Circular dichroism spectra of DNA quadruplexes [d(G(5)T(5))](4) as formed with G(4) and T(4) tetrads and [d(G(5)T(5)). d(A(5)C(5))]2 as formed with Watson-Crick-like (G-C)(2) and (T-A)(2) tetrads

We utilize electrophoresis and find that a thermally treated equimolar mixture of the oligonucleotide d(G(5)T(5)) and its complementary oligonucleotide d(A(5)C(5)) exhibits either two bands or a single band in one lane, depending on the conditions of the incubation solutions. The thermally treated d(G(5)T(5)) solution loaded in a different lane exhibits a single band of the parallel quadruplex [d(G(5)T(5))](4), which is composed of homocyclic hydrogen-bonded G(4) and T(4) tetrads previously proposed. For the thermally treated equimolar mixture of d(G(5)T(5)) and d(A(5)C(5)), the fast band is assigned to a Watson-Crick d(G(5)T(5)). d(A(5)C(5)) duplex, so that the slow band with the same low mobility as that of [d(G(5)T(5))](4) may be assigned to either [d(G(5)T(5))](4) itself or a [d(G(5)T(5)). d(A(5)C(5))](2) quadruplex. If the latter compound is true, this may be the antiparallel quadruplex composed of the heterocyclic hydrogen-bonded G-C-G-C and T-A-T-A tetrads proposed previously. After removing these three bands for the duplex and two kinds of hypothetical quadruplexes, we electrophoretically elute the corresponding compounds in the same electrophoresis buffer using an electroeluter. The eluted compounds are ascertained to be stable by electrophoresis. The circular dichroism (CD) and UV absorption spectra measured for the three isolated compounds are found to be clearly different. For the electrophoretic elution of the hypothetical [d(G(5)T(5))](4) quadruplex, the result of the molecularity of n = 4 obtained from the CD melting curve analysis provides further support for the formation of the parallel [d(G(5)T(5))](4) quadruplex already proposed. For the thermally treated equimolar mixture of d(G(5)T(5)) and d(C(5)A(5)), the fast band with a molecularity of n = 2 corresponds to the Watson-Crick duplex, d(G(5)T(5)). d(A(5)C(5)). The slow band with a molecularity of n = 4 indicates the antiparallel quadruplex [d(G(5)T(5)). d(A(5)C(5))](2), whose observed CD and UV spectra are different from those of [d(G(5)T(5))](4). By electrophoresis, after reannealing the eluted compound [d(G(5)T(5)). d(A(5)C(5))](2), a distinct photograph showing the band splitting of this quadruplex band into the lower duplex and upper quadruplex bands is not possible; but by a transilluminator, we occasionally observe this band splitting with the naked eye. The linear response polarizability tensor calculations for the thus determined structures of the [d(G(5)T(5))](4) quadruplex, the McGavin-like [d(G(5)T(5)). d(A(5)C(5))](2) quadruplex, and the Watson-Crick d(G(5)T(5)). d(A(5)C(5)) duplex are found to qualitatively predict the observed CD and UV spectra.     

Variation in 16S-23S rRNA intergenic spacer regions in Photobacterium damselae: a mosaic-like structure

Phenotypically, Photobacterium damselae subsp. piscicida and P. damselae subsp. damselae are easily distinguished. However, their 16S rRNA gene sequences are identical, and attempts to discriminate these two subspecies by molecular tools are hampered by their high level of DNA-DNA similarity. The 16S-23S rRNA internal transcribed spacers (ITS) were sequenced in two strains of Photobacterium damselae subsp. piscicida and two strains of P. damselae subsp. damselae to determine the level of molecular diversity in this DNA region. A total of 17 different ITS variants, ranging from 803 to 296 bp were found, some of which were subspecies or strain specific. The largest ITS contained four tRNA genes (tDNAs) coding for tRNA(Glu(UUC)), tRNA(Lys(UUU)), tRNA(Val(UAC)), and tRNA(Ala(GGC)). Five amplicons contained tRNA(Glu(UUC)) combined with two additional tRNA genes, including tRNA(Lys(UUU)), tRNA(Val(UAC)), or tRNA(Ala(UGC)). Five amplicons contained tRNA(Ile(GAU)) and tRNA(Ala(UGC)). Two amplicons contained tRNA(Glu(UUC)) and tRNA(Ala(UGC)). Two different isoacceptor tRNA(Ala) genes (GGC and UGC anticodons) were found. The five smallest amplicons contained no tRNA genes. The tRNA-gene combinations tRNA(Glu(UUC))-tRNA(Val(UAC))-tRNA(Ala(UGC)) and tRNA(Glu(UUC))-tRNA(Ala(UGC)) have not been previously reported in bacterial ITS regions. The number of copies of the ribosomal operon (rrn) in the P. damselae chromosome ranged from at least 9 to 12. For ITS variants coexisting in two strains of different subspecies or in strains of the same subspecies, nucleotide substitution percentages ranged from 0 to 2%. The main source of variation between ITS variants was due to different combinations of DNA sequence blocks, constituting a mosaic-like structure.     

