Search results for: Amino%20acids

Authors: R. Razavizadeh, F. Adabavazeh, M. Rezaee Chermahini

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Salinity is one of the most widespread agricultural problems in arid and semi-arid areas that limits the plant growth and crop productivity. In this study, the salt stress effects on protein, reducing sugar, proline contents and antioxidant enzymes activities of Carum copticum L. under in vitro conditions were studied. Seeds of C. copticum were cultured in Murashige and Skoog (MS) medium containing 0, 25, 50, 100 and 150 mM NaCl and calli were cultured in MS medium containing 1 μM 2, 4-dichlorophenoxyacetic acid, 4 μM benzyl amino purine and different levels of NaCl (0, 25, 50, 100 and 150 mM). After NaCl treatment for 28 days, the proline and reducing sugar contents of shoots, roots and calli increased significantly in relation to the severity of the salt stress. The highest amount of proline and carbohydrate were observed at 150 and 100 mM NaCl, respectively. The reducing sugar accumulation in shoots was the highest as compared to roots, whereas, proline contents did not show any significant difference in roots and shoots under salt stress. The results showed significant reduction of protein contents in seedlings and calli. Based on these results, proteins extracted from the shoots, roots and calli of C. copticum treated with 150 mM NaCl showed the lowest contents. The positive relationships were observed between activity of antioxidant enzymes and the increase in stress levels. Catalase, ascorbate peroxidase and superoxide dismutase activity increased significantly under salt concentrations in comparison to the control. These results suggest that the accumulation of proline and sugars, and activation of antioxidant enzymes play adaptive roles in the adaptation of seedlings and callus of C. copticum to saline conditions.

Keywords: Antioxidant enzymes, Carum copticum, organic solutes, salt stress.

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Authors: Sadegh Lotfieblisofla, Arash Khodabakhshi

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Tissue plasminogen activator (tPA) as a serine protease plays an important role in the fibrinolytic system and the dissolution of fibrin clots in human body. The production of this drug in plants such as tobacco could reduce its production costs. In this study, expression of tPA gene and protein targeting to different plant cell compartments, using various signal peptides has been investigated. For high level of expression, Kozak sequence was used after CaMV35S in the beginning of the gene. In order to design the final construction, Extensin, KDEL (amino acid sequence including Lys-Asp-Glu-Leu) and SP (γ-zein signal peptide coding sequence) were used as leader signals to conduct this protein into apoplast, endoplasmic reticulum and cytosol spaces, respectively. Cloned human tPA gene under the CaMV (Cauliflower mosaic virus) 35S promoter and NOS (Nopaline Synthase) terminator into pBI121 plasmid was transferred into tobacco explants by Agrobacterium tumefaciens strain LBA₄₄₀₄. The presence and copy number of genes in transgenic tobacco was proved by Southern blotting. Enzymatic activity of the rt-PA protein in transgenic plants compared to non-transgenic plants was confirmed by Zymography assay. The presence and amount of rt-PA recombinant protein in plants was estimated by ELISA analysis on crude protein extract of transgenic tobacco using a specific antibody. The yield of recombinant tPA in transgenic tobacco for SP, KDEL, Extensin signals were counted 0.50, 0.68, 0.69 microgram per milligram of total soluble proteins.

Keywords: Recombinant tissue plasminogen activator, plant cell comportment, leader signals, transgenic tobacco.

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Authors: Abu Salim Mustafa

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Rv3873 is a relatively large size protein (371 amino acids in length) and its gene is located in the immunodominant genomic region of difference (RD)1 that is present in the genome of Mycobacterium tuberculosis but deleted from the genomes of all the vaccine strains of Bacillus Calmette Guerin (BCG) and most other mycobacteria. However, when tested for cellular immune responses using peripheral blood mononuclear cells from tuberculosis patients and BCG-vaccinated healthy subjects, this protein was found to be a major stimulator of cell mediated immune responses in both groups of subjects. In order to further identify the sequence of immunodominant epitopes and explore their Human Leukocyte Antigen (HLA)-restriction for epitope recognition, 24 peptides (25-mers overlapping with the neighboring peptides by 10 residues) covering the sequence of Rv3873 were synthesized chemically using fluorenylmethyloxycarbonyl chemistry and tested in cell mediated immune responses. The results of these experiments helped in the identification of an immunodominant peptide P9 that was recognized by people expressing varying HLA-DR types. Furthermore, it was also predicted to be a promiscuous binder with multiple epitopes for binding to HLA-DR, HLA-DP and HLA-DQ alleles of HLA-class II molecules that present antigens to T helper cells, and to HLA-class I molecules that present antigens to T cytotoxic cells. In addition, the evaluation of peptide P9 using an immunogenicity predictor server yielded a high score (0.94), which indicated a greater probability of this peptide to elicit a protective cellular immune response. In conclusion, P9, a peptide with multiple epitopes and ability to bind several HLA class I and class II molecules for presentation to cells of the cellular immune response, may be useful as a peptide-based vaccine against tuberculosis.

