Effect of the Partial Substitution of Soy Proteins by Highly Methyl-esterified Pectin on Chemical and Sensory Characteristics of Sausages

Pectin can be used as a natural emulsifier in food formulations. In this study, textured soybean protein (TSP), used as an emulsifier in commercial sausages, was partially replaced by a mixture containing pectin and isolated soybean proteins, which were either extruded (EXT) or not extruded (MIX), and the chemical and sensory characteristics of samples were evaluated after 60 days of storage at 4 degrees C. Responses such as oxidation measured by PV and TBARS, hardness, color, pH and sensory characteristics were compared with those of a commercial sausage (CON). The mixture containing highly methyl-esterified pectin, textured soybean proteins and isolated soybean proteins, as emulsifier agent, reduced the hardness (EXT: 21.69 +/- 0.98 and MIX: 20.17 +/- 2.76 N) and the pH (EXT: 5.46 +/- 0.03 and MIX: 5.29 +/- 0.01) of the samples and increased the concentration of peroxides (EXT: 0.10 +/- 0.01 and MIX: 0.15 +/- 0.01 meq/kg) when compared with samples formulated only with TSP (28.57 +/- 2.54 N, pH of 6.92 +/- 0.04 and PV = 0.07 +/- 0.01 meq/kg). These effects were likely caused by the anionic character of the emulsifier. However, no sensory difference was observed between the sausages containing highly methyl-esterified pectin, textured soybean proteins and isolated soybean proteins submitted to the extrusion process (EXT) and the control sausages, suggesting that the formulation proposed in this study can be a potential alternative for the further development of sausages that have functional properties or are free of artificial additives.

Increased class A scavenger receptor and glomerular lipid precede mesangial matrix expansion in the bGH mouse model

Objective: Elevated neutral lipid content and mRNA expression of class A scavenger receptor (SRA) have been found in the renal cortex of the bovine growth hormone (bGH) mouse model of progressive glomerulosclerosis (GS). We hypothesize that the increased expression of SRA precedes glomerular scarring in this model. Design: Real time RT-PCR and immunofluorescence were employed to measure SRA and collagen types I and IV in the bGH transgenic and control mice at 5 and 12 weeks (wk) of age to determine the chronology of change in SRA expression in relation to glomerular scarring. Alternative mechanisms for increasing glomerular lipid were assessed by measuring mRNA expression levels of low-density lipoprotein receptor (LDL-r), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), and ATP-binding cassette transporter A1 (ABCA1). In addition, the involvement of macrophages in early GS was assessed by CD68 mRNA expression in kidney cortex. Results: Both mRNA and protein levels of SRA were significantly increased in 5-wk bGH compared with control mice, whereas the expression of collagen I and IV was unaltered. Unchanged levels of LDL-r and HMGR mRNA indicate that neither regulated cholesterol uptake via LDL-r nor the cholesterol synthetic pathway played a role in the early lipid increase. The finding of increased ABCA1 expression was an indicator of excess intracellular lipid in the renal cortex of bGH mice at 5 wk. CD68 expression in bGH did not differ significantly from that of controls at 5 wk suggesting that cortical macrophage infiltration was not increased in bGH mice at this time point. Conclusion: An early increase in SRA mRNA and protein expression in the bGH kidney precedes glomerular scarring and is independent of macrophage influx. Published by Elsevier Ltd. on behalf of Growth Hormone Research Society.

Enhanced fibroblast adhesion and proliferation on electrospun fibers obtained from poly(isosorbide succinate-b-L-lactide) block copolymers

Block copolymers containing isosorbide succinate and L-lactic acid repeating units with different mass compositions were synthesized in two steps: bulk ring-opening copolymerization from L-lactide and poli(isosorbide succinate) (PIS) preoligomer, in the presence of tin(II) 2-ethylhexanoate as catalyst. followed by chain extension in solution by using hexamethylene diisocyanate. Poly(L-lactide) (PLLA) and a chain extension product from PIS were also obtained, for comparison. SEC, (1)H and (13)C NMR, MALDI-TOFMS, WAXD, DSC, TG, and contact angle measurements were used in their characterization. The incorporation of isosorbide succinate into PLLA main backbone had minor effect on the thermal stability and the T(g) of the products. However, it reduced the crystallinity and increased the surface energy in relation to PLLA. Nonwoven mats of the block copolymers and PLLA obtained by electrospinning technique were submitted to fibroblasts 3T3-L1 cell culture. The copolymers presented enhanced cell adhesion and proliferation rate as revealed by MTT assay and SEM images. (C) 2009 Elsevier Ltd. All rights reserved.

