Reaction centers from three herbicide resistant mutants of Rhodobacter sphaeroides 2.4.1: Kinetics of electron transfer reactions

Electron transfer rates were measured in RCs from three herbicide-resistant mutants with known amino acid changes to elucidate the structural requirements for last electron transfer. The three herbicide resistant mutants were IM(L229) (Ile-L229 Met), SP(L223) (Ser-L223 Pro) and YG(L222) (Tyr-L222 Gly). The electron transfer rate D⁺Q_A ^-Q_BD⁺Q_AQ_B (k _AB) is slowed 3 fold in the IM(L229) and YG(L222) RCs (pH 8). The stabilization of D⁺Q_AQ_B ^- with respect to D⁺Q_AQ_B ^- (pH 8) was found to be eliminated in the IM(L229) mutant RCs (G⁰ 0 meV), was partially reduced in the SP(L223) mutant RCs (G⁰=–30 meV), and was unaltered in the YG(L222) mutant RCs (G⁰=–60 meV), compared to that observed in the native RCs (G⁰=–60 meV). The pH dependences of the charge recombination rate D⁺Q_AQ_B ^-DQ_AQ_B (k _BD) and the electron transfer from Q_A ^- (k _QA ^-Q_A) suggest that the mutations do not affect the protonation state of Glu-L212 nor the electrostatic interactions of Q_B and Q_B ^- with Glu-L212. The binding affinities of UQ₁₀ for the Q_B site were found in order of decreasing values to be native IM(L229) > YG(L222) SP(L223). The altered properties of the mutant RCs are used to deduce possible structural changes caused by the mutations and are dicscussed in terms of photosynthetic efficiency of the herbicide resistant strains.Abbreviations Bchl bacteriochlorophyll - Bphe bacteriopheophytin - cholate 3,7,12-trihydroxycholanic acid - D donor (bacteriochlorophyll dimer) - EDTA ethylenediamine tetraacetic acid - Fe²⁺ non-heme iron atom - LDAO lauryl dimethylamine oxide - PS II photosystem II - Q_A and Q_B primary and secondary quinone acceptors - RC bacterial reaction center - Tris tris(hydroxymethyl)aminomethane - UQ₀ 2,3-dimethoxy-5-methyl benzoquinone - UQ₁₀ ubiquinone 50     

Spatio-temporal patterns of photosystem II activity and plasma-membrane proton flows in Chara corallina cells exposed to overall and local illumination

Pulse-amplitude modulated microfluorometry and an extracellular pH microprobe were used to examine light-induced spatial heterogeneity of photosynthetic and H⁺-transporting activities in cells of Chara corallina Klein ex Willd. Subcellular domains featuring different PSII photochemical activities were found to conform to alternate alkaline and acid zones produced near the cell surface, with peaks of PSII activity correlating with the position of acid zones. Buffers eliminated pH variations near the cell surface but did not destroy the variations in PSII photochemical yield (F/F_m). When a dark-adapted cell was exposed to actinic light, the PSII effective yield decreased within 5–15 min in the alkaline regions but rose after the initial decline in the acid regions. The light-induced decrease in F/F_m in the alkaline regions occurred prior to or synchronously with the steep rise in local pH. The kinetics of F/F_m, F_m, and F observed in alkaline regions under overall illumination of Chara cells were replaced by those typical of acid regions, when the illumination area size was restricted to 1.5–2 mm. The data show that photoinduced patterns in photosynthetic activity are not predetermined by the particular structural organization of alkaline and acid cell regions but are subject to dynamic changes.Abbreviations APW artificial pond water - a.u. arbitrary units - F_o and F_m minimal and maximal chlorophyll fluorescence yields in a dark-adapted cell - F and F_m actual (running) and maximal fluorescence yields in a cell exposed to actinic light - F/F_m(F_m–F)/F_m effective quantum yield of PSII photochemistry - pH_o pH of the medium near the cell surface - PSII photosystem II     

High misses after odd flashes in oxygen evolution in thoroughly dark-adapted thylakoids from pea and Chenopodium album

