Search results for: polyelectrolyte – protein complex

Authors: Sirikwan Ponprateep, Anchalee Tassanakajon, Vichien Rimphanitchayakit

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A Kazal-type serine proteinase inhibitor, SPIPm2, was abundantly expressed in the hemocytes and secreted into shrimp plasma has anti-viral property against white spot syndrome virus (WSSV). To discover the molecular mechanism of antiviral activity, the binding assay showed that SPIPm2 bind to the components of viral particle and shrimp hemocyte. From our previous report, viral target protein of SPIPm2 was identified, namely WSV477 using yeast two-hybrid screening. WSV477 is an early gene product of WSSV and involved in viral propagation. In this study, the co-immunoprecipitation technique and Tandem Mass Spectrometry (LC-MS/MS) was used to identify the target protein of SPIPm2 from shrimp hemocyte. The target protein of SPIPm2 was cyclophilin A. In vertebrate, cyclophilin A or peptidylprolyl isomerase A was reported to be the immune suppressor interacted with cyclosporin A involved in immune defense response. The recombinant cyclophilin A from Penaeus monodon (rPmCypA) was produced in E.coli system and purified using Ni-NTA column to confirm the protein-protein interaction. In vitro pull-down assay showed the interaction between rSPIPm2 and rPmCypA. To study the biological function of these proteins, the expression analysis of immune gene in shrimp defense pathways will be investigated after rPmCypA administration.

Keywords: cyclophilin A, protein-protein interaction, Kazal-type serine proteinase inhibitor, Penaeus monodon

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Authors: Waode Nurmayani, Nikmatul Riswanda

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Broilers are chickens which are kept with the most efficient time and hoped get a good body weight. All things are done, for example with the improvement of feed and use antibiotics. Feed cost is the most cost to be spent. Nearly 80% of the cost is spent just for buy feed. Earthworm (Lumbricus rubellus) is a good choice to reduce the cost of feed protein source. The Earthworm has a high crude protein content of about 48.5%-61.9%, rich with proline amino acid about 15% of the 62 amino acids. Not only about protein, this earthworm also has a role in disease prevention. Prevention of disease in livestock usual with use feed supplement. Earthworm (Lumbricus rubellus) is one of the natural materials used as feed. In addition, several types of earthworms that have been known to contain active substances about antibacterial pathogens namely Lumbricus rubellus. The earthworm could be used as an antibiotic because it contain the antibody of Lumbricine active substance. So that, this animal feed from Lumbricus rubellus could improve the performance of broilers. Bioactive of anti-bacterial is called Lumbricine able to inhibit the growth of pathogenic bacteria in the intestinal wall so that the population of pathogenic bacteria is reduced. The method of write in this scientific writing is divided into 3 techniques, namely data completion, data analysis, and thinking pan from various literature about earthworm (Lumbricus rubellus) as broiler feed. It is expected that innovation of feed material of earthworm (Lumbricus rubellus) could reduce the cost of protein feed and the use of chemical antibiotics.

Keywords: earthworm, broiler, protein, antibiotic

Procedia PDF Downloads 150

Authors: N. Mahmoudnia, M. Khormali

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This experiment was conducted to determine the apparent protein digestibility of poultry byproduct meal (PBPM) from two industrial poultry slaughter-houses on Ross 308 male broiler chickens in independent comparisons. The experiment consisted of seven dietary treatments and three replicates per treatment with three broiler chickens per replicate in a completely randomized design. Dietary treatments consisted of a control corn- soybean diet, and levels 3, 6, and 9% PBPM produced by slaughter-house 1 and levels 3, 6, and 9% PBPM produced by slaughter house 2. Chromic oxide was added to the experimental diets as an indigestible marker. The apparent protein digestibility of each diet were determined with two methods of sample collection of ileum and excreta in 21-28 d of age. The results this experiment showed that use of PBPM had no significant effect on the performance of broiler chicks during period of experiments. The apparent protein digestibility of PBPM groups was significantly higher than control group by excreta sampling procedure (P<0.05). Using of PBPM 2 significantly (P<0.05) decreased the apparent protein digestibility values based on ileum sampling procedure vs control (85.21 vs. 90.14).Based results of this experiment,it is possible to use of PBPM 1 in broiler chicken.

Keywords: poultry by-product meal, apparent protein digestibility, independed comparison, broiler chicken

Procedia PDF Downloads 485

Authors: Anna Trusek-Holownia, Andrzej Noworyta

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Using an enzyme of known specificity the hydrolysis of protein was carried out in a controlled manner. The aim was to obtain oligopeptides being the so-called active peptides or their direct precursors. An original way of expression of the protein hydrolysis kinetics was introduced. Peptide bonds contained in the protein were recognized as a diverse-quality substrate for hydrolysis by the applied protease. This assumption was positively verified taking as an example the hydrolysis of albumin by thermolysin. Peptide linkages for this system should be divided into at least four groups. One of them is a group of bonds non-hydrolyzable by this enzyme. These that are broken are hydrolyzed at a rate that differs even by tens of thousands of times. Designated kinetic constants were k'F = 10991.4 L/g.h, k'M = 14.83L/g.h, k'S about 10-1 L/g.h for fast, medium and slow bonds, respectively. Moreover, a procedure for unfolding of the protein, conducive to the improved susceptibility to enzymatic hydrolysis (approximately three-fold increase in the rate) was proposed.

Keywords: peptide bond hydrolysis, kinetics, enzyme specificity, biologically active peptides

Procedia PDF Downloads 432

Authors: H. Yousefnia, SJ. Ahmadi, S. Sajadi, S. Zolghadri, A. Bahrami-Samani, M. Bagherzadeh

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Nowadays, superparamagnetic iron oxide nanoparticles (SPIONs) as the multitask agents have showed advantageous characteristics. The aim of this study was the preparation and quality control of 153Sm-DTPA-DA-SPION complex. Samarium-153 was produced by neutron irradiation of the enriched 152Sm2O3 in a research reactor for 5 d. For radiolabeling purposes, 8 mg of the ligand was added to the vial containing 153SmCl3 and the mixture was sonicated 30 min, while pH was adjusted to 7-8. The radiochemical purity of the complex was checked by the ITLC method using NH4OH:MeOH:H2O (0.2:2:4) as the mobile phase. This new radiolabeled complex was prepared with a radiochemical purity of higher than 98% in 30 min at the optimized condition. The complex was kept at room temperature and in human serum at 37 °C for 48 h, showed no loss of 153Sm from the complex. Considering all of these features, this new radiolabeled complex can be considered as a good therapeutic agent; however, further studies on its biological behavior are still needed.

