Molecular evolution of rodent insulins

Several trees of amino acid sequences of rodent insulins were derived with the maximum-parsimony procedure. Possible orthologous and paralogous relationships were investigated. Except for a recent gene duplication in the ancestor of rat and mouse, there are no strong arguments for other paralogous relationships. Therefore, a tree in agreement with other biological data is the most reasonable one. According to this tree, the capacity to form zinc-binding hexamers was lost once in the ancestor of the hystricomorph rodents, followed by moderately increased evolutionary rates in the lineages to African porcupine and chinchilla but highly increased rates in at least three independent lines to other taxa of this suborder: guinea pig, cuis, and Octodontoidea (coypu and casiragua).      

Oxidation-reduction potentials of ferredoxin-NADP+ reductase and flavodoxin from Anabaena PCC 7119 and their electrostatic and covalent complexes.

The oxidation-reduction potentials of ferredoxin-NADP+ reductase and flavodoxin from the cyanobacterium Anabaena PCC 7119 were determined by potentiometry. The potentials at pH 7 for the oxidized flavodoxin/flavodoxin semiquinone couple (E2) and the flavodoxin semiquinone/hydroquinone couple (E1) were -212 mV and -436 mV, respectively. E1 was independent of pH above about pH 7, but changed by approximately -60 mV/pH below about pH 6, suggesting that the fully reduced protein has a redox-linked pKa at about 6.1, similar to those of certain other flavodoxins. E2 varied by -50 mV/pH in the range pH 5-8. The redox potential for the two-electron reduction of ferredoxin-NADP+ reductase was -344 mV at pH 7 (delta Em = -30 mV/pH). In the 1:1 electrostatic complex of the two proteins titrated at pH 7, E2 was shifted by +8 mV and E1 was shifted by -25 mV; the shift in potential for the reductase was +4 mV. The potentials again shifted following treatment of the electrostatic complex with a carbodiimide, to covalently link the two proteins. By comparison with the separate proteins at pH 7, E2 for flavodoxin shifted by -21 mV and E1 shifted by +20 mV; the reductase potential shifted by +2 mV. The potentials of the proteins in the electrostatic and covalent complexes showed similar pH dependencies to those of the individual proteins. Qualitatively similar changes occurred when ferredoxin-NADP+ reductase from Anabaena variabilis was complexed with flavodoxin from Azotobacter vinelandii. The shifts in redox potential for the complexes were used with previously determined values for the dissociation constant (Kd) of the electrostatic complex of the two oxidised proteins, in order to estimate Kd values for the interaction of the different redox forms of the proteins. The calculations showed that the electrostatic complexes, formed when the proteins differ in their redox states, are stronger than those formed when both proteins are fully oxidized or fully reduced.     

Overexpression of Flavodoxin in Bacteroids Induces Changes in Antioxidant Metabolism Leading to Delayed Senescence and Starch Accumulation in Alfalfa Root Nodules

