Cloning, overexpression, and purification of functional human purine nucleoside phosphorylase

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. A genetic deficiency due to mutations in the gene encoding for human PNP causes T-cell deficiency as the major physiological defect. Inappropriate activation of T-cells has been implicated in several clinically relevant human conditions such as transplant tissue rejection, psoriasis, rheumatoid arthritis, lupus, and T-cell lymphomas. Human PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation. In addition, bacterial PNP has been used as reactant in a fast and sensitive spectrophotometric method that allows both quantitation of inorganic phosphate (Pi) and continuous assay of reactions that generate P i such as those catalyzed by ATPases and GTPases. Human PNP may therefore be an important biotechnological tool for P i detection. However, low expression of human PNP in bacterial hosts, protein purification protocols involving many steps, and low protein yields represent technical obstacles to be overcome if human PNP is to be used in either high-throughput drug screening or as a reagent in an affordable P i detection method. Here, we describe PCR amplification of human PNP from a liver cDNA library, cloning, expression in Escherichia coli host, purification, and activity measurement of homogeneous enzyme. Human PNP represented approximately 42% of total soluble cell proteins with no induction being necessary to express the target protein. Enzyme activity measurements demonstrated a 707-fold increase in specific activity of cloned human PNP as compared to control. Purification of cloned human PNP was achieved by a two-step purification protocol, yielding 48 mg homogeneous enzyme from 1 L cell culture, with a specific activity value of 80 U mg -1. © 2002 Elsevier Science (USA). All rights reserved.

Spectrophotometric flow-injection determination of sulphite in white wines involving gas diffusion through a concentric tubular membrane

A flow-injection system is proposed for the spectrophotometric determination of sulphite in white wines. The method involves analyte conversion to SO2, gas diffusion through a Teflon® semi-permeable membrane, collection into an alkaline stream (pH 8), reaction with Malachite green (MG) and monitoring at 620 nm. With a concentric tubular membrane, the system design was simplified. Influence of reagent concentrations, pH of donor and acceptor streams, temperature, timing, surfactant addition and presence of potential interfering species of the wine matrix were investigated. A pronounced (ca. 100%) enhancement in sensitivity was noted by adding cetylpyridinium chloride (CPC). The proposed system is robust and baseline drift is not observed during 4 h operating periods. Only 400 μL of sample and 0.32 mg MG are required per determination. The system handles 30 samples per hour, yielding precise results (r.s.d. < 0.015 for 1.0 - 20.0 mg L-1 SO2) in agreement with those obtained by an alternative procedure.

Faixas de controle de plantas daninhas e seus reflexos no crescimento de plantas de eucalipto

Two field trial were conducted in Eucalyptus grandis W. Hill ex Maiden x Eucalyptus urophylla S.T. Blake crops at Três Lagoas, Mato Grosso do Sul State and Brotas, São Paulo State, Brazil, to evaluate the effects of weed control in strip of different width on the Eucalyptus plant growth. The experiments consisted of two groups treatments: group 1 - with constant width control strips being kept during the first 12 months, i.e., at 0, 25, 50, 100, 125 and 150cm on each side of the Eucalyptus row and, group 2: with increasing width control strips being adopted along the experimental period, i.e., at 25 to 150, 25-50-150, 50 to 150, 50-125-150, 100-125-150, 100 tot 150 and 125 to 150 cm on each side of the Eucalyptus row during the first 12 months. At 49 moths after planting it was verified that the Eucalyptus plants that had grown both in the constant and increasing width control strips, equal to or higher than 100 and 50 cm, respectively, showed higher steam diameter, plant height, volume and annual increment average of wood. These results led to the conclusion that the minimum control strip width should be 100 cm on each side of the Eucalyptus row in order to keep the crop free of weed interference.

Influence of activation modes on diametral tensile strength of dual-curing resin cements.

