Histology and histopathology Vol.24, nº9 (2009)
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- PublicationOpen AccessMethylation of histone H3 lysine 27 associated with apoptosis in osteosarcoma cells induced by staurosporine(Murcia : F. Hernández, 2009) Cheng, Ming-Fang; Lee, Chian-Her; Hsia, Kan-Tai; Huang, , Guo-Shu; Lee, Herng-ShengThe relationship between histone methylation and apoptosis, programmed cell death, is beginning to be explored. The objective of this study was to investigate the effects of staurosporine, a PKC inhibitor on the methylation of histone H3 in osteosarcoma cells. Following stimulation by staurosporine in vitro of G292 cells, a human osteosarcoma cell line with fibroblast-like phenotype, methylation of histone H3 was evaluated by western blotting and immunocytochemistry. G292 cells revealed the expression of cleaved PARP after incubation with staurosporine for 3 hours. Monomethyl lysine (K) 27 was induced by staurosporine at a concentration of 1, but no monomethyl K4 or K9 in histone H3 was seen. Dimethyl and trimethyl histone H3 K27 were also identified. There was no expression of dimethyl or trimethyl histone H3 K4 and K9. Expression of monomethyl histone H3 K27 was dose-dependent. The morphologic changes of apoptosis induced by staurosporine were observed under microscopy. Immunocytochemistry of monomethyl histone H3 K27 showed a weak signal in controls, a strong signal in staurosporine-treated tumor cells and a denser signal in the apoptotic cells. Our studies demonstrated that monomethyl histone H3 lysine 27 is expressed in staurosporine-induced apoptotic osteosarcoma cells. The findings may provide novel bridge information between the epigenetic episodes and apoptotic process
- PublicationOpen AccessMgSO4 treatment preserves the ischemia-induced reduction in S-100 protein without modification of the expression of endothelial tight junction molecules(Murcia : F. Hernández, 2009) Goñi-de-Cerio, Felipe; Alvarez, Antonia; Alvarez, Francisco J.; Rey-Santano, Maria C.; Alonso-Alconada, Daniel; Mielgo, Victoria E.; Gastiasoro, Elena; Hilario, EnriqueThe aim of this work was to evaluate the effect of magnesium sulphate (MgSO4 ) administration on blood-brain barrier (BBB) permeabilization after cerebral hypoxia-ischemia (HI) induced by partial occlusion of the umbilical cord of premature fetal lambs. We also characterized BBB dysfunction in terms of the levels of expression of a panel of BBB proteins; Occludin, Claudin, Zona Occludens-1, Zonula Occludens-2, VE-cadherin and ß-catenin. Lambs were assigned to: Control group: non-injured animals, 0 h post-partial cord occlusion (0h-PCO) group: animals subjected to 60 min HI and sacrificed just after the insult, 3h-PCO group: HI injured animals resuscitated and managed on ventilation for 3 hours and MgSO4 group: animals which received a dose of 400 mg/kg MgSO4 after the HI event and managed on ventilation for 3 hours. Brains were fixed and blocks processed for S-100 protein immunohistochemistry. Other brains were dissociated and processed for S-100 and BBB protein immunochemistry for analysis by flow cytometry. The percentage of S-100 positive cells was found to be dramatically reduced in all studied brain tissues in the 3h-PCO group with respect to the other groups. No differences were found in the percentage or mean intensity of BBB protein immunolabeled cells among the groups. In the MgSO4 group, the percentage of S-100 positive cells 3 h after the HI event was similar to the control group. These results suggest that MgSO4 treatment preserves the ischemia-induced reduction in S- 100 protein without modification in the expression of endothelial tight junction molecules. We speculate that MgSO4 treatment confers neuroprotection by restoration of blood brain permeability in hypoxia-ischemia.
