The antioxidative function of eicosapentaenoic acid in a marine bacterium, Shewanella marinintestina IK-1

When the eicosapentaenoic acid (EPA)-deficient mutant strain IK-1Delta8 of the marine EPA-producing Shewanella marinintestina IK-1 was treated with various concentrations of hydrogen peroxide (H(2)O(2)), its colony-forming ability decreased more than that of the wild type. Protein carbonylation, induced by treating cells with 0.01 mM H(2)O(2) under bacteriostatic conditions, was enhanced only in cells lacking EPA. The amount of cells recovered from the cultures was decreased more significantly by the presence of H(2)O(2) for cells lacking EPA than for those producing EPA. Treatment of the cells with 0.1 mM H(2)O(2) resulted in much lower intracellular concentrations of H(2)O(2) being consistently detected in cells with EPA than in those without EPA. These results suggest that cellular EPA can directly protect cells against oxidative damage by shielding the entry of exogenously added H(2)O(2) in S. marinintestina IK-1.     

Oxidative stress reduces transintestinal transports and (Na+, K+) -ATPase activity in rat jejunum

Because oxidative stress is a component of gastrointestinal injury, we investigated the effect of H(2)O(2) on transintestinal transport using isolated rat jejunum incubated in vitro. Millimolar concentrations of H(2)O(2) inhibited all the tested parameters without inducing any cytotoxic effect. Electrophysiological experiments indicated that H(2)O(2) decreases significantly both short circuit current and transepithelial electrical potential difference without affecting transepithelial resistance. The possibility that H(2)O(2) could influence (Na+, K+) -ATPase activity was explored using isolated basolateral membranes. Besides H(2)O(2), free radicals (O(2)(*-), HO*) were generated using different iron-dependent and independent systems; (Na+, K+) -ATPase activity was inhibited after membrane exposure to all ROS tested. The inhibition was prevented by allopurinol, superoxide dismutase or desferrioxamine. Western blot analysis showed a decreased expression of the alpha(1)-subunit of (Na+, K+) -ATPase. We conclude that H(2)O(2) may be a modulator of jejunal ion and water transport by multiple mechanisms, among which a significant inhibition of the basolateral (Na+, K+) -ATPase.     

Free radical oxidation (autoxidation) of alkenones and other lipids in cells of Emiliania huxleyi

Cells of the coccolithophorid Emiliania huxleyi strain CS-57 grown under an atmosphere of air+0.5% CO(2) showed oxidative damage after 10 days growth with concomitant and major changes to the lipid composition. The fatty acid profile was strongly altered and lacked appreciable amounts of the polyunsaturated fatty acids (PUFA: C(18:5), C(18:3) and C(22:6)) typical of healthy cells. Oxidation products of these PUFA could not be detected, but monounsaturated fatty acids proved to be good indicators of oxidative processes. The presence (after NaBH(4)-reduction) of a high proportion of 11-hydroxyoctadec-cis-9-enoic and 8-hydroxyoctadec-cis-9-enoic acids showed that the degradation of oleic acid involved mainly free radical oxidation processes (70-75% autoxidation and 20-25% photooxidation). We also detected large amounts of degradation products of the oxidation product 9,10-epoxyoctadecanoic acid including diols, methoxyhydrins and chlorohydrins. These oxidative effects were found in all the lipid classes examined. Products included significant amounts of chlorophyll side-chain autooxidation products Z- and E-3,7,11,15-tetramethylhexadec-3-ene-1,2-diols and Z-and E-3,7,11,15-tetramethylhexadec-2-ene-1,4-diols, while phytyldiol was present in relatively low proportions. Delta(5)-3beta,7-epimeric unsaturated steroidal diols arising from the autooxidation of the Delta(5) double bond of epi-brassicasterol and minor amounts of Delta(4)-3beta,6-diols were also detected. Long-chain unsaturated ketone (alkenone) content per cell was much higher in the presence of 0.5% CO(2) likely due to carbon storage under these conditions. The proportions of di- and tri-unsaturated alkenones was relatively stable throughout the growth cycle in the absence of additional CO(2), but not when grown with 0.5% CO(2). The detection of characteristic alkenone autoxidation products in cells grown under these latter conditions allowed us to attribute the significant increase in index observed to the involvement of free radical oxidation processes.     