In Photosystem II (PS II), water is oxidized to molecular oxygen and plastoquinone is reduced to plastoquinol. The oxidation of water requires the accumulation of four oxidizing equivalents, through the so-called S-states of the oxygen evolving complex; the production of plastoquinol requires the accumulation of two reducing equivalents on a bound plastoquinone, Q_B. It has been generally believed that during the flash-induced transition of each of the S-states (S_n S_n+1, where n=0, 1, 2 and 3), a certain small but equal fraction of the PS II reaction centers are unable to function and, thus, miss being turned over. We used thoroughly dark-adapted thylakoids from peas (Pisum sativum) and Chenopodium album (susceptible and resistant to atrazine) starting with 100% of the oxygen evolving complex in the S₁ state. Thylakoids were illuminated with saturating flashes, providing a double hit parameter of about 0.07. Our experimental data on flashnumber dependent oscillations in the amount of oxygen per flash fit very well with a binary pattern of misses: 0, 0.2, 0, 0.4 during S₀ S₁, S₁ S₂, S₂ S₃ and S₃ S₀ transitions. Addition of 2 mM ferricyanide appears to shift this pattern by one flash. These results are consistent with the bicycle model recently proposed by V. P. Shinkarev and C. A. Wraight (Oxygen evolution in photosynthesis: From unicycle to bicycle, 1993, Proc Natl Acad Sci USA 90: 1834–1838), where misses are due to the presence of P⁺ or Q_A ^- among the various equilibrium states of PS II centers.Abbreviations miss parameter - double hit parameter - PS II Photosystem II - Q_A primary one-electron acceptor of PS II, a plastoquinone molecule - Q_B secondary plastoquinone two-electron acceptor of PS II - S-states (S_n, where n=0, 1, 2, 3 or 4) redox states of the oxygen evolving complex     

Stereochemistry and Stereoelectronics of Azines. 13. Conformational Effects on the Quadrupolarity of Azines. An Ab Initio Quantum-Mechanical Study of a Lateral Synthon

The quadrupole moment of formaldazine, H₂C=N-N=CH₂, has been studied for the trans structure (Ð(C-N-N-C) = = 180) and a series of gauche structures ( > 120). Restricted Hartree-Fock theory, second-order Møller-Plesset theory, and quadratic CI theory have been used in conjunction with the basis sets 6-31G^*, 6-31G**, 6-311G** and 6-311++G**. Formaldazine is a quadrupolar molecule with primitive quadrupole moment tensor components of Q _xx = -22.4, Q _yy = -20.4 and Q _zz = -25.6 DÅ at the theoretical level QCISD/6-311++G**. The examination of the theoretical level dependency shows that the reliable computation of a quadrupole moment requires the use of a flexible basis set. A large part of the component Q _zz = -25.6 DÅ is due to the -system and compares, on a per electron basis, with the Q _zz value of benzene. Conformational changes of the azines in the range 120° < < 180 have but a minute effect on the energy and are associated with only minor electronic relaxation. These conformational changes alter the quadrupole moment tensor components less than Q _xx = +0.4, Q _yy = +1.6 and Q _zz = -1.0 DÅ at QCISD/6-311++G**//QCISD/6-31G*. The direction of these changes is explained by consideration of the rotation of the CN--systems and a small reduction of the CN bond polarity in the gauche structures. The Q _zz component of formaldazine is representative of the quadrupole moment tensor component along the direction of the C ₂ axis of the azine bridge as such. Hence, the results of this study suggest that azines can engage in strong quadrupole-quadrupole interactions and can be employed as lateral synthons in crystal engineering. Electronic Supplementary Material available.     

Isolation and characterization of a Forssman antigen-binding lectin from velvet bean (Mucuna derringiana) seeds

A Forssman antigen (GalNAc1-3GalNAc1-3Gal1-4Gal1-4Glc1-1Cer)-binding lectin has been purified from velvet bean (Mucuna derringiana) seeds by a combination of affinity chromatography and reversed phase HPLC. This lectin agglutinates both native and trypsin-treated sheep erythrocytes as well as trypsinized rabbit erythrocytes, but neither native rabbit nor human erythrocytes, irrespective of blood group type. SDS-PAGE and gel filtration chromatography reveal the lectin to be a homodimer consisting of two 54 kDa subunits linked by non-covalent bonds. The results obtained by quantitative precipitation, haemagglutination inhibition and TLC overlay assays indicate that theMucuna lectin specifically recognizes Forssman antigen and Forssman disaccharide (GalNAc1-3GalNAc)-related structures. Abbreviations: The abbreviations and the trivial names used are: AH, 6-aminohexyl; BSA, bovine serum albumin; Cer, ceramide; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; PBS, 10mm phosphate-buffered saline, pH 7,2, containing 0.15m NaCl; PMSF, phenyl methyl sulfonyl fluoride; SDS, sodium dodecyl sulphate; TFA, trifluoroacetic acid; TBS, 20mm tris-buffered saline, pH 7.2; TLC, thin-layer chromatography; A disaccharide, GalNAc1-3Gal; A trisaccharide, GalNAc1-3[Fuc1-2]Gal; Forssman disaccharide, GalNAc1-3GalNAc; CDH (ceramide dihexoside or lactosyl ceramide) Gal1-4Glc1-1Cer (LacCer); CTH (ceramide trihexoside or globotriosyl ceramide), Gal1-4Gal1-4Glc1-1Cer (GbOse₃Cer or Gb₃); globoside (globotetraosyl ceramide), GalNAc1-3Gal1-4Gal1-4Glc1-1Cer (GbOse₄Cer or Gb₄); Forssman antigen (globopentaosyl ceramide), GalNAc1-3GalNAc1-3Gal1-4Gal1-4Glc1-1Cer (GbOse₅Cer).     

