Quercetin restores plasma nitrite and nitroso species levels in renovascular hypertension

Quercetin has antioxidants properties which may increase nitric oxide (NO) bioavailability. However, the effects of quercetin on NO status have been poorly studied. We evaluated whether quercetin improves the plasma levels of NO metabolites in two-kidney one-clip (2K1C) hypertensive rats and assessed its effect on endothelial function. Sham-operated and 2K1C rats were treated with quercetin (10 mg(-1) kg(-1) day(-1) by gavage) or vehicle for 3 weeks. Systolic blood pressure (SBP) was monitored weekly. Vascular responses to acetylcholine (Ach) and sodium nitroprusside (SNP) were assessed in hindquarter vascular bed. Plasma nitrate levels were assessed by Griess reagent and plasma nitrite and nitroso species (S, N-nitroso species) were assessed by ozone- based chemiluminescence. Aortic NADPH oxidase activity and superoxide production were evaluated. While quercetin had no effects in control normotensive rats (P > 0.05), it significantly reduced SBP in 2K1C rats (P < 0.05). At the end of treatment, plasma nitrate levels were similar in all experimental groups (P > 0.05). However, plasma nitrite and the nitroso species levels were significantly lower in 2K1C rats when compared with controls (P < 0.05). Quercetin treatment restored plasma nitrite and nitroso species levels to those found in the sham-vehicle group (P < 0.05). While quercetin treatment induced no significant changes in responses to SNP (P > 0.05), it restored the vascular responses to Ach. Quercetin significantly attenuated 2K1C-hypertension-induced increases in NADPH oxidase activity and vascular superoxide production (P < 0.05). These results suggest that the antihypertensive effects of quercetin were associated with increased NO formation and improved endothelial function, which probably result from its antioxidant effects.

In LipL32, the major leptospiral lipoprotein, the C terminus is the primary immunogenic domain and mediates interaction with collagen wand plasma fibronectin

LipL32 is the major leptospiral outer membrane lipoprotein expressed during infection and is the immunodominant antigen recognized during the humoral immune response to leptospirosis in humans. In this study, we investigated novel aspects of LipL32. In order to define the immunodominant domains(s) of the molecule, subfragments corresponding to the N-terminal, intermediate, and C-terminal portions of the UpL32 gene were cloned and the proteins were expressed and purified by metal affinity chromatography. Our immunoblot results indicate that the C-terminal and intermediate domains of LipL32 are recognized by sera of patients with laboratory-confirmed leptospirosis. An immunoglobulin M response was detected exclusively against the LipL32 C-terminal fragment in both the acute and convalescent phases of illness. We also evaluated the capacity of LipL32 to interact with extracellular matrix (ECM) components. Dose-dependent, specific binding of LipL32 to collagen type IV and plasma fibronectin was observed, and the binding capacity could be attributed to the C-terminal portion of this molecule. Both heparin and gelatin could inhibit LipL32 binding to fibronectin in a concentration-dependent manner, indicating that the 30-kDa heparin-binding and 45-kDa gelatin-binding domains of fibronectin are involved in this interaction. Taken together, our results provide evidence that the LipL32 C terminus is recognized early in the course of infection and is the domain responsible for mediating interaction with ECM proteins.

Long-Term Maternal Separation Differentially Alters Serum Corticosterone Levels and Blood Neutrophil Activity in A/J and C57BL/6 Mouse Offspring

Objectives: In this work, we searched for maternal separation effects on serum corticosterone levels and blood neutrophil activity in adult male A/J and C57BL/6 mouse offspring. Methods: 40 male A/J mice and 40 male C57BL/6 mice were divided within each strain into two groups. Mice in the maternal separation group were separated from their mothers (1 h/day) on postnatal days 0-13. Mice in the control group were left undisturbed. On postnatal day 45, blood was drawn from all mice and used to assess neutrophil activity by flow cytometry and serum corticosterone levels by radioimmunoassay. Results: The results showed that each mouse strain responded differently to maternal separation, but in both cases, serum corticosterone levels were affected. In both strains, adult mice that experienced maternal separation showed lower serum corticosterone levels than control mice. In relation to control mice kept together with their mothers, the levels of serum corticosterone were 72.7 and 36.36% lower in A/J and C57BL/6 mice submitted to maternal separation, respectively. The current findings showed that maternal separation increased neutrophil activity in mice after reaching adulthood. The observed effects, although in the same direction, differed between A/J and C57BL/6 mice. Maternal separation increased both the percentage and intensity of phagocytosis in C57BL/6 mice, but had no effects on A/J mice. Furthermore, maternal separation increased basal and propidium iodide-labeled Staphylococcus aureus-induced oxidative burst in A/J mice but did not affect oxidative burst in C57BL/6 mice. Finally, phorbol myristate acetate-induced oxidative burst increased in both strains. Conclusion: These results indicate that early maternal separation increases innate immunity, most likely by modifying hypothalamus-pituitary-adrenal axis activity. This suggests that maternal separation is a good model for stress which produces long-term neuroimmune changes whatever the animal species and strain used. Copyright (C) 2011 S. Karger AG, Basel

