Search results for: recombinant protein

Authors: Vernie C. Convicto, Henry P. Aringa, Wilson I. Barredo

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This study presents a conformational model of the helical structures of globular protein particularly ferritin in the framework of white noise path integral formulation by using Associated Legendre functions, Bessel and convolution of Bessel and trigonometric functions as modulating functions. The model incorporates chirality features of proteins and their helix-turn-helix sequence structural motif.

Keywords: globular protein, modulating function, white noise, winding probability

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Authors: B. Coşgun, O. Ozturk

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Canola is a specific edible type of rapeseed, developed in the 1970s, which contains about 40 percent oil. This research was carried out to determine the yield and some quality characteristics of some winter canola cultivars during the 2010-2011 vegetation period in Central Anatolia of Turkey. In this research; Oase, Dante, Californium, Excalibur, Elvis, ES Hydromel, Licord, Orkan, Vectra, Nelson, Champlain and NK Petrol winter canola varieties were used as material. The field experiment was set up in a “Randomized Complete Block Design” with three replications on 21 September 2010. In this research; seed yield, oil content, protein content, oil yield and protein yield were examined. As a result of this research; seed yield, oil content, oil yield and protein yield (except protein content) were significant differences between the cultivars. The highest seed yield (6348 kg ha-1) was obtained from the NK Petrol, while the lowest seed yield (3949 kg ha-1) was determined from the Champlain cultivar was obtained. The highest oil content (46.73%) was observed from Oase and the lowest value was obtained from Vectra (41.87%) cultivar. The highest oil yield (2950 kg ha-1) was determined from NK Petrol while the least value (1681 kg ha-1) was determined from Champlain cultivar. The highest protein yield (1539.3 kg ha-1) was obtained from NK Petrol and the lowest protein yield (976.5 kg ha-1) was obtained from Champlain cultivar. The main purpose of the cultivation of oil crops, to increase the yield of oil per unit area. According the result of this research, NK Petrol cultivar which ranks first with regard to both seed yield and oil yield between cultivars as the most suitable winter canola cultivar of local conditions.

Keywords: rapeseed, cultivar, seed yield, crude oil ratio, crude protein ratio, crude oil yield, crude protein yield

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Authors: Ali Taheri Mirghaed, Sara Ahani, Ashkan Zargar, Seyyed Morteza Hoseini

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Diseases are the major challenges in intensive aquaculture that cause significant annual losses. Antibiotic-therapy is a common way to control bacterial disease in fish, and oxytetracycline (OTC) is the only oral antibiotic in aquaculture approved FDA. OTC has been found to have negative effects on fish, such as oxidative stress and immune-suppression, thus, it is necessary to mitigate such effects. Medicinal herbs have various benefits on fish, including antioxidant, immunostimulant, and anti-microbial effects. Therefore, we hypothesized if dietary ginger meal (GM) interacts with dietary OTC by monitoring plasma protein fractions in rainbow trout. The study was conducted as a 2 × 2 factorial design, including diets containing 0 and 1% GM and 0 and 1.66 % OTC (corresponding to 100 mg/kg fish biomass per day). After ten days treating the fish (60 g individual weight) with these feeds, blood samples were taken from al treatments (n =3). Plasma was separated by centrifugation, and protein fractions were determined by electrophoresis. The results showed that OTC and GM had interaction effects on total protein (P<0.001), albumin (P<0.001), alpha-1 fraction (P=0.010), alpha-2 fraction (P=0.001), beta-2 fraction (P=0.014), and gamma fraction (P<0.001). Beta-1 fraction was significantly (P=0.030) affected by dietary GM. GM decreased plasma total protein, albumin, and beta-2 but increased beta-1 fraction. OTC significantly decreased total protein (P<0.001), albumin (P=0.001), alpha-2 fraction (P<0.001), beta-2 fraction (P=0.004), and gamma fraction (P<0.001) but had no significant effects on alpha-1 and beta-1 fractions. Dietary GM inhibited/suppressed the effects of dietary OTC on the plasma total protein and protein fractions. In conclusion, adding 1% GM to diet can mitigate the negative effects of dietary OTC on plasma proteins. Thus, GM may boost health of rainbow trout during the period of medication with OTC.

Keywords: ginger, plasma protein electrophoresis, dietary additive, rainbow trout

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Authors: Jai Myung Yang, Na Young Kim, Sung Ho Shin

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The adverse reactions of current live smallpox vaccines and potential use of smallpox as a bioterror weapon have heightened the development of new effective vaccine for this infectious disease. In the present study, DNA vaccine vector was produced which was optimized for expression of the vaccinia virus L1 antigen in the mouse model. A plasmid IgM-tL1R, which contains codon-optimized L1R gene, was constructed and fused with an IgM signal sequence under the regulation of a SV40 enhancer. The expression and secretion of recombinant L1 protein was confirmed in vitro 293 T cell. Mice were administered the DNA vaccine by electroporation and challenged with vaccinia virus. We observed that immunization with IgM-tL1R induced potent neutralizing antibody responses and provided complete protection against lethal vaccinia virus challenge. Isotyping studies reveal that immunoglobulin G2 (IgG2) antibody predominated after the immunization, indicative of a T helper type 1 response. Our results suggest that an optimized DNA vaccine, IgM-tL1R, can be effective in stimulating anti-vaccinia virus immune response and provide protection against lethal orthopoxvirus challenge.

