Claudin-4 Deficiency Results in Urothelial Hyperplasia and Lethal Hydronephrosis

Claudin (Cld)-4 is one of the dominant Clds expressed in the kidney and urinary tract, including selective segments of renal nephrons and the entire urothelium from the pelvis to the bladder. We generated Cldn4 ^−/− mice and found that these mice had increased mortality due to hydronephrosis of relatively late onset. While the renal nephrons of Cldn4 ^−/− mice showed a concomitant diminution of Cld8 expression at tight junction (TJ), accumulation of Cld3 at TJ was markedly enhanced in compensation and the overall TJ structure was unaffected. Nonetheless, Cldn4 ^−/− mice showed slightly yet significantly increased fractional excretion of Ca²⁺ and Cl⁻, suggesting a role of Cld4 in the specific reabsorption of these ions via a paracellular route. Although the urine volume tended to be increased concordantly, Cldn4 ^−/− mice were capable of concentrating urine normally on dehydration, with no evidence of diabetes insipidus. In the urothelium, the formation of TJs and uroplaques as well as the gross barrier function were also unaffected. However, intravenous pyelography analysis indicated retarded urine flow prior to hydronephrosis. Histological examination revealed diffuse hyperplasia and a thickening of pelvic and ureteral urothelial layers with markedly increased BrdU uptake in vivo. These results suggest that progressive hydronephrosis in Cldn4 ^−/− mice arises from urinary tract obstruction due to urothelial hyperplasia, and that Cld4 plays an important role in maintaining the homeostatic integrity of normal urothelium.     

Helminths collected from imported pet murids, with special reference to concomitant infection of the golden hamsters with three pinworm species of the genus Syphacia (Nematoda: oxyuridae)

A total of 210 individuals of 13 species belonging to 4 subfamilies of Muridae imported into Japan as pets were examined; 5 species of Syphacia (Nematoda: Oxyuridae), Aspiculuris tetraptera (Nematoda: Heteroxynematidae), and Rodentolepis nana (Cestoidea: Hymenolepididae) were collected. Concurrent infection with 3 pinworm species, Syphacia mesocriceti, Syphacia stroma, and Syphacia peromysci, was recorded for the first time in the golden hamster, Mesocricetus auratus. Syphacia mesocriceti was also identified in the desert hamster, Phodopus roborovskii, and S. peromysci was recovered from the fat-tailed gerbil, Pachyuromys duprasi, and the Cairo spiny mouse, Acomys cahirinus. From the pygmy mouse, Mus minutoides, an undetermined species closely resembling Syphacia megaloon and Syphacia ohtaorum, both parasitic in Mus spp., was collected. Females of another undetermined Syphacia sp. were observed in the greater Egyptian gerbil, Gerbillus pyramidum. All of the host-Syphacia associations, except S. mesocriceti in the golden hamsters, were recorded for the first time. It is suggested that overlapping breeding situations provided the opportunity for host switching by the pinworms.     

Torulaspora maleeae sp. nov., a novel ascomycetous yeast species from Japan and Thailand

Nine strains of a new Torulaspora species were isolated from natural samples collected in Japan and Thailand including one strain obtained from a leaf of Rhizophora stylosa (NBRC 11061T), one strain from soil (NBRC 11062), six strains from mosses (ST-14, ST-266, ST-510, ST-511, ST-513 and ST-581) and one strain from sediment in mangrove forest (RV-51). On the basis of morphological, biochemical, physiological and chemotaxonomic characteristics, and the sequence analyses of the D1/D2 domain of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) (ITS1-5.8S rRNA gene-ITS2) region, the nine strains were found to represent a single novel species of the genus Torulaspora, which were named Torulaspora maleeae sp. nov. The type strain is NBRC 11061T (BCC 25515T=CBS 10694T). In the phylogenetic trees based on the sequences of the D1/D2 domain of the LSU rRNA gene, T. maleeae showed a close relationship with the five recognized species of the genus Torulaspora, Torulaspora delbrueckii, Torulaspora franciscae, Torulaspora globosa, Torulaspora microellipsoides and Torulaspora pretoriensis. Torulaspora maleeae differed from the five recognized species of the genus Torulaspora by six to 12 nucleotide substitutions (1.1-2.1%) in the D1/D2 domain of the LSU rRNA gene and by 6.4-11.7% nucleotide substitutions in the ITS (ITS1-5.8S rRNA gene-ITS2) region.     

Differential dephosphorylation of the FcRgamma immunoreceptor tyrosine-based activation motif tyrosines with dissimilar potential for activating Syk

