Efficient targeting of plant disease resistance loci using NBS profiling

The conserved sequences in the nucleotide-binding sites of the nucleotide-binding site-leucine-rich repeat (NBS-LRR) class of disease resistance (R) genes have been used for PCR-based R-gene isolation and subsequent development of molecular markers. Here we present a PCR-based approach (NBS profiling) that efficiently targets R genes and R-gene analogs (RGAs) and, at the same time, produces polymorphic markers in these genes. In NBS profiling, genomic DNA is digested with a restriction enzyme, and an NBS-specific (degenerate) primer is used in a PCR reaction towards an adapter linked to the resulting DNA fragments. The NBS profiling protocol generates a reproducible polymorphic multilocus marker profile on a sequencing gel that is highly enriched for R genes and RGAs. NBS profiling was successfully used in potato with several restriction enzymes, and several primers targeted to different conserved motifs in the NBS. Across primers and enzymes, the NBS profiles contained 50–90% fragments that were significantly similar to known R-gene and RGA sequences. The protocol was similarly successful in other crops (including tomato, barley, and lettuce) without modifications. NBS profiling can thus be used to produce markers tightly linked to R genes and R-gene clusters for genomic mapping and positional cloning and to mine for new alleles and new sources of disease resistance in available germplasm.Communicated by H.F. Linskens     

Development of a high throughput screening method to test flavour-forming capabilities of anaerobic micro-organisms

AIM: Development of a fast, automated and reliable screening method for screening of large collections of bacterial strains with minimal handling time. METHODS AND RESULTS: The method is based on the injection of a small headspace sample (100 microl) from culture vials (2 ml) in 96-well format directly into the mass spectrometry (MS). A special sample tray has been developed for liquid media, and anaerobically grown cultures. In principle, all volatile components can be measured, but a representative mass fragment has to be obtained in the MS. Representative masses for 3-methylbutanal, 2-methylpropanal and benzaldehyde are 58, 72 and 105, respectively. In 1 day over 1500 samples could be analysed and the coefficient of variation for the response was <5%. CONCLUSION: Screening of 72 strains belonging to the genus Lactococcus in quadruple on the production of the key-flavour compound 3-methylbutanal illustrated the effectiveness of the method. Furthermore, knowledge of the biochemistry and physiology of 3-methylbutanal formation was used to optimize the composition of the growth medium to enhance 3-methylbutanal production, and thereby improve the screening. SIGNIFICANCE AND IMPACT OF THE STUDY: A commonly used method to control flavour formation in fermented food products is the selection of bacterial strains, which are able to produce the desired flavour compounds. As large collections of strains are available for such screenings, studying biodiversity of micro-organisms on the level of metabolic routes is strongly facilitated by highly automated high throughput screening methods for measuring enzyme activities or production of metabolites. Therefore, this method will be a useful tool for selecting flavour-producing strains and for enhancing starter culture development.     

Yeast epiarginase regulation,an enzyme-enzyme activity control: identification of residues of ornithine carbamoyltransferase and arginase responsible for enzyme catalytic and regulatory activities

In the presence of ornithine and arginine, ornithine carbamoyltransferase (OTCase) and arginase form a one-to-one enzyme complex in which the activity of OTCase is inhibited whereas arginase remains catalytically active. The mechanism by which these nonallosteric enzymes form a stable complex triggered by the binding of their respective substrates raises the question of how such a cooperative association is induced. Analyses of mutations in both enzymes identify residues that are required for their association, some of them being important for catalysis. In arginase, two cysteines at the C terminus of the protein are crucial for its epiarginase function but not for its catalytic activity and trimeric structure. In OTCase, mutations of putative ornithine binding residues, Asp-182, Asn-184, Asn-185, Cys-289, and Glu-256 greatly reduced the affinity for ornithine and impaired the interaction with arginase. The four lysine residues located in the SMG loop, Lys-260, Lys-263, Lys-265, and Lys-268, also play an important role in mediating the sensitivity of OTCase to ornithine and to arginase and appear to be involved in transducing and enhancing the signal given by ornithine for the closure of the catalytic domain.     

Enhanced flavour formation by combination of selected lactococci from industrial and artisanal origin with focus on completion of a metabolic pathway

Combinations of lactococcal strains from various origins with divers properties were developed as new starters for new dairy products. Flavour formation by such tailor-made cultures was studied. In some cases, a strongly enhanced flavour was observed. For instance, the combination of B1157 and SK110 strains in milk resulted in a very strong chocolate-like flavour. B1157 produces only a moderate chocolate-like flavour, whereas SK110 alone fails to produce this flavour. Headspace gas chromatography results corroborate the organoleptic evaluations. High levels of branched-chain aldehydes were found when B1157 and SK110 were grown together. The enzyme activities involved in this pathway were studied; both strains contain transaminase activity. Although B1157 had a very high amino acid decarboxylating activity, its release of amino acids from milk protein was limited. SK110 was strongly limited in decarboxylating activity, although this strain is very active in proteolysis. By combining these strains, the substrates released by SK110 can directly be used by the other strain, resulting in the completion of the whole flavour-formation pathway. This opens new avenues for the preparation of tailor-made cultures.     

