Fatty acid and peptide profiles in plasma membrane and membrane rafts of PUFA supplemented RAW264.7 macrophages

The eukaryotic cell membrane possesses numerous complex functions, which are essential for life. At this, the composition and the structure of the lipid bilayer are of particular importance. Polyunsaturated fatty acids may modulate the physical properties of biological membranes via alteration of membrane lipid composition affecting numerous physiological processes, e.g. in the immune system. In this systematic study we present fatty acid and peptide profiles of cell membrane and membrane rafts of murine macrophages that have been supplemented with saturated fatty acids as well as PUFAs from the n-3, the n-6 and the n-9 family. Using fatty acid composition analysis and mass spectrometry-based peptidome profiling we found that PUFAs from both the n-3 and the n-6 family have an impact on lipid and protein composition of plasma membrane and membrane rafts in a similar manner. In addition, we found a relation between the number of bis-allyl-methylene positions of the PUFA added and the unsaturation index of plasma membrane as well as membrane rafts of supplemented cells. With regard to the proposed significance of lipid microdomains for disease development and treatment our study will help to achieve a targeted dietary modulation of immune cell lipid bilayers.

Proteome analysis of human plasma and amniotic fluid by Off-Gel isoelectric focusing followed by nano-LC-MS/MS

This paper presents a comparative proteomic analysis of human maternal plasma and amniotic fluid (AF) samples from the same patient at term of pregnancy in order to find specific AF proteins as markers of premature rupture of membranes, a complication frequently observed during pregnancy. Maternal plasma and the corresponding AF were immunodepleted in order to remove the six most abundant proteins before the systematic analysis of their protein composition. The protein samples were then fractionated by IEF Off-Gel electrophoresis (OGE), digested and analyzed with nano-LC-MS/MS separation, revealing a total of 73 and 69 proteins identified in maternal plasma and AF samples, respectively. The proteins identified in AF have been compared to those identified in the mother plasma as well as to the reference human plasma protein list reported by Anderson et al. (Mol. Cell. Proteomics 2004, 3, 311-326). This comparison showed that 26 proteins were exclusively present in AF and not in plasma among which 10 have already been described to be placenta or pregnancy specific. As a further validation of the method, plasma proteins fractionated by OGE and analysed by nano-LC-MS/MS have been compared to the Swiss 2-D PAGE reference map by reconstructing a map that matches 2-D gel and OGE experimental data. This representation shows that 36 of 49 reference proteins could be identified in both data sets, and that isoform shifts in pI are well conserved in the OGE data sets.

Down-Regulating γ-gliadins in bread wheat leads to non-specific increases in other gluten proteins and has no major effect on dough gluten strength.

Background Gliadins are a major component of gluten proteins but their role in the mixing of dough is not well understood because their contribution to wheat flour functional properties are not as clear as for the glutenin fraction. Methodology/Principal Findings Transgenic lines of bread wheat with γ-gliadins suppressed by RNAi are reported. The effects on the gluten protein composition and on technological properties of flour were analyzed by RP-HPLC, by sodium dodecyl sulfate sedimentation (SDSS) test and by Mixograph analysis. The silencing of γ-gliadins by RNAi in wheat lines results in an increase in content of all other gluten proteins. Despite the gluten proteins compensation, in silico analysis of amino acid content showed no difference in the γ-gliadins silenced lines. The SDSS test and Mixograph parameters were slightly affected by the suppression of γ-gliadins. Conclusions/Significance Therefore, it is concluded that γ-gliadins do not have an essential functional contribution to the bread-making quality of wheat dough, and their role can be replaced by other gluten proteins

One Hundred Years of Grain Omics: Identifying the Glutens That Feed the World.

Glutens, the storage proteins in wheat grains, are a major source of protein in human nutrition. The protein composition of wheat has therefore been an important focus of cereal research. Proteomic tools have been used to describe the genetic diversity of wheat germplasms from different origins at the level of polymorphisms in alleles encoding glutenin and gliadin, the two main proteins of gluten. More recently, proteomics has been used to understand the impact of specific gluten proteins on wheat quality. Here we review the impact of proteomics on the study of gluten proteins as it has evolved from fractionation and electrophoretic techniques to advanced mass spectrometry. In the postgenome era, proteomics is proving to be essential in the effort to identify and understand the interactions between different gluten proteins. This is helping to fill in gaps in our knowledge of how the technological quality of wheat is determined by the interaction between genotype and environment. We also collate information on the various storage protein alleles identified and their prevalence, which makes it possible to infer the effects of wheat selection on grain protein content. We conclude by reviewing the more recent use of transgenesis aimed at improving the quality of gluten.

