Complex Mix Of Proteins Research Articles

Apolipoprotein E enhance survival of effector memory regulatory T lymphocytes by regulating caspase-dependent apoptosis and lipid metabolism.

Abstract Mechanisms underlying the pleiotropic anti-inflammatory effect of HDL remain incompletely understood. Regulatory T cells (Tregs) are essential for maintaining immune homeostasis in cardiovascular diseases (CVD) and metabolic disorders. We recently show Tregs, particularly effector memory Tregs (emTregs), bind/internalize HDL, which promotes their survival by limiting caspase-dependent apoptosis. As HDL contain a complex mix of proteins and lipids, we investigated which components of HDL mediate this process and found HDL proteins were the most critical. Using reconstituted HDL mimic vesicles containing recombinant HDL proteins and POPC, we found that APOE, but none of the APOA proteins, significantly decreased caspase expression in emTregs similarly to total HDL. Neither APOE nor HDL had any effect on naïve Tregs. APOE-depleted HDL did not rescue emTregs, confirming the critical role of APOE. Because Tregs have distinct metabolic requirements, we next investigated the effect of HDL, APOE3-POPC and APOE-depleted HDL on lipid metabolism in emTregs. APOE3-POPC and HDL significantly increased the concentration of long chain (14:0 20:0) and polyunsaturated fatty acids (FA, 18:3 22:6) in emTregs. Our data also suggested the involvement of de novo FA synthesis, as APOE3-POPC and HDL increased the concentration of palmitate (16:0), the earlier precursor of this pathway. Further, treatment of Tregs with the fatty acid synthase inhibitor C75 abolished HDL survival effect. Our results show that HDL, specifically APOE, promote emTregs survival by regulating de novo FA synthesis and caspase-dependent apoptosis. HDL-APOE levels seem to be dysregulated in CVD and obesity, suggesting this mechanism may contribute to HDL protective role

Uruguayan wheat proteins: their relationship with traditional parameters and how are they affected by genotype and environment

Wheat (Triticum aestivum L.) is important in Uruguay both as food as well as crop. To achieve an attractive product for consumers and profitable for farmers, grain components should be able to produce high-quality bread. The most used device to characterize bread quality in wheat and flour is the Alveograph. The key component is gluten, a complex mix of proteins. In order to understand the relationship between gluten component proteins and alveographic parameters, as well as how they are affected by genotype and environment, sixteen genotypes were grown in two locations with differential nitrogen management. Traditional parameters were determined: protein content and alveogram. Gluten components were studied through size exclusion hplc. Rheological parameters presented a clear relationship with the hplc ones. It was possible to verify that to reach good extensibility, high protein content was needed; to obtain a strong dough, it was necessary to have a large number of high molecular weight proteins, and to get good resistance to extension, the polymeric proteins should have high molecular weight. Among the studied genotypes and environments, the first ones had a higher contribution to variability in extensibility and strength, while the latter explained most of the variability in resistance to extension. An equilibrium between genotype and environment is needed to achieve a dough with balanced characteristics.

Immunotherapy using algal-produced Ara h 1 core domain suppresses peanut allergy in mice.

SummaryPeanut allergy is an IgE‐mediated adverse reaction to a subset of proteins found in peanuts. Immunotherapy aims to desensitize allergic patients through repeated and escalating exposures for several months to years using extracts or flours. The complex mix of proteins and variability between preparations complicates immunotherapy studies. Moreover, peanut immunotherapy is associated with frequent negative side effects and patients are often at risk of allergic reactions once immunotherapy is discontinued. Allergen‐specific approaches using recombinant proteins are an attractive alternative because they allow more precise dosing and the opportunity to engineer proteins with improved safety profiles. We tested whether Ara h 1 and Ara h 2, two major peanut allergens, could be produced using chloroplast of the unicellular eukaryotic alga, Chlamydomonas reinhardtii. C. reinhardtii is novel host for producing allergens that is genetically tractable, inexpensive and easy to grow, and is able to produce more complex proteins than bacterial hosts. Compared to the native proteins, algal‐produced Ara h 1 core domain and Ara h 2 have a reduced affinity for IgE from peanut‐allergic patients. We further found that immunotherapy using algal‐produced Ara h 1 core domain confers protection from peanut‐induced anaphylaxis in a murine model of peanut allergy.

Treatment: special conditions: Co-existing heart disease: coronary artery disease, myocardial infarction, heart failure

Capillary isoelectric focusing (cIEF) coupled to mass spectrometry (cIEF-MS) offers a potentially very powerful analytical tool for the investigation of physiological samples. The high resolving capabilities of cIEF in combination with the high sensitivity and enhanced structural information provided by MS is highly desirable for the analysis of complex samples. However a major limitation of the technique has always been the requirement to desalt samples prior to cIEF analysis. Such desalting normally occurs off-line and therefore adds complexity and the possibility of sample loss or contamination. In this study we demonstrate the use of a modified cIEF protocol which enables samples containing physiological levels of salts to be desalted on-line, within the cIEF capillary. This new technique is very fast and efficient, allowing the direct analysis of a physiologically derived fluid that contains a complex mix of proteins, such as human cerebrospinal fluid by cIEF-MS in a single step experiment.

