pH optimum of the photosystem II H₂O oxidation reaction: effects of PsbO, the manganese-stabilizing protein, Cl- retention, and deprotonation of a component required for O₂ evolution activity

Hydroxide ion inhibits Photosystem II (PSII) activity by extracting Cl(-) from its binding site in the O(2)-evolving complex (OEC) under continuous illumination [Critchley, C., et al. (1982) Biochim. Biophys. Acta 682, 436]. The experiments reported here examine whether two subunits of PsbO, the manganese-stabilizing protein, bound to eukaryotic PSII play a role in protecting the OEC against OH(-) inhibition. The data show that the PSII binding properties of PsbO affect the pH optimum for O(2) evolution activity as well as the Cl(-) affinity of the OEC that decreases with an increasing pH. These results suggest that PsbO functions as a barrier against inhibition of the OEC by OH(-). Through facilitation of efficient retention of Cl(-) in PSII [Popelkova, H., et al. (2008) Biochemistry 47, 12593], PsbO influences the ability of Cl(-) to resist OH(-)-induced release from its site in the OEC. Preventing inhibition by OH(-) allows for normal (short) lifetimes of the S(2) and S(3) states in darkness [Roose, J. L., et al. (2011) Biochemistry 50, 5988] and for maximal steady-state activity by PSII. The data presented here indicate that activation of H(2)O oxidation occurs with a pK(a) of ～6.5, which could be a function of deprotonation of one or more amino acid residues that reside near the OEC active site on the D1 and CP43 intrinsic subunits of the PSII reaction center.     

Review of the fauna of Rotatoria,Cladocera, and Copepoda of the basin of the Anadyr’ River

The zooplankton composition is studied in the thermokarst, glacial and meteorite lakes, channels, former riverbeds, and hollows in the basin of Anadyr’. We found 174 taxa: 78, Rotatoria, 55, Cladocera, and 41, Copepoda. The most diverse is the lake fauna: 51 taxa of Rotatoria, 48, Cladocera, and 37, Copepoda. The thermokarst Lake Maiorskoe hosts 68 taxa: 31, Rotatoria, 14, Cladocera, and 23, Copepoda, wheras the cold ultraoligotrophic Lake El’gygytgyn features only one species of Cyclop of the group scutifer Cyclops neymanae Strel., and Rotatoria and Cladocera are present as allochtonous forms. The Copepoda illustrate the relations of the Anadyr’ fauna with those of Europe, North America, and Japan.     

Crystal structure of the O intermediate of the Leu93→Ala mutant of bacteriorhodopsin

The lifetime of the O intermediate of bacteriorhodopsin (BR) is extended by a factor of ～250 in the Leu93‐to‐Ala mutant (BR_L93A). To clarify the structural changes occurring in the last stage of the proton pumping cycle of BR, we crystallized BR_L93A into a hexagonal P622 crystal. Diffraction data from the unphotolyzed state showed that the deletion of three carbon atoms from Leu93 is compensated by the insertion of four water molecules in the cytoplasmic vicinity of retinal. This insertion of water is suggested to be responsible for the blue‐shifted λ_max (540 nm) of the mutant. A long‐lived substate of O with a red‐shifted λ_max (～565 nm) was trapped when the crystal of BR_L93A was flash‐cooled after illumination with green light. This substate (O_slow) bears considerable similarity to the M intermediate of native BR; that is, it commonly shows deformation of helix C and the FG loop, downward orientation of the side chain of Arg82, and disruption of the Glu194/Glu204 pair. In O_slow, however, the main chain of Lys216 is less distorted and retinal takes on the 13‐cis/15‐syn configuration. Another significant difference is seen in the pH dependence of the structure of the proton release group, the pK_a value of which is suggested to be much lower in O_slow than in M. Proteins 2012;. © 2012 Wiley Periodicals, Inc.     

Characterization of the Fourth α Isoform of the Na,K-ATPase

The Na,K-ATPase is a major ion transport protein found in higher eukaryotic cells. The enzyme is composed of two subunits, α and β, and tissue-specific isoforms exist for each of these, α1, α2 and α3 and β1, β2 and β3. We have proposed that an additional α isoform, α4, exists based on genomic and cDNA cloning. The mRNA for this gene is expressed in rats and humans, exclusively in the testis, however the expression of a corresponding protein has not been demonstrated. In the current study, the putative α4 isoform has been functionally characterized as a novel isoform of the Na,K-ATPase in both rat testis and in α4 isoform cDNA transfected 3T3 cells. Using an α4 isoform-specific polyclonal antibody, the protein for this novel isoform is detected for the first time in both rat testis and in transfected cell lines. Ouabain binding competition assays reveal the presence of high affinity ouabain receptors in both rat testis and in transfected cell lines that have identical K _D values. Further studies of this high affinity ouabain receptor show that it also has high affinities for both Na⁺ and K⁺. The results from these experiments definitively demonstrate the presence of a novel isoform of the Na,K-ATPase in testis. Received: 4 December 1998/Revised: 1 February 1999     

Does copper-d-penicillamine catalyze the dismutation of O2−?

