CK2-mediated phosphorylation of a type II regulatory subunit of cAMP-dependent protein kinase from the mollusk Mytilus galloprovincialis

Two isoforms of regulatory (R) subunit of cAMP-dependent protein kinase (PKA), named R(myt1) and R(myt2), were identified so far in the sea mussel Mytilus galloprovincialis. Out of them, only R(myt2) was phosphorylated in vitro by casein kinase 2 (CK2) using GTP as phosphate donor. CK2 catalytic subunit (CK2alpha) itself was sufficient to phosphorylate R(myt2), but phosphorylation was enhanced by the presence of the regulatory subunit CK2beta. Even in the absence of CK2, R(myt2) was phosphorylated to a certain extent when it was incubated with GTP. This basal phosphorylation was partially abolished by the known inhibitors apigenin and emodin, which suggests the presence of a residual amount of endogenous CK2 in the preparation of purified R subunit. CK2-mediated phosphorylation significantly decreases the ability of R(myt2) to inhibit PKA catalytic (C) subunit activity in the absence of cAMP. On the other hand, the sequence of several peptides obtained from the tryptic digestion of R(myt2) showed that mussel protein contains the signature sequence common to all PKA family members, within the "phosphate binding cassette" (PBC) A and B. Moreover, the degree of identity between the sequences of peptides from R(myt2), as a whole, and those from type II R subunits was 68-75%, but the global identity percentage with type I R subunits was only about 30%, so that R(myt2) can be classified as a type II R subunit.     

Study of laser created ZRO2 and hydroxyapatite/ZrO2 films for implantology

Thin films of ZrO2 and hydroxyapatite/ZrO2 were created by excimer laser ablation on Ti6Al4V substrates. ZrO2 layers were fabricated in vacuum by KrF laser at various substrate temperatures and hydroxyapatite (HA) layers were fabricated in water vapor ambient by ArF laser and in water vapor/argon ambient by KrF excimer laser. Film properties were evaluated by XRD, SEM and WDX methods. The test of mechanical adhesion was proceeded on ZrO2 films. XRD analysis proved the presence of amorphous or crystalline HA in the deposited films. SEM method demonstrated smooth surface covered by droplets for both HA and ZrO2 films. Ca/P ratio of the HA films is higher than that of the natural HA and is within the range of 2.8-3.0. The HA/ZrO2 and ZrO2 samples were tested in vitro for cytotoxicity. The best results were received by the HA/ZrO2 samples in the test of cytotoxicity. Fibroblasts cultivating with HA/ZrO2 samples exhibited subconfluent and confluent growth and showed fibronectin homogenously.     

Density‐related effects on the infectivity and aggressiveness of a sterilising smut in a wild population of Digitaria sanguinalis

Understanding host–pathogen evolutionary dynamics needs characterisation and quantification of processes occurring at many spatiotemporal scales. With this aim, the effects of smut on a naturally infected population of the summer annual Digitaria sanguinalis were followed for 4 years in an uncropped field. The main purpose of the study was to quantify the effects of within‐population density on the infectivity and the aggressiveness of the pathogen in a range of densities that occurred naturally. The infectivity‐related variable measured was the proportion of smutted plants at the end of each growing season; proportions were analysed using a generalised linear model with a binomial distribution considering the year, the density and their interaction as effects. The aggressiveness‐related variables chosen were the number of smutted inflorescences per plant and per area, obtained over the last 2 years; they were analysed by means of ancova considering disease status (seeded or smutted), year, density and all the interactions between them. Although the disease is monocyclic, results showed clearly that infectivity increased with plant density. The number of inflorescences per plant was 1.5 times higher in smutted plants than in healthy plants throughout the range of densities. This variable declined when density increased, but as the infectivity increased at a higher rate, the aggressiveness also increased with density. The surprising results on infectivity are discussed in the context of current knowledge of plant–pathogen interaction dynamics, as well as neighbour effects on pathogen aggressiveness. Moreover, the results could be useful to develop weed biological control strategies.     

Enhancing yeast cell viability after dehydration by modification of the lipid profile

In the present study, we analysed metabolite features during the dehydration-rehydration process for different yeast species genetically closely related to S. cerevisiae, in order to determine whether metabolites might play a role in cell viability. We ranked the species S. cerevisiae, S. paradoxus, S. kudriavzevii, L. kluyveri, N. castellii, S. mikatae, S. bayanus, and S. servazzii according to their viability rate after the dehydration-rehydration process, and showed that desiccation tolerance across the species did not correlate with the intracellular content of trehalose or glycogen. Cell lipid composition was also investigated during this process, to see whether the content of triacylglycerols and phosphatidylcholine showed significant variations across the species. The increase of phosphatidylcholine level increase in both S. paradoxus and S. bayanus cells grown in supplemented media enhanced both their cell viability after stress imposition and lipid storage.     

