Phloem‐specific resistance in Brassica oleracea against the whitefly Aleyrodes proletella

The cabbage whitefly [Aleyrodes proletella L. (Hemiptera: Aleyrodidae)] is becoming a serious pest in Brassica oleracea L. (Brassicaceae) crops. However, almost nothing is known about the interaction of this insect with its host plants. Previous studies have shown differences in the natural occurrence of adults, eggs, and nymphs on the closely related B. oleracea cultivars Christmas Drumhead and Riviera grown in the field. In this study, we aimed to identify the nature of these differences and to gain insight into the resistance mechanisms against A. proletella. We used no‐choice experiments on field‐ and greenhouse‐grown plants to show that the differences between the two cultivars are mainly based on antibiosis (traits that reduce herbivore performance) and not on antixenosis (traits that deter herbivory). This was further supported by laboratory choice experiments that indicated little or no discrimination between the two cultivars based on plant volatiles. We showed that resistance is dependent on plant age, that is, resistance increased during plant development, and is mainly independent of environmental factors. Analysis of probing behaviour revealed that the resistance trait affects A. proletella at the phloem level and that morphological differences between the two cultivars are most likely not involved. We suggest that compounds present in the phloem reduce sap ingestion by the whitefly and that this explains the observed resistance.     

Two new miR‐16 targets: caprin‐1 and HMGA1, proteins implicated in cell proliferation

Background information. miRNAs (microRNAs) are a class of non‐coding RNAs that inhibit gene expression by binding to recognition elements, mainly in the 3′ UTR (untranslated region) of mRNA. A single miRNA can target several hundred mRNAs, leading to a complex metabolic network. miR‐16 (miRNA‐16), located on chromosome 13q14, is involved in cell proliferation and apoptosis regulation; it may interfere with either oncogenic or tumour suppressor pathways, and is implicated in leukaemogenesis. These data prompted us to search for and validate novel targets of miR‐16. Results. In the present study, by using a combined bioinformatics and molecular approach, we identified two novel putative targets of miR‐16, caprin‐1 (cytoplasmic activation/proliferation‐associated protein‐1) and HMGA1 (high‐mobility group A1), and we also studied cyclin E which had been previously recognized as an miR‐16 target by bioinformatics database. Using luciferase activity assays, we demonstrated that miR‐16 interacts with the 3′ UTR of the three target mRNAs. We showed that miR‐16, in MCF‐7 and HeLa cell lines, down‐regulates the expression of caprin‐1, HMGA1a, HMGA1b and cyclin E at the protein level, and of cyclin E, HMGA1a and HMGA1b at the mRNA levels. Conclusions. Taken together, our data demonstrated that miR‐16 can negatively regulate two new targets, HMGA1 and caprin‐1, which are involved in cell proliferation. In addition, we also showed that the inhibition of cyclin E expression was due, at least in part, to a decrease in its mRNA stability.     

Further Insights into the Roles of GTP and the C Terminus of the Hepatitis C Virus Polymerase in the Initiation of RNA Synthesis

The hepatitis C virus (HCV) NS5b protein is an RNA-dependent RNA polymerase essential for replication of the viral RNA genome. In vitro and presumably in vivo, NS5b initiates RNA synthesis by a de novo mechanism. Different structural elements of NS5b have been reported to participate in RNA synthesis, especially a so-called “β-flap” and a C-terminal segment (designated “linker”) that connects the catalytic core of NS5b to a transmembrane anchor. High concentrations of GTP have also been shown to stimulate de novo RNA synthesis by HCV NS5b. Here we describe a combined structural and functional analysis of genotype 1 HCV-NS5b of strains H77 (subtype 1a), for which no structure has been previously reported, and J4 (subtype 1b). Our results highlight the linker as directly involved in lifting the first boundary to processive RNA synthesis, the formation of the first dinucleotide primer. The transition from this first dinucleotide primer state to processive RNA synthesis requires removal of the linker and of the β-flap with which it is shown to strongly interact in crystal structures of HCV NS5b. We find that GTP specifically stimulates this transition irrespective of its incorporation in neosynthesized RNA.     

Cellular and biochemical features of skeletal muscle in obese Yucatan minipigs

Objective: To examine cellular and biochemical features of skeletal muscle in response to dietary‐induced obesity in a novel Yucatan minipig model of childhood obesity. Research Methods and Procedures: From 4 to 16 months of age, minipigs were fed either a recommended human‐type diet (NF; n = 4) or were overfed a western‐type diet with saturated fat and high‐glycemic index carbohydrates (OF, n = 4). Muscle samples (biceps femoris) were histochemically stained for the identification of intramuscular adipocytes, intramyocellular lipid aggregates (oil red O), and myofiber types (myosin ATPase, succinate dehydrogenase). Gene expressions and/or activities of factors involved in lipogenesis, lipolysis, or energetic metabolism were quantified in muscle. Results: Cross‐sectional areas of myofibers paralleled pig body weight (r = 0.86, p < 0.01). The size of intramuscular adipocytes, the relative proportion of oil red O‐stained fibers, and total muscle lipid content tended (p ≤ 0.10) to increase in response to OF diet. Hormone‐sensitive lipase, carnitine palmityl transferase‐I, and uncoupling protein 2 mRNA levels were lower (p < 0.05) in OF pigs than in NF pigs. Activities of β‐hydroxyacyl‐coenzyme A dehydrogenase and citrate synthase assessing post‐carnitine palmityl transferase I events and the proportion of oxidative myofibers were not altered by OF diet. Activity and gene expression of fatty acid synthase were lower (p < 0.02) in OF pigs than in NF pigs. Discussion: Overfeeding in Yucatan minipigs reduced the expression levels of three catabolic steps in skeletal muscle that are involved also in the etiology of human obesity.     

