Effects of conformational activation of integrin alpha 1I and alpha 2I domains on selective recognition of laminin and collagen subtypes

Collagen receptor integrins alpha 1 beta 1 and alpha 2 beta 1 can selectively recognize different collagen subtypes. Here we show that their alpha I domains can discriminate between laminin isoforms as well: alpha 1I and alpha 2I recognized laminin-111, -211 and -511, whereas their binding to laminin-411 was negligible. Residue Arg-218 in alpha1 was found to be instrumental in high-avidity binding. The gain-of-function mutation E318W makes the alpha 2I domain to adopt the "open" high-affinity conformation, while the wild-type alpha 2I domain favors the "closed" low-affinity conformation. The E318W mutation markedly increased alpha 2I domain binding to the laminins (-111, -211 and -511), leading us to propose that the activation state of the alpha 2 beta 1 integrin defines its role as a laminin receptor. However, neither wild-type nor alpha 2IE318W domain could bind to laminin-411. alpha 2IE318W also bound tighter to all collagens than alpha 2I wild-type, but it showed reduced ability to discriminate between collagens I, IV and IX. The corresponding mutation, E317A, in the alpha 1I domain transformed the domain into a high-avidity binder of collagens I and IV. Thus, our results indicate that conformational activation of integrin alpha 1I and alpha 2I domains leads to high-avidity binding to otherwise disfavored collagen subtypes.     

Reactions of permanganyl chloride with allene in low-temperature matrices

The light-induced (546 nm) reaction of MnO₃Cl with allene has been investigated in low-temperature argon matrices at 11 K. IR spectroscopic studies in combination with isotopic enrichment experiments (¹⁸O, D) and DFT calculations (B3LYP/LanL2DZ) allowed the identification of (O)₂MnCl(OCCH₂CH₂) (1), and (O)₂MnCl(H₂COCCH₂) (2) as the products. Possible ways for their formation are first of all discussed qualitatively in the context of the literature available, and then quantitatively with the background of DFT data (B3LYP/6-311G(d)) obtained for starting materials, products, transition states and intermediates. The most reasonable interpretation involves two-state reactivity.     

The kisspeptin/gonadotropin-releasing hormone pathway and molecular signaling of puberty in fish

The mechanisms underlying the initiation of puberty in fish are poorly understood, and whether the Kiss1 receptor (Kiss1r; previously designated G protein-coupled receptor 54; GPR54) and its ligands, kisspeptins, play a significant role, as has been established in mammals, is not yet known. We determined (via real-time PCR) temporal patterns of expression in the brain of kiss1r, gnrh2, and gnrh3 and a suite of related genes in the hypothalamo-pituitary-gonadal (HPG) axis and analyzed them against the timing of gonadal germ cell development in male and female fathead minnow (Pimephales promelas). Full- or partial-length cDNAs for kiss1r (736 bp), gnrh2 (698 bp), and gnrh3 (804 bp) cloned from fathead minnow were found to be expressed only in the brain, testis, and ovary of adult fish. Localization of kiss1r, gnrh2, and gnrh3 within the brain provided evidence for their physiological roles and a likely hypophysiotropic role for GnRH3 in this species (which, like other cyprinids, does not appear to express gnrh1). In both sexes, kiss1r expression in the brain increased at the onset of puberty and reached maximal expression in males when spermatagonia type B appeared in the testis and in females when cortical alveolus-stage oocytes first appeared in the ovary, the timings of which differed for the two sexes. However, kiss1r expression was considerably lower during more advanced stages of spermatogenesis and oogenesis. The expression of kiss1r closely aligned with that of the gnrh genes (gnrh3 in particular), suggesting the Kiss1r/kisspeptin system in fish has a similar role in puberty to that occurring in mammals, and this hypothesis was supported by the induction of gnrh3 (2.25-fold) and kiss1r (1.5-fold) in early-mid pubertal fish injected with mammalian kisspeptin-10 (2 nmol/g wet weight). An intriguing finding, and contrasting that in mammals, was an elevated expression of esr1, ar, and cyp19a2 (genes involved in sex steroid signaling) in the brain at the onset of puberty, and in females slightly in advance of the elevation in the expression of kiss1r.     