Keywords: Mycobacterium tuberculosis, Rv3873, peptides, vaccine

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Authors: Abdellali Kelil, Shengrui Wang, Ryszard Brzezinski

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The literature reports a large number of approaches for measuring the similarity between protein sequences. Most of these approaches estimate this similarity using alignment-based techniques that do not necessarily yield biologically plausible results, for two reasons. First, for the case of non-alignable (i.e., not yet definitively aligned and biologically approved) sequences such as multi-domain, circular permutation and tandem repeat protein sequences, alignment-based approaches do not succeed in producing biologically plausible results. This is due to the nature of the alignment, which is based on the matching of subsequences in equivalent positions, while non-alignable proteins often have similar and conserved domains in non-equivalent positions. Second, the alignment-based approaches lead to similarity measures that depend heavily on the parameters set by the user for the alignment (e.g., gap penalties and substitution matrices). For easily alignable protein sequences, it's possible to supply a suitable combination of input parameters that allows such an approach to yield biologically plausible results. However, for difficult-to-align protein sequences, supplying different combinations of input parameters yields different results. Such variable results create ambiguities and complicate the similarity measurement task. To overcome these drawbacks, this paper describes a novel and effective approach for measuring the similarity between protein sequences, called SAF for Substitution and Alignment Free. Without resorting either to the alignment of protein sequences or to substitution relations between amino acids, SAF is able to efficiently detect the significant subsequences that best represent the intrinsic properties of protein sequences, those underlying the chronological dependencies of structural features and biochemical activities of protein sequences. Moreover, by using a new efficient subsequence matching scheme, SAF more efficiently handles protein sequences that contain similar structural features with significant meaning in chronologically non-equivalent positions. To show the effectiveness of SAF, extensive experiments were performed on protein datasets from different databases, and the results were compared with those obtained by several mainstream algorithms.

Keywords: Protein, Similarity, Substitution, Alignment.

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Authors: N. Umasuthan, S. D. N. K. Bathige, W. S. Thulasitha, I. Whang, J. Lee

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The MyD88 is an evolutionarily conserved host-expressed adaptor protein that is essential for proper TLR/ IL1R immune-response signaling. A previously identified complete cDNA (1626 bp) of OfMyD88 comprised an ORF of 867 bp encoding a protein of 288 amino acids (32.9 kDa). The gDNA (3761 bp) of OfMyD88 revealed a quinquepartite genome organization composed of 5 exons (with the sizes of 310, 132, 178, 92 and 155 bp) separated by 4 introns. All the introns displayed splice signals consistent with the consensus GT/AG rule. A bipartite domain structure with two domains namely death domain (24-103) coded by 1^st exon, and TIR domain (151-288) coded by last 3 exons were identified through in silico analysis. Moreover, homology modeling of these two domains revealed a similar quaternary folding nature between human and rock bream homologs. A comprehensive comparison of vertebrate MyD88 genes showed that they possess a 5-exonic structure.In this structure, the last three exons were strongly conserved, and this suggests that a rigid structure has been maintained during vertebrate evolution.A cluster of TATA box-like sequences were found 0.25 kb upstream of cDNA starting position. In addition, putative 5'-flanking region of OfMyD88 was predicted to have TFBS implicated with TLR signaling, including copies of NFkB1, APRF/ STAT3, Sp1, IRF1 and 2 and Stat1/2. Using qPCR technique, a ubiquitous mRNA expression was detected in liver and blood. Furthermore, a significantly up-regulated transcriptional expression of OfMyD88 was detected in head kidney (12-24 h; >2-fold), spleen (6 h; 1.5-fold), liver (3 h; 1.9-fold) and intestine (24 h; ~2-fold) post-Fla challenge. These data suggest a crucial role for MyD88 in antibacterial immunity of teleosts.

Keywords: MyD88, Innate immunity, Flagellin, Genomic analysis.

