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PubMed2004

Electrochemical methods for the preparation of gold-coated TiO2 nanoparticles with variable coverages.

We report here the first electrochemical methods to prepare elemental Au0-coated TiO2 nanoparticles with controllable coverages. First, Au substrates were cycled in a deoxygenated aqueous solution containing 0.1 N HCl and 1 mM TiO2 nanoparticles from -0.28 to +1.22 V versus Ag/AgCl at 500 mV/s with different numbers of scans. The durations at the cathodic and anodic vertexes were 10 and 5 s, respectively. After this process, positively charged Au-coated TiO2 nanoparticles were formed in the solutions. Then a cathodic overpotential of 0.6 V from the open circuit potential of ca. 0.82 V versus Ag/AgCl was applied under sonication to synthesize elemental Au0-coated TiO2 nanoparticles. The coverage of Au shells in the elemental Au-coated TiO2 nanoparticles is varied from 10% to 95% by increasing the number of scans from 10 to 50 in preparing the positively charged Au-coated TiO2 nanoparticles. The extremely high coverage of 95% in this study is notable, as compared with other methods to prepare Au-coated TiO2 nanoparticles.

Journal Article↗
PubMed2005

Electrodeposition of manganese and molybdenum mixed oxide thin films and their charge storage properties.

Manganese and molybdenum mixed oxides in a thin film form were deposited anodically on a platinum substrate by cycling the electrode potential between 0 and +1.0 V vs Ag/AgCl in aqueous manganese(II) solutions containing molybdate anion (MoO(4)2-). A possible mechanism for the film formation is as follows. First, electrooxidation of Mn2+ ions with H2O yields Mn oxide and protons. Then, the protons being accumulated near the electrode surface react with MoO(4)2- to form polyoxomolybdate through a dehydrated condensation reaction (by protonation and dehydration). The condensed product coprecipitates with the Mn oxide. Cyclic voltammetry of the Mn/Mo oxide film-coated electrode in aqueous 0.5 M Na2SO4 solution exhibited a pseudocapacitive behavior with higher capacitance and better rate capability than that of the pure Mn oxide prepared similarly, most likely as a result of an increase in electrical conductivity of the film. Electrochemical quartz crystal microbalance and X-ray photoelectron spectroscopy clearly demonstrated that the observed pseudocapacitive behavior results from reversible extraction/insertion of hydrated protons to balance the charge upon oxidation/reduction of Mn3+/Mn4+ in the film.

Journal Article↗
PubMed1989

Glycolysis-induced discordance between glucose metabolic rates measured with radiolabeled fluorodeoxyglucose and glucose.

We have developed an autoradiographic method for estimating the oxidative and glycolytic components of local CMRglc (LCMRglc), using sequentially administered [18F]fluorodeoxyglucose (FDG) and [14C]-6-glucose (GLC). FDG-6-phosphate accumulation is proportional to the rate of glucose phosphorylation, which occurs before the divergence of glycolytic (GMg) and oxidative (GMo) glucose metabolism and is therefore related to total cerebral glucose metabolism GMt: GMg + GMo = GMt. With oxidative metabolism, the 14C label of GLC is temporarily retained in Krebs cycle-related substrate pools. We hypothesize that with glycolytic metabolism, however, a significant fraction of the 14C label is lost from the brain via lactate production and efflux from the brain. Thus, cerebral GLC metabolite concentration may be more closely related to GMo than to GMt. If true, the glycolytic metabolic rate will be related to the difference between FDG- and GLC-derived LCMRglc. Thus far, we have studied normal awake rats, rats with limbic activation induced by kainic acid (KA), and rats visually stimulated with 16-Hz flashes. In KA-treated rats, significant discordance between FDG and GLC accumulation, which we attribute to glycolysis, occurred only in activated limbic structures. In visually stimulated rats, significant discordance occurred only in the optic tectum.

Animals↗
PubMed1997

Regulation of citrulline recycling in nitric oxide-dependent neurotransmission in the murine proximal colon.

