Partial characterization and anticoagulant activity of a heterofucan from the brown seaweed Padina gymnospora

The brown algae Padina gymnospora contain different fucans. Powdered algae were submitted to proteolysis with the proteolytic enzyme maxataze. The first extract of the algae was constituted of polysaccharides contaminated with lipids, phenols, etc. Fractionation of the fucans with increasing concentrations of acetone produced fractions with different proportions of fucose, xylose, uronic acid, galactose, and sulfate. One of the fractions, precipitated with 50% acetone (v/v), contained an 18-kDa heterofucan (PF1), which was further purified by gel-permeation chromatography on Sephadex G-75 using 0.2 M acetic acid as eluent and characterized by agarose gel electrophoresis in 0.05 M 1,3 diaminopropane/acetate buffer at pH 9.0, methylation and nuclear magnetic resonance spectroscopy. Structural analysis indicates that this fucan has a central core consisting mainly of 3-ß-D-glucuronic acid 1-> or 4-ß-D-glucuronic acid 1 ->, substituted at C-2 with alpha-L-fucose or ß-D-xylose. Sulfate groups were only detected at C-3 of 4-alpha-L-fucose 1-> units. The anticoagulant activity of the PF1 (only 2.5-fold lesser than low molecular weight heparin) estimated by activated partial thromboplastin time was completely abolished upon desulfation by solvolysis in dimethyl sulfoxide, indicating that 3-O-sulfation at C-3 of 4-alpha-L-fucose 1-> units is responsible for the anticoagulant activity of the polymer.

The extracellular matrix provides directional cues for neuronal migration during cerebellar development

Normal central nervous system development relies on accurate intrinsic cellular programs as well as on extrinsic informative cues provided by extracellular molecules. Migration of neuronal progenitors from defined proliferative zones to their final location is a key event during embryonic and postnatal development. Extracellular matrix components play important roles in these processes, and interactions between neurons and extracellular matrix are fundamental for the normal development of the central nervous system. Guidance cues are provided by extracellular factors that orient neuronal migration. During cerebellar development, the extracellular matrix molecules laminin and fibronectin give support to neuronal precursor migration, while other molecules such as reelin, tenascin, and netrin orient their migration. Reelin and tenascin are extracellular matrix components that attract or repel neuronal precursors and axons during development through interaction with membrane receptors, and netrin associates with laminin and heparan sulfate proteoglycans, and binds to the extracellular matrix receptor integrins present on the neuronal surface. Altogether, the dynamic changes in the composition and distribution of extracellular matrix components provide external cues that direct neurons leaving their birthplaces to reach their correct final location. Understanding the molecular mechanisms that orient neurons to reach precisely their final location during development is fundamental to understand how neuronal misplacement leads to neurological diseases and eventually to find ways to treat them.

Transient outward potassium current and Ca2+ homeostasis in the heart: beyond the action potential

Normal central nervous system development relies on accurate intrinsic cellular programs as well as on extrinsic informative cues provided by extracellular molecules. Migration of neuronal progenitors from defined proliferative zones to their final location is a key event during embryonic and postnatal development. Extracellular matrix components play important roles in these processes, and interactions between neurons and extracellular matrix are fundamental for the normal development of the central nervous system. Guidance cues are provided by extracellular factors that orient neuronal migration. During cerebellar development, the extracellular matrix molecules laminin and fibronectin give support to neuronal precursor migration, while other molecules such as reelin, tenascin, and netrin orient their migration. Reelin and tenascin are extracellular matrix components that attract or repel neuronal precursors and axons during development through interaction with membrane receptors, and netrin associates with laminin and heparan sulfate proteoglycans, and binds to the extracellular matrix receptor integrins present on the neuronal surface. Altogether, the dynamic changes in the composition and distribution of extracellular matrix components provide external cues that direct neurons leaving their birthplaces to reach their correct final location. Understanding the molecular mechanisms that orient neurons to reach precisely their final location during development is fundamental to understand how neuronal misplacement leads to neurological diseases and eventually to find ways to treat them.