Sinorhizobium meliloti cells were engineered to overexpress Anabaena variabilis flavodoxin, a protein that is involved in the response to oxidative stress. Nodule natural senescence was characterized in alfalfa (Medicago sativa) plants nodulated by the flavodoxin-overexpressing rhizobia or the corresponding control bacteria. The decline of nitrogenase activity and the nodule structural and ultrastructural alterations that are associated with nodule senescence were significantly delayed in flavodoxin-expressing nodules. Substantial changes in nodule antioxidant metabolism, involving antioxidant enzymes and ascorbate-glutathione cycle enzymes and metabolites, were detected in flavodoxin-containing nodules. Lipid peroxidation was also significantly lower in flavodoxin-expressing nodules than in control nodules. The observed amelioration of the oxidative balance suggests that the delay in nodule senescence was most likely due to a role of the protein in reactive oxygen species detoxification. Flavodoxin overexpression also led to high starch accumulation in nodules, without reduction of the nitrogen-fixing activity.Symbiotic nodules have a limited functional life that varies among different legume species. Nodule senescence is the sequence of structural, molecular, biochemical, and physiological events taking place in the process that a mature and functional nodule undergoes leading to the loss of the nitrogen-fixing activity and culminating in cell death of symbiotic tissue (Swaraj and Bishnoi, 1996; Puppo et al., 2005; Van de Velde et al., 2006).Various models have been proposed to explain the mechanisms that trigger the process of natural or stress-induced nodule senescence. However, it is generally accepted that a senescence-inducing signal from the plant causes a decrease in antioxidant levels and thus an increase in reactive oxygen species (ROS) up to a point of no return. Numerous studies have shown that ROS and antioxidant systems are involved in natural (Lucas et al., 1998; Evans et al., 1999; Hernández-Jiménez et al., 2002; Puppo et al., 2005) as well as induced (Dalton et al., 1993; Becana et al., 2000; Hernández-Jiménez et al., 2002; Matamoros et al., 2003) nodule senescence. Nitrogen fixation is very sensitive to ROS, and nitrogenase activity drastically decreases during nodule senescence (Dalton et al., 1986).Antioxidant systems that protect cells from oxidative damage have been described in symbiotic nodules (Dalton et al., 1986, 1993; Evans et al., 1999; Becana et al., 2000; Matamoros et al., 2003; Puppo et al., 2005). These include the enzymes superoxide dismutase (SOD), catalase, and peroxidase. Another enzymatic system associated with ROS detoxification is the ascorbate-glutathione pathway, which includes ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR), and glutathione reductase (GR; Dalton et al., 1986, 1992; Noctor and Foyer 1998; Becana et al., 2000). Ascorbate and reduced glutathione (GSH) in this pathway can also scavenge superoxide and hydrogen peroxide.During nodule senescence, several ultrastructural alterations in the nodule tissues and cells have been observed (Lucas et al., 1998; Hernández-Jiménez et al., 2002; Puppo et al., 2005, and refs. therein; Van de Velde et al., 2006). Cytosol becomes electron dense, altered vesicles proliferate, and eventually the cytosol undergoes lysis. The number of peroxisomes increases, mitochondria form complex elongated structures, and symbiosomes change in size and shape and fuse during natural and induced senescence of nodules (Hernández-Jiménez et al., 2002). Damage of the symbiosome membrane is also detected (Puppo et al., 2005; Van de Velde et al., 2006).A strategy of delayed nodule senescence could lead to increased nitrogen fixation and legume productivity. Delayed nodule senescence together with enhanced sustainability under field conditions are among the key aims of legume improvement programs (Puppo et al., 2005). An interesting approach proposed to achieve delayed senescence is to induce nodulation in legumes using rhizobial strains with modified redox capacity (Zahran, 2001).The protein flavodoxin contains a FMN group acting as a redox center transferring electrons at low potentials (Pueyo et al., 1991; Pueyo and Gómez-Moreno, 1991). The FMN cofactor of flavodoxin can exist in three different redox states: oxidized, one-electron-reduced semiquinone, and two-electron-reduced hydroquinone. This property confers high versatility to flavodoxins in electron transport systems (Simondsen and Tollin, 1980; McIver et al., 1998). To date, flavodoxin has not been described in plants, as flavodoxin-encoding genes were lost during the transition of algae to plants (Zurbriggen et al., 2007) and, consequently, no homologs have been identified in the sequenced genome of Arabidopsis (Arabidopsis thaliana; Arabidopsis Genome Initiative, 2000). Flavodoxin is present as a constitutive or inducible protein in different microorganisms (Klugkist et al., 1986). In the nitrogen-fixing cyanobacterium Anabaena variabilis PCC 7119, flavodoxin is expressed under conditions of limited iron availability, replacing ferredoxin in the photosynthetic electron transport from PSI to NADP⁺ and in nitrogenase reduction (Sandmann et al., 1990). Reversible electron transfer from flavodoxin to NADP⁺ is catalyzed by ferredoxin NADP⁺ reductase in different pathways of oxidative metabolism (Arakaki et al., 1997). In its reduced state, flavodoxin might be able to react with ROS and revert to its original redox state in the presence of an appropriate electron source. This could probably occur without the associated molecular damage that metallic complexes in catalases or SODs suffer (Keyer et al., 1995). The presence of flavodoxin has not been documented to date in the symbiotic bacterium Sinorhizobium meliloti. In Escherichia coli, however, flavodoxin induction is linked to the oxidative stress-responsive regulon soxRS (Zheng et al., 1999). It has been suggested that flavodoxin and ferredoxin (flavodoxin) NADP⁺ reductase might be induced and have a role in reestablishing the cell redox balance under oxidative stress conditions (Liochev et al., 1994). The properties of flavodoxin suggest that its presence in the cell may have a facilitating effect on ROS detoxification. In fact, an increase in the amount of flavodoxin has been observed in some bacterial species subjected to oxidative stress (Zheng et al., 1999; Yousef et al., 2003; Singh et al., 2004), and transgenic tobacco (Nicotiana tabacum) plants expressing flavodoxin in chloroplasts show enhanced tolerance to a broad range of stresses related to oxidative damage (Tognetti et al., 2006, 2007a, 2007b).In this work, Sinorhizobium meliloti was transformed with the A. variabilis flavodoxin gene and used to nodulate alfalfa (Medicago sativa) plants. The effects of flavodoxin expression on nodulation dynamics, on nodule development and senescence processes, and on nitrogen-fixing activity were analyzed. Mechanistic insights suggesting putative roles for flavodoxin in protection from ROS and the induced delay of nodule senescence are likewise discussed.     

