Additional materials of Myanmarpithecus yarshensis (Amphipithecidae, Primates) from the middle Eocene Pondaung Formation

Myanmarpithecus yarshensis is an amphipithecid primate from the middle Eocene Pondaung Formation in Myanmar. It was previously known based on maxillary fragments with P⁴–M³ and mandibular fragments with C–P₃ and M_2–3. This study reports new materials for the genus, including a humeral head fragment, a lingual fragment of the right M², a lingual fragment of the right M³, and a left I¹. These new materials were collected from approximately the same point, and likely belonged to the same individual. The upper molar morphology and size of the new materials show similarity to those of the type specimen, indicating that the new materials can be assigned to M. yarshensis. The humeral head is the first postcranial element that is associated with dental materials for amphipithecids. The morphological similarity between the previously reported larger humerus and this specimen confirms the assignment of the former specimen to Amphipithecidae and suggests common locomotor adaptations in the family. The upper central incisor is large relative to the molar fragments, but is within the variation among extant platyrrhines. The tooth is spatulate-shaped and high crowned, and lacks the mesial process, indicating similarity to I¹ of haplorhines and clear differences from that of adapoids. It has been suggested that amphipithecids, including Myanmarpithecus, have affinities with notharctine adapoids, but the morphology of I¹ does not support the notharctine hypothesis of the Amphipithecidae.     

