Selective flowers to attract and enhance Telenomus laeviceps (Hymenoptera: Scelionidae): a released biocontrol agent of Mamestra brassicae (Lepidoptera: Noctuidae).

The importance of the right food source for the survival and reproduction of certain insect species is well documented. In the case of biocontrol agents, this is even more important in order to reach a high predation or parasitation performance. The egg parasitoid Telenomus laeviceps (Förster, 1861) (Hymenoptera: Scelionidae) is a promising candidate for mass release as a biological control agent of the cabbage moth Mamestra brassicae (Linnaeus, 1758) (Lepidoptera: Noctuidae). However, adult T. laeviceps need a sugar-rich food source to increase their parasitation performance and produce a good amount of female offspring. Released biocontrol agents were shown to benefit from conservation biocontrol, which includes the provision of selected flowers as nectar resources for beneficial insects. In Switzerland, Centaurea cyanus L. (Asteraceae), Fagopyrum esculentum Moench (Polygonaceae) and Vicia sativa L. (Fabaceae) are successfully implemented in the field to attract and promote natural enemies of different cabbage pests. In this study, we investigated the potential of these selected flowers to attract and promote T. laeviceps under laboratory conditions. In Y-tube olfactometer experiments, we first tested whether the three nectar providing plant species are attractive to T. laeviceps. Furthermore, we assessed their effects on survival and parasitation performance of adult T. laeviceps. We found that flowers of F. esculentum and C. cyanus were attractive in contrast to V. sativa. Also fecundity and the number of female offspring produced were higher for females kept on F. esculentum and C. cyanus than on V. sativa. In contrast, survival was similar on all treatments. Our findings present a further key step towards the implementation of T. laeviceps as a biocontrol agent.

Bioavailability of detomidine administered sublingually to horses as an oromucosal gel.

The objective of the study was to determine the absorption, bioavailability and sedative effect of detomidine administered to horses as an oromucosal gel compared to intravenous and intramuscular administration of detomidine injectable solution. The study was open and randomized, with three sequences crossover design. Nine healthy horses were given 40 µg/kg detomidine intravenously, intramuscularly or administered under the tongue with a 7-day wash-out period between treatments. Blood samples were collected before and after drug administration for the measurement of detomidine concentrations in serum. The effects of the route of administration on heart rate and rhythm were evaluated and the depth of sedation assessed. Mean (±SD) bioavailability of detomidine was 22% (±5.3%) after sublingual administration and 38.2% (±7.9%) after intramuscular administration. The sedative effects correlated with detomidine concentrations regardless of the route of administration. We conclude that less detomidine is absorbed when given sublingually than when given intramuscularly, because part of it does not reach the circulation. Sublingual administration of detomidine oromucosal gel at 40 µg/kg produces safe sedation in horses. Slow absorption leads to fewer and less pronounced adverse effects than the more rapid absorption after intramuscular injection.

Diffusion MRI Microstructural Abnormalities at Term-Equivalent Age Are Associated with Neurodevelopmental Outcomes at 3 Years of Age in Very Preterm Infants.

BACKGROUND AND PURPOSE: Microstructural white matter abnormalities on DTI using Tract-Based Spatial Statistics at term-equivalent age are associated with cognitive and motor outcomes at 2 years of age or younger. However, neurodevelopmental tests administered at such early time points are insufficiently predictive of mild-moderate motor and cognitive impairment at school age. Our objective was to evaluate the microstructural antecedents of cognitive and motor outcomes at 3 years' corrected age in a cohort of very preterm infants. MATERIALS AND METHODS: We prospectively recruited 101 very preterm infants (<32 weeks' gestational age) and performed DTI at term-equivalent age. The Differential Ability Scales, 2nd ed, Verbal and Nonverbal subtests, and the Bayley Scales of Infant and Toddler Development, 3rd ed, Motor subtest, were administered at 3 years of age. We correlated DTI metrics from Tract-Based Spatial Statistics with the Bayley Scales of Infant and Toddler Development, 3rd ed, and the Differential Ability Scales, 2nd ed, scores with correction for multiple comparisons. RESULTS: Of the 101 subjects, 84 had high-quality DTI data, and of these, 69 returned for developmental testing (82%). Their mean (SD) gestational age was 28.4 (2.5) weeks, and birth weight was 1121.4 (394.1) g. DTI metrics were significantly associated with Nonverbal Ability in the corpus callosum, posterior thalamic radiations, fornix, and inferior longitudinal fasciculus and with Motor scores in the corpus callosum, internal and external capsules, posterior thalamic radiations, superior and inferior longitudinal fasciculi, cerebral peduncles, and corticospinal tracts. CONCLUSIONS: We identified widespread microstructural white matter abnormalities in very preterm infants at term that were significantly associated with cognitive and motor development at 3 years' corrected age.