Conformation of the circular dumbbell d〈pCGC-TT-GCG-TT〉: Structure determination and molecular dynamics

Summary The circular DNA decamer 5-dpCGC-TT-GCG-TT-3 was studied in solution by means of NMR spectroscopy and molecular dynamics in H₂O. At a temperature of 269 K, a 50/50 mixture of two dumbbell structures (denoted L2L2 and L2L4) is present. The L2L2 form contains three Watson-Crick C-G base pairs and two two-residue loops in opposite parts of the molecule. On raising the temperature from 269 K to 314 K, the L2L4 conformer becomes increasingly dominant (95% at 314 K). This conformer has a partially disrupted G(anti)-C(syn) closing base pair in the 5-GTTC-3 loop with only one remaining (solvent-accessible) hydrogen bond between NH of the cytosine dC(1) and O6 of the guanine dG(8). The opposite 5-CTTG-3 loop remains stable. The two conformers occur in slow equilibrium (rate constant 2–20 s^–1). Structure determination of the L2L2 and L2L4 forms was performed with the aid of a full relaxation matrix approach (IRMA) in combination with restrained MD. Torsional information was obtained from coupling constants. Coupling constant analysis (³J_HH, ³J_HP, ³J_CP) gave detailed information about the local geometry around backbone torsion angles , , and , revealing a relatively high flexibility of the 5-GTTC-3 loop. The values of the coupling constants are virtually temperature-independent. Weakly constrained molecular dynamics in solvent was used to sample the conformational space of the dumbbell. The relaxation matrices from the MD simulation were averaged over r^–3 to predict dynamic NOE volumes. In order to account for the 1:1 conformational mixture of L2L2 and L2L4 present at 271 K, we also included S² factors and r^–6 averaging of the r^–3-averaged relaxation matrices. On matrix averaging, the agreement of NOE volumes with experiment improved significantly for protons located in the thermodynamically less stable 5-GTTC-3 loop. The difference in stability of the 5-CTTG-3 and 5-GTTC-3 loops is mainly caused by differences in the number of potential hydrogen bonds in the minor groove and differences in stacking overlap of the base pairs closing the minihairpin loops. The syn conformation for dC(1), favored at high temperature, is stabilized by solvation in the major groove. However, the conformational properties of the dC(1) base, as deduced from R-factor analysis and MD simulations, include a large flexibility about torsion angle .     

Elevated concentration of TNF-α induces trophoblast differentiation in mouse blastocyst outgrowths

Elevated expression of tumour necrosis factor- (TNF-) is associated with adverse pregnancy outcome. This study has examined the expression of TNF- and its receptors (TNF-Rs) by mouse blastocysts and blastocyst outgrowths from day 4 to 9.5 of pregnancy and investigated the effects of elevated TNF- on the inner cell mass (ICM) and trophoblast cells of blastocyst outgrowths. RT-PCR demonstrated TNF- mRNA expression from day 7.5 to 9.5, TNF-R1 from day 6.5 to 9.5 and TNF-R2 from day 5.5 to 7.5 of pregnancy, and in situ hybridisation revealed the trophoblast giant cells (TGCs) of the early placenta as the site of TNF- expression. Day 4 blastocysts were cultured in a physiologically high concentration of TNF- (100 ng/ml) for 72 h to the outgrowth stage and then compared to blastocysts cultured in media alone. TNF--treated blastocyst outgrowths exhibited a significant reduction in ICM cells (mean ± SD 23.90±10.42 vs 9.37±7.45, t-test, P<0.0001) with no significant change in the numbers of trophoblast cells (19.97±8.14 vs 21.73±7.79, t-test, P=0.39). Within the trophoblast cell population, the TNF--treated outgrowths exhibited a significant increase in multinucleated cells (14.10±5.53 vs 6.37±5.80, t-test, P<0.0001) and a corresponding significant decrease in mononucleated cells (5.87±3.60 vs 15.37±5.87, t-test, P<0.0001). In summary, this study describes the expression of TNF- and its receptors during the peri-implantation period in the mouse. It also reports that elevated TNF- restricts ICM proliferation in the blastocyst and changes the ratio of mononucleated to multinucleated trophoblast cells. These findings suggest a mechanism by which increased expression of TNF- during trophoblast differentiation may be detrimental to pregnancy.This work was supported by the National Health and Medical Research Council of Australia     

