Histology and histopathology Vol.23, nº4 (2008)

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The role of nitric oxide in remodeling of capillary network in rat interscapular brown adipose tissue after long-term cold acclimation
(Murcia : F. Hernández, 2008) Korac, A.; Buzadzic, B.; Petrovic, V.; Vasilijevic, A.; Jankovic, A.; Micunovic, K.; Korac, B.
Cold exposure has been shown to increase blood flow in interscapular brown adipose tissue (IBAT). The aim of the present study was to evaluate the role of the L-arginine-nitric oxide (•NO) pathway on IBAT capillary network remodeling and its possible correlation with superoxide anion radical (O2 •–). In the rats that received L-arginine (2.25%) or NG-nitro-L-arginine methyl ester (L-NAME, 0.01%) as a drinking liquid and maintained at room (22±1°C) or low (4±1°C) temperature for 45 days, IBAT capillaries were analyzed by stereology and observed by light and electron microscopy. Additionally, endothelial •NO synthase (eNOS) expression, nitrotyrosine immunoreactivity and both copper zinc superoxide dismutase (CuZnSOD) enzyme activity and immunohistochemical localization were examined. Stereological analyses of IBAT show that the capillary volume density, as well as capillary-tobrown adipocytes ratio, are increased in cold. L-arginine treatment increases, while L-NAME decreases both parameters, compared to respective controls. Those changes were accompanied by capillary dilatation observed by light and electron microscopy. The activity of CuZnSOD is lower in control cold-acclimated rats, as well as in both L-arginine-treated groups, when compared to control animals acclimated to room temperature. L-NAME treatment attenuates the effects both of cold and L-arginine on CuZnSOD and increases immunopositivity for CuZnSOD in room temperatureacclimated rats. Our results show that •NO induces remodeling of the IBAT capillary network by angiogenesis, and presumably that interaction with O2 •– has a role in that modulation. The increased eNOS expression accompanied by an increased nitrotyrosine immunoreaction observed in both L-arginine-treated groups compared to corresponding controls strengthens this hypothesis.
Open Access
Aluminium exposure induces Alzheimer s disease-like histopathological alterations in mouse brain
(Murcia : F. Hernández, 2008) Rodella, L.F.; Ricci, F.; Borsani, E.; Stacchiotti, A.; Foglio, E.; Favero, G.; Rezzani, R.; Mariani, C.; Bianchi, R.
Aluminium (Al) is a neurotoxic metal and Al exposure may be a factor in the aetiology of various neurodegenerative diseases such as Alzheimer’s disease (AD). The major pathohistological findings in the AD brain are the presence of neuritic plaques containing ßamyloid (Aß) which may interfere with neuronal communication. Moreover, it has been observed that GRP78, a stress-response protein induced by conditions that adversely affect endoplasmic reticulum (ER) function, is reduced in the brain of AD patients. In this study, we investigated the correlation between the expression of Aß and GRP78 in the brain cortex of mice chronically treated with aluminium sulphate. Chronic exposure over 12 months to aluminium sulphate in drinking water resulted in deposition of Aß similar to that seen in congophilic amyloid angiopathy (CAA) in humans and a reduction in neuronal expression of GRP78 similar to what has previously been observed in Alzheimer’s disease. So, we hypothesise that chronic Al administration is responsible for oxidative cell damage that interferes with ER functions inducing Aß accumulation and neurodegenerative damage.
