Toward Système International d'Unité-traceable protein quantification: from amino acids to proteins

Here we present a demonstration of the proof of principle that absolute concentration of a protein within a mixture of other proteins can be measured with SI traceability. The method used was based on tryptic digestion of a protein followed by quantification using double exact matching isotope dilution mass spectrometry (IDMS) of the peptides released. To provide full SI traceability to measurements of protein concentration we demonstrated a method of SI traceable peptide quantification in which the peptide standards used were quantified by an amino acid analysis method that incorporated double exact matching IDMS and amino acid standards of known purity. The concentration of the protein was therefore determined based upon the concentration of tryptic peptides, which in turn had been quantified based upon amino acid standards. This allowed fully SI-traceable measurements of protein concentration to be made. Important caveats in the implementation of this approach are also discussed and examples of how these can have detrimental effects on the measurements are shown.     

Small- and large-scale conformational changes of adenylate kinase: a molecular dynamics study of the subdomain motion and mechanics

Adenylate kinase, an enzyme that catalyzes the phosphoryl transfer between ATP and AMP, can interconvert between the open and catalytically potent (closed) forms even without binding ligands. Several aspects of the enzyme elasticity and internal dynamics are analyzed here by atomistic molecular dynamics simulations covering a total time span of 100 ns. This duration is sufficiently long to reveal a partial conversion of the enzyme that proceeds through jumps between structurally different substates. The intra- and intersubstates contributions to the enzyme's structural fluctuations are analyzed and compared both in magnitude and directionality. It is found that, despite the structural heterogeneity of the visited conformers, the generalized directions accounting for conformational fluctuations within and across the substates are mutually consistent and can be described by a limited set of collective modes. The functional-oriented nature of the consensus modes is suggested by their good overlap with the deformation vector bridging the open and closed crystal structures. The consistency of adenylate kinase's internal dynamics over timescales wide enough to capture intra- and intersubstates fluctuations adds elements in favor of the recent proposal that the free (apo) enzyme possesses an innate ability to sustain the open/close conformational changes.     

Induction of efficient energy dissipation in the isolated light-harvesting complex of Photosystem II in the absence of protein aggregation

Under excess illumination, the Photosystem II light-harvesting antenna of higher plants has the ability to switch into an efficient photoprotective mode, allowing safe dissipation of excitation energy into heat. In this study, we show induction of the energy dissipation state, monitored by chlorophyll fluorescence quenching, in the isolated major light-harvesting complex (LHCII) incorporated into a solid gel system. Removal of detergent caused strong fluorescence quenching, which was totally reversible. Singlet-singlet annihilation and gel electrophoresis experiments suggested that the quenched complexes were in the trimeric not aggregated state. Both the formation and recovery of this quenching state were inhibited by a cross-linker, implying involvement of conformational changes. Absorption and CD measurements performed on the samples in the quenched state revealed specific alterations in the spectral bands assigned to the red forms of chlorophyll a, neoxanthin, and lutein 1 molecules. The majority of these alterations were similar to those observed during LHCII aggregation. This suggests that not the aggregation process as such but rather an intrinsic conformational transition in the complex is responsible for establishment of quenching. 77 K fluorescence measurements showed red-shifted chlorophyll a fluorescence in the 690-705 nm region, previously observed in aggregated LHCII. The fact that all spectral changes associated with the dissipative mode observed in the gel were different from those of the partially denatured complex strongly argues against the involvement of protein denaturation in the observed quenching. The implications of these findings for proposed mechanisms of energy dissipation in the Photosystem II antenna are discussed.     

Use of methylumbeliferyl-derivative substrates for lipase activity characterization

Lipases and esterases have been recognized as very useful biocatalysts because of their wide-ranging versatility in industrial applications, their stability, low cost, and non-requirement for added cofactors. The physical properties of lipidic substrates, typically water insoluble, have determined a great difficulty in studying lipolytic enzymes. A method for fast and simple detection of lipolytic activity, based on the use of 4-methylumbelliferone (MUF)-derivative substrates was developed. The system has been used for the detection of lipase activity either from microbial colonies, cell culture suspensions, or from proteins separated on SDS-polyacrylamide or isoelectric focusing gels. The use of MUF-derivative substrates has also been extended to the quantitative determination of lipolytic activity from a variety of assays including optimum pH and temperature determination, growth dependency, kinetics or stability studies, or residual activity quantification after treatment with potential inhibitors. The method has shown to be a useful tool for the characterization of a variety of lipases from microbial origin, including those cloned in heterologous hosts.     

