Search results for: glucosamine-6-phosphate synthase

Authors: Anna Höfer, Johannes Herrmann, Patrick Meybohm, Christopher Lotz

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Mitochondria are pivotal for energy supply and regulation of cellular functions. Deficiencies of mitochondrial metabolism have been implicated in diverse stressful conditions including infections. Platelets are key mediators for thrombo-inflammation during development and resolution of acute respiratory distress syndrome (ARDS). Previous data point to an exhausted platelet phenotype in critically-ill patients with coronavirus 19 disease (COVID-19) impacting the course of disease. The objective of this work was to characterize platelet mitochondrial metabolism in patients suffering from COVID-19 ARDSA longitudinal analysis of platelet mitochondrial metabolism in 24 patients with COVID-19 induced ARDS compared to 35 healthy controls (ctrl) was performed. Blood samples were analyzed at two time points (t1=day 1; t2=day 5-7 after study inclusion). The activity of mitochondrial citrate synthase was photometrically measured. The impact of oxidative stress on mitochondrial permeability was assessed by a photometric calcium-induced swelling assay and the activity of superoxide dismutase (SOD) by a SOD assay kit. The amount of protein carbonylation and the activity of mitochondria complexes I-IV were photometrically determined. Levels of interleukins (IL)-1α, IL-1β and tumor necrosis factor (TNF-) α were measured by a Multiplex assay kit. Median age was 54 years, 63 % were male and BMI was 29.8 kg/m2. SOFA (12; IQR: 10-15) and APACHE II (27; IQR: 24-30) indicated critical illness. Median Murray Score was 3.4 (IQR: 2.8-3.4), 21/24 (88%) required mechanical ventilation and V-V ECMO support in 14/24 (58%). Platelet counts in ARDS did not change during ICU stay (t1: 212 vs. t2: 209 x109/L). However, mean platelet volume (MPV) significantly increased (t1: 10.6 vs. t2: 11.9 fL; p<0.0001). Citrate synthase activity showed no significant differences between ctrl and ARDS patients. Calcium induced swelling was more pronounced in patients at t1 compared to t2 and to ctrl (50µM; t1: 0.006 vs. ctrl: 0.016 ΔOD; p=0.001). The amount of protein carbonylation as marker for irreversible proteomic modification constantly increased during ICU stay and compared to ctrl., without reaching significance. In parallel, superoxid dismutase activity gradually declined during ICU treatment vs. ctrl (t2: - 29 vs. ctrl.: - 17 %; p=0.0464). Complex I analysis revealed significantly stronger activity in ARDS vs. ctrl. (t1: 0.633 vs. ctrl.: 0.415 ΔOD; p=0.0086). There were no significant differences in complex II, III or IV activity in platelets from ARDS patients compared to ctrl. IL-18 constantly increased during the observation period without reaching significance. IL-1α and TNF-α did not differ from ctrl. However, IL-1β levels were significantly elevated in ARDS (t1: 16.8; t2: 16.6 vs. ctrl.: 12.4 pg/mL; p1=0.0335, p2=0.0032). This study reveals new insights in platelet mitochondrial metabolism during COVID-19 caused ARDS. it data point towards enhanced platelet activity with a pronounced turnover rate. We found increased activity of mitochondria complex I and evidence for enhanced oxidative stress. In parallel, protective mechanisms against oxidative stress were narrowed with elevated levels of IL-1β likely causing a pro-apoptotic environment. These mechanisms may contribute to platelet exhaustion in ARDS.

Keywords: acute respiratory distress syndrome (ARDS), coronavirus 19 disease (COVID-19), oxidative stress, platelet mitochondrial metabolism

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Authors: S. Ghalem, M. Mesmoudi, I. Daoudand, H. Allali

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The nitrogen-containing bisphosphonates (N-BPs) are well established as the treatments of choice for disorders of excessive bone resorption, myeloma and bone metastases, and osteoporosis. They inhibit farnesyl pyrophosphate synthase (FFPS), a key enzyme in the mevalonate pathway, resulting in inhibition of the prenylation of small GTP-binding proteins in osteoclasts and disruption of their cytoskeleton, adhesion/spreading, and invasion of cancer cells. A very few examples for synthesis of α-amino bisphosphonates based on several amino acids are known from the literature. In the present work, esters of aminoacid react with ketophsophonate (or their analog acid or acyl) to afford the desired products, α-iminophosphonates. The reaction of imine with dimethyl phosphate in the presence of catalytic amount of I2 give ester of α-aminobisphosphonate as sole product in good yield. Finally, we used computational docking methods to predict how several α-aminobisphosphonates bind to FPPS and how R and X influence. Pamidronate, β-aminobisphosphonate already marketed, was used as reference. These results are of interest since they represent a new and simple way to sythesize α-aminobisphosphonates with a free COOH group increased by R2 functionalisable and opening up the possibility of using the molecular docking to facilitate the design of other, novel FFPS inhibitors.

Keywords: drug research, cancer, α-amino bisphosphonates, molecular docking

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Authors: Chia-Ying Yen, Chin-Yuan Hsu

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The lifespans of queen honeybees (Apis mellifera) are much longer than those of worker bees. The expression, concentration, and activity of mitochondrial energy-utilized molecules decreased with age in the trophocytes and oenocytes of worker bees, but they are unknown in queen bees. In this study, the expression, concentration, and activity of mitochondrial energy-utilized molecules were evaluated in the trophocytes and oenocytes of young and old queen bees by biochemical techniques. The results showed that mitochondrial density and mitochondrial membrane potential; nicotinamide adenine dinucleotide (NAD+), nicotinamide adenine dinucleotide reduced form (NADH), and adenosine triphosphate (ATP) levels; the NAD+/NADH ratio; and relative expression of NADH dehydrogenase 1 and ATP synthase normalized against mitochondrial density were not significantly different between young and old queen bees. These findings reveal that mitochondrial energy utilization maintains a young status in the trophocytes and oenocytes of old queen bees and that trophocytes and oenocytes have aging-delaying mechanisms and can be used to study cellular longevity.