- PublicationOpen AccessA review of ERGIC-53, Its structure, functions, regulation and relations with diseases(Murcia : F. Hernández, 2009) Yong Ci Zhang; Yuan Zhou; Chun Zheng Yang; Dong Sheng XiongERGIC-53 is a type I transmembrane protein. It includes an N-terminal signal sequence, a carbohydrate recognition domain, which is calciumdependent and pH-sensitive, a stalk region, a transmembrane domain, and a short cytoplasmic domain; ERGIC-53 mainly acts as a receptor of a limited number of glycoprotein and transports them from ER to ERGIC and Golgi, meanwhile it has a secondary glycoprotein quality control function. Recent research has revealed that UPR, heat shock and VIPL may regulate the expression of ERGIC-53. F5F8D, and some ER storage diseases have relationship with ERGIC-53.
- PublicationOpen AccessPolarized endocytic transport ,The roles of Rab11 and Rab11-FIPs in regulating cell polarity(Murcia : F. Hernández, 2009) Jing, Jian; Prekeris, RytisEndocytic transport plays a vital role in the establishment and maintenance of cell polarity. Many studies have demonstrated that endosome-dependent protein targeting is required for polarization of epithelial cells and neurons. Endocytic transport regulates several highly polarized cellular events, such as cell motility and division. Rab11 GTPase has been shown to be a master regulator of protein transport via recycling endosomes, and many recent studies have focused on the molecular machinery that mediates Rab11-dependent endocytic protein transport in polarized cells. This mini-review describes the recent advances in identifying and characterizing the role of Rab11 and its effector proteins that play important roles in polarized endocytic sorting and transport.
- PublicationOpen AccessInvolvement of resistance to apoptosis in the pathogenesis of endometriosis(Murcia : F. Hernández, 2009) Nasu, K.; Yuge, A.; Tsuno, A.; Nishida, M.; Narahara, H.Endometriosis, a disease affecting 3-10% of women of reproductive age, is characterized by the ectopic growth of endometrial tissue. Increasingly, endometriosis is also becoming recognized as a condition in which ectopic endometrial cells exhibit abnormal proliferative and apoptotic regulation in response to appropriate stimuli. Apoptosis plays a critical role in maintaining tissue homeostasis and represents a normal function to eliminate excess or dysfunctional cells. Accumulated evidence suggests that, in healthy women, endometrial cells expelled during menstruation do not survive in ectopic locations because of programmed cell death, while decreased apoptosis may lead to the ectopic survival and implantation of these cells, resulting in the development of endometriosis. Both the inability of endometrial cells to transmit a ‘death’ signal and the ability of endometrial cells to avoid cell death have been associated with increased expression of anti-apoptotic factors and decreased expression of pre-apoptotic factors. This paper is a review of the recent literature focused on the differential expression of apoptosisassociated molecules in the normal endometria of women without endometriosis, and in the eutopic and ectopic endometria of women with endometriosis. The role of apoptosis in the pathogenesis of endometriosis and the basic and clinical research on the current medical treatment for endometriosis from the view of apoptosis will be discussed.
- PublicationOpen AccessUseful DNA typing using AmpFlSTR® Identifiler® Kit for formaldehyde-fixed paraffin-embedded (FFPE) tissues in early gastric cancer patient with lymph node metastasis(Murcia : F. Hernández, 2009) Hisako Motani-Saitoh; Hiroyuki Inoue; Tohru Tanizawa; Yoshihiro Nabeya; Daisuke Yajima; Mutsumi Hayakawa; Yayoi Sato; Yukio Nakatan; Hisahiro Matsubara; Hirotaro IwaseAfter distal gastrectomy in a patient with early gastric cancer, 27 regional lymph nodes around the stomach were evaluated for the existence of metastasis. There was a 0IIa+IIc type tumor 2.0x1.5cm in size in the gastric angle of the lesser curvature according to the Japanese Classification of Gastric Carcinoma (JCGC). Histologically, the lesion extended no deeper than the muscularis mucosae. The cancer stage was so early that no metastasis was expected to occur but a lymph node with metastasis was found in one lymph node along the common anterior hepatic artery (station No.8a). This histological type was a little different from that of a primary tumor. The doctor began to suspect that the lymph node with metastasis might have been from another patient by mistake. Therefore, DNA typing using the AmpFlSTR® Identifiler® kit was performed in formaldehyde-fixed paraffin-embedded (FFPE) tissues: 2 parts of gastric mucosa without cancer, one part of gastric mucosa with cancer, 4 lymph nodes without metastasis, and the lymph node station No.8a with metastasis. STR typing was successful in 6~14 STR loci and amelogenin gene, and the detected STR type was the same in all samples. Compared with the STR type using DNA from the patient’s blood, the lymph node station No.8a was from the same patient. The lymph node with metastasis turned out to be not from another patient. Therefore, we suggest that DNA typing using the AmpFlSTR® Identifiler® Kit for FFPE samples is useful in such clinical cases.
