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Acute alcohol administration is harmful especially for the developing nervous system, where it induces massive apoptotic neurodegeneration leading to alcohol-related disorders of newborn infants. Neuroprotection against ethanol-induced apoptosis may save neurons and reduce the consequences of maternal alcohol consumption. Previously we have shown that taurine protects immature cerebellar neurons in the internal granular layer of cerebellum from ethanol-induced apoptosis. Now we describe a similar protective action for taurine in the external layer of cerebellum of 7-day-old mice. 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 0 h and 2.5 g/kg at 2 h) to the ethanol and ethanol + taurine groups. The ethanol + taurine group also received subcutaneously two injections of taurine (1 g/kg each, 1 h before the first dose of ethanol and 1 h after the second dose of ethanol). To verify 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 8 h after the first ethanol injection. Ethanol induced apoptosis in the cerebellar external granular layer. Taurine treatment significantly reduced the number of activated caspase-3-immunoreactive and TUNEL-positive cells. Taurine has thus a neuroprotective antiapoptotic action in the external granular layer of the cerebellum, preserving a number of neurons from ethanol-induced apoptosis.

The ability to utilize oxygen has been shown to affect a wide variety of physiological factors often considered beneficial for survival. As the ability to learn can be seen as one of the core factors of survival in mammals, we studied whether selective breeding for endurance running, an indication of aerobic capacity, also has an effect on learning. Rats selectively bred over 23 generations for their ability to perform forced treadmill running were trained in an appetitively motivated discrimination-reversal classical conditioning task, an alternating T-maze task followed by a rule change (from a shift-win to stay-win rule) and motor learning task. In the discrimination-reversal and T-maze tasks, the high-capacity runner (HCR) rats outperformed the low-capacity runner (LCR) rats, most notably in the phases requiring flexible cognition. In the Rotarod (motor-learning) task, the HCR animals were overall more agile but learned at a similar rate with the LCR group as a function of training. We conclude that the intrinsic ability to utilize oxygen is associated especially with tasks requiring plasticity of the brain structures implicated in flexible cognition., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Atherosclerosis is a complex disease with hundreds of genes influencing its progression. In addition, the phenotype of the disease varies significantly depending on the arterial bed., Methodology/principal Findings: We characterized the genes generally involved in human advanced atherosclerotic (AHA type V-VI) plaques in carotid and femoral arteries as well as aortas from 24 subjects of Tampere Vascular study and compared the results to non-atherosclerotic internal thoracic arteries (n=6) using genome-wide expression array and QRT-PCR. In addition we determined genes that were typical for each arterial plaque studied. To gain a comprehensive insight into the pathologic processes in the plaques we also analyzed pathways and gene sets dysregulated in this disease using gene set enrichment analysis (GSEA). According to the selection criteria used (>3.0 fold change and p-value <0.05), 235 genes were up-regulated and 68 genes down-regulated in the carotid plaques, 242 genes up-regulated and 116 down-regulated in the femoral plaques and 256 genes up-regulated and 49 genes down-regulated in the aortic plaques. Nine genes were found to be specifically induced predominantly in aortic plaques, e.g., lactoferrin, and three genes in femoral plaques, e.g., chondroadherin, whereas no gene was found to be specific for carotid plaques. In pathway analysis, a total of 28 pathways or gene sets were found to be significantly dysregulated in atherosclerotic plaques (false discovery rate [FDR] <0.25)., Conclusions: This study describes comprehensively the gene expression changes that generally prevail in human atherosclerotic plaques. In addition, site specific genes induced only in femoral or aortic plaques were found, reflecting that atherosclerotic process has unique features in different vascular beds.