Keywords: aging, longevity, mitochondrial energy, queen bees

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Authors: Arif İsmailov, Naila Hasanova, Gunay Orujalieva

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Homocysteine (HCY) is an indicator of prognostic value in monitoring regenerative processes in osteoporosis and osteoporotic fractures. The osteoporosis is known to be a serious health and economic problem, especially for women in the postmenopausal period. The study was carried out on patients 45-83 years old divided into 3 groups: group I – 14 patients with osteoporosis , group II – 15 patients with non-osteoporotic fractures, group III – 25 patients with osteoporotic fractures. The control group consisted of practically healthy 14 people. A blood sample was taken at 3 stages to monitor the dynamics of HCY level: on the 1st day before treatment, on the 10th day of treatment and 1 month after it. Blood levels of Hcy were determined at a wavelength of 450 nm by the ELİSA(Cloud Clone Corp.Elisa kits,USA). The statistical evaluation was performed by using SPSS 26.0 program (IBM SPSS Inc., USA).The results showed that on the 1st day before the treatment HCY concentration was statistically increased 2.7 times(PU = 0.108) in group I, 5.6 times (PU <0.001) in group II and 6.5 times (PU <0.001) in group III compared to the control group. Thus, the average value of HCY in group I was 1.76 ± 0.56 μg/ml; in group II – 3.57 ± 0.62 μg/ml; in group III – 4.2 ± 0.50 μg/ml. HCY level increases more sharply after fractures,especially in osteoporotic patients. In treatment period Vitamin D plays an important role in synthesis of the Cystathionine β‐synthase enzyme, which regulates HCY metabolism. Increased Hcy levels could lead to an increase in the risk of fracture through the interference in collagen cross-linking.

Keywords: homocysteine, osteoporosis, osteoporotic fractures, Vitamin D

Procedia PDF Downloads 27

Authors: Eun Young Choi, So Hui Choe, Jin Yi Hyeon, Ji Young Jin, Bo Ram Keum, Jong Min Lim, Hyung Rae Cho, Kwang Keun Cho, In Soon Choi

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This research analyzed the effect of β-glucan that is expected to alleviate the production of inflammatory mediator in macrophagocyte, which was processed by the lipopolysaccharide (LPS) of Escherichia, a pathogen related to allergy. The incubated layer was used for nitric oxide (NO) analysis. The DNA-binding activation of the small unit of NF-κB was measured using ELISA-based kit. In RAW264.7 cells that were vitalized by E.coli LPS, β-glucan inhibited both the combatant and rendering phases of inducible NO synthase (iNOS)-derived NO. β-glucan increased the expression of heme oxygenase-1 (HO-1) in the cell that was stimulated by E.coli LPS, and HO-1 activation was inhibited by SnPP. This shows that NO production induced by LPS is related to the inhibition effect of β-glucan. The phosphorylation of JNK and p38 induced by LPS were not influenced by β-glucan, and IκB-α decomposition was not influenced either. Instead, β-glucan remarkably inhibited the phosphorylation of STAT1 that was induced by E.coli LPS. Overall, β-glucan inhibited the production of NO in macrophagocyte that was vitalized by E.coli LPS through HO-1 induction and STAT1 pathways inhibition in this research. As the host inflammation reaction control by β-glucan weakens the progress of allergy, β-glucan can be used as an effective treatment method.

Keywords: β-glucan, lipopolysaccharide (LPS), nitric oxide (NO), RAW264.7 cells, STAT1

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Authors: Maryam Saeidi, Ehsan Motamedian, Seyed Abbas Shojaosadati

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Zymomonas mobilis is known as an example of the uncoupled growth phenomenon. This microorganism also has a unique metabolism that degrades glucose by the Entner–Doudoroff (ED) pathway. In this paper, a genome-scale metabolic model including 434 genes, 757 reactions and 691 metabolites was reconstructed to simulate uncoupled growth and study its effect on flux distribution in the central metabolism. The model properly predicted that ATPase was activated in experimental growth yields of Z. mobilis. Flux distribution obtained from model indicates that the major carbon flux passed through ED pathway that resulted in the production of ethanol. Small amounts of carbon source were entered into pentose phosphate pathway and TCA cycle to produce biomass precursors. Predicted flux distribution was in good agreement with experimental data. The model results also indicated that Z. mobilis metabolism is able to produce biomass with maximum growth yield of 123.7 g (mol glucose)-1 if ATP synthase is coupled with growth and produces 82 mmol ATP gDCW-1h-1. Coupling the growth and energy reduced ethanol secretion and changed the flux distribution to produce biomass precursors.

Keywords: genome-scale metabolic model, Zymomonas mobilis, uncoupled growth, flux distribution, ATP dissipation

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Authors: Yomna T. Abdou, Hala F. Zaki, Sanaa A. Kenawy

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Naringenin is a flavanone, a type of flavonoid, found in fruits such as grapefruit, oranges, and tomatoes, was found to possess antioxidant, anti-inflammatory and antitumor effects. The present study was conducted to investigate the protective effect of naringenin on iodoacetamide-induced ulcerative colitis (UC) in rats. Male Wistar rats were pretreated with sulfasalazine (300 mg/kg, p.o.) as standard anti-inflammatory drug or naringenin (50 mg/kg, p.o.) for 7 consecutive days then UC was induced by intracolon administration of 0.1 ml (2%) iodoacetamide dissolved in 1% methylcelluose. One week later, animals were scarificed and the colonic tissues were dissected. Colon inflammation was evident by elevation in colon tumor necrosis factor alpha (TNFα) and interleukin-8 (IL-8) as well as inducible nitric oxide synthase (iNOS) enzyme, prostaglandin- E2 (PG-E2) and myeloperoxidase (MPO) activities. Additionally, oxidative stress was manifested by increased colon lipoperoxidation (MDA), glutathione (GSH) depletion, elevated nitric oxide (NO) content and glutathione peroxidase (GPx) activity. Pretreatment with naringenin largely mitigated these alterations. The present study reinforces the hypothetical use of naringenin as an anti-inflammatory complement to conventional UC treatment and could be considered in the dietary prevention of intestinal inflammation and related disorders.

