Protein kinase C modulation in apoptotic rat thymocytes: an ultrastructural analysis

Numerous events in the cell, such as gene expression, cell growth and metabolism are regulated by signal transduction pathways involving protein kinase C (PKC). Recent data indicate that a PKC-dependent mechanism also underlies the apoptotic death of cells induced by glucocorticoid hormones. In this report we have analysed the changes of PKC during dexamethasone-induced apoptosis in thymocytes by means of immunocytochemical and immunochemical analysis. The data obtained show an increase and intracellular movement of protein kinase C, which is translocated to the nucleus and linked to the nuclear matrix during the apoptotic process.     

2-D DIGE analysis of UV-C radiation-responsive proteins in globe artichoke leaves

Plants respond to ultraviolet stress inducing a self-defence through the regulation of specific gene family members. The UV acclimation is the result of biochemical and physiological processes, such as enhancement of the antioxidant enzymatic system and accumulation of UV-absorbing phenolic compounds (e.g. flavonoids). Globe artichoke is an attractive species for studying the protein network involved in UV stress response, being characterized by remarkable levels of inducible antioxidants. Proteomic tools can assist the evaluation of the expression patterns of UV-responsive proteins and we applied the difference in-gel electrophoresis (DIGE) technology for monitoring the globe artichoke proteome variation at four time points following an acute UV-C exposure. A total of 145 UV-C-modulated proteins were observed and 119 were identified by LC-MS/MS using a ～144,000 customized Compositae protein database, which included about 19,000 globe artichoke unigenes. Proteins were Gene Ontology (GO) categorized, visualized on their pathways and their behaviour was discussed. A predicted protein interaction network was produced and highly connected hub-like proteins were highlighted. Most of the proteins differentially modulated were chloroplast located, involved in photosynthesis, sugar metabolisms, protein folding and abiotic stress. The identification of UV-C-responsive proteins may contribute to shed light on the molecular mechanisms underlying plant responses to UV stress.     

Signaling from Ras to Rac and beyond: not just a matter of GEFs

Members of a family of intracellular molecular switches, the small GTPases, sense modifications of the extracellular environment and transduce them into a variety of homeostatic signals. Among small GTPases, Ras and the Rho family of proteins hierarchically and/or coordinately regulate signaling pathways leading to phenotypes as important as proliferation, differentiation and apoptosis. Ras and Rho-GTPases are organized in a complex network of functional interactions, whose molecular mechanisms are being elucidated. Starting from the simple concept of linear cascades of events (GTPase-->activator--> GTPase), the work of several laboratories is uncovering an increasingly complex scenario in which upstream regulators of GTPases also function as downstream effectors and influence the precise biological outcome. Furthermore, small GTPases assemble into macromolecular machineries that include upstream activators, downstream effectors, regulators and perhaps even final biochemical targets. We are starting to understand how these macromolecular complexes work and how they are regulated and targeted to their proper subcellular localization. Ultimately, the acquisition of a cogent picture of the various levels of integration and regulation in small GTPase-mediated signaling should define the physiology of early signal transduction events and the pathological implication of its subversion.     

Inhibition of adenylate cyclase by transglutaminase-catalyzed reactions in pigeon erythrocyte ghosts

We report the occurrence in pigeon erythrocytes of a soluble Ca2+-dependent transglutaminase (TGase) activity. The effect of the erythrocyte ghost protein modifications, determined by TGase-catalyzed reactions, on adenylate cyclase, phospholipid methyltransferase I and II activities and on the lipidic matrix fluidity of the membrane was investigated by using a purified guinea pig liver TGase preparation. The results showed a significant inhibitory effect of such modifications both on the basal and on the variously stimulated (by NaF, Gpp(NH)p alone or in the presence of 1-isoproterenol) adenylate cyclase activity. By contrast, both the phospholipid methylation and the fluidity of the lipidic matrix of the membrane were unaffected by TGase-mediated reactions. These data suggest a new possible inhibitory mechanism of the cyclic AMP synthesis which might be triggered by the enhancement of the cytosolic Ca2+ concentration.     

