Integral role of GDF-9 and BMP-15 in ovarian function

The oocyte plays an important role in regulating and promoting follicle growth, and thereby its own development, by the production of oocyte growth factors that predominantly act on supporting granulosa cells via paracrine signaling. Genetic studies in mice demonstrated critical roles of two key oocyte-derived growth factors belonging to the transforming growth factor-β (TGF-β) superfamily, growth and differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15), in ovarian function. The identification of Bmp15 and Gdf9 gene mutations as the causal mechanism underlying the highly prolific or infertile nature of several sheep strains in a dosage-sensitive manner also highlighted the crucial role these two genes play in ovarian function. Similarly, large numbers of mutations in the GDF9 and BMP15 genes have been identified in women with premature ovarian failure and in mothers of dizygotic twins. The purpose of this article is to review the genetic studies of GDF-9 and BMP-15 mutations identified in women and sheep, as well as describing the various knockout and overexpressing mouse models, and to summarize the molecular and biological functions that underlie the crucial role of these two oocyte factors in female fertility.     

Evidence for osteocyte regulation of bone homeostasis through RANKL expression

Osteocytes embedded in bone have been postulated to orchestrate bone homeostasis by regulating both bone-forming osteoblasts and bone-resorbing osteoclasts. We find here that purified osteocytes express a much higher amount of receptor activator of nuclear factor-κB ligand (RANKL) and have a greater capacity to support osteoclastogenesis in vitro than osteoblasts and bone marrow stromal cells. Furthermore, the severe osteopetrotic phenotype that we observe in mice lacking RANKL specifically in osteocytes indicates that osteocytes are the major source of RANKL in bone remodeling in vivo.     

Brain‐derived neurotrophic factor,corticotropin‐releasing factor,and hypothalamic neuronal histamine interact to regulate feeding behavior

Brain‐derived neurotrophic factor (BDNF), corticotropin‐releasing factor (CRF), and hypothalamic neuronal histamine are anorexigenic substances within the hypothalamus. This study examined the interactions among BDNF, CRF, and histamine during the regulation of feeding behavior in rodents. Food intake was measured after treatment with BDNF, α‐fluoromethyl histidine (FMH; a specific suicide inhibitor of histidine decarboxylase that depletes hypothalamic neuronal histamine), or CRF antagonist. We measured food intake in wild‐type mice and mice with targeted disruption of the histamine H1 receptor (H1KO mice) after central BDNF infusion. Furthermore, we investigated CRF content and histamine turnover in the hypothalamus after BDNF treatment, and conversely, BDNF content in the hypothalamus after histamine treatment. We used immunohistochemical staining for histamine H1 receptors (H1‐R) in BDNF neurons. BDNF‐induced feeding suppression was partially attenuated in rats pre‐treated with FMH or a CRF antagonist, and in H1KO mice. BDNF treatment increased CRF content and histamine turnover in the hypothalamus. Histamine increased BDNF content in the hypothalamus. Immunohistochemical analysis revealed that H1‐Rs were expressed on BDNF neurons in the ventromedial nucleus of the hypothalamus. These results indicate that CRF and hypothalamic neuronal histamine mediate the suppressive effects of BDNF on feeding behavior and body weight.     

Rho-kinase negatively regulates thyroid hormone-stimulated osteocalcin synthesis in osteoblasts

Evidence is accumulating that Rho-associated kinase (Rho-kinase) plays important roles not only in vascular smooth muscle cell contraction, but also in a variety of cellular functions, including bone metabolism. In the present study, we investigated the involvement of Rho-kinase in the osteocalcin synthesis induced by triiodothyronine (T₃) in osteoblast-like MC3T3-E1 cells. T₃ time-dependently induced phosphorylation of myosin phosphatase targeting subunit (MYPT-1), a substrate of Rho-kinase. Y27632, a specific inhibitor of Rho-kinase, attenuated the MYPT-1 phosphorylation induced by T₃. T₃-stimulated osteocalcin release was significantly enhanced by Y27632. Fasudil, another Rho-kinase inhibitor, amplified the osteocalcin release induced by T₃. T₃-stimulated osteocalcin release was significantly augmented in Rho-knockdown cells with Rho A-siRNA. Y27632 and fasudil also increased the mRNA expression level of osteocalcin induced by T₃. These results strongly suggest that T₃ stimulates the activation of Rho-kinase in osteoblasts, which functions as a negative regulator of T₃-stimulated osteocalcin synthesis.     

Activin A is essential for Feeder‐free culture of human induced pluripotent stem cells

Feeder‐free culture of human induced pluripotent stem (hiPS) cells is necessary for their clinical application to avoid adverse effects of foreign proteins. hiPS cells were cultured with combinations of activin (A), CHIR99021 (C), basic fibroblast growth factor (F), and leukemia inhibitory factor (L) under feeder‐free conditions. Culture was terminated after 12 passages or when the cell morphology changed from pluripotency. Pluripotency was analyzed by alkaline phosphatase (ALP) staining and immunostaining with antibodies to Oct3/4, Nanog, SSEA4, and TRA‐1‐60. SB431542 (SB), an activin inhibitor, was added to the culture, and the morphology of the cells was observed. hiPS cells cultured with A, AC, and ACL after 12 passages were positive for ALP staining. Oct3/4 was positive in hiPS cells cultured with A, AC, and ACL. hiPS cells were positive for Nanog when cultured with A and AC; however, Nanog signal was weaker in cells cultured with ACL. SSEA4 was positive in hiPS cells cultured with A and AC but almost negative in those cultured with ACL. hiPS cells were positive for TRA‐1‐60 when cultured with A, AC, and ACL. hiPS cells lose their undifferentiated morphology at six passages when cultured with A + SB, five passages with AC + SB, and nine passages with ACL. We conclude that feeder‐free culture of hiPS cells requires A or AC to maintain pluripotency. J. Cell. Biochem. 114: 584–588, 2013. © 2012 Wiley Periodicals, Inc.     

