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.

Apical leakage after root canal filling with an experimental calcium hydroxide gutta-percha point

This study was an in vitro analysis of the apical seal of root canals filled with an experimental calcium hydroxide gutta-percha point. One hundred and ten recently extracted human permanent teeth with a single root were used. The root canals were biomechanically prepared, and some received a dressing of calcium hydroxide for 7 days. Root canals not dressed were filled by the lateral condensation technique with either regular gutta-percha or calcium hydroxide gutta-percha points. The dressed root canals were filled with the same sealers and regular gutta-percha points. The specimens were placed into a 2% methylene blue dye solution under vacuum, and the apical leakage was linearly evaluated. The experimental groups with calcium hydroxide dressing and the one with calcium hydroxide gutta-percha points exhibited significantly (p < 0.05) less leakage than the groups filled with regular gutta-percha points. The results obtained indicated that calcium hydroxide guttapercha points produced an improvement in the apical sealing quality of the root canal filling. Copyright © 1996 by The American Association of Endodontists.

Frequent p53 mutations and occasional loss of chromosome 4 in invasive bladder carcinoma induced by N-butyl-N-(4-hydroxybutil)nitrosamine in B6D2F1 mice

B6D2F1 mice (45/group) were treated with N-butyl-N-(4- hydroxybutyl)nitrosamine (BBN) or uracil as follows: Group 1 received 0.05% BBN in drinking water for the entire experiment, Group 2 received 5 mg of BBN by gastric gavage in 0.1 mL of 20% ethanol twice per week for 10 wk, Group 3 received a 2.5% uracil-containing diet for the entire experiment, and Group 4 was controls (received 0.1 mL of 20% ethanol by gavage twice per week for 10 wk). The surviving mice in Group 1 were killed after week 26 and those in the other groups after week 30. By week 15, three of 11 Group 1 and one of 15 Group 2 mice had bladder carcinoma. By 26 and 30 wk, respectively, invasive carcinomas were observed in 33 of 34 and six of 21 mice in Groups 1 and 2 and renal pelvic carcinomas in 11 of 34 and three of 21 mice in Groups 1 and 2. Four of 19 uracil-treated mice had bladder nodular hyperplasia. By polymerase chain reaction-single-strand conformation polymorphism and sequence analyses, 16 of 20 and two of five bladder carcinomas from Groups 1 and 2, respectively, showed mutations in the p53 gene. Ha-ras mutation was present in one case. Loss of heterozygosity analysis with simple-sequence length polymorphism markers for chromosome 4 showed that 10 of 21, two of 15, and nine of 13 mice in Groups 1-3, respectively, had heterozygous or homozygous deletions. B6D2F1 mice are therefore susceptible to the urothelial carcinogenic effects of BBN and develop frequent p53 mutations and chromosome 4 deletions. Chromosome 4 deletions were also seen with uracil.

Elevation of boiling point of coffee extract

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

Non-transposed three-phase line analyses with a single real transformation matrix

In transmission line transient analyses, a single real transformation matrix can obtain exact modes when the analyzed line is transposed. For non-transposed lines, the results are not exact. In this paper, non-symmetrical and non transposed three-phase line samples are analyzed with a single real transformation matrix application (Clarke's matrix). Some interesting characteristics of this matrix application are: single, real, frequency independent, line parameter independent, identical for voltage and current determination. With Clarke's matrix use, mathematical simplifications are obtained and the developed model can be applied directly in programs based on time domain. This model works without convolution procedures to deal with phase-mode transformation. In EMTP programs, Clarke's matrix can be represented by ideal transformers and the frequency dependent line parameters can be represented by modified-circuits. With these representations, the electrical values at any line point can be accessed for phase domain or mode domain using the Clarke matrix or its inverse matrix. For symmetrical and non-transposed lines, the model originates quite small errors. In addition, the application of the proposed model to the non-symmetrical and non-transposed three phase transmission lines is investigated. ©2005 IEEE.