In metallic restorations, the polymerization of dual-curing resin cements depends exclusively on chemical activation. The effect of the lack of photoactivation on the strength of these cements has been rarely studied. This study evaluated the influence of activation modes on the diametral tensile strength (DTS) of dual-curing resin cements. Base and catalyst pastes of Panavia F, Variolink II, Scotchbond Resin Cement, Rely X and Enforce were mixed and inserted into cylindrical metal moulds (4 x 2 mm). Cements were either: 1) not exposed to light (chemical activation = self-cured groups) or 2) photoactivated through mylar strips (chemical and photo-activation = dual-cured groups) (n = 10). After a 24 h storage in 37 masculineC distilled water, specimens were subjected to compressive load in a testing machine. A self-curing resin cement (Cement-It) and a zinc phosphate cement served as controls. Comparative analyses were performed: 1) between the activation modes for each dual-curing resin cement, using Students t test; 2) among the self-cured groups of the dual-curing resin cements and the control groups, using one-way ANOVA and Tukeys test (alpha = 0.05). The dual-cured groups of Scotchbond Resin Cement (53.3 MPa), Variolink II (48.4 MPa) and Rely X (51.6 MPa) showed higher DTS than that of self-cured groups (44.6, 40.4 and 44.5 MPa respectively) (p < 0.05). For Enforce (48.5 and 47.8 MPa) and Panavia F (44.0 and 43.3 MPa), no significant difference was found between the activation modes (p > 0.05). The self-cured groups of all the dual-curing resin cements presented statistically the same DTS as that of Cement-It (44.1 MPa) (p > 0.05), and higher DTS than that of zinc phosphate (4.2 MPa). Scotchbond Resin Cement, Variolink II and Rely X depended on photoactivation to achieve maximum DTS. In the absence of light, all the dual-curing resin cements presented higher DTS than that of zinc phosphate and statistically the same as that of Cement-It (p > 0.05).

Quantitative analysis of potentially toxic metals in alginates for dental use

Alginate is one the materials most employed in practice to make dental impressions. Substances like zinc, cadmium and lead silicate, which are included in several alginate brands with the aim of improving their physical, chemical and mechanical properties, are a source of serious concern as regards their toxicity. The most serious chronic effect of oral exposure to cadmium is renal toxicity. Assimilation of lead has deleterious effects on the gastrointestinal tract, hematopoietic system, cardiovascular system, central and peripheral nervous systems, kidneys, immune system, and reproductive system. Chronic oral exposures to zinc have resulted in hypochromic and microcyte anemia in some individuals. The aim of the present study was to measure the cadmium, lead and zinc contents of seven brands of alginate for dental use on sale in Brazil. The samples were weighed and placed in the Teflon cups of a closed-system microwave oven. Aqua regia (4mL concentrated HCI:HNO3, 3:1 v/v) and hydrofluoric acid (2mL concentrated HF) were added to the samples, which were then subjected to heating. The samples were then cooled to room temperature and diluted to 25 mL in deionized water in a volumetric glass flask. The samples were diluted in duplicate and analyzed against a reagent blank. The analyses were performed in an atomic absorption flame spectrophotometer. Neither lead nor cadmium was detected. Zinc contents ranged from 0.001% to 1.36% by weight. The alginates exhibited low contents of the metals under study and gave no cause for concern regarding toxicity; even so, it is advisable to monitor potentially toxic materials continually and to analyze their plasmatic levels in the professionals working with them.

Emprego do reagente de fenton como agente coagulante na remoção de substâncias húmicas de água por meio da flotação por ar dissolvido e filtração

Fenton 's reagent was used as coagulant agent to treat water with high true color (100 ± 5 HU) caused by the introduction of humic substances extracted from peat, using dissolved air flotation. The pair value of coagulant dosage x coagulation pH was optimized to posterior construction of coagulation diagrams, reaching apparent color removal efficiency slightly superior to 60%. It was tried to simulate a treatment with complete cycle, carrying out an experiment with sand filtration after flotation, obtaining an effluent with excellent quality, presenting remnant apparent color, turbidity and absorbance of 253.7 nm less or equal to 2 HU, 0.40 TU and 0.009 cm -1, respectively, and residual total iron < 0.005 mg/L and DOC < 0.001 mg/L.

