Proton pumping by NADH:ubiquinone oxidoreductase. A redox driven conformational change mechanism?

The modular evolutionary origin of NADH:ubiquinone oxidoreductase (complex I) provides useful insights into its functional organization. Iron-sulfur cluster N2 and the PSST and 49 kDa subunits were identified as key players in ubiquinone reduction and proton pumping. Structural studies indicate that this 'catalytic core' region of complex I is clearly separated from the membrane. Complex I from Escherichia coli and Klebsiella pneumoniae was shown to pump sodium ions rather than protons. These new insights into structure and function of complex I strongly suggest that proton or sodium pumping in complex I is achieved by conformational energy transfer rather than by a directly linked redox pump.     

Observations concerning the quinol oxidation site of the cytochrome bc1 complex

A direct hydrogen bond between ubiquinone/quinol bound at the QO site and a cluster-ligand histidine of the iron-sulfur protein (ISP) is described as a major determining factor explaining much experimental data on position of the ISP ectodomain, electron paramagnetic resonance (EPR) lineshape and midpoint potential of the iron-sulfur cluster, and the mechanism of the bifurcated electron transfer from ubiquinol to the high and low potential chains of the bc1 complex.     

Ischemic preconditioning in a rodent hepatocyte model of liver hypothermic preservation injury

Ischemic preconditioning (IPC) is a phenomenon of protection in various tissues from normothermic ischemic injury by previous exposure to short cycles of ischemia-reperfusion. The ability of IPC to protect hepatocytes from a model of hypothermic transplant preservation injury was tested in this study. Rat hepatocytes were subjected to 30min of warm ischemia (37 degrees C) followed by 24 or 48h of hypothermic (4 degrees C) storage in UW solution and subsequent re-oxygenation at normothermia for 1h. Studies were performed with untreated control cells and cells treated with IPC (10min anoxia followed by 10min re-oxygenation, 1 cycle). Hepatocytes exposed to IPC prior to warm ischemia released significantly less LDH and had higher ATP concentrations, relative to untreated ischemic hepatocytes. IPC significantly reduced LDH release after 24h of cold storage before reperfusion and after 48h of cold storage and after 60min of warm re-oxygenation, relative to the corresponding untreated hepatocytes. ATP levels were also significantly higher when IPC was used prior to the warm and cold ischemia-re-oxygenation protocols. In parallel studies, IPC increased new protein synthesis and lactate after cold storage and reperfusion compared to untreated cells but no differences in the patterns of protein banding were detected on electrophoresis between the groups. In conclusion, IPC significantly improves hepatocyte viability and energy metabolism in a model of hypothermic preservation injury preceded by normothermic ischemia. These protective effects on viability may be related to enhanced protein and ATP synthesis at reperfusion.     

Measurement of ATP production and respiratory chain enzyme activities in mitochondria isolated from small muscle biopsy samples

A set of methods suitable for assessment of respiratory chain function in mitochondria isolated from 25mg of muscle is described. This set of methods includes determination of the mitochondrial ATP production rate (MAPR) and the activities of the respiratory chain complexes I, I+III, II+III, and IV and citrate synthase. MAPR is determined with an optimized version of a luminometric method previously described. The optimized method measures 50-220% higher activities than the original method. The highest MAPRs are recorded using the substrate combinations glutamate+succinate and N,N,N(1),N(1)-tetramethyl-1,4-phenyldiamine+ascorbate. The respiratory chain complex activities are determined with standard spectrophotometric methods, adapted to an automated photometer. The sensitivity in the determination of complex I, I+III, and II+III activities was increased considerably by pretreating the samples with saponin. The set of methods was evaluated on double biopsy samples from five healthy volunteers and showed coefficients of variation between 7 and 14% when citrate synthase was used as reference base. All of the various measures of mitochondrial function showed high correlation coefficients to each other (r=0.84-0.98; p<0.01). It is concluded that the set of methods is suitable for diagnosis of mitochondrial disorders in adults and small children.     

Molecular cloning, expression, purification, and mass spectrometric characterization of 3C-like protease of SARS coronavirus

Severe acute respiratory syndrome (SARS) is an acute respiratory illness, which has broken out in China. It has been known that SARS coronavirus (SARS_CoV) is a novel human coronavirus and is responsible for SARS infection. Belonging to one of the major proteins associated with SARS_CoV, SARS 3C-like protease (SARS_3CL(pro)) functions as a cysteine protease engaging in the proteolytic cleavage of the viral precursor polyprotein to a series of functional proteins required for coronavirus replication and is considered as an appealing target for designing anti-SARS agents. To facilitate the studies regarding the functions and structures of SARS_3CL(pro), in this report the synthetic genes encoding 3CL(pro) of SARS_CoV were assembled, and the plasmid was constructed using pQE30 as vector and expressed in Escherichia coli M15 cells. The highly yielded ( approximately 15mg/L) expressed protease was purified by use of NTA-Ni(2+) affinity chromatography and FPLC system, and its sequence was determined by LC/MS with the residue coverage of 46.4%.     