Nanoscale complexity of phospholipid monolayers investigated by near-field scanning optical microscopy.

Near-field scanning optical microscopy of phospholipid monolayers doped with fluorescent lipid analogs reveals previously undescribed features in various phases, including a concentration gradient at the liquid-expanded/liquid-condensed domain boundary and weblike structures in the solid-condensed phase. Presumably, the web structures are grain boundaries between crystalline solid lipid. These structures are strongly modulated by the addition of low concentrations of cholesterol and ganglioside GM1 in the monolayer.

Biophysical approaches to membrane protein structure determination.

Recently, there have been several technical advances in the use of solution and solid-state NMR spectroscopy to determine the structures of membrane proteins. The structures of several isolated transmembrane (TM) helices and pairs of TM helices have been solved by solution NMR methods. Similarly, the complete folds of two TM beta-barrel proteins with molecular weights of 16 and 19 kDa have been determined by solution NMR in detergent micelles. Solution NMR has also provided a first glimpse at the dynamics of an integral membrane protein. Structures of individual TM helices have also been determined by solid-state NMR. A combination of NMR with site-directed spin-label electron paramagnetic resonance or Fourier transform IR spectroscopy allows one to assemble quite detailed protein structures in the membrane.

Membrane insertion and lateral mobility of synthetic amphiphilic signal peptides in lipid model membranes.

Amphiphilic signal sequences with the potential to form alpha-helices with a polar, charged face and an apolar face are common in proteins which are imported into mitochondria, in the PTS permeases of bacteria, and in bacterial rhodopsins. Synthetic peptides of such sequences partition into the surface region of lipid membranes where they can adopt different secondary structures. A finely controlled balance of electrostatic and hydrophobic interactions determines the 'affinity' of amphiphilic signal peptides for lipid membranes, as well as the structure, orientation and depth of penetration of these peptides in lipid bilayer membranes. The ability of an individual peptide to associate with lipid bilayer membranes in several different modes is, most likely, a general feature of amphiphilic signal peptides and is reflected in several common physical properties of their amino acid sequences.

Formation of supported planar bilayers by fusion of vesicles to supported phospholipid monolayers.

A technique for the production of supported phospholipid bilayers by adsorption and fusion of small unilamellar vesicles to supported phospholipid monolayers on quartz is described. The physical properties of these supported bilayers are compared with those of supported bilayers which are prepared by Langmuir-Blodgett deposition or by direct vesicle fusion to plain quartz slides. The time courses of vesicle adsorption, fusion and desorption are followed by total internal reflection fluorescence microscopy and the lateral diffusion of the lipids in the adsorbed layers by fluorescence recovery after photobleaching. Complete supported bilayers can be formed with phosphatidylcholine vesicles at concentrations as low as 35 microM. However, the adsorption, fusion and desorption kinetics strongly depend on the used lipid, NaCl and Ca2+ concentrations. Asymmetric negatively charged supported bilayers can be produced by incubating a phosphatidylcholine monolayer with vesicles composed of 80% phosphatidylcholine and 20% phosphatidylglycerol. Adsorbed vesicles can be removed by washing with buffer. The measured fluorescence intensities after washing are consistent with single supported bilayers. The lateral diffusion experiments confirm that continuous extended bilayers are formed by the monolayer-fusion technique. The measured lateral diffusion coefficient of NBD-labeled phosphatidylethanolamine is (3.6 +/- 0.5) x 10(-8) cm2/s in supported phosphatidylcholine bilayers, independent of the method by which the bilayers were prepared.

pH-dependent self-association of influenza hemagglutinin fusion peptides in lipid bilayers.

We have recently designed a host-guest peptide system that allows us to quantitatively measure the energetics of interaction of viral fusion peptides with lipid bilayers. Here, we show that fusion peptides of influenza hemagglutinin reversibly associate with one another at membrane surfaces above critical surface concentrations, which range from one to five peptides per 1000 lipids in the systems that we investigated. It is further demonstrated by using circular dichroism and Fourier transform infrared spectroscopy that monomeric peptides insert into the bilayers in a predominantly alpha-helical conformation, whereas self-associated fusion peptides adopt predominantly antiparallel beta-sheet structures at the membrane surface. The two forms are readily interconvertible and the equilibrium between them is determined by the pH and ionic strength of the surrounding solution. Lowering the pH favors the monomeric alpha-helical conformation, whereas increasing the ionic strength shifts the equilibrium towards the membrane-associated beta-aggregates. The binding data are interpreted in terms of a cooperative binding model that yields free energies of insertion and free energies of self-association for each of the peptides studied at pH 7.4 and pH 5. At pH 5 and 35 mM ionic strength, the insertion energy of the 20 residue influenza hemagglutinin fusion peptide is -7.2 kcal/mol and the self-association energy is -1.9 kcal/mol. We propose that self-association of fusion peptides could be a major driving force for recruiting a small number of hemagglutinin trimers into a fusion site.