Computation of threshold conditions for epidemiological models and global stability of the disease-free equilibrium (DFE)

One goal of this paper is to give an algorithm for computing a threshold condition for epidemiological systems arising from compartmental deterministic modeling. We calculate a threshold condition T(0) of the parameters of the system such that if T(0)<1 the disease-free equilibrium (DFE) is locally asymptotically stable (LAS), and if T(0)>1, the DFE is unstable. The second objective, by adding some reasonable assumptions, is to give, depending on the model, necessary and sufficient conditions for global asymptotic stability (GAS) of the DFE. In many cases, we can prove that a necessary and sufficient condition for the global asymptotic stability of the DFE is R(0)< or =1, where R(0) is the basic reproduction number [O. Diekmann, J.A. Heesterbeek, Mathematical Epidemiology of Infectious Diseases: Model Building, Analysis and Interpretation, Wiley, New York, 2000]. To illustrate our results, we apply our techniques to examples taken from the literature. In these examples we improve the results already obtained for the GAS of the DFE. We show that our algorithm is relevant for high dimensional epidemiological models.     

Effect of storage xyloglucans on peritoneal macrophages

Xyloglucans from seeds of Copaifera langsdorffii (XGC), Hymenaea courbaril (XGJ) and Mucuna sloanei (XGM) were obtained from milled and defatted cotyledons by aqueous extraction at 25 degrees C. The resulting fractions contained Glc, Xyl and Gal in molar ratios of 2.5: 1.5: 1.0 (XGC), 3.8: 2.6: 1.0 (XGJ) and 2.5: 1.6: 1.0 (XGM). HPSEC-MALLS/RI analysis showed that each polysaccharide fraction was homogeneous; M(w) values were 1.6 x 10(5), 2.0 x 10(5) and 1.5 x 10(5)g/mol, respectively. The effect of the xyloglucans on the production of O(2)*(-) and NO* and on the recruitment of macrophages to the mouse peritoneum was evaluated. All polysaccharides promoted an increase in the number of peritoneal macrophages in a dose-dependent manner. The largest increase, of 576% in comparison to the control group, was elicited by XGJ at 200 mg/kg. The effect of XGC, XGJ and XGM on O(2)*(-) production, in the presence or absence of phorbol 12-myristate 13-acetate (PMA), was not statistically significant. For NO(.) production, the lowest concentration of XGC (10 microg/ml) gave rise to an increase of 262% when compared to the control group; the effect was dose-dependent, reaching 307% at 50 microg/ml. On the other hand, XGJ at a concentration of 50 microg/ml enhanced NO* production by 92%. XGM did not affect NO* production significantly. The results indicate that xyloglucans from C. langsdorffii, H. courbaril and M. sloanei have immunomodulatory activity.     

Intermonomer electron transfer in the bc1 complex dimer is controlled by the energized state and by impaired electron transfer between low and high potential hemes

The cytochrome bc(1) complex (commonly called Complex III) is the central enzyme of respiratory and photosynthetic electron transfer chains. X-ray structures have revealed the bc(1) complex to be a dimer, and show that the distance between low potential (b(L)) and high potential (b(H)) hemes, is similar to the distance between low potential hemes in different monomers. This suggests that electron transfer between monomers should occur at the level of the b(L) hemes. Here, we show that although the rate constant for b(L)-->b(L) electron transfer is substantial, it is slow compared to the forward rate from b(L) to b(H), and the intermonomer transfer only occurs after equilibration within the first monomer. The effective rate of intermonomer transfer is about 2-orders of magnitude slower than the direct intermonomer electron transfer.     

A divergent synthesis of [1-14C]-mono-E isomers of fatty acids

A convenient synthesis of [1-14C]-mono-trans fatty acid using olefin inversion as a key-step is described. This methodology allows for a facile synthesis of [1-14C]-labelled mono-trans analogues of oleic, linoleic and linolenic acids. As an example, only eleven steps were necessary to obtain the [1-14C]-mono-E isomers of linolenic acid from its commercial all-Z form. In the first step, Barton's decarboxylation procedure yielded a bromo intermediate. Epoxidation of this compound resulted in the formation of three monoepoxides, which could be separated by HPLC. After identification by 1H NMR and MS, the pure monoepoxides were then subjected to inversion consisting of a stereospecific deoxygenation followed by a beta-elimination step. Finally, the labelling was introduced by substitution of the bromine by a [14C]-cyano group followed by hydrolysis.     

