Search results for: potential protein

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

Procedia PDF Downloads 252

Authors: Mohamad Ammar Ayass, Natalya Griko, Victor Pashkov, Wanying Cao, Kevin Zhu, Jin Zhang, Lina Abi Mosleh

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Respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent for coronavirus disease 2019 (COVID-19). Early evidence pointed at the angiotensin-converting enzyme 2 (ACE-2) expressed on the epithelial cells of the lung as the main entry point of SARS-CoV-2 into the cells. The viral entry is mediated by the binding of the Receptor Binding Domain (RBD) of the spike protein that is expressed on the surface of the virus to the ACE-2 receptor. As the number of SARS-CoV-2 variants continues to increase, mutations arising in the RBD of SARS-CoV-2 may lead to the ineffectiveness of RBD targeted neutralizing antibodies. To address this limitation, the objective of this study is to develop a combination of aptamers that target different regions of the RBD, preventing the binding of the spike protein to ACE-2 receptor and subsequent viral entry and replication. A safe and innovative biomedical tool was developed to inhibit viral infection and reduce the harms of COVID-19. In the present study, DNA aptamers were developed against a recombinant trimer S protein using the Systematic Evolution of Ligands by Exponential enrichment (SELEX). Negative selection was introduced at round number 7 to select for aptamers that bind specifically to the RBD domain. A series of 9 aptamers (ADI2010, ADI2011, ADI201L, ADI203L, ADI205L, ADIR68, ADIR74, ADIR80, ADIR83) were selected and characterized with high binding affinity and specificity to the RBD of the spike protein. Aptamers (ADI25, ADI2009, ADI203L) were able to bind and pull down endogenous spike protein expressed on the surface of SARS-CoV-2 virus in COVID-19 positive patient samples and determined by liquid chromatography- tandem mass spectrometry analysis (LC-MS/MS). LC-MS/MS data confirmed that aptamers can bind to the RBD of the spike protein. Furthermore, results indicated that the combination of the 9 best aptamers inhibited the binding of the purified trimer spike protein to the ACE-2 receptor found on the surface of Vero E6 cells. In the same experiment, the combined aptamers displayed a better neutralizing effect than antibodies. The data suggests that the selected aptamers could be used in therapy to neutralize the effect of the SARS-CoV-2 virus by inhibiting the interaction between the RBD and ACE-2 receptor, preventing viral entry into target cells and therefore blocking viral replication.

Keywords: aptamer, ACE-2 receptor, binding inhibitor, COVID-19, spike protein, SARS-CoV-2, treatment

Procedia PDF Downloads 171

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: 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

Procedia PDF Downloads 295

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: Deepika Saini, Sandeep Jain, Ajay Kumar

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The quinoline scaffold is one of the most widely studied in the discovery of derivatives with various heterocyclic moieties due to its potential antimalarial activities. In the present study, a chalcone series of quinoline derivatives clubbed with pyrazole were synthesized to evaluate their antimalarial property by in vitro schizont maturation inhibition assay against both chloroquine sensitive, 3D7 and chloroquine resistant, RKL9 strain of Plasmodium falciparum. Further, top five compounds were studied for in vivo preclinical study for antimalarial potential against P. berghei in Swiss albino mice. To understand the mechanism of synthesized analogues, they were screened computationally by molecular docking techniques. Compounds were docked into the active site of a protein receptor, Plasmodium falciparum Cysteine Protease Falcipain-2. The compounds were successfully synthesized, and structural confirmation was performed by FTIR, 1H-NMR, mass spectrometry and elemental analysis. In vitro study suggested that the compounds 5b, 5g, 5l, 5s and 5u possessed best antimalarial activity and further tested for in vivo screening. Compound 5u (CH₃ on both rings) with EC₅₀ 0.313 & 0.801 µg/ml against CQ-S & CQ-R strains of P. falciparum respectively and 78.01% suppression of parasitemia. The molecular docking studies of the compounds helped in understanding the mechanism of action against falcipain-2. The present study reveals the binding signatures of the synthesized ligands within the active site of the protein, and it explains the results from in vitro study in their EC₅₀ values and percentage parasitemia.

