Search results for: enzyme immobilization

Authors: Timea I. Hajnal-Jafari, Simonida S. Đurić, Dragana R. Stamenov

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Microbial inoculants from the group of symbiotic-nitrogen-fixing rhizobia are well known and widely used in production of legumes. On the other hand, nonsymbiotic plant growth promoting rhizobacteria (PGPR) are not commonly used in practice. The objective of this study was to examine the effects of soybean inoculation with symbiotic and nonsymbiotic bacteria on plant growth and seed yield of soybean. Microbiological activity in rhizospheric soil was also determined. The experiment was set up using a randomized block system in filed conditions with the following treatments: control-no inoculation; treatment 1-Bradyrhizobium japonicum; treatment 2-Azotobacter sp.; treatment 3-Bacillus sp..In the flowering stage of growth (FS) the number of nodules per plant (NPP), root length (RL), plant height (PH) and weight (PW) were measured. The number of pod per plant (PPP), number of seeds per pod (SPP) and seed weight per plant (SWP) were recorded at the end of vegetation period (EV). Microbiological analyses of soil included the determination of total number of bacteria (TNB), number of fungi (FNG), actinomycetes (ACT) and azotobacters (AZB) as well as the activity of the dehydrogenase enzyme (DHA). The results showed that bacterial inoculation led to the formation of root nodules regardless of the treatments with statistically no significant difference. Strong nodulation was also present in control treatment. RL and PH were positively influenced by inoculation with Azotobacter sp. and Bacillus sp., respectively. Statistical analyses of the number of PPP, SPP, and SWP showed no significant differences among investigated treatments. High average number of microorganisms were determined in all treatments. Most abundant were TNB (log No 8,010) and ACT (log No 6,055) than FNG and AZB with log No 4,867 and log No 4,025, respectively. The highest DHA activity was measured in the FS of soybean in treatment 3. The application of nonsymbiotic bacteria in soybean production can alleviate initial plant growth and help the plant to better overcome different stress conditions caused by abiotic and biotic factors.

Keywords: bacteria, inoculation, soybean, microbial activity

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Authors: Sara Arafeh, Kovriguine Evguine

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Cytochromes P450 are enzymes that require a supply of electrons to catalyze the synthesis of steroid hormones, fatty acids, and prostaglandin hormone. Cytochrome P450 Oxidoreductase (CYPOR), a membrane bound enzyme, provides these electrons in its open conformation. CYPOR has two cytosolic domains (FAD domain and FMN domain) and an N-terminal in the membrane. In its open conformation, electrons flow from NADPH, FAD, and finally to FMN where cytochrome P450 picks up these electrons. In the closed conformation, cytochrome P450 does not bind to the FMN domain to take the electrons. It was found that when the cytosolic domains are isolated, CYPOR could not bind to cytochrome P450. This suggested that the membrane environment is important for CYPOR function. This project takes the initiative to better understand the dynamics of CYPOR in its full length. Here, we determine the distance between specific sites in the FAD and FMN binding domains in CYPOR by Forster Resonance Energy Transfer (FRET) and Ultrafast TA spectroscopy with and without NADPH. The approach to determine these distances will rely on labeling these sites with red and infrared fluorophores. Mimic membrane attachment is done by inserting CYPOR in lipid nanodiscs. By determining the distances between the donor-acceptor sites in these domains, we can observe the open/closed conformations upon reducing CYPOR in the presence and absence of cytochrome P450. Such study is important to better understand CYPOR mechanism of action in various endosomal membranes including hepatic CYPOR which is vital in plasma cholesterol homeostasis. By investigating the conformational cycles of CYPOR, we can synthesize drugs that would be more efficient in affecting the steroid hormonal levels and metabolism of toxins catalyzed by Cytochrome P450.

Keywords: conformational cycle of CYPOR, cytochrome P450, cytochrome P450 oxidoreductase, FAD domain, FMN domain, FRET, Ultrafast TA Spectroscopy

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Authors: Agatha Swasti Ayuning Tyas

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Hyperglycemia in Diabetes Mellitus (DM) is an important factor in cellular and vascular damage, which is caused by activation of C Protein Kinase, polyol and hexosamine track, and production of Advanced Glycation End-Products (AGE). Those mentioned before causes the accumulation of Reactive Oxygen Species (ROS). Oxidative stress increases the expression of proinflammatory factors IL-6 as one of many signs of endothelial disfunction. Genistein in soy milk has a high immunomodulator potential. Goat milk contains amino acids which have antioxidative potential. Fermented kefir has an anti-inflammatory activity which believed will also contribute in potentiating goat milk and soy milk. This study is a quasi-experimental posttest-only research to 30 Wistar mice. This study compared the levels of IL-6 between healthy Wistar mice group (G1) and 4 DM Wistar mice with intervention and grouped as follows: mice without treatment (G2), mice treated with 100% goat milk kefir (G3), mice treated with combination of 50% goat milk kefir and 50% soy milk kefir (G4), and mice treated with 100% soy milk kefir (G5). DM animal models were induced with Streptozotocin & Nicotinamide to achieve hyperglycemic condition. Goat milk kefir and soy milk kefir are given at a dose of 2 mL/kg body weight/day for four weeks to intervention groups. Blood glucose was analyzed by the GOD-POD principle. IL-6 was analyzed by enzyme-linked sandwich ELISA. The level of IL-6 in DM untreated control group (G2) showed a significant difference from the group treated with the combination of 50% goat milk kefir and 50% soy milk kefir (G3) (p=0,006) and the group treated with 100% soy milk kefir (G5) (p=0,009). Whereas the difference of IL-6 in group treated with 100% goat milk kefir (G3) was not significant (p=0,131). There is also synergism between glucose level and IL-6 in intervention groups treated with combination of 50% goat milk kefir and 50% soy milk kefir (G3) and the group treated with 100% soy milk kefir (G5). Combination of 50 % goat milk kefir and 50% soy milk kefir and administration of 100% soy milk kefir alone can control the level of IL-6 remained low in DM Wistar mice induced with streptozocin and nicotinamide.

Keywords: diabetes mellitus, goat milk kefir, soy milk kefir, interleukin 6

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Authors: B. Sadepovich Maikanov, Z. Shabanbayevich Adilbekov, R. Husainovna Mustafina, L. Tyulegenovna Auteleyeva