Effects of spinal or general anesthesia on F₂-isoprostanes and isofurans during ischemia/reperfusion of the leg in patients undergoing knee replacement surgery

General and spinal anesthesia are used extensively in orthopedic surgery. However, these methods of anesthesia may result in different amounts of oxygen being delivered to the patient. Ischemia/reperfusion injury after release of the tourniquet initiates free radical-mediated oxidative stress. F₂-isoprostanes are reliable markers of in vivo lipid peroxidation. However, under conditions of high oxygen tension, isofurans are formed. We aimed to compare plasma isofurans and F₂-isoprostanes in spinal versus general anesthesia in patients undergoing knee-replacement surgery in a randomized, blinded study. Thirty-nine patients were randomized to spinal (SA; n = 19) or general anesthesia (GA; n = 20). Blood was collected before anesthesia, and a tourniquet was then applied to the limb during surgery. After release of the tourniquet, blood samples were collected at 30 min, 2 h, and 24 h for measurement of plasma F₂-isoprostanes and isofurans by gas chromatography–mass spectrometry. The two groups were comparable in age and body mass index. Plasma F₂-isoprostanes were significantly lower in the GA patients compared with the SA patients (p = 0.045). In contrast, the GA patients had significantly elevated plasma isofurans (p = 0.032). Increased isofurans during GA compared with SA are likely to reflect increased oxidative stress due to elevated oxygen concentrations during GA. Further studies are required to assess the implications of these findings on perioperative outcomes.     

Pivalic acid acts as a starter unit in a fatty acid and antibiotic biosynthetic pathway in Alicyclobacillus, Rhodococcus and Streptomyces

A biosynthetic pathway using pivalic acid as a starter unit was found in three bacterial species, Alicyclobacillus acidoterrestris, Rhodococcus erythropolis and Streptomyces avermitilis. When deuterium-labelled pivalic acid was added to A. acidoterrestris and R. erythropolis nutrient media it was incorporated into fatty acids to give rise to tert-butyl fatty acids (t-FAs). In addition, in R. erythropolis, pivalic acid was transformed into two starter units, i.e. isobutyric and 2-methylbutyric acid, which served as precursors of corresponding iso-even FAs and anteiso-FAs. In S. avermitilis the biosynthesis also yielded all three branched FAs; apart from this pathway, both pivalic and 2-methylbutyric acids were incorporated into the antibiotic avermectin.     

ORF-selector ESPRIT: a second generation library screen for soluble protein expression employing precise open reading frame selection

Here we present ORF-selector ESPRIT, a 9-fold enhanced version of our technology for screening incremental truncation libraries to identify soluble high yielding constructs of challenging proteins. Gene fragments are truncated at both termini to access internal domains and the resulting reading frame problem is addressed by an unbiased, intein-based open reading frame selection yielding only in-frame DNA inserts. This enriched library is then subcloned into a standard high-level expression plasmid where tens of thousands of constructs can be assayed in a two-step process using colony- and liquid-handling robots to isolate rare highly expressing clones useful for production of multi milligram quantities of purifiable proteins. The p85α protein was used to benchmark the system resulting in isolation of all known domains, either alone or in tandem. The human kinase IKK1 was then screened resulting in purification of a predicted internal domain. This strategy provides an integrated, facile route to produce soluble proteins from challenging and poorly understood target genes at quantities compatible with structural biology, screening applications and immunisation studies. The high genetic diversity that can be sampled opens the way to study more diverse systems including multisubunit complexes.     

Proteomic analysis of polypeptides captured from blood during extracorporeal albumin dialysis in patients with cholestasis and resistant pruritus

Albumin dialysis using the molecular adsorbent recirculating system (MARS) is a new therapeutic approach for liver diseases. To gain insight into the mechanisms involved in albumin dialysis, we analyzed the peptides and proteins absorbed into the MARS strong anion exchange (SAX) cartridges as a result of the treatment of patients with cholestasis and resistant pruritus. Proteins extracted from the SAX MARS cartridges after patient treatment were digested with two enzymes. The resulting peptides were analyzed by multidimensional liquid chromatography coupled to tandem mass spectrometry. We identified over 1,500 peptide sequences corresponding to 144 proteins. In addition to the proteins that are present in control albumin-derived samples, this collection includes 60 proteins that were specific to samples obtained after patient treatment. Five of these proteins (neutrophil defensin 1 [HNP-1], secreted Ly-6/uPAR-related protein 1 [SLURP1], serum amyloid A, fibrinogen alpha chain and pancreatic prohormone) were confirmed to be removed by the dialysis procedure using targeted selected-reaction monitoring MS/MS. Furthermore, capture of HNP-1 and SLURP1 was also validated by Western blot. Interestingly, further analyses of SLURP1 in serum indicated that this protein was 3-fold higher in cholestatic patients than in controls. Proteins captured by MARS share certain structural and biological characteristics, and some of them have important biological functions. Therefore, their removal could be related either to therapeutic or possible adverse effects associated with albumin dialysis.