Mechanism of arsenate resistance in the ericoid mycorrhizal fungus Hymenoscyphus ericae

Arsenate resistance is exhibited by the ericoid mycorrhizal fungus Hymenoscyphus ericae collected from As-contaminated mine soils. To investigate the mechanism of arsenate resistance, uptake kinetics for arsenate (H(2)AsO(4)(-)), arsenite (H(3)AsO(3)), and phosphate (H(2)PO(4)(-)) were determined in both arsenate-resistant and -non-resistant H. ericae. The uptake kinetics of H(2)AsO(4)(-), H(3)AsO(3), and H(2)PO(4)(-) in both resistant and non-resistant isolates were similar. The presence of 5.0 microM H(2)PO(4)(-) repressed uptake of H(2)AsO(4)(-) and exposure to 0.75 mM H(2)AsO(4)(-) repressed H(2)PO(4)(-) uptake in both H. ericae. Mine site H. ericae demonstrated an enhanced As efflux mechanism in comparison with non-resistant H. ericae and lost approximately 90% of preloaded cellular As (1-h uptake of 0.22 micromol g(-1) dry weight h(-1) H(2)AsO(4)(-)) over a 5-h period in comparison with non-resistant H. ericae, which lost 40% of their total absorbed H(2)AsO(4)(-). As lost from the fungal tissue was in the form of H(3)AsO(3). The results of the present study demonstrate an enhanced H(3)AsO(3) efflux system operating in mine site H. ericae as a mechanism for H(2)AsO(4)(-) resistance. The ecological significance of this mechanism of arsenate resistance is discussed.     

Substrate-specific interactions with the heme-bound oxygen molecule of nitric-oxide synthase

We report the characterization by resonance Raman spectroscopy of the oxygenated complex (Fe(II)O(2)) of nitric-oxide synthases of Staphylococcus aureus (saNOS) and Bacillus subtilis (bsNOS) saturated with N(omega)-hydroxy-l-arginine. The frequencies of the nu(Fe-O) and nu(O-O) modes were 530 and 1135 cm(-), respectively, in both the presence and absence of tetrahydrobiopterin. On the basis of a comparison of these frequencies with those of saNOS and bsNOS saturated with l-arginine (nu(Fe-O) at 517 cm(-1) and nu(O-O) at 1123 cm(-1)) and those of substrate-free saNOS (nu(Fe-O) at 517 and nu(O-O) at 1135 cm(-1)) (Chartier, F. J. M., Blais, S. P., and Couture, M. (2006) J. Biol. Chem. 281, 9953-9962), we propose two models that account for the frequency shift of nu(Fe-O) (but not nu(O-O)) upon N(omega)-hydroxy-l-arginine binding as well as the frequency shift of nu(O-O) (but not nu(Fe-O)) upon l-arginine binding. The implications of these substrate-specific interactions with respect to catalysis by NOSs are discussed.     

Reconstitution of vacuolar-type rotary H+-ATPase/synthase from Thermus thermophilus

