Functional and structural roles of residues in the third extramembrane segment of adrenal cytochrome b561

Several residues in the third extramembrane segment (EM3) of adrenal cytochrome b(561) have been proposed to be involved in this cytochrome's interaction with ascorbate, but there has been no systematic evaluation of residues in the segment. We used alanine scanning mutagenesis to assess the functional and structural roles of the EM3 residues and several adjacent residues (residues 70-85) in the bovine cytochrome. Each alanine mutant was expressed in a bacterial system, solubilized with detergent, and affinity-purified. The recombinant proteins contained approximately two hemes per monomer and, except for R74A, retained basic functionality (≥ 94% reduced by 20 mM ascorbate). Equilibrium spectrophotometric titrations with ascorbate were used to analyze the α-band line shape and amplitude during reduction of the high- and low-potential heme centers (b(H) and b(L), respectively) and the midpoint ascorbate concentrations for the b(H) and b(L) transitions (C(H) and C(L), respectively). Y73A and K85A markedly narrowed the b(H) α-band peak; other mutants had weaker effects or no effect on b(H) or b(L) spectra. Relative changes in C(H) for the mutants were larger than changes in C(L), with 1.5-2.9-fold increases in C(H) for L70A, L71A, Y73A, R74A, N78A, and K85A. The amounts of functional b(H) and b(L) centers in additional Arg74 mutants, assessed by ascorbate titration and EPR spectroscopy, declined in concert in the following order: wild type > R74K > R74Q > R74T and R74Y > R74E. The results of this first comprehensive experimental test of the proposed roles of EM3 residues have identified residues with a direct or indirect impact on ascorbate interactions, on the environment of the b(H) heme center, and on formation of the native b(H)-b(L) unit. Surprisingly, no individual EM3 residue was by itself indispensable for the interaction with ascorbate, and the role of the segment appears to be more subtle than previously thought. These results also support our topological model of the adrenal cytochrome, which positions b(H) near the cytoplasmic side of the membrane.     

Cyclooxygenase reaction mechanism of PGHS--evidence for a reversible transition between a pentadienyl radical and a new tyrosyl radical by nitric oxide trapping

Incubation of prostaglandin H synthase-1 (PGHS-1) under anaerobic conditions with peroxide and arachidonic acid leads to two major radical species: a pentadienyl radical and a radical with a narrow EPR spectrum. The proportions of the two radicals are sensitive to temperature, favoring the narrow radical species at 22 °C. The EPR characteristics of this latter radical are somewhat similar to the previously reported narrow-singlet tyrosine radical NS1a and are insensitive to deuterium labeling of AA. To probe the origin and structure of this radical, we combined EPR analysis with nitric oxide (NO) trapping of tyrosine and substrate derived radicals for both PGHS-1 and -2. Formation of 3-nitrotyrosine in the proteins was analyzed by immunoblotting, whereas NO adducts to AA and AA metabolites were analyzed by mass spectrometry and by chromatography of ¹⁴C-labeled products. The results indicate that both nitrated tyrosine residues and NO-AA adducts formed upon NO trapping. The predominant NO-AA adduct was an oxime at C11 of AA with three conjugated double bonds, as indicated by absorption at 275 nm and by mass spectral analysis. This adduct amounted to 10% and 20% of the heme concentration of PGHS-1 and -2, respectively. For PGHS-1, the yield of NO-AA adduct matched the yield of the narrow radical signal obtained in parallel EPR experiments. High frequency EPR characterization of this narrow radical, reported in an accompanying paper, supports assignment to a new tyrosyl radical, NS1c, rather than an AA-based radical. To reconcile the results from EPR and NO-trapping studies, we propose that NS1c is in equilibrium with an AA pentadienyl radical, and that the latter reacts preferentially with NO.     

Multidimensional prognostic indices for use in COPD patient care. A systematic review

Background

A growing number of prognostic indices for chronic obstructive pulmonary disease (COPD) is developed for clinical use. Our aim is to identify, summarize and compare all published prognostic COPD indices, and to discuss their performance, usefulness and implementation in daily practice.

Methods

We performed a systematic literature search in both Pubmed and Embase up to September 2010. Selection criteria included primary publications of indices developed for stable COPD patients, that predict future outcome by a multidimensional scoring system, developed for and validated with COPD patients only. Two reviewers independently assessed the index quality using a structured screening form for systematically scoring prognostic studies.

Results

Of 7,028 articles screened, 13 studies comprising 15 indices were included. Only 1 index had been explored for its application in daily practice. We observed 21 different predictors and 7 prognostic outcomes, the latter reflecting mortality, hospitalization and exacerbation. Consistent strong predictors were FEV₁ percentage predicted, age and dyspnoea. The quality of the studies underlying the indices varied between fairly poor and good. Statistical methods to assess the predictive abilities of the indices were heterogenic. They generally revealed moderate to good discrimination, when measured. Limitations: We focused on prognostic indices for stable disease only and, inevitably, quality judgment was prone to subjectivity.

Conclusions

We identified 15 prognostic COPD indices. Although the prognostic performance of some of the indices has been validated, they all lack sufficient evidence for implementation. Whether or not the use of prognostic indices improves COPD disease management or patients'' health is currently unknown; impact studies are required to establish this.     