Open Access
Autoimmune glomerulonephritis induced in congenic mouse strain carrying telomeric region of chromosome 1 derived from MRL-MpJ
(Murcia : F. Hernández, 2008) Ichii, Osamu; Konno, Akihiro; Sasaki, Nobuya; Endoh, Daiji; Hashimoto, Yoshiharu; Kon, Yasuhiro
In lupus erythematosus-prone mice, including the BXSB, NZW and NZB strains, telomeric regions of chromosome 1 (Chr.1) contain major glomerulonephritis susceptibility loci such as Bxs3, Sle1, and Nba2. To assess whether strain MRL, a model for lupus erythematosus, had glomerulonephritis susceptibility loci on Chr.1, we created B6.MRLc1(82- 100) congenic mice carrying MRL/MpJ Chr.1 (82- 100cM) based on the C57BL/6 background and investigated renal pathology. From 6 months of age, B6.MRLc1 (82-100) showed the onset of diseases such as splenomegaly due to proliferation of CD3- or B220- positive cells, glomerular damage, and an increased serum anti-dsDNA antibody concentration, and these were earlier and severer in females. The score for glomerular damage was higher in B6.MRLc1(82-100) mice over 12 months old than in C57BL/6 or even in wild-type MRL/MpJ. Immune-complex depositions were demonstrated on glomerular basement membrane in B6.MRLc1(82-100) by immunohistochemistry and electron microscopy. For the percentage of IgG1- positive glomeruli, B6.MRLc1 (82-100) had significantly higher values than C57BL/6. In evaluations of clinical parameters, serum levels of blood urea nitrogen and the anti-dsDNA antibody in B6.MRLc1(82- 100) were significantly higher than those in C57BL/6. In conclusion, B6.MRLc1(82-100) clearly developed autoimmune-mediated glomerulonephritis, and we demonstrated that MRL Chr.1 contained a novel glomerulonephritis susceptibility locus. We named this locus Mag (MRL autoimmune glomerulonephritis) and it provided new insights into the genetic basis and pathogenesis of lupus nephritis.
Open Access
Current strategies in the search for low penetrance genes in cancer
(Murcia : F. Hernández, 2008) Milne, Roger L.; Benítez, Javier
The genetic etiology of most cancers remains largely unclear and it has been hypothesised that common genetic variants with modest effects on disease susceptibility cause the bulk of this unexplained risk. Case-control association studies are considered the most effective strategy to identify these low-penetrance genes. While traditionally, such studies have focused on putative functional single nucleotide polymorphisms (SNPs) in candidate genes, a more comprehensive approach can now be taken, as a result of a number of recent developments: the mapping of the human genome, including the identification of almost ten million SNPs; and the development of high-throughput genotyping technologies that enable hundreds of thousands of SNPs to be genotyped in a single reaction, in multiple subjects and at an affordable cost. All common genomic variation can be captured by genotyping SNPs in gene-, pathway- or genome-widebased strategies and these are now being applied to many diseases, including cancer. We present an outline of each of these approaches, including recent published examples, and discuss a number of challenges that remain to be addressed.
Open Access
Isolation of high quality protein samples from punches of formalin fixed and paraffin embedded tissue blocks
(Murcia : F. Hernández, 2008) Kroll, J.; Becker, K.F.; Kuphal, S.; Hein, R.; Hofstädter, F.; Bosserhoff, A.K.
In general, it is believed that the extraction of proteins from formalin-fixed paraffin embedded samples is not feasible. However, recently a new technique was developed, presenting the extraction of non-degraded, full length proteins from formalin fixed tissues, usable for western blotting and protein arrays. In the study presented here, we applied this technique to punch biopsies of formalin fixed tissues embedded in paraffin to reduce heterogeneity of the tissue represented in sections, and to ensure analysing mainly defined cellular material. Successful extraction was achieved even from very small samples (0.7 mm3). Additionally, we were able to detect highly glycosylated proteins and protein modification, such as phosphorylation. Interestingly, with this technique it is feasible to extract high quality proteins from 14 year old samples. In summary, the new technique makes a great pool of material now usable for molecular analysis with high throughput tools.
Open Access
Mandibular bone alterations of ovariectomized rats under vitamin D insufficiency
(Murcia : F. Hernández, 2008) Said, Faika; Ghoul-Mazgar, Sonia; Ruhin, Blandine; Abdellaoui, Mohieddine; Chlaghmia, Faycel; Safta, Sihem; Guezguez, Leila; Saidane-Mosbahi, Dalila; Khemiss, Fathia
Experimental osteoporosis was studied in mandible bone by means of ovariectomy and vitamin D insufficiency. METHODS: 42 female Wistar rats were divided into the following four groups: (1) ovariectomized rats maintained in 12h day-night light conditions (ov-l), (2) ovariectomized rats maintained in 24h dark light conditions (ov-ob), (3) sham-operated rats maintained in 12h day-night light conditions (ch-l) and (4) sham-operated rats maintained in 24 h dark conditions (ch-ob). 12 weeks later the animals were sacrificed, the mandibles were excised, cleaned and weighed, the right side of the mandibles were histologically examined and the left side of the mandibles were prepared for mineral phase analysis by X-ray diffraction. Immunohistochemical analysis was performed to detect apoptotic cells by anti-PARP p85 antibody. RESULTS: In group 2, the weight of mandibles significantly decreased. Chondroid areas were observed in ovariectomized groups and polarized light observation validated the collagen distribution disturbance in these groups (groups 1 and 2). Apoptotic osteoblasts were localized in groups 1, 2 and 4. They were numerous in group 2. The mineral phase analysis did not find differences between the groups. CONCLUSION: This study validates a new model of osteoporotic animal associating estrogens deficiency and vitamin D insufficiency where matrix synthesis and osteoblast biology are altered, but not biomineralization.