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This study involves detailed information about contaminants of honey in the Republic of Kazakhstan. The requirements of the technical regulation ‘Requirements to safety of honey and bee products’ and GOST 19792-2001 were taken into account in this research. Contamination of honey by antibiotics wqs determined by the IEA (immune-enzyme analysis), Ridder analyzer and Tecna produced test systems. Voltammetry (TaLab device) was used to define contamination by salts of heavy metals and gamma-beta spectrometry, ‘Progress BG’ system, with preliminary ashing of the sample of honey was used to define radioactive contamination. This article pointed out that residues of chloramphenicol were detected in 24% of investigated products, in 22% of them –streptomycin, in 7.3% - sulfanilamide, in 2.4% - tylosin, and in 12% - combined contamination was noted. Geographically, the greatest degree of contamination of honey with antibiotics occurs in the Northern Kazakhstan – 54.4%, and Southern Kazakhstan - 50%, and the lowest in Central and Eastern Kazakhstan with 30% and 25%, respectively. Generally, pollution by heavy metals is within acceptable limits, but the contamination from lead is highest in the Akmola region. The level of radioactive cesium and strontium is also within acceptable concentrations. The highest radioactivity in terms of cesium was observed in the East Kazakhstan region - 49.00±10 Bq/kg, in Akmola, North Kazakhstan and Almaty - 12.00±5, 11.05±3 and 19.0±8 Bq/kg, respectively, while the norm is 100 Bq/kg. In terms of strontium, the radioactivity in the East Kazakhstan region is 25.03±15 Bq/kg, while in Akmola, North Kazakhstan and Almaty regions it is 12.00±3, 10.2±4 and 1.0±2 Bq/kg, respectively, with the norm of 80 Bq/kg. This accumulation is mainly associated with the environmental degradation, feeding and treating of bees. Moreover, in the process of collecting nectar, external substances can penetrate honey. Overall, this research determines factors and reasons of honey contamination.

Keywords: antibiotics, contamination of honey, honey, radionuclides

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Authors: Kwangman Park, Jinhee Kang, Suhee Kim, Dohyeon Yu, Kyoungseong Choi, Jinho Park

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Statement of the Problem: Diarrhea is a major cause of death in young calves. This causes great economic damage to the livestock industry. These diarrhea cause dehydration, decrease blood flow, lower the pH and degrade enzyme function. In the past, serum screening was not possible in the field. However, now with the spread of portable serum testing devices, it is now possible to conduct tests directly on field. Thus, accurate serological changes can be identified and used in the field of large animals. Methodology and Theoretical Orientation: The test groups were calves from 1 to 44 days old. The status of the feces was divided into four grade to determine the severity of diarrhea (grade 0,1,2,3). Grade 0, 1 is considered to have no diarrhea. Grade 2, 3 is considered to diarrhea positive group. One or more viruses were detected in this group. Diarrhea negasitive group consisted of 57 calves (Asan=30, Samrye=27). Diarrhea positive group consisted of 34 calves (Kimje=27, Geochang=7). The feces of all calves were analyzed by PCR Test. Blood sample was measured using an automatic blood analyzer(i-STAT, Abbott inc. Illinois, US). Calves were divided into 3 groups according to age. Group 1 is 1 to 14 days old. Group 2 is 15 to 28 days old. Group 3 is more than 28 days old. Findings: Diarrhea caused an increase in HCT due to dehydration. The difference from normal was highest in 15 to 28 days old (p < 0.01). At all ages, bicarbonate decreased compared to normal, and therefore pH decreased. Similar to HCT, the largest difference was observed between 15 and 28 days (p < 0.01). The pCO₂ decreases to compensate for the decrease in pH. Conclusion and Significance: At all ages, HCT increases, and bicarbonate, pH, and pCO₂ decrease in diarrhea calves. The calf from 15 days to 28 days shows the most difference from normal. Over 28 days of age, weight gain and homeostasis ability increase, diarrhea is seen in the stool, there are fewer hematologic changes than groups below 28 days of age.

Keywords: calves, diarrhea, hematological changes, i-STAT

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Authors: Simona Moravcova, Jiri Novotny, Zdenka Bendova

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The pineal gland of the vertebrate brain is a circumventricular organ which serves as a major neuroendocrine gland with the primary function of rhythmic secretion of neurohormone melatonin under the control of the hypothalamic suprachiasmatic nucleus (SCN). Soon after the onset of the darkness, the activity of the key rate-limiting enzyme for melatonin synthesis, arylalkylamine N-acetyltransferase (AANAT), raises due to the increased release of norepinephrine from sympathetic neurons terminating on the parenchymal cells where it binds to β-adrenergic receptors. Melatonin codes the length of the night, and it is well recognized for its anti-inflammatory effects. However, to our knowledge, less is known about the effect of the immune system on the melatonin biosynthesis and the precise role of the STAT3 in the signaling pathway leading to the expression of AANAT. Lipopolysaccharide (LPS) is the essential component in the outer surface membrane of gram-negative bacteria and acts as a strong stimulator of natural and innate immunity. STAT3 acts as an important factor in immune response. Here we investigated the effect of LPS on the components of the melatonergic synthetic pathway in the pineal gland. The experiments were performed both in vivo and in vitro. The changes in AANAT activity were determined by radioenzymatic assay. PCR analyses were carried out to detect aa-nat, icer, spi-3 and stat3 gene expression. From our results, it is apparent that the high basal level of phosphorylated forms of STAT3 can be elevated after systemic as well as in vitro administration of LPS. Our experiments have shown that LPS reduces melatonin synthesis, nevertheless, the activity of AANAT was increased. Moreover, the basal level of phosphorylated STAT3 counteracts β-adrenergic receptor-mediated aa-nat gene expression and sustains its own and spi-3 gene expression. In conclusion, LPS can affect immunomodulators such as melatonin in the pineal gland.

Keywords: AANAT, lipopolysaccharide, pineal gland, rat, STAT3

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Authors: M. Kouyate, M. Sangare, S. Samake, S. Keita, H. G. Kim, D. H. Geschwind

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Background & Objectives: Human genetic studies can be expensive, even unaffordable, in developing countries, partly due to the sequencing costs. Our aim is to pilot the use of bioinformatics tools to guide scientifically valid, locally relevant, and economically sound autism genetic research in Mali. Methods: The following databases, NCBI, HGMD, and LSDB, were used to identify hot point mutations. Phenotype, transmission pattern, theoretical protein expression in the brain, the impact of the mutation on the 3D structure of the protein) were used to prioritize selected autism genes. We used the protein database, Modeller, and clustal W. Results: We found Mef2c (Gly27Ala/Leu38Gln), Pten (Thr131IIle), Prodh (Leu289Met), Nme1 (Ser120Gly), and Dhcr7 (Pro227Thr/Glu224Lys). These mutations were associated with endonucleases BseRI, NspI, PfrJS2IV, BspGI, BsaBI, and SpoDI, respectively. Gly27Ala/Leu38Gln mutations impacted the 3D structure of the Mef2c protein. Mef2c protein sequences across species showed a high percentage of similarity with a highly conserved MADS domain. Discussion: Mef2c, Pten, Prodh, Nme1, and Dhcr 7 gene mutation frequencies in the Malian population will be very informative. PCR coupled with restriction enzyme digestion can be used to screen the targeted gene mutations. Sanger sequencing will be used for confirmation only. This will cut down considerably the sequencing cost for gene-to-gene mutation screening. The knowledge of the 3D structure and potential impact of the mutations on Mef2c protein informed the protein family and altered function (ex. Leu38Gln). Conclusion & Future Work: Bio-informatics will positively impact autism research in Mali. Our approach can be applied to another neuropsychiatric disorder.