重寄生真菌盾壳霉pH信号通路中Pal相关基因的功能鉴定

【目的】研究pH信号通路(Pal)在重寄生真菌盾壳霉与寄主核盘菌互作过程中的作用。【方法】从盾壳霉全基因组信息中分析获得了6个Pal相关基因CmpalA、CmpalB、CmpalC、CmpalF、CmpalH和CmpalI的全编码序列和氨基酸序列,通过PEG介导的原生质转化技术获得了CmpalA、CmpalB、CmpalC、CmpalF和CmpalH等5个基因的敲除突变体,分析这些敲除突变体与野生型在菌落培养性状、重寄生能力、降解草酸能力、产生抗真菌物质能力等方面的差异。【结果】与野生型相比,在pH 6–8的条件下,5个Pal相关基因敲除突变体的菌丝生长受到显著抑制,这说明缺失Pal相关基因使盾壳霉对高pH值环境更加敏感。菌核重寄生试验发现5个Pal相关基因敲除突变体的重寄生能力均显著低于野生型。qRT-PCR试验结果表明,敲除Pal相关基因之后导致重寄生相关酶基因Cmch1、Cmg1和Cmsp1的表达量显著降低,而且pH信号通路下游的CmpacC基因的表达量也显著降低。Pal相关基因敲除突变体在pH 6条件下对草酸盐的降解能力显著高于野生型,同时这5个突变体在pH 8条件下产生抗真菌物质能力也显著高于野生型。【结论】pH信号通路相关基因的缺失影响盾壳霉对环境pH的响应。pH信号通路在盾壳霉与核盘菌互作中发挥重要作用,不仅影响盾壳霉的重寄生作用,而且还影响盾壳霉的草酸降解作用和抗真菌作用。     

Effects of Triton X-100 and Quillaya Saponin on the ex situ bioremediation of a chronically polychlorobiphenyl-contaminated soil

The possibility of enhancing the ex situ bioremediation of a chronically polychlorinated biphenyl (PCB)-contaminated soil by using Triton X-100 or Quillaya Saponin, a synthetic and a biogenic surfactant, respectively, was studied. The soil, which contained about 350 mg/kg of PCBs and indigenous aerobic bacteria capable of growing on biphenyl or on monochlorobenzoic acids, was amended with inorganic nutrients and biphenyl, saturated with water and treated in aerobic batch slurry- and fixed-phase reactors. Triton X-100 and Quillaya Saponin were added to the reactors at a final concentration of 10 g/l at the 42nd day of treatment, and at the 43rd and 100th day, respectively. Triton X-100 was not metabolised by the soil microflora and it exerted inhibitory effects on the indigenous bacteria. Quillaya Saponin, on the contrary, was readily metabolised by the soil microflora. Under slurry-phase conditions, Triton X-100 negatively influenced the soil bioremediation process by affecting the availability of the chlorobenzoic acid degrading indigenous bacteria, whereas Quillaya Saponin slightly enhanced the biological degradation and dechlorination of the soil PCBs. In the fixed-phase reactors, where both the surfactant availability and the mixing of the soil were lower, Triton X-100 did not exert inhibitory effects on the soil biomass and enhanced significantly the soil PCB depletion, whereas Quillaya Saponin did not influence the bioremediation process. Received: 28 April 1998 / Received last revision: 15 July 1998 / Accepted: 29 July 1998     

Estimated comparative integration hotspots identify different behaviors of retroviral gene transfer vectors

Integration of retroviral vectors in the human genome follows non random patterns that favor insertional deregulation of gene expression and may cause risks of insertional mutagenesis when used in clinical gene therapy. Understanding how viral vectors integrate into the human genome is a key issue in predicting these risks. We provide a new statistical method to compare retroviral integration patterns. We identified the positions where vectors derived from the Human Immunodeficiency Virus (HIV) and the Moloney Murine Leukemia Virus (MLV) show different integration behaviors in human hematopoietic progenitor cells. Non-parametric density estimation was used to identify candidate comparative hotspots, which were then tested and ranked. We found 100 significative comparative hotspots, distributed throughout the chromosomes. HIV hotspots were wider and contained more genes than MLV ones. A Gene Ontology analysis of HIV targets showed enrichment of genes involved in antigen processing and presentation, reflecting the high HIV integration frequency observed at the MHC locus on chromosome 6. Four histone modifications/variants had a different mean density in comparative hotspots (H2AZ, H3K4me1, H3K4me3, H3K9me1), while gene expression within the comparative hotspots did not differ from background. These findings suggest the existence of epigenetic or nuclear three-dimensional topology contexts guiding retroviral integration to specific chromosome areas.