Distinct mechanisms underlie adaptation of proximal tubule Na+/H+ exchanger isoform 3 in response to chronic metabolic and respiratory acidosis

The Na+/H+ exchanger isoform 3 (NHE3) is essential for HCO3- reabsorption in renal proximal tubules. The expression and function of NHE3 must adapt to acid-base conditions. The goal of this study was to elucidate the mechanisms responsible for higher proton secretion in proximal tubules during acidosis and to evaluate whether there are differences between metabolic and respiratory acidosis with regard to NHE3 modulation and, if so, to identify the relevant parameters that may trigger these distinct adaptive responses. We achieved metabolic acidosis by lowering HCO3- concentration in the cell culture medium and respiratory acidosis by increasing CO2 tension in the incubator chamber. We found that cell-surface NHE3 expression was increased in response to both forms of acidosis. Mild (pH 7.21 +/- 0.02) and severe (6.95 +/- 0.07) metabolic acidosis increased mRNA levels, at least in part due to up-regulation of transcription, whilst mild (7.11 +/- 0.03) and severe (6.86 +/- 0.01) respiratory acidosis did not up-regulate NHE3 expression. Analyses of the Nhe3 promoter region suggested that the regulatory elements sensitive to metabolic acidosis are located between -466 and -153 bp, where two consensus binding sites for SP1, a transcription factor up-regulated in metabolic acidosis, were localised. We conclude that metabolic acidosis induces Nhe3 promoter activation, which results in higher mRNA and total protein level. At the plasma membrane surface, NHE3 expression was increased in metabolic and respiratory acidosis alike, suggesting that low pH is responsible for NHE3 displacement to the cell surface.

Extensive structural change of the envelope protein of dengue virus induced by a tuned ionic strength: conformational and energetic analyses

The Dengue has become a global public health threat, with over 100 million infections annually; to date there is no specific vaccine or any antiviral drug. The structures of the envelope (E) proteins of the four known serotype of the dengue virus (DENV) are already known, but there are insufficient molecular details of their structural behavior in solution in the distinct environmental conditions in which the DENVs are submitted, from the digestive tract of the mosquito up to its replication inside the host cell. Such detailed knowledge becomes important because of the multifunctional character of the E protein: it mediates the early events in cell entry, via receptor endocytosis and, as a class II protein, participates determinately in the process of membrane fusion. The proposed infection mechanism asserts that once in the endosome, at low pH, the E homodimers dissociate and insert into the endosomal lipid membrane, after an extensive conformational change, mainly on the relative arrangement of its three domains. In this work we employ all-atom explicit solvent Molecular Dynamics simulations to specify the thermodynamic conditions in that the E proteins are induced to experience extensive structural changes, such as during the process of reducing pH. We study the structural behavior of the E protein monomer at acid pH solution of distinct ionic strength. Extensive simulations are carried out with all the histidine residues in its full protonated form at four distinct ionic strengths. The results are analyzed in detail from structural and energetic perspectives, and the virtual protein movements are described by means of the principal component analyses. As the main result, we found that at acid pH and physiological ionic strength, the E protein suffers a major structural change; for lower or higher ionic strengths, the crystal structure is essentially maintained along of all extensive simulations. On the other hand, at basic pH, when all histidine residues are in the unprotonated form, the protein structure is very stable for ionic strengths ranging from 0 to 225 mM. Therefore, our findings support the hypothesis that the histidines constitute the hot points that induce configurational changes of E protein in acid pH, and give extra motivation to the development of new ideas for antivirus compound design.

Monoalkyl carbonates in carbonated alcoholic beverages

The presence of monoethyl carbonate (MEC) in beer and sparkling wine is demonstrated for the first time, as well as the formation of this species in drinks prepared with a distilled beverage and a carbonated soft drink. A capillary electrophoresis (CE) equipment with two capacitively coupled contactless conductivity detector ((CD)-D-4) was used to identify and quantify this species. The concentrations of MEC in samples of lager beer and rum and cola drink were, respectively, 1.2 and 4.1 mmol/l, which agree with the levels of ethanol and CO2 available in these products. Previous results about the kinetics of the reaction suggest that only a small amount of MEC should be formed after the ingredients of a drink are mixed. However, in all three cases (whisky and club soda: rum with cola; gin and tonic water), MEC was quickly formed, which was attributed to the low pH of the drinks. (C) 2012 Elsevier Ltd. All rights reserved.