Keywords: bioinformatics, endonucleases, autism, Sanger sequencing, point mutations

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Authors: Leo Paul Vaurs, Sonia Heaven, Charles Banks

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Municipal solid waste (MSW) is a virtually unlimited source of lignocellulosic material in the form of a waste paper/cardboard mixture which can be converted into fermentable sugars via cellulolytic enzyme hydrolysis in a biorefinery. The extraction of the lignocellulosic fraction and its preparation, however, are energy and water demanding processes. The waste water generated is a rich organic liquor with a high Chemical Oxygen Demand that can be partially cleaned while generating biogas in an Upflow Anaerobic Sludge Blanket bioreactor and be further re-used in the process. In this work, an experiment was designed to determine the critical contaminant concentrations in water affecting either anaerobic digestion or enzymatic hydrolysis by simulating multiple water re-circulations. It was found that re-using more than 16.5 times the same water could decrease the hydrolysis yield by up to 65 % and led to a complete granules desegregation. Due to the complexity of the water stream, the contaminant(s) responsible for the performance decrease could not be identified but it was suspected to be caused by sodium, potassium, lipid accumulation for the anaerobic digestion (AD) process and heavy metal build-up for enzymatic hydrolysis. The experimental data were incorporated into a Water Pinch technology based model that was used to optimize the water re-utilization in the modelled system to reduce fresh water requirement and wastewater generation while ensuring all processes performed at optimal level. Multiple scenarios were modelled in which sub-process requirements were evaluated in term of importance, operational costs and impact on the CAPEX. The best compromise between water usage, AD and enzymatic hydrolysis yield was determined for each assumed contaminant degradations by anaerobic granules. Results from the model will be used to build the first MSW based biorefinery in the USA.

Keywords: anaerobic digestion, enzymatic hydrolysis, municipal solid waste, water optimization

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Authors: Hinda Berghiche, Hamida Benradia, Noureddine Soltani

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Pesticides are widely used in crop production and are known to induce a major contamination of ecosystems especially in aquatic environments. The leaching of a large amount of pollutants derived from agricultural activities (fertilizers, pesticides) might contaminate rivers which diverse into the likes and estuarine and coastal environments affecting several organisms such as crustacean species. In this context, there is searched for new selective insecticides with minimal toxic effects on the environment and human health such as growth insect regulators (GIRs). The current study aimed to examine the impact of novaluron (CE 20%), a potent benzoylphenylurea derivative insecticide on mosquito larvae, against non-target shrimp, Palaemon adspersus (Decapoda, Palaemonidae). The compound was tested at two concentrations (0.91 mg/L and 4.30 mg/L) corresponding respectively to the LC50 and LC90 determined against fourth-instar larvae of Culiseta longiareolata (Diptera, Culicidae). The molting hormone titer was determined in the haemolymph by an enzyme-immunoassay, while chitin was measured in peripheral integument at different stages during the molting cycle. Under normal conditions, the haemolymphatic ecdysteroid concentrations increased during the molting cycle to reach peak at stage D. In the treated series, we note absence of the peak at stage D and an increase at stages B, C and D as compared to the controls. Concerning the chitin amounts, we observe an increase from stage A to stage C followed by a decrease at stage D. Exposition of shrimps to novaluron resulted in a significant decrease of values at all molting stages with a dose-response effect. Thus, the insecticide can present secondary effects on this non-target arthropod species.

Keywords: toxicology, novaluron, crustacean, palaemon adspersus, ecdysteroids, cuticle, chitin

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Authors: Medina Kadiri

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Blue-green otherwise called cyanobacteria, produce an array of biotoxins grouped into five categories notably hapatotoxins, neurotoxins, cytotoxins, dermatotoxins, and irritant toxins. Microcystins which are examples of hepatotoxins produced by blue-green algae Microcystins comprise the most common group of the cyanobacterial toxins. Blue-green algae flourish in aquatic environments, whether marine, brackish or freshwater, producing blooms in different forms such as microscopic, mats, or unsightly odoriferous scums. Microcystins biotoxins cause a plethora of animal and human hazards such as liver damage/cirrhosis and cancer, kidney damage, dermatitis, tinnitus, gastroenteritis, sore throat, nausea, myalgia, neurological problems, respiratory irritation and death. Water samples were collected from coastal regions of Nigeria in March 2014, June 2014, October 2014 and January 2015 and analyzed with Enzyme Linked Immunosorbent Assay (ELISA) kits. Microcystin biotoxin was recorded in all sites both during dry and wet seasons. The range of microcystins found was 0.000041-There was a seasonal trend of increasing microcystin concentrations from March till Octobers and a decrease thereafter. Generally in the oceanic waters, microcystin levels were highest at Cross Rivers in March and January, Barbeach in June and Lekki in October. In the adjoining riverine ecosystems, on the other hand, the highest concentrations of microcystin were observed at Akwa Ibom in March, June and October and in Bayelsa in January. Continuous monitoring and screening of coastal water bodies is suggested to minimize the health risks of cyanobacterial biotoxins to coastal communities of Nigeria.

Keywords: biotoxins, harmful algae, marine, microcystin, Nigeria

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Authors: David Polcari, Samuel C. Perry, Loredano Pollegioni, Matthias Geissler, Janine Mauzeroll

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ᴅ-serine acts as an endogenous co-agonist for N-methyl-ᴅ-aspartate receptors in neuronal synapses. This makes it a key component in the development and function of a healthy brain, especially given its role in several neurodegenerative diseases such as Alzheimer’s disease and dementia. Despite such clear research motivations, the primary site and mechanism of ᴅ-serine release is still currently unclear. For this reason, we are developing a biosensor for the detection of ᴅ-serine utilizing a microelectrode in combination with a ᴅ-amino acid oxidase enzyme, which produces stoichiometric quantities of hydrogen peroxide in response to ᴅ-serine. For the fabrication of a biosensor with good selectivity, we use a permselective poly(meta-phenylenediamine) film to ensure only the target molecule is reacted, according to the size exclusion principle. In this work, we investigated the effect of the electrodeposition conditions used on the biosensor’s response time and selectivity. Careful optimization of the fabrication process allowed for enhanced biosensor response time. This allowed for the real time sensing of ᴅ-serine in a bulk solution, and also provided in means to map the efflux of ᴅ-serine in real time. This was done using scanning electrochemical microscopy (SECM) with the optimized biosensor to measure localized release of ᴅ-serine from an agar filled glass capillary sealed in an epoxy puck, which acted as a model system. The SECM area scan simultaneously provided information regarding the rate of ᴅ-serine flux from the model substrate, as well as the size of the substrate itself. This SECM methodology, which provides high spatial and temporal resolution, could be useful to investigate the primary site and mechanism of ᴅ-serine release in other biological samples.

