Cells and axons that supply direct afferent input to the medial nucleus of the trapezoid body are described. Afferents were intracellularly labeled in brainstem tissue slices of two rodent and two bat species. The main afferents are calyciferous axons from globular bushy cells of the ventral cochlear nucleus. Calyciferous axons were highly consistent across species, projecting directly from the cochlear nucleus, across the midline in the trapezoid body, to the contralateral medial nucleus of the trapezoid body. Within the target nucleus, a typical axon turned sharply away from horizontal to form a large ending, the calyx of Held, around the soma of a single principal cell. Three groups of calyciferous axons were classified based on the path taken from bend to calyx. In subjects younger than four weeks, single axons often formed two calyces, each on a different cell. These calyx pairs were often found on adjacent or vertically aligned cells. In older animals, calyx pairs were more closely aligned, but fewer double calyx axons were seen. A secondary focus of this study was the system of thin collateral branches that characterizes calyciferous axons in all species. The projection patterns of these collaterals suggest that calyciferous axons may provide ascending input to periolivary cell groups with descending projections. In addition to calyciferous afferents, labeled cells that provide input to the medial nucleus of the trapezoid body from adjacent periolivary cell groups are described. Also described is a type of afferent that descends from the level of the lateral lemniscus to the medial nucleus of the trapezoid body.

Plant NADPH oxidases (Rboh, for respiratory burst oxidase homolog) produce reactive oxygen species that are key regulators of various cellular events including plant innate immunity. Rbohs possess a highly conserved cytoplasmic N-terminal region containing two EF-hand motifs that regulate Rboh activity. Rice (Oryza sativa) RbohB (OsRbohB) is regulated by the direct binding of a small GTPase (Rac1) to this regulatory region as well as by Ca 2؉ binding to the EF-hands. Here, we present the atomic structure of the N-terminal region of OsRbohB. The structure reveals that OsRbohB forms a unique dimer stabilized by swapping the EF-hand motifs. We identified two additional EF-hand-like motifs that were not predicted from sequence data so far. These EF-hand-like motifs together with the swapped EF-hands form a structure similar to that found in calcineurin B. We observed conformational changes mediated by Ca 2؉ binding to only one EF-hand. Structure-based in vitro pulldown assays and NMR titration experiments defined the OsRac1 binding interface within the coiled-coil region created by swapping the EF-hands. In addition, we demonstrate a direct intramolecular interaction between the N and C terminus, and that the complete N-terminal cytoplasmic region is required for this interaction. The structural features and intramolecular interactions characterized here might be common elements shared by Rbohs that contribute to the regulation of reactive oxygen species production. (Fig. 1A) (3, 8). Although these motifs are absent from the mammalian gp91 phox , they are present in non-phagocytic NADPH oxidases such as Nox5, Duox1, and Duox2 (9,10).Rboh is stimulated directly by Ca 2ϩ , likely through the N-terminal region (11). Recently, it has been reported that Ca 2ϩ activated the Arabidopsis NADPH oxidase, RHD2 (AtRbohC), to produce ROS at the growing tip of root hairs. Moreover, Ca 2ϩ binding to EF-hand motifs is required for activation of ROS production by RHD2 (12). Recent work has also demonstrated that calcium-dependent protein kinases activate potato RbohB (StRbohB) protein by phosphorylation of the N-terminal region (13), and that Ca 2ϩ binding and phosphorylation synergistically activate ROS production by AtRbohD (14). These results demonstrate that the extended N-terminal region plays a key role in the regulation of Rboh.OsRac1, a Rac GTPase located in the plasma membrane, functions as a positive regulator of NADPH oxidase activity and is involved in defense responses in rice (15,16). In fact, constitutively active and dominant-negative forms of OsRac1 were found to activate and suppress ROS production, respectively. Direct interaction between the constitutively active OsRac1 and the N-terminal region of OsRbohB was verified by yeast two-hybrid analysis, in vitro pulldown assays, NMR titration experiments, and in vivo fluorescence resonance energy trans- 81-45-508-7365; E-mail: shimizu@tsurumi.yokohama-cu.ac.jp. 2 The abbreviations used are: ROS, reactive oxygen species; GST, glutathione S-transferase; GMP-P(NH)P, gu...

In this study we present direct evidence of axonal projections from both the medial and lateral nuclei of the trapezoid body to the medial superior olive. Projections were traced by intracellularly labeling cells and axons in a tissue slice preparation of two rodent species, Mus musculus and Meriones unguiculatus and two bat species, Eptesicus fuscus and Pteronotus parnellii. The main axon of most principal cells in the medial nucleus of the trapezoid body gives off one or more collateral branches which arborize within the medial superior olive. These collateral axons form small bouton-like swellings which primarily contact somata within the central cell column in the medial superior olive. Likewise, labeled elongate and multipolar cells of the lateral nucleus of the trapezoid body send axons to both the medial and lateral superior olives. These axons also form perisomatic contacts in both target nuclei. These two sets of projections may relay ascending input to the medial superior olive and the lateral superior olive; the medial nucleus of the trapezoid body is known to relay input from the contralateral ventral cochlear nucleus, and the lateral nucleus of the trapezoid body may relay input from the ipsilateral ventral cochlear nucleus. These projections offer two routes for indirect, possibly inhibitory input to reach the medial superior olive from both cochlear nuclei. These indirect, inhibitory pathways may parallel the direct excitatory projections the medial superior olive receives from each cochlear nucleus.

