ATDC5: An excellent in vitro model cell line for skeletal development

The ATDC5 cell line is derived from mouse teratocarcinoma cells and characterized as a chondrogenic cell line which goes through a sequential process analogy to chondrocyte differentiation. Thus, it is regarded as a promising in vitro model to study the factors that influence cell behaviors during chondrogenesis. It also provides insights in exploring signaling pathways related to skeletal development as well as interactions with innovative materials. To date, over 200 studies have utilized ATDC5 to obtain lots of significant findings. In this review, we summarized the literature of ATDC5 related studies and emphasized the application of ATDC5 in chondrogenesis. In addition, the general introduction of ATDC5 including its derivation and characterization is covered in this article. J. Cell. Biochem. 114: 1223–1229, 2013. © 2012 Wiley Periodicals, Inc.     

Efficient auto-excision of a selectable marker gene from transgenic citrus by combining the Cre/loxP system and ipt selection

Key message

A highly efficient Cre-mediated deletion system, offering a good alternative for producing marker-free transgenic plants that will relieve public concerns regarding GMOs, was first developed in citrus.

Abstract

The presence of marker genes in genetically modified crops raises public concerns regarding their safety. The removal of marker genes can prevent the risk of their flow into the environment and hasten the public’s acceptance of transgenic products. In this study, a new construct based on the Cre/loxP site-recombination system was designed to delete marker genes from transgenic citrus. In the construct, the selectable marker gene isopentenyltransferase gene (ipt) from Agrobacterium tumefaciens and the Cre recombinase gene were flanked by two loxP recognition sites in the direct orientation. The green fluorescent protein (gfp) reporter gene for monitoring the transformation of foreign genes was located outside of the loxP sequences. Transformation and deletion efficiencies of the vector were investigated using nopaline synthase gene (NosP) and CaMV 35S promoters to drive expression of Cre. Analysis of GFP activity showed that 28.1 and 13.6 % transformation efficiencies could be obtained by NosP- and CaMV 35S-driven deletions, respectively. Molecular analysis demonstrated that 100 % deletion efficiency was observed in the transgenic plants. The complete excision of the marker gene was found in all deletion events driven by NosP and in 81.8 % of deletion events driven by CaMV 35S. The results showed that Cre/loxP-mediated excision was highly efficient and precise in citrus. This approach provides a reliable strategy for auto-deletion of selectable marker genes from transgenic citrus to produce marker-free transgenic plants.     

Effects of 12‐Hour Rotating Shifts on Menstrual Cycles of Photoelectronic Workers in Taiwan

12 h rotating shifts are common in high‐tech industries in Taiwan. The aim of this longitudinal study was to evaluate the effect of the disruption of circadian rhythms by the shift schedule on menstrual cycle length (MCL) and regularity of female workers at an optoelectronic company in Taiwan. We recruited females who worked rotating shifts in a clean room environment as the shift‐work group and female office workers who worked normal business hours as the comparison group. Every participant recorded their MCL for each menstruation cycle up to eight consecutive months prospectively and provided demographic characteristics, reproductive history, and menstrual characteristics. We collected data on 1,135 and 117 menstruation cycles in the shift‐work (n=280) and comparison groups (n=49). Whereas the two groups had similar group means for MCL and number of menstrual bleeding days, the prevalence of menstrual cycle irregularity (cycles<25 or>35 days) was higher in the shift‐work group (p=0.04). Univariate and multivariate logistic regression analyses demonstrated that rotating shift work was an independent predictor of menstrual cycle irregularity (odds ratio=1.71, 95% confidence interval: 1.03–2.88) after adjusting for shift‐work history, employment duration, coffee consumption, and pre‐employment menstrual cycle irregularity. Although further study is required to confirm our findings plus to explore prevention and control measures, our data indicate rotating shift work can increase the risk of MCL irregularity.     

Converting paddy fields to Lei bamboo (Phyllostachys praecox) stands affected soil nutrient concentrations, labile organic carbon pools, and organic carbon chemical compositions

Background and aims

Land-use change often markedly alters soil carbon (C) and nitrogen (N) pool sizes with implications for climate change and soil sustainability. The objective of this research was to study the effect of converting paddy fields to Lei bamboo (Phyllostachys praecox) stands on soil C and N and other nutrient pools as well as the chemical structure of soil organic C (SOC) in the soil profile.

Methods

Soils (Anthrosols) from four adjacent paddy field–bamboo forest pairs with a known land-use history were sampled from Lin’an County, Zhejiang Province. Soil water soluble organic C (WSOC), hot water soluble organic C (HWSOC), microbial biomass C (MBC), readily oxidizable C (ROC), water soluble organic N (WSON), and other soil chemical and physical properties were determined. Soil organic C functional group compositions were determined by ¹³C-nuclear magnetic resonance (NMR).

