Effect of Food Restriction and Intense Physical Training on Estrous Cyclicity and Plasma Leptin Concentrations in Rats

Intense physical training and dietary energy restriction have been associated with consequences such as nutritional amenorrhea. We investigated the effects of intense physical training, food restriction or the combination of both strategies on estrous cyclicity in female rats, and the relationship between leptin ad these effects. Twenty-seven female Wistar rats were distributed into four groups: SF: sedentary, fed ad libitum; SR: sedentary subjected to 50% food restriction (based on the food intake of their fed counterparts); TF: trained (physical training on a motor treadmill with a gradual increase in speed and time), fed ad libitum; TR; trained with 50% food restriction. We analysed estrous cyclicity, plasma leptin and estradiol as well as chemical composition of the carcass, body weight variation. and weight of ovaries and perirenal adipose tissue. Data demonstrate that physical training alone was not responsible for significant modifications in either carcass chemical composition or reproductive function. Food restriction reduced leptin levels in all animals and interrupted the estrous cyclicity in some animals, but only the combination of food restriction and physical training was capable of interrupting the estrous cyclicity in all animals. Leptin was not directly related to estrous cyclicity. From our findings, it may be concluded that there is an additive or synergistic effect of energy intake restriction and energy expenditure by intense physical training on estrous cyclicity. Leptin appears to be one among others factors related to estrous cycle, but it probably acts indirectly.

Aging and calorie restriction modulate yeast redox state, oxidized protein removal, and the ubiquitin-proteasome system

The ubiquitin-proteasome system governs the half-life of most cellular proteins. Calorie restriction (CR) extends the maximum life span of a variety of species and prevents oxidized protein accumulation. We studied the effects of CR on the ubiquitin-proteasome system and protein turnover in aging Saccharomyces cerevisiae. CR increased chronological life span as well as proteasome activity compared to control cells. The levels of protein carbonyls, a marker of protein oxidation, and those of polyubiquitinated proteins were modulated by CR. Controls, but not CR cells, exhibited a significant increase in oxidized proteins. In keeping with decreased proteasome activity, polyubiquitinated proteins were increased in young control cells compared to time-matched CR cells, but were profoundly decreased in aged control cells despite decreased proteasomal activity. This finding is related to a decreased polyubiquitination ability due to the impairment of the ubiquitin-activating enzyme in aged control cells, probably related to a more oxidative microenvironment. CR preserves the ubiquitin-proteasome system activity. Overall, we found that aging and CR modulate many aspects of protein modification and turnover. (C) 2011 Elsevier Inc. All rights reserved.

Exercise training and caloric restriction prevent reduction in cardiac Ca(2+)-handling protein profile in obese rats

Previous studies show that exercise training and caloric restriction improve cardiac function in obesity. However, the molecular mechanisms underlying this effect on cardiac function remain unknown. Thus, we studied the effect of exercise training and/or caloric restriction on cardiac function and Ca(2+) handling protein expression in obese rats. To accomplish this goal, male rats fed with a high-fat and sucrose diet for 25 weeks were randomly assigned into 4 groups: high-fat and sucrose diet, high-fat and sucrose diet and exercise training, caloric restriction, and exercise training and caloric restriction. An additional lean group was studied. The study was conducted for 10 weeks. Cardiac function was evaluated by echocardiography and Ca(2+) handling protein expression by Western blotting. Our results showed that visceral fat mass, circulating leptin, epinephrine, and norepinephrine levels were higher in rats on the high-fat and sucrose diet compared with the lean rats. Cardiac nitrate levels, reduced/oxidized glutathione, left ventricular fractional shortening, and protein expression of phosphorylated Ser(2808)-ryanodine receptor and Thr(17-)phospholamban were lower in rats on the high-fat and sucrose diet compared with lean rats. Exercise training and/or caloric restriction prevented increases in visceral fat mass, circulating leptin, epinephrine, and norepinephrine levels and prevented reduction in cardiac nitrate levels and reduced: oxidized glutathione ratio. Exercise training and/or caloric restriction prevented reduction in left ventricular fractional shortening and in phosphorylation of the Ser(2808)-ryanodine receptor and Thr(17)-phospholamban. These findings show that exercise training and/or caloric restriction prevent cardiac dysfunction in high-fat and sucrose diet rats, which seems to be attributed to decreased circulating neurohormone levels. In addition, this nonpharmacological paradigm prevents a reduction in the Ser(2808)-ryanodine receptor and Thr(17-)phospholamban phosphorylation and redox status. (Hypertension. 2010;56:629-635.)