Molecular model of an alpha-helical prion protein dimer and its monomeric subunits as derived from chemical cross-linking and molecular modeling calculations

Prions are the agents of a series of lethal neurodegenerative diseases. They are composed largely, if not entirely, of the host-encoded prion protein (PrP), which can exist in the cellular isoform PrP^C and the pathological isoform PrP^Sc. The conformational change of the α-helical PrP^C into β-sheet-rich PrP^Sc is the fundamental event of prion disease. The transition of recombinant PrP from a PrP^C-like into a PrP^Sc-like conformation can be induced in vitro by submicellar concentrations of SDS. An α-helical dimer was identified that might represent either the native state of PrP^C or the first step from the monomeric PrP^C to highly aggregated PrP^Sc. In the present study, the molecular structure of these dimers was analyzed by introducing covalent cross-links using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. Inter- and intramolecular bonds between directly neighboured amino groups and carboxy groups were generated. The bonds formed in PrP dimers of recombinant PrP (90-231) were identified by tryptic digestion and subsequent mass spectrometric analysis. Intra- and intermolecular cross-links between N-terminal glycine and three acidic amino acid side chains in the globular part of PrP were identified, showing the N-terminal amino acids (90-124) are not as flexible as known from NMR analysis. When the cross-linked sites were used as structural constraint, molecular modeling calculations yielded a structural model for PrP dimer and its monomeric subunit, including the folding of amino acids 90-124 in addition to the known structure. Molecular dynamics of the structure after release of the constraint indicated an intrinsic stability of the domain of amino acids 90-124.     

Monitoring the Bacterial Population Dynamics in Sourdough Fermentation Processes by Using PCR-Denaturing Gradient Gel Electrophoresis

Four sourdoughs (A to D) were produced under practical conditions by using a starter mixture of three commercially available sourdough starters and a baker's yeast constitutively containing various species of lactic acid bacteria (LAB). The sourdoughs were continuously propagated until the composition of the LAB flora remained stable. Two LAB-specific PCR-denaturing gradient gel electrophoresis (DGGE) systems were established and used to monitor the development of the microflora. Depending on the prevailing ecological conditions in the different sourdough fermentations, only a few Lactobacillus species were found to be competitive and became dominant. In sourdough A (traditional process with rye flour), Lactobacillus sanfranciscensis and a new species, L. mindensis, were detected. In rye flour sourdoughs B and C, which differed in the process temperature, exclusively L. crispatus and L. pontis became the predominant species in sourdough B and L. crispatus, L. panis, and L. frumenti became the predominant species in sourdough C. On the other hand, in sourdough D (corresponding to sourdough C but produced with rye bran), L. johnsonii and L. reuteri were found. The results of PCR-DGGE were consistent with those obtained by culturing, except for sourdough B, in which L. fermentum was also detected. Isolates of the species L. sanfranciscensis and L. fermentum were shown by randomly amplified polymorphic DNA-PCR analysis to originate from the commercial starters and the baker's yeast, respectively.     

A Conceptual Framework for Using Mussels as Biomonitors in Whole Effluent Toxicity

As a main source of direct and continuous input of pollutants in the aquatic ecosystem, studying the effects of effluents on receiving ecosystems has a high ecological relevance. While ecological risk assessment procedures are usually based on a chemical-based single component approach, their application for complex mixtures and effluents is less straightforward. A chemical-based approach has to rely on the knowledge of what chemicals are present in effluents, their potential toxicity, how all of these individual chemicals interact and what their individual and combined contribution to the mixture is. Whole effluent toxicity (WET) testing, however, is an integrative tool that measures the toxic effect of an effluent as a whole and accounts for uncharacterized sources of toxicity and for interactions. This paper addresses the use of transplanted bivalves, i.e., caged mussels, as a biomonitoring tool in WET testing with special reference to field situations in both freshwater and marine environments. We indicate how endpoints at different levels of biological organization within exposed mussels can give an integrative overview of effects. Finally, we will provide a framework for future research using mussels and discuss a multitude of instream responses for routine, efficient and cost-effective active biomonitoring applications.     

