Structural and functional characterization of ferredoxin-NADP+-oxidoreductase using knock-out mutants of Arabidopsis

In Arabidopsis thaliana, the chloroplast-targeted enzyme ferredoxin-NADP+-oxidoreductase (FNR) exists as two isoforms, AtLFNR1 and AtLFNR2, encoded by the genes At5g66190 and At1g20020, respectively. Both isoforms are evenly distributed between the thylakoids and soluble stroma, and they are separated by two-dimensional electrophoresis in four distinct spots, suggesting post-translational modification of both isoforms. To reveal the functional specificity of AtLFNR1, we have characterized the T-DNA insertion mutants with an interrupted At5g66190 gene. Absence of AtLFNR1 resulted in a reduced size of the rosette with pale green leaves, which was accompanied by a low content of chlorophyll and light-harvesting complex proteins. Also the photosystem I/photosystem II (PSI/PSII) ratio was significantly lower in the mutant, but the PSII activity, measured as the F(V)/F(M) ratio, remained nearly unchanged and the excitation pressure of PSII was lower in the mutants than in the wild type. A slow re-reduction rate of P700 measured in the mutant plants suggested that AtLFNR1 is involved in PSI-dependent cyclic electron flow. Impaired function of FNR also resulted in decreased capacity for carbon fixation, whereas nitrogen metabolism was upregulated. In the absence of AtLFNR1, we found AtLFNR2 exclusively in the stroma, suggesting that AtLFNR1 is required for membrane attachment of FNR. Structural modeling supports the formation of a AtLFNR1-AtLFNR2 heterodimer that would mediate the membrane attachment of AtLFNR2. Dimer formation, in turn, might regulate the distribution of electrons between the cyclic and linear electron transfer pathways according to environmental cues.     

Removal of microcystin-LR by strains of metabolically active probiotic bacteria

The ability of specific strains of probiotic bacteria to remove the cyanobacterial peptide toxin microcystin-LR from aqueous solutions was assessed. Lactobacillus rhamnosus strains GG and LC-705, Bifidobacterium longum 46, Bifidobacterium lactis 420 and Bifidobacterium lactis Bb12 were shown to be the most effective in toxin removal among 11 tested strains. The highest removal percentage of microcystin-LR was 58.1%, observed with B. lactis Bb12 (toxin concentration 100 microg L(-1), 10(10) CFU mL(-1), 37 degrees C, 24 h). Freshly cultured bacteria were shown to be more efficient in microcystin removal than lyophilized or nonviable bacteria. Removal of microcystin-LR was shown to be dependent on both temperature and bacterial concentration. It is concluded that some of the tested strains have good potential in removing microcystins from aqueous solutions.     

Can genetically based clines in plant defence explain greater herbivory at higher latitudes?

Greater plant defence is predicted to evolve at lower latitudes in response to increased herbivore pressure. However, recent studies question the generality of this pattern. In this study, we tested for genetically based latitudinal clines in resistance to herbivores and underlying defence traits of Oenothera biennis. We grew plants from 137 populations from across the entire native range of O. biennis. Populations from lower latitudes showed greater resistance to multiple specialist and generalist herbivores. These patterns were associated with an increase in total phenolics at lower latitudes. A significant proportion of the phenolics were driven by the concentrations of two major ellagitannins, which exhibited opposing latitudinal clines. Our analyses suggest that these findings are unlikely to be explained by local adaptation of herbivore populations or genetic variation in phenology. Rather greater herbivory at high latitudes can be explained by latitudinal clines in the evolution of plant defences.     

Model structures of alpha-2 adrenoceptors in complex with automatically docked antagonist ligands raise the possibility of interactions dissimilar from agonist ligands