1. We investigated the contribution of nitric oxide (NO) to inhibitory neuromuscular transmission in murine proximal colon and the possibility that citrulline is recycled to arginine to maintain the supply of substrate for NO synthesis. 2. Intracellular microelectrode recordings were made from circular smooth muscle cells in the presence of nifedipine and atropine (both 1 microM). Electrical field stimulation (EFS, 0.3-20 Hz) produced inhibitory junction potentials (i.j.ps) composed of an initial transient hyperpolarization (fast component) followed by a slow recovery to resting potential (slow component). 3. L-Nitro-arginine-methyl ester (L-NAME, 100 microM) selectively abolished the slow component of i.j.ps. The effects of L-NAME were reversed by L-arginine (0.2-2 mM) but not by D-arginine (2 mM). Sodium nitroprusside (an NO donor, 1 microM) reversibly hyperpolarized muscle cells. This suggests that NO mediates the slow component of i.j.ps. 4. L-Citrulline (0.2 mM) also reversed the effects of L-NAME, and this action was maintained during sustained exposures to L-citrulline (0.2 mM). This may reflect intraneuronal recycling of L-citrulline to L-arginine. 5. Higher concentrations of L-citrulline (e.g. 2 mM) had time-dependent effects. Brief exposure (15 min) reversed the effects of L-NAME, but during longer exposures (30 min) the effects of L-NAME gradually returned. In the continued presence of L-citrulline, L-arginine (2 mM) readily restored nitrergic transmission, suggesting that during long exposures to high concentrations of L-citrulline, the ability to generate arginine from citrulline was reduced. 6. Aspartate (2 mM) had no effect on i.j.ps, the effects of L-NAME, or the actions of L-citrulline in the presence of L-NAME, L-Citrulline (0.2-2 mM) alone had no effect on i.j.ps under control conditions. 7. S-methyl-L-thiocitrulline (10 microM), a novel NOS inhibitor, blocked the slow component of i.j.ps. The effects of this inhibitor were reversed by L-arginine (2 mM), but not by L-citrulline (2 mM). 8. These results suggest that i.j.ps in the murine colon result from release of multiple inhibitory neurotransmitters. NO mediates a slow component of enteric inhibitory neurotransmission. Recycling of L-citrulline to L-arginine may sustain substrate concentrations in support of NO synthesis and this pathway may be inhibited when concentrations of L-citrulline are elevated.

Animals↗
PubMed2005

Bioelectronic detector with monoamine oxidase for halitosis monitoring.

Methyl mercaptan (MM) is known as one of the major chemicals of halitosis (bad breath). In this study, a bioelectronic gas sensor (bio-detector) for gaseous MM was developed and was applied to measure halitosis in breath. The bio-detector consisted of a Clark-type dissolved oxygen electrode, a monoamine oxidase type-A (MAO-A) immobilized membrane and a reaction unit that had liquid and gaseous compartments separated by a hydrophobic porous polytetrafluoroethylene (PTFE) diaphragm membrane. The tip of the electrode covered with MAO-A membrane was placed into the liquid compartment as touching to the PTFE diaphragm membrane. In order to amplify the bio-detector output, a substrate regeneration cycle caused by coupling the monooxygenase with l-ascorbic acid as reducing reaction with reagent system, was applied. The results of MM vapor measurements showed the calibration range of the bio-detector for MM vapor was from 0.087 to 11.5 ppm (correlation coefficient: 0.993) and included the human sense of smell level 5 (0.2 ppm). The bio-detector had good selectivity being attributed to enzyme specificity was obtained for several substances (trimethyl amine, ammonia, dimethyl sulfide, etc.). The bio-detector was applied for halitosis measurement. Expired gases in five subjects were sampled every hour and the concentrations of MM in the expired gases were monitored. The output of bio-detector showed behaviour of halitosis level changes in a day such as increasing with passage of time and decreasing after eating.

Biosensing Techniques↗
PubMed1981

Maximum activities and properties of glucose 6-phosphatase in muscles from vertebrates and invertebrates.

1. The maximum catalytic activities of glucose 6-phosphatase were measured in a large number of muscles from vertebrates and invertebrates. The activities range from less than 0.1 to 8.0 mumol/min per g fresh wt. at 30 degrees C: the highest activity, observed in the flight muscle of the wasp (Vespa vulgaris), is similar to that in rat liver. The hydrolytic activity was shown to be specific towards glucose 6-phosphate. 2. The pH optimum was 6.8 and the Km was approx. 0.6 mM (flight muscle of a moth). 3. Almost all of the glucose 6-phosphatase activity from extracts of the flight muscle of a moth and the pectoral muscle of a pigeon were recovered in the cytosolic fraction (i.e. 150,000 g supernatant). 4. During development of the locust (Schistocerca gregaria), the activity of the phosphatase in the flight muscle increased during the first 3 days after the final moult. 5. The activity of glucose 6-phosphatase from insect and avian muscle was separated from that of non-specific phosphatase on a Bio-Gel P-100 column. 6. For the activities from 63 muscles, there was a strong positive correlation between those of glucose 6-phosphatase and hexokinase, but no correlation between the activities of glucose 6-phosphatase and fructose bisphosphatase. It is suggested that the role of glucose 6-phosphate in muscle is either to produce glucose from glucose 6-phosphate derived from glycogen or to provide the enzymic basis for a substrate ("futile") cycle between glucose and glucose 6-phosphatase in muscle to improve the sensitivity of the mechanism that regulates the rate of glucose phosphorylation.

