Alteration of cyanobacterial glutamine synthetase activity in vivo in response to light and NH 4 +

Extractable glutamine synthetase activity of the cyanobacterium Anabaena cylindrica was reduced by approximately 50% when N₂-fixing cultures were treated with 10 mM NH ₄ ⁺ or were placed in darkness. The deactivated enzyme could be rapidly reactivated (within 5 min) by adding 40 mM 2-mercaptoethanol to the biosynthetic reaction mixture. The enzyme could also be reactivated in vivo by replacing the culture in light or by removing NH ₄ ⁺ . When the enzyme was deactivated by simultaneously adding NH ₄ ⁺ and placing the culture in darkness, reactivation occurred on reillumination and removal of NH ₄ ⁺ . The removal of NH ₄ ⁺ in darkness did not result in reactivation. On in vitro reactivation of glutamine synthetase from dark or NH ₄ ⁺ -treated cultures the maximum glutamine synthetase activity observed frequently exceeded that of glutamine synthetase extracted from untreated cultures. Anacystis nidulans showed a similar type of reversible dark deactivation to A. cylindrica but Plectonema boryanum and a Nostoc did not. With A. cylindrica, a direct positive correlation between the size of the intracellular pool of glutamate and biosynthetic glutamine synthetase activity occurred during light/dark shifts, and on treatment with NH ₄ ⁺ . The changes in activity of glutamine synthetase in A. cylindrica in response to light resemble in some respects the light modulation of enzymes of the oxidative and reductive pentose phosphate pathways noted in cyanobacteria by others.     

Expression of the mosquitocidal cryIVB gene under the control of different promoters in Bacillus sphaericus 2362 and acrystalliferous Bacillus thuringiensis subsp. israelensis c4Q2-72

The 3.7 kb XbaI fragment harbouring the cryIVB gene which encoded a 130 kDa mosquitocidal toxin protein from Bacillus thuringiensis subsp. israelensis (B.t.i.) was placed downstream to the cat-86 gene promoter (P _cat-86, spore stage specific expression) or bgaB gene promoter (P _bgaB , vegetative stage specific expression). The constructs were subcloned into pBC16 to obtain pBTC3 and pBTC6, respectively. Both plasmids and the other construct, pBTC1 were successfully transferred into B. thuringiensis subsp. israelensis c4Q2-72 and B. sphaericus 2362. Western blot analysis showed that P _bgaB in front of P _cryIVB could enable cells to produce a 130 kDa protein from the vegetative stage (4 h) whereas those with P _cat-86 could not. The positive detection of 130 kDa crystal protein during the vegetative stage (4 h) by Western blot analysis indicated the vegetative-stage-specific expression of P _bgaB , while the 130 kDa crystal protein produced from cryIVB gene under control of P _cat-86 was detected only at 48 h. The strong activity of P _bgaB , together with P _cryIVB within pBTC6 in both bacterial hosts was also shown by the toxicity assay against Aedes aegypti larvae (B.t.i. c4Q2-72, 5.6 ± 3.6 × 10² c.f.u./ml; B. sphaericus 2362, 5.4 ± 2.5 × 10² c.f.u./ml) which were 100-fold and 10-fold more toxic to such larvae when compared with pBTC3 (P _cat-86 together with P _cryIVB ) and pBTC1 (contained only its self promoter) in the same bacterial host strains, respectively. The plasmid pBTC6 is not stable in either Bacillus host.     

Purification of cytochrome a 1 c 1 from Nitrobacter agilis and characterization of nitrite oxidation system of the bacterium

