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 AccessGli-similar (Glis) Krüppel-like zinc finger proteins: insights into their physiological functions and critical roles in neonatal diabetes and cystic renal disease(Murcia : F. Hernández, 2010) Hong Soon, Kang; ZeRuth, Gary; Lichti-Kaiser, Kristin; Vasanth, Shivakumar; Yin, Zhengyu; Kim, Yong-Sik; Jetten, Anton M.GLI-similar (Glis) 1-3 proteins constitute a subfamily of the Krüppel-like zinc finger transcription factors that are closely related to the Gli family. Glis1-3 play critical roles in the regulation of a number of physiological processes and have been implicated in several pathologies. Mutations in GLIS2 have been linked to nephronophthisis, an autosomal recessive cystic kidney disease. Loss of Glis2 function leads to renal atrophy and fibrosis that involves epithelialmesenchymal transition (EMT) of renal tubule epithelial cells. Mutations in human GLIS3 have been implicated in a syndrome characterized by neonatal diabetes and congenital hypothyroidism (NDH) and in some patients accompanied by polycystic kidney disease, glaucoma, and liver fibrosis. In addition, the GLIS3 gene has been identified as a susceptibility locus for the risk of type 1 and 2 diabetes. Glis3 plays a key role in pancreatic development, particularly in the generation of ß-cells and in the regulation of insulin gene expression. Glis2 and Glis3 proteins have been demonstrated to localize to the primary cilium, a signaling organelle that has been implicated in several pathologies, including cystic renal diseases. This association suggests that Glis2/3 are part of primary cilium-associated signaling pathways that control the activity of Glis proteins. Upon activation in the primary cilium, Glis proteins may translocate to the nucleus where they subsequently regulate gene transcription by interacting with Glis-binding sites in the promoter regulatory region of target genes. In this review, we discuss the current knowledge of the Glis signaling pathways, their physiological functions, and their involvement in several human pathologies.
- 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 AccessRenal expression of organic anion transporter Oat5 in rats and mice exhibits the female-dominant sex differences(Murcia : F. Hernández, 2010) Breljak, Davorka; Ljubojević, Marija; Balen, Daniela; Žlender, Vilim; Brzica, Hrvoje; Micek, Vedran; Kušan, Marija; Anzai, Naohiko; Sabolić, IvánThe organic anion transporter 5 (Oat5, Slc22a19) was previously localized to the brush-border of proximal tubule (PT) S3 segment in rat and mouse kidneys. Here we report on sex hormone-regulated expression of Oat5 in rat kidneys, after reinvestigating: a) expression of its mRNA by end-point and real time RT-PCR in the tissue, b) abundance of its protein by Western blotting (WB) in isolated membranes, and c) immunolocalization in tissue cryosections. In untreated male (M) and female (F) adult rats, the expression of Oat5 mRNA was predominant in the outer stripe (OS), exhibiting sex differences (M
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