The catalysis of nucleotide polymerization by compounds of divalent lead

Summary Soluble lead salts and a number of lead-containing minerals catalyze the formation of oligonucleotides from nucleoside 5-phosphorimidazolides. The effectiveness of lead compounds correlates strongly with their solubility. Under optimal conditions we were able to obtain 18% of pentamer and higher oligomers from ImpA. Reactions involving ImpU gave smaller yields.Abbreviations A adenosine - U uridine - Im imidazole - MeIm 1-methyl-imidazole - EDTA ethylenediaminetetraacetic acid - pA adenosine 5-phosphate - pU uridine 5-phosphate - Ap adenosine cyclic 2:3-phosphate - ATP adenosine 5-triphosphate - AppA P₁,P₂-diadenosine 5-diphosphate - pNp (N = A,U) nucleotide 2(3), 5-diphosphate - ImpA adenosine 5-phosphoreimidazolide - ImpU uridine 5-phosphorimidazolide - A ²pA adenylyl-[25]-adenosine - A ³pA adenylyl-[35]-adenosine - pA ²pA 5-phospho-adenylyl-[25]-adenosine - pA ³pA 5-phospho-adenylyl-[35]-adenosine - pUpU 5-phospho-uridylyl-uridine - pApU 5-phospho-adenylyl-uridine - pUpA 5-phospho-uridylyladenine - (pA)n (n, 2,3,4,) oligoadenylates with 5 terminal phosphate - ImpApA 5-phosphorimidazolide of adenylyl adenosine - (pA) ₅₊ pentamer and higher oligoadenylates with 5 terminal phosphate - (Ap)nA (n = 2,3,4) oligoadenylates without terminal phosphates In the following we do not specify the nature of the internucleotide linkageIn the following we do not specify the nature of the internucleotide linkage     

Synthesis of Aminoethyl Glycosides of the Carbohydrate Chains of Glycolipids Gb3, Gb4, and Gb5

4-O-Glycosylation of 2-azidoethyl 2,3,6-tri-O-benzoyl-4-O-(2,3,6-tri-O-benzoyl--D-galactopyranosyl)--D-glucopyranoside with ethyl 2,3,4,6-tetra-O-benzyl- and ethyl 3-O-acetyl-2,4,6-tri-O-benzyl-1-thio--D-galactopyranoside in the presence of methyl trifluoromethanesulfonate led to trisaccharide 2-azidoethyl (2,3,4,6-tetra-O-benzyl--D-galactopyranosyl)-(14)-(2,3,6-tri-O-benzoyl--D-galactopyranosyl)-(14)-2,3,6-tri-O-benzoyl--D-glucopyranoside and its 3"-O-acetylated analogue, 2-azidoethyl (3-O-acetyl-2,4,6-tri-O-benzyl--D-galactopyranosyl)-(14)-(2,3,6-tri-O-benzoyl--D-galactopyranosyl)-(14)-2,3,6-tri-O-benzoyl--D-glucopyranoside in yields of 85 and 83%, respectively. Deacetylation of the latter compound and subsequent glycosylation with 4-trichloroacetamidophenyl 3,4,6-tri-O-acetyl-2-deoxy-1-thio-2-trichloroacetamido--D-galactopyranoside and 4-trichloroacetamidophenyl 4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-1-thio-2-trichloroacetamido--D-galactopyranoside in dichloromethane in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid resulted in the corresponding selectively protected derivatives of the tetrasaccharide GalNAc(13)Gl(14)Gal(14)Glc-OH₂CH₂N₃ and the pentasaccharide Gal(13)GalNAc(13)Gl(14)Gal(14)Glc-OH₂CH₂N₃ in 88 and 73% yields, respectively. Removal of O-protecting groups, substitution of acetyl group for the N-trichloroacetyl group, and reduction of the aglycone azide group resulted in the target 2-aminoethyl globo-tri-, -tetra-, and -pentasaccharide, respectively.     

Adenosine-5′-phosphosulfate (APS) as sulfate donor for assimilatory sulfate reduction in Rhodospirillum rubrum

Crude extracts of Rhodospirillum rubrum catalyzed the formation of acid-volatile radioactivity from (³⁵S) sulfate, (³⁵S) adenosine-5-phosphosulfate, and (³⁵S) 3-phosphoadenosine-5-phosphosulfate. An enzyme fraction similar to APS-sulfotransferases from plant sources was purified 228-fold from Rhodospirillum rubrum. It is suggested here that this enzyme is specific for adenosine-5-phosphosulfate, because the purified enzyme fraction metabolized adenosine-5-phosphosulfate, however, only at a rate of 1/10 of that with adenosine-5-phosphosulfate. Further, the reaction with 3-phosphoadenosine-5-phosphosulfate was inhibited with 3-phosphoadenosine-5-phosphate whereas this nucleotide had no effect on the reaction with adenosine-5-phosphosulfate. For this activity with adenosine-5-phosphosulfate the name APS-sulfotransferase is suggested. This APS-sulfotransferase needs thiols for activity; good rates were obtained with either dithioerythritol or reduced glutathione; other thiols like cysteine, 2-3-dimercaptopropanol or mercaptoethanol are less effective. The electron donor methylviologen did not catalyze this reaction. The pH-optimum was about 9.0; the apparent K _m for adenosine-5-phosphosulfate was determined to be 0.05 mM with this so far purified enzyme fraction. Enzyme activity was increased with K₂SO₄ and Na₂SO₄ and was inhibited by 5-AMP. These properties are similar to assimilatory APS-sulfotransferases from spinach and Chlorella.Abbreviations APS adenosine-5-phosphosulfate - PAPS 3-phosphoadenosine-5-phosphosulfate - 5-AMP adenosine-5-monophosphate - 3-AMP adenosine-3-monophosphate - 3-5-ADP 3-phosphoadenosine-5-phosphate (PAP) - DTE dithiorythritol - GSH reduced glutathione - BAL 2-3-dimercaptopropanol     