Keywords: ᴅ-serine, enzymatic biosensor, microelectrode, scanning electrochemical microscopy

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Authors: Patricia M. B. Saint-Vincent, Anna Furches, Stephanie Galanie, Erica Teixeira Prates, Piet Jones, Nancy Engle, David Kainer, Wellington Muchero, Daniel Jacobson, Timothy J. Tschaplinski

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Uridine diphosphate-glycosyltransferases (UGTs) are enzymes that catalyze sugar transfer to a variety of plant metabolites. UGT substrates, which include plant secondary metabolites involved in lignification, demonstrate new activities and incorporation when glycosylated. Knowledge of UGT function, substrate specificity, and enzyme products is important for plant engineering efforts, especially related to increasing plant biomass through lignification. UGTs in Populus trichocarpa, a biofuel feedstock, and model woody plant, were selected from a pool of gene candidates using rapid prioritization strategies. A functional genomics workflow, consisting of a metabolite genome-wide association study (mGWAS), expression of synthetic codon-optimized genes, and high-throughput biochemical assays with mass spectrometry-based analysis, was developed for determining the substrates and products of previously-uncharacterized enzymes. A total of 40 UGTs from P. trichocarpa were profiled, and the biochemical assay results were compared to predicted mGWAS connections. Assay results confirmed seven of 11 leaf mGWAS associations and demonstrated varying levels of substrate specificity among candidate UGTs. P. trichocarpa UGT substrate processing confirms the role of these newly-characterized enzymes in lignan, flavonoid, and phytohormone metabolism, with potential implications for cell wall biosynthesis, nitrogen uptake, and biotic and abiotic stress responses.

Keywords: Populus, metabolite-gene associations, GWAS, bio feedstocks, glycosyltransferase

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Authors: Azza EL-Medany, Jamila EL-Medany

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Lung fibrosis is a common side effect of the chemotherapeutic agent, bleomycin. Current evidence suggests that reactive oxygen species may play a key role in the development of lung fibrosis. The present work studied the effect of green tea extract on bleomycin–induced lung fibrosis in rats. Animals were divided into three groups: (1) Saline control group; (2) bleomycin group in which rats were injected with bleomycin (15mg/kg,i.p.) three times a week for four weeks; (3) bleomycin and green tea group in which green tea extract was given to rats (100mg/kg/day, p.o) a week prior to bleomycin and daily during bleomycin injections for 4 weeks until the end of the experiment. Bleomycin–induced pulmonary injury and lung fibrosis that was indicated by increased lung hydroxyproline content, elevated nitric oxide synthase, myeoloperoxidase (MPO), platelet activating factor (PAF), tumor necrosis factor α (TNF_α), transforming growth factor 1β (TGF1β) and angiotensin converting enzyme (ACE) activity in lung tissues. On the other hand, bleomycin induced a reduction in reduced glutathione concentration (GSH). Moreover, bleomycin resulted in a severe histological changes in lung tissues revealed as lymphocytes and neutrophils infiltration, increased collagen deposition and fibrosis. Co-administration of bleomycin and green tea extract reduced bleomycin–induced lung injury as evaluated by the significant reduction in hydroxyproline content, nitric oxide synthase activity, levels of MPO, PAF, TNF-α, and ACE in lung tissues. Furthermore, green tea extract ameliorated bleomycin– induced reduction in GSH concentration. Finally, histological evidence supported the ability of green tea extract to attenuate bleomycin–induced lung fibrosis and consolidation. Thus, the finding of the present study provides that green tea may serve as a novel target for potential therapeutic treatment of lung fibrosis.

Keywords: bleomycin, lung fibrosis, green tea, oxygen species

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Authors: Kabange Kasumbwe, Viresh Mohanlall, Bharti Odhav, Venu Narayanaswamy

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Coumarins are naturally occurring plant metabolites known for their pharmacological properties such as anticoagulant, antimicrobial, anticancer, antioxidant, anti-inflammatory and antiviral properties. The pharmacological and biochemical properties and curative applications of coumarins depend on the substitution around the coumarin core structure. In the present study, seven halogenated coumarins CMRN1-CMRN7 were synthesized and evaluated for their anticancer activity. The cytotoxicity potential of the test compounds was evaluated against UACC62 (Melanoma), MCF-7 (Breast cancer) and PBM (Peripheral Blood Mononuclear) cell lines using MTT assay keeping doxorubicin as standard drug. The apoptotic potential of the coumarin compounds was evaluated against UACC62 (Melanoma) cell by assessing their morphological changes, membrane change, mitochondria membrane potential; pro-apoptotic changes were investigated using the AnnexinV-PI staining, JC-1, caspase-3 enzyme kits respectively on flow cytometer. The synthetic coumarin has strongly suppressed the cell proliferation of UACC-62 (Melanoma) and MCF-7 (Breast) Cancer cells, the higher toxicity of these compounds against UACC-62 (Melanoma) and MCF-7 (Breast) were CMRN3, CMRN4, CMRN5, CMRN6. However, compounds CMRN1, CMRN2, and CMRN7 had no significant inhibitory effect. Furthermore the active compounds CMRN3, CMRN4, CMRN5, CMRN6 exerted antiproliferative effects through apoptosis induction against UACC-62 (Melanoma), suggesting their potential could be considered as attractive lead molecules in the future for the development of potential anticancer agents since one of the important criteria in the development of therapeutic drugs for cancer treatment is to have high selectivity and less or no side-effects on normal cells and these compounds had no inhibitory effect against the PBMC cells.

Keywords: coumarin, MTT, apoptosis, cytotoxicity

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Authors: Enlin Lo, Ioannis Dogaris, George Philippidis

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Succinic acid is a compound used for manufacturing lacquers, resins, and other coating chemicals. It is also used in the food and beverage industry as a flavor additive. It is predominantly manufactured from petrochemicals, but it can also be produced by fermentation of sugars from renewable feedstocks, such as plant biomass. Bio-based succinic acid has great potential in becoming a platform chemical (building block) for commodity and high-value chemicals. In this study, the production of bio-based succinic acid from sweet sorghum was investigated. Sweet sorghum has high fermentable sugar content and can be cultivated in a variety of climates. In order to avoid competition with food feedstocks, its non-edible ‘bagasse’ (the fiber part after extracting the juice) was targeted. Initially, various conditions of pretreating sweet sorghum bagasse (SSB) were studied in an effort to remove most of the non-fermentable components and expose the cellulosic fiber containing the fermentable sugars (glucose). Concentrated (83%) phosphoric acid was utilized at temperatures 50-80 oC for 30-60 min at various SSB loadings (10-15%), coupled with enzymatic hydrolysis using commercial cellulase (Ctec2, Novozymes) enzyme, to identify the conditions that lead to the highest glucose yields for subsequent fermentation to succinic acid. As the pretreatment temperature and duration increased, the bagasse color changed from light brown to dark brown-black, indicating decomposition, which ranged from 15% to 72%, while the theoretical glucose yield is 91%. With Minitab software statistical analysis, a model was built to identify the optimal pretreatment condition for maximum glucose released. The projected theoretical bio-based succinic acid production is 23g per 100g of SSB, which will be confirmed with fermentation experiments using the bacterium Actinobacillus succinogenes.