Keywords: DNA vaccine, electroporation, L1R, vaccinia virus

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Authors: Khaled Khaladi, Rafika Bibi, Hind Mokrane, Boubekeur Nadjemi

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Sorghum (Sorghum bicolor (L.) Moench) is an important cereal crop grown in the semi-arid tropics of Africa and Asia due to its drought tolerance. Sorghum grain has protein content varying from 6 to 18%, with an average of 11%, Sorghum proteins can be broadly classified into prolamin and non-prolamin proteins. Kafirins, the major storage proteins, are classified as prolamins, and as such, they contain high levels of proline and glutamine and are soluble in non-polar solvents such as aqueous alcohols. Kafirins account for 77 to 82% of the protein in the endosperm, whereas non-prolamin proteins (namely, albumins, globulins, and glutelins) make up about 30% of the proteins. To optimize the extraction of sorghum proteins, several variables were examined: detergent type and concentration, reducing agent type and concentration, and buffer pH and concentration. Samples were quantified and characterized by RP-HPLC.

Keywords: sorghum, protein extraction, detergent, food science

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Authors: Fadilah Aleanizy

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Colicins are a family of bacterial toxins that kill Escherichia coli and other closely related species. The mode of action of colicins involves binding to an outer membrane receptor and translocation across the cell envelope, leading to cytotoxicity through specific targets. The mechanism of colicin cytotoxicity includes a non-specific endonuclease activity or depolarization of the cytoplasmic membrane by pore-forming activity. For Group A colicins, translocation requires an interaction between the N-terminal domain of the colicin and a series of membrane- bound and periplasmic proteins known as the Tol system (TolB, TolR, TolA, TolQ, and Pal and the active domain must be translocated through the outer membranes. Protein-protein interactions are intrinsic to virtually every cellular process. The transient protein-protein interactions of the colicin include the interaction with much more complicated assemblies during colicin translocation across the cellular membrane to its target. The potassium release assay detects variation in the K+ content of bacterial cells (K+in). This assays is used to measure the effect of pore-forming colicins such as ColA on an indicator organism by measuring the changes of the K+ concentration in the external medium (K+out ) that are caused by cell killing with a K+ selective electrode. One of the goals of this work is to employ a quantifiable in-vivo method to spot which Tol protein are more implicated in the interaction with colicin A as it is translocated to its target.

Keywords: K+ efflux, Colicin A, Tol-proteins, E. coli

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Authors: Elaheh Jooybar, Mohammad J. Abdekhodaie, Marcel Karperien, Pieter J. Dijkstra

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In this study, hyaluronic acid (HA) microgels were developed for the goal of protein delivery. First, a hyaluronic acid-tyramine conjugate (HA-TA) was synthesized with a degree of substitution of 13 TA moieties per 100 disaccharide units. Then, HA-TA microdroplets were produced using a water in oil emulsion method and crosslinked in the presence of horseradish peroxidase (HRP) and hydrogen peroxide (H₂O₂). Loading capacity and the release kinetics of lysozyme and BSA, as model proteins, were investigated. It was shown that lysozyme, a cationic protein, can be incorporated efficiently in the HA microgels, while the loading efficiency for BSA, as a negatively charged protein, is low. The release profile of lysozyme showed a sustained release over a period of one month. The results demonstrated that the HA-TA microgels are a good carrier for spatial delivery of cationic proteins for biomedical applications.

Keywords: microgel, inverse emulsion, protein delivery, hyaluronic acid, crosslinking

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Authors: Nichole Haag, Kimberly Velk, Tyler McCune, Chun Wu

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Methicillin/multiple-resistant Staphylococcus aureus (MRSA) are infectious bacteria that are resistant to common antibiotics. A previous in silico study in our group has identified a hypothetical protein SAV1226 as one of the potential drug targets. In this study, we reported the bioinformatics characterization, as well as cloning, expression, purification and kinetic assays of hypothetical protein SAV1226 from methicillin/vancomycin-resistant Staphylococcus aureus Mu50 strain. MALDI-TOF/MS analysis revealed a low degree of structural similarity with known proteins. Kinetic assays demonstrated that hypothetical protein SAV1226 is neither a domain of an ATP dependent dihydroxyacetone kinase nor of a phosphotransferase system (PTS) dihydroxyacetone kinase, suggesting that the function of hypothetical protein SAV1226 might be misannotated on public databases such as UniProt and InterProScan 5.

Keywords: Methicillin-resistant Staphylococcus aureus, dihydroxyacetone kinase, essential genes, drug target, phosphoryl group donor

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Authors: Zarmina Gillani, Nuzhat Huma, Aysha Sameen, Mulazim Hussain Bukhari

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Whey is an excellent food ingredient owing to its high nutritive value and its functional properties. However, composition of whey varies depending on composition of milk, processing conditions, processing method, and its whey protein content. The aim of this study was to prepare a whey powder from raw whey and to determine the influence of different processing temperatures (160 and 180 °C) on the physicochemical, functional properties during storage of 180 days and on whey protein denaturation. Results have shown that temperature significantly (P < 0.05) affects the pH, acidity, non-protein nitrogen (NPN), protein total soluble solids, fat and lactose contents. Significantly (p < 0.05) higher foaming capacity (FC), foam stability (FS), whey protein nitrogen index (WPNI), and a lower turbidity and solubility index (SI) were observed in whey powder processed at 160 °C compared to whey powder processed at 180 °C. During storage of 180 days, slow but progressive changes were noticed on the physicochemical and functional properties of whey powder. Reverse phase-HPLC analysis revealed a significant (P < 0.05) effect of temperature on whey protein contents. Denaturation of β-Lactoglobulin is followed by α-lacalbumin, casein glycomacropeptide (CMP/GMP), and bovine serum albumin (BSA).

