Breeding biology and the evolution of dynamic sexual dichromatism in frogs.

Dynamic sexual dichromatism is a temporary colour change between the sexes and has evolved independently in a wide range of anurans, many of which are explosive breeders wherein males physically compete for access to females. Behavioural studies in a few species indicate that dynamic dichromatism functions as a visual signal in large breeding aggregations; however, the prevalence of this trait and the social and environmental factors underlying its expression are poorly understood. We compiled a database of 178 anurans with dynamic dichromatism that include representatives from 15 families and subfamilies. Dynamic dichromatism is common in two of the three subfamilies of hylid treefrogs. Phylogenetic comparative analyses of 355 hylid species (of which 95 display dynamic dichromatism) reveal high transition rates between dynamic dichromatism, ontogenetic (permanent) dichromatism and monochromatism reflecting the high evolutionary lability of this trait. Correlated evolution in hylids between dynamic dichromatism and forming large breeding aggregations indicates that the evolution of large breeding aggregations precedes the evolution of dynamic dichromatism. Multivariate phylogenetic logistic regression recovers the interaction between biogeographic distribution and forming breeding aggregations as a significant predictor of dynamic dichromatism in hylids. Accounting for macroecological differences between temperate and tropical regions, such as seasonality and the availability of breeding sites, may improve our understanding of ecological contexts in which dynamic dichromatism is likely to arise in tropical lineages and why it is retained in some temperate species and lost in others.

Sexual selection predicts brain structure in dragon lizards.

Phenotypic traits such as ornaments and armaments are generally shaped by sexual selection, which often favours larger and more elaborate males compared to females. But can sexual selection also influence the brain? Previous studies in vertebrates report contradictory results with no consistent pattern between variation in brain structure and the strength of sexual selection. We hypothesize that sexual selection will act in a consistent way on two vertebrate brain regions that directly regulate sexual behaviour: the medial preoptic nucleus (MPON) and the ventromedial hypothalamic nucleus (VMN). The MPON regulates male reproductive behaviour whereas the VMN regulates female reproductive behaviour and is also involved in male aggression. To test our hypothesis, we used high-resolution magnetic resonance imaging combined with traditional histology of brains in 14 dragon lizard species of the genus Ctenophorus that vary in the strength of precopulatory sexual selection. Males belonging to species that experience greater sexual selection had a larger MPON and a smaller VMN. Conversely, females did not show any patterns of variation in these brain regions. As the volumes of both these regions also correlated with brain volume (BV) in our models, we tested whether they show the same pattern of evolution in response to changes in BV and found that the do. Therefore, we show that the primary brain nuclei underlying reproductive behaviour in vertebrates can evolve in a mosaic fashion, differently between males and females, likely in response to sexual selection, and that these same regions are simultaneously evolving in concert in relation to overall brain size.

Effects of fenofibrate on renal function in patients with type 2 diabetes mellitus: the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) Study.

AIMS/HYPOTHESIS: Fenofibrate caused an acute, sustained plasma creatinine increase in the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) and Action to Control Cardiovascular Risk in Diabetes (ACCORD) studies. We assessed fenofibrate's renal effects overall and in a FIELD washout sub-study. METHODS: Type 2 diabetic patients (n = 9,795) aged 50 to 75 years were randomly assigned to fenofibrate (n = 4,895) or placebo (n = 4,900) for 5 years, after 6 weeks fenofibrate run-in. Albuminuria (urinary albumin/creatinine ratio measured at baseline, year 2 and close-out) and estimated GFR, measured four to six monthly according to the Modification of Diet in Renal Disease Study, were pre-specified endpoints. Plasma creatinine was re-measured 8 weeks after treatment cessation at close-out (washout sub-study, n = 661). Analysis was by intention-to-treat. RESULTS: During fenofibrate run-in, plasma creatinine increased by 10.0 µmol/l (p < 0.001), but quickly reversed on placebo assignment. It remained higher on fenofibrate than on placebo, but the chronic rise was slower (1.62 vs 1.89 µmol/l annually, p = 0.01), with less estimated GFR loss (1.19 vs 2.03 ml min(-1) 1.73 m(-2) annually, p < 0.001). After washout, estimated GFR had fallen less from baseline on fenofibrate (1.9 ml min(-1) 1.73 m(-2), p = 0.065) than on placebo (6.9 ml min(-1) 1.73 m(-2), p < 0.001), sparing 5.0 ml min(-1) 1.73 m(-2) (95% CI 2.3-7.7, p < 0.001). Greater preservation of estimated GFR with fenofibrate was observed with baseline hypertriacylglycerolaemia (n = 169 vs 491 without) alone, or combined with low HDL-cholesterol (n = 140 vs 520 without) and reductions of ≥ 0.48 mmol/l in triacylglycerol over the active run-in period (pre-randomisation) (n = 356 vs 303 without). Fenofibrate reduced urine albumin concentrations and hence albumin/creatinine ratio by 24% vs 11% (p < 0.001; mean difference 14% [95% CI 9-18]; p < 0.001), with 14% less progression and 18% more albuminuria regression (p < 0.001) than in participants on placebo. End-stage renal event frequency was similar (n = 21 vs 26, p = 0.48). CONCLUSIONS/INTERPRETATION: Fenofibrate reduced albuminuria and slowed estimated GFR loss over 5 years, despite initially and reversibly increasing plasma creatinine. Fenofibrate may delay albuminuria and GFR impairment in type 2 diabetes patients. Confirmatory studies are merited. TRIAL REGISTRATION: ISRCTN64783481.