Cytochrome a ₁ c ₁ was highly purified from Nitrobacter agilis. The cytochrome contained heme a and heme c of equimolar amount, and its reduced form showed absorption peaks at 587, 550, 521, 434 and 416 nm. Molecular weight per heme a of the cytochrome was estimated to be approx. 100,000–130,000 from the amino acid composition. A similar value was obtained by determining the protein content per heme a. The cytochrome molecule was composed of three subunits with molecular weights of 55,000, 29,000 and 19,000, respectively. The 29 kd subunit had heme c.Hemes a and c of cytochrome a ₁ c ₁ were reduced on addition of nitrite, and the reduced cytochrome was hardly autoxidizable. Exogenously added horse heart cytochrome c was reduced by nitrite in the presence of cytochrome a ₁ c ₁; K _m values of cytochrome a ₁ c ₁ for nitrite and N. agilis cytochrome c were 0.5 mM and and 6 M, respectively. V _max was 1.7 mol ferricytochrome c reduced/min·mol of cytochrome a ₁ c ₁ The pH optimum of the reaction was about 8. The nitrite-cytochrome c reduction catalyzed by cytochrome a ₁ c ₁ was 61% and 88% inhibited by 44M azide and cyanide, respectively. In the presence of 4.4 mM nitrate, the reaction was 89% inhibited. The nitrite-cytochrome c reduction catalysed by cytochrome a ₁ c ₁ was 2.5-fold stimulated by 4.5 mM manganous chloride. An activating factor which was present in the crude enzyme preparation stimulated the reaction by 2.8-fold, and presence of both the factor and manganous ion activated the reaction by 7-fold.Cytochrome a ₁ c ₁ showed also cytochrome c-nitrate reductase activity. The pH optimum of the reaction was about 6. The nitrate reductase activity was also stimulated by manganous ions and the activating factor.     

Interactions between carbon dioxide and oxygen in the photosynthesis of three species of marine red macroalgae

Summary

Red algae have the highest known selectivity factor (S_rel) for CO₂ over O₂ of ribulose bisphosphate carboxylase-oxygenase (RUBISCO). This allows the prediction that a red alga relying on diffusive supply of CO₂ to RUBISCO from air-equilibrated solution should have less O₂ inhibition of photosynthesis than would an otherwise similar non-red alga with a lower S_rel of RUBISCO. Furthermore, RUBISCO shows an increased S_rel values at low temperatures. The prediction that O ₂inhibition of photosynthesis should be small for marine red algae relying on diffusive CO₂ entry growing in the North Sea with an annual temperature range of 4–16°C was tested in O₂ electrode experiments at 12°C. Phycodrys rubens and Plocamium cartilagineum, which rely on diffusive CO₂ entry showed, as predicted, only a small inhibition at lower inorganic C concentrations. Palmaria palmata, which has a CO₂ concentrating mechanism, had the expected negligible O ₂ inhibition of photosynthesis at any inorganic C concentration except (non-significantly) for saturating inorganic C.     

Kinetics of nitrite oxidation in two Nitrobacter species grown in nitrite-limited chemostats

The influence of growth rate, the presence of acetate and variation in the dissolved oxygen concentration on the kinetics of nitrite oxidation was studied in suspensions of intact cells of Nitrobacter winogradskyi and Nitrobacter hamburgensis. The cells were grown in nitrite-limited chemostats at different dilution rates under chemolithotrophic and mixotrophic conditions. Growth of N. hamburgensis in continuous culture was dependent on the presence of acetate. Acetate hardly affected the maximal nitrite oxidation rate per cell (V _max), but displayed a distinctly negative effect on the saturation constants for nitrite oxidation (K _m ) of both Nitrobacter species. This effect was reversible; when acetate was removed from the suspensions the K _m -values for nitrite oxidation returned to their original values. A reduction of the dissolved oxygen concentration from 100% to 18% air saturation slightly decreased the V _max of chemolithotrophically grown N. winogradskyi cells, whereas a 2.3 fold increase was observed with mixotrophically grown cells of N. hamburgensis. It is suggested that the large variation in K _m encountered in field samples could be due to this observed phenotypic variability. The V _max per cell is not a constant, but apparently is dependent on growth rate and environmental conditions. This implies that potential nitrite oxidation activity and numbers of cells are not necessarily related. Considering their kinetic characteristics, it is unlikely that N. hamburgensis is able to compete succesfully with N. winogradskyi for limiting amounts of nitrite under mixotrophic conditions. However, at reduced partial oxygen tensions, N. hamburgensis may become the better competitor.     