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Authors: P. Slepicka, N. Slepickova Kasalkova, I. Michaljanicova, O. Nedela, Z. Kolska, V. Svorcik

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Polymers exposed to laser or plasma treatment or modified with different wet methods which enable the introduction of nanoparticles or biologically active species, such as amino-acids, may find many applications both as biocompatible or anti-bacterial materials or on the contrary, can be applied for a decrease in the number of cells on the treated surface which opens application in single cell units. For the experiments, two types of materials were chosen, a representative of non-biodegradable polymers, polyethersulphone (PES) and polyhydroxybutyrate (PHB) as biodegradable material. Exposure of solid substrate to laser well below the ablation threshold can lead to formation of various surface structures. The ripples have a period roughly comparable to the wavelength of the incident laser radiation, and their dimensions depend on many factors, such as chemical composition of the polymer substrate, laser wavelength and the angle of incidence. On the contrary, biopolymers may significantly change their surface roughness and thus influence cell compatibility. The focus was on the surface treatment of PES and PHB by pulse excimer KrF laser with wavelength of 248 nm. The changes of physicochemical properties, surface morphology, surface chemistry and ablation of exposed polymers were studied both for PES and PHB. Several analytical methods involving atomic force microscopy, gravimetry, scanning electron microscopy and others were used for the analysis of the treated surface. It was found that the combination of certain input parameters leads not only to the formation of optimal narrow pattern, but to the combination of a ripple and a wrinkle-like structure, which could be an optimal candidate for cell attachment. The interaction of different types of cells and their interactions with the laser exposed surface were studied. It was found that laser treatment contributes as a major factor for wettability/contact angle change. The combination of optimal laser energy and pulse number was used for the construction of a surface with an anti-cellular response. Due to the simple laser treatment, we were able to prepare a biopolymer surface with higher roughness and thus significantly influence the area of growth of different types of cells (U-2 OS cells).

Keywords: Polymer treatment, laser, periodic pattern, cell response.

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Authors: Parisa Shirzadeh

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Drug delivery systems in which drugs are traditionally used, multi-stage and at specified intervals by patients, do not meet the needs of the world's up-to-date drug delivery. In today's world, we are dealing with a huge number of recombinant peptide and protean drugs and analogues of hormones in the body, most of which are made with genetic engineering techniques. Most of these drugs are used to treat critical diseases such as cancer. Due to the limitations of the traditional method, researchers sought to find ways to solve the problems of the traditional method to a large extent. Following these efforts, controlled drug release systems were introduced, which have many advantages. Using controlled release of the drug in the body, the concentration of the drug is kept at a certain level, and in a short time, it is done at a higher rate. Graphene is a natural material that is biodegradable, non-toxic, natural and wide surfaces of graphene plates makes it more effective to modify graphene than carbon nanotubes. Graphene oxide is often synthesized using concentrated oxidizers such as sulfuric acid, nitric acid, and potassium permanganate based on Hummer method. graphene oxide is very hydrophilic and easily dissolves in water and creates a stable solution. Graphene oxide (GO) has been modified by chitosan (CS) covalently, developed for control release of doxorubicin (DOX). In this study, GO is produced by the hummer method under acidic conditions. Then, it is chlorinated by oxalyl chloride to increase its reactivity against amine. After that, in the presence of CS, the amino reaction was performed to form amide transplantation, and the DOX was connected to the carrier surface by π-π interaction in buffer phosphate. GO, GO-CS, and GO-CS-DOX were characterized by FT-IR and TGA to recognize new functional groups which show the new bonding of CS to GO, RAMA and SEM to recognize size of layers that show changing in size and number of layers. The ability to load and release is determined by UV-Visible spectroscopy. The loading result showed a high capacity of DOX absorption (99%) and pH dependence identified as a result of DOX release from GO-CS nanosheet at pH 5.3 and 7.4, which show a fast release rate in acidic conditions.

Keywords: Graphene oxide, chitosan, nanosheet, controlled drug release, doxorubicin.

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

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Rice bran is normally used as a raw material for rice bran oil production or sold as feed with a low price. Conventionally, the protein in defatted rice bran was extracted using alkaline extraction and acid precipitation, which involves in chemical usage and lowering some nutritious component. This study was conducted in order to extract of rice bran protein concentrate (RBPC) from defatted rice bran using enzymes and employing polysaccharides in a precipitating step. The properties of RBPC obtained will be compared 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 1:0.016) exhibited the highest protein (27.55%) and yield (6.84%). 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 capacity and foaming 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 e.g. healthy drink with high protein and fiber. In addition, the basic knowledge of functional property of rice bran protein concentrate was obtained, which can be used to appropriately select the application of this value-added product from rice bran.

Keywords: Alginate, carrageenan, rice bran, rice bran protein.