Foam behavior of biological media

Summary The surface tension and foaminess of (a) unlimited, (b) substrate limited, and (c) oxygen transfer limited growth media of Hansenula polymorpha were measured using methanol, ethanol or glucose as a substrate.The time dependence of can be described by the Avrami-Überreiter relationship: log (2.3 log V)=n log t+log b, where V = (_O – _eq/(_t – _eq, and _O, _t and _eq are at t_M=0, t_M=t and t_M (equilibrium value).The constants n and b are functions of the fermentation time t_F as long as the growth is unlimited but they are constant in the state of limited growth. With glucose substrate, the foaminess can be presented as a definite function of the time, t_DG, which is necessary to attain _eq. With alcohol as a substrate no definite (t_DG) function was found.Symbols b constant in Eq. (1) - n constant in Eq. (1) - S substrate concentration - T temperature - t_M time h (measured from the beginning of the determination of the surface tension ) - t_F cultivation time h (measured from the time of inoculation) - t_DG time (min) necessary to attain the equilibrium surface tension ) - X dry biomass concentration (gl^–1) - V (_O – _eq)/(_t – _eq) - V_S equilibrium volume of the foam (cm³) - V_G volumetric gas flow rate during the estimation of (cm³ s^–1) - vvm volumetric gas flow rate with regard to the volume of the medium (min^–1) - w_SG superficial gas velocity (cm s^–1) - _m maximum specific growth rate (h^–1) - V_S/V_G foaminess (s) - surface tension, mMm^–1 (milli Newton m^–1) - _O at t_M=0 - _eq equilibrium surface tension ( at t_M) - _t at t_M=t - HP probes from Hansenula polymorpha cultivation - NLG non limited growth - OTLG oxygen transfer limited growth - SLG substrate limited growth     

Three-dimensional structures of photosynthetic reaction centers

In this article, the three-dimensional structures of photosynthetic reaction centers (RCs) are presented mainly on the basis of the X-ray crystal structures of the RCs from the purple bacteria Rhodopseudomonas (Rp.) viridis and Rhodobacter (Rb.) sphaeroides. In contrast to earlier comparisons and on the basis of the best-defined Rb. sphaeroides structure, a number of the reported differences between the structures cannot be confirmed. However, there are small conformational differences which might provide a basis for the explanation of observed spectral and functional discrepancies between the two species.A particular focus in this review is on the binding site of the secondary quinone (Q_B), where electron transfer is coupled to the uptake of protons from the cytoplasm. For the discussion of the Q_B site, a number of newlydetermined coordinate sets of Rp. viridis RCs modified at the Q_B site have been included. In addition, chains of ordered water molecules are found leading from the cytoplasm to the Q_B site in the best-defined structures of both Rp. viridis and Rb. sphaeroides RCs.Abbreviations B_A accessory bacteriochlorophyll in the active branch - B_B accessory bacteriochlorophyll in the inactive branch - D primary electron donor (special pair) - D_L special pair bacteriochorophyll bound by the L subunit - D_M special pair bacteriochorophyll bound by the M subunit - Q_A primary electron acceptor quinone - Q_B secondary electron acceptor quinone - RC reaction center - Rb. Rhodobacter - Rp. Rhodopseudomonas - _A bacteriopheophytin in the active branch - _B bacteriopheophytin in the inactive branch     

