Isolation of small-subunit rRNA for stable isotopic characterization

Small-subunit ribosomal RNA (SSU rRNA) has several characteristics making it a good candidate biomarker compound: it is found in bacteria, archaea and eukaryotes; it is quickly degraded extracellularly, hence SSU rRNA extracted from a sample probably derives from the currently active population; it includes both conserved and variable regions, allowing the design of capture probes at various levels of phylogenetic discrimination; and rRNA sequences from uncultured species can be classified by comparison with the large and growing public database. Here we present a method for isolation of specific classes of rRNAs from mixtures of total RNA, employing biotin-labelled oligonucleotide probes and streptavidin-coated paramagnetic beads. We also show that the stable carbon isotope composition of Escherichia coli total RNA and SSU rRNA reflects that of the growth substrate for cells grown on LB, M9 glucose and M9 acetate media. SSU rRNA is therefore a promising biomarker for following the flow of carbon, and potentially nitrogen, in natural microbial populations. Some possible applications are discussed.     

LRRK2 Transport Is Regulated by Its Novel Interacting Partner Rab32

Leucine-rich repeat kinase 2 (LRRK2) is a multi-domain 280 kDa protein that is linked to Parkinson''s disease (PD). Mutations especially in the GTPase and kinase domains of LRRK2 are the most common causes of heritable PD and are also found in sporadic forms of PD. Although the cellular function of LRRK2 is largely unknown there is increasing evidence that these mutations cause cell death due to autophagic dysfunction and mitochondrial damage. Here, we demonstrate a novel mechanism of LRRK2 binding and transport, which involves the small GTPases Rab32 and Rab38. Rab32 and its closest homologue Rab38 are known to organize the trans-Golgi network and transport of key enzymes in melanogenesis, whereas their function in non-melanogenic cells is still not well understood. Cellular processes such as autophagy, mitochondrial dynamics, phagocytosis or inflammatory processes in the brain have previously been linked to Rab32. Here, we demonstrate that Rab32 and Rab38, but no other GTPase tested, directly interact with LRRK2. GFP-Trap analyses confirmed the interaction of Rab32 with the endogenous LRRK2. In yeast two-hybrid experiments we identified a predicted coiled-coil motif containing region within the aminoterminus of LRRK2 as the possible interacting domain. Fluorescence microscopy demonstrated a co-localization of Rab32 and LRRK2 at recycling endosomes and transport vesicles, while overexpression of a constitutively active mutant of Rab32 led to an increased co-localization with Rab7/9 positive perinuclear late endosomes/MVBs. Subcellular fractionation experiments supported the novel role of Rab32 in LRRK2 late endosomal transport and sorting in the cell. Thus, Rab32 may regulate the physiological functions of LRRK2.     

Divergent ecological strategies determine different impacts on community production by two successful non-native seaweeds

The consequences of plant introductions into ecosystems are frequently reported from terrestrial environments, but little is known about the effects on ecosystem functioning caused by non-native primary producers in marine systems. In this study we explored the effects of the invasion by the two filamentous red algae Heterosiphonia japonica and Bonnemaisonia hamifera on the primary production of seaweed communities by using single and mixed cultures of non-native and native red algae. The experiments were conducted both in the presence and absence of herbivores. Biomass production of the invaded community increased more than four times in mixed cultures with H. japonica, while introduction by B. hamifera had no significant effect. The different impact on community production could be explained by differences in life history strategies between the invaders; H. japonica grew considerably faster than the native seaweeds which directly increased the community production, while B. hamifera showed a relatively slow growth rate and therefore had no effect. From previous studies it is known that B. hamifera produces a highly deterrent, but also costly, chemical defence. The assessment of survival and growth of a native generalist herbivore further corroborated that the biomass produced by B. hamifera constitutes a very low-quality food, whereas the performance of herbivores on a diet of H. japonica was comparable to that on native algal diets. In summary, this study demonstrates that successful invaders belonging to the same functional group (filamentous red algae) may have distinctly different impacts on productivity in the recipient community, depending on their specific life history traits.     

Species constancy depends on plot size – a problem for vegetation classification and how it can be solved

Question: While it is well known that species richness depends on plot size, it is not generally recognised that the same must be true for constancy. Accordingly, many authors use varying plot sizes when classifying vegetation based on the comparison of constancies between groups of plots. We ask whether the constancy‐area relationship follows a general rule, how strong the effect of plot sizes is on constancies, and if it is possible to correct constancies for area. Location: For empirical evaluation, we use data from plant communities in the Czech Republic, Sweden and Russia. Methods: To assess the potential influence of differences in plot size on constancies, we develop a mathematical model. Then, we use series of nested plot species richness data from a wide range of community types (herbaceous and forest) to determine the parameters of the derived function and to test how much the shape of the constancy‐area relationship depends on taxa or vegetation types. Results: Generally, the constancy‐area relationship can be described by C (A)=1?(1?C₀)^{(A/A0)^d}, with C being constancy, A area, C₀ known constancy on a specific area A₀, and d a damping parameter accounting for spatial autocorrelation. As predicted by this function, constancies in plant communities always varied from values near 0% to near 100% if plot sizes were changed sufficiently. For the studied vegetation types, a two‐ to fourfold increase in plot size resulted in a change of conventional constancy classes, i.e. an increase of constancy by 20% or more. Conclusions: Vegetation classification, which largely relies on constancy values, irrespective of whether traditional or modern fidelity definitions are used, is strongly prone to distorting scale effects when relevés of different plot sizes are combined in studies. The constancy‐area functions presented allow an approximate transformation of constancies to other plot sizes but are flawed by idiosyncrasies in taxa and vegetation types. Thus, we conclude that the best solution for future surveys is to apply uniform plot sizes within a few a priori delimited formations and to determine diagnostic species only within these formations. Finally, we suggest that more detailed analyses of constancy‐area relationships can contribute to a better understanding of species‐area relationships because the latter are the summation of the first for all species.     

Genetically acquired diabetes: adipocyte guanine nucleotide regulatory protein expression and adenylate cyclase regulation.

Adipocyte membranes from diabetic (db/db) animals showed marked elevations in the levels of alpha-subunits for Gi-1 which were almost twice those found in membranes from their normal, lean littermates. In contrast, no apparent differences were noted for levels of the alpha-subunits of Gi-2 and Gi-3, the 42 and 45 kDa forms of Gs and for G-protein beta-subunits. Adenylate cyclase specific activity was similar in membranes from both normal and diabetic animals under basal conditions and also when stimulated by optimal concentrations of either NaF or forskolin. In contrast, the ability of isoprenaline, glucagon and secretin to stimulate adenylate cyclase activity was greater in membranes from normal animals compared with membranes from diabetic animals. Receptor-mediated inhibition of adenylate cyclase, as assessed using PGE1 and nicotinate, was similar using membranes from both sources, but PIA (phenylisopropyladenosine) was a slightly more effective inhibitor in membranes from diabetic animals. A doubling in the expression of Gi-1 thus appears to have little discernible effect upon the inhibitory regulation of adenylate cyclase.