Salivary protein profiles and sensitivity to the bitter taste of caffeine

The interindividual variation in the sensitivity to bitterness is attributed in part to genetic polymorphism at the taste receptor level, but other factors, such as saliva composition, might be involved. In order to investigate this, 2 groups of subjects (hyposensitive, hypersensitive) were selected from 29 healthy male volunteers based on their detection thresholds for caffeine, and their salivary proteome composition was compared. Abundance of 26 of the 255 spots detected on saliva electrophoretic patterns was significantly different between hypo- and hypersensitive subjects. Saliva of hypersensitive subjects contained higher levels of amylase fragments, immunoglobulins, and serum albumin and/or serum albumin fragments. It also contained lower levels of cystatin SN, an inhibitor of protease. The results suggest that proteolysis occurring within the oral cavity is an important perireceptor factor associated to the sensitivity to the bitter taste of caffeine.     

Endostatin's heparan sulfate-binding site is essential for inhibition of angiogenesis and enhances in situ binding to capillary-like structures in bone explants

The functional role of endostatin's affinity for heparan sulfates was addressed using an ex vivo bone angiogenesis model. Capillary-like sprouts showed prominent expression of collagen XVIII/endostatin. Outgrowth of endothelial cells was not altered in the absence of collagen XVIII but inhibited by the addition of recombinant endostatin. Mutant non-heparan sulfate binding endostatin and the collagen XV endostatin homologue were ineffective. The ability of mutant endostatin to bind to capillary structures was reduced when compared to endostatin. Endostatin-XV completely failed to bind to endothelial cells. Our data indicate that endostatin's angiostatic function is heparan sulfate-dependent, and that in situ-binding of endostatin to endothelial cells is increased by heparan sulfates.     

Seasonal diet changes in elephant and impala in mopane woodland

Elephant and impala as intermediate feeders, having a mixed diet of grass and browse, respond to seasonal fluctuations of forage quality by changing their diet composition. We tested the hypotheses that (1) the decrease in forage quality is accompanied by a change in diet from more monocots in the wet season to more dicots in the dry season and that that change is more pronounced and faster in impala than in elephant; (2) mopane (Colophospermum mopane), the most abundant dicot species, is the most important species in the elephant diet in mopane woodland, whereas impala feed relatively less on mopane due to the high condensed tannin concentration; and (3) impala on nutrient-rich soils have a diet consisting of more grass and change later to diet of more browse than impala on nutrient-poor soils. The phosphorus content and in vitro digestibility of monocots decreased and the NDF content increased significantly towards the end of the wet season, whereas in dicots no significant trend could be detected. We argue that this decreasing monocot quality caused elephant and impala to consume more dicots in the dry season. Elephant changed their diet gradually over a 16-week period from 70% to 25% monocots, whereas impala changed diets rapidly (2?C4?weeks) from 95% to 70% monocots. For both elephants and impala, there was a positive correlation between percentage of monocots and dicots in the diet and the in vitro digestibility of these forage items. Mopane was the most important dicot species in the elephant diet and its contribution to the diet increased significantly in the dry season, whereas impala selected other dicot species. On nutrient-rich gabbroic soils, impala ate significantly more monocots than impala from nutrient-poor granitic soils, which was related to the higher in vitro digestibility of the monocots on gabbroic soil. Digestibility of food items appears to be an important determinant of diet change from the wet to the dry season in impala and elephants.     

Functional characterization of an anti-estradiol antibody by site-directed mutagenesis and molecular modelling: modulation of binding properties and prominent role of the V(L) domain in estradiol recognition

The high-affinity monoclonal anti-estradiol antibody 9D3 presents a specificity defect towards estradiol-3-sulphate and 3-glucuronide conjugates incompatible with use in direct immunoassays. The corresponding single-chain variable fragment (scFv), cloned and produced in E. coli, exhibited a 10-fold lower affinity for estradiol (K(a)=1.2 x 10(9) M (-1)) and a slightly increased specificity defect for the 3-position. Site-directed mutagenesis revealed critical residues involved in estradiol recognition and produced mutants exhibiting up to a 3-fold increase of the binding affinity for estradiol and up to a 2-fold decrease of the cross-reactivity with estradiol-3-sulphate. A comparative model of the antibody 9D3-estradiol complex was built in which the estradiol D-ring is buried into the binding pocket while the 3-, 6- and 7-positions are solvent exposed, agreeing with the lack of specificity for these three positions. Two potential alternative orientations of the A-ring, one close to CDR H3 and L2 loops, and the other one close to CDR H2 and L3 loops, have been considered for the docking of estradiol, none of which could be unambiguously privileged taking into account data from cross-reactivity measurements, photolabelling and mutagenesis studies. For both orientations, estradiol is stabilized by hydrogen bonding of the 17beta-OH group with TyrL36, His89 and GlnH35 in the first case, or TyrL36, only, in the second case and by van der Waals contacts from TyrL91 with alpha- or beta-face of estradiol, respectively, and from ValH95 and GlyH97 with the opposite face. To elucidate the molecular basis of antibody 9D3 specificity, as compared with that of another anti-estradiol antibody 15H11, single variable domains (V(H) and V(L)) and scFv hybrids have been constructed. The binding activity of V(L)9D3 as well as the specificity of the V(L)9D3/V(H)15H11 hybrid, both similar to antibody 9D3, revealed a prominent role of V(L) in estradiol recognition. These findings establish premises for antibody engineering to reduce cross-reactivity, especially with estradiol-3-conjugates.