Template-directed reactions of aminonucleosides

Summary We have studied the reactions between adenosine 5-phosphorimidazolide and 9-(2-amino-2-deoxyxylofuranosyl) adenine (I) or 3-methylamino-3-deoxyadenosine (II), both with and without a poly (U) template. We find that both amino compounds react much more rapidly than does adenosine, in the absence of a template. The rate of reaction is greatly enhanced by a poly (U) template in the case of I, but the enhancement is slight in the case of II.Abbreviations A adenosine - xylo A_NH2 9-(2-amino-2-deoxy--D-xylofuranosyl) adenine - A_NHMe 3-methylamino-3-deoxyadenosine - ImpA adenosine 5-phosphorimidazolide - A³ pA adenylyl-[35]-adenosine - A² pA adenylyl-[25]-adenosine - U_NPA adenylyl-[52]-2-amino-2-deoxyuridine - xylo A_NPA 9-[adenylyl-(52)-2-amino-2-deoxy--D-xylofuranosyl]adenine - A_(NMe)pA adenylyl-[53]-3-methylamino-3-deoxyadenosine - pA adenosine 5phosphate - AppA P₁, P₂-diadenosine 5pyrophosphate - (pA)_n n = 2, 3 [2-5]-linked oligomers of pA - A² pA² pA [2-5]-linked trinucleoside diphosphate of A - poly (U) polyuridylic acid     

Polymerization of nucleotide analogues I: Reaction of nucleoside 5′-phosphorimidazolides with 2′-amino-2′-deoxyuridine

Summary 2-Amino-2-deoxyuridine reacts efficiently with nucleoside 5-phosphorimidazolides in aqueous solution. The dinucleoside monophosphate analogues were obtained in yields exceeding 80% under conditions in which little reaction occurs with the natural nucleosides.In a similar way, the 5-phosphorimidazolide of 2-amino-2-deoxyuridine undergoes self-condensation in aqueous solution to give a complex mixture of oligomers.The phosphoramidate bond in the dinucleoside monophosphate analogues is stable for several days at room temperature and pH 7. The mechanisms of their hydrolysis under acidic and alkaline conditions are described.Abbreviations A adenosine - C cytidine - G guanosine - U uridine - T thymidine - U_{N 3} 2-azido-2-deoxyuridine - U_{NH 2} 2-amino-2-deoxyuridine - ImpA adenosine 5-phosphorimidazolide - ImpU uridine 5-phosphorimidazolide - ImpU_{N 3} 2-azido-2-deoxyuridine 5-phosphorimidazolide - ImpU_{NH 2} 2-amino-2-deoxyuridine 5-phosphorimidazolide - pA adenosine 5-phosphate - pU uridine 5-phosphate - pU_{N 3} 2-azido-2-deoxyuridine 5-phosphate - pU_{NH 2} 2-amino-2-deoxyuridine 5-phosphate - UpA uridylyl-[35]-adenosine - UpU uridylyl-[35]-uridine - U_NpA adenylyl-[52]-2-amino-2-deoxy-uridine - U_NpU uridylyl-[52]-2-amino-2-deoxyuridine (pU_N)_n n=2,3,4 [25]-linked oligomers of pU_{NH 2} poly(A) polyadenylic acid - Im imidazole - MeIm l-methylimidazole     

Observations on the reduction of non-activated carboxylates by Clostridium formicoaceticum with carbon monoxide or formate and the influence of various viologens

Crude extracts or supernatants of broken cells of Clostridium formicoaceticum reduce unbranched, branched, saturated and unsaturated carboxylates at the expense of carbon monoxide to the corresponding alcohols. The presence of viologens with redox potentials varying from E ₀=-295 to-650 mV decreased the rate of propionate reduction. The more the propionate reduction was diminished the more formate was formed from carbon monoxide. The lowest propionate reduction and highest formate formation was observed with methylviologen. The carbon-carbon double bond of E-2-methyl-butenoate was only hydrogenated when a viologen was present. Formate as electron donor led only in the presence of viologens to the formation of propanol from propionate. The reduction of propionate at the expense of a reduced viologen can be followed in cuvettes. With respect to propionate Michaelis Menten behavior was observed. Experiments are described which lead to the assumption that the carboxylates are reduced in a non-activated form. That would be new type of biological reduction.Non-standard abbreviations glc Gas liquid chromatography - HPLC high performance liquid chromatography - RP reverse phase; Mediators (the figures in parenthesis of the mediators are redox potentials E ₀ in mV) - CAV²⁺ carbamoylmethylviologen, 1,1-carbamoyl-4,4-dipyridinium dication (E ₀=-296 mV) - BV²⁺ benzylviologen, 1,1-dibenzyl-4,4-dipyridinium dication (E ₀=-360 mV) - MV methylviologen, 1,1-dimethyl-4,4-dipyridinium-dication (E ₀=-444 mV) - DMDQ²⁺ dimethyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-ethylendication (E ₀=-514 mV) - TMV²⁺ tetramethylviologen, 1,1,4,4-tetramethyl-4,4-dipyridinium dication (E ₀=-550 mV) - PDQ²⁺ propyldiquat, 2,2-dipyridino-1,1-propenyl dication (E ₀=-550 mV) - DMPDQ²⁺ dimethylpropyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-propenyl dication (E ₀=-656 mV) - PN productivity number=mmol product (obtained by the uptake of one pair of electrons) x (biocatalyst (dry weight) kg)^-1×h^-1     