Keywords: iodoacetamide, naringenin, sulfasalazine, ulcerative colitis

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Authors: Zi-Han Li, Lu Fang, Liang Wu, Dong-Yuan Chang, Manyuan Dong, Liang Ji, Qi Zhang, Ming-Hui Zhao, Sydney C. W. Tang, Lemin Zheng, Min Chen

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Uncoupling of mitochondrial respiration by chemical uncouplers has proven effective in ameliorating obesity, insulin resistance, and hyperglycemia, which were risk factors for diabetic nephropathy (DN). Recently, we found that ANXA1 could improve mitochondrial function to mitigate DN progression. However, the underlying mechanism is not fully clear yet. Here, we identified uncoupling protein 1 (UCP1), an inner membrane protein of mitochondria, as a key to mitochondrial homeostasis improved by ANXA1. Specifically, ANXA1 attenuated mitochondrial dysfunction via appropriately upregulating UCP1 by stabilizing its transcription factor GATA binding protein 3 (GATA3) by combining it with thioredoxin. Moreover, specific overexpression of UCP1 in the renal cortex rescued renal injuries in diabetic Anxa1-KO mice. UCP1 deletion aggravated renal injuries in HFD/STZ-induced diabetic mice. Mechanistically, UCP1 reduced mitochondrial fission through the aristaless-related homeobox (ARX)/cardiolipin synthase 1 (CRLS1) pathway. Therapeutically, CL316243, a UCP1 agonist, could attenuate established DN in db/db mice. This work established an alternative principle to harness the power of uncouplers for the treatment of DN.

Keywords: diabetic nephropathy, uncoupling protein 1, mitochondrial homeostasis, cardiolipin metabolism

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Authors: Yantao Li, Jun Zheng

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Food poison caused by consumption of contaminated food, especially seafood, is one of most serious public health threats worldwide. Vibrio parahaemolyticus is emerging bacterial pathogen and the leading cause of human gastroenteritis associated with food poison, especially in the southern coastal region of China. To successfully cause disease in host, bacterial pathogens need to overcome the host-derived stresses encountered during infection. One of the toxic chemical species elaborated by the host is nitric oxide (NO). NO is generated by acidified nitrite in the stomach and by enzymes of the inducible NO synthase (iNOS) in the host cell, and is toxic to bacteria. Bacterial pathogens have evolved some mechanisms to battle with this toxic stress. Such mechanisms include genes to sense NO produced from immune system and activate others to detoxify NO toxicity, and genes to repair the damage caused by toxic reactive nitrogen species (RNS) generated during NO toxic stress. However, little is known about the NO resistance in V. parahaemolyticus. In this study, a transposon coupled with next generation sequencing (Tn-seq) technology will be utilized to identify genes for NO resistance in V. parahaemolyticus. Our strategy will include construction the saturating transposon insertion library, transposon library challenging with NO, next generation sequencing (NGS), bioinformatics analysis and verification of the identified genes in vitro and in vivo.

Keywords: vibrio parahaemolyticus, nitric oxide, tn-seq, virulence

Procedia PDF Downloads 243

Authors: Zi-Han Li, Lu Fang, Liang Wu, Dong-Yuan Chang, Manyuan Dong, Liang Ji, Qi Zhang, Ming-Hui Zhao, Sydney C.W. Tang, Lemin Zheng, Min Chen

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Uncoupling of mitochondrial respiration by chemical uncouplers has proven effective in ameliorating obesity, insulin resistance, and hyperglycemia, which were risk factors for diabetic nephropathy (DN). Recently, it was found that annexin A1(ANXA1) could improve mitochondrial function to mitigate DN progression. However, the underlying mechanism is not fully clear yet. Here, it was identified that uncoupling protein 1 (UCP1), an inner membrane protein of mitochondria, as a key to mitochondrial homeostasis improved by ANXA1. Specifically, ANXA1 attenuated mitochondrial dysfunction via appropriately upregulating UCP1 by stabilizing its transcription factor GATA binding protein 3 (GATA3) through combining with thioredoxin. Moreover, specific overexpression of UCP1 in renal cortex rescued renal injuries in diabetic Anxa1-KO mice. UCP1 deletion aggravated renal injuries in HFD/STZ-induced diabetic mice. Mechanistically, UCP1 reduced mitochondrial fission through the aristaless-related homeobox (ARX)/cardiolipin synthase 1 (CRLS1) pathway. Therapeutically, CL316243, a UCP1 agonist, could attenuate established DN in db/db mice. This work established a novel principle to harness the power of uncouplers for the treatment of DN.

Keywords: diabetic nephropathy, uncoupling protein 1, mitochondrial homeostasis, cardiolipin metabolism

Procedia PDF Downloads 44

Authors: Y. S. Mohamed, L. A. Ahmed, H. A. Salem, A. M. Agha

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Liver Fibrosis is one of the most serious conditions that affect the Egyptian society. In the present study, the effect of nicorandil was investigated in experimentally-induced liver fibrosis by bile duct ligation in rats. Nicorandil (3mg/kg/day) was given orally 24 h after bile duct ligation for 14 days till the end of the experiment. Nicorandil group showed a significant improvement in liver function tests (ALT and ALP) as well as a significant decrease in oxidative stress biomarkers (TBARS and GSH), area of fibrosis and activity of hepatic stellate cells as indicated by decreased expression of alpha smooth muscle actin.Moreover, nicorandil treatment decreased HSCs proliferation due to its inhibitory effects on protein kinase C(PKC) and Platelet derived growth factor (PDGF) . Oral administration of either glibenclamide (10 mg/kg/day)(a KATP channel blocker) or L-NAME (30 mg/kg/day) (an inhibitor of nitric oxide synthase) blocked the protective effects of nicorandil. However, nicorandil and L-NAME treated group showed more or less results similar to that of untreated bile duct ligated group. In conclusion, nicorandil was effective against the development of bile duct ligated-induced liver fibrosis in rats where activation of the NO pathway plays an important role in the protective effect nicorandil.

