Controlled unfolding and refolding of a single sodium-proton antiporter using atomic force microscopy

Single-molecule force-spectroscopy was employed to unfold and refold single sodium-proton antiporters (NhaA) of Escherichia coli from membrane patches. Although transmembrane alpha-helices and extracellular polypeptide loops exhibited sufficient stability to individually establish potential barriers against unfolding, two helices predominantly unfolded pairwise, thereby acting as one structural unit. Many of the potential barriers were detected unfolding NhaA either from the C-terminal or the N-terminal end. It was found that some molecular interactions stabilizing secondary structural elements were directional, while others were not. Additionally, some interactions appeared to occur between the secondary structural elements. After unfolding ten of the 12 helices, the extracted polypeptide was allowed to refold back into the membrane. After five seconds, the refolded polypeptide established all secondary structure elements of the native protein. One helical pair showed a characteristic spring like "snap in" into its folded conformation, while the refolding process of other helices was not detected in particular. Additionally, individual helices required characteristic periods of time to fold. Correlating these results with the primary structure of NhaA allowed us to obtain the first insights into how potential barriers establish and determine the folding kinetics of the secondary structure elements.     

Identification of functionally important residues of Arp2/3 complex by analysis of homology models from diverse species

We constructed homology models from the crystal structure of bovine Arp2/3 complex and sequences from six phylogenetically diverse species (Arabidopsis thaliana, Caenorhabditis elegans, Dictyostelium discoideum, Drosophila melanogaster, Saccharomyces cerevisiae, Schizosaccharomyces pombe) representing over 800 million years of evolution and used conserved surface residues to search for functionally important structural elements. The folds of the seven subunits and their core residues are well conserved, as well as residues at subunit interfaces. Only 45% of solvent-exposed surface residues are conserved and only 15% are identical across the seven species. Arp residues expected to interact with nucleotide and with the first and second actin subunits in a daughter filament are conserved and similar to actin. Arp residues required to form an Arp dimer differ from actin and may contribute to the dissociated state of the Arps in the unactivated complex. Conserved patches of surface residues guided us to candidate sites for nucleation promoting factors to interact with Arp3, Arp2, and ARPC3. Other conserved residues were used with experimental constraints to propose how residues on the subunits ARPC1, ARPC2, ARPC4 and ARPC5 might interact with the mother filament at branch junctions.     

Neuronal A1 receptors mediate increase in extracellular kynurenic acid after local intrastriatal adenosine infusion

The naturally occurring purine nucleoside adenosine has pronounced anticonvulsant and neuroprotective properties and plays a neuromodulatory role in the CNS. Kynurenic acid (KYNA) is an astrocyte-derived, endogenous neuroinhibitory compound, which shares several of adenosine's properties. In a first attempt to examine possible interactions between these two biologically active molecules, adenosine was focally applied into the striatum of freely moving rats by reverse microdialysis, and changes in extracellular KYNA were monitored over time. A 2-h infusion of adenosine increased KYNA levels in a dose-dependent manner, with 10 mm of adenosine causing a twofold elevation within 1 h. This effect was reversible and was effectively blocked by coinfusion of the specific A1 adenosine receptor antagonist 8-cyclopentyltheophylline (100 microm). In contrast, coinfusion of adenosine with MSX-3 (100 microm), an A2A receptor antagonist, did not affect the adenosine-induced increase in KYNA levels. Local striatal perfusion with the A1 receptor agonist N6-cyclopentyladenosine (100 microm) mimicked the effect of adenosine, whereas perfusion with the A2A receptor agonist CGS-21680 (100 microm) was ineffective. Finally, we tested the effect of adenosine (10 mm) on extracellular KYNA in striata that had been injected with quinolinate (60 nmol/1 microL) 7 days earlier. In this neuron-depleted tissue, perfusion with adenosine failed to affect extracellular KYNA levels. These data demonstrate that adenosine is capable of raising extracellular KYNA in the rat striatum by interacting with postsynaptic neuronal A1 receptors. This mechanism may result in a synergism between the neurobiological effects of adenosine and KYNA.     