The EBSD spatial resolution of a Timepix-based detector in a tilt-free geometry.

Performing EBSD with a horizontal sample and a parallel EBSD detector sensor, enables safer specimen movements for data collection of large specimen areas and improves the longitudinal spatial resolution. The collection of electron backscattering patterns (EBSPs) at normal incidence to the electron beam has been revisited via the use of a direct electron detection (DED) sensor. In this article we present a fully operational DED EBSD detection system in this geometry, referred to as the tilt-free geometry. A well-defined Σ=3[101]{121} twin boundary in a Molybdenum bicrystal was used to measure the physical spatial resolution of the EBSD detector in this tilt-free geometry. In this study, two separate methods for estimating the spatial resolution of EBSD, one based on a pattern quality metric and the other on a normalised cross correlation coefficient were used. The spatial resolution was determined at accelerating voltages of 8 kV, 10 kV, 12 kV, 15 kV and 20 kV ranging from ~22-38 nm using the pattern quality method and ~31-46 nm using the normalised cross correlation method.

Acute effects of 5-methyltetrahydrofolate on endothelial function and asymmetric dimethylarginine in patients with chronic heart failure.

BACKGROUND AND AIMS: Folic acid enhances endothelial function in vascular disease states but its effects in chronic heart failure (CHF) are largely unknown. We studied the acute effects of i.v. methyltetrahydrofolate (5MTHF), the active metabolite of folic acid, on endothelial function and asymmetric dimethylarginine (ADMA) in CHF patients. METHODS AND RESULTS: Twenty two CHF patients and 22 controls received one of the following three-step infusions (1h per each step) in a randomized, parallel group, placebo-control study: (1) active treatment (saline, 5MTHF, and 5MTHF+the endothelial nitric oxide inhibitor N(G)-monomethyl l-arginine, LNMMA); or (2) placebo (salinex3). Endothelium-dependent vasodilatation was assessed by pulse-wave analysis (salbutamol-mediated changes in augmentation index, AIx). 5MTHF did not exert any significant effects on endothelium-dependent vasodilatation both in controls [DeltaAIx post-salbutamol baseline -7.6% (-24.8/-4.1) vs. 5MTHF -5.5% (-16.7/-3.6), medians and interquartile range, and CHF patients [-1.8% (-17.3/+1.3) vs. -2.4% (-3.8/-1.2)]. However, a significant reduction in ADMA concentrations was observed in both groups [controls baseline 0.68micromol/L (0.64/0.77) vs. 5MTHF 0.65 (0.57/0.74); CHF baseline 0.76 (0.63/0.82) vs. 5MTHF 0.69 (0.66/0.71), P=0.05 for both vs. baseline and placebo. These effects persisted during co-infusion with LNMMA. CONCLUSION: 5MTHF did not affect endothelial function but significantly reduced serum ADMA concentrations both in CHF patients and controls. This suggests a direct effect of 5MTHF on ADMA metabolism.

Simultaneous measurement of urinary metanephrines and catecholamines by liquid chromatography with tandem mass spectrometric detection.