Effects of temperature and CO2 enrichment on kinetic properties of NADP+-malate dehydrogenase in two ecotypes of Barnyard grass (Echinochloa crus-galli (L.) Beauv.) from contrasting climates

Summary The apparent energy of activation (E _a), Michaelis-Menten constant (K _mfor oxaloacetate), V _max/K _mratios and specific activities of NADP⁺-malate dehydrogenase (NADP⁺-MDH; EC 1.1.1.82) were analyzed in plants of Barnyard grass from Québec (QUE) and Mississippi (MISS) acclimated to two thermoperiods 28/22°C, 21/15°C, and grown under two CO₂ concentrations, 350 l l^-1 and 675 l l^-1. E _avalues of NADP⁺-MDH extracted from QUE plants were significantly lower than those of MISS plants. K _mvalues and V _max/K _mratios of the enzyme from both ecotypes were similar over the range of 10–30°C but reduced V _max/K _mratios were found for the enzyme of QUE plants at 30 and 40°C assays. MISS plants had higher enzyme activities when measured on a chlorophyll basis but this trend was reversed when activities were expressed per fresh weight leaf or per leaf surface area. Activities were significantly higher in plants of both populations acclimated to 22/28°C. CO₂ enrichment did not modify appreciably the catalytic properties of NADP⁺-MDH and did not have a compensatory effect upon catalysis or enzyme activity under cool acclimatory conditions. NADP⁺-MDH activities were always in excess of the amount required to support observed rates of CO₂ assimilation and these two parameters were significantly correlated. The enhanced photosynthetic performance of QUE plants under cold temperature conditions, as compared to that of MISS plants, cannot be attributed to kinetic differences of NADP⁺-malate dehydrogenase among these ecotypes.     

On the integration of subthreshold inputs from Perforant Path and Schaffer Collaterals in hippocampal CA1 pyramidal neurons

Using a realistic model of a CA1 hippocampal pyramidal neuron, we make experimentally testable predictions on the roles of the non-specific cation current, I _h , and the A-type Potassium current, I _A , in modulating the temporal window for the integration of the two main excitatory afferent pathways of a CA1 neuron, the Schaffer Collaterals and the Perforant Path. The model shows that the experimentally observed increase in the dendritic density of I _h and I _A could have a major role in constraining the temporal integration window for these inputs, in such a way that a somatic action potential (AP) is elicited only when they are activated with a relative latency consistent with the anatomical arrangement of the hippocampal circuitry.     

The chloroplast cytochrome b 6 f complex can exist in monomeric and dimeric states

The cytochrome b ₆ f complex isolated from spinach chloroplast membranes can be resolved into two forms, a monomeric and a dimeric form, by centrifugation on sucrose gradients. The conversion of the dimeric form of the complex into the monomeric form could be prevented by cross-linking with the homobifunctional reagent, dithiobis(succinimidylpropionate) but not by cross-linking with disuccinimidyltartrate or glutaraldehyde. SDS-PAGE analyses of the monomeric and dimeric forms of the cytochrome complex showed the presence of specific cross-linked products in each respective form of the complex. For example, the monomeric form contained a cross-linked product of cytochrome f, cytochrome b ₆ f and subunit IV while the dimeric form contained a cross-linked dimer of cytochrome b ₆ f. The presence of the former in the isolated cytochrome b ₆ f complex prepared by the method of Hurt and Hauska (Eur J Biochem 117: 591–599, 1981) indicates the presence of the monomer in his preparation.Abbreviations DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DSP dithiobis(succinimidylpropionate) - DST disuccinimidyltartrate     

The role of the membrane bound cytochromes of b- and c-type in the electron transport chain of Rhodobacter capsulatus