Background: Proprotein convertase subtilisin/kexin (PCSK) enzymes cleave proproteins into mature end products. Previously, MBTPS1 and PCSK9 have been shown to regulate cholesterol metabolism and LDL receptor recycling, whereas FURIN and PCSK5 have been suggested to inactivate lipases and regulate inflammation in atherosclerosis. Here, we systematically analyzed the expression of PCSKs and their targets in advanced atherosclerotic plaques., Methods and Results: Microarray and quantitative real-time PCR experiments showed that FURIN (42.86 median fold, p = 2.1e-8), but no other PCSK, is universally overexpressed in the plaques of different vascular regions. The mRNA expression screen of PCSK target proteins in plaques identified many known factors, but it also identified the significant upregulation of the previously overlooked furin-processed B cell activating cytokines APRIL (TNFSF13, 2.52 median fold, p = 3.0e-5) and BAFF (TNFSF13B, 2.97 median fold, p = 7.6e-6). The dysregulation of FURIN did not associate with its htSNPs or the previously reported regulatory SNP (-229, rs4932178) in the promoter. Immunohistochemistry experiments showed the upregulation of FURIN in the plaque lymphocytes and macrophages where it was co-expressed with BAFF/TNFSF13B and APRIL/TNFSF13., Conclusions: Our data unequivocally show that FURIN is the primary PCSK that is dysregulated in the immune cells of advanced human atherosclerotic plaques, which implies a role for this enzyme in plaque pathology. Therefore, drugs that inhibit FURIN in arteries may modulate the course of this disease., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

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.

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.

Objective: The single nucleotide polymorphism (SNP) rs2995300 in the metalloproteinase-disintegrin gene ADAM8 has been shown to affect the areas of complicated coronary plaques and the risk of fatal myocardial infarction (MI) in men. This study was set up to further investigate the role of ADAM8 in MI., Aim: To investigate the possible association of the ADAM8 SNPs rs2995300 and rs2275725 with ADAM8 mRNA levels, serum soluble ADAM8 (sADAM8) concentrations, and MI risk., Methods: Samples from the Finnish cardiovascular study (FINCAVAS, N=2156) and the angiography and genes study (ANGES, N=1000) were genotyped. Serum sADAM8 concentrations were determined with ELISA (N=443). ADAM8 mRNA levels in atherosclerotic plaques were analysed from the tampere vascular study (TVS, N=53) samples., Results: A significantly increased MI risk for carriers of the rs2995300C allele and the rs2275725 A allele was revealed in the meta-analysis of the ANGES and FINCAVAS patient data (OR=1.42, P<0.001 and OR=1.43, P<0.001). The risk increase was comparable to that caused by smoking in these cohorts. The risk allele carriers also had higher sADAM8 serum concentrations., Conclusions: The risk alleles of the investigated ADAM8 SNPs were associated with elevated sADAM8 serum levels and MI risk. The present results implicate ADAM8 in the development of CVDs and suggest its prognostic and therapeutic potential., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Our previous study demonstrated that preconditioning by 3-times repetitive mild hypoxia significantly augmented expression of mitochondrial thioredoxin-2 (Trx-2) at 3 h after subsequent acute severe hypoxia in rat hippocampus. However, it was unclear whether this augmentation was due to build up of Trx-2 by mild hypoxia before severe hypoxia or by modification of reaction to severe hypoxia itself. To answer on this question we study the expression level during and after preconditioning without subsequent severe hypoxia. Trx-2 expression was studied by immunocytochemistry 3 h and 24 h after first session and 3 h and 24 h after last session of 3-times (spaced at 24 h) mild hypobaric hypoxia (360 Torr, 2h). At 3 h after 1-time hypoxia (first session of 3-time hypoxia) the total number of Trx-2-immunoreactive cells (Nt) was significantly decreased in contrast with control in CA2, CA3 and DG. The number of cells with intensive expression of Trx-2 (Ni) was reduced in CA1 and CA3. At 24 h after the same 1-time hypoxia Nt was lower than in control and at 3 h time-point in all hippocampal areas studied (CA1, CA2, CA3 and DG); Ni was decreased only compared to control in CA1 and CA3. At 3 h after last session of 3-times hypoxia Nt and Ni were significantly down regulated in comparison with control only in CA1. At 24 h after it Nt was significantly decreased compared to control in CA1, CA2 and CA3 (in DG the decrease was not statistically significant) but in all areas was higher than at 24 h after 1-time hypoxia. Dynamics of Nt changes from 3-hours after single to 24-hours after triple moderate hypoxia had the wave phase character. These findings indicate that Trx-2 expression in most areas of hippocampus was decreased to 24 h after 3-time mild hypoxia. Thus the augmentation of Trx-2 expression in hippocampal neurons of preconditioned animals in response to subsequent severe hypoxia is caused obviously not by Trx-2 accumulation during preconditioning sessions but by modification of reaction to severe impact.

