Search results for: protein kinase C alpha

Authors: Yuting Wu, Faraz Choudhury, Daniel Benjamin, James Whalin, Joshua Blatz, Leon Shohet, Michael Sussman, Mark Richards

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Plasma-Induced Modification of Biomolecules (PLIMB) has been developed as a technology, which, together with mass spectrometry, measures three-dimensional structural characteristics of proteins. This technique uses hydroxyl radicals generated by atmospheric-pressure plasma discharge to react with the solvent-accessible side chains of protein in an aqueous solution. In this work, we investigate the three-dimensional structure of hemoglobin and myoglobin using PLIMB. Additional modifications to these proteins, such as oxidation, fragmentations, and conformational changes caused by PLIMB are also explored. These results show that PLIMB, coupled with mass spectrometry, is an effective way to determine solvent access to hemoproteins. Furthermore, we show that many factors, including pH and the electrical parameters used to generate the plasma, have a significant influence on solvent accessibility.

Keywords: plasma, hemoglobin, myoglobin, solvent access

Procedia PDF Downloads 195

Authors: Alexander A. Osmolovskiy, Anastasia V. Orekhova, Daria M. Bednenko, Yelyzaveta Boiko

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Micromycetes from Aspergillus genus can produce proteases capable of promoting proteolysis of hemostasis proteins or, along with hydrolytic activity, to show the ability to convert proenzymes of this system activating them into an active form. At the same time, practical medicine needs specific activators for quantitation of the level of some plasma enzymes, especially protein C and factor X, the lack of which leads to the development of thromboembolic diseases. Thus, some micromycetes of the genus Aspergillus were screened for the ability to synthesize extracellular proteases with promising activity for designing anti-thrombotic and diagnostic preparations. Such standard methods like salting out, electrophoresis, isoelectrofocusing were used for isolation, purification and study of physicochemical properties of proteases. Enzyme activity was measured spectrophotometrically fibrin as a substrate of the reaction and chromogenic peptide substrates of different proteases of the human hemostasis system. As a result of the screening, four active producers were selected: Aspergillus janus 301, A. flavus 1, A. terreus 2, and A. ochraceus L-1. The enzyme of A. janus 301 showed the greatest fibrinolytic activity (around 329.2 μmol Tyr/(ml × min)). The protease produced by A. terreus 2 had the highest plasmin-like activity (54.1 nmol pNA/(ml × min)), but fibrinolytic activity was lower than A. janus 301 demonstrated (25.2 μmol Tyr/(ml × min)). For extracellular protease of micromycete A. flavus a high plasmin-like activity was also shown (39.8 nmol pNA / (ml × min)). Moreover, according to our results proteases one of the fungi - A. terreus 2 were able to activate protein C of human plasma - the key factor of the human anticoagulant hemostasis system. This type of activity was 39.8 nmol pNA/(ml × min)). It was also shown that A. ochraceus L-1 could produce extracellular proteases with protein C and factor X activator activities (65.9 nmol pNA/(ml × min) and 34.6 nmol pNA/(ml × min) respectively). The maximum accumulation of the proteases falls on the 4th day of cultivation. Using isoelectrofocusing was demonstrated that the activation of both proenzymes might proceed via limited proteolysis induced by proteases of A. ochraceus L-1. The activatory activity of A. ochraceus L-1 proteases toward essential hemostatic proenzymes, protein C and X factor may be useful for practical needs. It is well known that similar enzymes, activators of protein C and X factor isolated from snake venom, South American copperhead Agkistrodon contortrix contortrix and Russell’s viper Daboia russelli russeli, respectively, are used for the in vitro diagnostics of the functional state of these proteins in blood plasma. Thus, the proteases of Aspergillus genus can be used as cheap components for enzyme thrombolytic preparations.

Keywords: anti-trombotic drugs, fibrinolysis, diagnostics, proteases, micromycetes

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Authors: Akhil Raj Pushparajan, Ranjit Ramachandran, Jijimole Gopi Reji, Ajay Kumar Ramakrishnan

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Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is one of the leading causes of death by an infectious disease. In spite of the availability of effective drugs and a vaccine, TB is a major health concern and was declared a global emergency by the World Health Organization (WHO). The success of intracellular pathogens like Mtb depends on its ability to overcome the challenging environment in the host. Gene regulation controlled by transcriptional regulators (TRs) plays a crucial role for the bacteria to adapt to the host environment. In vitro studies on gene regulatory mechanisms during dormancy and reactivation have provided insights into the adaptations employed by Mtb to survive in the host. Here we present our efforts to functionally characterize Rv1019, a putative TR of Mtb H37Rv which was found to be present at significantly varying levels during dormancy and reactivation in vitro. The expression of this protein in the dormancy-reactivation model was validated by qRT-PCR and western blot. By DNA- protein interaction studies and reporter assays we found that under normal laboratory conditions of growth this protein behaves as an auto-repressor and tetracycline was found to abrogate this repression by interfering with its ability to bind DNA. Further, by cDNA analysis, we found that this TR is co-transcribed with its downstream genes Rv1020 (mfd) and Rv1021 (mazG) which are involved in DNA damage response in Mtb. Constitutive expression of this regulator in the surrogate host M. smegmatis showed downregulation of the orthologues of downstream genes suggested that Rv1019 could negatively regulate these genes. Our finds also show that M. smegmatis expressing Rv1019 is sensitive to DNA damage suggests the role of this protein in regulating DNA damage response induced by oxidative stress. Because of its role in regulating DNA damage response which may help in the persistence of Mtb, Rv1019 could be used as a prospective target for therapeutic intervention to fight TB.

Keywords: auto-repressor, DNA repair, mycobacterium smegmatis, mycobacterium tuberculosis, tuberculosis

Procedia PDF Downloads 139

Authors: Wael El-Deeb

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The present study aimed to investigate the diagnostic importance of acute phase proteins in urinary tract infection (UTI) in cattle. We describe the clinical, bacteriological and biochemical findings in 99 lactating cows. Blood and urine samples from diseased (n=84) and control healthy cows (n=15) were submitted to laboratory investigations. The urine analysis revealed hematuria and pyuria in UTI group. The isolated bacteria were E.coli (43/84) Corynebacterium spp, (31/84), Proteus spp. (6/84) and Streptococcus spp (4/84). The concentrations of Haptoglobin (Hp), serum amyloid A (SAA), α1-Acid glycoprotein (AGP), fibrinogen (Fb), total protein, albumen, and globulin were higher in cows with UTI when compared to healthy ones. Fifty-one of 84 cows with UTI were successfully treated. The levels of Hp, SAA, AGP, total protein, and globulin were associated with the odds of treatment failure. Conclusively, acute phase proteins could be used as diagnostic and prognostic biomarkers in cows with UTI.