Detection of the TLR4 1196C > T Polymorphism by Mismatched-Polymerase Chain Reaction Using Plasmid DNA as Internal Control in Restriction Fragment Length Polymorphism Assays

Background: Restriction fragment length polymorphism (RFLP) is a common molecular assay used for genotyping, and it requires validated quality control procedures to prevent mistyping caused by impaired endonuclease activity. We have evaluated the usefulness of a plasmid-based internal control in RFLP assays. Results: Blood samples were collected from 102 individuals with acute myocardial infarction (AMI) and 108 non-AMI individuals (controls) for DNA extraction and laboratory analyses. The 1196C> T polymorphism in the toll-like receptor 4 (TLR4) gene was amplified by mismatched-polymerase chain reaction (PCR). Amplicons and pBluescript II SK-plasmid were simultaneously digested with endonuclease HincII. Fragments were separated on 2% agarose gels. Plasmid was completely digested using up to 55.2 nmL/L DNA solutions and 1 mu L PCR product. Nevertheless, plasmid DNA with 41.4 nM or higher concentrations was incompletely digested in the presence of 7 mL PCR product. In standardized conditions, TLR4 1196C> T variant was accurately genotyped. TLR4 1196T allele frequency was similar between AMI (3.1%) and controls (2.0%, p = 0.948). TLR4 SNP was not associated with AMI in this sample population. In conclusion, the plasmid-based control is a useful approach to prevent mistyping in RFLP assays, and it is validate for genetic association studies such as TLR4 1196C> T.

In vitro Fluoride Release and Tensile Bond Strength of a Polymeric Intra-Buccal Bioadhesive

The intra-buccal polymeric bioadhesive systems that can stay adhered to the oral soft tissues for drug programmed release, with the preventive and/or therapeutic purpose has been employed for large clinical situations. A system based on hydroxypropyl methyl cellulose/Carbopol 934`/magnesium stearate (HPMC/Cp/StMg) was developed having the sodium fluoride as active principle. This kind of system was evaluated according to its resistance to the removal by means of physical test of tensile strength. Swine buccal mucosa extracted immediately after animals` sacrifice was employed as substrate for the physical trials, to obtain 16 test bodies. Artificial saliva with or without mucin was used to involve the substrate/bioadhesive system sets during the trials. Artificial salivas viscosity was determined by means of Brookfield viscometer, showing the artificial saliva with mucin 10.0 cP, and the artificial saliva without mucin 7.5 cP. The tensile strength assays showed the following averages: for the group ""artificial saliva with mucin"" - 12.89 Pa, and for the group ""without mucin"" - 12.35 Pa. Statistical analysis showed no significant difference between the assays for both artificial salivas, and it was possible to conclude that the variable mucin did not interfered with the bioadhesion process for the polymeric devices. The device was able to release fluoride in a safe, efficient and constant way up to 8 hours.

Relative dispersions of intra-albumin transit times across rat and elasmobranch perfused livers, and implications for intra- and inter-species scaling of hepatic clearance using microsomal data

