Sperm count in Swedish clinical stage I testicular cancer patients following adjuvant treatment.

BACKGROUND: Little is known regarding sperm production following adjuvant treatment in testicular cancer (TC) clinical stage I (CS I) patients. PATIENTS AND METHODS: A total of 182 TC patients aged 18-50 years were prospectively included during 2001-2006 at any given time within 5 years of orchiectomy. Semen samples were delivered postorchiectomy but before further treatment, 6, 12, 24, 36 and 60 months (T0-T60) after completed therapy. Total sperm number (TSN) and sperm concentration (SC) were used as measurements of testicular function. Four groups according to treatment modality were identified; Radiotherapy; To a total dose of 25.2 Gy to the infradiaphragmal paraaortic and ipsilateral iliac lymph nodes (RT, N = 70), one cycle of adjuvant BEP (bleomycin, etoposide, cisplatin, 5 day regimen) (BEP, N = 62), one cycle of adjuvant carboplatin AUC 7 (Carbo, N = 22), and patients managed by surveillance (SURV, N = 28). RESULTS: In the cross-sectional analysis, a significant but transient drop in mean TSN and mean SC (T0-T60) was seen at T6 after radiotherapy. Apart from a significant increase in mean SC at T12 compared with baseline, no significant differences were observed in the other treatment groups. In 119 patients delivering 3 or more samples, values in TSN and SC were rather stable over time. Azoospermic patients (N = 11) were observed in most treatment groups except for in the BEP group. During follow-up, one azoospermic patient belonging to the Carbo group became normospermic. CONCLUSIONS: No clinically significant long-term effect on TSN or SC associated with adjuvant treatment in TC CSI patients was found. However, as patients may have low sperm counts before orchiectomy as well as after adjuvant treatment, we offer sperm banking before orchiectomy as assisted reproductive measures may be necessary regardless of treatment given.

Tryptophan Metabolism Along the Kynurenine Pathway Downstream of Toll-like Receptor Stimulation in Peripheral Monocytes.

Tryptophan degradation along the kynurenine pathway is of central importance for the immune function. Toll-like receptors (TLRs), representing the first line of immune defence against pathogens, are expressed in various cell types. The most abundant expression is found on monocytes, macrophages and dendritic cells. The aim of this study was to investigate whether stimulation with different TLR ligands induces the kynurenine pathway in human peripheral monocytes. Cell supernatants were analysed using a liquid chromatography/mass spectrometry to measure kynurenine, kynurenic acid (KYNA), quinolinic acid (QUIN) and tryptophan. Stimulation of TLR-2, TLR-3, TLR-4, TLR-7/8 and TLR-9 was found to induce the production of kynurenine, but only stimulation of TLR-3 increased levels of further downstream metabolites, such as KYNA and QUIN. Stimulation of TLR-1, TLR-5 and TLR-6 did not induce the kynurenine pathway. Taken together, this study provides novel evidence demonstrating that TLR activation induces a pattern of downstream tryptophan degradation along the kynurenine pathway in monocytes. The results of this study may implicate that TLRs can be used as new drug targets for the regulation of aberrant tryptophan metabolism along this pathway, a potential therapeutic strategy that may be of importance in several disorders.

Repeated LPS Injection Induces Distinct Changes in the Kynurenine Pathway in Mice.