Winter habitat–space use in a large arctic herbivore facing contrasting forage abundance

We analysed how changes in resource levels influence foraging trade-offs in late winter by wild Svalbard reindeer. Forage plants, and particularly lichens, were less abundant at the overgrazed Brøggerhalvøya compared with the neighbouring Sarsøyra. Strong interactions occurred between habitat selection, home range size, and feeding crater selection. At Brøggerhalvøya, radiocollared females generally selected productive habitat (high summer NDVI; Normalised Difference Vegetation Index). “Immigrants” at Sarsøyra (dispersed from Brøggerhalvøya in early winter) had similar habitat preferences, probably due to past experience. In contrast, “residents” at Sarsøyra were more influenced by abiotic conditions, using habitat with low NDVI, but selecting for high-quality forage (lichens) when cratering. This suggests more quality-based selection at the expense of quantity when forage abundance increases. Habitat–space use relationships also differed between the animal categories, as home range size decreased with availability of preferred habitat. Thus, changes in forage abundance can strongly influence winter habitat–space use interactions in predator-free systems.     

Isolation of a new butanol-producing Clostridium strain: High level of hemicellulosic activity and structure of solventogenesis genes of a new Clostridium saccharobutylicum isolate

New isolates of solventogenic bacteria exhibited high hemicellulolytic activity. They produced butanol and acetone with high selectivity for butanol (about 80% of butanol from the total solvent yield). Their 16S rDNA sequence was 99% identical to that of Clostridium saccharobutylicum. The genes responsible for the last steps of solventogenesis and encoding crotonase, butyryl-CoA dehydrogenase, electron-transport protein subunits A and B, 3-hydroxybutyryl-CoA dehydrogenase, alcohol dehydrogenase, CoA-transferase (subunits A and B), acetoacetate decarboxylase, and aldehyde dehydrogenase were identified in the new C. saccharobutylicum strain Ox29 and cloned into Escherichia coli. The genes for crotonase, butyryl-CoA dehydrogenase, electron-transport protein subunits A and B, and 3-hydroxybutyryl-CoA dehydrogenase composed the bcs-operon. A monocistronic operon containing the alcohol dehydrogenase gene was located downstream of the bcs-operon. Genes for aldehyde dehydrogenase, CoA-transferase (subunits A and B), and acetoacetate decarboxylase composed the sol-operon. The gene sequences and the gene order within the sol- and bcs-operons of C. saccharobutylicum Ox29 were most similar to those of Clostridium beijerinckii. The activity of some of the bcs-operon genes, expressed in heterologous E. coli, was determined.     

Southern Ocean deep-sea isopod species richness (Crustacea, Malacostraca): influences of depth, latitude and longitude

We examined deep-sea epibenthic sledge isopod data from the Atlantic sector of the Southern Ocean (SO) (depth range=742–5,191 m). Samples were taken during the expeditions EASIZ II (ANT XV-3) in 1998 and ANDEEP I and II (ANT XIX3/4) in 2002. A total of 471 isopod species were recorded from 28 sites. The species richness of the epibenthic sledge samples was highly variable (6–82 species). Species richness was highest at site 131-3 in 3,053 m depth in the north-eastern Weddell Sea. The highest numbers of species were sampled in the middle depth range and lower species richness was found in the shallower and deeper parts of the study area. Depth is suggested to explain isopod species richness better than both latitude and longitude. Between 58°S and 65°S, the number of species ranged from 9 to 82 (mean=35.9). Further south in the Weddell Sea, between 73°S and 74°S, species richness was lower and the number of species ranged from 6 to 35 (mean=19.2). With regard to longitude, the highest species richness (up to 82 species) was found between 50°W and 60°W in the area of the South Shetland Islands and around the Antarctic Peninsula, while numbers did not exceed 50 species in the eastern Weddell Sea. The haul length, ranging from 807 to 6,464 m, was positively correlated with depth; however, there was no linear relationship between haul length and species richness. We therefore suggest that depth was the most important factor explaining isopod species richness. However, only 28 sites were visited and the statistical power is thus limited. Sampling in the deep sea is expensive and time consuming and as yet this is the best isopod data set available from the Atlantic sector of the SO. Future expeditions are therefore important to better explain the current patterns of benthic diversity in Antarctica.