Keywords: cows, urinary, infections, haptoglobin, serum Amyloid A

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Authors: A. Alsoqeer

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The establishment, dry matter production and feeding value of six perennial grasses were measured over two growing seasons in a field experiments. The experiments were conducted at the Agricultural and Veterinary Medicine Research Station, Faculty of Agriculture and Veterinary Medicine, Qassim University, Kingdom of Saudi Arabia in 2009 and 2010 seasons. The six perennial grasses were: creeping bluegrass (Bothriochloa insculpta cv. Bisset), digit grass (Digitaria smutsi), Jarra digit grass (Digitaria milanjiana), panic (Panicum coloratum cv. Bambatsii), Sabi grass (Urochloa mosambicensis) and setaria (Setaria sphacelata cv. Kazungula). The experimental design used was a completely randomized block design with four replications. The results revealed significant differences among plant species of all agronomic characters and quality traits in the first year, while in the second year, plant species differed significantly for quality traits only. D. smutsi had a superior performance for all agronomic characters, however, it had the lowest values in protein content in the two years comparing with other genotypes. D. milanjiana and U. mosambicensis showed high values in dry matter yield and protein content in the first year, but showed a very poor performance in the second year because most of plants were die due to the low temperatures in the winter. These two species appear to be suitable for annual cultivation. The other species tolerate the cold winter and were a highly productive in the second year.

Keywords: dry mater yield, grass species, cuts, quality traits, crude protein content

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Authors: Tamphasana Wairokpam

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Inflammatory myopathies (IMs) have long been recognised as a heterogenous family of myopathies with acute, subacute, and sometimes chronic presentation and are potentially treatable. Necrotizing autoimmune myopathies (NAM) are a relatively new subset of myopathies. Patients generally present with subacute onset of proximal myopathy and significantly elevated creatinine kinase (CK) levels. It is being increasingly recognised that there are limitations to the independent diagnostic utility of muscle biopsy. Immunohistochemistry tests may reveal important information in these cases. The traditional classification of IMs failed to recognise NAM as a separate entity and did not adequately emphasize the diversity of IMs. This review and case report on NAM aims to highlight the heterogeneity of this entity and focus on the distinct clinical presentation, biopsy findings, specific auto-antibodies implicated, and available treatment options with prognosis. This article is a meta-analysis of literatures on NAM and a case report illustrating the clinical course, investigation and biopsy findings, antibodies implicated, and management of a patient with NAM. The main databases used for the search were Pubmed, Google Scholar, and Cochrane Library. Altogether, 67 publications have been taken as references. Two biomarkers, anti-signal recognition protein (SRP) and anti- hydroxyl methylglutaryl-coenzyme A reductase (HMGCR) Abs, have been found to have an association with NAM in about 2/3rd of cases. Interestingly, anti-SRP associated NAM appears to be more aggressive in its clinical course when compared to its anti-HMGCR associated counterpart. Biopsy shows muscle fibre necrosis without inflammation. There are reports of statin-induced NAM where progression of myopathy has been seen even after discontinuation of statins, pointing towards an underlying immune mechanism. Diagnosisng NAM is essential as it requires more aggressive immunotherapy than other types of IMs. Most cases are refractory to corticosteroid monotherapy. Immunosuppressive therapy with other immunotherapeutic agents such as IVIg, rituximab, mycophenolate mofetil, azathioprine has been explored and found to have a role in the treatment of NAM. In conclusion,given the heterogeneity of NAM, it appears that NAM is not just a single entity but consists of many different forms, despite the similarities in presentation and its classification remains an evolving field. A thorough understanding of underlying mechanism and the clinical correlation with antibodies associated with NAM is essential for efficacious management and disease prognostication.

Keywords: inflammatory myopathies, necrotising autoimmune myopathies, anti-SRP antibody, anti-HMGCR antibody, statin induced myopathy

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Authors: P. M. Ikonić, T. A. Tasić, L. S. Petrović, S. B. Škaljac, M. R. Jokanović, V. M. Tomović, B. V. Šojić, N. R. Džinić, A. M. Torbica, B. B. Ikonić

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The aim of the study was to determine how different ripening processes (traditional vs. industrial) influenced the proteolysis in traditional Serbian dry-fermented sausage Petrovská klobása. The obtained results indicated more intensive pH decline (0.7 units after 9 days) in industrially ripened products (I), what had a positive impact on drying process and proteolytic changes in these samples. Thus, moisture content in I sausages was lower at each sampling time, amounting 24.7% at the end of production period (90 days). Likewise, the process of proteolysis was more pronounced in I samples, resulting in higher contents of non-protein nitrogen (NPN) and free amino acids nitrogen (FAAN), as well as in faster and more intensive degradation of myosin (≈220 kDa), actin (≈45 kDa) and other polypeptides during processing. Consequently, the appearance and accumulation of several protein fragments were registered.

Keywords: dry-fermented sausage, Petrovská klobása, proteolysis, ripening process

Procedia PDF Downloads 333

Authors: Hien Thi Thu Le, Nuno Rafael Candeias, Olli Yli-Harja, Meenakshisundaram Kandhavelu

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Purinergic receptor 1 (P2Y1R) is the potential therapeutic target for inducing prostate cancer (PCa) cell death. Recently, 1-indolinoalkyl 2-phenolic derivative, HIC, was identified as a P2Y1R agonist that increases apoptosis and inhibits cell proliferation of PCa. However, the biological effects of HIC have not been extensively studied at the molecular level. In the present study, we have investigated the anticancer effects of HIC and the molecular mechanisms underlying in PCa cells. Half maximal inhibitory concentration (IC₅₀) of HIC was measured as 15.98 μM and 15.64 μM for DU145 and PC3 cells, respectively. In addition, we found that HIC inhibited cell growth and metastasis of PC3 and DU145 cells colonies, spheroid areas, and migrated cells. RNA seq analysis revealed significant changes of over 3000 genes (p value < 0.05) upon HIC treatment in PC3 and DU145 cells. Genes involved in DNA damage, apoptosis, cell cycle arrest at G1/S phase were modulated by HIC treatment. MAPK and NF-κB protein array revealed the increased expression of ERK1/2, JNK1/2, p53 phosphorylation, and p53 protein. ERK1/2 and JNK1/2 activations are known to increase the stabilization of p53, a tumor suppressor protein, which is required to arrest the cell cycle at G1/S phase and cause cell death of PCa cells. Overall, our results suggest that HIC can serve as a multi-dimensional chemotherapeutic agent possessing strong cytotoxic, anti-cancer, and anti-metastasis against PCa growth.

