Kinetic characterization of hypophosphatasia mutations with physiological substrates

We have analyzed 16 missense mutations of the tissue-nonspecific AP (TNAP) gene found in patients with hypophosphatasia. These mutations span the phenotypic spectrum of the disease, from the lethal perinatal/infantile forms to the less severe adult and odontohypophosphatasia. Site-directed mutagenesis was used to introduce a sequence tag into the TNAP cDNA and eliminate the glycosylphosphatidylinositol (GPI)-anchor recognition sequence to produce a secreted epitope-tagged TNAP (setTNAP). The properties of GPI-anchored TNAP (gpiTNAP) and setTNAP were found comparable. After introducing each single hypophosphatasia mutation, the setTNAP and mutant TNAP cDNAs were expressed in COS-1 cells and the recombinant flagged enzymes were affinity purified. We characterized the kinetic behavior, inhibition, and heat stability properties of each mutant using the artificial substrate p-nitrophenylphosphate (pNPP) at pH 9.8. We also determined the ability of the mutants to metabolize two natural substrates of TNAP, that is, pyridoxal-5'-phosphate (PLP) and inorganic pyrophosphate (PPi), at physiological pH. Six of the mutant enzymes were completely devoid of catalytic activity (R54C, R54P, A94T, R206W, G317D, and V365I), and 10 others (A16V, A115V, A160T, A162T, E174K, E174G, D277A, E281K, D361V, and G439R) showed various levels of residual activity. The A160T substitution was found to decrease the catalytic efficiency of the mutant enzyme toward pNPP to retain normal activity toward PPi and to display increased activity toward PLP. The A162T substitution caused a considerable reduction in the pNPPase, PPiase, and PLPase activities of the mutant enzyme. The D277A mutant was found to maintain high catalytic efficiency toward pNPP as substrate but not against PLP or PPi. Three mutations ( E174G, E174K, and E281K) were found to retain normal or slightly subnormal catalytic efficiency toward pNPP and PPi but not against PLP. Because abnormalities in PLP metabolism have been shown to cause epileptic seizures in mice null for the TNAP gene, these kinetic data help explain the variable expressivity of epileptic seizures in hypophosphatasia patients.

Interval training at 95% and 100% of the velocity at VO2 max: effects on aerobic physiological indexes and running performance

The objective of this study was to analyze the effect of two different high-intensity interval training (HIT) programs on selected aerobic physiological indices and 1500 and 5000 m running performance in well-trained runners. The following tests were completed (n = 17): (i) incremental treadmill test to determine maximal oxygen uptake (VO2max), running velocity associated with VO2 max (VVO2max), and the velocity corresponding to 3.5 mmol/L of blood lactate concentration (vOBLA); (ii) submaximal constant-intensity test to determine running economy (RE); and (iii) 1500 and 5000 m time trials on a 400 m track. Runners were then randomized into 95% vVO(2max) or 100% vVO(2max) groups, and undertook a 4 week training program consisting of 2 HIT sessions (performed at 95% or 100% vVO(2max), respectively) and 4 submaximal run sessions per week. Runners were retested on all parameters at the completion of the training program. The VO2 max values were not different after training for both groups. There was a significant increase in post-training vVO(2 max), RE, and 1500 in running performance in the 100% vVO(2 max) group. The vOBLA and 5000 m running performance were significantly higher after the training period for both groups. We conclude that vOBLA and 5000 m running performance can be significantly improved in well-trained runners using a 4 week training program consisting of 2 HIT sessions (performed at 95% or 100% vVO(2max)) and 4 submaximal run sessions per week. However, the improvement in vVO(2 max), RE, and 1500 in running performance seems to be dependent on the HIT program at 100% vVO(2 max).

