Search results for: protein expression

Authors: Patrick Nwabueze Okechukwu, Ima Nirwana Soelaiman, Gabriele Anisah Ruth Froemming, Mohd Yusri Idorus, Norazlina Mohamed

Abstract:

Supplementation of palm vitamin E has been reported to prevent loss of bone density in ovariectomised female rats. The mechanism by which palm vitamin E exerts these effects is still unknown. We hypothesized that palm vitamin E may act by preventing the protein expression changes. Two dimensional poly acyrilamide gel electrophoresis (2-D PAGE) and PD Quest software genomic solutions Investigator (proteomics) was used to analyze the differential protein expression profile in femoral and humeri bones harvested from three groups of rats; sham-operated rats (SO), ovariectomised rats (Ovx) and ovariectomised rats supplemented for 2 months with palm vitamin E. The results showed that there were over 300 valued spot on each of the groups PVE and OVX as compared to about 200 in SO. Comparison between the differential protein expression between OVX and PVE groups showed that ten spots were down –regulated in OVX but up-regulated in PVE. The ten differential spots were separately named P1-P10. The identification and understanding of the pathway of the differential protein expression among the groups is ongoing and may account for the molecular mechanism through which palm vitamin E exert its anti-osteoporotic effect.

Keywords: Palm vitamin E, ovariectomised, osteoporosis protein expression, 2-d-page.

Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 1894

Authors: Ming-Lun Chiang, Hsi-Chia Chen, Chieh Wu, Yu-Ting Tseng, Ming-Ju Chen

Abstract:

In this study, three strains of Vibrio parahaemolyticus (690, BCRC 13023 and BCRC 13025) were subjected to acid adaptation at pH 5.5 for 90 min. The survival of acid-adapted and non-adapted V. parahaemolyticus strains under simulated gastric condition and their protein expression profiles were investigated. Results showed that acid adaptation increased the survival of the test V. parahaemolyticus strains after exposure to simulated gastric juice (pH 3). Additionally, acid adaptation also affected the protein expression in these V. parahaemolyticus strains. Nine proteins, identified as atpA, atpB, DnaK, GroEL, OmpU, enolase, fructose-bisphosphate aldolase, phosphoglycerate kinase and triosephosphate isomerase, were induced by acid adaptation in two or three of the test strains. These acid-adaptive proteins may play important regulatory roles in the acid tolerance response (ATR) of V. parahaemolyticus.

Keywords: Acid adaptation, protein expression, simulated gastric juice, Vibrio parahaemolyticus

Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 1588

Authors: S. Banerjee, A. Apte Deshpande, N. Mandi, S. Padmanabhan

Abstract:

We report a novel fusion tag for expressing recombinant proteins in E. coli. The fusion tag is the C-terminus part of the human GMCSF gene comprising 45 amino acids, which aid in over expression of otherwise non expressible genes. Expression of hIFN a2b with this fusion tag also escapes the requirement of rare codons for expression. This is also a first report of a small fusion tag of human origin having affinity to heparin sepharose column facilitating the purification of fusion protein.

Keywords: fusion tag, bacterial expression, rare codons, human GMCSF

Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 1897

Authors: Masaki Yamaguchi, Akira Ito, Noriaki Okamoto, Yoshinori Kawabe, Masamichi Kamihira

Abstract:

Heat-inducible gene expression vectors are useful for hyperthermia-induced cancer gene therapy, because the combination of hyperthermia and gene therapy can considerably improve the therapeutic effects. In the present study, we developed an enhanced heat-inducible transgene expression system in which a heat-shock protein (HSP) promoter and tetracycline-responsive transactivator were combined. When the transactivator plasmid containing the tetracycline-responsive transactivator gene was co-transfected with the reporter gene expression plasmid, a high level of heat-induced gene expression was observed compared with that using the HSP promoter without the transactivator. In vitro evaluation of the therapeutic effect using HeLa cells showed that heat-induced therapeutic gene expression caused cell death in a high percentage of these cells, indicating that this strategy is promising for cancer gene therapy.

Keywords: Inducible gene expression, Gene therapy, Hyperthermia, Heat shock protein, Tetracycline transactivator.

Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 2135

Authors: Fawzi Irshaid, Fatiha Dilmi, Khaled Tarawneh, Raji Hadeth, Adnan Jaran, Ahad Al-Khatib

Abstract:

P16/INK4A is tumor suppressor protein that plays a critical role in cell cycle regulation. Loss of P16 protein expression has been implicated in pathogenesis of many cancers, including lymphoma. Therefore, we sought to investigate if loss of P16 protein expression is associated with lymphoma and/or any specific lymphoma subtypes (Hodgkin-s lymphoma (HL) and nonHodgkin-s lymphoma (NHL)). Fifty-five lymphoma cases consisted of 30 cases of HL and 25 cases of NHL, with an age range of 3 to 78 years, were examined for loss of P16 by immunohistochemical technique using a specific antibody reacting against P16. In total, P16 loss was seen in 33% of all lymphoma cases. P16 loss was identified in 47.7% of HL cases. In contrast, only 16% of NHL showed loss of P16. Loss of P16 was seen in 67% of HL patients with 50 years of age or older, whereas P16 loss was found in only 42% of HL patients with less than 50 years of age. P16 loss in HL is somewhat higher in male (55%) than in female (30%). In subtypes of HL, P16 loss was found exclusively in all cases of lymphocyte depletion, lymphocyte predominance and unclassified cases, whereas P16 loss was seen in 39% of mixed cellularity and 29% of nodular sclerosis cases. In low grade NHL patients, P16 loss was seen in approximately one-third of cases, whereas no or very rare of P16 loss was found in intermediate and high grade cases. P16 loss did not show any correlation with age or gender of NHL patients. In conclusion, the high rate of P16 loss seen in our study suggests that loss of P16 expression plays a critical role in the pathogenesis of lymphoma, particularly with HL.

Keywords: B-cells, immunostaining, P16 protein, Reed-Sternberg cells, tumors.

Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 1665

Authors: Uun Yanuhar

Abstract:

The purpose of research was to know the role of immunogenic protein of 49 kDa from V.alginolyticus which capable to initiate molecule expression of MHC Class II in receptor of Cromileptes altivelis. The method used was in vivo experimental research through testing of immunogenic protein 49 kDa from V.alginolyticus at Cromileptes altivelis (size of 250 - 300 grams) using 3 times booster by injecting an immunogenic protein in a intramuscular manner. Response of expressed MHC molecule was shown using immunocytochemistry method and SEM. Results indicated that adhesin V.alginolyticus 49 kDa which have immunogenic character could trigger expression of MHC class II on receptor of grouper and has been proven by staining using immunocytochemistry and SEM with labeling using antibody anti MHC (anti mouse). This visible expression based on binding between epitopes antigen and antibody anti MHC in the receptor. Using immunocytochemistry, intracellular response of MHC to in vivo induction of immunogenic adhesin from V.alginolyticus was shown.

Keywords: C.altivelis, immunogenic, MHC, V.alginolyticus.

Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 1237

Authors: Chan-Jung Liang, Shu-Huei Wang, Pei-Jhen Wu, Jaw-Shiun Tsai, Chau-Chung Wu, Yuh-Lien Chen

Abstract:

Heme oxygenase-1 (HO-1), an enzyme degrading heme to carbon monoxide, iron, and biliverdin, has been recognized as playing a crucial role in cellular defense against stressful conditions, not only related to heme release. In the present study, the effects of TNF-a on the expression of heme oxygenase-1 (HO-1) in human aortic endothelial cells (HAECs) as well as the related mechanisms were investigated. 10 ng/mL TNF-α treatment significantly increased HO-1 expression after 6h, then a further increase at 12h and declined at 24h. Treatment with 2 ng/mL of TNF-a after 12 h resulted in a significant increase in HO-1 expression, which peaked at 10 ng/mL, then declined at 20 ng/mL. TNF-α induced HO-1 expression and then HO-1 expression reduced  vascular cell adhesion molecule-1 (VCAM-1) expression. Phosphorylation studies of ERK1/2, JNK, and p38, three subgroups of mitogen-activated protein kinases (MAPKs) demonstrated TNF-α-induced ERK1/2, JNK, and p38 phosphorylation. The increase in HO-1 expression in response to TNF-α treatment was affected by pretreatment with SP600125 (a JNK inhibitor) and SB203580 (a p38 inhibitor), not with PD98059 (an ERK1/2 inhibitor). The expression of HO-1 was stronger in aortas of TNF-α-treated apo-E deficient mice when compared with control mice. These results suggest that low dose of TNF-α treatment notably induced HO-1 expression was mediated through JNK/p38 phosphorylation and may have a protective potential in cardiovascular diseases and inflammatory response through the regulation of HO-1 expression.