Keywords: prostate cancer, P2Y1 receptor, apoptosis, metastasis

Procedia PDF Downloads 133

Authors: Manal Kamel, Sara Maher, Hanan El Baz, Faten Salah, Omar Sayyouh, Zeinab Demerdash

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In the recent COVID 19 pandemic, experts of public health have emphasized testing, tracking infected people, and tracing their contacts as an effective strategy to reduce the spread of the virus. Development of rapid and sensitive diagnostic assays to replace reverse transcription polymerase chain reaction (RT-PCR) is mandatory..Our innovative test strip relying on the application of nanoparticles conjugated to recombinant nanobodies for SARS-COV-2 spike protein (S1) & angiotensin-converting enzyme 2 (that is responsible for the virus entry into host cells) for rapid detection of SARS-COV-2 spike protein (S1) in saliva or sputum specimens. Comparative tests with RT-PCR will be held to estimate the significant effect of using COVID 19 nanobodies for the first time in the development of lateral flow test strip. The SARS-CoV-2 S1 (3 ng of recombinant proteins) was detected by our developed LFIA in saliva specimen of COVID-19 Patients No cross-reaction was detected with Middle East respiratory syndrome coronavirus (MERS-CoV) or SARS- CoV antigens..Our developed system revealed 96 % sensitivity and 100% specificity for saliva samples compared to 89 % and 100% sensitivity and specificity for nasopharyngeal swabs. providing a reliable alternative for the painful and uncomfortable nasopharyngeal swab process and the complexes, time consuming PCR test. An increase in testing compliances to be expected.

Keywords: COVID 19, diagnosis, LFIA, nanobodies, ACE2

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Authors: Rana Bilal, Faiz-Ul-Hassan, Moazzam Jameel

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There is a dire need to replace high-cost protein with more economical protein to overcome animal protein shortage in developing nations especially countries like Pakistan. In conjunction with these efforts, the current study was planned to evaluate the effects of various dried distillery yeast sludge (DYS) levels on the immune level, egg quality, and performance of layers by replacing soybean meal. The study was designed with two hundred layers of Hy-Line variety. Distillery yeast sludge was dried and ground for 2 mm mesh size and after this proximate and mineral analysis was determined. Five isocaloric and isonitrogeneous feeds were given containing C (control), 5, 10, 15, 20% distillery yeast sludge by replacing soybean meal. The trial was performed in the completely randomized design with five treatments, 4 replicates and 10 hen per replicate. Results demonstrated that feed intake, egg production, feed conversion ratio decreased (P < 0.05) with the increased dietary DYS. However, statistically significant decrease (P < 0.05) was found in hens having DYS20 diet than control. Layers on Diets C, DYS5 and DYS10 exerted a higher immune level than DYS15 and DYS20 diets. Egg weight, eggshell weight, eggshell thickness, egg albumen height as well as haugh unit score were affected significantly by the increased level of DYS. In general, results of this study demonstrated that inclusion of DYS up to 10% showed no adverse effects on health and performance of layers.

Keywords: egg quality, immunity, layers, performance

Procedia PDF Downloads 235

Authors: Samar Saadeldin Abdelmotalab Omer, Ikram Mohamed Eltayeb Elsiddig, Saad Mohammed Hussein Ayoub

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A variety of herbal medicinal plants are known to confer beneficial effects in regards to modification of cardiovascular ri’=sk factors. The anti-hypercholesterolaemic and antioxidant activities of the crude ethanolic extracts of Citrus reticulate fruit peel, Zingiber officinale rhizome and Sesamum indicum seed extracts have been demonstrated. These plants are assumed to possess biologically active principles, which impart their pharmacologic activities. GC-MS analysis of the ethanolic extracts was carried out to identify the active principles and their percentages of occurrence in the analytes. Analysis of the extracts was carried out using (GS-MS QP) type Schimadzu 2010 equipped with a capillary column RTX-50 (restec), (length 30mm, diameter 0.25mm, and thickness 0.25mm). Helium was used as a carrier gas, the temperature was programmed at 200°C for 5 minutes at a rate of 15ml/minute, and the extracts were injected using split injection mode. The identification of different components was achieved from their Mass Spectra and Retention time, compared with those in the NIST library. The results revealed the presence of 80 compounds in Sudanese locally grown C. reticulata fruit peel extract, most of which were monoterpenoid compounds including Limonene (3.03%), Alpha & Gamma - terpinenes (2.61%), Linalool (1.38%), Citral (1.72%) which are known to have profound antioxidant effects. The Sesquiterpenoids Humulene (0.26%) and Caryophyllene (1.97%) were also identified, the latter known to have profound anti-anxiety and anti-depressant activity in addition to the beneficiary effects in lipid regulation. The analysis of the locally grown S. indicum oily and water soluble portions of seed extract revealed the presence of a total of 64 compounds with considerably high percentage of the mono-unsaturated fatty acid ester methyl oleate (66.99%) in addition to methyl stearate (9.35%) and palmitate (15.71%) of oil portion, whereas, plant sterols including Gamma-sitosterol (13.5%), fucosterol (2.11%) and stigmasterol (1.95%) in addition to gamma-tocopherol (1.16%) were detected in extract water-soluble portion. The latter indicate various principles known to have valuable pharmacological benefits including antioxidant activities and beneficiary effects on intestinal cholesterol absorption and regulation of serum cholesterol levels. Z. officinale rhizome extract analysis revealed the presence of 93 compounds, the most abundant were alpha-zingeberine (16.5%), gingerol (9.25%), alpha-sesquiphellandrene (8.3%), zingerone (6.78%), beta-bisabolene (4.19%), alpha-farnesene (3.56%), ar-curcumene (3.29%), gamma-elemene (1.25%) and a variety of other compounds. The presence of these active principles reflected on the activity of the extract. Activity could be assigned to a single or a combination of two or more extract components. GC-MS analysis concluded the occurrence of compounds known to possess antioxidant activity and lipid profile effects.

Keywords: gas chromatography, indicum, officinale, reticulata

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Authors: Shalini Singh, Sanjay K. Srivastava, Govind, Mukhtar. A. Khan, P. K. Singh

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Nanotechnology is revolutionizing the development of biosensors. Nanomaterials and nanofabrication technologies are increasingly being used to design novel biosensors. Sensitivity and other attributes of biosensors can be improved by using nanomaterials with unique chemical, physical, and mechanical properties in their construction. Silicon is a promising biomaterial that is non-toxic and biodegradable and can be exploited in chemical and biological sensing. Present study demonstrated the streptavidin–biotin interaction on silicon surfaces with different topographies such as flat and nanostructured silicon (nanowires) surfaces. Silicon nanowires with wide range of surface to volume ratio were prepared by electrochemical etching of silicon wafer. The large specific surface of silicon nanowires can be chemically modified to link different molecular probes (DNA strands, enzymes, proteins and so on), which recognize the target analytes, in order to enhance the selectivity and specificity of the sensor device. The interaction of streptavidin with biotin was carried out on 3-aminopropyltriethoxysilane (APTS) functionalized silicon surfaces. Fourier Transform Infrared Spectroscopy (FTIR) and X-ray Photoelectron Spectroscopy (XPS) studies have been performed to characterize the surface characteristics to ensure the protein attachment. Silicon nanowires showed the enhance protein attachment, as compared to flat silicon surface due to its large surface area and good molecular penetration to its surface. The methodology developed herein could be generalized to a wide range of protein-ligand interactions, since it is relatively easy to conjugate biotin with diverse biomolecules such as antibodies, enzymes, peptides, and nucleotides.