Keywords: biomass, cellulose, enzymatic hydrolysis, fermentation, pretreatment, succinic acid

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Authors: Yong Hwang, Kang Yeol Suh, Yundeok Jang, Tae Hoon Kim

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Introduction: Rhabdomyolysis is a syndrome characterized by muscle necrosis and the release of intracellular muscle constituents into the circulation. Acute kidney injury is a potential complication of severe rhabdomyolysis and the prognosis is substantially worse if renal failure develops. We try to identify the factors that were predictive of AKI in severe trauma patients with rhabdomyolysis. Methods: This retrospective study was conducted at the emergency department of a level Ⅰ trauma center. Patients enrolled that initial creatine phosphokinase (CPK) levels were higher than 1000 IU with acute multiple trauma, and more than 18 years older from Oct. 2012 to June 2016. We collected demographic data (age, gender, length of hospital day, and patients’ outcome), laboratory data (ABGA, lactate, hemoglobin. hematocrit, platelet, LDH, myoglobin, liver enzyme, and BUN/Cr), and clinical data (Injury Mechanism, RTS, ISS, AIS, and TRISS). The data were compared and analyzed between AKI and Non-AKI group. Statistical analyses were performed using IMB SPSS 20.0 statistics for Window. Results: Three hundred sixty-four patients were enrolled that AKI group were ninety-six and non-AKI group were two hundred sixty-eight. The base excess (HCO3), AST/ALT, LDH, and myoglobin in AKI group were significantly higher than non-AKI group from laboratory data (p ≤ 0.05). The injury severity score (ISS), revised Trauma Score (RTS), Abbreviated Injury Scale 3 and 4 (AIS 3 and 4) were showed significant results in clinical data. The patterns of CPK level were increased from first and second day, but slightly decreased from third day in both group. Seven patients had received hemodialysis treatment despite the bleeding risk and were survived in AKI group. Conclusion: We recommend that HCO3, CPK, LDH, and myoglobin should be checked and be concerned about ISS, RTS, AIS with injury mechanism at the early stage of treatment in the emergency department.

Keywords: acute kidney injury, emergencies, multiple trauma, rhabdomyolysis

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Authors: Ouchemoukh Salim, Amessis-Ouchemoukh Nadia, Guenaoui Nawel, Moumeni Lynda, Zaidi Hicham, Otmani Amar, Sadou Dyhia

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Honey is a hive food rich in carbohydrates and water and it also has a lot of nutrients (enzymes, minerals, organic acids, phytochemicals...). It is used in different nutritional and therapeutic fields. Algerian honey was studied for its physicochemical parameters, nutritional values (moisture, brix, pH, electrical conductivity, and amounts of HMF, proteins, proline, total phenolic compounds and flavonoids) and some biological activities (antioxidant, anti-inflammatory and enzymatic anti-browning). The antioxidant activities of the samples were estimated using different methods (ABTS, DPPH free radicals scavenging, reducing power, and chelating ferrous activity). All honeys were acidic (3.45≤pH≤4.65). The color varied from mimosa yellow to dark brown. The specific rotation was levorotatory in most honey samples, and the electrical conductivity, hydroxymethylfurfural, and proline values agreed with the international honey requirements. For anti-inflammatory activity, the results showed that the inhibiting capacity of the denaturation of the BSA of the honey analyzed varied from 15 to 75 % with a maximum of activity at the concentration of 0,5 mg/ml. All honey exhibited enzymatic anti-browning on different slices of fruits. In fact, the results showed that the controls have the greatest browning unit compared to the honeys studied and PPO and POD enzymes had the lowest enzyme activity. High significant correlations were found between the color of honey, its antioxidant content and its biological activities (antioxidant, anti-inflammatory and enzymatic anti-browning). The dark color of honey is a good indicator of the best biological properties, therefore, the best nutritional and therapeutic values.

Keywords: honey, physico-chemical parameters, bioactive compounds, biological properties

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Authors: Younmin Shin, Jin Kyu Kim, Mirim Jin, Jeong June Choi

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Demilitarized Zone (DMZ) is a peace region located between South and North Korea border to avoid accidental armed conflict. Because human accessing to the area was forced to be prohibited for more than 60 years, DMZ is one of the cleanest land keeping wild lives as nature itself in South Korea. In this study, we evaluated the biological efficacies of plants (SS, PC, and AR) inhabiting in DMZ for the development of functional cosmetics. First, we tested the cytotoxicity of plant extracts in keratinocyte and melanocyte, which are the major cell components of skin. By 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay with the cell lines, we determined the safety concentrations of the extracts for the efficacy tests. Next, we assessed the anti-wrinkle cosmetic function of SS by demonstrating that SS treatment decreased the expression of Matrix metalloproteinase-1 (MMP-1) in UV-irradiated keratinocytes via real-time PCR. The suppressive effect of SS was greatly potentiated by combination with other DMZ-inhabiting plants, PC and AR. The expression of tyrosinase, which is one the main enzyme that producing melanin in melanocyte, was also down-regulated by the DMZ-inhabiting SS extract. Wound healing activity was also investigated by in vitro test with HaCat cell line, a human fibroblast cell line. All the natural materials extracted form DMZ habiting plants accelerated the recovery of the cells. These results suggested that DMZ is a treasure island of functional plants and DMZ-inhabiting natural products are warranted to develop functional cosmetic materials. This study was carried out with the support of R&D Program for Forest Science Technology (Project No. 2017027A00-1819-BA01) provided by Korea Forest Service (Korea Forestry Promotion Institute).

Keywords: anti-wrinkle, Demilitarized Zone, functional cosmetics, whitening

Procedia PDF Downloads 121

Authors: Sekena H. Abd El-Aziem, Heba A. M. Abd El-Kader, Sally S. Alam, Othman E. Othman

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Growth hormone (GH) is an anabolic hormone synthesized and secreted by the somatotroph cells of the anterior lobe of the pituitary gland in a circadian and pulsatile manner, the pattern of which plays an important role in postnatal longitudinal growth and development, tissue growth, lactation, reproduction as well as protein, lipid and carbohydrate metabolism. The aim of this study was to detect the genetic polymorphism of GH gene in five camel breeds reared in Egypt; Sudany, Somali, Mowaled, Maghrabi and Falahy, using PCR-RFLP technique. Also this work aimed to identify the single nucleotide polymorphism between different genotypes detected in these camel breeds. The amplified fragment of camel GH at 613-bp was digested with the restriction enzyme MspI and the result revealed the presence of three different genotypes; CC, CT and TT in tested breeds and significant differences were recorded in the genotype frequencies between these camel breeds. The result showed that the Maghrabi breed that is classified as a dual purpose camels had higher frequency for allele C (0.75) than those in the other tested four breeds. The sequence analysis declared the presence of a SNP (C→T) at position 264 in the amplified fragment which is responsible for the destruction of the restriction site C^CGG and consequently the appearance of two different alleles C and T. The nucleotide sequences of camel GH alleles T and C were submitted to nucleotide sequences database NCBI/Bankit/GenBank and have accession numbers: KP143517 and KP143518, respectively. It is concluded that only one SNP C→T was detected in GH gene among the five tested camel breeds reared in Egypt and this nucleotide substitution can be used as a marker for the genetic biodiversity between camel breeds reared in Egypt. Also, due to the possible association between allele C and higher growth rate, we can used it in MAS for camels and enter the camels possess this allele in breeding program as a way for enhancement of growth trait in camel breeds reared in Egypt.