Enzyme catalysis via control of activation entropy: site-directed mutagenesis of 6,7-dimethyl-8-ribityllumazine synthase

6,7-Dimethyl-8-ribityllumazine synthase (lumazine synthase) catalyses the penultimate step in the biosynthesis of riboflavin. In Bacillus subtilis, 60 lumazine synthase subunits form an icosahedral capsid enclosing a homotrimeric riboflavin synthase unit. The ribH gene specifying the lumazine synthase subunit can be expressed in high yield. All amino acid residues exposed at the surface of the active site cavity were modified by PCR assisted mutagenesis. Polar amino acid residues in direct contact with the enzyme substrates, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate, could be replaced with relative impunity with regard to the catalytic properties. Only the replacement of Arg127, which forms a salt bridge with the phosphate group of 3,4-dihydroxy-2-butanone 4-phosphate, reduced the catalytic rate by more than one order of magnitude. Replacement of His88, which is believed to assist in proton transfer reactions, reduced the catalytic activity by about one order of magnitude. Surprisingly, the activation enthalpy deltaH of the lumazine synthase reaction exceeds that of the uncatalysed reaction. On the other hand, the free energy of activation deltaG of the uncatalysed reaction is characterised by a large entropic term (TdeltaS) of -37.8 kJmol(-1), whereas the entropy of activation (TdeltaS) of the enzyme-catalysed reaction is -6.7 kJmol(-1). This suggests that the rate enhancement by the enzyme is predominantly achieved by establishing a favourable topological relation of the two substrates, whereas acid/base catalysis may play a secondary role.     

Thermodynamic and EPR studies of slowly relaxing ubisemiquinone species in the isolated bovine heart complex I

Previously, we investigated ubisemiquinone (SQ) EPR spectra associated with NADH-ubiquinone oxidoreductase (complex I) in the tightly coupled bovine heart submitochondrial particles (SMP). Based upon their widely differing spin relaxation rate, we distinguished SQ spectra arising from three distinct SQ species, namely SQ(Nf) (fast), SQ(Ns) (slow), and SQ(Nx) (very slow). The SQ(Nf) signal was observed only in the presence of the proton electrochemical gradient (deltamu(H)(+)), while SQ(Ns) and SQ(Nx) species did not require the presence of deltamu(H+). We have now succeeded in characterizing the redox and EPR properties of SQ species in the isolated bovine heart complex I. The potentiometric redox titration of the g(z,y,x)=2.00 semiquinone signal gave the redox midpoint potential (E(m)) at pH 7.8 for the first electron transfer step [E(m1)(Q/SQ)] of -45 mV and the second step [E(m2)(SQ/QH(2))] of -63 mV. It can also be expressed as [E(m)(Q/QH(2))] of -54 mV for the overall two electron transfer with a stability constant (K(stab)) of the SQ form as 2.0. These characteristics revealed the existence of a thermodynamically stable intermediate redox state, which allows this protein-associated quinone to function as a converter between n=1 and n=2 electron transfer steps. The EPR spectrum of the SQ species in complex I exhibits a Gaussian-type spectrum with the peak-to-peak line width of approximately 6.1 G at the sample temperature of 173 K. This indicates that the SQ species is in an anionic Q(-) state in the physiological pH range. The spin relaxation rate of the SQ species in isolated complex I is much slower than the SQ counterparts in the complex I in situ in SMP. We tentatively assigned slow relaxing anionic SQ species as SQ(Ns), based on the monophasic power saturation profile and several fold increase of its spin relaxation rate in the presence of reduced cluster N2. The current study also suggests that the very slowly relaxing SQ(Nx) species may not be an intrinsic complex I component. The functional role of SQ(Ns) is further discussed in connection with the SQ(Nf) species defined in SMP in situ.     

Patterning, prestress, and peeling dynamics of myocytes

As typical anchorage-dependent cells myocytes must balance contractility against adequate adhesion. Skeletal myotubes grown as isolated strips from myoblasts on micropatterned glass exhibited spontaneous peeling after one end of the myotube was mechanically detached. Such results indicate the development of a prestress in the cells. To assess this prestress and study the dynamic adhesion strength of single myocytes, the shear stress of fluid aspirated into a large-bore micropipette was then used to forcibly peel myotubes. The velocity at which cells peeled from the surface, V(peel), was measured as a continuously increasing function of the imposed tension, T(peel), which ranges from approximately 0 to 50 nN/ micro m. For each cell, peeling proved highly heterogeneous, with V(peel) fluctuating between 0 micro m/s ( approximately 80% of time) and approximately 10 micro m/s. Parallel studies of smooth muscle cells expressing GFP-paxillin also exhibited a discontinuous peeling in which focal adhesions fractured above sites of strong attachment (when pressure peeled using a small-bore pipette). The peeling approaches described here lend insight into the contractile-adhesion balance and can be used to study the real-time dynamics of stressed adhesions through both physical detection and the use of GFP markers; the methods should prove useful in comparing normal versus dystrophic muscle cells.     

