Raman optical activity and circular dichroism reveal dramatic differences in the influence of divalent copper and manganese ions on prion protein folding

The binding of divalent copper ions to the full-length recombinant murine prion protein PrP23-231 at neutral pH was studied using vibrational Raman optical activity (ROA) and ultraviolet circular dichroism (UV CD). The effect of the Cu2+ ions on PrP structure depends on whether they are added after refolding of the protein in water or are present during the refolding process. In the first case ROA reveals that the hydrated alpha-helix is lost, with UV CD revealing a drop from approximately 25% to approximately 18% in the total alpha-helix content. The lost alpha-helix could be that comprising residues 145-156, located within the region associated with scrapie PrP formation. In the second case, ROA reveals the protein's structure to be almost completely disordered/irregular, with UV CD revealing a drop in total alpha-helix content to approximately 5%. Hence, although Cu2+ binding takes place exclusively within the unfolded/disordered N-terminal region, it can profoundly affect the structure of the folded/alpha-helical C-terminal region. This is supported by the finding that refolding in the presence of Cu2+ of a mutant in which the first six histidines associated with copper binding to the N-terminal region are replaced by alanine has a similar alpha-helix content to the metal-free protein. In contrast, when the protein is refolded in the presence of divalent manganese ions, ROA indicates the alpha-helix is reinforced, with UV CD revealing an increase in total alpha-helix content to approximately 30%. The very different influence of Cu2+ and Mn2+ ions on prion protein structure may originate in the different stability constants and geometries of their complexes.     

Reference gene selection for quantitative real-time PCR normalization: Application in the caprine mammary gland

In dairy animals, gene expression analysis has become increasing key to understand the biological processes occurring in mammary gland development that shape future milk potential. Selecting high-stability reference genes is crucial to interpret real-time qPCR data. This study investigated the expression stability of five top-ranked candidate reference genes in the goat mammary gland through three assays comparing different experimental conditions (physiological states, sample types and experimental treatments). The expression stability of genes including β-actin, glyceraldehyde-3-phosphate dehydrogenase, 18S rRNA, cyclophilin A and ribosomal protein large P0 was analyzed. Normalization for each experimental condition expression data revealed a different reference gene. Nevertheless, in our various assays, genes encoding for ribosomal proteins, 18S rRNA and RPLP0 presented the best expression stability. This result has been confirmed using a combined analysis of stability on the three assays. All genes showed the same distribution within and among the three assays and a different distribution between Ct variability and GeNorm normalization. In addition, the application on Catenin B1 expression using an inappropriate reference gene confirmed erroneous variations in interpretation. To conclude, there is no single ideal reference gene for caprine mammary gland studies and we recommend using a panel of top-ranked reference genes, including RPLP0, at the beginning of each experiment to validate the most stable(s) gene(s).     

Schwartz Formula: Is One k-Coefficient Adequate for All Children?

Background/Objective

Plasma-creatinine-based equations to estimate the glomerular filtration rate are recommended by several clinical guidelines. In 2009, Schwartz et al. adapted the traditional Schwartz equation to children and adolescents but did not find different k-coefficients between children and adolescents (k = 36.5 for all patients). We reevaluated the coefficient of the 2009-Schwartz formula according to sex and age in a pediatric population.

Patients/Methods

We used linear mixed-effects models to reestimate the 2009-Schwartz k-coefficient in 360 consecutive French subjects aged 1 to 18 years referred to a single centre between July 2003 and July 2010 (965 measurements). We assessed the agreement between the estimated glomerular filtration rate obtained with the new formula (called Schwartz-Lyon) and the rate measured by inulin clearance. We then compared this agreement to the one between the measured glomerular filtration rate and 2009-Schwartz formula, first in the French then in a Swedish cohort.