Keywords: FTIR, silicon nanowires, streptavidin-biotin, XPS

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Authors: M. Dehestani, F. Kamali, M. Klantari Pour, L. Zeidabadi-Nejad

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Chemical bonding between polyethylene glycol (PEG) with pharmaceutical proteins called pegylation is one of the most effective methods of improving the pharmacological properties. The covalent attachment of polyethylene glycol (PEG) to proteins will increase their pharmacologic properties. For the formation of a combination of pegylated protein should first be activated PEG and connected to the protein. Interferons(IFNs) are a family of cytokines which show antiviral effects in front of the biological and are responsible for setting safety system. In this study, the nature and properties of the interaction between active positions of IFNs and polyethylene glycol have been investigated using molecular dynamics simulation. The main aspect of this theoretical work focuses on the achievement of valuable data on the reaction pathways of PEG-IFNs and the transition state energy. Our results provide a new perspective on the interactions, chemical properties and reaction pathways between IFNs and PEG.

Keywords: interaction, interferons, molecular dynamics simulation, polyethylene glycol

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Authors: Soomal Hamza, Uzma Imran

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Organochlorine pesticides bio-accumulate into the fat of fish, birds, and animals through which it enters the human food cycle. Due to their persistence and stability in the environment, many health impacts are associated with them, most of which are carcinogenic in nature. In this study, the level of organochlorine pesticides has been detected in Keenjhar Lake and remediated using Rhizoremediation technique. 14 OC pesticides namely, Aldrin, Deldrin, Heptachlor, Heptachlor epoxide, Endrin, Endosulfun I and II, DDT, DDE, DDD, Alpha, Beta, Gamma BHC and two plants namely, Water Hyacinth and Slvinia Molesta were used in the system using pot experiment which processed for 11 days. A consortium was inoculated in both plants to increase its efficiency. Water samples were processed using liquide-liquid extraction. Sediments and roots samples were processed using Soxhlet method followed by clean-up and Gas Chromatography. Delta-BHC was the predominantly found in all samples with mean concentration (ppb) and standard deviation of 0.02 ± 0.14, 0.52 ± 0.68, 0.61 ± 0.06, in Water, Sediments and Roots samples respectively. The highest levels were of Endosulfan II in the samples of water, sediments and roots. Water Hyacinth proved to be better bioaccumulaor as compared to Silvinia Molesta. The pattern of compounds reduction rate by the end of experiment was Delta-BHC>DDD > Alpha-BHC > DDT> Heptachlor> H.Epoxide> Deldrin> Aldrin> Endrin> DDE> Endosulfun I > Endosulfun II. Not much significant difference was observed between the pots with the consortium and pots without the consortium addition. Phytoremediation is a promising technique, but more studies are required to assess the bioremediation potential of different aquatic plants and plant-endophyte relationship.

Keywords: aquatic plant, bio remediation, gas chromatography, liquid liquid extraction

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Authors: Sujata Sharma, Avinash Singh, Lovely Gautam, Pradeep Sharma, Mau Sinha, Asha Bhushan, Punit Kaur, Tej P. Singh

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Peptidyl-tRNA hydrolase (Pth) Pth is an essential bacterial enzyme that catalyzes the release of free tRNA and peptide moeities from peptidyl tRNAs during stalling of protein synthesis. In order to design inhibitors of Pth from Pseudomonas aeruginosa (PaPth), we have determined the structures of PaPth in its native state and in the bound states with two compounds, amino acylate-tRNA analogue (AAtA) and 5-azacytidine (AZAC). The peptidyl-tRNA hydrolase gene from Pseudomonas aeruginosa was amplified by Phusion High-Fidelity DNA Polymerase using forward and reverse primers, respectively. The E. coliBL21 (λDE3) strain was used for expression of the recombinant peptidyl-tRNA hydrolase from Pseudomonas aeruginosa. The protein was purified using a Ni-NTA superflow column. The crystallization experiments were carried out using hanging drop vapour diffusion method. The crystals diffracted to 1.50 Å resolution. The data were processed using HKL-2000. The polypeptide chain of PaPth consists of 194 amino acid residues from Met1 to Ala194. The centrally located β-structure is surrounded by α-helices from all sides except the side that has entrance to the substrate binding site. The structures of the complexes of PaPth with AAtA and AZAC showed the ligands bound to PaPth in the substrate binding cleft and interacted with protein atoms extensively. The residues that formed intermolecular hydrogen bonds with the atoms of AAtA included Asn12, His22, Asn70, Gly113, Asn116, Ser148, and Glu161 of the symmetry related molecule. The amino acids that were involved in hydrogen bonded interactions in case of AZAC included, His22, Gly113, Asn116, and Ser148. As indicated by fittings of two ligands and the number of interactions made by them with protein atoms, AAtA appears to be a more compatible with the structure of the substrate binding cleft. However, there is a further scope to achieve a better stacking than that of O-tyrosyl moiety because it is not still ideally stacked. These observations about the interactions between the protein and ligands have provided the information about the mode of binding of ligands, nature and number of interactions. This information may be useful for the design of tight inhibitors of Pth enzymes.

Keywords: peptidyl tRNA hydrolase, Acinetobacter baumannii, Pth enzymes, O-tyrosyl

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Authors: Lebogang L. R. Mphahlele, Bruce B. Sithole

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Chicken meat is the highest consumed meat in south Africa, with a per capita consumption of >33 kg yearly. Hence, South Africa produces over 250 million kg of waste chicken feathers each year, the majority of which is landfilled or incinerated. The discarded feathers have caused environmental pollution and natural protein resource waste. Therefore, the valorisation of waste chicken feathers is measured as a more environmentally friendly and cost-effective treatment. Feather contains 91% protein, the main component being beta-keratin, a fibrous and insoluble structural protein extensively cross linked by disulfide bonds. Keratin is usually converted it into nanofibers via electrospinning for a variety of applications. keratin nanofiber composites have many potential biomedical applications for their attractive features, such as high surface-to-volume ratio and very high porosity. The application of nanofibers in the biomedical wound dressing requires antimicrobial properties for materials. One approach is incorporating inorganic nanoparticles, among which silver nanoparticles played an important alternative antibacterial agent and have been studied against many types of microbes. The objective of this study is to combine synthetic polymer, chicken feather keratin, and antibacterial nanoparticles to develop novel electrospun antibacterial nanofibrous composites for possible wound dressing application. Furthermore, this study will converting a two-dimensional electrospun nanofiber membrane to three-dimensional fiber networks that resemble the structure of the extracellular matrix (ECM)

