Search

Abstract Background Acute ethanol administration leads to massive apoptotic neurodegeneration in the developing central nervous system. We studied whether taurine is neuroprotective in ethanol-induced apoptosis in the mouse cerebellum during the postnatal period. Methods The mice were divided into three groups: ethanol-treated, ethanol+taurine-treated and controls. Ethanol (20% solution) was administered subcutaneously at a total dose of 5 g/kg (2.5 g/kg at time 1 h and 2.5 g/kg at 3 h) to the ethanol and ethanol+taurine groups. The ethanol+taurine group also received two injections of taurine (1 g/kg each, at time zero and at 4 h). To estimate apoptosis, immunostaining for activated caspase-3 and TUNEL staining were made in the mid-sagittal sections containing lobules I-X of the cerebellar vermis at 12 or 8 hours after the first taurine injection. Changes in the blood taurine level were monitored at each hour by reverse-phase high-performance liquid chromatography (HPLC). Results Ethanol administration induced apoptosis of Purkinje cells on P4 in all cerebellar lobules, most extensively in lobules IX and X, and on P7 increased the number of activated caspase-3-immunoreactive and TUNEL-positive cells in the internal layer of the cerebellum. Administration of taurine significantly decreased the number of activated caspase-3-immunoreactive and TUNEL-positive cells in the internal layer of the cerebellum on P7, but had no effect on Purkinje cells in P4 mice. The high initial taurine concentration in blood of the ethanol+taurine group diminished dramatically during the experiment, not being different at 13 h from that in the controls. Conclusions We conclude that the neuroprotective action of taurine is not straightforward and seems to be different in different types of neurons and/or requires prolonged maintenance of the high taurine concentration in blood plasma.

ADAM metalloproteinase disintegrins have emerged as the major proteinase family that mediates ectodomain shedding, the proteolytic release of extracellular domains from their membrane-bound precursors. Recent gene-manipulation studies have established the role of ADAM-mediated shedding in mammalian physiology and, in addition, raised the issue of functional redundancy among ADAM sheddases. ADAM sheddases activate, for example, growth factors and cytokines, thus regulating signalling pathways that are important in development and pathological processes such as cancer. The recent studies have also begun to elucidate the substrate specificity and the mechanisms that control ADAM-mediated shedding events that regulate, for example, growth-factor and Notch signalling, and the processing of the amyloid precursor protein.

Background. A disintegrin and metalloproteinase-8 (ADAM8) is a potential surrogate of inflammation which has recently been associated with myocardial infarction. We evaluated in a rat cardiac transplantation model whether ischemia-reperfusion injury alone (IRI) or with early regional myocardial infarction (MI) would suffice to induce inflammatory myocardial remodeling and ADAM8 expression. Material and methods. Isogenic heterotopic cardiac transplantation after cardiac arrest was performed to 48 Fischer 344 rats to induce ischemia-reperfusion injury (IRI), of which 27 rats also underwent ligation of the left anterior coronary artery (LAD) of the heart to yield MI. Histology was performed at 0.5, 24 and 48 h after transplantation. ADAM8 was evaluated by qRT-PCR after graft harvesting. Results. After 0.5 and 48 h respectively, edematous intramyocardial artery nuclei and periadventitial inflammation were more prominent in MI after transplantation, as compared with IRI alone and Controls (57.0 vs 40.0 and 5.0; 1.9 vs 1.1 and 0.9, point score units, p < 0.05, respectively). The expression of ADAM-8 was increased in MI as compared with Controls (1.9 vs 1.0, 1.9 fold increase) at 48 h. In grafts with MI, ADAM8 was localized using immunohistochemistry to the vicinity of the area corresponding to the developing infarction as well as in intramyocardial arteries remote to the infarction area. Conclusions. Remote histopathological changes of ischemic cardiac grafts are associated with increased expression of ADAM8 thus emphasizing a global myocardial impact of MI. [ABSTRACT FROM AUTHOR]