Vacuolar-type rotary H(+)-ATPase/synthase (V(o)V(1)) from Thermus thermophilus, composed of nine subunits, A, B, D, F, C, E, G, I, and L, has been reconstituted from individually isolated V(1) (A(3)B(3)D(1)F(1)) and V(o) (C(1)E(2)G(2)I(1)L(12)) subcomplexes in vitro. A(3)B(3)D and A(3)B(3) also reconstituted with V(o), resulting in a holoenzyme-like complexes. However, A(3)B(3)D-V(o) and A(3)B(3)-V(o) did not show ATP synthesis and dicyclohexylcarbodiimide-sensitive ATPase activity. The reconstitution process was monitored in real time by fluorescence resonance energy transfer (FRET) between an acceptor dye attached to subunit F or D in V(1) or A(3)B(3)D and a donor dye attached to subunit C in V(o). The estimated dissociation constants K(d) for V(o)V(1) and A(3)B(3)D-V(o) were ～0.3 and ～1 nm at 25 °C, respectively. These results suggest that the A(3)B(3) domain tightly associated with the two EG peripheral stalks of V(o), even in the absence of the central shaft subunits. In addition, F subunit is essential for coupling of ATP hydrolysis and proton translocation and has a key role in the stability of whole complex. However, the contribution of the F subunit to the association of A(3)B(3) with V(o) is much lower than that of the EG peripheral stalks.     

Chemical and biological characterization of technetium(I) and Rhenium(I) tricarbonyl complexes with dithioether ligands serving as linkers for coupling the Tc(CO)(3) and Re(CO)(3) moieties to biologically active molecules

The organometallic precursor (NEt(4))(2)[ReBr(3)(CO)(3)] was reacted with bidendate dithioethers (L) of the general formula H(3)C-S-CH(2)CH(2)-S-R (R = -CH(2)CH(2)COOH, CH(2)-C&tbd1;CH) and R'-S-CH(2)CH(2)-S-R' (R' = CH(3)CH(2)-, CH(3)CH(2)-OH, and CH(2)COOH) in methanol to form stable rhenium(I) tricarbonyl complexes of the general composition [ReBr(CO)(3)L]. Under these conditions, the functional groups do not participate in the coordination. As a prototypic representative of this type of Re compounds, the propargylic group bearing complex [ReBr(CO(3))(H(3)C-S-CH(2)CH(2)-S-CH(2)C&tbd1;CH)] Re2 was studied by X-ray diffraction analysis. Its molecular structure exhibits a slightly distorted octahedron with facial coordination of the carbonyl ligands. The potentially tetradentate ligand HO-CH(2)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(2)-OH was reacted with the trinitrato precursor [Re(NO(3))(3)(CO)(3)](2-) to yield a cationic complex [Re(CO)(3)(HO-CH(2)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(2)-OH)]NO(3) Re8 which shows the coordination of one hydroxy group. Re8 has been characterized by correct elemental analysis, infrared spectroscopy, capillary electrophoresis, and X-ray diffraction analysis. Ligand exchange reaction of the carboxylic group bearing ligands H(3)C-S-CH(2)CH(2)-S-CH(2)CH(2)-COOH and HOOC-CH(2)-S-CH(2)CH(2)-S-CH(2)-COOH with (NEt(4))(2)[ReBr(3)(CO)(3)] in water and with equimolar amounts of NaOH led to complexes in which the bromide is replaced by the carboxylic group. The X-ray structure analysis of the complex [Re(CO)(3)(OOC-CH(2)-S-CH(2)CH(2)-S-CH(2)-COOH)] Re6 shows the second carboxylic group noncoordinated offering an ideal site for functionalization or coupling a biomolecule. The no-carrier-added preparation of the analogous (99m)Tc(I) carbonyl thioether complexes could be performed using the precursor fac-[(99m)Tc(H(2)O)(3)(CO)(3)](+), with yields up to 90%. The behavior of the chlorine containing (99m)Tc complex [(99m)TcCl(CO)(3)(CH(3)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(3))] Tc1 in aqueous solution at physiological pH value was investigated. In saline, the chromatographically separated compound was stable for at least 120 min. However, in chloride-free aqueous solution, a water-coordinated cationic species Tc1a of the proposed composition [(99m)Tc(H(2)O)(CO)(3)(CH(3)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(3))](+) occurred. The cationic charge of the conversion product was confirmed by capillary electrophoresis. By the introduction of a carboxylic group into the thioether ligand as a third donor group, the conversion could be suppressed and thus the neutrality of the complex preserved. Biodistribution studies in the rat demonstrated for the neutral complexes [(99m)TcCl(CO)(3)(CH(3)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(3))] Tc1 and [(99m)TcCl(CO)(3)(CH(2)-S-CH(2)CH(2)-S-CH(2)-C&tbd1;CH)] Tc2 a significant initial brain uptake (1.03 +/- 0.25% and 0.78 +/- 0.08% ID/organ at 5 min. p.i.). Challenge experiments with glutathione clearly indicated that no transchelation reaction occurs in vivo.     