Diabetes and mitochondrial bioenergetics: alterations with age

Several studies have been carried out to evaluate the alterations in mitochondrial functions of diabetic rats. However, some of the results reported are controversial, since experimental conditions, such as aging, and/or strain of animals used were different. The purpose of this study was to evaluate the metabolic changes in liver mitochondria, both in the presence of severe hyperglycaemia (STZ-treated rats) and mild hyperglycaemia (Goto-Kakizaki (GK) rats). Moreover, metabolic alterations were evaluated both at initial and at advanced states of the disease. We observed that both models of type 1 and type 2 diabetes presented alterations on respiratory chain activity. Because of continual severe hyperglycaemia, 9 weeks after the induction of diabetes, the respiratory function declined in STZ-treated rats, as observed by membrane potential and respiratory ratios (RCR, P/O, and FCCP-stimulated respiration) assessment. In contrast, GK rats of 6 months age presented increased respiratory ratios. To localize which respiratory complexes are affected by diabetes, enzymatic respiratory chain activities were evaluated. We observed that succinate dehydrogenase and cytochrome c oxidase activities were significantly augmented both in STZ-treated rats and GK rats of 6 months age. Moreover, H(+)-ATPase activity was also significantly increased in STZ-treated rats with 3 weeks of diabetes and in GK rats of 6 months age as compared to controls. Therefore, these results clearly suggest that both animal models of diabetes present some metabolic adjustments in order to circumvent the deleterious effects promoted by the high glucose levels typical of the disease.     

Impaired neutrophil function in patients with pulmonary tuberculosis and its normalization in those undergoing specific treatment,except the HIV-coinfected cases

Our study investigated whether the respiratory burst (RB) of polymorphonuclear neutrophils from tuberculosis (TB) patients was related with the disease severity or treatment, as well as the circulating levels of TNF-alpha. The sample comprised 57 patients with moderate (n=21) or advanced disease (n=36, 13 of them with HIV coinfection, TB-HIV) and 12 controls. Patients were newly diagnosed (n=27) or under treatment (moderate=14, advanced=10, TB-HIV=6). Cytometric analysis showed that untreated patients had a depressed RB in response to Candida albicans, being more pronounced in the advanced group and nearly absent in TB-HIV cases. A recovered RB was observed in treated patients, except for the TB-HIV cases that continued to show a poor response. TNF-alpha serum levels were increased in untreated patients, mostly in the advanced and TB-HIV groups, and showed an inverse and significant correlation with the RB. Disease severity and anti-TB therapy exerted negative and positive influences on the reactive oxygen intermediates production, respectively.     

Endotoxic activity of lipopolysaccharides isolated from emergent potential cystic fibrosis pathogens

Improved antimicrobial therapies against the classical spectrum of pathogenic bacteria which colonise the lungs of cystic fibrosis (CF) patients has resulted in improved life expectancy and quality of life. Bacterial species that are resistant to a broad range of antibiotics including Stenotrophomonas maltophilia and Alcaligenes xylosoxidans have now emerged as potential new pathogens to fill the niche. At present, it is unclear from clinical data whether these microbes are commensal or pathogenic. In this study we have quantified the inflammatory potential of lipopolysaccharide (LPS) from eight species of Gram-negative organisms which have been cultured with increasing frequency from CF patients. Inflammatory responses induced by LPS from whole human blood and a human-derived monocyte cell line (THP-1) were assessed. Enzyme-linked immunosorbent assays were used to detect interleukin-6, interleukin-8, and tumour necrosis factor alpha (TNF). A bioassay was also used to assess TNF activity. With the exception of S. maltophilia, LPS extracted from all of the bacteria tested upregulated, by varying degrees, expression of each of the proinflammatory cytokines assayed. This study represents the first comprehensive report of the endotoxic potential of a new wave of microbes which are associated with CF.     

The Bacillus subtilis ctaB paralogue, yjdK, can complement the heme A synthesis deficiency of a CtaB-deficient mutant

Heme A is a prosthetic group in many respiratory oxidases. It is synthesised from heme B (protoheme IX) with heme O as an intermediate. In Bacillus subtilis two genes required for heme A synthesis, ctaA and ctaB, have been identified. CtaB is the heme O synthase and CtaA is involved in the conversion of heme O to heme A. A ctaB paralogue, yjdK, has been identified through the B. subtilis genome sequencing project. In this study we show that when carried on a low copy number plasmid, the yjdK gene can complement a ctaB deletion mutant with respect to heme A synthesis. Our results indicate that YjdK has heme O synthase activity. We therefore suggest that yjdK be renamed as ctaO.