Knockdown of IGF‐binding protein 7 inhibits transformation of the endometrial gland in an in vitro model

Uterine endometrial glands and their secretory products are critical for the implantation and survival of the peri‐implantation embryo, and for the establishment of uterine receptivity. We previously reported that insulin‐like growth factor binding protein 7 (IGFBP7) is abundantly expressed in uterine glandular epithelial cells during the secretory phase of the menstrual cycle. In the present study, we used a cultured glandular epithelial cell line of human (EM1) to investigate the significance of IGFBP7 in the function of endometrial glands. EM1 cells formed a mesh‐like structure on Matrigel, which was accompanied by elevated levels of intracellular cyclic AMP. However, these morphological changes were blocked by treatment with protein kinase A (PKA) inhibitor (H89). IGFBP7 knockdown using specific short interference RNA (siRNA) inhibited the formation of the mesh‐like structure on Matrigel. Cyclic AMP analogs, dibutyryl‐cAMP, and N⁶‐phenyl‐cAMP induced the expression of leukemia inhibitory factor (LIF) which is essential for the onset of implantation. Enhanced LIF expression was suppressed by IGFBP7 siRNA treatment. Western blot analysis revealed that IGFBP7 knockdown results in the aberrant, constitutive expression of the MAPK signaling pathway. These results suggest that IGFBP7 regulates morphological changes of glandular cells by interfering with the normal PKA and MAPK signaling pathways that are associated with the transformation and/or differentiation of endometrial glands. Mol. Reprod. Dev. 77: 265–272, 2010. © 2009 Wiley‐Liss, Inc.     

Beneficial effect of preconditioning on ischemia-reperfusion injury in the rat bladder in vivo

We investigated the effect of preconditioning on ischemia-reperfusion injury in the rat bladder. Rat abdominal aorta was clamped with a small clip to induce ischemia-reperfusion injury in the bladder. Twelve-week-old male SD rats were divided into three groups; sham-operated control (Cont), 30 min ischemia-60 min reperfusion (IR) and three times of 5 min ischemia and then 30 min ischemia-60 min reperfusion (PC) groups. The bladder functions were estimated by cystometric and functional studies. Contractile response curves to increasing concentrations of carbachol were constructed in the absence and presence of various concentrations of subtype selective muscarinic antagonists, i.e. atropine (non-selective), pirenzepine (M1 selective), methoctramine (M2 selective), and 4-DAMP (M1/M3 selective). We also measured tissue levels of malonaldehyde (MDA) and examined possible histological changes in these rats' bladders. Preconditioning partially prevented the reduction of bladder dysfunction induced by ischemia-reperfusion. Estimation of the pA2 values for atropine, pirenzepine, methoctramine, and 4-DAMP indicates that the carbachol-induced contractile response in bladder dome is mediated through the M3 receptor subtype in all groups. The MDA concentration in the IR group was significantly larger than that of the control group, and preconditioning significantly reduced MDA production in the bladder. In histological studies, the ischemia-reperfusion with or without preconditioning caused infiltration of leukocytes and rupture of microcirculation in the regions of submucosa and smooth muscle without a corresponding sloughing of mucosal cells. Our data indicate that preconditioning has a beneficial effect on ischemia-reperfusion injury in the rat bladder.     

Larval development of the oriental lancelet, Branchiostoma belcheri, in laboratory mass culture

We are successfully maintaining a laboratory colony of the lancelet Branchiostoma belcheri bred in the laboratory. Based on living individuals in this mass culture, morphological characteristics from the seven-day larval to benthic juvenile stages have been studied. Most striking was that later larval development of B. belcheri showed great individual variation even in a rather stable culture environment. Metamorphosis first occurred on 60 days post fertilization (dpf) and was continuously observed throughout the present study up to 100 dpf. Morphological traits such as the number of primary gill slits and body size at the start of metamorphosis are apparently affected by culture condition. Body size measured in the largest individuals showed nearly linear growth at 0.087 mm/day. The variability found in larval development calls for caution when developmental stages and chronological ages are compared between populations. However, the developmental flexibility of this animal also raises the possibility that growth and sexual maturation could be controlled artificially in captivity.     