Sulfide oxidation at halo-alkaline conditions in a fed-batch bioreactor

A biotechnological process is described to remove hydrogen sulfide (H(2)S) from high-pressure natural gas and sour gases produced in the petrochemical industry. The process operates at halo-alkaline conditions and combines an aerobic sulfide-oxidizing reactor with an anaerobic sulfate (SO(4) (2-)) and thiosulfate (S(2)O(3) (2-)) reducing reactor. The feasibility of biological H(2)S oxidation at pH around 10 and total sodium concentration of 2 mol L(-1) was studied in gas-lift bioreactors, using halo-alkaliphilic sulfur-oxidizing bacteria (HA-SOB). Reactor operation at different oxygen to sulfide (O(2):H(2)S) supply ratios resulted in a stable low redox potential that was directly related with the polysulfide (S(x) (2-)) and total sulfide concentration in the bioreactor. Selectivity for SO(4) (2-) formation decreased with increasing S(x) (2-) and total sulfide concentrations. At total sulfide concentrations above 0.25 mmol L(-1), selectivity for SO(4) (2-) formation approached zero and the end products of H(2)S oxidation were elemental sulfur (S(0)) and S(2)O(3) (2-). Maximum selectivity for S(0) formation (83.3+/-0.7%) during stable reactor operation was obtained at a molar O(2):H(2)S supply ratio of 0.65. Under these conditions, intermediary S(x) (2-) plays a major role in the process. Instead of dissolved sulfide (HS(-)), S(x) (2-) seemed to be the most important electron donor for HA-SOB under S(0) producing conditions. In addition, abiotic oxidation of S(x) (2-) was the main cause of undesirable formation of S(2)O(3) (2-). The observed biomass growth yield under SO(4) (2-) producing conditions was 0.86 g N mol(-1) H(2)S. When selectivity for SO(4) (2-) formation was below 5%, almost no biomass growth was observed.     

Integrating two physiological approaches helps relate respiration to growth of Pinus radiata

Correlation methods originating in the growth and maintenance paradigm (GMP) are traditionally used to calculate a 'growth coefficient' (g) or the 'growth potential' (1/g) of entire plants. The enthalpy balance approach is usually applied to plant organs and relies on determination of both CO(2) release and O(2) reduction to provide a measure of instantaneous rates of enthalpic growth (R(SG)DeltaH(B)). Aspects of both the approaches to explore physiological mechanisms that govern enthalpic growth (variation in rates of CO(2) release versus rates of O(2) reduction) were combined. Respiration and growth rates of apical buds of Pinus radiata were affected strongly by canopy position, and moderately by branching order. A linear relation between enthalpic growth and CO(2) respiration explained 69% of the observed variation. Despite faster rates of growth, enthalpic growth potential (1/g(H)) was comparatively low in the upper canopy. Low enthalpic growth potential entailed comparatively low enthalpy conversion efficiency (eta(H), ratio of R(SG)DeltaH(B) to R(CO(2)) DeltaH(CO(2)); proportional to CO(2):O(2) and to carbon conversion efficiency epsilon) at large R(SG)DeltaH(B). Maximizing enthalpic growth requires a large capacity for O(2) reduction. Relations between R(SG)DeltaH(B) and eta(H) could be described by hyperbolae using two parameters. One parameter, P(1), is equivalent to enthalpic growth potential (1/g(H)).     

Transcranial Doppler estimation of cerebral blood flow and cerebrovascular conductance during modified rebreathing.