Keywords: whey powder, temperature, denaturation, reverse phase, HPLC

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Authors: Indra Gonzalez Ojeda, Tatiana Quinones, Manuel Rosario, Igor Lednev, Juan Lopez Garriga

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Amyloid fibrils are stable aggregates of misfolded protein associated with many neurodegenerative disorders. It has been shown that hydrogen sulfide (H2S), inhibits the fibrillation of lysozyme through the formation of trisulfide (S-S-S) bonds. However, the overall mechanism remains elusive. Here, the concentration dependence of H2S effect was investigated using Atomic force microscopy (AFM), non-resonance Raman spectroscopy, Deep-UV Raman spectroscopy and circular dichroism (CD). It was found that small spherical aggregates with trisulfide bonds and a unique secondary structure were formed instead of amyloid fibrils when adding concentrations of 25 mM and 50 mM of H2S. This could indicate that H2S might serve as a protecting agent for the protein. However, further characterization of these aggregates and their trisulfide bonds is needed to fully unravel the function H2S has on protein fibrillation.

Keywords: amyloid fibrils, hydrogen sulfide, protein folding, raman spectroscopy

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Authors: Bin Liu

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As one of the most important tasks in protein sequence analysis, protein remote homology detection has been studied for decades. Currently, the profile-based methods show state-of-the-art performance. Position-Specific Frequency Matrix (PSFM) is widely used profile. However, there exists noise information in the profiles introduced by the amino acids with low frequencies. In this study, we propose a method to remove the noise information in the PSFM by removing the amino acids with low frequencies called Top frequency profile (TFP). Three new matrix transformation methods, including Autocross covariance (ACC) transformation, Tri-gram, and K-separated bigram (KSB), are performed on these profiles to convert them into fixed length feature vectors. Combined with Support Vector Machines (SVMs), the predictors are constructed. Evaluated on two benchmark datasets, and experimental results show that these proposed methods outperform other state-of-the-art predictors.

Keywords: protein remote homology detection, protein fold recognition, top frequency profile, support vector machines

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Authors: Suchao Donpudsa, Suwattana Visetnan, Anchalee Tassanakajon, Vichien Rimphanitchayakit

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The Single Wap Domain (SWD) is a type III crustin antimicrobial peptide whose function is to defense the host animal against the bacterial infection by means of antimicrobial and antiproteinase activities. A study of LvSWD from Litopenaeus vannamei is reported herein about its activities and function against bacteria, particularly the Vibrio parahaemolyticus AHPND (VPAHPND) that causes acute hepatopancreatic necrosis disease. The over-expressed mature recombinant (r)LvSWD exhibits antimicrobial activity against both Gram-positive and Gram-negative bacteria, especially VPAHPND. With four times the MIC of rLvSWD, the treated post larval shrimp infected by VPAHPND is able to survive longer with the 50% survival rate as long as 78 h as compared to 36 h of the infected shrimp without rLvSWD. To a certain extent, we have demonstrated that the rLvSWD can be applied to protect the post larval shrimp.

Keywords: crustin, Litopenaeus vannamei, Vibrio parahaemolyticus AHPND, antimicrobial activity

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Authors: Athar Hussain Memon

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Objectives: We tried to determine the frequency of raised C-reactive protein (CRP) in patients with type 2 diabetes mellitus. Patients and Methods: This cross-sectional descriptive study of six months study was conducted at Liaquat University Hospital Hyderabad from March 2013 to August 2013. All diabetic patients of ≥35 years age of either gender for >01 year duration visited at OPD were evaluated for C-reactive protein and their glycemic status by hemoglobin A1c. The data was analyzed in SPSS and the frequency and percentage were calculated. Results: During six month study period, total 100 diabetic patients were evaluated for C-reactive protein. The majority of patients were from urban areas 75/100 (75%). The mean ±SD for age of patients with diabetes mellitus was 51.63±7.82. The mean age ±SD of patient with raised CRP was 53±7.21. The mean ±SD for HbA1c in patients with raised CRP is 9.55±1.73. The mean random blood sugar level in patients with raised CRP was 247.42 ± 6.62. The majority of subjects were of 50-69 years of age group with female predominance (p=0.01) while the CRP was raised in 70 (70%) patients in relation to age (p=0.02) and gender (p=0.01), respectively. Both HbA1c and CRP were raised in 64.9% (p=0.04) in patients with type 2 diabetes mellitus. The mean ±SD of CRP was 5.8±1.21 while for male and female individuals with raised CRP was 3.52±1.22 and 5.7±1.63, respectively. Conclusions: The raised CRP was observed in patients with type 2 diabetes mellitus.

