Yongmei Xi

Implications of Rice Agriculture for Wild Birds in China

Abstract. Data on wild birds in rice fields in China are scarce. The potential significance of Chinese rice fields, which represent about 6% of the worlds wetland area, is considerable but whether this potential is met is largely unknown. In this review, traditional and modern Chinese rice agriculture are compared, including detailing historical changes and their implications for wild birds. Traditional practices, with one crop each year and long periods of fallow flooding, provide greater benefit to biodiversity and species such as the Crested Ibis (Nipponia nippon). The method and alternatives, such as rice-fish, duck-rice and swidden agriculture, are contrasted with modern techniques which, through associated water regimes and chemical use, have been implicated in the decline of biodiversity and of species such as the Black-faced Spoonbill (Platalea minor). Agrochemical use is particularly pertinent because China is likely to have been the worlds largest pesticide consumer since the mid-1990s, with use greatest in rice (Oryza sativa). However, few studies have measured the direct effects of agro-chemicals on wild birds in China. The most detailed information on birds in Chinas rice fields comes from charismatic species such as the Crested Ibis and Red-crowned Crane (Grus japonensis). Preliminary data from possibly the first systematic bird survey of a Chinese rural county are presented. More detailed and widespread studies of the implications of rice agriculture to wild birds in China are required.

Alterations of circadian clockworks during differentiation and apoptosis of rat ovarian cells

Ovarian development is related to cell proliferation, differentiation, and apoptosis of granulosa cells and luteal cells under the control of various modulators, including follicle-stimulating hormone (FSH), luteinizing hormone (LH), and growth factors. In the present study, the expression of clock genes and the related regulation mechanism were analyzed in different ovarian cell types during differentiation and apoptosis. The authors focused on the circadian expression of Per2 as a core clock gene for the maintenance of circadian rhythms. By using a real-time monitoring system of the Per2 promoter activity, the circadian oscillation was analyzed in the granulosa and luteal cells from preantral follicles, antral follicles, and corpora lutea of immature Per2 promoter–destabilized luciferase transgenic rats that were primed with diethylstilbestrol, equine chorionic gonadotropin (eCG), and/or human CG. In addition, transcript levels of Per2, Bmal1, Clock, and Nampt were quantified by quantitative polymerase chain reaction (qPCR). Immunohistochemical studies revealed strong circadian rhythmicity of PER2 protein in the luteal cells, but apparently little rhythmicity in granulosa cells of both preantral and antral follicles. In vitro monitoring of promoter activity showed generation of several oscillations in luteal cells after exposure to dexamethasone (DXM), whereas oscillatory amplitudes of immature and mature granulosa cells were rapidly attenuating. The circadian rhythm of the Bmal1 transcript levels, but not the Per2 transcript, was very weak in the granulosa cells, as compared with that in luteal cells. Granulosa cells gained a strong circadian rhythm ability of the Per2 promoter activity after stimulation with FSH for 3 days. In contrast, LH had little effect on the circadian rhythm before stimulation of granulosa cells with FSH, probably owing to lack of LH receptor. In luteal cells, induction of apoptosis by inhibiting progesterone synthesis resulted in deregulation of Per2 circadian oscillation. Transcript levels of Bmal1 and Clock, but not Per2 and Nampt, were significantly decreased in apoptotic luteal cells. The Bmal1 transcript level was particularly reduced. Consequently, these results strongly suggest the circadian clockwork alters in ovarian cells during follicular development, luteinization, and apoptosis, and expression of Bmal1 may be related to the switch-on and switch-off of the circadian oscillation. (Author correspondence: mhattori@agr.kyushu-u.ac.jp)

Could the effect of modeled microgravity on osteogenic differentiation of human mesenchymal stem cells be reversed by regulation of signaling pathways