LC-MS/MS quantitation of plasma progesterone in cattle

Quantitation of progesterone (P(4)) in biological fluids is often performed by radioimmunoassay (RIA), whereas liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) has been used much less often. Due to its autoconfirmatory nature, LC-MS/MS greatly minimizes false positives and interference. Herein we report and compare with RIA an optimized LC-MS/MS method for rapid, efficient, and cost-effective quantitation of P(4) in plasma of cattle with no sample derivatization. The quantitation of plasma P(4) released from three nonbiodegradable, commercial, intravaginal P(4)-releasing devices (IPRD) over 192 h in six ovariectomized cows was compared in a pairwise study as a test case. Both techniques showed similar P(4) kinetics (P > 0.05) whereas results of P(4) quantitation by RIA were consistently higher compared with LC-MS/MS (P < 0.05) due to interference and matrix effects. The LC-MS/MS method was validated according to the recommended analytical standards and displayed P(4) limits of detection (LOD) and quantitation (LOQ) of 0.08 and a 0.25 ng/mL, respectively. The high selective LC-MS/MS method proposed herein for P(4) quantitation eliminates the risks associated with radioactive handling; it also requires no sample derivatization, which is a common requirement for LC-MS/MS quantitation of steroid hormones. Its application to multisteroid assays is also viable, and it is envisaged that it may provide a gold standard technique for hormone quantitation in animal reproductive science studies. (C) 2011 Elsevier Inc. All rights reserved.

Effect of Recombinant Bovine Somatotropin on Plasma Concentrations of Insulin-like Growth Factor I, Insulin and Membrane Integrity of Bull Spermatozoa

Contents This study aimed to evaluate the effect of the exogenous recombinant bovine somatotropin (rbST) on plasma concentrations of insulin-like growth factor I (IGF-I), insulin and semen quality of bulls. Twenty bulls (Aberdeen Angus and Brangus) were divided by breed into two groups. Placebo group was injected with NaCl 0.9% (s.c.) and treatment group with rbST (s.c., 500 mg) at days 0 and 14 of the experiment. Immediately after semen collection, blood samples were taken on days 0, 14, 28, 42 and 56 of the experiment. Semen was also collected on day 70 of the experiment. Evaluation of sperm motility was performed at pre-freezing and post-thawing stage, whereas assessment of sperm membrane integrity was performed after freezing and thawing. Analysis of data revealed that the effect of treatment and treatment-by-collection day on plasma concentrations of IGF-I and insulin was not significant. However, mean plasma concentrations of IGF-I and insulin were affected (p < 0.0001) by days of blood sampling. Effect of treatment and treatment-by-collection day on motility of spermatozoa was similar (p > 0.05) at pre-freezing and post-thawing stage. Intactness of plasmalemma and tail membrane of spermatozoa at post-thawing stage was higher (p < 0.05) in rbST-treated group than in control. In conclusion, rbST did not affect plasma concentrations of IGF-I and insulin, however, it did improve post-thaw sperm membrane integrity.

Validation of non-fluorescent methods to reliably detect acrosomal and plasma membrane integrity of common marmoset (Callithrix jacchus) sperm

