HPLC shadowing: Artifacts in peptide characterization monitored by RIA

High performance liquid chromatography (HPLC), a valuable tool for characterization of peptides, is frequently used in combination with sensitive radioimmunoassays (RIA). The shadow phenomenon, representing carry-over of the peptide from previous application of the standard, can appear to result in the presence of endogenous peptide in the test sample when none is actually there. With delta sleep-inducing peptide (DSIP), we found the shadowing to be as high as 10%, although it was only 1% with ¹²⁵I-Tyr-DSIP. Thus, when HPLC-RIA systems are used for identification of peptides, caution must be used to avoid false positive results.     

Gap junction remodeling and altered connexin43 expression in the failing human heart

Gap junctions (GJ) are important determinants of cardiac conduction and the evidence has recently emerged that altered distribution of these junctions and changes in the expression of their constituent connexins (Cx) may lead to abnormal coupling between cardiomyocytes and likely contribute to arrhythmogenesis. However, it is largely unknown whether changes in the expression and distribution of the major cardiac GJ protein, Cx43, is a general feature of diverse chronic myocardial diseases or is confined to some particular pathophysiological settings. In the present study, we therefore set out to investigate qualitatively and quantitatively the distribution and expression of Cx43 in normal human myocardium and in patients with dilated (DCM), ischemic (ICM), and inflammatory cardiomyopathies (MYO). Left ventricular tissue samples were obtained at the time of cardiac transplantation and investigated with immunoconfocal and electron microscopy. As compared with the control group, Cx43 labeling in myocytes bordering regions of healed myocardial infarction (ICM), small areas of replacement fibrosis (DCM) and myocardial inflammation (MYO) was found to be highly disrupted instead of being confined to the intercalated discs. In all groups, myocardium distant from these regions showed an apparently normal Cx43 distribution at the intercalated discs. Quantitative immunoconfocal analyis of Cx43 in the latter myocytes revealed that the Cx43 area per myocyte area or per myocyte volume is significantly decreased by respectively 30 and 55% in DCM, 23 and 48% in ICM, and by 21 and 40% in MYO as compared with normal human myocardium. In conclusion, focal disorganization of GJ distribution and down-regulation of Cx43 are typical features of myocardial remodeling that may play an important role in the development of an arrhythmogenic substrate in human cardiomyopathies.     

Short-term effects of temperature enhancement on growth and reproduction of alpine grassland species

In order to study the effects of temperature enhancement on alpine calcareous grassland species, a warming experiment was carried out in the Berchtesgaden National Park (Southeast Germany, Northern Calcareous Alps) between 2002 and 2004. The study was conducted in stands of the Carex sempervirens and the Carex firma communities; the two most widespread grassland types in the alpine zone of the Northern Calcareous Alps. The temperature of the vegetation stand and the upper soil was passively enhanced using open top chambers (OTCs). The construction of the OTCs was appropriate since temperature was clearly increased while water conditions (humidity, soil water content) were not changed.

By comparing manipulated (temperature enhancement) with non-manipulated plots, the effects of warming on growth and reproduction of selected key species were studied. To test if vegetation response to temperature enhancement is at least partly due to increases in nutrient availability, soil solution concentrations of nitrate and ammonium were analysed.

We found that most of the studied plant species are sensitive to temperature enhancement. Growth and/or reproduction of 12 of the 14 studied species were significantly stimulated by warming. Only two species showed no response; none of the species experienced decreases in growth or reproduction. Dwarf shrubs and graminoids showed a stronger response than herbaceous perennials. A significant effect of warming on nutrient availability could not be detected. The observed response of vegetation is therefore mainly caused by direct and not by indirect temperature effects.     

Secondary production and richness of native and non-native macroinvertebrates are driven by human-altered shoreline morphology in a large river

Animals living in extreme environments with predictable seasonality may have important life history events correlated to favourable periods. These animals pass critical life stages in protected habitats, especially during early life, often receiving parental care. It is thus hypothesized that juveniles rely on protective microhabitats provided by their parents, becoming independent only during favourable seasons. Semi-terrestrial crayfish Parastacus pugnax inhabit burrows in highly seasonal and predictable environments, thus being well suited to test this hypothesis. Following marked burrows and individual crayfish we examined the life history patterns of P. pugnax in their natural environment to test the predictions that (i) burrowing activity is higher during the wet season, (ii) reproductive events occur during favourable seasons and (iii) juveniles only disperse after reaching larger sizes. There was little or no burrowing activity during the dry season, when soil was more compact, but burrows became wider and had more openings during the wet season. After hatching, juveniles cohabited with adults for at least 4 months during the dry season. During this period juveniles grew considerably, starting independent lives during the wet season. These results suggest that the prolonged parent-offspring cohabitation evolved in response to the predictable seasonal variations in the crayfish habitat.     

Molecular mechanics of silk nanostructures under varied mechanical loading

Spider dragline silk is a self-assembling tunable protein composite fiber that rivals many engineering fibers in tensile strength, extensibility, and toughness, making it one of the most versatile biocompatible materials and most inviting for synthetic mimicry. While experimental studies have shown that the peptide sequence and molecular structure of silk have a direct influence on the stiffness, toughness, and failure strength of silk, few molecular-level analyses of the nanostructure of silk assemblies, in particular, under variations of genetic sequences have been reported. In this study, atomistic-level structures of wildtype as well as modified MaSp1 protein from the Nephila clavipes spider dragline silk sequences, obtained using an in silico approach based on replica exchange molecular dynamics and explicit water molecular dynamics, are subjected to simulated nanomechanical testing using different force-control loading conditions including stretch, pull-out, and peel. The authors have explored the effects of the poly-alanine length of the N. clavipes MaSp1 peptide sequence and identify differences in nanomechanical loading conditions on the behavior of a unit cell of 15 strands with 840-990 total residues used to represent a cross-linking β-sheet crystal node in the network within a fibril of the dragline silk thread. The specific loading condition used, representing concepts derived from the protein network connectivity at larger scales, have a significant effect on the mechanical behavior. Our analysis incorporates stretching, pull-out, and peel testing to connect biochemical features to mechanical behavior. The method used in this study could find broad applications in de novo design of silk-like tunable materials for an array of applications.     

