Phospholipase C-beta 2 as a mammalian taste signaling marker is expressed in the multiple gustatory tissues of medaka fish, Oryzias latipes

Phospholipase C-beta 2 (PLC-beta 2) is a key enzyme in mammalian taste signal transduction. To analyze the taste system in fishes at molecular level, we cloned mfplc-beta 2 as a medaka fish homologue of PLC-beta 2. In situ hybridization analysis revealed that mfplc-beta 2 is expressed in the lip and branchial region where chemosensory tissues are distributed. Immunohistochemical detection of nerve fibers near the mfplc-beta 2 positive cells suggests the characteristic of peripheral sensory cells. These results suggest that mfplc-beta 2 is expressed in the gustatory sensory cells of medaka. This may provide a molecular basis for the taste reception at multiple tissues in fish species.     

Simple and rapid PCR method for identification of streptococcal species relevant to animal infections based on 23S rDNA sequence

A PCR identification system targeting 23S rDNA sequences for the identification of eight streptococcal species relevant to animal infections (Streptococcus agalactiae, S. bovis, S. canis, S. dysgalactiae, S. equi, S. porcinus, S. suis and S. uberis) was developed. This system consists of two PCR reactions, A and B, in which seven and eight primers, respectively, are used simultaneously, and was designed so that each amplification product indicates a species by its size. A total of 111 cultures, including the type strain of eight species, could be successfully identified and differentiated as individual species, except for the cross reactivity between S. bovis and S. equinus. The developed PCR system can complete the identification procedure for eight streptococcal species through two tube reactions per isolate, and, therefore, might provide a rapid, simple and accurate diagnostic tool for veterinary laboratories.     

Fugu immunoglobulin D: a highly unusual gene with unprecedented duplications in its constant region

We have isolated and characterized the cDNA that encodes IgD of fugu (Takifugu rubripes). Though the splicing of 1 with the 1 domain was similar to those reported for teleost IgDs, highly unusual and unprecedented domain duplications were found in the constant region of the fugu IgD. The structure of the fugu IgD is like VDJ-1-(1-2-3-4-5-6)₂-7-m1-m2. Genomic sequence analysis of the fugu IgD gene supported the results of cDNA sequencing that the first six domains in the constant region are duplicated. Such a novel duplication pattern has not been reported in any other vertebrates. However, IgD secretory domains could not be identified in this study. The deduced amino acid sequence of the fugu IgD constant region showed high identity (35–55%) to the sequences of previously reported teleost IgDs. Gene expression analyses based on RT-PCR demonstrated that the IgD gene is preferentially expressed in presumptive lymphoid tissues; moreover, in situ hybridization showed that IgD-positive cells are distributed throughout the spleen and head kidney. The expression pattern is similar to that of IgM, corroborating the hypothesis that IgD plays an important role in the humoral immune system of this species.The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession numbers AB159481 and AB159482.     

Synovial sarcoma of the thyroid. Report of a case with aspiration cytology findings and gene analysis

BACKGROUND: Synovial sarcoma, generally known as a soft tissue tumor, can also occur in the head and neck region, including the thyroid gland. Cytologic findings are important to differentiate the tumor from other types of neoplasms arising in the thyroid gland. CASE: A 60-year-old man complained of hoarseness. A palpable neck tumor was detected, and a computed tomography scan showed a thyroid tumor accompanied by destruction of the thyroid and cricoid cartilage. The results of a preoperative fine needle aspiration biopsy showed numerous spindle cells with pale cytoplasm and oval nuclei with fine, granular chromatin, all of which suggested a medullary carcinoma. The extirpated thyroid tissue weighed approximately 120 g, and a grayish white, elastic, solid tumor (6.8 x 6.5 cm) was present in the left lobe. Histologically, fasciculation of spindle cells that had proliferated solidly and densely was observed. Also, the expression of a chimera gene, SYT-SSX, was detected in the tumor tissue. CONCLUSION: Synovial sarcoma of the thyroid is extremely rare, and its diagnosis by fine needle aspiration biopsy is generally considered very difficult. The detailed cytologic findings observed here might be helpful with the differential diagnosis of thyroid neoplasms.     