Reversible pH-dependent conformational change of reconstituted influenza hemagglutinin.

Fusion between influenza virus and cell membranes is mediated by a major acid-induced conformational change of the spike glycoprotein of the viral envelope, hemagglutinin (HA). The conformational change of HA is commonly believed to be a kinetically controlled irreversible process, although the experimental evidence for this is controversial. Here we show by polarized infrared spectroscopy that the previously described acid-induced inclination of HA reconstituted in supported phospholipid bilayers is reversible in the absence, but irreversible in the presence, of bound target membranes. We also demonstrate reversible pH-dependent changes in the capability of reconstituted HA to bind target membranes. These results support a thermodynamically controlled mechanism of the conformational change of HA and provide new insight into the understanding of the energetics of influenza-mediated membrane fusion.

Structural changes in a secretory phospholipase A2 induced by membrane binding: a clue to interfacial activation?

Activation of phospholipase A2 (PLA2) upon binding to phospholipid assemblies is poorly understood. X-ray crystallography revealed little structural change in the enzyme upon binding of monomeric substrate analogs, whereas small conformational changes in PLA2 complexed with substrate micelles and an inhibitor were found by NMR. The structure of PLA2 bound to phospholipid bilayers is not known. Here we uncover by FTIR spectroscopy a splitting in the alpha-helical region of the amide I absorbance band of PLA2 upon binding to lipid bilayers. We provide evidence that a higher frequency component, which is only observed in the membrane-bound enzyme, is a property of more flexible helices. Formation of flexible helices upon interaction with the membrane is likely to contribute to PLA2 activation.

New approach for atomic force microscopy of membrane proteins. The imaging of cholera toxin.

We demonstrate that supported synthetic phospholipid bilayers, which are stabilized by lateral cross-linking in both leaflets, can be used for specimen preparation for atomic force microscopy of purified membrane proteins with high stability and excellent reproducibility under water or low-salt buffer. A bilayer containing 1,2-dipentacosa-10,12-diynoyl-phosphatidylcholine and 20 mol % ganglioside (GM1) was transferred onto the surface of mica from a Langmuir trough. Cholera toxin, both the B-subunit and the complete molecular randomly bound to the gangliosides, were imaged by atomic force microscopy in solution with a resolution of better than 2 nm. The pentameric structure of the B-subunit oligomers was well resolved. This result indicates that, with this preparation procedure, other membrane proteins may be studied at intermediate to high resolution under physiologically relevant conditions without the need for crystallization.

Peptide mimics of SNARE transmembrane segments drive membrane fusion depending on their conformational plasticity.

SNARE proteins are essential for different types of intracellular membrane fusion. Whereas interaction between their cytoplasmic domains is held responsible for establishing membrane proximity, the role of the transmembrane segments in the fusion process is currently not clear. Here, we used an in vitro approach based on lipid mixing and electron microscopy to examine a potential fusogenic activity of the transmembrane segments. We show that the presence of synthetic peptides representing the transmembrane segments of the presynaptic soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) synaptobrevin II (also referred to as VAMP II) or syntaxin 1A, but not of an unrelated control peptide, in liposomal membranes drives their fusion. Liposome aggregation by millimolar Ca(2+) concentrations strongly potentiated the effect of the peptides; this indicates that juxtaposition of the bilayers favours their fusion in the absence of the cytoplasmic SNARE domains. Peptide-driven fusion is reminiscent of natural membrane fusion, since it was suppressed by lysolipid and involved both bilayer leaflets. This suggests transient presence of a hemifusion intermediate followed by complete membrane merger. Structural studies of the peptides in lipid bilayers performed by Fourier transform infrared spectroscopy indicated mixtures of alpha-helical and beta-sheet conformations. In isotropic solution, circular dichroism spectroscopy showed the peptides to exist in a concentration-dependent equilibrium of alpha-helical and beta-sheet structures. Interestingly, the fusogenic activity decreased with increasing stability of the alpha-helical solution structure for a panel of variant peptides. Thus, structural plasticity of transmembrane segments may be important for SNARE protein function at a late step in membrane fusion.