Simple, rapid and stable sperm evaluation methods which have been optimized for common marmoset (Callithrix jacchus) are critical for studies involving collection and evaluation of sperm in the field. This is particularly important for new species groups such as Callitrichidae where the sperm have been little studied. Of this family, C jacchus is the best known, and has been chosen as a model species for other members of the genus Callithrix. The fundamental evaluation parameters for sperm of any species are viability and acrosomal status. Semen samples were collected by penile vibratory stimulation. To evaluate sperm plasma membrane integrity, Eosin-Nigrosin was tested here for the common marmoset sperm to be used under field conditions. Further, a non-fluorescent stain for acrosome, the ""Simple"" stain, developed for domestic and wild cats, was tested on common marmoset sperm. This was compared with a fluorescent staining, Fluorescein isothiocyanate-Pisum sativum agglutinin (FITC-PSA), routinely used and validated for common marmoset at the German Primate Centre to evaluate acrosomal integrity. Results obtained with the ""Simple"" stain showed a marked differentiation between sperm with intact and non-intact acrosome both with and without ionophore treatment and closely correlated with results obtained with FITC-PSA. Temperature had no effect on the results with the ""Simple"" stain and the complete processing is simple enough to be carried out under field conditions. These findings indicated that the ""Simple"" stain and Eosin-Nigrosin provide rapid and accurate results for C. jacchus sperm and that those methods can be reliably used as field tools for sperm evaluation for this species. (c) 2008 Elsevier Inc. All rights reserved.

Exercise training accelerates the removal from plasma of LDL-like nanoemulsion in moderately hypercholesterolemic subjects

Objective: Exercise training improves plasma lipid profile and diminishes risk of coronary heart disease. Previously, we showed that training increases LDL plasma clearance, as tested by an artificial LDL-like nanoemulsion method, presumably by increasing LDL receptor activity. In this study, we investigated whether training could also improve LDL clearance in hypercholesterolemic subjects (HCh) that are exposed to increased risk of cardiovascular events. Methods: Twenty sedentary HCh and 20 normolipidemic (NL) sedentary volunteers were divided into four groups: 12 HCh submitted to 4-month training program, 8 HCh with no exercise program, 12 NL submitted to 4-month training and 8 NL with no exercise program. An LDL-like nanoemulsion labeled with 14C-cholesteryl ester was injected intravenously into all subjects and plasma samples were collected during 24h after injection to determine the fractional clearance rate (FCR, in h-1) by compartmental analysis. The study was performed on the first and on the last day of the 4-month study period. Results: In both, trained HCh and NL groups, training increased nanoemulsion FCR by 36% (0.0443 +/- 0.0126; 0.0602 +/- 0.0187, p=0.0187 and 0.0503 +/- 0.0203; 0.0686 +/- 0.0216, p=0.0827, respectively). After training, LDL cholesterol diminished in both HCh and NL groups. In HCh, but not in NL group, LDL susceptibility to oxidation decreased, but oxidized LDL was unchanged. In both non-trained groups FCR was the same for the last and the 4-month previous evaluation. Conclusion: In HCh, exercise training increased the removal of LDL as tested by the nanoemulsion, and this probably accounted for decreased LDL cholesterol and diminished LDL susceptibility to oxidation. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Effects of sperm concentration and straw volume on motion characteristics and plasma, acrosomal, and mitochondrial membranes of equine cryopreserved spermatozoa

The advantages of using cryopreserved semen in equine reproduction are well known. During cryopreservationl spermatozoa undergo many changes that lead to a decrease in fertility. There is no agreement on the ideal sperm dose and concentration to maximize fertility rates. Thus, the objectives of this experiment were to evaluate sperm motion by computer-assisted analysis (CASA), sperm membrane integrity and function with fluorescence probes of cryopreserved sperm at three concentrations: 100 (C100), 200 (C200) and 400 x 10(6) sperm/mL (C400), and two straw volumes (0.50 and 0.25 mL). There was no interaction between sperm concentration and storage volume (P > .05). Sperm motion characteristics were influenced by concentration (C100 > C200 > C400; P < .05). Curvilinear velocity (VCL) in 0.25-mL straws had higher average values (P < .05). Membrane integrity and function were not changed by straw volume (P > .05). However, sperm concentration changed the percentage of cells with intact plasma membrane (C100 > C200 > C400; P < .05) and the percentage of cells with high mitochondrial membrane potential (C100 = C200; P > .05 and C400 < C100 and C200; P < .05). According to this experiment, the best freeing method was that involving 100 x 10(6) sperm/mL, regardless of straw volume.

