Effects of different levels of protein intake and physical training on growth and nutritional status of young rats

This study aimed to investigate the effects of physical training, and different levels of protein intake in the diet, on the growth and nutritional status of growing rats. Newly-weaned Wistar rats (n=48) were distributed into six experimental groups: three of them were subjected to physical swim training (1 h per day. 5 d per week, for 4 wk, after 2 wk of familiarization) and the other three were considered as controls (non-trained). Each pair of groups, trained and non-trained, received diets with a different level of protein in their composition: 14%. 21% or 28%. The animals were euthanized at the end of the training period and the following analyses were performed: proteoglycan synthesis as a biomarker of bone and cartilage growth, IGF-I (insulin-like growth factor-I) assay as a biomarker of growth and nutritional status. total RNA and protein concentration and protein synthesis measured in vivo using a large-dose phenylalanine method. As a main finding, increased dietary protein, combined with physical training, was able to improve neither tissue protein synthesis nor muscle growth. In addition, cartilage and bone growth seem to be deteriorated by the lower and the higher levels of protein intake. Our data allow us to conclude that protein enhancement in the diet, combined with physical exercise, does not stimulate tissue protein synthesis or muscle mass growth. Furthermore, physical training, combined with low protein intake, was not favorable to bone development in growing animals

Recovery and upgrading bovine rumen protein by extrusion: effect of lipid content on protein disulphide cross-linking, solubility and molecular weight

Bovine rumen protein with two levels of residual lipids (1.9 per cent or 3.8 per cent) was subjected to thermoplastic extrusion under different temperatures and moisture contents. Protein solubility in different buffers, disulphide cross-linking and molecular weight distribution were determined on the extrudates. After extrusion, samples with 1.9 per cent residual lipids content had a higher concentration of protein insoluble by undetermined forces, irrespective of feed moisture and processing temperature used. Lipid content of 3.8 per cent in the feed material resulted in more protein participating in the extrudate network through non-covalent interactions (hydrophobic and electrostatic) and disulphide bonds. A small dependency of the extrusion process on moisture and temperature and a marked dependency on lipid content, especially phospholipid, was observed, Electrophoresis under non-reducing conditions showed that protein extrusion with low feed moisture promoted high molecular breakdown inside the barrel, probably due to intense shear force, and further protein aggregation at the die end

Effect of protein intake on bone and muscle mass in the elderly

The aging process is frequently characterized by an involuntary loss of muscle (sarcopenia) and bone (osteoporosis) mass. Both chronic diseases are associated with decreased metabolic rate, increased risk of falls fracture, and, as a result, increased morbidity and loss of independence in the elderly. The quality and quantity of protein intake affects bone and muscle mass in several ways and there is evidence that increased essential amino acid or protein availability can enhance muscle protein synthesis and anabolism, as well as improve bone homeostasis in older subjects. A thorough evaluation of renal function is important, since renal function decreases with age. Finally, protein and calcium intake should be considered in the prevention or treatment of the chronic diseases osteoporosis and sarcopenia

Consumption of a flaxseed-rich food is not more effective than a placebo in alleviating the climacteric symptoms of postmenopausal women

Our objective in this work was to test the effects of daily intake of bread produced with partially defatted ground flaxseed on the climacteric symptoms and endometrial thickness of postmenopausal women. A double-blind, placebo-controlled, randomized clinical trial was performed with 38 women who had been postmenopausal for 1–10 y and consumed 2 slices of bread containing 25 g of flaxseed (46 mg lignans) or wheat bran (<1 mg lignans; control) every day for 12 consecutive weeks. The outcome variables were the daily number of hot flashes, the Kupperman Menopausal Index (KMI), and endometrial thickness. The plasma lipid profile (total cholesterol and HDL, LDL, and VLDL cholesterol fractions and triglycerides) and the hormones estradiol, follicle-stimulating hormone, thyroid-stimulating hormone, and free thyroxine also were measured. Food intake was evaluated by means of 2 24-h recalls, before and after the treatment. Twenty patients in the study group and 18 in the control group completed the study. The general characteristics did not differ between the 2 groups at the start of the study. Both had significant, but similar, reductions in hot flashes and KMI after 3 mo of treatment. Moreover, endometrial thickness was not affected in either group. Our findings clearly show that although flaxseed is safe, its consumption at this level (46 mg lignans/d) is no more effective than placebo for reducing hot flashes and KMI

The expression of genes coding for distinct types of glycine-rich proteins varies according to the biology of three metastriate ticks, Rhipicephalus (Boophilus) microplus, Rhipicephalus sanguineus and Amblyomma cajennense