Keywords: antimalarial activity, chalcone, docking, quinoline

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Authors: Xiaolin Li, Terence Turney, John Forsythe, Bryce Feltis, Paul Wright, Vinh Truong, Will Gates

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Clay-hydrogel nanocomposites have attracted great attention recently, mainly because of their enhanced mechanical properties and ease of fabrication. Moreover, the unique platelet structure of clay nanoparticles enables the incorporation of bioactive molecules, such as proteins or drugs, through ion exchange, adsorption or intercalation. This study seeks to improve the mechanical and rheological properties of a novel hydrogel system, copolymerized from a tetrapodal polyethylene glycol (PEG) thiol and a linear, triblock PEG-PPG-PEG (PPG: polypropylene glycol) α,ω-bispropynoate polymer, with the simultaneous incorporation of various amounts of Na-saturated, montmorillonite clay (MMT) platelets (av. lateral dimension = 200 nm), to form a bioactive three-dimensional network. Although the parent hydrogel has controlled swelling ability and its PEG groups have good affinity for the clay platelets, it suffers from poor mechanical stability and is currently unsuitable for potential applications. Nanocomposite hydrogels containing 4wt% MMT showed a twelve-fold enhancement in compressive strength, reaching 0.75MPa, and also a three-fold acceleration in gelation time, when compared with the parent hydrogel. Interestingly, clay nanoplatelet incorporation into the hydrogel slowed down the rate of its dehydration in air. Preliminary results showed that protein binding by the MMT varied with the nature of the protein, as horseradish peroxidase (HRP) was more strongly bound than bovine serum albumin. The HRP was no longer active when bound, presumably as a result of extensive structural refolding. Further work is being undertaken to assess protein binding behaviour within the nanocomposite hydrogel for potential diabetic wound healing applications.

Keywords: hydrogel, nanocomposite, small molecule, wound healing

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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: 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: Jae Young Song, In-Ohk Ouh, Seyeon Park, Byeong Sul Kang, Soo Dong Cho, In-Soo Cho

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Canine parvovirus (CPV) is a major pathogen of diarrhea disease in dogs. CPV type 2 has three of antigenic variants such as 2a, 2b, and 2c. CPV constructs a small non-enveloped, icosahedral capsid that contains single-stranded DNA. It has capsids that two largely overlapping virion proteins (VP), VP1 (82 kDa), and VP2 (65 kDa). Baculoviruses are insect pathogens that regulate insect populations in nature and are being successfully used to control insect pests. The proteins produced in the baculovirus-expression system are used for instance for functional studies, vaccine preparations, or diagnostics. The vaccines produced by baculovirus-expression system showed elicitation of antibodies. The recombinant baculovirus infected SF9 cells showed broken shape. The recombinant VP2 proteins from cell pellet or supernatant were confirmed by western blotting. The result showed that the recombinant VP2 protein bands were appeared at 65 kDa molecular weight in both cell pellet and supernatant of infected SF9 cell. These results indicated that the recombinant baculovirus infected SF9 cell express the recombinant VP2 protein successfully. In addition, the expressed recombinant VP2 protein is secreted from cell to supernatant. The baculovirus expression system can be used to produce the VP2 protein of CPV 2c. In addition, the secretion property of the expression of VP2 protein may decrease the cost of production, because it can be skipped the cell breaking step. The produced VP2 protein could be used for vaccine and the agent of diagnostic tests. This study provides the foundation of the production of CPV 2c vaccine and the diagnostic agent.

Keywords: baculovirus, canine parvovirus 2c, dog, Korea

Procedia PDF Downloads 123

Authors: Mohammed Alasel, Michael Keusgen

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Gold is a noble metal; in its nano-scale level (e.g. spherical nanoparticles), the conduction electrons are triggered to collectively oscillate with a resonant frequency when certain wavelengths of electromagnetic radiation interact with its surface; this phenomenon is known as surface plasmon resonance (SPR). SPR is responsible for giving the gold nanoparticles its intense red color depending mainly on its size, shape and distance between nanoparticles. A decreased distance between gold nanoparticles results in aggregation of them causing a change in color from red to blue. This aggregation enables gold nanoparticles to serve as a sensitive biosensoric indicator. In the proposed work, gold nanoparticles were modified with two proteins: i) Borrelia antigen, variable lipoprotein surface-exposed protein (VlsE), and ii) protein A. VlsE antigen induces a strong antibody response against Lyme disease and can be detected from early to late phase during the disease in humans infected with Borrelia. In addition, it shows low cross-reaction with the other non-pathogenic Borrelia strains. The high specificity of VlsE antigen to anti-Borrelia antibodies, combined simultaneously with the high specificity of protein A to the Fc region of all IgG human antibodies, was utilized to develop a rapid test for serological point of care diagnosis of borreliosis in human serum. Only in the presence of anti-Borrelia antibodies in the serum probe, an aggregation of gold nanoparticles can be observed, which is visible by a concentration-dependent colour shift from red (low IgG) to blue (high IgG). Experiments showed it is clearly possible to distinguish between positive and negative sera samples using a simple suspension of the two-protein modified gold nanoparticles in a very short time (30 minutes). The proposed work showed the potential of using such modified gold nanoparticles generally for serological diagnosis. Improved specificity and reduced assay time can be archived in applying increased salt concentrations combined with decreased pH values (pH 5).

