Effects of Temperature on the Kinetic Isotope Effects for Proton and Hydride Transfers in the Active Site Variant of Choline Oxidase Ser101Ala

Choline oxidase catalyzes the oxidation of choline to glycine betaine. The reaction includes betaine aldehyde as an intermediate. FAD is reduced by the alcohol substrate, betaine aldehyde intermediate and oxidized by molecular oxygen to give hydrogen peroxide. In this study, the Ser101Ala variant of choline oxidase was prepared to elucidate the contribution of the hydroxyl group of Ser101 in the proton and hydride transfer reactions for proper preorganization and reorganization of the active site towards quantum mechanical tunneling. The thermodynamic parameters associated with the enzyme-catalyzed OH and CH bond cleavages and the temperature dependence of the associated solvent and substrate kinetic isotope effects were investigated using a stopped-flow spectrophotometer. The proton and hydride transfer have been shown to be occurring via quantum tunneling in CHO-S101A enzyme

Biochemical and Biophysical Studies of Heme Transport Proteins: HtaA, HtaB, and ChtB from the Corynebacterium diphtheriae

Many pathogenic bacteria require iron for their survival and virulence; in most cases hemin is the main iron source. Pathogens have developed sophisticated heme uptake mechanisms in order to maintain the homeostasis and remain as infectious agents. Corynebacterium diphtheria can obtain hemin during human infection through series of conserved domains (CR) of DxtR-regulated and heme-transport-associated (hta) proteins: HtaA, HtaB, ChtA, ChtB and ChtC. HtaA includes two conserved regions (CR1 and CR2) while the other proteins include a single CR domain. These proteins orchestrate the heme transport to the HmuT protein, a lipoprotein which delivers heme to the ABC membrane transporter HmuUV. Homology modeling of HtaA-CR2, HtaB and ChtB based on amino acid sequence indicated that these proteins have a novel structure. Two tyrosines and one histidine residue are fully conserved in all CR domains. Mutations of these conserved amino acids to alanine significantly lowered the heme binding in comparison to the wild-type proteins. Reconstitution of HtaA-CR2 after removal of the heme with butanone extraction method gave a different form of the protein. UV-visible absorption spectra and resonance Raman spectra data are consistent with heme ligation with an axial tyrosine including a histidine hydrogen-binding partner in HtaA-CR2, HtaB and ChtB. HtaA-CR2 is highly stable to thermal unfolding; the protein was also stable to chemical unfolding using GdnHCl or GdnSCN (up to 4 M at 25oC) . For HtaA-CR2, unfolding could be observed at 37 oC as a single process at high concentrations of denaturant (6.8 – 7.4 M GdnHCl). In contrast, HtaA-CR2 apoproteins (WT, as well as the Y361A and H412A mutants) unfolded readily with low denaturant concentrations (~ 1.3 M GdnHCl). HtaB shows significantly lower stability; a half-life of 330 min was observed in the presence of 6.6 M GdnHCl at 37 oC for HtaA-CR2 and a half-life of 39 min was observed in the presence of 4.0 M GdnHCl at 25 oC for HtaB; ChtB was very similar to HtaB. For HtaB and ChtB, the high amino acid sequence similarity and identity, similar biophysical characteristics and gene deletion studies suggest that these proteins may function interchangeably during the heme uptake process

The Effects of Coenzyme Q10 on Inflammation Markers in Streptozotocin-Induced Diabetic Rats

