Proteomic analysis of formalin-fixed prostate cancer tissue

Proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tissue would enable retrospective biomarker investigations of this vast archive of pathologically characterized clinical samples that exist worldwide. These FFPE tissues are, however, refractory to proteomic investigations utilizing many state of the art methodologies largely due to the high level of covalently cross-linked proteins arising from formalin fixation. A novel tissue microdissection technique has been developed and combined with a method to extract soluble peptides directly from FFPE tissue for mass spectral analysis of prostate cancer (PCa) and benign prostate hyperplasia (BPH). Hundreds of proteins from PCa and BPH tissue were identified, including several known PCa markers such as prostate-specific antigen, prostatic acid phosphatase, and macrophage inhibitory cytokine-1. Quantitative proteomic profiling utilizing stable isotope labeling confirmed similar expression levels of prostate-specific antigen and prostatic acid phosphatase in BPH and PCa cells, whereas the expression of macrophage inhibitory cytokine-1 was found to be greater in PCa as compared with BPH cells.     

Ca2+-selective transient receptor potential V channel architecture and function require a specific ankyrin repeat

Transient receptor potential (TRP) proteins form cation-conducting ion channels with currently 28 known genes encoding TRP channel monomers in mammals. These monomers are thought to coassemble to form homo- or heterotetrameric channels, but the signals governing their assembly are unknown. Within the TRPV subgroup, TRPV5 and TRPV6 show exclusive calcium selectivity and play an important role in calcium uptake. To identify signals that mediate assembly of functional TRPV6, we screened domains for self-association using co-immunoprecipitation, sucrose gradient centrifugation, bacterial two-hybrid assays, and patch clamp analysis. Of the two identified interaction domains within the N-terminal region, we showed that the first domain encompassing the third ankyrin repeat is the stringent requirement for physical assembly of TRPV6 subunits and when transferred to an unrelated protein enables its interaction with TRPV6. Deletion of this repeat or mutation of critical residues within this repeat rendered nonfunctional channels that do not co-immunoprecipitate or form tetramers. Suppression of dominant-negative inhibitors of TRPV6-specific currents was achieved by deletion of ankyrin (ANK) 3. We propose that the third ANK repeat initiates a molecular zippering process that proceeds past the fifth ANK repeat and creates an intracellular anchor that is necessary for functional subunit assembly.     

Tryptophan Cu(I)–π interaction fine-tunes the metal binding properties of the bacterial metallochaperone CusF

The periplasmic metallochaperone CusF coordinates Cu(I) and Ag(I) through a unique site consisting of a Met₂His motif as well as a Cu(I)–π interaction between a nearby tryptophan, W44, and the metal ion. Through mutational analyses we investigate here the role that W44 in CusF plays in metal coordination. Nuclear magnetic resonance spectra show that the specificity of CusF for Cu(I) and Ag(I) is not altered by mutation of W44. X-ray absorption spectroscopy studies reveal that W44 protects the bound Cu(I) from oxidation as well as from adventitious ligands. Competition assays demonstrate that W44 does not significantly contribute to the affinity of CusF for metal, but that substitution of W44 by methionine, which forms a fourth Cu(I) ligand, substantially increases the affinity. These studies indicate that W44 is important in maintaining a moderate-affinity and solvent-shielded three-coordinate environment for Cu(I), which has implications for the function of CusF as a metallochaperone. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Engaging Transformation: Using Seasonal Rounds to Anticipate Climate Change

Human Ecology - Seasonal rounds are deliberative articulations of a community’s sociocultural relations with their ecological system. The process of visualizing seasonal rounds informs...     