Keywords: hepatic stellate cells, nicorandil, nitric oxide donor, liver fibrosis

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Authors: Gizem Buldum, Alexander Bismarck, Athanasios Mantalaris

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Cellulose is the most abundant biopolymer on earth and it is currently being utilised in a multitude of industrial applications. Over the last 30 years, attention has been paid to the bacterial cellulose (BC), since BC exhibits unique physical, chemical and mechanical properties when compared to plant-based cellulose, including high purity and biocompatibility. Although Acetobacter xylinum is the most efficient producer of BC, it’s long doubling time results in insufficient yields of the cellulose production. This limits widespread and continued use of BC. In this study, E. coli BL21 (DE3) or E. coli HMS cells are selected as host organisms for the expression of bacterial cellulose synthase operon (bcs) of A.xylinum. The expression system is created based on pET-Duet1 and pCDF plasmid vectors, which carry bcs operon. The results showed that all bcs genes were successfully transferred and expressed in E.coli strains. The expressions of bcs proteins were shown by SDS and Native page analyses. The functionality of the bcs operon was proved by congo red binding assay. The effect of culturing temperature and the inducer concentration (IPTG) on cell growth and plasmid stability were monitored. The percentage of plasmid harboring cells induced with 0.025 mM IPTG was obtained as 85% at 22˚C in the end of 10-hr culturing period. It was confirmed that the high output cellulose production machinery of A.xylinum can be transferred into other organisms.

Keywords: bacterial cellulose, biopolymer, recombinant expression system, production

Procedia PDF Downloads 362

Authors: Saroj Poudel, Joshua Mancini, Douglas Pike, Jennifer Timm, Alexei Tyryshkin, Vikas Nanda, Paul Falkowski

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On the early Earth, protein-metal complexes likely harvested energy from a reduced environment. These complexes would have been precursors to the metabolic enzymes of ancient organisms. Hydrogenase is an essential enzyme in most anaerobic organisms for the reduction and oxidation of hydrogen in the environment and is likely one of the earliest evolved enzymes. To attempt to reinvent a precursor to modern hydrogenase, we computationally designed a short thirteen amino acid peptide that binds the often-required catalytic transition metal Nickel in hydrogenase. This simple complex can achieve hundreds of hydrogen evolution cycles using light energy in a broad range of temperature and pH. Biophysical and structural investigations strongly indicate the peptide forms a di-nickel active site analogous to Acetyl-CoA synthase, an ancient protein central to carbon reduction in the Wood-Ljungdahl pathway and capable of hydrogen evolution. This work demonstrates that prior to the complex evolution of multidomain enzymes, early peptide-metal complexes could have catalyzed energy transfer from the environment on the early Earth and enabled the evolution of modern metabolism

Keywords: hydrogenase, prebiotic enzyme, metalloenzyme, computational design

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Authors: Anusha Bhardwaj, Shekhar Shinde

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Nitric oxide (NO•) has been documented in research papers as one of the most versatile player in the therapeutics. It is identified as a biological multifunctional messenger molecule which is synthesized by the action of nitric oxide synthase (NOS) enzyme from L-arginine. The protective and the toxic effect in conjunction form the complete picture of the biological function of nitric oxide in humans. The dual nature is because of various factors such as concentration of NO, the isoform of NOS involved, type of cells in which it is synthesized, reaction partners like proteins, reactive oxygen intermediates, prosthetic groups, thiols etc., availability of the substrate L-arginine, intracellular environment in which NO is produced and generation of guanosine 3, 5’- cyclic monophosphate (cGMP). Activation of NOS through infection or trauma leads to one or more systemic effects including enhanced immune activity against invading pathogens, vaso/bronchodilatation in the cardiovascular and respiratory systems and altered neurotransmission which can be protective or toxic. Hence, NO affects the balance between healthy signaling and neurodegeneration in the brain. In lungs, it has beneficial effects on the function of airways as a bronchodilator and acts as the neurotransmitter of bronchodilator nerves. Whereas, on the other hand, NO may have deleterious effects by amplifying the asthmatic inflammatory response and also act as a vasodilator in the airways by increasing plasma exudation. But NOS Inhibitors and NO donors hamper the signalling pathway and hence a therapeutic application of NO is compromised.

Keywords: nitric oxide, multifunctional, dual nature, therapeutic applications

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Authors: Hae-Jun Lee, Ji-Sun Shin, Kyung-Tae Lee

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Potentilla species (Rosasease) have been used in traditional medicine to treat different ailment, disease or malady. In this study, we investigated the anti-inflammatory effects of ethanol extracts of NUTT (EPP) in lipopolysaccharide (LPS)-induced Raw 264.7 macrophages and septic mice. EPP suppressed LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production in LPS-induced Raw 264.7 macrophages. Consistent with these observations, EPP reduced the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) by downregulation of their promoter activities. EPP inhibited tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) at production and mRNA levels. Molecularly, EPP attenuated the LPS-induced transcriptional activity, and DNA-binding activity of nuclear factor-κB (NF-κB), and this was associated with a decrease of translocation and phosphorylation of p65 NF-κB by inhibiting the inhibitory κB-α (IκB-α) degradation and IκB kinase-α/β (IKK-α/β) phosphorylation. Furthermore, EPP suppressed the LPS-induced activation of activator protein-1 (AP-1) by reducing the expression of c-Fos and c-Jun in nuclear. EPP also reduced the phosphorylation of mitogen-activated protein kinase (MAPK), such as p38 MAPK and c-Jun N-terminal kinase/stress-activated protein kinase (JNK). In a sepsis model, pretreatment with EPP reduced the LPS-induced lethality. Collectively, these results suggest that the anti-inflammatory effects of EPP were associated with the suppression of NF-κB and AP-1 activation, and support its possible therapeutic role for the treatment of sepsis.

