Folding units govern the cytochrome c alkaline transition

The alkaline transition of cytochrome c is a model for protein structural switching in which the normal heme ligand is replaced by another group. Stopped flow data following a jump to high pH detect two slow kinetic phases, suggesting two rate-limiting structure changes. Results described here indicate that these events are controlled by the same structural unfolding reactions that account for the first two steps in the reversible unfolding pathway of cytochrome c. These and other results show that the cooperative folding-unfolding behavior of protein foldons can account for a variety of functional activities in addition to determining folding pathways.     

Recombinant equine cytochrome c in Escherichia coli: high-level expression,characterization, and folding and assembly mutants

To promote studies of cytochrome c (Cyt c) ranging from apoptosis to protein folding, a system for facile mutagenesis and high-level expression is desirable. This work used a generally applicable strategy for improving yields of heterologously expressed protein in Escherichia coli. Starting with the yeast Cyt c plus heme lyase construct of Pollock et al. [Pollock, W. B., Rosell, F. I., Twitchett, M. B., Dumont, M. E., and Mauk, A. G. (1998) Biochemistry 37, 6124-6131], an E. coli-based system was designed that consistently produces high yields of recombinant eucaryotic (equine) Cyt c. Systematic changes to the ribosome binding site, plasmid sequence, E. coli strain, growth temperature, and growth duration increased yields from 2 to 3 mg/L to as much as 105 mg/L. Issues related to purification, fidelity of heme insertion, equilibrium stability, and introduction and analysis of mutant forms are described. As an example, variants tailored for folding studies are discussed. These remove known pH-dependent kinetic folding barriers (His26 and His33 and N-terminus), reveal an additional kinetic trap at higher pH due to some undetermined residue(s), and show how a new barrier can be placed at different points in the folding pathway in order to trap and characterize different folding intermediates. In addition, destabilizing glycine mutants in the N-terminal helix are shown to affect the fractional yield of a heme inverted Cyt c isoform.     

Urinary Biomarkers at Early ADPKD Disease Stage

Background

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by a decline in renal function at late disease stage when the majority of functional renal parenchyma is replaced by cystic tissue. Thus, kidney function, assessed by estimated glomerular filtration rate (eGFR) does not well represent disease burden in early disease. Here, we investigated various urinary markers for tubular injury and their association with disease burden in ADPKD patients at early disease course.

Methods

ADPKD patients between 18 and 40 years with an eGFR greater or equal to 70 ml per min per 1.73m² were eligible for this cross-sectional study. Urinary Neutrophil Gelatinase-Associated Lipocalin (NGAL), Kidney Injury Molecule-1 (KIM-1), and Uromodulin (UMOD) were investigated by Enzyme-Linked Immunosorbent Assay. Clara Cell Protein 16 (CC16) was investigated by Latex Immuno Assay. Cryoscopy was performed to assess urine osmolality and Urinary Albumin-to-Creatinine Ratio (UACR) was calculated. The association and the predictive properties of the markers on eGFR and height adjusted total kidney volume (htTKV) was evaluated using multiple regression analysis, incorporating different control variables for adjustment. Internal bootstrapping validated the obtained results.

Results

In 139 ADPKD patients (age 31 ±7 years, mean eGFR of 93 ± 19 ml per min per 1.73 m²) the total kidney volume was negatively correlated with eGFR and UMOD and positive associated with age, UACR, KIM-1 and urine osmolality after adjustment for possible confounders. Urine osmolality and htTKV were also associated with eGFR, whereas no association of CC16, NGAL and UMOD with eGFR or htTKV was found.

Conclusion

UACR and urinary KIM-1 are independently associated with kidney size but not with renal function in our study population. Urine osmolality was associated with eGFR and kidney volume following adjustment for multiple confounders. Despite statistical significance, the clinical value of our results is not yet conceivable. Further studies are needed to evaluate the property of the aforementioned biomarkers to assess disease state at early ADPKD stage.     