The role of virus infection in a simple phytoplankton zooplankton system

Many planktonic species show spectacular bursts ("blooms") in population density. Though viral infections are known to cause behavioural and other changes in phytoplankton and other aquatic species, yet their role in regulating the phytoplankton population is still far from being understood. To study the role of viral diseases in the planktonic species, we model the phytoplankton-zooplankton system as a prey-predator system. Here the prey (phytoplankton) species is infected with a viral disease that divides the prey population into susceptible and infected classes, with the infected prey being more vulnerable to predation by the predator (zooplankton). The dynamical behaviour of the system is investigated from the point of view of stability and persistence both analytically and numerically. The model shows that infection can be sustained only above a threshold of force of infection, and, there exists a range in the infection rate where this system shows "bloom"-like stable limit cycle oscillations. The time series of natural "blooms" with different types of irregular oscillations can arise in this model simply from a biologically realistic feature, i.e., by the random variation of the epidemiological parameter (rate of infection) in the infected prey population. The difference in mean strength of infection alone can lead to the different types of patterns observed in natural planktonic blooms.     

Impact of herbivores on nitrogen cycling: contrasting effects of small and large species

Herbivores are reported to slow down as well as enhance nutrient cycling in grasslands. These conflicting results may be explained by differences in herbivore type. In this study we focus on herbivore body size as a factor that causes differences in herbivore effects on N cycling. We used an exclosure set-up in a floodplain grassland grazed by cattle, rabbits and common voles, where we subsequently excluded cattle and rabbits. Exclusion of cattle lead to an increase in vole numbers and a 1.5-fold increase in net annual N mineralization at similar herbivore densities (corrected to metabolic weight). Timing and height of the mineralization peak in spring was the same in all treatments, but mineralization in the vole-grazed treatment showed a peak in autumn, when mineralization had already declined under cattle grazing. This mineralization peak in autumn coincides with a peak in vole density and high levels of N input through vole faeces at a fine-scale distribution, whereas under cattle grazing only a few patches receive all N and most experience net nutrient removal. The other parameters that we measured, which include potential N mineralization rates measured under standardized laboratory conditions and soil parameters, plant biomass and plant nutrient content measured in the field, were the same for all three grazing treatments and could therefore not cause the observed difference. When cows were excluded, more litter accumulated in the vegetation. The formation of this litter layer may have added to the higher mineralization rates under vole grazing, through enhanced nutrient return through litter or through modification of microclimate. We conclude that different-sized herbivores have different effects on N cycling within the same habitat. Exclusion of large herbivores resulted in increased N annual mineralization under small herbivore grazing.     

Delineating the effects of a plant trait on interactions among associated insects

Plant traits can affect ecological interactions between plants, herbivores, and predators. Our study tests whether reduced leaf wax in peas alters the interaction between the pea aphid ( Acyrthosiphon pisum), a foliar-foraging predator (a lady beetle, Hippodamia convergens) and a ground-foraging predator (a ground beetle, Poecilus scitulus). We performed a 2×2×2 factorial experiment in which wax level, presence of H. convergens, and presence of P. scitulus were manipulated. Experimental arenas consisted of a cage surrounding three pea plants. One plant in each cage was stocked with 15 pea aphids. In greenhouse and field cage experiments, we assessed the effect of each factor and their interactions on aphid density. As in previous studies, H. convergens foraged for aphids more effectively on reduced wax peas than on normal peas. Other interactions among H. convergens, P. scitulus , and A. pisum were the same on both types of peas. We consider how aphid movement, plant growth, and a high frequency of predation by P. scitulus on H. convergens influenced pea aphid density.     