Noncrystalline uric acid inhibits proteoglycan and glycosaminoglycan synthesis in distal tubular epithelial cells (MDCK)

Hyperuricemia is associated with renal stones, not only consisting of uric acid (UrAc) but also of calcium oxalate (CaOx). Glycosaminoglycans (GAGs) are well-known inhibitors of growth and aggregation of CaOx crystals. We analyzed the effect of noncrystalline UrAc on GAG synthesis in tubular distal cells. MDCK (Madin-Darby canine kidney) cells were exposed to noncrystalline UrAc (80 µg/mL) for 24 h. GAGs were labeled metabolically and characterized by agarose gel electrophoresis. The expression of proteoglycans and cyclooxygenase 2 (COX-2) was assessed by real-time PCR. Necrosis, apoptosis and prostaglandin E2 (PGE2) were determined by acridine orange, HOESCHT 33346, and ELISA, respectively. CaOx crystal endocytosis was evaluated by flow cytometry. Noncrystalline UrAc significantly decreased the synthesis and secretion of heparan sulfate into the culture medium (UrAc: 2127 ± 377; control: 4447 ± 730 cpm) and decreased the expression of perlecan core protein (UrAc: 0.61 ± 0.13; control: 1.07 ± 0.16 arbitrary units), but not versican. Noncrystalline UrAc did not induce necrosis or apoptosis, but significantly increased COX-2 and PGE2 production. The effects of noncrystalline UrAc on GAG synthesis could not be attributed to inflammatory actions because lipopolysaccharide, as the positive control, did not have the same effect. CaOx was significantly endocytosed by MDCK cells, but this endocytosis was inhibited by exposure to noncrystalline UrAc (control: 674.6 ± 4.6, CaOx: 724.2 ± 4.2, and UrAc + CaOx: 688.6 ± 5.4 geometric mean), perhaps allowing interaction with CaOx crystals. Our results indicate that UrAc decreases GAG synthesis in MDCK cells and this effect could be related to the formation of UrAc and CaOx stones.

Effect of activation of canonical Wnt signaling by the Wnt-3a protein on the susceptibility of PC12 cells to oxidative and apoptotic insults

Wnt proteins are involved in tissue development and their signaling pathways play an important role during embryogenesis. Wnt signaling can promote cell survival, which is beneficial for neurons, but could also lead to tumor development in different tissues. The present study investigated the effects of a Wnt protein on the susceptibility of a neural tumor cell line (PC12 cells) to the cytotoxic compounds ferrous sulfate (10 mM), staurosporine (100 and 500 nM), 3-nitropropionic acid (5 mM), and amyloid β-peptide (Aβ25-35; 50 µM). Cells (1 x 10(6) cells/mL) were treated with the Wnt-3a recombinant peptide (200 ng/mL) for 24 h before exposure to toxic insults. The Wnt-3a protein partially protected PC12 cells, with a 6-15% increase in cell viability in the presence of toxic agents, similar to the effect measured using the MTT and lactate dehydrogenase cell viability assays. The Wnt-3a protein increased protein expression of β-catenin by 52% compared to control. These findings suggest that Wnt signaling can protect neural cells against apoptosis induced by toxic agents, which are relevant to the pathogenesis of Alzheimer’s and Huntington’s diseases.

Heparan sulphate, its derivatives and analogues share structural characteristics that can be exploited, particularly in inhibiting microbial attachment

Heparan sulphate (HS) and the related polysaccharide, heparin, exhibit conformational and charge arrangement properties, which provide a degree of redundancy allowing several seemingly distinct sequences to exhibit the same activity. This can also be mimicked by other sulphated polysaccharides, both in overall effect and in the details of interactions and structural consequences of interactions with proteins. Together, these provide a source of active compounds suitable for further development as potential drugs. These polysaccharides also possess considerable size, which bestows upon them an additional useful property: the capability of disrupting processes comprising many individual interactions, such as those characterising the attachment of microbial pathogens to host cells. The range of involvement of HS in microbial attachment is reviewed and examples, which include viral, bacterial and parasitic infections and which, in many cases, are now being investigated as potential targets for intervention, are identified.

