Monitoring sodium caseinate and whey protein isolate interaction with mild preheat treatment using ultrasound spectroscopy and confocal laser scanning microscopy.

Ultrasound spectroscopy and confocal laser scanning microscopy (CLSM) methods were developed to visualize the interaction between sodium caseinate (SC) and whey protein isolate (WPI) with a mild preheat treatment (57°C, 10 min) followed by adding glucono-δ-lactone (GDL). Ultrasonic velocity changes during incubation at 25°C after adding GDL for four kinds of mixtures (no-treated SC plus no-treated WPI, preheated SC plus no-treated WPI, no-treated SC plus preheated WPI and preheated SC plus preheated WPI) were monitored. The results reveal that the mild preheating treatment of the proteins affected the timing of the increase in compressibility of each system. CLSM observation with individualized dyes which have different maxima of excitation and emission wavelengths, showed the preheated SC plus no-treated WPI mixture had a slightly coarse structure and the highest correlation coefficient, suggesting the highest colocalization of the SC and WPI among the four kinds of mixed-protein systems. Furthermore, the scanning electron microscopy (SEM) observation suggests that there are some differences among the gels, namely, preheated WPI leads to the formation of developed three-dimensional gel networks with filamentous structures, whereas SC promotes the formation of cluster-like crowded networks composed of more fine aggregated particles instead of developed filamentous structures. These results demonstrated that although SC is known as a heat-stable protein, pretreated SC could lead to an increase of the collaboration with WPI in the presence of GDL. This finding anticipated the possibility creating a food material with another texture using a milk-protein mixed system.

Characterization of the effect of dry-heat treatment on the gelation of alkaline dried egg whites using dynamic viscoelastic measurement and ultrasound spectroscopy.

In the present study, the mechanism for gelation of dried egg whites (DEWs) samples, which were differentially pretreated with dry-heat treatment and alkaline pH, were investigated using dynamic viscoelastic measurements and ultrasound spectroscopy for gels formed with at high protein concentrations. Rheological measurements showed that DEW gels with dry-heat treatments have a higher dynamic complex modulus than DEW gels without dry-heat treatments. Furthermore, ultrasonic attenuation analyses showed that subjecting DEWs to dry-heat treatment and alkaline pH induced the formation of an increased number of DEW protein "soluble aggregates" compared with unheated and neutral DEWs. Our data suggests that pretreatment of DEW samples led to partially unfolded DEW proteins with more "soluble aggregates", which were strongly correlated with the formation of gels with a more homogeneous and rigid texture. Understanding their structural properties at a molecular detail would enable desirable textural modifications for the development of new food products.

Characterization of the gelation and resulting network of a mixed-protein gel derived from sodium caseinate and ovalbumin in the presence of glucono-δ-lactone.

We investigated mixed-protein gels made from sodium caseinate and ovalbumin at different ratios with use of the acidification agent glucono-δ-lactone. Dynamic viscoelastic measurements revealed that increasing the ovalbumin content decreased the mechanical properties of the gel but accelerated onset time of the phase transition. Ultrasound spectroscopy during gelation revealed that the relative velocity gradually decreased, whereas the ultrasonic attenuation increased during the whole acidification process until gelation was complete, although these changes were much smaller than those observed with heat-induced gelation. Confocal laser scanning microscopy along with scanning electron microscopy revealed that although uniform mixing of sodium caseinate and ovalbumin was observed, sodium caseinate is likely to mainly lead formation of the gel network, and the porosity of the resulting gel network depends on the ratio of these two components. The results demonstrate that confocal laser scanning microscopy is a useful tool for analyzing both the networks within mixed-protein gels and the contribution of each protein to the network and gelation.

Primitive Extracellular Lipid Components on the Surface of the Charophytic Alga Klebsormidium flaccidum and Their Possible Biosynthetic Pathways as Deduced from the Genome Sequence.

