Cleavage of the carboxyl tail from the G3 domain of aggrecan but not versican and identification of the amino acids involved in the degradation

Aggrecan, a major structural proteoglycan in cartilage, contains three globular domains, G1, G2, and G3, as well as sequences for glycosaminoglycan modification. A large number of proteases are implicated in aggrecan cleavage in normal metabolism, aging, and arthritis. These proteases are known to cleave at the IGD, KS, and CS domains. Here we report for the first time evidence of cleavage at a novel site, the carboxyl tail of aggrecan. Results from deletion mutants of the tail indicated that the likely cleavage sites were two consensus sequences, RRLXK and RSPR, present in the aggrecan analogs of many species. This was confirmed by site-directed mutagenesis. A construct containing two G3 domains (G3G3) was also found to cleave between the G3 duplicates. When G3 tail was linked to a glycosaminoglycan-modifying sequence, it was protected from cleavage. Furin inhibitor also reduced the levels of tail cleavage. The carboxyl tails of chicken and human versican were not cleaved, despite the presence of the consensus sequence. Our studies indicate that the basic amino acids present in the tail play an important role in cleavage, and this mechanism is specific to aggrecan.     

Application of 13C NMR to investigate the transformations and biodegradation of organic materials by wood- and soil-feeding termites, and a coprophagous litter-dwelling dipteran larva

Solid-state 13C nuclear magnetic resonance spectroscopy has been used to characterize the C in samples of the food (wood), gut contents and faeces from the wood-feeding termite, Microcerotermes parvus; soil in the guts and mound material from the soil-feeding termite, Thoracotermes macrothorax; and the food and faeces from the litter-feeding, coprophagous larvae of the dipteran fly, Bibio marci. Spectra from the wood-feeding termite indicated preferential loss of polysaccharide and accumulation of lignin with some modification to the O-aromatic-C and methoxyl-C (O-methyl-C) components during passage through the gut. Spectra for the soil-feeding termite indicated little change in the distribution of 13C between resonances following passage through the gut, except for some evidence of preferential polysaccharide loss. Interpretation of the spectra from these organisms was restricted by the relatively low C content of the soils and mound material, and by the large contribution to the NMR spectra from the gut tissue rather than the gut contents. Spectra for the litter-feeding dipteran larvae indicated preferential feeding on the polysaccharide-rich component of the litter and then overall loss of polysaccharide-C and accumulation of both aromatic-C and methoxyl-C in the gut. These changes were greater for the second passage than for the first passage through the gut, suggesting that principally mechanical and physical changes occurred initially and that chemical digestion was prevalent during the second passage.     

Application of ARDRA and PLFA analysis in characterizing the bacterial communities of the food,gut and excrement of saprophagous larvae of<Emphasis Type="Italic">Penthetria holosericea</Emphasis> (<Emphasis Type="Italic">Diptera: Bibionidae</Emphasis>): a pilot study

Amplified ribosomal DNA restriction analysis (ARDRA) was used to compare the bacterial communities of the food, the gut sections (ceca, anterior and posterior midgut, hindgut) and the excrement of the litter feeding bibionid larvae of Penthetria holosericea. For universal eubacterial primers ARDRA patterns were complex with only minor differences among samples. Taxon specific primers were also applied to characterize the samples. Fragment composition was transformed to presence/absence binary data and further analyzed. Cluster analysis revealed that bacterial communities of gut highly resembled each other with the exception of the ceca. ARDRA patterns of consumed leaves clustered together with the intact leaves but differed from those of the excrement. ARDRA results were compared with microbial community structure based on phospholipid fatty acid (PLFA) fingerprints. The cluster analysis of PLFA (presence/absence binary) data resulted in a pattern similar to the ARDRA data. The PCA analysis of PLFA relative content separated microbial communities into five groups: (1) anterior and posterior midgut, (2) hindgut, (3) ceca, (4) consumed and intact litter, (5) excrement. Both methods indicated that conditions in the larval gut result in formation of a specific microbial community which differs from both the food and excrement ones. Particularly ceca--(blind appendages, harbor very specific microbial community) are divided from the rest of the gut by perithropic membrane.     

The effect of litter type and macrofauna community on litter decomposition and organic matter accumulation in post-mining sites

Field microcosms consisting of mineral soil (spoil substrate) and two types of litter taken either from an unreclaimed site with spontaneously developed vegetation (mostly Salix caprea) or from an alder plantation (a mixture of Alnus glutinosa and A. incana) were exposed in spontaneously developed or reclaimed sites at a post-mining heap near Sokolov (Czech Republic) for one year. The litter types differed remarkably in C:N ratio which was 29 for spontaneous litter and 14 for alder litter. The two microcosm types were either accessible or not accessible to soil macrofauna. The effect of macrofauna exclusion on soil mixing was complex and depended on litter quality and the site that determined soil fauna composition. In reclaimed sites where macrofauna was dominated by saprophags, mainly earthworms, the macrofauna access increased soil mixing. In sites where predators dominated, the macrofauna exclusion probably suppressed fragmentation and mixing activity of the mesofauna.     