- PublicationOpen AccessVoluntary oral feeding of rats not requiring a very high fat diet is a clinically relevant animal model of non-alcoholic fatty liver disease (NAFLD)(Murcia : F. Hernández, 2009) Tipoe, G.L.; Ho, C.T.; Liong, Emily C.; Leung, T.M.; Lau, T.Y.H.; Fung, M.L.; Nanji, A.A.Animal models used to study the pathogenesis of non-alcoholic fatty liver disease (NAFLD) are, in general, either genetically altered, or fed with a diet that is extremely high in fat or carbohydrates. Recent findings support the role of oxidative stress, lipid peroxidation and inflammation as probable causative factors. We hypothesize that not only the amount of dietary fat, but the quality of fat is also important in inducing NAFLD. Based on previous observations that female rats fed a diet comprising unsaturated fatty acids are susceptible to liver injury, we proposed that female rats fed with a diet containing fish oil and dextrose would develop pathological and biochemical features of NAFLD. We fed a highly unsaturated fat diet (30% fish oil) to female SpragueDawley rats (180-200g), consumed ad libitum for 8 weeks (NAFLD; n=6-8 ). Control animals (CF; n=6-8) were fed with an isocaloric regular rat chow. At killing, blood and liver samples were collected for serum alanine aminotransferase (ALT), histology and molecular analysis. Each histological sample was evaluated for fatty liver (graded from 0 to 4+ according to the amount of fatty change), necrosis (number of necrotic foci (no./mm2 ) and inflammation (cells per mm2 ). The amount of collagen formation was estimated based on the amount of Sirius Red staining. Reverse transcriptase polymerase chain reaction (RT-PCR) was carried out for tumor necrosis factor alpha (TNF-α), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), adiponectin, glutathione peroxidase (GPx), superoxide dismutase (Cu/Zn SOD) and catalase (CAT). Western Blot analysis was done for cyclooxygenases-2 (COX-2), inducible nitric oxide synthase (iNOS) and nitrotyrosine. Electrophoretic mobility shift assay was performed for nuclear factor-kappa B (NF-κB) activity. NAFLD rats had a significantly higher serum ALT level, amount of collagen formation, fatty liver, necrosis and inflammation when compared with the chow-fed control rats. mRNA and protein levels of NF-κB regulated genes, which included TNF-alpha, COX-2 and iNOS were also significantly (p<0.01; p<0.01; p<0.05 respectively) upregulated in the NAFLD group when compared with the chow-fed control rats. mRNA levels of antioxidants CAT and GPX were reduced by 35% and 50% respectively in the NAFLD group. However, Cu/Zn SOD mRNA was similar in both groups. The mRNA level of adiponectin was also reduced in NAFLD group. NF-κB activity was markedly increased in the NAFLD rats (p<0.01). The level of oxidative stress, represented by the formation of nitrotyrosine, was significantly elevated in the NAFLD rats (p<0.01). We conclude that NAFLD rats demonstrated several features of NAFLD, which included fatty liver, inflammation, necrosis, increased oxidative stress, an imbalance between pro and antioxidant enzymes mRNAs, reduced adiponectin levels and upregulation of pro-inflammatory mediators. We propose that female rats fed with a diet containing highly unsaturated fatty acids are an extremely useful model for the study of NAFLD.
