Adenosine receptors may be important determinants of intrinsic ischemic tolerance. Genetically modified mice were used to examine effects of global A1 adenosine receptor (A1AR) knockout (KO) on ...function and ischemic tolerance in perfused mouse hearts. Baseline contractile function and heart rate were unaltered by A1AR KO, which was shown to abolish the negative chronotropic effects of 2-chloroadenosine (A1AR-mediated) without altering A2 adenosine receptor–mediated coronary dilation. Tolerance to 25 minutes global normothermic ischemia (followed by 45 minutes reperfusion) was significantly limited by A1AR KO, with impaired contractile recovery (reduced by ≈25%) and enhanced lactate dehydrogenase (LDH) efflux (increased by ≈100%). Functional effects of A1AR KO involved worsened systolic pressure development with little to no change in diastolic dysfunction. In contrast, cardiac specific A1AR overexpression enhanced ischemic tolerance with a primary action on diastolic dysfunction. Nonselective receptor agonism (10 μmol/L 2-chloroadenosine) protected wild-type and also A1AR KO hearts (albeit to a lesser extent), implicating protection via subtypes additional to A1ARs. However, A1AR KO abrogated effects of 2-chloroadenosine on ischemic contracture and diastolic dysfunction. These data are the first demonstrating global deletion of the A1AR limits intrinsic myocardial resistance to ischemia. Data indicate the function of intrinsically activated A1ARs appears primarily to be enhancement of postischemic contractility and limitation of cell death.
Multiple sclerosis (MS) is a complex autoimmune disorder of the CNS with both genetic and environmental contributing factors. Clinical symptoms are broadly characterized by initial onset, and ...progressive debilitating neurological impairment. In this study, RNA from MS chronic active and MS acute lesions was extracted, and compared with patient matched normal white matter by fluorescent cDNA microarray hybridization analysis. This resulted in the identification of 139 genes that were differentially regulated in MS plaque tissue compared to normal tissue. Of these, 69 genes showed a common pattern of expression in the chronic active and acute plaque tissues investigated (
P
value<0.0001,
ρ=0.73, by Spearman's
ρ analysis); while 70 transcripts were uniquely differentially expressed (≥1.5-fold) in either acute or chronic active tissues. These results included known markers of MS such as the
myelin basic protein (
MBP) and
glutathione S-transferase (
GST)
M1, nerve growth factors, such as nerve
injury-induced protein 1 (
NINJ1), X-ray and excision DNA repair factors (
XRCC9 and
ERCC5) and X-linked genes such as the ribosomal protein,
RPS4X. Primers were then designed for seven array-selected genes, including
transferrin (
TF),
superoxide dismutase 1 (
SOD1),
glutathione peroxidase 1 (
GPX1),
GSTP1,
crystallin,
alpha-B (
CRYAB),
phosphomannomutase 1 (
PMM1) and
tubulin β-5 (
TBB5), and real time quantitative (Q)-PCR analysis was performed. The results of comparative Q-PCR analysis correlated significantly with those obtained by array analysis (
r=0.75,
P
value<0.01, by Pearson's bivariate correlation). Both chronic active and acute plaques shared the majority of factors identified suggesting that quantitative, rather than gross qualitative differences in gene expression pattern may define the progression from acute to chronic active plaques in MS.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Adenosine deaminase (ADA) may be multifunctional, regulating adenosine levels and adenosine receptor (AR) agonism, and potentially modifying AR functionality. Herein we assess effects of ADA (and ...A1AR) deficiency on AR-mediated responses and ischaemic tolerance.
Normoxic function and responses to 20 or 25 min ischaemia and 45 min reperfusion were studied in isolated hearts from wild-type mice and from mice deficient in ADA and/or A1ARs.
Neither ADA or A1AR deficiency significantly modified basal contractility, although ADA deficiency reduced resting heart rate (an effect abrogated by A1AR deficiency). Bradycardia and vasodilation in response to AR agonism (2-chloroadenosine) were unaltered by ADA deficiency, while A1AR deficiency eliminated the heart rate response. Adenosine efflux increased 10- to 20-fold with ADA deficiency (at the expense of inosine). Deletion of ADA improved outcome from 25 min ischaemia, reducing ventricular diastolic pressure (by 45%; 21+/-4 vs. 38+/-3mm Hg) and lactate dehydrogenase (LDH) efflux (by 40%; 0.12+/-0.01 vs. 0.21+/-0.02 U/g/min ischaemia), and enhancing pressure development (by 35%; 89+/-6 vs. 66+/-5mm Hg). Similar protection was evident after 20 min ischaemia, and was mimicked by the ADA inhibitor EHNA (5 microM). Deletion of ADA also enhanced tolerance in A1AR deficient hearts, though effects on diastolic pressure were eliminated.
