A new kinetochore component CENP-W interacts with the polycomb-group protein EZH2 to promote gene silencing.

Polycomb recessive complex 2 (PRC2) plays a central roles in chromatin compaction and remodeling. EZH2, the catalytic subunit of PRC2, is frequently overexpressed in many human tumors. Together with another essential core component, SUZ12, EZH2 trimethylates histone H3 on lysine 27 (H3K27me3). CENP-W was originally identified as a putative oncogene overexpressed in various human tumors, and later characterized as an essential factor for the formation of functional kinetochore during mitosis. In this study, we found that CENP-W associates with EZH2 to subsequently enhance the protein stability of EZH2. Chromatin immunoprecipitation revealed that ectopically expressed CENP-W bound the promoter of EZH2 target genes to enhance EZH2-mediated transcriptional repression, possibly by facilitating the recruitment of EZH2 to its target genes. Collectively, this study suggests CENP-W is a novel kinetochore component that may be involved in the EZH2-mediated silencing machinery.

CSN5/JAB1 interacts with the centromeric components CENP-T and CENP-W and regulates their proteasome-mediated degradation.

The CENP-T·CENP-W complex is a recently identified inner centromere component that plays crucial roles in the formation of a functional kinetochore involved in cell division during mitosis. Using yeast two-hybrid screening, we identified an interaction between CENP-T and CSN5, the fifth component of the COP9 signalosome and a key modulator of the cell cycle and cancer. Co-immunoprecipitation revealed that CSN5 directly interacts with both CENP-T and CENP-W. Ectopically expressed CSN5 promoted the ubiquitin- and proteasome-dependent degradation of CENP-T·CENP-W. The formation of a CENP-T·CENP-W complex greatly enhanced the stabilities of the respective proteins, possibly by blocking CSN5-mediated degradation. Furthermore, dysregulation of CSN5 induced severe defects in the recruitment of CENP-T·CENP-W to the kinetochore during the prophase stage of mitosis. Thus, our results indicate that CSN5 regulates the stability of the inner kinetochore components CENP-T and CENP-W, providing the first direct link between CSN5 and the mitotic apparatus, highlighting the role of CSN5 as a multifunctional cell cycle regulator.

New centromeric component CENP-W is an RNA-associated nuclear matrix protein that interacts with nucleophosmin/B23 protein.

CENP-W was originally identified as a putative oncogene, cancer-upregulated gene 2 (CUG2) that was commonly up-regulated in many cancer tissues. Recently, CENP-W has also been identified as a new centromeric component that interacts with CENP-T. As a complex with CENP-T, CENP-W plays crucial roles in assembly of the functional kinetochore complex. In this study, the subnuclear localization of CENP-W was extensively analyzed using various approaches. We found that ectopically expressed CENP-W primarily accumulated in the nucleolus and remained substantially associated with the nucleolus in stable cells. The following fractionation study also showed that CENP-W is associated with RNA as well as DNA. Moreover, a considerable amount of CENP-W was found in the nuclear mesh-like structure, nuclear matrix, possibly indicating that CENP-W participates in diverse subnuclear activities. Finally, biochemical affinity binding analysis revealed that CENP-W specifically interacts with the nucleolar phosphoprotein, nucleophosmin (B23). Depletion of cellular B23 by siRNA treatment induced a dramatic decrease of CENP-W stability and severe mislocalization during prophase. Our data proposed that B23 may function in the assembly of the kinetochore complex by interacting with CENP-W during interphase.

New cell line development for antibody-producing Chinese hamster ovary cells using split green fluorescent protein.

BACKGROUND: The establishment of high producer is an important issue in Chinese hamster ovary (CHO) cell culture considering increased heterogeneity by the random integration of a transfected foreign gene and the altered position of the integrated gene. Fluorescence-activated cell sorting (FACS)-based cell line development is an efficient strategy for the selection of CHO cells in high therapeutic protein production. RESULTS: An internal ribosome entry site (IRES) was introduced for using two green fluorescence protein (GFP) fragments as a reporter to both antibody chains, the heavy chain and the light chain. The cells co-transfected with two GFP fragments showed the emission of green fluorescence by the reconstitution of split GFP. The FACS-sorted pool with GFP expression had a higher specific antibody productivity (q(Ab)) than that of the unsorted pool. The q(Ab) was highly correlated with the fluorescence intensity with a high correlation coefficient, evidenced from the analysis of median GFP and q(Ab) in individual selected clones. CONCLUSIONS: This study proved that the fragment complementation for split GFP could be an efficient indication for antibody production on the basis of high correlation of q(Ab) with reconstitution of GFP. Taken together, we developed an efficient FACS-based screening method for high antibody-producing CHO cells with the benefits of the split GFP system.

Improvement of atopic dermatitis-like skin lesions by Platycodon grandiflorum fermented by Lactobacillus plantarum in NC/Nga mice.