Abstract Microgravity (MG) results in a reduction in bone formation. Bone formation involves osteogenic differentiation from mesenchymal stem cells (hMSCs) in bone marrow. We modeled MG to determine its effects on osteogenesis of hMSCs and used activators or inhibitors of signaling factors to regulate osteogenic differentiation. Under osteogenic induction, MG reduced osteogenic differentiation of hMSCs and decreased the expression of osteoblast gene markers. The expression of Runx2 was also inhibited, whereas the expression of PPARγ2 increased. MG also decreased phosphorylation of ERK, but increased phosphorylation of p38MAPK. SB203580, a p38MAPK inhibitor, was able to inhibit the phosphorylation of p38MAPK, but did not reduce the expression of PPARγ2. Bone morphogenetic protein (BMP) increased the expression of Runx2. Fibroblast growth factor 2 (FGF2) increased the phosphorylation of ERK, but did not significantly increase the expression of osteoblast gene markers. The combination of BMP, FGF2 and SB203580 significantly reversed the effect of MG on osteogenic differentiation of hMSCs. Our results suggest that modeled MG inhibits the osteogenic differentiation and increases the adipogenic differentiation of hMSCs through different signaling pathways. Therefore, the effect of MG on the differentiation of hMSCs could be reversed by the mediation of signaling pathways.

Proteomic analysis of human bone marrow mesenchymal stem cells transduced with human telomerase reverse transcriptase gene during proliferation

Abstract.  Objectives: Previous studies have reported immortalization and tumorigenicity of human mesenchymal stem cells (hMSCs) transduced with exogenous human telomerase reverse transcriptase (hTERT). We also have established a line of hMSCs transduced with hTERT (hTERT–hMSCs) and we have cultured these cells for 290 population doublings (PDs) during which they demonstrated a large proliferation potential but with no tumorigenicity. The aim of this study was to investigate the protein expression profile of hTERT–hMSCs with two‐dimensional gel electrophoresis and peptide mass fingerprinting by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry, to be able to analyse the effects of exogenous hTERT on protein expression in hMSCs. Materials and methods: We generated proteome maps of primary hMSCs and hTERT–hMSCs at PD 95 and PD 275. Results: A total of 1543 ± 145 protein spots in gels of primary MSCs at PD 12, 1611 ± 186 protein spots in gels of hTERT–hMSCs at PD 95 and 1451 ± 126 protein spots in gels of hTERT–hMSCs at 275 PD were detected. One hundred of these were successfully identified, including 20 which were differentially expressed. Conclusions: The results suggest that sustaining levels of prohibitin and p53 expression along with differential expression of proteins in hTERT–hMSCs provide an insight into lack of transforming activity of hTERT–hMSCs during cell proliferation.

Preclinical transplantation and safety of HS/PCs expanded from human umbilical cord blood

AIM To expand hematopoietic/progenitor stem cells (HS/PCs) from umbilical cord blood (UCB) and prepare the HS/PC product, and analyze preclinical transplantation and safety of HS/PC product. METHODS Human bone marrow-derived mesenchymal stem cells (MSCs) were used as feeder cells to expand HS/PCs from UCB in a serum-free culture system. The proliferation potential of HS/PCs was analyzed. The expanded HS/PCs were suspended in the L-15 medium to prepare the HS/PC product. The contamination of bacteria, fungi and mycoplasmas, the infection of exogenous virus, the concentration of bacterial endotoxin, and the SCF residual in HS/PC product were determined. Finally, cells from the HS/PC product with or without bone marrow-derived mesenchymal stem cells (BM-MSCs) were transplanted into the irradiated NOD/SCID mice to determine the in vivo engraftment potential. RESULTS After co-culture for 10 d, the total nuclear cells (TNCs) increased 125-fold, and CD34(+) cells increased 43-fold. The granulocyte-macrophage colony- forming cells (GM-CFCs) and erythroid colony-forming cells (E-CFCs) increased 3.3- and 4.7-fold respectively. The expanded cells were collected and prepared as the expanded product of HS/PCs by re-suspending cells in L-15 medium. For preclinical safety, the HS/PC product was analysed for contamination by bacteria, fungi and mycoplasmas, the bacterial endotoxin concentration and the SCF content. The results showed that the HS/PC product contained no bacteria, fungi or mycoplasmas. The bacterial endotoxin concentration was less than the detection limit of 6 EU/mL, and residual SCF was 75 pg/mL. Based on clinical safety, the HS/PC product was qualified for clinical transplantation. Finally, the HS/PC product was transplanted the irradiated mice where it resulted in rapid engraftment of hematopoietic cells. CONCLUSION HSPC product prepared from UCB in the serum-free culture system with hMSCs as feeder cells should be clinically safe and effective for clinical transplantation.