It is recognized that vascular dispersion in the liver is a determinant of high first-pass extraction of solutes by that organ. Such dispersion is also required for translation of in-vitro microsomal activity into in-vivo predictions of hepatic extraction for any solute. We therefore investigated the relative dispersion of albumin transit times (CV2) in the livers of adult and weanling rats and in elasmobranch livers. The mean and normalized variance of the hepatic transit time distribution of albumin was estimated using parametric non-linear regression (with a correction for catheter influence) after an impulse (bolus) input of labelled albumin into a single-pass liver perfusion. The mean +/- s.e. of CV2 for albumin determined in each of the liver groups were 0.85 +/- 0.20 (n = 12), 1.48 +/- 0.33 (n = 7) and 0.90 +/- 0.18 (n = 4) for the livers of adult and weanling rats and elasmobranch livers, respectively. These CV2 are comparable with that reported previously for the dog and suggest that the CV2 Of the liver is of a similar order of magnitude irrespective of the age and morphological development of the species. It might, therefore, be justified, in the absence of other information, to predict the hepatic clearances and availabilities of highly extracted solutes by scaling within and between species livers using hepatic elimination models such as the dispersion model with a CV2 of approximately unity.

Quantifying the influence of intra-aggregate concentration gradients on solute transport

The physical nonequilibrium of solute concentration resulting from preferential now of soil water has often led to models where the soil is partitioned into two regions: preferential flow paths, where solute transport occurs mainly by advection, and the remaining region, where significant solute transport occurs through diffusive exchange with the flow paths. These two-region models commonly ignore concentration gradients within the regions. Our objective was to develop a simple model to assess the influence of concentration gradients on solute transport and to compare model results with experiments conducted on structured materials. The model calculates the distribution of solutes in a single spherical aggregate surrounded by preferential now paths and subjected to alternating boundary conditions representing either an exchange of solutes between the two regions (a wet period) or no exchange but redistribution of solutes within the aggregate (a dry period). The key parameter in the model is the aggregate radius, which defines the diffusive time scales. We conducted intermittent leaching experiments on a column of packed porous spheres and on a large (300 mm long by 216 mm diameter) undisturbed field soil core to test the validity of the model and its application to field soils. Alternating wet and dry periods enhanced leaching by up to 20% for this soil, which was consistent with the model's prediction, given a fitted equivalent aggregate radius of 1.8 cm, If similar results are obtained for other soils, use of alternating wet and dry periods could improve management of solutes, for example in salinity control and in soil remediation.

Changes in intra-abdominal pressure during postural and respiratory activation of the human diaphragm

In humans, when the stability of the trunk is challenged in a controlled manner by repetitive movement of a limb, activity of the diaphragm becomes tonic but is also modulated at the frequency of limb movement. In addition, the tonic activity is modulated by respiration. This study investigated the mechanical output of these components of diaphragm activity. Recordings were made of costal diaphragm, abdominal, and erector spinae muscle electromyographic activity; intra-abdominal, intrathoracic, and transdiaphragmatic pressures; and motion of the rib cage, abdomen, and arm. During limb movement the diaphragm and transversus abdominis were tonically active with added phasic modulation at the frequencies of both respiration and limb movement. Activity of the other trunk muscles was not modulated by respiration. Intra-abdominal pressure was increased during the period of limb movement in proportion to the reactive forces from the movement. These results show that coactivation of the diaphragm and abdominal muscles causes a sustained increase in intra-abdominal pressure, whereas inspiration and expiration are controlled by opposing activity of the diaphragm and abdominal muscles to vary the shape of the pressurized abdominal cavity.

In vivo measurement of the effect of intra-abdominal pressure on the human spine

In humans, intra-abdominal pressure (IAP) is elevated during many everyday activities. This experiment aimed to investigate the extent to which increased IAP-without concurrent activity of the abdominal or back extensor muscles-produces an extensor torque. With subjects positioned in side lying on a swivel table with its axis at L3, moments about this vertebral level were measured when IAP was transiently increased by electrical stimulation of the diaphragm via the phrenic nerve. There was no electromyographic activity in abdominal and back extensor muscles. When IAP was increased artificially to similar to 15% of the maximum IAP amplitude that could be generated voluntarily with the trunk positioned in flexion, a trunk extensor moment (similar to6 Nm) was recorded. The size of the effect was proportional to the increase in pressure. The extensor moment was consistent with that predicted from a model based on measurements of abdominal cross-sectional area and IAP moment arm. When IAP was momentarily increased while the trunk was flexed passively at a constant velocity, the external torque required to maintain the velocity was increased. These results provide the first in vivo data of the amplitude of extensor moment that is produced by increased IAP. Although the net effect of this extensor torque in functional tasks would be dependent on the muscles used to increase the IAP and their associated flexion torque, the data do provide evidence that IAP contributes, at least in part, to spinal stability. (C) 2001 Elsevier Science Ltd. All rights reserved.

