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.     

A New Real-Time PCR for the Detection of Plasmodium ovale wallikeri

It has been proposed that ovale malaria in humans is caused by two closely related but distinct species of malaria parasites: P. ovale curtisi and P. ovale wallikeri. We have extended and optimized a Real-time PCR assay targeting the parasite’s small subunit ribosomal RNA (ssrRNA) gene to detect both these species. When the assay was applied to 31 archival blood samples from patients diagnosed with P. ovale, it was found that the infection in 20 was due to P. ovale curtisi and in the remaining 11 to P. ovale wallikeri. Thus, this assay provides a useful tool that can be applied to epidemiological investigations of the two newly recognized distinct P. ovale species, that might reveal if these species also differ in their clinical manifestation, drugs susceptibility and relapse periodicity. The results presented confirm that P. ovale wallikeri is not confined to Southeast Asia, since the majority of the patients analyzed in this study had acquired their P. ovale infection in African countries, mostly situated in West Africa.     

Pectolytic clostridia isolated from sugar beet pulp silages in Italy

Twenty-eight strains of pectolytic clostridia were isolated from sugar beet pulp silages. Seventeen non-pigmented strains were presumed to be Clostridium acetobutylicum ; the remaining 11 pigmented strains were similar to Cl. felsineum. The addition of molasses to sugar beet pulps favoured the growth of other bacteria, particularly lactic acid organisms, whereas pectolytic clostridia were only occasionally found. The pectolytic clostridia promoted the structure loss of simulated silages. The use of molasses in sugar beet pulp ensiling was suggested to prevent texture loss of the ensiled mass.     

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.