Causal mechanisms underlying host specificity in bat ectoparasites

In parasites, host specificity may result either from restricted dispersal capacity or from fixed coevolutionary host-parasite adaptations. Knowledge of those proximal mechanisms leading to particular host specificity is fundamental to understand host-parasite interactions and potential coevolution of parasites and hosts. The relative importance of these two mechanisms was quantified through infection and cross-infection experiments using mites and bats as a model. Monospecific pools of parasitic mites (Spinturnix myoti and S. andegavinus) were subjected either to individual bats belonging to their traditional, native bat host species, or to another substitute host species within the same bat genus (Myotis). The two parasite species reacted differently to these treatments. S. myoti exhibited a clear preference for, and had a higher fitness on, its native host, Myotis myotis. In contrast, S. andegavinus showed no host choice, although its fitness was higher on its native host M. daubentoni. The causal mechanisms mediating host specificity can apparently differ within closely related host-parasite systems.     

Conformational insights on the molecular recognition processes of carbohydrate molecules by proteins and enzymes: A 3D view by using NMR

This review focuses, in a non-exhaustive manner, on the essential structural and conformational features of protein-carbohydrate interactions and on some applications of NMR spectroscopy to deal with this topic from different levels of complexity.     

Resource partitioning and interspecific interactions among sympatric rain forest arboreal mammals of the Western Ghats, India

Resource partitioning in a community of diurnal arboreal mammalsconsisting of the lion-tailed macaque Macaca silenus, bonnetmacaque (BM) Macaca radiata, Nilgiri langur Semnopithecus johnii,and the Indian giant squirrel Ratufa indica of the Western Ghats,southern India, was studied. Differences in their diet, verticalstratification, food resource niche breadth, niche overlap,and behavioral interactions were examined. Resource partitioningwas through differential habitat use, resource use, and verticalstratification. Of the four species, the BM was not a residentspecies and made frequent forays into evergreen forest fromthe adjacent deciduous forest during the flowering season ofCullenia exarillata and fruiting season of Ficus microcarpa.The macaques had narrower niches, and the langur and the squirrelhad wider niches. Niche overlap was highest between the twomacaques. Overlap among the study species was particularly pronouncedduring the flowering of C. exarillata. There was significantcorrelation between niche overlap and intolerance among thestudy species. Certain species pairs showed little or no mutualintolerance despite high overlap. Cooperative interactions suchas alarm calls occurred more frequently among the resident species.Interaction matrices revealed an underlying pattern of interspecificdominance hierarchy, with the BM dominating over the other threespecies. Our study suggests that the BM do not coexist withthe other three because of high overlap with its congener andlow occurrence of cooperative interactions.     

Computation of non-covalent interactions in TNF proteins and interleukins

The roles played by the non-covalent interactions have been investigated for a set of six TNF proteins and nine Interleukins. The stabilizing residues have been identified by a consensus approach using the concepts of available surface area, medium and long-range interactions and conservation of amino acid residues. The cation-pi interactions have been computed based on a geometric approach such as distance and energy criteria. We identified an average of 1 energetically significant cation-pi interactions in every 94 residues in TNF proteins and 1 in every 62 residues in Interleukins. In TNF proteins, the cationic groups Lys preferred to be in helix while Arg preferred to be in strand regions while in Interleukins the Arg residues preferred to be in helix and Lys preferred to be in strand regions. From the available surface area calculations, we found that, almost all the cation and pi residues in TNF proteins and Interleukins were either in buried or partially buried regions and none of them in the exposed regions. Medium and long-range interactions were predominant in both TNF proteins and Interleukins. It was observed that the percentage of stabilizing centers were more in TNF proteins as compared to the Interleukins, while the percentage of conserved residues were more in Interleukins than in TNF proteins. In the stabilizing residues Lys was observed to be a stabilizing residue in both TNF proteins and Interleukins. Among the aromatic group, Phe was seen to be a stabilizing residue in both TNF and Interleukins. We suggest that this study on the computation of cation-pi interactions in TNF proteins and Interleukins would be very helpful in further understanding the structure, stability and functional similarity of these proteins.