Molecular basis for the differences in lipid and lipoprotein binding properties of human apolipoproteins E3 and E4

Human apolipoprotein (apo) E4 binds preferentially to very low-density lipoproteins (VLDLs), whereas apoE3 binds preferentially to high-density lipoproteins (HDLs), resulting in different plasma cholesterol levels for the two isoforms. To understand the molecular basis for this effect, we engineered the isolated apoE N-terminal domain (residues 1-191) and C-terminal domain (residues 192-299) together with a series of variants containing deletions in the C-terminal domain and assessed their lipid and lipoprotein binding properties. Both isoforms can bind to a phospholipid (PL)-stabilized triolein emulsion, and residues 261-299 are primarily responsible for this activity. ApoE4 exhibits better lipid binding ability than apoE3 as a consequence of a rearrangement involving the segment spanning residues 261-272 in the C-terminal domain. The strong lipid binding ability of apoE4 coupled with the VLDL particle surface being ～60% PL-covered is the basis for its preference for binding VLDL rather than HDL. ApoE4 binds much more strongly than apoE3 to VLDL but less strongly than apoE3 to HDL(3), consistent with apoE-lipid interactions being relatively unimportant for binding to HDL. The preference of apoE3 for binding to HDL(3) arises because binding is mediated primarily by interaction of the N-terminal helix bundle domain with the resident apolipoproteins that cover ～80% of the HDL(3) particle surface. Thus, the selectivity in the binding of apoE3 and apoE4 to HDL(3) and VLDL is dependent upon two factors: (1) the stronger lipid binding ability of apoE4 relative to that of apoE3 and (2) the differences in the nature of the surfaces of VLDL and HDL(3) particles, with the former being largely covered with PL and the latter with protein.     

Deuterium kinetic isotope effects in heterotetrameric sarcosine oxidase from Corynebacterium sp. U-96: the anionic form of the substrate in the enzyme-substrate complex is a reactive species

Heterotetrameric sarcosine oxidase is a flavoprotein that catalyses the oxidative demethylation of sarcosine. It is thought that the dehydrogenated substrate is the anionic form of sarcosine. To verify this assumption, the rate of flavin-adenine dinucleotide (FAD) reduction (k(red)) was analysed using protiated and deuterated sarcosine (N-methyl-d(3)-Gly) at various pH values using stopped-flow method. By increasing the pH from 6.2 to 9.8, k(red) increased for both substrates and reached a plateau, but the pK(a) value (reflecting the ionization of the enzyme-substrate complex) was 6.8 and 7.1 for protiated and deuterated sarcosine, respectively, and the kinetic isotope effect of k(red) decreased from approximately 19 to 8, indicating deprotonation of the bound sarcosine. The k(red)/K(d) (K(d), sarcosine dissociation constant) increased with increasing pH and reached a plateau. The pK (reflecting the ionization of free enzyme or free sarcosine) was 7.0 for both substrates, suggesting deprotonation of the βLys358 residue, which has a pK(a) of 6.7, as the pK(a) of the free sarcosine amine proton was determined to be approximately 10.1. These results indicate that the amine proton of sarcosine is transferred to the unprotonated Lys residue in the enzyme-substrate complex.     

Land-use intensification systematically alters the size structure of aquatic communities in the Neotropics

Land-use and land-cover transitions can affect biodiversity and ecosystem functioning in a myriad of ways, including how energy is transferred within food-webs. Size spectra (i.e. relationships between body size and biomass or abundance) provide a means to assess how food-webs respond to environmental stressors by depicting how energy is transferred from small to larger organisms. Here, we investigated changes in the size spectrum of aquatic macroinvertebrates along a broad land-use intensification gradient (from Atlantic Forest to mechanized agriculture) in 30 Brazilian streams. We expected to find a steeper size spectrum slope and lower total biomass in more disturbed streams due to higher energetic expenditure in physiologically stressful conditions, which has a disproportionate impact on large individuals. As expected, we found that more disturbed streams had fewer small organisms than pristine forest streams, but, surprisingly, they had shallower size spectrum slopes, which indicates that energy might be transferred more efficiently in disturbed streams. Disturbed streams were also less taxonomically diverse, suggesting that the potentially higher energy transfer in these webs might be channelled via a few efficient trophic links. However, because total biomass was higher in pristine streams, these sites still supported a greater number of larger organisms and longer food chains (i.e. larger size range). Our results indicate that land-use intensification decreases ecosystem stability and enhances vulnerability to population extinctions by reducing the possible energetic pathways while enhancing efficiency between the remaining food-web linkages. Our study represents a step forward in understanding how land-use intensification affects trophic interactions and ecosystem functioning in aquatic systems.     