Impedimetric immunosensor for electronegative low density lipoprotein (LDL(-)) based on monoclonal antibody adsorbed on (polyvinyl formal)-gold nanoparticles matrix

Monoclonal antibodies (MAb) have been commonly applied to measure LDL in vivo and to characterize modifications of the lipids and apoprotein of the LDL particles. The electronegative low density lipoprotein (LDL(-)) has an apolipoprotein B-100 modified at oxidized events in vivo. In this work, a novel LDL-electrochemical biosensor was developed by adsorption of anti-LDL(-) MAb on an (polyvinyl formal)-gold nanoparticles (PVF-AuNPs)-modified gold electrode. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to characterize the recognition of LDL-. The interaction between MAb-LDL(-) leads to a blockage in the electron transfer of the [Fe(CN)(6)](4-)/K(4)[Fe(CN)(6)](3-) redox couple, which may could result in high change in the electron transfer resistance (R(CT)) and decrease in the amperometric responses in CV analysis. The compact antibody-antigen complex introduces the insulating layer on the assembled surface, which increases the diameter of the semicircle, resulting in a high R(CT), and the charge transferring rate constant k(0) decreases from 18.2 x 10(-6) m/s to 4.6 x 10(-6) m/s. Our results suggest that the interaction between MAb and lipoprotein can be quantitatively assessed by the modified electrode. The PVF-AuNPs-MAb system exhibited a sensitive response to LDL(-), which could be used as a biosensor to quantify plasmatic levels of LDL(-). (C) 2011 Elsevier B.V. All rights reserved.

Capacity of the extracellular matrix of the bone marrow to induce proliferation of myeloid cells in vitro in model of protein malnutrition in mice

The aim of this study was to verify the capacity of the extracellular matrix (ECM) obtained from bone marrow of malnourished mice to sustain survival and to induce the proliferation of myeloid cells. We also verified the capacity of the tests to interact with in vitro hematopoietic cytokines. Male ""Swiss"" mice were submitted to protein malnutrition with a diet contents of 4% casein until they lost 20% of the original weight, while the group-control was kept with a diet content of 14% of casein. The bone marrow was extracted with 1.0 mg of aprotinin/mL in PBS. The proliferation tests were carried out with myeloid cell line FDCP-1, by the colorimetric method of reduction of the MTT. The obtained ECM from nourished and undernourished mice induced cellular proliferation in vitro. Tests performed with Il-3 and GM-CSF cytokines in a concentration of 10 and 500 rho g/mL displayed synergic and regulatory effects respectively. The ECM obtained from the malnourished group submitted to the binding to GM-CSF demonstrated higher cellular proliferation than the ECM obtained from the control group (p<0.05). The results suggest that the alterations in the composition of ECM of bone marrow caused by malnutrition might lead to modification of the GM-CSF activity modulation.

Influence of gamma radiation onto polymeric matrix with papain

Papain is a proteolytic enzyme that has been widely used as debridement agent for scars and wound healing treatment. However, papain presents low stability, which limits its use to extemporaneous or short shelf-life formulations. The purpose of this study was to entrap papain into a polymeric matrix in order to obtain a drug delivery system that could be used as medical device. Since these systems must be sterile, gamma radiation is an interesting option and presents advantages in relation to conventional agents: no radioactive residues are formed: the product can be sterilized inside the final packaging and has an excellent reliability. The normative reference for the establishment of the sterilizing dose determines 25 kGy as the inactivation dose for viable microorganisms. A silicone dispersion was selected to prepare membranes containing 2% (w/w) papain. Irradiated and non-irradiated membranes were simultaneously assessed in order to verify whether gamma radiation interferes with the drug-releasing profile. Results showed that irradiation does not affect significantly papain release and its activity. Therefore papain shows radioresistance in the irradiation conditions applied. In conclusion, gamma radiation can be easily used as sterilizing agent without affecting the papain release profile and its activity onto the biocompatible device is studied. (C) 2009 Elsevier Ltd. All rights reserved.

