SR proteins preferentially associate with mRNAs in the nucleus and facilitate their export to the cytoplasm

Different classes of RNA are exported to the cytoplasm by distinct mechanisms. Each class of RNA forms distinct complexes with nuclear proteins prior to its export to the cytoplasm. In our attempt to obtain comprehensive information of protein factors that specifically associate with mRNAs in the nucleus, we performed in vivo UV-crosslinking analysis after microinjection of various RNAs into Xenopus oocyte nucleus. We found a group of proteins preferentially crosslinked to mRNAs. Immunoprecipitation experiments revealed that some of the crosslinked signals corresponded to SR (serine/arginine-rich) proteins, a family of essential RNA-binding proteins involved in pre-mRNA splicing. It was previously suggested that some members of SR protein family are involved in export of a specific intronless mRNA, histone H2A mRNA and some spliced mRNAs. However, it is still to be clarified if SR proteins are involved in export of general mRNAs, especially general intronless mRNAs that do not contain specific RNA export elements. When we microinjected an antibody against SR proteins into the nucleus, export of mRNAs was severely inhibited, regardless of whether the mRNAs were produced via pre-mRNA splicing or not, whereas export of other RNAs was not affected. These results unequivocally showed that SR proteins are involved in export of both general intronless and spliced mRNAs.     

Development of a particle agglutination assay system for detecting Japanese encephalitis virus-specific human IgM,using hydroxyapatite-coated nylon beads

Japanese encephalitis virus-specific IgM is a reliable indicator for serodiagnosis of Japanese encephalitis. A particle agglutination (PA) assay system was developed to detect anti-Japanese encephalitis virus IgM in human serum samples. The newly developed PA assay consisted of hydroxyapatite-coated nylon beads and V-bottom 96-well microplates. Hydroxyapatite-coated nylon beads were coated with Japanese encephalitis virus antigens. Japanese encephalitis virus antigen-coated, hydroxyapatite-coated nylon beads agglutinated in the IgM-captured wells when anti-Japanese encephalitis virus IgM-positive serum samples were used. A button pattern was formed at the bottom of the wells when anti-Japanese encephalitis virus IgM-negative serum samples were used. Thirty anti-Japanese encephalitis virus IgM-positive serum samples from Japanese encephalitis-confirmed cases were tested by the PA assay. All these serum samples were determined to be Japanese encephalitis virus IgM-positive. IgM titers determined by the PA assay corresponded to those determined by enzyme-linked immunosorbent assay. The titers were consistent in two independent PA assays. These results indicate that the newly developed PA assay is a reliable method for detecting anti-Japanese encephalitis virus IgM in human serum samples and that this assay will be a suitable diagnostic system especially in rural areas of Asia.     

Survey of atopic dermatitis and skin barrier functions in Japanese and Chinese school students]

BACKGROUND: The comparative studies of the prevalence of atopic dermatitis and skin barrier functions in Japanese and Chinese were performed. METHODS: Clinical investigations were performed in 68 elementary school students in Lhasa, Tibet Autonomous Region and 67 students in Yixing, Jiangsu Province in China, and 99 students in Nishinomiya, Hyogo in Japan. Transepidermal water loss (TEWL) and capacitance were measured. Questionary survey about bathing frequency was also performed for students in Lhasa, Yixing and Osaka. RESULT: The prevalence rate of atopic dermatitis was 0% in Lhasa, 2.63% in Yixing, 4.26% in Nishinomiya. TEWL of students in Nishinomiya was higher than that in Yixing and Lhasa. Capacitance of students in Lhasa was lower than that in Nishinomiya and Yixing. The frequency of taking a bath in Lhasa was about 2.2 times per month and fewer than that in Nishinomiya and Yixing. CONCLUSION: There was tendency that the prevalence of atopic dermatitis increased according to increase of TEWL. It was thought that more investigations are necessary whether the development of habitat and environment influence the prevalence of atopic dermatitis and skin barrier function.     