Keywords: chicken feather keratin, nanofibers, nanoparticles, nanocomposites, wound dressing

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Authors: Hanaa A. S. Oraby, Amal A. M. Hassan, Mahmoud M. Sakr, Atef A. A. Haiba

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Applying the profiling approach for the assessment of proteomic variations due to genetic modification of the Egyptian tomato cultivar "Edkawy", three complementary approaches were used. These methods are amino acids analysis, gel electrophoresis, and Gas chromatography coupled with mass spectrometry (GC/MS). The results of the present study Show evidence of proteomic variations between both modified tomato and its non-modified counterpart. Amino acids concentrations, and the protein patterns separation on the 1D SDS-PAGE were not similar in the case of transformed tomato compared to that of the non-transformed counterpart. These detected differences are most likely derived from the process of transformation. Results also revealed that the efficiency of GC/MS approach to identify a mixture of unknown proteins is limited. GC/MS analysis was only able to identify few number of protein molecules. Therefore, more advanced and specific technologies like MALDI-TOF-MS are recommended to be employed.

Keywords: GMOs, unintended effects, proteomic variations, 1D SDS-PAGE, GC/MS

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Authors: M. Cherki, I. Taouam, A. Amiri, F. Hmimid, T. Ould Bellahcen

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The Moroccan coasts represent an important wealth of red algae which have an economic interest. Among these algae, the Gelidium sesquipedale, which is exploited industrially for its richness in agar. The aim of this study is to establish a general overview of the macronutrient composition of Gelidium sesquipedale and to compare this composition according to three factors: the harvest site (El Jadida, Casablanca and Mohammadia), the harvest depth (coast and depth) and the drying process (open air and oven). Proteins, lipids, and carbohydrates are measured by different methods. The analysis of results show that the protein concentrations of the El Jadida and Mohammadia samples are significantly higher than that of Casablanca (0.026 ± 0.0007 µg/µg DW 0.024 ± 0.001 µg/µg DW and 0.006 ± 0.0007 µg/µg DW, p < 0.05 respectively). However, Casablanca samples are significantly richer in total sugars (0.023 ± 0.002 µg/µg DW, p < 0.05) and less rich in reducing sugars (0.0001 ± 0.00001 µg/µg DW, p < 0.05) compared to other samples. The lipid concentrations of the samples from the three harvest sites do not show any significant difference. With respect to depth, only total protein and total sugar concentrations were significantly higher in the coast versus depth samples (0.035 ± 0.004 µg/µg DW vs. 0.026 ± 0.0007 µg/µg DW and 0.035 ± 0.006 µg/µg DW vs. 0.012 ± 0.005 µg/µg DW p < 0.05 respectively). For the drying process, protein, total sugars and lipid concentrations were significantly higher in open air samples compared to oven samples (0.006 ± 0.0007 µg/µg DW). vs 0.004 ± 0.0003 µg/µg DW, 0.023 ± 0.002 µg/µg DW vs 0.007 ± 0.002 µg/µg DW and 8% vs 4% p < 0.05 respectively). Our results demonstrate that the chemical composition of Gelidium sesquipedale varies according to the harvest region. In addition, samples harvested on the coast and dried in the open air are the richest in macronutrients.

Keywords: biochemical composition, drying, depth, Gelidium sesquipedale, red algae, region

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Authors: Kai Pai Huang

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Background: There are several types of plasma cell neoplasms including multiple myeloma, plasmacytoma, lymphoplasmacytic lymphoma, and monoclonal gammopathy of undetermined significance (MGUS) are found in our lab. Today, we want to compare with two cases using the method of capillary electrophoresis. Method: Serum is prepared and electrophoresis is performed at alkaline PH in a capillary using the Sebia® Capillary 2. Albumin and globulins are detected by the detector which is located in the cathode of the capillary and the signals are transformed to peaks. Serum was treated with beta-mercaptoethanol which reducing the polymerized immunoglobulin to monomer immunoglobulin to clarify two M-protein are secreted from the same plasma cell clone in bone marrow. Result: Case 1: A 78-year-old female presenting dysuria, oliguria and leg edema for several months. Laboratory data showed proteinuria, leukocytosis, results of high serum IgA and lambda light chain. A renal biopsy found amyloid fibrils in the glomerular mesangial area. Serum protein electrophoresis shows a major monoclonal peak in the β region and minor small peak in gamma region, and the immunotyping studies for serum showed two IgA/λ type. Case 2: A 55-year-old male presenting abdominal distension and low back pain for more than one month. Laboratory data showed T12 T8 compression fracture, results of high serum IgM and kappa light chain. Bone marrow aspiration showed the cells from the bone marrow are B cells with monotypic kappa chain expression. Bone marrow biopsy found this is lymphoplasmacytic lymphoma (Waldenstrom macroglobulin). Serum protein electrophoresis shows a monoclonal peak in the β region and the immunotyping studies for serum showed IgM/κ type. Conclusion: Plasma cell neoplasm can be diagnosed by many examinations. Among them, using capillary electrophoresis by a lab can separate several types of gammopathy and the quantification of a monoclonal peak can be used to evaluate the patients’ prognosis or treatment.

Keywords: plasma cell neoplasm, capillary electrophoresis, serum protein electrophoresis, immunotyping

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Authors: Sulhee Lee, Geon Kim, Young-Seo Park

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Unlike the traditional milk sterilization methods (LTLT, HTST, or UHT), pulsed electric field (PEF) technology is a non-thermal pasteurization process. This technology minimizes energy required for heat treatment in food processing, changes in sensory properties, and physical losses. In this study, structural changes of bovine milk proteins, the amount of immunoproteins such as IgG, and their storability by PEF treatment were examined. When the changes of protein content in PEF-treated milk were examined using HPLC, the amounts of α-casein and β-lactoglobulin were reduced over 40% each, whereas those of κ-casein and β-casein did not change. The amount of α-casein in HTST milk was reduced to 50%. When PEF was applied to milk at the energy level of 250 kJ, the amounts of IgG, IgA, β-lactoglobulin (β-LG), lactoferrin, and α-lactalbumin (α-LA) decreased by 43, 41, 35, 63, and 45%, respectively. When milk was sterilized by LTLT process followed by PEF process at the level of 150 kJ, the concentrations of IgG, IgA, β-LG, lactoferrin, and α-LA were 56.6, 10.6, 554, 2.8 and 660.1 μg/mL, respectively. When the bovine milk was sterilized by LTLT process followed by PEF process at the energy level of 180 kJ, storability of immunoproteins of milk was the highest and the concentrations of IgG, IgA, and β-LG decreased by 79.5, 6.5, and 134.5 μg/mL, respectively, when compared with the initial concentrations of those proteins. When bovine milk was stored at 4℃ after sterilization through HTST sterilizer followed by PEF process at the energy level of 200 kJ, the amount of lactoferrin decreased 7.3% after 36 days of storage, whereas that of lactoferrin of raw milk decreased 16.4%. Our results showed that PEF treatment did not change the protein structure nor induce protein denaturation in milk significantly when compared with LTLT or HTST sterilization. Also, LTLT or HTST process in combination with PEF were more effective than LTLT only or HTST only process in the conservation of immunoproteins in bovine milk.

