Nutrient conservation strategies in native Andean‐Patagonian forests

Abstract. Nutrient conservation in vegetation affects rates of litter decomposition and soil nutrient availability. Although resorption has been traditionally considered one of the most important plant strategies to conserve nutrients in temperate forests, long leaf life‐span and low nutrient requirements have been postulated as better indicators. We aimed at identifying nutrient conservation strategies within characteristic functional groups of NW Patagonian forests on Andisols. We analysed C‐, N‐, P‐, K‐ and lignin‐concentrations in mature and senescent leaves of ten native woody species within the functional groups: broad‐leaved deciduous species, broad‐leaved evergreens and conifers. We also examined mycorrhizal associations in all species. Nutrient concentration in mature leaves and N‐ resorption were higher in broad‐leaved deciduous species than in the other two functional groups. Conifers had low mature leaf nutrient concentrations, low N‐resorption and high lignin/N ratios in senescent leaves. P‐ and K‐resorptions did not differ among functional groups. Broad‐leaved evergreens exhibited a species‐dependent response. Nitrogen in mature leaves was positively correlated with both N resorption and soil N‐fertility. Despite the high P‐retention capacity of Andisols, N appeared to be the more limiting nutrient, with most species being proficient in resorbing N but not P. The presence of endomycorrhizae in all conifers and the broad‐leaved evergreen Maytenus boaria, ectomycorrhizae in all Nothofagus species (four deciduous, one evergreen), and cluster roots in the broad‐leaved evergreen Lomatia hirsuta, would be possibly explaining why P is less limiting than N in these forests.     

Dimer–monomer equilibrium of human thymidylate synthase monitored by fluorescence resonance energy transfer

An ad hoc bioconjugation/fluorescence resonance energy transfer (FRET) assay has been designed to spectroscopically monitor the quaternary state of human thymidylate synthase dimeric protein. The approach enables the chemoselective engineering of allosteric residues while preserving the native protein functions through reversible masking of residues within the catalytic site, and is therefore suitable for activity/oligomerization dual assay screenings. It is applied to tag the two subunits of human thymidylate synthase at cysteines 43 and 43′ with an excitation energy donor/acceptor pair. The dimer–monomer equilibrium of the enzyme is then characterized through steady‐state fluorescence determination of the intersubunit resonance energy transfer efficiency.     

Legionella pneumophila in Cooling Towers: Fluctuations in Counts, Determination of Genetic Variability by Pulsed-Field Gel Electrophoresis (PFGE), and Persistence of PFGE Patterns

The concentrations of Legionella pneumophila in cooling towers may vary considerably over short periods of time, producing significant fluctuations throughout the year. Despite genetic variability, in small geographical areas the same indistinguishable pulsed-field gel electrophoresis patterns may be shared among different cooling towers and persist over time.     

Heterogeneous phenotype of human glioblastoma: In vitro study

Glioblastomas (GBMs) are the most lethal primary brain tumours. Increasing evidence shows that brain tumours contain the population of stem cells, so‐called cancer stem cells (CSCs). Stem cell marker CD133 was reported to identify CSC population in GBM. Further studies have indicated that CD133 negative cells exhibiting similar properties and are able to initiate the tumour, self‐renew and undergo multilineage differentiation. GBM is a highly heterogeneous tumour and may contain different stem cell populations with different functional properties. We characterized five GBM cell lines, established from surgical samples, according to the marker expression, proliferation and differentiation potential. CD133 positive cell lines showed increased proliferation rate in neurosphere condition and marked differentiation potential towards neuronal lineages. Whereas two cell lines low‐expressing CD133 marker showed mesenchymal properties in vitro, that is high proliferation rate in serum condition and differentiation in mesenchymal cell types. Further, we compared therapy resistance capacity of GBM cell lines treated with hydroxyurea. Our results suggest that CSC concept is more complex than it was believed before, and CD133 could not define entire stem cell population within GBM. At least two different subtypes of GBM CSCs exist, which may have different biological characteristics and imply different therapeutic strategies. Copyright © 2013 John Wiley & Sons, Ltd.     

Engineering crops, a deserving venture.