Background: Ticks secrete a cement cone composed of many salivary proteins, some of which are rich in the amino acid glycine in order to attach to their hosts' skin. Glycine-rich proteins (GRPs) are a large family of heterogeneous proteins that have different functions and features; noteworthy are their adhesive and tensile characteristics. These properties may be essential for successful attachment of the metastriate ticks to the host and the prolonged feeding necessary for engorgement. In this work, we analyzed Expressed Sequence Tags (ESTs) similar to GRPs from cDNA libraries constructed from salivary glands of adult female ticks representing three hard, metastriate species in order to verify if their expression correlated with biological differences such as the numbers of hosts ticks feed on during their parasitic life cycle, whether one (monoxenous parasite) or two or more (heteroxenous parasite), and the anatomy of their mouthparts, whether short (Brevirostrata) or long (Longirostrata). These ticks were the monoxenous Brevirostrata tick, Rhipicephalus (Boophilus) microplus, a heteroxenous Brevirostrata tick, Rhipicephalus sanguineus, and a heteroxenous Longirostrata tick, Amblyomma cajennense. To further investigate this relationship, we conducted phylogenetic analyses using sequences of GRPs from these ticks as well as from other species of Brevirostrata and Longirostrata ticks. Results: cDNA libraries from salivary glands of the monoxenous tick, R. microplus, contained more contigs of glycine-rich proteins than the two representatives of heteroxenous ticks, R. sanguineus and A. cajennense (33 versus, respectively, 16 and 11). Transcripts of ESTs encoding GRPs were significantly more numerous in the salivary glands of the two Brevirostrata species when compared to the number of transcripts in the Longirostrata tick. The salivary gland libraries from Brevirostrata ticks contained numerous contigs significantly similar to silks of true spiders (17 and 8 in, respectively, R. microplus and R. sanguineus), whereas the Longirostrata tick contained only 4 contigs. The phylogenetic analyses of GRPs from various species of ticks showed that distinct clades encoding proteins with different biochemical properties are represented among species according to their biology. Conclusions: We found that different species of ticks rely on different types and amounts of GRPs in order to attach and feed on their hosts. Metastriate ticks with short mouthparts express more transcripts of GRPs than a tick with long mouthparts and the tick that feeds on a single host during its life cycle contain a greater variety of these proteins than ticks that feed on several hosts.

AtPIN: Arabidopsis thaliana Protein Interaction Network

Background: Protein-protein interactions (PPIs) constitute one of the most crucial conditions to sustain life in living organisms. To study PPI in Arabidopsis thaliana we have developed AtPIN, a database and web interface for searching and building interaction networks based on publicly available protein-protein interaction datasets. Description: All interactions were divided into experimentally demonstrated or predicted. The PPIs in the AtPIN database present a cellular compartment classification (C(3)) which divides the PPI into 4 classes according to its interaction evidence and subcellular localization. It has been shown in the literature that a pair of genuine interacting proteins are generally expected to have a common cellular role and proteins that have common interaction partners have a high chance of sharing a common function. In AtPIN, due to its integrative profile, the reliability index for a reported PPI can be postulated in terms of the proportion of interaction partners that two proteins have in common. For this, we implement the Functional Similarity Weight (FSW) calculation for all first level interactions present in AtPIN database. In order to identify target proteins of cytosolic glutamyl-tRNA synthetase (Cyt-gluRS) (AT5G26710) we combined two approaches, AtPIN search and yeast two-hybrid screening. Interestingly, the proteins glutamine synthetase (AT5G35630), a disease resistance protein (AT3G50950) and a zinc finger protein (AT5G24930), which has been predicted as target proteins for Cyt-gluRS by AtPIN, were also detected in the experimental screening. Conclusions: AtPIN is a friendly and easy-to-use tool that aggregates information on Arabidopsis thaliana PPIs, ontology, and sub-cellular localization, and might be a useful and reliable strategy to map protein-protein interactions in Arabidopsis. AtPIN can be accessed at http://bioinfo.esalq.usp.br/atpin.

