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 AccessImmunohistochemical localization of galectins-1 and -3 and monitoring of tissue galectin-binding sites during tubular regeneration after renal ischemia reperfusion in the rat(2010) Vansthertem, David; Cludts, Stéphanie; Nonclercq, Denis; Gossiaux, Annabel; Saussez, Sven; Legrand, Alexandre; Gabius, H.J.; Toubeau, GérardEndogenous lectins act as effectors of cellular activities such as growth regulation, migration and adhesion. In this study, we report the histochemical detection of galectins and their binding sites in rat kidneys after ischemic injury (35 min) with regard to renal regeneration. In this context, we have shown in a previous publication (Vansthertem et al., 2008) that extrarenal cells (CD44+, vimentin +) could be involved in this process of tubular restoration. In controls, galectin-1 is expressed by fusiform-shaped cells within cortical and medullar interstitium. Two days after ischemia, the number of positive interstitial cells increased temporarily within OSOM in the vicinity of altered tubules to later reach control level. After ischemia, we identified a population of galectin-3 (+), CD44 (+), and vimentin (+) interstitial round cells located in the outer stripe of outer medulla (OSOM) in the vicinity of necrotic tubules, but also in the lumen of adjacent blood vessels. The immunocytochemical characteristics of theses cells, along with their distribution within OSOM, suggest the involvement of a unique cell population during kidney regeneration. On the other hand, the distribution and density of binding sites for galectins within OSOM were not modified after ischemia and remained similar to controls. Altogether, our observations suggest that galectin-3 may be involved in the complex process of kidney regeneration following ischemia/reperfusion injury.
- 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.
- 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
- 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 AccessThe metastasis-associated gene MTA3 is downregulated in advanced endometrioid adenocarcinomas(Murcia : F. Hernández, 2010) Brüning, Ansgar; Jückstock, Julia; Blankenstein, Thomas; Makovitzky, Josef; Kunze, Sussane; Mylonas, I.The metastasis-associated gene MTA3 has an important function in invasion and metastasis of human cancer cells. Therefore, the aim of this study was to investigate the expression of this protein in endometrial adenocarcinomas and to analyse potential correlations between this nuclear transcription factor and estrogen receptors in endometrial adenocarcinomas. Additionally, we evaluated whether MTA3 might be a prognostic parameter in endometrioid adenocarcinomas. Endometrioid adenocarcinomas were obtained from 200 patients and immunohistochemically analysed for MTA3 and estrogen receptor alpha and beta (ER-alpha and ERbeta) expression. Overall, endometrioid adenocarcinomas of histological differentiation grade 3 demonstrated a significantly lower expression of MTA3 compared to carcinomas of histological grade 1 and 2 (p<0.05). MTA3 expression is reduced in endometrioid adenocarcinomas of poor differentiation, though without any correlation to ER-alpha and ER-beta expression. Furthermore, the expression of MTA3 did not affect progression-free, cause-specific and overall survival. Overall, MTA3 did not constitute an independent prognostic factor in this study, suggesting that MTA3 is not a useful marker to assess and identify high-risk patients with endometrial adenocarcinomas. Still, the downregulation of MTA3 predispose this cell type to be of high metastatic potential after malignant transformation, playing an essential, but as yet unknown role in human endometrial carcinogenesis.
- PublicationOpen AccessWhen one plus one equals more than two - a novel stain for renal biopsies is a combination of two classical stains(Murcia : F. Hernández, 2010) Brodsky, Sergey V.; Albaward, Alia; Satoskar, Anjali A.; Nadasdy, Gyongyi; Nadasdy, TiborHistologic evaluation of renal biopsies includes multiple ancillary stains, including Periodic acid-Schiff ’s (PAS) and Masson’s trichrome (Trichrome). Herein we report an innovative doublestain, derived from two standard stains (PAS and Trichrome). This novel stain not only has advantages of both ancestor stains, but became more distinguishable and colorful, when basement membranes stain darkviolet, whereas the interstitial collagen remains blue. This allows the pathologist immediate estimation of the amount of collagen, tubular atrophy and the degree of interstitial fibrosis in one section. Using computer-based analysis, we confirmed that our innovative double-stain highlights interstitial collagen better than Trichrome stain alone. We strongly recommend renal pathologists to try this innovative stain in their practice.
- PublicationOpen AccessHistopathological and biochemical changes in rat thyroid following acute exposure to hexavalent chromium(Murcia : F. Hernández, 2010) Mahmood, Tariq; Zia Quresh, Irfan; Javed Iqbal, MuhammadChromium in hexavalent form is highly toxic and a known carcinogen, although its effects on thyroid structure and function are relatively unexplored. Workers in an industrial environment can be, at times, exposed to this form of chromium. The present study was, therefore, designed using laboratory rats as a model system to investigate the effect on thyroid structure and function following two acute intraperitoneal doses of 30 mg/kg b.w. potassium dichromate administered within 48 hours. The results showed that hypothalamic chromium concentration increased (p<0.05) while thyroid chromium concentration decreased (p<0.01).The excretion of chromium in urine increased (p<0.05). The treated thyroid sections revealed hyperplasia. Follicles were disorganized, clustered and collapsed, while some of them were fused. Interfollicular spaces widened. Morphometrical analysis showed significantly (p<0.001) increased number of follicles whereas the follicular size significantly decreased (p<0.001). Nuclei were regressed (p<0.001); nuclear shapes were irregular; round, oval and shrunken. The membrane on the apical as well as the basal lamina side showed disruption. Colloid retraction within the follicles was noticeable in some sections stained with Periodic acid Schiff (PAS). Serum free tetra-iodothyronine (FT4) and free tri-iodothyronine (FT3) levels decreased (p<0.01 and p<0.001, respectively), while serum thyroid stimulating hormone (TSH) concentration increased (p<0.01). Ultrastructural analysis showed disrupted basal laminae of the follicles, regressed nuclei and disrupted cell organelles. Acridine orange stained thyroid cells demonstrated excessive dead cells, whereas DNA fragmentation assay demonstrated percent decrease of hypothalamic, pituitary and thyroidal total DNA.
- PublicationOpen AccessThe relationship between pregnancy-associated plasma protein-A (PAPP-A) and human intervertebral disc degeneration(Murcia : F. Hernández, 2010) Gruber, H.E.; Buchanan, Laura; Ingram, Jane A.; Zinchenko, Natalia; Norton, H. James; Hanley Jr, Edward N.Pregnancy-associated plasma protein-A (PAPP-A), a metalloproteinase expressed by a number of cell types, has the important role of cleaving insulin-like growth factor (IGF)-binding protein-2, -4 and -5 in the extracellular matrix and thus freeing up IGF and making it available to cells. The objective of the present study was to utilize immunocytochemical analysis to determine the proportion of PAPP-A-positive cells in a large group of disc specimens which covered the spectrum of changes from relatively healthy Thompson grade II discs to extremely degenerate grade V discs. Work was approved by our institutional human subjects review board. Seventy-two intervertebral disc specimens were assessed for immunocytochemical localization of PAPP-A and the proportion of positive cells determined in the outer annulus, inner annulus and nucleus pulposus. The percentage of PAPP-A positive cells in both the outer and inner annulus correlated significantly with increasing stages of disc degeneration in a fashion which was not dependent upon subject age. There was no significant difference in the percentage of PAPP-A positive cells in the inner annulus of herniated vs nonherniated sites, or in the outer annulus of herniated vs non-herniated sites. Data reported here point to the importance of additional work to elucidate the role of PAPP-A in intervertebral disc aging and degeneration.