The cell surface-expressed gamma chain of the high affinity receptor for IgE (FcepsilonRI) can be phosphorylated on two tyrosine residues of the immunoreceptor tyrosine-based activation motif (ITAM), leading to recruitment and activation of spleen tyrosine kinase (Syk), a kinase that is essential for mast cell signaling and allergic responses. However, it is not known whether preferential phosphorylation or dephosphorylation of the two individual FcRgamma tyrosines (the N-terminal Tyr47 and C-terminal Tyr58) could regulate Syk activation. Herein we report that phosphorylation of only Tyr58 was able to elicit Syk phosphorylation and a weak rise in intracellular calcium, suggesting that Tyr58 phosphorylation may be distinctively important for Syk activation. In vitro and in vivo studies revealed that both Tyr47 and Tyr58 could be similarly phosphorylated. However, mass spectrometric analysis of the phosphorylated FcepsilonRgamma from bone marrow-derived mast cells showed that phosphorylation at Tyr47 was at least 2-fold greater than at Tyr58. This suggested that, once phosphorylated, Tyr58 is preferentially dephosphorylated. In vitro studies demonstrated more efficient dephosphorylation of Tyr58 (by the receptor-associated phosphatases SHP-1 and SHP-2) than of Tyr47. Analysis of Syk binding to wild type and mutant phosphorylated FcepsilonRI revealed that mutation at Tyr58 almost completely ablated Syk binding, whereas mutation at Tyr47 moderately reduced Syk binding. The findings argue for a novel regulatory mechanism, where dephosphorylation of phospho-Tyr58 is likely to promote the down-regulation of Syk activation and suppression of mast cell responses.     

Quantitative analysis of EGFR affinity to immobilized glycolipids by surface plasmon resonance

EGF-induced activation of EGFR tyrosine kinase is known to be inhibited by ganglioside GM3, its dimer, and other mimetics. However, details of the interaction, such as kinetic properties, have not yet been clarified. The direct interaction is now defined by the surface plasmon resonance (SPR) technique. To determine the affinity of EGFR for lyso-GM3 or lyso-GM3 mimetic, these glycolipid ligands were covalently immobilized onto a sensor chip, and binding affinities were investigated. Results of these studies confirmed the direct interaction of lyso-GM3 or its mimetic with EGFR. A strong interaction between EGFR and lyso-GM3 or its mimetic was indicated by increased binding of EGFR to glycolipid-immobilized surface, in an EGFR dose-dependent manner.     

Protein kinase B (PKB/Akt) is required for the completion of meiosis in mouse oocytes

Akt, also known as protein kinase B, is implicated in many cellular processes. Akt is phosphorylated at two residues, Thr308 and Ser473. Thr308-phosphorylated Akt is present in pericentriolar materials, while localization of Ser473-phosphorylated Akt was similar to that of microtubules in metaphase oocytes. Spindles were shorter and aberrant in oocytes injected with Thr308- or Ser473-phosphorylated Akt antibodies. Specifically, Thr308- and Ser473-phosphorylated Akts function individually and are both necessary to assemble the metaphase II (MII) spindle. Moreover, the functions of Thr308- and Ser473-phosphorylated Akts differ in MII oocytes. Although oocytes exhibited second polar body (PB2) emission after the injection of a peptide for Thr308, the chromosomal alignment and microtubular organization were aberrant. In contrast, the injection of a peptide for Ser473 caused a failure of PB2 emission. These results suggest that Thr308- and Ser473-phosphorylated Akts are individually involved in fertilization to complete meiosis, including different roles (i.e., Ser473-phosphorylated Akts are involved in PB2 emission, whereas Thr308-phosphorylated Akts regulate the organization of microtubules).     

YIPF5 and YIF1A recycle between the ER and the Golgi apparatus and are involved in the maintenance of the Golgi structure

Yip1p/Yif1p family proteins are five-span transmembrane proteins localized in the Golgi apparatus and the ER. There are nine family members in humans, and YIPF5 and YIF1A are the human orthologs of budding yeast Yip1p and Yif1p, respectively. We raised antisera against YIPF5 and YIF1A and examined the localization of endogenous proteins in HeLa cells. Immunofluorescence, immunoelectron microscopy and subcellular fractionation analysis suggested that YIPF5 and YIF1A are not restricted to ER exit sites but also localized in the ER-Golgi intermediate compartment (ERGIC) and some in the cis-Golgi at steady state. Along with ERGIC53, YIPF5 and YIF1A remained in the cytoplasmic punctate structures after brefeldin A treatment, accumulated in the ERGIC and the cis-Golgi after treatment with AlF₄^- and accumulated in the ER when ER to Golgi transport was inhibited by Sar1(H79G). These results supported the localization of YIPF5 and YIF1A in the ERGIC and the cis-Golgi, and strongly suggested that they are recycling between the ER and the Golgi apparatus. Analysis by blue native PAGE and co-immunoprecipitation showed that YIPF5 and YIF1A form stable complexes of three different sizes. Interestingly, the knockdown of YIPF5 or YIF1A caused partial disassembly of the Golgi apparatus suggesting that YIPF5 and YIF1A are involved in the maintenance of the Golgi structure.     

Identification of the P-TEFb complex-interacting domain of Brd4 as an inhibitor of HIV-1 replication by functional cDNA library screening in MT-4 cells

We conducted a phenotypic cDNA screening using a T cell line-based assay to identify human genes that render cells resistant to human immunodeficiency virus type 1 (HIV-1). We isolated potential HIV-1 resistance genes, including the carboxy terminal domain (CTD) of bromodomain-containing protein 4 (Brd4). Expression of GFP-Brd4-CTD was tolerated in MT-4 and Jurkat cells in which HIV-1 replication was markedly inhibited. We provide direct experimental data demonstrating that Brd4-CTD serves as a specific inhibitor of HIV-1 replication in T cells. Our method is a powerful tool for the identification of host factors that regulate HIV-1 replication in T cells.