Agronomic effectiveness of cationic phosphate impurities present in superphosphate fertilizers as affected by soil pH

The general concept that low-water-soluble phosphorus (P) fertilizers should be more agronomically effective when applied to acidic soils was developed based on sources containing mainly calcium (Ca)-P compounds, but it may not hold true for sources with different chemical composition. To obtain information related to this issue, two important iron (Fe)-potassium (K)-P compounds present in superphosphates [Fe 3 KH 8 (PO 4 ) 6·6H 2 O, H8, and Fe 3 KH 14 (PO 4 ) 8·4H 2 O, H14] were prepared and characterized. These P sources were used to provide 30 and 60 mg P kg -1 as neutral ammonium citrate (NAC)+H 2 O-soluble P. Reagent-grade monocalcium phosphate (MCP) was used as a standard P source with high water solubility with an additional rate of 120 mg P kg -1 included. Also, mixtures of both Fe-K-P compounds and MCP were prepared to provide 0, 25, 50, 75, and 100% of the total P as MCP. All sources were applied to a clayey loamy acid soil (pH 5.3) classified as Rhodic Kanhapludult. The soil was incubated at two rates (0 and 10 g kg -1 ) of lime, which resulted in pH 5.4 and 6.8. Upland rice was cultivated to maturity. The H14 compound confirmed to be a highly effective source of P for the rice plants at both soil pH, as opposed to the H8, which was poorly effective when applied alone. When mixed with water-soluble P (WSP), the H8 was able to provide P to the plants with the maximum yield of upland rice reached with 54.8 and 80.5% of WSP for pH 5.4 and 6.8, respectively. The high agronomic performance of the H14 compound clearly indicates that this low-water-soluble P source cannot be deemed as ineffective at high soil pH. Copyright © Taylor & Francis Group, LLC.

A multicommuted flow-based system for hydrogen peroxide determination by chemiluminescence detection using a photodiode

A simple, rapid, and automated assay for hydrogen peroxide in pharmaceutical samples was developed by combining the multicommutation system with a chemiluminescence (CL) detector. The detection was performed using a spiral flow-cell reactor made from polyethylene tubing that was positioned in front of a photodiode. It allows the rapid mixing of CL reagent and analyte and simultaneous detection of the emitted light. The chemiluminescence was based on the reaction of luminol with hydrogen peroxide catalyzed by hexacyanoferrate(III). The feasibility of the flow system was ascertained by analyzing a set of pharmaceutical samples. A linear response within the range of 2.2-210 μmol l-1 H2O2 with a LD of 1.8 μmol l-1 H2O2 and coefficient of variations smaller than 0.8% for 1.0×10-5 mol l-1 and 6.8×10-5 mol l-1 hydrogen peroxide solutions (n=10) were obtained. Reagents consumption of 90 μg of luminol and 0.7 mg of hexacyanoferrate(III) per determination and sampling rate of 200 samples per hour were also achieved. Copyright © Taylor & Francis Group, LLC.

Comparative studies of the sample decomposition of green and roasted coffee for determination of nutrients and data exploratory analysis

The contents of some nutrients in 35 Brazilian green and roasted coffee samples were determined by flame atomic absorption spectrometry (Ca, Mg, Fe, Cu, Mn, and Zn), flame atomic emission photometry (Na and K) and Kjeldahl (N) after preparing the samples by wet digestion procedures using i) a digester heating block and ii) a conventional microwave oven system with pressure and temperature control. The accuracy of the procedures was checked using three standard reference materials (National Institute of Standards and Technology, SRM 1573a Tomato Leaves, SRM 1547 Peach Leaves, SRM 1570a Trace Elements in Spinach). Analysis of data after application of t-test showed that results obtained by microwave-assisted digestion were more accurate than those obtained by block digester at 95% confidence level. Additionally to better accuracy, other favorable characteristics found were lower analytical blanks, lower reagent consumption, and shorter digestion time. Exploratory analysis of results using Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA) showed that Na, K, Ca, Cu, Mg, and Fe were the principal elements to discriminate between green and roasted coffee samples. ©2007 Sociedade Brasileira de Química.