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.

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.

Human embryonic stem cell (hESC) differentiation in embryoid bodies (EBs) provides a valuable tool to study the interplay of different germ layers and their influence on cell differentiation. The gene expression of the developing EBs has been shown in many studies, but the protein expression and the spatial composition of different germ layers in human EBs have not been systematically studied. The aim of the present work was to study the temporal and spatial organisation of germ layers based on the expression of mesoderm (Brachyury T), endoderm (AFP) and ectoderm (SOX1) markers during the early stages of differentiation in eight hESC lines. Tissue multi-array technology was applied to study the protein expression of a large number of EBs. According to our results, EB formation and the organisation of germ layers occurred in a similar manner in all the lines. During 12 days of differentiation, all the germ layer markers were present, but no obvious distinct trajectories were formed. However, older EBs were highly organised in structure. Pluripotency marker OCT3/4 expression persisted unexpectedly long in the differentiating EBs. Cavity formation was observed in the immunocytological sections, and caspase-3 expression was high, suggesting a role of apoptosis in hESC differentiation and/or EB formation. The expression of Brachyury T was notably low in all the lines, also those with the best cardiac differentiation capacity, while the expression of SOX1 was higher in some lines, suggesting that the neural differentiation propensity may be detectable already in the early stages of EB differentiation.

Corticosteroid hormones are important intrinsic factors that not only mediate the response to stress but also largely contribute to the main physiological processes. The biological actions of these steroids involve, first of all, the activation of specific receptors, namely mineralocorticoid (MR) and glucocorticoid (GR) receptors. These two receptor types govern a flexible and well-balanced mechanism that leads to the often opposing changes in the cell. The hippocampus is the central part of the extrahypothalamic feedback loop in the control of the hypothalamic-pituitary-adrenal (HPA) axis activity. The coexpression of both MR and GR in the hippocampus serves a coordinated response to corticosteroids in the hippocampal neurons, thereby mediating the neuronal excitability, stress response, and behavioral adaptation. Each receptor type reveals distinct ontogenetic pattern over the postnatal period. This review addresses the issues relating to postnatal development of the HPA axis and especially the hippocampal expression of the GR proteins in intact and prenatally stressed rats., (Copyright 2010 Elsevier Inc. All rights reserved.)

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.

Genetic and experimental evidence points to a critical involvement of the atypical mammalian orphan receptor DAX-1 in reproductive development and steroidogenesis. Unlike conventional nuclear receptors, DAX-1 appears not to function as a DNA-bound transcription factor. Instead, it has acquired the capability to act as a transcriptional corepressor of steroidogenic factor 1 (SF-1). The interplay of DAX-1 and SF-1 is considered a central, presumably ligand-independent element of adrenogonadal development and function that requires tight regulation. This raises a substantial interest in identifying its modulators and the regulatory signals involved. Here, we uncover molecular mechanisms that link DAX-1 to the ubiquitin modification system via functional interaction with the E3 ubiquitin ligase RNF31. We demonstrate that RNF31 is coexpressed with DAX-1 in steroidogenic tissues and participates in repressing steroidogenic gene expression. We provide evidence for the in vivo existence of a corepressor complex containing RNF31 and DAX-1 at the promoters of the StAR and CYP19 genes. Our data suggest that RNF31 functions to stabilize DAX-1, which might be linked to DAX-1 monoubiquitination. In conclusion, RNF31 appears to be required for DAX-1 to repress transcription, provides means to regulate DAX-1 in ligand-independent ways, and emerges as a relevant coregulator of steroidogenic pathways governing physiology and disease.

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.

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.

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.

Acute ethanol administration causes extensive apoptosis throughout the nervous system. We studied the protective effect of taurine on alcohol-induced apoptosis in the cerebellum of developing mice. Taurine rescued a part of immature neurons by markedly reducing caspase-3 immunoreactivity and the number of TUNEL-positive cells in most cerebellar lobules.

Glycosylation of proteins and lipids is important in cellular communication and maintenance of tissues. B3GTL (beta3-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.