Results

In Schwartz-Lyon formula, k was estimated at 32.5 in boys <13 years and all girls and at 36.5 in boys aged ≥13 years. The performance of this formula was higher than that of 2009-Schwartz formula in children <13 years. This was first supported by a statistically significant reduction of the overestimation of the measured glomerular filtration rate in both cohorts, by better 10% and 30% accuracies, and by a better concordance correlation coefficient.

Conclusions

The performance and simplicity of Schwartz formula are strong arguments for its routine use in children and adolescents. The specific coefficient for children aged <13 years further improves this performance.     

Evolutionary origins of members of a superfamily of integral membrane cytochrome c biogenesis proteins

We have analyzed the relationships of homologues of the Escherichia coli CcmC protein for probable topological features and evolutionary relationships. We present bioinformatic evidence suggesting that the integral membrane proteins CcmC (E. coli; cytochrome c biogenesis System I), CcmF (E. coli; cytochrome c biogenesis System I) and ResC (Bacillus subtilis; cytochrome c biogenesis System II) are all related. Though the molecular functions of these proteins have not been fully described, they appear to be involved in the provision of heme to c-type cytochromes, and so we have named them the putative Heme Handling Protein (HHP) family (TC #9.B.14). Members of this family exhibit 6, 8, 10, 11, 13 or 15 putative transmembrane segments (TMSs). We show that intragenic triplication of a 2 TMS element gave rise to a protein with a 6 TMS topology, exemplified by CcmC. This basic 6 TMS unit then gave rise to two distinct types of proteins with 8 TMSs, exemplified by ResC and the archaeal CcmC, and these further underwent fusional or insertional events yielding proteins with 10, 11 and 13 TMSs (ResC homologues) as well as 15 TMSs (CcmF homologues). Specific evolutionary pathways taken are proposed. This work provides the first evidence for the pathway of appearance of distantly related proteins required for post-translational maturation of c-type cytochromes in bacteria, plants, protozoans and archaea.     

Functional interaction between PARP-1 and PARP-2 in chromosome stability and embryonic development in mouse

The DNA damage-dependent poly(ADP-ribose) polymerases, PARP-1 and PARP-2, homo- and heterodimerize and are both involved in the base excision repair (BER) pathway. Here, we report that mice carrying a targeted disruption of the PARP-2 gene are sensitive to ionizing radiation. Following alkylating agent treatment, parp-2(-/-)-derived mouse embryonic fibroblasts exhibit increased post-replicative genomic instability, G(2)/M accumulation and chromosome mis-segregation accompanying kinetochore defects. Moreover, parp-1(-/-)parp-2(-/-) double mutant mice are not viable and die at the onset of gastrulation, demonstrating that the expression of both PARP-1 and PARP-2 and/or DNA-dependent poly(ADP-ribosyl) ation is essential during early embryogenesis. Interestingly, specific female embryonic lethality is observed in parp-1(+/-)parp-2(-/-) mutants at E9.5. Meta phase analyses of E8.5 embryonic fibroblasts highlight a specific instability of the X chromosome in those females, but not in males. Together, these results support the notion that PARP-1 and PARP-2 possess both overlapping and non-redundant functions in the maintenance of genomic stability.     

Nest covering in plovers: How modifying the visual environment influences egg camouflage

Camouflage is one of the most widespread antipredator defences, and its mechanistic basis has attracted considerable interest in recent years. The effectiveness of camouflage depends on the interaction between an animal's appearance and its background. Concealment can therefore be improved by changes to an animal's own appearance, by behaviorally selecting an optimal background, or by modifying the background to better match the animal's own appearance. Research to date has largely focussed on the first of these mechanisms, whereas there has been little work on the second and almost none on the third. Even though a number of animal species may potentially modify their environment to improve individual‐specific camouflage, this has rarely if ever been quantitatively investigated, or its adaptive value tested. Kittlitz's plovers (Charadrius pecuarius) use material (stones and vegetation) to cover their nests when predators approach, providing concealment that is independent of the inflexible appearance of the adult or eggs, and that can be adjusted to suit the local surrounding background. We used digital imaging and predator vision modeling to investigate the camouflage properties of covered nests, and whether their camouflage affected their survival. The plovers' nest‐covering materials were consistent with a trade‐off between selecting materials that matched the color of the eggs, while resulting in poorer nest pattern and contrast matching to the nest surroundings. Alternatively, the systematic use of materials with high‐contrast and small‐pattern grain sizes could reflect a deliberate disruptive coloration strategy, whereby high‐contrast material breaks up the telltale outline of the clutch. No camouflage variables predicted nest survival. Our study highlights the potential for camouflage to be enhanced by background modification. This provides a flexible system for modifying an animal's conspicuousness, to which the main limitation may be the available materials rather than the animal's appearance.     