Ca2+-dependent nuclear contraction in the heliozoon Actinophrys sol

Ca2+-dependent contractility was found to exist in the nucleus of the heliozoon protozoan Actinophrys sol. Upon addition of Ca2+ ([Ca2+]free = 2.0 x 10(-3) M), diameters of isolated and detergent-extracted nuclei became reduced from 16.5+/-1.7 microm to 11.0+/-1.3 microm. The threshold level of [Ca2+]free for the nuclear contraction was 2.9 x 10(-7) M. The nuclear contraction was not induced by Mg2+, and was not inhibited by colchicine or cytochalasin B. Contracted nuclei became expanded when Ca2+ was removed by EGTA; thus cycles of contraction and expansion could be repeated many times by alternating addition of Ca2+ and EGTA. The Ca2+-dependent nuclear contractility remained even after high salt treatment, suggesting a possible involvement of nucleoskeletal components in the nuclear contraction. Electron microscopy showed that, in the relaxed state, filamentous structures were observed to spread in the nucleus to form a network. After addition of Ca2+, they became aggregated and constructed a mass of thicker filaments, followed by re-distribution of the filaments spread around inside of the nucleus when Ca2+ was removed. These results suggest that the nuclear contraction is induced by Ca2+-dependent transformation of the filamentous structures in the nucleus.     

Home range structure and inter-group competition for land of Japanese macaques in evergreen and deciduous forests

The per capita home range area of Japanese macaques,Macaca fuscata, is significantly smaller in evergreen forest than in deciduous forest, though a corresponding difference in food resource utilization patterns has never been described. The present study compared the home range utilization pattern of Japanese macaques living in two habitats: the Yakushima population inhabits an evergreen forest, while the Kinkazan population inhabits a deciduous forest. We found that in the Yakushima population, (1) food density was higher; (2) inter-feeding bout sites distance was shorter; (3) daily travel distance was shorter; (4) home range size was smaller; and (5) the unit value of the main home range was higher, than in the Kinkazan population. Yakushima groups utilized a small home range area intensively, compared to Kinkazan groups. We also found that a Yakushima group shared 24% of its main home range with neighboring groups, though a Kinkazan group shared only 10% with other groups. It is supposed that food distribution affects daily ranging pattern, and ultimately the social relationships between groups in Japanese macaques.     

Role of side-edge site of sphingomyelinase from Bacillus cereus

Bacillus cereus sphingomyelinase (Bc-SMase) belongs to the Mg(2+)-dependent neutral sphingomyelinase (nSMase) which hydrolyzes sphingomyelin (SM) to produce phosphocholine and ceramide, and acts as an extracellular hemolysin. Bc-SMase has two metal ion-binding sites in a long horizontal cleft across the molecule, with one Mg(2+) in the central region of the cleft and one divalent metal ion at the side-edge of the cleft. The role of the Mg(2+) at the side-edge of the long horizontal cleft in Bc-SMase remains unresolved. The replacement of Asn-57, Glu-99, and Asp-100 located in close proximity to Mg(2+) at the side-edge with alanine resulted in a striking reduction in binding to and hydrolysis of sphingomyelin in membranes of sheep erythrocytes or SM-liposomes but that of Phe-55 did not. However, the replacement of these residues had little effect on the enzymatic activity. N57A, E99A, and D100A contained 2 mol of Mg(2+) per mol of protein, and the wild type and F55A contained 3 mol. A crystal analysis showed that N57A with Mg(2+) had no metal ion at the side-edge. These results indicate that the Mg(2+) at the side-edge of Bc-SMase plays an important role in the binding to membranes.     

24(S)-hydroxycholesterol induces neuronal cell death through necroptosis, a form of programmed necrosis

24(S)-Hydroxycholesterol (24S-OHC) produced by cholesterol 24-hydroxylase expressed mainly in neurons plays an important physiological role in the brain. Conversely, it has been reported that 24S-OHC possesses potent cytotoxicity. The molecular mechanisms of 24S-OHC-induced cell death have not yet been fully elucidated. In this study, using human neuroblastoma SH-SY5Y cells and primary cortical neuronal cells derived from rat embryo, we characterized the form of cell death induced by 24S-OHC. SH-SY5Y cells treated with 24S-OHC exhibited neither fragmentation of the nucleus nor caspase activation, which are the typical characteristics of apoptosis. 24S-OHC-treated cells showed necrosis-like morphological changes but did not induce ATP depletion, one of the features of necrosis. When cells were treated with necrostatin-1, an inhibitor of receptor-interacting serine/threonine kinase 1 (RIPK1) required for necroptosis, 24S-OHC-induced cell death was significantly suppressed. The knockdown of RIPK1 by transfection of small interfering RNA of RIPK1 effectively attenuated 24S-OHC-induced cell death. It was found that neither SH-SY5Y cells nor primary cortical neuronal cells expressed caspase-8, which was regulated for RIPK1-dependent apoptosis. Collectively, these results suggest that 24S-OHC induces neuronal cell death by necroptosis, a form of programmed necrosis.