Keywords: diabetes mellitus, C-reactive protein, hemoglobin A1c, diabetes and metabolism

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Authors: Navneet Kaur, S. D. Tiwari

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Ferritin is a protein which present in the blood of mammals. It maintains the need of iron inside the body. It has an antiferromagnetic iron core, 7-8 nm in size, which is encapsulated inside a protein cage. The thickness of this protein shell is about 2-3 nm. This protein shell reduces the interaction among particles and make ferritin a model superparamagnet. The major composition of ferritin core is mineral ferrihydrite. The molecular formula of ferritin core is (FeOOH)8[FeOOPO3H2]. In this study, we discuss the phase transition of ferritin. We characterized ferritin using x-ray diffractometer, transmission electron micrograph, thermogravimetric analyzer and vibrating sample magnetometer. It is found that ferritin core is amorphous in nature with average particle size of 8 nm. The thermogravimetric and differential thermogravimetric analysis curves shows mass loss at different temperatures. We heated ferritin at these temperatures. It is found that ferritin core starts decomposing after 390^o C. At 1020^o C, the ferritin core is finally converted to alpha phase of iron oxide. Magnetization behavior of final sample clearly shows the iron oxyhydroxide core is completely converted to alpha iron oxide.

Keywords: Antiferromagnetic, Ferritin, Phase, Superparamagnetic

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Authors: Michal Štros, Eva Polanská, Šárka Pospíšilová

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HMGB1 is an architectural protein in chromatin, acting also as a signaling molecule outside the cell. Recent reports from several laboratories provided evidence that a number of both the intracellular and extracellular functions of HMGB1 may depend on redox-sensitive cysteine residues of the protein. MALDI-TOF analysis revealed that mild oxidization of HMGB1 resulted in a conformational change of the protein due to formation of an intramolecular disulphide bond by opposing Cys23 and Cys45 residues. We have demonstrated that redox state of HMGB1 could significantly modulate the ability of the protein to bind and bend DNA. We have also shown that reduced HMGB1 could easily displace histone H1 from DNA, while oxidized HMGB1 had limited capacity for H1 displacement. Using microscale thermophoresis (MST) we have further studied mechanism of HMGB1 interaction with histone H1 in free solution or when histone H1 was bound to DNA. Our MST analysis indicated that reduced HMGB1 exhibited in free solution > 1000 higher affinity of for H1 (KD ~ 4.5 nM) than oxidized HMGB1 (KD <10 M). Finally, we present a novel mechanism for the HMGB1-mediated modulation of histone H1 binding to DNA.

Keywords: HMGB1, histone H1, redox state, interaction, cross-linking, DNA bending, DNA end-joining, microscale thermophoresis

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Authors: Zoe M. G. Skalkos, Sam N. Dowland, James U. Van Dyke, Camilla. M. Whittington

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Syngnathid fishes (seahorses, pipefishes, and seadragons) are unique because embryos develop on or in the male in a specialised brooding structure. Many seahorse species are endangered or vulnerable, while others are popular in the ornamental fish trade. Seahorses are capable of nutrient provisioning (patrotrophy) of lipids during pregnancy via their fully enclosed brood pouch. Protein is vital for gene regulation and tissue growth during embryogenesis. We tested the hypothesis that protein is paternally transported to developing embryos during pregnancy in the Australian Pot-bellied seahorse, Hippocampus abdominalis. We compared the dry masses and nitrogen content in recently fertilised H. abdominalis embryos and newborns. We calculated an updated patrotrophy index, 1.34, but without a significant difference in dry mass between the two developmental stages. There was, however, a significant increase in total protein content from recently fertilised embryos to neonates. This suggests paternal protein transport is essential for H. abdominalis embryogenesis because protein yolk reserves are depleted by embryonic metabolism, and supplementation is required. This study is the first to provide evidence for paternal protein transport during pregnancy in seahorses. It furthers our understanding of the paternal influence on embryonic development in male pregnancy and how a protein-deficient diet during pregnancy may limit the allocation of resources to embryos, reducing offspring fitness. This research contributes to a deeper understanding of the fundamental reproductive biology of seahorses, which can help improve conservation and farming production outcomes.

Keywords: brood pouch, embryonic provisioning, nitrogen, parentotrophy, paternal investment, reproduction

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Authors: K. Cartelier, D. Aime, V. Vernoud, J. Buitink, J. M. Prosperi, K. Gallardo, C. Le Signor

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Legumes can produce protein-rich seeds without nitrogen fertilizer through root symbiosis with nitrogen-fixing rhizobia. Rich in lysine, these proteins are used for human nutrition and animal feed. However, the instability of seed protein yield and quality due to environmental fluctuations limits the wider use of legumes such as pea. Breeding efforts are needed to optimize and stabilize seed nutritional value, which requires to identify the genetic determinism of seed protein plasticity in response to the environment. Towards this goal, we have studied the plasticity of protein content and composition of seeds from a collection of 200 Medicago truncatula ecotypes grown under four controlled conditions (optimal, drought, and winter/spring sowing). A quantitative analysis of one-dimensional protein profiles of these mature seeds was performed and plasticity indices were calculated from each abundant protein band. Genome-Wide Association Studies (GWAS) from these data identified major GWAS hotspots, from which a list of candidate genes was obtained. A Gene Ontology Enrichment Analysis revealed an over-representation of genes involved in several amino acid metabolic pathways. This led us to propose that environmental variations are likely to modulate amino acid balance, thus impacting seed protein composition. The selection of candidate genes for controlling the plasticity of seed protein composition was refined using transcriptomics data from developing Medicago truncatula seeds. The pea orthologs of key genes were identified for functional studies by mean of TILLING (Targeting Induced Local Lesions in Genomes) lines in this crop. We will present how this study highlighted mechanisms that could govern seed protein plasticity, providing new cues towards the stabilization of legume seed quality.