Effect of castration on renal glycosaminoglycans and their urinary excretion in male and female rats with chronic renal failure

Glycosaminoglycans (GAGs) participate in a variety of processes in the kidney, and evidence suggests that gender-related hormones participate in renal function. The aim of this study was to analyze the relationship of GAGs, gender, and proteinuria in male and female rats with chronic renal failure (CRF). GAGs were analyzed in total kidney tissue and 24-h urine of castrated (c), male (M), and female (F) Wistar control (C) rats (CM, CMc, CF, CFc) and after 30 days of CRF induced by 5/6 nephrectomy (CRFM, CRFMc, CRFF, CRFFc). Total GAG quantification and composition were determined using agarose and polyacrylamide gel electrophoresis, respectively. Renal GAGs were higher in CF compared to CM. CRFM presented an increase in renal GAGs, heparan sulfate (HS), and proteinuria, while castration reduced these parameters. However, CRFF and CRFFc groups showed a decrease in renal GAGs concomitant with an increase in proteinuria. Our results suggest that, in CRFM, sex hormones quantitatively alter GAGs, mainly HS, and possibly the glomerular filtration barrier, leading to proteinuria. The lack of this response in CRFMc, where HS did not increase, corroborates this theory. This pattern was not observed in females. Further studies of CRF are needed to clarify gender-dependent differences in HS synthesis.

Streptozotocin-induced diabetes mellitus affects lysosomal enzymes in rat liver

It has been previously shown that dextran sulfate administered to diabetic rats accumulates in the liver and kidney, and this could be due to a malfunction of the lysosomal digestive pathway. The aim of the present study was to evaluate the expression and activities of lysosomal enzymes that act upon proteins and sulfated polysaccharides in the livers of diabetic rats. Diabetes mellitus was induced by streptozotocin in 26 male Wistar rats (12 weeks old), while 26 age-matched controls received only vehicle. The livers were removed on either the 10th or the 30th day of the disease, weighed, and used to evaluate the activity, expression, and localization of lysosomal enzymes. A 50-60% decrease in the specific activities of cysteine proteases, especially cathepsin B, was observed in streptozotocin-induced diabetes mellitus. Expression (mRNA) of cathepsins B and L was also decreased on the 10th, but not on the 30th day. Sulfatase decreased 30% on the 30th day, while glycosidases did not vary (or presented a transitory and slight decrease). There were no apparent changes in liver morphology, and immunohistochemistry revealed the presence of cathepsin B in hepatocyte granules. The decrease in sulfatase could be responsible for the dextran sulfate build-up in the diabetic liver, since the action of sulfatase precedes glycosidases in the digestive pathway of sulfated polysaccharides. Our findings suggest that the decreased activities of cathepsins resulted from decreased expression of their genes, and not from general lysosomal failure, because the levels of glycosidases were normal in the diabetic liver.

Promoting inflammatory lymphangiogenesis by vascular endothelial growth factor-C (VEGF-C) aggravated intestinal inflammation in mice with experimental acute colitis