Evaluación de la calidad funcional y sensorial en cultivares de Triticum aestivum ssp. vulgare y ssp. spelta en cultivo ecológico

En esta Tesis Doctoral se ha estudiado la influencia del cultivar sobre el comportamiento reológico y panadero de cinco cultivares de trigo sembrados en el mismo año y en el mismo ambiente, en condiciones de cultivo ecológico. Tres de ellos eran de trigo panadero (Triticum aestivum ssp. vulgare), ‘Bonpain’, ‘Craklin’ y ‘Sensas’ y los otros dos de trigo espelta (Triticum aestivum ssp. spelta), ‘Espelta Álava’ y ‘Espelta Navarra’. Actualmente, el alohexaploide trigo panadero (2n=6x=42 genomio AABBDD) supone en torno al 90% del trigo cultivado en el mundo. En cambio, el cultivo del trigo alohexaploide espelta (2n=6x=42 genomio AABBDD) se limita a pequeñas regiones de Europa y de América del Norte. En España, el cultivo de trigo espelta se ha mantenido durante años ligado a la región de Asturias, aunque en la actualidad su cultivo está empezando a diversificarse hacia otras regiones. Esto se debe, fundamentalmente, a su potencial nutricional y a su adaptabilidad a condiciones de agricultura sostenible. El reciente resurgimiento de la espelta en productos de panificación, se debe, en gran parte, a la percepción del consumidor de que se trata de un producto ”más saludable” y “más natural” y con menor requerimiento de insumos que los trigos modernos. A medida que el consumo de alimentos a base de harina de espelta aumenta, se plantea la necesidad de evaluar su calidad harino-panadera, nutricional y sensorial en comparación con los productos elaborados con variedades de trigo común. Se caracterizaron las gluteninas de alto peso molecular (HMW) y las puroindolinas de los cinco cultivares. Se evaluó la calidad del grano, la reología de sus masas y se analizó la calidad instrumental y sensorial de sus panes. Para tal fin se ha puesto a punto un protocolo de panificación adecuado a las características particulares de los trigos espelta y se ha propuesto para el análisis sensorial de los panes un protocolo de selección, entrenamiento y validación de jueces. Teniendo en cuenta la composición en gluteninas HMW de los cultivares, se comprobó su influencia en el volumen de sedimentación y en la fuerza panadera. La composición en puroindolinas se vió reflejada en el parámetro dureza del endospermo. Los resultados indicaron que hay diferencias entre trigo panadero y trigo espelta en parámetros como, la tenacidad y el equilibrio de sus masas, la capacidad de absorción de agua de la harina y el comportamiento de la masa durante el amasado. Los trigos espeltas mostraron menor valor en el tiempo en alcanzar la presión máxima y la tolerancia al amasado, mientras que presentaron valores superiores en el decaimiento a los 250 y 450 segundos respectivamente. Respecto a la calidad de los panes elaborados, los trigos espeltas tenían mayor elasticidad en la miga y mayores valores en el área y en el diámetro de sus alveolos. Estas diferencias en la estructura y textura de la miga fueron también detectadas a nivel sensorial por el panel de jueces. Mediante el perfil sensorial descriptivo, se determinó que uno de los dos panes elaborado con trigo espelta (‘Espelta Navarra’) fue el pan más complejo considerando conjuntamente los atributos de aroma y flavor. En este trabajo no se apreciaron diferencias entre ambos tipos de trigo ni en el contenido en proteína, ni en minerales, ni en la viscosidad de su almidón. ABSTRACT In this Doctoral Thesis, the influence of various cultivars on rheological and baking behavior was studied. Five wheat cultivars were used, all planted in the same year and same organic farming environment. Three were bread wheat (Triticum aestivum ssp. vulgare), 'Bonpain', 'Craklin' and 'Sensas' and the other two were spelt wheat (Triticum aestivum ssp. spelta) , 'Espelta Álava' and 'Espelta Navarra' . Currently, the allohexaploid bread wheat (2n=6x=42 genome AABBDD) represents about 90% of global wheat production. On the other hand, allohexaploid spelt wheat (2n=6x=42 genome AABBDD) is merely produced in small areas of Europe and North America. For many years, the cultivation of spelt wheat in Spain was limited to the region of Asturias, although nowadays its production has begun to spread into other regions. This is owing to its nutritional potential and adaptability to conditions of sustainable agriculture. The recent resurgence of spelt in baking products is mainly due to consumers perception of it, as "healthier" and "more natural", and to the fewer agricultural input requirements compared to modern wheat products. As the consumption of foods made from spelt flour increases, there is a need to assess its baking, nutritional and sensory quality, compared to products made with common varieties of wheat. High molecular weight glutenins and puroindolines from the five cultivars were characterized. The quality of the grain and the rheology of the dough were evaluated and the instrumental and sensory quality of its breads were analyzed. To this end it a baking protocol was appropriately developed to the particular characteristics of spelt wheat and a selection protocol was proposed for the sensory analysis of breads, after proper training and validation of judges. Considering the HMW glutenin composition of the cultivars, the influence on the sedimentation volume and the baking strength was proven. The composition of puroindolines was reflected in the endosperm hardness parameter. The results show that there are differences between bread wheat and spelt wheat on parameters such as the tenacity and tenacity/elasticity ratio of their masses, the water absorption capacity of the flour and the behavior of the dough during kneading. The values for total time to reach maximum pressure and tolerance to mixing were lower for spelt wheat, and higher values were found for the drop at 250 s and 450 s. Regarding the quality of manufactured bread, spelt wheat had the greatest elasticity of the crumb and higher values in the area and diameter of the cells. These differences in the structure and texture of the crumb were also noticed at a sensory level by the panel of judges. It was determined by a descriptive sensory profile that one of the two loaves of bread made with spelt ('Espelta Navarra') was the most complex in the sense of its attributes of scents and flavors altogether. In this study, no differences were appreciated between the two types of wheat or the protein composition, or minerals or viscosity of the starch.

