Separation and partitioning of Green Fluorescent Protein from Escherichia coli homogenate in poly(ethylene glycol)/sodium-poly(acrylate) aqueous two-phase systems

The partitioning of Green Fluorescent Protein (GFP) in poly(ethylene glycol)/Na-poly(acrylate) aqueous two-phase systems (PEG/NaPA-ATPS) has been investigated. The aqueous two-phase systems are formed by mixing the polymers with a salt and a protein solution. The protein partitioning in the two-phase system was investigated at 25 degrees C. The concentration of the GFP was measured by fluorimetry. It was found that the partitioning of GFP depends on the salt type, pH and concentration of PEG. The data indicates that GFP partitions more strongly to the PEG phase in presence of Na2SO4 relative to NaCl. Furthermore, the GFP partitions more to the PEG phase at higher pH. The partition to the PEG phase is strongly favoured in systems with larger tie-line lengths (i.e. systems with higher polymer concentrations). The molecular weight of PEG is important since the partition coefficient (K) of GFP gradually decreases with increasing PEG size, from K ca. 300-400 for PEG 400 to K equal to 1.19 for PEG 8000. A separation process was developed where GFP was separated from a homogenate in two extraction steps: the GFP is first partitioned to the PEG phase in a PEG 3000/NaPA 8000 system containing 3 wt% Na2SO4, where the K value of GFP was 8. The GFP is then re-extracted to a salt phase formed by mixing the previous top-phase with a Na2SO4 solution. The K-value of GFP in this back-extraction was 0.22. The total recovery based on the start material was 74%. (c) 2008 Elsevier B.V. All rights reserved.

Protein partitioning in poly(ethylene glycol)/sodium polyacrylate aqueous two-phase systems

The partition of hemoglobin, lysozyme and glucose-6-phospate dehydrogenase (G6PDH) in a novel inexpensive aqueous two-phase system (ATPS) composed by poly(ethylene glycol) (PEG) and sodium polyacrylate (NaPA) has been studied. The effect of NaCl and Na2SO4, pH and PEG molecular size on the partitioning has been studied. At high pH (above 9), hemoglobin partitions strongly to the PEG-phase. Although some precipitation of hemoglobin occurs, high recovery values are obtained particularly for lysozyme and G6PDH. The partitioning forces are dominated by the hydrophobic and electrochemical (salt) effects, since the positively charged lysozyme and negatively charged G6PDH partitions to the non-charged PEG and the strongly negatively charged polyacrylate enriched phase, respectively. (c) 2007 Elsevier B.V. All rights reserved.

Bioavailability of dietary sodium copper chlorophyllin and its effect on antioxidant defence parameters of Wistar rats

BACKGROUND: There has been growing interest in sodium copper chlorophyllin (Cu-Chl) as a food colourant and supplement owing to its beneficial biological activities. Studies have revealed that this green pigment inhibits experimental carcinogenesis and interacts with proteins and genotoxic agents. Health-related activities have also been associated with the prevention of lipid peroxidation. However, intestinal absorption of this pigment has been considered insignificant, raising questions of whether eventual biological properties are related to pre- or post-absorptive actions. In this study, intestinal absorption of Cu-Chl and its appearance in serum and organs were estimated by high-performance liquid chromatography analysis in rat feeding experiments. The effect of ingested Cu-Chl on lipid peroxidation was analysed by measuring thiobarbituric acid-reactive substances and antioxidant enzyme activities in hepatic and brain tissues of oxidative stress-induced rats. RESULTS: The two main components of commercial Cu-Chl, namely Cu-chlorin e(6) and Cu-chlorin e(4), showed different digestive behaviours, and only Cu-chlorin e4 was found in serum, liver and kidneys. Antioxidant activity in vivo could be observed in brain and seemed to be related to in situ protection but not to antioxidant enzyme modulation. CONCLUSION: As at least one of the major components of Cu-Chl is effectively absorbed, further pharmacolkinetic studies are encouraged to access absorption rates and the role of ingested copper chlorophyllins in mammals. (C) 2009 Society of Chemical Industry

Manufacture of low-sodium Minas fresh cheese: Effect of the partial replacement of sodium chloride with potassium chloride

We investigated the effect of sodium reduction by partial substitution of sodium chloride (NaCl) with potassium chloride (KCl) on the manufacture of Minas fresh cheese during 21 d of refrigerated storage. Four treatments of low-sodium Minas fresh cheese were manufactured, with partial replacement of NaCl by KCl at 0, 25, 50, and 75% (wt/wt), respectively. The cheeses showed differences in the content of moisture, ash, protein, salt, and lipid contents, as well as on the extent of proteolysis and hardness throughout the storage period. However, no difference was observed among treatments within each storage day tested. The partial substitution of NaCl by KCl decreased up to 51.8% the sodium concentration of the cheeses produced. The consumer test indicated that it is possible to manufacture a low-sodium Minas fresh cheese that is acceptable to consumers by partial substitution of NaCl by KCl at 25% (wt/wt) in the salting step.