Keywords: Heme oxygenase-1 inflammation, endothelial cells, mitogen-activated protein kinases (MAPKs).

Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 5737

Authors: Ngo Thanh Xuan, Mai Thi Hang, Vu Nguyen Thanh

Abstract:

A. niger XP isolated from Vietnam produces very low amount of acidic phytase with optimal pH at 2.5 and 5.5. The phytase production of this strain was successfully improved through gene cloning and expression. A 1.4 - kb DNA fragment containing the coding region of the phyA gene was amplified by PCR and inserted into the expression vector pPICZαA with a signal peptide α- factor, under the control of AOX1 promoter. The recombined plasmid was transformed into the host strain P. pastoris KM71H and X33 by electroporation. Both host strains could efficiently express and secret phytase. The multicopy strains were screened for over expression of phytase. All the selected multicopy strains of P. pastoris X33 were examined for phytase activity, the maximum phytase yield of 1329 IU/ml was obtained after 4 days of incubation in medium BMM. The recombinant protein with MW of 97.4 KW showed to be the only one protein secreted in the culture broth. Multicopy transformant P. pastoris X33 supposed to be potential candidate for producing the commercial preparation of phytase.

Keywords: Aspergillus niger XP, cloning, over expression, Pichia pastoris, phyA , phytase.

Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 2211

Authors: Tzyy Rong Jinn, Nguyen Tiep Khac, Tzong Yuan Wu

Abstract:

Highly pathogenic avian influenza (HPAI) H5N1 viruses have created demand for a cost-effective vaccine to prevent a pandemic of the disease. Here, we report that Trichoplusia ni (T. ni) larvae can act as a cost-effective bioreactor to produce recombinant HA5 (rH5HA) proteins as an potential effective vaccine for chickens. To facilitate the recombinant virus identification, virus titer determination and access the infected larvae, we employed the internal ribosome entry site (IRES) derived from Perina nuda virus (PnV, belongs to insect picorna like Iflavirus genus) to construct a bi-cistronic baculovirus expression vector that can express the rH5HA protein and enhanced green fluorescent protein (EGFP) simultaneously. Western blot analysis revealed that the 70 kDa rH5HA protein and partially cleaved products (40 kDa H5HA1) were generated in T. ni larvae infected with recombinant baculovirus carrying the H5HA gene. These data suggest that the baculovirus-larvae recombinant protein expression system could be a cost-effective platform for H5N1 vaccine production.

Keywords: Avian Influenza, baculovirus, hemagglutinin, Trichoplusia ni larvae

Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 1816

Authors: Nazar Zaki, Safaai Deris, Hany Alashwal

Abstract:

Detecting protein-protein interactions is a central problem in computational biology and aberrant such interactions may have implicated in a number of neurological disorders. As a result, the prediction of protein-protein interactions has recently received considerable attention from biologist around the globe. Computational tools that are capable of effectively identifying protein-protein interactions are much needed. In this paper, we propose a method to detect protein-protein interaction based on substring similarity measure. Two protein sequences may interact by the mean of the similarities of the substrings they contain. When applied on the currently available protein-protein interaction data for the yeast Saccharomyces cerevisiae, the proposed method delivered reasonable improvement over the existing ones.

Keywords: Protein-Protein Interaction, support vector machine, feature extraction, pairwise alignment, Smith-Waterman score.

Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 1936

Authors: Fiona Browne, Huiru Zheng, Haiying Wang, Francisco Azuaje

Abstract:

Recent years have seen a growing trend towards the integration of multiple information sources to support large-scale prediction of protein-protein interaction (PPI) networks in model organisms. Despite advances in computational approaches, the combination of multiple “omic" datasets representing the same type of data, e.g. different gene expression datasets, has not been rigorously studied. Furthermore, there is a need to further investigate the inference capability of powerful approaches, such as fullyconnected Bayesian networks, in the context of the prediction of PPI networks. This paper addresses these limitations by proposing a Bayesian approach to integrate multiple datasets, some of which encode the same type of “omic" data to support the identification of PPI networks. The case study reported involved the combination of three gene expression datasets relevant to human heart failure (HF). In comparison with two traditional methods, Naive Bayesian and maximum likelihood ratio approaches, the proposed technique can accurately identify known PPI and can be applied to infer potentially novel interactions.

Keywords: Bayesian network, Classification, Data integration, Protein interaction networks.

Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 1615

Authors: Maria E. Manioudaki, Panayiota Poirazi

Abstract:

Yeast cells live in a constantly changing environment that requires the continuous adaptation of their genomic program in order to sustain their homeostasis, survive and proliferate. Due to the advancement of high throughput technologies, there is currently a large amount of data such as gene expression, gene deletion and protein-protein interactions for S. Cerevisiae under various environmental conditions. Mining these datasets requires efficient computational methods capable of integrating different types of data, identifying inter-relations between different components and inferring functional groups or 'modules' that shape intracellular processes. This study uses computational methods to delineate some of the mechanisms used by yeast cells to respond to environmental changes. The GRAM algorithm is first used to integrate gene expression data and ChIP-chip data in order to find modules of coexpressed and co-regulated genes as well as the transcription factors (TFs) that regulate these modules. Since transcription factors are themselves transcriptionally regulated, a three-layer regulatory cascade consisting of the TF-regulators, the TFs and the regulated modules is subsequently considered. This three-layer cascade is then modeled quantitatively using artificial neural networks (ANNs) where the input layer corresponds to the expression of the up-stream transcription factors (TF-regulators) and the output layer corresponds to the expression of genes within each module. This work shows that (a) the expression of at least 33 genes over time and for different stress conditions is well predicted by the expression of the top layer transcription factors, including cases in which the effect of up-stream regulators is shifted in time and (b) identifies at least 6 novel regulatory interactions that were not previously associated with stress-induced changes in gene expression. These findings suggest that the combination of gene expression and protein-DNA interaction data with artificial neural networks can successfully model biological pathways and capture quantitative dependencies between distant regulators and downstream genes.

Keywords: gene modules, artificial neural networks, yeast, stress

Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 1464

Authors: Rezvan Najafi, Korosh Heydari, Massoud Saidijam

Abstract:

5-FU is a chemotherapeutic agent that has been used in colorectal cancer (CRC) treatment. However, it is usually associated with the acquired resistance, which decreases the therapeutic effects of 5-FU. miR-200c is involved in chemotherapeutic drug resistance, but its mechanism is not fully understood. In this study, the effect of inhibition of miR-200c in sensitivity of HCT-116 CRC cells to 5-FU was evaluated. HCT-116 cells were transfected with LNA-anti- miR-200c for 48 h. mRNA expression of miR-200c was evaluated using quantitative real- time PCR. The protein expression of phosphatase and tensin homolog (PTEN) and E-cadherin were analyzed by western blotting. Annexin V and propidium iodide staining assay were applied for apoptosis detection. The caspase-3 activation was evaluated by an enzymatic assay. The results showed LNA-anti-miR-200c inhibited the expression of PTEN and E-cadherin protein, apoptosis and activation of caspase 3 compared with control cells. In conclusion, these results suggest that miR-200c as a prognostic marker can overcome to 5-FU chemoresistance in CRC.

Keywords: Colorectal cancer, miR-200c, 5-FU resistance, E-cadherin, PTEN.

Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 916

Authors: Fawzi Irshaid, Adnan Jaran, Fatiha Dilmi, Khaled Tarawneh, Raji Hadeth, Ahad Al-Khatib

Abstract:

The aim of this study was to estimate the frequency of EBV infection in Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL) occurring in Jordanian patients. A total of 55 patients with lymphoma were examined in this study. Of 55 patients, 30 and 25 were diagnosed as HL and NHL, respectively. The four HL subtypes were observed with the majority of the cases exhibited the mixed cellularity (MC) subtype followed by the nodular sclerosis (NS). The high grade was found to be the commonest subtype of NHL in our sample, followed by the low grade. The presence of EBV virus was detected by immunostating for expression of latent membrane protein-1 (LMP-1). The frequency of LMP-1 expression occurred more frequent in patients with HL (60.0%) than in patients with NHL (32.0%). The frequency of LMP-1 expression was also higher in patients with MC subtype (61.11%) than those patients with NS (28.57%). No age or gender difference in occurrence of EBV infection was observed among patient with HL. By contrast, the prevalence of EBV infection in NHL patients aged below 50 was lower (16.66%) than in NHL patients aged 50 or above (46.15%). In addition, EBV infection was more frequent in females with NHL (38.46%) than in male with NHL (25%). In NHL cases, the frequency of EBV infection in intermediate grade (60.0%) was high when compared with frequency of low (25%) or high grades (25%). In conclusion, analysis of LMP-1 expression indicates an important role for this viral oncogene in the pathogenesis of EBV-associated malignant lymphomas. These data also support the previous findings that people with EBV may develop lymphoma and that efforts to maintain low lymphoma should be considered for people with EBV infection.

Keywords: Hodgkin lymphoma, Epstein Barr virus, hematoxylin, infection, LMP-1 expression.

Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 1463

Authors: Hong Ding, Fatiha Benslimane, Isra Marei, Chris R. Triggle

Abstract:

Hyperglycaemia is a key factor that contributes to the development of diabetes-related microvascular disease and a major risk factor for endothelial dysfunction. In the current study, we have explored glucose-induced abnormal intracellular calcium (Ca2+ i) homeostasis in mouse microvessel endothelial cells (MMECs) in high glucose (HG) (40mmol/L) versus control (low glucose, LG) (11 mmol/L). We demonstrated that the exposure of MMECs to HG for 3 days did not change basal Ca2+ i, however, there was a significant increase of acetylcholine-induced Ca2+ entry. Western blots illustrated that exposure to HG also increased STIM1 (Stromal Interaction Molecule 1), but not Orai1 (the pore forming subunit), protein expression levels. Although the link between HG-induced changes in STIM1 expression, enhanced Ca2+ entry and endothelial dysfunction requires further study, the current data are suggestive that targeting these pathways may reduce the impact of HG on endothelial function.

Keywords: store-operated calcium entry, hyperglycaemia, STIM1, endothelial dysfunction

Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 1709

Authors: H. Sakamoto, Y. Nakagawara, S. Oguri

Abstract:

Drought stress is a critical environmental factor that adversely affects crop productivity and quality. Because of their immobile nature, plants have evolved mechanisms to sense and respond to drought stress. We identified a novel locus of Arabidopsis, designated DRA1 (drought responsive ankyrin1), whose disruption leads to increased drought-stress tolerance. DRA1 encodes a transmembrane protein with an ankyrin-repeat motif that has been implicated in diverse cellular processes such as signal transduction. RT-PCR analysis revealed that there were at least two splicing variants of DRA1 transcripts in wild-type plants. In response to drought stress, the levels of DRA1 transcripts retaining second and third introns were increased, whereas these introns were removed under unstressed conditions. These results suggest that DRA1 protein may negatively regulate plant drought tolerance and that the expression of DRA1is regulated in response to drought stress by alternative splicing.

Keywords: Alternative splicing, ankyrin repeat, Arabidopsis, drought tolerance.

Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 1764

Authors: H. Sakamoto, Y. Nakagawara, S. Oguri

Abstract:

Drought stress is a critical environmental factor that adversely affects crop productivity and quality. Because of their immobile nature, plants have evolved mechanisms to sense and respond to drought stress. We identified a novel locus of Arabidopsis, designated DRA1 (drought responsive ankyrin1), whose disruption leads to increased drought-stress tolerance. DRA1 encodes a transmembrane protein with an ankyrin-repeat motif that has been implicated in diverse cellular processes such as signal transduction. RT-PCR analysis revealed that there were at least two splicing variants of DRA1 transcripts in wild-type plants. In response to drought stress, the levels of DRA1 transcripts retaining second and third introns were increased, whereas these introns were removed under unstressed conditions. These results suggest that DRA1 protein may negatively regulate plant drought tolerance and that the expression of DRA1is regulated in response to drought stress by alternative splicing.

Keywords: Alternative splicing, ankyrin repeat, Arabidopsis, drought tolerance.

Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 1866

Authors: Sadegh Lotfieblisofla, Arash Khodabakhshi

Abstract:

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.

Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 714

Authors: Maysam Mard-Soltani, Mohamad Javad Rasaee, Saeed Khalili, Abdol Karim Sheikhi, Mehdi Hedayati

Abstract:

Production of specific antibody responses against hTSH is a cumbersome process due to the high identity between the hTSH and the other members of the glycoprotein hormone family (FSH, LH and HCG) and the high identity between the human hTSH and host animals for antibody production. Therefore, two polyclonal antibodies were purified against two recombinant proteins. Four possible ELISA tests were designed based on these antibodies. These ELISA tests were checked against hTSH and other glycoprotein hormones, and their sensitivity and specificity were assessed. Bioinformatics tools were used to analyze the immunological properties. After the immunogen region selection from hTSH protein, c terminal of B hTSH was selected and applied. Two recombinant genes, with these cut pieces (first: two repeats of C terminal of B hTSH, second: tetanous toxin+B hTSH C terminal), were designed and sub-cloned into the pET32a expression vector. Standard methods were used for protein expression, purification, and verification. Thereafter, immunizations of the white New Zealand rabbits were performed and the serums of them were used for antibody titration, purification and characterization. Then, four ELISA tests based on two antibodies were employed to assess the hTSH and other glycoprotein hormones. The results of these assessments were compared with standard amounts. The obtained results indicated that the desired antigens were successfully designed, sub-cloned, expressed, confirmed and used for in vivo immunization. The raised antibodies were capable of specific and sensitive hTSH detection, while the cross reactivity with the other members of the glycoprotein hormone family was minimum. Among the four designed tests, the test in which the antibody against first protein was used as capture antibody, and the antibody against second protein was used as detector antibody did not show any hook effect up to 50 miu/l. Both proteins have the ability to induce highly sensitive and specific antibody responses against the hTSH. One of the antibody combinations of these antibodies has the highest sensitivity and specificity in hTSH detection.

Keywords: hTSH, bioinformatics, protein expression, cross reactivity.

Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 1199

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

Abstract:

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

Keywords: Dihydroxyacetone kinase, essential genes, Methicillin-resistant Staphylococcus aureus, drug target.

Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 1769

Authors: Yanchao Wang, Rajshekhar Sunderraman

Abstract:

This paper presents an algebraic approach to optimize queries in domain-specific database management system for protein structure data. The approach involves the introduction of several protein structure specific algebraic operators to query the complex data stored in an object-oriented database system. The Protein Algebra provides an extensible set of high-level Genomic Data Types and Protein Data Types along with a comprehensive collection of appropriate genomic and protein functions. The paper also presents a query translator that converts high-level query specifications in algebra into low-level query specifications in Protein-QL, a query language designed to query protein structure data. The query transformation process uses a Protein Ontology that serves the purpose of a dictionary.

Keywords: Domain-Specific Data Management, Protein Algebra, Protein Ontology, Protein Structure Data.

Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 1542

Authors: Anna Trusek-Holownia, Andrzej Noworyta

Abstract:

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 APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 2643

Authors: N. Baspinar, A. Basoglu, O. Ozdemir, C. Ozel, F. Terzi, O. Yaman

Abstract:

Current research is targeting new molecular mechanisms that underlie non-alcoholic fatty liver disease (NAFLD) and associated metabolic disorders like non-alcoholic steatohepatitis (NASH). Forty New Zealand White rabbits have been used and fed a high protein (HP) and energy diet based on grains and containing 11.76 MJ/kg. Boron added to 3 experimental groups’ drinking waters (30 mg boron/L) as boron compounds. Biochemical analysis including boron levels, and nuclear magnetic resonance (NMR) based metabolomics evaluation, and mRNA expression of peroxisome proliferator-activated receptor (PPAR) family was performed. LDLcholesterol concentrations alone were decreased in all the experimental groups. Boron levels in serum and feces were increased. Content of acetate was in about 2x higher for anhydrous borax group, at least 3x higher for boric acid group. PPARα mRNA expression was significantly decreased in boric acid group. Anhydrous borax attenuated mRNA levels of PPARγ, which was further suppressed by boric acid. Boron supplementation decreased the degenerative alterations in hepatocytes. Except borax group other boron groups did not have a pronounced change in tubular epithels of kidney. In conclusion, high protein and energy diet leads hepatocytes’ degenerative changes which can be prevented by boron supplementation. Boric acid seems to be more effective in this situation.

Keywords: High protein and energy diet, boron, metabolomic, transcriptomic.

Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 1997

Authors: Hany Alashwal, Safaai Deris, Razib M. Othman

Abstract:

Predicting protein-protein interactions represent a key step in understanding proteins functions. This is due to the fact that proteins usually work in context of other proteins and rarely function alone. Machine learning techniques have been applied to predict protein-protein interactions. However, most of these techniques address this problem as a binary classification problem. Although it is easy to get a dataset of interacting proteins as positive examples, there are no experimentally confirmed non-interacting proteins to be considered as negative examples. Therefore, in this paper we solve this problem as a one-class classification problem using one-class support vector machines (SVM). Using only positive examples (interacting protein pairs) in training phase, the one-class SVM achieves accuracy of about 80%. These results imply that protein-protein interaction can be predicted using one-class classifier with comparable accuracy to the binary classifiers that use artificially constructed negative examples.

Keywords: Bioinformatics, Protein-protein interactions, One-Class Support Vector Machines

Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 1988

Authors: Hany Alashwal, Safaai Deris, Razib M. Othman

Abstract:

The protein domain structure has been widely used as the most informative sequence feature to computationally predict protein-protein interactions. However, in a recent study, a research group has reported a very high accuracy of 94% using hydrophobicity feature. Therefore, in this study we compare and verify the usefulness of protein domain structure and hydrophobicity properties as the sequence features. Using the Support Vector Machines (SVM) as the learning system, our results indicate that both features achieved accuracy of nearly 80%. Furthermore, domains structure had receiver operating characteristic (ROC) score of 0.8480 with running time of 34 seconds, while hydrophobicity had ROC score of 0.8159 with running time of 20,571 seconds (5.7 hours). These results indicate that protein-protein interaction can be predicted from domain structure with reliable accuracy and acceptable running time.

Keywords: Bioinformatics, protein-protein interactions, support vector machines, protein features.

Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 1917

Authors: Prasad Senadheera, Younousse Saidi, Frans JM Maathuis

Abstract:

Rice seed expression (cDNA) library in the Lambda Zap 11® phage constructed from the developing grain 10-20 days after flowering was transformed into yeast for functional complementation assays in three salt sensitive yeast mutants S. cerevisiae strain CY162, G19 and Axt3K. Transformed cells of G19 and Axt3K with pYES vector with cDNA inserts showed enhance tolerance than those with empty pYes vector. Sequencing of the cDNA inserts revealed that they encode for the putative proteins with the sequence homologous to rice putative protein PROLM24 (Os06g31070), a prolamin precursor. Expression of this cDNA did not affect yeast growth in absence of salt. Axt3k and G19 strains expressing the PROLM24 were able to grow upto 400 mM and 600 mM of NaCl respectively. Similarly, Axt3k mutant with PROLM24 expression showed comparatively higher growth rate in the medium with excess LiCl (50 mM). The observation that expression of PROLM24 rescued the salt sensitive phenotypes of G19 and Axt3k indicates the existence of a regulatory system that ameliorates the effect of salt stress in the transformed yeast mutants. However, the exact function of the cDNA sequence, which shows partial sequence homology to yeast UTR1 is not clear. Although UTR1 involved in ferrous uptake and iron homeostasis in yeast cells, there is no evidence to prove its role in Na+ homeostasis in yeast cells. Absence of transmembrane regions in Os06g31070 protein indicates that salt tolerance is achieved not through the direct functional complementation of the mutant genes but through an alternative mechanism.