Nutritive value and physical characteristics of Xaraes palisadegrass as affected by grazing strategy

The aim of this study was to ascertain whether the defoliation frequency based on a fixed rest period would generate variable sward structural and physiological conditions at each subsequent grazing event. The relative importance of the physiological age was established in comparison with the chronological age in the determination of the forage nutritive value of Xaraes palisadegrass [Brachiaria brizantha (Hochst ex A. RICH.) STAPF. cv. Xaraes]. Two grazing frequencies were defined by light interception (LI) at initiation of grazing (95% LI - ""target grazing"" [TG] or 100% LI - ""delayed grazing"" [DG]) and one based on chronological time, grazing every 28 days (28-d). Forage produced under the TG schedule was mostly leaves (93%) with a higher concentration of crude protein (CP; 138 g/kg in the whole forage), a lower concentrations of neutral detergent fibre (NDF) in the stems (740 g/kg), and higher in vitro dry matter digestibility (IVDMD) of the leaves (690 g/kg), compared to the other treatments. Lower grazing frequency strategies (DG and 28-d) resulted in forage with higher proportions of stems (10 and 9%, respectively). Strategies based on light interception did not produce pre-graze forage with a uniform nutritive value, as the indicators varied across grazing cycles. The treatment based on fixed days of rest did not result in uniformity.

Polyelectrolyte-protein complexation driven by charge regulation

The interplay between the biocolloidal characteristics (especially size and charge), pH, salt concentration and the thermal energy results in a unique collection of mesoscopic forces of importance to the molecular organization and function in biological systems. By means of Monte Carlo simulations and semi-quantitative analysis in terms of perturbation theory, we describe a general electrostatic mechanism that gives attraction at low electrolyte concentrations. This charge regulation mechanism due to titrating amino acid residues is discussed in a purely electrostatic framework. The complexation data reported here for interaction between a polyelectrolyte chain and the proteins albumin, goat and bovine alpha-lactalbumin, beta-lactoglobulin, insulin, k-casein, lysozyme and pectin methylesterase illustrate the importance of the charge regulation mechanism. Special attention is given to pH congruent to pI where ion-dipole and charge regulation interactions could overcome the repulsive ion-ion interaction. By means of protein mutations, we confirm the importance of the charge regulation mechanism, and quantify when the complexation is dominated either by charge regulation or by the ion-dipole term.

Proteasome Inhibition Represses Unfolded Protein Response and Nox4, Sensitizing Vascular Cells to Endoplasmic Reticulum Stress-Induced Death

Background: Endoplasmic reticulum (ER) stress has pathophysiological relevance in vascular diseases and merges with proteasome function. Proteasome inhibition induces cell stress and may have therapeutic implications. However, whether proteasome inhibition potentiates ER stress-induced apoptosis and the possible mechanisms involved in this process are unclear. Methodology/Principal Findings: Here we show that proteasome inhibition with MG132, per se at non-lethal levels, sensitized vascular smooth muscle cells to caspase-3 activation and cell death during ER stress induced by tunicamycin (Tn). This effect was accompanied by suppression of both proadaptive (KDEL chaperones) and proapoptotic (CHOP/GADD153) unfolded protein response markers, although, intriguingly, the splicing of XBP1 was markedly enhanced and sustained. In parallel, proteasome inhibition completely prevented ER stress-induced increase in NADPH oxidase activity, as well as increases in Nox4 isoform and protein disulfide isomerase mRNA expression. Increased Akt phosphorylation due to proteasome inhibition partially offset the proapoptotic effect of Tn or MG132. Although proteasome inhibition enhanced oxidative stress, reactive oxygen species scavenging had no net effect on sensitization to Tn or MG132-induced cell death. Conclusion/Relevance: These data indicate unfolded protein response-independent pathways whereby proteasome inhibition sensitizes vascular smooth muscle to ER stress-mediated cell death. This may be relevant to understand the therapeutic potential of such compounds in vascular disease associated with increased neointimal hyperplasia.

Comparison of C-reactive protein and serum amyloid A protein in septic shock patients

Septic shock is a severe inflammatory state caused by an infectious agent. Our purpose was to investigate serum amyloid A (SAA) protein and C-reactive protein (CRP) as inflammatory markers of septic shock patients. Here we evaluate 29 patients in postoperative period, with septic shock, in a prospective study developed in a surgical intensive care unit. All eligible patients were monitored over a 7-day period by sequential organ failure assessment (SOFA) score, daily CRP, SAA, and lactate measurements. CRP and SAA strongly correlated up to the fifth day of observation but were not good predictors of mortality in septic shock. Copyright (C) 2008.