His Hormone, Her Oogenesis: How Male Malaria Mosquitoes Trigger Female Egg Development

A female mosquito needs two things to reproduce: your blood and a visit from a male mosquito. The blood provides the nutrient-rich meal she will use to feed her developing eggs. The male provides not only sperm, but also a complex mix of proteins and other molecules, secreted from the so-called male accessory glands (MAGs) and deposited along with the sperm during mating. The secretions form a gelatinous “mating plug,” which blocks access to the uterus. Anopheles oocytes after blood feeding. The oocyte, which is surrounded by epithelial cells (blue) and muscle cells (green), accumulates lipids (red) in the cytoplasm. This accumulation depends on the interaction between a female protein, MISO, and the ... Both the blood meal and copulation provide necessary signals for the female to begin development of eggs in her ovary. While much is known about the events triggered by feeding, relatively little is known about how mating stimulates oogenesis. A new study by Francesco Baldini, Flaminia Catteruccia, and colleagues shows that a hormone delivered by males stimulates production of a protein in females, and together the pair induces expression of multiple genes to increase egg development. Working with the malaria-carrying mosquito Anopheles gambiae, the authors had previously shown that a protein called mating-induced stimulator of oogenesis (MISO) was upregulated in the uterus, or atrium, shortly after mating. They began the current work by using RNA interference to block MISO expression in mated, blood-fed females. They found that oogenesis specifically was impaired, decreasing egg development in treated insects to the level found in virgin females. Those egg follicles that did develop did so slowly, suggesting that they were unable to accumulate lipids at the normal rate. This, the authors found, was due to decreased expression of the lipid transporter lipophorin, whose levels were again similar to those seen in virgin females, as a consequence of loss of MISO. Previous studies have suggested that MAG secretions control aspects of behavior and physiology in females after mating and that one key component of the secretions is the steroid hormone 20-hydroxy-ecdysone (20E), which has multiple effects in female mosquitoes. Control and MISO-deficient females had equivalent levels of 20E in the mating plug, but it dispersed from the plug much more slowly in the females with reduced MISO. This suggested that the two might interact, a hypothesis confirmed when the authors showed that 20E and MISO co-immunoprecipitated. Loss of circulating 20E led to reduced expression of several of its gene targets, including the yolk protein precursor vitellogeninand the ecdysone receptor (EcR), which acts as nuclear receptor for the hormone. In control mosquitoes, an increase in 20E led to an increase in production of MISO, an effect that could be blocked by loss of its receptor EcR. The picture that emerges is that male-supplied 20E binds to EcR to induce the female to produce MISO, which it then binds as well. This complex then promotes transcription of a variety of oogenic genes, leading to increased lipid accumulation in oocytes and promoting egg development. The elucidation of this pathway may have important consequences in the fight against malaria, which yearly kills over half a million people worldwide. Introduction of males deficient in 20E synthesis, for instance, could reduce reproduction in females that mate with them. Furthermore, the lipid accumulation that occurs during oogenesis reduces the ability of the female mosquitos to destroy the malaria parasites they carry; thus, blocking this pathway could reduce the deadliness of a bite from these troublesome insects. Baldini F, Gabrieli P, South A, Valim C, Mancini F, et al. (2013) The Interaction between a Sexually Transferred Steroid Hormone and a Female Protein Regulates Oogenesis in the Malaria Mosquito Anopheles gambiae. doi:10.1371/journal.pbio.1001695

Metabolomics reveals increased isoleukotoxin diol (12,13-DHOME) in human plasma after acute Intralipid infusion

Intralipid is a fat emulsion that is regularly infused into humans and animals. Despite its routine use, Intralipid infusion can cause serious adverse reactions, including immunosuppression. Intralipid is a complex mix of proteins, lipids, and other small molecules, and the effect of its infusion on the human plasma metabolome is unknown. We hypothesized that untargeted metabolomics of human plasma after an Intralipid infusion would reveal novel insights into its effects. We infused Intralipid and saline into 10 healthy men in a double-blind, placebo-controlled experiment and used GC/MS, LC/MS, and NMR to profile the small-molecule composition of their plasma before and after infusion. Multivariate statistical analysis of the 40 resulting plasma samples revealed that after Intralipid infusion, a less-well-characterized pathway of linoleic acid metabolism had resulted in the appearance of (9Z)-12,13-dihydroxyoctadec-9-enoic acid (12,13-DHOME, P < 10(-3)), a leukotoxin that has powerful physiological effects and is known to inhibit the neutrophil respiratory burst. Intralipid infusion caused increased plasma 12,13-DHOME. Given that 12,13-DHOME is known to directly affect neutrophil function, we conclude that untargeted metabolomics may have revealed a hitherto-unknown mechanism of intralipid-induced immunosuppression.

