Flexible mating: cross-pollination affects sex-expression in a marine clonal plant

Many functionally hermaphroditic plants have evolved mechanisms to reduce interference between the sex functions and to optimize reproductive output. In addition to physical mechanisms such as the spatial (herkogamy) and temporal (dichogamy) separation of male and female functions, plasticity in sex expression by means of mate-recognition (flexible mating) could be important in plants with variable access to cross-pollen. This applies particularly to clonal plants because of their modular growth form. We experimentally tested for the effects of pollen source and vegetative neighbourhood on instantaneous sex ratio and seed production in the self-compatible clonal marine angiosperm Zostera marina L. To this end, we exposed the (monoecious) flowering shoots to self and cross-pollen and to neighbourhoods of their own and a mix of foreign vegetative shoots. Flowering shoots that had been exposed to cross-pollen showed (1) a significantly lower female/male ratio at peak flowering, evidence for mate-recognition, and (2) a significantly higher seed set by the end of the season. Both effects were independent of the genetic composition of their vegetative neighbourhood. The results suggest that Z. marina maintains a cryptic self-incompatibility system not previously described for angiosperms with sub-aqueous pollination. In Z. marina, and possibly other self-compatible clonal plant species, mate-recognition could be a means of increasing the out-crossing probability for flowering shoots with central positions within their clone.     

Oxidized LDL autoantibodies are related to apolipoprotein E phenotype,independently of postprandial change in plasma triglycerides and LDL size,among patients with angiographically verified coronary artery disease and healthy controls

OBJECTIVE: Oxidized low-density lipoprotein (LDL) autoantibodies (oxLDLab), apolipoprotein E (apoE) phenotype, postprandial triglyceride changes and LDL size are suggested to be risk factors for coronary artery disease (CAD). Our aim was to study the interaction between these new risk factors among patients with CAD and healthy controls. METHODS: oxLDLab from 31 men with angiographically verified CAD and 31 healthy men were analyzed by enzyme-linked immunosorbent assay. Isoelectric focusing and immunoblotting were used for apoE phenotyping. Triglyceride level was measured after 12 h of fasting and 3, 5 and 7 h after a high-fat meal. Nondenaturing gradient gel electrophoresis was used to separate LDL particles according to size. RESULTS: oxLD- Lab levels increased according to apoE phenotype in the following order: E2 < E3 < E4 (p = 0.004, ANOVA). The postprandial response of triglycerides, the size of LDL particles and the concentration of LDL and high-density lipoprotein (HDL) cholesterol did not differ between apoE phenotypes, and the use of these variables as covariates did not change the statistically significant difference in oxLDLab levels between apoE phenotypes (p = 0.01, ANCOVA). oxLDLab levels did not differ between the patient and control groups. CONCLUSION: We found an association between apoE allele epsilon2 and decreased levels of oxLDLab, which was independent of the postprandial response of triglycerides, the size of LDL particles and plasma LDL and HDL cholesterol levels. The mechanism by which apoE affects oxidation of LDL remains unknown.     

(E)-beta-ocimene and myrcene synthase genes of floral scent biosynthesis in snapdragon: function and expression of three terpene synthase genes of a new terpene synthase subfamily