Open Access
Expression of acyl-CoA synthetase 5 in human epidermis
(Murcia : F. Hernández, 2008) Gaisa, N.T.; Köster, J.; Reinartz, A.; Ertmer, K.; Ehling, J.; Raupach, K.; Perez-Bouza, A.; Knüchel, R.; Gassler, N.
The human epidermis is characterized by a constant renewal of keratinocytes embedded in a matrix enriched with lipids. Numerous proteins involved in lipid metabolism are found in human epidermis, especially in keratinocytes. Long-chain acyl-CoA derivatives, which are catalyzed by human ACSL5, are important metabolites in several biochemical pathways, including ceramide de novo synthesis. The aim of the present study was to investigate expression of acyl-CoA synthetase isoform 5 (ACSL5) in human epidermis by an in situ, as well as a molecular approach. We show that ACSL5 mRNA and protein are found in human epidermis, as well as in non-differentiated and differentiated HaCaT cells. Keratinocytes of stratum spinosum are the main source for ACSL5 expression in both meshed facial or abdominal skin and ridged skin of upper or lower extremities including TUNEL-positive cells in upper cellular layers. Single keratinocytes of chronic solar-exposed meshed facial epidermis occasionally display a stronger ACSL5 immunostaining. In conclusion, our study indicates that epidermal ACSL5 expression might be involved in differentiation and the stress response of keratinocytes.
Open Access
Characterization by immunocytochemistry of ionic channels in Helix aspersa suboesophageal brain ganglia neurons
(Murcia : F. Hernández, 2008) Azanza, M.J.; Pérez-Castejon, C.; Pes, N.; Pérez-Bruzón, R.N.; Aisa, J.; Junquera, C.; Maestú, C.; Lahoz, M.; Martínez Ciriano, M.C.; Vera Gil, A.; Del Moral, A.
The aim of this work was to characterize several ionic channels in nervous cells of the suboesophageal visceral, left and right parietal, and left and right pleural brain ganglia complex of the snail Helix aspersa by immunocytochemistry. We have studied the immunostaining reaction for a wide panel of eleven polyclonal antibodies raised against mammal antigens as follows: voltage-gated-Na+ channel; voltagegated- delayed-rectifier-K+ channel; SK2-smallconductance- Ca2+-dependent-K+ channel apamin sensitive; SK3 potassium channel; charybdotoxinsensitive voltage-dependent potassium channel; BKCamaxi- conductance-Ca2+-dependent-K+ channel; hyperpolarization-activated cyclic nucleotide-gated potassium channel 4; G-protein-activated inwardly rectifying potassium channel GIRK2 and voltage-gatedcalcium of L, N and P/Q type channels. Our results show positive reaction in neurons, but neither in glia cells nor in processes in the Helix suboesophageal ganglia. Our results suggest the occurrence of molecules in Helix neurons sharing antigenic determinants with mammal ionic channels. The reaction density and distribution of immunoreactive staining within neurons is specific for each one of the antisera tested. The studies of colocalization of immunoreaction, on alternate serial sections of the anterior right parietal ganglion, have shown for several recognized mapped neurons that they can simultaneously be expressed among two and seven different ionic protein channels. These results are considered a key structural support for the interpretation of Helix aspersa neuron electrophysiological activity.
Open Access
Evidence for a potential tumor suppressor role for the Na,K-ATPase B1-subunit
(Murcia : F. Hernández, 2008) Inge, Landon J.; Rajasekaran, Sigrid A.; Yoshimoto, Koji; Mischel, Paul S.; McBride, William; Landaw, Elliot; Rajasekaran, Ayyappan K.