Association of Nonribosomal Nucleolar Proteins in Ribonucleoprotein Complexes during Interphase and Mitosis

rRNA precursors are bound throughout their length by specific proteins, as the pre-rRNAs emerge from the transcription machinery. The association of pre-rRNA with proteins as ribonucleoprotein (RNP) complexes persists during maturation of 18S, 5.8S, and 28S rRNA, and through assembly of ribosomal subunits in the nucleolus. Preribosomal RNP complexes contain, in addition to ribosomal proteins, an unknown number of nonribosomal nucleolar proteins, as well as small nucleolar RNA-ribonucleoproteins (sno-RNPs). This report describes the use of a specific, rapid, and mild immunopurification approach to isolate and analyze human RNP complexes that contain nonribosomal nucleolar proteins, as well as ribosomal proteins and rRNA. Complexes immunopurified with antibodies to nucleolin—a major nucleolar RNA-binding protein—contain several distinct specific polypeptides that include, in addition to nucleolin, the previously identified nucleolar proteins B23 and fibrillarin, proteins with electrophoretic mobilities characteristic of ribosomal proteins including ribosomal protein S6, and a number of additional unidentified proteins. The physical association of these proteins with one another is mediated largely by RNA, in that the complexes dissociate upon digestion with RNase. Complexes isolated from M-phase cells are similar in protein composition to those isolated from interphase cell nuclear extracts. Therefore, the predominant proteins that associate with nucleolin in interphase remain in RNP complexes during mitosis, despite the cessation of rRNA synthesis and processing in M-phase. In addition, precursor rRNA, as well as processed 18S and 28S rRNA and candidate rRNA processing intermediates, is found associated with the immunopurified complexes. The characteristics of the rRNP complexes described here, therefore, indicate that they represent bona fide precursors of mature cytoplasmic ribosomal subunits.