Keywords: anti-inflammation, activator protein-1, nuclear factor κB, Potentilla paradoxa Nutt

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Authors: Ming-Tzu Tsai, Jui-Ting Hsu, Chia-Chieh Lin, Feng-Tsai Chiang, Cheng-Chin Huang

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Far infrared (FIR), an invisible and short electromagnetic waves ranges from 6-14 μm also defines as the “growth ray.” Although the mechanism of FIR is still unknown, most data have suggested that FIR could accelerate the skin microcirculation by elevating the blood flow and nitric-oxide (NO) synthesis. In this present work, the effect of FIR irradiation and endothelial cell growth supplement (ECGS) on human umbilical vascular endothelial cells (HUVECs) was evaluated. To understand whether the cell viability and NO production of HUVECs affected by NO, cells with/without ECGS were treated in the presence or absence of L-NAME, an eNOS inhibitor. For FIR exposure, FIR-emitted ceramic powders consisted of a variety of well-mixed metal oxides were developed. The results showed that L-NAME did had a strong effect on the inhibition of NO production, especially in the ECGS-treated group. However, the cell viability of each group was rarely affected in the presence of L-NAME. Cells with the incubation of ECGS showed much higher cell viability compared to the control. Moreover, NO production of HUVECs exposed to FIR irradiation was significantly inhibited in the presence of L-NAME. It suggested that NO could play a role modulating the downstream signals of HUVECs during FIR exposure.

Keywords: far-infrared irradiation (FIR), nitric oxide (NO), endothelial nitric oxide synthase (eNOS), endothelial cell growth supplement (ECGS)

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Authors: Mehmet Ali Kisaçam, P. Sema Temizer Ozan, Ayşe Doğan, Gonca Ozan, F. Sarper Türker

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Cardiovascular disease which is one of the most common health problems worldwide has crucial importance because of its’ morbidity and mortality rates. Nitric oxide synthase and arginase use L-arginine as a substrate and produce nitric oxide (NO), citrulline and urea, ornithine respectively. Endothelial dysfunction is characterized by reduced bioavailability of vasodilator and anti-inflammatory molecule NO. The purpose of the study to assess endothelial function via arginase activity and NO levels in patients undergoing coronary artery bypass grafting (CABG) surgery. The study was conducted on 26 patients (14 male, 12 female) undergoing CABG surgery. Blood samples were collected from the subjects before surgery, after the termination and after 24 hours of the surgery. Arginase activity and NO levels measured in collected samples spectrophotometrically. Arginase activity decreased significantly in subjects after the termination of the surgery compared to before surgery data. 24 hours after the surgery there wasn’t any significance in arginase activity as it compared to before surgery and after the termination of the surgery. On the other hand, NO levels increased significantly in the subject after the termination of the surgery. However there was no significant increase in NO levels after 24 hours of the surgery, but there was an insignificant increase compared to before surgery data. The results indicate that after the termination of the surgery vascular and endothelial function improved and after 24 hours of the surgery arginase activity and NO levels returned to normal.

Keywords: arginase, bypass, cordiopulmonary, nitric oxide

Procedia PDF Downloads 183

Authors: Suphaket Saenthaweesuk, Nutiya Somparn, Atcharaporn Thewmore

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The present study aims to investigate the effects of Cymbopogon citratus, Stapf (CS) or lemon grass ethanol extract on antioxidant and vascular disorders parameters in rat. The CS ethanol extract was screened for its phytochemical contents and antioxidant activity in vitro. Moreover, the extract was studied in rats to evaluate its effects in vivo. Rats were orally administered with CS at 1,000 mg/kg/day for 30 days. Phytochemical screening of CS extract indicated the presence of tannins, flavonoids and phenolic compounds. The extract contained phenolic compounds 1,400.10 ± 0.47 mg of gallic acid equivalents per gram CS extract. The free radical scavenging activity assessed by DPPH assay gave IC50 of 168.77 ± 3.32µg/mL, which is relatively lower than that of BHT with IC50 of 12.34 ± 1.14 µg/mL. In the animals, the protein expression of antioxidant enzymes, γ-glutamylcysteine ligase (γ-GCL) in liver was significantly increased. This was consistent with elevation of serum catalase (CAT) and superoxide dismutase (SOD) activities. However, Protein expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule (ICAM-1) and endothelial nitric oxide synthase (eNOS) in heart and aorta were not differenced from normal control. Taken together, the present study provides evidence that CCS water extract exhibits direct antioxidant properties and can induce cytoprotective enzymes in vivo.

Keywords: antioxidant, Cymbopogon citratus Stapf, VCAM-1, γ-glutamylcysteine ligase

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Authors: Hu-Jiang Shi, Li-Juan Zhu

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The implication of 5-hydroxytryptamine 2C receptor (5-HT2CR) in affective behaviors is a topic of debate, and the underlying mechanisms remain largely unclear. Here, we elucidate that the interaction between hippocampal neuronal nitric oxide synthase (nNOS) and carboxy-terminal PDZ ligand of nNOS (CAPON) contributes to the disruption of hippocampal excitation-inhibition (E/I) balance, which is responsible for the anxiety- and depressive-like behaviors caused by chronic stress-related 5-HT2CR signaling deficiency. In detail, activation or inhibition of 5-HT2CR by CP809101 or SB242084 modulates nNOS-CAPON interaction by influencing intracellular Ca²⁺ release. Notably, the dissociation of nNOS-CAPON abolishes SB242084-induced anxiety- and depressive-like behaviors, as well as the reduction in extracellular signal-regulated kinase (ERK)/cAMP-response element binding protein (CREB)/synapsin signaling and SNARE complex assembly. Furthermore, nNOS-CAPON blockers restore the impairments caused by SB242084, including the reduction in SNARE assembly-mediated γ-aminobutyric acid (GABA) vesicle release and a consequent shift of the E/I balance toward excitation in CA3 pyramidal neurons. Conclusively, our findings disclose the regulatory role of 5-HT2CR in anxiety- and depressive-like behaviors and highlight the hippocampal nNOS-CAPON coupling-triggered E/I imbalance as a pivotal cellular event underpinning the behavioral consequences of 5-HT2CR inhibition.