Clinical transcranial Doppler assessment of cerebral vasomotor reactivity (CVMR) uses linear regression of cerebral blood flow velocity (CBFV) vs. end-tidal CO(2) (Pet(CO(2))) under steady-state conditions. However, the cerebral blood flow (CBF)-Pet(CO(2)) relationship is nonlinear, even for moderate changes in CO(2). Moreover, CBF is increased by increases in arterial blood pressure (ABP) during hypercapnia. We used a modified rebreathing protocol to estimate CVMR during transient breath-by-breath changes in CBFV and Pet(CO(2)). Ten healthy subjects (6 men) performed 15 s of hyperventilation followed by 5 min of rebreathing, with supplemental O(2) to maintain arterial oxygen saturation constant. To minimize effects of changes in ABP on CVMR estimation, cerebrovascular conductance index (CVCi) was calculated. CBFV-Pet(CO(2)) and CVCi-Pet(CO(2)) relationships were quantified by both linear and nonlinear logistic regression. In three subjects, muscle sympathetic nerve activity was recorded. From hyperventilation to rebreathing, robust changes occurred in Pet(CO(2)) (20-61 Torr), CBFV (-44 to +104% of baseline), CVCi (-39 to +64%), and ABP (-19 to +23%) (all P < 0.01). Muscle sympathetic nerve activity increased by 446% during hypercapnia. The linear regression slope of CVCi vs. Pet(CO(2)) was less steep than that of CBFV (3 vs. 5%/Torr; P = 0.01). Logistic regression of CBF-Pet(CO(2)) (r(2) = 0.97) and CVCi-Pet(CO(2)) (r(2) = 0.93) was superior to linear regression (r(2) = 0.91, r(2) = 0.85; P = 0.01). CVMR was maximal (6-8%/Torr) for Pet(CO(2)) of 40-50 Torr. In conclusion, CBFV and CVCi responses to transient changes in Pet(CO(2)) can be described by a nonlinear logistic function, indicating that CVMR estimation varies within the range from hypocapnia to hypercapnia. Furthermore, quantification of the CVCi-Pet(CO(2)) relationship may minimize the effects of changes in ABP on the estimation of CVMR. The method developed provides insight into CVMR under transient breath-by-breath changes in CO(2).     

Light-harvesting function of carotenoids in photo-synthesis: the roles of the newly found 1(1)Bu- state

This minireview article highlights the energetics and the dynamics of the 1(1)B(u)(-) and 3(1)A(g)(-) states of carotenoids discovered very recently. Those "hidden" covalent states have been revealed by measurements of resonance-Raman excitation profiles of crystalline carotenoids. The dependence of the energies of the low-lying singlet states, including the 1(1)B(u)(+), 3(1)A(g)(-), 1(1)B(u)(-), and 2(1)A(g)(-) states, on the number of conjugated double bonds (n) is in agreement with the extrapolation of those state energies calculated by Tavan and Schulten for shorter polyenes (P. Tavan and K. Schulten, Journal of Chemical Physics, 1986, vol. 85, pp. 6602-6609). It has also been shown that the internal-conversion processes among those singlet states take place in accord with the state ordering, i.e., 1(1)B(u)(+) --> 1(1)B(u)(-) --> 2(1)A(g)(-) --> 1(1)A(g)(-) (the ground state) for carotenoids having n = 9 and 10, whereas 1(1)B(u)(+) --> 3(1)A(g)(-) --> 1(1)B(u) (-) --> 2(1)A(g)(-) --> 1(1)A(g)(-) for carotenoids having n = 11-13. Radiative transitions of 1(1)B(u)(+) --> 2(1)A(g)(-) and 1(1)B(u)(-) --> 2(1)A(g)(-) as well as a branching into the triplet manifold of 1(1)B(u)(-) --> 1(3)A(g) --> 1(3)B(u) have also been found. Those low-lying singlet states of all-trans carotenoids can facilitate multiple channels of singlet-energy transfer to bacteriochlorophyll in the LH2 antenna complexes of purple photosynthetic bacteria. Thus, the newly found 1(1)B(u)(-) and 3(1)A(g)(-) states of carotenoids need to be incorporated into the picture of carotenoid-to-bacteriochlorophyll singlet-energy transfer.     

Studies on Chemical Constituents of Essential Oil from Leaves of Jiang-Zhang

Jiang-Zhang is a Physiological type of Cinnamomum porrectum (Roxb.) Kosterm. The essential oil of leaves of Jiang-Zhang can be extracted by steam distillation, with yields of 0.5–0.8%. It contained dtral (α-,β-citral) 64.11% and can be used in aromatic and medicinal industries. We used the methods of GC, IR, GC/MS/DS and prepared derivative and 47 chemical constituents were identified, as follows: α-thujene (0.06 %), α-pinene (2.42%), camphene (1.26%), sabinene (0.21%),β- pinene (1.38%) myrcene(0.38%), α-phellandrene (0.32%),△3-carene (0.01%), p-cymene(0.21%),α-limonene (1.57%), 1 8-cineole (0.82 % ), β-phellandrene (0.10 %), cis-linalool oxide(0.07 % ), linalool (8.43 % ) epicamphor(0.26 % ), camphor(1.10 %), borneol( 1.07 % ), β-citral (neral) (28.28 % ), geraniol (0.25%), nerol (0.47%), α-citral (geranial) (35.83%), methyl citronellate (0.12%), n-undecane (0.18), safrole (0.02), methyl geranate (0.23%), geranyl formate (0.09%), α-copaene (0.12%), trans-methyl cinnamate (0.02%), n-dodecane (0.20), β-elemene (0.12%), caryophyllene (4.67%), α-guaiene (0.04), β-guaiene (0.06%), β-selinene (0.97%), azulene (0.38%), β-cubebene (0.30%), n-pentadecane (0.05%), β-gurjunene (0.04 %), epi-β-santalene (0.46 %), aremophliene (0.05 % ), alloaromadendrene (0.03 % ), α-elemene (0.26%), trans-β-farnesene (0.04%), (z)-β-farnesene (0.30%), r-elemene (0.03%), β-bisaboiol(0.41%), cedrol (0.16%), respectively.     