BACKGROUND: The measurement of catecholamines and metanephrines in urine is an important diagnostic test in biochemical screening for phaeochromocytoma. Tandem mass spectrometry (MSMS) has the potential to be used in a profiling method for simultaneous assay of these analytes. METHODS: Optimal conditions were established for the MSMS detection of catecholamines (noradrenalin, adrenalin and dopamine) and metanephrines (normetanephrine and metanephrine), including commercially available isotopically labelled compounds for use as internal standards. Chromatographic separation of all five polar biogenic amines was achieved under solvent conditions that were compatible with MSMS and multiple reaction monitoring. Several types of solid-phase extraction cartridge were used to investigate clean-up conditions for urine, and acid-hydrolysates of urine, prior to LC-MSMS. RESULTS: Total catecholamines and metanephrines from acid-hydrolysed urines, or free catecholamines and free metanephrines from native urines, were complexed with diphenyl-boronate and recovered in high yield from polymer cartridges after elution with formic acid. Direct injection of eluates into the LC-MSMS system allowed quantitation of catecholamines and metanephrines with a run time of 6 min per sample. Biogenic amine concentrations for patient urines and quality assurance programme samples, and assay imprecision, were similar to values obtained with high-performance liquid chromatography methods, which used electrochemical detection. In normal urines, the ratio of free to total catecholamines was around three-fold higher than the ratio of free to total metanephrines. CONCLUSION: The assay of urinary catecholamines and metanephrines can be achieved simultaneously using one LC-MSMS method, which is rapid and reduces labour and consumable costs for routine application.

Bile acid synthesis by cultured rabbit hepatocytes: stimulation by three lipoprotein fractions.

Bile acid and cholesterol synthesis were measured in monolayer cultures of rabbit hepatocytes maintained in a defined culture medium. In the absence of lipoproteins, bile acid synthesis and secretion were correlated with cholesterol synthesis and were increased 245% by mevalonolactone (10 mM) and inhibited 45% by lovastatin (50 micrograms/ml) over 24 h. When included in the culture medium, normal rabbit plasma low-density and high-density lipoproteins increased bile acid synthesis and secretion by up to 140% of values obtained without lipoproteins in hepatocytes from normal or cholestyramine-fed rabbits. Three cholesterol-rich lipoprotein fractions (beta-very low density, low density and high density) also were isolated from rabbits fed 1% cholesterol for 14 days. When added to rabbit hepatocyte cultures, each fraction markedly increased hepatocellular cholesterol content, stimulated bile acid synthesis and secretion in a dose-dependent manner, and inhibited cholesterol synthesis from radioactive acetate. These data indicate that three different lipoprotein fractions can provide cholesterol for uptake and subsequent breakdown to bile acids by cultured rabbit hepatocytes.

Bile acid synthesis and secretion by rabbit hepatocytes in primary monolayer culture: comparison with rat hepatocytes.

Rabbit hepatocytes isolated after liver perfusion with collagenase were maintained in primary monolayer culture for periods up to 96 h. Bile acid synthesis and secretion was measured by capillary gas-liquid chromatography and by a rapid enzymatic-bioluminescence assay. As expected from the bile acid profile of rabbit gallbladder bile, cholic acid was the only bile acid synthesized in detectable amounts and was produced at a linear rate of 170 pmol/h per mg cell protein from 24 to 96 h in culture. Ketoconazole (20 microM) inhibited cholic acid synthesis and secretion by 78%, whereas the bile acids chenodeoxycholic acid (100 microM), deoxycholic acid (100 microM) or lithocholic acid (2 microM) had no effect. When rat hepatocytes were cultured under identical conditions, the rate of bile acid synthesis was found to be only 12 pmol/h per mg cell protein, a value in agreement with previous work. The large difference in rates of bile acid synthesis between rabbit and rat hepatocytes may be due to rapid loss of cytochrome P-450 from rat hepatocytes when placed in monolayer culture. Although reportedly active in cholesterol 7 alpha-hydroxylation, form 4 cytochrome P-450 levels in rabbit hepatocytes did not correlate with rates of bile acid synthesis.

Measurement of plasma LDL cholesterol in patients with diabetes.

OBJECTIVE: To assess the accuracy of plasma LDL cholesterol concentrations estimated by the Friedewald formula and a direct immunoseparation method by comparison with a reference ultracentrifugation procedure in patients with diabetes. RESEARCH DESIGN AND METHODS: Fasting plasma samples with triglyceride concentrations < 4.5 mmol/l were collected from 100 patients with diabetes (28 type I and 72 type II) and LDL cholesterol concentrations were compared by the three methods. RESULTS: LDL cholesterol values determined by the reference beta-quantitation procedure were highly correlated with both the Friedewald formula (r = 0.96) and a direct immunoseparation method (r = 0.92). Calculated (Friedewald) LDL cholesterol coincided with the reference method with < 10% error in 74% of the total diabetic group (82% of type I and 68% of type II diabetic patients). However, agreement between the direct LDL cholesterol and reference methods was significantly less (P = 0.02), with only 44% of patients having an error of < 10% (52% of type I and 41% of type II diabetic patients). The direct immunoseparation method for LDL cholesterol showed a positive bias with increasing triglyceride concentrations, particularly for patients with type II diabetes. CONCLUSIONS: In the group of diabetic patients studied with plasma triglyceride concentrations < 4.5 mmol/l, the Friedewald formula provided an accurate estimation of LDL cholesterol. The direct immunoseparation method significantly overestimated LDL cholesterol at triglyceride levels between 2 and 4.5 mmol/l.

