Kinetic modifications of C4 PEPC are qualitatively convergent, but larger in Panicum than in Flaveria

C4 photosynthesis results from a set of anatomical features and biochemical components that act together to concentrate CO2 within the leaf and boost productivity. This complex trait evolved independently many times, resulting in various realizations of the phenotype, but in all C4 plants the primary fixation of atmospheric carbon is catalyzed by phosphoenolpyruvate carboxylase. Comparisons of C4 and non-C4 PEPC from a few closely related species suggested that the enzyme was modified to meet the demands of the C4 cycle. However, very few C4 groups have been investigated, hampering general conclusions. To test the hypothesis that distant C4 lineages underwent convergent biochemical changes, we compare the kinetic variation between C4 and non-C4 PEPC from a previously assessed young lineage (Flaveria, Asteraceae) with those from an older lineage found within the distantly related grass family (Panicum). Despite the evolutionary distance, the kinetic changes between the non-C4 and C4 PEPC are qualitatively similar, with a decrease in sensitivity for inhibitors, an increased specificity (kcat/Km) for bicarbonate, and a decreased specificity (kcat/Km) for PEP. The differences are more pronounced in the older lineage Panicum, which might indicate that optimization of PEPC for the C4 context increases with evolutionary time

The coproporphyrin ferrochelatase of Staphylococcus aureus: mechanistic insights into a regulatory iron binding site

The majority of characterised ferrochelatase enzymes catalyse the final step of classical haem synthesis, inserting ferrous iron into protoporphyrin IX. However, for the recently-discovered coproporphyrin-dependent pathway, ferrochelatase catalyses the penultimate reaction where ferrous iron is inserted into coproporphyrin III. Ferrochelatase enzymes from the bacterial phyla Firmicutes and Actinobacteria have previously been shown to insert iron into coproporphyrin, and those from Bacillus subtilis and Staphylococcus aureus are known to be inhibited by elevated iron concentrations. The work herein reports a Km (coproporphyrin III) for S. aureus ferrochelatase of 1.5 µM and it is shown that elevating the iron concentration increases the Km for coproporphyrin III, providing a potential explanation for the observed iron-mediated substrate inhibition. Together, structural modelling, site-directed mutagenesis, and kinetic analyses confirm residue Glu271 as being essential for the binding of iron to the inhibitory regulatory site on S. aureus ferrochelatase, providing a molecular explanation for the observed substrate inhibition patterns. This work therefore has implications for how haem biosynthesis in S. aureus is regulated by iron availability

Iridium-catalysed ortho-directed deuterium labelling of aromatic esters – an experimental and theoretical study on directing group chemoselectivity

Herein we report a combined experimental and theoretical study on the deuterium labelling of benzoate ester derivatives, utilizing our developed iridium N-heterocyclic carbene/phosphine catalysts. A range of benzoate esters were screened, including derivatives with electron-donating and -withdrawing groups in the para- position. The substrate scope, in terms of the alkoxy group, was studied and the nature of the catalyst counter-ion was shown to have a profound effect on the efficiency of isotope exchange. Finally, the observed chemoselectivity was rationalized by rate studies and theoretical calculations, and this insight was applied to the selective labelling of benzoate esters bearing a second directing group

Understanding Maori 'lived' culture to determine cultural connectedness and wellbeing.

Maori tribal authorities have sought to measure the wellbeing of their people as a baseline for determining the extent to which their economic, social, and cultural goals are being achieved. In recent years, data from government-administered social surveys and/or censuses have become a significant source of information. Using the tribal authority of Te Runanga o Ngai Tahu (TRONT) as a case study, this paper explores and compares data concerning Ngai Tahu wellbeing contained in two recently completed TRONT reports: the Ngai Tahu State of the Nation 2015 report (a quantitative study derived from government-administered survey data); and, the preliminary findings from the Ngai Tahu Whenua Project (a qualitative study undertaken by TRONT). Both studies present similar results regarding levels of tribal economic wellbeing, however, they show different results in regards to levels of cultural wellbeing. The qualitative study reveals reasonably high levels of cultural engagement among participants. Conversely, the quantitative study demonstrates reasonably low levels of cultural engagement. The difference is explained in each study’s approach to understanding culture. The quantitative study viewed culture as engagement in ‘static’ cultural practices, whereas the qualitative study viewed Maori culture as a ‘lived’ set of deep networks and connections between individuals, their whanau (extended family), and places of symbolic cultural importance (particularly land and water). It is argued that measuring ‘lived’ culture would provide a better means of ascertaining cultural wellbeing. It is suggested that a useful means of measuring Maori lived culture would be to determine the quality and depth of relational networks

Five Glutamic Acid Residues in the C-Terminal Domain of the ChlD Subunit Play a Major Role in Conferring Mg2+ Cooperativity upon Magnesium Chelatase

Magnesium chelatase catalyzes the first committed step in chlorophyll biosynthesis by inserting a Mg2+ ion into protoporphyrin IX in an ATP-dependent manner. The cyanobacterial (Synechocystis) and higher-plant chelatases exhibit a complex cooperative response to free magnesium, while the chelatases from Thermosynechococcus elongatus and photosynthetic bacteria do not. To investigate the basis for this cooperativity, we constructed a series of chimeric ChlD proteins using N-terminal, central, and C-terminal domains from Synechocystis and Thermosynechococcus. We show that five glutamic acid residues in the C-terminal domain play a major role in this process

The ChlD subunit links the motor and porphyrin binding subunits of magnesium chelatase