Keywords: pulsed electric field, bovine milk, immunoproteins, sterilization

Procedia PDF Downloads 436

Authors: Aysegul Alyamac, Sukru Gulec

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Ankaferd Blood Stopper (ABS) is a plant extract used to stop bleeding caused by injuries and surgical interventions. ABS also involved in wound healing of intestinal mucosal damage due to oxidative stress and inflammation. Inflammatory Bowel Disease (IBD) is a common chronic disorder of the gastrointestinal tract that causes abdominal pain, diarrhea, and gastrointestinal bleeding, and increases the risk of colon cancer. Inflammation is an essential factor in the development of IBD. The various studies have been performed about the physiological effects of ABS; however, ABS dependent mechanism on colonic inflammation has not been elucidated. Thus, the protective effect of ABS on colonic inflammation was investigated in this study. The Caco-2 and RAW 264.7 murine macrophage cells were used as a model of in vitro colonic inflammation. RAW 264.7 cells were treated with lipopolysaccharide (LPS) for 12 hours to induce the inflammation, and a conditional medium was obtained. Caco-2 cells were treated with 15 µl/ml ABS for 4 hours, then incubated with conditional medium and the cells also were incubated with 15 µl/ml ABS and conditional medium together for 4 hours. Tumor necrosis factor alpha (TNF-α) protein levels were targeted in testing inflammatory condition and its level was significantly increased (25 fold, p<0.001) compared to the control group by using Enzyme-Linked Immunosorbent Assay (ELISA) method. The COX-2 mRNA level was used as a marker gene to show the possible anti-inflammatory effect of ABS in Caco-2 cells. RAW cells-derived conditional medium significantly (3.3 fold, p<0.001) induced cyclooxygenase-2 (COX-2) mRNA levels in Caco-2 cells. The pretreatment of Caco-2 cells caused a significant decrease (3.3 fold, p<0.001) in COX-2 mRNA levels relative to conditional medium given group. Furthermore, COX-2 mRNA level was significantly reduced (4,7 fold, p<0.001) in ABS and conditional medium treated group. These results suggest that ABS might have an anti-inflammatory effect in vitro.

Keywords: Ankaferd blood stopper, CaCo-2, colonic inflammation, RAW 264.7

Procedia PDF Downloads 146

Authors: Yuchien Lin

Abstract:

This study attempted to investigate the changes in electroencephalography (EEG) and emotion regulation following eight-week inward-attention meditation training program. The subjects were 24 adults without meditation experiences divided into meditation and control groups. The quantitatively analyzed changes in psychophysiological parameters during inward-attention meditation, and evaluated the emotion scores assessed by the State-Trait Anxiety Inventory (STAI), the Positive and Negative Affect Schedule (PANAS), and the Emotion Regulation Scale (ERS). The results were found: (1) During meditation, significant EEG increased for theta-band activity in the frontal and the bilateral temporal areas, for alpha-band activity in the left and central frontal areas, and for gamma-band activity in the left frontal and the left temporal areas. (2) The meditation group had significantly higher positive affect in posttest than in pretest. (3) There was no significant difference in the changes of EEG spectral characteristics and emotion scores in posttest and pretest for the control group. In the present study, a unique meditative concentration task with a constant level of moderate mental effort focusing on the center of brain was used, so as to enhance frontal midline theta, alpha, and gamma-band activity. These results suggest that this mental training allows individual reach a specific mental state of relaxed but focused awareness. The gamma-band activity, in particular, enhanced over left frontoparietal area may suggest that inward-attention meditation training involves temporal integrative mechanisms and may induce short-term and long-term emotion regulation abilities.

Keywords: meditation, EEG, emotion regulation, gamma activity

Procedia PDF Downloads 215

Authors: Elif Gencturk, Ekin Yurdakul, Ahmet Y. Celik, Senol Mutlu, Kutlu O. Ulgen

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Yeast cells are generally used as a model system of eukaryotes due to their complex genetic structure, rapid growth ability in optimum conditions, easy replication and well-defined genetic system properties. Thus, yeast cells increased the knowledge of the principal pathways in humans. During fermentation, carbohydrates (hexoses and pentoses) degrade into some toxic by-products such as 5-hydroxymethylfurfural (5-HMF or HMF) and furfural. HMF influences the ethanol yield, and ethanol productivity; it interferes with microbial growth and is considered as a potent inhibitor of bioethanol production. In this study, yeast single cell behavior under HMF application was monitored by using a continuous flow single phase microfluidic platform. Microfluidic device in operation is fabricated by hot embossing and thermo-compression techniques from cyclo-olefin polymer (COP). COP is biocompatible, transparent and rigid material and it is suitable for observing fluorescence of cells considering its low auto-fluorescence characteristic. The response of yeast cells was recorded through Red Fluorescent Protein (RFP) tagged Nop56 gene product, which is an essential evolutionary-conserved nucleolar protein, and also a member of the box C/D snoRNP complexes. With the application of HMF, yeast cell proliferation continued but HMF slowed down the cell growth, and after HMF treatment the cell proliferation stopped. By the addition of fresh nutrient medium, the yeast cells recovered after 6 hours of HMF exposure. Thus, HMF application suppresses normal functioning of cell cycle but it does not cause cells to die. The monitoring of Nop56 expression phases of the individual cells shed light on the protein and ribosome synthesis cycles along with their link to growth. Further computational study revealed that the mechanisms underlying the inhibitory or inductive effects of HMF on growth are enriched in functional categories of protein degradation, protein processing, DNA repair and multidrug resistance. The present microfluidic device can successfully be used for studying the effects of inhibitory agents on growth by single cell tracking, thus capturing cell to cell variations. By metabolic engineering techniques, engineered strains can be developed, and the metabolic network of the microorganism can thus be manipulated such that chemical overproduction of target metabolite is achieved along with the maximum growth/biomass yield.  