Keywords: GWAS, Medicago truncatula, plasticity, seed, storage proteins

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Authors: Rohit Singh Dangi, Ravi Kant Pal, Monica Sundd

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Acyl-CoA binding protein (ACBP) is a housekeeping protein which functions as an intracellular carrier of acyl-CoA esters. Given the fact that the amastigote stage (blood stage) of Leishmania depends largely on fatty acids as the energy source, of which a large part is derived from its host, these proteins might have an important role in its survival. In Leishmania major, genome sequencing suggests the presence of six ACBPs, whose function remains largely unknown. For functional and structural characterization, one of the ACBP genes was cloned, and the protein was expressed and purified heterologously. Acyl-CoA ester binding and stoichiometry were analyzed by isothermal titration calorimetry and Dynamic light scattering. Our results shed light on high affinity of ACBP towards longer acyl-CoA esters, such as myristoyl-CoA to arachidonoyl-CoA with single binding site. To understand the binding mechanism & dynamics, Nuclear magnetic resonance assignments of this protein are being done. The protein's crystal structure was determined at 1.5Å resolution and revealed a classical topology for ACBP, containing four alpha-helical bundles. In the binding pocket, the loop between the first and the second helix (16 – 26AA) is four residues longer from other extensively studied ACBPs (PfACBP) and it curls upwards towards the pantothenate moiety of CoA to provide a large tunnel space for long acyl chain insertion.

Keywords: acyl-coa binding protein (ACBP), acyl-coa esters, crystal structure, isothermal titration, calorimetry, Leishmania

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Authors: Juan Huang, Nanqu Huang, Jingshan Shi, Yu Qiu

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Objectives: There are pathophysiological connections between type 2 diabetes mellitus (T2DM) and Alzheimer's disease (AD), and research on drugs with hypoglycemic and beta-amyloid (Aβ)-clearing effects have great therapeutic potential for AD. Dendrobium nobile Lindl. Alkaloids (DNLA) as one of the active compounds of Dendrobium nobile Lindl. In this study, we attempted to verify the hypoglycemic effect and investigate the effects of DNLA on the amyloid precursor protein (APP) metabolic pathway of the hippocampus in db/db mice. Methods: 4-weeks-old male C57BL/KsJ mice were the control group. And the same age and sexuality db/db mice were: model, DNLA-L (20 mg/kg), DNLA-M (40 mg/kg), and DNLA-H (80 mg/kg). After, mice were treated with different concentrations of DNLA for 17 weeks. The fasting blood glucose (FBG) was detected by glucose oxidase assay every week from the 4th to last week. The protein expression of β-amyloid 1-42 (Aβ1-42), β-site amyloid precursor protein-cleaving enzyme 1 (BACE1), and APP were examined by Western blotting. Results: The concentration of FBG and the protein expression of Aβ1-42, BACE1, and APP were increased in the hippocampus of the model group. Moreover, DNLA not only significantly decreased the concentration of FBG but also reduced the protein expressions of Aβ1-42, BACE1 and APP in the hippocampus of db/db mice in a dose-dependent manner. Conclusions: DNLA can decrease the protein expressions of Aβ1-42 in the hippocampus of db/db mice, and the mechanism may be involved in the APP metabolic pathway.

Keywords: Alzheimer's disease, type 2 diabetes mellitus, β-site amyloid precursor protein-cleaving enzyme 1, traditional Chinese medicines, beta-amyloid

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Authors: Li Yuchen, Wu Qingxin, Jin Yuxing, Yang Qian

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Interleukin-11 (IL-11), a well-known anti-inflammatory factor, helps to protect against intestinal epithelium damage caused by physical or chemical factors. However, little is known about the role of IL-11 during viral infection. Herein, high mRNA and protein levels of IL-11 were found in epithelial cells and jejunum of piglets during porcine epidemic diarrhea virus (PEDV) infection, and IL-11 expression was positively correlated with the level of viral infection. Pretreatment with recombinant porcine IL-11 (pIL-11) suppressed PEDV replication in Vero E6 cells, while IL-11 knockdown promoted viral infection. Furthermore, pIL-11 inhibited viral infection by preventing PEDV-mediated apoptosis of cells through activating the IL-11/STAT3 signal pathway. Conversely, application of a STAT3 phosphorylation inhibitor significantly antagonized the anti-apoptosis function of pIL-11 and counteracted its inhibition of PEDV. Our data suggested that that IL-11 is a novel PEDV-inducible cytokine, and its production enhances the anti-apoptosis ability of epithelial cells against PEDV infection. The potential uses of IL-11 as a novel therapeutic against devastating viral diarrhea in piglets deserves more attention and study.