Weekly self-monitoring and treatment adjustment benefit patients with partly controlled and uncontrolled asthma: an analysis of the SMASHING study

Background

Internet-based self-management has shown to improve asthma control and asthma related quality of life, but the improvements were only marginally clinically relevant for the group as a whole. We hypothesized that self-management guided by weekly monitoring of asthma control tailors pharmacological therapy to individual needs and improves asthma control for patients with partly controlled or uncontrolled asthma.

Methods

In a 1-year randomised controlled trial involving 200 adults (18-50 years) with mild to moderate persistent asthma we evaluated the adherence with weekly monitoring and effect on asthma control and pharmacological treatment of a self-management algorithm based on the Asthma Control Questionnaire (ACQ). Participants were assigned either to the Internet group (n = 101) that monitored asthma control weekly with the ACQ on the Internet and adjusted treatment using a self-management algorithm supervised by an asthma nurse specialist or to the usual care group (UC) (n = 99). We analysed 3 subgroups: patients with well controlled (ACQ ≤ 0.75), partly controlled (0.75>ACQ ≤ 1.5) or uncontrolled (ACQ>1.5) asthma at baseline.

Results

Overall monitoring adherence was 67% (95% CI, 60% to 74%). Improvements in ACQ score after 12 months were -0.14 (p = 0.23), -0.52 (p < 0.001) and -0.82 (p < 0.001) in the Internet group compared to usual care for patients with well, partly and uncontrolled asthma at baseline, respectively. Daily inhaled corticosteroid dose significantly increased in the Internet group compared to usual care in the first 3 months in patients with uncontrolled asthma (+278 μg, p = 0.001), but not in patients with well or partly controlled asthma. After one year there were no differences in daily inhaled corticosteroid use or long-acting β₂-agonists between the Internet group and usual care.

Conclusions

Weekly self-monitoring and subsequent treatment adjustment leads to improved asthma control in patients with partly and uncontrolled asthma at baseline and tailors asthma medication to individual patients'' needs.

Trial registration

Current Controlled Trials ISRCTN79864465     

Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

Background  

The assembly and spatial organization of enzymes in naturally occurring multi-protein complexes is of paramount importance for the efficient degradation of complex polymers and biosynthesis of valuable products. The degradation of cellulose into fermentable sugars by Clostridium thermocellum is achieved by means of a multi-protein "cellulosome" complex. Assembled via dockerin-cohesin interactions, the cellulosome is associated with the cell surface during cellulose hydrolysis, forming ternary cellulose-enzyme-microbe complexes for enhanced activity and synergy. The assembly of recombinant cell surface displayed cellulosome-inspired complexes in surrogate microbes is highly desirable. The model organism Lactococcus lactis is of particular interest as it has been metabolically engineered to produce a variety of commodity chemicals including lactic acid and bioactive compounds, and can efficiently secrete an array of recombinant proteins and enzymes of varying sizes.