Disseminated Atypical Mycobacteriosis

Two cases of disseminated infection caused by Mycobacterium intracellulare were reported and discussed. In the first case, the patient was a fifty-seven-year-old male who complained of general fatigue, weight loss, and fever. Biopsy of the right inguinal lymph nodes and the liver revealed infiltration by histiocytes engulfing many acid-fast bacilli. At autopsy an egg-sized abscess was found is the region of the right Iliac lymph nodes. Histological examination showed histiocytic infiltration in the abscess wall, neighboring lymph nodes, liver, and spleen. In the second case, the patient was a four-year-old boy, who had persistent fever and splenomegaly. Splenectomy was performed and histological examination of the spleen revealed multiple nodular infiltration by swollen histiocytes with many acid-fast bacilli in their cytoplasm. The bone marrow aspirates and liver tissue obtained in the necropsy also showed many histiocytes containing many acid-fast bacilli. The authors emphasized the importance of paying special attention to atypical mycobacteriosis in feverish patients having lesions with a proliferation of histiocytes.     

Age-related changes in gene expression patterns of matrix metalloproteinases and their collagenous substrates in mandibular condylar cartilage in rats

Matrix metalloproteinases (MMPs) have been implicated in physiological cartilage matrix remodelling as well as in pathological and invasive extracellular matrix remodelling of tissue. Age-related changes in the gene expression patterns of MMPs in mandibular condylar cartilages (MCCs) were analysed. We examined the gene expression patterns of Mmp-8 and -13 and their substrates, Col1a1, Col2a1 and Col10a1, in MCC of growing and ageing rats. Temporomandibular joints of male Wistar rats aged 4, 8, 16 and 32 weeks were subjected to in situ hybridization analysis. Histologically, MMCs showed characteristics of growth plate cartilage at ages 4 and 8 weeks, and more closely resembled articular cartilage thereafter. Mmp-8 was expressed in the cells in all cartilaginous cell layers at ages 4 and 8 weeks, and then was localized only in the mature cells at ages 16 and 32 weeks. Whereas Mmp-13 expression was limited to the lowermost hypertrophic chondrocytes in the growth stage, mature chondrocytes instead of hypertrophic chondrocytes expressed Mmp-13 in adult non-hypertrophic MCC. Because Mmp-8 and -13 expression overlapped with Col2a1 and Col10a1, chondrocytes could play a pivotal role in degradation as well as production of the cartilaginous matrix in MCC.     

Involvement of cAMP response element-binding protein activation in salivary secretion.

OBJECTIVE: Saliva secretion is mediated by cAMP and the calcium signaling pathway in salivary acinar cells. The PKA signaling pathway plays an important role in protein secretion through the activation of cAMP, in fluid secretion through the elevation of intracellular calcium and in the activation of cAMP response element-binding protein (CREB), which is involved in these signaling cascades. In this study, we investigated whether the activation of CREB plays a part in the salivary secretion in mice. METHODS: We examined CREB activation by assessing phosphorylation at the serine-133 position using Western blotting. RESULTS: Carbachol (a muscarinic acetylcholine agonist) and isoproterenol (a beta-adrenergic agonist) markedly activated CREB in parotid acinar cells. Carbachol and isoproterenol-induced CREB phosphorylation was blocked by atropine (a muscarinic acetylcholine antagonist) and propranolol (a beta-adrenergic antagonist), respectively. The PKA inhibitor H89 inhibited CREB activation, but the PLC inhibitor U73122 did not. Moreover, carbachol- and isoproterenol-stimulated amylase secretion from parotid acinar cells was inhibited by H89 and adenoviral dominant-negative CREB. CONCLUSION: These results indicate that the muscarinic and beta-adrenergic activation of CREB was mediated through the PKA pathway and that CREB is involved in protein secretion from parotid acinar cells.     

Balloon cell nevus of the soft palate: an immunohistochemical and ultrastructural study

An ultrastructural analysis of oral balloon cell nevus of intramucosal type complemented with an immunohistochemical study was performed for the first time. The lesion was composed of large balloon cells with an admixture of small nevus cells and melanophages at the periphery. Balloon cells showed cytoplasmic accumulation of vacuoles of varying sizes and the presence of microgranular and vacuolated melanosomes were found. Residual cytoplasm contained no identifiable organelles. A spectrum of transitional forms between balloon cells and conventional nevus cells with microvacuoles was readily observed. Both cells exhibited intense immunoreactivity to multiple melanocytic markers. Ballooning phenomenon was not evident in melanophages containing a large amount of melanosome complex. It can be inferred, from the present and previous observations, that progressive vacuolization of melanosomes in nevomelanocytes may be responsible for the formation of peculiar ballooning appearance, suggesting an aberrant melanogenesis.     