Keywords: Interleukin-11, Porcine epidemic diarrhea virus, STAT3, anti-apoptosis

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Authors: Shahnaz Ashrafpour, Tahereh Tohidi Moghadam, Bijan Ranjbar

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Identifying the nature of protein-nanoparticle interactions and favored binding sites is an important issue in functional characterization of biomolecules and their physiological responses. Herein, interaction of silver nanoparticles with lysozyme as a model protein has been monitored via fluorescence spectroscopy. Formation of complex between the biomolecule and silver nanoparticles (AgNPs) induced a steady state reduction in the fluorescence intensity of protein at different concentrations of nanoparticles. Tryptophan fluorescence quenching spectra suggested that silver nanoparticles act as a foreign quencher, approaching the protein via this residue. Analysis of the Stern-Volmer plot showed quenching constant of 3.73 µM−1. Moreover, a single binding site in lysozyme is suggested to play role during interaction with AgNPs, having low affinity of binding compared to gold nanoparticles. Unfolding studies of lysozyme showed that complex of lysozyme-AgNPs has not undergone structural perturbations compared to the bare protein. Results of this effort will pave the way for utilization of sensitive spectroscopic techniques for rational design of nanobiomaterials in biomedical applications.

Keywords: nanocarrier, nanoparticles, surface plasmon resonance, quenching fluorescence

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Authors: Srishti Sinha, Deepti Tikariha, Kallol K. Ghosh

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Over the past few decades protein-surfactant interactions have been a subject of extensive studies as they are of great importance in wide variety of industries, biological, pharmaceutical and cosmetic systems. Protein-surfactant interactions have been explored the effect of surfactants on structure of protein in the form of solubilization and denaturing or renaturing of protein. Globular proteins are frequently used as functional ingredients in healthcare and pharmaceutical products, due to their ability to catalyze biochemical reactions, to be adsorbed on the surface of some substance and to bind other moieties and form molecular aggregates. One of the most widely used globular protein is bovine serum albumin (BSA), since it has a well-known primary structure and been associated with the binding of many different categories of molecules, such as dyes, drugs and toxic chemicals. Protein−surfactant interactions are usually dependent on the surfactant features. Most of the research has been focused on single-chain surfactants. More recently, the binding between proteins and dimeric surfactants has been discussed. In present study interactions of one dimeric surfactant Butanediyl-1,4-bis (dimethylhexadecylammonium bromide) (16-4-16, 2Br-) and the corresponding single-chain surfactant cetyl trimethylammonium bromide (CTAB) with bovine serum albumin (BSA) have been investigated by surface tension and spectrofluoremetric methods. It has been found that the bindings of all gemini surfactant to BSA were cooperatively driven by electrostatic and hydrophobic interactions. The gemini surfactant carrying more charges and hydrophobic tails, showed stronger interactions with BSA than the single-chain surfactant.

Keywords: bovine serum albumin, gemini surfactants, hydrophobic interactions, protein surfactant interaction

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Authors: K. Azimzadeh, S. Rasouli

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The present study investigates whether malondialdehyde (MDA) and heat shock protein-27 (HSP-27) are altered in sheep with cystic echinococcosis (CE). For this purpose, forty parasitized and thirty healthy sheep were selected based on severe cystic form observation in liver and lack of blood parasite along with no cystic conformation in carcass respectively. The results revealed a significant decrease (p<0.01) in albumin (Alb) and total plasma protein (TPP) and a significant increase (p<0.01) in HSP-27, MDA, total bilirubin and unconjugated bilirubin in the infected group compared with healthy ones.The results indicate low levels of TPP and Alb reveal liver damage in suffered sheep and MDA elevation demonstrates oxidative stress in infected group. In addition, HSP-27 enhancement may attribute to disease-induced stress conditions.

Keywords: malondialdehyde, heat shock protein-27, Echinococcosis, blood parasites

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Authors: A. N.Ukwuani, A. K. Abdulfatah

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G. crenata is used locally for the treatment of fractured bones, wound healing and inflammatory conditions. In vitro and in vivo antioxidant activity of hydromethanolic extracts of the leaves of G. crenata were assessed. The phytochemical analysis shows the presence of phenols, flavonoids, saponins, cardiac glycosides and tannins. An in vitro quantitative analysis of phenols, flavonoids and tannins respectively were (164±1.20, 199±0.88 and 88.67±0.88 mg/100g FW). In vivo studies of hydromethanolic extract demonstrated a dose dependent increase in hepatic superoxide dismutase (1.14±0.14, 2.13±0.11, 2.55±0.11 U/mg Protein) with improvement in hepatic glutathione (6.98±0.42, 8.91±0.37, 11.07±0.46 µM/mg Protein) and Catalase (4.47±0.05, 6.24±0.02, 7.17±0.04 U/mg Protein) and Total protein (6.18±0.08, 6.69±0.18, 7.27±0.16 mg/ml) respectively at 100-300mg/kg body weight Grewia crenata leaves when compared to the control and standard drug. It can be concluded from the present findings of that G. crenata leaves possess antioxidant potential.

