Endothelin receptor type A expression defines a distinct cardiac subdomain within the heart field and is later implicated in chamber myocardium formation

The avian and mammalian heart originates from two distinct embryonic regions: an early differentiating first heart field and a dorsomedially located second heart field. It remains largely unknown when and how these subdivisions of the heart field divide into regions with different fates. Here, we identify in the mouse a subpopulation of the first (crescent-forming) field marked by endothelin receptor type A (Ednra) gene expression, which contributes to chamber myocardium through a unique type of cell behavior. Ednra-lacZ/EGFP-expressing cells arise in the ventrocaudal inflow region of the early linear heart tube, converge to the midline, move anteriorly along the outer curvature and give rise to chamber myocardium mainly of the left ventricle and both atria. This movement was confirmed by fluorescent dye-labeling and transplantation experiments. The Ednra-lacZ/EGFP-expressing subpopulation is characterized by the presence of Tbx5-expressing cells. Ednra-null embryonic hearts often demonstrate hypoplasia of the ventricular wall, low mitotic activity and decreased Tbx5 expression with reciprocal expansion of Tbx2 expression. Conversely, endothelin 1 stimulates ERK phosphorylation and Tbx5 expression in the early embryonic heart. These results indicate that early Ednra expression defines a subdomain of the first heart field contributing to chamber formation, in which endothelin 1/Ednra signaling is involved. The present finding provides an insight into how subpopulations within the crescent-forming (first) heart field contribute to the coordination of heart morphogenesis through spatiotemporally defined cell movements.     

C-type lectins do not act as functional receptors for filovirus entry into cells

Cellular C-type lectins have been reported to facilitate filovirus infection by binding to glycans on filovirus glycoprotein (GP). However, it is not clearly known whether interaction between C-type lectins and GP mediates all the steps of virus entry (i.e., attachment, internalization, and membrane fusion). In this study, we generated vesicular stomatitis viruses pseudotyped with mutant GPs that have impaired structures of the putative receptor binding regions and thus reduced ability to infect the monkey kidney cells that are routinely used for virus propagation. We found that infectivities of viruses with the mutant GPs dropped in C-type lectin-expressing cells, parallel with those in the monkey kidney cells, whereas binding activities of these GPs to the C-type lectins were not correlated with the reduced infectivities. These results suggest that C-type lectin-mediated entry of filoviruses requires other cellular molecule(s) that may be involved in virion internalization or membrane fusion.     

Nifedipine prevents vascular endothelial dysfunction in a mouse model of obesity and type 2 diabetes, by improving eNOS dysfunction and dephosphorylation

The effect of calcium channel blockers (CCBs) on type 2 diabetes is still unclear. The present study was undertaken to examine the efficacy of nifedipine, a dihydropyridine CCB, on obesity, glucose intolerance and vascular endothelial dysfunction in db/db mice (a mouse model of obesity and type 2 diabetes). db/db mice, fed high-fat diet (HFD) were treated with vehicle, nifedipine (10 mg kg(-1) day(-1)) or hydralazine (5 mg kg(-1) day(-1)) for 4 weeks, and the protective effects were compared. Although nifedipine and hydralazine exerted similar blood pressure lowering in db/db mice, neither affected body weight, fat weight, and glucose intolerance of db/db mice. However, nifedipine, but not hydralazine, significantly improved vascular endothelial function in db/db mice, being accompanied by more attenuation of vascular superoxide by nifedipine than hydralazine. These protective effects of nifedipine were attributed to the attenuation of eNOS uncoupling as shown by the prevention of vascular endothelial nitric oxide synthase (eNOS) dimer disruption, and the prevention of dihydrofolate reductase (DHFR) downregulation, the key enzyme responsible for eNOS uncoupling. Moreover, nifedipine, but not hydralazine, significantly prevented the decreases in phosphorylation of vascular akt and eNOS in db/db mice. Our work provided the first evidence that nifedipine prevents vascular endothelial dysfunction, through the inhibition of eNOS uncoupling and the enhancement of eNOS phosphorylation, independently of blood pressure-lowering effect. We propose that nifedipine may be a promising therapeutic agent for cardiovascular complications in type 2 diabetes.     