Keywords: 5-HT2CR, anxiety, depression, nNOS-CAPON coupling, excitation-inhibition balance, neurotransmitter release

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Authors: Saleh N. Maodaa

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Perinatal exposure to nicotine imbalances the redox status in newborns. This study investigated the effect of Anethum graveolens (dill) extract on oxidative stress and tissue injury in the liver and kidney of mice newborns exposed to nicotine perinatally. Pregnant mice received nicotine (0.25 mg/kg) on gestational day 12 to day 5 after birth and/or A. graveolens extract on a gestational day 1 to day 15 after birth. Newborn mice exposed to nicotine showed multiple histopathological alterations in the kidney and liver, including inflammatory cell infiltration and degenerative changes. Nicotine exposure increased hepatic and renal reactive oxygen species (ROS), lipid peroxidation, tumor necrosis factor (TNF-_), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS) (p < 0.001), and decreased antioxidant defenses (p < 0.001). A. graveolens supplementation significantly prevented liver and kidney injury, suppressed ROS generation (p < 0.001), lipid peroxidation (p < 0.001), and inflammatory response (p < 0.001), and enhanced antioxidant defenses. In addition, A. graveolens upregulated hepatic and renal Nrf2 and HO-1 mRNA and increased HO-1 activity in normal and nicotine-exposed mice. In conclusion, A. graveolens protects against perinatal nicotine-induced oxidative stress, inflammation, and tissue injury in the liver and kidney of newborn mice. A. graveolens upregulated hepatic and renal Nrf2/HO-1 signaling and enhanced antioxidant defenses in mice.

Keywords: dill, oxidative stress, cytokines, nicotine

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Authors: Nafiseh Noormohammadi, Ahmad Sobhani Najafabadi

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Hypericins (hypericin and pseudohypericin) are natural napthodianthrone derivatives produced by Hypericum perforatum (St. John’s Wort), which have many medicinal properties such as antitumor, antineoplastic, antiviral, and antidepressant activities. Production and accumulation of hypericin in the plant are influenced by both genetic and environmental conditions. Despite the existence of different high-throughput data on the plant, genetic dimensions of hypericin biosynthesis have not yet been completely understood. In this research, 21 high-quality RNA-seq data on different parts of the plant were integrated into metabolic data to reconstruct a coexpression network. Results showed that a cluster of 30 transcripts was correlated with total hypericin. The identified transcripts were divided into three main groups based on their functions, including hypericin biosynthesis genes, transporters, detoxification genes, and transcription factors (TFs). In the biosynthetic group, different isoforms of polyketide synthase (PKSs) and phenolic oxidative coupling proteins (POCPs) were identified. Phylogenetic analysis of protein sequences integrated into gene expression analysis showed that some of the POCPs seem to be very important in the biosynthetic pathway of hypericin. In the TFs group, six TFs were correlated with total hypericin. qPCR analysis of these six TFs confirmed that three of them were highly correlated. The identified genes in this research are a rich resource for further studies on the molecular breeding of H. perforatum in order to obtain varieties with high hypericin production.

Keywords: hypericin, St. John’s Wort, data mining, transcription factors, secondary metabolites

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Authors: Ghibeche Abderrahmane

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To examine whether genes associated with cellular defense against oxidative stress are associated with insulin sensitivity, patients with type 2 diabetes (n=7) and age-matched (n=5) and young (n=9) control subjects underwent a euglycemic-hyperinsulinemic clamp for 120 min. Muscle samples were obtained before and after the clamp and analyzed for heat shock protein (HSP)72 and heme oxygenase (HO)-1 mRNA, intramuscular triglyceride content, and the maximal activities of β-hyroxyacyl-CoA dehydrogenase (β-HAD) and citrate synthase (CS). Basal expression of both HSP72 and HO-1 mRNA were lower (P < 0.05) by 33 and 55%, respectively, when comparing diabetic patients with age-matched and young control subjects, with no differences between the latter groups. Both basal HSP72 (r = 0.75, P < 0.001) and HO-1 (r = 0.50,P < 0.05) mRNA expression correlated with the glucose infusion rate during the clamp. Significant correlations were also observed between HSP72 mRNA and both β-HAD (r = 0.61, P < 0.01) and CS (r = 0.65, P < 0.01). HSP72 mRNA was induced (P < 0.05) by the clamp in all groups. Although HO-1 mRNA was unaffected by the clamp in both the young and age-matched control subjects, it was increased (P < 0.05) ∼70-fold in the diabetic patients after the clamp. These data demonstrate that genes involved in providing cellular protection against oxidative stress are defective in patients with type 2 diabetes and correlate with insulin-stimulated glucose disposal and markers of muscle oxidative capacity. The data provide new evidence that the pathogenesis of type 2 diabetes involves perturbations to the antioxidant defense mechanism within skeletal muscle.