Intra- and extracellular osmotic regulation in the hololimnetic Caridea and Anomura: a phylogenetic perspective on the conquest of fresh water by the decapod Crustacea

We investigate extra- and intracellular osmoregulatory capability in two species of hololimnetic Caridea and Anomura: Macrobrachium brasiliense, a palaemonid shrimp, and Aegla franca, an aeglid anomuran, both restricted to continental waters. We also appraise the sharing of physiological characteristics by the hololimnetic Decapoda, and their origins and role in the conquest of fresh water. Both species survive salinity exposure well. While overall hyperosmoregulatory capability is weak in A. franca and moderate in M. brasiliense, both species strongly hyporegulate hemolymph [Cl(-)] but not osmolality. Muscle total free amino acids (FAA) increase slowly but markedly in response to the rapid rise in hemolymph osmolality consequent to hyperosmotic challenge: 3.5-fold in A. franca and 1.9-fold in M. brasiliense. Glycine, taurine, arginine, alanine and proline constitute a parts per thousand 85% of muscle FAA pools in fresh water; taurine, arginine, alanine each contribute a parts per thousand 22% in A. franca, while glycine predominates (70%) in M. brasiliense. These FAA also show the greatest increases on salinity challenge. Muscle FAA titers correlate strongly (R = 0.82) with hemolymph osmolalities across the main decapod sub/infraorders, revealing that marine species with high hemolymph osmolalities achieve isosmoticity of the intra- and extracellular fluids partly through elevated intracellular FAA concentrations; freshwater species show low hemolymph osmolalities and exhibit reduced intracellular FAA titers, consistent with isosmoticity at a far lower external osmolality. Given the decapod phylogeny adopted here and their multiple, independent invasions of fresh water, particularly by the Caridea and Anomura, our findings suggest that homoplastic strategies underlie osmotic and ionic homeostasis in the extant freshwater Decapoda.

Intra- and interspecific divergence in the nuclear sequences of the clock gene period in species of the Drosophila buzzatii cluster

We have measured nucleotide variation in the CLOCK/CYCLE heterodimer inhibition domain (CCID) of the clock X-linked gene period in seven species belonging to the Drosophila buzzatii cluster, namely D. buzzatii, Drosophila koepferae, Drosophila antonietae, Drosophila serido, Drosophila gouveai, Drosophila seriema and Drosophila borborema. We detected that the purifying selection is the main force driving the sequence evolution in period, in agreement with the important role of CCID in clock machinery. Our survey revealed that period provides valuable phylogenetic information that allowed to resolve phylogenetic relationships among D. gouveai, D. borborema and D. seriema, which composed a polytomic clade in preliminary studies. The analysis of patterns of intraspecific variation revealed two different lineages of period in D. koepferae, probably reflecting introgressive hybridization from D. buzzatii, in concordance with previous molecular data.

Subpopulations of corticotrophs in the sheep pituitary during late gestation: Effects of development and placental restriction