Background. Dilatation of the ascending aorta (AA) is affected by extra-cellular matrix modifications and inflammation. A disintegrin and metalloproteases (ADAMs) may reveal differences between AA and ascending aortic dissection (AD). We characterized the inflammatory histology of AD and AA and examined the role of ADAM8 and -15 in these diseases. Material and methods. Aortic wall histology and immunohistochemistry for leukocytes, T- and B-lymphocytes, plasma cells, macrophages, endothelial cells, smooth muscle cells, cell proliferation, elastase and Van-Gieson-staining were performed to 40 consecutive patients that underwent surgery for AA or AD. The expressions of ADAM8 and -15 mRNA and proteins were evaluated using QRT-PCR and immunohistochemistry. Results. Thirty-four patients were enrolled, of which 29 had AA and five had AD of the ascending aorta. B-cells throughout the aortic wall and intimal plasma cells were more numerous during AD as compared with AA ( p < 0.05). The gene expressions for ADAM8 and -15 were notably lower in AA as compared with AD. The median for down-regulation of ADAM8 and -15 in AA was -2.7 and −1.8, respectively. ADAM8 and -15 were mainly found in the media layer in patients with AD. Two of the patients with AA and increased ADAMs developed AD of the remaining aorta. Conclusions. The involvement of ADAM8 and -15 together with inflammation consisting of B-cells may indicate active remodelling of the aortic wall leading to AD. [ABSTRACT FROM AUTHOR]

Background and aims. Carbonic anhydrases (CA) play a central role in osteoclast function and bone remodeling by catalyzing the formation of bicarbonate and proton from carbon dioxide. According to previous histochemical studies, advanced atherosclerotic plaques share similarities with bone. However, whether CAs are expressed in plaques is not known. Methods and results. Whole genome expression array of arterial samples ( n = 24) confirmed that several genes indicating osteoblastogenesis and osteoclastogenesis were up-regulated in plaques when compared to control vessel samples from internal thoracic arteries ( n = 6), including CA2 and CA12, expression of which was also verified with quantitative reverse transcription polymerase chain reaction (RT-PCR). In atherosclerotic plaques there was 11.6-fold ( P < 0.0001) and 11.4-fold ( P < 0.0001) up-regulation of CA2 and CA12, compared to controls, respectively. According to quantitative PCR, CA2 expression was elevated in carotid (12.3-fold, P < 0.0001), femoral (13.2-fold, P < 0.01), and aortic plaques (7.5-fold, P < 0.0001). CA12 expression was elevated in carotid (11.6-fold, P < 0.0001), femoral (11.5-fold, P < 0.01), and aortic plaques (9.7-fold, P < 0.0001). CAII, CAXII, and CD68 and tartrate-resistant acid phosphatase (TRAP), a marker of osteoclast-like cells, were found to be co-localized in multinucleated giant cells in the atherosclerotic plaques using immunohistochemistry and double-staining immunofluorescence analysis. Conclusions. The present findings provide evidence for the involvement of CAs in advanced atherosclerosis in osteoclast-like cells of monocyte-macrophage lineage. [ABSTRACT FROM AUTHOR]

Objective. We aimed to characterize the expression of indoleamine 2,3-dioxygenase (IDO) or IDO-induced tryptophan degradation-dependent pathways, which may lead to suppression of T cells and possible protection against atherosclerosis. Methods and results. Expression of IDO and IDO-related pathway components was analyzed in advanced human atherosclerotic plaques ( n = 24) and in non-atherosclerotic arteries ( n = 6). Up-regulation of IDO and genes related to the IDO pathway was found to be pronounced in atherosclerotic plaques. Immunohistochemistry demonstrated IDO protein in the atheromatous core and co-distribution with monocyte-macrophages (CD68-positive cells). In gene-set enrichment analysis, the IDO pathway revealed a significant (false discovery rate (FDR) = 0.07) regulatory T cell, fork-head box protein 3 (FoxP3)-initiated CD28-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4)-inducible T cell co-stimulator (ICOS)-driven pathway leading to activation of IDO expression in antigen-presenting cells (APCs). Expression of these IDO pathway genes varied between 2.1- and 16.8-fold as compared to control tissues ( P < 0.05 for all). Conclusions. IDO and the IDO-related pathway are important mediators of the immunoinflammatory responses in advanced atherosclerosis offering new viable therapeutic targets for the development of antiatherogenic immunosuppressive therapies. [ABSTRACT FROM AUTHOR]

