Modulation of cytochrome oxidase activity by inorganic and organic phosphate.

The activity of cytochrome oxidase reconstituted into phospholipid vesicles has been studied as a function of orthophosphate, ATP and inositol hexakisphosphate concentrations. The respiratory-control ratio was found to be quite sensitive to these compounds and was inversely related to the anion concentration. This effect is related to a phosphate-dependent decrease in the rate constant for ferrocytochrome c oxidation observed in the presence of ionophores. The data cannot be interpreted simply on the basis of ionic strength, which is known to limit cytochrome c binding to cytochrome oxidase, since cytochrome oxidase-containing vesicles responded differently to phosphate depending on the energization state of the phospholipid membrane.     

Reconstitution of cytochrome c oxidase in phospholipid vesicles containing polyvinylic polymers.

Cytochrome c oxidase was reconstituted in phospholipid vesicles in the presence of highly hydrophobic poly(vinyl alkanoate) polymers. Electron-microscopy observations demonstrated that polymer interaction with the lipid phase induces vesicles to adopt smaller diameters than those typical of standard proteoliposomes. Functional characterization of these polymer-proteoliposome structures indicates that the reconstitution of the enzyme proceeds efficiently without causing either scrambling of the protein orientation in the membrane or loss of respiratory control. A clear dependence of respiratory control ratio on vesicle size was also demonstrated, which is in agreement with a previous model proposed for control of activity of cytochrome c oxidase vesicles [Brunori, Sarti, Colosimo, Antonini, Malatesta, Jones & Wilson (1985) EMBO J. 4, 2365-2368].     

Do changes in cardiovascular risk factors explain changes in mortality from stroke in Finland?

OBJECTIVES--To estimate the extent to which the changes in the main cardiovascular risk factors (blood pressure, smoking, and serum cholesterol concentration) can explain the observed changes in mortality from stroke in Finland during the past 20 years. DESIGN--Predicted changes in mortality from cerebrovascular disease mortality were calculated by a proportional hazards model from data obtained in cross sectional population surveys in 1972, 1977, 1982, 1987, and 1992. Predicted changes were compared with the observed changes in mortality statistics. SETTING--North Karelia and Kuopio provinces, Finland. SUBJECTS--16,741 men and 16,389 women aged 30-59 randomly selected from the national population register, of whom 14,054 men and 14,546 women participated. MAIN OUTCOME MEASURES--Levels of risk factors and predicted and observed changes in mortality from cerebrovascular disease. RESULTS--The observed changes in diastolic blood pressure, total serum cholesterol concentration, and smoking in the population from 1972 to 1992 predicted a 44% fall in mortality from stroke in men and changes in diastolic blood pressure and smoking predicted a 34% fall in women. The observed fall in mortality from stroke was 66% in men and 60% in women. CONCLUSIONS--Two thirds of the fall in mortality from stroke in men and half in women can be explained by changes in the three main cardiovascular risk factors.     

Nitric oxide reacts with the ferryl-oxo catalytic intermediate of the CuB-lacking cytochrome bd terminal oxidase

Cytochrome bd is a bacterial respiratory oxidase carrying three hemes but no copper. We show that nitric oxide (NO) reacts with the intermediate F of cytochrome bd from Azotobacter vinelandii: (i) with a 1:1 stoichiometry, (ii) rapidly (k=1.2 +/- 0.1 x 10(5)M(-1)s(-1) at 20 degrees C), and (iii) yielding the oxidized enzyme with nitrite bound to heme d at the active site. Unexpectedly, the NO reaction mechanism of this catalytic intermediate in the Cu(B)-lacking cytochrome bd appears similar to that of beef heart cytochrome c oxidase, where Cu(B) was proposed to play a key role.     

Reactive oxygen species formation is not enhanced by exposure to UMTS 1950 MHz radiation and co‐exposure to ferrous ions in Jurkat cells

This study was designed to assess if radiofrequency (RF) radiation induces oxidative stress in cultured mammalian cells when given alone or in combination with ferrous ions (FeSO₄). For this purpose the production of reactive oxygen species (ROS) was measured by flow cytometry in human lymphoblastoid cells exposed to 1950 MHz signal used by the third generation wireless technology of the Universal Mobile Telecommunication System (UMTS) at Specific Absorption Rate of 0.5 and 2.0 W/kg. Short (5–60 min) or long (24 h) duration exposures were carried out in a waveguide system under strictly controlled conditions of both dosimetry and environment. Cell viability was also measured after 24 h RF exposure using the Resazurin and Neutral Red assays. Several co‐exposure protocols were applied to test if RF radiation is able to alter ROS formation induced by FeSO₄ (RF given before or concurrently to FeSO₄). The results obtained indicate that non‐thermal RF exposures do not increase spontaneous ROS formation in any of the experimental conditions investigated. Consistent with the lack of ROS production, no change in cell viability was observed in Jurkat cells exposed to RF radiation for 24 h. Similar results were obtained when co‐exposures were considered: combined exposures to RF radiation and FeSO₄ did not increase ROS formation induced by the chemical treatment alone. In contrast, in cultures treated with FeSO₄ as positive control, a dose‐dependent increase in ROS formation was recorded, validating the sensitivity of the method employed. Bioelectromagnetics 30:525–535, 2009. © 2009 Wiley‐Liss, Inc.     

