Mechanism of sulfide-quinone reductase investigated using site-directed mutagenesis and sulfur analysis

Biological sulfide oxidation is a reaction occurring in all three domains of life. One enzyme responsible for this reaction in many bacteria has been identified as sulfide:quinone oxidoreductase (SQR). The enzyme from Rhodobacter capsulatus is a peripherally membrane-bound flavoprotein with a molecular mass of approximately 48 kDa, presumably acting as a homodimer. In this work, SQR from Rb. capsulatus has been modified with an N-terminal His tag and heterologously expressed in and purified from Escherichia coli. Three cysteine residues have been shown to be essential for the reductive half-reaction by site-directed mutagenesis. The catalytic activity has been nearly completely abolished after mutation of each of the cysteines to serine. A decrease in fluorescence on reduction by sulfide as observed for the wild-type enzyme has not been observed for any of the mutated enzymes. Mutation of a conserved valine residue to aspartate within the third flavin-binding domain led to a drastically reduced substrate affinity, for both sulfide and quinone. Two conserved histidine residues have been mutated individually to alanine. Both of the resulting enzymes exhibited a shift in the pH dependence of the SQR reaction. Polysulfide has been identified as a primary reaction product using spectroscopic and chromatographic methods. On the basis of these data, reaction mechanisms for sulfide-dependent reduction and quinone-dependent oxidation of the enzyme and for the formation of polysulfide are proposed.     

Soluble TNF-like cytokine (TL1A) production by immune complexes stimulated monocytes in rheumatoid arthritis

TNF-like cytokine (TL1A) is a newly identified member of the TNF superfamily of ligands that is important for T cell costimulation and Th1 polarization. However, despite increasing information about its functions, very little is known about expression of TL1A in normal or pathological states. In this study, we report that mononuclear phagocytes appear to be a major source of TL1A in rheumatoid arthritis (RA), as revealed by their strong TL1A expression in either synovial fluids or synovial tissue of rheumatoid factor (RF)-seropositive RA patients, but not RF-/RA patients. Accordingly, in vitro experiments revealed that human monocytes express and release significant amounts of soluble TL1A when stimulated with insoluble immune complexes (IC), polyethylene glycol precipitates from the serum of RF+/RA patients, or with insoluble ICs purified from RA synovial fluids. Monocyte-derived soluble TL1A was biologically active as determined by its capacity to induce apoptosis of the human erythroleukemic cell line TF-1, as well as to cooperate with IL-12 and IL-18 in inducing the production of IFN-gamma by CD4(+) T cells. Because RA is a chronic inflammatory disease with autoimmune etiology, in which ICs, autoantibodies (including RF), and various cytokines contribute to its pathology, our data suggest that TL1A could be involved in its pathogenesis and contribute to the severity of RA disease that is typical of RF+/RA patients.     

Cu(II) ion interaction with teicoplanin-vancomycin’s analog

Teicoplanin, a member of the “last chance” antibiotic family has a similar structure and the same mechanism of action as parent drug vancomycin, which is proved to be an effective binder of Cu(II) ions. However, the potentiometric and spectroscopic studies (UV-visible, CD, NMR) have shown that the modification of the N-terminal structure of the peptide backbone in teicoplanin affects considerably the binding ability towards Cu(II) ions. While vancomycin forms almost instantly the stable 3 N complex species involving the N-terminal and two amide nitrogen donors, in case of teicoplanin only two nitrogen donors derived from the N-terminal amino group and adjacent peptide bond are coordinated to Cu(II) ion within the whole pH range studied. The major factor influencing the binding mode is most likely the structure of the N-terminus of the peptide unit in the antibiotic ligand.     

Pralidoxime in Acute Organophosphorus Insecticide Poisoning—A Randomised Controlled Trial

Background

Poisoning with organophosphorus (OP) insecticides is a major global public health problem, causing an estimated 200,000 deaths each year. Although the World Health Organization recommends use of pralidoxime, this antidote''s effectiveness remains unclear. We aimed to determine whether the addition of pralidoxime chloride to atropine and supportive care offers benefit.

Methods and Findings

We performed a double-blind randomised placebo-controlled trial of pralidoxime chloride (2 g loading dose over 20 min, followed by a constant infusion of 0.5 g/h for up to 7 d) versus saline in patients with organophosphorus insecticide self-poisoning. Mortality was the primary outcome; secondary outcomes included intubation, duration of intubation, and time to death. We measured baseline markers of exposure and pharmacodynamic markers of response to aid interpretation of clinical outcomes. Two hundred thirty-five patients were randomised to receive pralidoxime (121) or saline placebo (114). Pralidoxime produced substantial and moderate red cell acetylcholinesterase reactivation in patients poisoned by diethyl and dimethyl compounds, respectively. Mortality was nonsignificantly higher in patients receiving pralidoxime: 30/121 (24.8%) receiving pralidoxime died, compared with 18/114 (15.8%) receiving placebo (adjusted hazard ratio [HR] 1.69, 95% confidence interval [CI] 0.88–3.26, p = 0.12). Incorporating the baseline amount of acetylcholinesterase already aged and plasma OP concentration into the analysis increased the HR for patients receiving pralidoxime compared to placebo, further decreasing the likelihood that pralidoxime is beneficial. The need for intubation was similar in both groups (pralidoxime 26/121 [21.5%], placebo 24/114 [21.1%], adjusted HR 1.27 [95% CI 0.71–2.29]). To reduce confounding due to ingestion of different insecticides, we further analysed patients with confirmed chlorpyrifos or dimethoate poisoning alone, finding no evidence of benefit.

