Convergent synthesis of functionalized derivatives of stage-specific embryonic antigens 3 & 4.

Stage-specific embryonic antigens (SSEAs) are carbohydrate markers that have diverse roles in embryonic development. However, the exact roles of SSEAs remain unclear. To obtain mechanistic insights into their roles, we aimed to develop functionalized SSEA glycan analogs via chemical synthesis. Herein, we report a convergent synthetic approach for SSEA-3 and SSEA-4 analogs using readily available versatile building blocks. A key step, namely the stereoselective glycosylation of a common tetrasaccharide acceptor, was successfully achieved using a 4-O-Bn Gal donor for SSEA-3 and a Neu-Gal donor for SSEA-4, which were previously developed by our group. The obtained SSEA-3 and SSEA-4 glycans were further functionalized with biotin and deuterated lipid for applications in biological studies. Thus, the findings of this study will facilitate further research on SSEAs.

Transcription of DNA duplex containing deoxypseudouridine and deoxypseudoisocytidine, and inhibition of transcription by triplex forming oligonucleotide that recognizes the modified duplex.

We developed new DNA triplexes that contain four base triads T-A·T, A-ψ·CBr, G-PIC·YO, and C-G·Py+, where CBr, YO, Py, ψ, and PIC are 5-bromocytosine, 5-methyl-4-pyrimidone, 2-aminopyridine, the aglycons of deoxypseudouridine, and deoxypseudoisocytidine, respectively. DNA duplex incorporating T-A, A-ψ, G-PIC, and C-G, and triplex forming oligonucleotide incorporating T, CBr, YO, and Py formed the triplex as evaluated by Tm measurements. The triplex formation was successfully applied to the inhibition of transcription of the DNA duplex incorporating T7-promoter sequence modified by the above modified bases.

Mice with defects in HB-EGF ectodomain shedding show severe developmental abnormalities.

Heparin-binding EGF-like growth factor (HB-EGF) is first synthesized as a membrane-anchored form (proHB-EGF), and its soluble form (sHB-EGF) is released by ectodomain shedding from proHB-EGF. To examine the significance of proHB-EGF processing in vivo, we generated mutant mice by targeted gene replacement, expressing either an uncleavable form (HBuc) or a transmembrane domain-truncated form (HBdeltatm) of the molecule. HB(uc/uc) mice developed severe heart failure and enlarged heart valves, phenotypes similar to those in proHB-EGF null mice. On the other hand, mice carrying HBdeltatm exhibited severe hyperplasia in both skin and heart. These results indicate that ectodomain shedding of proHB-EGF is essential for HB-EGF function in vivo, and that this process requires strict control.

Clinical significance of heparin-binding epidermal growth factor-like growth factor and a disintegrin and metalloprotease 17 expression in human ovarian cancer.

PURPOSE: Lysophosphatidic acid, which is enriched in the peritoneal fluid of ovarian cancer patients, plays a key role in the progression of ovarian cancer. Lysophosphatidic acid can generate epidermal growth factor receptor (EGFR) signal transactivation involving processing of EGFR ligands by ADAM (a disintegrin and metalloprotease) family metalloproteases. We aimed to investigate the clinical significance of EGFR ligands and ADAM family in the lysophosphatidic acid-induced pathogenesis of ovarian cancer. EXPERIMENTAL DESIGN: We examined the expression of EGFR ligands and ADAM family members in 108 patients with normal ovaries or ovarian cancer, using real-time PCR, immunohistochemistry, and in situ hybridization, and analyzed the clinical roles of these molecules. Statistical analyses of these data were done using the Mann-Whitney test, Kaplan-Meier method, or Spearman's correlation analysis. RESULTS: Large differences in expression were found for heparin-binding EGF-like growth factor (HB-EGF) and other EGFR ligands and for ADAM 17 and other ADAM family members. HB-EGF expression was significantly increased in advanced ovarian cancer compared with that in normal ovaries (P < 0.01). HB-EGF expression was significantly associated with the clinical outcome (P < 0.01). ADAM 17 expression was significantly enhanced in both early and advanced ovarian cancer compared with that in normal ovaries (both P < 0.01), although it had no clinical significance in the progression-free survival. HB-EGF expression was significantly correlated with ADAM 17 expression (gamma = 0.437, P < 0.01). CONCLUSIONS: Our findings suggest that HB-EGF and ADAM 17 contribute to the progression of ovarian cancer and that HB-EGF plays a pivotal role in the aggressive behavior of a tumor in ovarian cancer.

Heparin-binding EGF-like growth factor is a promising target for ovarian cancer therapy.