Recombinant expression, localization and in vitro inhibition of midgut cysteine peptidase (Sl-CathL) from sugarcane weevil, Sphenophorus levis

A cDNA coding for a digestive cathepsin L, denominated Sl-CathL, was isolated from a cDNA library of Sphenophorus levis larvae, representing the most abundant EST (10.49%) responsible for proteolysis in the midgut. The open reading frame of 972 bp encodes a preproenzyme similar to midgut cathepsin L-like enzymes in other coleopterans. Recombinant Sl-CathL was expressed in Pichia pastoris, with molecular mass of about 42 kDa. The recombinant protein was catalytically activated at low pH and the mature enzyme of 39 kDa displayed thermal instability and maximal activity at 37 degrees C and pH 6.0. Immunocytochemical analysis revealed Sl-CathL production in the midgut epithelium and secretion from vesicles containing the enzyme into the gut lumen, confirming an important role for this enzyme in the digestion of the insect larvae. The expression profile identified by RT-PCR through the biological cycle indicates that Sl-CathL is mainly produced in larval stages, with peak expression in 30-day-old larvae. At this stage, the enzyme is 1250-fold more expressed than in the pupal fase, in which the lowest expression level is detected. This enzyme is also produced in the adult stage, albeit in lesser abundance, assuming the presence of a different array of enzymes in the digestive system of adults. Tissue-specific analysis revealed that Sl-CathL mRNA synthesis occurs fundamentally in the larval midgut, thereby confirming its function as a digestive enzyme, as detected in immunolocalization assays. The catalytic efficiency of the purified recombinant enzyme was calculated using different substrates (Z-Leu-Arg-AMC, Z-Arg-Arg-AMC and Z-Phe-Arg-AMC) and rSl-CathL exhibited hydrolysis preference for Z-Leu-Arg-AMC (k(cat)/K-m = 37.53 mM S-1), which is similar to other insect cathepsin L-like enzymes. rSl-CathL activity inhibition assays were performed using four recombinant sugarcane cystatins. rSl-CathL was strongly inhibited by recombinant cystatin CaneCPI-4 (K-i = 0.196 nM), indicating that this protease is a potential target for pest control. (C) 2011 Elsevier Ltd. All rights reserved.

Antioxidative responses of cell suspension cultures of two Coffea arabica varieties to low aluminum levels at pH 5.8

The effects of aluminum (Al) on the activities of antioxidant enzymes and ferritin expression were studied in cell suspension cultures of two varieties of Coffea arabica, Mundo Novo and Icatu, in medium with pH at 5.8. The cells were incubated with 300 µM Al3+, and the Al speciation as Al3+ was 1.45% of the mole fraction. The activities of superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST) were increased in Mundo Novo, whereas glutathione reductase (GR) and guaiacol peroxidase (GPOX) activities remained unchanged. SOD, GR, and GST activities were increased in Icatu, while CAT activity was not changed, and GPOX activity decreased. The expression of two ferritin genes (CaFer1 and CaFer2) were analyzed by Real-Time PCR. Al caused a downregulation of CaFER1 expression and no changes of CaFER2 expression in both varieties. The Western blot showed no alteration in ferritin protein levels in Mundo Novo and a decrease in Icatu. The differential enzymes responses indicate that the response to Al is variety-dependent.

Prediction of apparent protein digestibility of ingredients and diets by in vitro pH-stat degree of protein hydrolysis with species-specific enzymes for juvenile Pacific white shrimp Litopenaeus vannamei