Deficiency of ADA does not alter sensitivities of cardiovascular A1 or A2ARs (despite markedly elevated adenosine), but significantly improves ischaemic tolerance. Conversely, A1AR deficiency impairs ischaemic tolerance. Effects of ADA deficiency on diastolic pressure appear solely A1AR-dependent while other ARs or processes additionally contribute to improved contractile recovery and reduced cell death.
Elasmobranch fishes are an ancient group of vertebrates which have high potential as model species for research into evolutionary physiology and genomics. However, no comparative studies have ...established suitable reference genes for quantitative PCR (qPCR) in elasmobranchs for any physiological conditions. Oxygen availability has been a major force shaping the physiological evolution of vertebrates, especially fishes. Here we examined the suitability of 9 reference candidates from various functional categories after a single hypoxic insult or after hypoxia preconditioning in epaulette shark (Hemiscyllium ocellatum).
Epaulette sharks were caught and exposed to hypoxia. Tissues were collected from 10 controls, 10 individuals with single hypoxic insult and 10 individuals with hypoxia preconditioning (8 hypoxic insults, 12 hours apart). We produced sequence information for reference gene candidates and monitored mRNA expression levels in four tissues: cerebellum, heart, gill and eye. The stability of the genes was examined with analysis of variance, geNorm and NormFinder. The best ranking genes in our study were eukaryotic translation elongation factor 1 beta (eef1b), ubiquitin (ubq) and polymerase (RNA) II (DNA directed) polypeptide F (polr2f). The performance of the ribosomal protein L6 (rpl6) was tissue-dependent. Notably, in one tissue the analysis of variance indicated statistically significant differences between treatments for genes that were ranked as the most stable candidates by reference gene software.
Our results indicate that eef1b and ubq are generally the most suitable reference genes for the conditions and tissues in the present epaulette shark studies. These genes could also be potential reference gene candidates for other physiological studies examining stress in elasmobranchs. The results emphasise the importance of inter-group variation in reference gene evaluation.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The well-documented age-related change in ischemic tolerance may result from impaired adenosine-mediated cardioprotection. Additionally, ischemia itself may potentially modify adenosine signalling, ...contributing to the post-ischemic phenotype. This study investigates age- and ischemia-dependent changes in adenosine receptor transcript levels (Adora) for the A
1, A
2A, A
2B, and A
3 receptor subtypes in mouse myocardium. Hearts from young (2–4 months) and moderately aged (16–18 months) mice were subjected to 20-min ischemia and 45-min reperfusion. Ischemic tolerance was impaired in aged hearts, which recovered less than 30% ventricular pressure development (compared with ∼70% in young hearts), and lost 2-fold higher levels of lactate dehydrogenase during reperfusion (reflecting cellular disruption). Real-time PCR analyses revealed an age-related decline in Adora3 levels and induction of Adora2B. Curiously, this effect was mimicked by ischemia, which acutely reduced Adora3 levels and induced Adora2B in young (but not old) hearts. In contrast, in aged hearts ischemia selectively reduced levels of Adora1 transcript (∼2-fold) without altering transcript levels for the other receptors. These results demonstrate selective modulation of cardioprotective adenosine receptor transcription by both aging and ischemia. Reduced A
3 adenosine receptor transcription may contribute to impaired ischemic tolerance in aged hearts, whereas changes in Adora transcription induced by ischemia may impact on the post-ischemic phenotype at later time points.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Aged hearts exhibit reduced tolerance to ischemia-reperfusion, together with altered structure and post-ischemic remodelling. The molecular bases of such changes are unclear. Using cDNA microarrays ...and quantitative RT-PCR we characterized shifts in gene expression patterns with aging in normoxic and post-ischemic (20
min global ischemia, 60
min reperfusion) murine hearts (young: 2–4 months; aged: 16–18 months). We identified an age-associated up-regulation of transcripts involved in cell death, oxygen transport and metabolism in normoxic hearts. Down-regulated transcripts were involved in transporter activity, protein binding and hydrolase activity, changes in MAPK, WNT and TGF-β signalling with aging were also observed. Ischemic stress generated a much greater degree of contractile impairment and cellular damage in aged vs. young hearts. This was associated with a substantially modified transcriptional response, with selective changes in Ca
