Pectinases in Aqueous Extracts of Ditylenchus dipsaci

Aqueous extracts of a population of Ditylenchus dipsaci isolated from onion and maintained monoxenically on onion callus contained endo-polygalacturonase (endo-PG) and endo-pectinmethyltranseliminase (endo-PMTE). In viscometric tests pH 4.2 and 4.0 were optimal for degradation of sodium polypectate and pectin N.F., respectively, by endo-PG. Endo-PMTE reduced viscosity of pectin N.F. optimally at pH 8.5 or above. Activity was dependent on CaCl₂. Pectinmethylesterase activity was not detected in water, NaCl, or sucrose extracts of these nematodes. The extracts macerated potato tuber tissue, onion cotyledonary tissue, and strips of onion epidermis from the ventral surface of onion bulb scales at pH 4.2, 5.3, and 6.2. Pectin could not be localized with hydroxylamine-ferric chloride reagent in macerated tissues treated for 24 hr with active extract.     

PSM/SH2-B distributes selected mitogenic receptor signals to distinct components in the PI3-kinase and MAP kinase signaling pathways

The Pro-rich, PH, and SH2 domain containing mitogenic signaling adapter PSM/SH2-B has been implicated as a cellular partner of various mitogenic receptor tyrosine kinases and related signaling mechanisms. Here, we report in a direct comparison of three peptide hormones, that PSM participates in the assembly of distinct mitogenic signaling complexes in response to insulin or IGF-I when compared to PDGF in cultured normal fibroblasts. The complex formed in response to insulin or IGF-I involves the respective peptide hormone receptor and presumably the established components leading to MAP kinase activation. However, our data suggest an alternative link from the PDGF receptor via PSM directly to MEK1/2 and consequently also to p44/42 activation, possibly through a scaffold protein. At least two PSM domains participate, the SH2 domain anticipated to link PSM to the respective receptor and the Pro-rich region in an association with an unidentified downstream component resulting in direct MEK1/2 and p44/42 regulation. The PDGF receptor signaling complex formed in response to PDGF involves PI 3-kinase in addition to the same components and interactions as described for insulin or IGF-I. PSM associates with PI 3-kinase via p85 and in addition the PSM PH domain participates in the regulation of PI 3-kinase activity, presumably through membrane interaction. In contrast, the PSM Pro-rich region appears to participate only in the MAP kinase signal. Both pathways contribute to the mitogenic response as shown by cell proliferation, survival, and focus formation. PSM regulates p38 MAP kinase activity in a pathway unrelated to the mitogenic response.     

Optimization of heterologous production of the polyketide 6-MSA in Saccharomyces cerevisiae

Polyketides are a group of natural products that have gained much interest due to their use as antibiotics, cholesterol lowering agents, immunosuppressors, and as other drugs. Many organisms that naturally produce polyketides are difficult to cultivate and only produce these metabolites in small amounts. It is therefore of general interest to transfer polyketide synthase (PKS) genes from their natural sources into heterologous hosts that can over-produce the corresponding polyketides. In this study we demonstrate the heterologous expression of 6-methylsalicylic acid synthase (6-MSAS), naturally produced by Penicillium patulum, in the yeast Saccharomyces cerevisiae. In order to activate the PKS a 4'-phosphopantetheinyl transferase (PPTase) is required. We therefore co-expressed PPTases encoded by either sfp from Bacillus subtilis or by npgA from Aspergillus nidulans. The different strains were grown in batch cultures. Growth and product concentration were measured and kinetic parameters were calculated. It was shown that both PPTases could be efficiently used for activation of PKS's in yeast as good yields of 6-MSA were obtained with both enzymes.     

Temperature acclimation in a biochemical model of photosynthesis: a reanalysis of data from 36 species

The Farquhar et al. model of C(3) photosynthesis is frequently used to study the effect of global changes on the biosphere. Its two main parameters representing photosynthetic capacity, V(cmax) and J(max), have been observed to acclimate to plant growth temperature for single species, but a general formulation has never been derived. Here, we present a reanalysis of data from 36 plant species to quantify the temperature dependence of V(cmax) and J(max) with a focus on plant growth temperature, i.e. the plants' average ambient temperature during the preceding month. The temperature dependence of V(cmax) and J(max) within each data set was described very well by a modified Arrhenius function that accounts for a decrease of V(cmax) and J(max) at high temperatures. Three parameters were optimized: base rate, activation energy and entropy term. An effect of plant growth temperature on base rate and activation energy could not be observed, but it significantly affected the entropy term. This caused the optimum temperature of V(cmax) and J(max) to increase by 0.44 degrees C and 0.33 degrees C per 1 degrees C increase of growth temperature. While the base rate of V(cmax) and J(max) seemed not to be affected, the ratio J(max) : V(cmax) at 25 degrees C significantly decreased with increasing growth temperature. This moderate temperature acclimation is sufficient to double-modelled photosynthesis at 40 degrees C, if plants are grown at 25 degrees C instead of 17 degrees C.     