Aquafeed production faces global issues related to availability of feed ingredients. Feed manufacturers require greater flexibility in order to develop nutritional and cost-effective formulations that take into account nutrient content and availability of ingredients. The search for appropriate ingredients requires detailed screening of their potential nutritional value and variability at the industrial level. In vitro digestion of feedstuffs by enzymes extracted from the target species has been correlated with apparent protein digestibility (APD) in fish and shrimp species. The present study verified the relationship between APD and in vitro degree of protein hydrolysis (DH) with Litopenaeus vannamei hepatopancreas enzymes in several different ingredients (n = 26): blood meals, casein, corn gluten meal, crab meal, distiller`s dried grains with solubles, feather meal, fish meals, gelatin, krill meals, poultry by-product meal, soybean meals, squid meals and wheat gluten. The relationship between APD and DH was further verified in diets formulated with these ingredients at 30% inclusion into a reference diet. APD was determined in vivo (30.1 +/- 0.5 degrees C, 32.2 +/- 0.4%.) with juvenile L vannamei (9 to 12 g) after placement of test ingredients into a reference diet (35 g kg(-1) CP: 8.03 g kg(-1) lipid; 2.01 kcal g(-1)) with chromic oxide as the inert marker. In vitro DH was assessed in ingredients and diets with standardized hepatopancreas enzymes extracted from pond-reared shrimp. The DH of ingredients was determined under different assay conditions to check for the most suitable in vitro protocol for APD prediction: different batches of enzyme extracts (HPf5 or HPf6), temperatures (25 or 30 degrees C) and enzyme activity (azocasein): crude protein ratios (4 U: 80 mg CP or 4 U: 40 mg CP). DH was not affected by ingredient proximate composition. APD was significantly correlated to DH in regressions considering either ingredients or diets. The relationships between APD and DH of the ingredients could be suitably adjusted to a Rational Function (y = (a + bx)/(1 + cx + dx2), n = 26. Best in vitro APD predictions were obtained at 25 degrees C, 4 U: 80 mg CP both for ingredients (R(2) = 0.86: P = 0.001) and test diets (R(2) = 0.96; P = 0.007). The regression model including all 26 ingredients generated higher prediction residuals (i.e., predicted APD - determined APD) for corn gluten meal, feather meal. poultry by-product meal and krill flour. The remaining test ingredients presented mean prediction residuals of 3.5 points. A model including only ingredients with APD>80% showed higher prediction precision (R(2) = 0.98: P = 0.000004; n = 20) with average residual of 1.8 points. Predictive models including only ingredients from the same origin (e.g., marine-based, R(2) = 0.98; P = 0.033) also displayed low residuals. Since in vitro techniques have been usually validated through regressions against in vivo APD, the DH predictive capacity may depend on the consistency of the in vivo methodology. Regressions between APD and DH suggested a close relationship between peptide bond breakage by hepatopancreas digestive proteases and the apparent nitrogen assimilation in shrimp, and this may be a useful tool to provide rapid nutritional information. (C) 2009 Elsevier B.V. All rights reserved.

Characterization of depolymerized residues from extremely low acid hydrolysis (ELA) of sugarcane bagasse cellulose: Effects of degree of polymerization, crystallinity and crystallite size on thermal decomposition

Sugarcane bagasse cellulose was subjected to the extremely low acid (ELA) hydrolysis in 0.07% H2SO4 at 190, 210 and 225 degrees C for various times. The cellulose residues from this process were characterized by TGA, XRD, GPC, FIR and SEM. A kinetic study of thermal decomposition of the residues was also carried out, using the ASTM and Kissinger methods. The thermal studies revealed that residues of cellulose hydrolyzed at 190, 210 and 225 degrees C for 80,40 and 8 min have initial decomposition temperature and activation energy for the main decomposition step similar to those of Avicel PH-101. XRD studies confirmed this finding by showing that these cellulose residues are similar to Avicel in crystallinity index and crystallite size in relation to the 110 and 200 planes. FTIR spectra revealed no significant changes in the cellulose chemical structure and analysis of SEM micrographs demonstrated that the particle size of the cellulose residues hydrolyzed at 190 and 210 degrees C were similar to that of Avicel. (C) 2011 Elsevier B.V. All rights reserved.

Comparison of Ozone and Chlorine in Low Concentrations as Sanitizing Agents of Chicken Carcasses in the Water Immersion Chiller