It has been reported (M. Younes and U. Weser, 1977, Biochem. Biophys. Res. Commun.78, 1247–1253; E. Lengfelder and E. F. Elstner, 1978, Hoppe-Seyler's Z. Physiol. Chem.359, 751–757) that the complex [Cu(I)₈Cu(II)₆(D-penicillamine)₁₂Cl]^5?-efficiently catalyzes the dismutation of O₂^? and that this activity is resistant to both EDTA and CN^?. However, careful study has demonstrated that this complex is unable to catalyze the dismutation of O₂^?, but that it slowly decomposes to simpler copper complexes which are active. Moreover, the activity which is observed is suppressed by EDTA or by Chelex 100 treatment.     

A comparison of the retention of pathogenic Escherichia coli?O157 by sprouts,leaves and fruits

The retention (binding to or association with the plant) of Escherichia coli by cut leaves and fruits after vigorous water washing was compared with that by sprouts. Retention by fruits and leaves was similar but differed from retention by sprouts in rate, effect of wounding and requirement for poly-β,1-6-N-acetyl-D-glucosamine. Escherichia coli was retained by cut ends of lettuce leaves within 5 min while more than 1 h was required for retention by the intact epidermis of leaves and fruits, and more than 1 day for sprouts. Retention after 5 min at the cut leaf edge was specific for E. coli and was not shown by the plant-associated bacteria Agrobacterium tumefaciens and Sinorhizobium meliloti. Escherichia coli was retained by lettuce, spinach, alfalfa, bean, tomato, Arabidopsis thaliana, cucumber, and pepper leaves and fruits faster than by sprouts. Wounding of leaves and fruits but not sprouts increased bacterial retention. Mutations in the exopolysaccharide synthesis genes yhjN and wcaD reduced the numbers of bacteria retained. PgaC mutants were retained by cut leaves and fruits but not by sprouts. There was no significant difference in the retention of an O157 and a K12 strain by fruits or leaves. However, retention by sprouts of O157 strains was significantly greater than K12 strains. These findings suggest that there are differences in the mechanisms of E coli retention among sprouts, and leaves and fruits.     

Chitinozoan zonation of the Late Ordovician and the Early Silurian of the island of Anticosti, Québec, Canada

A reappraisal of chitinozoan distribution across the Ordovician-Silurian boundary on the Island of Anticosti has led to the recognition of a new zone, the Ancyrochitina ellisbayensis biozone, in the uppermost part of the Ellis Bay Formation. This biozone lies between the well defined Upper Ordovician Spinachitina taugourdeaui biozone and the lowest Silurian (Rhuddanian) Plectochitina nodifera biozone of the Becscie Formation. The occurrence of such diagnostic species as P. nodifera, Belonechitina postrobusta, Conochitina electa and Ancyrochitina ramosaspina in the Lower Silurian of Anticosti points to a close similarity to faunas in Estonia and north Latvia and indicates an age ranging from the Parakidograptus acuminatus to the Coronograptus cyphus in terms of graptolite zones. The chitinozoan biozonation harmonizes with that based on conodonts and, to a lesser extent, with the known graptolite faunal succession. Five new species: Ancyrochitina ellisbayensis sp. nov., Clathrochitina postconcinna sp. nov., Conochitina gunriveris sp. nov., Clathrochitina perexilis sp. nov., Bursachitina basiconcava sp. nov. and three species in open nomenclature are described.     

Lithocarpic acids O–S,five homo-cycloartane derivatives from the cupules of Lithocarpus polystachyus

Chemical investigation of the cupules of Lithocarpus polystachyus resulted in the identification of four 3,4-seco-homo-cycloartane and one homo-cycloartane derivatives named lithocarpic acids O–S. Their structures were determined based on extensive 1D/2D NMR, IR, MS spectroscopic analyses and chemical methods. Lithocarpic acid O (1) exhibited inhibitory activities on mouse and human isozymes of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) with IC₅₀ values of 0.49 and 1.1 μM, respectively.     

The Synthesis of Dimethyl Esters of dl-O,O′-Dimethylfukiic Acid and dl-O,O′-Dimethylepifukiic Acid

The synthesis of dimethyl esters of dl-O,O′-dimethylfukiic acid (11) and dl-O,O′-dimethylepifukiic acid (12) are described.     