Primary Succession of Soil Rotifers in Clays of Brown Coal Post‐Mining Dumps

Changes in rotifer soil communities along a primary succession chronosequence was studied on brown coal post mining areas near Sokolov, NW part of the Czech Republic. The chronosequence of unreclaimed plots was 2, 11, 14, 20, 43 years old. The rotifers were extracted from soil samples using a modification of the Baermann funnel method with combined light and temperature gradients. In total, 34 taxa of soil rotifers were identified throughout the study. The most common species were Encentrum arvicola, Adineta vaga, A. steineri, Habrotrocha rosa, H. elegans, H. filum, Macrotrachela quadricornifera and M. nana. Rotifer abundance varied from 4 ± 2 · 10³ to 516 ± 488 · 10³ individuals m^–2. Species number per sample increased with age of the plot (r = 0.45, P = 0.003). The most important environmental variables which significantly affected rotifer community were wood cover, sodium concentration and age of the plot. Pioneer plant species occupied 2 and 11 year old plots, 14–20 year old plots were covered by Salix caprea shrubs and a forest formed by Betula pendula and Populus tremuloides developed on the 43 year old plot. Some species were ubiquitous and present throughout the chronosequence (Macrotrachela quadricornifera). Among the pioneer species were Encentrum incisum, Habrotrocha rosa and Macrotrachela papillosa, 14–20 years old plots were preferred by Adineta vaga, E. arvicola, H. filum and M. nana, while the oldest plot was dominated by Adineta steineri and Encentrum mucronatum. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

The effect of vegetation patterns on oviposition habitat preference: A driving mechanism in terrestrial chironomid (Diptera: Chironomidae) succession?

Relationships between changes of two species of terrestrial chironomids(Smittia cf. aterrima andBryophaenocladius cf. virgo) and changes of vegetation cover during succession were studied with respect to plant — chironomid interaction in particular stages of chironomid life cycle. Several vegetation types forming vegetation mosaics in suburban fallow were distinguished according to their spatial architecture (height; density etc.). Abundance of chironomid larvae in particular type of vegetation and an area covered by these vegetation types were studied for three years. During one year the effect of vegetation types on density of aerial drift, oviposition habitat preference and adult emergence was studied in detail. Aerial drift was studied using pan traps, oviposition using soil sampling and adult emergence using emergence trap. Main results were similar for both species. Chironomid larvae were abundant in soil in initial years of succession. They were concentrated in patches with open and low height vegetation. Later their abundance decreased corresponding to a reduction of open and low vegetation areas. Greater density of aerial drift and oviposition preference was observed in areas of open and low vegetation. The success of larval development agreed with oviposition preference for winter larval generation only. The less preferred habitats seemed to be more suitable for summer generation. The results indicated that successional changes of terrestrial chironomids can be explained by the effect of vegetation cover on oviposition habitat preference.     

Nutrient limitation drives response of <Emphasis Type="Italic">Calamagrostis epigejos</Emphasis> to arbuscular mycorrhiza in primary succession

Little is known about the functioning of arbuscular mycorrhizal (AM) symbiosis over the course of primary succession, where soil, host plants, and AM fungal communities all undergo significant changes. Over the course of succession at the studied post-mining site, plant cover changes from an herbaceous community to the closed canopy of a deciduous forest. Calamagrostis epigejos (Poaceae) is a common denominator at all stages, and it dominates among AM host species. Its growth response to AM fungi was studied at four distinctive stages of natural succession: 12, 20, 30, and 50 years of age, each represented by three spatially separated sites. Soils obtained from all 12 studied sites were γ-sterilized and used in a greenhouse experiment in which C. epigejos plants were (1) inoculated with a respective community of native AM fungi, (2) inoculated with reference AM fungal isolates from laboratory collection, or (3) cultivated without AM fungi. AM fungi strongly boosted plant growth during the first two stages but not during the latter two, where the effect was neutral or even negative. While plant phosphorus (P) uptake was generally increased by AM fungi, no contribution of mycorrhizae to nitrogen (N) uptake was recorded. Based on N:P in plant biomass, we related the turn from a positive to a neutral/negative effect of AM fungi on plant growth, observed along the chronosequence, to a shift in relative P and N availability. No functional differences were found between native and reference inocula, yet root colonization by the native AM fungi decreased relative to the reference inoculum in the later succession stages, thereby indicating shifts in the composition of AM fungal communities reflected in different functional characteristics of their members.     