A herpes simplex virus 1 recombinant lacking the glycoprotein G coding sequences is defective in entry through apical surfaces of polarized epithelial cells in culture and in vivo

During infection of a new host, the first surfaces encountered by herpes simplex viruses are the apical membranes of epithelial cells of mucosal surfaces. These cells are highly polarized, and the protein composition of their apical and basolateral membranes are very different, so that different viral entry pathways have evolved for each surface. To determine whether the viral glycoprotein G (gG) is specifically required for efficient infection of a particular surface of polarized cells, apical and basal surfaces were infected with wild-type virus or a gG deletion mutant. After infection of polarized cells in culture, the gG− virus was deficient in infection of apical surfaces but was able to infect cells through basal membranes, replicate, and spread into surrounding cells. The gG-dependent step in apical infection was a stage beyond attachment. After in vivo infection of apical surfaces of epithelial cells of nonscarified mouse corneas, infection by glycoprotein C− or gG− virus was considerably reduced as compared with that observed after infection with wild-type virus. In contrast, when corneas were scarified, allowing virus access to other cell surfaces, the gG and glycoprotein C deletion mutants infected eyes as efficiently as wild-type viruses. A secondary mutation allowing infection of apical surfaces by gG− virus arose readily during passage of the virus in nonpolarized cells, indicating that either the gG-dependent step of apical infection can be bypassed or that another viral protein can acquire the same function.

Male fertility defects in mice lacking the serine protease inhibitor protease nexin-1

Understanding infertility and sterility requires knowledge of the molecular mechanisms underlying sexual reproduction. We have found that male mice deficient for the gene encoding the protease inhibitor protease nexin-1 (PN-1) show a marked impairment in fertility from the onset of sexual maturity. Absence of PN-1 results in altered semen protein composition, which leads to inadequate semen coagulation and deficient vaginal plug formation upon copulation. Progressive morphological changes of the seminal vesicles also are observed. Consistent with these findings, abnormal PN-1 expression was found in the semen of men displaying seminal dysfunction. The data demonstrate that the level of extracellular proteolytic activity is a critical element in controlling male fertility.

Cytoplasmic dynein is associated with slow axonal transport.

Neuronal function is dependent on the transport of materials from the cell body to the synapse via anterograde axonal transport. Anterograde axonal transport consists of several components that differ in both rate and protein composition. In fast transport, membranous organelles are moved along microtubules by the motor protein kinesin. The cytoskeleton and the cytomatrix proteins move in the two components of slow transport. While the mechanisms underlying slow transport are unknown, it has been hypothesized that the movement of microtubules in slow transport is generated by sliding. To determine whether dynein, a motor protein that causes microtubule sliding in flagella, may play a role in slow axonal transport, we identified the transport rate components with which cytoplasmic dynein is associated in rat optic nerve. Nearly 80% of the anterogradely moving dynein was associated with slow transport, whereas only approximately 15% of the dynein was associated with the membranous organelles of anterograde fast axonal transport. A segmental analysis of the transport of dynein through contiguous regions of the optic nerve and tract showed that dynein is associated with the microfilaments and other proteins of slow component b. Dynein from this transport component has the capacity to bind microtubules in vitro. These results are consistent with the hypothesis that cytoplasmic dynein generates the movement of microtubules in slow axonal transport. A model is presented to illustrate how dynein attached to the slow component b complex of proteins is appropriately positioned to generate force of the correct polarity to slide microtubules down the axon.

The effects of formulation and processing on surface characteristics and functional properties of dairy powders