Capacitance and resistance of the bilayer lipid membrane formed of phosphatidylcholine and cholesterol

Capacity and electric resistance of lipid membranes composed of lecithin and cholesterol were determined. The components were chosen for the study because they were present in biological membranes. Capacitance of the lecithin and cholesterol membranes amounts to 0.38 and 0.61 microF/cm(2), and resistance to 1.44(10(4)and 2.12(10(6)Omega cm(2), respectively. A 1:1 complex appears as a result of lecithin-cholesterol membrane formation. Parameters of the membrane formed of the lecithin-cholesterol complex were determined: surface concentration (Gamma(3)), capacitance (C(3)), and conductance (R;(3)(-1), as well as the stability constant (K) of the complex. The mean values of those magnitudes are as follows: 4.265(10(-6)mol/m(2), 0.54 microF/cm(2), 1.381(10(-6)Omega(-1)cm(-2)and 3.748(10(7), respectively.     

Biochemical and biophysical evidence for gamma 2 subunit association with neuronal voltage-activated Ca2+ channels.

A novel gene (Cacng2; gamma(2)) encoding a protein similar to the voltage-activated Ca(2+) channel gamma(1) subunit was identified as the defective gene in the epileptic and ataxic mouse, stargazer. In this study, we analyzed the association of this novel neuronal gamma(2) subunit with Ca(2+) channels of rabbit brain, and the function of the gamma(2) subunit in recombinant neuronal Ca(2+) channels expressed in Xenopus oocytes. Our results showed that the gamma(2) subunit and a closely related protein (called gamma(3)) co-sedimented and co-immunoprecipitated with neuronal Ca(2+) channel subunits in vivo. Electrophysiological analyses showed that gamma(2) co-expression caused a significant decrease in the current amplitude of both alpha(1B)(alpha(1)2.2)-class (36.8%) and alpha(1A)(alpha(1)2.1)-class (39.7%) Ca(2+) channels (alpha(1)beta(3)alpha(2)delta). Interestingly, the inhibitory effects of the gamma(2) subunit on current amplitude were dependent on the co-expression of the alpha(2)delta subunit. In addition, co-expression of gamma(2) or gamma(1) also significantly decelerates the activation kinetics of alpha(1B)-class Ca(2+) channels. Taken together, these results suggest that the gamma(2) subunit is an important constituent of the neuronal Ca(2+) channel complex and that it down-regulates neuronal Ca(2+) channel activity. Furthermore, the gamma(2) subunit likely contributes to the fine-tuning of neuronal Ca(2+) channels by counterbalancing the effects of the alpha(2)delta subunit.     

Hydroperoxide-dependent epoxidation of unsaturated fatty acids in the broad bean (Vicia faba L.)

Incubation of linoleic acid with the 105,000g particle fraction of the homogenate of the broad bean (Vicia faba L.) led to the formation of the following products: 13(S)-hydroxy-9(Z),11(E)-octadecadienoic acid, 9,10-epoxy-12(Z)-octadecenoic acid (9(R),10(S)/9(S)/10(R), 80/20), 12,13-epoxy-9(Z)-octadecenoic acid (12(S),13(R)/12(R)/13(S), 64/36), and 9,10-epoxy-13(S)-hydroxy-11(E)-octadecenoic acid (9(S),10(R)/9(R),10(S), 91/9). Oleic acid incubated with the enzyme preparation in the presence of 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid or cumene hydroperoxide was converted into 9,10-epoxyoctadecanoic acid (9(R),10(S)/9(S),10(R), 79/21). Two enzyme activities were involved in the formation of the products, an omega 6-lipoxygenase and a hydroperoxide-dependent epoxygenase. The lipoxygenase, but not the epoxygenase, was inhibited by low concentrations of 5,8,11,14-eicosatetraynoic acid and nordihydroguaiaretic acid. In contrast, the epoxygenase, but not the lipoxygenase, was readily inactivated in the presence of 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid. Studies with 18O2-labeled 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid showed that the epoxide oxygens of 9,10-epoxyoctadecanoic acid and of 9,10-epoxy-13(S)-hydroxy-11(E)-octadecenoic acid were derived from hydroperoxide and not from molecular oxygen.     