The prepartum surge in fetal plasma cortisol is essential for the normal timing of parturition in sheep and may result from an increase in the ratio of ACTH to proopiomelanocortin (POMC) in the fetal circulation. In fetuses subjected to experimental induction of placental restriction, the prepartum surge in fetal cortisol is exaggerated, whereas pituitary POMC mRNA levels are decreased, and in vitro, unstimulated ACTH secretion is elevated in corticotrophs nonresponsive to CRH. We therefore investigated the changes in the relative proportions of cells expressing POMC, ACTH, and the CRH type 1 receptor (CRHR1) shortly before birth and during chronic placental insufficiency. Placental restriction (PR) was induced by removal of the majority of placental attachment sites in five ewes before mating. Pituitaries were collected from control and PR fetal sheep at 140 d (control, n = 4; PR, n = 4) and 144 d (control, n = 6; PR, n = 4). Pituitary sections were labeled with specific antisera raised against POMC, ACTH, and CRHR1. Three major subpopulations of corticotrophs were identified that expressed POMC + ACTH + CRHR1, ACTH + CRHR1, or POMC only. The proportion of pituitary corticotrophs expressing POMC + ACTH + CRHR1 decreased (P < 0.05) between 140 (control, 60 +/- 1%; PR, 66 +/- 4%) and 144 (control, 45 +/- 2%; PR, 56 +/- 6%) d. A significantly higher (P < 0.05) proportion of corticotrophs expressed POMC + ACTH + CRHR1 in the pituitary of the PR group compared with controls. This study is the first to demonstrate subpopulations of corticotrophs in the fetal sheep pituitary that differentially express POMC, ACTH, and CRHR1 and the separate effects of gestational age and placental restriction on these subpopulations of corticotrophs.

Intra-bone marrow injection of mesenchymal stem cells improves the femur bone mass of osteoporotic female rats

The effect of intra-bone injection of differentiated rat bone marrow mesenchymal stem cells (BMMSCs) into the femur of osteoporotic female rats was studied. Osteoporosis was induced in Wistar female rats by bilateral ovariectomy. Then, 0.75 million BMMSCs isolated from healthy rats were injected into the femurs of osteoporotic rats. Histomorphometric analysis and histology clearly revealed improvements in the treated group as compared to untreated group. In 2 months, the femurs of treated rats, unlike untreated rats, showed trabecular bone percentage almost similar to the femurs from control healthy rats. To confirm the origin of newly formed bone, the experiment was repeated with BMMSCs isolated from green fluorescent protein transgenic rats. Confocal microscopy demonstrated green fluorescent protein-positive cells at the surface of trabecular bone of the treated rats. We investigated in vitro osteogenic differentiation of BMMSCs isolated from osteoporotic rats by studying alkaline phosphatase activity, collagen synthesis, and the ability to form mineralized nodules. Osteoporotic BMMSCs showed less differentiation capabilities as compared to those isolated from healthy rats. The results clearly demonstrated the importance of BMMSCs in osteoporosis and that the disease can be treated by injection of BMMSCs.

Dietary salt restriction increases plasma lipoprotein and inflammatory marker concentrations in hypertensive patients

Background: Dietary salt restriction has been reported to adversely modify the plasma lipoprotein profile in hypertensive and in normotensive subjects. We investigated the effects of the low sodium intake (LSI) on the plasma lipoprotein profile and on inflammation and thrombosis biomarkers during the fasting and postprandial periods. Methods: Non-obese, non-treated hypertensive adults (n=41) were fed strictly controlled diets. An initial week on a control diet (CID, Na=160 mmol/day) was followed by 3 weeks on LSI (Na=60mmol/day). At admission and on the last day of each period, the 24-h ambulatory blood pressure was monitored and blood was drawn after an overnight fasting period and after a fat-rich test meal. Results: The dietary adherence was confirmed by 24-h urinary sodium excretion. Fasting triglyceride (TG), chylomicron-cholesterol, hsC-reactive protein (CRP), tumor necrosis factor-a (TNF-alpha). interleukin-6 (IL-6) concentrations, renin activity, aldosterone, insulin, and homeostasis model assessment insulin resistance (HOMA-IR) Values were higher, but non-esterified fatty acids (NEFA) were lower on LSI than on CD. For LSI, areas under the curve (AUC) of TG, chylomicron-cholesterol, apoB and the cholesterol/apoB ratio were increased, whereas AUC-NEFA was lowered. LSI did not modify body weight, hematocrit, fasting plasma cholesterol, glucose, adiponectin, leptin, fibrinogen and factor VII (FVII), and AUC of lipoprotein lipase and of lipoprotein remnants. Conclusion: LSI induced alterations in the plasma lipoproteins and in inflammatory markers that are common features of the metabolic syndrome. (C) 2008 Elsevier Ireland Ltd. All rights reserved.