Objective. Previously, we scanned all 23,000 human genes for differential expression between normal and atherosclerotic tissues and found the involvement of ADAM8. Methods. We investigated the expression of ADAM8 mRNA and protein level in human atherosclerotic tissues and non-atherosclerotic internal thoracic arteries as well as the association of ADAM8 2662 T/G single nucleotide polymorphism (SNP) with the extent of coronary atherosclerosis and with the risk of fatal myocardial infarction. Results. ADAM8 mRNA was up-regulated in carotid, aortic, and femoral atherosclerotic plaques (n=24) when compared with non-atherosclerotic arteries. ADAM8 protein expression was increased in advanced atherosclerotic plaques as compared to control vessels wherein it was localized to macrophages and smooth muscle cells The G allele carriers of the ADAM8 2662 T/G SNP had significantly larger areas of fibrotic, calcified, and complicated plaques in coronary arteries (P=0.027, P=0.011, and P=0.011, respectively) and significantly higher occurrence of myocardial infarction (MI) (P=0.004) and fatal pre-hospital MI (P=0.003) than did the TT homozygotes. Conclusion. ADAM8 is a promising candidate to be involved in atherosclerosis, and its 2662 T/G allelic variant significantly associates with advanced atherosclerotic lesion areas and MI. [ABSTRACT FROM AUTHOR]

Aim. The differentiation efficiencies of human embryonic stem cell (hESC) lines differ from each other. To assess this in more detail we studied the cardiac differentiation of eight hESC lines derived in the same laboratory. Results. Substantial variation in growth and in the ability to form beating areas was seen between the different hESC lines; line HS346 gave the best efficiency (9.4%), while HS293 did not differentiate into beating colonies at all. Nine germ layer and differentiation markers were quantified during early differentiation in four hESC lines. The expression levels of Brachyury T, MESP1 and NKX2.5 were highest in the most efficient cardiac line (HS346). A systematic characterization of the beating cells revealed proper cardiac marker expression, electrophysiological activity, and pharmacological response. Conclusions. The hESC lines derived in the same laboratory varied considerably in their potential to differentiate into beating cardiomyocytes. None of the expression markers could clearly predict cardiac differentiation potential, although the expression of early cardiomyogenic genes was upregulated in the best cardiac line. The proper cardiomyocyte characteristics and pharmacological response indicate that these cells could be used as a model for human cardiomyocytes in pharmacological and toxicological analyses when investigating new heart medications or cardiac side-effects. [ABSTRACT FROM AUTHOR]

Background and aims. The expression of disintegrin and metalloprotease ADAM-9, ADAM-15, and ADAM-17 has been associated with cell-cell, cell-platelet, and cell-matrix interactions and inflammation. They are possibly implicated in the pathophysiology of atherosclerosis. Methods and results. Whole-genome expression array and quantitative real-time polymerase chain reaction (PCR) analysis confirmed that ADAM-9, ADAM-15, and ADAM-17 are upregulated in advanced human atherosclerotic lesions in samples from carotid, aortic, and femoral territories compared to samples from internal thoracic artery (ITA) free of atherosclerotic plaques. Western analysis indicated that the majority of these ADAMs were in the catalytically active form. ADAM-9, ADAM-15, and ADAM-17-expressing cells were shown to co-localize with CD68-positive cells of monocytic origin in the atherosclerotic plaques using immunohistochemistry and double-staining immunofluorescence analysis. Co-localization was demonstrated in all vascular territories. In the carotid territory, cells expressing the ADAMs co-distributed also with smooth muscle cells and, in femoral territory, with CD31-positive endothelial cells, indicating that the ADAM expression pattern depends on vascular bed territory. Conclusions. Present findings provide strong evidence for the involvement of catalytically active ADAM-9, ADAM-15, and ADAM-17 in advanced atherosclerosis, most notably associated with cells of monocytic origin. [ABSTRACT FROM AUTHOR]