Effects of resource level and habitat type on behavioral and morphological plasticity in Eurasian perch

Spatial and temporal heterogeneity in the environment is a common feature affecting many natural populations. For example, both the resource levels and optimal habitat choices of individuals likely change over time. One way for organisms to cope with environmental variation is to display adaptive plasticity in traits such as behavior and morphology. Since trait plasticity is hypothesized to be a prerequisite for character divergence, studies of mechanisms behind such plasticity are warranted. In this study, we looked at the interaction of two potentially important environmental variables on behavioral and morphological plasticity in Eurasian perch (Perca fluviatilis L.). More specifically, the plastic responses in activity and morphology of perch exposed to different resource levels and simulated habitat types were studied in an aquarium experiment. The resource level experienced had a large influence on plasticity in both activity and morphology. Behavioral adaptations have been thought to mediate morphological transitions, and we suggest that the morphological response to the resource level was mediated by differences in activity and growth rates. The habitat type also affected morphological plasticity but to a lesser extent, and there was no effect on activity from habitat type. Based on these results, we suggest that it is essential to include several environmental factors acting in concert when studying mechanisms behind trait plasticity. We also propose that variation in resource levels might play a key role in fostering trait plasticity in at least fish populations, while other environmental variables such as divergent habitat complexities and prey types might be less influential. Dynamics in resource levels and optimal habitat choices might thus be important factors influencing character divergence in natural populations.     

An ana2/ctp/mud complex regulates spindle orientation in Drosophila neuroblasts

Drosophila neural stem cells, larval brain neuroblasts (NBs), align their mitotic spindles along the apical/basal axis during asymmetric cell division (ACD) to maintain the balance of self-renewal and differentiation. Here, we identified a protein complex composed of the tumor suppressor anastral spindle 2 (Ana2), a dynein light-chain protein Cut up (Ctp), and Mushroom body defect (Mud), which regulates mitotic spindle orientation. We isolated two ana2 alleles that displayed spindle misorientation and NB overgrowth phenotypes in larval brains. The centriolar protein Ana2 anchors Ctp to centrioles during ACD. The centriolar localization of Ctp is important for spindle orientation. Ana2 and Ctp localize Mud to the centrosomes and cell cortex and facilitate/maintain the association of Mud with Pins at the apical cortex. Our findings reveal that the centrosomal proteins Ana2 and Ctp regulate Mud function to?orient the mitotic spindle during NB asymmetric division.     

Protein kinase CK2 links polyamine metabolism to MAPK signalling in Drosophila

MAPK signalling is a complex process not only requiring the core components Raf, MEK and Erk, but also many proteins like the scaffold protein KSR and several kinases to specifically localize, modulate and fine-tune the outcome of the pathway in a cell context specific manner. In mammals, protein kinase CK2 was shown to bind to the scaffold protein KSR and to phosphorylate Raf proteins at a conserved serine residue in the negative-charge regulatory (N−) region, thereby facilitating maximal activity of the MAPK signalling pathway. In this work we show that in Drosophila CK2 is also bound to KSR. However, despite the presence of a corresponding serine residue in the N-region of DRaf, CK2-mediated phosphorylation of DRaf takes place on a serine residue at the N-terminus and is required for Erk activation. Previous work identified polyamines as regulators of CK2 kinase activity. The main cellular source of polyamines is the catabolism of amino acids. Evidence is provided that phosphorylation of DRaf by CK2 is modulated by polyamines, with spermine being the most potent inhibitor of the reaction. We suggest that CK2 is able to monitor intracellular polyamine levels and translates this information to modulate MAPK signalling.     

Simplified Intestinal Microbiota to Study Microbe-Diet-Host Interactions in a Mouse Model

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Epitope-specific human influenza antibody repertoires diversify by B cell intraclonal sequence divergence and interclonal convergence

We generated from a single blood sample five independent human mAbs that recognized the Sa antigenic site on the head of influenza hemagglutinin and exhibited inhibitory activity against a broad panel of H1N1 strains. All five Abs used the V(H)3-7 and J(H)6 gene segments, but at least four independent clones were identified by junctional analysis. High-throughput sequence analysis of circulating B cells revealed that each of the independent clones were members of complex phylogenetic lineages that had diversified widely using a pattern of progressive diversification through somatic mutation. Unexpectedly, B cells encoding multiple diverging lineages of these clones, including many containing very few mutations in the Ab genes, persisted in the circulation. Conversely, we noted frequent instances of amino acid sequence convergence in the Ag combining sites exhibited by members of independent clones, suggesting a strong selection for optimal binding sites. We suggest that maintenance in circulation of a wide diversity of somatic variants of dominant clones may facilitate recognition of drift variant virus epitopes that occur in rapidly mutating virus Ags, such as influenza hemagglutinin. In fact, these Ab clones recognize an epitope that acquired three glycosylation sites mediating escape from previously isolated human Abs.