Mice deficient for Id2, a negative regulator of basic helix–loop–helix (bHLH) transcription factors, exhibit a defect in lactation due to impaired lobuloalveolar development during pregnancy, similar to the mice lacking the CCAAT enhancer binding protein (C/EBP) β. Here, we show that Id2 is a direct target of C/EBPβ. Translocation of C/EBPβ into the nucleus, which was achieved by using a system utilizing the fusion protein between C/EBPβ and the ligand-binding domain of the human estrogen receptor (C/EBPβ-ERT), demonstrated the rapid induction of endogenous Id2 expression. In reporter assays, transactivation of the Id2 promoter by C/EBPβ was observed and, among three potential C/EBPβ binding sites found in the 2.3 kb Id2 promoter region, the most proximal element was responsible for the transactivation. Electrophoretic mobility shift assay (EMSA) identified this element as a core sequence to which C/EBPβ binds. Chromatin immunoprecipitation (ChIP) furthermore confirmed the presence of C/EBPβ in the Id2 promoter region. Northern blotting showed that Id2 expression in C/EBPβ-deficient mammary glands was reduced at 10 days post coitus (d.p.c.), compared with that in wild-type mammary glands. Thus, our data demonstrate that Id2 is a direct target of C/EBPβ and provide insight into molecular mechanisms underlying mammary gland development during pregnancy.
Streptolysin S (SLS) is a serum-extractable and oxygen-stable hemolysin produced by Group A Streptococcus. A SLS-deficient mutant in which transposon Tn 916 was inserted in a locus distinct from the sag gene cluster [Nizet et al. (2000) Infect. Immun. 68, 4245-4254] was obtained by filter mating of the transposon-harbouring Enterococcus faecalis strain and Streptococcus pyogenes BL(T). This mutant, N22, had completely lost the hemolytic activity, in consequence of insertion of a single Tn 916 into a hitherto-unknown lantibiotic gene cluster composed of 10 open reading frames. The arrangement and sequence of this lantibiotic gene cluster were similar to those of nisin and subtilin, and so we designated this new lantibiotic as streptin. The bactericidal activity of streptin was abolished on treatment with trypsin or proteinase K. The different host range and nucleotide sequence clearly distinguished streptin from streptococcins. Streptin was not hemolytic and its bacteriocin activity was independent of carrier oligonucleotides effective for SLS. The fact that N22 also lost the anti-bacterial activity against indicator streptococci reveals that the factor(s) required for lantibiotic formation plays an important role in SLS formation as well.
Cisplatin plays an important role in the therapy for human head and neck cancers. However, cancer cells develop cisplatin resistance, leading to difficulty in treatment and poor prognosis. To analyze cisplatin-resistant mechanisms, a cisplatin-resistant cell line, IMC-3CR, was established from the IMC-3 human maxillary cancer cell line. Flow cytometry revealed that, compared with IMC-3 cells, cisplatin more dominantly induced cell cycle G2/M arrest rather than apoptosis in IMC-3CR cells. That fact suggests that IMC-3CR cells avoid cisplatin-induced apoptosis through induction of G2/M arrest, which allows cancer cells to repair damaged DNA and survive. In the present study, we specifically examined Poly(rC)-Binding Protein 4 (PCBP4), which reportedly induces G2/M arrest. Results showed that suppression of PCBP4 by RNAi reduced cisplatin-induced G2/M arrest and enhanced apoptosis in IMC-3CR cells, resulting in the reduction of cisplatin resistance. In contrast, overexpression of PCBP4 in IMC-3 cells induced G2/M arrest after cisplatin treatment and enhanced cisplatin resistance. We revealed that PCBP4 combined with Cdc25A and suppressed the expression of Cdc25A, resulting in G2/M arrest. PCBP4 plays important roles in the induction of cisplatin resistance in human maxillary cancers. PCBP4 is a novel molecular target for the therapy of head and neck cancers, especially cisplatin-resistant cancers.
