ANGPTL3 blockade with a human monoclonal antibody reduces plasma lipids in dyslipidemic mice and monkeys

Angiopoietin-like protein 3 (ANGPTL3) is a circulating protein synthesized exclusively in the liver that inhibits LPL and endothelial lipase (EL), enzymes that hydrolyze TGs and phospholipids in plasma lipoproteins. Here we describe the development and testing of a fully human monoclonal antibody (REGN1500) that binds ANGPTL3 with high affinity. REGN1500 reversed ANGPTL3-induced inhibition of LPL activity in vitro. Intravenous administration of REGN1500 to normolipidemic C57Bl/6 mice increased LPL activity and decreased plasma TG levels by ≥50%. Chronic administration of REGN1500 to dyslipidemic C57Bl/6 mice for 8 weeks reduced circulating plasma levels of TG, LDL-cholesterol (LDL-C), and HDL-cholesterol (HDL-C) without any changes in liver, adipose, or heart TG contents. Studies in EL knockout mice revealed that REGN1500 reduced serum HDL-C through an EL-dependent mechanism. Finally, administration of a single dose of REGN1500 to dyslipidemic cynomolgus monkeys caused a rapid and pronounced decrease in plasma TG, nonHDL-C, and HDL-C. REGN1500 normalized plasma TG levels even in monkeys with a baseline plasma TG greater than 400 mg/dl. Collectively, these data demonstrate that neutralization of ANGPTL3 using REGN1500 reduces plasma lipids in dyslipidemic mice and monkeys, and thus provides a potential therapeutic agent for treatment of patients with hyperlipidemia.     

Specific Activation of the Plant P-type Plasma Membrane H+-ATPase by Lysophospholipids Depends on the Autoinhibitory N- and C-terminal Domains

Eukaryotic P-type plasma membrane H⁺-ATPases are primary active transport systems that are regulated at the post-translation level by cis-acting autoinhibitory domains, which can be relieved by protein kinase-mediated phosphorylation or binding of specific lipid species. Here we show that lysophospholipids specifically activate a plant plasma membrane H⁺-ATPase (Arabidopsis thaliana AHA2) by a mechanism that involves both cytoplasmic terminal domains of AHA2, whereas they have no effect on the fungal counterpart (Saccharomyces cerevisiae Pma1p). The activation was dependent on the glycerol backbone of the lysophospholipid and increased with acyl chain length, whereas the headgroup had little effect on activation. Activation of the plant pump by lysophospholipids did not involve the penultimate residue, Thr-947, which is known to be phosphorylated as part of a binding site for activating 14-3-3 protein, but was critically dependent on a single autoinhibitory residue (Leu-919) upstream of the C-terminal cytoplasmic domain in AHA2. A corresponding residue is absent in the fungal counterpart. These data indicate that plant plasma membrane H⁺-ATPases evolved as specific receptors for lysophospholipids and support the hypothesis that lysophospholipids are important plant signaling molecules.     

Metal Fluoride Inhibition of a P-type H+ Pump: STABILIZATION OF THE PHOSPHOENZYME INTERMEDIATE CONTRIBUTES TO POST-TRANSLATIONAL PUMP ACTIVATION*

The plasma membrane H⁺-ATPase is a P-type ATPase responsible for establishing electrochemical gradients across the plasma membrane in fungi and plants. This essential proton pump exists in two activity states: an autoinhibited basal state with a low turnover rate and a low H⁺/ATP coupling ratio and an activated state in which ATP hydrolysis is tightly coupled to proton transport. Here we characterize metal fluorides as inhibitors of the fungal enzyme in both states. In contrast to findings for other P-type ATPases, inhibition of the plasma membrane H⁺-ATPase by metal fluorides was partly reversible, and the stability of the inhibition varied with the activation state. Thus, the stability of the ATPase inhibitor complex decreased significantly when the pump transitioned from the activated to the basal state, particularly when using beryllium fluoride, which mimics the bound phosphate in the E2P conformational state. Taken together, our results indicate that the phosphate bond of the phosphoenzyme intermediate of H⁺-ATPases is labile in the basal state, which may provide an explanation for the low H⁺/ATP coupling ratio of these pumps in the basal state.     