A simple and inexpensive trap-tube sampler for zooplankton collection in shallow waters

The paper describes a new type of zooplankton sampler, which combines the concepts of the Pennak core sampler and the Schindler-Patalas plankton trap. The new sampler, called Trap Tube Sampler, consists of a PVC water pipe (1.5–2.0 m, long; 10 cm diameter) provided, at the bottom end, of a filtering unit and closing mechanism which alternatively closes the mouth of the tube and the mouth of the filtering unit. The new device is particularly suitable for collecting samples from the entire water column in shallow vegetated water bodies, fish ponds and mesocosm tanks.     

Assembly of an FtsZ Mutant Deficient in GTPase Activity Has Implications for FtsZ Assembly and the Role of the Z Ring in Cell Division

FtsZ, the ancestral homologue of eukaryotic tubulins, assembles into the Z ring, which is required for cytokinesis in prokaryotic cells. Both FtsZ and tubulin have a GTPase activity associated with polymerization. Interestingly, the ftsZ2 mutant is viable, although the FtsZ2 mutant protein has dramatically reduced GTPase activity due to a glycine-for-aspartic acid substitution within the synergy loop. In this study, we have examined the properties of FtsZ2 and found that the reduced GTPase activity is not enhanced by DEAE-dextran-induced assembly, indicating it has a defective catalytic site. In the absence of DEAE-dextran, FtsZ2 fails to assemble unless supplemented with wild-type FtsZ. FtsZ has to be at or above the critical concentration for copolymerization to occur, indicating that FtsZ is nucleating the copolymers. The copolymers formed are relatively stable and appear to be stabilized by a GTP-cap. These results indicate that FtsZ2 cannot nucleate assembly in vitro, although it must in vivo. Furthermore, the stability of FtsZ-FtsZ2 copolymers argues that FtsZ2 polymers would be stable, suggesting that stable FtsZ polymers are able to support cell division.     

Blood trace metals in a sporadic amyotrophic lateral sclerosis geographical cluster

Amyotrophic lateral sclerosis (ALS) is a fatal disorder with unknown etiology, in which genetic and environmental factors interplay to determine the onset and the course of the disease. Exposure to toxic metals has been proposed to be involved in the etiology of the disease either through a direct damage or by promoting oxidative stress. In this study we evaluated the concentration of a panel of metals in serum and whole blood of a small group of sporadic patients, all living in a defined geographical area, for which acid mine drainage has been reported. ALS prevalence in this area is higher than in the rest of Italy. Results were analyzed with software based on artificial neural networks. High concentrations of metals (in particular Se, Mn and Al) were associated with the disease group. Arsenic serum concentration resulted lower in ALS patients, but it positively correlated with disease duration. Comet assay was performed to evaluate endogenous DNA damage that resulted not different between patients and controls. Up to now only few studies considered geographically well-defined clusters of ALS patients. Common geographical origin among patients and controls gave us the chance to perform metallomic investigations under comparable conditions of environmental exposure. Elaboration of these data with software based on machine learning processes has the potential to be extremely useful to gain a comprehensive view of the complex interactions eventually leading to disease, even in a small number of subjects.     

<Emphasis Type="Italic">Lecanicillium attenuatum</Emphasis> isolates affecting the invasive cypress aphid (<Emphasis Type="Italic">Cinara cupressi</Emphasis>) in Chile

The cypress aphid (Cinara cupressi) is listed among the hundred most important invasive pests in the world. In Chile, it was first detected in 2003 and currently is present throughout the country. In the course of a survey of their natural enemies in Chile, three strains of entomopathogenic fungi were isolated. The isolates were identified and tested against the aphid in laboratory experiments. Two further entomopathogenic fungi (ARSEF 5126 and 5128), formulated in the mycoinsecticides Vertalec^® and Mycotal^®, were used as reference strains. The three Chilean isolates were identified genomically as Lecanicillium attenuatum and were pathogenic to third-instar nymphs. The isolate ARSEF 13279 yielded the lowest overall lethal concentration (LC₅₀), 3 × 10⁵ conidia ml^?1 at four days post-inoculation, and the shortest lethal time (LT₅₀), 3.7 days after inoculation with 10⁶ conidia ml^?1. The results indicate that the isolates have considerable potential as microbial control agents of the invasive cypress aphid.