Animals↗
PubMed2006

Chlamydial ribonucleotide reductase: tyrosyl radical function in catalysis replaced by the FeIII-FeIV cluster.

Ribonucleotide reductase (RNR) from Chlamydia trachomatis is a class I RNR composed of proteins R1 and R2. In protein R2, the tyrosine residue harboring the radical that is necessary for catalysis in other class I RNRs is replaced by a phenylalanine. Active C. trachomatis RNR instead uses the Fe(III)-Fe(IV) state of the iron cluster in R2 as an initiator of catalysis. The paramagnetic Fe(III)-Fe(IV) state, identified by (57)Fe substitution, becomes electron spin resonance detectable in samples that are frozen during conditions of ongoing catalysis. Its amount depends on the conditions for catalysis, such as incubation temperature and the R1/R2 ratio. The results link induction of the Fe(III)-Fe(IV) state with enzyme activity of chlamydial RNR. Based on these observations, a reaction scheme is proposed for the iron site. This scheme includes (i) an activation cycle involving reduction and an oxygen reaction in R2 and (ii) a catalysis cycle involving substrate binding and turnover in R1.

Bacterial Proteins↗
PubMed1994

The development of mitochondrial medicine.

Primary defects in mitochondrial function are implicated in over 100 diseases, and the list continues to grow. Yet the first mitochondrial defect--a myopathy--was demonstrated only 35 years ago. The field's dramatic expansion reflects growth of knowledge in three areas: (i) characterization of mitochondrial structure and function, (ii) elucidation of the steps involved in mitochondrial biosynthesis, and (iii) discovery of specific mitochondrial DNA. Many mitochondrial diseases are accompanied by mutations in this DNA. Inheritance is by maternal transmission. The metabolic defects encompass the electron transport complexes, intermediates of the tricarboxylic acid cycle, and substrate transport. The clinical manifestations are protean, most often involving skeletal muscle and the central nervous system. In addition to being a primary cause of disease, mitochondrial DNA mutations and impaired oxidation have now been found to occur as secondary phenomena in aging as well as in age-related degenerative diseases such as Parkinson, Alzheimer, and Huntington diseases, amyotrophic lateral sclerosis and cardiomyopathies, atherosclerosis, and diabetes mellitus. Manifestations of both the primary and secondary mitochondrial diseases are thought to result from the production of oxygen free radicals. With increased understanding of the mechanisms underlying the mitochondrial dysfunctions has come the beginnings of therapeutic strategies, based mostly on the administration of antioxidants, replacement of cofactors, and provision of nutrients. At the present accelerating pace of development of what may be called mitochondrial medicine, much more is likely to be achieved within the next few years.

Aging↗
PubMed1998

GroEL under heat-shock. Switching from a folding to a storing function.

Chaperonin GroEL from Escherichia coli, together with its cochaperonin GroES, are proteins involved in assisting the folding of polypeptides. GroEL is a tetradecamer composed of two heptameric rings, which enclose a cavity where folding takes place through multiple cycles of substrate and GroES binding and release. GroEL and GroES are also heat-shock proteins, their synthesis being increased during heat-shock conditions to help the cell coping with the thermal stress. Our results suggest that, as the temperature increases, GroEL decreases its protein folding activity and starts acting as a "protein store." The molecular basis of this behavior is the loss of inter-ring signaling, which slows down GroES liberation from GroEL and therefore the release of the unfolded protein from the GroEL cavity. This behavior is reversible, and after heat-shock, GroEL reverts to its normal function. This might have a physiological meaning, since under thermal stress conditions, it may be inefficient for the cell to fold thermounstable proteins that are prone to denaturation.

Adenosine Triphosphatases↗
PubMed2004

Hsp105alpha suppresses Hsc70 chaperone activity by inhibiting Hsc70 ATPase activity.