(1) The electron transport system of heterotrophically dark-grown Rhodobacter capsulatus was investigated using the wild-type strain MT1131 and the phototrophic non-competent (Ps^-) mutant MT-GS18 carrying deletions of the genes for cytochrome c ₁ and b of the bc ₁ complex and for cytochrome c ₂. (2) Spectroscopic and thermodynamic data demonstrate that deletion of both bc ₁ complex and cyt. c ₂ still leaves several haems of c- and b-type with Em_7.0 of +265 mV and +354 mV at 551–542 nm, and +415 mV and +275 mV at 561–575 nm, respectively. (3) Analysis of the oxidoreduction kinetic patterns of cytochromes indicated that cyt. b ₄₁₅ and cyt. b ₂₇₅ are reduced by either ascorbate-diaminodurene or NADH, respectively. (4) Growth on different carbon and nitrogen sources revealed that the membrane-bound electron transport chain of both MT1131 and MT-GS18 strains undergoes functional modifications in response to the composition of the growth medium used. (5) Excitation of membrane fragments from cells grown in malate minimal medium by a train of single turnover flashes of light led to a rapid oxidation of 32% of the membrane-bound c-type haem complement. Conversely, membranes prepared from peptone/yeast extract grown cells did not show cyt. c photooxidation. These results are discussed within the framework of an electron transport chain in which alternative pathways bypassing both the cyt. c ₂ and bc ₁ complex might involve high-potential membrane bound haems of b- and c-type.Abbreviations AA antimycin A - CCCP carbonylcyanide m-chlorophenyl hydrazone - CN^- cyanide - DAD diaminodurene - Q₂H₂ ubiquinol-2 - Q-pool ubiquinone-10 pool - RC photochemical reaction center     

Cellular localization of cytochrome c 553 in the N2-fixing cyanobacterium Anabaena variabilis

The in situ location of the electron carrier protein cytochrome C ₅₅₃ (cyt c ₅₅₃) has been investigated in both vegetative cells and heterocysts of the cyanobacterium Anabaena variabilis ATCC 29413 using the antibody-gold technique, carried out as a post-ernbedding immunoelectron microscopy procedure. When using a rabbit polyclonal anti-cyt c ₅₅₃ specific antiserum an intense labelling, associated mainly with the cell periphery (cytoplasmic membrane and periplasmic area), was seen in both heterocysts and vegetative cells. The selective release of most of the cellular cyt c ₅₅₃ during a Tris-EDTA treatment confirms a periplasmic localization of this protein in A. variabilis. The results indicate that most of cyt c ₅₅₃ is located in the periplasmic space. The roles ascribed to this protein in both respiration and photosynthesis in cyanobacteria are discussed.Abbreviations Cyt c ₅₅₃ cytochrome c ₅₅₃ - PBS phosphate buffered saline (20 mM sodium phosphate, 0.9% NaCl, pH 7.4) - PMSF phenylmethylsulfonyl fluoride Recipient of a Research Fellowship of the Alexander von Humboldt Foundation (Bonn, FRG) for a leave to the University of Konstanz.     

Genes determining leucine aminopeptidase and mildew resistance from the ornamental apple, ‘White Angel’

Mildew resistance in the ornamental apple White Angel was found to be determined by complementary genes. The gene R _w was found to be necessary for the expression of resistance controlled by the resistance gene Pl _w . The close linkage between the isoenzyme gene, Lap-2, for leucine aminopeptidase and P1 _w was confirmed. The efficiency of Lap-2 as a marker in screening for mildew resistance is limited, as it cannot account for susceptible plants with the r _w r _w P1 _w p1 _w genotype. It has, however, an important role to play in combining resistance genes from different sources. The genotypes of White Angel (R _w r _w , Pl _w pl _w , Lap-2an), Jester (R _w r _w , p1 _w p _w , Lap-2an) Katja (R _w r _w ,p1 _w p1 _w , Lap-2an) and Gloster 69 (r _w r _w , p1 _w p1 _w , Lap-2an) were determined. It also appeared that R _w might influence Lap-2 activity in young seedlings.     