Glycogen synthase kinase 3beta phosphorylates Alzheimer's disease-specific Ser396 of microtubule-associated protein tau by a sequential mechanism

Phosphorylation of tau on S(396) was suggested to be a key step in the development of neurofibrillary pathology in Alzheimer's disease brain [Bramblett, G. T., Goedert, M., Jacks, R., Merrick, S. E., Trojanowski, J. Q., and Lee, V. M.-Y. (1993) Neuron 10, 1089-1099]. GSK3beta phosphorylates Ser(396) of tau in the brain by a mechanism which is not clear. In this study, when HEK-293 cells were cotransfected with tau and GSK3beta, GSK3beta co-immunoprecipitated with tau and phosphorylated tau on S(202), T(231), S(396), and S(400) but not on S(262), S(235), and S(404). Blocking phosphorylation on T(231), S(235), S(396), S(400), or S(404) did not prevent the subsequent phosphorylation on S(202) by GSK3beta. These data suggest that GSK3beta directly phosphorylates tau on S(202) (without requiring prephosphorylation). However, preventing phosphorylation on S(235), S(400), and S(404) prevented GSK3beta-dependent phosphorylation of T(231), S(396), and S(400), respectively. This indicates that phosphorylation of T(231), S(396), and S(400) by GSK3beta depends on a previous phosphorylation of S(235), S(400), and S(404), respectively. To examine S(396) phosphorylation, we analyzed phosphorylation of S(396), S(400), and S(404). Blocking phosphorylation of S(404) prevented the subsequent GSK3beta-dependent phosphorylation of both S(400) and S(396). When phosphorylation of S(404) was allowed but S(400) blocked, GSK3beta failed to phosphorylate S(396). Thus, GSK3beta phosphorylates S(396) by a two-step mechanism. In the first step, GSK3beta phosphorylates S(400) of previously S(404)-phosphorylated tau. This event primes tau for second-step phosphorylation of S(396) by GSK3beta. We conclude that GSK3beta phosphorylates tau directly at S(202) but requires the previous phosphorylation on S(235) to phosphorylate T(231). Phosphorylation of S(396), on the other hand, occurs sequentially. Once a priming kinase phosphorylates S(404), GSK3beta sequentially phosphorylates S(400) and then S(396).     

End-stacking of copper cationic porphyrins on parallel-stranded guanine quadruplexes

Nucleic acids that contain multiple sequential guanines assemble into guanine quadruplexes (G-quadruplexes). Drugs that induce or stabilize G-quadruplexes are of interest because of their potential use as therapeutics. Previously, we reported on the interaction of the Cu(2+) derivative of 5,10,15,20-tetrakis(1-methyl-4-pyridyl)-21H,23H-porphine (CuTMpyP4), with the parallel-stranded G-quadruplexes formed by d(T(4)G( n )T(4)) (n = 4 or 8) (Keating and Szalai in Biochemistry 43:15891-15900, 2004). Here we present further characterization of this system using a series of guanine-rich oligonucleotides: d(T(4)G( n )T(4)) (n = 5-10). Absorption titrations of CuTMpyP4 with all d(T(4)G( n )G(4)) quadruplexes produce approximately the same bathochromicity (8.3 +/- 2 nm) and hypochromicity (46.2-48.6%) of the porphyrin Soret band. Induced emission spectra of CuTMpyP4 with d(T(4)G( n )T(4))(4) quadruplexes indicate that the porphyrin is protected from solvent. Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry revealed a maximum porphyrin to quadruplex stoichiometry of 2:1 for the shortest (n = 4) and longest (n = 10) quadruplexes. Electron paramagnetic resonance spectroscopy shows that bound CuTMpyP4 occupies magnetically noninteracting sites on the quadruplexes. Consistent with our previous model for d(T(4)G(4)T(4)), we propose that two CuTMpyP4 molecules are externally stacked at each end of the run of guanines in all d(T(4)G( n )T(4)) (n = 4-10) quadruplexes.     