Flavonoids such as luteolin, fisetin and apigenin are inhibitors of interleukin-4 and interleukin-13 production by activated human basophils

BACKGROUND: We have previously shown that fisetin, a flavonol, inhibits IL-4 and IL-13 synthesis by allergen- or anti-IgE-antibody-stimulated basophils. This time, we investigated the inhibition of IL-4 and IL-13 production by basophils by other flavonoids and attempted to determine the fundamental structure of flavonoids related to inhibition. We additionally investigated whether flavonoids suppress leukotriene C4 synthesis by basophils and IL-4 synthesis by T cells in response to anti-CD3 antibody. METHODS: Highly purified peripheral basophils were stimulated for 12 h with anti-IgE antibody alone or anti-IgE antibody plus IL-3 in the presence of various concentrations of 18 different kinds of flavones and flavonols. IL-4 and IL-13 concentrations in the supernatants were then measured. Leukotriene C4 synthesis was also measured after basophils were stimulated for 1 h in the presence of flavonoids. Regarding the inhibitory activity of flavonoids on IL-4 synthesis by T cells, peripheral blood mononuclear cells were cultured with flavonoids in anti-CD3-antibody-bound plates for 2 days. RESULTS: Luteolin, fisetin and apigenin were found to be the strongest inhibitors of both IL-4 and IL-13 production by basophils but did not affect leukotriene C4 synthesis. At higher concentrations, these flavonoids suppressed IL-4 production by T cells. Based on a hierarchy of inhibitory activity, the basic structure for IL-4 inhibition by basophils was determined. CONCLUSIONS: Due to the inhibitory activity of flavonoids on IL-4 and IL-13 synthesis, it can be expected that the intake of flavonoids, depending on the quantity and quality, may ameliorate allergic symptoms or prevent the onset of allergic diseases.     

A possible role of two hydrophobic amino acids in antigen recognition by synovial T cells in rheumatoid arthritis

Synovial T cells play a crucial role in the pathogenesis of rheumatoid arthritis (RA) synovitis. We have quantitatively analyzed the T cell receptor (TcR) variable (V) region gene repertoire of freshly isolated synovial fluid (SF) T cells, comparing it with that of peripheral blood (PB) T cells in RA. The TcR V gene repertoire of PB and SF T cells in RA and osteoarthritis was heterogeneous. In contrast, Vail in SF was expressed to a greater degree in three of five RA patients, and increased levels of Vp6, 1-3 were found in the SF of four of six RA, compared with paired PB. Of note, Vβ6, 1–3 was universally used in four RA patients with a disease duration of less than 10 years, irrespective of their HLA-DR types. This was in contrast to two other RA patients, suffering for more than 20 years, who showed different Vα and Vβ usages. β-chain sequence analysis in RA patients with a preference for Vβ6, 1–3 has shown that a few clones dominated in SF, whereas polyclonality was observed in PB. These findings suggest oligoclonal expansion of T cells in response to specific antigen(s) in the SF of these patients with RA of relatively short duration. Concomitant use of two hydrophobic amino acids, leucine and valine, in the Dβ region was noticeable among the predominant SF clones. These two amino acids might directly contact a peptide specific for the induction of synovitis in RA patients. TcR-directed therapy may, therefore, be useful for the treatment of early RA synovitis.     

Correlation between the number of apoptotic cells and expression of the apoptosis-related antigens Fas, Ley and bcl-2 protein in non-Hodgkin's lymphomas

The relationship between the number of apoptotic cells and the expression of apoptosis-related antigens was examined In 56 cases of non-Hodgkin's lymphomas and in 10 cases of reactive hyperplastic lymph nodes (RHL). Apoptosis was visually quantified by the in situ end-labeling (ISEL) method, and the expression of Fas, Le^y antigens and bcl-2 protein was examined by Immunohistochemistry. The expression of Le^y antigen was observed in germinal centers of RHL and 45% of non-Hodgkin's lymphomas. The apoptotic cell count (AC) in follicular lymphomas was significantly less than that in diffuse lymphomas. The distribution pattern of apoptotic cells In follicular lymphomas was inverse to that in RHL. In follicular lymphomas, AC was lower in follicles than in inter-follicular areas. In contrast, AC was higher in follicles than in Interfollicular areas in RHL. Le^y antigen-positive lymphomas showed a significantly higher AC than the negative cases. The Fas antigen-positive lymphomas showed a higher AC than the negative cases. However, AC in bcl-2 protein-positive and negative cases was not significantly different. These results suggest that Le^y and Fas antigens appear to be involved in the apoptotic tendency of tumor cells in non-Hodgkin's lymphomas, whereas bcl-2 does not necessarily.