Photoinhibition of photosynthesis represents a mechanism for the long-term regulation of photosystem II

The obligate shade plant, Tradescantia albiflora Kunth grown at 50 mol photons · m^–2 s^–1 and Pisum sativum L. acclimated to two photon fluence rates, 50 and 300 mol · m^–2 · s^–1, were exposed to photoinhibitory light conditions of 1700 mol · m^–2 · s^–1 for 4 h at 22° C. Photosynthesis was assayed by measurement of CO₂-saturated O₂ evolution, and photosystem II (PSII) was assayed using modulated chlorophyll fluorescence and flash-yield determinations of functional reaction centres. Tradescantia was most sensitive to photoinhibition, while pea grown at 300 mol · m^–2 · s^–1 was most resistant, with pea grown at 50 mol · m^–2 · s^–1 showing an intermediate sensitivity. A very good correlation was found between the decrease of functional PSII reaction centres and both the inhibition of photosynthesis and PSII photochemistry. Photoinhibition caused a decline in the maximum quantum yield for PSII electron transport as determined by the product of photochemical quenching (q_p) and the yield of open PSII reaction centres as given by the steady-state fluorescence ratio, F_vF_m, according to Genty et al. (1989, Biochim. Biophys. Acta 990, 81–92). The decrease in the quantum yield for PSII electron transport was fully accounted for by a decrease in F_vF_m, since q_p at a given photon fluence rate was similar for photoinhibited and noninhibited plants. Under lightsaturating conditions, the quantum yield of PSII electron transport was similar in photoinhibited and noninhibited plants. The data give support for the view that photoinhibition of the reaction centres of PSII represents a stable, long-term, down-regulation of photochemistry, which occurs in plants under sustained high-light conditions, and replaces part of the regulation usually exerted by the transthylakoid pH gradient. Furthermore, by investigating the susceptibility of differently lightacclimated sun and shade species to photoinhibition in relation to q_p, i.e. the fraction of open-to-closed PSII reaction centres, we also show that irrespective of light acclimation, plants become susceptible to photoinhibition when the majority of their PSII reaction centres are still open (i.e. primary quinone acceptor oxidized). Photoinhibition appears to be an unavoidable consequence of PSII function when light causes sustained closure of more than 40% of PSII reaction centres.Abbreviations F_o and F_o minimal fluorescence when all PSII reaction centres are open in darkness and steady-state light, respectively - F_m and F_m maximal fluorescence when all PSII reaction centres are closed in darkand light-acclimated leaves, respectively - F_v variable fluorescence - (F_m-F_o) under steady-state light con-ditions - F_s steady-state fluorescence in light - Q_A the primary,stable quinone acceptor of PSII - q_Ne non-photochemical quench-ing of fluorescence due to high energy state - (pH); q_Ni non-photochemical quenching of fluorescence due to photoinhibition - q_p photochemical quenching of fluorescence To whom correspondence should be addressedThis work was supported by the Swedish Natural Science Research Council (G.Ö.) and the award of a National Research Fellowship to J.M.A and W.S.C. We thank Dr. Paul Kriedemann, Division of Forestry and Forest Products, CSIRO, Canberra, Australia, for helpful discussions.     

Edge preference of retinal and tectal neurons in common toads (Bufo bufo) in response to worm-like moving stripes: the question of behaviorally relevant ‘position indicators’