Circulating glucocorticoids, of which their concentration is largely under the control of the hypothalamic-pituitary adrenal (HPA) axis, acting through the glucocorticoid receptors (GR) regulate a large variety of pivotal functions of the organism such as growth, development, immune- and stress-response. The main mechanism of regulation of the HPA axis activity is via negative feedback at all levels of the HPA axis itself as well as at the extra-hypothalamic level, a central part of which is the hippocampus. During neonatal development, the HPA axis of rats undergoes a period of hyporesponsiveness (SHRP)-when most stress stimuli fail to induce stress-response. Here, we describe the pattern of GR proteins expression in the hippocampal area of the rat brain during postnatal development and in adulthood. We demonstrated that the GR protein, of which its expression level is gradually enhanced in the hippocampus during postnatal life, exists in three different molecular sized forms. A larger molecular form was expressed at rather high levels at all studied time periods. A second smaller variant of GR was transiently expressed during the first one and a half weeks that corresponds with SHRP and then appeared again only in the adulthood. By the end of SHRP on PD 13, third smallest protein form of GR started to be detected in the hippocampal area. Thus, it remains to be disclosed in the nearest future, how the hippocampal GR isoforms may be involved in regulation of the neonatal HPA axis hyporesponsiveness as well as in functions of this system during the ensuing period of the brain maturation.

The thioredoxin system, composed of thioredoxin (Trx) and thioredoxin reductase (TrxR), emerges as one of the most important thiol-based systems involved in the maintenance of the cellular redox balance. Thioredoxin-like-1 (TXL-1) is a highly conserved protein comprising an N-terminal Trx domain and a C-terminal domain of unknown function. Here we show that TXL-1 is a substrate for the cytosolic selenoprotein TrxR-1. In situ hybridization experiments demonstrates high expression of Txl-1 mRNA in various areas of central nervous system and also in some reproductive organs. Glucose deprivation, but not hydrogen peroxide treatment, reduced the levels of endogenous TXL-1 protein in HEK-293 cell line. Conversely, overexpression of TXL-1 protects against glucose deprivation-induced cytotoxicity. Taken together, the finding that Txl-1 mRNA is highly expressed in tissues which use glucose as a primary energy source and the modulation of TXL-1 levels upon glucose deprivation indicate that TXL-1 might be involved in the cellular response to sugar starvation stress.

Severe hypoxia results in functional and structural injury of the brain. A preconditioning with repetitive episodes of mild hypoxia considerably ameliorates neuronal resistance to subsequent severe hypoxia. Activation of endogenous antioxidants including Cu, Zn-depending superoxide dismutase (Cu, Zn-SOD) (EC.1.15.1.1) is one of the main cell defense mechanisms against oxidative stress induced by hypoxia. Alterations of expression and enzyme activity of Cu, Zn-SOD 3 and 24h after severe hypobaric hypoxia in forebrain structures of preconditioned and non-preconditioned rats were investigated. We found that hypoxia without preconditioning suppressed the Cu, Zn-SOD enzyme activity at 3h time-point but preconditioning essentially modified the reaction to severe hypoxia by increasing the expression and activity of Cu, Zn-SOD during early stages of reoxygenation crucial for apoptosis initiation.

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.

The aim of this work was to study effects of mild preconditioning hypobaric hypoxia (380 Torr for 2 h, repeated 3 or 6 times spaced at 24 h) on brain NGFI-A immunoreactivity and passive avoidance (PA) behavior in rats exposed to severe hypoxia (160 Torr for 3 h). Severe hypobaric hypoxia produced extensive neuronal loss in hippocampal CA1, while the preceding hypoxic preconditioning had clear protective effect on neuronal viability of vulnerable hippocampal cells. Besides, the hypoxic preconditioning prevented impairment of acquisition and retention of PA caused by severe hypoxia. The six-trial hypobaric preconditioning was more effective in protection against PA learning deficits in severe hypoxia exposed rats than the three-trial preconditioning. The preconditioning up-regulated severe hypoxia-suppressed neocortical and hippocampal expression of NGFI-A, suggesting a possible role for NGFI-A in the neuroprotective mechanisms activated by hypoxic preconditioning.

