Reduced metal transferrin binding in neurological diseases

By employing G75 gel-filtration chromotography, it has been demonstrated that human plasma gallium speciation (and by implication, Al speciation) is bimodal. Normally, gallium was predominantly bound to a high molecular weight fraction which was presumably transferrin. Literature reviews and experimental work throughout this thesis provided evidence to support this idea. An aluminium-transferrin species was assumed to be relatively non-toxic and a protective function for this complex has been suggested. A second, low molecular weight species of gallium was observed and its identity has been suggested to be citrate. The results of this thesis support the concept citrate was a gallium binding ligand present in the plasma, but there was another species (tentatively identified as phosphate) which bound gallium to a much greater degree than did citrate in the majority of samples studied. The consequence of a low molecular weight species of aluminium is the possibility that this leads to a more rapid, uncontrolled deposition of the metal in the brain compared to a transferrin mediated mechanism. Plasma speciation studies in Alzheimer's disease, Parkinson's disease, Down's syndrome, and neonates has revealed an altered ratio of the two gallium species found in control subjects. In all groups there was an increase in the potentially more neurotoxic low molecular weight species. These observations have led to a suggested mechanism of accumulation of metals in the brain, which is known to occur in the first three groups. Possible pathogenic mechanisms are described. The results can also offer an explanation to the reported increased sensitivity to the toxic effects of aluminium in the neonate. Speciation studies on normal plasma has shown the balance between high and low molecular weight species of gallium to be influenced by many physiological factors. There appears to be a fine equilibrium between both species which can be altered without any great difficulty. Therefore, in the diseased groups studied, it is possible that there are subtle biochemical changes within the circulatory system to affect the equilibrium which results in an increased low molecular weight species of aluminium. Furthermore, it has been demonstrated that there is a group of normal controls with no clinical signs of Alzheimer's or Parkinson's disease which have reduced transferrin binding. This indicates there is a population of healthy people who are at risk to the development of either disease.

The functional effects of ceramide on the monocyte CD36 scavenger receptor and NADPH oxidase

Atherosclerosis is the principal cause of death in the United States, Europe and much of Asia. During the last decade, inflammation has been suggested to play a key role in the development of atherosclerosis. Reactive oxygen species (ROS) released during inflammation additionally oxidize LDL, which is subsequently taken up in an unregulated way through scavenger receptors on macrophages to form foam cells, the hallmark of atherosclerotic lesions. Previous work has shown that the lipid ceramide, which is found in aggregated LDL and in atherosclerotic plaques, decreases intracellular peroxide most likely through reducing NADPH oxidase activity. Ceramide is an important component of membrane microdomains called lipid rafts which are important for membrane protein function. Endogenous ceramide enhances lipid raft f'ormation and alters theirs composition. NADPH oxidase membrane subunits cytochrome b558 (which includes gp91) strongly associates with lipid rafts Therefore present study investigated whether short chain ceramides reduce NADPH oxidase in U937 monocytes by disrurting the membrane component of NADPH oxidase. Results showed that C2 ceramide alters the distribution of raft marker, flottillin and the raft environment. NADPH oxidase membrane component gp9J phox and cytosolic component p47 phox were identified in rafts. C2 ceramide reduces both gp91 and p47 phox in rafts, which leads to the decrease of peroxide production by NADPH oxidase. Ceramide is also an important second messenger involved in many different signaling pathways associated with atherogenesis from the activation of sphingomyelinase (SMase). It has been reported that SMase enhances LDL receptor mediated LDL endocytosis. However, no study has been done to investigate the effect of ceramide on scavenger receptors such as CD36 and oxidized LDL (OxLDL) uptake. CD36 is the major recertor far OxLDL. Reduced CD36 expression results in less foam cell formation and less atherosclerotic lesion without disrupting the clearance of OxLDL from plasma. This thesis shows that ceramides significantly reduce CD36 surface expression on U937 monocytes, macrophages and human primary monocytes. This effect is seen using both synthetic short chain ceramide and SMase catalysed long chain ceramide treatment. To investigate whether the effect of ceramide on CD36 is functional, OxLOL uptake was measured in ceramide treated cells. Ceramide reduces the uptake of OxLOL by both U937 monocytes and PMA-differentiated macrophages. The mechanism of ceramide reduction of CD36 expression was studied by measuring the surface antigen using flow cytometry and fluorescence microscopy, whole cellular CD36 expression and shedding of C036 by Western blotting of cell lysates and cell culture supernatants and mRNA level of CD36 using RT-PCR. Ceramide reduces shedding of CD36, activates mRNA expression of CD36 and induces intracellular CD36 accumulation probably through retaining the receptor inside cells. In summary, ceramides modulate several of the processes involved in LOL oxidation and uptake by CD36 receptors on monocytes/macrophages in a way which may protect against atherosclerosis.