Keywords: iron nanoparticles, preparation, quality control, 153Sm

Procedia PDF Downloads 326

Authors: Maria Magdy Danial Riad

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The objective of this study was to investigate the effect of different protein sources with different degradability ratios during late gestation of ewes on colostrum composition and its IgG concentration, body weight change of dams, and birth weight of their lambs. Objectives: 35 multiparous native crossbred ewes (BW= 59±2.5kg) were randomly allocated to five dietary treatments (7 ewes / treatment) for 2 months prior to lambing. Methods: Experimental diets were isonitrogenous (12.27% CP) and isocaloric (2.22 Mcal ME/kg DM). In diet I (the control), solvent extract soybeans (SESM 33% RUP of CP), II feed grade urea (FGU 31% RUP), III slow release urea (SRU 31% RUP). As sources of undegradable protein, extruded expeller SBM-EESM 40 (37% RUP) and extruded expeller SBM-EESM 60 (41% RUP) were used in groups IV and V, respectively. Results showed no significant effect on feed intake, crude protein (CP), metabolizable energy (ME), and body condition score (BCS). Ewes fed the 37% RUP diet gained more (p<0.05) weight compared with ewes fed the 31% RUP diet (5.62 vs. 2.5kg). Ewes in EESM 60 had the highest levels of fat, protein, total solid, solid not fat, and immunoglobulin and the lowest in urea N content (P< 0.05) in colostrum during the first 24hrs after lambing. Conclusions: Protein source and RUP levels in ewes’ diets had no significant effect (P< 0.05) on lambs’ birth weight and ewes' blood biochemical parameters. Increasing the RUP content of diet during late gestation resulted in an increase in colostrum constituents and its IgG level but had no effect on ewes’ performance and their lambs’ outcome.

Keywords: colostrum, ewes, lambs output, pregnancy, undegradable protein

Procedia PDF Downloads 46

Authors: Sima Shakoorjavan, Dawid Stawski, Somaye Akbari

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In this study, great attempts have been made to initially modify polylactide (PLA) nonwoven surface with poly(amidoamine) (PAMMA) dendritic polymer to create amine active sites on PLA surface through aminolysis reaction. Further, layer-by-layer deposition of four layers of two weak polyelectrolytes, including PAMAM as polycation and polyacrylic acid (PAA) as polyanion on activated PLA, was monitored with turbidity analysis of waste-polyelectrolytes after each deposition step. The FTIR-ATR analysis confirmed the successful introduction of amine groups into PLA polymeric chains through the emerging peak around 1650 cm⁻¹ corresponding to N-H bending vibration and a double wide peak at around 3670-3170 cm⁻¹ corresponding to N-H stretching vibration. The adsorption-desorption behavior of (PAMAM) and poly (PAA) deposition was monitored by turbidity test. Turbidity results showed the desorption and removal of the previously deposited layer (second and third layers) upon the desorption of the next layers (third and fourth layers). Also, the importance of proper rinsing after aminolysis of PLA nonwoven fabric was revealed by turbidity test. Regarding the sample with insufficient rinsing process, higher desorption and removal of ungrafted PAMAM from aminolyzed-PLA surface into PAA solution was detected upon the deposition of the first PAA layer. This phenomenon can be due to electrostatic attraction between polycation (PAMAM) and polyanion (PAA). Moreover, the successful layer deposition through LBL was confirmed by the staining test of acid red 1 through spectrophotometry analysis. According to the results, layered PLA with four layers with PAMAM as the top layer showed higher dye absorption (46.7%) than neat (1.2%) and aminolyzed PLA (21.7%). In conclusion, the complicated adsorption-desorption behavior of dendritic polycation and linear polyanion systems was observed. Although desorption and removal of previously adsorbed layers occurred upon the deposition of the next layer, the remaining polyelectrolyte on the substrate is sufficient for the adsorption of the next polyelectrolyte through electrostatic attraction between oppositely charged polyelectrolytes. Also, an increase in dye adsorption confirmed more introduction of PAMAM onto PLA surface through LBL.

Keywords: surface modification, layer-by-layer technique, weak polyelectrolytes, adsorption-desorption behavior

Procedia PDF Downloads 57

Authors: N. Mahmoudnia, M. Khormali

Abstract:

This experiment was conducted to determine the apparent protein digestibility of poultry by- product meal (PBPM) from two industrial poultry slaughter houses on Ross 308 male broiler chickens in independed comparisons. The experiment consisted of seven dietary treatments and three replicates per treatment with three broiler chickens per replicate in a completely randomized design. Dietary treatments consisted of a control corn- soybean diet, and levels 3, 6 and 9% PBPM produced by slaughter house 1 and levels 3, 6 and 9% PBPM produced by slaughter house 2. Chromic oxide was added to the experimental diets as indigestible marker. The apparent protein digestibility of each diet were determined with two methods of sample collection of ileum and excreta in 21-28 d of age. The results this experiment showed that use of PBPM had no significantly effect on performance of broiler chicks during period of experiments. The apparent protein digestibility of PBPM groups was significantly higher than control group by excreta sampling procedure (P<0.05). Using of PBPM 2 significantly (P<0.05) decreased the apparent protein digestibility values based on ileum sampling procedure vs control ( 85.21 vs 90.14).Based results of this experiment,it is possible to use of PBPM 1 in broiler chicken.