Molecular Basis of the Recognition of Nephronectin by Integrin ��8��1

Integrin α8β1 interacts with a variety of Arg-Gly-Asp (RGD)-containing ligands in the extracellular matrix. Here, we examined the binding activities of α8β1 integrin toward a panel of RGD-containing ligands. Integrin α8β1 bound specifically to nephronectin with an apparent dissociation constant of 0.28 ± 0.01 nm, but showed only marginal affinities for fibronectin and other RGD-containing ligands. The high-affinity binding to α8β1 integrin was fully reproduced with a recombinant nephronectin fragment derived from the RGD-containing central “linker” segment. A series of deletion mutants of the recombinant fragment identified the LFEIFEIER sequence on the C-terminal side of the RGD motif as an auxiliary site required for high-affinity binding to α8β1 integrin. Alanine scanning mutagenesis within the LFEIFEIER sequence defined the EIE sequence as a critical motif ensuring the high-affinity integrin-ligand interaction. Although a synthetic LFEIFEIER peptide failed to inhibit the binding of α8β1 integrin to nephronectin, a longer peptide containing both the RGD motif and the LFEIFEIER sequence was strongly inhibitory, and was ∼2,000-fold more potent than a peptide containing only the RGD motif. Furthermore, trans-complementation assays using recombinant fragments containing either the RGD motif or LFEIFEIER sequence revealed a clear synergism in the binding to α8β1 integrin. Taken together, these results indicate that the specific high-affinity binding of nephronectin to α8β1 integrin is achieved by bipartite interaction of the integrin with the RGD motif and LFEIFEIER sequence, with the latter serving as a synergy site that greatly potentiates the RGD-driven integrin-ligand interaction but has only marginal activity to secure the interaction by itself.Integrins are a family of adhesion receptors that interact with a variety of extracellular ligands, typically cell-adhesive proteins in the extracellular matrix (ECM).² They play mandatory roles in embryonic development and the maintenance of tissue architectures by providing essential links between cells and the ECM (1). Integrins are composed of two non-covalently associated subunits, termed α and β. In mammals, 18 α and 8 β subunits have been identified, and combinations of these subunits give rise to at least 24 distinct integrin heterodimers. Based on their ligand-binding specificities, ECM-binding integrins are classified into three groups, namely laminin-, collagen- and RGD-binding integrins (2, 3), of which the RGD-binding integrins have been most extensively investigated. The RGD-binding integrins include α5β1, α8β1, αIIbβ3, and αV-containing integrins, and have been shown to interact with a variety of ECM ligands, such as fibronectin and vitronectin, with distinct binding specificities.The α8 integrin subunit was originally identified in chick nerves (4). Integrin α8β1 is expressed in the metanephric mesenchyme and plays a crucial role in epithelial-mesenchymal interactions during the early stages of kidney morphogenesis. Disruption of the α8 gene in mice was found to be associated with severe defects in kidney morphogenesis (5) and stereocilia development (6). To date, α8β1 integrin has been shown to bind to fibronectin, vitronectin, osteopontin, latency-associated peptide of transforming growth factor-β1, tenascin-W, and nephronectin (also named POEM) (7–13), among which nephronectin is believed to be an α8β1 integrin ligand involved in kidney development (10).Nephronectin is one of the basement membrane proteins whose expression and localization patterns are restricted in a tissue-specific and developmentally regulated manner (10, 11). Nephronectin consists of five epidermal growth factor-like repeats, a linker segment containing the RGD cell-adhesive motif (designated RGD-linker) and a meprin-A5 protein-receptor protein-tyrosine phosphatase μ (MAM) domain (see Fig. 3A). Although the physiological functions of nephronectin remain only poorly understood, it is thought to play a role in epithelial-mesenchymal interactions through binding to α8β1 integrin, thereby transmitting signals from the epithelium to the mesenchyme across the basement membrane (10). Recently, mice deficient in nephronectin expression were produced by homologous recombination (14). These nephronectin-deficient mice frequently displayed kidney agenesis, a phenotype reminiscent of α8 integrin knock-out mice (14), despite the fact that other RGD-containing ligands, including fibronectin and osteopontin, were expressed in the embryonic kidneys (9, 15). The failure of the other RGD-containing ligands to compensate for the deficiency of nephronectin in the developing kidneys suggests that nephronectin is an indispensable α8β1 ligand that plays a mandatory role in epithelial-mesenchymal interactions during kidney development.Open in a separate windowFIGURE 3.Binding activities of α8β1 integrin to nephronectin and its fragments. A, schematic diagrams of full-length nephronectin (NN) and its fragments. RGD-linker and RGD-linker (GST), the central RGD-containing linker segments expressed in mammalian and bacterial expression systems, respectively; PRGDV, a short RGD-containing peptide modeled after nephronectin and expressed as a GST fusion protein (see Fig. 4A for the peptide sequence). The arrowheads indicate the positions of the RGD motif. B, purified recombinant proteins were analyzed by SDS-PAGE in 7–15% gradient (left and center panels) and 12% (right panels) gels, followed by Coomassie Brilliant Blue (CBB) staining, immunoblotting with an anti-FLAG mAb, or lectin blotting with PNA. The quantities of proteins loaded were: 0.5 μg (for Coomassie Brilliant Blue staining) and 0.1 μg (for blotting with anti-FLAG and PNA) in the left and center panels;1 μg in the right panel. C, recombinant proteins (10 nm) were coated on microtiter plates and assessed for their binding activities toward α8β1 integrin (10 nm) in the presence of 1 mm Mn²⁺. The backgrounds were subtracted as described in the legend to Fig. 2. The results represent the mean ± S.D. of triplicate determinations. D, titration curves of α8β1 integrin bound to full-length nephronectin (NN, closed squares), the RGD-linker segments expressed in 293F cells (RGD-linker, closed triangles) and E. coli (RGD-linker (GST), open triangles), the MAM domain (MAM, closed diamonds), and the PRGDV peptide expressed as a GST fusion protein in E. coli (PRGDV (GST), open circles). The assays were performed as described in the legend to Fig. 2B. The results represent the means of duplicate determinations.Although ligand recognition by RGD-binding integrins is primarily determined by the RGD motif in the ligands, it is the residues outside the RGD motif that define the binding specificities and affinities toward individual integrins (16, 17). For example, α5β1 integrin specifically binds to fibronectin among the many RGD-containing ligands, and requires not only the RGD motif in the 10th type III repeat but also the so-called “synergy site” within the preceding 9th type III repeat for fibronectin recognition (18). Recently, DiCara et al. (19) demonstrated that the high-affinity binding of αVβ6 integrin to its natural ligands, e.g. foot-and-mouth disease virus, requires the RGD motif immediately followed by a Leu-Xaa-Xaa-Leu/Ile sequence, which forms a helix to align the two conserved hydrophobic residues along the length of the helix. Given the presence of many naturally occurring RGD-containing ligands, it is conceivable that the specificities of the RGD-binding integrins are dictated by the sequences flanking the RGD motif or those in neighboring domains that come into close proximity with the RGD motif in the intact ligand proteins. However, the preferences of α8β1 integrin for RGD-containing ligands and how it secures its high-affinity binding toward its preferred ligands remain unknown.In the present study, we investigated the binding specificities of α8β1 integrin toward a panel of RGD-containing cell-adhesive proteins. Our data reveal that nephronectin is a preferred ligand for α8β1 integrin, and that a LFEIFEIER sequence on the C-terminal side of its RGD motif serves as a synergy site to ensure the specific high-affinity binding of nephronectin to α8β1 integrin.     