Structure and thermal properties of yttrium alumino-phosphate glasses

The structure and thermal properties of yttrium alumino-phosphate glasses, of nominal composition (Y2O3)(0.31-z)(Al2O3)(z)(P2O5)(0.69) with 0 less than or similar to z less than or similar to 0.31, were studied by using a combination of neutron diffraction, Al-27 and P-31 magic angle spinning nuclear magnetic resonance, differential scanning calorimetry and thermal gravimetric analysis methods. The Vickers hardness of the glasses was also measured. The data are compared to those obtained for pseudo-binary Al2O3-P2O5 glasses and the structure of all these materials is rationalized in terms of a generic model for vitreous phosphate materials in which Y3+ and Al3+ act as modifying cations that bind only to the terminal (non-bridging) oxygen atoms of PO4 tetrahedra. The results are used to help elucidate the phenomenon of rare-earth clustering in phosphate glasses which can be reduced by substituting Al3+ ions for rare-earth R3+ ions at fixed modifier content.

Structure, Morphology, and Electrochemical Properties of Transition Metal Oxide, Hydroxide, and Phosphate Nanomaterials for Energy Storage

Energy storage technologies are crucial for efficient utilization of electricity. Supercapacitors and rechargeable batteries are of currently available energy storage systems. Transition metal oxides, hydroxides, and phosphates are the most intensely investigated electrode materials for supercapacitors and rechargeable batteries due to their high theoretical charge storage capacities resulted from reversible electrochemical reactions. Their insulating nature, however, causes sluggish electron transport kinetics within these electrode materials, hindering them from reaching the theoretical maximum. The conductivity of these transition metal based-electrode materials can be improved through three main approaches; nanostructuring, chemical substitution, and introducing carbon matrices. These approaches often lead to unique electrochemical properties when combined and balanced.

Ethanol-mediated solvothermal synthesis we developed is found to be highly effective for controlling size and morphology of transition metal-based electrode materials for both pseudocapacitors and batteries. The morphology and the degree of crystallinity of nickel hydroxide are systematically changed by adding various amounts glucose to the solvothermal synthesis. Nickel hydroxide produced in this manner exhibited increased pseudocapacitance, which is partially attributed to the increased surface area. Interestingly, this morphology effect on cobalt doped-nickel hydroxide is found to be more effective at low cobalt contents than at high cobalt contents in terms of improving the electrochemical performance.

Moreover, a thin layer of densely packed nickel oxide flakes on carbon paper substrate was successfully prepared via the glucose-assisted solvothermal synthesis, resulting in the improved electrode conductivity. When reduced graphene oxide was used for conductive coating on as-prepared nickel oxide electrode, the electrode conductivity was only slightly improved. This finding reveals that the influence of reduced graphene oxide coating, increasing the electrode conductivity, is not that obvious when the electrode is already highly conductive to begin with.

We were able to successfully control the interlayer spacing and reduce the particle size of layered titanium hydrogeno phosphate material using our ethanol-mediated solvothermal reaction. In layered structure, interlayer spacing is the key parameter for fast ion diffusion kinetics. The nanosized layered structure prepared via our method, however, exhibited high sodium-ion storage capacity regardless of the interlayer spacing, implying that interlayer space may not be the primary factor for sodium-ion diffusion in nanostructured materials, where many interstitials are available for sodium-ion diffusion.