Effects of the ΔF508 mutation on the structure,function, and folding of the first nucleotide-binding domain of CFTR

The fatal autosomal recessive disease cystic fibrosis (CF) is caused by mutations in the gene which encodes the cystic fibrosis transmembrane conductance regulator (CFTR). Many of these disease-causing mutations, including the deletion of F508 (F508) which accounts for approximately 70% of the disease alleles, occur in one of the two consensus nucleotide binding sequences. Peptide studies have directly demonstrated that the N-terminal nucleotide binding sequences bind adenine nucleotides. Structurally, circular dichroism spectropolarimetry indicates that this region of CFTR assumes a -stranded structure in solution. The F508 mutation causes a diminution in the amount of -stranded structure and a concomitant increase in the amount of random coil structure present, indicating that either the mutant peptide has a different native structure or that the conformational equilibrium is shifted toward a more disordered form. Furthermore, the mutant peptide is more sensitive to denaturation, indicating that F508 is a stability, or protein-folding mutant. Here we review these results and discuss their implications for interpreting the behavior of F508in situ and for the rational design of new CF drugs.     

The possession of coli surface antigen CS6 by enterotoxigenicEscherichia coli of serogroups O25, O27, O148, and O159: a possible colonization factor?

A total of 134 enterotoxigenicEscherichia coli (ETEC) of serogroups O25, O27, O148, and O159 were tested in the enzyme-linked immunosorbent assays for the colonization factor antigens I (CFA/I), CFA/II (coli surface antigens CS1, 2 and 3) and putative colonization factor (PCF) 8775 (CS4, 5 and 6). CS6 was detected without CS4 or CS5 in 94% of the strains of serogroup O25, 86% of strains of serogroup O27, 87% of strains of serogroup O148, and 29% of strains of serogroup, O159. The frequency with which CS6 occurs in ETEC of common serotypes without the antigens CS4 or CS5 suggests that it might be a colonization factor.     

Do the higher oxidation states of the photosynthetic O2-evolving system contain bound H2O?

A modified mass spectrometer was used to determine whether the higher oxidation states of the photosynthetic O2-evolving system contain substrate water that is not freely exchangeable with the external medium. Our data indicated that the higher oxidation states contain no appreciable bound, non-exchangeable H2O. This suggests that H2O oxidation takes place via a rapid, concerted, all-or-none mechanism rather than by a mechanism involving stable, partially oxidized, H2O-derived intermediates. These findings set definite constraints on possible mechanisms of O2 evolution.     

Cloning,characterisation and expression of the α-tubulin genes of the leech,Hirudo medicinalis

We have isolated two α-tubulin cDNAs from the leech, Hirudo medicinalis. Both encode putative proteins of 451 amino-acids which differ from each other at only two positions. Southern blotting suggests that there are only two α-tubulin genes in the leech. The genes contain two introns and, because of the extremely high homology of the nucleotide sequence from the second intron to the end of the genes, we have inferred that a gene conversion event about 9.5 million years ago has homogenised the Hirudo α-tubulin sequences. Using in situ hybridisation to tissue sections, we have shown that the two genes are probably expressed in all neurons of the leech ganglia and that their spatial distribution remains unchanged during neuronal regeneration. The deduced amino-acid sequences of the leech α-tubulins show that they have greatest similarity to those from a platyhelminth, echiuran and mollusc with rather less to arthropod α-tubulins. The protein sequences of the leech α-tubulins have been compared with representatives of those from across all phyla to determine if any specific feature labels certain isotypes of tubulin for neuronal expression.     

Formation of the Si-B bond: insertion reactions of silylenes into B-X(X = F,Cl, Br,O, and N) bonds

The insertion reactions of the silylene H₂Si with H₂BXH_n-1 (X?=?F, Cl, Br, O, N; n?=?1, 1, 1, 2, 3) have been studied by DFT and MP2 methods. The calculations show that the insertions occur in a concerted manner, forming H₂Si(BH₂)(XH_n-1). The essences of H₂Si insertions with H₂BXH_n-1 are the transfers of the σ electrons on the Si atom to the positive BH₂ group and the electrons of X into the empty p orbital on the Si atom in H₂Si. The order of reactivity in vacuum shows the barrier heights increase for the same-family element X from up to down and the same-row element X from right to left in the periodic table. The energies relating to the B-X bond in H₂BXH_n-1, and the bond energies of Si-X and Si-B in H₂Si(BH₂)(XH_n-1) may determine the preference of insertions of H₂Si into B-X bonds for the same-column element X or for the same-row element X. The insertion reactions in vacuum are similar to those in solvents, acetone, ether, and THF. The barriers in vacuum are lower than those in solvents and the larger polarities of solvents make the insertions more difficult to take place. Both in vacuum and in solvents, the silylene insertions are thermodynamically exothermic.

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Graphical Abstract The insertion process of H₂Si and H₂BXH_n-1(X?=?F, Cl, Br, O, and N; n?=?1, 1 , 1, 2, 3).