Genes involved in systemic and arterial bed dependent atherosclerosis--Tampere Vascular study.

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.

Taurine protects cerebellar neurons of the external granular layer against ethanol-induced apoptosis in 7-day-old mice.

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.

Selective breeding for endurance running capacity affects cognitive but not motor learning in rats.

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.

Proprotein convertases in human atherosclerotic plaques: the overexpression of FURIN and its substrate cytokines BAFF and APRIL.

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.

One- and three-time mild hypobaric hypoxia modifies expression of mitochondrial thioredoxin-2 in hippocampus of rat.

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.

Myocardial infarction induces early increased remote ADAM8 expression of rat hearts after cardiac arrest.

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.

A disintegrin and metalloprotease -8 and -15 and susceptibility for ascending aortic dissection.

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.

Common variation in the ADAM8 gene affects serum sADAM8 concentrations and the risk of myocardial infarction in two independent cohorts.

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.

Neuroprotection by taurine in ethanol-induced apoptosis in the developing cerebellum.

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.

Carbonic anhydrases II and XII are up-regulated in osteoclast-like cells in advanced human atherosclerotic plaques-Tampere Vascular Study.

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.

Postnatal ontogeny of the glucocorticoid receptor in the hippocampus.

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.

Spatial and temporal expression pattern of germ layer markers during human embryonic stem cell differentiation in embryoid bodies.

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.

Activation of indoleamine 2,3-dioxygenase-induced tryptophan degradation in advanced atherosclerotic plaques: Tampere vascular study.

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.

ADAM8 and its single nucleotide polymorphism 2662 T/G are associated with advanced atherosclerosis and fatal myocardial infarction: Tampere vascular study.

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.

ADAM-9, ADAM-15, and ADAM-17 are upregulated in macrophages in advanced human atherosclerotic plaques in aorta and carotid and femoral arteries--Tampere vascular study.

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.

E3 ubiquitin ligase RNF31 cooperates with DAX-1 in transcriptional repression of steroidogenesis.

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.

Taurine protects immature cerebellar granullar neurons against acute alcohol administration.

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.

Substantial variation in the cardiac differentiation of human embryonic stem cell lines derived and propagated under the same conditions--a comparison of multiple cell lines.

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.

Murine ortholog of the novel glycosyltransferase, B3GTL: primary structure, characterization of the gene and transcripts, and expression in tissues.

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.

Expression of glucocorticoid receptors in the hippocampal region of the rat brain during postnatal development.

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.