Floristic heterogeneity between forested sites in Kibale National Park, Uganda: insights into the fine-scale determinants of density in a large-bodied frugivorous primate

1.  Despite a long history of research on the influence of fruit availability on the population density of large-bodied vertebrate frugivores, operational understanding of the factors regulating density in these taxa remains elusive. We propose that fruit resources can be distinguished from one another on the basis of their functional role for the animals in question, and that such a classification system can aid in identifying the most influential determinants of frugivore density.
2.  We compared the availability of several resource classes between two sites in Kibale National Park, Uganda separated by only 12 km yet differing threefold in density of chimpanzees ( Pan troglodytes ).
3.  We categorized plant species used for fruit by chimpanzees according to their availability relative to habitat-wide fruit productivity, and by their tendency towards inter-individual fruiting synchrony. We predicted that the site of high chimpanzee density would support a higher density of food plant species tending to produce crops during periods of high habitat-wide productivity [high fruit abundance (HFA foods)] and of those tending to fruit synchronously among individuals during times of low habitat-wide availability (sLFA foods). The first food class should provide chimpanzees with a high nutrient density (and thus promote population growth), whereas the second should provide stable subsistence during lean periods and thus a temporally consistent resource base.
4.  Counter to our prediction, only sLFA resources were more abundant at the site of high chimpanzee density than at the site of low density. We suggest that sLFA resources are most important in influencing density of large-bodied frugivores.     

Direct Regulation of Prokaryotic Kir Channel by Cholesterol

Our earlier studies have shown that channel activity of Kir2 subfamily of inward rectifiers is strongly suppressed by the elevation of cellular cholesterol. The goal of this study is to determine whether cholesterol suppresses Kir channels directly. To achieve this goal, purified prokaryotic Kir (KirBac1.1) channels were incorporated into liposomes of defined lipid composition, and channel activity was assayed by ⁸⁶Rb⁺ uptake. Our results show that ⁸⁶Rb⁺ flux through KirBac1.1 is strongly inhibited by cholesterol. Incorporation of 5% (mass cholesterol/phospholipid) cholesterol into the liposome suppresses ⁸⁶Rb⁺ flux by >50%, and activity is completely inhibited at 12–15%. However, epicholesterol, a stereoisomer of cholesterol with similar physical properties, has significantly less effect on KirBac-mediated ⁸⁶Rb⁺ uptake than cholesterol. Furthermore, analysis of multiple sterols suggests that cholesterol-induced inhibition of KirBac1.1 channels is mediated by specific interactions rather than by changes in the physical properties of the lipid bilayer. In contrast to the inhibition of KirBac1.1 activity, cholesterol had no effect on the activity of reconstituted KscA channels (at up to 250 μg/mg of phospholipid). Taken together, these observations demonstrate that cholesterol suppresses Kir channels in a pure protein-lipid environment and suggest that the interaction is direct and specific.Inwardly rectifying potassium channels (Kir) are known to play critical roles in the regulation of multiple cellular functions including membrane excitability, heart rate, and vascular tone (1–3). Kir channels are classified into seven subfamilies (Kir1–7) identified by distinct biophysical properties and sensitivities to different regulators (2). Our earlier studies have shown that Kir2 channels, one of the major subfamilies of Kir that are responsible for maintaining membrane potential in a variety of cell types, are strongly suppressed by the elevation of membrane cholesterol (4, 5). Cholesterol-induced suppression of Kir2 was first observed in aortic endothelial cells (4), in which resting K⁺ conductance is dominated by Kir2.1 and Kir2.2 channels (6), and then when channels were heterologously expressed in Chinese hamster ovary cells (5, 7). Furthermore, the same effect was observed ex vivo in endothelial cells and bone marrow-derived progenitor cells isolated from hypercholesterolemic pigs (8, 9).In terms of the mechanism, the first insights came from comparing the effects of cholesterol and of its chiral analogue, epicholesterol. Although the two sterols are known to have almost identical effects on the biophysical properties of the lipid bilayer (10, 11), their impact on Kir activity is completely different; partial substitution of endogenous cholesterol with epicholesterol resulted in significant increase in Kir current in endothelial cells (4). These observations suggest that specific sterol-protein interactions may be involved in the cholesterol sensitivity of Kir2 channels. However, in the complex environment of the plasma membrane, cholesterol may interact not only with the channels themselves but also with other proteins, which in turn may regulate the activity of the channels. In the cellular environment, therefore, it is impossible to discriminate between direct channel-cholesterol interactions and indirect effects. Moreover, it is impossible to define the actual concentrations of cholesterol in any given membrane compartment. To quantitatively test direct cholesterol-protein interactions, it is necessary to examine sensitivity of pure Kir channels to membrane cholesterol in a membrane of defined lipid composition. To date, only the cytoplasmic domains of several mammalian Kir channels have been purified (Kir2.1, Kir3.1, and Kir3.2) (12–15). We therefore concentrate in this study on the effect of cholesterol on two bacterial K⁺ channels that differ in the level of their homology to mammalian Kir channels, KirBac1.1 and KcsA. KirBac channels have high sequence homology with mammalian Kirs (e.g. 52% homology between KirBac1.1 and Kir2.1; see Fig. 7A) and have now been extensively used as structural models of mammalian Kir channels (3, 16, 17). The sequence similarity between KcsA and mammalian K channels lies mainly in the transmembrane domain (18). The overall sequence homology of KcsA to mammalian Kir channels is relatively low (e.g. 22% homology between KcsA and Kir2.1; see Fig. 7A), with an entirely different cytoplasmic domain structure.Open in a separate windowFIGURE 7.Cholesterol has no effect on KcsA-mediated ⁸⁶Rb⁺ uptake. A, time courses of ⁸⁶Rb⁺ uptake into liposomes reconstituted with 50 μg of cholesterol/mg of PL and as compared with liposomes containing no cholesterol (control). Both batches of liposomes contained 5 μg of KcsA/mg of PL. Blank liposomes contain no protein. The points represent averages of three independent experiments (means ± S.D.). B, normalized time courses of ⁸⁶Rb⁺ uptake in liposomes incorporating 50, 150, and 250 μg of cholesterol/mg of PL. C, maximal uptake of ⁸⁶Rb⁺ after 240 s in liposomes containing 10, 25, 50, 100, 150, 200, and 250 μg of cholesterol/mg of PL normalized to control (means ± S.D. of 3–5 independent experiments; *, p < 0.05). DPM, disintegrations per minute.Here we show that, similarly to Kir2 channels, prokaryotic Kir channels incorporated into liposomes are strongly suppressed by an increase in membrane cholesterol. Furthermore, the sensitivity of prokaryotic Kir to cholesterol is stereo-selective to cholesterol optical analogues. In contrast, KscA channels are insensitive to membrane cholesterol. These observations suggest that cholesterol directly suppresses Kir channels.     