Structural studies on membrane-embedded influenza hemagglutinin and its fragments.

The mechanism of influenza virus hemagglutinin (HA)-mediated membrane fusion has been inferred in part from studies examining pH-induced structural changes in soluble HA derivatives lacking the viral membrane anchor and, sometimes, the fusion peptide (the C- and N-terminal residues of the HA2 chain, respectively). To reconcile structure-based mechanisms of HA-mediated membrane fusion with structural implications of functional studies performed on membrane-embedded HA, we have undertaken attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopic analyses of membrane-embedded HA (strain X:31) and its fragments reconstituted into supported lipid bilayers. The fragments correspond to proteolytic products with the majority of the HA1 chain and, in some cases, the fusion peptide removed (THA2 and THA2F-, respectively). In combination with R18 fluorescence dequenching to monitor the functional implications of HA1 subunit removal, we have assessed the influence of pH and target membrane presentation on the secondary structures, orientations relative to the membrane, and dynamics of these molecules. We find that X:31 HA is more tilted towards the plane of the membrane under fusion than under resting conditions, that the fitting of HA depends on the presence of the HA1 chain, that the residues connecting the membrane-inserted fusion peptide with the crystallographically determined coiled coil probably adopt an alpha-helical conformation, and that several changes in the secondary structure and the amide H/D exchange kinetics occur as a result of acidification and target membrane presentation, which can be interpreted as small changes and a release of strain in the static and dynamic structure of membrane-bound HA. THA2 mediatcs fusion, but less efficiently and with less pH-selectivity than HA.

pH-induced conformational changes of membrane-bound influenza hemagglutinin and its effect on target lipid bilayers.

Influenza virus hemagglutinin (HA) has served as a paradigm for both pH-dependent and -independent viral membrane fusion. Although large conformational changes were observed by X-ray crystallography when soluble fragments of HA were subjected to fusion-pH conditions, it is not clear whether the same changes occur in membrane-bound HA, what the spatial relationship is between the conformationally changed HA and the target and viral membranes, and in what way HA perturbs the target membrane at low pH. We have taken a spectroscopic approach using an array of recently developed FTIR techniques to address these questions. Difference attenuated total reflection FTIR spectroscopy was employed to reveal reversible and irreversible components of the pH-induced conformational change of the membrane-bound bromelain fragment of HA, BHA. Additional proteolytic fragments of BHA were produced which permitted a tentative assignment of the observed changes to the HA1 and HA2 subunits, respectively. The membrane-bound HA1 subunit undergoes a reversible conformational change, which most likely involves the loss of a small proportion of beta-sheet at low pH. BHA was found to undergo a partially reversible tilting motion relative to the target membrane upon exposure to pH 5, indicating a previously undescribed hinge near the anchoring point to the target membrane. Time-resolved amide H/D exchange experiments revealed a more dynamic (tertiary) structure of membrane-bound BHA and its HA2, but not its HA1, subunit. Finally BHA and, to a lesser degree, HA1 perturbed the lipid bilayer of the target membrane at the interface, as assessed by spectral changes of the lipid ester carbonyl groups. These results are discussed in the context of a complementary study of HA that was bound to viral membranes through its transmembrane peptide (Gray C, Tamm LK, 1997, Protein Sci 6:1993-2006). A distinctive role for the HA1 subunit in the conformational change of HA becomes apparent from these combined studies.

Outer membrane protein A of E. coli folds into detergent micelles, but not in the presence of monomeric detergent.

Outer membrane protein A (OmpA) of Escherichia coli is a beta-barrel membrane protein that unfolds in 8 M urea to a random coil. OmpA refolds upon urea dilution in the presence of certain detergents or lipids. To examine the minimal requirements for secondary and tertiary structure formation in beta-barrel membrane proteins, folding of OmpA was studied as a function of the hydrophobic chain length, the chemical structure of the polar headgroup, and the concentration of a large array of amphiphiles. OmpA folded in the presence of detergents only above a critical minimal chain length of the apolar chain as determined by circular dichroism spectroscopy and a SDS-PAGE assay that measures tertiary structure formation. Details of the chemical structure of the polar headgroup were unimportant for folding. The minimal chain length required for folding correlated with the critical micelle concentration in each detergent series. Therefore, OmpA requires preformed detergent micelles for folding and does not adsorb monomeric detergent to its perimeter after folding. Formation of secondary and tertiary structure is thermodynamically coupled and strictly dependent on the interaction with aggregated amphiphiles.

Secondary structure of a mitochondrial signal peptide in lipid bilayer membranes.

