Isolated lymphoid follicles are not IgA inductive sites for recombinant Salmonella

In this study, we investigated whether isolated lymphoid follicles (ILF) play a role in the regulation of intestinal IgA antibody (Ab) responses. The transfer of wild type (WT) bone marrow (BM) to lymphotoxin-alpha-deficient (LTalpha(-/-)) mice resulted in the formation of mature ILF containing T cells, B cells, and FDC clusters in the absence of mesenteric lymph nodes and Peyer's patches. Although the ILF restored total IgA Abs in the intestine, antigen (Ag)-specific IgA responses were not induced after oral immunization with recombinant Salmonella expressing fragment C of tetanus toxin. Moreover, Ag-specific cell proliferation was not detected in the ILF. Interestingly, no IgA anti-LPS Abs were detected in the fecal extracts of LTalpha(-/-) mice reconstituted with WT BM. On the basis of these findings, ILF can be presumed to play a role in the production of IgA Abs, but lymphoid nodules are not inductive sites for the regulation of Ag-specific intestinal IgA responses to recombinant Salmonella.     

Identification and characterization of zebrafish semaphorin 6D

The semaphorin gene family contains a large number of secreted and transmembrane proteins; some function as repulsive and attractive cues of axon guidance during development. Here, we report cloning and characterization of zebrafish transmembrane semaphorin gene, semaphorin 6D (sema6D). Sema6D is expressed predominantly in the nervous system during embryogenesis, as determined by in situ hybridization. We also found that Sema6D binds Plexin-A1 in vitro, but not other Plexins. It induces the repulsion of dorsal root ganglion axons, but not sympathetic axons. Consequently, Sema6D might use Plexin-A1 as a receptor to repel specific types of axons during development.     

Parkin interacts with LIM Kinase 1 and reduces its cofilin-phosphorylation activity via ubiquitination

Mutations in the PARKIN (PARK2) gene have been found in the majority of early-onset familial Parkinson's disease (PD) patients with autosomal recessive juvenile parkinsonism (ARJP). Parkin protein functions as an ubiquitin (E3) ligase that targets specific proteins for degradation in the 26S proteasome. Here, based on a mass spectrometry analysis of the human dopaminergic neuroblastoma-derived cell line SH-SY5Y that over-expresses parkin, we found that parkin may suppress cofilin phosphorylation. LIM Kinase 1 (LIMK1) is the upstream protein that phosphorylates cofilin, an actin depolymerizing protein. Thus, we postulated a possible connection between parkin and LIMK1. Our studies in other cell lines, using co-transfection assays, demonstrated that LIMK1 and parkin bind each other. LIMK1 also interacted with previously known parkin interactors Hsp70 and CHIP. Parkin enhanced LIMK1-ubiquitination in the human neuroblastoma-derived BE(2)-M17 cell line, but not in the human embryonic kidney-derived HEK293 cell line. In fact, parkin-over-expression reduced the level of LIMK1-induced phosphocofilin in the BE(2)-M17 cells but not in the HEK293 cells. Additionally, in simian kidney-derived COS-7 cells, parkin-over-expression reduced LIMK1-induced actin filament accumulation. LIMK1 in cultured cells regulates parkin reversibly: LIMK1 did not phosphorylate parkin but LIMK1 overexpression reduced parkin self-ubiquitination in vitro and in HEK293 cells. Furthermore, in the cells co-transfected with parkin and p38, LIMK1 significantly decreased p38-ubiquitination by parkin. These findings demonstrate a cell-type dependent functional interaction between parkin and LIMK1 and provide new evidence that links parkin and LIMK1 in the pathogenesis of familial PD.     

Genetic relationships among salamanders of the genus Hynobius (Amphibia, Caudata) from Korea and southwestern Japan

We performed allozyme analysis for three Korean (Hynobius leechii, H. quelpaertensis, and H. yangi) and three Japanese (H. nebulosus, H. tsuensis, and H. dunni) salamanders to clarify their interspecific relationships using H. naevius as an outgroup. The genetic distances (Nei's D) within ingroup species ranged from 0.11 to 0.78 with a mean of 0.33. In the NJ and CONTML trees, monophyly of the ingroup was not supported and Korean H. quelpaertensis and H. leechii diverged first from the remaining species, which together formed a weakly supported clade. Korean H. yangi, long identified as H. leechii, was closer to Japanese H. nebulosus (D=0.108) and H. tsuensis (D=0.138) than to Korean H. leechii (D=0.197) and H. quelpaertensis (D=0.305). Hynobius tsuensis and H. nebulosus were very close (D=0.108) despite their different breeding habits. A geohistorical hypothesis is proposed to explain the divergence of the six species.     

