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.

Stability for impulsive control systems

In this paper we extend the notion of the control Lyapounov pair of functions and derive a stability theory for impulsive control systems. The control system is a measure driven differential inclusion that is partly absolutely continuous and partly singular. Some examples illustrating the features of Lyapounov stability are provided.

Protein deficiency during pregnancy and lactation impairs glucose-induced insulin secretion but increases the sensitivity to insulin in weaned rats

We studied glucose homeostasis in rat pups from darns fed on a normal-protein (170 g/kg) (NP) diet or a diet containing 60 g protein/kg (LP) during fetal life and the suckling period. At birth, total serum protein, serum albumin and serum insulin levels were similar in both groups. However, body weight and serum glucose levels in LP rats were lower than those in NP rats. At the end of the suckling period (28 d of age), total serum protein, serum albumin and serum insulin were significantly lower and the liver glycogen and serum free fatty acid levels were significantly higher in LP rats compared with NP rats. Although the fasting serum glucose level was similar in both groups, the area under the blood glucose concentration curve after a glucose load was higher for NP rats (859 (SEM 58) mmol/l per 120 min for NP rats v. 607 (SEM 52) mmol/l per 120 min for LP rats; P < 0.005). The mean post-glucose increase in insulin was higher for NP rats (30 (SEM 4.7) nmol/l per 120 min for NP rats v. 17 (SEM 3.9) nnol/l per 120 min for LP rats; P < 0.05). The glucose disappearance rate for NP rats(0.7 (SEM 0.1) %/min) was lower than that for LP rats (1.6 (SEM 0.2) %/min; P < 0.001). Insulin secretion from isolated islets (1 h incubation) in response to 16.7 mmol glucose/l was augmented 14-fold in NP rats but only 2.6-fold in LP rats compared with the respective basal secretion (2.8 mmol/l; P <0.001). These results indicate that in vivo as well as in vitro insulin secretion in pups from dams maintained on a LP diet is reduced. This defect may be counteracted by an increase in the sensitivity of target tissues to insulin.

Thermodynamic models for water sorption by grape skin and pulp

The enthalpy-entropy compensation theory was applied to water sorption for grapes of Italy variety. The moisture sorption isotherms were analyzed using the static gravimetric method at 35, 40, 50, 60, 70 and 75 degrees C. For isotherms construction, the skin and pulp of the grape were used separately and it was possible to observe significant differences. The GAB equation was fitted to the experimental data, using direct nonlinear regression analysis; the agreement between experimental and calculated values was satisfactory. The net isosteric heat or enthalpy of water sorption, determined from the equilibrium sorption data, showed a different behavior when compared with other works, as it was obtained for skin and pulp separately. Plots of Delta h vs Delta S for skin and pulp provided the isokinetic temperatures T-Bs = 423.2 +/- 27.6 K and T-Bp = 424.5 +/- 25.3 K, respectively, indicating an enthalpy-controlled desorption process over the whole range of moisture content considered.

The effects of ice-water storage on blood gas and acid-base measurements

Objective: To determine the effects of storage of arterial and venous blood samples in ice water on blood gas and acid-base measurements.Design: Prospective, in vitro, laboratory study.Setting: School of veterinary medicine.Subjects: Six healthy dogs.Measurements and main results: Baseline measurements of partial pressure of oxygen (PO2), partial pressure of carbon dioxide (PCO2), pH, hemoglobin concentration (tHb), oxyhemoglobin saturation, and oxygen content (ContO(2)) were made. Bicarbonate (HCO3) and standard base excess (SBE) were calculated. Arterial and venous blood samples were separated into 1 and 3 mL samples, anaerobically transferred into 3 mL plastic syringes, and stored in ice water for 6 hours. Measurements were repeated at 15, 30 minutes, and 1, 2, 4, and 6 hours after baseline measurements. Arterial (a) PO2 increased significantly from baseline after 30 minutes of storage in the 1 mL samples and after 2 hours in the 3 mL samples. Venous (v) PO2 was significantly increased from baseline after 4 hours in the 1 mL samples and after 6 hours in the 3 mL samples. The pHa significantly decreased after 2 hours of storage in the 1 mL samples and after 4 hours in the 3 mL samples. In both the 1 and 3 mL samples, pHv decreased significantly only after 6 hours. Neither the arterial nor the venous PCO2 values changed significantly in the 1 mL samples and increased only after 6 hours in the 3 mL samples. No significant changes in tHb, ContO(2), SBE, or HCO3 were detected.Conclusions: the PO2 of arterial and venous blood increased significantly when samples were stored in plastic syringes in ice water. These increases are attributable to the diffusion of oxygen from and through the plastic of the syringe into the blood, which occurred at a rate that exceeded metabolic consumption of oxygen by the nucleated cells.

Highly swollen liquid crystals as new reactors for the synthesis of nanomaterials

Synthesis and self-assembly of nanomaterials can be controlled by the properties of soft matter. on one hand, dedicated nanoreactors such as reverse microemulsions or miniemulsions can be designed. on the other hand, direct shape control can be provided by the topology of liquid crystals that confine the reacting medium within a specific geometry. In the first case, the preparation of micro- or miniemulsions generally requires energetic mechanical stirring. The second approach uses thermodynamically stable systems, but it remains usually limited to binary (water + surfactant) systems. We report the preparation of different families of materials in highly ordered quaternary mediums that exhibit a liquid crystal structure with a high cell parameter. They were prepared with the proper ratios of salted water, nonpolar solvent, surfactant. and cosurfactants that form spontaneously swollen hexagonal phases. These swollen liquid crystals can be prepared from all classes of surfactants (cationic, anionic, and nonionic). They contain a regular network of parallel cylinders, whose diameters can be swollen with a nonpolar solvent, that are regularly spaced in a continuous aqueous salt solution. We demonstrate in the present report that both aqueous and organic phases can be used as nanoreactors for the preparation of materials. This property is illustrated by various examples such as the synthesis of platinum nanorods prepared in the aqueous phase or zirconia needles or the photo- or gamma-ray-induced polymerization of polydiacetylene in the organic phase. In all cases, materials can be easily extracted and their final shapes are directed by the structure-directing effect imposed by the liquid crystal.

