Autolysis and ageing of Penicillium chrysogenum under carbon starvation: respiration and glucose oxidase production

During the exponential growth phase of Penicillium chrysogenum NCAIM 00237 the effective conversion of glucose and O2 to gluconate and H2O2 by glucose oxidase (GOX) was the most likely source of intracellular ROS measured. In glucose-supplemented autolysing cultures, the increased of intracellular ROS concentration was attributed to respiration in the absence of any significant GOX activity. The induction of GOX and catalase by glucose and H2O2 was clearly age-dependent in P. chrysogenum. In ageing cryptic growth phase cultures, superoxide dismutase and cyanide-resistant respiration were the major elements of antioxidative defence but these activities were insufficient to prevent the progressive accumulation of ROS and the concomitant decrease in cell vitality.     

Hematological indices and oxidative stress biomarkers response to the starvation of Clarias gariepinus

Starvation effects for five weeks on energy reserves, oxidative stress and hematological indices in Nile catfish Clarias gariepinus was studied. The low protein level in starved fish may result from the lowering effect of prolonged starvation on protein synthesis rather than due to its degenerating protein. Moreover, the elevated level of serum amino acids may promote gluconeogenesis in liver. In addition, the lipid depletion in starved fish may be related to the preferential uses of lipids as an energy to starve fish. Also, unchanged glycemic level may introduce a potent evidence for the presence of active gluconeogenesis, depending on both amino and fatty acids precursors. Also, kidney and liver showed disturbances in metabolites associated with oxidative damage such as elevations in total peroxide, carbonyl protein and DNA fragmentation; these may cause dysfunction to these organs after five weeks of starvation. Total peroxide, carbonyl protein and DNA fragmentation were significantly increased in gills, liver and kidney by 29.9, 30.9 and 30.5; 83.6, 84.6 and 53.7; 82.4, 43.3 and 75.7%, respectively. Starvation induced severe anemia and loss of body weight in the fish. However, white muscle did not show any oxidative damage after five weeks of starvation.     

Autolysis and aging of Penicillium chrysogenum cultures under carbon starvation: Chitinase production and antifungal effect of allosamidin

In carbon-depleted cultures of Penicillium chrysogenum, age-related chitinases were shown to play a crucial role in both autolysis and fragmentation as indicated by in vivo enzyme inhibition experiments using allosamidin. This pseudotrisaccharide even hindered significantly the outgrowth of new hyphal tips from the surviving yeastlike fragments after glucose supplementation. The antifungal effect of allosamidin on autolyzing P. chrysogenum mycelia was fungistatic rather than fungicidal. In growing hyphae, membrane-bound microsomal chitinase zymogen(s) were detected, which may be indicative of some compartmentalization of these hydrolases. Later, during autolysis, no zymogenic chitinase was detected in any enzyme fraction studied, including microsomes. These observations may explain the different sensitivity of growing and autolyzing mycelia to allosamidin. Chitinases taking part in the age-related fragmentation of hyphae and the outgrowth of surviving hyphal fragments seem to be potent targets for future antifungal drug research.     

一个新的产黄青霉谷胱甘肽转移酶基因的克隆和鉴定

以产黄青霉(Penicillium chrysogenum Thom)cDNA为模板,克隆得到一个新的谷胱甘肽转移酶基因PcgstB,其开放阅读框长651bp,编码216个氨基酸的蛋白质。与已知序列进行BLASTp比较显示,该蛋白具有保守的GST结构域,与烟曲霉GstB的序列一致性最高,达65%。将PcgstB与原核表达载体pTrc99A连接得到表达质粒pTrc-gstB,转化大肠杆菌DH5α,经IPTG诱导后获得以可溶形式表达的重组PcGstB蛋白。以1-chloro-2,4-dinitrobenzene(CDNB)为底物检测,确认该蛋白具有GST活性。     

Intrachromosomal recombination between direct repeats in Penicillium chrysogenum : gene conversion and deletion events

Recombination between direct repeats has been studied in Penicillium chrysogenum using strain TD7-88 (lys⁻ pyr⁺), which contains two inactive copies of the lys2 gene separated by 4.5 kb of DNA (including the pyrG gene) in its genome. Gene conversion leading to products with the lys⁺ pyr⁺ phenotype was observed at a frequency of 1 in 3.2 × 10³ viable spores. Two types of deletion events giving rise to lys⁺ pyr⁻ and lys⁻ pyr⁻ phenotypes were obtained with different frequencies. Southern analysis revealed that gene conversion occurs mainly as a result of crossing over events that remove the BamHI frameshift mutation present in one of the repeats. In lys⁻ pyr⁻ recombinants, the deletion events do not affect the frameshift mutation in the BamHI site, while lys⁺ pyr⁻ recombinants showed repair of the BamHI frameshift mutation and the genotype of the parental non-disrupted strain was restored. In summary, deletion events in P. chrysogenum tend to favor the restoration of the phenotype and genotype characteristic of the parental non-disrupted strain. Received: 9 November 1998 / Accepted: 14 April 1999     

Cell death and reactive oxygen species metabolism during accelerated ageing of soybean axes

In this paper, Glycine max L. seeds under accelerated ageing condition (40°C and 100% relative humidity) were used as experimental material to study the relationships between seed viability and cell death, production and scavenging of reactive oxygen species (ROS) during accelerated ageing. Water content of seeds gradually increased, while the final germination percentage, germination rate of seeds and fresh weight of seedlings produced decreased with increasing accelerated ageing time. The accelerated ageing time (T ₅₀) when final seed germination decreased to 50% was about 10.5 days. During the period of accelerated ageing, the viability of root cells was lost gradually as manifested by the increase in staining with Evans blue. The respiration rate of seeds, ^·O₂ production rate, and H₂O₂ content of axes increased, peaked at the 10 days of accelerated ageing, and then decreased. Activities of superoxide dismutase, ascorbate peroxidase, catalase, and glutathione reductase of axes decreased; and malondialdehyde contents of axes markedly increased. A sceme to explain relationships between seed vigor, cell death, and production and scavenging of ROS during accelerated ageing was suggested. This text was submitted by the authors in English.