Plasma profiling reveals human fibulin-1 as candidate marker for renal impairment

There is a need for reliable and sensitive biomarkers for renal impairments to detect early signs of kidney toxicity and to monitor progression of disease. Here, antibody suspension bead arrays were applied to profile plasma samples from patients with four types of kidney disorders: glomerulonephritis, diabetic nephropathy, obstructive uropathy, and analgesic abuse. In total, 200 clinical renal-associated cases and control plasma samples from different cohorts were profiled. Parallel plasma protein profiles were obtained using biotinylated and nonfractionated samples and a selected set of 94 proteins targeted by 129 antigen-purified polyclonal antibodies. Out of the analyzed target proteins, human fibulin-1 was detected at significantly higher levels in the glomerulonephritis patient group compared to the controls and with elevated levels in patient samples for all other renal disorders investigated. Two polyclonal antibodies and one monoclonal antibody directed toward separate, nonoverlapping epitopes showed the same trend in the discovery cohorts. A technical verification using Western blot analysis of selected patient plasma confirmed the trends toward higher abundance of the target protein in disease samples. Furthermore, a verification study was carried out in the context of glomerulonephritis using an independent case and control cohort, and this confirmed the results from the discovery cohort, suggesting that plasma levels of fibulin-1 could serve as a potential indicator to monitor kidney malfunction or kidney damage.     

The Role of <Emphasis Type="Italic">Habitus</Emphasis> in the Maintenance of Traditional Noongar Plant Knowledge in Southwest Western Australia

We examine the role that habitus, an individual’s or group’s dispositions, has played in the retention of traditional ecological knowledge among the Noongar people of south-western Australia. We sought to determine if current plant knowledge reflects Noongar habitus or, alternatively, the use of fall-back species that were important due to the intermittency of agricultural employment and the social exclusion of Aboriginal people up until at least the 1960s in Western Australia. We compared the seasonal availability of Noongar food plant resources currently known by Noongar Elders to those described at the time of European settlement. We used non-metric Multidimensional Scaling (nMDS) and multivariate statistics to compare the seasonal availability of plant resources with the seasonal availability of work prior to the introduction of civil rights for Aboriginal Australians in the 1960s. We show that the seasonal pattern of plant knowledge has changed little since settlement and that there was no significant relationship between the seasonal availability of work and plant knowledge. This result suggests that prior to 1960 Noongars maintained a reasonably traditional round of seasonal activities involving traditional plant use. We suggest that Noongar habitus guided their response to the colonising culture and helped preserve traditional ecological knowledge.     

Management strategies for controlling endemic and seasonal mouse parvovirus infection in a barrier facility

Despite improved diagnostic and rederivation capabilities, research facilities still struggle to manage parvovirus infections (e.g., mouse parvovirus (MPV) and minute virus of mice) in mouse colonies. Multi-faceted approaches are needed to prevent adventitious organisms such as MPV from breaching a barrier facility. In this article, the authors document recent changes to the Salk Institute's animal care program that were intended to help manage mouse parvovirus in the barrier facility. Specifically, the Institute started to use a new disinfectant and to give mice irradiated feed. The authors found an association between these modifications and a reduction in MPV incidence and prevalence in endemically infected colonies. These data suggest that using irradiated feed and appropriate disinfectants with contemporary management practices can be an effective plan for eradicating or controlling MPV infection in a research facility. The authors recommend further study of the environmental risk factors for parvovirus infection and of potential biological interactions associated with the use of irradiated feed.     

Aerosols transmit prions to immunocompetent and immunodeficient mice

Prions, the agents causing transmissible spongiform encephalopathies, colonize the brain of hosts after oral, parenteral, intralingual, or even transdermal uptake. However, prions are not generally considered to be airborne. Here we report that inbred and crossbred wild-type mice, as well as tga20 transgenic mice overexpressing PrP(C), efficiently develop scrapie upon exposure to aerosolized prions. NSE-PrP transgenic mice, which express PrP(C) selectively in neurons, were also susceptible to airborne prions. Aerogenic infection occurred also in mice lacking B- and T-lymphocytes, NK-cells, follicular dendritic cells or complement components. Brains of diseased mice contained PrP(Sc) and transmitted scrapie when inoculated into further mice. We conclude that aerogenic exposure to prions is very efficacious and can lead to direct invasion of neural pathways without an obligatory replicative phase in lymphoid organs. This previously unappreciated risk for airborne prion transmission may warrant re-thinking on prion biosafety guidelines in research and diagnostic laboratories.     