The de novo synthesis and expression of sulfo sLex glycan on vascular endothelial glycoproteins has a central role in the initiation of inflammatory reactions, serving as a putative ZIP code for organ-specific trafficking of leukocytes into sites of inflammation. The synthesis of sulfo sLex requires energy carrying donors, CMP-sialic acid (CMP-SA), GDP-fucose (GDP-Fuc), and adenosine 3'-phosphate 5'-phosphosulphate (PAPS) for donation of SA, Fuc, and sulfate, respectively. These donors are synthesized in the nucleus or cytosol and translocated into Golgi by specific transporters where corresponding transferase and proteins as well as enzymatic activities increase on inflammatory stimuli. Here we analyze the transcriptional coregulation of CMP-SA, GDP-Fuc, and PAPS transporters with in situ hybridization and real-time PCR in acute inflammation using kidney and heart allografts as model systems. Our results indicate that these three transporters display coordinated transcriptional regulation during the induction of the sulfo sLex glycan biosynthesis. With in silico analysis, the data generated with 230 human Affymetrix U133A gene chips indicated that the coregulated expression of CMP-SA and GDP-Fuc transporters was not common. Taken together our results suggest that inflammation-induced transcriptional regulation exists for Golgi membrane transporters required for the synthesis of the inflammation-inducible ZIP code sulfo sLex glycans.

The impact of severe hypoxia and preconditioning on the expression of the mitochondrial antioxidant thioredoxin-2 (Trx-2) in rat hippocampus (CA1, CA2, CA3 fields, and dentate gyrus) and neocortex was studied by immunocytochemistry. The preconditioning consisted of three trials of mild hypobaric hypoxia (360 Torr, 2 hr) spaced at 24 hr. The last trial was followed by severe hypobaric hypoxia (180 Torr, 3 hr) 24 hr later. Both in hippocampus and in neocortex, severe hypobaric hypoxia resulted in enhanced Trx-2 expression at 3 hr, followed by a slight decline in Trx-2 levels, which nevertheless remained increased at 24 hr elsewhere except for the CA1 region. The preconditioning considerably augmented severe hypoxia-induced Trx-2 immunoreactivity, affecting both the number of immunoreactive cells and the intensity of immunostaining. The findings suggest a role for Trx-2 in the formation of brain hypoxic/ischemic tolerance accomplished by the preconditioning.

Induction of endogenous antioxidants is one of the key molecular mechanisms of cell resistance to hypoxia/ischemia. The effect of severe hypoxia on the expression of cytosolic antioxidant thioredoxin-1 (Trx) in hippocampus and neocortex was studied in preconditioned and non-preconditioned rats. The preconditioning consisted of three trials of mild hypobaric hypoxia (360 Torr, 2 h) spaced at 24 h. Twenty-four hours after the last trial rats were subjected to severe hypobaric hypoxia (180 Torr, 3 h). Trx expression was studied by immunocytochemistry. In hippocampus severe hypobaric hypoxia rapidly induced Trx expression, which remained elevated still at 24 h. In neocortex the enhanced expression appeared only at 24 h. The preconditioning significantly augmented severe hypoxia-induced Trx-immunoreactivity at 3 h but not at 24 h. These findings point out that Trx contributes to mechanisms of brain tolerance to hypobaric hypoxia, especially in early periods after the exposure.

Mammalian germ cells are endowed with a complete set of thioredoxins (Trx), a class of redox proteins located in specific structures of the spermatid and sperm tail. We report here the characterization, under normal and pathological conditions, of a novel thioredoxin with a germ line-restricted expression pattern, named spermatocyte/spermatid-specific thioredoxin-3 (SPTRX-3). The human SPTRX-3 gene maps at 9q32, only 50 kb downstream from the TRX-1 gene from which it probably originated as genomic duplication. Therefore, human SPTRX-3 protein comprises a unique thioredoxin domain displaying high homology with the ubiquitously expressed TRX-1. Among the tissues investigated, Sptrx-3 mRNA is found exclusively in the male germ cells at pachytene spermatocyte and round spermatid stages. Light and electron microscopy show SPTRX-3 protein to be predominately located in the Golgi apparatus of pachytene spermatocytes and round and elongated spermatids, with a transient localization in the developing acrosome of round spermatids. In addition, increased levels of SPTRX-3, possibly caused by overexpression, are observed in morphologically abnormal human spermatozoa from infertile men. In addition, SPTRX-3 is identified as a novel postobstruction autoantigen. In this report, we propose that SPTRX-3 can be used as a specific marker for diverse sperm and testis pathologies. SPTRX-3 is the first thioredoxin specific to the Golgi apparatus, and its function within this organelle might be related to the post-translational modification of proteins required for germ cell-specific functions, such as acrosomal biogenesis.