Klebsormidium flaccidum is a charophytic alga living in terrestrial and semiaquatic environments. K. flaccidum grows in various habitats, such as low-temperature areas and under desiccated conditions, because of its ability to tolerate harsh environments. Wax and cuticle polymers that contribute to the cuticle layer of plants are important for the survival of land plants, as they protect against those harsh environmental conditions and were probably critical for the transition from aquatic microorganism to land plants. Bryophytes, non-vascular land plants, have similar, but simpler, extracellular waxes and polyester backbones than those of vascular plants. The presence of waxes in terrestrial algae, especially in charophytes, which are the closest algae to land plants, could provide clues in elucidating the mechanism of land colonization by plants. Here, we compared genes involved in the lipid biosynthetic pathways of Arabidopsis thaliana to the K. flaccidum and the Chlamydomonas reinhardtii genomes, and identified wax-related genes in both algae. A simple and easy extraction method was developed for the recovery of the surface lipids from K. flaccidum and C. reinhardtii. Although these algae have wax components, their surface lipids were largely different from those of land plants. We also investigated aliphatic substances in the cell wall fraction of K. flaccidum and C. reinhardtii. Many of the fatty acids were determined to be lipophilic monomers in K. flaccidum, and a Fourier transform infrared spectroscopic analysis revealed that their possible binding mode was distinct from that of A. thaliana. Thus, we propose that K. flaccidum has a cuticle-like hydrophobic layer composed of lipids and glycoproteins, with a different composition from the cutin polymer typically found in land plant cuticles.

Utilization of dietary urea nitrogen is stimulated by D-mannitol feeding in rabbits.

The effect of D-mannitol as an indigestible sugar on apparent digestibility of nutrients, nitrogen (N) utilization and growth performance, have been investigated in growing rabbits fed a urea-containing diet. In experiment 1, 40-day-old male rabbits were fed a commercial diet with urea 10 g/kg with added glucose or D-mannitol 60 g/kg each. Thereafter, in experiment 2 the rabbits aged 55 days were fed a commercial diet with or without urea 10 g/kg. Feed restriction at 55 g/kg(0.75)/day was carried out in both experiments. Addition of D-mannitol to the urea-containing diet decreased urinary N excretion, which consequently increased N retention and ratios of retained N to consumed N or apparently absorbed N. Addition of urea alone to the diet increased daily N intake and urinary N excretion but did not affect N retention. Weight gain and feed efficiency were improved by D-mannitol feeding, whereas feeding with urea alone did not affect these parameters. Utilization of dietary urea was enhanced in growing rabbits fed the urea-containing diet with D-mannitol, resulting in improvement of N retention and growth performance.

ß-Casein aids in the formation of a sodium caprate-induced ß-lactoglobulin B gel.

The effects of sodium caprate on the gelation of ß-lactoglobulin B and a ß-lactoglobulin B/ß-casein mixture at ambient temperature were investigated using ultrasonic spectroscopy and rheology. A 12% ß-lactoglobulin B solution gelled in the presence of 3.6% sodium caprate. Conversely, sodium caprate did not induce the formation of a gel when ß-casein was in isolation, regardless of the protein concentration. Although a 6% ß-lactoglobulin B/1.8% sodium caprate solution did not form a gel, a gel was formed when 6% ß-casein was added to a mixture containing 6% ß-lactoglobulin B and 3.6% sodium caprate. This gel showed comparable rheological properties to that of a gel containing 12% ß-lactoglobulin B. The results clearly indicated that ß-casein aids in the gelation of a ß-lactoglobulin B/sodium caprate mixture, when the concentration of ß-lactoglobulin B is insufficient to allow for gelation. It appears that ß-casein self-aggregation is also inhibited. Therefore, it could be concluded that ß-casein can be used as a texture modifier for ß-lactoglobulin gelation induced by sodium caprate.

Gluten gel and film properties in the presence of cysteine and sodium alginate.