Hsp105alpha is a mammalian member of the HSP105/110 family, a diverged subgroup of the HSP70 family. Hsp105alpha associates with Hsp70/Hsc70 as complexes in vivo and regulates the chaperone activity of Hsp70/Hsc70 negatively in vitro and in vivo. In this study, we examined the mechanisms by which Hsp105alpha regulates Hsc70 chaperone activity. Using a series of deletion mutants of Hsp105alpha and Hsc70, we found that the interaction between Hsp105alpha and Hsc70 was necessary for the suppression of Hsc70 chaperone activity by Hsp105alpha. Furthermore, Hsp105alpha and deletion mutants of Hsp105alpha that interacted with Hsc70 suppressed the ATPase activity of Hsc70, with the concomitant appearance of ATPase activity of Hsp105alpha. As the ATPase activity of Hsp70/Hsc70 is essential for the efficient folding of nonnative protein substrates, Hsp105alpha is suggested to regulate the substrate binding cycle of Hsp70/Hsc70 by inhibiting the ATPase activity of Hsp70/Hsc70, thereby functioning as a negative regulator of the Hsp70/Hsc70 chaperone system.

Adenosine Triphosphatases↗
PubMed2006

Amide hydrogen exchange reveals conformational changes in hsp70 chaperones important for allosteric regulation.

Hsp70 chaperones assist protein folding processes by a nucleotide-driven cycle of substrate binding and release. Although structural information is available for the isolated nucleotide-binding (NBD) and substrate-binding domains (SBD) in the high affinity conformation, the low affinity conformations and the conformational changes associated with mutual allosteric regulation remained largely enigmatic. By using amide hydrogen exchange in combination with mass spectrometry, we analyzed the Escherichia coli Hsp70 homologue DnaK as full-length protein and its individual domains in the nucleotide-free and ATP-bound conformation. We found a surprising degree of flexibility in both domains. The comparison of the full-length protein with the isolated domains demonstrates a mutual stabilization of both domains. This protection from solvent was most pronounced and in addition was nucleotide-dependent in the lowerbeta-sheet of the SBD and the loop that connects the last beta-strand with helix alphaA. Interestingly, the linker region, which connects NBD and SBD and which is close to the protected loop in the SBD, is solvent-exposed in the absence of nucleotide and completely protected from hydrogen exchange in the presence of ATP. Peptide binding to DnaK.ATP reverts the ATP-induced conformational changes in the linker and selected parts of the NBD. Our data outline a pathway for allosteric interdomain control and suggest an important role of the linker and the base of helix alphaA.

Adenosine Triphosphate↗
PubMed1967

Studies of the mitochondrial energy-transfer system of brown adipose tissue.

An investigation of the mechanisms of norepinephrine action and heat production in brown adipose tissue from newborn rabbits has been carried out. Data obtained with the use of biochemical techniques has been correlated with morphological data from electron microscopy. Norepinephrine was found to stimulate the respiration of brown fat in vitro. Inhibitors of glycolysis abolish this effect, whereas inhibitors of oxidative phosphorylation do not, at least not to the same extent. Brown fat is readily permeable to added Krebs cycle intermediates. Substrate level phosphorylation, but no electron transport-coupled phosphorylation, could be demonstrated in isolated mitochondria. It is suggested that the rate of fatty acid oxidation is limited by the availability of phosphate acceptor systems which break down ATP formed at the substrate level and thus provide ADP for further substrate level phosphorylation. The theory of respiratory control by the action of reesterification of fatty acids is discussed in the light of these findings. Under the electron microscope, brown fat mitochondria are characterized by their large size, tightly packed cristae, and by the different types of granules in the matrix. No elementary particles are seen when the mitochondria are examined by the negative-staining technique. The absence of electron transport-coupled phosphorylation together with the apparent absence of elementary particles seems to be of particular significance.

Adenine Nucleotides↗
PubMed1992

Oxygen consumption, energy expenditure, and substrate utilization after cardiac surgery in children.

OBJECTIVE: To determine the oxygen consumption (VO2), resting energy expenditure, and substrate utilization after cardiac surgery in children. DESIGN: Prospective, observational, cross-sectional study with factorial design. SETTING: Pediatric ICU at a university hospital. PATIENTS: Twenty-six consecutive children during the first 3 days after open-heart surgery. INTERVENTIONS: Patients were mechanically ventilated and received routine therapeutic interventions. MEASUREMENTS AND MAIN RESULTS: VO2, resting energy expenditure, and substrate utilization were determined by indirect calorimetry. Cardiac index was calculated using the Fick equation from the measured VO2 and the arterial-mixed venous oxygen content difference, and this cardiac index value was compared with a simultaneous cardiac index value that was measured by thermodilution whenever possible. There were excellent correlation and agreement between cardiac index measurements by Fick equation and thermodilution, indicating accurate VO2 measurements. VO2 was consistent with predicted values in healthy resting children. Resting energy expenditure was consistent with the predicted basal metabolic rate. The mean caloric intake was 19% of the mean energy expenditure. The respiratory quotient was 0.74 +/- 0.05. The substrate utilization showed a shift toward fat oxidation and either gluconeogenesis or impaired carbohydrate utilization. CONCLUSIONS: Cardiovascular surgery in children does not significantly alter resting energy expenditure but influences the substrate utilization. Perioperative hormonal stress responses and therapeutically administered catecholamines may explain the shift toward fat oxidation.