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.

Thioredoxins compose a growing family of proteins that participate in different cellular processes via redox-mediated reactions. We report here the cloning, developmental expression, and location of murid Sptrx-2. Mouse and rat SPTRX-2 proteins display a high homology to their human ortholog in the thioredoxin and NDP kinase domains, and the coding genes are located at syntenic positions. Northern blotting and in situ hybridization confirmed the testis-specific expression of murine Sptrx-2 mRNA, mostly in round spermatids. Immunohistochemical analysis of the 19 steps of rat spermiogenesis showed that SPTRX-2 expression becomes prominent in the cytoplasmic lobe of step 15-18 spermatids and diminishes in step 19 just before spermiation. However, in the spermatid tail, SPTRX-2 immunoreactivity increased from step 15 to 19 and was confined to the principal piece. By immunogold electron microscopy, SPTRX-2 was first detected scattered throughout the cytoplasm of the axoneme in step 14-15 spermatids, but began to be incorporated by step 16 into the fibrous sheath (FS). During steps 17-18, the labeling increased over the ribs and columns of the assembled FS. It peaked in step 19 and remained in the FS of epididymal spermatozoa. Immunoblots of isolated FS obtained from spermatozoa confirmed that SPTRX-2 is an integral component of the FS and a post-obstruction autoantigen in vasectomized rats. Our data indicate that SPTRX-2 incorporation into the FS lags well behind FS assembly, suggesting it is required during the final stages of sperm tail maturation in the testis and/or epididymis, where extensive disulfide bonding of FS proteins occurs.

Aint was originally identified on the basis of its interaction in vitro with the aryl hydrocarbon nuclear receptor translocator (Arnt). Arnt is a common heterodimerization partner in the basic helix-loop-helix (bHLH)-PER-ARNT-SIM (PAS) protein family and is involved in diverse biological functions. These include xenobiotic metabolism, hypoxic response, and circadian rhythm. In addition, Arnt has a crucial role during development. Aint is a member of a growing family of transforming acidic coiled-coil (TACC) proteins and is the murine homologue of human TACC3. Here we report the spatiotemporal expression of Tacc3 mRNA and protein in embryonic, postnatally developing, and adult mouse tissues using in situ hybridization and immunocytochemistry. Tacc3 mRNA was highly expressed in proliferating cells of several organs during murine development. However, the only adult tissues expressing high levels were testis and ovary. Immunocytochemistry revealed that Tacc3 is a nuclear protein. Our results suggest that Tacc3 has an important role in murine development, spermatogenesis, and oogenesis.

Dioxins, e.g. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), use the aryl hydrocarbon receptor (AHR)/aryl hydrocarbon receptor nuclear translocator (ARNT) receptor complex to mediate their toxic actions. In addition to interaction with environmental pollutants, several transcription factors, steroid receptors, and growth factors are capable interacting with the AHR/ARNT complex, which suggests a constitutive role for the receptor complex. The testis has been reported to be among the most sensitive organs to TCDD exposure. Our experiments revealed a complex distribution of AHR and ARNT mRNAs and proteins in rat and human testis. AHR and ARNT immunoreactivities could be detected in the nuclei of interstitial and tubular cells. The incubation of seminiferous tubules in a serum-free culture medium resulted in up-regulation of AHR mRNA, which could be depressed by adding FSH to the culture medium. Furthermore, the incubation of tubular segments with a solution of 1 or 100 nM TCDD resulted in a 2- to 3-fold increase in apoptotic cells. Thus, up-regulation of AHR in cultured tubular segments and consecutive depression by FSH suggest a role for AHR in controlled cell death during spermatogenesis. We suggest that AHR and ARNT mediate effects by direct action on testicular cells in the rat and human testis.

