Human Adenosine A2A Receptor Binds Calmodulin with High Affinity in a Calcium-Dependent Manner

Understanding how ligands bind to G-protein-coupled receptors and how binding changes receptor structure to affect signaling is critical for developing a complete picture of the signal transduction process. The adenosine A2A receptor (A2AR) is a particularly interesting example, as it has an exceptionally long intracellular carboxyl terminus, which is predicted to be mainly disordered. Experimental data on the structure of the A2AR C-terminus is lacking, because published structures of A2AR do not include the C-terminus. Calmodulin has been reported to bind to the A2AR C-terminus, with a possible binding site on helix 8, next to the membrane. The biological meaning of the interaction as well as its calcium dependence, thermodynamic parameters, and organization of the proteins in the complex are unclear. Here, we characterized the structure of the A2AR C-terminus and the A2AR C-terminus-calmodulin complex using different biophysical methods, including native gel and analytical gel filtration, isothermal titration calorimetry, NMR spectroscopy, and small-angle X-ray scattering. We found that the C-terminus is disordered and flexible, and it binds with high affinity (K_d = 98 nM) to calmodulin without major conformational changes in the domain. Calmodulin binds to helix 8 of the A2AR in a calcium-dependent manner that can displace binding of A2AR to lipid vesicles. We also predicted and classified putative calmodulin-binding sites in a larger group of G-protein-coupled receptors.     

Responses of ground vegetation species to clear-cutting in a boreal forest: aboveground biomass and nutrient contents during the first 7 years

Rapid growth of ground vegetation following clear-cutting is important to site productivity because vegetation retains nutrients in the ecosystem and can decrease nutrient leaching prior to stand re-establishment. Aboveground biomass, nutrient contents (N, P, K and Ca) and species composition of ground vegetation were determined 1 year before and for 7 years after clear-cutting of a mixed forest dominated by Norway spruce [Picea abies (L.) H. Karst.] in eastern Finland. The biomass of the feather mosses [Pleurozium schreberi Brid. and Hylocomium splendens (Hedw.) B. S.& G.] and the dwarf shrubs (Vaccinium myrtillus L. and V. vitis-idaea L.), which had dominated the ground vegetation in the mature forest, significantly decreased after clear-cutting. However, with the exception of H. splendens, these species had recovered within 3–5 years. The biomass of Deschampsia flexuosa (L.) Trin. considerably increased soon after clear-cutting, and Epilobium angustifolium L. appeared 3–5 years after cutting. These species contributed to the retention of nutrients not simply because of their biomass but also because of higher nutrient concentrations in their tissues. Total biomass and nutrient contents of the ground vegetation exceeded those of the pre-cutting levels. The proportion of ground vegetation biomass and nutrient contents represented by mosses decreased after cutting, while V. myrtillus, although reduced after cutting, remained a marked nutrient sink. The results suggest that H. splendens is the most sensitive species to cutting, but the biomass of P. schreberi, V. myrtillus and V. vitis-idaea return to initial levels soon after clear-cutting as do the nutrient contents of ground vegetation.     

Changes in the Above- and Below-ground Biomass and Nutrient Pools of Ground Vegetation After Clear-cutting of a Mixed Boreal Forest

Ground vegetation may act as a sink for nutrients after clear-cutting and thus decrease leaching losses. Biomass and nutrient (N, P, K, Ca) pools of ground vegetation (mosses, roots and above-ground parts of field layer) were determined one year before and five years after clear-cutting of a Norway spruce (Picea abies (L.) H. Karst.) dominated boreal mixed forest stand in eastern Finland (63°51′ N, 28°58′ E). Before clear-cutting the average biomass of ground vegetation was 5307 kg ha⁻¹, with nutrient contents of 46.9 kg N ha⁻¹1, 4.1 kg P ha⁻¹1, 16.2 kg K ha⁻¹1 and 13.9 kg Ca ha⁻¹1. The biomass and nutrient pools decreased after clear-cutting being lowest in the second year, the biomass decreasing by 46–65% in the cut plots. The nutrient pools decreased as follows: N 54–72%, P 36–68%, K 51–71% and Ca 57–74%. The decrease in ground vegetation nutrient uptake, and the observed reduced depth of rooting may decrease nutrient retention after clear-cutting and decomposing dead ground vegetation is a potential source of leached nutrients. These negative effects of clear-cutting on the nutrient binding capacity of ground vegetation was short-lived since the total biomass and nutrient pools returned to pre-cutting levels or were even greater by the end of the 5-year study period.     