Location of single line-to-ground faults on distribution feeders using voltage measurements

This paper proposes a dedicated algorithm for lation of single line-to-ground faults in distribution systems. The proposed algorithm uses voltage and current phasors measured at the substation level, voltage magnitudes measured at some buses of the feeder, a database containing electrical, operational and topological parameters of the distribution networks, and fault simulation. Voltage measurements can be obtained using power quality devices already installed on the feeders or using voltage measurement devices dedicated for fault location. Using the proposed algorithm, likely faulted points that are located on feeder laterals geographically far from the actual faulted point are excluded from the results. Assessment of the algorithm efficiency was carried out using a 238 buses real-life distribution feeder. The results show that the proposed algorithm is robust for performing fast and efficient fault location for sustained single line-to-ground faults requiring less than 5% of the feeder buses to be covered by voltage measurement devices. © 2006 IEEE.

Thermal evaporation: Representation of rise in boiling point of grapefruit juice

The rise in boiling point of grapefruit juice was experimentally measured at soluble solids concentrations in the range of 9.3-60.6 °Brix and pressures between °6.0 × 103 and 9.0 × 104 Pa. Different approaches to represent experimental data, including the Dhring's rule, the Antoine equation and empirical models proposed in the literature were tested. In the range of 9.3-29.0 °Brix, the rise in boiling point was nearly independent of pressure, varying only with juice concentration. Considerable deviations of this behavior began to occur at concentrations higher than 29.0 °Brix. Experimental data could be best predicted by adjusting an empirical model, which consisted of a single equation that takes into account the dependence of rise in boiling point on pressure and concentration. © SAGE Publications 2007.

A single multiplex SNaPshot reaction of 42 SNPs to classify admixture populations into mitochondrial DNA haplogroups

SNaPshot minisequencing reaction is in increasing use because of its fast detection of many polymorphisms in a single assay. In this work we described a highly sensitive single nucleotide polymorphisms (SNPs) typing method with detection of 42 mitochondrial DNA (mtDNA) SNPs in a single PCR and SNaPshot multiplex reaction in order to allow haplogroup classification in Latin American admixture population. We validated the panel typing 160 Brazilian individuals. DNA was extracted from blood spotted on filter paper using Chelex protocol. Forty SNPs were selected targeting haplogroup-specific mutations in Europeans, Africans and Asians (only precursors of Native Americans haplogroups A2, B2, C1, and D1) and two non-coding SNPs were chosen to increase the power of discrimination between individuals (SNPs positions 16,519 and 16,362). It was done using a modified version of a previously published multiplex SNaPshot minisequencing reaction established to resolve European haplogroups, adding SNPs targeting Africans (L0, L1, L2, L3, and L*) and Asians (A, B, C, and D) haplogroups based on SNPs described at PhyloTree.org build 2. PCR primers were designed using PerlPrimer software and checked with the Autodimer program. Thirty-three primer-pairs were used to amplify 42 SNPs. Using this panel, we were able to successfully classify 160 individuals into their correct haplogroups. Complete SNP profiles were obtained from 10. pg of total DNA. We conclude that it is possible to build and genotype more than 40 mtDNA SNPs in a single multiplex PCR and SNaPshot reaction, with sensitivity and reliability, resolving haplogroup classification in admixture populations. © 2011.

Point vibration measurements for the detection of shallow-buried objects

A major UK initiative, entitled 'Mapping the Underworld', is seeking to address the serious social, environmental and economic consequences arising from an inability to locate accurately and comprehensively the buried utility service infrastructure without resorting to extensive excavations. Mapping the Underworld aims to develop and prove the efficacy of a multi-sensor device for accurate remote buried utility service detection, location and, where possible, identification. One of the technologies to be incorporated in the device is low-frequency vibro-acoustics, and application of this technique for detecting buried infrastructure is currently being investigated. Here, the potential for making a number of simple point vibration measurements in order to detect shallow-buried objects, in particular plastic pipes, is explored. Point measurements can be made relatively quickly without the need for arrays of surface sensors, which can be expensive, time-consuming to deploy, and sometimes impractical in congested areas. At low frequencies, the ground behaves as a simple single-degree-of-freedom (mass-spring) system with a well-defined resonance, the frequency of which will depend on the density and elastic properties of the soil locally. This resonance will be altered by the presence of a buried object whose properties differ from the surrounding soil. It is this behavior which can be exploited in order to detect the presence of a buried object, provided it is buried at a sufficiently shallow depth. The theoretical background is described and preliminary measurements are made both on a dedicated buried pipe rig and on the ground over a domestic waste pipe. Preliminary findings suggest that, for shallow-buried pipes, a measurement of this kind could be a quick and useful adjunct to more conventional methods of buried pipe detection. © 2012 Elsevier Ltd. All rights reserved.