Gamma-secretase activity is present in rafts but is not cholesterol-dependent

Cholesterol has been claimed to be involved in the generation and/or accumulation of amyloid beta protein (Abeta). However, the underlying molecular mechanisms have not been fully elucidated yet. Here, we have investigated the effect of membrane cholesterol content on gamma-secretase activity using Chinese hamster ovary cells stably expressing beta-amyloid precursor protein (APP) and either wild-type or N141I mutant-type presenilin 2. Cholesterol was acutely depleted from the isolated membrane by methyl-beta-cyclodextrin, and Abeta production was assessed in a cell-free assay system. Reduced cholesterol did not significantly alter the amounts of Abeta produced by either total cell membranes or cholesterol-rich low-density membrane domains. Even its extremely low levels in the latter domains did not affect Abeta production. This indicates that the membrane cholesterol content does not directly modulate the activity of gamma-secretase. To ascertain that gamma-secretase resides in cholesterol-rich membrane domains, low-density membrane domains were further fractionated with BCtheta (biotinylated theta-toxin nicked with subtilisin Carlsberg protease), which has recently been shown to bind selectively to rafts of intact cells. The membrane domains purified with BCtheta did indeed produce Abeta. These observations indicate that the gamma-cleavage required for generating Abeta occurs in rafts, but its activity is virtually cholesterol-independent.     

Secretory proprotein convertases PACE4 and PC6A are heparin-binding proteins which are localized in the extracellular matrix. Potential role of PACE4 in the activation of proproteins in the extracellular matrix

PACE4, PC6 and furin are potent subtilisin-like proprotein convertases (SPCs) which are responsible for the activation of transforming growth factor-beta (TGFbeta)-related factors such as bone morphogenetic proteins. Heparan sulfate proteoglycan within the extracellular matrix (ECM) is known to regulate the biological activity of various differentiation factors including TGFbeta-related molecules. PACE4 binds tightly to heparin and its heparin-binding region was found to be a cationic stretch of amino acids between residues 743 and 760. Furthermore, PACE4 was detected in the extracellular material fraction of the HEK293 cells, defined as the material remaining on the culture plate following the removal of the cells from the plate. PACE4 bound to the extracellular fraction was selectively dislodged by heparin into the culture medium. Heparin has no inhibitory activity against PACE4. Similarly, PC6A is also able to bind to heparin, whereas soluble furin does not. In human placenta, PACE4 is mainly present in syncytiotrophoblasts and can be released by heparin. These results suggest that PACE4 and PC6 are unique SPC family proteases that anchor heparan sulfate proteoglycans at the ECM. The interaction between PACE4 and heparan sulfate proteoglycans might play an important role in the delicate spatiotemporal regulation of TGFbeta-related factors' biological activity.     

Usage of tautomycetin,a novel inhibitor of protein phosphatase 1 (PP1), reveals that PP1 is a positive regulator of Raf-1 in vivo

Protein phosphatase type 1 (PP1), together with protein phosphatase 2A (PP2A), is a major eukaryotic serine/threonine protein phosphatase involved in regulation of numerous cell functions. Although the roles of PP2A have been studied extensively using okadaic acid, a well known inhibitor of PP2A, biological analysis of PP1 has remained restricted because of lack of a specific inhibitor. Recently we reported that tautomycetin (TC) is a highly specific inhibitor of PP1. To elucidate the biological effects of TC, we demonstrated in preliminary experiments that treatment of COS-7 cells with 5 microm TC for 5 h inhibits endogenous PP1 by more than 90% without affecting PP2A activity. Therefore, using TC as a specific PP1 inhibitor, the biological effect of PP1 on MAPK signaling was examined. First, we found that inhibition of PP1 in COS-7 cells by TC specifically suppresses activation of ERK, among three MAPK kinases (ERK, JNK, and p38). TC-mediated inhibition of PP1 also suppressed activation of Raf-1, resulting in the inactivation of the MEK-ERK pathway. To examine the role of PP1 in regulation of Raf-1, we overexpressed the PP1 catalytic subunit (PP1C) in COS-7 cells and found that PP1C enhanced activation of Raf-1 activity, whereas phosphatase-dead PP1C blocked Raf-1 activation. Furthermore, a physical interaction between PP1C and Raf-1 was also observed. These data strongly suggest that PP1 positively regulates Raf-1 in vivo.