Ser-474 is the major target of insulin-mediated phosphorylation of protein kinase B beta in primary rat adipocytes.

The mechanism of activation for protein kinase B (PKB), an important target for insulin signaling, has been scarcely investigated in primary cells. In this study, we have characterized the insulin-induced phosphorylation and activation of PKB beta in primary rat adipocytes. Insulin stimulation resulted in a translocation of PKB beta from cytosol to membranes, and phosphorylation and activation of PKB beta. Phosphoamino acid analysis and phosphopeptide mapping demonstrated that the phosphorylation occurred mainly on serines, also when using calyculin A, and that these were localized within one major phosphopeptide. Radiosequencing showed that the radioactivity was released in Cycle No. 7. In addition, the peptide was specifically immunoprecipitated from a tryptic digest of PKB beta using the anti-phospho-PKB (Ser-473) antibody. Taken together, these results show that rat adipocyte PKB beta mainly is phosphorylated on Ser-474 in response to insulin stimulation, in contrast to previous studies in human embryonic kidney (HEK) 293 cells demonstrating, in addition, phosphorylation of Thr-309.     

Oils for Increased Efficacy of Metarhizium anisopliae to Control Whiteflies

The efficacy of Metarhizium anisopliae in combination with sublethal concentrations of oils and potassium-oleate for biological control of whiteflies was tested under controlled conditions. Three commercial products (Biola ® , Naturen ® , Neudosan ® ) and five experimental formulations of plant oils were tested. The efficacy of M. anisopliae against Trialeurodes vaporariorum and Bemisia tabaci without additives was about 50%. At 1/20 of their recommended dosages, all compounds tested significantly increased the efficacy of M. anisopliae for the control of T. vaporariorum , with the formulated sunflower oil Biola ® giving the highest synergistic effect, reaching nearly 100% control. Not only was the level of control increased but also the speed of action was improved, resulting in a higher reliability of control. Three of seven additives showed no effects on the viability of conidia on the leaf surface, whereas the formulations of the other oils and oleates reduced the longevity of spores. The synergistic effect of Biola resulted from the more even distribution of M. anisopliae conidia on leaves and insects. Other positive effects of oils on the efficacy of M. anisopliae are discussed in relation to an extended spectrum of environmental conditions and pests to be controlled.     

Lectin activity of Lentinus edodes

The hemagglutinating activity of submerged mycelium and culture liquid for four strains of Lentinus edodes (Berk.) Sing [L. edodes (Berk.) Pegler] was studied in the search for lectins. The hemagglutinating activity of culture liquid was substantially higher, compared with mycelium. The carbohydrate-binding capacity of the agglutinins was established, and the lectin activity of extracts from mycelia grown on several agar media was elucidated in relation to fruiting. The lectin activity of L. edodes was examined at different morphogenetic steps: mycelium, brown mycelial film, primordium, and fruiting body. Hemagglutination titers at the brown film step were higher than in the mycelium, whereas activity at the primordial and fruiting bodies steps decreased. Lectins seem to be involved in the formation of hyphal aggregates of brown mycelial film.     

Massive parallel analysis of the binding specificity of histone-like protein HU to single- and double-stranded DNA with generic oligodeoxyribonucleotide microchips