Angiogenesis and lymphangiogenesis are thought to play a role in the pathogenesis of inflammatory bowel diseases (IBD). However, it is not understood if inflammatory lymphangiogenesis is a pathological consequence or a productive attempt to resolve the inflammation. This study investigated the effect of lymphangiogenesis on intestinal inflammation by overexpressing a lymphangiogenesis factor, vascular endothelial growth factor-C (VEGF-C), in a mouse model of acute colitis. Forty eight-week-old female C57BL/6 mice were treated with recombinant adenovirus overexpressing VEGF-C or with recombinant VEGF-C156S protein. Acute colitis was then established by exposing the mice to 5% dextran sodium sulfate (DSS) for 7 days. Mice were evaluated for disease activity index (DAI), colonic inflammatory changes, colon edema, microvessel density, lymphatic vessel density (LVD), and VEGFR-3mRNA expression in colon tissue. When acute colitis was induced in mice overexpressing VEGF-C, there was a significant increase in colonic epithelial damage, inflammatory edema, microvessel density, and neutrophil infiltration compared to control mice. These mice also exhibited increased lymphatic vessel density (73.0±3.9 vs 38.2±1.9, P<0.001) and lymphatic vessel size (1974.6±104.3 vs 1639.0±91.5, P<0.001) compared to control mice. Additionally, the expression of VEGFR-3 mRNA was significantly upregulated in VEGF-C156S mice compared to DSS-treated mice after induction of colitis (42.0±1.4 vs 3.5±0.4, P<0.001). Stimulation of lymphangiogenesis by VEGF-C during acute colitis promoted inflammatory lymphangiogenesis in the colon and aggravated intestinal inflammation. Inflammatory lymphangiogenesis may have pleiotropic effects at different stages of IBD.

Partial purification and characterization of a bacteriocin produced by Enterococcus faecium 130 isolated from mozzarella cheese

Lactic acid bacteria are important in foods as potential probiotics and also due to the ability to produce antimicrobial compounds that can contribute for biopreservation. In this work, the bacteriocin produced by the food isolate Enterococcus faecium 130 was partially purified and characterized. The compound was active against Gram-positive bacteria, including Listeria monocytogenes. It was produced after 4 days of storage at a broad temperature range (4 to 37 °C); it was stable at pH ranging from 2 to 10 with no loss of activity after heating at 100 °C for 15 minutes. Bacteriocin was partially purified by the adsorption-desorption technique, and the analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a molecular mass of 3.5 to 6.5 kDa. These data encourage studies on application of this bacteriocin in food systems as an additional hurdle to microbial growth.

A statistical approach to define some tofu processing conditions

The aim of this work was to make tofu from soybean cultivar BRS 267 under different processing conditions in order to evaluate the influence of each treatment on the product quality. A fractional factorial 2(5-1) design was used, in which independent variables (thermal treatment, coagulant concentration, coagulation time, curd cutting, and draining time) were tested at two different levels. The response variables studied were hardness, yield, total solids, and protein content of tofu. Polynomial models were generated for each response. To obtain tofu with desirable characteristics (hardness ~4 N, yield 306 g tofu.100 g-1 soybeans, 12 g proteins.100 g-1 tofu and 22 g solids.100 g-1 tofu), the following processing conditions were selected: heating until boiling plus 10 minutes in water bath, 2% dihydrated CaSO4 w/w, 10 minutes coagulation, curd cutting, and 30 minutes draining time.

Iron enriched Saccharomyces cerevisiae maintains its fermenting power and bakery properties

Iron is an essential micronutrient in the metabolism of almost all living organisms; however, its deficiency is well documented especially in pregnant women and in children. Iron salts as a dietary supplement have low bioavailability and can cause gastrointestinal discomforts. Iron enriched yeasts can provide a supplementation of this micronutrient to the diet because this mineral has a better bioavailability when bonded to yeast cell macromolecules. These yeasts can be used as feed supplement for human and animals and also as baker's yeast. Baker's yeast Saccharomyces cerevisiae was cultivated in a reactor employing yeast media supplemented with 497 mg ferrous sulfate.L-1, and the resultant biomass incorporated 8 mg Fe.g-1 dry matter. This biomass maintained its fermenting power regarding both water displace measurement through carbonic dioxide production and bakery characteristics. The bread produced using the yeast obtained by cultivation in yeast media supplemented with iron presented six times more iron than the bread produced using the yeast obtained by cultivation without iron supplementation.