Synthesis and characterization of phosphorylcholine-substituted chitosans soluble in physiological pH conditions

A polymer analogous synthesis involving the reductive amination of phosphorylcholine (PC)-glyceraldehyde with primary amines of deacetylated chitosan (M-w approximate to 57000 g mol(-1)) was used to prepare phosphorylcholine-substituted chitosans (PC-CH) with a degree of substitution (DS) ranging from similar to 11 to similar to 53 mol% PC-substituted glucosamine residues. The PC-CH derivatives were characterized by H-1 NMR spectroscopy, FTIR spectroscopy, and multiangle laser light scattering gel permeation chromatography (MALLS-GPC). The pKa of the PC-substituted amine groups (pKa approximate to 7.20) was determined by H-1 NMR titration. The PC-CH samples (1.0 g L-1) were shown to be nontoxic using an MTT assay performed with human KB cells. Aqueous solutions of PC-CH samples (4.0 g L-(1)) of DS g 22 mol% PC-substituted glucosamine residues remained clear, independently of pH (4.0 < pH < 11.0). The remarkable water solubility and nontoxicity displayed by the new PC-CH samples open up new opportunities in the design of chitosan-based biomaterials and nanoparticles.

Energy and protein requirements for maintenance and growth of Boer crossbred kids

Meat production by goats has become an important livestock enterprise in several parts of the world. Nonetheless, energy and protein requirements of meat goats have not been defined thoroughly. The objective of this study was to determine the energy and protein requirements for maintenance and growth of 34 3/4 Boer x 1/4 Saanen crossbred, intact male kids (20.5 +/- 0.24 kg of initial BW). The baseline group was 7 randomly selected kids, averaging 21.2 +/- 0.36 kg of BW. An intermediate group consisted of 6 randomly selected kids, fed for ad libitum intake, that were slaughtered when they reached an average BW of 28.2 +/- 0.39 kg. The remaining kids (n = 21) were allocated randomly on d 0 to 3 levels of DMI (treatments were ad libitum or restricted to 70 or 40% of the ad libitum intake) within 7 slaughter groups. A slaughter group contained 1 kid from each treatment, and kids were slaughtered when the ad libitum treatment kid reached 35 kg of BW. Individual body components (head plus feet, hide, internal organs plus blood, and carcass) were weighed, ground, mixed, and subsampled for chemical analyses. Initial body composition was determined using equations developed from the composition of the baseline kids. The calculated daily maintenance requirement for NE was 77.3 +/- 1.05 kcal/kg(0.75) of empty BW (EBW) or 67.4 +/- 1.04 kcal/kg(0.75) of shrunk BW. The daily ME requirement for maintenance (118.1 kcal/g(0.75) of EBW or 103.0 kcal/kg(0.75) of shrunk BW) was calculated by iteration, assuming that the heat produced was equal to the ME intake at maintenance. The partial efficiency of use of ME for NE below maintenance was 0.65. A value of 2.44 +/- 0.4 g of net protein/kg(0.75) of EBW for daily maintenance was determined. Net energy requirements for growth ranged from 2.55 to 3.0 Mcal/kg of EBW gain at 20 and 35 kg of BW, and net protein requirements for growth ranged from 178.8 to 185.2 g/kg of EBW gain. These results suggest that NE and net protein requirements for growing meat goats exceed the requirements previously published for dairy goats. Moreover, results from this study suggest that the N requirement for maintenance for growing goats is greater than the established recommendations.

Effect of thermal stress on physiological parameters, feed intake and plasma thyroid hormones concentration in Alentejana, Mertolenga, Frisian and Limousine cattle breeds

The aim of the present study was to assess the heat tolerance of animals of two Portuguese (Alentejana and Mertolenga) and two exotic (Frisian and Limousine) cattle breeds, through the monitoring of physiological acclimatization reactions in different thermal situations characterized by alternate periods of thermoneutrality and heat stress simulated in climatic chambers. In the experiment, six heifers of the Alentejana, Frisian and Mertolenga breeds and four heifers of the Limousine breed were used. The increase in chamber temperatures had different consequences on the animals of each breed. When submitted to heat stress, the Frisian animals developed high thermal polypnea (more than 105 breath movements per minute), which did not prevent an increase in the rectal temperature (from 38.7 degrees C to 40.0 degrees C). However, only a slight depression in food intake and in blood thyroid hormone concentrations was observed under thermal stressful conditions. Under the thermal stressful conditions, Limousine animals decreased food intake by 11.4% and blood triiodothyronine (T3) hormone concentration decreased to 76% of the level observed in thermoneutral conditions. Alentejana animals had similar reactions. The Mertolenga cattle exhibited the highest capacity for maintaining homeothermy: under heat stressful conditions, the mean thermal polypnea increased twofold, but mean rectal temperature did not increase. Mean food intake decreased by only 2% and mean T3 blood concentration was lowered to 85,6% of the concentration observed under thermoneutral conditions. These results lead to the conclusion that the Frisian animals had more difficulty in tolerating high temperatures, the Limousine and Alentejana ones had an intermediate difficulty, and the Mertolenga animals were by far the most heat tolerant.