Population dynamics of wireworms (Coleoptera,Elateridae) in arable land after abandonment

The community of elaterid larvae of three sites (field and two fallows), representing different stages of secondary succession, were studied using soil sampling from 1986 to 1993. All three sites were abandoned arable land: a field cultivated until 1991, a fallow I abandoned in 1986, and a fallow II abandoned about 1976. The fallow II was used as a meadow after abandonment and was regularly mown until 1985, when cultivation stopped. Six species of Elateridae larvae were found at all three study sites. In the field, Agriotes obscurus, Athous niger, Athous subsuscus, Dalopius marginatus and Athous vittatus were found, A. niger and A. obscurus being the most abundant species. During cultivation, larval densities were very low, however, larval abundance increased up to 8.8 ± 8.3 ind. m^-2 when cultivation stopped. Only small A. obscurus larvae were found during cultivation, whereas larval size increased after abandonment. In fallow I A. obscurus, A. niger, A. subsuscus, D. marginatus, and Agrypnus murinus were found and the average annual abundance fluctuated between 0.8 ± 1.4 to 40.8 ± 10.9 ind. m^-2 with A. obscurus being the most abundant species. Fallow II supported the highest densities of wireworms from all plots studied (71.2±35.2 to 280.0±24.8 ind. m^-2). A. obscurus, A. niger, A. subsuscus and D. marginatus were found in fallow II. The abundance of all larval Elateridae as well as the dominant species A. obscurus decreased during the study period, while simultaneously the occurrence of small sized A. obscurus larvae decreased. The frequency of cultivation and time elapsed since last cultivation appeared to be the most important factors affecting elaterid occurrence in the field and fallow I. In fallow II, the decrease in abundance correlates with the accumulation of a dense litter layer, which may correspond with soil surface structure, plant community changes or predator pressure.     

Screening for microbial markers in Miocene sediment exposed during open-cast brown coal mining

Viable microorganisms were found in Miocene lacustrine clays of the cypris formation excavated from 200-m below the surface as spoil during open-cast brown coal mining (Sokolov Brown Coal Basin, North-Western Bohemia, Czech Republic). Both saprotrophic microfungi of the genera Penicillium, Verticillium, Cladosporium and Aspergillus as well as heterotrophic bacteria were isolated from an intact sediment cores. Heterotrophic bacteria were classified by the MIS Sherlock System as representatives of genera Nocardiopsis, Arthrobacter, Micrococcus, Kocuria, Rothia, Clavibacter, Bacillus, Paenibacillus, Brevibacillus, Microbacterium, Acinetobacter and Pseudomonas. A bacterium found among the strains had an atypical fatty acids profile enriched by branched fatty acids and polyunsaturated fatty acid (18:36) and gave no MIS Sherlock match. Phospholipid fatty acids analysis indicates a relatively high (535 pmol g⁻¹) but inhomogeneously distributed viable microbial biomass. Fatty acids analyses of non-fractioned lipids (representing viable, storage and dead biomass; 8390  pmol  g⁻¹) detected rich and homogenous profiles with fungal, bacterial and actinomycetal markers but no protozoan and algal fatty acids markers.     

Understory vegetation in reclaimed and unreclaimed post-mining forest stands

In the Sokolov coal mining district of the Czech Republic, spoil heaps are reclaimed by forest plantations, which are planted directly into the rough substrate (alkaline tertiary clay). We compared the understory that spontaneously developed in seven types of forest stands: one type was unreclaimed stands (spontaneously overgrown by Betula pendula and Salix caprea) and six were plantations, each dominated by one tree genus (Alnus, Larix, Picea, Pinus, Quercus, and Tilia). The age of the stands ranged from 22 to 33 years. The cover of understory plant species in each stand was estimated, and 16 other environmental and community variables were quantified. The number of plant species was highest in Quercus, Larix, and unreclaimed stands, and was negatively correlated with forest canopy cover and with the cover of the understory dominant, the grass Calamagrostis epigejos. Understory composition differed considerably among the types of forest stands and was significantly explained by the measured environmental variables. Forward selection in redundancy analysis indicated that the most important variable driving understory composition was thickness of the fermentation layer, which is clearly connected with soil development. Environmental variables, including fermentation layer, were also affected by the type of forest stand. Therefore, all of the explained variability in understory composition could be attributed to the type of forest stand, which apparently affected the understory by its impact on soil formation. However, the most favorable soil conditions were not favorable for understory development, as they supported mainly C. epigejos, which suppressed other species. Our study also showed that even in the absence of reclamation measures, mining sites can be successfully restored due to spontaneous succession.