Association of Genetic Variants at 3q22 with Nephropathy in Patients with Type 1 Diabetes Mellitus

Diabetic nephropathy (DN) is the primary cause of morbidity and mortality in patients with type 1 diabetes mellitus (T1DM) and affects about 30% of these patients. We have previously localized a DN locus on chromosome 3q with suggestive linkage in Finnish individuals. Linkage to this region has also been reported earlier by several other groups. To fine map this locus, we conducted a multistage case-control association study in T1DM patients, comprising 1822 cases with nephropathy and 1874 T1DM patients free of nephropathy, from Finland, Iceland, and the British Isles. At the screening stage, we genotyped 3072 tag SNPs, spanning a 28 Mb region, in 234 patients and 215 controls from Finland. SNPs that met the significance threshold of p < 0.01 at this stage were followed up by a series of sample sets. A genetic variant, rs1866813, in the noncoding region at 3q22 was associated with increased risk of DN (overall p = 7.07 × 10⁻⁶, combined odds ratio [OR] of the allele = 1.33). The estimated genotypic ORs of this variant in all Finnish samples suggested a codominant effect, resulting in significant association, with a p value of 4.7 × 10⁻⁵ (OR = 1.38; 95% confidence interval = 1.18–1.62). Additionally, an 11 kb segment flanked by rs62408925 and rs1866813, two strongly correlated variants (r² = 0.95), contains three elements highly conserved across multiple species. Independent replication will clarify the role of the associated variants at 3q22 in influencing the risk of DN.     

Redox properties of the oxygen-detoxifying flavodiiron protein from the human parasite Giardia intestinalis

Flavodiiron proteins (FDPs) are enzymes identified in prokaryotes and a few pathogenic protozoa, which protect microorganisms by reducing O₂ to H₂O and/or NO to N₂O. Unlike most prokaryotic FDPs, the protozoan enzymes from the human pathogens Giardia intestinalis and Trichomonas vaginalis are selective towards O₂. UV/vis and EPR spectroscopy showed that, differently from the NO-consuming bacterial FDPs, the Giardia FDP contains an FMN with reduction potentials for the formation of the single and the two-electron reduced forms very close to each other (E₁ = −66 ± 15 mV and E₂ = −83 ± 15 mV), a condition favoring destabilization of the semiquinone radical. Giardia FDP contains also a non-heme diiron site with significantly up-shifted reduction potentials (E₁ = +163 ± 20 mV and E₂ = +2 ± 20 mV). These properties are common to the Trichomonas hydrogenosomal FDP, and likely reflect yet undetermined subtle structural differences in the protozoan FDPs, accounting for their marked O₂ specificity.     

Control of Respiration by Cytochrome c Oxidase in Intact Cells: ROLE OF THE MEMBRANE POTENTIAL*

Metabolic control analysis was applied to intact HepG2 cells. The effect on the control coefficient of cytochrome c oxidase (CcOX) over cell respiration of both the electrical (Δψ) and chemical (ΔpH) component of the mitochondrial transmembrane proton electrochemical gradient (Δμ_H⁺) was investigated. The overall O₂ consumption and specific CcOX activity of actively phosphorylating cells were titrated with cyanide under conditions in which Δψ and ΔpH were selectively modulated by addition of ionophores. In the absence of ionophores, CcOX displayed a high control coefficient (C_IV = 0.73), thus representing an important site of regulation of mitochondrial oxidative phosphorylation. A high control coefficient value (C_IV = 0.85) was also measured in the presence of nigericin, i.e. when Δψ is maximal, and in the presence of nigericin and valinomycin (C_IV = 0.77), when Δμ_H⁺ is abolished. In contrast, CcOX displayed a markedly lower control coefficient (C_IV = 0.30) upon addition of valinomycin, when Δψ is converted into ΔpH. These results show that Δψ is responsible for the tight control of CcOX over respiration in actively phosphorylating cells.     

Single-nucleotide polymorphisms in the IL2RA gene are associated with age at diagnosis in late-onset Finnish type 1 diabetes subjects

The onset of type 1 diabetes can occur at any age, with as many as half of all cases diagnosed after age 15. Despite this wide distribution in age at diagnosis, most genetic studies focus on cases diagnosed in childhood or during early adulthood. To better understand the genetics of late-onset type 1 diabetes, we collected a Finnish case/control cohort with all cases diagnosed between ages 15 and 40. We genotyped 591 probands and 1,538 control subjects at regions well established as susceptibility loci in early onset type 1 diabetes. These loci were then tested for disease association and age-at-diagnosis effects. Using logistic regression, we found that single-nucleotide polymorphisms (SNPs) at the INS, PTPN22, and IFIH1 loci were associated with late-onset disease (OR (95%CI)?=?0.57(0.47–0.69), p?=?2.77?×?10^?9; OR (95%CI)?=?1.50 (1.27–1.78), p?=?3.98?×?10^?6; and OR (95%CI)?=?0.81(0.71–0.93), p?=?0.0028, respectively). In contrast, a disease association was not detected for two SNPs at the IL2RA locus (rs11594656 and rs41295061). Despite this, we did find an independent age-at-diagnosis effect for each IL2RA SNP using a multivariate Cox proportional hazards model (p?=?0.003, 0.002, respectively). Taken together, polymorphisms at the IL2RA locus were a major determinant of age at diagnosis in our cohort with an effect at par with the HLA-DQ2/DQ8 genotype as measured by hazard ratios. These findings suggest that the IL2RA locus controls both the susceptibility to disease and its time of occurrence. Thus, we believe the IL2/IL2R axis represents a potential therapeutic target for delaying the onset of disease.