Plant transformation has had a deep impact on several aspects of basic and applied research. Genetic transformation has offered new opportunities compared to traditional breeding practises since it allows the integration into a host genome of specific sequences leading to a strong reduction of the casualness of gene transfer. One of the first target areas was plant protection against pests, pathogens and environmental stresses while the recent plant engineering programs are aimed at increasing food quality, in particular at increasing nutritional characteristics of food crops. Moreover, transgenic plants, tissue or cell cultures represent an attractive biological system for producing heterologous proteins since they offer economic and qualitative benefits. High yield production can be obtained and large-scale commercial production will take advantage of the existing infrastructure for crop cultivation, processing and storage. There are also qualitative benefits since protein synthesis secretion and post-translational modifications are similar in plants and animal cells. There are no human viral pathogens harboured by plants: thus, especially for pharmaceuticals, plants represent the safer production system. Plant transformation has become an essential instrument also for basic research, in particular for the functional characterisation of genes identified by sequencing of whole genomes. Large collections of insertion mutants have been obtained in the model plant Arabidopsis to provide a high level of genome saturation that means 95% chance of inactivating any gene at least once. To instil greater public confidence in modern plant biotechnology recent advances have already been made to overcome the potential risks for human health and environment.     

Characterization of a promoter up mutation in the -35 region of the promoter of the primer for ColE1 replication

Summary A point mutation in the -35 region of the promoter of the primer for initiation of DNA replication in the plasmid pMB1 was characterized. This base change causes a promoter up phenotype. The analysis of a second mutant obtained by site-directed mutagenesis allowed the exclusion of a role in the phenotype for the potential intrastrand secondary structure as well as for the methylation state of the DNA in the promoter region. The promoter up phenotype is concluded to be due to a change in the primary structure of the — 35 element with the consequent production of a better cluster of hydrogen bond donors and acceptors for the RNA polymerase.     

Ca2+ phospholipid-dependent and independent phosphorylation of synthetic peptide substrates by protein kinase C

Several synthetic peptides reproducing fragments of protamines have been used as model substrates for Ca2+/phospholipid-dependent protein kinase C, tested both in the absence of any effector (basal conditions) and upon activation by either Ca2+ and phosphatidylserine (or diacylglycerol) or limited proteolysis. Only the peptide Arg4-Tyr-Gly-Ser-Arg6-Tyr [Ga(52-65)] shares the unique property of protamines of being readily phosphorylated even under basal conditions. Optimal activity in the absence of effectors is observed with Tris/HCl buffer pH 7.5; Pipes and Hepes are less effective at pH 7.5, and at pH 6.5 basal phosphorylation is reduced. Under the best conditions for basal phosphorylation of Ga(52-65), its derivative with ornithine replaced for arginine and those corresponding to its C-terminal fragments Gly-Ser-Arg6-Tyr [Ga(57-65)] and Gly-Ser-Arg3 [Ga(57-61)], as well as the peptides Pro-Arg5-Ser2-Arg-Pro-Val-Arg [Th(1-12)], Arg4-Tyr-Arg2-Ser-Thr-Val-Ala [Th(13-23)] and Arg2-Leu-Ser2-Leu-Arg-Ala are not significantly affected though all of them, like histones, are more or less readily phosphorylated upon activation of protein kinase C by Ca2+/phosphatidylserine. The peptide Ser2-Arg-Pro-Val-Arg [Th(7-12)] however, corresponding to the C-terminal part of Th(1-12), is not phosphorylated even in the presence of activators. Limited proteolysis can roughly mimic the Ca2+/phosphatidylserine effect inducing however different extents of activation depending on the nature of the peptide substrates. Our results support the following two conclusions. Basal phosphorylation by protein kinase C in the absence of any effector requires peptide substrates whose target residue(s) are included between two extended arginyl blocks and is also dependent on pH and nature of the buffer. Peptides having extended clusters of either arginyl or ornithyl residues on the C-terminal side of serine are also readily phosphorylated, but they need activation of protein kinase by either Ca2+/phosphatidylserine or limited proteolysis. The same is true of peptides having basic residues only on the N-terminal side, or even on both sides but in limited number.     

Mammalian DNA polymerase alpha: a replication-competent holoenzyme form from calf thymus

Calf thymus DNA polymerase alpha, like the replication-specific DNA polymerase III holoenzyme of Escherichia coli, can be isolated as a distinct complex. A specific multiprotein form of the polymerase alpha, a form designated replication-competent (RC) holoenzyme, consists of a complex of a polymerase-primase core and at least six other polypeptides. The RC holoenzyme can efficiently replicate several naturally occurring templates, including the genomic DNA of the porcine circovirus (PCV). The DNA of this virion consists of a single-stranded circle with a defined replication origin, and its replication requires the cellular DNA replication machinery. It might therefore provide an invaluable opportunity to investigate chromosomal replication mechanisms, analogous to the way that studies on E. coli bacteriophage DNA replication elucidated host DNA replication mechanisms. Calf RC holoenzyme alpha selectively initiates PCV DNA replication in vitro at a site that possibly represents a consensus sequence of cellular DNA replication origins. The cell-free PCV replication system will be exploited for the in vitro dissection and reconstitution of the RC holoenzyme and the functional analysis of its component polypeptides.