Tenascin-C is a hexabrachion-shaped matricellular protein with a very restricted expression in normal musculoskeletal tissues, but it is expressed abundantly during regenerative processes of these tissues and embryogenesis. To examine the importance of mechanical stress for the regulation of tenascin-C expression in the muscle-tendon unit, the effects of various states of mechanical loading (inactivity by cast-immobilization and three-varying intensities of subsequent re-activity by treadmill running) on the expression of tenascin-C were studied using immunohistochemistry and mRNA in situ hybridization at the different locations of the muscle-tendon unit of the rat gastrocnemius muscle, the Achilles tendon complex. This muscle-tendon unit was selected as the study site, because the contracting activity of the gastrocnemius-soleus muscle complex, and thus the mechanical loading-induced stimulation, is easy to block by cast immobilization. Tenascin-C was expressed abundantly in the normal myotendinous and myofascial junctions, as well as around the cells and the collagen fibers of the Achilles tendon. Tenascin-C expression was not found in the normal skeletal muscle, although it was found in blood vessels within the muscle tissue. Following the removal of the mechanical loading-induced stimulation on the muscle-tendon unit by cast immobilization for 3 weeks, the immonoreactivity of tenascin-C substantially decreased or was completely absent in the regions expressing tenascin-C normally. Restitution of the mechanical loading by removing the cast and allowing free cage activity for 8 weeks resulted in an increase in tenascin-C expression, but it could not restore the expression of tenascin-C to the normal level (in healthy contralateral leg). In response to the application of a more strenuous mechanical loading stimulus after the removal of the cast (after 8 weeks of low- and high-intensity treadmill running), the expression of tenascin-C was markedly increased and reached the level seen in the healthy contralateral limb. Tenascin-C was abundantly expressed in myotendinous and myofascial junctions and in the Achilles tendon, but even the most strenuous mechanical loading (high-intensity treadmill running) could not induce the expression of tenascin-C in the skeletal muscle. This was in spite of the marked immobilization-induced atrophy of the previously immobilized skeletal muscle, which had been subjected to intensive stress during remobilization. mRNA in situ hybridization analysis confirmed the immunohistochemical results for the expression of tenascin-C in the study groups. In summary, this study shows that mechanical loading regulates the expression of tenascin-C in an apparently dose-dependent fashion at sites of the muscle-tendon unit normally expressing tenascin-C but can not induce de novo synthesis of tenascin-C in the skeletal muscle without accompanying injury to the tissue. Our results suggest that tenascin-C provides elasticity in mesenchymal tissues subjected to heavy tensile loading.

The basic helix-loop-helix (bHLH-PAS) proteins aryl hydrocarbon receptor nuclear translocator (Arnt) and Arnt2 are transcriptional regulators that function as dimerizing partners for several bHLH-PAS proteins and also some nonrelated partners. They are involved in various biological functions, including regulation of developmental genes. In earlier studies, the developmental expression of Arnt was reported to be almost ubiquitous, whereas Arnt2 expression has been shown to be more limited, comprising neuronal tissues as the main site of expression. Here we provide a detailed description of the expression of Arnt and Arnt2 mRNA in mouse tissues during embryonic and early postnatal development. Arnt and also Arnt2 transcripts, in contrast to earlier reports, are shown to be expressed more widely during development yet show a temporally and spatially specific pattern.

Thioredoxins (Trx) are a class of small multifunctional redox-active proteins found in all organisms. Recently, we reported the cloning of a mitochondrial thioredoxin, Trx2, from rat heart. To investigate the biological role of Trx2 we have isolated the human homologue, hTrx2, and generated HEK-293 cells overexpressing Trx2 (HEK-Trx2). Here, we show that HEK-Trx2 cells are more resistant toward etoposide. In addition, HEK-Trx2 are more sensitive toward rotenone, an inhibitor of complex I of the respiratory chain. Finally, overexpression of Trx2 confers an increase in mitochondrial membrane potential, DeltaPsi(m). Treatment with oligomycin could both reverse the effect of rotenone and decrease the membrane potential suggesting that Trx2 interferes with the activity of ATP synthase. Taken together, these results suggest that Trx2 interacts with specific components of the mitochondrial respiratory chain and plays an important role in the regulation of the mitochondrial membrane potential.