The objectives of this thesis were to (i) study the effect of increasing protein concentration in milk protein concentrate (MPC) powders on surface composition and sorption properties; (ii) examine the effect of increasing protein content on the rehydration properties of MPC; (iii) study the physicochemical properties of spraydried emulsion-containing powders having different water and oil contents; (iv) analyse the effect of protein type on water sorption and diffusivity properties in a protein/lactose dispersion, and; (v) characterise lactose crystallisation and emulsion stability of model infant formula containing intact or hydrolysed whey proteins. Surface composition of MPC powders (protein contents 35 - 86 g / 100 g) indicated that fat and protein were preferentially located on the surface of powders. Low protein powder (35 g / 100 g) exhibited lactose crystallisation, whereas powders with higher protein contents did not, due to their high protein: lactose ratio. Insolubility was evident in high protein MPCs and was primarily related to insolubility of the casein fraction. High temperature (50 °C) was required for dissolution of high protein MPCs (protein content > 60 g / 100 g). The effect of different oil types and spray-drying outlet temperature on the physicochemical properties of the resultant fat-filled powders was investigated and showed that increasing outlet temperature reduced water content, water activity and tapped bulk density, irrespective of oil type, and increased solvent-extractable free fat for all oil types and onset of glass transition (Tg) and crystallisation (Tcr) temperature. Powder dispersions of protein/lactose (0.21:1), containing either intact or hydrolysed whey protein (12 % degree of hydrolysis; DH), were spray-dried at pilot scale. Moisture sorption analysis at 25 °C showed that dispersions containing intact whey protein exhibited lactose crystallisation at a lower relative humidity (RH). Dispersions containing hydrolysed whey protein had significantly higher (P < 0.05) water diffusivity. Finally, a spray-dried model infant formula was produced containing hydrolysed or intact whey as the protein with sunflower oil as the fat source. Reconstituted, hydrolysed formula had a significantly (P < 0.05) higher fat globule size and lower emulsion stability than intact formula. Lactose crystallisation in powders occurred at higher RH for hydrolysed formula. In conclusion, this research has shown the effect of altering the protein type, protein composition, and oil type on the surface composition and physical properties of different dairy powders, and how these variations greatly affect their rehydration characteristics and storage stability.

Effect of foliar application of Cu, Zn, and Mn on yield and quality indicators of winter wheat grain

Micronutrients are part of many crucial physiological plant processes. The combined application of N and micronutrients helps in obtaining grain yield with beneficial technological and consumer properties. The main micronutrients needed by cereals include Cu, Mn, and Zn. The subject of this study was to determine yield, quality indicators (protein content and composition, gluten content, grain bulk density, Zeleny sedimentation index, and grain hardness), as well as mineral content (Cu, Zn, Mn, Fe) in winter wheat grain ( Triticum aestivum L.) fertilized by foliar micronutrient application. A field experiment was carried out at the Educational and Experimental Station in Tomaszkowo, Poland. The application of mineral fertilizers (NPK) supplemented with Cu increased Cu content (13.0%) and ω, α/β, and γ (18.7%, 4.9%, and 3.4%, respectively) gliadins in wheat grain. Foliar Zn fertilization combined with NPK increased Cu content (14.9%) as well as high (HMW) and low molecular weight (LMW) glutenins (38.8% and 6.7%, respectively). Zinc fertilization significantly reduced monomeric gliadin content and increased polymeric glutenin content in grain, which contributed in reducing the gliadin:glutenin ratio (0.77). Mineral fertilizers supplemented with Mn increased Fe content in wheat grain (14.3%). It also significantly increased protein (3.8%) and gluten (4.4%) content, Zeleny sedimentation index (12.4%), and grain hardness (18.5%). Foliar Mn fertilization increased the content of ω, α/β, and γ gliadin fractions (19.9%, 9.5%, and 2.1%, respectively), as well as HMW and LMW glutenins (18.9% and 4.5%, respectively). Mineral NPK fertilization, combined with micronutrients (Cu + Zn + Mn), increased Cu and Zn content in grain (22.6% and 17.7%, respectively). The content of ω, α/β, and γ gliadins increased (20.3%, 10.5%, and 12.1%, respectively) as well as HMW glutenins (7.9%).

Oral food perception and polyphenol-rich foods acceptance - the importance of knowing individuals saliva characteristics for promoting consumption

Polyphenols are widely present in fruits, vegetables, cereals and beverages. Their study gained scientific interest because of their beneficial effects on health. Although there is currently no official dietary recommendation for polyphenol intake, health professionals recommend the consumption of 5-8 daily portions of fruits and vegetables. This is not always achieved and, despite possible causes associated to practical schedule difficulties, the aversive bitter and astringent sensations associated to polyphenols may also lead to avoidance. As such, a better understanding on mechanisms responsible for differences among people, in polyphenol oral perception, is needed for promoting healthier choices. Saliva has been linked to polyphenol consumption. We have previously observed, in animal models, changes in salivary proteome induced by tannin-enriched diets. Moreover, differences in astringency perception were attributed to differences in salivary protein composition. In a recent experiment, we observed differences among individuals with dissimilar tannic-acid perception: people with high sensitivity for the oral sensations elicited by tannins have higher amounts of salivary cystatins and lower capacity to maintain their levels after tannic-acid ingestion. Additionally, and similarly to previous studies, salivary amylase was observed to be involved in tannin perception. In this presentation, oral cavity characteristics influencing the perception of polyphenol-containing foods will be discussed.