Involvement of Na+/H+ exchanger in the calcium signaling in epimastigotes of Trypanosoma cruzi

We studied the effect of Na(+) extracellular on Ca(2+) mobilization from intracellular store evoked by carbachol in Trypanosoma cruzi. We report that slow component of Ca(2+) signaling evoked by agonist is dependent on extracellular Na(+) but not on InsP(3) increase. Moreover, this Ca(2+) signaling progressively increased when pH of the medium changed from 7.0 to 7.8. In addition, we found that it was regulated by PKC. The agonist was also able to induce the alkalinization of the acidic compartment, and both Ca(2+) signaling and alkalinization were inhibited by the EIPA-inhibitor of the Na(+)/H(+) exchanger. These results demonstrated the alkalinization of acidic vacuoles and PKC are involved in the triggering of the epimastigote Ca(2+) signaling.     

Mitochondrial nitric oxide localization in H9c2 cells revealed by confocal microscopy.

This study shows the presence of all three nitric oxide synthases (NOSs) and NOS activity in H9c2 cells cultured under non-stimulated conditions. By using the 4,5 diaminofluoresceindiacetate (DAF-2DA) fluorimetric nitric oxide (NO(*)) detection system we observed NO(*) production in H9c2 cells. As revealed by confocal microscopy, NO(*) fluorescence colocalizes in mitochondria labeled with Mito-Tracker Red CM-H(2)Xros. Upon stimulation with acetylcholine (Ach), which increased NOS activity by 75%, the colocalization coefficient C(green) value, calculated as Pearson's correlation, increased from 0.07 to 0.10, demonstrating an augmented presence of NO(*) in mitochondria. Conversely, the presence of NO(*) in mitochondria decreased following cells pretreatment with l-MonoMethylArginine (L-NMMA), a competitive inhibitor of NOS activity, as indicated by the reduction of the C(green) value to 0.02. This work confirms that the presence of NO(*) in mitochondria can be modulated in response to different fluxes of NO(*).     

Extent of over-expression of hepatocyte growth factor receptor in colorectal tumours is dependent on the choice of normaliser

For reliable results from quantitative RT-PCR, the starting quantity of total RNA and other parameters need to be controlled. Most studies do this by normalising their results to a single reference gene. This study quantified the mRNA expression of three putative reference genes (ubiquitin C, cyclophilin E, and porphobilinogen deaminase) and the target gene hepatocyte growth factor receptor (HGFR) in matched colorectal tumour and normal mucosa samples. Each of the putative reference genes was found to be significantly over-expressed in the tumour samples compared to the normal samples. When HGFR expression was normalised to each of these reference genes using the 2 (-DeltaDeltaC(T)) method of relative quantification, the number of tumour samples in which HGFR was found to be over-expressed varied from 30% to 63% depending on the reference gene chosen for normalisation. This shows that normalising to a single reference gene without prior validation is inappropriate.     

The cell membrane-shielding function of eicosapentaenoic acid for Escherichia coli against exogenously added hydrogen peroxide

The colony-forming ability of catalase-deficient Escherichia coli mutant genetically modified to produce eicosapentaenoic acid (EPA) showed less decrease than in a control strain producing no EPA, when treated with 0.3mM hydrogen peroxide (H(2)O(2)) under non-growth conditions. H(2)O(2)-induced protein carbonylation was enhanced in cells lacking EPA. The amount of fatty acids was decreased more significantly for cells lacking EPA than for those producing EPA. Much lower intracellular concentrations of H(2)O(2) were detected for cells with EPA than those lacking EPA. These results suggest that cellular EPA can directly protect cells against oxidative damage by shielding the entry of exogenously added H(2)O(2).     

Molecular cloning and functional expression of a sodium bicarbonate cotransporter from guinea-pig parotid glands

We recently found that the concentration of HCO3- in guinea-pig saliva is very similar to that of human saliva; however, the entity that regulates HCO3- transport has not yet been fully characterized. In order to investigate the mechanism of HCO3- transport, we identified, cloned, and characterized a sodium bicarbonate (Na(+)/HCO3- cotransporter found in guinea-pig parotid glands (gpNBC1). The gpNBC1 gene encodes a 1079-amino acid protein that has 95% and 96% homology with human and mouse parotid NBC1, respectively. Oocytes expressing gpNBC1 were exposed to HCO3- or Na(+)-free solutions, which resulted in a marked change in membrane potentials (V(m)), suggesting that gpNBC1 is electrogenic. Likewise, a gpNBC1-mediated pH recovery was observed in gpNBC1 transfected human hepatoma cells; however, in the presence of 4, 4-diisothiocyanostilbene-2,2-disulfonic acid, a specific NBC1 inhibitor, such changes in V(m) and pH(i) were not observed. Together, the data show that the cloned guinea-pig gene is a functional, as well as sequence homologue of human NBC1.     