Our ethanol-mediated solvothermal reaction was also effective for synthesis of NaTi2(PO4)3 nanoparticles with uniform size and morphology, well connected by a carbon nanotube network. This composite electrode exhibited high capacity, which is comparable to that in aqueous electrolyte, probably due to the uniform morphology and size where the preferable surface for sodium-ion diffusion is always available in all individual particles.

Fundamental understandings of the relationship between electrode microstructures and electrochemical properties discussed in this dissertation will be important to design high performance energy storage system applications.

Functional analysis of the 5′ flanking region of the human G6PC3 gene: regulation of promoter activity by glucose, pyruvate, AMP kinase and the pentose phosphate pathway

G6PC3 is a widely expressed isoform of glucose-6-phosphatase, found in many foetal and adult tissues. Mutations in this gene cause developmental abnormalities and severe neutropenia due to abolition of glucose recycling between the cytoplasm and endoplasmic reticulum. Low G6PC3 expression as a result of promoter polymorphisms or dysregulation could produce similar outcomes. Here we investigated the regulation of human G6PC3 promoter activity. HeLa and H4IIE cells were transiently transfected with G6PC3 promoter coupled to the firefly luciferase gene, and promoter activity was measured by dual luciferase assay. Activity was highest in a 453 bp segment of the G6PC3 promoter, from − 455 to − 3 relative to the transcriptional start site. This promoter was unresponsive to glucostatic hormones. Its activity increased significantly between 1 and 5.5 mM glucose, and was not elevated further by glucose concentrations up to 25 mM. Pyruvate increased its activity, but β-hydroxybutyrate and sodium acetate did not. Promoter activity was reduced by inhibitors of hexokinase, glyceraldehyde phosphate dehydrogenase and the oxidative branch of the pentose phosphate pathway, but not by a transketolase inhibitor. Deletion of two adjacent Enhancer-boxes (− 274 to − 279 and − 299 to − 304) reduced promoter activity and abolished the glucose effect, suggesting they could function as a glucose response element. Deletion of an additional downstream 140 bp (− 140 to − 306) restored activity, but not the glucose response, suggesting the presence of repressor elements in this region. 5-Aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR) reduced promoter activity, showing dependence on AMP-kinase. Regulation of the G6PC3 promoter is thus radically different to that of the hepatic isoform, G6PC. It is sensitive to carbohydrate, but not to fatty acid metabolites, and at much lower physiological concentrations. Based on these findings, we speculate that reduced G6PC3 expression could occur during hypoglycemic episodes in vivo, which are common in utero and in the postnatal period. If such episodes lower G6PC3 expression they could place the foetus or infant at risk of impaired immune function and development, and this possibility requires further examination both in vitro and in vivo.

Evolution of protein bound Maillard reaction end-products and free Amadori compounds in low lactose milk in presence of fructosamine oxidase I

Thermal treatments and storage influence milk quality, particularly in low lactose milk as the higher concentration of reducing sugars can lead to the increased formation of the Maillard reaction products (MRPs). The control of the Amadori products (APs) formation is the key step to mitigate the Maillard reaction (MR) in milk. The use of fructosamine oxidases, (Faox) provided promising results. In this paper, the effects of Faox I were evaluated by monitoring the concentration of free and bound MRPs in low lactose milk during shelf life. Results showed that the enzyme reduced the formation of protein-bound MRPs down to 79% after six days at 37 °C. Faox I lowered the glycation of almost all the free amino acids resulting effective on basic and polar amino acids. Data here reported corroborate previous findings on the potentiality of Faox enzymes in controlling the early stage of the MR in foods.

Vitamins Reverse Endothelial Dysfunction Through Regulation of eNOS and NAD(P)H Oxidase Activities