Atopic dermatitis (AD) is characterized as a multi-factorial inflammatory skin disease that has been increasing worldwide. Previously, we demonstrated that FPG, which is Platycodon grandiflorum (PG) fermented by Lactobacillus plantarum (LP), increases the level of interferon (IFN)-gamma in mouse splenocytes in vitro. In this study, we investigated the effects of FPG in an animal model of AD, with a particular emphasis on its effects on T helper (Th)1 and Th2 immune responses. To assess the potential use of FPG for the inhibition of AD, we established a model of AD-like skin lesions in NC/Nga mice. Immunoglobulin isotypes (Igs) and Th1/Th2 cytokines in the sera and spleens of AD-like mice were examined. In addition, histological examination was also performed. AD symptoms in skin lesions improved following oral administration of FPG. IgE secretion was significantly down-regulated, and this was accompanied by decreased levels of interleukin (IL)-4 and IgG1 and increased serum levels of IL-12p40 and IgG2a in FPG-treated animals. In splenocytes, the production of the Th1 cytokines IL-12p40 and IFN-gamma was up-regulated, while the levels of the Th2 cytokines IL-4 and 5 were down-regulated by FPG treatment. These results suggest that FPG inhibits the development of AD-like skin lesions in NC/Nga mice by suppressing the Th2 cell response and increasing the Th1 cell responses. Our results indicate that FPG is safe and effective for the prevention of AD-like skin lesions.

Impact of preoperative ultrasonography and fine-needle aspiration of axillary lymph nodes on surgical management of primary breast cancer.

PURPOSE: To evaluate the accuracy of preoperative ultrasonography (US) and US-guided fine-needle aspiration (US-FNA) for detecting axillary lymph node (ALN) metastasis. PATIENTS AND METHODS: We retrospectively reviewed 382 breast cancer patients with clinically negative ALN who underwent US and/or US-FNA for ALN. US-FNA of ALN was performed in 121 patients with suspicious findings on US. The diagnostic performance of US alone or with the addition of US-FNA for detecting ALN metastasis was calculated on the basis of final pathologic reports of ALN surgery. RESULTS: Among a total of 382 patients, 129 had metastatic ALNs while 253 exhibited no signs of axillary metastasis on final pathology. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of axillary US alone were 56.6% (73/129), 81.0% (205/253), 60.3% (73/121), and 78.5% (205/261), respectively. Addition of US-FNA resulted in sensitivity, specificity, PPV, and NPV of 39.5% (51/129), 95.7% (242/253), 82.3% (51/62), and 75.6% (242/320), respectively. Excluding complete responders to neoadjuvant chemotherapy, specificity and PPV after adding US-FNA were increased to 99.6% (242/243) and 98.1% (51/52), respectively. The sensitivity and specificity of ALN metastasis were similar between the palpable and nonpalpable breast cancer groups; however, after adding US-FNA, NPV was increased in the nonpalpable breast cancer group compared with the palpable breast cancer group (p = 0.0398). By including preoperative axillary US and US-FNA, 16.2% (62/382) of all breast cancer patients were able to avoid unnecessary sentinel lymph node biopsy (SLNB). CONCLUSIONS: The combination of axillary US and US-FNA is useful in preoperative work-up of breast cancer patients and provides valuable information for planning proper breast cancer management.

Inhibitory effect of Platycodon grandiflorum on T(H)1 and T(H)2 immune responses in a murine model of 2,4-dinitrofluorobenzene-induced atopic dermatitis-like skin lesions.

BACKGROUND: Platycodon grandiflorum is a traditional Asian medicine that is used to treat pulmonary and respiratory allergic disorders. OBJECTIVE: to investigate the effects of P grandiflorum in vivo in an animal model of atopic dermatitis (AD), with particular emphasis on its effects on T(H)1 and T(H)2 immune responses. METHODS: we established a model of AD-like skin lesions in NC/Nga mice. After oral administration of P grandiflorum, we measured cytokine and immunoglobulin profiles along with histologic examination of skin. RESULTS: P grandiflorum was nontoxic in a 2,4-dinitrofluorobenzene-induced model of AD-like skin lesions in NC/Nga mice. AD symptoms in skin lesions improved after oral administration of P grandiflorum. IgE secretion was significantly downregulated in P grandiflorum-treated animals, accompanied by decreased levels of interleukin (IL) 4 and IgG1 and increased serum levels of IL-12p40 and IgG2a. In isolated splenocytes, the production of the T(H)1 cytokines IL-12p40 and interferon-γ was upregulated by P grandiflorum, whereas the levels of the T(H)2 cytokines IL-4 and IL-5 were downregulated in a mouse model of AD-like skin lesions. CONCLUSIONS: these results suggest that P grandiflorum inhibits the development of AD-like skin lesions in NC/Nga mice by suppressing the T(H)2 cell response and increasing the T(H)1 cell responses. Our results indicate that P grandiflorum is safe and effective as a natural herbal medicine for the treatment of AD-like skin lesions.

Newly identified tumor-associated role of human Sharpin.