Antagonist binding to alpha-2 adrenoceptors (alpha2-ARs) is not well understood. Structural models were constructed for the three human alpha2-AR subtypes based on the bovine rhodopsin X-ray structure. Twelve antagonist ligands (including covalently binding phenoxybenzamine) were automatically docked to the models. A hallmark of agonist binding is the electrostatic interaction between a positive charge on the agonist and the negatively charged side chain of D3.32. For antagonist binding, ion-pair formation would require deviations of the models from the rhodopsin structural template, e.g., a rotation of TM3 to relocate D3.32 more centrally within the binding cavity, and/or creation of new space near TM2/TM7 such that antagonists would be shifted away from TM5. Thus, except for the quinazolines, antagonist ligands automatically docked to the model structures did not form ion-pairs with D3.32. This binding mode represents a valid alternative, whereby the positive charge on the antagonists is stabilized by cation-pi interactions with aromatic residues (e.g., F6.51) and antagonists interact with D3.32 via carboxylate-aromatic interactions. This binding mode is in good agreement with maps derived from a molecular interaction library that predicts favorable atomic contacts; similar interaction environments are seen for unrelated proteins in complex with ligands sharing similarities with the alpha2-AR antagonists.     

Jararhagin-derived RKKH peptides induce structural changes in alpha1I domain of human integrin alpha1beta1

Integrin alpha(1)beta(1) is one of four collagen-binding integrins in humans. Collagens bind to the alphaI domain and in the case of alpha(2)I collagen binding is competitively inhibited by peptides containing the RKKH sequence and derived from the metalloproteinase jararhagin of snake venom from Bothrops jararaca. In alpha(2)I, these peptides bind near the metal ion-dependent adhesion site (MIDAS), where a collagen (I)-like peptide is known to bind; magnesium is required for binding. Published structures of the ligand-bound "open" conformation of alpha(2)I differs significantly from the "closed" conformation seen in the structure of apo-alpha(2)I near MIDAS. Here we show that two peptides, CTRKKHDC and CARKKHDC, derived from jararhagin also bind to alpha(1)I and competitively inhibit collagen I binding. Furthermore, calorimetric and fluorimetric measurements show that the structure of the complex of alpha(1)I with Mg(2+) and CTRKKHDC differs from structure in the absence of peptide. A comparison of the x-ray structure of apo-alpha(1)I ("closed" conformation) and a model structure of the alpha(1)I ("open" conformation) based on the closely related structure of alpha(2)I reveals that the binding site is partially blocked to ligands by Glu(255) and Tyr(285) in the "closed" structure, whereas in the "open" structure helix C is unwound and these residues are shifted, and the "RKKH" peptides fit well when docked. The "open" conformation of alpha(2)I resulting from binding a collagen (I)-like peptide leads to exposure of hydrophobic surface, also seen in the model of alpha(1)I and shown experimentally for alpha(1)I using a fluorescent hydrophobic probe.     

Plants express a lipid transfer protein with high similarity to mammalian sterol carrier protein-2

This is the first report describing the cloning and characterization of sterol carrier protein-2 (SCP-2) from plants. Arabidopsis thaliana SCP-2 (AtSCP-2) consists of 123 amino acids with a molecular mass of 13.6 kDa. AtSCP-2 shows 35% identity and 56% similarity to the human SCP-2-like domain present in the human D-bifunctional protein (DBP) and 30% identity and 54% similarity to the human SCP-2 encoded by SCP-X. The presented structural models of apo-AtSCP-2 and the ligand-bound conformation of AtSCP-2 reveal remarkable similarity with two of the structurally known SCP-2s, the SCP-2-like domain of human DBP and the rabbit SCP-2, correspondingly. The AtSCP-2 models in both forms have a similar hydrophobic ligand-binding tunnel, which is extremely suitable for lipid binding. AtSCP-2 showed in vitro transfer activity of BODIPY-phosphatidylcholine (BODIPY-PC) from donor membranes to acceptor membranes. The transfer of BODIPY-PC was almost completely inhibited after addition of 1-palmitoyl 2-oleoyl phosphatidylcholine or ergosterol. Dimyristoyl phosphatidic acid, stigmasterol, steryl glucoside, and cholesterol showed a moderate to marginal ability to lower the BODIPY-PC transfer rate, and the single chain palmitic acid and stearoyl-coenzyme A did not affect transfer at all. Expression analysis showed that AtSCP-2 mRNA is accumulating in most plant tissues. Plasmids carrying fusion genes between green fluorescent protein and AtSCP-2 were transformed with particle bombardment to onion epidermal cells. The results from analyzing the transformants indicate that AtSCP-2 is localized to peroxisomes.     