Keywords: gold nanoparticles, gold aggregation, serological diagnosis, protein A, lyme borreliosis

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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: Rahul Shrivastava, Manish Tripathi, Mohmmad Yasir, Shailesh Singh

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Life style changes and depletion in atmospheric ozone layer in recent decades lead to increase in skin cancer including both melanoma and nonmelanomas. Natural products which were obtained from different plant species have the potential of anti skin cancer activity. In regard of this, present study focuses the potential effect of Glycosmis pentaphylla against anti skin cancer activity. Different Phytochemical constituents which were present in the roots of Glycosmis pentaphylla were identified and were used as ligands after sketching of their structures with the help of ACD/Chemsketch. These ligands are screened for their anticancer potential with proteins which are involved in skin cancer effects with the help of pyrx software. After performing docking studies, results reveal that Noracronycine secondary metabolite of Glycosmis pentaphylla shows strong affinity of their binding energy with Ribosomal S6 Kinase 2 (2QR8) protein. Ribosomal S6 Kinase 2 (2QR8) has an important role in the cell proliferation and transformation mediated through by N-terminal kinase domain and was induced by the tumour promoters such as epidermal growth factor. It also plays a key role in the neoplastic transformation of human skin cells and in skin cancer growth. Noracronycine interact with THR-493 and MET-496 residue of Ribosomal S6 Kinase 2 protein with binding energy ΔG = -8.68 kcal/mole. Thus on the basis of this study we can say that Noracronycine which present in roots of Glycosmis pentaphylla can be used as lead compound against skin cancer.

Keywords: glycosmis pentaphylla, pyrx, ribosomal s6 kinase, skin cancer

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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: 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: Chaoran Liu

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Bioactive polysaccharides and polysaccharide-protein complex derived from mushrooms and fungi have a wide range of immunomodulatory activity with low side-effects and have therefore the potential to be developed as an adjuvant in cancer therapies. Mushrooms sclerotium is rich in polysaccharides and the polysaccharides isolated from the sclerotium of Polyporus rhinocerus have shown potent in vivo and in vitro immunomodulatory effects. Macrophages are considered to be an important component of the innate immune response against bacterial infection and cancer. To better understanding the immunomodulatory effects and its underlying mechanisms of sclerotial water-soluble polysaccharides extracted from P. rhinocerus on macrophages, the objectives of this study are to purify the water-soluble novel sclerotial polysaccharides and to characterize the structure and properties as well as to study the detailed molecular mechanisms of the in vitro immunomodulating effects in murine macrophages. The hot water-soluble fraction PRW from the sclerotium of P. rhinocerus was obtained using solvent extraction. PRW was further fractionated by membrane ultrafiltration to a give a fraction (PRW1) with molecular mass less than 50 kDa. PRW1 was characterized to be a polysaccharide-protein complex composed of 45.7% polysaccharide and 44.2% protein. The chemical structure of the carbohydrate moiety of PRW1 was elucidated by GC and FTIR to be mainly beta-D-glucan with trace amount of galactose and mannose. The immunomodulatory effects of PRW1 on murine RAW 264.7 macrophages were demonstrated in terms of the increase in nitric oxide production and cytokine production. Mechanistically, PRW1 initiates ERK phosphorylation to activate macrophages within 15 min and significantly improves the expression level of inducible NOS (iNOS) from 6 h after treatment. In summary, this study indicates that PRW1 is a potent immunomodulatory agent for macrophages and suggests that mushroom sclerotia from Polyporus rhinocerus requires for further investigation in cancer research.