- PublicationOpen AccessAtorvastatin induces thrombomodulin expression in the aorta of cholesterol-fed rabbits and in TNFa-treated human aortic endothelial cells(Murcia : F. Hernández, 2009) Lin, Shing-Jong; Hsieh, Fang-Yu; Chen, Yung-Hsiang; Lin, Chia-Chi; Kuan, I-I; Wang, Shu-Huei; Wu, Chau-Chung; Chien, Hsiung-Fei; Lin, Fen-Yen; Chen, Yuh-LienExpression of functionally active thrombomodulin (TM) on endothelial cells is critical for vascular thromboresistance. 3-Hydroxyl-3-methyl coenzyme A reductase inhibitors (statins) can protect the vasculature from inflammation and atherosclerosis caused by cholesterol-dependent and cholesterolindependent mechanisms. In the present study, the effects of atorvastatin on TM expression in the aorta of cholesterol-fed rabbits and in TNFa-treated human aortic endothelial cells (HAECs) were investigated. When rabbits were fed a 0.5% cholesterol diet with and without supplementation with atorvastatin for 9 weeks, the neointimal area in the thoracic aorta of the atorvastatin-treated group was significantly reduced and there was significant induction of TM protein expression. In HAECs, TNFa treatment decreased the expression of TM in a time- and dose-dependent manner and atorvastatin pretreatment upregulated the expression of TM mRNA and protein in HAECs with or without TNFa treatment. Atorvastatin also inhibited monocyte adhesion to control and TNFa-treated HAECs via TM expression. ERK1/2 phosphorylation was significantly reduced by 24 h pretreatment with atorvastatin, whereas TNFa increased the phosphorylation of the MAPKs, p38, JNK, and ERK1/2. Blocking the transcriptional activation of NF-kB and nuclear translocation of NF-kB p65 prevented the TNFa-induced downregulation of TM. Atorvastatin regulated TM expression in control and TNFa-treated HAECs by inhibiting the activation of ERK and NF-kB. The increase in endothelial TM activity in response to atorvastatin constitutes an important pleiotropic effect of this commonly used compound and may be of clinical significance in cardiovascular disorders in which deficient endothelial TM plays a pathophysiological role.
- PublicationOpen AccessUltrastructure and lectin cytochemistry of secretory cells in lingual glands of the Japanese quail (Coturnix coturnix japonica)(Murcia : F. Hernández, 2009) Capacchietti, M.; Sabbieti, M.G.; Agas, D.; Materazzi, S.; Menghi, Giovanna; Marchetti, L.In the present study, as continuation of our previous research, Japanese quail (Coturnix coturnix japonica) lingual glands were investigated by means of transmission electron microscopy (TEM) to understand the cytoarchitecture and the subcellular sugar distribution within the different secretory structures. Indeed, glycosidic residues were visualized by applying an indirect technique of binding and the terminal sialoglycoconjugate sequences were characterized by employing sialidase digestion combined with lectin affinity. The ultrastructural analysis revealed an unusual cytoarchitecture of the caudal portion of anterior lingual gland that was composed of both secretory cells, filled with granules, and non-secretory cells, filled with mitochondria. Conversely, the posterior lingual gland was composed of secretory units of lingual glands only containing mucous cells filled with secretory granules with a variable morphology, including bipartite features characterized by an electron-lucent matrix and one or more electron-dense areas. Actual findings further supported that the quail lingual glands produce sialoglycoconjugates characterized by a heterogeneous composition. In conclusion, the cytological characteristics and the carbohydrate composition of quail lingual glands suggest that, analogously to mammal salivary glands, avian lingual glands could also be involved in several functions that can be correlated with the occurrence of sialic acids.