Alteration of Ca(2+)-ATPase activity in the homogenate, plasma membrane and microsomes of the salivary glands of streptozotocin-induced diabetic rats

Diabetes has been implicated in the dryness of the mouth, loss of taste sensation, sialosis, and other disorders of the oral cavity, by impairment of the salivary glands. The aim of the present study was to examine the plasma membrane, microsomal, and homogenate Ca(2+)-ATPase activity in the rat submandibular and parotid salivary glands of streptozotocin-induced diabetes. We have also examined the influence of the acidosis state oil this parameter. Diabetes was induced by an intraperitoneal injection of streptozotocin and acidosis was induced by daily injection of NH(4)Cl. At 15 and 30 days after diabetes induction, the animals were euthanized and the submandibular and parotid salivary glands were removed and analyzed. Ca(2+)-ATPase (total, independent, and dependent) was determined in the homo-enate, microsomal, and plasma membranes of the salivary glands of diabetic and control rats. Calcium concentration was also determined in the glands and showed to be hi-her in the diabetic animals. Ca(2+)-ATPase activity was found to be reduced in all cell fractions studied in the diabetic animals compared with control. Similar results were obtained for the submandibular salivary glands of acidotic animals; however in the parotid salivary glands it was found an increase in the enzyme activity. Copyright (c) 2009 John Wiley & Sons, Ltd.

Daily variations in human plasma fluoride concentrations

This study investigated the variations in human plasma fluoride concentrations ([F]) and sought to determine the causes. Five subjects (27-33 years old) received a low-F diet during the 5 days of the study. Plasma samples and urine were collected every 3 h from 8 a.m. to 8 p.m. F, PTH, Ca and P were analyzed with the electrode, by chemiluminescence, AAS and colorimetry, respectively. A trend for the plasma [F] was found. The peak [F], 0.55 +/- 0.11 mu mol L(-1), occurred at 11 a.m. and the lowest [F], 0.50 +/- 0.06 mu mol L(-1) occurred between 5 and 8 p.m. Plasma [F] were positively correlated with urinary F excretion rates and with serum PTH levels, but not with the Ca or P levels. Serum PTH levels were positively correlated with urinary F excretion rates and negatively correlated with plasma Ca. The results suggest that the renal system seems to control the daily fluctuations in plasma [F]. (c) 2008 Elsevier B.V. All rights reserved.

Kinetics of fluoride removal from plasma and bone of rats after chronic intake of fluoride

This study evaluated the kinetics of fluoride in plasma, femur surface and the whole femur of rats, after chronic exposure to different water fluoride levels was interrupted. Four groups of Wistar rats received drinking water containing 0, 5, 15 or 50 mu g F/ml for 60 days (n = 50/group). The animals were euthanized immediately after exposure to fluoride or after 7, 30, 90 or 180 days (n = 10/subgroup). Plasma and femurs were collected. Fluoride on the femur surface, whole femur and plasma was analyzed with an electrode. Data were analyzed using ANOVA and Tukey`s test (p < 0.05). The increase in plasma fluoride levels was significant only for the 50 mu g F/ml group at 0 and 7 days. Regarding bone surface and whole bone, for most groups, significant increases in fluoride concentrations were observed with the increase in water fluoride concentrations at each time of euthanasia. For fluoride doses up to 15 mu g F/ml, femur surface fluoride levels were reestablished 180 days after the exposure was discontinued, which Was not valid for whole femur or for higher fluoride doses. We found a different kinetics of fluoride in plasma,femur surface and the whole femur of rats after chronic exposure to fluoride is interrupted. Copyright 2008 Prous Science, S.A.U. or its licensors. All rights reserved.

Effect of Fluoridated Water on Plasma Insulin Levels and Glucose Homeostasis in Rats with Renal Deficiency

Glucose intolerance in fluorosis areas and when fluoride is administered for the treatment of osteoporosis has been reported. Controlled fluoridation of drinking water is regarded as a safe and effective measure to control dental caries. However, the effect on glucose homeostasis was not studied so far. The aim of this study was to evaluate the effect of the intake of fluoridated water supply on glucose metabolism in rats with normal and deficient renal function. Male Sprague-Dawley rats were divided into eight groups of four rats. Renal insufficiency was induced in four groups (NX) which received drinking water containing 0, 1, 5, and 15 ppm F (NaF) for 60 days. Four groups with simulated surgery acted as controls. There were no differences in plasma glucose concentration after a glucose tolerance test between controls and NX rats and among rats with different intakes of fluoride. However, plasma insulin level increased as a function of fluoride concentration in drinking water, both in controls and in NX rats. It is concluded that the consumption of fluoridated water from water supply did not affect plasma glucose levels even in cases of animals with renal disease. However, a resistance to insulin action was demonstrated.