Nε-Modified lysine containing inhibitors for SIRT1 and SIRT2

Sirtuins catalyze the NAD⁺ dependent deacetylation of N^ε-acetyl lysine residues to nicotinamide, O′-acetyl-ADP-ribose (OAADPR) and N^ε-deacetylated lysine. Here, an easy-to-synthesize Ac-Ala-Lys-Ala sequence has been used as a probe for the screening of novel N^ε-modified lysine containing inhibitors against SIRT1 and SIRT2. N^ε-Selenoacetyl and N^ε-isothiovaleryl were the most potent moieties found in this study, comparable to the widely studied N^ε-thioacetyl group. The N^ε-3,3-dimethylacryl and N^ε-isovaleryl moieties gave significant inhibition in comparison to the N^ε-acetyl group present in the substrates. In addition, the studied N^ε-alkanoyl, N^ε-α,β-unsaturated carbonyl and N^ε-aroyl moieties showed that the acetyl binding pocket can accept rather large groups, but is sensitive to even small changes in electronic and steric properties of the N^ε-modification. These results are applicable for further screening of N^ε-acetyl analogues.     

Intrinsic Properties of So-Called Dormant Probiotic Bacteria, Determined by Flow Cytometric Viability Assays

Plate counting and four culture-independent flow cytometric assays were used to determine the viability and intrinsic properties of three probiotic strains during storage. The strains showed reduction in plate counts but were able to maintain esterase activity, intact cytoplasmic membrane, and pH gradient. The apparently uncultivable probiotic cells were active and stress resistant.     

Crystal structure of Streptococcus mutans pyrophosphatase: a new fold for an old mechanism

BACKGROUND: Streptococcus mutans pyrophosphatase (Sm-PPase) is a member of a relatively uncommon but widely dispersed sequence family (family II) of inorganic pyrophosphatases. A structure will answer two main questions: is it structurally similar to the family I PPases, and is the mechanism similar? RESULTS: The first family II PPase structure, that of homodimeric Sm-PPase complexed with metal and sulfate ions, has been solved by X-ray crystallography at 2.2 A resolution. The tertiary fold of Sm-PPase consists of a 189 residue alpha/beta N-terminal domain and a 114 residue mixed beta sheet C-terminal domain and bears no resemblance to family I PPase, even though the arrangement of active site ligands and the residues that bind them shows significant similarity. The preference for Mn2+ over Mg2+ in family II PPases is explained by the histidine ligands and bidentate carboxylate coordination. The active site is located at the domain interface. The C-terminal domain is hinged to the N-terminal domain and exists in both closed and open conformations. CONCLUSIONS: The active site similiarities, including a water coordinated to two metal ions, suggest that the family II PPase mechanism is "analogous" (not "homologous") to that of family I PPases. This is a remarkable example of convergent evolution. The large change in C-terminal conformation suggests that domain closure might be the mechanism by which Sm-PPase achieves specificity for pyrophosphate over other polyphosphates.     

Genetic effects of supportive stockings on native pikeperch populations in boreal lakes--cases, three different outcomes

The genetic consequences and gene flow of pikeperch (Sander lucioperca) stocking were assessed in three boreal lakes based on admixture model analysis and comparison of the pre- and post-release patterns of genetic variability at 9 DNA microsatellite loci in the recipient populations. In two out of the three cases, the releases of fish from foreign populations caused significant changes in the genetic structure of the recipient population. The largest changes were observed in Lake Ouluj?rvi, where the post-release sample was almost identical to the released Lake Vanajanselk? population, and about 90% of the catch was composed of the released population. The genetic composition of Lake Lohjanj?rvi pikeperch also shifted markedly towards that of the released Lake Vanajanselk? population, and about half of the later catch was of released Vanajanselk? origin. In Lake Vanajanselk?, in contrast, releases of pikeperch from lakes Painio and Averia had only a small impact on the genetic structure of the pikeperch population. These results indicate that the current stocking practices create an effective artificial gene flow that may strongly shape and reduce the genetic differentiation among the remaining native pikeperch populations. A common feature of all three cases was the lack of prior appraisal of the potential genetic and ecological risks in relation to the expected benefits of the release programmes.