Antioxidant vitamins C and E have protective properties in genetic hypertension associated with enhanced oxidative stress. This study investigated whether vitamins C and/or E modulate vascular function by regulating enzymatic activities of endothelial nitric oxide synthase (eNOS) and NAD(P)H oxidase using thoracic aortas of 20- to 22-week-old male spontaneously hypertensive rats (SHR) and their matched normotensive counterparts, Wistar-Kyoto rats (WKY). SHR aortas had impaired relaxant responses to acetylcholine but not to sodium nitroprusside, despite an 2-fold increase in eNOS activity and NO release. The levels of superoxide anion (O2 ), a potent NO scavenger, and NAD(P)H oxidase activity were also 2-fold higher in SHR aortas. Mechanical but not pharmacological inactivation of endothelium (by rubbing and 100 mol/L L-NAME, respectively) significantly abrogated O2 in both strains. Treatments of SHR aortas with NAD(P)H oxidase inhibitors, namely diphenyleneiodinium and apocynin, significantly diminished O2 production. The incubation of SHR aortas with different concentrations of vitamin C (10 to 100 mol/L) and specifically with high concentrations of vitamin E (100 mol/L) improved endothelial function, reduced superoxide production as well as NAD(P)H oxidase activity, and increased eNOS activity and NO generation in SHR aortas to the levels observed in vitamin C- and E-treated WKY aortas. Our results reveal endothelial NAD(P)H oxidase as the major source of vascular O2 in SHR and also show that vitamins C and E are critical in normalizing genetic endothelial dysfunction through regulation of eNOS and NAD(P)H oxidase activities.

Nitric oxide synthase and NAD(P)H oxidase modulate coronary endothelial cell growth

Reactive oxygen species (ROS) including nitric oxide (NO) and superoxide anion (O2-) are associated with cell migration, proliferation and many growth-related diseases. The objective of this study was to determine whether there was a reciprocal relationship between rat coronary microvascular endothelial cell (CMEC) growth and activity/expressions (mRNA and protein) of endothelial NO synthase (eNOS) and NAD(P)H oxidase enzymes. Proliferating namely, 50% confluent CMEC possessed approximately three-fold increased activity and expression of both enzymes compared to 100% confluent cells. Treatment of CMEC with an inhibitor of eNOS (L-NAME, 100M) increased cell proliferation as assessed via three independent methods i.e. cell counting, determination of total cellular protein levels and [3H]thymidine incorporation. Similarly, treatment of CMEC with pyrogallol (0.3-3 mM), a superoxide anion (O2-)- generator, also increased CMEC growth while spermine NONOate (SpNO), a NO donor, significantly reduced cell growth. Co-incubation of CMEC with a cell permeable superoxide dismutase mimetic (Mn-III-tetrakis-4-benzoic acid-porphyrin; MnTBAP) plus either pyrogallol or NO did not alter cell number and DNA synthesis thereby dismissing the involvement of peroxynitrite (OONO-) in CMEC proliferation. Specific inhibitors of NAD(P)H oxidase but not other ROS-generating enzymes including cyclooxygenase and xanthine oxidase, attenuated cell growth. Transfection of CMEC with antisense p22-phox cDNA, a membrane-bound component of NAD(P)H oxidase, resulted in substantial reduction in [3H]thymidine incorporation, total cellular protein levels and expression of p22-phox protein. These data demonstrate a cross-talk between CMEC growth and eNOS and NAD(P)H oxidase enzyme activity and expression, thus suggesting that the regulation of these enzymes may be critical in preventing the initiation and/or progression of coronary atherosclerosis.

Analysis of β-tricalcium phosphate granules prepared with different formulations by nano-computed tomography and scanning electron microscopy

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PPARα regulates endothelial progenitor cell maturation and myeloid lineage differentiation through a NADPH oxidase-dependent mechanism in mice

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Chronic intermittent hypoxia increases rat sternohyoid muscle NADPH oxidase expression with attendant modest oxidative stress