Keywords: poultry by-product meal, apparent protein digestibility, independed comparison, broiler chicken

Procedia PDF Downloads 594

Authors: A. Mirzaei, S. Zolghadri, M. Athari-Allaf, H. Yousefnia, A. R. Jalilian

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Rheumatoid arthritis is the most common autoimmune disease, leading to the destruction of the joints. The aim of this study was the preparation of 90Y-chitosan complex as a novel agent for radiosynovectomy. The complex was prepared in the diluted acetic acid solution. At the optimized condition, the radiochemical purity of higher than 99% was obtained by ITLC method on Whatman No. 1 and by using a mixture of methanol/water/acetic acid (4:4:2) as the mobile phase. The complex was stable in acidic media (pH=3) and its radiochemical purity was above 98% even after 48 hours. The biodistribution data in rats showed that there was no significant leakage of the injected activity even after 48 h. Considering all of the excellent features of the complex, 90Y-chitosan can be used to manipulate synovial inflammation effectively.

Keywords: chitosan, Y-90, radiosynovectomy, biodistribution

Procedia PDF Downloads 478

Authors: Neha Bhadauria, Indu Gaur, Shilpi Shilpi, Susmita Goswami, Prabir K. Paul

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The aerial surface of plants colonized by Human Enteric Pathogens ()has been implicated in outbreaks of enteric diseases in humans. Practice of organic farming primarily using animal dung as manure and sewage water for irrigation are the most significant source of enteric pathogens on the surface of leaves, fruits and vegetables. The present work aims to have an insight into the molecular mechanism of interaction of Human Enteric Pathogens or their metabolites with cell wall receptors in plants. Tomato plants grown under aseptic conditions at 12 hours L/D photoperiod, 25±1°C and 75% RH were inoculated individually with S. fonticola and K. pneumonia. The leaves from treated plants were sampled after 24 and 48 hours of incubation. The cell wall and cytoplasmic proteins were extracted and isocratically separated on 1D SDS-PAGE. The sampled leaves were also subjected to formaldehyde treatment prior to isolation of cytoplasmic proteins to study protein-protein interactions induced by Human Enteric Pathogens. Protein bands extracted from the gel were subjected to MALDI-TOF-TOF MS analysis. The foremost interaction of Human Enteric Pathogens on the plant surface was found to be cell wall bound receptors which possibly set ups a wave a critical protein-protein interaction in cytoplasm. The study revealed the expression and suppression of specific cytoplasmic and cell wall-bound proteins, some of them being important components of signaling pathways. The results also demonstrated HEP induced rearrangement of signaling pathways which possibly are crucial for adaptation of these pathogens to plant surface. At the end of the study, it can be concluded that controlling the over-expression or suppression of these specific proteins rearrange the signaling pathway thus reduces the outbreaks of food-borne illness.

Keywords: cytoplasmic protein, cell wall-bound protein, Human Enteric Pathogen (HEP), protein-protein interaction

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Authors: E. Obreque-Slier, V. Contreras-Cortez, R. López-Solís

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Astringency is a drying, roughing, and sometimes puckering sensation that is experienced on the various oral surfaces during or immediately after tasting foods. This sensation has been closely related to the interaction and precipitation between salivary proteins and polyphenols, specifically flavanols or proanthocyanidins. In addition, the type and concentration of proanthocyanidin influences significantly the intensity of the astringency and consequently the protein/proanthocyanidin interaction. However, most of the studies are based on the interaction between saliva and highly complex polyphenols, without considering the effect of monomeric proanthoancyanidins present in different foods. The aim of this study was to evaluate the effect of different monomeric proanthocyanidins on the diffusion and precipitation of salivary proteins. Thus, solutions of catechin, epicatechin, epigallocatechin and gallocatechin (0, 2.0, 4.0, 6.0, 8.0 and 10 mg/mL) were mixed with human saliva (1: 1 v/v). After incubation for 5 min at room temperature, 15 µL aliquots of each mix were dotted on a cellulose membrane and allowed to dry spontaneously at room temperature. The membrane was fixed, rinsed and stained for proteins with Coomassie blue. After exhaustive washing in 7% acetic acid, the membrane was rinsed once in distilled water and dried under a heat lamp. Both diffusion area and stain intensity of the protein spots were semiqualitative estimates for protein-tannin interaction (diffusion test). The rest of the whole saliva-phenol solution mixtures of the diffusion assay were centrifuged, and 15-μL aliquots from each of the supernatants were dotted on a cellulose membrane. The membrane was processed for protein staining as indicated above. The blue-stained area of protein distribution corresponding to each of the extract dilution-saliva mixtures was quantified by Image J 1.45 software. Each of the assays was performed at least three times. Initially, salivary proteins display a biphasic distribution on cellulose membranes, that is, when aliquots of saliva are placed on absorbing cellulose membranes, and free diffusion of saliva is allowed to occur, a non-diffusible protein fraction becomes surrounded by highly diffusible salivary proteins. In effect, once diffusion has ended, a protein-binding dye shows an intense blue-stained roughly circular area close to the spotting site (non-diffusible fraction) (NDF) which becomes surrounded by a weaker blue-stained outer band (diffusible fraction) (DF). Likewise, the diffusion test showed that epicatechin caused the complete disappearance of DF from saliva with 2 mg/mL. Also, epigallocatechin and gallocatechin caused a similar effect with 4 mg/mL, while catechin generated the same effect at 8 mg/mL. In the precipitation test, the use of epicatechin and gallocatechin generated evident precipitates at the bottom of the Eppendorf tubes. In summary, the flavanol type differentially affects the diffusion and precipitation of saliva, which would affect the sensation of astringency perceived by consumers.

Keywords: astringency, polyphenols, tannins, tannin-protein interaction

Procedia PDF Downloads 191

Authors: Homayun Ghasemi, Mojtaba Yousefirad, Mozhgan Farzamisepehr

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Heavy metals are among soil pollutant resources that in case of accumulation in the soil and absorption by the plant, enter into the food chain and poison the plants or the people who consume those plants. This research was performed in order to examine the role of cadmium as a heavy metal in the activity of catalase and peroxidase as well as protein concentration in Trifolium resupinatum L. based on a randomized block design with three repetitions. The used treatments included consumption of Cd (NO3)2 at four levels, namely, 0, 100, 200, and 300 ppm. The plants under study were treated for 10 days. The results of the study showed that catalase activity decreased by the increase of cadmium. Moreover, peroxidase activity increased by an increase inthe consumption of cadmium. The analysis of protein level showed that plantlet protein decreased in high cadmium concentrations. The findings also demonstrated that cadmium concentration in roots was higher than in shoots.