Snapdragon flowers emit two monoterpene olefins, myrcene and (E)-beta-ocimene, derived from geranyl diphosphate, in addition to a major phenylpropanoid floral scent component, methylbenzoate. Emission of these monoterpenes is regulated developmentally and follows diurnal rhythms controlled by a circadian clock. Using a functional genomics approach, we have isolated and characterized three closely related cDNAs from a snapdragon petal-specific library that encode two myrcene synthases (ama1e20 and ama0c15) and an (E)-beta-ocimene synthase (ama0a23). Although the two myrcene synthases are almost identical (98%), except for the N-terminal 13 amino acids, and are catalytically active, yielding a single monoterpene product, myrcene, only ama0c15 is expressed at a high level in flowers and contributes to floral myrcene emission. (E)-beta-Ocimene synthase is highly similar to snapdragon myrcene synthases (92% amino acid identity) and produces predominantly (E)-beta-ocimene (97% of total monoterpene olefin product) with small amounts of (Z)-beta-ocimene and myrcene. These newly isolated snapdragon monoterpene synthases, together with Arabidopsis AtTPS14 (At1g61680), define a new subfamily of the terpene synthase (TPS) family designated the Tps-g group. Members of this new Tps-g group lack the RRx(8)W motif, which is a characteristic feature of the Tps-d and Tps-b monoterpene synthases, suggesting that the reaction mechanism of Tps-g monoterpene synthase product formation does not proceed via an RR-dependent isomerization of geranyl diphosphate to 3S-linalyl diphosphate, as shown previously for limonene cyclase. Analyses of tissue-specific, developmental, and rhythmic expression of these monoterpene synthase genes in snapdragon flowers revealed coordinated regulation of phenylpropanoid and isoprenoid scent production.     

Structure of the large subunit of class Ib ribonucleotide reductase from Salmonella typhimurium and its complexes with allosteric effectors

The three-dimensional structure of the large subunit of the first member of a class Ib ribonucleotide reductase, R1E of Salmonella typhimurium, has been determined in its native form and together with three allosteric effectors. The enzyme contains the characteristic ten-stranded alpha/beta-barrel with catalytic residues at a finger loop in its center and with redox-active cysteine residues at two adjacent barrel strands. Structures where the redox-active cysteine residues are in reduced thiol form and in oxidized disulfide form have been determined revealing local structural changes. The R1E enzyme differs from the class Ia enzyme, Escherichia coli R1, by not having an overall allosteric regulation. This is explained from the structure by differences in the N-terminal domain, which is about 50 residues shorter and lacks the overall allosteric binding site. R1E has an allosteric substrate specificity regulation site and the binding site for the nucleotide effectors is located at the dimer interface similarly as for the class Ia enzymes. We have determined the structures of R1E in the absence of effectors and with dTTP, dATP and dCTP bound. The low affinity for ATP at the specificity site is explained by a tyrosine, which hinders nucleotides containing a 2'-OH group to bind.     

The 1.15A crystal structure of the Staphylococcus aureus methionyl-aminopeptidase and complexes with triazole based inhibitors

Methionyl aminopeptidases (MetAPs) represent a unique class of protease that are responsible for removing the N-terminal methionine residue from proteins and peptides. There are two major classes of MetAPs (type I and type II) described and each class can be subdivided into two subclasses. Eukaryotes contain both the type I and type II MetAPs, whereas prokaryotes possess only the type I enzyme. Due to the physiological importance of these enzymes there is considerable interest in inhibitors to be used as antiangiogenic and antimicrobial agents. Here, we describe the 1.15A crystal structure of the Staphylococcus aureus MetAP-I as an apo-enzyme and its complexes with various 1,2,4-triazole-based derivatives at high-resolution. The protein has a typical "pita-bread" fold as observed for the other MetAP structures. The inhibitors bind in the active site with the N1 and N2 atoms of the triazole moiety complexing two divalent ions. The 1,2,4-triazols represent a novel class of potent non-peptidic inhibitors for the MetAP-Is.     

Efficient conformational sampling of local side-chain flexibility

Side-chain flexibility of ligand-binding sites needs to be considered in the rational design of novel inhibitors. We have developed a method to generate conformational ensembles that efficiently sample local side-chain flexibility from a single crystal structure. The rotamer-based approach is tested here for the S1' pocket of human collagenase-1 (MMP-1), which is known to undergo conformational changes in multiple side-chains upon binding of certain inhibitors. First, a raw ensemble consisting of a large number of conformers of the S1' pocket was generated using an exhaustive search of rotamer combinations on a template crystal structure. A combination of principal component analysis and fuzzy clustering was then employed to successfully identify a core ensemble consisting of a low number of representatives from the raw ensemble. The core ensemble contained geometrically diverse conformers of stable nature, as indicated in several cases by a relative energy lower than that of the minimised template crystal structure. Through comparisons with X-ray crystallography and NMR structural data we show that the core ensemble occupied a conformational space similar to that observed under experimental conditions. The synthetic inhibitor RS-104966 is known to induce a conformational change in the side-chains of the S1' pocket of MMP-1 and could not be docked in the template crystal structure. However, the experimental binding mode was reproduced successfully using members of the core ensemble as the docking target, establishing the usefulness of the method in drug design.     