A fast and efficient method for the study of caffeine levels in energy drinks using micellar electrokinetic chromatography (MEKC)

Energy drinks are becoming popular in Brazil and in the world due to their stimulant properties. Caffeine is present in energy drinks with the aim of stimulating the central nervous system and intensifying brain activity. On the other hand, the ingestion of high doses of caffeine can cause undesirable symptoms such as anxiety and tachycardia. Therefore, it is necessary to monitor the caffeine content added to energy drinks to guarantee that the levels in the final product are in accordance with the labeling and within the legislation limits. The goal of this work was to validate a fast, efficient, and low-cost method for the determination of caffeine in energy drinks by micellar electrokinetic chromatography (MEKC). A total of seven brands were analyzed, each in three lots. The electrolyte was prepared with 50 mmol.L-1 of sodium dodecyl sulfate (SDS) and 10 mmol.L-1 of sodium carbonate (pH 11.0). The mean concentration of caffeine ranged from 122.8 to 318.6 mg.L-1. None of the brands had caffeine levels above the maximum limit. Considering the interval of confidence (95%), 72% of the samples had less caffeine than the amount informed on the product label.

Effect of high and low molecular weight glutenin subunits, and subunits of gliadin on physicochemical parameters of different wheat genotypes

Identification of functional properties of wheat flour by specific tests allows genotypes with appropriate characteristics to be selected for specific industrial uses. The objective of wheat breeding programs is to improve the quality of germplasm bank in order to be able to develop wheat with suitable gluten strength and extensibility for bread making. The aim of this study was to evaluate 16 wheat genotypes by correlating both glutenin subunits of high and low molecular weight and gliadin subunits with the physicochemical characteristics of the grain. Protein content, sedimentation volume, sedimentation index, and falling number values were analyzed after the grains were milled. Hectoliter weight and mass of 1000 seeds were also determined. The glutenin and gliadin subunits were separated using polyacrylamide gel in the presence of sodium dodecyl sulfate. The data were evaluated using variance analysis, Pearson's correlation, principal component analysis, and cluster analysis. The IPR 85, IPR Catuara TM, T 091015, and T 091069 genotypes stood out from the others, which indicate their possibly superior grain quality with higher sedimentation volume, higher sedimentation index, and higher mass of 1000 seeds; these genotypes possessed the subunits 1 (Glu-A1), 5 + 10 (Glu-D1), c (Glu-A3), and b (Glu-B3), with exception of T 091069 genotype that possessed the g allele instead of b in the Glu-B3.

Evaluation of iron bioavailability in a mixture of cereals, seeds, and grains ("Human Ration")

Iron bioavailability was evaluated in three mixtures of cereals, seeds, and grains ("Human Ration"): light, regular, and homemade provided to rats. The animals received an iron depletion diet for 21 days, followed by a repletion diet containing 12 mg·kg-1 of iron for 14 days. The hemoglobin regeneration efficiency and the relative biological value did not differ between the light mixture and control group. The iron bioavailability of the light mixture of cereals, seeds, and grains and the control group were 99.99±27.62 and 80.02±36.63, respectively, while the regular and homemade mixtures of cereals, seeds, and grains showed lower iron bioavailability, 50.12±35.53 and 66.66±15.44, respectively; the iron content of the diet with light cereal mixture light was statistically similar to that of the control (ferrous sulfate 99.99±27.62). The high content of tannin (202.81±19.53 mg·100-1) in the diet with the regular cereal mixture may have contributed to its low iron bioavailability. The higher intake of soluble fiber by the animals fed the light mixture (21.15±0.92 g) was moderately correlated (r=0.5712, p=0.0018) with the concentration of propionate in the caecal bulk (65.49±11.08 µmol/g). The short chain fatty acids produced by soluble fiber fermentation, associated with the low-content of tannin may have improved iron solubility and absorption in the light cereal mixture diet. The iron bioavailability in the light mixture of cereals, seeds, and grains was similar to that of ferrous sulfate.