The sterol-binding protein Kes1/Osh4p is a regulator of polarized exocytosis

Oxysterol-binding protein (OSBP)-related protein Kes1/ Osh4p is implicated in nonvesicular sterol transfer between membranes in Saccharomyces cerevisiae. However, we found that Osh4p associated with exocytic vesicles that move from the mother cell into the bud, where Osh4p facilitated vesicle docking by the exocyst tethering complex at sites of polarized growth on the plasma membrane. Osh4p formed complexes with the small GTPases Cdc42p, Rho1p and Sec4p, and the exocyst complex subunit Sec6p, which was also required for Osh4p association with vesicles. Although Osh4p directly affected polarized exocytosis, its role in sterol trafficking was less clear. Contrary to what is predicted for a sterol-transfer protein, inhibition of sterol binding by the Osh4p Y97F mutation did not cause its inactivation. Rather, OSH4(Y97F) is a gain-of-function mutation that causes dominant lethality. We propose that in response to sterol binding and release Osh4p promotes efficient exocytosis through the co-ordinate regulation of Sac1p, a phosphoinositide 4-phosphate (PI4P) phosphatase, and the exocyst complex. These results support a model in which Osh4p acts as a sterol-dependent regulator of polarized vesicle transport, as opposed to being a sterol-transfer protein.     

A postmortem study of the corpus callosum in the common minke whale (Balaenoptera acutorostrata)

Cetaceans diverged from terrestrial mammals approximately 53 mya and have evolved independently since then. During this time period, they have developed a complex nervous system with many adaptations to the marine environment. This study used stereological methods to estimate the total number and diameter of the myelinated fibers in the corpus callosum of the common minke whale (Balaenoptera acutorostrata) (n= 4). The total number of callosal fibers was estimated to 55.3 × 10⁶ (range: 49.0 × 10⁶–59.1 × 10⁶). Despite large variations of the callosal area (350–950 mm²), there was little variation in total fiber number. The fibers with diameters ranging from 0.822 to 1.14 μm were the most frequent, which is similar to results obtained in the human brain using the same method. There was no systematic distribution of large‐, middle‐, or small‐sized fibers along the rostrocaudal axis of the corpus callosum. This study indicated that the corpus callosum of the common minke whale is small and has few fibers compared to terrestrial mammals.     

Getting the measure of eutrophication in the Baltic Sea: towards improved assessment principles and methods

The eutrophication status of the entire Baltic Sea is classified using a multi-metric indicator-based assessment tool. A total of 189 areas are assessed using indicators where information on reference conditions (RefCon), and acceptable deviation (AcDev) from reference condition could be combined with national monitoring data from the period 2001?C2006. Most areas (176) are classified as ??affected by eutrophication?? and only two open water areas and 11 coastal areas are classified as ??unaffected by eutrophication??. The classification is made by application of the recently developed HELCOM Eutrophication Assessment Tool (HEAT), which is described in this paper. The use of harmonized assessment principles and the HEAT tool allows for direct comparisons between different parts of the Baltic Sea despite variations in monitoring activities. The impaired status of 176 areas is directly related to nutrient enrichment and elevated loads from upstream catchments. Baltic Sea States have implemented nutrient management strategies since years which have reduced nutrient inputs. However, eutrophication is still a major problem for large parts of the Baltic Sea. The 2007 Baltic Sea Action Plan is projected to further reduce nutrient inputs aiming for a Baltic Sea unaffected by eutrophication by 2021.     

Enzyme kinetic studies of histone demethylases KDM4C and KDM6A: towards understanding selectivity of inhibitors targeting oncogenic histone demethylases

To investigate ligand selectivity between the oncogenic KDM4C and tumor repressor protein KDM6A histone demethylases, KDM4C and KDM6A were enzymatically characterized, and subsequently, four compounds were tested for inhibitory effects. 2,4-dicarboxypyridine and (R)-N-oxalyl-O-benzyltyrosine (3) are both known to bind to a close KDM4C homolog and 3 binds in the part of the cavity that accommodates the side chain in position 11 of histone 3. The inhibition measurements showed significant selectivity between KDM4C and KDM6A. This demonstrates that despite very similar active site topologies, selectivity between Jumonji family histone demethylases can be obtained even with small molecule ligands.