Keywords: catalase, heavy metal, peroxidase, protein

Procedia PDF Downloads 242

Authors: Vytautas Galvanauskas, Rimvydas Simutis, Donatas Levisauskas, Vykantas Grincas, Renaldas Urniezius

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The paper deals with model-based development and implementation of efficient control strategies for recombinant protein synthesis in fed-batch E.coli cultivation processes. Based on experimental data, a kinetic dynamic model for cultivation process was developed. This model was used to determine substrate feeding strategies during the cultivation. The proposed feeding strategy consists of two phases – biomass growth phase and recombinant protein production phase. In the first process phase, substrate-limited process is recommended when the specific growth rate of biomass is about 90-95% of its maximum value. This ensures reduction of glucose concentration in the medium, improves process repeatability, reduces the development of secondary metabolites and other unwanted by-products. The substrate limitation can be enhanced to satisfy restriction on maximum oxygen transfer rate in the bioreactor and to guarantee necessary dissolved carbon dioxide concentration in culture media. In the recombinant protein production phase, the level of substrate limitation and specific growth rate are selected within the range to enable optimal target protein synthesis rate. To account for complex process dynamics, to efficiently exploit the oxygen transfer capability of the bioreactor, and to maintain the required dissolved oxygen concentration, adaptive control algorithms for dissolved oxygen control have been proposed. The developed model-based control strategies are useful in scale-up of cultivation processes and accelerate implementation of innovative biotechnological processes for industrial applications.

Keywords: adaptive algorithms, model-based control, recombinant E. coli, scale-up of bioprocesses

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Authors: Victor Prevost, Solene Landerneau, Michel Duhamel, Joel Sternheimer, Olivier Gallet, Pedro Ferrandiz, Marwa Mokni

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The search for homologous proteins is one of the ongoing challenges in biology and bioinformatics. Traditionally, a pair of proteins is thought to be homologous when they originate from the same ancestral protein. In such a case, their sequences share similarities, and advanced scientific research effort is spent to investigate this question. On this basis, we propose the Protein-Wave Alignment Tool (”P-WAT”) developed within the framework of the France Relance 2030 plan. Our work takes into consideration the mass-related wave aspect of protein biosynthesis, by associating specific frequencies to each amino acid according to its mass. Amino acids are then regrouped within their mass category. This way, our algorithm produces specific alignments in addition to those obtained with a common amino acid coding system. For this purpose, we develop the ”P-WAT” original algorithm, able to address large protein databases, with different attributes such as species, protein names, etc. that allow us to align user’s requests with a set of specific protein sequences. The primary intent of this algorithm is to achieve efficient alignments, in this specific conceptual frame, by minimizing execution costs and information loss. Our algorithm identifies sequence similarities by searching for matches of sub-sequences of different sizes, referred to as primers. Our algorithm relies on Boolean operations upon a dot plot matrix to identify primer amino acids common to both proteins which are likely to be part of a significant alignment of peptides. From those primers, dynamic programming-like traceback operations generate alignments and alignment scores based on an adjusted PAM250 matrix.

Keywords: protein, alignment, homologous, Genodic

Procedia PDF Downloads 107

Authors: Friday A. Ogori, Ojotu M. Eke, Oneh J. Abu, Abraham T. Girgih

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B.aeqygtiaca del (Balanites aegyptiaca del) seed protein concentrate (APC) was hydrolyzed using different enzymes such as pepsin+pancreatin (PP), Alcalase (Alca), and Flavourzyme (Flav). The Alca has higher yield (100%) when compared to PP (83.23%) and Flav (62.90%). The hydrophobic amino acid and Sulphur containing amino acid (40.19%, 7.04%) in PP hydrolysate were higher compared to Alcalase (38.92%, 6.69%), Flavourenzyme (37.43%,6.35%), and APC (39.97%, 6.95%) samples. The PP has stronger DPPH, Hydroxyl radical quenching, Ferric reducing activity, and linoleic acid peroxidation activity, followed by the protein concentrate (APC) and Alcalase (Alca), while Flavourenzyme (Flav) derived hydrolysate was least in scavenging and inhibiting radical peroxidation properties. Flavourenzyme derived hydrolysate had stronger Ferric reducing antioxidant potential and metal chelating property. The result showed that the Alcalase hydrolysate has promising peptide yield, and PP hydrolysate had excellent amino acid residues and good in-vitro antioxidant potentials and could be a preferred ingredients in the nutraceutical and functional food emerging industries.

Keywords: balanites aegyptiaca del, protein concentrate, protein hydrolysates, enzymatic hydrolysis, antioxidants

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Authors: A. Gilewska, J. Masternak, K. Kazimierczuk, L. Turlej, J. Wietrzyk, B. Barszcz

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Platinum-based drugs are now widely used as chemotherapeutic agents. However the platinum complexes show the toxic side-effects: i) the development of platinum resistance; ii) the occurrence of severe side effects, such as nephro-, neuro- and ototoxicity; iii) the high toxicity towards human fibroblast. Therefore the development of new anticancer drugs containing different transition-metal ions, for example, ruthenium, rhodium, iridium is a valid strategy in cancer treatment. In this paper, we reported the synthesis, spectroscopic, structural and biological properties of complexes of ruthenium, rhodium, and iridium containing N,N-chelating ligand (2,2’-bisimidazole). These complexes were characterized by elemental analysis, UV-Vis and IR spectroscopy, X-ray diffraction analysis. These complexes exhibit a typical pseudotetrahedral three-legged piano-stool geometry, in which the aromatic arene ring forms the seat of the piano-stool, while the bidentate 2,2’-bisimidazole (ligand) and the one chlorido ligand form the three legs of the stool. The spectroscopy data (IR, UV-Vis) and elemental analysis correlate very well with molecular structures. Moreover, the cytotoxic activity of the complexes was carried out on human cancer cell lines: LoVo (colorectal adenoma), MV-4-11 (myelomonocytic leukaemia), MCF-7 (breast adenocarcinoma) and normal healthy mouse fibroblast BALB/3T3 cell lines. To predict a binding mode, a potential interaction of metal complexes with calf thymus DNA (CT-DNA) and protein (BSA) has been explored using UV absorption and circular dichroism (CD). It is interesting to note that the investigated complexes show no cytotoxic effect towards the normal BALB/3T3 cell line, compared to cisplatin, which IC₅₀ values was determined as 2.20 µM. Importantly, Ru(II) displayed the highest activity against HL-60 (IC₅₀ 4.35 µM). The biological studies (UV-Vis and circular dichroism) suggest that arene-complexes could interact with calf thymus DNA probably via an outside binding mode and interact with protein (BSA).