Chronic intermittent hypoxia (CIH) causes upper airway muscle dysfunction. We hypothesized that the superoxide generating NADPH oxidase (NOX) is upregulated in CIH-exposed muscle causing oxidative stress. Adult male Wistar rats were exposed to intermittent hypoxia (5% O2 at the nadir for 90 s followed by 210 s of normoxia), for 8 h per day for 14 days. The effect of CIH exposure on the expression of NOX subunits, total myosin and 4-hydroxynonenal (4-HNE) protein adducts in sternohyoid muscle was determined by western blotting and densitometry. Sternohyoid protein free thiol and carbonyl group contents were determined by 1D electrophoresis using specific fluorophore probes. Aconitase and glutathione reductase activities were measured as indices of oxidative stress. HIF-1α content and key oxidative and glycolytic enzyme activities were determined. Contractile properties of sternohyoid muscle were determined ex vivo in the absence and presence of apocynin (putative NOX inhibitor). We observed an increase in NOX 2 and p47 phox expression in CIH-exposed sternohyoid muscle with decreased aconitase and glutathione reductase activities. There was no evidence, however, of increased lipid peroxidation or protein oxidation in CIH-exposed muscle. CIH exposure did not affect sternohyoid HIF-1α content or aldolase, lactate dehydrogenase, or glyceraldehyde-3-phosphate dehydrogenase activities. Citrate synthase activity was also unaffected by CIH exposure. Apocynin significantly increased sternohyoid force and power. We conclude that CIH exposure upregulates NOX expression in rat sternohyoid muscle with concomitant modest oxidative stress but it does not result in a HIF-1α-dependent increase in glycolytic enzyme activity. Constitutive NOX activity decreases sternohyoid force and power. Our results implicate NOX-dependent reactive oxygen species in CIH-induced upper airway muscle dysfunction which likely relates to redox modulation of key regulatory proteins in excitation-contraction coupling.

Phosphate Regulated Proteins Of Xanthomonas Citri Subsp. Citri: A Proteomic Approach.

Xanthomonas citri subsp. citri (X. citri) is the causative agent of the citrus canker, a disease that affects several citrus plants in Brazil and across the world. Although many studies have demonstrated the importance of genes for infection and pathogenesis in this bacterium, there are no data related to phosphate uptake and assimilation pathways. To identify the proteins that are involved in the phosphate response, we performed a proteomic analysis of X. citri extracts after growth in three culture media with different phosphate concentrations. Using mass spectrometry and bioinformatics analysis, we showed that X. citri conserved orthologous genes from Pho regulon in Escherichia coli, including the two-component system PhoR/PhoB, ATP binding cassette (ABC transporter) Pst for phosphate uptake, and the alkaline phosphatase PhoA. Analysis performed under phosphate starvation provided evidence of the relevance of the Pst system for phosphate uptake, as well as both periplasmic binding proteins, PhoX and PstS, which were formed in high abundance. The results from this study are the first evidence of the Pho regulon activation in X. citri and bring new insights for studies related to the bacterial metabolism and physiology. Biological significance Using proteomics and bioinformatics analysis we showed for the first time that the phytopathogenic bacterium X. citri conserves a set of proteins that belong to the Pho regulon, which are induced during phosphate starvation. The most relevant in terms of conservation and up-regulation were the periplasmic-binding proteins PstS and PhoX from the ABC transporter PstSBAC for phosphate, the two-component system composed by PhoR/PhoB and the alkaline phosphatase PhoA.

Reduced Insulin Clearance And Lower Insulin-degrading Enzyme Expression In The Liver Might Contribute To The Thrifty Phenotype Of Protein-restricted Mice.

Nutrient restriction during the early stages of life usually leads to alterations in glucose homeostasis, mainly insulin secretion and sensitivity, increasing the risk of metabolic disorders in adulthood. Despite growing evidence regarding the importance of insulin clearance during glucose homeostasis in health and disease, no information exists about this process in malnourished animals. Thus, in the present study, we aimed to determine the effect of a nutrient-restricted diet on insulin clearance using a model in which 30-d-old C57BL/6 mice were exposed to a protein-restricted diet for 14 weeks. After this period, we evaluated many metabolic variables and extracted pancreatic islet, liver, gastrocnemius muscle (GCK) and white adipose tissue samples from the control (normal-protein diet) and restricted (low-protein diet, LP) mice. Insulin concentrations were determined using RIA and protein expression and phosphorylation by Western blot analysis. The LP mice exhibited lower body weight, glycaemia, and insulinaemia, increased glucose tolerance and altered insulin dynamics after the glucose challenge. The improved glucose tolerance could partially be explained by an increase in insulin sensitivity through the phosphorylation of the insulin receptor/protein kinase B and AMP-activated protein kinase/acetyl-CoA carboxylase in the liver, whereas the changes in insulin dynamics could be attributed to reduced insulin secretion coupled with reduced insulin clearance and lower insulin-degrading enzyme (IDE) expression in the liver and GCK. In summary, protein-restricted mice not only produce and secrete less insulin, but also remove and degrade less insulin. This phenomenon has the double benefit of sparing insulin while prolonging and potentiating its effects, probably due to the lower expression of IDE in the liver, possibly with long-term consequences.