EVALUATION of COVERING MATERIALS IN INDIVIDUAL SHELTERS and ITS EFFECTS on PHYSIOLOGICAL RESPONSES and PERFORMANCE of DAIRY CALVES

In Tropical regions, the animal performance is often affected by climate conditions. This study aimed to evaluate covering materials in individual shelters, normally used to house dairy calves, and its influence on the calves physiology and performance. The design used was completely randomized, with a 2x3 factorial arrangement to compare the averages of 5% through the Tukey's test, i.e., both genders-and three types of covering in the shelters (Z - zinc; AC - asbestos cement; and WPAC - white-painted asbestos cement). Parameters evaluated included daily weight gain (DWG), dry matter intake (DMI), feed conversion (FC), rectal temperature (RT), and respiratory frequency (RF). Results showed significant differences (P < 0.05) among males (1.04kg/day) and females (0.74kg/day) for DWG and interaction between gender and treatment (P < 0.05) for zinc covering (0.562kg/day for females and 1.120kg/day for males). Significant differences were also observed in FI of animals housed under shelters with the covering of zinc (48.35kgDM/day for females and 96.91 kgDM/day for males). There were no significant differences (P > 0.05) in the FC and the RT, and there were significant differences (P < 0.05) for RF in the Z treatments (56.9 mov.min(-1)), WPAC (62.2 mov.min(-1)) and FC (70.25 mov.min-1). It was concluded that different covering materials did not affect performance and dry matter intake of dairy calves. However, the animals' physiology of thermoregulation was altered by the different covering materials used in individual shelters.

Mineral requirements for growth of Moxoto goats grazing in the semi-arid region of Brazil

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

EFFECTS of 14-WEEK SWIMMING TRAINING PROGRAM on THE PSYCHOLOGICAL, HORMONAL, and PHYSIOLOGICAL PARAMETERS of ELITE WOMEN ATHLETES

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Changes in physiological and stroking parameters during interval swims at the slope of the d-t relationship

The slope of the distance-time relationship from maximal 200 and 400 in bouts (S(200-400)) has been increasingly employed for setting training intensities in swimming. However, physiological and mechanical responses at this speed are poorly understood. Thus, this study investigated blood lactate, heart rate (HR), stroke rate (SR), stroke length (SL) and RPE responses to an interval swimming set at S(200-400) in trained swimmers. In a 50-m pool, twelve athletes (16.5 +/- 1.2 yr, 176 +/- 7 cm, 68.4 +/- 5.4 kg, and 7.8 +/- 2.5% body fat) performed maximal 200 and 400 m crawl trials for S(200-400) determination (1.28 +/- 0.05 m/s). Thereafter, swimmers were instructed to perform 5 x 400 in at this speed with 1.5 min rest between repetitions. Three athletes Could not complete the set (exhaustion at 21.0 +/- 3.1 min). For the remaining swimmers (total set duration = 32.0 +/- 1.3 min) significant increases) (p < 0.05) in blood lactate (5.7 +/- 0.8-7.9 +/- 2.4 mmol/l), SR (29.6 +/- 3.2-32.1 +/- 4.1 cycles/min), HR (169 +/- 11-181 +/- 8 bpm) and RPE (13.3 +/- 1.6-16.3 +/- 2.6) were observed through the IS. Conversely, SL decreased significantly (p < 0.05) from the first to the fifth repetition (2.48 +/- 0.22-2.31 +/- 0.24 m/cycle). These results suggest that interval swimming at S(200-400) represents an intense physiological, mechanical and perceptual stimulus that can be sustained for a prolonged period by most athletes. (C) 2008 Sports Medicine Australia. Published by Elsevier Ltd. All rights reserved.

Maintenance requirements for methionine and cysteine, and threonine for poultry

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Physiological Responses and Characteristics of Table Tennis Matches Determined in Official Tournaments

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

CATHODIC BEHAVIOUR of ANODIZED TITANIUM IN SIMULATED PHYSIOLOGICAL CONDITIONS

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)