Basal Metabolism↗
PubMed2000

Asymmetries in the nucleosome core particle at 2.5 A resolution.

The 2.5 A X-ray crystal structure of the nucleosome core particle presented here provides significant additions to the understanding of the nucleosome, the fundamental unit of chromatin structure. Extensions are made to the structure of the N-terminal histone tails and details are provided on hydration and ion binding. The structure is composed of twofold symmetric molecules, native chicken histone octamer cores and the DNA palindrome, which were expected to form a perfectly twofold symmetric nucleosome core particle. In fact, the result is asymmetric owing to the binding of the DNA to the protein surface and to the packing of the particles in the crystal lattice. An analysis is made of the asymmetries by comparisons both within the nucleosome core particle and to the structure of the histone octamer core of the nucleosome.

Amino Acid Sequence↗
PubMed2004

Experimentally biased model structure of the Hsc70/auxilin complex: substrate transfer and interdomain structural change.

A model structure of the Hsc70/auxilin complex has been constructed to gain insight into interprotein substrate transfer and ATP hydrolysis induced conformational changes in the multidomain Hsc70 structure. The Hsc70/auxilin system, which is a member of the Hsp70/Hsp40 chaperone system family, uncoats clathrin-coated vesicles in an ATP hydrolysis-driven process. Incorporating previous results from NMR and mutant binding studies, the auxilin J-domain was docked into the Hsc70 ATPase domain lower cleft using rigid backbone/flexible side chain molecular dynamics, and the Hsc70 substrate binding domain was docked by a similar procedure. For comparison, J-domain and substrate binding domain docking sites were obtained by the rigid-body docking programs DOT and ZDOCK, filtered and ranked by the program ClusPro, and relaxed using the same rigid backbone/flexible side chain dynamics. The substrate binding domain sites were assessed in terms of conserved surface complementarity and feasibility in the context of substrate transfer, both for auxilin and another Hsp40 protein, Hsc20. This assessment favors placement of the substrate binding domain near D152 on the ATPase domain surface adjacent to the J-domain invariant HPD segment, with the Hsc70 interdomain linker in the lower cleft. Examining Hsc70 interdomain energetics, we propose that long-range electrostatic interactions, perhaps due to a difference in the pKa values of bound ATP and ADP, could play a major role in the structural change induced by ATP hydrolysis. Interdomain electrostatic interactions also appear to play a role in stimulation of ATPase activity due to J-domain binding and substrate binding by Hsc70.

Adenosine Triphosphate↗
PubMed2002

Effect of steroids on energy expenditure and substrate oxidation in women with Crohn's disease.

OBJECTIVE: Patients with Crohn's disease (CD) have increased energy expenditure and fat oxidation. Steroids, commonly used to treat flare-up of CD, induce weight gain. This study was designed to evaluate the effects of prednisone and budesonide on energy expenditure and substrate oxidation in patients with CD. METHODS: Twenty-nine women with CD and 10 healthy controls were studied. Ten patients received prednisone (0.75-1.0 mg/kg/day), nine received budesonide (9 mg/ day), and 10 did not receive steroids. Resting energy expenditure and substrate oxidation were measured by indirect calorimetry in a fasting state and after a standard diet. RESULTS: In the fasting state, resting energy expenditure was higher in patients without steroids than in the controls. Lipid oxidation was lower (p < 0.01) in patients with prednisone (0.46 +/- 0.39 mg/kg/min) than in patients with budesonide (0.97 +/- 0.28 mg/kg/min) and without steroids (1.06 +/- 0.32 mg/kg/min), but was similar with control subjects (0.47 +/- 0.20 mg/kg/min). Postprandially, lipid oxidation was lower (p < 0.01) in patients with prednisone (0.32 +/- 0.23 mg/kg/min) than in patients with budesonide (0.75 +/- 0.20 mg/kg/min), without steroids (0.82 +/- 0.23 mg/kg/min), and controls (0.58 +/- 0.15 mg/kg/min). Protein oxidation was significantly higher in patients with prednisone than in the other subjects. CONCLUSIONS: In women with CD, prednisone decreases lipid oxidation and increases protein oxidation. These effects are not observed with budesonide and may contribute to the weight gain and side effects commonly observed with prednisone. A low-fat/high-protein diet could be proposed during a course of prednisone.

Adult↗