Constraints on prostaglandin biosynthesis in tissues

The formation of prostaglandins by prostaglandin H synthase can be limited by the availability of the fatty acid substrate or the hydroperoxide activator and also by a self-catalyzed inactivation associated with the oxygenation reaction. Each pmol of synthase appeared able to form only about 1300 pmol of prostaglandin from arachidonate before it was inactivated. This extent of synthesis was not diminished when substrate fatty acid was complexed with cytosolic proteins even though the velocity of the oxygenation reaction was greatly decreased by the lower availability of substrate acid. When the availability of hydroperoxide activator was decreased by added glutathione peroxidase, the extent of oxygenation per mol of synthase was decreased irrespective of the amount of cytosolic protein present. Approximately 65% of the total prostaglandin synthesis by homogenates was suppressed with a glutathione peroxidase to prostaglandin H synthase ratio of about 90. The remaining prostaglandin synthetic activity was more resistant, being completely suppressed only when the ratio of peroxidase to synthase exceeded 750. The overall ratio of glutathione peroxidase (peroxide-removing) capacity to prostaglandin synthetic (peroxide-forming) capacity in selected tissues ranged from over 1800 in rat liver to less than 30 in leukocytes. A comparison between the daily urinary output of prostaglandin metabolites and tissue prostaglandin synthetic capacity suggested that prostaglandin H synthase inactivation along with glutathione peroxidase suppression of the extent of prostaglandin synthase may be important in limiting prostaglandin biosynthesis within cells.     

Tyrosine phosphorylation of myosin heavy chain during skeletal muscle differentiation: an integrated bioinformatics approach

Background  

Previously it has been shown that insulin-mediated tyrosine phosphorylation of myosin heavy chain is concomitant with enhanced association of C-terminal SRC kinase during skeletal muscle differentiation. We sought to identify putative site(s) for this phosphorylation event.     

Effect of Anionic Salt and Highly Fermentable Carbohydrate Supplementations on Urine pH and on Experimentally Induced Hypocalcaemia in Cows

The objective of this experiment was to determine the effect of dietary grain on calcium homeostasis. Six rumen-fistulated dairy cows with 3 or more previous lactations and no history of parturient paresis were randomly assigned to a sequence of diets in a crossover study with 4 periods of 10 days each. Dietary treatments were: A control ration consisting of wrap grass silage alone (1), the control ration supplemented with ammonium chloride and ammonium sulphate salt solution (2), control ration following a period with supplementation (3) and control ration supplemented with increasing amounts of barley from 4 to 10 kg/cow per day, expected to produce subclinical rumen acidosis (4). Daily intake of the diets was adjusted to 14 kg DM/cow per day. On day 11, the calcium-regulating mechanisms in cows were challenged until recumbency by a standardized intravenous EDTA infusion and cows were left to recover spontaneously. Anion supplementation and the feeding of highly fermentable carbohydrate lowered urine pH below 7.0 due to subclinical acidosis. During spontaneous recovery from EDTA induced hypocalcaemia, the cows more quickly regained a whole blood free calcium concentration of 1.00 mmol/L if they had most recently been supplemented with either anionic salts or with increasing amounts of barley, as compared to the basic ration. It is concluded that so-called slug-feeding or 'steaming up' with highly fermentable carbohydrates before parturition in milk fever susceptible cows enhanced calcium homeostasis similar to the effect seen in cows on anionic diets.     

Peroxidase self-inactivation in prostaglandin H synthase-1 pretreated with cyclooxygenase inhibitors or substituted with mangano protoporphyrin IX

Self-inactivation imposes an upper limit on bioactive prostanoid synthesis by prostaglandin H synthase (PGHS). Inactivation of PGHS peroxidase activity has been found to begin with Intermediate II, which contains a tyrosyl radical. The structure of this radical is altered by cyclooxygenase inhibitors, such as indomethacin and flurbiprofen, and by replacement of heme by manganese protoporphyrin IX (forming MnPGHS-1). Peroxidase self-inactivation in inhibitor-treated PGHS-1 and MnPGHS-1 was characterized by stopped-flow spectroscopic techniques and by chromatographic and mass spectrometric analysis of the metalloporphyrin. The rate of peroxidase inactivation was about 0.3 s(-)1 in inhibitor-treated PGHS-1 and much slower in MnPGHS-1 (0.05 s(-)1); as with PGHS-1 itself, the peroxidase inactivation rates were independent of peroxide concentration and structure, consistent with an inactivation process beginning with Intermediate II. The changes in metalloporphyrin absorbance spectra during inactivation of inhibitor-treated PGHS-1 were similar to those observed with PGHS-1 but were rather distinct in MnPGHS-1; the kinetics of the spectral transition from Intermediate II to the next species were comparable to the inactivation kinetics in each case. In contrast to the situation with PGHS-1 itself, significant amounts of heme degradation occurred during inactivation of inhibitor-treated PGHS-1, producing iron chlorin and heme-protein adduct species. Structural perturbations at the peroxidase site (MnPGHS-1) or at the cyclooxygenase site (inhibitor-treated PGHS-1) thus can influence markedly the kinetics and the chemistry of PGHS-1 peroxidase inactivation.     

Comparison of the properties of prostaglandin H synthase-1 and -2

Biosynthesis of prostanoid lipid signaling agents from arachidonic acid begins with prostaglandin H synthase (PGHS), a hemoprotein in the myeloperoxidase family. Vertebrates from humans to fish have two principal isoforms of PGHS, termed PGHS-1 and-2. These two isoforms are structurally quite similar, but they have very different pathophysiological roles and are regulated very differently at the level of catalysis. The focus of this review is on the structural and biochemical distinctions between PGHS-1 and-2, and how these differences relate to the functional divergence between the two isoforms.