A generic hexadeoxyribonucleotide microchip has been applied to test the DNA-binding properties of HU histone-like bacterial protein, which is known to have a low sequence specificity. All 4096 hexamers flanked within 8mers by degenerate bases at both the 3′- and 5′-ends were immobilized within the 100 × 100 × 20 mm polyacrylamide gel pads of the microchip. Single-stranded immobilized oligonucleotides were converted in some experiments to the double-stranded form by hybridization with a specified mixture of 8mers. The DNA interaction with HU was characterized by three type of measurements: (i) binding of FITC-labeled HU to microchip oligonucleotides; (ii) melting curves of complexes of labeled HU with single-stranded microchip oligonucleotides; (iii) the effect of HU binding on melting curves of microchip double-stranded DNA labeled with another fluorescent dye, Texas Red. Large numbers of measurements of these parameters were carried out in parallel for all or many generic microchip elements in real time with a multi-wavelength fluorescence microscope. Statistical analysis of these data suggests some preference for HU binding to G/C-rich single-stranded oligonucleotides. HU complexes with double-stranded microchip 8mers can be divided into two groups in which HU binding either increased the melting temperature (T_m) of duplexes or decreased it. The stabilized duplexes showed some preference for presence of the sequence motifs AAG, AGA and AAGA. In the second type of complex, enriched with A/T base pairs, the destabilization effect was higher for longer stretches of A/T duplexes. Binding of HU to labeled duplexes in the second type of complex caused some decrease in fluorescence. This decrease also correlates with the higher A/T content and lower T_m. The results demonstrate that generic microchips could be an efficient approach in analysis of sequence specificity of proteins.     

Production and specificity of polyclonal antibodies against soluble proteins from the arbuscular mycorrhizal fungus Glomus intraradices

Rabbit polyclonal antibodies were produced against a soluble protein fraction from a vesicle and spore mixture of the arbuscular mycorrhizal fungus (AMF) Glomus intraradices. The protocol for isolation of vesicles and spores from plant roots was optimized to minimize debris contamination. Protein extract purification and preparation for immunization was adapted to increase protein content and immunogenicity. Active antisera were produced starting from the second boost immunization. Antibodies obtained were specific for surface antigens of AMF and revealed different patterns of soluble protein antigens in G. intraradices, G. constrictum and an unidentified Glomus species. Accepted: 6 December 2000     

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues¹. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest. The amber mutant is then expressed in E. coli together with genes encoding an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii. Using this system, the photo activatable amino acid analog p-benzoylphenylalanine (Bpa) is incorporated at the amber codon. Cells are then irradiated with ultraviolet light to covalently link the Bpa residue to proteins that are located within 3-8 Å. Photocrosslinking is performed in combination with pulse-chase labeling and immunoprecipitation of the protein of interest in order to monitor changes in protein-protein interactions that occur over a time scale of seconds to minutes. We optimized the procedure to study the assembly of a bacterial virulence factor that consists of two independent domains, a domain that is integrated into the outer membrane and a domain that is translocated into the extracellular space, but the method can be used to study many different assembly processes and biological pathways in both prokaryotic and eukaryotic cells. In principle interacting factors and even specific residues of interacting factors that bind to a protein of interest can be identified by mass spectrometry.     

Pore water N:P and NH4 +:NO3 ? alter the response of Phragmites australis and Glyceria maxima to extreme nutrient regimes

Phragmites australis and Glyceria maxima are fast-growing littoral grasses often competing for similar wetland habitats. Eutrophication affects their competitiveness, but the outcome is not generally predictable due to the complexity of interrelated factors. We hypotheses that pore water N:P and NH₄ ⁺:NO₃ ^? modify their growth and metabolic responses to the trophic status of the habitat. The hypothesis was tested under standardized conditions of long-term sand cultures. Application of N?+?P up to extreme levels in combination with N:P?<?10 and NH₄ ⁺:NO₃ ^??<?1 triggered positive growth response in both species. In contrast, similar N levels applied in N:P?>?90 and NH₄ ⁺:NO₃ ^??=?4 caused lower productivity, changes in resource allocation, morphology and metabolic relations (e.g. high shoot density, low shoot diameters and heights, reduced root and rhizome growth). Observed signs of stress resembled the factors associated with the reed retreat at the die-back sites. Unbalanced N levels obviously alter plant susceptibility to stresses (altering, e.g. ventilation efficiency, plant anchorage or below-ground storage capacity). The positive effect of sufficient P supply was pronounced in Glyceria. It might therefore favour Glyceria in competition with Phragmites at highly fertile habitats rich in P.