Application of HN(C)N to rapid estimation of 1J(N-C(alpha)) coupling constants correlated to psi torsion angles in proteins: implication to structural genomics

We recently described a triple resonance experiment, HN(C)N, for sequential correlation of H(N) and 15N atoms in (15N, 13C) labeled proteins [J. Biomol. NMR. 20 (2001) 135]. Here, we describe an approach based on this experiment for estimation of one bond N-C(alpha) J-couplings in medium size labeled proteins, which seem to show good correlations with psi torsion angles along the protein backbone. The approach uses the ratio of the intensities of the sequential and diagonal peaks in the F(2)-F(3) planes of the HN(C)N spectrum. The reliability of the approach has been demonstrated using a short peptide wherein the coupling constants have been measured by the present method and also independently from peak splittings in HSQC spectra. The two results agree within 10%. The applicability of the procedure to proteins has been demonstrated using doubly labeled FK506 binding protein (FKBP, molecular mass approximately 12 kDa). Coupling constant estimates have been obtained for 62 out of 100 non-proline residues and they show a correlation with psi torsion angles, as has been reported before. This semi-quantitative application of HN(C)N extends the significance of the experiment especially, in the context of structural genomics, since the single experiment, not only provides a great enhancement in the speed of resonance assignment, but also provides quantitative structural information.     

Three-person game facilitates indirect reciprocity under image scoring

Reputation building plays an important role in the evolution of reciprocal altruism when the same individuals do not interact repeatedly because, by referring to reputation, a reciprocator can know which partners are cooperative and can reciprocate with a cooperator. This reciprocity based on reputation is called indirect reciprocity. Previous studies of indirect reciprocity have focused only on two-person games in which only two individuals participate in a single interaction, and have claimed that indirectly reciprocal cooperation cannot be established under image scoring reputation criterion where the reputation of an individual who has cooperated (defected) becomes good (bad). In this study, we specifically examine three-person games, and reveal that indirectly reciprocal cooperation can be formed and maintained stably, even under image scoring, by a nucleus shield mechanism. In the nucleus shield, reciprocators are a shield that keeps out unconditional defectors, whereas unconditional cooperators are the backbone of cooperation that retains a good reputation among the population.     

The evolution of n-player cooperation-threshold games and ESS bifurcations

An evolutionary game of individuals cooperating to obtain a collective benefit is here modelled as an n-player Prisoner's Dilemma game. With reference to biological situations, such as group foraging, we introduce a threshold condition in the number of cooperators required to obtain the collective benefit. In the simplest version, a three-player game, complex behaviour appears as the replicator dynamics exhibits a catastrophic event separating a parameter region allowing for coexistence of cooperators and defectors and a region of pure defection. Cooperation emerges through an ESS bifurcation, and cooperators only thrive beyond a critical point in cost-benefit space. Moreover, a repelling fixed point of the dynamics acts as a barrier to the introduction of cooperation in defecting populations. The results illustrate the qualitative difference between two-player games and multiple player games and thus the limitations to the generality of conclusions from two-player games. We present a procedure to find the evolutionarily stable strategies in any n-player game with cost and benefit depending on the number of cooperators. This was previously done by Motro [1991. Co-operation and defection: playing the field and the ESS. J. Theor. Biol. 151, 145-154] in the special cases of convex and concave benefit functions and constant cost.     

KCNE3 is an inhibitory subunit of the Kv4.3 potassium channel

The mammalian Kv4.3 potassium channel is a fast activating and inactivating K+ channel widely distributed in mammalian tissues. Kv4.3 is the major component of various physiologically important currents ranging from A-type currents in the CNS to the transient outward potassium conductance in the heart (I(to)). Here we show that the KCNE3 beta-subunit has a strong inhibitory effect on current conducted by heterologously expressed Kv4.3 channels. KCNE3 reduces the Kv4.3 current amplitude, and it slows down the channel activation and inactivation as well as the recovery from inactivation. KCNE3 also inhibits currents generated by Kv4.3 in complex with the accessory subunit KChIP2. We find the inhibitory effect of KCNE3 to be specific for Kv4.3 within the Kv4 channel family. Kv4.3 has previously been shown to interact with a number of beta-subunits, but none of the described subunit-interactions exert an inhibitory effect on the Kv4.3 current.