Leaf beetle grazing does not induce willow trichome defence in the coppicing willow Salix viminalis

Abstract 1 Willows are frequently attacked and defoliated by adult leaf beetles (Phratora vulgatissima L.) early in the season and the plants are then attacked again when new larvae emerge. The native willow Salix cinerea has previously been shown to respond to adult grazing by producing new leaves with an increased trichome density. Subsequent larval feeding was reduced on new leaves. This type of induced plant response may reduce insect damage and could potentially be utilized for plant protection in agricultural systems. 2 Here, we investigated if the willow species most commonly used for biomass production in short rotation coppice, Salix viminalis, also responds to adult beetle grazing by increasing trichome density. Larval performance and feeding behaviour on plants previously exposed to adult beetles was compared with that on undefoliated control plants in a greenhouse. 3 We found an overall decrease in trichome density within all the plants (i.e. trichome density was lower on new leaves compared to that for older basal leaves on S. viminalis). However, leaves of beetle defoliated plants had a higher trichome density compared to control plants. Larval growth and feeding was not affected by this difference between treatments. Larvae appeared to remove trichomes when feeding on S. viminalis, a behaviour that might explain the lack of difference between treatments.     

Crystal structure of cod liver class I alcohol dehydrogenase: substrate pocket and structurally variable segments.

The structural framework of cod liver alcohol dehydrogenase is similar to that of horse and human alcohol dehydrogenases. In contrast, the substrate pocket differs significantly, and main differences are located in three loops. Nevertheless, the substrate pocket is hydrophobic like that of the mammalian class I enzymes and has a similar topography in spite of many main-chain and side-chain differences. The structural framework of alcohol dehydrogenase is also present in a number of related enzymes like glucose dehydrogenase and quinone oxidoreductase. These enzymes have completely different substrate specificity, but also for these enzymes, the corresponding loops of the substrate pocket have significantly different structures. The domains of the two subunits in the crystals of the cod enzyme further differ by a rotation of the catalytic domains by about 6 degrees. In one subunit, they close around the coenzyme similarly as in coenzyme complexes of the horse enzyme, but form a more open cleft in the other subunit, similar to the situation in coenzyme-free structures of the horse enzyme. The proton relay system differs from the mammalian class I alcohol dehydrogenases. His 51, which has been implicated in mammalian enzymes to be important for proton transfer from the buried active site to the surface is not present in the cod enzyme. A tyrosine in the corresponding position is turned into the substrate pocket and a water molecule occupies the same position in space as the His side chain, forming a shorter proton relay system.     

Cloning of an Aeromonas hydrophila type IV pilus biogenesis gene cluster: complementation of pilus assembly functions and characterization of a type IV leader peptidase/N-methyltransferase required for extracellular protein secretion

Aeromonas hydrophila secretes several extracellular proteins that are associated with virulence including an enterotoxin, a protease, and the hole-forming toxin, aerolysin. These degradative enzymes and toxins are exported by a conserved pathway found in many Gram-negative bacteria. In Pseudomonas aeruginosa this export pathway and type IV pilus biogenesis are dependent on the product of the pilD gene. PilD is a bifunctional enzyme that processes components of the extracellular secretory pathway as well as a type IV prepilin. An A. hydrophila genomic library was transferred into a P. aeruginosa pilD mutant that is defective for type IV pilus biogenesis. The A. hydrophila pilD homologue, tapD , was identified by its ability to complement the pilD mutation in P. aeruginosa . Transconjugants containing tapD were sensitive to the type IV pilus-specific phage, PO4. Sequence data revealed that tapD is part of a cluster of genes ( tapABCD ) that are homologous to P. aeruginosa type IV pilus biogenesis genes ( pilABCD ). We showed that TapB and TapC are functionally homologous to P. aeruginosa PilB and PilC, the first such functional complementation of pilus assembly demonstrated between bacteria that express type IV pili. In vitro studies revealed that TapD has both endopeptidase and N -methyltransferase activities using P. aeruginosa prepilin as substrate. Furthermore, we show that tapD is required for extracellular secretion of aerolysin and protease, indicating that tapD may play an important role in the virulence of A. hydrophila     

Extensive MHC class II DRB3 diversity in African and European cattle

Genetic deversity at the highly polymorphic BoLA-DRB3 locus was investigated by DNA sequence analyses of 18 African cattle from two breeds representing the two subspecies of cattle, Bos primigenius indicus and Bos primigenius taurus. Yhe polymorphism was compared with that found in a sample ofd 32 European cattle from four breeds, all classified as B. p. taurus. Particularly extensive genetic diversity was found among African cattle, in which as many as 18 alleles were recognized in this small random sample of animals from two breeds. The observed similarity in allele frequency distribution between the two African populations, N'Dama and Zebu cattle, is consistent with the recent recognition of gene flow between B. p. indicus and B. P taurus cattle in Africa. A total of 30 DRB3 alleles were documented and as many as 26 of these were classified as major allelic types showing at least five amino acid substitutions compared with other major types. The observation of extensive genetic diversity at MHC loci in cattle, as well as in other farm animals, provides a compelling argument against matin-type preferences as a primary cause in maintaining major histocompatibility complex diversity, since the reproduction of these animals has been controlled by humans for many generations.The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been given the accession numbers X87641-X87670     

Proteolytic cleavage of recombinant two-chain factor VIII during cell culture production is mediated by protease(s) from lysed cells. The use of pulse labelling directly in production medium