The aim of this study was to investigate the effects of the use of chlorine or ozone as sanitizing agents in the water of chicken immersion chilling, using the residual levels usually applied in Brazil (1.5 ppm), comparing the effects of these treatments on the microbiological, physicochemical, and sensory characteristics of carcasses. Chicken carcasses were chilled in water (4 degrees C) with similar residual levels of ozone and chlorine until reaching temperatures below 7 degrees C (around 45 min). The stability of carcasses was assessed during 15 days of storage at 2 +/- 1 degrees C. Microbiological, surface color (L*, a*, b* parameters), pH value, lipid oxidation (thiobarbituric acid reactive substances index), and sensory evaluation (on a 9-point hedonic scale for odor and appearance) analyses were carried out. The presence of Salmonella was not detected, coagulase-positive staphylococci counts were below 10(2) CFU/ml of rinse fluid, and Escherichia coil and total coliform counts were below 10(5) CFU/ml of rinse fluid until the end of the storage period for both treatments. Psychrotrophic microorganism counts did not differ (P > 0.05) between chlorine and ozone treatments, and both values were near 10(9) CFU/ml of rinse fluid after 15 days at 4 +/- 1 degrees C. pH values did not differ between treatments (P > 0.05) or during the storage period (P > 0.05). In addition, neither chlorine nor ozone treatment showed differences (P > 0.05) in the lipid oxidation of carcasses; however, the thiobarbituric acid reactive substances index of both treatments increased (P <= 0.05) during the storage period, reaching values of approximately 0.68 mg of malonaldehyde per kg. Samples from both treatments did not differ (P > 0.05) in their acceptance scores for odor and overall appearance, but in the evaluation of color, ozone showed an acceptance score significantly higher (P <= 0.05) than that for the chlorine treatment. In general, under the conditions tested, ozone showed results similar to the results for chlorine in the disinfection of chicken carcasses in the immersion chilling, which may indicate its use as a substitute for chlorine in poultry slaughterhouses.

Dendrimers/TiO2 nanoparticles layer-by-layer films as extended gate FET for pH detection

The layer-by-layer (LbL) technique combined with field-effect transistor (FET) based sensors has enabled the production of pH-sensitive platforms with potential application in biosensors. A variation of the FET architecture, so called separative extended gate FET (SEGFET) devices, are promise as an alternative to conventional ion sensitive FET (ISFET). SEGFET configuration exhibits the advantage of combining the field-effect concept with organic and inorganic materials directly adsorbed on the extended gate, allowing the test of new pH-sensitive materials in a simple and low cost way. In this communication, poly(propylene imine) dendrimer (PPI) and TiO2 nanoparticles (TiO2-np) were assembled onto gold-covered substrates via layer-by-layer technique to produce a low cost SEGFET pH sensor. The sensor presented good pH sensitivity, ca. 57 mV pH(-1), showing that our strategy has potential advantages to fabricate low cost pH-sensing membranes. (C) 2012 Elsevier B.V. All rights reserved.

Aniline-1,4-benzoquinone as a model system for the characterization of products from aniline oligomerization in low acidic media

Increase in the pH medium of aniline polymerization is used for giving products of different morphologies, which are often wrongly attributed to PANI chains. Infrared and Raman spectroscopic data, supported by quantum chemical calculations, show that aniline-1,4-benzoquinone (AnBzq) is a model system for the characterization of the products of aniline oligomerization in low acidic media. The Raman spectra excited at different laser lines reveal the bichromophoric nature of AnBzq, whose absorption bands at 550 nm and 440 nm can be attributed to pi-pi* transitions of the delocalized benzoquinone and amino-phenyl moieties, respectively. (C) 2012 Elsevier B.V. All rights reserved.

Effect of low fluoride acidic dentifrices on dental remineralization

This study evaluated the capacity of fluoride acidic dentifrices (pH 4.5) to promote enamel remineralization using a pH cycling model, comparing them with a standard dentifrice (1,100 µgF/g). Enamel blocks had their surface polished and surface hardness determined (SH). Next, they were submitted to subsurface enamel demineralization and to post-demineralization surface hardness analysis. The blocks were divided into 6 experimental groups (n=10): placebo (without F, pH 4.5, negative control), 275, 412, 550, 1,100 µgF/g and a standard dentifrice (positive control). The blocks were submitted to pH cycling for 6 days and treatment with dentifrice slurries twice a day. After pH cycling, surface and cross-sectional hardness were assessed to obtain the percentage of surface hardness recovery (%SHR) and the integrated loss of subsurface hardness (ΔKHN). The results showed that %SHR was similar among acidic dentifrices with 412, 550, 1,100 µgF/g and to the positive control (Tukey's test; p>0.05). For ΔKHN, the acidic dentifrice with 550 µg F/g showed a better performance when compared with the positive control. It can be concluded that acidic dentifrice 550 µgF/g had similar remineralization capacity to that of positive control.