Multivariate-activity mining for molecular quasi-species in a glutathione transferase mutant library

A library of recombinant glutathione transferases (GSTs) generated by shuffling of DNA encoding human GST M1-1 and GST M2-2 was screened with eight alternative substrates, and the activities were subjected to multivariate analysis. Assays were made in lysates of bacteria in which the GST variants had been expressed. The primary data showed clustering of the activities in eight-dimensional substrate-activity space. For an incisive analysis, the rows of the data matrix, corresponding to the different enzyme variants, were individually scaled to unit length, thus accounting for different expression levels of the enzymes. The columns representing the activities with alternative substrates were subsequently individually normalized to unit variance and a zero mean. By this standardization, the data were adjusted to comparable orders of magnitude. Three molecular quasi-species were recognized by multivariate K-means and principal component analyses. Two of them encompassed the parental GST M1-1 and GST M2-2. A third one diverged functionally by displaying enhanced activities with some substrates and suppressed activities with signature substrates for GST M1-1 and GST M2-2. A fourth cluster contained mutants with impaired functions and was not regarded as a quasi-species. Sequence analysis of representatives of the mutant clusters demonstrated that the majority of the variants in the diverging novel quasi-species were structurally similar to the M1-like GSTs, but distinguished themselves from GST M1-1 by a Ser to Thr substitution in the active site. The data show that multivariate analysis of functional profiles can identify small structural changes influencing the evolution of enzymes with novel substrate-activity profiles.     

Methyl Jasmonate-Induced Monoterpenes in Scots Pine and Norway Spruce Tissues Affect Pine Weevil Orientation

In large parts of Europe, insecticide-free measures for protecting conifer plants are desired to suppress damage by the pine weevil Hylobius abietis (L.). Treatment with methyl jasmonate (MeJA), a chemical elicitor already used in crop production, may enhance expression of chemical defenses in seedlings in conifer regenerations. However, in a previous experiment, MeJA treatment resulted in substantially better field protection for Scots pine (Pinus sylvestris L.) than for Norway spruce (Picea abies (L.) Karst.). Hypothesizing that the variations may be at least due partly to volatiles released by MeJA-treated seedlings and their effects on pine weevil orientation, we examined tissue extracts of seedlings (from the same batches as previously used) by two-dimensional GC-MS. We found that the MeJA treatment increased contents of the monoterpene (?)-β-pinene in phloem (the weevil’s main target tissue) of both tree species, however, the (?)-β-pinene/(?)-α-pinene ratio increased more in the phloem of P. sylvestris. We also tested the attractiveness of individual monoterpenes found in conifer tissues (needles and phloem) for pine weevils using an arena with traps baited with single-substance dispensers and pine twigs. Trap catches were reduced when the pine material was combined with a dispenser releasing (?)-β-pinene, (+)-3-carene, (?)-bornyl acetate or 1,8-cineole. However, (?)-α-pinene did not have this effect. Thus, the greater field protection of MeJA-treated P. sylvestris seedlings may be due to the selective induction of increases in contents of the deterrent (?)-β-pinene, in contrast to strong increases in both non-deterrent (?)-α-pinene and the deterrent (?)-β-pinene in P. abies seedlings.     

Timing of Male Sex Pheromone Biosynthesis in a Butterfly – Different Dynamics under Direct or Diapause Development

The life history traits and behavior of the butterfly Pieris napi are well-known, as the species is often used as a model organism for evolutionary and ecological studies. The species has two or more generations per year in the major part of its temperate distribution, and as different selection pressures affect the different generations, both behavioral and physiological seasonal polyphenisms have been shown previously. Here, we explored the dynamics of male sex pheromone production. The two generations are shown to have significantly different scent compositions early in life; the direct developers--who have shorter time for pupal development--need the first 24 hr of adult life after eclosion to synthesize the sex pheromone citral (geranial and neral 1:1)--whereas the diapausing individuals who have spent several months in the pupal stage eclose with adult scent composition. Resource allocation and biosynthesis also were studied in greater detail by feeding butterflies (13)C labeled glucose either in the larval or adult stage, and recording incorporation into geranial, neral, and other volatiles produced. Results demonstrate that the pheromone synthesized by newly eclosed adult males is based on materials ingested in the larval stage, and that adult butterflies are able to synthesize the pheromone components geranial and neral and the related alcohols also from adult intake of glucose. In summary, our study shows that time-stress changes the timing in biosynthesis of the complete pheromone between generations, and underpins the importance of understanding resource allocation and the physiological basis of life history traits.     