Abundant brachiopods on a tropical, upwelling-influenced shelf (Southeast Brazilian Bight, South Atlantic)

Rhynchonelliform brachiopods were diverse and often dominant benthos of tropical seas in the Paleozoic. In contrast, they are believed to be rare in open habitats of modern oceans, especially at low latitudes. This study documents numerous occurrences of rhynchonelliform brachiopods on a modern tropical shelf, particularly in areas influenced by upwelling. Extensive sampling of the outer shelf and coastal bays of the Southeast Brazilian Bight revealed dense populations of terebratulid brachiopods (>10(3) individuals /m(2) of seafloor) between 24 and 26 S. on the outer shelf, brachiopods are more abundant than bivalves and gastropods combined. However, brachiopod diversity is low: only four species belonging to the genera Bouchardia, Terebratulina, Argyrotheca, and Platidia were identified among over 16000 examined specimens. Brachiopods occur preferentially on carbonate bottoms and include two substrate-related associations: Bouchardia (40-70% CaCO3, weight content) and Terebratulina-Argyrotheca (70-95% CaCO3). All four species display a broad bathymetric range that contrasts with a narrow depth tolerance postulated for many Paleozoic rhynchonelliforms. The most abundant populations occur in the depth range between 100 and 200 m, and coincide with zones of shelf-break upwelling, where relatively colder and nutrient-rich water masses of the South Atlantic Central Water are brought upward by cyclonic meanders of the South Brazil Current (a western boundary current that flows poleward along the coast of Brazil). This is consistent with previous biological and paleontological studies that suggest upwelling may play a role in sustaining brachiopod-dominated benthic associations. The presence of abundant brachiopods in the open habitats of the tropical shelf of the western South Atlantic contrasts with current understanding of their latitudinal distribution and points to major gaps in our knowledge of their present-day biogeography. The ecological importance of rhynchonelliform brachiopods in modern oceans and their role as producers of biogenic sedimentary particles may be underestimated.

Effect of continuous and pulsed therapeutic ultrasound in the appearance of local recurrence of mammary cancer in rats

Purpose: Ultrasound (US) therapy is an elect rot hermotherapeutic modality that uses US energy to provoke physical and chemical alterations. US therapy has been widely used in physical therapy. However in clinical practice, it is contra-indicated in cancer patients due to the possibility of exacerbating tumor growth.Methods: Sixty-eight female Sprague-Dawley rats bred in UNIFAE vivarium were studied. At 50 days of age, 7, 12-dimetylbenz(a)anthracene (7, 12-DMBA) was administered to 35 rats by gastric gavage to induce mammary cancer After 90 days the mammary glands of the rats belonging to the group with mammary cancer induction and stimulated by US were removed. Animals received either continuous or pulsed US. US waves were generated at a frequency of 1 MHz during 10 days, with an intensity dose of 0.5 W in the continuous group, and 0.9 W (duty cycle: 20%) in the pulsed group.Results: Among the rats treated with continuous US, 44.4% developed local recurrence, while among the rats treated with pulsed US, 22.2% had local tumor recurrence (p < 0.05). No evidence of distant metastases was shown in any of the rats studied.Conclusion: The use ofcontinuous and pulsed therapeutic US promoted the development of local recurrence of mammary cancer in female Sprague-Dawley rats in the postoperative period.

Evaluation of the microscopic observation drug susceptibility assay for detection of Mycobacterium tuberculosis resistance to pyrazinamide

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Tratamento de esgoto e produção de água de reúso com o emprego de filtros de areia

In this research it was studied a system composed by the anaerobic filter combined with a sand filter for the wastewater treatment. For this, three anaerobic filters were operated with hydraulic detention time of nine hours which had the effluent disposed over four sand filters in different frequencies of application. on the first sand filter, 50 L.m(-2) were applied once a day. on the second, the third and the fourth filters, the same load was disposed in twice, three and four times a day, distributed between 9 a. m. and 4 p. m. The system presented a final effluent suitable for the COD and BOD legislation maximum limit to be discharged into water body (Decreto Paulista no 8,468/1976), showing the viability of dispose a higher quantity of effluent then the one suggested by NBR 13969/1997, which guides the constructive aspects for this kind of treatment.

Estudo populacional de gambás, Didelphis albiventris (Mammalia, Didelphidae), em um pequeno fragmento florestal

Population study of white-eared-opossum, Didelphis albiventris (Mammalia, Didelphidae), in a small forest fragment. The white-eared-opossums are habitat generalist animals and occur in high abundance in forest fragments. Therefore these animals are important to understand the dynamics of this landscape. The population of Didelphis albiventris in a 7 ha stational semidecidual forest fragment and its agricultural surroundings was studied during one year. The population abundance, age structure and sex ratio were estimated monthly. Two movements between the fragment and the surrounding area were detected. The population dynamics showed an annual pattern with a high peak during the dry season.