Automated in situ measurement of cell-specific antibody secretion and laser-mediated purification for rapid cloning of highly-secreting producers

Cloning of highly-secreting recombinant cells is critical for biopharmaceutical manufacturing, but faces numerous challenges including the fact that secreted protein does not remain associated with the producing cell. A fundamentally new approach was developed combining in situ capture and measurement of individual cell protein secretion followed by laser-mediated elimination of all non- and poorly-secreting cells, leaving only the highest-secreting cell in a well. Recombinant cells producing humanized antibody were cultured serum-free on a capture matrix, followed by staining with fluorescently-labeled anti-human antibody fragment. A novel, automated, high-throughput instrument (called LEAP) was used to image and locate every cell, quantify the cell-associated and secreted antibody (surrounding each cell), eliminate all undesired cells from a well via targeted laser irradiation, and then track clone outgrowth and stability. Temporarily sparing an island of helper cells around the clone of interest improved cloning efficiency (particularly when using serum-free medium), and helper cells were easily eliminated with the laser after several days. The in situ nature of this process allowed several serial sub-cloning steps to be performed within days of one another, resulting in rapid generation of clonal populations with significantly increased and more stable, homogeneous antibody secretion. Cell lines with specific antibody secretion rates of > 50 pg/cell per day (in static batch culture) were routinely obtained as a result of this cloning approach, often times representing up to 20% of the clones screened.     

Avian vision and the evolution of egg color mimicry in the common cuckoo

Coevolutionary arms races are a potent force in evolution, and brood parasite-host dynamics provide classical examples. Different host-races of the common cuckoo, Cuculus canorus, lay eggs in the nests of other species, leaving all parental care to hosts. Cuckoo eggs often (but not always) appear to match remarkably the color and pattern of host eggs, thus reducing detection by hosts. However, most studies of egg mimicry focus on human assessments or reflectance spectra, which fail to account for avian vision. Here, we use discrimination and tetrachromatic color space modeling of bird vision to quantify egg background and spot color mimicry in the common cuckoo and 11 of its principal hosts, and we relate this to egg rejection by different hosts. Egg background color and luminance are strongly mimicked by most cuckoo host-races, and mimicry is better when hosts show strong rejection. We introduce a novel measure of color mimicry-"color overlap"-and show that cuckoo and host background colors increasingly overlap in avian color space as hosts exhibit stronger rejection. Finally, cuckoos with better background color mimicry also have better pattern mimicry. Our findings reveal new information about egg mimicry that would be impossible to derive by the human eye.     

Investigation of the heterogeneity of xyloglucans from the cell walls of apple

Cell-wall material from parenchymatous tissues of apple was sequentially extracted with 50mm NaOH at 1°, m KOH at 1° and 20°, and 4m KOH at 20°, to leave a residue of α-cellulose. From the 4m KOH-soluble fraction, a crude xyloglucan was isolated by anion-exchange chromatography, and further resolved into seven xyloglucans by borate anion-exchange chromatography. The relative amounts of the xyloglucans, in order of elution, were 2.7:1.3:29.7:1.0:3.2:1.2:10.3. The structural features of five of the xyloglucans were determined by methylation analysis. These results show that apple xyloglucans exhibit heterogeneity.