The price of informality : how informal finance schemes defaulted in China, 1989–2015.

The default of a large number of informal finance schemes in China has caused enormous financial losses, and therefore has potential social and political significance. Analysing 354 defaulted schemes from 1989 to 2015, this study defines how they differ from other types of informal finance. It also produces an ideal-type representation of the default process and concludes that the default results from greed, increasing financial pressure at the individual level and private enterprises’ restricted access to state bank loans at the institutional level. China’s financial system should be more flexible in order to prevent further financial losses through informal financial relations

Metabolic adaptation to a high-fat diet is associated with a change in the gut microbiota

Objective The gut microbiota, which is considered a causal factor in metabolic diseases as shown best in animals, is under the dual influence of the host genome and nutritional environment. This study investigated whether the gut microbiota per se, aside from changes in genetic background and diet, could sign different metabolic phenotypes in mice. Methods The unique animal model of metabolic adaptation was used, whereby C57Bl/6 male mice fed a high-fat carbohydrate-free diet (HFD) became either diabetic (HFD diabetic, HFD-D) or resisted diabetes (HFD diabetes-resistant, HFD-DR). Pyrosequencing of the gut microbiota was carried out to profile the gut microbial community of different metabolic phenotypes. Inflammation, gut permeability, features of white adipose tissue, liver and skeletal muscle were studied. Furthermore, to modify the gut microbiota directly, an additional group of mice was given a glucooligosaccharide (GOS)-supplemented HFD (HFD+GOS). Results Despite the mice having the same genetic background and nutritional status, a gut microbial profile specific to each metabolic phenotype was identified. The HFD-D gut microbial profile was associated with increased gut permeability linked to increased endotoxaemia and to a dramatic increase in cell number in the stroma vascular fraction from visceral white adipose tissue. Most of the physiological characteristics of the HFD-fed mice were modulated when gut microbiota was intentionally modified by GOS dietary fibres. Conclusions The gut microbiota is a signature of the metabolic phenotypes independent of differences in host genetic background and diet

The PGM3 gene encodes the major phosphoribomutase in the yeast Saccharomyces cerevisiae

The phosphoglucomutases (PGM) Pgm1, Pgm2, and Pgm3 of the yeast Saccharomyces cerevisiae were tested for their ability to interconvert ribose-1-phosphate and ribose-5-phosphate. The purified proteins were studied in vitro with regard to their kinetic properties on glucose-1-phosphate and ribose-1-phosphate. All tested enzymes were active on both substrates with Pgm1 exhibiting only residual activity on ribose-1-phosphate. The Pgm2 and Pgm3 proteins had almost equal kinetic properties on ribose-1-phosphate, but Pgm2 had a 2000 times higher preference for glucose-1-phosphate when compared to Pgm3. The in vivo function of the PGMs was characterized by monitoring ribose-1-phosphate kinetics following a perturbation of the purine nucleotide balance. Only mutants with a deletion of PGM3 hyper-accumulated ribose-1-phosphate. We conclude that Pgm3 functions as the major phosphoribomutase in vivo. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved

A generic HTS assay for kinase screening: Validation for the isolation of an engineered malate kinase

<div><p>An end-point ADP/NAD⁺ acid/alkali assay procedure, directly applicable to library screening of any type of ATP-utilising/ADP producing enzyme activity, was implemented. Typically, ADP production is coupled to NAD⁺ co-enzyme formation by the conventional addition of pyruvate kinase and lactate dehydrogenase. Transformation of enzymatically generated NAD⁺ into a photometrically active alkali derivative product is then achieved through the successive application of acidic/alkali treatment steps. The assay was successfully miniaturized to search for malate kinase activity in a structurally-guided library of LysC aspartate kinase variants comprising 6,700 clones. The screening procedure enabled the isolation of nine positive variants showing novel kinase activity on (L)-malate, the best mutant, LysC V115A:E119S:E434V exhibited strong substrate selectivity for (L)-malate compared to (L)-aspartate with a (<i>k</i>_cat/<i>K</i>_m)_malate/(<i>k</i>_cat/<i>K</i>_m)_aspartate ratio of 86. Double mutants V115A:E119S, V115A:E119C and E119S:E434V were constructed to further probe the origins of stabilising substrate binding energy gains for (L)-malate due to mutation. The introduction of less sterically hindering side-chains in engineered enzymes carrying E119S and V115A mutations increases the effective volume available for substrate binding in the catalytic pocket. Improved binding of the (L)-malate substrate may be assisted by less hindered movement of the Phe184 aromatic side-chain. Additional favourable long-range electostatic effects on binding arising from the E434V surface mutation are conditionally dependent upon the presence of the V115A mutation close to Phe184 in the active-site.</p></div

Screening assay set-up.

<p>(A) Absorbance spectra of NADH and NAD⁺ co-enzyme solutions, at an initial concentration of 0.5 mM, during the sequential acid/alkali treatment. UV-visible spectra were recorded on solutions of the pure co-enzyme forms (blue curves), after acid (red curves) and alkali treatment (green curves). (B) Absorbance at 360 nm, determined in microplate format, as a function of NAD⁺ alkali derivative concentration assuming the total conversion of the NAD⁺ co-enzyme initially present. (C) NAD⁺ alkali derivative calibration curve, determined in microplate format, using solutions mimicking a reaction medium, typically containing inactivated enzyme crude extract, aspartate, ATP and a varying NADH/NAD⁺ co-enzyme ratio at a fixed final total concentration of the reduced and oxidised forms of 1.5 mM.</p

Principle of the end-point alkali assay method for the high-throughput screening of kinase activity.

<p>Principle of the end-point alkali assay method for the high-throughput screening of kinase activity.</p

Calculated (L)-malate electrostatic binding interaction energies in the V115A:E119S and V115A:E119S:E434V Ec-LysC mutant ternary complexes with Mg-ADP.

<p>Calculated (L)-malate electrostatic binding interaction energies in the V115A:E119S and V115A:E119S:E434V Ec-LysC mutant ternary complexes with Mg-ADP.</p

Utilisation of additional (L)-malate binding energy gained by LysC mutation in stabilising enzyme-substrate and enzyme transition-state complexes.

<p>Utilisation of additional (L)-malate binding energy gained by LysC mutation in stabilising enzyme-substrate and enzyme transition-state complexes.</p

Molecular model of a ternary complex of the V115A, E119S Lys-C mutant with (L)-malate and Mg-ADP.

<p>Cartoon representations of the dimer subunits are coloured grey and green. (L)-malate and Mg- ADP are shown as van der Waals spheres coloured according to element type: carbon, grey; nitrogen, blue; oxygen, red; phosphorus, orange; magnesium, ochre Side-chain atoms in the E434 residues at the enzyme surface are highlighted as orange-coloured van der Waals spheres. E434 lies approximately 27Å from the (L)-malate substrate molecule bound in one of the two active sites.</p