The immune system has been recognized as a potential contributor to psychiatric disorders. In animals, lipopolysaccharide (LPS) is used to induce inflammation and behaviors analogous to some of the symptoms in these disorders. Recent data indicate that the kynurenine pathway contributes to LPS-induced aberrant behaviors. However, data are inconclusive regarding optimal LPS dose and treatment strategy. Here, we therefore aimed to evaluate the effects of single versus repeated administration of LPS on the kynurenine pathway. Adult C57BL6 mice were given 0.83 mg/kg LPS as a single or a repeated injection (LPS + LPS) and sacrificed after 24, 48, 72, or 120 h. Mice receiving LPS + LPS had significantly elevated brain kynurenine levels at 24 and 48 h, and elevated serum kynurenine at 24, 48 and 72 h. Brain kynurenic acid and quinolinic acid were significantly increased at 24 and 48 h in mice receiving LPS + LPS, whereas serum kynurenic acid levels were significantly decreased at 24 h. The increase of brain kynurenic acid by LPS + LPS was likely unrelated to the higher total dose as a separate group of mice receiving 1.66 mg/kg LPS as single injection 24 h prior to sacrifice did not show increased brain kynurenic acid. Serum quinolinic acid levels were not affected by LPS + LPS compared to vehicle. Animals given repeated injections of LPS showed a more robust induction of the kynurenine pathway in contrast to animals receiving a single injection. These results may be valuable in light of data showing the importance of the kynurenine pathway in psychiatric disorders.

Reduction of plasma aldosterone and arterial stiffness in obese pre- and stage1 hypertensive subjects after aerobic exercise.

Obesity-related hypertension is associated with increased activity of the renin-angiotensin-aldosterone system (RAAS), increasing arterial stiffness. Aerobic exercise decreases pulse wave velocity (PWV), therefore a treatment option for hypertension and obesity. Assess RAAS activity and PWV before and after 4 weeks of aerobic training in unmedicated, pre-to-stage-1 hypertensives. Ten obese subjects (52±3.2 years, body mass index=33.5±1.4) performed 30 min of aerobic exercise on a treadmill 3 days per week at 65% of peak oxygen consumption (VO2peak). Descriptive characteristics, systolic and diastolic blood pressure (SBP and DBP), PWV, and a blood draw was performed at baseline, following the 4-week control and training interventions. No differences in descriptive characteristics during the control period were observed, however, a significant decrease in plasma aldosterone (ALDO) (255.4±75 to 215.8±66 pg ml(-1), P=0.001), SBP (140±12 to 136±10.4 mm Hg; P=0.02), DBP (89±4.2 to 85±6.3 mm Hg; P=0.03) and central PWV (11.2±0.6 to 9.8±0.8 m s(-1); P=0.04) was shown pre-to-post exercise training. Four weeks of moderate-intensity aerobic training in obese, hypertensives decreases plasma ALDO independently of body weight and is significantly correlated to decreases in PWV reductions.

Spectrum of chronic lung disease in a population of newborns with extremely low gestational age.

AIMS: To determine how the ability to oxygenate the blood develops after birth in infants of extremely low gestational age (ELGANs) and to find risk factors for chronic lung disease. METHOD: A prospective, population-based, cohort study was undertaken in one tertiary-care centre. The alveolar-arterial oxygen pressure difference (AaDO(2)) was monitored. RESULTS: Of 41 survivors, 21 had a period of normal lung function in the first week of life, after which oxygenation deteriorated. Low gestational age and low Apgar score at 5 min were found to be strong and independent predictors of AaDO(2) in the first month of life. Mechanical ventilation did not appear as a risk factor. Lung function at 36 weeks of gestation and duration of oxygen treatment could be better predicted by the severity of lung disease in the first month than by gestational age at birth. CONCLUSIONS: Difficulty in oxygenation was a general observation in ELGANs and not only a particular subset. Gestational age and Apgar score were independent predictors of the degree of difficulty over the first month of life. As oxygenation failure often developed after a few days, the process may be possible to treat or prevent once the pathogenesis is known.

Comparative linkage mapping of the Grey coat colour gene in horses.

Grey horses are born coloured, turn progressively grey and often develop melanomas late in life. Grey shows an autosomal dominant inheritance and the locus has previously been mapped to horse chromosome 25 (ECA25), around the TXN gene. We have now developed eight new single nucleotide polymorphisms (SNPs) associated with genes on ECA25 using information on the linear order of genes on human chromosome 9q, as well as the human and mouse coding sequences. These SNPs were mapped in relation to the Grey locus using more than 300 progeny from matings between two Swedish Warmblood grey stallions and non-grey mares. Grey was firmly assigned to an interval with flanking markers NANS and ABCA1. This corresponds to a region of approximately 6.9 Mb on human chromosome 9q. Furthermore, no recombination was observed between Grey, TGFBR1 and TMEFF1, the last two being 1.4 Mb apart in human. There are no obvious candidate genes in this region and none of the genes has been associated with pigmentation disorders or melanoma development, suggesting that the grey phenotype is caused by a mutation in a novel gene.