Polymorphism of N-stearoylethanolamine: differential scanning calorimetric,vibrational spectroscopic (FTIR), and crystallographic studies

Based on a series of physicochemical properties (differential scanning calorimetry, powder X-ray crystallographic studies and Fourier-transform infra red spectroscopic analysis) determined for N-stearoylethanolamine (NSEA) (C18:0) at different temperatures, evidence has been given that this compound can exist in (at least) three polymorphic forms. Powder X-ray crystallography clearly demonstrates the presence of three distinct molecular packings at distinct temperatures while spectral changes in the vibrational spectra reveal that the geometry of the CH(2)z.sbnd;CO functional group of the molecule is affected during the polymorphic transitions. Rationalization of the thermal physicochemical behavior of NSEA in terms of molecular packing is also proposed. It supposes rearrangement of the hydrocarbon chains upon heating of the molecule.     

A despecialization step underlying evolution of a family of serine proteases

In the trypsin superfamily of serine proteases, non-trypsin-like primary specificities have arisen in only two monophyletic descendent subbranches. We have recreated an ancestor to one of these subbranches (granzyme) using phylogenetic inference, gene synthesis, and protein expression. This ancestor has two unusual properties. First, it has broad primary specificity encompassing the entire repertoire of novel primary specificities found in its descendents. Second, unlike extant members that have narrow primary specificities, the ancestor exhibits tolerance to mutational changes in primary specificity-conferring residues-that is, structural plasticity. Molecular modeling and mutagenesis studies indicate that these unusual properties are due to a particularly wide substrate binding pocket. These two crucial properties of the ancestor not only distinguish it from its extant descendents but also from the trypsin-like proteases that preceded it. This indicates that a despecialization step, characterized by broad specificity and structural plasticity, underlies evolution of new primary specificities in this protease superfamily.     

Changes in glycolytic activity of Lactococcus lactis induced by low temperature

The effects of low-temperature stress on the glycolytic activity of the lactic acid bacterium Lactococcus lactis were studied. The maximal glycolytic activity measured at 30 degrees C increased approximately 2.5-fold following a shift from 30 to 10 degrees C for 4 h in a process that required protein synthesis. Analysis of cold adaptation of strains with genes involved in sugar metabolism disrupted showed that both the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) subunit HPr and catabolite control protein A (CcpA) are involved in the increased acidification at low temperatures. In contrast, a strain with the PTS subunit enzyme I disrupted showed increased acidification similar to that in the wild-type strain. This indicates that the PTS is not involved in this response whereas the regulatory function of 46-seryl phosphorylated HPr [HPr(Ser-P)] probably is involved. Protein analysis showed that the production of both HPr and CcpA was induced severalfold (up to two- to threefold) upon exposure to low temperatures. The las operon, which is subject to catabolite activation by the CcpA-HPr(Ser-P) complex, was not induced upon cold shock, and no increased lactate dehydrogenase (LDH) activity was observed. Similarly, the rate-limiting enzyme of the glycolytic pathway under starvation conditions, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was not induced upon cold shock. This indicates that a factor other than LDH or GAPDH is rate determining for the increased glycolytic activity upon exposure to low temperatures. Based on their cold induction and involvement in cold adaptation of glycolysis, it is proposed that the CcpA-HPr(Ser-P) control circuit regulates this factor(s) and hence couples catabolite repression and cold shock response in a functional and mechanistic way.     

Structural insights into the acidophilic pH adaptation of a novel endo-1,4-β-xylanase from Scytalidium acidophilum

In this study, the crystal structure of a novel endo-1,4-β-xylanase from Scytalidium acidophilum, XYL1, was solved at 1.9 Å resolution. This is one of the few solved crystal structures of acidophilic proteins. The enzyme has the overall fold typical to family 11 xylanases. Comparison of this structure with other homologous acidophilic, neutrophilic and alkalophilic xylanases provides additional insights into the general features involved in low pH adaptation (stability and activity). Several sequence and structure modifications appeared to be responsible for the acidophilic characteristic: (a) the presence of an aspartic acid H bonded to the acid/base catalyst (b) the nature of specifically conserved residues in the active site (c) the negative potential at the surface (d) the decreased number of salt bridges and H bonds in comparison with highly alkaline enzymes.     

Specific recommendations (or lack thereof) for older patients with cardiovascular disease in the current European Society of Cardiology guidelines

Due to population ageing, the number of older and frail patients with cardiovascular disease is increasing. In the current guidelines of the European Society of Cardiology specific recommendations for this older population are missing or scarce, probably due to limited evidence concerning diagnosis and treatment of cardiovascular disease in older patients. Moreover, recommendations on shared decision making, palliative care and advanced care planning are also essential in these guidelines. In this article we evaluate the current European of Society of Cardiology guidelines (2013–2020) to determine whether specific recommendations for older patients have been included.