Coordinate changes in the low density lipoprotein receptor activity of liver and mononuclear cells in the rabbit.

In the rabbit, dietary cholesterol downregulates the hepatic LDL receptor and concomitant treatment with 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors partly restores its expression. The aim of this study was to determine whether the LDL receptor activity of circulating mononuclear cells would reflect the changes seen in liver. New Zealand White rabbits were fed for 3 weeks either a normal diet or diets containing 0.25% (w/w) cholesterol, 0.25% cholesterol plus 22 mg/kg per day pravastatin or 0.25% cholesterol plus 6 mg/kg per day simvastatin. Dietary cholesterol increased plasma cholesterol 8.9-fold, liver membrane cholesterol 1.8-fold and bile cholesterol saturation 2.3-fold, and decreased the LDL receptor activities of liver and mononuclear cells by 69% and 58%, respectively. In the cholesterol-fed rabbit, pravastatin decreased plasma cholesterol by 55%, liver membrane cholesterol by 29% and bile cholesterol saturation by 23%, and increased liver and mononuclear cell LDL receptor activities by 120% and 77%, respectively. Similarly, simvastatin decreased plasma cholesterol by 74%, liver membrane cholesterol by 24% and bile cholesterol saturation by 38%, and increased liver and mononuclear cell LDL receptor activities by 80% and 62%, respectively. Liver and mononuclear cell LDL receptor activities were directly correlated (r = 0.73, P < 0.005) and both activities were inversely correlated with plasma cholesterol concentration in a log-linear fashion (r = -0.70, P < 0.005 and r = -0.69, P < 0.01, respectively). The LDL receptor activity of mononuclear cells therefore reflected the hepatic LDL receptor activity in these rabbits.

Regulation of bile acid pool size and plasma lipid levels in the SHR/N-corpulent rat: influence of the level of caloric intake.

In the obese progeny of the SHR/N-cp strain of the rat the bile acid pool was at least twice as large as that in their lean littermates even when only 6 weeks old. The composition of the pool remained unchanged in the obese females, but in their male counterparts the proportion of cholic acid was significantly increased. Cholestyramine feeding reduced the pool size by 26% in the obese rats, but a similar effect also occurred in the lean animals. The obese rats consumed about 60% more food per day than their lean littermates. When obese females were pair-fed to the intake of their lean controls from 6 to 11 weeks of age, the bile acid pool remained significantly enlarged, although not to the same extent as in the obese rats fed ad lib. Plasma cholesterol levels were reduced but remained significantly higher than the levels in the lean animals. The marked hypertriglyceridemia exhibited by the obese rats fed ad libitum did not develop in their pair-fed counterparts. In contrast, there was a comparatively smaller reduction in plasma cholesterol and triglyceride levels in the obese rats fed cholestyramine. Hepatic steatosis persisted in the pair-fed animals as well as in those given cholestyramine. Restricting caloric intake significantly reduced the body weight gain of the obese rats but had little effect on the extent of their corpulence. These studies show that at least some of the characteristics of this congenic strain, including hypertriglyceridemia and hepatic and intestinal hypertrophy, are due mainly to excess dietary intake.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclic fluctuations in fasting serum bile acid levels detected with a sensitive enzyme/bioluminescent assay.

A sensitive two-step bioluminescent assay for total serum bile acids was developed using commercially available enzymes. In the first step, the bile acids present in 10 microL of alkali-treated serum were oxidised at pH 9.5 by high purity 3 alpha-hydroxysteroid dehydrogenase to form NADH. Then, NADH was quantitated at pH 6.5 under optimal conditions for bioluminescence using FMN:NADH oxidoreductase and luciferase from Photobacterium fischeri. The enzyme/bioluminescent assay correlated well with gas-liquid chromatography and radioimmunoassay methods. Assay of fasting sera in eight healthy subjects revealed cyclic fluctuations in bile acid concentrations which were inversely related to gallbladder volume. These results provide biochemical evidence for interdigestive partial gallbladder emptying as a normal physiological process.

Lipoprotein profiling by high performance gel chromatography.