Regulation of pituitary corticotropin releasing factor type-1 receptor mRNA binding proteins by modulation of the hypothalamic-pituitary-adrenal axis.

Long-term adrenalectomy (ADX) is associated with marked down-regulation of pituitary corticotropin releasing factor type-1 receptors (CRF-R1) but normal CRF-R1 mRNA levels, suggesting that regulation of receptor levels occurs at post-transcriptional sites. We have reported that adrenal cytosolic proteins, which bind to cis elements in the 5' leader sequence (5'LS) of the rat angiotensin II type 1a receptor (AT(1a)R) mRNA, participate in the regulation of AT(1a)R density by inhibiting AT(1a)R mRNA translation. In this study, we examined anterior pituitary cytosolic proteins that form RNA protein complexes (RPC) with the 5'LS of the CRF-R1 and the AT(1a)R. Competition studies and ultraviolet-crosslinking analysis suggest that formation of CRF-R1 and AT(1a)R 5'LS RPC require at least some proteins that are common to both receptor mRNAs. Pituitaries isolated from male Sprague-Dawley rats six days after ADX showed significant (P < 0.05) increases of 2.9-fold in CRF-R1 5'LS RNA binding protein (BP) activity compared to pituitaries from sham-operated rats; this effect of ADX was prevented by glucocorticoid replacement. By contrast, no differences in the number of pituitary AT(1a)R binding sites or pituitary AT(1a)R 5'LS BP activity were observed between sham and ADX rats, indicating that the effect of ADX on RPC formation was specific for CRF-R1 mRNA. Addition of pituitary cytosolic extracts inhibited in vitro translation of CRF-R1 mRNA by 79% under conditions that had no significant effects on the translation of a control mRNA. The data suggest that CRF-R1 translation is regulated by modulation of the HPA axis through pituitary cytosolic proteins binding to the CRF-R1 5'LS.

The second generation of the International Equine Gene Mapping Workshop half-sibling linkage map.

A low-density, male-based linkage map was constructed as one of the objectives of the International Equine Gene Mapping Workshop. Here we report the second generation map based on testing 503 half-sibling offspring from 13 sire families for 344 informative markers using the CRIMAP program. The multipoint linkage analysis localized 310 markers (90%) with 257 markers being linearly ordered. The map included 34 linkage groups representing all 31 autosomes and spanning 2262 cM with an average interval between loci of 10.1 cM. This map is a milestone in that it is the first map with linkage groups assigned to each of the 31 automosomes and a single linkage group to all but three chromosomes.

Molecular characterization and mutational screening of the PRKAG3 gene in the horse.

The PRKAG3 gene encodes a muscle-specific isoform of the regulatory gamma subunit of AMP-activated protein kinase (AMPK). A major part of the coding PRKAG3 sequence was isolated from horse muscle cDNA using reverse-transcriptase (RT)-PCR analysis. Horse-specific primers were used to amplify genomic fragments containing 12 exons. Comparative sequence analysis of horse, pig, mouse, human, Fugu, and zebrafish was performed to establish the exon/intron organization of horse PRKAG3 and to study the homology among different isoforms of AMPK gamma genes in vertebrates. The results showed conclusively that the three different isoforms (gamma1, gamma2, and gamma3) were established already in bony fishes. Seven single nucleotide polymorphisms (SNPs), five causing amino acid substitutions, were identified in a screening across horse breeds with widely different phenotypes as regards muscle development and intended performance. The screening of a major part of the PRKAG3 coding sequence in a small case/control material of horses affected with polysaccharide storage myopathy did not reveal any mutation that was exclusively associated with this muscle storage disease. The breed comparison revealed several potentially interesting SNPs. One of these (Pro258Leu) occurs at a residue that is highly conserved among AMPK gamma genes. In an SNP screening, the variant allele was only found in horse breeds that can be classified as heavy (Belgian) or moderately heavy (North Swedish Trotter, Fjord, and Swedish Warmblood) but not in light horse breeds selected for speed or racing performance (Standardbred, Thoroughbred, and Quarter horse) or in ponies (Icelandic horses and Shetland pony). The results will facilitate future studies of the possible functional significance of PRKAG3 polymorphisms in horses.