Wheat flour has an ability of forming dough by mixing with water, which exhibits a rheological property required for making bread. The major protein is gluten, which is a valuable protein material for food industry. In this study, gluten protein gels and films were formed with cysteine and sodium alginate. Adding cysteine improved gel and film properties (stress relaxation behavior, bending strength). The gel containing 0.01 M cysteine had a longer relaxation time and was more rigid than the gel without cysteine. Although adding sodium alginate to the gluten suspension containing cysteine improved the water-holding ability and homogeneity of the gel network, the film from this gel was more brittle than the gluten film with cysteine alone. Microstructural observations of the gels and films with scanning electron microscopy suggested that water evaporation was more heterogeneous from the gel containing sodium alginate than from the gel with cysteine alone. Fourier transform-infrared (FT-IR) analysis during film formation suggested that the presence of cysteine encourages interaction between gluten molecules and results in intermolecular beta-sheet formation in earlier stages than in the no additive condition. FT-IR results also suggested that the combined effect of sodium alginate and cysteine on the protein secondary structure was remarkably different from that of cysteine alone. Our results suggest that addition of a suitable amount of cysteine (0.01 M) and heat treatment to 80 degrees C during gluten gel and film formation induces a homogenous network in the gel and film by regulating disulfide-sulfide interactions.

Determination of asymmetric dimethylarginine, an endogenous nitric oxide synthase inhibitor, in umbilical blood.

Endothelial cells produce nitric oxide (NO), a potent vasodilator. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NO synthase. Little is known about the potential physiological roles of ADMA in a perinatal setting. This study measures concentrations of ADMA in umbilical blood using enzyme-linked immunosorbent assay and those of NO as nitrite/nitrate (NOx(-)) using the Griess assay. Their relationship to the degree of prematurity and maternal clinical condition is examined. Results show that ADMA concentrations in umbilical blood from control newborns were about twice as high as those of lactating women, healthy children, and healthy adults. Umbilical blood NOx(-) concentrations from control newborns were about half of those of lactating women, healthy children, and healthy adults. Consequently, the levels of ADMA relative to NOx(-) were about 4-fold higher in umbilical blood from control newborns than in blood from lactating women, healthy children, and healthy adults. Furthermore, the umbilical blood ADMA concentrations and the ratios of ADMA to NOx(-) in newborns were higher according to their birth prematurity and lower birth weight. The umbilical ADMA concentrations were independent of the delivery mode and maternal preeclampsia. We infer that the high ADMA levels play physiological roles in maintaining vascular tone and blood redistribution to vital organs during birth, thereby favoring the circulatory transition from fetal to neonatal life.

High levels of growth factors in human breast milk.

We found very high concentrations of vascular endothelial growth factor, hepatic growth factor, and epidermal growth factor in early breast milk samples obtained from healthy mothers of term infants. This is the first report of simultaneous measurements of three major gastrointestinal trophic substances in human milk.

Characterization of beta-lactoglobulin A gelation in the presence of sodium caprate by ultrasound spectroscopy and electron microscopy.

The effect of sodium caprate (a fatty acid salt) on the formation of beta-lactoglobulin A gels was studied at constant temperature (30 or 35 degrees C) using ultrasonic spectroscopy. During incubation at these temperatures, ultrasonic attenuation increased with the addition of sodium caprate, and reached a plateau after 5-7 h of incubation. Comparing beta-lactoglobulin A with and without sodium caprate, a decrease in net ultrasonic velocity was observed. These results suggested that aggregation occurred during incubation with sodium caprate, and the sample showed an increase in compressibility. Transmission electron microscopy with negative staining showed the formation of filamentous aggregates of beta-lactoglobulin A at around 3-4.5 h of incubation with sodium caprate. These results demonstrated that sodium caprate induced the formation of structures with unique gel properties compared to those formed by heating beta-lactoglobulin in the presence of NaCl alone.

Leukotriene D(4) enhances the function of endothelin-1-primed fibroblasts.