Pyrazines as marking volatiles in philanthine and nyssonine wasps (Hymenoptera: Sphecidae)

Males of several sphecid wasps apply volatile secretion on specific sites, perches. The odor is supposed to act in premating behavior as an attracting pheromone. There are strong indications that the scenting material is produced by the mandibular glands. One philanthine species,Philanthus triangulum, and three nyssonine ones,Argogorytes fargei, A. mystaceus, andNysson spinosus, the latter cleptoparasite onArgogorytes wasps, have been analyzed by gas chromatography-mass spectrometry. The major compound in the mandibular gland secretion of the four species was tentatively identified as 2,5-dimethyl-3-isopentylpyrazine. A few other pyrazines not yet identified were also found. Preliminary tests withP. triangulum show that alkylpyrazines influence male behavior.     

Chemocommunication in Phyllonorycter ulmifoliella (HBN.) (Lepidoptera: Gracillariidae): Periodicity, Sex Pheromone, and Inhibitors

(Z)-10-Tetradecenyl acetate (Z10-14:OAc) from abdominal tip extracts of virgin females of the tentiform leafminer moth Phyllonorycter ulmifoliella (Lepidoptera: Gracillariidae) was identified by gas chromatography and mass spectrometry. The biological activity of the component was confirmed by field tests with synthetic compounds. As a sex pheromone component this ester is novel both in the family Gracillariidae and in the superfamily Gracillarioidea. Field trapping of P. ulmifoliella with synthetic Z10-14: OAc at dosages of 1 and 0.2 mg/dispenser led to catches of approximately 9000 and 3000 male moths, respectively. The attractivity of the Z10-14:OAc was strongly inhibited by a 10% admixture of either (Z)-9-tetradecenyl acetate (Z9-14:OAc), (E)-9-tetradecenyl acetate (E9-14:OAc), or (E)-11-tetradecenyl acetate (E11-14:OAc). Addition of 10% (E)-10-tetradecenyl acetate (E10-14:OAc) to the sex pheromone reduced attractivity, but significantly less than the inhibitors previously mentioned. The pheromone releasing (or “calling”) behavior of virgin P. ulmifoliella females was recorded under laboratory conditions. Calling activity started about half an hour before lightson and the maximum number of calling females was registered half an hour after the start of photophase. A high level of pheromone releasing activity lasted for about 2 hr and ceased about 5 hr after the start of photophase. Chemocommunication activity in the light period of day is assumed to be an adaptation which allows this phyllonoryctid to avoid inhibitors emitted as pheromones by many other species. A scheme of probable interactions by means of semiochemicals between P. ulmifoliella and other lepidopterans is presented and the appearance of Z10-14: OAc as a sex pheromone component in Lepidoptera during evolution of the order is discussed.     

Electrophysiological and Behavioral Responses to Chocolate Volatiles in Both Sexes of the Pyralid Moths <Emphasis Type="Italic">Ephestia cautella</Emphasis> and <Emphasis Type="Italic">Plodia interpunctella</Emphasis>

Volatiles from chocolate mediate upwind flight behavior in Ephestia cautella and Plodia interpunctella. We used gas chromatography with electroantennographic detection and found 12 active compounds derived from three different chocolate types, i.e., plain, nut-containing, and rum-flavored. Eight of the compounds were identified with mass spectrometry, and the activity of three compounds, ethyl vanillin, nonanal, and phenylacetaldehyde (PAA), was subsequently confirmed in both electrophysiological and behavioral assays. In the electroantennogram experiment, PAA and nonanal were consistently eliciting responses in both species and sexes. Ethyl vanillin was active in males of both species, and also in P. interpunctella females. E. cautella females showed no antennal activity in response to ethyl vanillin. All three volatiles were attractive to E. cautella males and P. interpunctella females in a flight tunnel. E. cautella females were significantly attracted only to ethyl vanillin. P. interpunctella males were attracted to PAA. Ethyl vanillin is a novel insect attractant, whereas both nonanal and phenylacetaldehyde mediate behavior in many insect species. A final experiment revealed that a blend of the three volatiles was required to induce landing in the flight tunnel bioassay, and that the landing rate was dependent on dose. The three-component blend attracted both sexes of P. interpunctella and females of E. cautella, whereas E. cautella males were not attracted.     