Lipid Metabolism in Subclinical Hypothyroidism: Plasma Kinetics of Triglyceride-Rich Lipoproteins and Lipid Transfers to High-Density Lipoprotein Before and After Levothyroxine Treatment

Background: Subclinical hypothyroidism (SCH) has been associated with atherosclerosis, but the abnormalities in plasma lipids that can contribute to atherogenesis are not prominent. The aim of this study was to test the hypothesis that patients with normocholesterolemic, normotriglyceridemic SCH display abnormalities in plasma lipid metabolism not detected in routine laboratory tests including abnormalities in the intravascular metabolism of triglyceride-rich lipoproteins, lipid transfers to high-density lipoprotein (HDL), and paraoxonase 1 activity. The impact of levothyroxine (LT4) treatment and euthyroidism in these parameters was also tested. Methods: The study included 12 SCH women and 10 matched controls. Plasma kinetics of an artificial triglyceride-rich emulsion labeled with radioactive triglycerides and cholesteryl esters as well as in vitro transfer of four lipids from an artificial donor nanoemulsion to HDL were determined at baseline in both groups and after 4 months of euthyroidism in the SCH group. Results: Fractional clearance rates of triglycerides (SCH 0.035 +/- 0.016 min(-1), controls 0.029 +/- 0.013 min(-1), p=0.336) and cholesteryl esters (SCH 0.009 +/- 0.007 min(-1), controls 0.009 +/- 0.009 min(-1), p=0.906) were equal in SCH and controls and were unchanged by LT4 treatment and euthyroidism in patients with SCH, suggesting that lipolysis and remnant removal of triglyceride-rich lipoproteins were normal. Transfer of triglycerides to HDL (SCH 3.6 +/- 0.48%, controls 4.7 +/- 0.63%, p=0.001) and phospholipids (SCH 16.2 +/- 3.58%, controls 21.2 +/- 3.32%, p=0.004) was reduced when compared with controls. After LT4 treatment, transfers increased and achieved normal values. Transfer of free and esterified cholesterol to HDL, HDL particle size, and paraoxonase 1 activity were similar to controls and were unchanged by treatment. Conclusions: Although intravascular metabolism of triglyceride-rich lipoproteins was normal, patients with SCH showed abnormalities in HDL metabolism that were reversed by LT4 treatment and achievement of euthyroidism.

Lsa21, a novel leptospiral protein binding adhesive matrix molecules and present during human infection

Background: It has been well documented over past decades that interaction of pathogens with the extracellular matrix (ECM) plays a primary role in host cell attachment and invasion. Adherence to host tissues is mediated by surface-exposed proteins expressed by the microorganisms during infection. The mechanisms by which pathogenic leptospires invade and colonize the host remain poorly understood since few virulence factors contributing to the pathogenesis of the disease have been identified. Whole-genome sequencing analysis of L. interrogans allowed identification of a repertoire of putative leptospiral surface proteins. Results: Here, we report the identification and characterization of a new leptospiral protein that exhibits extracellular matrix-binding properties, called as Lsa21 (leptospiral surface adhesin, 21 kDa). Compatible with its role in adhesion, the protein was shown to be surface-exposed by indirect immunofluorescence. Attachment of Lsa21 to laminin, collagen IV, and plasma fibronectin was specific and dose dependent. Laminin oxidation by sodium metaperiodate reduced the protein-laminin interaction in a concentration-dependent manner, indicating that laminin sugar moieties are crucial for this interaction. The gene coding for Lsa21 is present in pathogenic strains belonging to the L. interrogans species but was not found in the saprophytic L. biflexa serovar Patoc strain Patoc 1. Loss of gene expression occurs upon culture attenuation of pathogenic strains. Environmental factors such as osmolarity and temperature affect Lsa21 expression at the transcriptional level. Moreover, anti-Lsa21 serum labeled liver and kidney tissues of human fatal cases of leptospirosis. Conclusion: Our data suggest a role of Lsa21 in the pathogenesis of leptospirosis.

Sensitization Prevalence, Antibody Cross-Reactivity and Immunogenic Peptide Profile of Api g 2, the Non-Specific Lipid Transfer Protein 1 of Celery

