Histology and histopathology Vol.25,nº11 (2010)
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- PublicationOpen AccessHow can mammalian Rab small GTPases be comprehensively analyzed?: Development of new tools to comprehensively analyze mammalian Rabs in membrane traffic(Murcia : F. Hernández, 2010) Fukuda, MitsunoriSmall GTPase Rabs constitute the largest family of membrane trafficking proteins that are conserved in all eukaryotic cells. The number of different Rab isoforms in multicellular organisms is usually greater than that in unicellular organisms (e.g., approximately 60 different Rabs in each species of mammals investigated versus approximately 10 Rabs in yeasts). The expansion of Rab isoforms in mammals is often regarded as due to the acquisition of specialized membrane trafficking events in the specialized cell types of higher eukaryotes. However, because of their large numbers the precise function of most mammalian Rab isoforms is still unknown. The recent development of new tools for comprehensive analysis (e.g., Rab panels) has paved the way for systematic investigation of the involvement of mammalian Rab isoforms in specialized membrane trafficking events. The tools include collections of enhanced green fluorescent protein (EGFP)-tagged mouse and human Rabs, FLAG-tagged Rabs, glutathione S-transferase (GST)-tagged Rabs, Gal4-binding domain (GBD)-tagged Rabs, Tre- 2/Bub2/Cdc16 (TBC) domain-containing Rab-GTPaseactivating proteins (GAPs), and small interfering RNAs. EGFP-Rabs are used to screen for Rabs that are localized on specific organelles and regulate their transport, and GST-Rabs and GBD-Rabs are used to screen for novel Rab effectors by GST pull-down assays and yeast two-hybrid assays, respectively. Combined use of these tools now makes it possible to efficiently determine the function of mammalian Rab isoforms in membrane traffic. This article reviews the development of new tools for systematic analysis of Rab proteins and their application to Rab-mediated membrane traffic.
- PublicationOpen AccessReduced UVB-induced corneal damage caused by reactive oxygen and nitrogen species and decreased changes in corneal optics after trehalose treatment(Murcia: F. Hernández, 2010) Cejkova, Jitka; Cejka, Čestmír; Ardan, T.; Širc, Jakub; Michálek, Jiří; Luyckx, JacquesTrehalose, a nonreducing disaccharide of glucose, produced and stored in many lower and higher organisms, although not in mammals, is synthetized as a stress responsive factor when cells are exposed to various environmental stress conditions. Recently, trehalose has been implicated in various situations in mammals. The aim of this paper was to examine whether trehalose might decrease the damage of the rabbit cornea evoked by UVB rays. During irradiation with UVB rays, consisiting of a daily dose of 0.5 J/cm2 for four days, trehalose was applied in eye drops on the right eye and buffered saline on the left eye. One day after the end of irradiation the animals were sacrificed and the corneas examined spectrophotometrically for light absorption. Another group of corneas similarly treated were examined morphologically and immunohistochemically. Corneal thickness (hydration) was measured using a Pachymeter. The results show that compared to buffered saline, trehalose treated corneas displayed fewer corneal disturbances during UVB irradiation. The increases in corneal hydration and light absorption were less pronounced and intracorneal inflammation and corneal neovascularization were suppressed. Nitric oxide synthases that generate nitric oxide were less expressed in the cornea, and formation of cytotoxic peroxynitrite (demonstrated by nitrotyrosine residues) was decreased. The expression of the antioxidant aldehyde dehydrogenase3A1 was less inhibited in the corneal epithelium, and apoptotic corneal epithelial cell death (detected by immunostaining for active caspase-3) was greatly diminished. In conclusion, trehalose reduced UVB-induced damage caused by reactive oxygen and nitrogen species and decreased changes in the corneal optics.
