Nitrogen catabolite repression modulates the production of aromatic thiols characteristic of Sauvignon Blanc at the level of precursor transport

The free thiols 3-mercapto-hexanol (3MH) and its acetate, practically absent from musts, are liberated by yeast during fermentation from a cysteinylated precursor [S-3-(hexan-1-ol)-l-cysteine (Cys-3MH)] present in the grape must and contribute favorably to the flavor of Sauvignon white wines. Production of 3MH is increased when urea is substituted for diammonium phosphate (DAP) as the sole nitrogen source on a synthetic medium. On grape must, complementation with DAP induces a decrease of 3MH production. This observation is reminiscent of nitrogen catabolite repression (NCR). The production of 3MH is significantly lower for a gap1Delta mutant compared with the wild type, during fermentation of a synthetic medium containing Cys-3MH as the precursor and urea as the sole nitrogen source. Mutants isolated from an enological strain with a relief of NCR on GAP1 produce significantly higher amounts of 3MH on synthetic medium than the parental strain. These phenotypes were not confirmed on grape must. It is concluded that on synthetic medium, Cys-3MH enters the cell through at least one identified transporter, GAP1p, whose activity is limiting the release of volatile thiols. On grape must, the uptake of the precursor through GAP1p is not confirmed, but the effect of addition of DAP, eventually prolonging NCR, is shown to decrease thiol production.     

Fysieke fixatie en slimme technologie in Vlaamse woonzorgcentra: Resultaten van een surveybevraging bij zorg- en beleidspersoneel over visie en gebruik

Aim

The STAFF-project investigates in what way ‘smart technology’ can offer an alternative for physical restraints in nursing homes. A survey is realized aimed at gaining more insight into the vision on and the use of physical restraints and ‘smart technology’.

Method

Two partly overlapping structured questionnaires were developed and sent to nursing home staff in Flanders (Belgium). One hundred fifty six administrators (managers or assistant-managers) and 238 caregiving staff (nurses, nursing aids, paramedical staff and other) completed the online questionnaire.

Results

In general there is a low acceptability of physical restraint use, however, a more nuanced picture of acceptability is present depending on the specific motivation for using physical restraints and on the specific means of physical restraints. About half of the administrators say they use smart technology in the nursing home. The two main reasons for not applying (yet) smart technology are ‘too high price for smart technology’ and ‘inadequate infrastructure of the nursing home’. All respondents underscore the importance of multiple strategies to diminish the use of physical restraints in nursing homes.

Conclusion

Physical restraint use is a complex theme and needs a nuanced analysis and management. This study shows that there is still room for improvement in diminishing the use of physical restraints and that nursing homes in Flanders are open to use smart technology.

     

Stability of a host-vector system based on complementation of an essential gene in Escherichia coli.

Antibiotic selection is the most common selection system for plasmid-containing bacteria. This technique, nevertheless, can be a source of problems during the expression of heterologous genes in Escherichia coli. We have developed an alternative selection system based on the complementation of a chromosomal auxotrophic (dapD2) mutation by the corresponding wild type gene carried on a plasmid. We show that the system effectively selects for the presence of plasmid on solid and liquid medium. In addition, we have observed a loss of viability associated with high levels of gene expression and accumulation of a heterologous protein, but the selective power and improved intrinsic stability of the dap+ plasmid, compared to a beta-lactamase (bla) based vector, excludes overgrowth of the culture by plasmidless cells.     

Use of synthetic oligonucleotides in gene isolation and manipulation

Great progress has occurred in the techniques of synthesis of DNA molecules of defined sequences in terms of speed, length of the obtained oligonucleotides, and automation of the processes. Corresponding progress also occurred in the ways of using synthetic DNA in molecular biology and recombinant DNA research. Screening of cloned DNA sequence banks with long, unique oligonucleotides, provided a new approach to isolate the genes for proteins which are present in very small quantity. This technique can present considerable advantages over the more classical use of mixtures of oligonucleotides, in reducing the number of potentially positive clones on a primary screen, and enabling cloning with a minimum of amino acid sequence data. Synthetic oligonucleotides also provide the basis of a set of techniques for site-directed mutagenesis of DNA sequences. This allows the possibility of engineering the structure of particular proteins, and the properties of new variants can be tested by expressing the protein in a heterologous host. An example of this approach is the production of variants of human alpha 1-antitrypsin. A variant where valine replaces the methionine at the active site is equally active as an antielastase, but no longer susceptible to oxidative inactivation. A second variant, where arginine replaces the methionine, now functions as an antithrombin, but no longer inhibits elastase. Total gene synthesis is now feasible for larger and larger genes, and some of the recent strategies of whole gene synthesis are presented.     