Keywords: ruthenium(II) complex, rhodium(III) complex, iridium(III) complex, biological activity

Procedia PDF Downloads 124

Authors: Thanusha D. Abeywickrama, Inoka C. Perera, Genji Kurisu

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Tuberculosis, considered being as the ninth leading cause of death worldwide, cause from a single infectious agent M. tuberculosis and the drug resistance nature of this bacterium is a continuing threat to the world. Therefore TB preventing treatment is expanding, where this study designed to analyze the regulatory mechanism of GntR transcriptional regulator gene Rv0792c, which lie between several genes codes for some hypothetical proteins, a monooxygenase and an oxidoreductase. The gene encoding Rv0792c was cloned into pET28a and expressed protein was purified to near homogeneity by Nickel affinity chromatography. It was previously reported that the protein binds within the intergenic region (BS region) between Rv0792c gene and monooxygenase (Rv0793). This resulted in binding of three protein molecules with the BS region suggesting tight control of monooxygenase as well as its own gene. Since monooxygenase plays a key role in metabolism, this gene may have a global regulatory role. The natural ligand for this regulator is still under investigation. In relation to the Rv0792 protein structure, a Circular Dichroism (CD) spectrum was carried out to determine its secondary structure elements. Percentage-wise, 17.4% Helix, 21.8% Antiparallel, 5.1% Parallel, 12.3% turn and 43.5% other were revealed from CD spectrum data under room temperature. Differential Scanning Calorimetry (DSC) was conducted to assess the thermal stability of Rv0792, which the melting temperature of protein is 57.2 ± 0.6 °C. The graph of heat capacity (Cp) versus temperature for the best fit was obtained for non-two-state model, which concludes the folding of Rv0792 protein occurs through stable intermediates. Peak area (∆HCal ) and Peak shape (∆HVant ) was calculated from the graph and ∆HCal / ∆HVant was close to 0.5, suggesting dimeric nature of the protein.

Keywords: CD spectrum, DSC analysis, GntR transcriptional regulator, protein structure

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Authors: Malgorzata Krysiak, Anna Wegrzyn, Maciej Garstka, Radoslaw Mazur

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Under constantly fluctuating environmental conditions, the thylakoid membrane protein network evolved the ability to dynamically respond to changing biotic and abiotic factors. One of the most important protective mechanism is rearrangement of the chlorophyll-protein (CP) complexes, induced by protein phosphorylation. In a temperate climate, low temperature is one of the abiotic stresses that heavily affect plant growth and productivity. The aim of this study was to determine the role of LHCII antenna complex phosphorylation in the dark-chilling response. The study included an experimental model based on dark-chilling at 4 °C of detached chilling sensitive (CS) runner bean (Phaseolus coccineus L.) and chilling tolerant (CT) garden pea (Pisum sativum L.) leaves. This model is well described in the literature as used for the analysis of chilling impact without any additional effects caused by light. We examined changes in thylakoid membrane protein phosphorylation, interactions between phosphorylated LHCII (P-LHCII) and CP complexes, and their impact on the dynamics of photosystem II (PSII) under dark-chilling conditions. Our results showed that the dark-chilling treatment of CS bean leaves induced a substantial increase of phosphorylation of LHCII proteins, as well as changes in CP complexes composition and their interaction with P-LHCII. The PSII photochemical efficiency measurements showed that in bean, PSII is overloaded with light energy, which is not compensated by CP complexes rearrangements. On the contrary, no significant changes in PSII photochemical efficiency, phosphorylation pattern and CP complexes interactions were observed in CT pea. In conclusion, our results indicate that different responses of the LHCII phosphorylation to chilling stress take place in CT and CS plants, and that kinetics of LHCII phosphorylation and interactions of P-LHCII with photosynthetic complexes may be crucial to chilling stress response. Acknowledgments: presented work was financed by the National Science Centre, Poland grant No.: 2016/23/D/NZ3/01276

Keywords: LHCII, phosphorylation, chilling stress, pea, runner bean

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Authors: Austin Jiang, Richard M. Salmon, Nicholas W. Morrell, Wei Li

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BMP10 is highly expressed in the developing heart and plays essential roles in cardiogenesis. BMP10 deletion in mice results in embryonic lethality due to impaired cardiac development. In adults, BMP10 expression is restricted to the right atrium, though ventricular hypertrophy is accompanied by increased BMP10 expression in a rat hypertension model. However, reports of BMP10 activity in the circulation are inconclusive. In particular it is not known whether in vivo secreted BMP10 is active or whether additional factors are required to achieve its bioactivity. It has been shown that high-affinity binding of the BMP10 prodomain to the mature ligand inhibits BMP10 signaling activity in C2C12 cells, and it was proposed that prodomain-bound BMP10 (pBMP10) complex is latent. In this study, we demonstrated that the BMP10 prodomain did not inhibit BMP10 signaling activity in multiple endothelial cells, and that recombinant human pBMP10 complex, expressed in mammalian cells and purified under native conditions, was fully active. In addition, both BMP10 in human plasma and BMP10 secreted from the mouse right atrium were fully active. Finally, we confirmed that active BMP10 secreted from mouse right atrium was in the prodomain-bound form. Our data suggest that circulating BMP10 in adults is fully active and that the reported vascular quiescence function of BMP10 in vivo is due to the direct activity of pBMP10 and does not require an additional activation step. Moreover, being an active ligand, recombinant pBMP10 may have therapeutic potential as an endothelial-selective BMP ligand, in conditions characterized by loss of BMP9/10 signaling.