Taurine Supplementation Reduces Blood Pressure And Prevents Endothelial Dysfunction And Oxidative Stress In Post-weaning Protein-restricted Rats.

Taurine is a sulfur-containing amino acid that exerts protective effects on vascular function and structure in several models of cardiovascular diseases through its antioxidant and anti-inflammatory properties. Early protein malnutrition reprograms the cardiovascular system and is linked to hypertension in adulthood. This study assessed the effects of taurine supplementation in vascular alterations induced by protein restriction in post-weaning rats. Weaned male Wistar rats were fed normal- (12%, NP) or low-protein (6%, LP) diets for 90 days. Half of the NP and LP rats concomitantly received 2.5% taurine supplementation in the drinking water (NPT and LPT, respectively). LP rats showed elevated systolic, diastolic and mean arterial blood pressure versus NP rats; taurine supplementation partially prevented this increase. There was a reduced relaxation response to acetylcholine in isolated thoracic aortic rings from the LP group that was reversed by superoxide dismutase (SOD) or apocynin incubation. Protein expression of p47phox NADPH oxidase subunit was enhanced, whereas extracellular (EC)-SOD and endothelial nitric oxide synthase phosphorylation at Ser 1177 (p-eNOS) were reduced in aortas from LP rats. Furthermore, ROS production was enhanced while acetylcholine-induced NO release was reduced in aortas from the LP group. Taurine supplementation improved the relaxation response to acetylcholine and eNOS-derived NO production, increased EC-SOD and p-eNOS protein expression, as well as reduced ROS generation and p47phox expression in the aortas from LPT rats. LP rats showed an increased aortic wall/lumen ratio and taurine prevented this remodeling through a reduction in wall media thickness. Our data indicate a protective role of taurine supplementation on the high blood pressure, endothelial dysfunction and vascular remodeling induced by post-weaning protein restriction. The beneficial vascular effect of taurine was associated with restoration of vascular redox homeostasis and improvement of NO bioavailability.

Two-component System Vicrk Regulates Functions Associated With Establishment Of Streptococcus Sanguinis In Biofilms.

Streptococcus sanguinis is a commensal pioneer colonizer of teeth and an opportunistic pathogen of infectious endocarditis. The establishment of S. sanguinis in host sites likely requires dynamic fitting of the cell wall in response to local stimuli. In this study, we investigated the two-component system (TCS) VicRK in S. sanguinis (VicRKSs), which regulates genes of cell wall biogenesis, biofilm formation, and virulence in opportunistic pathogens. A vicK knockout mutant obtained from strain SK36 (SKvic) showed slight reductions in aerobic growth and resistance to oxidative stress but an impaired ability to form biofilms, a phenotype restored in the complemented mutant. The biofilm-defective phenotype was associated with reduced amounts of extracellular DNA during aerobic growth, with reduced production of H2O2, a metabolic product associated with DNA release, and with inhibitory capacity of S. sanguinis competitor species. No changes in autolysis or cell surface hydrophobicity were detected in SKvic. Reverse transcription-quantitative PCR (RT-qPCR), electrophoretic mobility shift assays (EMSA), and promoter sequence analyses revealed that VicR directly regulates genes encoding murein hydrolases (SSA_0094, cwdP, and gbpB) and spxB, which encodes pyruvate oxidase for H2O2 production. Genes previously associated with spxB expression (spxR, ccpA, ackA, and tpK) were not transcriptionally affected in SKvic. RT-qPCR analyses of S. sanguinis biofilm cells further showed upregulation of VicRK targets (spxB, gbpB, and SSA_0094) and other genes for biofilm formation (gtfP and comE) compared to expression in planktonic cells. This study provides evidence that VicRKSs regulates functions crucial for S. sanguinis establishment in biofilms and identifies novel VicRK targets potentially involved in hydrolytic activities of the cell wall required for these functions.