Early Embryogenesis and Anterior-Posterior Axis Formation in the White-Tip Nematode Aphelenchoides besseyi (Nematoda: Aphelenchoididae)

We followed the early embryogenesis of Aphelenchoides besseyi from fertilization to the 4-cell stage under Nomarski optics and examined the chromosome number and structure by DAPI staining. After an oocyte is fertilized by a sperm, the eggshell forms and the male and female pronuclei are reconstructed. The male pronucleus moves toward the female pronucleus, which is located at the center of the egg. They meet, rotate 90°, and fuse. The embryo then divides unequally into a larger anterior AB cell and a smaller posterior P(1) cell. The site of sperm entry into the oocyte seems to become the future anterior pole of the embryo, and thus the formation of an anterior-posterior axis formation is the same as that for Bursaphelenchus xylophilus, but opposite to that for Caenorhabditis elegans. From immunostaining, the fertilizing sperm appears to bring the centrosome into the oocyte. The chromosome structure during the pronuclear meeting as observed by DAPI staining suggests that a haploid sperm (N = 3) fertilizes a haploid oocyte (N = 3) to form a diploid embryo (2N = 6) and that all chromosomes appear to be of a similar size. Unlike C. elegans does, the P(1) cell first divides anterior-posteriorly followed by the AB anterior-posteriorly. These divisions produced the 4-cell stage embryo with 4 cells arranged in a linear fashion, again in contrast to that for C. elegans or B. xylophilus configured in a rhomboid shape.     

Deformation of chlorin rings in the Photosystem II crystal structure

The crystal structure of Photosystem II (PSII) analyzed at a resolution of 1.9 ? revealed deformations of chlorin rings in the chlorophylls for the first time. We investigated the degrees of chlorin ring deformation and factors that contributed to them in the PSII crystal structure, using a normal-coordinate structural decomposition procedure. The out-of-plane distortion of the P(D1) chlorin ring can be described predominantly by a large "doming mode" arising from the axial ligand, D1-His198, as well as the chlorophyll side chains and PSII protein environment. In contrast, the deformation of P(D2) was caused by a "saddling mode" arising from the D2-Trp191 ring and the doming mode arising from D2-His197. Large ruffling modes, which were reported to lower the redox potential in heme proteins, were observed in P(D1) and Chl(D1), but not in P(D2) and Chl(D2). Furthermore, as P(D1) possessed the largest doming mode among the reaction center chlorophylls, the corresponding bacteriochlorophyll P(L) possessed the largest doming mode in bacterial photosynthetic reaction centers. However, the majority of the redox potential shift in the protein environment was determined by the electrostatic environment. The difference in the chlorin ring deformation appears to directly refer to the difference in "the local steric protein environment" rather than the redox potential value in PSII.     

Structure of the novel C-terminal domain of vacuolar protein sorting 30/autophagy-related protein 6 and its specific role in autophagy

Vacuolar protein sorting 30 (Vps30)/autophagy-related protein 6 (Atg6) is a common component of two distinct phosphatidylinositol 3-kinase complexes. In complex I, Atg14 links Vps30 to Vps34 lipid kinase and exerts its specific role in autophagy, whereas in complex II, Vps38 links Vps30 to Vps34 and plays a crucial role in vacuolar protein sorting. However, the molecular role of Vps30 in each pathway remains unclear. Here, we report the crystal structure of the carboxyl-terminal domain of Vps30. The structure is a novel globular fold comprised of three β-sheet-α-helix repeats. Truncation analyses showed that the domain is dispensable for the construction of both complexes, but is specifically required for autophagy through the targeting of complex I to the pre-autophagosomal structure. Thus, the domain is named the β-α repeated, autophagy-specific (BARA) domain. On the other hand, the N-terminal region of Vps30 was shown to be specifically required for vacuolar protein sorting. These structural and functional investigations of Vps30 domains, which are also conserved in the mammalian ortholog, Beclin 1, will form the basis for studying the molecular functions of this protein family in various biological processes.     

Human young children as well as adults demonstrate 'superior' rapid snake detection when typical striking posture is displayed by the snake

Humans as well as some nonhuman primates have an evolved predisposition to associate snakes with fear by detecting their presence as fear-relevant stimuli more rapidly than fear-irrelevant ones. In the present experiment, a total of 74 of 3- to 4-year-old children and adults were asked to find a single target black-and-white photo of a snake among an array of eight black-and-white photos of flowers as distracters. As target stimuli, we prepared two groups of snake photos, one in which a typical striking posture was displayed by a snake and the other in which a resting snake was shown. When reaction time to find the snake photo was compared between these two types of the stimuli, its mean value was found to be significantly smaller for the photos of snakes displaying striking posture than for the photos of resting snakes in both the adults and children. These findings suggest the possibility that the human perceptual bias for snakes per se could be differentiated according to the difference of the degree to which their presence acts as a fear-relevant stimulus.     