Keywords: bone morphogenetic protein 10 (BMP10), endothelial cell, signal transduction, transforming growth factor beta (TGF-B)

Procedia PDF Downloads 271

Authors: Jaturong Matidtor, Krisna R. Torrissen, Saengtong Pongjareankit, Sudaporn Tongsiri, Jiraporn Rojtinnakorn

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Low survival rate has being particular problem in nursery of marble goby juvenile. The aim of this study was to investigate effect of garlic extract on protein digestive pancreatic enzymes, trypsin (T) and chymotrypsin (C). The marble goby were reared with commercial feed mixed garlic extract at concentration of 0 (control), 0.3, 0.5, 1.0, 3.0 and 5.0% (w/w) for 6 weeks. Analysis of the digestive enzymes at 2 and 6 weeks was performed. Growth parameters; weight gain (WG), specific growth rate (SGR) and feed efficiency (FE), were identified. For T, C and T/C at 2 weeks, values of T and T/C ratio of 0.3% (w/w) group showed significant difference (p < 0.05) with the highest values of 17685.64± 11981.77 U/mg protein and of 51.64 ± 27.46 U/mg protein, respectively. For C at 2 weeks, 0% (w/w) group showed the highest values of 16191.76± 2225.56 U/mg protein. Whereas value of T, C and T/C ratio at 6 weeks, there was no significant difference (p > 0.05). For growth performance, it significantly increased in all garlic extract fed groups (0.3-5.0%, w/w), both at 2 and 6 weeks. At 2 weeks, values of WG and SGR of 0.5% (w/w) group showed the highest values of 71.51 ± 1.60%, and 3.85 ± 0.07%, respectively. For FE, 0.3% (w/w) group showed the highest value of 60.21 ± 6.51%. At 6 weeks, it illustrated that all growth parameters of 5.0% (w/w) group were the highest values; WG = 35.06 ± 5.66%, SGR = 2.14 ± 0.30%, and FE = 5.86 ± 0.68%. We suggested that garlic extract could be available for protein digestive enzyme and growth enhancement in marble goby nursery with artificial feed. This result will be high benefit for commercial aquaculture of marble goby.

Keywords: marble goby, nursery, garlic extract, digestive enzyme, growth

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Authors: Rizky Kusuma Cahyani

Abstract:

Hepatitis B is a liver inflammatory disease caused by hepatitis B virus (HBV). This infection can be prevented by vaccination which contains HBV surface protein (sHBsAg). However, vaccine supply is limited. Several attempts have been conducted to produce local sHBsAg. However, the purity degree and protein yield are still inadequate. Therefore optimization of HBsAg purification steps is required to obtain high yield with better purification fold. In this study, optimization of purification was done in 2 steps, precipitation using variation of NaCl concentration (0,3 M; 0,5 M; 0,7 M) and PEG (3%, 5%, 7%); ion exchange chromatography (IEC) using NaCl 300-500 mM elution buffer concentration.To determine HBsAg protein, bicinchoninic acid assay (BCA) and enzyme-linked immunosorbent assay (ELISA) was used in this study. Visualization of HBsAg protein was done by SDS-PAGE analysis. Based on quantitative analysis, optimal condition at precipitation step was given 0,3 M NaCl and PEG 3%, while in ion exchange chromatography step, the optimum condition when protein eluted with NaCl 500 mM. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicates that the presence of protein HBsAg with a molecular weight of 25 kDa (monomer) and 50 kDa (dimer). The optimum condition for purification of sHBsAg produced in Pichia pastoris gave a yield of 47% and purification fold 17x so that it would increase the production of hepatitis B vaccine to be more optimal.

Keywords: hepatitis B virus, HBsAg, hepatitis B surface antigen, Pichia pastoris, purification

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Authors: S. Bilal, S. A. Bhat, I. Hussain, J. D. Parrah, S. P. Ahmad, M. R. Mir

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Background: The wound healing involves a highly coordinated cascade of cellular and immunological response over a period including coagulation, inflammation, granulation tissue formation, epithelialization, collagen synthesis and tissue remodeling. Wounds in aged heal more slowly than those in younger, mainly because of comorbidities that occur as one age. The present study is about the influence of age on wound healing. 1x1cm^2 (100 mm) wounds were created on the back of the animal. The animals were divided into two groups; one group had animals in the age group of 3-9 months while another group had animals in the age group of 15-21 months. Materials and Methods: 24 clinically healthy rabbits in the age group of 3-21 months were used as experimental animals and divided into two groups viz A and B. All experimental parameters, i.e., Excision wound model, Measurement of wound area, Protein extraction and estimation, Protein extraction and estimation and DNA extraction and estimation were done by standard methods. Results: The parameters studied were wound contraction, hydroxyproline, glucosamine, protein, and DNA. A significant increase (p<0.005) in the hydroxyproline, glucosamine, protein and DNA and a significant decrease in wound area (p<0.005) was observed in the age group of 3-9 months when compared to animals of an age group of 15-21 months. Wound contraction together with hydroxyproline, glucosamine, protein and DNA estimations suggest that advanced age results in retarded wound healing. Conclusion: The decrease wound contraction and accumulation of hydroxyproline, glucosamine, protein and DNA in group B animals may be associated with the reduction or delay in growth factors because of the advancing age.

