Distribution and frequencies of PDS (SLC26A4) mutations in Pendred syndrome and nonsyndromic hearing loss associated with enlarged vestibular aqueduct: a unique spectrum of mutations in Japanese

Molecular diagnosis makes a substantial contribution to precise diagnosis, subclassification, prognosis, and selection of therapy. Mutations in the PDS (SLC26A4) gene are known to be responsible for both Pendred syndrome and nonsyndromic hearing loss associated with enlarged vestibular aqueduct, and the molecular confirmation of the PDS gene has become important in the diagnosis of these conditions. In the present study, PDS mutation analysis confirmed that PDS mutations were present and significantly responsible in 90% of Pendred families, and in 78.1% of families with nonsyndromic hearing loss associated with enlarged vestibular aqueduct. Furthermore, variable phenotypic expression by the same combination of mutations indicated that these two conditions are part of a continuous category of disease. Interestingly, the PDS mutation spectrum in Japanese, including the seven novel mutations revealed by this study, is very different from that found in Caucasians. Of the novel mutations detected, 53% were the H723R mutation, suggesting a possible founder effect. Ethnic background is therefore presumably important and should be noted when genetic testing is being performed. The PDS gene mutation spectrum in Japanese may be representative of those in Eastern Asian populations and its elucidation is expected to facilitate the molecular diagnosis of a variety of diseases.     

Renal glomerulogenesis in medaka fish, Oryzias latipes

We provide an overview of glomerulogenesis in medaka from the embryo to the adult by means of in situ hybridization with the wt1 gene as a marker as well as histology and three-dimensional images. The pronephric glomus starts to develop in the intermediate mesoderm during early somitogenesis, is completed before hatching, and persists throughout the lifetime of the fish. Within 5 days after hatching, mesonephric glomerulus formation begins in the caudomedial end of the pronephric sinus and duct area. The number of glomeruli reaches approximately 200-300 in each kidney within 2 months after hatching. wt1 expression during nephron maturation served as a marker for the formation of the mesenchymal condensate and the nephrogenic body. Existence of mesenchymal condensates and persistence of wt1 expression in the adult kidney suggest that the mesonephros retains precursor cells that may be capable of contributing to neoglomerulogenesis during adulthood. Developmental Dynamics 237:2342-2352, 2008. (c) 2008 Wiley-Liss, Inc.     

The maitotoxin-evoked Ca2+ entry into synaptosomes is enhanced by cholera toxin

Effects of the Gs protein-mediated adenylate cyclase facilitatory system on Ca2+ entry into synaptosomes were studied, using two specific toxins. A putative Ca2+-channel agonist, maitotoxin (MTX), increased the 45Ca2+ entry and [Ca2+]i, determined with Quin-II, into synaptosomes of the rat brainstem, which were not attenuated by nifedipine. However, another Ca2+-channel agonist, BAY K-8644 did not alter the 45Ca2+ entry nor [Ca2+]i. The MTX-induced increase of the 45Ca2+ entry was significantly enhanced by addition of dibutyryl cyclic adenosine monophosphate and by the pretreatment with cholera toxin. These findings support the view that stimulation of presynaptic receptors coupled to the Gs-adenylate cyclase system may lead to a facilitation of the release of neurotransmitters, through a cAMP dependent enhancement of the opening of the Ca2+ channels located on nerve terminals.     

Genomic structure and eight novel exonic polymorphisms of the human N-cadherin gene

 Analysis of the detailed genomic structure of human N-cadherin revealed that the 16-exon gene is more than 72 kb in length and that it consists of a mosaic of exons. Five repeated cadherin domains, a transmembrane domain, and a cytoplasmic domain are encoded by exons 4 to 13, 13 and 14, and 14 to 16, respectively. A search for molecular variants in the entire coding region in 96 Japanese individuals resulted in the identification of eight sequence polymorphisms including three CCT- or GCC-type trinucleotide repeat polymorphisms adjacent to the initiation codon and five other novel single-nucleoticle polymorphisms (SNPs) in the coding region. Three of the five SNPs accompanied an amino acid substitution: Ala118Thr, Ala826Thr, and Asn845Ser. Knowlege of the fine gene structure and eight novel polymorphisms will be useful for the genetic study of the role of N-cadherin in diseases involving cell adhesion in the brain and in cardiomyocytes. Received: January 23, 2002 / Accepted: March 12, 2002     

Prevention of endotoxin shock by an antibody against leukocyte integrin {beta}2 through inhibiting production and action of TNF

Septic shock remains a serious disorder associated with highmortality. Accumulating evidence indicates that TNF is a majorand essential mediator of endotoxin shock. We report here thatadministration of an antibody against CD18 dramatically reducedendotoxin-induced shock inrabbits as revealed by preventionof severe hypotension, metabolic acidosis and a pathologicalchange suggestive of disseminated intravascular coagulationwith concomitant inhibition of elevation of plasma TNF activity.The anti-CD18 antibody also inhibited the hypotension inducedby administering recombinant TNF. Furthermore, an antibody againsta ligand for CD18 complexes, intercellular adhesion molecule-1,also prevented TNF-induced shock as well as endotoxin shockinrabbits. These observations suggest that adhesion of leukocytesto endothelium may be of primary importance in the action ofTNF as well as in the production of TNF in vivo and that theantibody against adhesion molecules could be of therapeuticbenefit in life-threatening septic shock in humans.     