Molecular movement of the voltage sensor in a K channel

The X-ray crystallographic structure of KvAP, a voltage-gated bacterial K channel, was recently published. However, the position and the molecular movement of the voltage sensor, S4, are still controversial. For example, in the crystallographic structure, S4 is located far away (>30 A) from the pore domain, whereas electrostatic experiments have suggested that S4 is located close (<8 A) to the pore domain in open channels. To test the proposed location and motion of S4 relative to the pore domain, we induced disulphide bonds between pairs of introduced cysteines: one in S4 and one in the pore domain. Several residues in S4 formed a state-dependent disulphide bond with a residue in the pore domain. Our data suggest that S4 is located close to the pore domain in a neighboring subunit. Our data also place constraints on possible models for S4 movement and are not compatible with a recently proposed KvAP model.     

B7-H1 (programmed death-1 ligand) on dendritic cells is involved in the induction and maintenance of T cell anergy

In an effort to identify immunoregulatory molecules on dendritic cells (DC), we generated and screened for mAbs capable of modulating the T cell stimulatory function of DC. A particularly interesting mAb was mAb DF272. It recognizes monocyte-derived DC, but not blood monocytes or lymphocytes, and has profound immunomodulatory effects on DC. Treatment of DC with intact IgG or Fab of mAb DF272 enhanced their T cell stimulatory capacity. This effect on DC was accompanied by neither an up-regulation of costimulatory molecules such as B7.1 (CD80), B7.2 (CD86), and MHC class II molecules nor by an induction of cytokine production, including IL-1, TNF-alpha, IL-10, and IL-12. Moreover, the well-established inhibitory function of IL-10-treated DC could be reverted with mAb DF272. Even T cells, anergized because of stimulation with IL-10-treated DC, could be reactivated and induced to proliferate upon stimulation with mAb DF272-treated DC. Furthermore, mAb DF272-treated DC favored the induction of a type-1 cytokine response in T cells and inhibited IL-10 production. By using a retrovirus-based cDNA expression library generated from DC, we cloned and sequenced the mAb DF272-defined cell surface receptor and could demonstrate that it is identical with B7-H1 (programmed death-1 ligand), a recently identified new member of the B7 family of costimulatory molecules. Our results thus demonstrate that the mAb DF272-defined surface molecule B7-H1 represents a unique receptor structure on DC that might play a role in the induction and maintenance of T cell anergy.     

ATM is not required in somatic hypermutation of VH,but is involved in the introduction of mutations in the switch mu region

Class switch recombination (CSR) and somatic hypermutation (SHM) are mechanistically related processes that share common key factors such as activation-induced cytidine deaminase. We have previously shown a role for ATM (mutated in ataxia-telangiectasia) in CSR. In this paper we show that the frequency, distribution, and nature of base pair substitutions in the Ig variable (V) heavy chain genes in ataxia-telangiectasia patients are largely similar to those in normal donors, suggesting a normal SHM process. Characterization of the third complementarity-determining region in B cells from ataxia-telangiectasia patients also shows a normal V(D)J recombination process. SHM-like mutations could be identified in the switch (S) mu region (up to several hundred base pairs upstream of the S mu -S(alpha) breakpoints) in normal in vivo switched human B cells. In the absence of ATM, mutations can still be found in this region, but at less than half the frequency of that in normal donors. The latter mutations are mainly due to transitions (86% compared with 58% in controls) and are biased to A or T nucleotides. An ATM-dependent mechanism, different from that generating SHM in V genes, is therefore likely to be involved in introducing SHM-like mutations in the S region. ATM may thus be one of the factors that is not shared by the CSR and SHM processes.