Keywords: COP, HMF, ribosome biogenesis, thermoplastic microbioreactor, yeast

Procedia PDF Downloads 171

Authors: Hainan Chen, Jina Qing, Xiao Zhu, Ling Gao, Ampadu O. Jackson, Min Zhang, Kai Yin

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Objective: Editing macrophage activation to dampen inflammatory diseases by promoting the repolarization of inflammatory (M1) macrophages to anti-inflammatory (M2) macrophages is highly associated with mitochondrial respiration. Recent studies have suggested that mitochondrial apolipoprotein A-1 binding protein (APOA1BP) was essential for the cellular metabolite NADHX repair to NADH, which is necessary for the mitochondrial function. The exact role of APOA1BP in the repolarization of M1 to M2, however, is uncertain. Material and method: THP-1-derived macrophages were incubated with LPS (10 ng/ml) or/and IL-4 (100 U/ml) for 24 hours. Biochemical parameters of oxidative phosphorylation and M1/M2 markers were analyzed after overexpression of APOA1BP in cells. Results: Compared with control and IL-4-exposed M2 cells, APOA1BP was downregulated in M1 macrophages. APOA1BP restored the decline in mitochondrial function to improve metabolic and phenotypic reprogramming of M1 to M2 macrophages. Blocking oxidative phosphorylation by oligomycin blunts the effects of APOA1BP on M1 to M2 repolarization. Mechanistically, LPS triggered the hydration of NADH and increased its hydrate NADHX which inhibit cellular NADH dehydrogenases, a key component of electron transport chain for oxidative phosphorylation. APOA1BP decreased the level of NADHX via converting R-NADHX to biologically useful S-NADHX. The mutant of APOA1BP aspartate188, the binding site of NADHX, fail to repair oxidative phosphorylation, thereby preventing repolarization. Conclusions: Restoring mitochondrial function by increasing mitochondrial APOA1BP might be useful to improve the reprogramming of inflammatory macrophages into anti-inflammatory cells to control inflammatory diseases.

Keywords: inflammatory diseases, macrophage repolarization, mitochondrial respiration, apolipoprotein A-1 binding protein, NADHX, NADH

Procedia PDF Downloads 173

Authors: Zeiler M., Stadler D., Schmaderer C.

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Over the years of dialysis treatment, most patients experience significant weight loss. The primary emphasis in earlier research was the underlying mechanism of protein energy wasting and the subsequent malnutrition inflammation syndrome. In the interest to provide an effective and rapid solution for the patients, the aim of this study is identifying individual influences of their assumed reduced dietary intake, such as nausea, appetite loss and taste changes, and to determine whether the patients adhere to their nutrition guidelines. A prospective, controlled study with 38 end-stage renal disease patients was performed using a questionnaire to reflect their diet within the last 12 months. Thereby, the daily intake for the most important macro-and micronutrients was calculated to be compared with the individual KDQOI-guideline value, as well as controls matched in age and gender. The majority of the study population did not report symptoms commonly associated with dialysis, such as nausea or inappetence, and denied any change in dietary behavior since receiving renal replacement therapy. The patients’ daily intake of energy (3080kcal ± 1266) and protein (89,9g [53,4-142,0]) did not differ significantly from the controls (energy intake: 3233kcal ± 1046, p=0,597; protein intake: 103,7g [90,1-125,5], p=0,120). The average difference to the individual calculated KDQOI-guideline was +176,0kcal ± 1156 (p=0,357) for energy intake and -1,75g ± 45,9 (p=0,491) for protein intake. However, there was an observed imbalance in the distribution of macronutrients, with a preference for fats over proteins. The patients’ daily intake of sodium (5,4g [ 2,95-10,1]) was higher than in the controls (4,1g [2,04-5,99], p= 0,058) whereas both values for potassium (3,7g ± 1,84) and phosphorous (1,79g ± 0,91) went significantly below the controls’ values (potassium intake: 4,89g ± 1,74, p=0,014; phosphorous intake: 2,04g ± 0,64, p=0,038). Thus, the values exceeded the calculated KDQOI-recommendation by + 3,3g [0,63-7,90] (p<0,001) for sodium, +1,49g ± 1,84 (p<0,001) for potassium and +0,89g ± 0,91 (p<0,001) for phosphorous. Contrary to the assumption, the patients did not under-eat. Nevertheless, their diets did not align with the recommended values. These findings highlight the need for intervention and education among patients and that regular dietary monitoring could prevent unhealthy nutrition habits. The elaboration of individual references instead of standardized guidelines could increase the compliance to the advised diet so that interdisciplinary comorbidities do not develop or worsen.

Keywords: compliance, dialysis, end-stage renal disease, KDQOI, malnutrition, nutrition guidelines, questionnaire, salt intake

Procedia PDF Downloads 68

Authors: Ajith K. A. Gedara, Iva Chianella, Jose L. Endrino, Qi Zhang

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The olive seed is a by-product of the olive oil production industry. Biuret test and ferric chloride test revealed that water or alkali NaOH extractions of olive seed flour are rich in proteins and tannins. Both protein and tannins are well-known bio-based wood adhesives in the wood-based panel industry. In general, tannins-based adhesives show better mechanical and physical properties than protein wood adhesives. This paper explores different methods of extracting tannins from olive seed flour against the tannins yield and their applications as bio-based adhesives in wood-based panels. Once investigated, the physical and the mechanical properties of wood-based panels made using bio-adhesives based tannins extracted from olive seed flour revealed that the resulting products seemed to satisfy the Japanese Industrial Standards JIS A 5908:2015.

Keywords: bio-adhesives, olive seed flour, tannins, wood-based panels

Procedia PDF Downloads 152

Authors: Pawan Kumar Gupta, Sumana Ghosh

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Parkinson's Disease (PD) is a long-term neurodegenerative movement disorder of the central nervous system with vast symptoms related to the motor system. The common symptoms of PD are tremor, rigidity, bradykinesia/akinesia, and postural instability, but the clinical symptom includes other motor and non‐motor issues. The motor symptoms of the disease are consequence of death of the neurons in a region of the midbrain known as substantia nigra pars compacta, leading to decreased level of a neurotransmitter known as dopamine. The cause of this neuron death is not clearly known but involves formation of Lewy bodies, an abnormal aggregation or clumping of the protein alpha-synuclein in the neurons. Unfortunately, there is no cure for PD, and the management of this disease is challenging. Therefore, it is critical for a patient to be diagnosed at early stages. A limited choice of drugs is available to improve the symptoms, but those become less and less effective over time. Apart from that, with rapid growth in the field of science and technology, other methods such as multi-area brain stimulation are used to treat patients. In order to develop advanced techniques and to support drug development for treating PD patients, an accurate mathematical model is needed to explain the underlying relationship of dopamine secretion in the brain with the hand tremors. There has been a lot of effort in the past few decades on modeling PD tremors and treatment effects from a computational point of view. These models can effectively save time as well as the cost of drug development for the pharmaceutical industry and be helpful for selecting appropriate treatment mechanisms among all possible options. In this review paper, an effort is made to investigate studies on PD modeling and analysis and to highlight some of the key advances in the field over the past centuries with discussion on the current challenges.