Airway inflammation is accompanied by structural changes, termed remodeling, that lead to lung dysfunction over the long term. Although both endothelin-1 (ET-1) and cysteinyl leukotrienes (CysLTs) appear to be involved in airway remodeling in several lung diseases, how these molecules interact remains largely unknown. In this study, we examined the effects of leukotriene (LT) D(4) on the function of ET-1-primed fibroblasts. ET-1 at 10(-7) M up-regulated the expression of the CysLT receptors at both the mRNA and protein levels in human lung fibroblasts. LTD(4) enhanced matrix metalloproteinase-2 and pro-collagen production, and alpha-smooth muscle actin expression of ET-1-primed fibroblasts, but had little or no effect on unprimed fibroblasts. The CysLT1 receptor antagonist montelukast completely abrogated the effects of LTD(4). Our data suggested that LTD(4) may act as a precipitating factor during ET-1-mediated airway remodeling and that CysLT1 receptor antagonists may have a role in preventing aberrant extracellular matrix degradation.

Changes of plasma coenzyme Q10 levels in early infancy.

Rapid perfusion of oxygen in infants at birth may increase oxidative stress which has been incriminated in serious diseases including neonatal respiratory distress syndrome, chronic lung disease, and retinopathy of prematurity. Elucidating the antioxidant defense systems of neonates in clinical practice is important. Coenzyme Q(10) is a widely distributed, redox-active quinoid compound originally discovered as an essential part of the mitochondrial respiratory chain in mammals. Although coenzyme Q(10) is a powerful lipid antioxidant in vivo, few data pertain to plasma CoQ(10) levels in infants. This is the first paper to report plasma coenzyme Q(10) levels in preterm infants.

Concentrations of pentosidine, an advanced glycation end-product, in umbilical cord blood.

Advanced glycation end-products (AGEs) are formed over several weeks to months by non-enzymatic glycation and oxidation ("glycoxidation") reactions between carbohydrate-derived carbonyl groups and protein amino groups, known as the Maillard reaction. Pentosidine is one of the best-characterized AGEs and is accepted as a satisfactory marker for glycoxidation in vivo. The present study was intended to measure pentosidine concentrations in umbilical cord blood from newborns with various gestational ages using our recently established high-performance liquid chromatography method [Tsukahara, H. et al. (2003) Pediatr. Res. 54, 419-424]. Our study demonstrates, for the first time, that pentosidine is detected in most of the umbilical blood samples. This study also shows that the umbilical blood concentrations of pentosidine are considerably lower than normal adult values, but that they increase with gestation progression and fetal growth. Umbilical pentosidine concentrations were significantly elevated in newborns of mothers with preeclampsia compared to those of mothers without preeclampsia. We conclude that accumulation of AGEs and oxidative stress occurs in fetal tissues and organs in utero at the early stage of human life and that their accumulation is augmented in the maternal preeclampsic condition.

Oxidative stress in neonates: evaluation using specific biomarkers.

Increased oxidative stress has been implicated in pathogenesis of serious diseases in neonates. We measured urinary levels of 8-hydroxy-2'-deoxyguanosine (a marker of oxidative DNA damage), acrolein-lysine adduct (a marker of lipid peroxidation and oxidative protein damage), and nitrite/nitrate (a marker of endogenous nitric oxide formation) in one-month-old neonates to examine the status of oxidative stress and its relationship to the degree of prematurity and clinical condition in neonates. Study subjects comprised three groups: healthy term neonates, clinically stable preterm neonates requiring no supplemental oxygen, and clinically sick preterm neonates requiring supplemental oxygen and ventilator support. Urinary levels of 8-hydroxy-2'-deoxyguanosine and acrolein-lysine adduct were significantly higher in sick preterm neonates than those of stable preterm and healthy term neonates. In the sick preterm group, neonates developing active retinopathy showed significantly higher levels of acrolein-lysine adduct than the other neonates without retinopathy. There were no significant differences in both urinary markers of oxidative stress between stable preterm and healthy term neonates. The urinary nitrite/nitrate levels were not significantly different among the three groups, suggesting no difference in endogenous nitric oxide formation. Collectively, these results provide evidence of augmentation of oxidative damage to DNA, lipids and proteins, especially in clinically sick preterm neonates.