Revisiting the phylogeography and demography of European badgers (Meles meles) based on broad sampling,multiple markers and simulations

Although the phylogeography of European mammals has been extensively investigated since the 1990s, many studies were limited in terms of sampling distribution, the number of molecular markers used and the analytical techniques employed, frequently leading to incomplete postglacial recolonisation scenarios. The broad-scale genetic structure of the European badger (Meles meles) is of interest as it may result from historic restriction to glacial refugia and/or recent anthropogenic impact. However, previous studies were based mostly on samples from western Europe, making it difficult to draw robust conclusions about the location of refugia, patterns of postglacial expansion and recent demography. In the present study, continent-wide sampling and analyses with multiple markers provided evidence for two glacial refugia (Iberia and southeast Europe) that contributed to the genetic variation observed in badgers in Europe today. Approximate Bayesian computation provided support for a colonisation of Scandinavia from both Iberian and southeastern refugia. In the whole of Europe, we observed a decline in genetic diversity with increasing latitude, suggesting that the reduced diversity in the peripheral populations resulted from a postglacial expansion processes. Although MSVAR v.1.3 also provided evidence for recent genetic bottlenecks in some of these peripheral populations, the simulations performed to estimate the method''s power to correctly infer the past demography of our empirical populations suggested that the timing and severity of bottlenecks could not be established with certainty. We urge caution against trying to relate demographic declines inferred using MSVAR with particular historic or climatological events.     

Interference with the quorum sensing systems in a Vibrio harveyi strain alters the growth rate of gnotobiotically cultured rotifer Brachionus plicatilis

AIMS: To evaluate the effect of Vibrio harveyi strains on the growth rate of the gnotobiotically cultured rotifer Brachionus plicatilis, and to establish whether quorum sensing is involved in the observed phenomena. METHODS AND RESULTS: Gnotobiotic B. plicatilis sensu strictu, obtained by hatching glutaraldehyde-treated amictic eggs, were used as test organisms. Challenge tests were performed with 11 V. harveyi strains and different quorum sensing mutants derived from the V. harveyi BB120 strain. Brominated furanone [(5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone] as a quorum sensing inhibitor was tested in Brachionus challenge tests. Some V. harveyi strains, such as strain BB120, had a significantly negative effect on the Brachionus growth rate. In the challenge test with MM77, an isogenic strain of BB120 in which the two autoinducers (HAI-1 and AI-2) are both inactivated, no negative effect was observed. The effect of single mutants was the same as that observed in the BB120 strain. This indicates that both systems are responsible for the growth-retarding (GR) effect of the BB120 strain towards Brachionus. Moreover, the addition of an exogenous source of HAI-1 or AI-2 could restore the GR effect in the HAI-1 and AI-2 nonproducing mutant MM77. The addition of brominated furanone at a concentration of 2.5 mg l(-1) could neutralize the GR effect of some strains such as BB120 and VH-014. CONCLUSIONS: Two quorum sensing systems in V. harveyi strain BB120 (namely HAI-1 and AI-2-mediated) are necessary for its GR effect on B. plicatilis. With some other V. harveyi strains, however, growth inhibition towards Brachionus does not seem to be related to quorum sensing. SIGNIFICANCE AND IMPACT OF THE STUDY: Interference with the quorum sensing system might help to counteract the GR effect of some V. harveyi strains on Brachionus. However, further studies are needed to demonstrate the positive effect of halogenated furanone in nongnotobiotic Brachionus cultures and eventually, in other segments of the aquaculture industry.     

FTO-rs9939609 Polymorphism is a Predictor of Future Type 2 Diabetes: A Population-Based Prospective Study

Biochemical Genetics - The study aimed to evaluate the contribution of the FTO A/T polymorphism (rs9939609) to the prediction of the future type 2 diabetes (T2D). A population-based prospective...