Distribuição, tamanho populacional, hábitat e conservação do bicudinho-do-brejo Stymphalornis acutirostris Bornschein, Reinert e Teixeira, 1995 (Thamnophilidae)

The Marsh Antwren (Stymphalornis acutirostris) is restricted to the lowlands between Antonina Bay, in the coastal plain of the state of Paraná, and Itapocu river, in the northern coastal plain of the state of Santa Catarina (from 0 to c. 5 m a.s.l.). It doesn't occur continuously in this region, being found in eight populations that span over an total area of about 6,060 ha (= area of occupancy; 4,856.67 in Paraná and c. 1,200 in Santa Catarina). Nine habitat types used by the Marsh Antwren were defined, based on vegetation physiognomy, localization, dominancy of botanical species, dominant life-form and history of the region. Five of these are herbaceous (marshes), while four have an upper arboreal stratum and an herbaceous lower stratum with marsh plants. According to the classification criteria of the Brazilian vegetation proposed by the Radambrasil Project, they were classified as Pioneering Formation of Fluvial Influence, Pioneering Formation of Fluvial-marine Influence, and/or Pioneering Formation of Lacustrine Influence. They occur as patches or narrow strips ranging from 0.001 to 203.0 ha in the state of Paraná. They are found mainly in the interior of bays, in the lower courses of rivers that drain into bays, in alluvial plains, and between sand dunes in the coastal plain. Characteristic herbaceous species are cattail (Typha domingensis), bulrush (Scirpus californicus), Crinum salsum, Panicum sp. cf. P. mertensii, saw grass (Cladium mariscus) and Fuirena spp. Hibiscus pernambucensis is the characteristic bush species, and Calophyllum brasiliense, Tabebuia cassinoides, Annona glabra and Laguncularia racemosa are the characteristic arboreal species. The Marsh Antwren lives in herbaceous vegetation, but also uses bushes and branches of small tress. It has low flight capacity and a single flight of more than 25 m was never recorded. Territories of 0.25 ha were estimated in one kind of habitat (tidal marsh) (= 8 individuals per hectare) and of 3.2 ha in another one (saw grass marsh) (= 0.62 individual per hectare). The global population estimate is of about 17,700 mature individuals (13,700 in Paraná and 4,000 in Santa Catarina). The species is really under threat of extinction, mainly because of it's restricted geographical distribution and habitat loss by human activities and biological contamination caused by invasion of exotic grasses (Urochloa arrecta and Brachiaria mutica).

γ-Radiation induces apoptosis via sarcoplasmatic reticulum in guinea pig ileum smooth muscle cells

We investigated the effects of γ-radiation on cells isolated from the longitudinal smooth muscle layer of the guinea pig ileum, a relatively radioresistant tissue. Single doses (up to 50 Gy) reduced the amount of sarcoplasmatic reticulum and condensed the myofibrils, as shown by electron microscopy 3 days post-irradiation. After that, contractility of smooth muscle strips was reduced. Ca2+ handling was altered after irradiation, as shown in fura-2 loaded cells, with elevated basal intracellular Ca2+, reduced amount of intrareticular Ca2+, and reduced capacitive Ca2+ entry. Radiation also induced apoptosis, judged from flow cytometry of cells loaded with proprium iodide. Electron microscopy showed that radiation caused condensation of chromatin in dense masses around the nuclear envelope, the presence of apoptotic bodies, fragmentation of the nucleus, detachment of cells from their neighbors, and reductions in cell volume. Radiation also caused activation of caspase 12. Apoptosis was reduced by the administration of the caspase inhibitor Z-Val-Ala-Asp-fluoromethyl-ketone methyl ester (Z-VAD-FMK) during the 3 day period after irradiation, and by the chelator of intracellular Ca2+, 1,2-bis(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid (BAPTA), from 1 h before until 2 h after irradiation. BAPTA also reduced the effects of radiation on contractility, basal intracellular Ca2+, amount of intrareticular Ca2+, capacitative Ca2+ entry, and apoptosis. In conclusion, the effects of gamma radiation on contractility, Ca2+ handling, and apoptosis appear due to a toxic action of intracellular Ca2+. Ca2+-induced damage to the sarcoplasmatic reticulum seems a key event in impaired Ca2+ handling and apoptosis induced by γ-radiation. © 2008 Elsevier B.V. All rights reserved.