Differential Roles of Blocking Ions in KirBac1.1 Tetramer Stability

Potassium channels are tetrameric proteins that mediate K⁺-selective transmembrane diffusion. For KcsA, tetramer stability depends on interactions between permeant ions and the channel pore. We have examined the role of pore blockers on the tetramer stability of KirBac1.1. In 150 mm KCl, purified KirBac1.1 protein migrates as a monomer (∼40 kDa) on SDS-PAGE. Addition of Ba²⁺ (K_1/2 ∼ 50 μm) prior to loading results in an additional tetramer band (∼160 kDa). Mutation A109C, at a residue located near the expected Ba²⁺-binding site, decreased tetramer stabilization by Ba²⁺ (K_1/2 ∼ 300 μm), whereas I131C, located nearby, stabilized tetramers in the absence of Ba²⁺. Neither mutation affected Ba²⁺ block of channel activity (using ⁸⁶Rb⁺ flux assay). In contrast to Ba²⁺, Mg²⁺ had no effect on tetramer stability (even though Mg²⁺ was a potent blocker). Many studies have shown Cd²⁺ block of K⁺ channels as a result of cysteine substitution of cavity-lining M2 (S6) residues, with the implicit interpretation that coordination of a single ion by cysteine side chains along the central axis effectively blocks the pore. We examined blocking and tetramer-stabilizing effects of Cd²⁺ on KirBac1.1 with cysteine substitutions in M2. Cd²⁺ block potency followed an α-helical pattern consistent with the crystal structure. Significantly, Cd²⁺ strongly stabilized tetramers of I138C, located in the center of the inner cavity. This stabilization was additive with the effect of Ba²⁺, consistent with both ions simultaneously occupying the channel: Ba²⁺ at the selectivity filter entrance and Cd²⁺ coordinated by I138C side chains in the inner cavity.Potassium channels are expressed in many cell types and are key players in a wide range of physiological processes. One subset of potassium channels, the inward-rectifying potassium (Kir) channels, are functionally blocked by cytosolic cations such as Mg²⁺ and polyamines and contribute to the regulation of membrane excitability, cardiac rhythm, vascular tone, insulin release, and salt flow across epithelia (1–3). There are seven subfamilies of eukaryotic Kir channel genes. Among them, Kir1 encodes weak rectifiers, whereas Kir2 and Kir5 encode strong rectifiers; Kir3 encodes G-protein-regulated channels; and Kir6 encodes ATP-sensitive channels (4). Recently, a related bacterial family of genes (KirBac) has been identified (5, 6), and in 2003, the first member (KirBac1.1) was crystallized (7), providing a structural model for eukaryotic channels.The crystal structure of KirBac1.1 revealed a tetrameric pore structure similar to that seen in KcsA and a novel cytoplasmic domain (7, 8). The selectivity filter of both KirBac1.1 and KcsA consists of an extremely conserved pore loop followed by a central cavity, forming a transmembrane ion-selective permeation pore (7, 8). The linear arrangement of five oxygen rings (four from carbonyl oxygens and one from a Thr side chain) in the selectivity filter coordinates with ions, compensating for the energy barrier caused by K⁺ dehydration, thereby facilitating the rapid diffusion of K⁺ across the membrane (8–12). Two-thirds of the KirBac1.1 amino acid residues constitute the cytosolic domain that is highly conserved among the Kir subfamilies and form the cytosolic vestibule (13–16), which, together with the transmembrane pore, generates an 88-Å-long ion conduction pore (7).The prototypic potassium channel KcsA exists very stably as a tetramer, even in the harsh conditions of SDS-PAGE (17). In addition to protein-protein interaction between monomers, protein-lipid and protein-ion interactions play important roles in stabilizing the KcsA tetramer (17–20). The selectivity filter of KcsA, coordinated with K⁺ ions, can serve as a bridge between the four monomers to maintain the structure of the selectivity filter and the tetrameric architecture of the channel as a whole (11, 21). Blocking ions, such as Ba²⁺, also act as strong stabilizers (17). In the crystal structure of KcsA, Ba²⁺ occupies a site equivalent to the S4 K⁺-binding site within the selectivity filter (22). Other permeant ions (Rb⁺, Cs⁺, Tl⁺, and NH⁺₄) and strong blockers (Sr²⁺) can also contribute to the thermostability of the KcsA tetramer in SDS-PAGE (17). In contrast, impermeant ions such as Na⁺ and Li⁺ or weak blockers such as Mg²⁺ tend to destabilize the KcsA tetramer (17, 19).Like KcsA, KirBac1.1 purified using decylmaltoside or tridecylmaltoside is active and presumably stable as a tetramer in mild detergent solutions. However, in SDS-PAGE, KirBac1.1 migrates exclusively as a monomer (23). Because KcsA and KirBac1.1 are structurally similar in the transmembrane region of the pore, we hypothesized that permeant and blocking ions would also affect KirBac1.1 tetramer stability in SDS-PAGE. In the present work, the effects of blocking ions such as Ba²⁺ and Mg²⁺ on KirBac1.1 tetramer stability were examined to provide insight to the physical nature of their interaction with KirBac1.1, particularly in the selectivity filter and TM2 cavity. The data reveal important differences in the nature of the interaction of Mg²⁺ and Ba²⁺ with the channel as well as provide previously unavailable evidence for the nature of Cd²⁺ coordination within the channel.     