Keywords: age, wound healing, excision wound, hydroxyproline, glucosamine

Procedia PDF Downloads 655

Authors: Şevket Evci, Mehmet Akif Karsli

Abstract:

The aim of this study is to determine nutrient contents, in situ ruminal degradation kinetics and protein fractions of screenings bean (B), chick pea (ChP), red lentil (RL) and green lentil (GL) that is used as residue in grain legume packing industry. For this purpose, four samples of each legumes species-a total of 16 samples, collected from different parts of our country were utilized. Feedstuffs used in the experiment were incubated for 0, 2 4, 8, 12, 24, and 48 hours in the rumen of 3 ruminally cannulated Akkaraman rams as duplicate. The nutrient contents, in situ ruminal dry matter (DM), organic matter (OM) and crude protein (CP) degradabilities and fractions, and escape protein contents were evaluated. The highest OM and CP contents were observed in RL (P<0.05). Chick pea had the highest ether extract (EE) content and EE values were 3.47, 6.72, 2.26, 8.66 % for RL, B, GL and ChP, respectively (P<0.05). Crude fiber (CF), ADF, and NDF contents were the highest in RL and the lowest in ChP. CF values were 24.03, 10.80, 4.09 and 3.57 % for RL, GL, B and ChP (P<0.05). Acid detergent insoluble nitrogen content of samples did not differ. Escape protein content was the highest in RL and the lowest in B (P<0.05). After 48 h incubation, the lowest OM and CP degradabilities were observed in RL. While the highest OM degradability was seen in ChP the highest CP degradability was observed in B (P<0.05). The lowest water soluble OM and CP contents were observed in RL whereas the highest potentially degradable OM and CP contents were seen in B and ChP (P<0.05). Both rate of OM and CP degradations (k-1) did not differ among samples (P>0.05). In conclusion, it was noted that feedstuffs (GL, ChP and B) used in the experiment except RL had a greater ruminal degradibilities of both OM and CP and moreover, had a higher escape protein contents, except B. It was thought that these feedstuffs can be substituted with some of common protein sources used in animal nutrition.

Keywords: in situ, nutrient contents, ruminant, subsieve

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Authors: Lavinia Ruta, Ileana Farcasanu

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The yeast surface display (YSD) technique enables the expression of proteins on yeast cell surfaces, facilitating the identification and isolation of proteins with targeted binding properties, such as nanobodies. Nanobodies, derived from camelid species, are single-domain antibody fragments renowned for their high affinity and specificity towards target proteins, making them valuable in research and potentially in therapeutics. Their advantages include a compact size (~15 kDa), robust stability, and the ability to target challenging epitopes. The project endeavors to establish and validate a platform for producing Green Fluorescent Protein (GFP) and mCherry nanobodies using the yeast surface display method. mCherry, a prevalent red fluorescent protein sourced from coral species, is commonly utilized as a genetic marker in biological studies due to its vibrant red fluorescence. The GFP-nanobody, a single variable domain of heavy-chain antibodies (VHH), exhibits specific binding to GFP, offering a potent means for isolating and engineering fluorescent protein fusions across various biological research domains. Both GFP and mCherry nanobodies find specific utility in cellular imaging and protein analysis applications.

Keywords: YSD, nanobodies, GFP, Saccharomyces cerevisiae

Procedia PDF Downloads 52

Authors: Vineeta Kaushik, Manisha Goel

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Chaperones are proteins that help other proteins fold correctly, and are found in all domains of life i.e., prokaryotes, eukaryotes and archaea. Various comparative genomic studies have suggested that the archaeal protein folding machinery appears to be highly similar to that found in eukaryotes. In case of protein folding; slow rotation of peptide prolyl-imide bond is often the rate limiting step. Formation of the prolyl-imide bond during the folding of a protein requires the assistance of other proteins, termed as peptide prolyl cis-trans isomerases (PPIases). Cyclophilins constitute the class of peptide prolyl isomerases with a wide range of biological function like protein folding, signaling and chaperoning. Most of the cyclophilins exhibit PPIase enzymatic activity and play active role in substrate protein folding which classifies them as a category of molecular chaperones. Till date, there is not very much data available in the literature on archaeal cyclophilins. We aim to compare the structural and biochemical features of the cyclophilin protein from within the three domains to elucidate the features affecting their stability and enzyme activity. In the present study, we carry out in-silico analysis of the cyclophilin proteins to predict their conserved residues, sites under positive selection and compare these proteins to their bacterial and eukaryotic counterparts to predict functional divergence. We also aim to clone and express these proteins in heterologous system and study their biophysical characteristics in detail using techniques like CD and fluorescence spectroscopy. Overall we aim to understand the features contributing to the folding, stability and dynamics of the archaeal cyclophilin proteins.

Keywords: biophysical characterization, x-ray crystallography, chaperone-like activity, cyclophilin, PPIase activity

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Authors: Yasir Ali Arfat

Abstract:

Composite films based on fish protein isolate (FPI) and fish skin gelatin (FSG) blend incorporated with 50 and 100% (w/w, protein) basil leaf essential oil (BEO) in the absence and presence of 3% (w/w, protein) ZnO nanoparticles (ZnONP) were prepared and characterised. Tensile strength (TS) decreased, whilst elongation at break (EAB) increased as BEO level increased (p < 0.05). However, ZnONP addition resulted in higher TS but lower EAB (p < 0.05). The lowest water vapour permeability (WVP) was observed for the film incorporated with 100% BEO and 3% ZnONP (p < 0.05). BEO and ZnONP incorporation decreased transparency of FPI/FSG films (p < 0.05). FTIR spectra indicated that films added with BEO exhibited higher hydrophobicity. Both BEO and ZnONP had a marked impact on thermal stability of the films. Microstructural study revealed that presence of ZnONP prevented bilayer formation of film containing 100% BEO. FPI/FSG films incorporated with 100% BEO, especially in combination with ZnONP, exhibited strong antibacterial activity against food pathogenic and spoilage bacteria and thus could be used as an active food packaging material to ensure safety and to extend the shelf-life of packaged foods.