Cancer stem cells and human malignant melanoma

Cancer stem cells (CSC) have been identified in hematological malignancies and several solid cancers. Similar to physiological stem cells, CSC are capable of self-renewal and differentiation and have the potential for indefinite proliferation, a function through which they may cause tumor growth. Although conventional anti-cancer treatments might eradicate most malignant cells in a tumor, they are potentially ineffective against chemoresistant CSC, which may ultimately be responsible for recurrence and progression. Human malignant melanoma is a highly aggressive and drug-resistant cancer. Detection of tumor heterogeneity, undifferentiated molecular signatures, and increased tumorigenicity of melanoma subsets with embryonic-like differentiation plasticity strongly suggest the presence and involvement of malignant melanoma stem cells (MMSC) in the initiation and propagation of this malignancy. Here, we review these findings in the context of functional properties ascribed to melanocyte stem cells and CSC in other cancers. We discuss the association of deregulated signaling pathways, genomic instability, and vasculogenic mimicry phenomena observed in melanoma subpopulations in light of the CSC concept. We propose that a subset of MMSC may be responsible for melanoma therapy-resistance, tumor invasiveness, and neoplastic progression and that targeted abrogation of a MMSC compartment could therefore ultimately lead to stable remissions and perhaps cures of metastatic melanoma.     

Predicting RNA secondary structures with pseudoknots by MCMC sampling

The most probable secondary structure of an RNA molecule, given the nucleotide sequence, can be computed efficiently if a stochastic context-free grammar (SCFG) is used as the prior distribution of the secondary structure. The structures of some RNA molecules contain so-called pseudoknots. Allowing all possible configurations of pseudoknots is not compatible with context-free grammar models and makes the search for an optimal secondary structure NP-complete. We suggest a probabilistic model for RNA secondary structures with pseudoknots and present a Markov-chain Monte-Carlo Method for sampling RNA structures according to their posterior distribution for a given sequence. We favor Bayesian sampling over optimization methods in this context, because it makes the uncertainty of RNA structure predictions assessable. We demonstrate the benefit of our method in examples with tmRNA and also with simulated data. McQFold, an implementation of our method, is freely available from http://www.cs.uni-frankfurt.de/~metzler/McQFold.     

The Aspergillus fumigatus Protein GliK Protects against Oxidative Stress and Is Essential for Gliotoxin Biosynthesis

The function of a number of genes in the gliotoxin biosynthetic cluster (gli) in Aspergillus fumigatus remains unknown. Here, we demonstrate that gliK deletion from two strains of A. fumigatus completely abolished gliotoxin biosynthesis. Furthermore, exogenous H₂O₂ (1 mM), but not gliotoxin, significantly induced A. fumigatus gliK expression (P = 0.0101). While both mutants exhibited significant sensitivity to both exogenous gliotoxin (P < 0.001) and H₂O₂ (P < 0.01), unexpectedly, exogenous gliotoxin relieved H₂O₂-induced growth inhibition in a dose-dependent manner (0 to 10 μg/ml). Gliotoxin-containing organic extracts derived from A. fumigatus ATCC 26933 significantly inhibited (P < 0.05) the growth of the ΔgliK²⁶⁹³³ deletion mutant. The A. fumigatus ΔgliK²⁶⁹³³ mutant secreted metabolites, devoid of disulfide linkages or free thiols, that were detectable by reverse-phase high-performance liquid chromatography and liquid chromatography-mass spectrometry with m/z 394 to 396. These metabolites (m/z 394 to 396) were present at significantly higher levels in the culture supernatants of the A. fumigatus ΔgliK²⁶⁹³³ mutant than in those of the wild type (P = 0.0024 [fold difference, 24] and P = 0.0003 [fold difference, 9.6], respectively) and were absent from A. fumigatus ΔgliG. Significantly elevated levels of ergothioneine were present in aqueous mycelial extracts of the A. fumigatus ΔgliK²⁶⁹³³ mutant compared to the wild type (P < 0.001). Determination of the gliotoxin uptake rate revealed a significant difference (P = 0.0045) between that of A. fumigatus ATCC 46645 (9.3 pg/mg mycelium/min) and the ΔgliK⁴⁶⁶⁴⁵ mutant (31.4 pg/mg mycelium/min), strongly suggesting that gliK absence and the presence of elevated ergothioneine levels impede exogenously added gliotoxin efflux. Our results confirm a role for gliK in gliotoxin biosynthesis and reveal new insights into gliotoxin functionality in A. fumigatus.     

Hybrids of sugars and aromatics: A Pd-catalyzed modular approach to chromans and isochromans

Herein we describe the synthesis of highly substituted chromans and isochromans using carbohydrates as starting materials. The key step of our synthetic approach is the annelation of the benzene moiety via a highly efficient Pd-catalyzed domino reaction. This powerful approach led to a small library of highly substituted chromans and isochromans by making use of a variety of different diynes and bromoglycals. We investigated several Pd-catalysts in order to improve the yields and to enlarge the scope of the domino reaction. Furthermore, we elucidated the mechanistic picture of the reaction with isotope-labelling experiments. Most probably the reaction proceeds via an oxidative addition followed by two carbopalladation steps and a final cyclization reaction.