2+, WNT, NOTCH and G-protein coupled receptor signalling paths in aged vs. young hearts. Despite some common responses to ischemia in young and aged hearts (induction of heat shock protein transcripts), aging selectively modified ischemic responses of immediate early genes, and genes involved in modulating apoptosis and remodelling/angiogenesis. In summary, aging is associated with shifts in cardiovascular gene expression consistent with the phenotypic features of older hearts. Reduced tolerance with age may be related to modification of signalling (particularly WNT and TGF-β), and shifts in expression of immediate early genes, and genes important in control of cell death/survival, angiogenesis, and cardiac remodelling.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Abstract The adenosine receptor system has been attributed with a broad range of both physiological and so-called ‘retaliatory’ functions in the heart and vessels. Despite many years of research, the ...precise roles of adenosine within the cardiovascular system continue to be debated, and new functions are continually emerging. Adenosine acts via 4 known G-protein-coupled receptor (GPCR) sub-types: A1 , A2A , A2B , and A3 adenosine receptors (ARs). In addition to roles in cardiovascular control, these receptors may represent therapeutic targets, having been attributed with roles in modifying cell death and injury, inflammatory processes, and cardiac and vascular remodeling during/after ischemic or hypoxic insult. A number of models have been developed in which AR sub-types and adenosine handling enzymes have been genetically deleted or transgenically overexpressed in an attempt to more equivocally identify the regulatory functions of these proteins, to identify their potential value as therapeutic targets, and to uncover new regulatory functions of this receptor family. Findings generally support current dogma regarding cardioprotection via A1 and A3 ARs, and coronary vasoregulation via A2 AR sub-types. However, some outcomes are both novel and controversial. This review outlines AR-modified murine models currently under study from the perspective of cardiovascular phenotype.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
A method was developed to profile the major constituents of St John's wort extracts using highperformance liquid chromatography‐electrospray mass spectrometry (HPLC‐ESI‐MS). The objective was to ...simultaneously separate, identify and quantify hyperforin, hypericin, pseudohypericin, rutin, hyperoside, isoquercetrin, quercitrin and chlorogenic acid using HPLC‐MS. Quantification was performed using an external standardisation method with reference standards. The method consisted of two protocols: one for the analysis of flavonoids and glycosides and the other for the analysis of the more lipophilic hypericins and hyperforin. Both protocols used a reverse phase Luna phenyl hexyl column. The separation of the flavonoids and glycosides was achieved within 35 min and that of the hypericins and hyperforin within 9 min. The linear response range in ESI‐MS was established for each compound and all had linear regression coefficient values greater than 0.97. Both protocols proved to be very specific for the constituents analysed. MS analysis showed no other signals within the analyte peaks. The method was robust and applicable to alcoholic tinctures, tablet/capsule extracts in various solvents and herb extracts. The method was applied to evaluate the phytopharmaceutical quality of St John's wort preparations available in the UK in order to test the method and investigate if they contain at least the main constituents and at what concentrations.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
In an attempt to define genomic copy number changes associated with the development of basal cell carcinoma, we investigated 15 sporadic tumors by comparative genomic hybridization. With the ...incorporation of tissue microdissection and degenerate oligonucleotide primed-polymerase chain reaction we were able to isolate, and then universally amplify, DNA from the tumor type. This combined approach allows the investigation of chromosomal imbalances within a histologically distinct region of tissue. Using comparative genomic hybridization we have observed novel and recurrent chromosomal gains at 6p (47%), 6q (20%), 9p (20%), 7 (13%), and X (13%). In addition comparative genomic hybridization revealed regional loss on 9q in 33% of tested tumors encompassing 9q22.3 to which the putative tumor suppressor gene, Patched, has been mapped. The deletion of Patched has been indicated in the development of hereditary and sporadic basal cell carcinomas. The identification of these recurrent genetic aberrations suggests that basal cell carcinomas may not be as genetically stable as previously thought. Further investigation of these regions may lead to the identification of other genes responsible for basal cell carcinoma formation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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