Mixture Effects of Estrogenic Pesticides at the Human Estrogen Receptor α and β

Consumers of fruits and vegetables are frequently exposed to small amounts of hormonally active pesticides, some of them sharing a common mode of action such as the activation of the human estrogen receptor α (hERα) or β (hERβ). Therefore, it is of particular importance to evaluate risks emanating from chemical mixtures, in which the individual pesticides are present at human-relevant concentrations, below their corresponding maximum residue levels. Binary and ternary iso-effective mixtures of estrogenic pesticides at effect concentrations eliciting a 1 or 10% effect in the presence or absence of 17β-estradiol were tested experimentally at the hERα in the yeast-based estrogen screen (YES) assay as well as in the human U2-OS cell-based ERα chemical-activated luciferase gene expression (ERα CALUX) assay and at the hERβ in the ERβ CALUX assay. The outcome was then compared to predictions calculated by means of concentration addition. In most cases, additive effects were observed with the tested combinations in all three test systems, an observation that supports the need to expand the risk assessment of pesticides and consider cumulative risk assessment. An additional testing of mixture effects at the hERβ showed that most test substances being active at the hERα could also elicit additive effects at the hERβ, but the hERβ was less sensitive. In conclusion, effects of the same ligands at the hERα and the hERβ could influence the estrogenic outcome under physiological conditions.     

Die Fermentation von Sauerkraut. Das Experiment

This article explains the production of “Sauerkraut” (pickled cabbage) and delivers some background information to this subject. Furthermore there are shown some possibilities for the process and quality monitoring and control. Also it shows how to analyse and illustrate the Monod growth kinetic. It should work as a pattern for experiments in schools and universities.     

The multifunctional leucine-rich repeat receptor kinase BAK1 is implicated in Arabidopsis development and immunity

Plant receptor-like kinases (RLKs) are transmembrane proteins with putative N-terminal extracellular ligand-binding domains and C-terminal intracellular protein kinase domains. RLKs have been implicated in multiple physiological programs including plant development and immunity to microbial infection. Arabidopsis thaliana gene expression patterns support an important role of this class of proteins in biotic stress adaptation. Here, we provide a comprehensive survey of plant immunity-related RLK gene expression. We further document the role of the Arabidopsis Brassinosteroid Insensitive 1 (BRI1)-associated receptor kinase 1 (BAK1) in seemingly unrelated biological processes, such as plant development and immunity, and propose a role of this protein as an adaptor molecule that is required for proper functionality of numerous RLKs. This view is supported by the identification of an additional RLK, PEPR1, and its closest homolog, PEPR2 as BAK1-interacting RLKs.     

Do <Emphasis Type="Italic">Polylepis australis</Emphasis> trees tolerate herbivory? Seasonal patterns of shoot growth and its consumption by livestock

Browsing is one of the main factors determining survival, growth rate, woodland structure, and distribution of the high mountain tree Polylepis australis. This species has a substantial regrowth capacity, which may function as a mechanism to tolerate herbivory, but it is unknown to what extent it may compensate for the impact of herbivory. In 15 low-density tree stands subject to exclusion, moderate, and heavy livestock pressure, we selected 12 P. australis individuals <2 m tall, tagged four new shoots per tree and measured shoot length every month during a year. At the stand and at the tree level, we analyzed monthly dynamics of growth and browsing, and the annual output in terms of total browsing and total gross and net growth (not discounting and discounting consumption, respectively). In addition, we assessed the influence of stand, tree and microsite characteristics on growth and browsing patterns. Polylepis australis fully compensated for herbivory in terms of shoot gross growth at moderate, but not at heavy livestock pressure. In terms of net growth, this species did not fully compensate for herbivory at any stocking rate. We found a strong coupling between browsing and growth along the year, suggesting that regrowth attracts browsing, and browsing promotes regrowth. At the stand level, annual gross growth was not affected by habitat characteristics, while at the tree level, annual gross growth decreased on more rocky microsites for browsed but not for unbrowsed trees. We concluded that stocking densities should be limited to allow for a reasonable annual net growth, as its nitrogen rich leaves are a valuable food resource and P. australis forests provide important ecosystem services.