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.

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.

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.

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.

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)

TMD and GOHAI indices of Brazilian institutionalised and community-dwelling elderly

Considering the controversy in the literature regarding several aspects of temporomandibular dysfunction (TMD) in elderly populations and the absence of reliable data on elderly Brazilians in this field, this study consisted of an evaluation of TMD prevalence and the self-perception of oral health among institutionalised and community-dwelling elderly in Sao JosE dos Campos, Brazil.Two hundred and fifteen community-dwelling and 185 institutionalised elderly people were evaluated by the Helkimo anamnestic (Ai) and clinical dysfunction (Di) indices and answered a questionnaire using the Geriatric Oral Health Assessment Index (GOHAI).The major prevalence of TMD symptoms was for the Ai0 (symptom-free) group (69.5%), while the major prevalence of clinical signs was for the DiI (mild) group (56%). Women presented a higher AiII classification than men (chi(2) test, p = 0.049). Community-dwelling elderly presented a significantly lower Ai0 classification than the institutionalised ones (Two ratios equality test, p < 0.001). There was no relationship between the institutionalised status and the clinical dysfunction index for Di0 and DiIII classification (Two ratios equality test, p = 0.194 and 0.535 respectively). The institutionalised elderly presented greater (One-way anova = 0.005) self-perception of oral health (33.45) than did the community-dwelling group (32.66). There were only weak Pearson's correlations among the anamnestic (-33.0%) or clinical (-14.7%) findings by the TMD and GOHAI indices. Symptom-free (Ai0) institutionalised elderly presented better scores in all GOHAI dimensions and elderly representing an absence of clinical TMD signs (Di0) presented higher GOHAI physical dimension scores in both groups.The prevalence of TMD symptoms among this sample of elderly individuals was relatively low, self-perception of oral health was reasonable and a weak, inverse correlation was found between TMD signs and symptoms and elderly self-perception of oral health measured by the GOHAI index.

Ventrolateral periaqueductal gray lesion attenuates nociception but does not change anxiety-like indices or fear-induced antinociception in mice

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

Physiological and Stroke Parameters to Assess Aerobic Capacity in Swimming

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

Indices de atividade da doença inflamatória do intestino: indicadores clínicos e laboratoriais.

The quantification of the degree of activity of inflammatory bowel disease is assuming growing importance nowadays. The activity index of the disease can be attained by clinical and laboratorial indicators. For ulcerative colitis the mostly used clinical parameters are daily bowel movements and presence of bloody diarrhea whereas albumin, hemoglobin, ESR and positive acute phase protein measurements are the laboratory parameters. For Crohn's disease activity besides the daily bowel movements the presence of abdominal pain and discomfort sensation are also frequently used whereas the C-reactive protein is the most used laboratory test which is able to detect the disease reactivation even before the appearance of any clinical sign. The combinations of clinical signs with the laboratory tests earned the sympathy of the specialists and the set of ensembled indicators has been recognized by the author's name. In this sense, the classification of the ulcerative colitis activity originally proposed by Truelove and Witts deserves presently a wide acceptance whereas such agreement is still lacking for Crohn's disease activity. In the mean time, the Bristol index is clinically the most feasible, once the Crohn's disease activity index and the Van Hees index are considered too complex. However the latter indexes are still useful mainly for comparisons among multicentric data. It seems that the currently existing clinical signs used for Crohn's disease activity would be quantitatively improved by adding some easily made laboratory tests such as C-reactive protein.