Conclusions

Despite clear reactivation of red cell acetylcholinesterase in diethyl organophosphorus pesticide poisoned patients, we found no evidence that this regimen improves survival or reduces need for intubation in patients with organophosphorus insecticide poisoning. The reason for this failure to benefit patients was not apparent. Further studies of different dose regimens or different oximes are required.

Trial Registration

Controlled-trials.com ISRCTN55264358 Please see later in the article for Editors'' Summary     

Biochemical characterisation of the D60A mutant of the elongation factor 1α from the archaeon Sulfolobus solfataricus

The D60A mutant of the elongation factor (EF) 1α from Sulfolobus solfataricus (Ss), was obtained as heterologous expressed protein and characterised. This substitution was carried out in order to analyse the involvement of this evolutionally conserved amino acid position in the interaction between the elongation factor and guanosine nucleotides and in the coordination of magnesium ions. The expression system used produced a folded protein able to catalyse, although to a slightly lower extent with respect to the wild-type enzyme, protein synthesis in vitro and NaCl-dependent intrinsic GTPase activity. The affinity for guanosine nucleotides was almost identical to that exhibited by wild-type SsEF-1α; vice versa, the GDP exchange rate was one order of magnitude faster on the mutated elongation factor, a property partially restored when the exchange reaction was analysed in the presence of the magnesium ions chelating agent EDTA. Finally, the D60A substitution only a little affected the high thermal stability of the elongation factor. From a structural point of view, the analysis of the data reported confirmed that this conserved carboxyl group belongs to a protein region differentiating the GDP binding mode among elongation factors from different organisms.     

Method and strain rate dependence of Achilles tendon stiffness

Tendon stiffness is calculated by dividing changes in tendon force by tendon elongation. For this purpose, participants are commonly asked to perform a maximal muscle contraction (“active” method). Alternatively tendon elongation can be achieved by means of a passive joint rotation (“passive” method). The purpose of this study was to compare Achilles tendon stiffness obtained from both methods across different tendon strain rates. Twenty adults performed a series of ramped maximum isometric plantarflexions of different durations. Passive ankle rotations of different angular velocities were also performed. Achilles tendon stiffness was obtained from a combination of motion analysis, isokinetic dynamometry and ultrasonography and compared across methods at three strain rates. At all strain rates, tendon stiffness obtained from the active method was 6% greater compared to the passive method. In spite of this systematic bias, there was good agreement between the methods. Intraclass correlation coefficients were greater than 0.98, and more than 95% of data points fell into the 95% confidence intervals. This agreement will be acceptable in many research contexts. We also found a linear increase in tendon stiffness with increasing strain rate, which must be taken into consideration when interpreting or reporting tendon stiffness.     

Rho‐associated kinases in tumorigenesis: re‐considering ROCK inhibition for cancer therapy

The Rho‐associated (ROCK) serine/threonine kinases have emerged as central regulators of the actomyosin cytoskeleton, their main purpose being to promote contractile force generation. Aided by the discovery of effective inhibitors such as Y27632, their roles in cancer have been extensively explored with particular attention focused on motility, invasion and metastasis. Recent studies have revealed a surprisingly diverse range of functions of ROCK. These insights could change the way ROCK inhibitors might be used in cancer therapy to include the targeting of stromal rather than tumour cells, the concomitant blocking of ROCK and proteasome activity in K‐Ras‐driven lung cancers and the combination of ROCK with tyrosine kinase inhibitors for treating haematological malignancies such as chronic myeloid leukaemia. Despite initial optimism for therapeutic efficacy of ROCK inhibition for cancer treatment, no compounds have progressed into standard therapy so far. However, by carefully defining the key cancer types and expanding the appreciation of ROCK's role in cancer beyond being a cell‐autonomous promoter of tumour cell invasion and metastasis, the early promise of ROCK inhibitors for cancer therapy might still be realized.     

Begging and cowbirds: brood parasites make hosts scream louder

Avian brood parasites have evolved striking begging abilitythat often allows them to prevail over the host progeny in competitionfor parental resources. Host young are therefore selected bybrood parasites to evolve behavioral strategies that reducethe cost of parasitism. We tested the prediction that the intensityof nestling begging displays functioning to attract parentalcare increases across species with the frequency of parasitismby the brown-headed cowbird (Molothrus ater). This was expectedbecause host young should try to prevail over highly competitiveparasitic broodmates in scramble interactions, act more selfishlywhen frequency of parasitism is high because brood parasitesoften affect more severely host condition than conspecific broodmates,and discount the kin selection costs of subtracting resourcesto unrelated parasites. Across 31 North American host species,begging loudness positively covaried with parasitism rate inPasserines, and such effect was stronger in species with smallcompared with large clutches. Begging loudness increased withbrood parasitism and nest predation among the most suitablehost species. These results held after controlling for concomitantecological factors and for common ancestry effects. Our resultssupport the hypothesis that avian brood parasitism has playeda role in the evolution of begging behavior of host young.