Cell to cell contact through ICAM-1-LFA-1 and TNF-alpha synergistically contributes to GM-CSF and subsequent cytokine synthesis in DBA/2 mice induced by 1,3-beta-D-Glucan SCG.

SCG is a major 6-branched 1,3-beta-D-glucan in Sparassis crispa Fr. showing antitumor activity. We recently found that the splenocytes from naive DBA/1 and DBA/2 mice are potently induced by SCG to produce interferon- gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-12p70 (IL-12p70), and that GM-CSF plays a key biologic role among these cytokines. In this study, we investigated the contribution of cell-cell contact and soluble factors to cytokine induction by SCG in DBA/2 mice. Cell-cell contact involving intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1) was an essential step for the induction of GM-CSF and IFN-gamma by SCG but not for the induction of TNF-alpha or IL-12p70 by SCG. SCG directly induced adherent splenocytes to produce TNF-alpha and IL-12p70. GM-CSF was required for the induction of TNF-alpha by SCG, and in turn, TNF-alpha enhanced the release of GM-CSF and thereby augmented the induction of IL-12p70 and IFN-gamma by SCG. Neutralization of IL-12 significantly inhibited the induction of IFN-gamma by SCG. We concluded that induction of GM-CSF production by SCG was mediated through ICAM-1 and LFA-1 interaction, GM-CSF subsequently contributed to further cytokine induction by SCG, and reciprocal actions of the cytokines were essential for enhancement of the overall response to SCG in DBA/2 mice.     

Hepatocellular carcinoma arising in non-alcoholic steatohepatitis

The incidence and significance of hepatocellular carcinoma (HCC) in non-alcoholic steatohepatitis (NASH) has not been previously evaluated in detail. We recently experienced a case of NASH with multicentric HCC in a female patient. At the age of 58 years, the patient was diagnosed with non-insulin-dependent diabetes mellitus, treated by insulin therapy. The patient did not drink alcohol. She was negative for all serological markers of hepatitis B and C virus infection. Because of liver dysfunction, a needle biopsy was performed at the age of 62 years, and pathological findings, such as fatty change, Mallory's body, nuclear glycogen and pericellular fibrosis, suggested a diagnosis of NASH. Subsequently, four nodules were detected in the liver by imaging. Liver biopsies were performed from each nodule. One nodule was pathologically diagnosed as a pseudolymphoma, while three other nodules were moderately differentiated HCC (10 years after the diagnosis of non-alcoholic steatohepatitis), well-differentiated HCC (11 years later) and dysplastic nodule (11 years later), suggesting multicentric occurrence of HCC. This case suggests that HCC could be a late complication of NASH.     

Somatic activation of beta-catenin bypasses pre-TCR signaling and TCR selection in thymocyte development

Mutation or ablation of T cell factor 1 and lymphocyte enhancer factor 1 indicated involvement of the Wnt pathway in thymocyte development. The central effector of the Wnt pathway is beta-catenin, which undergoes stabilization upon binding of Wnt ligands to frizzled receptors. We report here that conditional stabilization of beta-catenin in immature thymocytes resulted in the generation of single positive T cells that lacked the alpha beta TCR and developed in the absence of pre-TCR signaling and TCR selection. Although active beta-catenin induced differentiation in the absence of TCRs, its action was associated with reduced proliferation and survival when compared to developmental changes induced by the pre-TCR or the alpha beta TCR.     

Culture of chondrocytes in fibroin-hydrogel sponge

Fibroin-hydrogel sponge and collagen gel were used as scaffold for in vitro cartilage regeneration. Fibroin-hydrogel sponge was formed by phase separation from freezed fibroin solution. Chondrocytes were harvested from proximal humerus, distal femur and proximal tibia of 4-week-old Japanese white rabbits and inoculated in the fibroin-hydrogel sponge and collagen gel. Those constructs were cultured in DMEM supplemented with 10% FCS and 50 ml L-ascorbate at 37 degrees C. Histological observation, measurement of sulfated glycosaminoglycan and cell density were carried out at 3, 7, and 14 days after the cultivation. Well-defined cartilage tissue can be seen both in the fibroin-hydrogel sponge and in the collagen gel. The matrix was intensely stained by safranin-O and showed a metachromatic reaction in both group. However, the quantity of sulfated glycosaminoglycan and cell density of the fibroin-hydrogel sponge group were increased more rapidly than these of the collagen gel group. Thus, the chondrocytes proliferated in the fibroin sponge without losing their differentiated phenotype. It is possible that culture environment in the fibroin sponge was suitable for chondrocytes regeneration.