Prevalence of a Septicemia Disease in the Crested Ibis (Nipponia nippon) in China

Abstract This study investigated six cases of septicemia in young crested ibises (Nipponia nippon). These birds all died with similar clinical signs, including sudden death, anorexia, diarrhea, and lameness. Immediately after death, the birds were necropsied; a blood sample was taken from heart and tissues were sampled from liver, lung, spleen, peritoneal mucus, and feces for bacteriologic examination. Anatomic observation showed that the main findings common to the sick birds were arthrocele, associated with congestion in the femur, tibiotarsus, and ventral side; swelling in the liver; hemorrhagic pericarditis; miliary tubercles in lung; and fibrous tubercles in the synovial capsule of the knee joint with suppurative abscesses. Through bacterial examination, the colonial type of Escherichia coli strain was represented prominently in cultures of the feces, heart blood, liver, lung, spleen, suppurative mucus of the synovial capsule, and peritoneal exudate. These symptoms suggested that the death of a number of endangered crested ibis within a short period was evidence of septicemia. The bacterial inoculation tests were also conducted using domestic pigeon, native chicken, and mice for the presence of and infection with E. coli. The study provided indications of the possible role of E. coli strains as bird pathogens and a potential risk in endangered species. Further work is needed to characterize E. coli strains and the toxin production in this bird. This disease occurrence also adds a note of caution to the continued efforts and interest in the reintroduction of the ibis back into its former wild ranges to ensure that formerly captive individuals do not transmit disease to the wild populations of its own or other sympatric species.

Inter embryonic (homo- or hetero-sexual) transfer of primordial germ cells between chicken embryos.

Chicken primordial germ cells (PGCs) collected from thecirculating blood in embryonic vessels at stage 13–15 were inter-embryonically, homo- or hetero-sexually,transferred to the blood vessels of recipient embryosat the same stage of development. Approximately 30%of the embryos treated with hetero-sexual transfer of PGCs had abnormal gonads, showing ovotestis likeorgans. In this case, some of these reversed gonadswere considered to be dependent upon the ratio of thenumber of PGCs from donor to recipient embryos. Oneof the treated embryos possessed completely reversedorgans. Therefore, the introduction of exogenousembryonic vessels was thought to be also useful forproducing transgened gonads.

Effects of ectopic expression of human telomerase reverse transcriptase on immortalization of feather keratinocyte stem cells

Normal somatic cells possess a finite life span owing to replicative senescence. Telomerase functions as a potential regulator of senescence in various cells. Expression level of human telomerase reverse transcriptase (hTERT) is correlated with telomerase activity and cellular immortalization. In this study, we investigated the effects of ectopic expression of hTERT on proliferation potential of chicken feather keratinocyte stem cells (FKSCs). We established FKSCs transduced with hTERT catalytic subunit fused with EGFP marker gene (hTERT-EGFP-FKSCs). hTERT-EGFP-FKSCs had the great potential of proliferation in vitro and expressed kerainocyte stem cell markers integrin beta1 and CD49c. Keratin 15 and keratin 19, as native FKSCs, were also detected in hTERT-EGFP-FKSCs. By the analysis of fluorescent RT-PCR, western blotting and TRAP assay, hTERT-EGFP-FKSCs were positive for telomerase activity, in comparison with native FKSCs showing no telomerase activity. We demonstrated that ectopic expression of hTERT could result in immortalization of FKSCs. Tumorigenecity of hTERT-EGFP-FKSCs were examined by soft agar assay and transplantation into NOD-SCID mice. Results showed that hTERT-EGFP-FKSCs sustained the cellular characteristics of native FKSCs and had no transforming activity. In vivo differentiation multipotentials of hTERT-EGFP-FKSCs were confirmed by transplantation into developing chicken embryos and in situ hybridization analysis. These data provide a novel framework for understanding human telomerase activity in different species and suggest a new insight for manipulating hTERT for therapeutic purposes in treating tissue injury and aging.