Solubilization of the ecdysone binding protein from anterior silk gland cell membranes of the silkworm, Bombyx mori

We previously provided preliminary evidence for the presence of a putative membrane ecdysone receptor (mEcR) anchored in the plasma membranes of anterior silk glands (ASGs) in Bombyx mori. This receptor may act in concert with the conventional EcR in 20E-dependent programmed cell death of these glands. We report here, for the first time, the solubilization of mEcR from ASG membranes using the zwitterionic detergent CHAPS in the presence of NaCl. Our results show by ligand binding assay that mEcR solubilized this way is functionally active and retains 75% of its native binding activity. We also defined experimental conditions that yielded protein/detergent complexes with partial binding activity, which makes it possible to purify the membrane-bound ecdysone binding protein.     

SAS-6 is a cartwheel protein that establishes the 9-fold symmetry of the centriole

Centrioles consist of nine-triplet microtubules arranged in rotational symmetry. This structure is highly conserved among various eukaryotic organisms and serves as the base for the ciliary axoneme. Recently, several proteins such as SAS-6 have been identified as essential to the early process of centriole assembly, but the mechanism that produces the 9-fold symmetry is poorly understood. In C. elegans and Drosophila, SAS-6 has been suggested to function in the formation of a centriolar precursor, a central tube that then assembles nine-singlet microtubules on its surface. However, the generality of the central tube is not clear because in many other organisms, the first structure appearing in the centriole assembly is not a tube but a flat amorphous ring or a cartwheel-a structure with a hub and nine radiating spokes. Here we show that in Chlamydomonas the SAS-6 protein localizes to the central part of the cartwheel and that a null mutant of SAS-6, bld12, lacks that part. Intriguingly, this mutant frequently has centrioles composed of 7, 8, 10, or 11 triplets in addition to 9-triplet centrioles. We presume that, in many organisms, SAS-6 is an essential component of the cartwheel, a structure that stabilizes the 9-triplet structure.     

Toll‐like receptor 5 is not essential for the promotion of secretory immunoglobulin A antibody responses to flagellated bacteria

Toll‐like receptor 5 recognizes bacterial flagellin, plays a critical role in innate immunity, and contributes to flagellin‐specific humoral immunity. Further, TLR5‐expressing dendritic cells play an important role in IgA synthesis in the intestine; however, the contribution of TLR5 to antigen (Ag)‐specific mucosal immunity remains unclear. Thus, whether TLR5 is essential for the induction of intestinal secretory (S)IgA antibody (Ab) responses against flagellin and bacterial Ags attached to the bacterial surface in response to an oral flagellated bacterium, Salmonella, was explored in this study. Our results indicate that when TLR5 knockout (TLR5^?/?) mice are orally immunized with recombinant Salmonella expressing fragment C of tetanus toxin (rSalmonella‐Tox C), tetanus toxoid (TT)‐ and flagellin (FliC)‐specific systemic IgG and intestinal SIgA Abs are elicited. The numbers of TT‐specific IgG Ab‐forming cells (AFCs) in the spleen and IgA AFCs in the lamina propria (LP) of TLR5^?/? mice were comparable to those in wild‐type mice. rSalmonella‐Tox C was equally disseminated in TLR5^?/? mice, TLR5^?/? mice lacking Peyer's patches (PPs), and wild‐type mice. In contrast, TLR5^?/? PP‐null mice failed to induce TT‐ and FliC‐specific SIgA Abs in the intestine and showed significantly reduced numbers of TT‐specific IgA AFCs in the LP. These results suggest that TLR5 is dispensable for the induction of flagellin and surface Ag‐specific systemic and mucosal immunity against oral flagellated bacteria. Rather, pathogen recognition, which occurs in PPs, is a prerequisite for the induction of mucosal immunity against flagellated bacteria.

     

Development of Chiral Building Blocks Having a Bicyclo[3.3.0]Octane Framework Using a Diastereomeric Resolution‐Selective Deprotection

A protocol is presented for an efficient and practical approach to the synthesis of enantiomerically pure bicyclo[3.3.0]octane derivatives from achiral C_s‐symmetric bicyclo[3.3.0]octane‐2,8‐dione using a diastereomeric resolution‐selective deprotection method. This method affords chiral building blocks having bicyclo[3.3.0]octane framework with the same site of diastereotopic carbonyl functional group. Chirality 27:364–369, 2015. © 2015 Wiley Periodicals, Inc.