Keywords: Grewia crenata, antioxidant, hydromethanolic extract, in vivo, in vitro

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Authors: Mei Yuin Joanne Wee, Rosli Md. Illias

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Cell surface display is the expression of a protein with an anchoring motif on the surface of the cell. This approach offers advantages when used in bioconversion in terms of easier purification steps and more efficient enzymatic reaction. A surface display system using ice nucleation protein (InaA) from Erwina ananas as an anchoring motif has been constructed to display xylanase (xyl) on the surface of Escherischia coli. The InaA was truncated so that it is made up of the N- and C-terminal domain (INPANC-xyl) and it has successfully directed xylanase to the surface of the cell. A study was also done on xylanase fused to two other ice nucleation proteins, InaK (INPKNC-xyl) and InaZ (INPZNC-xyl) from Pseudomonas syringae KCTC 1832 and Pseudomonas syringae S203 respectively. Surface localization of the fusion protein was verified using SDS-PAGE and Western blot on the cell fractions and all anchoring motifs were successfully displayed on the outer membrane of E. coli. Upon comparison, whole-cell activity of INPANC-xyl was more than six and five times higher than INPKNC-xyl and INPZNC-xyl respectively. Furthermore, the expression of INPANC-xyl on the surface of E. coli did not inhibit the growth of the cell. This is the first report of surface display system using ice nucleation protein, InaA from E. ananas. From this study, this anchoring motif offers an attractive alternative to the current surface display systems.

Keywords: cell surface display, Escherischia coli, ice nucleation protein, xylanase

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Authors: Sudarat Jiamyangyuen, Tipawan Thongsook, Riantong Singanusong, Chanida Saengtubtim

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Rice is a staple food as well as exported commodity of Thailand. Rice bran, a 10.5% constituent of rice grain, is a by-product of rice milling process. Rice bran is normally used as a raw material for rice bran oil production or sold as feed with a low price. Therefore, this study aimed to increase value of defatted rice bran as obtained after extracting of rice bran oil. Conventionally, the protein in defatted rice bran was extracted using alkaline extraction and acid precipitation, which results in reduction of nutritious components in rice bran. Rice bran protein concentrate is suitable for those who are allergenic of protein from other sources eg. milk, wheat. In addition to its hypoallergenic property, rice bran protein also contains good quantity of lysine. Thus it may act as a suitable ingredient for infant food formulations while adding variety to the restricted diets of children with food allergies. The objectives of this study were to compare properties of rice bran protein concentrate (RBPC) extracted from defatted rice bran using enzymes together with precipitation step using polysaccharides (alginate and carrageenan) to those of a control sample extracted using a conventional method. The results showed that extraction of protein from rice bran using enzymes exhibited the higher protein recovery compared to that extraction with alkaline. The extraction conditions using alcalase 2% (v/w) at 50 C, pH 9.5 gave the highest protein (2.44%) and yield (32.09%) in extracted solution compared to other enzymes. Rice bran protein concentrate powder prepared by a precipitation step using alginate (protein in solution: alginate 1:0.006) exhibited the highest protein (27.55%) and yield (6.62%). Precipitation using alginate was better than that of acid. RBPC extracted with alkaline (ALK) or enzyme alcalase (ALC), then precipitated with alginate (AL) (samples RBP-ALK-AL and RBP-ALC-AL) yielded the precipitation rate of 75% and 91.30%, respectively. Therefore, protein precipitation using alginate was then selected. Amino acid profile of control sample, and sample precipitated with alginate, as compared to casein and soy protein isolated, showed that control sample showed the highest content among all sample. Functional property study of RBP showed that the highest nitrogen solubility occurred in pH 8-10. There was no statically significant between emulsion capacity and emulsion stability of control and sample precipitated by alginate. However, control sample showed a higher of foaming and lower foam stability compared to those of sample precipitated with alginate. The finding was successful in terms of minimizing chemicals used in extraction and precipitation steps in preparation of rice bran protein concentrate. This research involves in a production of value-added product in which the double amount of protein (28%) compared to original amount (14%) contained in rice bran could be beneficial in terms of adding to food products eg. healthy drink with high protein and fiber. In addition, the basic knowledge of functional property of rice bran protein concentrate was obtained, which can be used to appropriately select the application of this value-added product from rice bran.

Keywords: alginate, carrageenan, rice bran, rice bran protein

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Authors: Yi Sun, George Ed Rainger, Steve P. Watson

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Background: Beyond the classical roles in haemostasis and thrombosis, platelets are important in the initiation and development of various thrombo-inflammatory diseases. In atherosclerosis and deep vein thrombosis, for example, platelets bridge monocytes with endothelium and form heterotypic aggregates with monocytes in the circulation. This can alter monocyte phenotype by inducing their activation, stimulating adhesion and migration. These interactions involve cell surface receptor-ligand pairs on both cells. This list is likely incomplete as new interactions of importance to platelet biology are continuing to be discovered as illustrated by our discovery of PEAR-1 binding to FcεR1α. Results: We have developed a highly sensitive avidity-based assay to identify novel extracellular interactions among 126 recombinantly-expressed platelet cell surface and secreted proteins involved in platelet aggregation. In this study, we will use this method to identify novel platelet-monocyte interactions. We aim to identify ligands for orphan receptors and novel partners of well-known proteins. Identified interactions will be studied in preliminary functional assays to demonstrate relevance to the inflammatory processes supporting atherogenesis. Conclusions: Platelet-monocyte interactions are essential for the development of thromboinflammatory disease. Up until relatively recently, technologies only allow us to limit our studies on each individual protein interaction at a single time. These studies propose for the first time to study the cell surface platelet-monocyte interactions in a systematic large-scale approach using a reliable screening method we have developed. If successful, this will likely to identify previously unknown ligands for important receptors that will be investigated in details and also provide a list of novel interactions for the field. This should stimulate studies on developing alternative therapeutic strategies to treat vascular inflammatory disorders such as atherosclerosis, DVT and sepsis and other clinically important inflammatory conditions.