Proteomics

Proteomics

The Potential Use of Enamel Matrix Derivative for In Situ Anterior Cruciate Ligament Tissue Engineering: A Translational In Vitro Investigation

Polyester scaffolds have been used as an alternative to autogenous tissues for the reconstruction of the anterior cruciate ligament (ACL). They are biocompatible and encourage tissue infiltration, leading to neoligament formation. However, rupture can occur, caused by abrasion of the scaffold against the bone tunnels through which it is implanted. Good early tissue induction is therefore considered essential to protect the scaffold from this abrasion. Enamel matrix derivative (EMD) is used clinically in the treatment of periodontal disease. It is a complex mix of proteins with growth factor-like activity, which enhances periodontal ligament fibroblast attachment, proliferation, and differentiation, leading to the regeneration of periodontal bone and ligament tissues. We hypothesized that EMD might, in a similar manner, enhance tissue induction around scaffolds used in ACL reconstruction. This preliminary investigation adopted a translational approach, modelling in vitro 3 possible clinical modes of EMD administration, to ascertain the suitability of each protocol for application in an animal model or clinically. Preliminary investigations in monolayer culture indicated that EMD had a significant dose-dependent stimulatory effect (p < 0.05, n = 6) on the proliferation of bovine primary synovial cells. However, pre-treating culture plates with EMD significantly inhibited cell attachment (p < 0.01, n = 6). EMD's effects on synovial cells, seeded onto ligament scaffolds, were then investigated in several in vitro experiments modelling 3 possible modes for clinical EMD administration (pre-, intra-, and post-operative). In the pre-operative model, EMD was adsorbed onto scaffolds before the addition of cells. In the intra-operative model, EMD and cells were added simultaneously to scaffolds in the culture medium. In the post-operative model, cells were pre-seeded onto scaffolds before EMD was administered. EMD significantly inhibited cell adhesion in the pre-operative model (p < 0.05, n = 6) and had no significant benefit in the intra-operative model. In the post-operative model, the addition of EMD to previously cell-seeded scaffolds significantly increased their total deoxyribonucleic acid content (p < 0.01, n = 5). EMD's stimulative effect on cell proliferation in vitro suggests that it may accelerate scaffold colonization by cells (and in turn tissue induction) in situ. However, its inhibitory effect on synovial cell attachment in vitro implies that it may only be suited to post-operative administration.

Quantity versus quality: Optimal methods for cell-free DNA isolation from plasma of pregnant women

Purpose: Methods to isolate cell-free fetal DNA from maternal plasma are critical in developing noninvasive fetal DNA testing strategies. Given that plasma consists of heterogeneous DNA-size fragments in a complex mix of proteins, recovery and analysis of this DNA are understandably inefficient. To facilitate recovery, we performed qualitative and quantitative analysis of DNA isolated from maternal plasma.Methods: DNA isolated from maternal blood (n = 15) was compared using five different DNA isolation protocols: two conventional, two column-based, and one magnetic-bead based. Purity and concentration of DNA recovered were determined with a NanoDrop spectrophotometer. Real-time polymerase chain reaction quantification of the β-globin and DYS1 loci was performed to determine total and fetal-specific genome equivalents, respectively.Results: DNA quality and quantity were different among the five methods tested. Although purity and concentration of total DNA were greatest with the conventional boiling-lysis approach, correct detection of a male fetus was achieved in only 62.5% of cases. DNA isolation using the magnetic beads yielded the highest quantity of total DNA (2018.83±4.09 GEq/mL), with 100% fetal DNA detection.Conclusions: Optimal plasma DNA recovery protocols must take into account DNA purity and concentration. We confirm that the magnetic-beads method provides a fast, simple, sensitive, and specific approach to purify plasma DNA. The resulting high-quality DNA facilitates efficient examination of fetal DNA sequences.

On-line desalting of physiologically derived fluids in conjunction with capillary isoelectric focusing-mass spectrometry

Capillary isoelectric focusing (cIEF) coupled to mass spectrometry (cIEF-MS) offers a potentially very powerful analytical tool for the investigation of physiological samples. The high resolving capabilities of cIEF in combination with the high sensitivity and enhanced structural information provided by MS is highly desirable for the analysis of complex samples. However a major limitation of the technique has always been the requirement to desalt samples prior to cIEF analysis. Such desalting normally occurs off-line and therefore adds complexity and the possibility of sample loss or contamination. In this study we demonstrate the use of a modified cIEF protocol which enables samples containing physiological levels of salts to be desalted on-line, within the cIEF capillary. This new technique is very fast and efficient, allowing the direct analysis of a physiologically derived fluid that contains a complex mix of proteins, such as human cerebrospinal fluid by cIEF-MS in a single step experiment.