A new nitrosyl ruthenium complex nitric oxide donor presents higher efficacy than sodium nitroprusside on relaxation of airway smooth muscle

Nitric oxide (NO) has been demonstrated to be the primary agent in relaxing airways in humans and animals. We investigated the mechanisms involved in the relaxation induced by NO-donors, ruthenium complex [Ru(terpy)(bdq)NO(+)](3+) (TERPY) and sodium nitroprusside (SNP) in isolated trachea of rats contracted with carbachol in an isolated organs chamber. For instance, we verified the contribution of K(+) channels, the importance of sGC/cGMP pathway, the influence of the extra and intracellular Ca(2+) sources and the contribution of the epithelium on the relaxing response. Additionally, we have used confocal microscopy in order to analyze the action of the NO-donors on cytosolic Ca(2+) concentration. The results demonstrated that both compounds led to the relaxation of trachea in a dependent-concentration way. However, the maximum effect (E(max)) of TERPY is higher than the SNP. The relaxation induced by SNP (but not TERPY) was significantly reduced by pretreatment with ODQ (sGC inhibitor). Only TERPY-induced relaxation was reduced by tetraethylammonium (K(+) channels blocker) and by pre-contraction with 75 mM KCl (membrane depolarization). The response to both NO-donors was not altered by the presence of thapsigargin (sarcoplasmic reticulum Ca(2+)-ATPase inhibitor). The epithelium removal has reduced the relaxation only to SNP, and it has no effect on TERPY. The both NO-donors reduced the contraction evoked by Ca(2+) influx, while TERPY have shown a higher inhibitory effect on contraction. Moreover, the TERPY was more effective than SNP in reducing the cytosolic Ca(2+) concentration measured by confocal microscopy. In conclusion, these results show that TERPY induces airway smooth muscle relaxation by cGMP-independent mechanisms, it involves the fluxes of Ca(2+) and K(+) across the membrane, it is more effective in reducing cytosolic Ca(2+) concentration and inducing relaxation in the rat trachea than the standard drug, SNP. (C) 2011 Elsevier B.V. All rights reserved.

Fluoride increases lead concentrations in whole blood and in calcified tissues from lead-exposed rats

Higher blood lead (BPb) levels have been reported in children living in communities that receive fluoride-treated water. Here, we examined whether fluoride co-administered with lead increases BPb and lead concentrations in calcified tissues in Wistar rats exposed to this metal from the beginning of gestation. We exposed female rats and their offspring to control water (Control Group), 100 mg/L of fluoride (F Group), 30 mg/L of lead (Pb Group), or 100 mg/L of fluoride and 30 mg/L of lead (F+ Pb Group) from 1 week prior to mating until offspring was 81 days old. Blood and calcified tissues (enamel, dentine, and bone) were harvested at day 81 for lead and fluoride analyses. Higher BPb concentrations were found in the F+ Pb Group compared with the Pb Group (76.7 +/- 11.0 mu g/dL vs. 22.6 +/- 8.5 mu g/dL, respectively: p <0.001). Two-to threefold higher lead concentrations were found in the calcified tissues in the F+ Pb Group compared with the Pb Group (all p <0.001). Fluoride concentrations were similar in the F and in the F+ Pb Groups. These findings show that fluoride consistently increases BPb and calcified tissues Pb concentrations in animals exposed to low levels of lead and suggest that a biological effect not yet recognized may underlie the epidemiological association between increased BPb lead levels in children living in water-fluoridated communities. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Fluoride ingestion from food items and dentifrice in 2-6-year-old Brazilian children living in a fluoridated area using a semiquantitative food frequency questionnaire

Objectives: The aim of this study was to evaluate the fluoride intake of 2-6-year-old Brazilian children using a semiquantitative food frequency questionnaire (FFQ) which also estimated fluoride intake from dentifrice. Methods: The FFQ was previously validated through application to 78 2-6-year-old Brazilian children and then administered to 379 children residing in an optimally fluoridated community in Brazil (Bauru, State of Sao Paulo). The FFQ was applied to the parents and used to estimate the food intake of the children. The constituents of the diet were divided into solids, water and other beverages. The fluoride content of the diet items was analyzed with the fluoride electrode. The questionnaire also estimated fluoride intake from dentifrice. Results: The average (+/- SD) fluoride intake from solids, water, other beverages and dentifrice was 0.008 +/- 0.005; 0.011 +/- 0.004; 0.009 +/- 0.014 and 0.036 +/- 0.028 mg F/kg body weight/day, respectively, totalizing 0.064 +/- 0.035 mg F/kg body weight/day. The dentifrice and the diet contributed with 56.3% and 43.7% of the daily fluoride intake, respectively. Among the children evaluated, 31.2% are estimated to have risk to develop dental fluorosis (intake > 0.07 mg F/kg body weight/day). Conclusions: The dentifrice was the main source of fluoride intake by the children evaluated. However, the fluoride concentration in food items also significantly contributed to the daily ingestion by 2-6-year-old children. The questionnaire used seems to be a promising alternative to duplicate diet to estimate the fluoride intake at this age range and may have potential to be used in broad epidemiological surveys.