Coronary calcification and cardiac events after percutaneous intervention in dialysis patients.

BACKGROUND: Previous studies have described increased vascular calcification in renal dialysis patients. The clinical significance of this finding with respect to outcomes after percutaneous coronary intervention in this population is unknown. METHODS: We analysed a prospective interventional database at a single tertiary center and identified 41 dialysis patients who underwent coronary angioplasty. All studies were reviewed for the presence of coronary calcium in the target and reference vessels and compared with respect to baseline clinical factors and cardiovascular outcomes. RESULTS: The mean ages for those with and without coronary calcification were 63.6 +/- 11.0 and 67.3 +/- 11.0, respectively, P = 0.30. The groups were similar in years on dialysis, diabetes, hypertension, smoking, and measures of calcium and phosphate balance. The total cholesterol, LDL-C, HDL-C, and triglycerides were 162.5 +/- 42.3 and 202.0 +/- 54.5, P = 0.02; 94.9 +/- 39.6 and 121.2 +/- 48.1, P = 0.18; 39.3 +/- 12.4 and 47.3 +/- 12.2, P = 0.15; 157.4 +/- 100.4 and 181.3 +/- 187.4, P = 0.15, for those with and without calcification, respectively. The composite of target vessel revascularization, myocardial infarction, or death was 47.4% and 77.3% for those with and without calcification, respectively, P = 0.06. The Cox proportional hazards model, controlling for years on dialysis, showed a significant, event-free survival in those with coronary calcium seen fluoroscopically, P = 0.05. CONCLUSIONS: In dialysis patients, coronary calcification identified in the target or reference vessels is associated with lower total cholesterol and favourable interventional outcomes.

Physical anchorage and orientation of equine linkage groups by FISH mapping BAC clones containing microsatellite markers.

A horse bacterial artificial chromosome (BAC) library was screened for 19 microsatellite markers from unassigned or non-oriented linkage groups. Clones containing 11 (AHT20, EB2E8, HMS45, LEX005, LEX014, LEX023, LEX044, TKY111, UCDEQ425, UCDEQ464 and VIASH21) of these were found, which were from eight different linkage groups. The BAC clones were used as probes in dual colour FISH to identify their precise chromosomal origin. The microsatellite markers are located on nine different horse chromosomes, four of which (ECA6, ECA25, ECA27 and ECA28) had no previously in situ assigned markers.

Combined angiotensin converting enzyme inhibition and angiotensin AT(1) receptor blockade up-regulates myocardial AT(2) receptors in remodeled myocardium post-infarction.

OBJECTIVES: In an ovine model of left ventricular (LV) remodeling after transmural anteroapical myocardial infarction (MI), we have previously demonstrated that the combination of angiotensin converting enzyme (ACE) inhibition and AT(1) receptor blockade is more effective at limiting LV remodeling than either therapy alone. We hypothesized that the beneficial effect of combined therapy is due in part to upregulation of AT(2) receptor levels. METHODS: Two days after transmural anteroapical MI by coronary ligation, 16 sheep were randomized to losartan (50 mg/day), ramipril (10 mg/day), ramipril+losartan (combined therapy), or no therapy. At 8 weeks after MI, radioligand receptor assay were deployed with homogenates from regional LV tissues. RESULTS: We found that AT receptors in normal sheep myocardium are predominantly of the AT(2) receptor subtype. Binding studies of remodeled myocardium 8 weeks later showed that the apparent maximum binding (B(max)) was increased from 23 to 48 fmol/mg protein only in animals with combined therapy. The AT(2)/AT(1) proportion was increased significantly in animals with combined therapy compared to infarcted controls (18.0 vs. 5.17). CONCLUSIONS: These results indicate that AT(2) receptor expression increased significantly during LV remodeling with combined therapy but not with either therapy alone. In combination with prior work demonstrating the effectiveness of combined therapy in limiting LV remodeling, this study is consistent with the hypothesis that AT(2) receptors play a cardioprotective role in LV remodeling after MI.