Keywords: euglycemic-hyperinsulinemic, HSP72, mRNA, diabete

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Authors: Seyed Davar Siadat, Samira Tarashi, Abolfazl Fateh, Arfa Moshiri

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Background: The global health issue of tuberculosis (TB) still affects patients in every country. TB control may not be as effective as it should be, especially when resistant strains are involved. In this regard, mycobacterial MTases play a major role in tuberculosis, but the mechanisms underlying their function have yet to be fully deciphered. Methods: Five resistant isolates of M.tb were accumulated. As a reference strain, M.tb H37Rv (ATCC 27249) was used. For this analysis, seven putative mycobacterial MTase genes (Rv0645c, Rv1694, Rv2966c, Rv3919c, Rv2756c, Rv1988, and Rv3263), as well as Rv1392 as SAM synthase, were selected. Comparing mutations and expression levels of MTases in different strains was accomplished by PCR-sequencing and qRT-PCR. The relative expression levels of these genes were calculated using the 2 -ΔΔCt method. Results: The Rv3919c gene (T to G in codon 341) and Rv1392 gene (G to A in codon 97) were the only mutations found in the INHR strain. In all sensitive and resistant isolates, Rv0645c, Rv3263, Rv2756c, and Rv2966c were overexpressed. However, the expression of Rv1988 and Rv3919c decreased in the sensitive strains, whereas the expression of Rv1694 increased. There was also a decreased expression of Rv1392 in the INHR isolate. Conclusion: The presence of mycobacterial MTases as well as resistance to antibiotics were found to be correlated in M.tb strains. Undoubtedly, there are some MTases that are associated with the virulence process. It is necessary to conduct additional studies to fully explore the impact of mycobacterial MTases within specific strains of M.tb to develop novel diagnostic and treatment strategies.

Keywords: mycobacterium tuberculosis, drug resistance, methyltransferases, s-adenosylmethionine

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Authors: Sadegh Lotfieblisofla, Arash Khodabakhshi

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

Keywords: tPA, recombinant, transgenic, tobacco

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Authors: Leila Karshenas, Hamid Reza Khodaei, Behnaz Mahdavi

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We know that nitric oxide (NO) plays an important role in all sexual activities of animals. It is made in body from NO synthase enzyme and L-arginin molecule. NO can bound with sulfur-iron complexes and because production of steroid sexual hormones is related to enzymes which have this complex, NO can change the activity of these enzymes. NO affects many cells including endothelial cells of veins, macrophages and mast cells. These cells are found in testis leydig cells and therefore are important source of NO in testis tissue. Minimizing damages to sperm at the time of sperm freezing and thawing is really important. The goal of this study was to determine the function of NO before freezing and its effects on quality and viability of sperms after thawing and incubation. 4 Holstein bulls were selected from the age of 4, and artificial insemination was done for 3 weeks (2 times a week). Treatments were 0, 10, 50 and 100 nm of sodium nitroprusside (SNP). Data analysis was performed by SAS98 program. Also, mean comparison was done using Duncan's multiple ranges test (P<0.05). Concentrations used was found to increase motility and viability of spermatozoa at 1, 2 and 3 hours after thawing significantly (P<0.05), but there was no significant difference at zero time. SNP levels reduced the amount of lipid peroxidation in sperm membrane, increased acrosome health and improved sample membranes especially in 50 and 100 nm treatments. According to results, adding SNP to semen diluents increases motility and viability of spermatozoa. Also, it reduces lipid peroxidation in sperm membrane and improves sperm function.

Keywords: sperm motility, nitric oxide, lipid peroxidation, spermatozoa

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Authors: Sachin K. Samuchiwal, Barbara Balestrieri, Amanda Paskavitz, Hannah Raff, Joshua A. Boyce

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IL-33, a recently discovered member of the IL-1 cytokine family, binds to the TLR/IL1R super family receptor ST2 and induces type 2 immune responses. IL-33 is constitutively expressed in structural cells at barrier sites such as skin, lung, and intestine, and also inducibly expressed by hematopoietic cells including macrophages. Stimulation of macrophages by Lipopolysaccharide (LPS) can induce de novo IL-33 expression, and also causes the production of prostaglandin-E2 (PGE2) via cyclooxygenase (COX)-2 and microsomal PGE2 synthase-1 (mPGES-1). Because PGE2 can regulate macrophage functions through both autocrine and paracrine mechanisms, the potential interplay of endogenous PGE2 on IL-33 production was explored. Bone-marrow derived murine macrophages (bmMF) that lack either mPGES-1 or EP2 receptor expression were stimulated with LPS in the absence or presence of exogenous PGE2 along with pharmacological agonists and antagonists. The study results demonstrate that endogenous PGE2 markedly enhances LPS-induced IL-33 production by bmMFs via EP2 receptors. Moreover, exogenous PGE2 can amplify LPS-induced IL-33 expression dominantly by EP2 and partly by EP4 receptors by a pathway involving cAMP and exchange protein activated by cAMP (EPAC), but not protein kinase A (PKA). Though both IL-33 production and PGE2 generation in response to LPS require activation of both p38 MAPK and NF-κB, PGE2 did not influence this activation. In conclusion, it is demonstrated that endogenous PGE2 signaling through EP2 and EP4 receptors is a prerequisite for LPS-induced IL-33 production in bmMFs and the underlying cAMP mediated pathway involves EPAC. Since IL-33 is a critical pro-inflammatory cytokine in various pathological disorders, this PGE2-EP2/EP4-cAMP mediated pathway can be exploited to intervene in IL-33 driven pathologies.

Keywords: bone marrow macrophages, EPAC, IL-33, PGE2

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Authors: Mohamed A. Morsy, Azza A. El-Sheikh, Abdulla Y. Al-Taher

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Resveratrol (RES) is a well-known polyphenol antioxidant. We have previously shown that testicular protective effect of RES against the anticancer drug methotrexate (MTX)-induced toxicity involves transporter-mediated mechanisms. Here, we investigated the effect of RES on MTX-induced nephrotoxicity. Rats were administered RES (10 mg/kg/day) for 8 days, with or without a single MTX dose (20 mg/kg i.p.) at day 4 of the experiment. MTX induced nephrotoxicity evident by significantly increase in serum blood urea nitrogen and creatinine compared to control, as well as distortion of kidney microscopic structure. MTX also significantly increased renal nitric oxide level, with induction of inducible nitric oxide synthase expression. MTX also significantly up-regulated fas ligand and caspase 3. Administering RES prior to MTX significantly improved kidney function and microscopic picture, as well as significantly decreased nitrosative and apoptotic markers compared to MTX alone. RES, but not MTX, caused significant increase in expression of breast cancer resistance protein (BCRP), an apical efflux renal transporter that participates in urinary elimination of both MTX and RES. Interestingly, concomitant MTX and RES caused further up-regulation of renal Bcrp compared to RES alone. Using Human BCRP ATPase assay, both RES and MTX exhibited dose-dependent increase in ATPase activity, with Km values of 0.52 ± 0.03 and 30.9 ± 4.2 µM, respectively. Furthermore, combined RES and MTX caused ATPase activity which was significantly less than maximum ATPase activity attained by the positive control; sulfasalazine (12.5 µM). In conclusion, RES exerted nephro-protection against MTX-induced toxicity through anti-nitrosative and anti-apoptotic effects, as well as via up-regulation of renal Bcrp.