Keywords: Polyporus rhinocerus, mushroom sclerotia, Polysaccharide-Protein Complex, macrophage activation

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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: Xian-Peng Jiang, Catherine C. Baucom, Toby Jiang, Robert L. Elliott

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HLA-G binds to the inhibitory receptors of uterine NK cells and plays an important role in protection of fetal cells from maternal NK lysis. HLA-G also mediates tumor escape, but the immunosuppressive role is often neglected. These studies have focused on the examination of HLA-G expression in human breast carcinoma and HLA-G immunosuppressive role in NK cytolysis. We examined HLA-G expression in breast cell lines by real time PCR, ELISA and immunofluorescent staining. We treated the breast cancer cell lines with anti-human HLA-G antibody or progesterone. Then, NK cytolysis was measured by using MTT assay. We find that breast carcinoma cell lines increase the expression of HLA-G mRNA and protein, compared to normal cells. Blocking HLA-G of the breast cancer cells by the antibody increases NK cytolysis. Progesterone upregulates HLA-G mRNA and protein of human breast cancer cell lines. The increased HLA-G expression suppresses NK cytolysis. In summary, human breast carcinoma overexpress HLA-G immunosuppressive molecules. Blocking HLA-G protein by antibody improves NK cytolysis. In contrast, upregulation of HLA-G expression by progesterone impairs NK cytolytic function. Thus, HLA-G is a new immunosuppressive checkpoint and potential cancer immunotherapeutic target.

Keywords: HLA-G, Breast carcinoma, NK cells, Immunosuppressive checkpoint

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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

Procedia PDF Downloads 589

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

Procedia PDF Downloads 367

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

Procedia PDF Downloads 267

Authors: Hung-Chieh Chen, Kuo-Hui Wang, Tsu-Lin Yeh

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Proteomics represent the performance of genomic complement proteins and the protein level on functional genomics. This study adopted proteomic strategies for comparing serum proteins among three groups: elite sprinter (sprint runner group, SR), long-distance runners (long-distance runner group, LDR), and the untrained control group (control group, CON). Purposes: This study aims to identify elite sprinters and long-distance runners’ serum protein and to provide a comparison of their serum proteome’ composition. Methods: Serum protein fractionations that separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by a quantitative nano-LC-MS/MS-based proteomic profiling. The one-way analysis of variance (ANOVA) and Scheffe post hoc comparison (α= 0.05) was used to determine whether there is any significant difference in each protein level among the three groups. Results: (1) After analyzing the 307 identified proteins, there were 26 unique proteins in the SR group, and 18 unique proteins in the LDR group. (2) For the LDR group, 7 coagulation function-associated proteins’ expression levels were investigated: vitronectin, serum paraoxonase/arylesterase 1, fibulin-1, complement C3, vitamin K-dependent protein, inter-alpha-trypsin inhibitor heavy chain H3 and von Willebrand factor, and the findings show the seven coagulation function-associated proteins were significantly lower than the group of SR. (3) Comparing to the group of SR, this study found that the LDR group’s expression levels of the 2 antioxidant proteins (afamin and glutathione peroxidase 3) were also significantly lower. (4) The LDR group’s expression levels of seven immune function-related proteins (Ig gamma-3 chain C region, Ig lambda-like polypeptide 5, clusterin, complement C1s subcomponent, complement factor B, complement C4-A, complement C1q subcomponent subunit A) were also significantly lower than the group of SR. Conclusion: This study identified the potential serum protein markers for elite sprinters and long-distance runners. The changes in the regulation of coagulation, antioxidant, or immune function-specific proteins may also provide further clinical applications for these two different track athletes.

Keywords: biomarkers, coagulation, immune response, oxidative stress

Procedia PDF Downloads 101

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 308

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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Authors: Vita Strazdina, Aleksandrs Jemeljanovs, Vita Sterna

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Not all proteins have the same nutritional value, since protein quality strongly depends on its amino acid composition and digestibility. The meat of game animals could be a high protein source because of its well-balanced essential amino acids composition. Investigations about biochemical composition of game meat such as wild boar (Sus scrofa scrofa), roe deer (Capreolus capreolus) and beaver (Castor fiber) are not very much. Therefore, the aim of the investigation was evaluate protein composition of game meat hunted in Latvia. The biochemical analysis, evaluation of connective tissue and essential amino acids in meat samples were done, the amino acids score were calculate. Results of analysis showed that protein content 20.88-22.05% of all types of meat samples is not different statistically. The content of connective tissue from 1.3% in roe deer till 1.5% in beaver meat allowed classified game animal as high quality meat. The sum of essential amino acids in game meat samples were determined 7.05–8.26g100g-1. Roe deer meat has highest protein content and lowest content of connective tissues among game meat hunted in Latvia. Concluded that amino acid score for limiting amino acids phenylalanine and tyrosine is high and shows high biological value of game meat.

Keywords: dietic product, game meat, amino acids, scores

Procedia PDF Downloads 298