Gas Exchange Responses to CO2 Concentration Instantaneously Elevated in Flag Leaves of Winter Wheat Cultivars Released in Different Years

Three winter wheat (Triticum aestivum L.) cultivars, representatives of those widely cultivated in Beijing over the past six decades, were grown in the same environmental conditions. Net photosynthetic rate (P _N) per unit leaf area and instantaneous water use efficiency (WUE) of flag leaves increased with elevated CO₂ concentration. With an increase in CO₂ concentration from 360 to 720 µmol mol^–1, P _N and WUE of Jingdong 8 (released in 1990s and having the highest yield) increased by 173 and 81 %, while those of Nongda 139 (released in 1970s) increased by 88 and 66 %, and Yanda 1817 (released in 1945, with lowest yield) by 76 and 65 %. Jingdong 8 had the highest P _N and WUE values under high CO₂ concentration, but Yanda 1817 showed the lowest P _N. Stomatal conductance (g _s) of Nongda 139 and Yanda 1817 declined with increasing CO₂ concentration, but g _s of Jingdong 8 firstly went down and then up as the CO₂ concentration further increased. Intercellular CO₂ concentration (C _i) of Jingdong 8 and Nongda 139 increased when CO₂ concentration elevated, while that of Yanda 139 increased at the first stage and then declined. Jingdong 8 had the lowest C _i of the three wheat cultivars, and Yanda 1817 had the highest C _i value under lower CO₂ concentrations. However, Jingdong 8 had the highest P _N and lowest C _i at the highest CO₂ concentration which indicates that its photosynthetic potential may be high.     

The mitochondrial ATP synthase of chlorophycean algae contains eight subunits of unknown origin involved in the formation of an atypical stator-stalk and in the dimerization of the complex

Mitochondrial F₁F _O -ATP synthase of Chlamydomonas reinhardtii and Polytomella sp. is a dimer of 1,600,000 Da. In Chlamydomonas the enzyme lacks the classical subunits that constitute the peripheral stator-stalk as well as those involved in the dimerization of the fungal and mammal complex. Instead, it contains eight novel polypeptides named ASA1 to 8. We show that homologs of these subunits are also present in the chlorophycean algae Polytomella sp. and Volvox carterii. Blue Native Gel Electrophoresis analysis of mitochondria from different green algal species also indicates that stable dimeric mitochondrial ATP synthases may be characteristic of all Chlorophyceae. One additional subunit, ASA9, was identified in the purified mitochondrial ATP synthase of Polytomella sp. The dissociation profile of the Polytomella enzyme at high-temperatures and cross-linking experiments finally suggest that some of the ASA polypeptides constitute a stator-stalk with a unique architecture, while others may be involved in the formation of a highly-stable dimeric complex. The algal enzyme seems to have modified the structural features of its surrounding scaffold, while conserving almost intact the structure of its catalytic subunits.     

Isolation,sequencing and mutational analysis of a gene cluster involved in nitrite reduction inParacoccus denitrificans

By using the gene encoding the C-terminal part of thecd ₁-type nitrite reductase ofPseudomonas stutzeri JM300 as a heterologous probe, the corresponding gene fromParacoccus denitrificans was isolated. This gene,nirS, codes for a mature protein of 63144 Da having high homology withcd ₁-type nitrite reductases from other bacteria. Directly downstream fromnirS, three othernir genes were found in the ordernirECF. The organization of thenir gene cluster inPa. denitrificans is different from the organization ofnir clusters in some Pseudomonads.nirE has high homology with a S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (uro'gen III methylase). This methylase is most likely involved in the hemed ₁ biosynthesis inPa. denitrificans. The third gene,nirC, codes for a small cytochromec of 9.3 kDa having high homology with cytochromec _55X ofPs. stutzeri ZoBell. The 4th gene,nirF, has no homology with other genes in the sequence databases and has no relevant motifs. Inactivation of either of these 4 genes resulted in the loss of nitrite and nitric oxide reductase activities but not of nitrous oxide reductase activity.nirS mutants lack thecd ₁-type nitrite reductase whilenirE, nirC andnirF mutants produce a small amount ofcd ₁-type nitrite reductase, inactive due to the absence of hemed ₁. Upstream from thenirS gene the start of a gene was identified which has limited homology withnosR, a putative regulatory gene involved in nitrous oxide reduction. A potential FNR box was identified between this gene andnirS.Abbreviations SDS sodium dodecyl sulfate - NBT nitroblue tetrazolium - PAGE polyacrylamide gel electrophoresis     