Summary Previous experiments have shown that during prey-catching behavior (orienting, snapping) in response to a worm-like moving stripe common toads.Bufo bufo (L.) exhibit a contrast-and direction-dependent edge preference. To a black (b) stripe moving against a white (w) background (b/w), they respond (R^*) preferably toward the leading (l) rather the trailing (t) edge (R _l ^* > R _t ^* ), thus displaying head preference. If the contrastdirection is reversed (w/b), the stripe's trailing edge is preferred (R _l ^* < R _t ^* ), hence showing tail preference. In the present study, neuronal activities of retinal classes R2 and R3 and tectal classes T5(2) and T7 have been extracellularly recorded in response to leading and trailing edges of a 3 ° × 30 ° stripe simulating a worm and traversing the centers of their excitatory receptive fields (ERF) horizontally at a constant angular velocity in variable movement direction (temporo-nasal or naso-temporal).The behavioral contrast-direction dependent edge preferences are best resembled by the responses (R) of prey-selective class T5(2) neurons (R_l R_t=101 for b/w, 0.31 for w/b) and T7 neurons (R_lR_t=61 for b/w, 0.41 for w/b); the T7 responses may be dendritic spikes. This property can be traced back to off-responses dominated retinal class R3 neurons (R_lR_t=61 for b/w, 0.51 for w/b), but not to class R2 (R_lR_t =1.21 for b/w and 0.91 for w/b). The respective edge preference phenomena are independent of the direction of movement.When stimuli were moved against a stationary black-white structured background, the head preference to the black stripe and the tail preference to the white stripe were maintained in class R3, T5(2), and T7 neurons. If the stripe traversed the ERF together with the structured background in the same direction at the same velocity, the responses of tectal class T5(2) and T7 neurons were strongly inhibited, particularly in the former. Responses of retinal R2 neurons in comparable situations could be reduced by about 50%, while class R3 neurons responded to both the stimulus and the moving background structure.The results support the concept that the prey feature analyzing system in toads applies principles of (i) parallel and (ii) hierarchial information processing. These are (i) divergence of retinal R3 neuronal output contributes to stimulus edge positioning and (in combination with R2 output) area evaluation intectal neurons and to stimulus area evaluation and (in combination with R4 output) sensitivity for moving background structures inpre tectal neurons; (ii) convergence of tectal excitatory and pretectal inhibitory inputs specify the property of prey-selective tectal T5(2) neurons which are known to project to bulbar/spinal motor systems.Abbreviations ERF excitatory receptive field - IRF inhibitory receptive field - N nasal - T temporal - R _w response to a worm-like stripe moving in the direction of its longer axis - R _A response to an antiworm-like stripe whose longer axis is oriented perpendicular to the direction of movement - R _l response to the leading edge of a worm-like moving stripe - R _t response to the trailing edge of a worm-like moving stripe - b/w black stimulus against a white background - w/b white stimulus against a black background - sm structured moving background - ss structured stationary background - u minimal structure width of a structured background consisting of rectangular black and white patches in random distribution - HRP horseradish peroxidase     

Characterization of second site mutations show that fast proton transfer to QB − is restored in bacterial reaction centers of Rhodobacter sphaeroides containing the Asp-L213 → Asn lesion

The structural basis for proton coupled electron transfer to Q_B in bacterial reaction centers (RCs) was studied by investigating RCs containing second site suppressor mutations (Asn M44 Asp, Arg M233 Cys, Arg H177 His) that complement the effects of the deleterious Asp L213 Asn mutation [DN(L213)]. The suppressor RCs all showed an increased proton coupled electron transfer rate k _AB ⁽²⁾(Q_A ^–Q_B ^– + H⁺ Q_AQ_BH^–) by at least 10³ (pH 7.5) and a recombination rate k _BD (D⁺Q_AQ_B ^– DQ_AQ_B) 15–40 times larger than the value found in DN(L213) RCs. Proton transfer was studied by measuring the dependence of k _AB ⁽²⁾ on the free energy for electron transfer (G_et). k _AB ⁽²⁾ was independent of G_et in DN(L213) RCs, but dependent on G_et in native and all suppressor RCs. This shows that proton transfer limits the k _AB ⁽²⁾ reaction with a rate of 0.1s^–1 in DN(L213) RCs but is not rate limiting and at least 10⁸-fold faster in native and 10⁵-fold faster in the suppressor RCs. The increased rate of proton transfer by the suppressor mutations are proposed to be due to: (i) a reduction in the barrier to proton transfer by providing a more negative electrostatic potential near Q_B ^–; and/or (ii) structural changes that permit fast proton transfer through the network of protonatable residues and water molecules near Q_B.     

The effect of humidity and light on cellular water relations and diffusion conductance of leaves ofTradescantia virginiana L.

Turgor (_p) and osmotic potential (_s) in epidermal and mesophyll cells, in-situ xylem water potential (-xyl) and gas exchange were measured during changes of air humidity and light in leaves ofTradescantia virginiana L., Turgor of single cells was determined using the pressure probe. Sap of individual cells was collected with the probe for measuring the freezing-point depression in a nanoliter osmometer. Turgor pressure was by 0.2 to 0.4 MPa larger in mesophyll cells than in epidermal cells. A water-potential gradient, which was dependent on the rate of transpiration, was found between epidermis and mesophyll and between tip and base of the test leaf. Step changes of humidity or light resulted in changes of epidermal and mesophyll turgor (_p-epi, _p-mes) and could be correlated with the transpiration rate. Osmotic potential was not affected by a step change of humidity or light. For the humidity-step experiments, stomatal conductance (g) increased with increasing epidermal turgor.g/_p-epi appeared to be constant over a wide range of epidermal turgor pressures. In light-step experiments this type of response was not found and stomatal conductance could increase while epidermal turgor decreased.Symbols E transpiration - g leaf conductance - w leaf/air vapour concentration difference - -epi water potential of epidermal cells - -mes water potential of mesophyll cells - -xyl water potential of xylem - _p-epi turgor pressure of epidermal cells - _p-mes turgor pressure of mesophyll cells - _s-epi osmotic potential of epidermal cells - _s-mes osmotic potential of mesophyll cells