Involvement of the phosphoinositide 3-kinase/protein kinase B signaling pathway in insulin/IGF-I-induced chondrogenesis of the mouse embryonal carcinoma-derived cell line ATDC5

The embryonal carcinoma-derived cell line, ATDC5, differentiates into chondrocytes in response to insulin/insulin-like growth factor-I (IGF-I) stimulation. In the present study, we examined whether insulin/IGF-I stimulation caused activation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB) pathway in ATDC5 cells. We also determined whether the insulin-stimulated differentiation of ATDC5 cells into chondrocytes could be mimicked by activation of the PKB pathway alone. ATDC5 cells produced phosphatidylinositol 3,4,5-trisphosphate and the pleckstrin homology domain of PKB was recruited to the plasma membrane in response to insulin stimulation. This was probably a result of activation of PI3K because the PI3K inhibitors, wortmannin and LY294002, inhibited both responses, although the effective concentrations were as high as 10 microM. Insulin stimulation caused the chondrogenic differentiation of ATDC5 cells as assessed by chondrogenic nodule staining with alcian blue. The addition of wortmannin or LY294002, PI3K inhibitors, suppressed the staining, and the suppression was reversible, indicating the effect of the inhibitors is not toxic. Finally, we exogenously expressed a constitutively-activated from of PKB (myristoylated PKB, myr-PKB) in ATDC5 cells, and found the chondrogenic differentiation of ATDC5 cells to form nodules occurred in the absence of insulin stimulation. The kinase-negative mutant of myr-PKB did not caused differentiation, indicating that kinase activity is required. These results support the hypothesis that the PI3K/PKB signaling pathway is involved in the chondrogenic differentiation of ATDC5 cells in response to insulin/IGF-I stimulation. This is the first report that demonstrates the involvement of phosphoinositide signaling in the induction of chondrogenesis from undifferentiated cells.     

Characterization of a donor mitochondrial DNA transmission bottleneck in nuclear transfer derived cow lineages

In embryos derived by nuclear-transfer (NT), fusion of donor cells with recipient oocytes resulted in varying patterns of mitochondrial DNA (mtDNA) transmission in NT animals. Distribution of donor cell mtDNA (D-mtDNA) found in offspring of NT-derived founders may also vary from donor cell and host embryo heteroplasmy to host embryo homoplasmy. Here we examined the transmission of mtDNA from NT cows to G(1) offspring. Eleven NT founder cows were produced by fusion of enucleated oocytes (Holstein/Japanese Black) with Jersey/ Holstein oviduct epithelial cells, or Holstein/Japanese Black cumulus cells. Transmission of mtDNA was analyzed by PCR mediated single-strand conformation polymorphism of the D-loop region. In six of seven animals sampled postmortem, heteroplasmy were detected in various tissues, while D-mtDNA could not be detected in blood or hair samples from four live animals. The average proportion of D-mtDNA detected in one NT cow was 7.6%, and those in other cows were <5%. Heteroplasmic NT cows (n = 6) generated a total 12 G(1) offspring. Four of 12 G(1) offspring exhibited high percentages of D-mtDNA populations (range 17-51%). The remaining eight G(1) offspring had slightly or undetectable D-mtDNA (<5%). Generally, a genetic bottleneck in the female germ-line should favor a homoplasmic state. However, proportions of some G(1) offspring maintained heteroplasmy with a much higher percentage of D-mtDNA than their NT dams, which may also reflect a segregation distortion caused by the proposed mitochondrial bottleneck. These results demonstrate that D-mtDNA in NT cows is transmitted to G(1) offspring with varying efficiencies.     

Aggregated ursolic acid, a natural triterpenoid, induces IL-1beta release from murine peritoneal macrophages: role of CD36

IL-1beta has been shown to play a pivotal role in the development of inflammatory disorders. We recently found that a natural triterpene, ursolic acid (UA), enhanced MIF release from nonstimulated macrophages. In this study, we examined the effects of UA on the production of several cytokines in resident murine peritoneal macrophages (pMphi). UA increased the protein release of IL-1beta, IL-6, and MIF, but not of TNF-alpha, in dose- and time-dependent manners. This triterpene also strikingly induced the activation of p38 MAPK and ERK1/2 together with that of upstream kinases. The release of UA-induced IL-1beta was significantly inhibited by the inhibitors of p38 MAPK, MEK1/2, ATP-binding cassette transporter, and caspase-1. Furthermore, UA induced intracellular ROS generation for IL-1beta production, which was suppressed by an antioxidant. Pretreatment with an anti-CD36 Ab significantly suppressed IL-1beta release, and surface plasmon resonance assay results showed that UA bound to CD36 on macrophages. In addition, the amount of IL-1beta released from UA-treated pMphi of CD36-deficient mice was markedly lower than that from those of wild-type mice. Interestingly, UA was found to aggregate in culture medium, and the aggregates were suggested to be responsible for IL-1beta production. In addition, i.p. administration of UA increased the levels of IL-1beta secretion and MPO activity in colonic mucosa of ICR mice. Taken together, our results indicate that aggregated UA is recognized, in part, by CD36 on macrophages for generating ROS, thereby activating p38 MAPK, ERK1/2, and caspase-1, as well as releasing IL-1beta protein via the ATP-binding cassette transporter.     