Capacity for Methane Oxidation in Landfill Cover Soils Measured in Laboratory-Scale Soil Microcosms

Laboratory-scale soil microcosms containing different soils were permeated with CH(inf4) for up to 6 months to investigate their capacity to develop a methanotrophic community. Methane emissions were monitored continuously until steady states were established. The porous, coarse sand soil developed the greatest methanotrophic capacity (10.4 mol of CH(inf4) (middot) m(sup-2) (middot) day(sup-1)), the greatest yet reported in the literature. Vertical profiles of O(inf2), CH(inf4), and methanotrophic potential in the soils were determined at steady state. Methane oxidation potentials were greatest where the vertical profiles of O(inf2) and CH(inf4) overlapped. A significant increase in the organic matter content of the soil, presumably derived from methanotroph biomass, occurred where CH(inf4) oxidation was greatest. Methane oxidation kinetics showed that a soil community with a low methanotrophic capacity (V(infmax) of 258 nmol (middot) g of soil(sup-1) (middot) h(sup-1)) but relatively high affinity (k(infapp) of 1.6 (mu)M) remained in N(inf2)-purged control microcosms, even after 6 months without CH(inf4). We attribute this to a facultative, possibly mixotrophic, methanotrophic microbial community. When purged with CH(inf4), a different methanotrophic community developed which had a lower affinity (k(infapp) of 31.7 (mu)M) for CH(inf4) but a greater capacity (V(infmax) of 998 nmol (middot) g of soil(sup-1) (middot) h(sup-1)) for CH(inf4) oxidation, reflecting the enrichment of an active high-capacity methanotrophic community. Compared with the unamended control soil, amendment of the coarse sand with sewage sludge enhanced CH(inf4) oxidation capacity by 26%; K(inf2)HPO(inf4) amendment had no significant effect, while amendment with NH(inf4)NO(inf3) reduced the CH(inf4) oxidation capacity by 64%. In vitro experiments suggested that NH(inf4)NO(inf3) additions (10 and 71 (mu)mol (middot) g of soil(sup-1)) inhibited CH(inf4) oxidation by a nonspecific ionic effect rather than by specific inhibition by NH(inf4)(sup+).     

Effect of arterial oxygenation on quadriceps fatigability during isolated muscle exercise

The effect of various levels of oxygenation on quadriceps muscle fatigability during isolated muscle exercise was assessed in six male subjects. Twitch force (Q(tw)) was assessed using supramaximal magnetic femoral nerve stimulation. In experiment 1, maximal voluntary contraction (MVC) and Q(tw) of resting quadriceps muscle were measured in normoxia [inspired O(2) fraction (Fi(O(2))) = 0.21, percent arterial O(2) saturation (Sp(O(2))) = 98.4%, estimated arterial O(2) content (Ca(O(2))) = 20.8 ml/dl], acute hypoxia (Fi(O(2)) = 0.11, Sp(O(2)) = 74.6%, Ca(O(2)) = 15.7 ml/dl), and acute hyperoxia (Fi(O(2)) = 1.0, Sp(O(2)) = 100%, Ca(O(2)) = 22.6 ml/dl). No significant differences were found for MVC and Q(tw) among the three Fi(O(2)) levels. In experiment 2, the subjects performed three sets of nine, intermittent, isometric, unilateral, submaximal quadriceps contractions (62% MVC followed by 1 MVC in each set) while breathing each Fi(O(2)). Q(tw) was assessed before and after exercise, and myoelectrical activity of the vastus lateralis was obtained during exercise. The percent reduction of twitch force (potentiated Q(tw)) in hypoxia (-27.0%) was significantly (P < 0.05) greater than in normoxia (-21.4%) and hyperoxia (-19.9%), as were the changes in intratwitch measures of contractile properties. The increase in integrated electromyogram over the course of the nine contractions in hypoxia (15.4%) was higher (P < 0.05) than in normoxia (7.2%) or hyperoxia (6.7%). These results demonstrate that quadriceps muscle fatigability during isolated muscle exercise is exacerbated in acute hypoxia, and these effects are independent of the relative exercise intensity.