Keywords: bionanocomposite, fish protein isolate, fish skin gelatin, basil essential oil, ZnO nanoparticles, antimicrobial packaging

Procedia PDF Downloads 465

Authors: Muhammad Ajmal

Abstract:

- Thermal processing and subsequent storage of ultra-heat treated (UHT) milk leads to alteration in protein profile, Maillard reaction and lipid oxidation. Concentration of carbohydrates in normal and flavored version of UHT milk is considerably different. Transition in protein profile, Maillard reaction and lipid oxidation in UHT flavored milk was determined for 90 days at ambient conditions and analyzed at 0, 45 and 90 days of storage. Protein profile, hydroxymethyl furfural, furosine, Nε-carboxymethyl-l-lysine, fatty acid profile, free fatty acids, peroxide value and sensory characteristics were determined. After 90 days of storage, fat, protein, total solids contents and pH were significantly less than the initial values determined at 0 day. As compared to protein profile normal UHT milk, more pronounced changes were recorded in different fractions of protein in UHT milk at 45 and 90 days of storage. Tyrosine content of flavored UHT milk at 0, 45 and 90 days of storage were 3.5, 6.9 and 15.2 µg tyrosine/ml. After 45 days of storage, the decline in αs1-casein, αs2-casein, β-casein, κ-casein, β-lactoglobulin, α-lactalbumin, immunoglobulin and bovine serum albumin were 3.35%, 10.5%, 7.89%, 18.8%, 53.6%, 20.1%, 26.9 and 37.5%. After 90 days of storage, the decline in αs1-casein, αs2-casein, β-casein, κ-casein, β-lactoglobulin, α-lactalbumin, immunoglobulin and bovine serum albumin were 11.2%, 34.8%, 14.3%, 33.9%, 56.9%, 24.8%, 36.5% and 43.1%. Hydroxy methyl furfural content of UHT milk at 0, 45 and 90 days of storage were 1.56, 4.18 and 7.61 (µmol/L). Furosine content of flavored UHT milk at 0, 45 and 90 days of storage intervals were 278, 392 and 561 mg/100g protein. Nε-carboxymethyl-l-lysine content of UHT flavored milk at 0, 45 and 90 days of storage were 67, 135 and 343mg/kg protein. After 90 days of storage of flavored UHT milk, the loss of unsaturated fatty acids 45.7% from the initial values. At 0, 45 and 90 days of storage, free fatty acids of flavored UHT milk were 0.08%, 0.11% and 0.16% (p<0.05). Peroxide value of flavored UHT milk at 0, 45 and 90 days of storage was 0.22, 0.65 and 2.88 (MeqO²/kg). Sensory analysis of flavored UHT milk after 90 days indicated that appearance, flavor and mouth feel score significantly decreased from the initial values recorded at 0 day. Findings of this investigation evidenced that in flavored UHT milk more pronounced changes take place in protein profile, Maillard reaction products and lipid oxidation as compared to normal UHT milk.

Keywords: UHT flavored milk , hydroxymethyl furfural, lipid oxidation, sensory properties

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Authors: Debbarma Asis, Guha Chandan

Abstract:

Tumor Protein-53 (P53) is one of the tumor suppressor proteins. P53 regulates the cell cycle that conserves stability by preventing genome mutation. It is named so as it runs as 53-kilodalton (kDa) protein on Polyacrylamide gel electrophoresis although the actual mass is 43.7 kDa. Experimental evidence has indicated that P53 cancer mutants loses tumor suppression activity and subsequently gain oncogenic activities to promote tumourigenesis. Tumor-specific DNA has recently been detected in the plasma of breast cancer patients. Detection of tumor-specific genetic materials in cancer patients may provide a unique and valuable tumor marker for diagnosis and prognosis. Commercially available MDA-468 breast cancer cell line was used for the proposed study.

Keywords: tumor protein (P53), cancer mutants, MDA-468, tumor suppressor gene

Procedia PDF Downloads 475

Authors: Yazun Jarrar

Abstract:

Smoking has effect on platelet aggregation and the activity of anti-platelet drugs. The chemical 20-hydroxyeicosatetraenoic acid (20-HETE) is a cardiotoxic arachidonic acid metabolite which increases platelet aggregation. In this study, we investigated the influence of cigarette smoking on 20-HETE levels and protein expression of 20-HETE producing enzyme CYP4A11 in isolated platelets from smoker and non-smoker volunteers. The protein expression and 20-HETE levels were analyzed using immunoblot and High-Performance Liquid Chromatography with Mass Spectrometry (HPL-MS) assays. The results showed that 20-HETE level was higher significantly among smokers than non-smokers (t-test, p-value<0.05). The protein expression of CYP4A11 was significantly higher (t-test, p-value<0.05) among the platelets of smokers. We concluded that cigarette smoking increased the level of platelet activator 20-HETE through increasing the protein expression of CYP4A11. These findings may increase the understanding of smoking-drug interaction during antiplatelets therapy.

Keywords: smoking, 20-HETE, CYP4A11, platelet

Procedia PDF Downloads 180

Authors: Aghasadeghi MR., Bahramali G., Sadat SM., Sadeghi SA., Yousefi M., Khodaei K., Ghorbani M., Sadat Larijani M.

Abstract:

Background:The emerging COVID-19 pandemic is a serious concernfor the public health worldwide. Despite the many mutations in the virus genome, it is important to find an effective vaccine against viral mutations. Therefore, in current study, we aimed at immunoinformatic evaluation of the virus proteins immunogenicity to design a preventive vaccine candidate, which could elicit humoral and cellular immune responses as well. Methods:Three antigenic regions are included;Spike, Membrane, and Nucleocapsid amino acid sequences were obtained, and possible fusion proteins were assessed andcompared by immunogenicity, structural features, and population coverage. The best fusion protein was also evaluated for MHC-I and MHC-II T-cell epitopes and the linear and conformational B-cell epitopes. Results: Among the four predicted models, the truncated Spike protein in fusion with M and N proteins is composed of 24 highly immunogenic human MHC class I and 29 MHC class II, along with 14 B-cell linear and 61 discontinues epitopes. Also, the selected protein has high antigenicity and acceptable population coverage of 82.95% in Iran and 92.51% in Europe. Conclusion: The data indicate that the truncated Spike-M-N SARS-CoV-2form which could be potential targets of neutralizing antibodies. The protein also has the ability to stimulate humoral and cellular immunity. The in silico study provided the fusion protein as a potential preventive vaccine candidate for further in vivo evaluation.

Keywords: SARS-CoV-2, immunoinformatic, protein, vaccine

Procedia PDF Downloads 217