Keywords: camel breeds in Egypt, GH, PCR-RFLP, SNPs

Procedia PDF Downloads 439

Authors: Carlos E. Rodríguez-Rodríguez, Karla Ruiz-Hidalgo, Kattia Madrigal-Zúñiga, Juan Salvador Chin-Pampillo, Mario Masís-Mora, Elizabeth Carazo-Rojas

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One of the main causes of contamination linked to agricultural activities is the spillage and disposal of pesticides, especially during the loading, mixing or cleaning of agricultural spraying equipment. One improvement in the handling of pesticides is the use of biopurification systems (BPS), simple and cheap degradation devices where the pesticides are biologically degraded at accelerated rates. The biologically active core of BPS is the biomixture, which is constituted by soil pre-exposed to the target pesticide, a lignocellulosic substrate to promote the activity of ligninolitic fungi and a humic component (peat or compost), mixed at a volumetric proportion of 50:25:25. Considering the known ability of lignocellulosic fungi to degrade a wide range of organic pollutants, and the high amount of lignocellulosic waste used in biomixture preparation, the bioaugmentation of biomixtures with these fungi represents an interesting approach for improving biomixtures. The present work aimed at evaluating the effect of the bioaugmentation of rice husk based biomixtures with the fungus Trametes versicolor in the removal of the insectice/nematicide carbofuran (CFN) and to optimize the composition of the biomixture to obtain the best performance in terms of CFN removal and mineralization, reduction in formation of transformation products and decrease in residual toxicity of the matrix. The evaluation of several lignocellulosic residues (rice husk, wood chips, coconut fiber, sugarcane bagasse or newspaper print) revealed the best colonization by T. versicolor in rice husk. Pre-colonized rice husk was then used in the bioaugmentation of biomixtures also containing soil pre-exposed to CFN and either peat (GTS biomixture) or compost (GCS biomixture). After spiking with 10 mg/kg CBF, the efficiency of the biomixture was evaluated through a multi-component approach that included: monitoring of CBF removal and production of CBF transformation products, mineralization of radioisotopically labeled carbofuran (14C-CBF) and changes in the toxicity of the matrix after the treatment (Daphnia magna acute immobilization test). Estimated half-lives of CBF in the biomixtures were 3.4 d and 8.1 d in GTS and GCS, respectively. The transformation products 3-hydroxycarbofuran and 3-ketocarbofuran were detected at the moment of CFN application, however their concentration continuously disappeared. Mineralization of 14C-CFN was also faster in GTS than GCS. The toxicological evaluation showed a complete toxicity removal in the biomixtures after 48 d of treatment. The composition of the GCS biomixture was optimized using a central composite design and response surface methodology. The design variables were the volumetric content of fungally pre-colonized rice husk and the volumetric ratio compost/soil. According to the response models, maximization of CFN removal and mineralization rate, and minimization in the accumulation of transformation products were obtained with an optimized biomixture of composition 30:43:27 (pre-colonized rice husk:compost:soil), which differs from the 50:25:25 composition commonly employed in BPS. Results suggest that fungal bioaugmentation may enhance the performance of biomixtures in CFN removal. Optimization reveals the importance of assessing new biomixture formulations in order to maximize their performance.

Keywords: bioaugmentation, biopurification systems, degradation, fungi, pesticides, toxicity

Procedia PDF Downloads 287

Authors: Shimayali Kaushal, Nitesh Priyadarshi, Nitin Kumar Singhal

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Food borne bacterial species have been identified as major pathogens in most of the severe pathogen-related diseases among humans which result in great loss to human health and food industry. Conventional methods like plating and enzyme-linked immune sorbent assay (ELISA) are time-consuming, laborious and require specialized instruments. Nanotechnology has emerged as a great field in case of rapid detection of pathogens in recent years. The AuNRs material has good electro-optical properties due to its larger light absorption band and scattering in surface plasmon resonance wavelength regions. By exploiting the sugar-based adhesion properties of microorganism, we can use the glycoconjugates capped gold nanorods as a potential nanobiosensor to detect the foodborne pathogen. In the present study, polyethylene glycol (PEG) coated gold nanorods (AuNRs) were prepared and functionalized with different types of carbohydrates and further characterized by UV-Visible spectrophotometry, dynamic light scattering (DLS), transmission electron microscopy (TEM). The reactivity of above said nano-biosensor was probed by lectin binding assay and also by different strains of foodborne bacteria by using spectrophotometric and microscopic techniques. Due to the specific interaction of probe with foodborne bacteria (Escherichia coli, Pseudomonas aeruginosa), our nanoprobe has shown significant and selective ablation of targeted bacteria. Our findings suggest that our nanoprobe can be an ideal candidate for selective optical detection of food pathogens and can reduce loss to the food industry.

Keywords: glyco-conjugates, gold nanorods, nanobiosensor, nanoprobe

Procedia PDF Downloads 114

Authors: Naso Isaiah Thanavisuth

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Introduction: Medical cannabis can be used for treatment, including anorexia, pain, inflammation, multiple sclerosis, Parkinson's disease, epilepsy, cancer, and metabolic syndrome-related disorders. However, medical cannabis leads to adverse effects (AEs), which is delta-9-tetrahydrocannabinol (THC). In previous studies, the major of THC metabolism enzymes are CYP2C9. Especially, the variation of CYP2C9 gene consist of CYP2C9*2 on exon 3 (C430T) (Arg144Cys) and CYP2C9*3 on exon 7 (A1075C) (Ile359Leu) to decrease enzyme activity. Notwithstanding, there is no data describing whether the variant of CYP2C9 genes are a pharmacogenetics marker for prediction of THC-induced AEs in Thai patients. Objective: We want to investigate the association between CYP2C9 gene and THC-induced AEs in Thai patients. Method: We enrolled 39 Thai patients with medical cannabis treatment consisting of men and women who were classified by clinical data. The quality of DNA extraction was assessed by using NanoDrop ND-1000. The CYP2C9*2 and *3 genotyping were conducted using the TaqMan real time PCR assay (ABI, Foster City, CA, USA). Results: All Thai patients who received the medical cannabis consist of twenty four (61.54%) patients who were female and fifteen (38.46%) were male, with age range 27- 87 years. Moreover, the most AEs in Thai patients who were treated with medical cannabis between cases and controls were tachycardia, arrhythmia, dry mouth, and nausea. Particularly, thirteen (72.22%) medical cannabis-induced AEs were female and age range 33 – 69 years. In this study, none of the medical cannabis groups carried CYP2C9*2 variants in Thai patients. The CYP2C9*3 variants (*1/*3, intermediate metabolizer, IM) and (*3/*3, poor metabolizer, PM) were found, three of thirty nine (7.69%) and one of thirty nine (2.56%) , respectively. Conclusion: This is the first study to confirm the genetic polymorphism of CYP2C9 and medical cannabis-induced AEs in the Thai population. Although, our results indicates that there is no found the CYP2C9*2. However, the variation of CYP2C9 allele might serve as a pharmacogenetics marker for screening before initiating the therapy with medical cannabis for prevention of medical cannabis-induced AEs.