SERS mapping in langmuir-blodgett films and single-molecule detection

Plasmon-enhanced spectroscopic techniques have expanded single-molecule detection (SMD) and are revolutionizing areas such as bio-imaging and single-cell manipulation. Surface-enhanced (resonance) Raman scattering (SERS or SERRS) combines high sensitivity with molecularfingerprint information at the single-molecule level. Spectra originating from single-molecule SERS experiments are rare events, which occur only if a single molecule is located in a hot-spot zone. In this spot, the molecule is selectively exposed to a significant enhancement associated with a high, local electromagnetic field in the plasmonic substrate. Here, we report an SMD study with an electrostatic approach in which a Langmuir film of a phospholipid with anionic polar head groups (PO 4 -) was doped with cationic methylene blue (MB), creating a homogeneous, two-dimensional distribution of dyes in the monolayer. The number of dyes in the probed area of the Langmuir-Blodgett (LB) film coating the Ag nanostructures established a regime in which single-molecule events were observed, with the identification based on direct matching of the observed spectrum at each point of the mapping with a reference spectrum for the MB molecule. In addition, advanced fitting techniques were tested with the data obtained from micro-Raman mapping, thus achieving real-time processing to extract the MB single-molecule spectra. © 2013 Society for Applied Spectroscopy.

A single chart with supplementary runs rules for monitoring the mean vector and the covariance matrix of multivariate processes

The MRMAX chart is a single chart based on the standardized sample means and sample ranges for monitoring the mean vector and the covariance matrix of multivariate processes. User's familiarity with the computation of these statistics is a point in favor of the MRMAX chart. As a single chart, the recently proposed MRMAX chart is very appropriate for supplementary runs rules. In this article, we compare the supplemented MRMAX chart and the synthetic MRMAX chart with the standard MRMAX chart. The supplementary and the synthetic runs rules enhance the performance of the MRMAX chart. © 2013 Elsevier Ltd.

Modernization of Customs and Implementation of Single Window Systems for Foreign Trade: Results of an International Seminar-Workshop

An international seminar-workshop entitled "Facilitation of trade and transport in Latin America: situation and outlook" was held at the headquarters of the Economic Commission for Latin America and the Caribbean (ECLAC) on 29 and 30 November 2005, organized jointly by the ECLAC Division of International Trade and Integration and the United Nations Conference on Trade and Development (UNCTAD). The event was attended by about 50 persons involved in customs modernization and/or the implementation of single window systems for foreign trade in 20 Ibero-American countries.The main purpose of the seminar-workshop was to exchange ideas, opinions and proposals concerning the efficient implementation of trade facilitation instruments. The conclusions reached at this event point to the need to seek convergence among the existing trade agreements associated with trade facilitation in Latin America. Customs modernization requires the re-design of processes and procedures in order to achieve interoperability among the systems, and single window systems for foreign trade can only be implemented successfully if clear political leadership is established with broad participation from both public and private organizations.

SNPs (Single Nucleotide Polymorphisms) em genes relacionados a absorção e translocação de Boro em Eucalyptus

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Investigation of single-strand conformational polymorphism of the TP53 gene in women with a family history of breast cancer

Breast cancer in families with germ line mutations in the TP53 gene has been described in the medical literature. Mutation screening for susceptibility genes should allow effective prophylactic and preventive measures. Using single-strand conformational polymorphism, we screened for mutations in exons 5, 6, 7 and 8 of gene TP53 in the peripheral blood of 8 young non-affected members (17 to 36 years old) of families with a history of breast cancer. Studies of this type on young patients (mean age, 25 years) are very rare in the literature. The identification of these mutations would contribute to genetic counseling of members of families with predisposition to breast cancer. The results obtained did not show any polymorphism indicating mutation. In our sample, the familial tumorigenesis is probably related to other gene etiologies.

Oxidative stress on cardiotoxicity after treatment with single and multiple doses of doxorubicin

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