RGD-dependent entry of coxsackievirus A9 into host cells and its bypass after cleavage of VP1 protein by intestinal proteases.

The recently reported nucleotide sequence of coxsackievirus A9 (CAV-9) showed that unlike other enteroviruses, CAV-9 has an insertion of about 17 amino acids at the C-terminal end of VP1 (K. H. Chang, P. Auvinen, T. Hyypi?, and G. Stanway, J. Gen. Virol. 70:3269-3280, 1989). This sequence includes the RGD (arginine-glycine-aspartic acid) motif which is known to be important in certain protein-protein interactions. We studied the inhibitory effect of RGD-containing peptides in the attachment of CAV-9 to African green monkey kidney cells. A peptide corresponding to the RRGDM sequence derived from the inserted segment of CAV-9 was found to block virus attachment effectively, and the inhibition was dose dependent. Substitution of glutamic acid for the homologous aspartic acid completely abolished the inhibitory effect, indicating great specificity of the action. During replication in the gut, all enteroviruses are exposed to host proteolytic enzymes. Exposure of CAV-9 to purified trypsin or human intestinal fluid resulted in selective cleavage of the VP1 capsid protein. Intact and trypsin-cleaved VP1 proteins gave identical N-terminal sequences, indicating that cleavage of VP1 takes place near the C terminus. Attachment of proteolytically cleaved infectious CAV-9 to green monkey kidney cells was not prevented by RGD-containing peptides, indicating that cleaved CAV-9 is able to bypass RGD-dependent entry. The altered receptor specificity of proteolytically cleaved viruses may have important consequences in the pathogenesis of enteric infections.     

Decomposition and nutrient release from logging residues after clear-cutting of mixed boreal forest

Elevated dissolved carbon (C), nitrogen (N) and phosphorus (P) concentrations are frequently observed in surface water soon after clear-cutting of boreal coniferous forests. It has been suggested that they originate from the fine logging residues whose decomposition may be accelerated as a result of changes in soil temperature and moisture conditions. In the present study, the decomposition rate and release of C, N, and P from Scots pine (Pinus sylvestris L.), Norway spruce (Picea abies Karsten) and silver birch (Betula pendula Roth.) logging residues (fine roots 2 mm, branches 10 mm and foliage) were investigated during three years with the litterbag method in a clear-cut area and in an adjacent Norway spruce dominated, mixed boreal forest in eastern Finland (63°51 N, 28°58 E, 220 m asl). The mass loss of the logging residues decreased in the order: foliage > roots > branches. Birch leaves were the only fraction that showed significantly higher losses of mass and C at the clear-cut plot than at the forest plot; otherwise there was no tendency for accelerated decomposition or mineralization at the clear-cut plot. After three years the initial C pool in the logging residues had declined by 33% and that of P by 49% but there was no net release of N as more N accumulated in roots and branches than was released from foliage. The results indicate that 1) logging residues release relatively large and rapid fluxes of CO₂ to the atmosphere 2) are potential source of elevated P in surface waters soon after clear-cutting 3) are not a net source of N immediately after clear-cutting.     

Development of ground vegetation biomass and nutrient pools in a clear-cut disc-plowed boreal forest