- PublicationOpen AccessExpression of mitotic kinases phospho-aurora A and aurora B correlates with clinical and pathological parameters in bladder neoplasms(Murcia : F. Hernández, 2010) Bufo, P.; Sanguedolce, Francesca; Tortorella, Simona; Cormio, Luigi; Carrieri, Giuseppe; Pannone, G.Aurora A and Aurora B are serine-threonine kinase proteins which have both been implicated in human carcinogenesis through development of aneuploidy and chromosomal instability. The aim of the study is to assess the correlation of both markers with clinical and pathological parameters in patients with bladder cancer of different grade and stage. A bladder cancer cell line was assessed for Aurora A and Aurora B expression by Western blotting. Furthermore, 85 consecutive cases of bladder neoplasms obtained by transurethral resection were quantitatively and qualitatively analysed by immunohistochemistry for Phospho-Aurora A and Aurora B expression. All cases were stratified in 4 groups according to intracellular localization (nuclear, cytoplasmic) of both markers. The association between each group and clinical and pathological parameters was assessed by statistical analysis. Higher expression of cytoplasmic PhosphoAurora A correlated significantly with poor histological differentiation (G3 vs. G1) and advanced stage (p<0.05); there was also high significant correlation between nuclear Aurora B and both grading (both G3 and G2 vs. G1) and mitotic index (p<0.05). No statistically significant association was found between protein levels detected in tumour and sex or age (p>0.05). To our knowledge, the present study is the first to highlight the existence of a statistical association between such markers and traditional prognostic factors in bladder cancer. These findings indicate that Aurora A and B could be involved in the tumorigenesis of bladder cancer, thus providing a basis for a target therapy approach by using specific anti-mitotic agents.
- PublicationOpen AccessOrganelle dynamics and membrane trafficking in apoptosis and autophagy(Murcia : F. Hernández, 2010) Cheng, Jade P.X.; Lane, Jon D.The accurate control of cell death is a vital aspect of development in metazoans and plays crucial roles in the prevention of disease. Apoptosis is the main form of regulated cell death in multicellular organisms, although there are other contributory pathways. During apoptosis, mammalian cells undergo dramatic changes in organelle structure ad organisation that define the apoptotic execution phase. Although the roles of apoptotic protease machinery (the caspases) in these rearrangements are quite well understood, the purpose of organelle disruption during cell death is not yet entirely appreciated. Indeed, recent evidence implicates caspase targeting of organellar proteins and subsequent organelle disruption upstream of apoptotic execution proper, suggesting the existence of pathways linking organelle damage to cell death. In this review, we describe the changes to the endomembrane system that are inherent during the apoptotic execution phase, and examine the evidence for endomembrane-mediated pathways towards apoptotic execution. We also discuss aspects of the molecular control of autophagy - an important contributor to a cell’s response to stress, and a membrane trafficking process whose regulation is linked to the apoptotic machinery at multiple levels.
- PublicationOpen AccessDistribution of CD105 and CD166 positive cells in the proximal epiphysis of developing rat humerus(Murcia : F. Hernández, 2010) Ozbey, Ozlem; Sahin, Zeliha; Acar, Nuray; Ustunel, IsmailThe expression of cell surface receptors, CD105 and CD166, are characteristic of mesenchymal stem cells in cartilage. However, there is limited data regarding their immunolocalization in the cartilage of developing rat epiphysis. The purpose of this study was to determine the presence of CD105 and CD 166 positive cells in the proximal epiphysis of developing rat humerus and specify their zonal distribution with age. The tissues of rat humerus were taken on embryonic day 15 (E15), embryonic day 19 (E19), postnatal day 10 (PN10), postnatal day 20 (PN20) and adult rats and studied for the immunolocalization of CD105 and CD166. Our results showed that CD105 and CD166 positive cells were scattered in early stages of development of humerus epiphysis. For E15, only the hypertrophic zone was positive, whereas for E19 almost all zones of the epiphysis were positively stained for these markers. For PN10 and PN20, the CD105 and CD166 positive cells were mainly localized on the surface of the articular cartilage. In adult articular cartilage the CD105 and CD166 positive cells were localized in the superficial and transitional zones and in the upper regions of the deep zone. Our study provides evidence that in the developing cartilage tissue the localization of CD105 and CD166 positive cells is both dynamic and stage dependent, which may imply the existence of stem cell-like cells in cartilage from an early age to adult.