The Na,K-ATPase, consisting of two essential subunits (a, ß), plays a critical role in the regulation of ion homeostasis in mammalian cells. Recent studies indicate that reduced expression of the ß1 isoform (NaK-ß1) is commonly observed in carcinoma and is associated with events involved in cancer progression. In this study, we present evidence that repletion of NaK-ß1 in Moloney sarcoma virustransformed Madin-Darby canine kidney cells (MSVMDCK), a highly tumorigenic cell line, inhibits anchorage independent growth and suppresses tumor formation in immunocompromised mice. Additionally, using an in vitro cell-cell aggregation assay, we showed that cell aggregates of NaK-ß1 subunit expressing MSVMDCK cells have reduced extracellular regulated kinase (ERK) 1/2 activity compared with parental MSV-MDCK cells. Finally, using immunohistochemistry and fully quantitative image analysis approaches, we showed that the levels of phosphorylated ERK 1/2 are inversely correlated to the NaK-ß1 levels in the tumors. These findings reveal for the first time that NaK-ß1 has a potential tumor-suppressor function in epithelial cells.
Open Access
Presence of perivenular elastic fibers in nonalcoholic steatohepatitis Fibrosis Stage III
(Murcia : F. Hernández, 2008) Nakayama, Hirofumi; Itoh, Hiroyuki; Kunita, Satoko; Kuroda, Naoto; Hiroi, Makoto; Matsuura, Hideo; Yasui, Wataru; Enzan, H.
Elastic fibers appear in extensive old fibrotic foci in general. We examined an association between hepatic fibrosis stage and the presence of perivenular elastic fibers in nonalcoholic steatohepatitis (NASH). A total of 48 liver needle biopsy specimens were used, taken from 48 cases with NASH. Fibrosis Stage (Brunt E, et al. Am. J. Gastroenterol. 1999) of the cases was as follows; six Fibrosis Stage I, twenty-two Fibrosis Stage II, and twenty Fibrosis Stage III. We examined Orcein stain sections in all of the liver needle biopsy specimens. In all twenty Fibrosis Stage III cases, perivenular elastic fiber bundles were observed. In contrast, perivenular elastic fibers were detected only in one of the six Fibrosis Stage I and two of the twenty-two Fibrosis Stage II cases. In liver needle biopsy specimens of NASH, detection of perivenular elastic fibers is useful in deciding Fibrosis Stage III.
Open Access
Osteopontin is histochemically detected by the AgNOR acid-silver staining
(Murcia : F. Hernández, 2008) Gaudin-Audrain, Christine; Gallois, Yves; Pascaretti-Grizon, Florence; Hubert, Laurent; Massin, Philippe; Baslé, Michael-Félix; Chappard, Daniel
Silver nitrate staining of decalcified bone sections is known to reveal osteocyte canaliculi and cement lines. Nucleolar Organising Regions (NOR) are part of the nucleolus, containing argyrophilic proteins (nucleoclin/C23, nucleophosmin/B23) that can be identified by silver staining at low pH. The aim of this study was to clarify the mechanism explaining why AgNOR staining also reveals osteocyte canaliculi. Human bone and kidney sections were processed for silver staining at light and electron microscopy with a modified method used to identify AgNOR. Sections were processed in parallel for immunohistochemistry with an antibody direct against osteopontin. Protein extraction was done in the renal cortex and decalcified bone and the proteins were separated by western blotting. Purified hOPN was also used as a control. Proteins were electro-transferred on polyvinylidene difluoride membranes and stained for AgNOR proteins. In bone, Ag staining identified AgNOR in cell nuclei, as well as in osteocyte canaliculi, cement and resting lines. In the distal convoluted tubules of the kidney, silver deposits were also observed in cytoplasmic granules on the apical side of the cells. Immunolocalization of osteopontin closely matched with all these locations in bone and kidney. Ag staining of membranes at low pH revealed bands for NOR proteins and 56 KDa (kidney), 60KDa (purified hOPN) and 75 KDa (bone) bands that corresponded to osteopontin. NOR proteins and osteopontin are proteins containing aspartic acid rich regions that can bind Ag. Staining protocols using silver nitrate at low pH can identify these proteins on histological sections or membranes.