Influence of point defects in KTaO3 on low-temperature dielectric relaxation

Substituted KTaO₃ ceramics were synthesized, sintered and studied using low-temperature microwave dielectric analysis and Raman spectroscopy. Because of a fundamentally different nature of aliovalent Mn- and isovalent Na-substitution mechanisms, significant differences in processing and dielectric properties were identified. The properties were correlated to the defect structure of the substituted KTaO₃ lattices. Characteristics of the induced polar domains were clearly different for the two substitutional mechanisms, which further reflects in a significantly different dielectric behavior. Linear response of changes in the Raman spectra corresponds to evidence of the formation of symmetry-breaking regions.     

BWR steam dryer for extended power uprate

A new steam dryer for extended power uprated conditions, based on a design with proven performance record, has been designed by Westinghouse with performance characteristics meeting all utility requirements. Extensive use of computational fluid dynamics (CFDs) has been made to design the new dryer intended for plants of the BWR 3000-type. In addition to numerical simulations, the analyses rely on previous knowledge, acquired from experimental work on steam flow characteristics in the BWR 3000 with its asymmetrically placed steam line nozzles. The new design is expected to both decrease vibration levels in the steam lines and to solve a water-level measurement problem. An extensive experimental verification of the new design is currently in progress.     

Influence of Fungal Infection and Wounding on Contents and Enantiomeric Compositions of Monoterpenes in Phloem of Pinus sylvestris

To identify chemical resistant markers induced by fungal or mechanical injury, young trees of Scots pine (Pinus sylvestris) were subjected to inoculations of blue stain fungi associated with the pine shoot beetles Tomicus piniperda and T. minor. Among the 20 trees selected for chemical analyses, 16 were divided into four groups: one as control and three were pretreated by wounding only, or by inoculation with either the blue stain fungus Leptographium wingfieldii or Ophiostoma canum. Four wk after pretreatment, all 16 pretreated trees were mass-inoculated with L. wingfieldii. The absolute and relative amounts, as well as the enantiomeric compositions of monoterpene hydrocarbons in the phloem, were determined via a small sample of the phloem before and after the pretreatment and mass inoculation, by using two-dimensional gas chromatography (2D GC) and GC-mass spectrometry (MS). After mass inoculation, the absolute amounts of most of the monoterpenes decreased in the phloem sampled >20 cm from the fungal infection, and were higher in the phloem sampled within the infected reaction zone. The relative amounts of both (−)-β-pinene and (−)-limonene increased in phloem samples taken >20 cm above the fungal inoculation in the preinoculated trees compared with phloem sampled from the remaining four control trees. The enantiomeric compositions of β-pinene and limonene changed, after fungal growth, at defined distances from the inoculation site: the proportion of the (−)-enantiomers was highest in the phloem sampled >20 cm from the fungal inoculation. Four wk after pretreatment, monoterpene production in the phloem at the site of inoculation was more enhanced by L. wingfieldii than by O. canum. However, the different virulence levels of the fungi did not affect the enantiomeric composition of the monoterpenes. The biosynthesis of monoterpene enantiomers is discussed in relation to induced pathogen resistance.     

Cognitive impairment in preclinical Alzheimer's disease: A meta-analysis.

To determine the size of the impairment across different cognitive domains in preclinical Alzheimer's disease (AD), a meta-analysis based on 47 studies involving 9,097 controls and 1,207 preclinical AD cases was conducted. There were marked preclinical deficits in global cognitive ability, episodic memory, perceptual speed, and executive functioning; somewhat smaller deficits in verbal ability, visuospatial skill, and attention; and no preclinical impairment in primary memory. Younger age (