Nitric oxide metabolites and adrenomedullin in human breast milk.

We found high concentrations of nitrite/nitrate (166-1460 micromol/l) in 43 milk samples obtained from 32 women during postpartum days 1-8, which indicates enhanced mammary nitric oxide (NO) secretion. We detected adrenomedullin (AM) (2.7-20.7 pmol/l) only in 9 of the 43 samples using highly specific immunoradiometric assay. It is uncertain whether the entire adrenomedullin is actively secreted in human milk.

High levels of urinary pentosidine, an advanced glycation end product, in children with acute exacerbation of atopic dermatitis: relationship with oxidative stress.

Pentosidine is an advanced glycation end product formed by sequential glycation and oxidation. The formation of pentosidine is increased in diseases associated with oxidative stress, such as inflammatory conditions. The aim of the present study was to determine the urinary concentration of pentosidine in atopic dermatitis (AD) and its relationship to the inflammatory status of AD. Urine samples of 32 children with AD and 30 age-matched healthy control subjects were assayed for pentosidine, pyrraline (another advanced glycation end product formed by nonoxidative glycation), and 8-hydroxy-2'-deoxyguanosine (8-OHdG) (an established marker of oxidative stress). Of these 3 markers, urinary concentrations of pentosidine were significantly higher in patients with acute exacerbation of AD than in healthy controls and patients with stable AD. Urinary concentrations of 8-OHdG were significantly higher in AD patients with and without acute exacerbation than in healthy controls. Urinary pentosidine levels correlated significantly with those of 8-OHdG when all data of healthy controls and AD patients were plotted. In patients with acute exacerbation of AD, both urinary pentosidine and 8-OHdG significantly decreased after 7 to 9 days of treatment. Our findings in patients with acute exacerbation of AD suggest that pentosidine levels are partly determined by the prevailing oxidative stress in these patients.

Oxidative stress and altered antioxidant defenses in children with acute exacerbation of atopic dermatitis.

The underlying mechanisms of skin inflammation in atopic dermatitis (AD) are not completely understood. The purpose of the present study was to examine the involvement of oxidative stress and antioxidant defenses in children with acute exacerbation of AD. We studied 13 children who were hospitalized for acute exacerbation of AD with purulent skin infection by Staphylococcal aureus (age, 1.5 to 10.0 years), and 28 age-matched healthy subjects (controls). Urine samples obtained from the patients on admission, on 2nd and 7th-9th hospital days, as well as from the controls were analyzed for 8-hydroxy-2'-deoxyguanosine (8-OHdG) (a marker of oxidative DNA damage), acrolein-lysine adducts (a marker of lipid peroxidation), bilirubin oxidative metabolites (BOM) (a marker of antioxidant activity of bilirubin under oxidative stress) and nitrite/nitrate (NO(x)(-)) (a marker of endogenous nitric oxide production). Of these, urinary concentrations of 8-OHdG, acrolein-lysine adducts and BOM, but not NO(x)(-), were significantly higher in AD children on admission than those in control subjects. Response to treatment was associated with significant falls in the concentrations of 8-OHdG and acrolein-lysine adducts. Urinary concentrations of acrolein-lysine adducts, but not 8-OHdG, were still significantly higher in AD patients on the 7th-9th hospital day relative to the control. Urinary BOM remained almost constant and significantly high in AD children during hospitalization. Our findings indicate that oxidative stress and altered antioxidant defenses are involved in the pathophysiology of acute exacerbation of AD, and that suppression of oxidative stress might be a potentially useful strategy for the treatment of AD.