Administration of fibroblast growth factor 2 in combination with bone marrow transplantation synergistically improves carbon-tetrachloride-induced liver fibrosis in mice

We previously reported that fibroblast growth factor 2 (FGF2) facilitated the differentiation of transplanted bone marrow cells (BMCs) into hepatocytes. Our earlier study also demonstrated that administration of FGF2 in combination with bone marrow transplantation (BMT) synergistically activated tumor necrosis factor-alpha signaling and significantly improved liver function and prognosis more than BMT alone. However, the way that it affected the extracellular matrix remained unclear. Here, we investigated the effect of FGF2 treatment together with BMT on liver fibrosis in mice treated with carbon tetrachloride (CCl₄). Transplantation of BMCs and concurrent treatment with FGF2 caused a statistically significant reduction in CCl₄-induced liver fibrosis that was accompanied by strong expression of matrix metalloproteinase 9 as compared with FGF2-only treatment or BMT alone. Moreover, in this process, the proliferation of bone-marrow-derived cells was accelerated without causing apoptosis. Thus, the administration of FGF2 in combination with BMT synergistically improves CCl₄-induced liver fibrosis in mice. This treatment has the potential of being an effective therapy for patients with liver cirrhosis. This study was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (nos. 16390211 and 16590597) and for translational research from the Ministry of Health, Labor and Welfare (H-trans-5 and H17-Special-015).     

Inhibition of reversed electron transfer and proton transport in the beef heart cytochrome bc1 complex by chemical modification

Chemical modification of the bovine heart cytochrome bc1 complex with N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) has been reported to inhibit the proton pumping activity without affecting the rate of electron transfer to ferricytochrome c. This study aims to examine the effect of EEDQ on energy-linked reversed electron transfer in the bc1 complex reconstituted into potassium-loaded phospholipid vesicles. Generation of a valinomycin-mediated potassium-diffusion potential induced the reduction of cytochrome b in the reconstituted bc1 complex in the presence of sodium ascorbate. The time course of the cytochrome b reduction was well correlated with that of the absorbance change of safranine, an optical probe for measuring membrane potential. Treatment of the bc1 complex with EEDQ caused a decrease in the potential-induced reduction of cytochrome b as well as in the proton translocation activity. But a significant loss in the ubiquinol-cytochrome c reducing activity was not observed in the EEDQ-treated bc1 complex. The time- and concentration-dependent effect of EEDQ on the reversed electron transfer was well correlated with that of the proton translocation activity of the bc1 complex. These findings strongly support the idea that the potential-induced reversal of electron transfer is coupled to the reverse flow of protons in the cytochrome bc1 complex.     

Synthesis and structure-activity relationship of novel RXR antagonists: orally active anti-diabetic and anti-obesity agents

A series of diazepinylbenzoic acid derivatives were synthesized and tested in the inhibition assay of the transactivation of RXR. Oral treatment of cyano derivatives (16f) was found to show anti-diabetic and anti-obesity effects in KK-A(y) mice.     

Comparison of conditions for mycelial growth of <Emphasis Type="Italic">Lepista sordida</Emphasis> causing fairy rings on <Emphasis Type="Italic">Zoysia matrella</Emphasis> turf to those on <Emphasis Type="Italic">Agrostis palustris</Emphasis> turf

Symptoms of fairy rings caused by Lepista sordida have been reported on Zoysiagrass (Zoysia spp.) turf maintained at fairway height (2 cm), but not on bentgrass (Agrostis spp.) maintained at putting green height (0.5 cm). The mycelia of this fungus inhabit primarily the upper 0–2 cm layer of the soil extending into the thatch. To compare conditions for the mycelial growth in Z. matrella turf to those in A. palustris turf, we examined the effects of nutrients, temperature, water potential, and pH in the field as well as in the laboratory. Greater growth of the mycelia was observed in medium that included hot water extracts from soil of the 0–1 cm zone in Z. matrella turf compared to that from A. palustris. The upper soil layer in Z. matrella turf contained more organic matter from clippings than that in A. palustris. The temperature and water potential of the 0–2 cm soil zone in Z. matrella turf were also more favorable for the mycelial growth. The soil pH values of this zone in Z. matrella turf were less favorable compared to A. palustris but within the range for accelerating mycelial growth. Part of this study was presented orally at the 46th meeting of the Mycological Society of Japan in 2002