Glycosylation of proteins and lipids is important in cellular communication and maintenance of tissues. B3GTL (β3-glycosyltransferase-like) is a novel glycosyltransferase that is found in multicellular animals ranging from mammals to insects and nematodes. The aim of this work was to identify and characterize the B3GTL gene in the mouse and to study its expression in various tissues. The murine gene codes for a protein which shares 84% amino acid sequence identity with its human ortholog, and contains all the primary structural features that characterize B3GTL proteins. The murine and human B3GTL genes share an identical exon/intron organization, and both genes utilize multiple polyadenylation signals. Their promoter regions show extensive conservation, implying that the two genes also share regulatory similarities. This notion was reinforced by Northern hybridization analysis of mouse tissues, which showed the tissue distribution of B3GTL mRNA to be similar to that previously found in human tissues, with the heart, kidney, and brain being major sites of expression in both species. The localization of B3GTL mRNA was studied by in situ hybridization in an extensive collection of mouse tissues, of which the granular cells of the olfactory bulb and the epithelium of the seminal vesicle displayed particularly strong signals. Together, these analyses indicate that the B3GTL mRNA is subject to strong tissue-specific and developmental regulation. The findings reported here make possible the design of a B3GTL "knock-out" mouse, provide a framework for analyzing the regulation of the gene, and provide an extensive catalog of tissues in which this novel protein acts. [ABSTRACT FROM AUTHOR]

Nuclear receptors are master regulators of metazoan gene expression with crucial roles during development and in adult physiology. Fushi tarazu factor 1 (FTZ-F1) subfamily members are ancient orphan receptors with homologues from Drosophila to human that regulate diverse gene expression programs important for developmental processes, reproduction and cholesterol homeostasis in an apparently ligand-independent manner. Thus, developmental and tissue-specific cofactors may be particularly important in modulating the transcriptional activities of FTZ-F1 receptors. In Drosophila, the homeodomain protein Fushi tarazu acts as a cofactor for FTZ-F1 (NR5A3), leading to the hypothesis that a similar type of homeodomain cofactor-nuclear receptor relationship might exist in vertebrates. In this study, we have identified and characterized the homeodomain protein Prox1 as a co-repressor for liver receptor homologue 1 (LRH1/NR5A2), a master regulator of cholesterol homeostasis in mammals. Our study suggests that interactions between LRH1 and Prox1 may fulfil roles both during development of the enterohepatic system and in adult physiology of the liver. [ABSTRACT FROM AUTHOR]

Examines the importance of mechanical stress for the regulation of tenascin-C expression in the muscle-tendon unit. Accounts on the signal transduction pathway that involves matrix metalloproteinases; Expression of tenascin-C in normal myotendinous and myofascial junction; Induction of a severe atrophy of the tissues.

Background A rat line developed by selective breeding for high alcohol sensitivity has blunted corticosterone responses to alcohol and stress. In the present study, we determined possible differences in adrenal activation after alcohol and motor performance testing between the alcohol-sensitive alcohol-nontolerant and alcohol-insensitive alcohol-tolerant rats. Methods The animals received ethanol (2 g/kg, intraperitoneally), and 30 min later they were subjected to a motor function test (i.e., normal selection test used in the breeding of the lines); the control animals for both rat lines received no treatment and minimal handling. Blood corticosterone and ACTH levels at the single time point were determined by radioimmunoassay, and adrenal activation was determined by in situ hybridization of the immediate early gene c-fos, nor1, nurr1, and NGFI-B mRNA expression. Results The alcohol nontolerant rats had lower corticosterone but normal ACTH levels after ethanol and motor testing. Adrenal early gene expression of all of the genes studied was strongly induced by the treatment in both rat lines, but the inductions of c-fos, nor1, and nurr1 were significantly lower in the alcohol-sensitive animals. Acute treatment with a high dose of ACTH also induced less adrenal gene expression in the alcohol-sensitive animals. Conclusions The results suggest that the reduced adrenal activation is associated with high alcohol sensitivity in a genetic animal model, which is in agreement with the human findings of alcohol insensitivity during glucocorticoid treatment. [ABSTRACT FROM AUTHOR]

