexin overexpression had no synergistic effect on MnP production. The possible function of these two chaperones in MnP maturation and production is discussed.     

Surface thermodynamics of pellet formation in Aspergillus niger

The fungal pellets were formed from the surface thermodynamic balance between fungal cell and liquid media. The Gibbs free energy of pellet formation of the initial culture media (–73 –81 ergs/cm2) were increased to –13 –46 ergs/cm2 at 48 h. FTIR analysis showed that factors inducing pellet formation simultaneously increased the cell wall hydrophobicity of Aspergillus niger.     

Fungal fractal morphology of pellet formation in Aspergillus niger

Mycelial fractal values were compared to the conventional fungal morphological parameters: average total mycelial length, average number of tips and average growth unit. The fractal values were between 1.47 to 1.3 for the various submerged culture conditions of Aspergillus niger. The average pellet diameter was 1.4 mm at the fractal value of 1.47. The mycelia with fractal values close to 1 were less branched and slim.     

4种黑曲霉基因组DNA提取方法的比较

目的:筛选适合提取曲霉DNA的方法.方法:比较2个菌落培养时间段(3d内和10d左右)提取DNA质量的差异;运用氯化苄法、石英砂+CTAB法、Biospin法和微波法分别提取黑曲霉基因组DNA,然后用直接电泳、浓度测定、PCR扩增等方法比较所提DNA的浓度和质量.结果:培养3d内的菌落提取的DNA纯度较高,无需纯化即可用于后续实验;4种方法制备的DNA均可用于PCR等后续实验,其中以石英砂+CTAB法提取的DNA纯度好,产率最高.结论石英砂+CTAB法是一种适用于曲霉DNA提取的简便方法.     

Effect of maltose on glucoamylase formation by Aspergillus niger

Low levels of glucoamylase are produced when Aspergillus niger is grown on sorbitol, but substitution of the latter by glucose, maltose, or starch results in greater formation of glucoamylase as measured by enzymatic activity. Both glucoamylase I and glucoamylase II are formed in a yeast extract medium; however, glucoamylase I appears to be the only form produced when ammonium chloride is the nitrogen source. Maltose or isomaltose (1.4 x 10(-4)m), but no other disaccharides or monosaccharides, dextrins, dextrans, or starches, stimulated glucoamylase formation when added to mycelia pregrown on sorbitol-ammonium salts. The induction of glucoamylase by maltose was independent of sulfate concentration but showed a dependency on low pH and the absence of utilizable carbon sources.     

Colonization,penetration and transformation of manganese oxide nodules by Aspergillus niger

In this study, the ability of the geoactive fungus Aspergillus niger to colonize and transform manganese nodules from the Clarion-Clipperton Zone in both solid and liquid media was investigated. Aspergillus niger was able to colonize and penetrate manganese nodules embedded in solid medium and effect extensive transformation of the mineral in both fragmented and powder forms, precipitating manganese and calcium oxalates. Transformation of manganese nodule powder also occurred in a liquid medium in which A. niger was able to remove the fine particles from suspension which were accumulated within the central region of the resulting mycelial pellets and transformed into manganese oxalate dihydrate (lindbergite) and calcium oxalate dihydrate (weddellite). These findings contribute to an understanding of environmental processes involving insoluble manganese oxides, with practical relevance to chemoorganotrophic mineral bioprocessing applications, and, to the best of our knowledge, represent the first demonstration of fundamental direct and indirect interactions between geoactive fungi and manganese nodules.     

Physical mapping of the mitochondrial DNA from Aspergillus niger

Abstract The mitochondrial DNA was isolated from Aspergillus niger WU-2223L, a citric acid-production strain, and characterized by restriction-endonuclease mapping. Cloned fragments which covered the total range of the mitochondrial DNA were assembled and utilized to construct the restriction-endonuclease map for nine restriction enzymes. This map showed that the mitochondrial DNA was a circular molecule of 32.6 kb.     