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Authors: U. K. Hulihalli, Shantveerayya

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Buckwheat (Fagopyrum esculentum Moench) is an annual crop belongs to family Poligonaceae. The cultivated buckwheat species are notable for their exceptional nutritive values. It is an important source of carbohydrates, fibre, macro, and microelements such as K, Ca, Mg, Na and Mn, Zn, Se, and Cu. It also contains rutin, flavonoids, riboflavin, pyridoxine and many amino acids which have beneficial effects on human health, including lowering both blood lipid and sugar levels. Rutin, quercetin and some other polyphenols are potent carcinogens against colon and other cancers. Buckwheat has significant nutritive value and plenty of uses. Cultivation of buckwheat in Sothern part of India is very meager. Hence, a study was planned with an objective to know the performance of buckwheat genotypes to different planting geometries and fertility levels. The field experiment was conducted at Main Agriculture Research Station, University of Agriculture Sciences, Dharwad, India, during 2017 Kharif. The experiment was laid-out in split-plot design with three replications having three planting geometries as main plots, two genotypes as sub plots and three fertility levels as sub-sub plot treatments. The soil of the experimental site was vertisol. The standard procedures are followed to record the observations. The planting geometry of 30*10 cm was recorded significantly higher seed yield (893 kg/ha⁻¹), stover yield (1507 kg ha⁻¹), clusters plant⁻¹ (7.4), seeds clusters⁻¹ (7.9) and 1000 seed weight (26.1 g) as compared to 40*10 cm and 20*10 cm planting geometries. Between the genotypes, significantly higher seed yield (943 kg ha⁻¹) and harvest index (45.1) was observed with genotype IC-79147 as compared to PRB-1 genotype (687 kg ha⁻¹ and 34.2, respectively). However, the genotype PRB-1 recorded significantly higher stover yield (1344 kg ha⁻¹) as compared to genotype IC-79147 (1173 kg ha⁻¹). The genotype IC-79147 was recorded significantly higher clusters plant⁻¹ (7.1), seeds clusters⁻¹ (7.9) and 1000 seed weight (24.5 g) as compared PRB-1 (5.4, 5.8 and 22.3 g, respectively). Among the fertility levels tried, the fertility level of 60:30 NP kg ha⁻¹ recorded significantly higher seed yield (845 kg ha^-1) and stover yield (1359 kg ha⁻¹) as compared to 40:20 NP kg ha^-1 (808 and 1259 kg ha⁻¹ respectively) and 20:10 NP kg ha^-1 (793 and 1144 kg ha⁻¹ respectively). Within the treatment combinations, IC 79147 genotype having 30*10 cm planting geometry with 60:30 NP kg ha⁻¹ recorded significantly higher seed yield (1070 kg ha⁻¹), clusters plant⁻¹ (10.3), seeds clusters⁻¹ (9.9) and 1000 seed weight (27.3 g) compared to other treatment combinations.

Keywords: Buckwheat, fertility levels, genotypes, geometry, polyphenols, rutin.

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Authors: Wisam H. Benamer, Ehab A. Elfallah, Mohamed A. Elshaari, Farag A. Elshaari

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A challenge for laboratories is to provide bacterial identification and antibiotic sensitivity results within a short time. Hence, advancement in the required technology is desirable to improve timing, accuracy and quality. Even with the current advances in methods used for both phenotypic and genotypic identification of bacteria the need is there to develop method(s) that enhance the outcome of bacteriology laboratories in accuracy and time. The hypothesis introduced here is based on the assumption that the chromosome of any bacteria contains unique sequences that can be used for its identification and typing. The outcome of a pilot study designed to test this hypothesis is reported in this manuscript. Methods: The complete chromosome sequences of several bacterial species were downloaded to use as search targets for unique sequences. Visual basic and SQL server (2014) were used to generate a complete set of 18-base long primers, a process started with reverse translation of randomly chosen 6 amino acids to limit the number of the generated primers. In addition, the software used to scan the downloaded chromosomes using the generated primers for similarities was designed, and the resulting hits were classified according to the number of similar chromosomal sequences, i.e., unique or otherwise. Results: All primers that had identical/similar sequences in the selected genome sequence(s) were classified according to the number of hits in the chromosomes search. Those that were identical to a single site on a single bacterial chromosome were referred to as unique. On the other hand, most generated primers sequences were identical to multiple sites on a single or multiple chromosomes. Following scanning, the generated primers were classified based on ability to differentiate between medically important bacterial and the initial results looks promising. Conclusion: A simple strategy that started by generating primers was introduced; the primers were used to screen bacterial genomes for match. Primer(s) that were uniquely identical to specific DNA sequence on a specific bacterial chromosome were selected. The identified unique sequence can be used in different molecular diagnostic techniques, possibly to identify bacteria. In addition, a single primer that can identify multiple sites in a single chromosome can be exploited for region or genome identification. Although genomes sequences draft of isolates of organism DNA enable high throughput primer design using alignment strategy, and this enhances diagnostic performance in comparison to traditional molecular assays. In this method the generated primers can be used to identify an organism before the draft sequence is completed. In addition, the generated primers can be used to build a bank for easy access of the primers that can be used to identify bacteria.

Keywords: Bacteria chromosome, bacterial identification, sequence, primer generation.

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