Analysis of the bacterial luciferase mobile loop by replica-exchange molecular dynamics

Bacterial luciferase contains an extended 29-residue mobile loop. Movements of this loop are governed by binding of either flavin mononucleotide (FMNH₂) or polyvalent anions. To understand this process, loop dynamics were investigated using replica-exchange molecular dynamics that yielded conformational ensembles in either the presence or absence of FMNH₂. The resulting data were analyzed using clustering and network analysis. We observed the closed conformations that are visited only in the simulations with the ligand. Yet the mobile loop is intrinsically flexible, and FMNH₂ binding modifies the relative populations of conformations. This model provides unique information regarding the function of a crystallographically disordered segment of the loop near the binding site. Structures at or near the fringe of this network were compatible with flavin binding or release. Finally, we demonstrate that the crystallographically observed conformation of the mobile loop bound to oxidized flavin was influenced by crystal packing. Thus, our study has revealed what we believe are novel conformations of the mobile loop and additional context for experimentally determined structures.     

<Emphasis Type="Italic">Dioscorea opposita</Emphasis> Thunb. α-mannosidase belongs to the glycosyl hydrolase family 38

α-Mannosidase (EC 3.2.1.24) was purified from ‘Iseimo’, a native variety of Dioscorea opposita Thunb. Before purification, we investigated the composition of a viscous polysaccharide that interferes with column chromatography procedures. The polysaccharide consisted mainly of mannose, and also contained uronic acid. We used the cationic detergent cetylpyridinium chloride (CPC) to remove the polysaccharide. CPC treatment decreased viscosity without affecting α-mannosidase activity. We purified α-mannosidase 2,650-fold. The optimal pH of the enzyme was 6.0 and the optimum temperature was 65°C. The K _m value for p-nitrophenyl-α-d-mannopyranoside was 0.35 ± 0.03 mM. Activity was inhibited by swainsonine but not kifunensine. The enzyme cleaved α-1,2 linkages preferentially to α-1,6 and α-1,3 linkages. The M _r of purified α-mannosidase was estimated to be 250–260 kDa by gel filtration and native-PAGE. Four polypeptides (86, 83, 80, and 28 kDa) were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is unclear whether the polypeptides are encoded by one gene or multiple genes. However, N-terminal sequence analysis suggested that post-translational cleavage and/or glycosylation resulted in the three large fragments, if these amino acids were encoded by the same gene. Homology searches and characterization of the enzyme’s properties indicated that Iseimo α-mannosidase belongs to the glycoside hydrolase family 38 proteins, and to the Class II mannosidase group.     

Soil and stream water acidification in a forested catchment in central Japan

Rapid industrialization in East Asia is causing adverse effects due to atmospheric deposition in terrestrial and freshwater ecosystems. Decreasing stream pH and alkalinity and increasing NO₃ ^? concentrations were observed throughout the 1990s in the forested Lake Ijira catchment in central Japan. We investigated these changes using data on atmospheric deposition, soil chemistry, stream water chemistry, and forest growth. Average atmospheric depositions (wet + dry) of 0.83, 0.57, and 1.37 kmol ha^?1 year^?1 for hydrogen, sulfur, and nitrogen, respectively, were among the highest levels in Japan. Atmospheric deposition generally decreased before 1994 and increased thereafter. The catchment was acid-sensitive; stream alkalinity was low (134 μmol_c l^?1) and pH in surface mineral soils decreased from 4.5 in 1990 to 3.9 in 2003. Stream NO₃ ^? concentration nearly doubled (from 22 to 42 μmol_c l^?1) from the late 1980s to the early 2000s. Stream NO₃ ^? concentration was controlled primarily by water temperature before 1996/1997 and by stream discharge thereafter. Stream NO₃ ^? concentrations decreased during the growing season before 1996/1997, but this seasonality was lost thereafter. The catchment became nitrogen-saturated (changing from stage 1 to 2) in 1996/1997, possibly because of declining forest growth rates due to the 1994 summer drought, defoliation of Japanese red pine by pine wilt disease, maturation of Japanese cedar stands, and stimulation of nitrogen mineralization and nitrification due to alkalinization of soils (increased exchangeable Ca²⁺ and soil pH) after the summer drought. Stream pH and alkalinity began decreasing in 1996/1997. The enhanced growing-season NO₃ ^? discharge since 1996/1997 appeared to be the major cause of stream acidification. Increased atmospheric deposition since 1994 may have contributed to this change.     