Dissolution parameters for sodium diclofenac-containing hypromellose matrix tablet

Sodium diclofenac (SD) release from dosage forms has been studied under different conditions. However, no dissolution method that is discriminatory enough to reflect slight changes in formulation or manufacturing process, and which could be effectively correlated with the biological properties of the dosage form, has been reported. This study sought to develop three different formulae of SD-containing matrix tablets and to determine the effect of agitation speed in its dissolution profiles. F1, F2 and F3 formulations were developed using hypromellose (10, 20 and 30%, respectively for F1, F2 and F3) and other conventional excipients. Dissolution tests were carried out in phosphate buffer pH 6.8 at 37 degrees C using apparatus 11 at 50, 75 or 100 rpm. Dissolution efficiency (DE), T(50) and T(90) were determined and plotted as functions of the variables agitation speed and hypromellose concentration. Regarding DE, F2 showed more sensitivity to variations in agitation speed than F1 and F3. Increasing hypromellose concentration reduced DE values, independent of agitation speed. Analysis of T(50) and T(90) suggests that F1 is less sensitive to variations in agitation speed than F2 and F3. Most discriminatory dissolution conditions were observed at 50 rpm. Results suggest that the comparison of dissolution performance of SD matrix tablets should take into account polymer concentration and agitation conditions. (C) 2009 Published by Elsevier B.V.

Development and In Vitro Evaluation of Propranolol Hydrochloride Controlled Release Tablets Using HPMC as Matrix Former

The purpose of this paper was to produce controlled-release matrices with 120 mg of propranolol hydrochloride (PHCl) employing hydroxypropyl methylcellulose (HPMC, Methocel (R) K100) as the gel forming barrier. Although this class of polymers has been commonly used for direct compression, with the intent of use reduced polymer concentrations to achieve controlled drug release, in this study tablets were produced by the wet granulation process. HPMC percentages ranged from 15-34 % and both soluble and non soluble diluents were tested in the 10 proposed tablet compositions. Dissolution testing of matrices was performed over a 12 h period in 1.2 pH medium (the first 2 h) and in pH 6.8 (10 h). Dissolution kinetic analysis was performed by applying Zero-order, First-order and Higuchi models with the aim of elucidating the drug release mechanism. All physical-chemical characteristics such as average weight, friability, hardness, diameter, height, and drug content were in accordance to the pharmacopeial specifications. Taking into account that PHCl is a very soluble drug, low concentrations (15 %) of HPMC were sufficient to reduce the drug release and to promote controlled release of PHCl, presenting good dissolution efficiencies, between 50 % and 63 %. The Higuchi model has presented the best fit to the 15 % HPMC formulations, indicating that the main release mechanism was diffusion. It could be concluded that the application of the wet granulation method reduced matrices erosion and promoted controlled release of the drug at low HPMC percentages.

Biochemical and biophysical characterization of lysozyme modified by PEGylation

PEGylation is a strategy that has been used to improve the biochemical properties of proteins and their physical and thermal stabilities. In this study, hen egg-white lysozyme (EC 3.2.1.17; LZ) was modified with methoxypolyethylene glycol-p-nitrophenyl carbonate (mPEG-pNP, MW 5000). This PEGylation of LZ produced conjugates that retained full enzyme activity with glycol chitosan, independent of degree of enzyme modification; its biological activity with the substrate Micrococcus lysodeikticus was altered according to its degree of modification. The conjugate obtained with a low degree of mPEG-pNP/NH(2) modification was studied by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), demonstrating a spectral peak at m/z 19,988 Da with 77% of its original enzymatic activity. Spectroscopic studies of Fourier transform infrared (FIR) and circular dichroism (CD) did not show any relevant differences in protein structure between the native and conjugate LZ. Studies of the effects of pH and temperature on PEGylated LZ indicated that the conjugate was active over a broad pH range, stable at 50 degrees C, and demonstrated resistance to proteolytic degradation. (C) 2010 Elsevier B.V. All rights reserved.

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.

Lercanidipine reduces matrix metalloproteinase-2 activity and reverses vascular dysfunction in renovascular hypertensive rats

Increased expression/activity of matrix metalloproteinases (MMPs), especially MMP-2, plays a role in the vascular alterations induced by hypertension, and increased oxidative stress is a major factor activating MMPs. Here, we hypothesized that lercanidipine, a calcium channel blocker, could attenuate the increases in oxidative stress and MMP-2 expression/activity in the two-kidney, one-clip (2K-1C) hypertensive rats. Sham-operated or 2K-1C hypertension rats were treated with lercanidipine 2.5 mg/kg/day (or vehicle) starting three weeks after hypertension was induced. Systolic blood pressure was monitored weekly. After five weeks of treatment, aortic rings were isolated to assess endothelium-dependent and independent relaxations. Quantitative morphometry of structural changes in the aortic wall were studied in hematoxylin/eosin sections. Aortic MMP-2 levels were determined by gelatin zymography. Aortic MMP-2/tissue inhibitor of metalloproteinases (TIMP)-2 mRNA levels were determined by quantitative real-time RT-PCR. Plasma thiobarbituric acid reactive substances concentrations were determined using a fluorometric method. Lercanidipine attenuated 2K-1C hypertension (224 12 versus 183 11 mm Hg in 2K-1C rats and 2K-1C + Lercandipine rats, respectively; P < 0.01) and prevented the reduction in endothelium-dependent vasorelaxation found in 2K-1C rats. Increased MMP-2 and Pro-MMP-2 levels were found in the aortas of 2K-1C rats (all P < 0.05). Lercandipine attenuated 2K-1C-induced increases in MMP-2 by more than 60% and blunted 2K-1C-induced increases in oxidative stress (both P < 0.001). While hypertension-induced significant aortic wall hypertrophy and approximately 9-fold increases in the ratio of MMP-2MMP-2 mRNA expression (both P < 0.05), lercandipine did not affect these changes. These results suggest that lercanidipine produces antihypertensive effects and reverses the endothelial dysfunction associated with 2K-1C hypertension, probably through mechanisms involving antioxidant effects leading to lower MMP-2 activation. (C) 2008 Elsevier B.V. All rights reserved.