During the production by mammalian cells of recombinant factor VIII from which the B domain was deleted (rFVIII), proteolytic cleavages in the C-terminal part of the heavy chain were observed (Kjalke et al., 1995). By radioactive pulse labelling it was investigated whether the cleavages took place inside the cells during protein synthesis or after release in the medium. The rFVIII-producing CHO (Chinese hamster ovary) cells were cultured in the presence of ³⁵S-methionine and then the cell lysate and the conditioned media were immunoprecipitated and analyzed by electrophoresis. By pulse labelling and chasing for various time periods, it was shown that the cleavages only took place after secretion of the protein from the cells. Adding cell lysate to uncleaved rFVIII caused cleavage of the heavy chain, as seen by loss of binding to a monoclonal antibody specific for intact rFVIII, indicating that the cleavage was performed by proteinase(s) released from the lysed cells. By incubating intact rFVIII with the multicatalytic proteinase (proteasome) present in cytoplasm and nucleus of eukaryotic cells, loss of binding to the monoclonal antibody was observed. This indicates that the multicatalytic proteinase, released from lysed rFVIII producing cells, could be responsible for the cleavage of rFVIII. Among several protease inhibitors tested, only bacitracin was found to diminish the extent of cleavage. Phosphatidylserine also protected rFVIII against cleavage, probably by binding to rFVIII. This revised version was published online in July 2006 with corrections to the Cover Date.     

Physical Activity,Obesity, and Cardiovascular Risk Factors in Children. The Belgian Luxembourg Child Study II

GUILLAUME, MICHÈLE, LEIF LAPIDUS, PER BJÖRNTORP, ANDRE LAMBERT. Physical activity, obesity, and cardiovascular risk factors in children. The Belgian Luxembourg Child Study II. Physical activity was measured in relation to cardiovascular (CV) risk factors in a randomly selected population of 1028 children from Province de Luxembourg in Belgium, a mainly rural area with a high prevalence of such risk factors among adults and children. Physical activity was estimated as participation in sport activities, a major indicator of leisure-time physical activity in schoolchildren, and physical inactivity was estimated as frequency and duration of television (TV) watching. Boys participated more frequently in sport activities than girls did (p=0. 001). A majority of the children watched TV daily. After age adjustment, bodyweight (girls, p<0. 012; boys, p<0. 027) and, in boys, body mass index (BMI) (p<0. 039) were related to days per week of TV watching. No significant relationships with other CV risk factors remained after adjustments for BMI. In analyses of independent contributions of age, TV watching, and sports activity on CV risk factors, age showed highly significant relationships. In boys, TV showed relationships with BMI (P<0. 04) and (borderline) with systolic blood pressure, independent of age and sports activity, whereas the latter was significantly related to subscapular skinfold (p<0. 04) and (borderline) with triceps skinfold and cholesterol. In girls, no significant independent contributions to risk factor associations were found. The father's education was directly associated with sports activities, whereas the mother being a housewife showed negative relationships to physical activity and positive to TV watching in their children, suggesting socioeconomic influence on the activity patterns of children. Furthermore, registrations suggested less physical activity in the most rural part of the area. It is concluded that children in this mainly rural area watch TV frequently. In boys, physical inactivity, measured both as TV watching and as registrations of sports activities, contributes independently to body fat mass. In girls, no contribution or weaker contributions of physical inactivity were found. This suggests that contributory factors leading to obesity might be different in girls and boys.     

Defective regulation of the cytosolic Ca2+ activity in parathyroid cells from patients with hyperparathyroidism

The parathyroid hormone (PTH) release and cytosolic Ca²⁺ activity were determined in normal bovine parathyroid cells and parathyroid cells obtained from patients with hyperparathyroidism (HPT). There was a sigmoid relation between the cytosolic Ca²⁺ activity and the extracellular calcium concentration between 0.5 and 6.0 mmol/l. The PTH release was inhibited in parallel with the rise in the cytosolic Ca²⁺ activity. Both the hormone release and the cytosolic Ca²⁺ activity were lower in cells from human adenomas and hyperplastic glands~ and in comparison with the bovine preparations these ceils had higher set points for the cytosolic Ca²⁺ activity and PTH release. There was a close correlation between the individual set points for the cytosolic Ca²⁺ activity and PTH release in a material containing both normal and pathological cells. The results indicate that the abnormal PTH release characteristic of HPT is due to a defective regulation of the cytosolic Ca²⁺ activity.     

Conformational and functional similarities between glutaredoxin and thioredoxins.

The tertiary structures of thioredoxin from Escherichia coli and bacteriophage T4 have been compared and aligned giving a common fold of 68 C alpha atoms with a root mean square difference of 2.6 A. The amino acid sequence of glutaredoxin has been aligned to those of the thioredoxins assuming that glutaredoxin has the same common fold. A model of the glutaredoxin molecule was built on a vector display using this alignment and the T4 thioredoxin tertiary structure. By comparison of the model with those of the thioredoxins, we have identified a molecular surface area on one side of the redox-active S-S bridge which we suggest is the binding area of these molecules for redox interactions with other proteins. This area comprises residues 33-34, 75-76 and 91-93 in E. coli thioredoxin; 15-16, 65-66 and 76-78 in T4 thioredoxin and 12-13, 59-60 and 69-71 in glutaredoxin. In all three molecules, this part of the surface is flat and hydrophobic. Charged groups are completely absent. In contrast, there is a cluster of charged groups on the other side of the S-S bridge which we suggest participates in the mechanisms of the redox reactions. In particular, a lysine residue close to an aromatic ring is conserved in all molecules.