Promyelocytic leukemia-nuclear body formation is an early event leading to retinoic acid-induced differentiation of neuroblastoma cells

Neuroblastoma is one of the most common cancers in children. Neuroblastoma differentiation is linked to the presence of the promyelocytic leukemia (PML) protein. Retinoic acid, a powerful differentiation-inducer in vitro , is a potent agent for the treatment of neuroblastoma. Using two different human neuroblastoma cell lines, SH-SY5Y and LA-N-5, we show here that PML protein leads to the formation of nuclear bodies (PML-NB) after only 1 h of retinoic acid treatment and that this formation is mediated by the extracellular signal-regulated kinase (ERK) pathway. Inhibition of protein kinase C also leads to formation of PML-NB via the ERK pathway. Both sumoylation and phosphorylation of PML in an ERK-dependent pathway are also required for formation of PML-NB. Finally, we show that PML-NB formation in neuroblastoma cells is associated with neurite outgrowth. These results support the proposal that the formation of PML-NB is correlated with the differentiation of neuroblastoma cells.     

D-Galactose uptake is nonfunctional in the conidiospores of Aspergillus niger

The majority of black Aspergilli (Aspergillus section Nigri), including Aspergillus niger, as well as many other Ascomycetes fail to germinate on d-galactose as a sole carbon source. Here, we provide evidence that the ability of A. niger to transport D-galactose is growth stage dependent, being absent in the conidiospores but present in the mycelia. Despite earlier claims, we could identify galactokinase activity in growing cells and all genes of the Leloir pathway (responsible for channelling D-galactose into the EMP pathway) are well induced on D-galactose (and also on lactose, D-xylose and L-arabinose) in the mycelial stage. Expression of all Leloir pathway genes was also detectable in conidiospores, although galE (encoding a galactokinase) and galD (encoding a galactose-1-phosphate uridylyl transferase) were expressed poorly. These results suggest that the D-galactose-negative phenotype of A. niger conidiospores may be due to the lack of inducer uptake.     

Rejoining of DNA strand breaks is an early nuclear event during the stimulation of quiescent lymphocytes

Activation of quiescent human peripheral blood lymphocytes or purified T cells by the mitogen, phytohemagglutinin (PHA), involves a rapid rejoining of DNA breaks present in the resting cells as detected by both nucleoid sedimentation analysis and rate of strand unwinding in alkali. Inhibitors of the enzyme ADP-ribosyltransferase (ADPRT) prevent activation of peripheral lymphocytes or T cells by PHA or concanavalin A in a dose-dependent manner, but only if present during the early stages. They do not affect subsequent proliferation if added later, nor do they inhibit the growth of lymphoblastoid cell lines. The inhibitors slow the rejoining of DNA breaks but do not affect the binding of mitogen to the cell surface or the early PHA-stimulated turnover of plasma membrane inositol phospholipids. DNA breaking and rejoining, regulated by ADPRT, may be involved in controlling gene expression during differentiation.     

Effect of manganese deficiency on plasma-membrane lipid composition and glucose uptake in Aspergillus niger

Abstract Plasma membranes were isolated by means of the concanavalin A technique from protoplasts of manganese deficient (< 10⁻⁸ M Mn²⁺) and sufficient (10⁻⁵ M Mn²⁺) grown mycelium. The membranes differed with respect to their quantitative contents of fatty acids, sterols and phospholipids. These changes did not influence the glucose transport system, as shown by kinetic investigations using intact mycelia.     

Metabolic effects of manganese deficiency in Aspergillus niger: evidence for increased protein degradation

The effect of manganese deficiency on macromolecule synthesis has been studied in a citric acid producing strain of Aspergillus niger: pulse labelling experiments showed that the synthesis of both protein and RNA was not influenced by the presence of manganese; however, increased protein degradation occurred under manganese deficiency. This was also reflected by the increased activity of an intracellular proteinase activity under these conditions. In replacement cultures addition of inhibitors of RNA, DNA or protein synthesis revealed that only emetine and cycloheximide (which both act at the ribosome) successfully antagonized the adverse effect of manganese ions on citric acid accumulation. Manganese deficiency was also characterized by a decreased portion of polysomes and 80 S ribosomes.