Performance of a generalist grasshopper on a C<Subscript>3</Subscript> and a C<Subscript>4</Subscript> grass: compensation for the effects of elevated CO<Subscript>2</Subscript> on plant nutritional quality

The increasing CO₂ concentration in Earths atmosphere is expected to cause a greater decline in the nutritional quality of C₃ than C₄ plants. As a compensatory response, herbivorous insects may increase their feeding disproportionately on C₃ plants. These hypotheses were tested by growing the grasses Lolium multiflorum C₃) and Bouteloua curtipendula C₄) at ambient (370 ppm) and elevated (740 ppm) CO₂ levels in open top chambers in the field, and comparing the growth and digestive efficiencies of the generalist grasshopper Melanoplus sanguinipes on each of the four plant × CO₂ treatment combinations. As expected, the nutritional quality of the C₃ grass declined to a greater extent than did that of the C₄ grass at elevated CO₂; protein levels declined in the C₃ grass, while levels of carbohydrates (sugar, fructan and starch) increased. However, M. sanguinipes did not significantly increase its consumption rate to compensate for the lower nutritional quality of the C₃ grass grown under elevated CO₂. Instead, these grasshoppers appear to use post-ingestive mechanisms to maintain their growth rates on the C₃ grass under elevated CO₂. Consumption rates of the C₃ and C₄ grasses were also similar, demonstrating a lack of compensatory feeding on the C₄ grass. We also examined the relative efficiencies of nutrient utilization from a C₃ and C₄ grass by M. sanguinipes to test the basis for the C₄ plant avoidance hypothesis. Contrary to this hypothesis, neither protein nor sugar was digested with a lower efficiency from the C₄ grass than from the C₃ grass. A novel finding of this study is that fructan, a potentially large carbohydrate source in C₃ grasses, is utilized by grasshoppers. Based on the higher nutrient levels in the C₃ grass and the better growth performance of M. sanguinipes on this grass at both CO₂ levels, we conclude that C₃ grasses are likely to remain better host plants than C₄ grasses in future CO₂ conditions.     

The Influences of I h on Temporal Summation in Hippocampal CA1 Pyramidal Neurons: A Modeling Study

Recent experimental and theoretical studies have found that active dendritic ionic currents can compensate for the effects of electrotonic attenuation. In particular, temporal summation, the percentage increase in peak somatic voltage responses invoked by a synaptic input train, is independent of location of the synaptic input in hippocampal CA1 pyramidal neurons under normal conditions. This independence, known as normalization of temporal summation, is destroyed when the hyperpolarization-activated current, I _h, is blocked [Magee JC (1999a), Nature Neurosci. 2: 508–514]. Using a compartmental model derived from morphological recordings of hippocampal CA1 pyramidal neurons, we examined the hypothesis that I _h was primarily responsible for normalization of temporal summation. We concluded that this hypothesis was incomplete. With a model that included I _h, the persistent Na⁺ current (I _NaP), and the transient A-type K⁺ current (I _A), however, we observed normalization of temporal summation across a wide range of synaptic input frequencies, in keeping with experimental observations.     

Co-function of C3-and C4-photosynthetic pathways in C3, C4 and C3-C4 intermediate Flaveria species