Sensitive detection of sodium in a flame using parametric four-wave mixing and seeded parametric four-wave mixing

Two-photon resonant parametric four-wave mixing and a newly developed variant called seeded parametric four-wave mixing are used to detect trace quantities of sodium in a flame. Both techniques are simple, requiring only a single laser to generate a signal beam at a different wavelength which propagates collinearly with the pump beam, allowing efficient signal recovery. A comparison of the two techniques reveals that seeded parametric four-wave mixing is more than two orders of magnitude more sensitive than parametric four-wave mixing, with an estimated detection sensitivity of 5 x 10(9) atoms/cm(3). Seeded parametric four-wave mixing is achieved by cascading two parametric four-wave mixing media such that one of the parametric fields generated in the first high-density medium is then used to seed the same four-wave mixing process in a second medium in order to increase the four-wave mixing gain. The behavior of this seeded parametric four-wave mixing is described using semiclassical perturbation theory. A simplified small-signal theory is found to model most of the data satisfactorily. However, an anomalous saturationlike behavior is observed in the large signal regime. The full perturbation treatment, which includes the competition between two different four-wave mixing processes coupled via the signal field, accounts for this apparently anomalous behavior.

Corrosion behaviour of AZ21, AZ501 and AZ91 in sodium chloride

The corrosion behaviour of AZ21, AZ501 and AZ91 was studied in 1 N NaCl at pH 11 by measuring electrochemical polarization curves, electrochemical AC impedance spectroscopy (EIS) and simultaneously measuring the hydrogen evolution rate and the: magnesium dissolution rate. The corrosion rates increased in the following order: AZ501 < AZ21 < AZ91. The: corrosion behaviour was related to alloy microstructure as revealed by optical and electron microscopy. The beta phase was very stable in the test solution and was an effective cathode. The beta phase served two roles, as a barrier and as a galvanic cathode. If the beta phase is present in the alpha matrix as intergranular precipitates with a small volume fraction, then the beta phase mainly serves as a galvanic cathode, and accelerates the corrosion of the alpha matrix. If the beta Fraction is high, then the beta phase may mainly act as an anodic barrier to inhibit the overall corrosion of the alloy. The composition and compositional distribution in the alpha phase is also crucial to the overall corrosion performance of dual phase alloys. Increasing the aluminum concentration in the alpha phase increases the anodic dissolution rate and also increases the cathodic hydrogen evolution rate. Increasing the zinc concentration in the alpha phase may have the opposite effect. (C) 1998 Elsevier Science Ltd. All rights reserved.

Neuronal sodium-channel alpha 1-subunit mutations in generalized epilepsy with febrile seizures plus

Generalized epilepsy with febrile seizures plus (GEFS+) is a familial epilepsy syndrome characterized by the presence of febrile and afebrile seizures. The first gene, GEFS1, was mapped to chromosome 19q and was identified as the sodium-channel beta1-subunit, SCN1B. A second locus on chromosome 2q, GEFS2, was recently identified as the sodium-channel alpha1-subunit, SCN1A. Single-stranded conformation analysis (SSCA) of SCN1A was performed in 53 unrelated index cases to estimate the frequency of mutations in patients with GEFS+. No mutations were found in 17 isolated cases of GEFS+. Three novel SCN1A mutations-D188V, V1353L, and I1656M-were found in 36 familial cases; of the remaining 33 families, 3 had mutations in SCN1B. On the basis of SSCA, the combined frequency of SCN1A and SCN1B mutations in familial cases of GEFS+ was found to be 17%.

Field-swept pulsed electron paramagnetic resonance of Cr3+-doped ZBLAN fluoride glass

Field-swept pulsed electron paramagnetic resonance (EPR) spectra of a ZBLAN fluoride glass doped with a low concentration of Cr3+ are obtained using echo-detected EPR and hole-burning free induction decay detection. We review the utility of the pulsed EPR technique in generating field-swept EPR spectra, as well as some of the distorting effects that are peculiar to the pulsed detection method. The application of this technique to Cr3+-doped ZBLAN reveals that much of the broad resonance extending from g(eff) = 5.1 to g(eff) = 1.97, characteristic of X-band continuous wave EPR of Cr3+ in glasses, is absent. We attribute this largely to the variation in nutation frequencies across the spectrum that result from sites possessing large fine structure interactions. The description of the spin dynamics of such sites is complicated and we discuss some possible approaches to the simulation of the pulsed EPR spectra.

Generalized epilepsy with febrile seizures plus: Mutation of the sodium channel subunit SCN1B

Generalized epilepsy with febrile seizures plus (GEFS(+)) is an important childhood genetic epilepsy syndrome with heterogeneous phenotypes, including febrile seizures (FS) and generalized epilepsies of variable severity. Forty unrelated GEFS(+) and FS patients were screened for mutations in the sodium channel beta-subunits SCN1B and SCN2B, and the second GEFS(+) family with an SCN1B mutation is described here. The family had 19 affected individuals: 16 with typical GEFS(+) phenotypes and three with other epilepsy phenotypes. Site-specific mutation within SCN1B remains a rare cause of GEFS(+), and the authors found no evidence to implicate SCN2B in this syndrome.