The Dietary Isoflavone Daidzein Reduces Expression of Pro-Inflammatory Genes through PPARα/γ and JNK Pathways in Adipocyte and Macrophage Co-Cultures

Obesity-induced inflammation caused by adipocyte-macrophage interactions plays a critical role in developing insulin resistance, and peroxisome proliferator-activated receptors (PPARs) regulate inflammatory gene expression in these cells. Recently, the soy isoflavone daidzein was reported to act as a PPAR activator. We examined whether daidzein affected adipocyte-macrophage crosstalk via the regulation of PPARs. Co-cultures of 3T3-L1 adipocytes and RAW264 macrophages, or palmitate-stimulated RAW264 macrophages were treated with daidzein in the presence or absence of specific inhibitors for PPARs: GW6471 (a PPARα antagonist), and GW9662 (a PPARγ antagonist). Inflammatory gene expression was then determined. Daidzein significantly decreased chemokine (C-C motif) ligand 2 (Ccl2, known in humans as monocyte chemo-attractant protein 1 (MCP1)) and interleukin 6 (Il6) mRNA levels induced by co-culture. In 3T3-L1 adipocytes, daidzein inversed the attenuation of adiponectin gene expression by co-culture, and these effects were inhibited by the PPAR-γ specific inhibitor. Daidzein also decreased Ccl2 and Il6 mRNA levels in RAW264 macrophages stimulated with palmitate or conditioned medium (CM) from hypertrophied 3T3-L1 adipocytes. This inhibitory effect on Il6 expression was abrogated by a PPAR-α inhibitor. Additionally, we examined the activation of nuclear factor-kappa B (NF-κB) and c-Jun N-terminal kinase (JNK) pathways and found that daidzein significantly inhibited palmitate-induced phosphorylation of JNK. Our data suggest that daidzein regulates pro-inflammatory gene expression by activating PPAR-α and -γ and inhibiting the JNK pathway in adipocyte and macrophage co-cultures. These effects might be favorable in improving adipose inflammation, thus, treatment of daidzein may be a therapeutic strategy for chronic inflammation in obese adipose tissue.     

Requisite Morphologic Interaction for Attachment between Ulva pertusa (Chlorophyta) and Symbiotic Bacteria

In order to understand the morphogenesis-inducing mechanism of Ulva pertusa by symbiotic bacteria, we observed the requisite conditions of bacteria for attachment to U. pertusa for algal morphogenesis. Non-morphogenesis-inducing bacterial mutants derived by ultraviolet irradiation did not attach onto the surface of this alga. Scanning electron microscopic observation during the process of morphogenesis in U. pertusa revealed a network-like structure formed on the algal surface within 1 week after application of bacteria. The bacteria attached onto the alga after 2 weeks of incubation. After this attachment process the morphologic change was observed in U. pertusa. Received August 7, 1997; accepted July 24, 1998.     

The cell sorting process of Xenopus gastrula cells involves the acto-myosin system and TGF-β signaling

We have previously shown that the cell sorting process of animal pole cells (AC) and vegetal pole cells (VC) from Xenopus gastrulae is considered to involve two steps: concentrification and polarization. In this study, we addressed the question of what specified the spatial relationship of the AC and VC clusters during the process. First, we examined the inhibitory or facilitatory treatment for myosin 2 activity during each of the two steps. The aggregates treated with Y27632 or blebbistatin during the concentrification step showed a cluster random arrangement, suggesting the prevention of the cell sorting by inhibition of myosin 2. Meanwhile, the treatment with a Rac1 inhibitor, NSC23766, during the same step resulted in promotion of the fusion of the AC clusters and the progression of the cell sorting, presumably by an indirect activation of myosin 2. On the other hand, the treatments with any of the three drugs during the polarization step showed that the two clusters did not appose, and their array remained concentric. Thus, the modulation of cell contraction might be indispensable to each of the two steps. Next, the activin/nodal TGF-β signaling was perturbed by using a specific activin receptor-like kinase inhibitor, SB431542. The results revealed a bimodal participation of the activin/nodal TGF-β signaling, i.e., suppressive and promotive effects on the concentrification and the polarization, respectively. Thus, the present in vitro system, which permits not only the cell contraction-mediated cell sorting but also the TGF-β-directed mesodermal induction such as cartilage formation, may fairly reflect the embryogenesis in vivo.     

Evaluation of the stability of standard reference genes of adipose-derived mesenchymal stem cells during in vitro proliferation and differentiation

Mesenchymal stem cells (MSCs) from a variety of sources are being used in pre-clinical and clinical studies. The choice of optimal source for treatment of diseases requires quantitative evaluation of self-renewal, proliferation and differentiation potencies of MSCs. For this purpose, quantitative real-time polymerase chain reaction (qRT-PCR) technique is used to determine the expression of genes. qRT-PCR requires the normalization of the gene expression levels by the use of reference genes in order to obtain accurate and reliable results. There is a limited number of studies focused on the selection of reference genes that are appropriate and reliable for MSCs. Thus, no single reference gene has yet been found for use in the in vitro proliferation and differentiation of MSCs. The aim of this study is to investigate the stability of the expression of widely used reference genes during the in vitro proliferation and differentiation of human adipose-derived mesenchymal stem cells (hASCs). For this purpose, 13 reference genes commonly used in MSC studies were selected. As a result, the expression stabilities of EF1α, RPLP0 and RPL13A genes were found to be high and were predicted to be suitable for use as reference genes for normalization in hASC studies. The GAPDH was identified as the gene with the lowest expression stability and evaluated to be an unsuitable reference gene for hASC differentiation studies. This piece of information could be crucial for the selection of appropriate reference genes and accurate measurement of gene expression in hASC studies.

     

Biocontrol of black scurf on potato by seed tuber treatment with Pythium oligandrum

The biological control activity of Pythium oligandrum against black scurf of potato caused by Rhizoctonia solani AG-3 was evaluated in field experiments after treatment of potato seed tubers with P. oligandrum. Seed tubers infected with black scurf sclerotia were dipped for a few seconds in a suspension of 10³, 10⁴ or 10⁵ mL^?1 P. oligandrum oospores and were then air-dried. Each level of P. oligandrum-treatment significantly reduced the disease rates of stolon at a level similar to that achieved by chemical control. When P. oligandrum populations adherent to the surface of seed tubers were determined, oospore counts on tubers treated with 10⁴ or 10⁵ oospores mL^?1 were about 540/cm² or about 22,000/cm² just after dipping and decreased to about 170/cm² or 2900/cm² after a 3-week incubation, respectively. Confocal laser scanning microscopic observation with an immuno-enzymatic staining procedure showed that P. oligandrum hyphae had colonized the sclerotia and established close contact by coiling around the R. solani hyphae present on the surface of seed tubers, in a manner similar to that observed in the dual-culture test. Quantification of R. solani DNA by PCR indicated that the R. solani population was reduced on the seed tubers treated with P. oligandrum compared to untreated tubers. Furthermore, the ability of P. oligandrum to induce resistance against black scurf was determined using a potato tuber disk assay. Treatment of tuber disks with the cell wall protein fraction of P. oligandrum enhanced the expression of defense-related genes such as 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase, lipoxygenase and basic PR-6 genes, and reduced disease severity upon challenge with R. solani compared with untreated controls. These results suggest that biocontrol mechanisms employed by P. oligandrum against black scurf involve both mycoparasitism and induced resistance.