The potential for C₄ photosynthesis was investigated in five C₃-C₄ intermediate species, one C₃ species, and one C₄ species in the genus Flaveria, using ¹⁴CO₂ pulse-¹²CO₂ chase techniques and quantum-yield measurements. All five intermediate species were capable of incorporating ¹⁴CO₂ into the C₄ acids malate and aspartate, following an 8-s pulse. The proportion of ¹⁴C label in these C₄ products ranged from 50–55% to 20–26% in the C₃-C₄ intermediates F. floridana Johnston and F. linearis Lag. respectively. All of the intermediate species incorporated as much, or more, ¹⁴CO₂ into aspartate as into malate. Generally, about 5–15% of the initial label in these species appeared as other organic acids. There was variation in the capacity for C₄ photosynthesis among the intermediate species based on the apparent rate of conversion of ¹⁴C label from the C₄ cycle to the C₃ cycle. In intermediate species such as F. pubescens Rydb., F. ramosissima Klatt., and F. floridana we observed a substantial decrease in label of C₄-cycle products and an increase in percentage label in C₃-cycle products during chase periods with ¹²CO₂, although the rate of change was slower than in the C₄ species, F. palmeri. In these C₃-C₄ intermediates both sucrose and fumarate were predominant products after a 20-min chase period. In the C₃-C₄ intermediates, F. anomala Robinson and f. linearis we observed no significant decrease in the label of C₄-cycle products during a 3-min chase period and a slow turnover during a 20-min chase, indicating a lower level of functional integration between the C₄ and C₃ cycles in these species, relative to the other intermediates. Although F. cronquistii Powell was previously identified as a C₃ species, 7–18% of the initial label was in malate+aspartate. However, only 40–50% of this label was in the C-4 position, indicating C₄-acid formation as secondary products of photosynthesis in F. cronquistii. In 21% O₂, the absorbed quantum yields for CO₂ uptake (in mol CO₂·[mol quanta]^-1) averaged 0.053 in F. cronquistii (C₃), 0.051 in F. trinervia (Spreng.) Mohr (C₄), 0.052 in F. ramosissima (C₃-C₄), 0.051 in F. anomala (C₃-C₄), 0.050 in F. linearis (C₃-C₄), 0.046 in F. floridana (C₃-C₄), and 0.044 in F. pubescens (C₃-C₄). In 2% O₂ an enhancement of the quantum yield was observed in all of the C₃-C₄ intermediate species, ranging from 21% in F. ramosissima to 43% in F. pubescens. In all intermediates the quantum yields in 2% O₂ were intermediate in value to the C₃ and C₄ species, indicating a co-function of the C₃ and C₄ cycles in CO₂ assimilation. The low quantum-yield values for F. pubescens and F. floridana in 21% O₂ presumably reflect an ineffcient transfer of carbon from the C₄ to the C₃ cycle. The response of the quantum yield to four increasing O₂ concentrations (2–35%) showed lower levels of O₂ inhibition in the C₃-C₄ intermediate F. ramosissima, relative to the C₃ species. This indicates that the co-function of the C₃ and C₄ cycles in this intermediate species leads to an increased CO₂ concentration at the site of ribulose-1,5-bisphosphate carboxylase/oxygenase and a concomitant decrease in the competitive inhibition by O₂.Abbreviations PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - RuBP ribulose-1,5-bisphosphate     

All-atom molecular dynamics simulations reveal significant differences in interaction between antimycin and conserved amino acid residues in bovine and bacterial bc1 complexes

Antimycin A is the most frequently used specific and powerful inhibitor of the mitochondrial respiratory chain. We used all-atom molecular dynamics (MD) simulations to study the dynamic aspects of the interaction of antimycin A with the Q_i site of the bacterial and bovine bc₁ complexes embedded in a membrane. The MD simulations revealed considerable conformational flexibility of antimycin and significant mobility of antimycin, as a whole, inside the Q_i pocket. We conclude that many of the differences in antimycin binding observed in high-resolution x-ray structures may have a dynamic origin and result from fluctuations of protein and antimycin between multiple conformational states of similar energy separated by low activation barriers, as well as from the mobility of antimycin within the Q_i pocket. The MD simulations also revealed a significant difference in interaction between antimycin and conserved amino acid residues in bovine and bacterial bc₁ complexes. The strong hydrogen bond between antimycin and conserved Asp-228 (bovine numeration) was observed to be frequently broken in the bacterial bc₁ complex and only rarely in the bovine bc₁ complex. In addition, the distances between antimycin and conserved His-201 and Lys-227 were consistently larger in the bacterial bc₁ complex. The observed differences could be responsible for a weaker interaction of antimycin with the bacterial bc₁ complex.     