Rad51 forms a helical filament on single-stranded DNA and promotes strand exchange between two homologous DNA molecules during homologous recombination. The Swi5-Sfr1 complex interacts directly with Rad51 and stimulates strand exchange. Here we describe structural and functional aspects of the complex. Swi5 and the C-terminal core domain of Sfr1 form an essential activator complex with a parallel coiled-coil heterodimer joined firmly together via two previously uncharacterized leucine-zipper motifs and a bundle. The resultant coiled coil is sharply kinked, generating an elongated crescent-shaped structure suitable for transient binding within the helical groove of the Rad51 filament. The N-terminal region of Sfr1, meanwhile, has an interface for binding of Rad51. Our data suggest that the snug fit resulting from the complementary geometry of the heterodimer activates the Rad51 filament and that the N-terminal domain of Sfr1 plays a role in the efficient recruitment of the Swi5-Sfr1 complex to the Rad51 filaments.

Cells in the medial nucleus of the trapezoid body were intracellularly labeled in brainstem tissue slices of two bat and two rodent species. The main cell type found in this nucleus, the principal cell, is an important link in the relay of ascending projections from the contralateral cochlear nucleus to the lateral superior olive, completing an essential pathway for sound localization. Principal cells are often viewed as a highly homogeneous group with a consistent morphology as well as a common function. Intracellular labeling has revealed a number of new axonal and dendritic features of principal cells. Some of these features vary widely from cell to cell, suggesting that the population of principal cells contains several morphologically distinct subgroups. Similar subsets of principal cells were recognized in all species examined. Five subgroups were distinguished on the basis of the position of dendritic fields. Although the dendrites of most labeled cells were confined to the medial nucleus of the trapezoid body, some principal cells had dendrites that spread outside the nucleus to one of several adjacent periolivary cell groups. Cells were also found that had dendrites that spread medially across the midline and into the contralateral medial nucleus of the trapezoid body. Axonal projections were used to distinguish two additional subgroups of principal cells. All principal cells project to the lateral superior olive and virtually all have one or more secondary projections. There are two subgroups with unusual collateral projections: one with collaterals that extended to the lateral lemniscus and one with recurrent collateral axons.

Abstract. Tubulointerstitial fibrosis is a common feature of many progressive renal diseases and is a main determinant that leads to an irreversible loss of renal function. In chronic cyclosporin A nephrotoxicity, we previously reported that inflammatory responses such as macrophage infiltration preceded interstitial fibrosis. This inflammation was accompanied by an elevation in renal nuclear factor κB (NF-κB) activity. Similar findings were obtained in chronic tacrolimus nephrotoxicity and obstructive nephropathy. Inhibition of NF-κB markedly attenuated renal inflammation and interstitial fibrosis in these models. Furthermore, administration of oral adsorbent (Kremezin ® ) significantly attenuated the increase in renal NF-κB activity and concomitantly reduced interstitial inflammation and renal fibrosis in chronic renal failure rats. Elimination of indoxyl sulfate by this adsorbent is likely involved in this mechanism since it is known that indoxyl sulfate activates NF-κB in renal tubular cells. It is suggested that strategy aiming at NF-κB inhibition is important to prevent the progression of renal fibrosis.

Background: Both BAG6 and Ubl4a are mediators of tail-anchored (TA) protein biogenesis and protein quality control.Results: The crystal structure of the BAG6-Ubl4a heterodimerization domain reveals a unique binding interface.Conclusion: The BAG-similar domain of BAG6 is completely distinct from other canonical BAG domains.Significance: Ubl4a forms a complex with BAG6, whereas its yeast homologue Get5 forms a homodimer.

The dystrophin glycoprotein complex, which connects the cell membrane to the basement membrane, is essential for a variety of biological events, including maintenance of muscle integrity. An O-mannose–type GalNAc-β1,3-GlcNAc-β1,4-(phosphate-6)-Man structure of α-dystroglycan (α-DG), a subunit of the complex that is anchored to the cell membrane, interacts directly with laminin in the basement membrane. Reduced glycosylation of α-DG is linked to some types of inherited muscular dystrophy; consistent with this relationship, many disease-related mutations have been detected in genes involved in O-mannosyl glycan synthesis. Defects in protein O-linked mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGnT1), a glycosyltransferase that participates in the formation of GlcNAc-β1,2-Man glycan, are causally related to muscle-eye-brain disease (MEB), a congenital muscular dystrophy, although the role of POMGnT1 in postphosphoryl modification of GalNAc-β1,3-GlcNAc-β1,4-(phosphate-6)-Man glycan remains elusive. Our crystal structures of POMGnT1 agreed with our previous results showing that the catalytic domain recognizes substrate O-mannosylated proteins via hydrophobic interactions with little sequence specificity. Unexpectedly, we found that the stem domain recognizes the β-linked GlcNAc of O-mannosyl glycan, an enzymatic product of POMGnT1. This interaction may recruit POMGnT1 to a specific site of α-DG to promote GlcNAc-β1,2-Man clustering and also may recruit other enzymes that interact with POMGnT1, e.g., fukutin, which is required for further modification of the GalNAc-β1,3-GlcNAc-β1,4-(phosphate-6)-Man glycan. On the basis of our findings, we propose a mechanism for the deficiency in postphosphoryl modification of the glycan observed in POMGnT1-KO mice and MEB patients.