Background: Celery (Apium graveolens) represents a relevant allergen source that can elicit severe reactions in the adult population. To investigate the sensitization prevalence and cross-reactivity of Api g 2 from celery stalks in a Mediterranean population and in a mouse model. Methodology: 786 non-randomized subjects from Italy were screened for IgE reactivity to rApi g 2, rArt v 3 (mugwort pollen LTP) and nPru p 3 (peach LTP) using an allergen microarray. Clinical data of 32 selected patients with reactivity to LTP under investigation were evaluated. Specific IgE titers and cross-inhibitions were performed in ELISA and allergen microarray. Balb/c mice were immunized with purified LTPs; IgG titers were determined in ELISA and mediator release was examined using RBL-2H3 cells. Simulated endolysosomal digestion was performed using microsomes obtained from human DCs. Results: IgE testing showed a sensitization prevalence of 25.6% to Api g 2, 18.6% to Art v 3, and 28.6% to Pru p 3 and frequent co-sensitization and correlating IgE-reactivity was observed. 10/32 patients suffering from LTP-related allergy reported symptoms upon consumption of celery stalks which mainly presented as OAS. Considerable IgE cross-reactivity was observed between Api g 2, Art v 3, and Pru p 3 with varying inhibition degrees of individual patients' sera. Simulating LTP mono-sensitization in a mouse model showed development of more congruent antibody specificities between Api g 2 and Art v 3. Notably, biologically relevant murine IgE cross-reactivity was restricted to the latter and diverse from Pru p 3 epitopes. Endolysosomal processing of LTP showed generation of similar clusters, which presumably represent T-cell peptides. Conclusions: Api g 2 represents a relevant celery stalk allergen in the LTP-sensitized population. The molecule displays common B cell epitopes and endolysosomal peptides that encompass T cell epitopes with pollen and plant-food derived LTP.

Expression and cellular localization of microRNA-29b and RAX, an activator of the RNA-dependent protein kinase (PKR), in the retina of streptozotocin-induced diabetic rats

Purpose: The apoptosis of retinal neurons plays a critical role in the pathogenesis of diabetic retinopathy (DR), but the molecular mechanisms underlying this phenomenon remain unclear. The purpose of this study was to investigate the cellular localization and the expression of microRNA-29b (miR-29b) and its potential target PKR associated protein X (RAX), an activator of the pro-apoptotic RNA-dependent protein kinase (PKR) signaling pathway, in the retina of normal and diabetic rats. Methods: Retinas were obtained from normal and diabetic rats within 35 days after streptozotocin (STZ) injection. In silico analysis indicated that RAX is a potential target of miR-29b. The cellular localization of miR-29b and RAX was assessed by in situ hybridization and immunofluorescence, respectively. The expression levels of miR-29b and RAX mRNA were evaluated by quantitative reverse transcription PCR (qRT-PCR), and the expression of RAX protein was evaluated by western blot. A luciferase reporter assay and inhibition of endogenous RAX were performed to confirm whether RAX is a direct target of miR-29b as predicted by the in silico analysis. Results: We found that miR-29b and RAX are localized in the retinal ganglion cells (RGCs) and the cells of the inner nuclear layer (INL) of the retinas from normal and diabetic rats. Thus, the expression of miR-29b and RAX, as assessed in the retina by quantitative RT-PCR, reflects their expression in the RGCs and the cells of the INL. We also revealed that RAX protein is upregulated (more than twofold) at 3, 6, 16, and 22 days and downregulated (70%) at 35 days, whereas miR-29b is upregulated (more than threefold) at 28 and 35 days after STZ injection. We did not confirm the computational prediction that RAX is a direct target of miR-29b. Conclusions: Our results suggest that RAX expression may be indirectly regulated by miR-29b, and the upregulation of this miRNA at the early stage of STZ-induced diabetes may have a protective effect against the apoptosis of RGCs and cells of the INL by the pro-apoptotic RNA-dependent protein kinase (PKR) signaling pathway.

Integration and visualization of systems biology data in context of the genome

Background: High-density tiling arrays and new sequencing technologies are generating rapidly increasing volumes of transcriptome and protein-DNA interaction data. Visualization and exploration of this data is critical to understanding the regulatory logic encoded in the genome by which the cell dynamically affects its physiology and interacts with its environment. Results: The Gaggle Genome Browser is a cross-platform desktop program for interactively visualizing high-throughput data in the context of the genome. Important features include dynamic panning and zooming, keyword search and open interoperability through the Gaggle framework. Users may bookmark locations on the genome with descriptive annotations and share these bookmarks with other users. The program handles large sets of user-generated data using an in-process database and leverages the facilities of SQL and the R environment for importing and manipulating data. A key aspect of the Gaggle Genome Browser is interoperability. By connecting to the Gaggle framework, the genome browser joins a suite of interconnected bioinformatics tools for analysis and visualization with connectivity to major public repositories of sequences, interactions and pathways. To this flexible environment for exploring and combining data, the Gaggle Genome Browser adds the ability to visualize diverse types of data in relation to its coordinates on the genome. Conclusions: Genomic coordinates function as a common key by which disparate biological data types can be related to one another. In the Gaggle Genome Browser, heterogeneous data are joined by their location on the genome to create information-rich visualizations yielding insight into genome organization, transcription and its regulation and, ultimately, a better understanding of the mechanisms that enable the cell to dynamically respond to its environment.