Multiobjective H2/H∞ synthetic gene network design based on promoter libraries

Some current promoter libraries have been developed for synthetic gene networks. But an efficient method to engineer a synthetic gene network with some desired behaviors by selecting adequate promoters from these promoter libraries has not been presented. Thus developing a systematic method to efficiently employ promoter libraries to improve the engineering of synthetic gene networks with desired behaviors is appealing for synthetic biologists.In this study, a synthetic gene network with intrinsic parameter fluctuations and environmental disturbances in vivo is modeled by a nonlinear stochastic system. In order to engineer a synthetic gene network with a desired behavior despite intrinsic parameter fluctuations and environmental disturbances in vivo, a multiobjective H₂/H_∞ reference tracking (H₂ optimal tracking and H_∞ noise filtering) design is introduced. The H₂ optimal tracking can make the tracking errors between the behaviors of a synthetic gene network and the desired behaviors as small as possible from the minimum mean square error point of view, and the H_∞ noise filtering can attenuate all possible noises, from the worst-case noise effect point of view, to achieve a desired noise filtering ability. If the multiobjective H₂/H_∞ reference tracking design is satisfied, the synthetic gene network can robustly and optimally track the desired behaviors, simultaneously.First, based on the dynamic gene regulation, the existing promoter libraries are redefined by their promoter activities so that they can be efficiently selected in the design procedure. Then a systematic method is developed to select an adequate promoter set from the redefined promoter libraries to synthesize a gene network satisfying these two design objectives. But the multiobjective H₂/H_∞ reference tracking design problem needs to solve a difficult Hamilton–Jacobi Inequality (HJI)-constrained optimization problem. Therefore, the fuzzy approximation method is employed to simplify the HJI-constrained optimization problem to an equivalent linear matrix inequality (LMI)-constrained optimization problem, which can be easily solved by selecting an adequate promoter set from the redefined promoter libraries using the LMI toolbox in Matlab.Based on the confirmation of in silico design examples, we can select an adequate promoter set from the redefined promoter libraries to achieve the multiobjective H₂/H_∞ reference tracking design. The proposed method can reduce the number of trial-and-error experiments in selecting an adequate promoter set for a synthetic gene network with desired behaviors. With the rapid increase of promoter libraries, this systematic method will accelerate progress of synthetic biology design.     

Carbon dioxide and methane fluxes in boreal peatland microcosms with different vegetation cover—effects of ozone or ultraviolet-B exposure

O₃ concentrations in the troposphere are rising and those in the stratosphere decreasing, the latter resulting in higher fluxes of solar ultraviolet-B (UV-B) radiation to the earth's surface. We assessed whether the fluxes of CO₂ and CH₄ are altered by enhanced UV-B radiation or elevated tropospheric O₃ concentrations in boreal peatland microcosms (core depth 40 cm, diameter 10.5 cm) with different vegetation cover. At the end of the UV-B experiment which lasted for a growing season, net CO₂ exchange (NEE) and dark ecosystem respiration (R _TOT) were sevenfold higher, and CH₄ efflux 12-fold higher, in microcosms with intact vegetation dominated by Eriophorum vaginatum L. and Sphagnum spp., compared to microcosms from which we removed E. vaginatum. Vegetation treatment had minor effects on CH₄ production and consumption potentials in the peat, suggesting that the large difference in CH₄ efflux is mainly due to efficient CH₄ transport via the aerenchyma of E. vaginatum. Ambient UV-B supplemented with 30% and elevated O₃ concentrations (100 and 200 ppb, for 7 weeks) significantly increased R _TOT in both vegetation treatments. Elevated O₃ concentrations reduced NEE over time, while UV-B had no clear effects on the fluxes of CO₂ or CH₄ in the cloudy summer of the study. Field experiments are needed to assess the significance of increasing UV-B radiation and elevated tropospheric O₃ concentration on peatland gas exchange in the long-term.