Age and gender interactions in physiological functional capacity: insight from swimming performance

Tanaka, Hirofumi, and Douglas R. Seals. Age and genderinteractions in physiological functional capacity: insight from swimming performance. J. Appl.Physiol. 82(3): 846-851, 1997.One experimentalapproach to studying the effects of aging on physiological functionalcapacity in humans is to analyze the peak exercise performance ofhighly trained athletes with increasing age. To gain insight into therelationships among age, gender, and exercise task duration with use ofthis model, we performed a 5-yr (1991-1995) retrospective analysisof top freestyle performance times from the US Masters SwimmingChampionships. Regression analysis showed that in both men and womenendurance swimming performance (i.e., 1,500 m) declined linearly frompeak levels at age 35-40 yr until ~70 yr of age, whereuponperformance declined exponentially thereafter. In both genders, thevariability among the top 10 winning times in each 5-yr age intervalincreased markedly with advancing age. Compared with the 1,500-mfreestyle, performance in the 50-m freestyle (short-duration task)showed only a modest decline until ages 75 and 80 yr in women and men,respectively. The rate and magnitude of the declines in both short- andlong-duration swimming performance with age were significantly(P < 0.05) greater in women than in men. In the women, the percent decline in swimming performance over a50-yr age period from the 19- to 24-yr to the 69- to 74-yr age groupsbecame progressively greater from the shortest distance (50 m) to thetwo longest distances (800 and 1,500 m), whereas in men, no differenceswere observed in the magnitude of performance decline with age amongthe five longest distance events (i.e., 100-1,500 m). The percentgender difference in performance throughout the age range studiedbecame progressively smaller (P < 0.05) with increasing distance from 50 m (19 ± 1%) to 1,500 m (11 ± 1%). The findings in this cross-sectional study indicate that from peak levels at age 35-40 yr, physiological functionalcapacity, as assessed by swimming performance, decreases linearly until ~70-80 yr of age, whereupon the decline becomes exponential.Moreover, the rate of decline with advancing age appears to beassociated with event duration and gender.

     

Novel Positively Charged Nanoparticle Labeling for In Vivo Imaging of Adipose Tissue-Derived Stem Cells

Stem cell transplantation has been expected to have various applications for regenerative medicine. However, in order to detect and trace the transplanted stem cells in the body, non-invasive and widely clinically available cell imaging technologies are required. In this paper, we focused on magnetic resonance (MR) imaging technology, and investigated whether the trimethylamino dextran-coated magnetic iron oxide nanoparticle -03 (TMADM-03), which was newly developed by our group, could be used for labeling adipose tissue-derived stem cells (ASCs) as a contrast agent. No cytotoxicity was observed in ASCs transduced with less than 100 µg-Fe/mL of TMADM-03 after a one hour transduction time. The transduction efficiency of TMADM-03 into ASCs was about four-fold more efficient than that of the alkali-treated dextran-coated magnetic iron oxide nanoparticle (ATDM), which is a major component of commercially available contrast agents such as ferucarbotran (Resovist), and the level of labeling was maintained for at least two weeks. In addition, the differentiation ability of ASCs labeled with TMADM-03 and their ability to produce cytokines such as hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE2), were confirmed to be maintained. The ASCs labeled with TMADM-03 were transplanted into the left kidney capsule of a mouse. The labeled ASCs could be imaged with good contrast using a 1T MR imaging system. These data suggest that TMADM-03 can therefore be utilized as a contrast agent for the MR imaging of stem cells.     

Coleoptile Senescence in Rice (Oryza sativa L.)

We investigated the cellular events associated with cell deathin the coleoptile of rice plants (Oryza sativa L.). Seeds germinatedunder submergence produced coleoptiles that were more elongatedthan those grown under aerobic conditions. Transfer of seedlingsto aerobic conditions was associated with coleoptile opening(i.e. splitting) due to death of specific cells in the sideof the organ. Another type of cell death occurred in the formationof lysigenous aerenchyma. Senescence of the coleoptile was alsonoted, during which discolouration of the chlorophyll and tissuebrowning were apparent. DNA fragmentation was observed by deoxynucleotidyltransferase-mediateddUTP nick end labelling (TUNEL) assay, and further confirmedby the appearance of oligonucleosomal DNA ladders in senescentcoleoptile cells. Two nucleases (Nuc-a and Nuc-b) were detectedby in-gel-assay from proteins isolated from coleoptiles. Nuc-a,commonly observed in three cell death phases required eitherCa²⁺or Mg²⁺, whereas Nuc-b which appeared during senescencerequired both Ca²⁺and Mg²⁺. Both nucleases were strongly inhibitedby Zn²⁺. Copyright 2000 Annals of Botany Company Aerenchyma, rice, cell death, coleoptile, fragmentation, nuclease, Oryza sativa, senescence, split, submergence, TUNEL