Growth hormone-releasing hormone (GHRH) is a well-known hypothalamic hormone that stimulates the synthesis and release of growth hormone (GH) as well as the proliferation of GH-producing cells in the anterior pituitary gland. Recent reports have shown GHRH synthesis in pituitary somatotroph adenomas, but GHRH immunoreactivity has not been demonstrated in the previous studies. In order to confirm the role of locally generated GHRH for the progression of somatotroph adenomas, we investigated the expression of GHRH in 25 pituitary somatotroph adenomas immunohistochemically using both conventional avidin-biotin-complex (ABC) method and novel catalyzed signal amplified (CSA) system.In addition, we investigated the expression of GHRH mRNA and GHRH receptor mRNA with in situ hybridization (ISH) using CSA system. The weak immunopositivity of GHRH were observed in only 2 adenomas (8.0%) out of 25 somatotroph adenomas using ABC method. In contrast, 15 adenomas (60.0%) out of 25 somatotroph adenomas were immunopositive for GHRH revealed by CSA system. The expression of GHRH mRNA was confirmed using CSA-ISH system in 13 adenomas (72.2%) out of 18 somatotroph adenomas. In 11 adenomas (61.1%) out of 18 somatotroph adenomas, the expression of GHRH receptor mRNA was demonstrated using CSA-ISH system. This is a first report that clarified histopathologically GHRH production in pituitary somatotroph adenomas. The demonstration of GHRH and its receptor expression is meaningful in clarifying the autocrinc or paracrine regulation of GHRH in GH production and progression of pituitary somatotroph adenomas. P-I-38Age-related changes of the microvasculature in the median eminence of rats. We have previously demonstrated that astroglial elements in the median eminence of aged rats (24 month-old rats), especially in the perivascular region in its outer layer, increased significantly using immunocytochemistry and computer-assisted analyzing system. According to aging. astrocytes in the perivascular region exhibited more intimate contact with the basement membrane of the perivascular space in the portal vessels compared to control groups (6 week-old rats). Therefore, age-related changes of astroglial elements are considered to affect on the microvasculature of the median eminence. In the present study, to clarify the effect of aging on the neuroendocrine system, the changes of microvasculature of the median eminence was investigated. The hypophyseal vascular plexus of aged and control rats were prepared for the injection of acrylic resin into the ascending aorta and examined with scanning electron microscope. In the saggital view, the plexuses of median eminence in the aged groups became thicker at the dorso-ventral level but these structures seemed to be not prominent change compared to those of control groups at the rostro-caudal level. The microvasculature of their capillaries increased in the diameter and exhibited to be more complex structure and they are considered to be sinusoid-like structure. In the anterior and posterior lobes, th...
Laser confocal microscopy is a powerful tool for histochemical and cytochemical studies. It enables us to detect the fluorescencelabeled molecules in the specimens by optical. Simultaneous staining of nuclei with appropriate fluorescent dyes as counterstaining greatly facilitates the identification of the localization of the label. Recently, a variety of nucleic acid binding fluorescent dyes have been developed. We screened 20 nucleic acid-specific dyes for use in nuclear staining in laser confocal microscopy with Kr/Ar laser. Results were evaluated in 1) fluorescent intensity, 2)DNA specificity, and 3)bleaching characteristics. Among green fluorochromes excited at 488 nm, YO-PRO-1, SYTOX Green, and SYBR Green l were suitable for nuclear DNA staining. Among red fluorochromes excited at 568 nm, propidium iodide stained nuclear DNA with simultaneous staining of cytoplasmic and nucleolar RNAs. Among far-red fluorochromes excited at 647 nm,TO-PRO-3 was specific for DNA, whereas TOTO-3 strongly co-stained cytoplasmic and nucleolar RNAs. Bleaching by laser illumination was retarded by mounting with media containing and-bleaching reagents such as DABCO and PPDA. Nuclear staining fluorescent dyes should be selected by comparing these characteristics. We evaluated both the number of chromosome 17 and nuclear DNA content on an identical nucleus by means of computer-controlled auto-scanning cytofluorometry in order to demonstrate the significance of the nuclei with 17-aneusomy among whole population of tumor cells in cases of human colorectal tumor. MATERIALS and METHODS We investigated a total of 14 lesions from surgically resected colorectal tumors. We prepared isolated cell smears from paraffin-embedded archival blocks, and memorized the position of the cells using a computer-controlled auto-scanning stage. Then, we evaluated nuclear DNA content and the number of chromosome 17 sequentially in the order of cell position data. We compared statistics of DNA profile between the nuclei with 17-disomy and those with 17-aneusomy. RESULTS and DISCUSSIONSWe detected an alteration of DNA profile in 3 diploid adenomas and 3 aneuploid carcinomas. In the 3 diploid adenomas, we found a minor population of cells having DNA aneuploid in the 17-aneusomy cells, and the existence of this population resulted in the significant difference between the nuclei with 17-disomy and 17-aneusomy. In the 3 aneuploid carcinomas, there was a remarkable alteration in the height of each peak of the DNA profile. We considered that an alteration in the constituent ratio of karyotypically different subpopulations among cytofluorometrically distinct subpopulations yielded the significant difference of DNA profile.
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