Keywords: Parkinson's disease, deep brain stimulation, tremor, modeling

Procedia PDF Downloads 140

Authors: Fatna Benmessaoud, Mohammed Cheikh, Vencent Velay, Vanessa Vidal, Farhad Rezai-Aria, Christine Boher

Abstract:

Ti-6Al-4V titanium alloy is one of the most widely used materials in aeronautical and aerospace industries. Because of its high specific strength, good fatigue, and corrosion resistance, this alloy is very suitable for moderate temperature applications. At room temperature, Ti-6Al-4V mechanical behavior is generally controlled by the behavior of alpha phase (beta phase percent is less than 8%). The plastic strain of this phase notably based on crystallographic slip can be hindered by various obstacles and mechanisms (crystal lattice friction, sessile dislocations, strengthening by solute atoms and grain boundaries…). The grains aspect of alpha phase (its morphology and texture) and the nature of its crystallographic lattice (which is hexagonal compact) give to plastic strain heterogeneous, discontinuous and anisotropic characteristics at the local scale. The aim of this work is to develop a multi-scale model for Ti-6Al-4V mechanical behavior using crystal plasticity approach; this multi-scale model is used then to investigate grains size, dispersion of grains size, crystallographic texture and slip systems activation effects on Ti-6Al-4V mechanical behavior under monotone quasi-static loading. Nine representative elementary volume (REV) are built for taking into account the physical elements (grains size, dispersion and crystallographic) mentioned above, then boundary conditions of tension test are applied. Finally, simulation of the mechanical behavior of Ti-6Al-4V and study of slip systems activation in alpha phase is reported. The results show that the macroscopic mechanical behavior of Ti-6Al-4V is strongly linked to the active slip systems family (prismatic, basal or pyramidal). The crystallographic texture determines which family of slip systems can be activated; therefore it gives to the plastic strain a heterogeneous character thus an anisotropic macroscopic mechanical behavior of Ti-6Al-4V alloy modeled. The grains size influences also on mechanical proprieties of Ti-6Al-4V, especially on the yield stress; by decreasing of the grain size, the yield strength increases. Finally, the grains' distribution which characterizes the morphology aspect (homogeneous or heterogeneous) gives to the deformation fields distribution enough heterogeneity because the crystallographic slip is easier in large grains compared to small grains, which generates a localization of plastic deformation in certain areas and a concentration of stresses in others.

Keywords: multi-scale modeling, Ti-6Al-4V alloy, crystal plasticity, grains size, crystallographic texture

Procedia PDF Downloads 157

Authors: J. Adeppa, S. Samanta, O. K. Raina

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Fasciolosis is a widespread economically important parasitic infection throughout the world caused by Fasciola hepatica and F. gigantica. In order to identify novel immunogen conferring significant protection against fasciolosis, currently, research has been focused on the defined antigens viz. glutathione S-transferase, fatty acid binding protein, cathepsin-L, fluke hemoglobin, paramyosin, myosin and F. hepatica- Kunitz Type Molecule. Among various antigens, GST which plays a crucial role in detoxification processes, i.e. phase II defense mechanism of this parasite, has a unique position as a novel vaccine candidate and a drug target in the control of this disease. For producing the antigens in large quantities and their purification to complete homogeneity, the recombinant DNA technology has become an important tool to achieve this milestone. RT- PCR was carried out using F. gigantica total RNA as template, and an amplicon of 657 bp GST gene was obtained. TA cloning vector was used for cloning of this gene, and the presence of insert was confirmed by blue-white selection for recombinant colonies. Sequence analysis of the present isolate showed 99.1% sequence homology with the published sequence of the F. gigantica GST gene of cattle origin (accession no. AF112657), with six nucleotide changes at 72, 74, 423, 513, 549 and 627th bp found in the present isolate, causing an overall change of 4 amino acids. The 657 bp GST gene was cloned at BamH1 and HindIII restriction sites of the prokaryotic expression vector pPROEXHTb in frame with six histidine residues and expressed in E. coli DH5α. Recombinant protein was purified from the bacterial lysate under non-denaturing conditions by the process of sonication after lysozyme treatment and subjecting the soluble fraction of the bacterial lysate to Ni-NTA affinity chromatography. Western blotting with rabbit hyper-immune serum showed immuno-reactivity with 25 kDa recombinant GST. Recombinant protein detected F. gigantica experimental as well as field infection in buffaloes by dot-ELISA. However, cross-reactivity studies on Fasciola gigantica GST antigen are needed to evaluate the utility of this protein in the serodiagnosis of fasciolosis.

Keywords: fasciola gigantic, fasciola hepatica, GST, RT- PCR

Procedia PDF Downloads 187

Authors: Nurcan Cetinkaya, Nadide Hulya Ozdemir

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The aim of the study was to investigate the effect of Saccharomyces cerevisiae (SC) live yeast culture on microbial protein supply to the small intestine in Kivircik male yearlings when fed with different ratio of forage and concentrate diets. Four Kivircik male yearlings with permanent rumen canula were used in the experiment. . The treatments were allocated to a 4x4 Latin square design. Diet I consisted of 70% alfalfa hay and 30% concentrate, Diet II consisted of 30% alfalfa hay and 70% concentrate, Diet I and II were supplemented with a SC. Daily urine was collected and stored at -20°C until analysis. Calorimetric methods were used for the determination of urinary allantoin and creatinin levels. The estimated microbial N supply to small intestine for Diets I, I+SC, II and II+SC were 2.51, 2.64, 2.95 and 3.43 g N/d respectively. Supplementation of Diets I and II with SC significantly affected the allantoin levels in µmol/W0. 75 (p<0.05). Mean creatinine values in µmol/W0. 75 and allantoin:creatinin ratios were not significantly different among diets. In conclusion, supplementation with SC live yeast culture had a significant effect on urinary allantoin excretion and microbial protein supply to small intestine in Kivircik yearlings fed with high concentrate Diet II (P<0.05). Hence urinary allantoin excretion may be used as a tool for estimating microbial protein supply in Kivircık yearlings. However, further studies are necessary to understand the metabolism of Saccharomyces cerevisiae live yeast culture with different forage: concentrate ratio in Kıvırcık Yearlings.

Keywords: allantoin, creatinin, Kivircik yearling, microbial nitrogen, Saccharomyces cerevisia

Procedia PDF Downloads 413