Tri-locus sequence data reject a “Gondwanan origin hypothesis” for the African/South Pacific crab genus Hymenosoma

Crabs of the family Hymenosomatidae are common in coastal and shelf regions throughout much of the southern hemisphere. One of the genera in the family, Hymenosoma, is represented in Africa and the South Pacific (Australia and New Zealand). This distribution can be explained either by vicariance (presence of the genus on the Gondwanan supercontinent and divergence following its break-up) or more recent transoceanic dispersal from one region to the other. We tested these hypotheses by reconstructing phylogenetic relationships among the seven presently-accepted species in the genus, as well as examining their placement among other hymenosomatid crabs, using sequence data from two nuclear markers (Adenine Nucleotide Transporter [ANT] exon 2 and 18S rDNA) and three mitochondrial markers (COI, 12S and 16S rDNA). The five southern African representatives of the genus were recovered as a monophyletic lineage, and another southern African species, Neorhynchoplax bovis, was identified as their sister taxon. The two species of Hymenosoma from the South Pacific neither clustered with their African congeners, nor with each other, and should therefore both be placed into different genera. Molecular dating supports a post-Gondwanan origin of the Hymenosomatidae. While long-distance dispersal cannot be ruled out to explain the presence of the family Hymenosomatidae on the former Gondwanan land-masses and beyond, the evolutionary history of the African species of Hymenosoma indicates that a third means of speciation may be important in this group: gradual along-coast dispersal from tropical towards temperate regions, with range expansions into formerly inhospitable habitat during warm climatic phases, followed by adaptation and speciation during subsequent cooler phases.     

Enhanced atherothrombotic formation after oxidative injury by FeCl3 to the common carotid artery in severe combined hyperlipidemic mice

Enhanced susceptibility to atherosclerosis from severe hypertriglyceridemia (HTG) resulting from lipoprotein lipase (LPL) deficiency has been demonstrated in our recent findings which employed a unique mouse model. In the present study we provide further evidence that severe HTG due to LPL deficiency also promotes an atherothrombotic response to arterial injury induced by ferric chloride in a severe combined hyperlipidemic mouse model. Methods and results: A mouse model (LPL^−/−XApoE^−/− double knockout, DKO) with severe combined hyperlipidemia was established by crossing ApoE and LPL-deficient mice. The common carotid arteries of ApoE knockout (EKO) and DKO mice were subjected to injury by ferric chloride, and the formation of arterial thrombosis together with various markers were compared in these lesions. DKO mice demonstrated significantly enhanced thrombus formation overlying atherosclerotic plaque after injury, which contained smooth muscle cells, macrophages, and neutral lipid. The area of neointima, mean intima/media ratios, and the percentage of luminal stenosis were significantly greater (P < 0.01) in DKO mice. Compared with EKO mice, the expression of von Willebrand factor (vWF) and plasminogen activator inhibitor type 1 (PAI-1) were increased in DKO mice. Conclusions: Severe combined hyperlipidemia promotes thrombosis after ferric chloride injury to atherosclerotic vessels and HTG plays a major role in the process.