Ovarian cancer is the most frequent cause of cancer death among all gynecologic cancers. We demonstrate here that lysophosphatidic acid (LPA)-induced ectodomain shedding of heparin-binding EGF-like growth factor (HB-EGF) is a critical to tumor formation in ovarian cancer. We found that among the epidermal growth factor receptor (EGFR) family of growth factors, HB-EGF gene expression in cancerous tissues and HB-EGF protein levels in patients' ascites fluid were significantly elevated. The human ovarian cancer cell lines SKOV3 and RMG-1 form tumors in nude mice. Tumor formation of these cells was enhanced by exogenous expression of pro-HB-EGF and completely blocked by pro-HB-EGF gene RNA interference or by CRM197, a specific HB-EGF inhibitor. Transfection with mutant forms of HB-EGF indicated that the release of soluble HB-EGF is essential for tumor formation. LPA, which is constitutively produced by ovarian cancer cells, induced HB-EGF ectodomain shedding in SKOV3 and RMG-1 cells, resulting in the transactivation of EGFR and the downstream kinase extracellular signal-regulated kinase/mitogen-activated protein kinase. LPA-induced transactivation was abrogated by HB-EGF gene RNA interference or by CRM197. Introduction of lipid phosphate phosphohydrolase, which hydrolyzes LPA, decreased the constitutive shedding of HB-EGF, EGFR transactivation, and the tumorigenic potential of SKOV3 and RMG-1 cells. These results indicate that HB-EGF is the primary member of the EGFR family of growth factors expressed in ovarian cancer and that LPA-induced ectodomain shedding of this growth factor is a critical step in tumor formation, making HB-EGF a novel therapeutic target for ovarian cancer.

Maternal deprivation in neonatal rats of different conditions affects growth rate, circadian clock, and stress responsiveness differentially.

Effects of periodic maternal deprivation (MD) were examined in rat pups on growth rate, circadian phase and period at weaning, and stress responsiveness in adulthood. MD was performed from postnatal day 1 to day 6 or day 7, with or without keeping ambient temperature at 37 degrees C and humidity at 70-80% during deprivation. Times of day and length of MD were also changed. Body weights were significantly reduced at weaning in MD12 (MD for 12 h) and MD6am (MD for 6 h in the morning) pups, whereas they were not changed in MD6pm (MD in the afternoon) and all MD3 groups. At 8 weeks old, body weight was still significantly lower in MD12 than the control, but not different from the control in other groups. Circadian phases of free-running locomotor rhythm at weaning were almost reversed in MD12, MD6am and MD6pm as compared with those in the control. Intermediate phase-shifts were observed in MD3Eam (3 h MD in the first quarter of the light phase; early am) and MD3Lam (late am; the second quarter), whereas no phase-shift was detected in MD3Epm (early pm; the third quarter) and MD3Lpm (late pm; the fourth quarter). Elevation of plasma corticosterone level after novelty exposure at 8 weeks old was more robustly in MD12 and MD3Lam than in the control, but the hormone response in MD3Lpm was not different from the control. Keeping ambient temperature at 37 degrees C during MD did not rescue the MD-induced body weight loss, but attenuated the phase-shifts of the circadian clock, and completely cancelled the stress-induced hormone response in MD12 rats. These findings indicate that MD in rat pups differentially affects growth rate, circadian clock, and stress responsiveness in adulthood, depending on time of day, length of MD and ambient temperature during MD.

Maternal deprivation in neonatal rats alters the expression of circadian system under light-dark cycles and restricted daily feeding in adulthood.

Effects of maternal deprivation (MD) with different conditions were examined on the circadian rhythms in plasma corticosterone and locomotor activity in adult rats under ad libitum and restricted daily feeding (RF), in which rats had free access to food for 2 h for 3 weeks. Three different types of MD were performed from postnatal day 1 (P1) to day 6 (P6); MD for 12 h/day (MD12), for 3 h/day in the morning (MD3am) and in the afternoon (MD3pm). Under ad libitum feeding, corticosterone levels at 08:00 h and 24:00 h were significantly increased in MD12 rats. A similar increase was detected in MD3am rats, but not in MD3pm rats. Under the RF, corticosterone levels at 08:00 h, 24:00 h, and 04:00 h were significantly increased in MD12 rats. Similar elevations were detected in MD3am rats, but not in MD3pm rats. Furthermore, prefeeding locomotor activities under the RF increased significantly in MD12 rats. A similar increase was observed in MD3am rats, but not in MD3pm rats. Keeping ambient temperature and humidity at 37 degrees C and 70-80% during MD attenuated the effects of MD on the corticosterone rhythmicities under RF as well as ad lib feeding. It is concluded that MD in the early postnatal period influences the expressions of circadian rhythms in plasma corticosterone and locomotor activity in adult rats. Not only the length but also the time of day of MD seems to be critical for these effects. Ambient temperature and/or humidity are suggested to be one of the factors by which MD exerts its effect.

Biologic significance of receptor-binding cancer antigen expressed on SiSo cells (RCAS1) as a pivotal regulator of tumor growth through angiogenesis in human uterine cancer.