In order to discover previously unidentified cancer-associated genes, we analyzed genome-wide differences in gene expression between tumor biopsies and normal tissues. Among those differentially regulated genes, we identified Sharpin (Shank-associated RH domain-interacting protein) as a commonly up-regulated gene in multiple human cancer types. Although rat Sharpin is reported to interact with Shank1, a multidomain scaffold protein localized in postsynaptic densities, its exact roles are unknown. Whereas human Sharpin homologue was primarily localized in the cytosol of cultured cells, they were detected in both cytosol and nucleus of the cells from ovarian and liver cancer tissues using immunohistochemical staining. In addition, Chinese ovary hamster cells over-expressing Sharpin exhibited enhanced cancer-specific phenotypes in multiple in vitro tumor assays. Taken together, the results suggest that Sharpin is not an inert scaffold protein, but may play tumor-associated roles during cancer biogenesis.

Sp1 and Sp3 mediate basal and serum-induced expression of human CENP-W.

Cancer-upregulated gene 2 (CUG2), which was named since it was originally identified as a putative oncogene up-regulated in various human cancers, was recently renamed CENP-W based on the new findings that it is a component of the centromeric complex playing a crucial role in the assembly of functional kinetochore complex during mitosis. To understand the transcriptional regulation of CENP-W, we analyzed its TATA-less promoter and identified a GC-rich putative Sp1 binding site located at -46 to -36 that was critical in CENP-W expression. Competitive electrophoretic gel mobility shift assay using mutated oligos and supershift assays with Sp1 and Sp3 antibodies demonstrated that both proteins specifically bound to this promoter region. Moreover, we found that CENP-W was highly induced by serum stimulation followed by serum deprivation, with Sp1 and Sp3 transcription factors involved in this transactivation. Taken together, our results suggest that Sp1 together with Sp3 may function as the main regulator of the basal and serum-induced transcription of CENP-W.

Clinical application of the BI-RADS final assessment to breast sonography in conjunction with mammography.

OBJECTIVE: The objective of our study was to report the results of classification of sonographic findings according to BI-RADS and to calculate the positive predictive value (PPV) for each BI-RADS assessment category. SUBJECTS AND METHODS: We prospectively classified 4,668 breast sonograms according to BI-RADS final assessment category. Suspicious sonographic findings were divided into major and minor suspicious findings. Category 1 was normal and category 2 was a benign finding such as cyst or nodule with uniform and intense hyperechogenicity. A nodule neither category 2 nor category 4 or 5 was defined as category 3. A nodule with one or more suspicious findings, not category 5, was defined as category 4. A nodule with two or more major suspicious findings was defined as category 5. RESULTS: Of the 4,668 cases, 321 cases failed to undergo follow-up of at least 1 year. The PPV was 0.1% in category 1 (3/2,191), 0% in category 2 (0/773), 0.8% in category 3 (6/737), 31.1% in category 4 (161/519), and 96.9% in category 5 (123/127). In palpable lesions (n = 751), the PPV was 2.2% in category 1 (2/93), 0.9% in category 3 (2/217), 54% in category 4 (107/198), and 98% in category 5 (98/100). In nonpalpable lesions (n = 3,596), the PPV was 0.05% in category 1 (1/2,098), 0.8% in category 3 (4/520), 16.8% in category 4 (54/321), and 92.6% in category 5 (25/27). CONCLUSION: As with mammography, placing sonographic lesions into BI-RADS categories is useful for predicting the presence of malignancy.

Cancer-upregulated gene 2 (CUG2), a new component of centromere complex, is required for kinetochore function.

We previously identified cancer-upregulated gene 2 (CUG2) as a commonly up-regulated gene in various human cancer tissues, especially in ovary, liver, and lung (Lee et al., 2007a). CUG2 was determined to be a nuclear protein that exhibited high proto-oncogenic activities when overexpressed in NIH3T3 mouse fibroblast cells. To identify other cellular functions of CUG2, we performed yeast two-hybrid screening and identified CENP-T, a component of CENP-A nucleosome complex in the centromere, as an interacting partner of CUG2. Moreover, CENP-A, the principle centromeric determinant, was also found in complex with CENP-T/CUG2. Immunofluorescent staining revealed the co-localization of CUG2 with human centromeric markers. Inhibition of CUG2 expression drastically affected cell viability by inducing aberrant cell division. We propose that CUG2 is a new component of the human centromeric complex that is required for proper chromosome segregation during mitosis.

Unconscious operant conditioning in the paradigm of brain-computer interface based on color perception.

This study investigate the mutual fine-tuning of ongoing EEG rhythmic features with RGB values controlling color shades of computer screen during neuro-feedback training. Fifteen participants had not been informed about the existence of neurofeedback loop (NF), but were guided only to look at the computer screen. It was found that during such unconscious NF training, a variety of color shades on the screen gradually changed from rather various types to the main one within the framework of color palette specified for each individual. This phenomenon was not observed in control experiments with simulated neuro-feedback. Individual color patterns induced on the screen during NF did not depend on the schema of connection between of EEG rhythms and RGB controller. It is suggested that the basic neurophysiological mechanism of described NF training consists of the directed selection of EEG patterns reinforced by comfortable color shades without conscious control.