Antioxidant treatment reduces matrix metalloproteinase-2-induced vascular changes in renovascular hypertension

Mounting evidence indicates that structural and functional vascular changes associated with two-kidney, one-clip (2K-1C) hypertension result, at least in part, from altered activity of matrix metalloproteinases (MMPs). Because MMPs are upregulated by increased formation of reactive oxygen species (ROS), we hypothesized that antioxidant approaches could attenuate the increases in MMP-2 expression/activity and the vascular dysfunction and remodeling associated with 2K-1C hypertension. Sham-operated or 2K-1C hypertensive rats were treated with tempol 18 mg/kg/day or apocyanin 25 mg/kg/day (or vehicle). Systolic blood pressure was monitored weekly. After 8 weeks of treatment, aortic rings were isolated to assess endothelium-dependent and -independent relaxation. Quantitative morphometry of structural changes in the aortic wall was studied in hematoxylin/eosin sections. Aortic and systemic ROS levels were measured using dihydroethidine and thiobarbituric acid-reactive substances, respectively. Aortic MMP-2 levels and activity were determined by gelatin and in situ zymography, fluorimetry, and immunohistochemistry. Tempol and apocyanin attenuated 2K-1C hypertension (181 +/- 20.8 and 192 +/- 17.6 mm Hg, respectively, versus 213 +/- 18 mm Hg in hypertensive controls; both p<0.05) and prevented the reduction in endothelium-dependent vasorelaxation found in 2K-1C rats. Tempol, but not apocyanin (p>0.05), prevented the vascular remodeling found in 2K-1C rats (all p<0.01). Tempol was more effective than apocyanin in attenuating hypertension-induced increases in oxidative stress (both p<0.05), MMP-2 levels, and MMP-2 activity in hypertensive rats (all p<0.05). Our results suggest that antioxidant approaches decrease MMP-2 upregulation and attenuate the vascular dysfunction and remodeling during 2K-1C hypertension. (C) 2009 Elsevier Inc. All rights reserved.

Effect of the dibenzylbutyrolactone lignan (-)-hinokinin on doxorubicin and methyl methanesulfonate clastogenicity in V79 Chinese hamster lung fibroblasts

The dibenzylbutyrolactone lignan (-)-hinokinin (HK) was obtained by partial synthesis from (-)-cubebin, isolated from the dry seeds of the pepper, Piper cubeba. In view of the trypanocidal activity of HK and its potential as a lead compound for drug development, evaluation of its possible genotoxic activity is required. We have tested HK for possible genotoxicity and evaluated the compound`s effect on the activity of the clastogens doxorubicin (DXR) and methyl methanesulfonate (MMS) in the micronucleus (MN) assay with Chinese hamster lung fibroblast V79 cells. HK alone did not induce MN, at concentrations up to 128 mu M. In combined treatments, HK reduced the frequency of MN induced by MMS. With respect to DXR, HK exerted a protective effect at lower concentrations, but at higher concentrations it potentiated DXR clastogenicity. (C) 2010 Elsevier B.V. All rights reserved.

Eudesmanolides and Methyl Ester Derivatives from Dimerostemma arnottii

The phytochemical investigation of Dimerostemma arnottii (Asteraceae) afforded, in addition to a known eudesmanolide, two unusual eudesmane methyl ester derivatives and a new eudesmanolide. Structural elucidation of the compounds was based on their ID and 2D NMR spectroscopic as well as HR-ESI-MS data. There is a remarkable similarity between the structures of the eudesmanes from D. arnottii and those previously encountered in other Dimerostemma species, which is in agreement with the results of a previous phylogenetic study based on molecular data. The chemotaxonomic relevance of the isolated compounds is briefly discussed.