Purification and characterization of tributyltin-binding protein type 2 from plasma of Japanese flounder, Paralichthys olivaceus

We used gel filtration chromatography, anion-exchange chromatography and polyacrylamide gel electrophoresis to purify tributyltin-binding protein type 2 (TBT-bp 2) from plasma of Japanese flounder (Paralichthys olivaceus) injected intraperitoneally with TBT (5.0 mg/kg body weight). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the molecular mass of TBT-bp 2 was approximately 48 kDa, and isoelectric focusing-polyacrylamide gel electrophoresis indicated that the isoelectric point was approximately 3.0. TBT-bp 2 contained 40% N-glycan. The complete cDNA nucleotide sequence and the genome sequence of TBT-bp 2 were determined by means of rapid amplification of cDNA ends of liver tissue of Japanese flounder and a genome-walking technique, respectively. The 216 amino acid sequence of TBT-bp 2 showed 47% identity to the sequences of puffer fish (Takifugu pardalis) saxitoxin- and tetrodotoxin-binding protein but only 27% similarity to the sequence of TBT-bp 1. Analysis of the motif sequence of the amino acid sequence and the structure of the gene encoding TBT-bp 2 suggested that this protein belongs to the lipocalin superfamily.     

Putative mechanism of the itch-scratch circle: repeated scratching decreases the cutaneous level of prostaglandin D2, a mediator that inhibits itching

In atopic dermatitis, scratching of the skin as a reaction to itching causes injury to the skin, which, in turn, further increases the itching resulting in the establishment of the so-called itch-scratch circle. We have shown that prostaglandin (PG) D2 plays an inhibitory role against pruritus in mice with atopic-like dermatitis; therefore, we examined the relationship between scratching and the cutaneous PGD2 level using an artificial scratching model with a wire brush. Mechanical scratching induced a temporary increase of the skin PGs levels (PGE2, PGD2, 6-ketoPGF1alpha, PGF2alpha). The skin PGD2 level and the ability of PGD2 production decreased at 48 h after repeated scratch, compared to that of normal skin, not so after single scratch. Immunohistochemical analysis and Western blotting revealed a decrease in the levels of cyclooxygenase-1 (COX-1) and hematopoietic PGD synthase in mechanically scratched skin. The reduced ability of the skin for PGD2 production following mechanical scratching could be caused by this decrease in the expression levels of COX-1 and PGD2 synthase. The results suggest that repeated scratching in mice decreases the ability of the skin to produce PGD2, which is an endogenous mediator that inhibits pruritus, resulting in the establishment of the itch-scratch circle.     

Combined application of Pseudomonas fluorescens strain LRB3W1 with a low dosage of benomyl for control of cabbage yellows caused by Fusarium oxysporum f. sp. conglutinans

An antagonistic bacterium, Pseudomonas fluorescens strain LRB3W1, inhibited the fungal growth of Fusarium oxysporum f. sp. conglutinans in vitro and suppressed cabbage yellows caused by F. oxysporum f. sp. conglutinans. Under glasshouse conditions, the bacterium survived at ca. 10⁶-10⁷ CFU g^-1 in the cabbage rhizosphere for 4 weeks after the initial application. The chemical fungicide, benomyl, did not suppress the disease severity at low concentration (1 or 10 µg mL^-1). However, the disease severity was decreased by the combined application of a low dosage of benomyl with strain LRB3W1. Combined application of a low dosage of benomyl with strain LRB3W1 was more effective than treatment with the bacterium alone. The survival of strain LRB3W1 was not influenced by the presence of benomyl. This combined use of the biocontrol agent, strain LRB3W1, and a fungicide, benomyl, should be an attractive approach for suppressing cabbage yellows in sustainable agriculture because of the reduced chemical dosages needed for disease management.     

Identification of the role of bone morphogenetic protein (BMP) and transforming growth factor‐β (TGF‐β) signaling in the trajectory of serotonergic differentiation in a rapid assay in mouse embryonic stem cells in vitro

The mechanism by which extracellular molecules control serotonergic cell fate remains elusive. Recently, we showed that noggin, which inactivates bone morphogenetic proteins (BMPs), induces serotonergic differentiation of mouse embryonic (ES) and induced pluripotent stem cells with coordinated gene expression along the serotonergic lineage. Here, we created a rapid assay for serotonergic induction by generating knock‐in ES cells expressing a naturally secreted Gaussia luciferase driven by the enhancer of Pet‐1/Fev, a landmark of serotonergic differentiation. Using these cells, we performed candidate‐based screening and identified BMP type I receptor kinase inhibitors LDN‐193189 and DMH1 as activators of luciferase. LDN‐193189 induced ES cells to express the genes encoding Pet‐1, tryptophan hydroxylase 2, and the serotonin transporter, and increased serotonin release without altering dopamine release. In contrast, TGF‐β receptor inhibitor SB‐431542 selectively inhibited serotonergic differentiation, without changing overall neuronal differentiation. LDN‐193189 inhibited expression of the BMP signaling target gene Id, and induced the TGF‐β target gene Lefty, whereas the opposite effect was observed with SB‐431542. This study thus provides a new tool to investigate serotonergic differentiation and suggests that inhibition of BMP type I receptors and concomitant activation of TGF‐β receptor signaling are implicated in serotonergic differentiation.