Open Access
Glycolytic enzyme Pgk1 is strongly expressed in the developing tooth germ of the mouse lower first molar
(Murcia : F. Hernández, 2008) Honda, Jun-ya; Kobayashi, Leyoshi; Kiyoshima, Tamotsu; Yamaza, Haruyoshi; Xie, Ming; Takahashi, Keiko; Enoki, Norio; Nagata, Kengo; Nakashima, Akihiko; Sakai, Hidetaka
This study examined detailed in situ expression patterns and possible functional roles of phosphoglycerate kinase 1 (Pgk1) gene in the developing tooth germ of the mouse lower first molar. The strong expression of Pgk1 mRNA was seen in the odontogenic epithelial cells and surrounding mesenchymal cells of the tooth germ from embryonic day 10.5 (E10.5) to E18.0. Western blotting analysis demonstrated that Pgk1 protein formed 84-kDa protein complex in these embryonic organs. The results of immunoprecipitation-western blotting also suggested this complex to be formed with glyceraldehyde-3- phosphate dehydrogenase (GAPDH). Moreover, the immunofluorescence expression of those proteins was shown to overlap each other in the tooth germ at E15.0. A strong immunofluorescence expression of both Pgk1 and GAPDH also corresponded to the in situ expression of those mRNAs. These results suggested that Pgk1 plays some functional roles in the development of tooth germ and other embryonic organs by forming protein complex with GAPDH.
Open Access
Tea polyphenols, their biological effects and potential molecular targets
(Murcia : F. Hernández, 2008) Chen, Di; Milacic, Vesna; Si Chen, Marina; Biao Wan, Sheng; Har Lam, Wai; Huo, Congde; Landis-Piwowar, Kristin R.; Cindy Cui, Qiuzhi; Wali, Anil; Hang Chan, Tak; Ping Dou, Q.
Tea is the most popular beverage in the world, second only to water. Tea contains an infusion of the leaves from the Camellia sinensis plant rich in polyphenolic compounds known as catechins, the most abundant of which is (-)-EGCG. Although tea has been consumed for centuries, it has only recently been studied extensively as a health-promoting beverage that may act to prevent a number of chronic diseases and cancers. The results of several investigations indicate that green tea consumption may be of modest benefit in reducing the plasma concentration of cholesterol and preventing atherosclerosis. Additionally, the cancer-preventive effects of green tea are widely supported by results from epidemiological, cell culture, animal and clinical studies. In vitro cell culture studies show that tea polyphenols potently induce apoptotic cell death and cell cycle arrest in tumor cells but not in their normal cell counterparts. Green tea polyphenols were shown to affect several biological pathways, including growth factor-mediated pathway, the mitogen-activated protein (MAP) kinasedependent pathway, and ubiquitin/proteasome degradation pathways. Various animal studies have revealed that treatment with green tea inhibits tumor incidence and multiplicity in different organ sites such as skin, lung, liver, stomach, mammary gland and colon. Recently, phase I and II clinical trials have been conducted to explore the anticancer effects of green tea in humans. A major challenge of cancer prevention is to integrate new molecular findings into clinical practice. Therefore, identification of more molecular targets and biomarkers for tea polyphenols is essential for improving the design of green tea trials and will greatly assist in a better understanding of the mechanisms underlying its anti-cancer activity.
Open Access
Role of OPG-RANKL-RANK axis on the vasculature
(Murcia : F. Hernández, 2008) Papadopouli, Aikaterini E.; Klonaris, Christos N.; Theocharis, Stamatios E.
Vascular calcification, a degenerative process considered in the past to be a passive procedure, has now been suggested to be related to ossification. Many proteins responsible for bone formation have been identified on the arterial wall. The OPG/RANKL/RANK axis, responsible for ossification and bone mineralization, seems to play a major role in vasculature and atherosclerosis. Mice lacking OPG gene present osteoporosis and arterial calcification, while overexpression of OPG gene leads to osteopetrosis. In the present review the latest knowledge related to the effects of the OPG/RANKL/RANK axis on vasculature, including atherosclerosis, will be analyzed. The clinical significance of circulating OPG and RANKL levels in vascular diseases will also be referred.
Open Access
Indices 4,5,6
(Murcia : F. Hernández, 2013-01-18)
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