The effect of preconditioning (PC) on the changes of nerve growth factor-induced gene A (NGFI-A) expression induced by severe hypobaric hypoxia was studied by in situ hybridization. A PC consisted of three trials of mild hypobaric hypoxia (360 Torr, 2 h) spaced at 24 h. The last trial was followed by severe hypoxia (SH; 180 Torr, 3 h) 24 h later. The PC hypoxia prevented the NGFI-A messenger RNA (mRNA) increase in the cortex, neostriatum, piriform cortex, amygdala and hippocampus detected 3 h after SH. The preconditioned SH caused a peak in NGFI-A mRNA expression at the 24 h time-point and thus abolished the dramatic decrease of the mRNA in vulnerable areas seen by 24 and 72 h after SH. The findings suggest a role of brain NGFI-A in the protective effect of hypoxic/ischemic PC., (Copyright 2002 Elsevier Science Ireland Ltd.)

TRAP220 (thyroid hormone receptor-associated protein) is a recently cloned nuclear receptor coactivator, which interacts with several nuclear receptors in a ligand-dependent manner and stimulates transcription by recruiting the TRAP mediator complex to hormone responsive promoter regions. TRAP220 has been shown to interact with thyroid hormone receptors, vitamin D receptors, peroxisome proliferator-activated receptors, retinoic acid receptors and oestrogen receptors. Thyroid hormone and retinoic acid play very important roles in brain development and they also influence adult brain. Using in situ hybridization we have examined expression of TRAP220 mRNA in the central nervous system during development and in adult rat and mouse brain. Expression of TRAP220 was seen already during early embryonic development in the epithelium of neural tube at E9 in mouse and at E12 in rat. At later stages of development the strongest signal was seen in different layers of cerebral neocortex, external germinal layer of cerebellum, differentiating fields of hippocampus and neuroepithelium, and a moderate signal was detected in basal ganglia, different areas of diencephalon and midbrain. In adult rat brain the signal was more restricted than during development. TRAP220 expression occurred mostly in the granular layer of cerebellar cortex, piriform cortex and hippocampal formation. The signal was found predominantly in neurons. Our work supports the assumption that TRAP220 plays an important role in growth and differentiation of central nervous system and may have a function in certain areas of adult brain.

Thioredoxins are proteins that participate in different cellular processes via redox-mediated reactions. For humans, we have recently described two novel members of this family that display a male germ cell specific expression pattern, named spermatid specific thioredoxin (Sptrx-1 and Sptrx-2 respectively). We report here the cloning and characterization of the mouse Sptrx-1 gene and protein, which are similar to those described for the human orthologue. The mouse Sptrx-1 open reading frame encodes for a protein of 462 aa composed of an N-terminal repetitive domain of a 15 residue motif followed by a C-terminal domain typical of thioredoxins. The mouse Sptrx-1 gene sequence is interrupted by only one intron of 525 bp located in the 5'-UTR, and using fluorescence in-situ hybridization we have mapped its chromosomal location to 17E1.2-1.3. Northern blot analysis identified the testis as the only tissue expressing mouse Sptrx-1 mRNA, and by in-situ hybridization we found a strong labelling in the testicular seminiferous tubules, mostly in the round spermatids. Affinity purified antibodies against human Sptrx-1 crossreacted well with the mouse protein confirming its expression in seminiferous tubules at the later stages of spermiogenesis. Recombinant mouse Sptrx-1 displayed protein disulphide reducing activity in an enzymatic assay coupled to NADPH and thioredoxin reductase. The availability of the mouse Sptrx-1 gene sequence is the first step towards the generation of knock-out mice, whose characterization will provide significant information regarding the in-vivo function of Sptrx-1 and its possible implication in several sperm anomalies.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a monogenic autosomal recessive disease caused by mutations in the AIRE gene. Here we have produced knock-out mice for the Aire gene. The Aire-/- mice develop normally; however, autoimmune features of APECED in Aire-/- mice are evident, including multiorgan lymphocytic infiltration, circulating autoantibodies and infertility. The distribution of B and T cells and thymic maturation as well as activation of T cells appear normal, while the TCR-Vbeta repertoire is altered in peripheral T cells of Aire-/- mice. When mice are challenged with immunization, the peripheral T cells of Aire-/- mice have a 3-5-fold increased proliferation. These findings suggest that the Aire gene is not necessary for normal T cell education and development, while a defect in immune response detected in challenged Aire-/- mice underlines the crucial role of AIRE/Aire in maintaining homeostatic regulation in the immune system.