Tomato defense to Oidium neolycopersici: dominant Ol genes confer isolate-dependent resistance via a different mechanism than recessive ol-2

Tomato powdery mildew caused by Oidium neolycopersici has become a globally important disease of tomato (Lycopersicon esculentum). To study the defense responses of tomato triggered by tomato powdery mildew, we first mapped a set of resistance genes to O. neolycopersici from related Lycopersicon species. An integrated genetic map was generated showing that all the dominant resistance genes (Ol-1, Ol-3, Ol-4, Ol-5, and Ol-6) are located on tomato chromosome 6 and are organized in three genetic loci. Then, near-isogenic lines (NIL) were produced that contain the different dominant Ol genes in a L. esculentum genetic background. These NIL were used in disease tests with local isolates of O. neolycopersici in different geographic locations, demonstrating that the resistance conferred by different Ol genes was isolate-dependent and, hence, may be race-specific. In addition, the resistance mechanism was analyzed histologically. The mechanism of resistance conferred by the dominant Ol genes was associated with hypersensitive response, which varies in details depending on the Ol-gene in the NIL, while the mechanism of resistance governed by the recessive gene ol-2 on tomato chromosome 4 was associated with papillae formation.     

A human SP-C promoter fragment targets α1-proteinase inhibitor gene expression to lung alveolar type II cells in transgenic mice

A 1.277 kb promoter fragment of the gene encoding one of the lung surfactant proteins, SP-C, was cloned from a human genomic library and characterized using the human 1-proteinase inhibitor (1PI) gene as reporter. Messenger RNA for human 1PI isolated from a single transgenic mouse line was detected solely in lung tissue. Using immunogold electron microscopy, accumulation of human 1PI was shown unambiguously to occur only in type II pulmonary cells and, in discrete amounts, in the alveolar lining fluid. The protein was secreted and glycosylated showing a molecular weight close to that of plasma-derived human 1PI.     

Point mutations modifying the thrombin inhibition kinetics and antithrombotic activity in vivo of recombinant hirudin

The hirudin variant HV2 was modified by in vitro site-specific mutagenesis of HV2 cDNA to generate HV2(Asn-47----Lys), HV2(Asn-47----Arg) and HV2(Lys-35----Thr, Asn-47----Lys). Residues 35 and 47 are positioned respectively within the finger and prothrombin-like domains of hirudin, both of which have been suggested as thrombin binding sites. The modified polypeptides were synthesized in Saccharomyces cerevisiae using a secretion vector and purified from culture supernatants. By analysis of the human alpha-thrombin:hirudin inhibition reaction in steady-state conditions it was shown that the dissociation constants for HV2(Lys-47) and HV2(Arg-47) were 5- to 14-fold lower than for unmodified HV2, whereas mutation of Lys-35 did not significantly alter the inhibition kinetics. Furthermore, HV2(Lys-47), whose sequence is identical to a natural hirudin variant, displayed enhanced anti-thrombotic activity in vivo, having a 100-fold lower ED50 compared to HV2 in the rabbit Wessler venous thrombosis model. These results support a role for the prothrombin-like domain in thrombin binding and, moreover, demonstrate that in vivo antithrombotic efficiency correlates with the dissociation constant of the inhibition reaction.     

The performance of DGT versus conventional soil phosphorus tests in tropical soils - An isotope dilution study

Aims

The aim of the present study was to compare lead accumulation and tolerance among heavy metal hyperaccumulating and non-hyperaccumulating metallophytes.

Methods

To this purpose, we compared Pb tolerance and accumulation in hydroponics among calamine and non-calamine populations of Silene vulgaris, Noccaea caerulescens, and Matthiola flavida. We established the effects of Ca on Pb tolerance and accumulation in M. flavida, and measured exchangeable soil Pb and Ca at two calamine sites.

Results

Results revealed that calamine populations of S. vulgaris and N. caerulescens were Pb hypertolerant, but the calamine M. flavida population was not. Pb hyperaccumulation capacity was exclusively found in one of the calamine N. caerulescens populations.

Conclusions

1) Pb hypertolerance is sometimes lacking in metallophyte populations from strongly Pb-enriched soil, probably due to a relatively high level of exchangeable soil Ca, 2) Ca effectively counteracts Pb uptake and Pb toxicity, 3) The tendency to hyperaccumulate Pb is a population-specific phenomenon in N. caerulescens, 4) Pb hypertolerance in N. caerulescens is not necessarily associated with a tendency to hyperaccumulate Pb, 5) apparent natural Pb hyperaccumulation in M. flavida is not reproducible in hydroponics, probably due to the absence of air-born contamination in laboratory experiments.