Nutrient leaching from forest substrate after clear-cutting and subsequent soil preparation is strongly influenced by the capacity of ground vegetation to sequester the released nutrients. We studied the rates and patterns of biomass and nutrient accumulation in ground vegetation growing on ridges, in furrows and on undisturbed surfaces for 2–5 years after disc-plowing in eastern Finland. The biomass of mosses on ridges remained significantly lower than that in furrows and on undisturbed surfaces. Field layer biomass on ridges and in furrows was significantly lower than on undisturbed surfaces throughout the study period. Field layer biomass increased more on ridges than in furrows. Root biomass on ridges and undisturbed surfaces was considerably higher than in furrows. Five years after disc-plowing, total biomass and nutrient pools for ridges (biomass 4,975 kg ha⁻¹, N 40 kg ha⁻¹, P 5 kg ha⁻¹, K 20 kg ha⁻¹ and Ca 18 kg ha⁻¹) and undisturbed surfaces (biomass 5,613 kg ha⁻¹, N 43 kg ha⁻¹, P 5 kg ha⁻¹, K 22 kg ha⁻¹ and Ca 18 kg ha⁻¹) were similar, but considerably lower for furrows (biomass 1,807 kg ha⁻¹, N 16 kg ha⁻¹, P 2 kg ha⁻¹, K 10 kg ha⁻¹ and Ca 6 kg ha⁻¹). Ridges covered 25% of the area, furrows 30 and 45% was undisturbed surfaces. Taking into account the proportion of each type of surface, values for the whole prepared clear-cut area were 4,312, 34, 4, 18 and 14 kg ha⁻¹ for biomass, N, P, K and Ca, respectively. Biomass and nutrient pools had not returned to uncut forest levels at the end of the 5-year study period. The results indicate that mosses and field layer vegetation respond differently to soil preparation, that the development of biomass on ridges, in furrows and on undisturbed surfaces proceeds at different rates, and that the biomass and nutrient uptake of ground vegetation remains below pre-site preparation levels for several years. However, ridges, which are known to be the most susceptible to leaching, revegetate rapidly. Responsible Editor: Tibor Kalapos.     

Bridge over troubled proline: assignment of intrinsically disordered proteins using (HCA)CON(CAN)H and (HCA)N(CA)CO(N)H experiments concomitantly with HNCO and i(HCA)CO(CA)NH

NMR spectroscopy is by far the most versatile and information rich technique to study intrinsically disordered proteins (IDPs). While NMR is able to offer residue level information on structure and dynamics, assignment of chemical shift resonances in IDPs is not a straightforward process. Consequently, numerous pulse sequences and assignment protocols have been developed during past several years, targeted especially for the assignment of IDPs, including experiments that employ H^N, H^α or ¹³C detection combined with two to six indirectly detected dimensions. Here we propose two new HN-detection based pulse sequences, (HCA)CON(CAN)H and (HCA)N(CA)CO(N)H, that provide correlations with ¹H^N(i ? 1), ¹³C′(i ? 1) and ¹⁵N(i), and ¹H^N(i + 1), ¹³C′(i) and ¹⁵N(i) frequencies, respectively. Most importantly, they offer sequential links across the proline bridges and enable filling the single proline gaps during the assignment. We show that the novel experiments can efficiently complement the information available from existing HNCO and intraresidual i(HCA)CO(CA)NH pulse sequences and their concomitant usage enabled >95 % assignment of backbone resonances in cytoplasmic tail of adenosine receptor A2A in comparison to 73 % complete assignment using the HNCO/i(HCA)CO(CA)NH data alone.     

Neuromuscular function during drop jumps in young and elderly males

The Hoffman reflex (H-reflex), indicating alpha-motoneuron pool activity, has been shown to be task – and in resting conditions – age dependent. How aging affects H-reflex activity during explosive movements is not clear at present. The purpose of this study was to examine the effects of aging on H-reflexes during drop jumps, and its possible role in drop jump performance. Ten young (26.8 ± 2.7 years) and twenty elderly (64.2 ± 2.7 years) subjects participated in the study. Maximal drop jump performance and soleus H-reflex response (H/M jump) 20 ms after ground contact were measured in a sledge ergometer. Maximal H-reflex, maximal M-wave, Hmax/Mmax-ratio and H-reflex excitability curves were measured during standing rest. Although in young the H-reflex response (Hmax/Mmax) was 6.5% higher during relaxed standing and 19.7% higher during drop jumps (H jump/M jump) than in the elderly group, these differences were not statistically significant. In drop jumps, the elderly subjects had lower jumping height (30.4%, p < 0.001), longer braking time (32.4%, p < 0.01), lower push-off force (18.0%, p < 0.05) and longer push-off time (31.0% p < 0.01). H jump/M jump correlated with the average push-off force (r = 0.833, p < 0.05) and with push-off time (r = ?0.857, p < 0.01) in young but not in the elderly. Correlations between H-reflex response and jumping parameters in young may indicate different jumping and activation strategies in drop jumps. However, it does not fully explain age related differences in jumping performance, since age related differences in H-reflex activity were non-significant.