Magnesium chelatase initiates chlorophyll biosynthesis, catalysing the MgATP2- dependent insertion of a Mg2+ ion into protoporphyin IX. The catalytic core of this large enzyme complex consists of three subunits: Bch/ChlI, Bch/ChlD and Bch/ChlH (in bacteriochlorophyll and chlorophyll producing species respectively). The D and I subunits are members of the AAA+ (ATPases associated with various cellular activities) superfamily of enzymes, and they form a complex that binds to H, the site of metal ion insertion. In order to investigate the physical coupling between ChlID and ChlH in vivo and in vitro , ChlD was FLAG-tagged in the cyanobacterium Synechocystis sp. PCC 6803 and co-immunoprecipitation experiments showed interactions with both ChlI and ChlH. Co-production of recombinant ChlD and ChlH in Escherichia coli yielded a ChlDH. Quantitative analysis using microscale thermophoresis (MST) showed magnesium-dependent binding ( K d 331 ± 58 nM) between ChlD and H. The physical basis for a ChlD-H interaction was investigated using chemical crosslinking coupled with mass spectrometry (XL-MS), together with modifications that either truncate ChlD or modify single residues. We found that the C-terminal integrin I domain of ChlD governs association with ChlH, the Mg2+ dependence of which also mediates the cooperative response of the Synechocystis chelatase to magnesium. Our work, showing the interaction site between the AAA+ motor and the chelatase domain of magnesium chelatase, will be essential for understanding how free energy from the hydrolysis of ATP on the AAA+ ChlI subunit is transmitted via the bridging subunit ChlD to the active site on ChlH

Inter-rater reliability of the EPUAP pressure ulcer classification system using photographs

Background. Many classification systems for grading pressure ulcers are discussed in the literature. Correct identification and classification of a pressure ulcer is important for accurate reporting of the magnitude of the problem, and for timely prevention. The reliability of pressure ulcer classification systems has rarely been tested. Aims and objectives. The purpose of this paper is to examine the inter-rater reliability of classifying pressure ulcers according to the European Pressure Ulcer Advisory Panel classification system when using pressure ulcer photographs.Design. Survey was among pressure ulcer experts.Methods. Fifty-six photographs were presented to 44 pressure ulcer experts. The experts classified the lesions as normal skin, blanchable erythema, pressure ulcer (four grades) or incontinence lesion. Inter-rater reliability was calculated.Results. The multirater-Kappa for the entire group of experts was 0.80 (P < 0.001).Various groups of experts obtained comparable results. Differences in classifications are mainly limited to 1 degree of difference. Incontinence lesions are most often confused with grade 2 (blisters) and grade 3 pressure ulcers (superficial pressure ulcers).Conclusions. The inter-rater reliability of the European Pressure Ulcer Advisory Panel classification appears to be good for the assessment of photographs by experts. The difference between an incontinence lesion and a blister or a superficial pressure ulcer does not always seem clear.Relevance to clinical practice. The ability to determine correctly whether a lesion is a pressure ulcer lesion is important to assess the effectiveness of preventive measures. In addition, the ability to make a correct distinction between pressure ulcers and incontinence lesions is important as they require different preventive measures. A faulty classification leads to mistaken measures and negative results. Photographs can be used as a practice instrument to learn to discern pressure ulcers from incontinence lesions and to get to know the different grades of pressure ulcers. The Pressure Ulcer Classification software package has been developed to facilitate learning

Amplitude Zeros in Radiative Decays of Scalar Particles

We study amplitude zeros in radiative decay processes with a photon or a gluon emission of all possible scalar particles(e.g. scalar leptoquarks) which may interact with the usual fermions in models beyond the standard model. For the decays with a photon emission, the amplitudes clearly exhibit the factorization property and the differential decay rates vanish at specific values of a certain variable which are determined only by the electric charges of the particles involved and independent of the particle masses and the various couplings. For the decays with a gluon emission, even though the zeros are washed away, the differential decay rates still have distinct minima. The branching ratios as a function of leptoquark masses are presented for the scalar leptoquark decays. We also comment on the decays of vector particles into two fermions and a photon.Comment: Revtex, 17 pages + 6 figures (available upon request), Preprint, OITS559. Several typos with tex file were correcte

The deubiquitinating enzyme Doa4p protects cells from DNA topoisomerase I poisons

DNA topoisomerase I (Top1p) catalyzes changes in DNA topology via the formation of an enzyme-DNA covalent complex that is reversibly stabilized by the antitumor drug, camptothecin (CPT). During S-phase, collisions with replication forks convert these complexes into cytotoxic DNA lesions that trigger cell cycle arrest and cell death. To investigate cellular responses to CPT-induced DNA damage, a yeast genetic screen identified conditional tah mutants with enhanced sensitivity to self-poisoning DNA topoisomerase I mutant (Top1T722Ap), which mimics the action of CPT. Mutant alleles of three genes, DOA4, SLA1 and SLA2, were recovered. A nonsense mutation in DOA4 eliminated the catalytic residues of the Doa4p deubiquitinating enzyme, yet retained the rhodanase domain. At 36 degrees C, this doa4-10 mutant exhibited increased sensitivity to CPT, osmotic stress, and hydroxyurea, and a reversible petite phenotype. However, the accumulation of pre-vacuolar class E vesicles that was observed in doa4Delta cells was not detected in the doa4-10 mutant. Mutations in SLA1 or SLA2, which alter actin cytoskeleton architecture, induced a conditional synthetic lethal phenotype in combination with doa4-10 in the absence of DNA damage. Here actin cytoskeleton defects coincided with the enhanced fragility of large-budded cells. In contrast, the enhanced sensitivity of doa4-10 mutant cells to Top1T722Ap was unrelated to alterations in endocytosis and was selectively suppressed by increased dosage of the ribonucleotide reductase inhibitor Sml1p. Additional studies suggest a role for Doa4p in the Rad9p checkpoint response to Top1p poisons. These findings indicate a functional link between ubiquitin-mediated proteolysis and cellular resistance to CPT-induced DNA damage