Autophagy-related Protein 8 (Atg8) Family Interacting Motif in Atg3 Mediates the Atg3-Atg8 Interaction and Is Crucial for the Cytoplasm-to-Vacuole Targeting Pathway

The autophagy-related protein 8 (Atg8) conjugation system is essential for the formation of double-membrane vesicles called autophagosomes during autophagy, a bulk degradation process conserved among most eukaryotes. It is also important in yeast for recognizing target vacuolar enzymes through the receptor protein Atg19 during the cytoplasm-to-vacuole targeting (Cvt) pathway, a selective type of autophagy. Atg3 is an E2-like enzyme that conjugates Atg8 with phosphatidylethanolamine. Here, we show that Atg3 directly interacts with Atg8 through the WEDL sequence, which is distinct from canonical interaction between E2 and ubiquitin-like modifiers. Moreover, NMR experiments suggest that the mode of interaction between Atg8 and Atg3 is quite similar to that between Atg8/LC3 and the Atg8 family interacting motif (AIM) conserved in autophagic receptors, such as Atg19 and p62. Thus, the WEDL sequence in Atg3 is a canonical AIM. In vitro analyses showed that Atg3 AIM is crucial for the transfer of Atg8 from the Atg8∼Atg3 thioester intermediate to phosphatidylethanolamine but not for the formation of the intermediate. Intriguingly, in vivo experiments showed that it is necessary for the Cvt pathway but not for starvation-induced autophagy. Atg3 AIM attenuated the inhibitory effect of Atg19 on Atg8 lipidation in vitro, suggesting that Atg3 AIM may be important for the lipidation of Atg19-bound Atg8 during the Cvt pathway.     

Chondroitin Sulfate Synthase-2/Chondroitin Polymerizing Factor Has Two Variants with Distinct Function

Chondroitin sulfate (CS) is a polysaccharide consisting of repeating disaccharide units of N-acetyl-d-galactosamine and d-glucuronic acid residues, modified with sulfated residues at various positions. To date six glycosyltransferases for chondroitin synthesis have been identified, and the complex of chondroitin sulfate synthase-1 (CSS1)/chondroitin synthase-1 (ChSy-1) and chondroitin sulfate synthase-2 (CSS2)/chondroitin polymerizing factor is assumed to play a major role in CS biosynthesis. We found an alternative splice variant of mouse CSS2 in a data base that lacks the N-terminal transmembrane domain, contrasting to the original CSS2. Here, we investigated the roles of CSS2 variants. Both the original enzyme and the splice variant, designated CSS2A and CSS2B, respectively, were expressed at different levels and ratios in tissues. Western blot analysis of cultured mouse embryonic fibroblasts confirmed that both enzymes were actually synthesized as proteins and were localized in both the endoplasmic reticulum and the Golgi apparatus. Pulldown assays revealed that either of CSS2A, CSS2B, and CSS1/ChSy-1 heterogeneously and homogeneously interacts with each other, suggesting that they form a complex of multimers. In vitro glycosyltransferase assays demonstrated a reduced glucuronyltransferase activity in CSS2B and no polymerizing activity in CSS2B co-expressed with CSS1, in contrast to CSS2A co-expressed with CSS1. Radiolabeling analysis of cultured COS-7 cells overexpressing each variant revealed that, whereas CSS2A facilitated CS biosynthesis, CSS2B inhibited it. Molecular modeling of CSS2A and CSS2B provided support for their properties. These findings, implicating regulation of CS chain polymerization by CSS2 variants, provide insight in elucidating the mechanisms of CS biosynthesis.     

Impairment of VEGF-A-stimulated lamellipodial extensions and motility of vascular endothelial cells by chondromodulin-I, a cartilage-derived angiogenesis inhibitor

Chondromodulin-I (ChM-I) is a cartilage-derived angiogenesis inhibitor that has been identified as inhibitory to the growth activity of vascular endothelial cells. In our present study, we demonstrate the anti-angiogenic activity of recombinant human ChM-I (rhChM-I) in mouse corneal angiogenesis and examine its action. We focus on the VEGF-A-induced migration of vascular endothelial cells, a critical regulatory step in angiogenesis. In a modified Boyden chamber assay, nanomolar concentrations of rhChM-I inhibited the chemotactic migration of human umbilical vein endothelial cells (HUVECs) induced by VEGF-A as well as by FGF-2 and IGF-I. The ChM-I action was found to be endothelial cell-specific and independent of cell adhesions. Time-lapse analysis further revealed that rhChM-I markedly reduces VEGF-A-stimulated motility of HUVECs and causes frequent alterations of the moving front due to the appearance of multiple transient protrusions. This action involved the inhibition of cell spreading and the disrupted reorganization of the actin cytoskeleton upon VEGF-A stimulation. Consistent with these observations, rhChM-I was found to significantly reduce the activity of Rac1/Cdc42 during cell spreading, and the VEGF-A-induced Rac1 activity but not its basal activity in quiescent cells. Taken together, our present data suggest that ChM-I impairs the VEGF-A-stimulated motility of endothelial cells by destabilizing lamellipodial extensions.