Determinants of mortality after myocardial infarction in patients with advanced renal dysfunction.

Previous studies using administrative data have shown high mortality in patients with renal failure requiring dialysis after acute myocardial infarction (AMI). There has been little investigation into the mortality after AMI in those with advanced renal disease who are not on dialysis therapy. We analyzed a prospective coronary care unit registry of 1,724 patients with ST segment elevation myocardial infarction admitted over an 8-year period at a single tertiary-care center. Those not on chronic dialysis therapy were stratified into groups based on corrected creatinine clearance, with cutoff values of 46.2, 63.1, and 81.5 mL/min/72 kg. Dialysis patients (n = 47) were considered as a fifth comparison group. Older age, black race, diabetes, hypertension, previous coronary disease, and heart failure were incrementally more common across increasing renal dysfunction strata. There were also graded increases in the relative risk for atrial and ventricular arrhythmias, heart block, asystole, development of pulmonary congestion, acute mitral regurgitation, and cardiogenic shock. Primary angioplasty, thrombolysis, and beta-blockers were used less often across the risk strata (P < 0.0001 for all trends). There was an early mortality hazard (age-adjusted relative risk, 8.76; P < 0.0001) for those with renal dysfunction but not on dialysis therapy for the first 60 months, followed by graded decrements in survival across increasing renal dysfunction strata. The excess mortality in this population appears to be mediated through arrhythmias, adverse hemodynamic events, and the lower use of mortality-reducing therapy.

Coordinate regulation of canine glomeruli and adrenal angiotensin receptors by dietary sodium manipulation.

BACKGROUND: This study evaluated the effects of dietary sodium manipulation in dogs on the regulation of canine angiotensin receptors (cAT1 and cAT2) in the kidney and adrenal. METHODS: Isolated glomeruli and membranes from renal medulla and the adrenal gland were used in radioligand binding assays from two groups of dogs: dogs maintained on low-sodium diet for two weeks followed by a high-sodium diet for two weeks (H), and dogs were maintained on the reverse schedule (L). RESULTS: Analysis of the binding data showed that dietary sodium manipulation had no significant effects on cAT1 and cAT2 receptor binding affinities in glomeruli, renal medulla, and adrenal tissues. In contrast, dietary sodium loading induced a marked increase in cAT1 receptor expression in both the glomeruli and adrenal compared with receptor expression in salt-restricted animals [H/L ratio: glomeruli (1.5), renal medulla (1.1), adrenal (1.6)] that inversely correlated with the activity of the plasma renin angiotensin system. Conversely, adrenal cAT2 receptor expression was regulated in an inverse manner in the H and L animal groups [H/L ratio: 0.7]. CONCLUSIONS: This study demonstrates that renal glomerular and adrenal AT1 receptors in the dog are coordinately down-regulated by dietary sodium restriction compared with sodium loading, which is distinctly different from the reciprocal regulation observed for rat AT1 receptors in these tissues. Collectively, these data suggest that postreceptor events in dogs are determinants of the aldosterone response observed during sodium restriction. These findings have important implications for the regulation of the renin-angiotensin system in humans, and suggest that coordinate regulation of AT1 receptors in the adrenal and glomeruli represent a negative feedback mechanism that when functioning normally prevents fluctuations of arterial blood pressure and development of arterial hypertension in response to changes in dietary sodium.

Mapping of 13 horse genes by fluorescence in-situ hybridization (FISH) and somatic cell hybrid analysis.