Interaction of bacteriophage lambda with its cell surface receptor: an in vitro study of binding of the viral tail protein gpJ to LamB (Maltoporin)

The cell surface receptor for bacteriophage Lambda is LamB (maltoporin). Responsible for phage binding to LamB is the C-terminal part, gpJ, of phage tail protein J. To study the interaction between LamB and gpJ, a chimera protein composed of maltose binding protein (MBP or MalE) connected to the C-terminal part of J (gpJ, amino acids 684-1131) of phage tail protein J of bacteriophage Lambda was expressed in Escherichia coli and purified to homogeneity. The interaction of the MBP-gpJ chimera protein with reconstituted LamB and its mutants LamB Y118G and the loop deletion mutant LamB Delta4+Delta6+Delta9v was studied using planar lipid bilayer membranes on a single-channel and multichannel level. Titration with the MBP-gpJ chimera blocked completely the ion current through reconstituted LamB when it was added to the cis side, the extracellular side of LamB with a half-saturation constant of approximately 6 nM in 1 M KCl. Control experiments with LamB Delta4+Delta6+Delta9v from which all major external loops had been removed showed similar blocking, whereas MBP alone caused no visible effect. Direct conductance measurement with His(6)-gpJ that contained a hexahistidyl tag (His(6) tag) at the N-terminal end of the protein for easy purification revealed no blocking of the ion current, requiring other measurements for the binding constant. However, when maltoporin was preincubated with His-gpJ, MBP-gpJ could not block the channel, which indicated that also His(6)-gpJ bound to the channel. High-molecular mass bands on SDS-PAGE and Western blots, confirming the planar lipid bilayer experiment results, also demonstrated stable complex formation between His(6)-gpJ and LamB or LamB mutants. The results revealed that phage Lambda binding includes not only the extracellular loops.     

Changes in heart rate variability of athletes during a training camp.

Heart rate variability (HRV) reflects regulatory processes of the cardiovascular system and reveals fractal characteristics. In this paper we investigated standard HRV parameters and scaling characteristics in ten athletes before, during, and after a 2-week training camp to assess the effects of short-term overtraining on cardiovascular control. High-resolution ECGs were recorded over 30 min under resting conditions 1 week before the training camp, after 1 week of training in the camp, and after 3-4 days of recovery. Standard HRV analysis was performed according to Task Force recommendations. Scaling characteristics were assessed, applying detrended fluctuation analysis (DFA). Standard HRV analysis showed significant changes in meanNN and rmssd during the training camp. DFA revealed three distinct regions of scale-invariance and significant alterations during the training camp. In conclusion, HRV might be used to monitor the training state in athletes.     

Engineering of betabellin 15D: Copper(II)-induced folding of a fibrillar β-sandwich protein

Summary The inverse protein-folding problem has been explored by designing de novo the betabellin target structure (a 64-residue β-sandwich protein), synthesizing a 32-residue peptide chain (HSLTAKIpkLTFSIAphTYTCAVpkYTAKVSH, wherep=DPro,k=DLys, andh=DHis) that might fold into this structure, and studying how its disulfide-bridged form (betabellin 15D) folds in 10 mM ammonium acetate with and without Cu²⁺. Circular dichroic spectropolarimetry indicated that at pH 5.8, 6.4, or 6.7 betabellin 15D exhibited β-sheet structure in the presence of Cu²⁺ but not in its absence. Electrospray mass spectrometry demonstrated that at pH 6.3 each molecule of betabellin 15D bound one or two Cu(II) ions. Electron microscopy showed that at pH 6.7 betabellin 15D formed short broad fibrils in the presence of Cu²⁺ but not in its absence. The observed width of the fibrils (7±2 nm) was consistent with the width (6.8nm) of a structural model of a fibril that contained two adjacent rows of betabellin 15D β-sandwiches joined lengthwise by multiple intersheet hydrogen bonds and widthwise by multiple Cu(II)-imidazole bonds. Electron paramagnetic resonance spectrometry revealed that some pairs of Cu(II) ions in a Cu(II)/betabellin 15D complex were magnetically coupled, which is consistent with the structural model of the Cu(II)/betabellin 15D fibril.     

Dasatinib, imatinib and staurosporine capture compounds - Complementary tools for the profiling of kinases by Capture Compound Mass Spectrometry (CCMS)

Capture Compound Mass Spectrometry (CCMS) is a platform technology for the functional isolation of subproteomes. Here we report the synthesis of two new kinase Capture Compounds (CCs) based on the tyrosine-kinase specific inhibitors dasatinib and imatinib and compare their interaction profiles to that of our previously reported staurosporine-CCs. CCs are tri-functional molecules: they comprise a sorting function (e.g. the small molecule or drug of interest) which interacts with target proteins, a photo-activatable reactivity function to covalently trap the interacting proteins, and a sorting function to isolate the CC-protein conjugates from complex biological samples for protein identification by liquid chromatography/mass spectrometry (LC-MS/MS). We present data of CCMS experiments from human HepG2 cells and compare the profiles of the kinases isolated with dasatinib, imatinib and staurosporine CC, respectively. Dasatinib and imatinib have a more selective kinase binding profile than staurosporine. Moreover, the new CCs allow isolation and identification of additional kinases, complementing the staurosporine CC. The family of kinase CCs will be a valuable tool for the proteomic profiling of this important protein class. Besides sets of expected kinases we identified additional specific interactors; these off-targets may be of relevance in the view of the pharmacological profile of dasatinib and imatinib.