Diferenciación molecular de Lemna aequinoctialis y L. Valdiviana (lemnaceae) por patrones proteicos

El estudio se llevó a cabo en el Laboratorio de Genética Molecular de la Universidad Nacional, Heredia, Costa Rica. Se utilizaron plantas acuáticas macrófitas de Lemna valdiviana y L. aequinoctialis previamente identificadas. Estas especies habitan en aguas ricas en sales disueltas y aguas servidas. Recientemente se les consideró como un indicador de la contaminación ocasionada por pesticidas y herbicidas en el agua. La composición proteica llega a niveles entre 38 y 43% de materia seca, importante para diversos usos. La extracción de proteínas totales, por medio de electroforesis discontinua en gel de poliacrilamida, se realizó en condiciones reductoras con SDS (SDS-PAGE). Los geles separadores empleados fueron al 8 y 10%, gel espaciador al 4% y la tinción se efectuó con azul commassie. Los diversos patrones proteicos reflejaron diferencias genéticas entre ambas especies que se pueden deber a las diferencias de hábitat y de reproducción.

Genetic control of wheat quality: interactions between chromosomal regions determining protein content and composition, dough rheology, and sponge and dough baking properties

While the genetic control of wheat processing characteristics such as dough rheology is well understood, limited information is available concerning the genetic control of baking parameters, particularly sponge and dough (S&D) baking. In this study, a quantitative trait loci (QTL) analysis was performed using a population of doubled haploid lines derived from a cross between Australian cultivars Kukri x Janz grown at sites across different Australian wheat production zones (Queensland in 2001 and 2002 and Southern and Northern New South Wales in 2003) in order to examine the genetic control of protein content, protein expression, dough rheology and sponge and dough baking performance. The study highlighted the inconsistent genetic control of protein content across the test sites, with only two loci (3A and 7A) showing QTL at three of the five sites. Dough rheology QTL were highly consistent across the 5 sites, with major effects associated with the Glu-B1 and Glu-D1 loci. The Glu-D1 5 + 10 allele had consistent effects on S&D properties across sites; however, there was no evidence for a positive effect of the high dough strength Glu-B1-al allele at Glu-B1. A second locus on 5D had positive effects on S&D baking at three of five sites. This study demonstrated that dough rheology measurements were poor predictors of S&D quality. In the absence of robust predictive tests, high heritability values for S&D demonstrate that direct selection is the current best option for achieving genetic gain in this product category.

Immediate, cumulative and residual effects of short- and long-term low plane of nutrition on milk protein and casein composition

Immediate and residual effects of two lengths of low plane of nutrition (PON) on the synthesis of milk protein and protein fractions were studied at the Mutdapilly Research Station, in south-east Queensland. Thirty-six multiparous Holstein-Friesian cows, between 46 and 102 days in milk (DIM) initially, were used in a completely randomised design experiment with three treatments. All cows were fed on a basal diet of ryegrass pasture (7.0 kg DM/cow.day), barley-sorghum concentrate mix (2.7 kg DM/cow.day) and a canola meal-mineral mix (1.3 kg DM/cow.day). To increase PON, 5.0 kg DM/cow.day supplemental maize and forage sorghum silage was added to the basal diet. The three treatments were (C) high PON (basal diet + supplemental silage); (L9) low PON (basal diet only) for a period of 9 weeks; and (L3) low PON (basal diet only) for a period of 3 weeks. The experiment comprised three periods (1) covariate – high PON, all groups (5 weeks), (2) period of low PON for either 3 weeks (L3) or 9 weeks (L9), and (3) period of high PON (all groups) to assess ability of cows to recover any production lost as a result of treatments (5 weeks). The low PON treatment periods for L3 and L9 were end-aligned so that all treatment groups began Period 3 together. Although there was a significant effect of L9 on yields of milk, protein, fat and lactose, and concentrations of true protein, whey protein and urea, these were not significantly different from L3. There were no residual effects of L3 or L9 on protein concentration or nitrogen distribution after 5 weeks of realimentation. There was no significant effect of low PON for 3 or 9 weeks on casein concentration or composition.