We report fluorescence in-situ hybridization (FISH) and somatic cell hybrid mapping data for 13 different horse genes (ANP, CD2, CLU, CRISP3, CYP17, FGG, IL1RN, IL10, MMP13, PRM1, PTGS2, TNFA and TP53). Primers for PCR amplification of intronic or untranslated regions were designed from horse-specific DNA or mRNA sequences in GenBank. Two different horse bacterial artificial chromosome (BAC) libraries were screened with PCR for clones containing these 13 Type I loci, nine of which were found in the libraries. BAC clones were used as probes in dual colour FISH to confirm their precise chromosomal origin. The remaining four genes were mapped in a somatic cell hybrid panel. All chromosomal assignments except one were in agreement with human-horse ZOO-FISH data and revealed new and more detailed information on the equine comparative map. CLU was mapped by synteny to ECA2 while human-horse ZOO-FISH data predicted that CLU would be located on ECA9. The assignment of IL1RN permitted analysis of gene order conservation between HSA2 and ECA15, which identified that an event of inversion had occurred during the evolution of these two homologous chromosomes.

Widespread origins of domestic horse lineages.

Domestication entails control of wild species and is generally regarded as a complex process confined to a restricted area and culture. Previous DNA sequence analyses of several domestic species have suggested only a limited number of origination events. We analyzed mitochondrial DNA (mtDNA) control region sequences of 191 domestic horses and found a high diversity of matrilines. Sequence analysis of equids from archaeological sites and late Pleistocene deposits showed that this diversity was not due to an accelerated mutation rate or an ancient domestication event. Consequently, high mtDNA sequence diversity of horses implies an unprecedented and widespread integration of matrilines and an extensive utilization and taming of wild horses. However, genetic variation at nuclear markers is partitioned among horse breeds and may reflect sex-biased dispersal and breeding.

Comparative analysis of amphibian and mammalian angiotensin receptors.

Amphibian angiotensin receptors (xAT receptors) share many similarities with mammalian type 1 angiotensin receptors (AT(1) receptors). Both xAT and AT(1) receptors belong to the super family of seven transmembrane spanning G protein-coupled receptors and share approximately 60% amino acid homology. Highly stable secondary structure in the 5' leader sequences and the presence of the mRNA destabilizing sequence (AUUUA) in the 3' untranslated region (3'UTR) of the xAT and AT(1) receptor mRNAs suggest similar mechanisms exist for regulating gene expression. Amphibian and mammalian AT receptors bind angiotensin with equivalent affinities but show marked differences in their affinities towards mammalian AT(1) receptor subtype selective non-peptide ligands. Both xAT and AT(1) receptors couple to G proteins and to the phospholipase C (PLC) signal transduction pathway. Mammalian AT(1) receptors play a key role in maintaining blood pressure and fluid homeostasis and there is considerable evidence that xAT receptors play a similarly important role in amphibians. This review focuses on the comparison of amphibian xAT receptors with mammalian AT(1) receptors in terms of their structure, pharmacology, signaling, and function.

Analysis of Cytosolic Proteins that Bind to the 5' Leader Sequence of the Angiotensin AT1 Receptor by RNA Electromobility Shift Assay.

Electromobility shift assays (EMSAs) provide a way to study proteinnucleic acid interactions. This method is based on the observation that the electrophoretic mobility of nucleic acids through polyacrylamide gels is retarded when bound to proteins. The mobility of nucleic acid-protein complexes are thus "shifted" with respect to the free nucleic acids. Typically, the nucleic acids are labeled with (32)P. Once the nucleic acid-protein complexes are separated from free radiolabeled nucleic acids, the electrophoresis is terminated and the gel dried. The radiolabeled nucleic acids in their free and complexed forms are visualized and quantified by phosphor autoradiography or by X-ray autoradiography. DNA-binding proteins are commonly identified by EMSA. EMSA also works well for studying purified RNA-binding proteins (1-3) and this technique is currently being developed for identifying unknown RNA-binding proteins.