BACKGROUND: The expression of receptor-binding cancer antigen expressed on SiSo cells (RCAS1) is related significantly to the overall survival of patients with various cancers. RCAS1 reportedly induces apoptotic cell death in peripheral lymphocytes, which may contribute to the escape of tumor cells from immune surveillance. RCAS1 expression also has been related to tumor invasiveness and size in uterine cervical cancer. To clarify whether RCAS1 exacerbates tumor progression, the authors investigated the association between RCAS1 expression and tumor growth potential. METHODS: The authors constructed small interfering ribonucleic acid (RNA) (siRNA) to target RCAS1. After transfection of siRNA and the RCAS1-encoding gene, growth of tumor cells was assessed in vitro and in vivo. The correlation between RCAS1 expression and angiogenesis was investigated in the transfected cells and in inoculated tumors from nude mice. In addition, the same association was investigated immunohistochemically with tissue samples from patients with uterine cervical cancer. RESULTS: Knockdown of RCAS1 expression by siRNA significantly suppressed the in vivo growth of SiSo and HOUA tumor cells (P < .005); however, in vitro cell growth was not affected significantly. Enhanced RCAS1 expression significantly promoted in vivo growth, but not in vitro growth, of tumors derived from COS-7 cells (P = .0039). Introduction of the RCAS1-encoding gene increased expression of vascular endothelial growth factor (VEGF). In uterine cervical cancer, RCAS1 expression was associated significantly with VEGF expression (P = .0407) and with microvessel density (P = .0108). CONCLUSIONS: RCAS1 may be a pivotal regulator of tumor growth through angiogenesis. Continued exploration of the biologic function of RCAS1 may allow the development of novel therapeutic strategies for uterine cancer.

Repeated exposures to daytime bright light increase nocturnal melatonin rise and maintain circadian phase in young subjects under fixed sleep schedule.

Effects of two different light intensities during daytime were examined on human circadian rhythms in plasma melatonin, core body temperature, and wrist activity under a fixed sleep schedule. Sleep qualities as indicated by polysomnography and subjective sleepiness were also measured. In the first week, under dim light conditions ( approximately 10 lx), the onset and peak of nocturnal melatonin rise were significantly delayed, whereas the end of melatonin rise was not changed. The peak level of melatonin rise was not affected. As a result, the width of nocturnal melatonin rise was significantly shortened. In the second week, under bright light conditions ( approximately 5,000 lx), the phases of nocturnal melatonin rise were not changed further, but the peak level was significantly increased. Core body temperature at the initial sleep phase was progressively elevated during the course of dim light exposure and reached the maximum level at the first night of bright light conditions. Subjective sleepiness gradually declined in the course of dim light exposure and reached the minimum level at the first day of bright light. These findings indicate that repeated exposures to daytime bright light are effective in controlling the circadian phase and increasing the peak level of nocturnal melatonin rise in plasma and suggest a close correlation between phase-delay shifts of the onset of nocturnal melatonin rise or body temperature rhythm and daytime sleepiness.

The stress- and inflammatory cytokine-induced ectodomain shedding of heparin-binding epidermal growth factor-like growth factor is mediated by p38 MAPK, distinct from the 12-O-tetradecanoylphorbol-13-acetate- and lysophosphatidic acid-induced signaling cascades.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a critical growth factor for a number of physiological and pathological processes. HB-EGF is synthesized as a membrane-anchored form (pro-HB-EGF), and pro-HB-EGF is cleaved at the cell surface to yield soluble HB-EGF by a mechanism called "ectodomain shedding." We show here that the ectodomain shedding of pro-HB-EGF in Vero cells is induced by various stress-inducing stimuli, including UV light, osmotic pressure, hyperoxidation, and translation inhibitors. The pro-inflammatory cytokine interleukin-1beta also stimulated the ectodomain shedding of pro-HB-EGF. An inhibitor of p38 MAPK (SB203580) or the expression of a dominant-negative (dn) form of p38 MAPK inhibited the stress-induced ectodomain shedding of pro-HB-EGF, whereas an inhibitor of JNK (SP600125) or the expression of dnJNK1 did not. 12-O-Tetradecanoylphorbol-13-acetate (TPA) and lysophosphatidic acid (LPA) are also potent inducers of pro-HB-EGF shedding in Vero cells. Stress-induced pro-HB-EGF shedding was not inhibited by the inhibitors of TPA- or LPA-induced pro-HB-EGF shedding or by dn forms of molecules involved in the TPA- or LPA-induced pro-HB-EGF shedding pathway. Reciprocally, SB203580 or dnp38 MAPK did not inhibit TPA- or LPA-induced pro-HB-EGF shedding. These results indicate that stress-induced pro-HB-EGF shedding is mediated by p38 MAPK and that the signaling pathway induced by stress is distinct from the TPA- or LPA-induced pro-HB-EGF shedding pathway.