The AAA+ ATPase ATAD3A Controls Mitochondrial Dynamics at the Interface of the Inner and Outer Membranes

Dynamic interactions between components of the outer (OM) and inner (IM) membranes control a number of critical mitochondrial functions such as channeling of metabolites and coordinated fission and fusion. We identify here the mitochondrial AAA⁺ ATPase protein ATAD3A specific to multicellular eukaryotes as a participant in these interactions. The N-terminal domain interacts with the OM. A central transmembrane segment (TMS) anchors the protein in the IM and positions the C-terminal AAA⁺ ATPase domain in the matrix. Invalidation studies in Drosophila and in a human steroidogenic cell line showed that ATAD3A is required for normal cell growth and cholesterol channeling at contact sites. Using dominant-negative mutants, including a defective ATP-binding mutant and a truncated 50-amino-acid N-terminus mutant, we showed that ATAD3A regulates dynamic interactions between the mitochondrial OM and IM sensed by the cell fission machinery. The capacity of ATAD3A to impact essential mitochondrial functions and organization suggests that it possesses unique properties in regulating mitochondrial dynamics and cellular functions in multicellular organisms.Mitochondria not only supply cells with the bulk of their ATP but also contribute to the fine regulation of metabolism, calcium homeostasis, and apoptosis (27). Coordination of these functions is dependent on the dynamic nature of mitochondria (5). These organelles constantly fuse and divide to form small spheres, short rods, or long tubules and are actively transported to specific subcellular locations. These processes are essential for mammalian development, and defects can lead to degenerative diseases and cancers (9, 17). In eukaryotes, these organellar gymnastics are controlled by numerous pathways that preserve proper mitochondrial morphology and function (30, 45). The best-understood mitochondrial process is the fusion and fission pathways, which rely on conserved GTPases, and their binding partners to regulate organelle connectivity (10, 18, 45). There are also evidences that dynamic interactions between the outer membrane (OM) and inner membrane (IM) exist for coordinated fusion and fission, channeling of metabolites, and protein transport, but proteins playing a role in these interactions have yet to be identified (34). In the present study, we provide a detailed biochemical and functional characterization of the mitochondrial AAA⁺ ATPase ATAD3A protein that is present exclusively in multicellular eukaryotes and which participates in the control of mitochondrial dynamics at the interface between the IMs and OMs. Proteins related to the Atad3A genes have been previously identified in proteomic surveys of mouse brain mitochondria (28) and liver mitochondrial inner membrane (8), as mitochondrial DNA-binding proteins (4, 21, 44) and as nuclear mRNA-associated proteins (6). The Atad3A protein has also been identified as a cell surface antigen in some human tumors (16). Functional genomics identified the Drosophila Atad3A ortholog (bor) as a major gene positively regulated by the TOR (for target of rapamycin) signaling pathway involved in cell growth and division (19). In our laboratory, we identified ATAD3A as a specific target for the Ca²⁺/Zn²⁺-binding S100B protein (B. Gilquin et al., unpublished data). We here show that ATAD3A is anchored into the mitochondrial IM at contact sites with the OM. The N-terminal domain of ATAD3A interacts with the inner surface of the OM and its C-terminal AAA ATPase domain localizes in a specific matrix compartment. Thanks to its simultaneous interaction with two membranes, ATAD3A regulates mitochondrial dynamics at the interface between the IMs and OMs and controls diverse cell responses ranging from cell growth, channeling of cholesterol, and mitochondrial fission.     

S-Benzylisothiourea derivatives as small-molecule inhibitors of indoleamine-2,3-dioxygenase

S-Benzylisothiourea 3a was discovered by its ability to inhibit indoleamine-2,3-dioxygenase (IDO) in our screening program. Subsequent optimization of the initial hit 3a lead to the identification of sub-μM inhibitors 3r and 10h, both of which suppressed kynurenine production in A431 cells. Synthesis and structure–activity relationship of S-benzylisothiourea analogues as small-molecule inhibitors of IDO are described.     

Synthesis and evaluation of opioid receptor-binding affinity of elaeocarpenine and its analogs

Both enantiomers of elaeocarpenine (1) and its analogs, 21, 22, 25, and 27, were synthesized from bicyclic aldehydes 8–10 via a flexible route previously established for total synthesis of grandisines, and their binding affinities for μ-, κ- and δ-opioid receptor subtypes were evaluated. We found that (9R)-1 exhibited higher affinity than (9S)-1 for all the subtypes, but the enantiomers showed little subtype selectivity. Analogs 21 having a pyrrolizidine skeleton and 27 having a stemona-type skeleton in place of the indolizidine unit of (9S)-1 showed μ-selective and μ-, κ-selective binding, respectively.