- PublicationOpen AccessPrognostic significance of AGR2 in pancreatic ductal adenocarcinoma(Murcia : F. Hernández, 2009) Riener, Marc-Oliver; Pilarsky, Christian; Gerhardt, Josefine; Grützmann, Robert; Fritzsche, F.R.; Bahra, Marcus; Weicher, Wilko; Kristiansen, GlenBackground/Aims: The human Anterior Gradient-2 (AGR2) is strongly upregulated in various human cancers, including pancreatic ductal adenocarcinomas (PDAC), but its prognostic value in PDAC has not yet been studied. Methods: We analysed 19 microdissected PDAC cases at the mRNA level, and also 148 cases at the protein level by immunohistochemistry based on tissue microarray, using a monoclonal AGR2 antibody, and statistical analyses were applied to test for prognostic associations. Results: Overexpression of AGR2 mRNA was found to be elevated in most pancreatic cell lines and in microdissected pancreatic cancer compared to microdissected normal ductal cells. AGR2 protein was expressed in 109/148 (73.7%) of PDAC, with a higher expression in female patients (p=0.040), whereas no significant associations with other clinical-pathological parameters were found. A prognostic value of AGR2 could not be demonstrated in univariate analyses. Conclusion: Although a prognostic value of AGR2 seems unlikely, further studies are warranted to investigate the biological role of AGR2 in pancreatic adenocarcinomas.
- PublicationOpen AccessThe expression of a novel cxcr4 gene in Xenopus embryo(Murcia : F. Hernández, 2009) Alonso, Edurne; Gómez-Santos, Laura; Sáez, F.J.; Madrid Cuevas, Juan FranciscoThe aim of the present work was to identify a homologue of zebrafish cxcr4b in Xenopus, which could be involved in primordial germ cell (PGC) guidance migration. Following a BLAST analysis, the clone gi 27519681, homologous to the zebrafish gene z-cxcr4b, was identified, inserted into pCMV-SPORT6 plasmid and cloned in Escherichia coli. Embryonic expression of x-cxcr4b was analyzed by RT-PCR. X-cxcr4b was weakly expressed maternally but sharply increased after the mid-blastula transition (MBT), declining significantly at stage 45 when PGCs migration is complete. In contrast, RT-PCR of isolated presumptive PGCs showed strong maternal expression at stage 8, which decreased by stage 10 post-MBT and was not detected at stage 14. Whole mount in situ hybridization of x-cxcr4b mRNA showed that this gene is expressed in neural and haematopoietic tissues, and should be linked to important processes during embryonic development of these organs. Although weak staining could be seen in some samples within the anterior endoderm, expression of x-cxcr4b was never coincident with that of Xpat mRNA, which labels PGCs restricted to the posterior endoderm. Therefore, maternal x-cxcr4b is specifically downregulated within PGCs at pre-migratory stages while it is expressed in other tissues.
- PublicationOpen AccessHistopathological changes of the hippocampus neurons in brain injury(Murcia : F. Hernández, 2009) Dong Ri Li; Takaki Ishikawa; Dong Zhao; Tomomi Michiue; Li Quan; Bao Li Zhu; Hitoshi MaedaThe glial fibrillary acidic protein (GFAP) is known as a peculiar marker of mature astrocytes of the central nervous system (CNS). However, we found distinct immunopositivity to a monoclonal anti-GFAP reagent in the hippocampus neurons in head injury fatalities. The present study investigated the neuronal and neuroglial GFAP-immunopositivity in the hippocampus in a series of head injury cases, which included acute and subacute/delayed deaths (n=17 and n=73, respectively), and acute cardiac death (n=13), delayed death due to multiple organ failure from nonhead injury (n=6), and pneumonia (n=9) cases were examined as controls. GFAP-immunopositivity in the neurons was frequently observed in CA4, CA3 and CA2 regions in cases of subacute/delayed head injury death that showed marked brain swelling accompanied by secondary brain stem hemorrhages, showing an inverse relationship to that in astrocytes. These findings suggest possible induction of GFAP or a related protein in hippocampus neurons depending on the severity of brain swelling following head injury.