Keywords: CYP2C9, medical cannabis, adverse effects, THC, P450

Procedia PDF Downloads 81

Authors: Leda Maria Costa Pereira, Maria Denise Lopes, Nucharin Songsasen

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Mammalian ovaries contain thousands of follicles that eventually degenerate or die after culture in vitro. Caspase-3 is a key enzyme that regulating cell death. Our objective was to examine the influence of anti-apoptotic drug Z-VAD-FMK (pan-caspase inhibitor) on in vitro viability of dog follicles within the ovarian cortex. Ovaries were obtained from prepubertal (age, 2.5–6 months) and adult (age, 8 months to 2 years) bitches and ovarian cortical fragments were recovered. The cortices were then incubated on 1.5% (w/v) agarose gel blocks within a 24-wells culture plate (three cortical pieces/well) containing Minimum Essential Medium Eagle - Alpha Modification (Alpha MEM) supplemented with 4.2 µg/ml insulin, 3.8 µg/ml transferrin, 5 ng/ml selenium, 2 mM L-glutamine, 100 µg/mL of penicillin G sodium, 100 µg/mL of streptomycin sulfate, 0.05 mM ascorbic acid, 10 ng/mL of FSH and 0.1% (w/v) polyvinyl alcohol in humidified atmosphere of 5% CO2 and 5% O2. The cortices were divided in six treatment groups: 1) 10 ng/mL EGF (EGF V0); 2) 10 ng/mL of EGF plus 1 mM Z-VAD-FMK (EGF V1); 3) 10 ng/mL of EGF and 10 mM Z-VAD-FMK (EGF V10); 4) 1 mM Z-VAD-FMK; 5) 10 mM Z-VAD-FMK and (6) no EGF and Z-VAD-FMK supplementation. Ovarian follicles within the tissues were processed for histology and assessed for follicle density, viability (based on morphology) and diameter immediately after collection (Control) or after 3 or 7 days of in vitro incubation. Comparison among fresh and culture treatment group was performed using ANOVA test. There were no differences (P > 0.05) in follicle density and viability among different culture treatments. However, there were differences in this parameter between culture days. Specifically, culturing tissue for 7 days resulted in significant reduction in follicle viability and density, regardless of treatments. We found a difference in size between culture days when these follicles were cultured using 10 mM Z-VAD-FMK or 10 ng/mL EGF (EGF V0). In sum, the finding demonstrated that Z-VAD-FMK at the dosage used in the present study does not provide the protective effect to ovarian tissue during in vitro culture. Future studies should explore different Z-VAD-FMK dosages or other anti-apoptotic agent, such as surviving in protecting ovarian follicles against cell death.

Keywords: anti apoptotic drug, bitches, follicles, Z-VAD-FMK

Procedia PDF Downloads 338

Authors: N. V. Kazmiruk, Y. R. Nartsissov

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Phosphate activated glutaminase (GA, E.C. 3.5.1.2) plays a key role in glutamine/glutamate homeostasis in mammalian brain, catalyzing the hydrolytic deamidation of glutamine to glutamate and ammonium ions. GA is mainly localized in mitochondria, where it has the catalytically active form on the inner mitochondrial membrane (IMM) and the other soluble form, which is supposed to be dormant. At present time, the exact localization of the membrane glutaminase active site remains a controversial and an unresolved issue. The first hypothesis called c-side localization suggests that the catalytic site of GA faces the inter-membrane space and products of the deamidation reaction have immediate access to cytosolic metabolism. According to the alternative m-side localization hypothesis, GA orients to the matrix, making glutamate and ammonium available for the tricarboxylic acid cycle metabolism in mitochondria directly. In our study, we used a multi-compartment kinetic approach to simulate metabolism of glutamate and glutamine in the astrocytic cytosol and mitochondria. We used physiologically important ratio between the concentrations of glutamine inside the matrix of mitochondria [Glnₘᵢₜ] and glutamine in the cytosol [Glncyt] as a marker for precise functioning of the system. Since this ratio directly depends on the mitochondrial glutamine carrier (MGC) flow parameters, key observation was to investigate the dependence of the [Glnmit]/[Glncyt] ratio on the maximal velocity of MGC at different initial concentrations of mitochondrial glutamate. Another important task was to observe the similar dependence at different inhibition constants of the soluble GA. The simulation results confirmed the experimental c-side localization hypothesis, in which the glutaminase active site faces the outer surface of the IMM. Moreover, in the case of such localization of the enzyme, a 3-fold decrease in ammonium production was predicted.

Keywords: glutamate metabolism, glutaminase, kinetic approach, mitochondrial membrane, multi-compartment modeling

Procedia PDF Downloads 94

Authors: Srishty Gulati, Anju Singh, Shrikant Kukreti

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The manifestation of structural polymorphism in DNA depends on the sequence and surrounding environment. Ample of folded DNA structures have been found in the cellular system out of which DNA hairpins are very common, however, are indispensable due to their role in the replication initiation sites, recombination, transcription regulation, and protein recognition. We enumerate this approach in our study, where the two base substitutions and change in temperature embark destabilization of DNA structure and misbalance the equilibrium between two structures of a sequence present at the overlapping region of the human COMT gene and MIR4761 gene. COMT and MIR4761 gene encodes for catechol-O-methyltransferase (COMT) enzyme and microRNAs (miRNAs), respectively. Environmental changes and errors during cell division lead to genetic abnormalities. The COMT gene entailed in dopamine regulation fosters neurological diseases like Parkinson's disease, schizophrenia, velocardiofacial syndrome, etc. A 19-mer deoxyoligonucleotide sequence 5'-AGGACAAGGTGTGCATGCC-3' (COMT19) is located at exon-4 on chromosome 22 and band q11.2 at the intersection of COMT and MIR4761 gene. Bioinformatics studies suggest that this sequence is conserved in humans and few other organisms and is involved in recognition of transcription factors in the vicinity of 3'-end. Non-denaturating gel electrophoresis and CD spectroscopy of COMT sequences indicate the formation of hairpin type DNA structures. Temperature-dependent CD studies revealed an unusual shift in the slipped DNA-Hairpin DNA equilibrium with the change in temperature. Also, UV-thermal melting techniques suggest that the two base substitutions on the complementary strand of COMT19 did not affect the structure but reduces the stability of duplex. This study gives insight about the possibility of existing structurally polymorphic transient states within DNA segments present at the intersection of COMT and MIR4761 gene.