High performance gel chromatography (HPGC) was used to separate lipoproteins on the basis of their size and to generate lipoprotein profiles for plasma collected from patients with different lipoprotein phenotypes. These profiles provided a direct measurement of low density lipoprotein (LDL)-cholesterol which was more precise than LDL-cholesterol values calculated by the Friedewald equation. In addition, LDL-cholesterol concentrations were obtained in patients with combined hyperlipidemia in whom LDL-cholesterol could not be accurately calculated by the Friedewald equation. The response of LDL-cholesterol to the drug gemfibrozil was reliably monitored and in addition changes in LDL particle size could be assessed from the LDL apolipoprotein B/cholesterol ratio. HPGC also assisted in the diagnosis of type III hyperlipidemia by revealing a characteristic lipoprotein profile. HPGC-derived lipoprotein profiles provided additional useful clinical information for combined hyperlipidemia (Fredrickson lipoprotein phenotypes IIb, III).

Nephelometric determination of total protein in cerebrospinal fluid and urine using benzalkonium chloride as precipitation reagent.

Four different protein precipitants, namely trichloroacetic acid, sulphosalicylic acid, benzethonium chloride and benzalkonium chloride, were used to estimate the total protein concentration in cerebrospinal fluid and urine by nephelometry. Protein determinations for 50 cerebrospinal fluid samples and 100 urine samples were compared with values obtained by a trichloroacetic acid-Ponceau S spectrophotometric method. All methods were correlated well, but total protein results using acid precipitants were usually lower, while results obtained using benzethonium chloride were generally higher, than results obtained by the trichloroacetic acid-Ponceau S method. Anomalous results were obtained for some urine samples with benzethonium chloride, but not with benzalkonium chloride. Assays using benzalkonium chloride as precipitating reagent showed good precision and closest agreement with the trichloroacetic acid-Ponceau S dye-binding method. The use of benzalkonium chloride as precipitant is recommended for automated cerebrospinal fluid and urine protein estimations by nephelometry.

Chenodeoxycholic acid administration monitored by serum bile acid profiles: a dose-response study.

Serum bile acid analysis was used to monitor the bile acid composition of bile in 16 healthy male volunteers before and during the oral administration of different doses of the gallstone-dissolving bile acid, chenodeoxycholic acid. Daily chenodeoxycholic acid ingestion increased the percentage of this bile acid in bile to a new steady-state level after two to three weeks. Doses of 125, 250, 500, and 750 mg/day significantly increased the proportion of chenodeoxycholic acid in bile from 41% to 55, 61, 78, and 79%, respectively, during the fourth week of ingestion. The results indicate that useful information concerning chenodeoxycholic acid-induced changes in the bile acid composition of bile can be obtained solely by serum bile acid analysis, and bile collection by duodenal intubation can be avoided.

Relationships between homocysteine and related amino acids in chronic hemodialysis patients.

AIMS: Homocysteine (Hcy) has emerged as an important risk factor for atherosclerotic disease. Elevated levels in chronic dialysis patients may contribute to high vascular mortality, but little is known about levels of related amino acids in this group. In an observational study in the clinical setting we sought to document these. METHODS: In 114 hemodialysis patients pre-dialysis total plasma homocysteine, vitamin B12 and red blood cell (RBC) folate concentrations were measured. In a subgroup of patients (n = 42), other plasma amino acids were measured pre- and post-dialysis. All patients were routinely taking oral folic acid supplements (1.2 mg per week). RESULTS: Elevated homocysteine concentrations were found in all patients (geometric mean 33.1 umol/l, range 13.8 - 69.2 umol/l, laboratory reference range (RR) 3-13 umol/l). RBC folate levels were high (1223 +/- 54.5 nmol/l mean +/- SE, RR 300 - 710 nmol/l) and inversely related to pre-dialysis plasma Hcy (r = -0.44, p < 0.001). Hcy levels were not related to vitamin B12 levels. A history of vascular disease was not associated with higher concentrations of Hcy. Hcy clearance on dialysis was substantial (mean Hcy reduction 33 +/- 14%). While plasma methionine levels were normal, serine levels were significantly lower than the reference range (59.3 +/- 2.39 umol/l (mean +/- SE, RR 70 - 195 umol/l)) and directly related to levels of glycine (r = 0.52, p < 0.001). Glycine levels were within normal range. Although overall levels were low, higher serine levels were related to elevated homocysteine (r = 0.42, p < 0.01). Dialytic loss of glycine, serine and methionine was moderate. CONCLUSION: An inverse association between RBC folate and homocysteine levels extended to 3 times the upper limit of normal for folate, suggesting a role for high dose folic acid supplementation in the treatment of renal-failure related hyperhomocysteinemia. Low serine levels are expected as it is primarily synthesized in the kidney. The direct relationship between serine and homocysteine is consistent with the reported lack of effect of serine supplements on high Hcy levels.