Keywords: methotrexate, resveratrol, nephrotoxicity, breast cancer resistance protein

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Authors: Anita K. Kovacs, Peter Hegyes, Zsolt Bozso, Gabor Toth

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Fluorination has been used extensively by the pharmaceutical industry as a strategy to improve the pharmacokinetics of drugs due to its effectiveness in increasing the potency of antimicrobial peptides (AMPs). Multiple-fluorinated indole-ring-containing tryptophan derivatives have the potential of having better antimicrobial activity than the widely used mono-fluorinated indole-ring containing tryptophan derivatives, but they are not available commercially. Therefore, our goal is to synthesize multiple-fluorinated indole-ring containing tryptophan derivatives to incorporate them into AMPs to enhance their antimicrobial activity. During our work, we are trying several methods (classical organic synthesis, enzymic synthesis, and solid phase peptide synthesis) for the synthesis of the said compounds, with mixed results. With classical organic synthesis (four different routes), we did not get the desired results. The reaction of serin with substituted indole in the presence of acetic anhydride led to racemic tryptophane; with the reaction of protected serin with indole in the presence of nickel complex was unsuccessful; the reaction of serin containing protected dipeptide with disuccinimidyl carbonate we achieved a tryptophane containing dipeptide, its chiral purity is being examined; the reaction of alcohol with substituted indole in the presence of copper complex was successful, but it was only a test reaction, we could not reproduce the same result with serine. The undergoing tryptophan-synthase method has shown some potential, but our work has not been finished yet. The successful synthesis of the desired multiple-fluorinated indole-ring-containing tryptophan will be followed by solid phase peptide synthesis in order to incorporate it into AMPs to enhance their antimicrobial activity. The successful completion of these phases will mean the possibility of manufacturing new, effective AMPs.

Keywords: halogenation, fluorination, tryptophan, enhancement of antimicrobial activity

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Authors: D. Bhowmik, Y. Tian, Q. Yin, F. Zhu

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Cyclic GMP-AMP synthase (cGAS), recognises cytoplasmic double-stranded DNA (dsDNA), indicative of bacterial and viral infections, as well as the leakage of self DNA by cellular dysfunction and stresses, to elicit the host's immune responses. Viruses also have developed numerous strategies to antagonize the cGAS-STING pathway. Kaposi's sarcoma-associated herpesvirus (KSHV) is a human DNA tumor virus that is the causative agent of Kaposi’s sarcoma and several other malignancies. To persist in the host, consequently causing diseases, KSHV must overcome the host innate immune responses, including the cGAS-STING DNA sensing pathway. We already found that ORF52 or KicGAS (KSHV inhibitor of cGAS), an abundant and basic gamma herpesvirus-conserved tegument protein, directly inhibits cGAS enzymatic activity. To better understand the mechanism, we have performed the biochemical and structural characterization of full-length KicGAS and various mutants in regarding binding to DNA. We observed that KicGAS is capable of self-association and identified the critical residues involved in the oligomerization process. We also characterized the DNA-binding of KicGAS and found that KicGAS cooperatively oligomerizes along the length of the double stranded DNA, the highly conserved basic residues at the c-terminal disordered region are crucial for DNA recognition. Deficiency in oligomerization also affects DNA binding. Thus DNA binding by KicGAS sequesters DNA and prevents it from being detected by cGAS, consequently inhibiting cGAS activation. KicGAS homologues also inhibit cGAS efficiently, suggesting inhibition of cGAS is evolutionarily conserved mechanism among gamma herpesvirus. These results highlight the important viral strategy to evade this innate immune sensor.

Keywords: Kaposi's sarcoma-associated herpesvirus, KSHV, cGAS, DNA binding, inhibition

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Authors: Mahima Dubey, Girish Chandel

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Minor millets are highly nutritious and climate resilient cereal crops. These features make them ideal candidates to excavate the physiology of the underlying mechanism. In an attempt to understand the basis of mineral nutrition in minor millets, a set of five Barnyard millet genotypes were analyzed for grain Fe and Zn content under contrasting Fe-Zn supply to identify genotypes proficient in tolerating mineral deficiency. This resulted in the identification of Melghat-1 genotype to be nutritionally superior with better ability to withstand deficiency. Expression analysis of several Nicotianamine synthase (NAS) genes showed that HvNAS1 and OsNAS2 genes were prominent in positively mediating mineral deficiency response in Barnyard millet. Further, strategic efforts were employed for fast-track identification of more effective orthologous NAS genes from Barnyard millet. This resulted in the identification of two genes namely EfNAS1 (orthologous to HvNAS1 of barley) and EfNAS2 (orthologous to OsNAS2 gene of rice). Sequencing and thorough characterization of these sequences revealed the presence of intact NAS domain and signature tyrosine and di-leucine motifs in their predicted proteins and thus established their candidature as functional NAS genes in Barnyard millet. Moreover, EfNAS1 showed structural superiority over previously known NAS genes and is anticipated to have role in more efficient metal transport. Findings of the study provide insight into Fe-Zn deficiency response and mineral nutrition in millets. This provides millets with a physiological edge over micronutrient deficient staple cereals such as rice in withstanding Fe-Zn deficiency and subsequently accumulating higher levels of Fe and Zn in millet grains.

Keywords: gene expression, micronutrient, millet, ortholog

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