Background: Coenzyme Q10 is a well-known cofactor in the mitochondrial electron transport chain required for ATP production. Coenzyme Q10 is recognized as an intracellular antioxidant that protects cell membrane phospholipids, mitochondrial membrane protein, and plasma low-density lipoprotein against oxidative damage caused by free radicals. Diabetes and its complications have been related to increased levels of free radicals and systemic proinflammatory cytokines and to an abnormal lipid profile. The aim of this study was to investigate the effects of coenzyme Q10 supplementation on some cytokine levels in streptozotocin-induced diabetic rats.Materials, Methods & Results: In this study, 38 healthy, adult male rats were used. The rats were divided into 5 groups. All animals were housed in separated cages during the four weeks. The animals in group 1 was fed standard rat pellets for 4 weeks. It was administered at 0.3 mL corn oil intraperitoneally daily for four weeks in group 2 animals. The animals in group 3 was injected intraperitoneally with 10 mg/kg CoQ10 daily for 4 weeks. Group 4 was made diabetic by subcutaneous injections of streptozotocin at dose of 40 mg/kg in 0.1 M citrate buffer (pH 4.5) single daily dose for two days and group 5 was made diabetic by subcutaneous injections of streptozotocin at dose of 40 mg/kg in 0.1 M citrate buffer (pH 4.5) single daily dose for two days and then was injected intraperitoneally with 10 mg/kg CoQ10 daily for 4 weeks. During the experiment, three animals from group 4 and one animals from group 5 were died due to streptozotocin-induced hypoglycemia. At the end of the study, blood samples were taken from all animals. In these blood samples, IL-4, IL-6, IL-10 and TNF-α plasma levels were determined with ELISA using sandwich enzyme-linked immunosorbent method via commercial kits. In this study, IL-4 level as an anti-inflammatory cytokine significantly decreased (P < 0.05) with diabetes induction compared to control group level. IL-10 level in diabetic group was statistically different (P < 0.05) from control group level. CoQ10 application to diabetic animals improved the falling in IL-10 level of diabetic group (P < 0.05). IL-6 and TNF-α levels in diabetic group significantly increased (P < 0.05) in parallel with each other compared to control group levels. The same parameters were reduced (P < 0.05) by CoQ10 application in diabetic animals.Discussion: In this study, the occurred changes in pro- and anti-inflammatory cytokines with experimentally induced diabetes are expected results and these results are consistent with some studies related diabetes. These results may be considered to hazardous effects and inflammation caused by diabetes on liver, pancreas and other tissues. CoQ10 suppressed the increments in plasma pro-inflammatory cytokine levels, whereas it restored the reducing in anti-inflammatory cytokine levels arising due to diabetes. The obtained results from this study after CoQ10 application supported similar studies used CoQ10 application against deleterious effects of diabetes in animals and humans. Therefore, it is possible to say that CoQ10 may play important role in regulation of imbalance between inflammation markers in diabetes conditions and further studies are needed to clear the beneficial effects of CoQ10 treatment on the other inflammation markers in diabetes status

Molecular Host-Nematode Interactions and Tuber Development

Potato, Solanum tuberosum, the most important non-grain food crop and essential crop globally, has been widely cultivated around the world for centuries. The significance of this plant is increasing due to high nutritional value of the tubers combined with the simplicity of its propagation. As a plant organ, tuber of potato, is mainly edible part of it and popular as nutrient for almost all nations. Tuberization in potato is a very complex biological occurrence affected by numerous ecological signals, genetics, plant nutrition and several different hormones. Many pests including nematodes limit potato tuber development that plant hormones play roles in nematode feeding cell formation. Parasitic nematodes, important pests which cause damage to plants, tubers, suck up nutrients from plants and weaken plant development and yield losses. Many genes involve in tuber development and plant response nematodes. The aim of this chapter is to demonstrate the new advances in the field of molecular host-nematode interactions and tuber development

Boron Stress Activates the General Amino Acid Control Mechanism and Inhibits Protein Synthesis

Boron is an essential micronutrient for plants, and it is beneficial for animals. However, at high concentrations boron is toxic to cells although the mechanism of this toxicity is not known. Atr1 has recently been identified as a boron efflux pump whose expression is upregulated in response to boron treatment. Here, we found that the expression of ATR1 is associated with expression of genes involved in amino acid biosynthesis. These mechanisms are strictly controlled by the transcription factor Gcn4 in response to boron treatment. Further analyses have shown that boron impaired protein synthesis by promoting phosphorylation of eIF2α in a Gcn2 kinase dependent manner. The uncharged tRNA binding domain (HisRS) of Gcn2 is necessary for the phosphorylation of eIF2α in the presence of boron. We postulate that boron exerts its toxic effect through activation of the general amino acid control system and inhibition of protein synthesis. Since the general amino acid control pathway is conserved among eukaryotes, this mechanism of boron toxicity may be of general importance