Keywords: membrane proteins, large-scale screening, platelets, recombinant expression

Procedia PDF Downloads 117

Authors: Yagahira E. Castro-Sesquen, Chloe Kim, Robert H. Gilman, David J. Sullivan, Peter C. Searson

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Diagnosis of severe malaria is particularly important in highly endemic regions since most patients are positive for parasitemia and treatment differs from non-severe malaria. Diagnosis can be challenging due to the prevalence of diseases with similar symptoms. Accurate diagnosis is increasingly important to avoid overprescribing antimalarial drugs, minimize drug resistance, and minimize costs. A nanoparticle-based assay for detection and quantification of Plasmodium falciparum histidine-rich protein 2 (HRP2) in urine and serum is reported. The assay uses magnetic beads conjugated with anti-HRP2 antibody for protein capture and concentration, and antibody-conjugated quantum dots for optical detection. Western Blot analysis demonstrated that magnetic beads allows the concentration of HRP2 protein in urine by 20-fold. The concentration effect was achieved because large volume of urine can be incubated with beads, and magnetic separation can be easily performed in minutes to isolate beads containing HRP2 protein. Magnetic beads and Quantum Dots 525 conjugated to anti-HRP2 antibodies allows the detection of low concentration of HRP2 protein (0.5 ng mL-1), and quantification in the range of 33 to 2,000 ng mL-1 corresponding to the range associated with non-severe to severe malaria. This assay can be easily adapted to a non-invasive point-of-care test for classification of severe malaria.

Keywords: HRP2 protein, malaria, magnetic beads, Quantum dots

Procedia PDF Downloads 299

Authors: Hao Cheng, Yingzhou Ni, Amr M. Bakry, Li Liang

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Functional foods containing bioactive nutrients offer benefits beyond basic nutrition and hence the possibility of delaying and preventing chronic diseases. However, many bioactive nutrients degrade rapidly under food processing and storage conditions. Encapsulation can be used to overcome these limitations. Food proteins have been widely used as carrier materials for the preparation of nano/micro-particles because of their ability to form gels and emulsions and to interact with polysaccharides. The mechanisms of interaction between bioactive nutrients and proteins must be understood in order to develop protein-based lipid-free delivery systems. Beta-lactoglobulin, a small globular protein in milk whey, exhibits an affinity to a wide range of compounds. Alfa-tocopherol, resveratrol and folic acid were respectively bound to the central cavity, the outer surface near Trp19–Arg124 and the hydrophobic pocket in the groove between the alfa-helix and the beta-barrel of the protein. Beta-lactoglobulin could thus bind the three bioactive nutrients simultaneously to form protein-multi-ligand complexes. Beta-casein, an intrinsically unstructured but major milk protein, could also interact with resveratrol and folic acid to form complexes. These results suggest the potential to develop milk-protein-based complex carrier systems for encapsulation of multiple bioactive nutrients for functional food application and also pharmaceutical and medical uses.

Keywords: milk protein, bioactive nutrient, interaction, protection

Procedia PDF Downloads 383

Authors: Daniel Zarzo Montes, Manuel Zarzo Castelló

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Proteins play a fundamental role in biology, but their structure is complex, and it is a challenge for teachers to conceptually explain the differences between their primary, secondary, tertiary, and quaternary structures. On the other hand, there are currently many computer programs to visualize the 3D structure of proteins, but they require advanced training and knowledge. Moreover, it becomes difficult to visualize the sequence of amino acids in these models, and how the protein conformation is reached. Given this drawback, a simple and instructive procedure is proposed in order to teach the protein structure to undergraduate and graduate students. For this purpose, insulin has been chosen because it is a protein that consists of 51 amino acids, a relatively small number. The methodology has consisted of the use of plastic atom models, which are frequently used in organic chemistry and biochemistry to explain the chirality of biomolecules. For didactic purposes, when the aim is to teach the biochemical foundations of proteins, a manipulative system seems convenient, starting from the chemical structure of amino acids. It has the advantage that the bonds between amino acids can be conveniently rotated, following the pattern marked by the 3D models. First, the 51 amino acids were modeled, and then they were linked according to the sequence of this protein. Next, the three disulfide bonds that characterize the stability of insulin have been established, and then the alpha-helix structure has been formed. In order to reach the tertiary 3D conformation of this protein, different interactive models available on the Internet have been visualized. In conclusion, the proposed methodology seems very suitable for biology and biochemistry students because they can learn the fundamentals of protein modeling by means of a manipulative procedure as a basis for understanding the functionality of proteins. This methodology would be conveniently useful for a biology or biochemistry laboratory practice, either at the pre-graduate or university level.

Keywords: protein structure, 3D model, insulin, biomolecule

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