Keywords: Rice seed expression, salt stress, prolamin, salinitytolerance, Oryza sativa

Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 1906

Authors: H. Sakamoto, S. Kurosawa, M. Suzuki, S. Oguri

Abstract:

In Arabidopsis, several genes encoding proteins with ankyrin repeats and transmembrane domains (AtANKTM) have been identified as mediators of biotic and abiotic stress responses. It has been known that the expression of an AtANKTM gene, At2g24600, is induced in response to abiotic stress and that there are four splicing variants derived from this locus. In this study, by RT-PCR and sequencing analysis, an unknown splicing variant of the At2g24600 transcript was identified. Based on differences in the predicted amino acid sequences, the five splicing variants are divided into three groups. The three predicted proteins are highly homologous, yet have different numbers of ankyrinrepeats and transmembrane domains. It is generally considered that ankyrin repeats mediate protein-protein interaction and that the number oftransmembrane domains affects membrane topology of proteins. The protein variants derived from the At2g24600 locus may have different molecular functions each other.

Keywords: Alternative splicing, ankyrin repeats, transmembrane domains, Arabidopsis.

Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 1917

Authors: Manpreet Singh, Parminder Kaur Wadhwa, Surinder Kaur

Abstract:

The drug discovery process starts with protein identification because proteins are responsible for many functions required for maintenance of life. Protein identification further needs determination of protein function. Proposed method develops a classifier for human protein function prediction. The model uses decision tree for classification process. The protein function is predicted on the basis of matched sequence derived features per each protein function. The research work includes the development of a tool which determines sequence derived features by analyzing different parameters. The other sequence derived features are determined using various web based tools.

Keywords: Sequence Derived Features, decision tree.

Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 1950

Authors: Waraporn Apiwatanapiwat, Pilanee Vaithanomsat, Phanu Somkliang, Taweesiri Malapant

Abstract:

This was the first document revealing the investigation of protein hydrolysate production optimization from J. curcas cake. Proximate analysis of raw material showed 18.98% protein, 5.31% ash, 8.52% moisture and 12.18% lipid. The appropriate protein hydrolysate production process began with grinding the J. curcas cake into small pieces. Then it was suspended in 2.5% sodium hydroxide solution with ratio between solution/ J. curcas cake at 80:1 (v/w). The hydrolysis reaction was controlled at temperature 50 °C in water bath for 45 minutes. After that, the supernatant (protein hydrolysate) was separated using centrifuge at 8000g for 30 minutes. The maximum yield of resulting protein hydrolysate was 73.27 % with 7.34% moisture, 71.69% total protein, 7.12% lipid, 2.49% ash. The product was also capable of well dissolving in water.

Keywords: Production, protein hydrolysate, Jatropha curcas cake, optimization.

Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 1954

Authors: Nazar Zaki, Safaai Deris

Abstract:

The amount of the information being churned out by the field of biology has jumped manifold and now requires the extensive use of computer techniques for the management of this information. The predominance of biological information such as protein sequence similarity in the biological information sea is key information for detecting protein evolutionary relationship. Protein sequence similarity typically implies homology, which in turn may imply structural and functional similarities. In this work, we propose, a learning method for detecting remote protein homology. The proposed method uses a transformation that converts protein sequence into fixed-dimensional representative feature vectors. Each feature vector records the sensitivity of a protein sequence to a set of amino acids substrings generated from the protein sequences of interest. These features are then used in conjunction with support vector machines for the detection of the protein remote homology. The proposed method is tested and evaluated on two different benchmark protein datasets and it-s able to deliver improvements over most of the existing homology detection methods.

Keywords: Protein homology detection; support vectormachine; string kernel.

Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 1391