An acidic milieu is required for sperm maturation and for keeping sperm quiescent during storage in the cauda epididymidis. Previous studies have implicated a Na+/H+ exchanger (NHE) in epididymal acidification together with carbonic anhydrase (CA) and vacuolar proton adenosine triphosphatase (H+-ATPase). The present studies were undertaken to discover whether the NHE isoform involved is NHE-3, which is known to mediate Na+ and HCO3– absorption in renal tubules. Using the reverse transcription polymerase chain reaction technique (RT-PCR), Northern blot analysis and in situ hybridization, NHE-3 mRNA was detected mainly in the cauda epididymis and to a lesser extent in other regions of the epididymis. Immunohistochemical studies showed that NHE-3 was present in the apical membranes of the epithelial principal cells and confirmed that its expression is strongest in the cauda region, decreasing towards the more proximal regions. Immunoblotting showed a similar expression pattern. These results demonstrate that NHE-3 is expressed in the rat epididymal duct with strongest expression in its cauda region. These findings are thus consistent with the possibility that NHE-3 in the epididymal duct is involved in luminal Na+ and/or HCO3– absorption, as in the renal proximal tubule, and thereby in the regulation of sperm motility and maturation. [ABSTRACT FROM AUTHOR]

Abstract Thioredoxins are a class of small redox-regulating proteins that have been implicated in the control of various aspects of cellular functions and seem to be one of the key regulators of signalling in the cellular responses to various stresses. Thioredoxin-2 (Trx2) is a novel mammalian thioredoxin which, in contrast to previously known cytosolic thioredoxin (Trx1), has been localized to the mitochondria. Trx2 is abundantly expressed in skeletal muscle, heart and adrenal gland, as well as in some other peripheral tissues with high metabolic activity. Using in situ hybridization and immunohistochemistry, we have studied the distribution and regulation of Trx2 expression in the rat brain. Trx2 mRNA and protein are highly expressed in the neurons in several brain regions, including the olfactory bulb, frontal cortex, hippocampus, some hypothalamic and thalamic nuclei, cerebellum and numerous brainstem nuclei. In addition, the Trx2 mRNA expression in paraventricular hypothalamic nucleus and reticular thalamic nucleus was found to be sensitive to peripheral glucocorticoids, as dexamethasone treatment caused significant elevation of Trx2 mRNA level in this area. No changes in other brain areas were observed after dexamethasone treatment. These findings implicate a significant regulatory and/or protective function of Trx2 in the nervous system. [ABSTRACT FROM AUTHOR]

Autoimmune-polyndocrinopathy-candidiasis-ecto-dermal dystrophy (APECED) is the only systemic autoimmune disease with a monogenic background known so far revealing no association with the major histocompatibility complex region. We have recently isolated the gene defective in this syndrome and characterized several different mutations in individuals with the disorder. The novel gene, AIRE, contains a putative bipartite nuclear targeting signal predicting a nuclear location of the corresponding protein. The presence of two PHD-type zinc finger domains as well as the newly described putative DNA-binding domain, SAND, in the amino acid sequence of the APECED protein implies that it may be involved in the regulation of gene expression. Using transient expression of AIRE cDNA in mammalian cells we demonstrate here the nuclear location of the APECED protein. Immunohistochemical staining of transfected cells revealed that most of the recombinant 58 kDa APECED protein is present in the form of nuclear dots. By double immuno-fluorescence labelling we further show that these APECED-containing structures and the previously described PML nuclear bodies are largely non-overlapping. The AIRE protein was also visualized in multiple human tissues: a subset of the cells in thymus, in spleen and in lymph node showed nuclear staining with APECED antiserum. Immunofluorescence labelling of peripheral blood mononuclear leukocytes also revealed a nuclear body-like staining pattern in a fraction of these cells. These data from both in vitro and ex vivo systems, together with the predicted structural features of the APECED protein, suggest that this protein is most probably involved in the regulation of gene expression. [ABSTRACT FROM AUTHOR]