Genetic Modification of Plant Hormones Induced by Parasitic Nematodes, Virus, Viroid, Bacteria, and Phytoplasma in Plant Growing

Plant hormones, such as auxin, play crucial roles in many plant developmental processes, which is crosstalk with gibberellin and strigolactone. The roles of hormones may vary in the biosynthesis of metabolisms. During the pathogen attack, including plant-parasitic nematodes, viroid, phytoplasma, virus, and bacteria, plant hormones are involved in several plant processes. Ethylene (ET), salicylate (SA), jasmonate (JA), and abscisic acid (ABA) primarily regulate synergistically or antagonistically against pathogens. Those pathogens—nematodes, bacteria, viroid, phytoplasma, and viruses regulate several plant hormones for successful parasitism, influencing the phytohormone structure and modifying plant development. Several genes are related to plant hormones that are involved in pathogens parasitism. In this chapter, how pathogens affect plant hormones in plants growing are discussed

Fruit softening: revisiting the role of pectin

Fruit softening is a major determinant of shelf life and commercial value. Here, we highlight recent work that revisits the role of pectin in fruit softening and primary cell wall structure. These studies demonstrate the importance of pectin and the link between its degradation and softening in fleshy fruits. Fruit softening, which is a major determinant of shelf life and commercial value, is the consequence of multiple cellular processes, including extensive remodeling of cell wall structure. Recently, it has been shown that pectate lyase (PL), an enzyme that degrades de-esterified pectin in the primary wall, is a major contributing factor to tomato fruit softening. Studies of pectin structure, distribution, and dynamics have indicated that pectins are more tightly integrated with cellulose microfibrils than previously thought and have novel structural features, including branches of the main polymer backbone. Moreover, recent studies of the significance of pectinases, such as PL and polygalacturonase, are consistent with a causal relationship between pectin degradation and a major effect on fruit softening

Pests, Diseases, Nematodes, and Weeds Management on Strawberries

Strawberry is an important crop for many features, including being rich in vitamins and minerals. In addition to fresh consumption, it has been appealing to a wide range of consumers in recent years. Its cultivation is in flat areas, slopes, and areas where other crops are limited. Many pests and diseases that are the main biotic stress factors cause significant crop losses in strawberry cultivation. The aim of this chapter is to reveal biotic stress factors and their management. Several plant-parasitic nematodes, fungal diseases, weeds, pests, virus diseases, and bacterial diseases are the main biotic stress factors in plant growing and fruit ripening. The preparation of this book chapter is based on previously published sources and researches and manuscripts. In this section, it is aimed to provide readers with new perspectives in terms of collecting data on nematodes, diseases, pests, weeds, and fruit ripening of strawberry plants. The effect and mechanism of those biotic stress factors on strawberry growing are discussed and revealed in this chapter

Disentangling pectic homogalacturonan and rhamnogalacturonan-I polysaccharides: Evidence for sub-populations in fruit parenchyma systems

The matrix polysaccharides of plant cell walls are diverse and variable sets of polymers influencing cell wall, tissue and organ properties. Focusing on the relatively simple parenchyma tissues of four fruits – tomato, aubergine, strawberry and apple – we have dissected cell wall matrix polysaccharide contents using sequential solubilisation and antibody-based approaches with a focus on pectic homogalacturonan (HG) and rhamnogalacturonan-I (RG-I). Epitope detection in association with anion-exchange chromatography analysis indicates that in all cases solubilized polymers include spectra of HG molecules with unesterified regions that are separable from methylesterified HG domains. In highly soluble fractions, RG-I domains exist in both HG-associated and non-HG-associated forms. Soluble xyloglucan and pectin-associated xyloglucan components were detected in all fruits. Aubergine glycans contain abundant heteroxylan epitopes, some of which are associated with both pectin and xyloglucan. These profiles of polysaccharide heterogeneity provide a basis for future studies of more complex cell and tissue systems