The secondary structure of the synthetic signal peptide of cytochrome c oxidase subunit IV (coxIV-25) has been measured by circular dichroism spectroscopy in different lipid environments. CoxIV-25 is polymorphic in membranes. It forms an amphiphilic alpha-helix both in negatively charged lipid bilayers (up to 49% helix) and in detergent micelles (up to 42% helix). In association with bilayers of the zwitterionic lipid phosphatidylcholine, coxIV-25 takes an aperiodic, unidentified structure. CoxIV-25 is also partially alpha-helical in bilayers of cardiolipin, mitochondrial lipid extracts and mixtures of synthetic phosphatidylcholine and phosphatidylglycerol.

Gender differences in children with ADHD, ODD, and co-occurring ADHD/ODD identified in a school population.

OBJECTIVE: To examine gender differences among children with disruptive behavior disorders (DBDs) from an ethnically diverse school sample. METHOD: From 2,984 children, children with attention-deficit/hyperactivity disorder, combined type (ADHD-C) (46 boys, 11 girls), oppositional defiant disorder (ODD) (59 boys, 35 girls), and co-occurring ADHD-C/ODD (76 boys, 27 girls), diagnosed by teacher-rated DSM-IV symptoms, were compared with each other and with 254 controls on teacher ratings of symptoms, social functioning and Achenbach Teacher's Report Form scales. RESULTS: Children with ADHD-C/ODD received the poorest ratings on all variables. In "pure" groups, children with ODD were rated as learning more, working harder, and being less inattentive than children with ADHD-C; only the ODD group showed more internalizing problems than controls. For ADHD-C and ODD groups, ratings of aggression and some individual symptoms were higher in boys than girls. Girls with ODD were rated as more appropriate and less inattentive, but unhappier and more socially impaired than boys with ODD. Overall, girls received higher peer dislike scores than boys. CONCLUSIONS: Comorbidity and gender issues affect the correlates of DBDs, with learning problems higher in ADHD-C, internalizing problems associated only with ODD, and greatest impairment for ADHD-C/ODD groups. Despite having similar or less behavioral dysfunction, girls with DBDs may have more social problems than boys with DBDs.

Experimental cross-validation of DSM-IV types of attention-deficit/hyperactivity disorder.

OBJECTIVE: To examine the discriminant validity of DSM-IV attention-deficit/hyperactivity disorder (ADHD) types by testing the hypothesis that types are associated with specific kinds of functional impairment and to compare overlap of DSM-IV and DSM-III-R ADHD. METHOD: Consecutive referrals (n = 692) to a pediatric subspecialty clinic for ADHD were classified into 1 of each of the 3 DSM-IV types of ADHD using parent and teacher checklist ratings of ADHD symptoms. The resulting types were compared on clinical correlates and on whether the children also met criteria for DSM-III-R ADHD. RESULTS: The validity of DSM-IV types was supported by dimension-specific impairment and other distinct correlates. Academic problems aggregated in the 2 types defined by extreme inattention, and externalizing problems aggregated in the 2 types defined by extreme hyperactivity. CONCLUSION: DSM-IV appeared superior to DSM-III-R in subcategorical homogeneity and in exhaustiveness (ability to classify all apparent cases).

Viral fusion peptides: a tool set to disrupt and connect biological membranes.

The structure and function of viral fusion peptides are reviewed. The fusion peptides of influenza virus hemagglutinin and human immunodeficiency virus are used as paradigms. Fusion peptides associated with lipid bilayers are conformationally polymorphic. Current evidence suggests that the fusion-promoting state is the obliquely inserted alpha-helix. Fusion peptides also have a tendency to self-associate into beta-sheets at membrane surfaces. Although the conformational conversion between alpha- and beta-states is reversible under controlled conditions, its physiological relevance is not yet known. The energetics of peptide insertion and self-association could be measured recently using more soluble "second generation" fusion peptides. Fusion peptides have been reported to change membrane curvature and the state of hydration of membrane surfaces. The combined results are built into a model for the mechanism by which fusion peptides are proposed to assist in biological membrane fusion.

[Cyclic nucleotides and creatine phosphokinase activity in children with cardiac arrhythmias]. / Tsiklicheskie nukleotidy i aktivnost' kreatinfosfokinazy u detei s narusheniiami ritma serdtsa.

Forty-one children with cardiac arrhythmias were examined. It was established that cardiac rhythm disorders in children were accompanied by an increase in the cGMP levels as well as a decrease in the cAMP content, CPK activity and the cAMP/cGMP ratio varying in relation to the form of arrhythmia and the nature of heart damage.