Keywords: base-substitution, catechol-o-methyltransferase (COMT), hairpin-DNA, structural polymorphism

Procedia PDF Downloads 99

Authors: A. Lubis, R. Widayanti, Z. Hikmah, A. Endaryanto, A. Harsono, A. Harianto, R. Etika, D. K. Handayani, M. Sampurna

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Neonatal lupus erythematous (NLE) is a rare disease marked by clinical characteristic and specific maternal autoantibody. Many cutaneous, cardiac, liver, and hematological manifestations could happen with affect of one organ or multiple. In this case, both babies were premature, low birth weight (LBW), small for gestational age (SGA) and born through caesarean section from a systemic lupus erythematous (SLE) mother. In the first case, we found a baby girl with dyspnea and grunting. Chest X ray showed respiratory distress syndrome (RDS) great I and echocardiography showed small atrial septal defect (ASD) and ventricular septal defect (VSD). She also developed anemia, thrombocytopenia, elevated C-reactive protein, hypoalbuminemia, increasing coagulation factors, hyperbilirubinemia, and positive blood culture of Klebsiella pneumonia. Anti-Ro/SSA and Anti-nRNP/sm were positive. Intravenous fluid, antibiotic, transfusion of blood, thrombocyte concentrate, and fresh frozen plasma were given. The second baby, male presented with necrotic tissue on the left ear and skin rashes, erythematous macula, athropic scarring, hyperpigmentation on all of his body with various size and facial haemorrhage. He also suffered from thrombocytopenia, mild elevated transaminase enzyme, hyperbilirubinemia, anti-Ro/SSA was positive. Intravenous fluid, methyprednisolone, intravenous immunoglobulin (IVIG), blood, and thrombocyte concentrate transfution were given. Two cases of neonatal lupus erythematous had been presented. Diagnosis based on clinical presentation and maternal auto antibody on neonate. Organ involvement in NLE can occur as single or multiple manifestations.

Keywords: neonatus lupus erythematous, maternal autoantibody, clinical characteristic, multiple organ manifestation

Procedia PDF Downloads 398

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 113

Authors: Avinash Vellore Sunder, Shikha Shah, Pramod P. Wangikar

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The enzymatic conversion of nitriles to carboxylic acids by nitrilases has gained significance in the green synthesis of several pharmaceutical precursors and fine chemicals. While nitrilases have been characterized from different sources, the industrial application requires the identification of nitrilases that possess higher substrate tolerance, wider specificity and better thermostability, along with the development of an efficient bioprocess for producing large amounts of nitrilase. To produce large amounts of nitrilase, we developed a fed-batch fermentation process on defined media for the high cell density cultivation of E. coli cells expressing the well-studied nitrilase from Alcaligenes fecalis. A DO-stat feeding approach was employed combined with an optimized post-induction strategy to achieve nitrilase titer of 2.5*105 U/l and 78 g/l dry cell weight. We also identified 16 novel nitrilase sequences from genome mining and analysis of substrate binding residues. The nitrilases were expressed in E. coli and their biocatalytic potential was evaluated on a panel of 22 industrially relevant nitrile substrates using high-throughput screening and HPLC analysis. Nine nitrilases were identified to exhibit high activity on structurally diverse nitriles including aliphatic and aromatic dinitriles, heterocyclic, -hydroxy and -keto nitriles. With fed-batch biotransformation, whole-cell Zobelia galactanivorans nitrilase achieved yields of 2.4 M nicotinic acid and 1.8 M isonicotinic acid from 3-cyanopyridine and 4-cyanopyridine respectively within 5 h, while Cupravidus necator nitrilase enantioselectively converted 740 mM mandelonitrile to (R)–mandelic acid. The nitrilase from Achromobacter insolitus could hydrolyze 542 mM iminodiacetonitrile in 1 h. The availability of highly active nitrilases along with bioprocesses for enzyme production expands the toolbox for industrial biocatalysis.

Keywords: biocatalysis, isonicotinic acid, iminodiacetic acid, mandelic acid, nitrilase

Procedia PDF Downloads 200

Authors: Ratmawati Malaka, Effendi Abustam, Kusumandari Indah Prahesti, Sudirman Baco

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Dangke is natural cheese from Enrekang South Sulawesi, Indonesia produced through aglutination buffalo milk, cow, goat or sheep using the sap of papaya (Carica papaya). Dangke has been widely known in South Sulawesi but this soft cheese product diversification by using passion fruit juice as milk clotting agents has not been used. Passion fruit juice has a high acidity with a pH of around 4 - 4.5 and has a proteolytic enzyme, so that it can be used to agglutinate milk. The purpose of this study was to investigate the nature Dangke using passion fruit juice as coagulate milk. Dangke made by 10 lt of raw milk by heating at a temperature of 73oC with coagulant passion fruit juice (7.5% and 10%), and added 1% salt. Curd clot and then be formed using a coconut shell, is then pressed until the cheese is compact. The cheese is then observed for 28 days ripening at a temperature of about 5 ° C. Dangke then studied to violence, pH, fat levels and microstructure. Hardness is determined using CD-shear Force, pH is measured using a pH meter Hanna, and fat concentrations were analyzed with methods of proximate. Microstructure viewed using a light microscope with magnification 1000 x. The results showed that the levels of clotting material very significant influence on hardness, pH, and lipid levels. Maturation increase the hardness but lower the pH, the level of fat soft cheese with an average Dangke respectively 21.4% and 30.5% on 7.5% addition of passion fruit juice and 10%. Dangke violence is increasing with the increasing maturation time (1.38 to 3.73 kg / cm), but Dangke pH was decreased by the increase in storage maturation (5.34 to 4.1). Microktrukture cheeses coagulated with 10% of the passion fruit are very firmer and compact with a full globular fat of 7.5%. But the sensory properties of the soft cheese similar in both treatment. The manufacturing process with the addition of coagulant passion fruit juice on making Dangke affect hardness, pH, fat content and microstructure during storage at 5 ° C for 1 d - 28 d.

Keywords: dangke, passion fruits, microstructure, cheese

Procedia PDF Downloads 385

Authors: Mohammad Alkhatatbeh, Nehad Ayoub, Nizar Mhaidat, Nesreen Saadeh, Lisa Lincz

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CD36 is involved in the development of atherosclerosis by enhancing macrophage endocytosis of oxidized-low density lipoproteins and foam cell formation. Soluble CD36 (sCD36) was found to be elevated in type 2 diabetic patients and was supposed to act as a marker of insulin resistance and atherosclerosis. In young subjects, sCD36 was associated with cardiovascular risk factors including obesity and hypertriglyceridemia. This study was conducted to further investigate the relationship between plasma sCD36 and cardiovascular risk factors among middle-aged patients with metabolic syndrome (MetS) and healthy controls. SCD36 concentrations were determined by enzyme-linked immunosorbent assays (ELISA) for 41 patients with MetS and 36 healthy controls. Data for other variables were obtained from patients' medical records. SCD36 concentrations were relatively low compared to most other studies and were not significantly different between the MetS group and controls (P-value=0.17). SCD36 was also not correlated with age, body mass index, glucose, lipid profile, serum electrolytes and blood counts. SCD36 was not significantly different between subjects with obesity, hyperglycemia, dyslipidemia, hypertension or cardiovascular disease and those without these abnormalities (P-value > 0.05). The inconsistency between results reported in this study and other studies may be unique to the study population or be a result of the lack of a reliable standardized method for determining absolute sCD36 concentrations. However, further investigations are required to assess CD36 tissue expression in the study population and to assess the accuracy of various commercially available sCD36 ELISA kits. Thus, the availability of a standardized simple sCD36 ELISA that could be performed in any basic laboratory would be more favorable to the specialized flow cytometry methods that detect CD36+ microparticles if it was to be used as a biomarker.

Keywords: metabolic syndrome, CD36, cardiovascular risk, obesity, type 2 diabetes mellitus

Procedia PDF Downloads 245