Functional polymorphisms in the promoter of the matrix metalloproteinase-9 (MMP-9) gene are not linked with significant plasma MMP-9 variations in healthy subjects

Background: Matrix metalloproteinase-9 (MMP-9) is involved in the degradation of the extracellular matrix during physiological and pathological processes. Two functional polymorphisms [C(-1562)T and microsatellite (CA)(13-25)] in the promoter region of the MMP-9 gene have been associated with several diseases. The aim of this study was to examine whether these MMP-9 polymorphisms and haplotypes are linked with plasma MMP-9 variations in healthy subjects. Methods: We studied 177 healthy male white volunteers (age range 20-55 years) who were non-smokers and not taking any medication. Genomic DNA was extracted from whole blood and genotypes for the C(-1562)T and the microsatellite (CA)(n) polymorphisms were determined. MMP-9 levels were measured in plasma samples by gelatin zymography. Results: The frequency of the alleles C and T for the C(-1562)T polymorphism were 90% and 10%, respectively. The frequency of the alleles with less than 21 CA repeats Q and with 21 repeats or higher (H) were 47% and 53%, respectively. We found no differences in plasma MMP-9 levels among the genotype groups or among different haplotypes (all p > 0.05). Conclusions: These findings suggest that functional polymorphisms in the promoter of the MMP-9 gene are not linked with significant plasma MMP-9 variations in healthy subjects.

Should we measure serum or plasma lead concentrations?

Project: Serum samples may not be appropriate to assess lead (Pb) concentrations because they may contain artificially higher Pb concentrations compared with those measured in plasma samples. Here, we compared Pb concentrations in serum versus heparin plasma separated from blood collected with or without vacuum. We have also examined the effects of sample standing time on Pb concentrations measured in serum, heparin plasma, and EDTA plasma. Procedure: We studied plasma and serum samples from twelve healthy subjects. Blood samples were collected via venous drainage phlebotomy with and without vacuum into trace metal free tubes containing no anticoagulants (serum), or lithium heparin, or EDTA (to obtain plasma). Variable sample standing times (0, 5, and 30 min) prior to centrifugation were allowed. Plasma and serum Pb and iron concentrations were determined by inductively coupled plasma mass spectrometry. Plasma and serum cell-free hemoglobin concentrations were measured. Results: Pb concentrations in serum and in heparin plasma from blood samples collected with or without vacuum were similar and not associated with significant changes in iron or hemoglobin concentrations. The sample standing time (up to 30 min) did not affect Pb concentrations in serum or in heparin plasma, which were approximately 50% lower than those found in EDTA plasma. Conclusions: Serum or heparin plasma separated from blood samples collected via venous phlebotomy with or without vacuum are appropriate medium to assess Pb concentrations, independently of the sample standing time. (C) 2010 Elsevier GmbH. All rights reserved.

Neonatal endotoxin exposure changes neuroendocrine, cardiovascular function and mortality during polymicrobial sepsis in adult rats

Our aim was to investigate whether neonatal LPS challenge may improve hormonal, cardiovascular response and mortality, this being a beneficial adaptation when adult rats are submitted to polymicrobial sepsis by cecal ligation and puncture (CLP). Fourteen days after birth, pups received an intraperitoneal injection of lipopolysaccharide (LPS; 100 mu g/kg) or saline. After 8-12 weeks, they were submitted to CLP, decapitated 4,6 or 24 h after surgery and blood was collected for vasopressin (AVP), corticosterone and nitrate measurement, while AVP contents were measured in neurohypophysis, supra-optic (SON) and paraventricular (PVN) nuclei. Moreover, rats had their mean arterial pressure (MAP) and heart rate (HR) evaluated, and mortality and bacteremia were determined at 24 h. Septic animals with neonatal LPS exposure had higher plasma AVP and corticosterone levels, and higher c-Fos expression in SON and PVN at 24 h after surgery when compared to saline treated rats. The LPS pretreated group showed increased AVP content in SON and PVN at 6 h, while we did not observe any change in neurohypophyseal AVP content. The nitrate levels were significantly reduced in plasma at 6 and 24 h after surgery, and in both hypothalamic nuclei only at 6 h. Septic animals with neonatal LPS exposure showed increase in MAP during the initial phase of sepsis, but HR was not different from the neonatal saline group. Furthermore, neonatally LPS exposed rats showed a significant decrease in mortality rate as well as in bacteremia. These data suggest that neonatal LPS challenge is able to promote beneficial effects on neuroendocrine and cardiovascular responses to polymicrobial sepsis in adulthood. (C) 2011 Elsevier B.V. All rights reserved.