New contributions to the determination of resonance frequencies of rectangular microstrip antennas by neural networks

This paper uses artificial neural networks (ANN) to compute the resonance frequencies of rectangular microstrip antennas (MSA), used in mobile communications. Perceptron Multi-layers (PML) networks were used, with the Quasi-Newton method proposed by Broyden, Fletcher, Goldfarb and Shanno (BFGS). Due to the nature of the problem, two hundred and fifty networks were trained, and the resonance frequency for each test antenna was calculated by statistical methods. The estimate resonance frequencies for six test antennas were compared with others results obtained by deterministic and ANN based empirical models from the literature, and presented a better agreement with the experimental values.

Lichen metabolites modulate hydrogen peroxide and nitric oxide in mouse macrophages

The activities of perlatolic acid (1), atranorin (2), and lecanoric acid (3) and their derivatives, such as orsellinates and β-methyl orsellinates obtained by alcoholysis, were assessed for stimulation of the release of hydrogen peroxide and nitric oxide in cultures of peritoneal macrophage cells from mice. The hydrogen peroxide production was estimated by oxidation of phenol red, while the Griess reagent was used to determine the nitric oxide production. 1 and 4-methoxy-ethyl orsellinate (XVII) were the compounds that induced the greatest release of H 2O 2, whereas n-pentyl orsellinate (IV), iso-propyl orsellinate (V), sec-butyl orsellinate (VI), and XVII induced a small release of NO. These results indicate that lichen products and their derivatives have potential immune-modulating activities. © 2009 Verlag der Zeitschrift für Naturforschung, Tübingen.

Endurance swimming periodized training in rats

The aim of this study was to develop an experimental protocol for endurance swimming periodization training in rats similar to high performance training in humans, and compare it to continuous training. Three groups of male Wistar rats (90 days old) were allocated to Sedentary Control (SC); Continuous Training (CT); and Periodized Experimental Training (PET) groups. PET and CT trained 5 days/week, over five weeks, CT: continuous training supporting a 5% body mass (bm) load for 40 min/day; PET: training subdivided into basic, specific, and taper periods, with overload changed daily (volume-intensity, continuous, and interval training). Total training overload was quantified (% bm X exercise time in training session) and equalized for the two trained groups. Glucose ([ 3H]2-deoxyglucose) uptake, incorporation to glycogen (synthesis), glucose oxidation (CO 2 production), and lactate production from [U- 14C]glucose by soleus muscle strips incubated in presence of insulin (100μU/mL) were evaluated 48h after the last training session. The load equivalent at 5.5mM blood lactate concentration ([La-5.5]) was determined in the incremental test. Lactate production was similar in all groups. PET presented higher glucose uptake (59%) than SC, and higher glycogen synthesis (51 and 22%) and glucose oxidation (147 and 178%) than SC and CT, respectively. CT presented higher glycogen synthesis rates (23%) than SC. Load [La-5.5] was similar between trained groups and higher than SC. PET presented higher values for glucose metabolism than CT and SC. These results open up new perspectives for studying training methods used in high performance sport through swimming exercise in rats.

Easiness of use and validity testing of VS-SENSE device for detection of abnormal vaginal flora and bacterial vaginosis

Accessing vaginal pH is fundamental during gynaecological visit for the detection of abnormal vaginal flora (AVF), but use of pH strips may be time-consuming and difficult to interpret. The aim of this study was to evaluate the VS-SENSE test (Common Sense Ltd, Caesarea, Israel) as a tool for the diagnosis of AVF and its correlation with abnormal pH and bacterial vaginosis (BV). The study population consisted of 45 women with vaginal pH ≥ 4.5 and 45 women with normal pH. Vaginal samples were evaluated by VS-SENSE test, microscopy and microbiologic cultures. Comparing with pH strips results, VS-SENSE test specificity was 97.8% and sensitivity of 91%. All severe cases of BV and aerobic vaginitis (AV) were detected by the test. Only one case with normal pH had an unclear result. Concluding, VS-SENSE test is easy to perform, and it correlates with increased pH, AVF, and the severe cases of BV and AV. Copyright © 2010 Gilbert G. G. Donders et al.