Results

Concentrations of soil available P, available K, and different N forms increased (P?<?0.05) by the land-use conversion. Higher concentrations of SOC and total N (TN) were observed in the subsoil (20–40 and 40–60 cm soil layers) but not in the topsoil (0–20 cm layer) in the bamboo stands than in the paddy fields. The storage of SOC and TN in the entire soil profile (0–60 cm) increased by 56.7 and 70.7 %, respectively, after the land-use change. The increases in the SOC stock of the three soil layers were 11.0, 14.3, and 9.5 Mg C ha^?1, respectively. The conversion decreased WSOC concentrations in the subsoil but increased the ROC concentration in the topsoil. Solid-state NMR spectroscopy of soil samples showed that the conversion increased (P?<?0.05) the O-alkyl C content while decreased the aromatic C content, alkyl C to O-alkyl C ratio (A/O-A), and aromaticity of SOC.

Conclusions

Conversion of paddy fields to bamboo stands increased soil nutrient availability, and SOC and TN stocks. Effects of land-use change on C pools and C chemistry of SOC varied among different soil layers in the profile. The impact of the land-use conversion on soil organic C pools was not restricted to the topsoil, but changes in the subsoil were equally large and should be accounted for.     

A core functional region of the RFP1 promoter from Chinese wild grapevine is activated by powdery mildew pathogen and heat stress

RING-finger proteins (RFP) function as ubiquitin ligases and play key roles in plant responses to biotic and abiotic stresses. However, little information is available on the regulation of RFP expression. Here, we isolate and characterize the RFP promoter sequence from the disease-resistant Chinese wild grape Vitis pseudoreticulata accession Baihe-35-1. Promoter-GUS fusion assays revealed that defense signaling molecules, powdery mildew infection, and heat stress induce VpRFP1 promoter activity. By contrast, the RFP1 promoter isolated from Vitis vinifera was only slightly induced by pathogen infection and heat treatment. By promoter deletion analysis, we found that the ?148 bp region of the VpRFP1 promoter was the core functional promoter region. We also found that, in Arabidopsis, VpRFP1 expressed under its own promoter activated defense-related gene expression and improved disease resistance, but the same construct using the VvRFP1 promoter slightly improve disease resistance. Our results demonstrated that the ?148 bp region of the VpRFP1 promoter plays a key role in response to pathogen and heat stress, and suggested that expression differences between VpRFP1 and VvRFP1 may be key for the differing disease resistance phenotypes of the two Vitis genotypes.     

Drought-induced H2O2 accumulation in subsidiary cells is involved in regulatory signaling of stomatal closure in maize leaves

Increasing H₂O₂ levels in guard cells in response to environmental stimuli are recently considered a general messenger involved in the signaling cascade for the induction of stomatal closure. But little is known as to whether subsidiary cells participate in the H₂O₂-mediated stomatal closure of grass plants. In the present study, 2-week-old seedlings of maize (Zea mays) were exposed to different degrees of soil water deficit for 3 weeks. The effects of soil water contents on leaf ABA and H₂O₂ levels and stomatal aperture were investigated using physiological, biochemical, and histochemical approaches. The results showed that even under well-watered conditions, significant amounts of H₂O₂ were observed in guard cells, whereas H₂O₂ concentrations in the subsidiary cells were negligible. Decreasing soil water contents led to a significant increase in leaf ABA levels associated with significantly enhanced O₂ ^? and H₂O₂ contents, consistent with reduced degrees of stomatal conductance and aperture. The significant increase in H₂O₂ appeared in both guard cells and subsidiary cells of the stomatal complex, and H₂O₂ levels increased with decreasing soil water contents. Drought-induced increase in the activity of antioxidative enzymes could not counteract the significant increase in H₂O₂ levels in guard cells and subsidiary cells. These results indicate that subsidiary cells participate in H₂O₂-mediated stomatal closure, and drought-induced H₂O₂ accumulation in subsidiary cells is involved in the signaling cascade regulating stomatal aperture of grass plants such as maize.     

Polyomavirus Large T Antigen Binds Symmetrical Repeats at the Viral Origin in an Asymmetrical Manner

Polyomaviruses have repeating sequences at their origins of replication that bind the origin-binding domain of virus-encoded large T antigen. In murine polyomavirus, the central region of the origin contains four copies (P1 to P4) of the sequence G(A/G)GGC. They are arranged as a pair of inverted repeats with a 2-bp overlap between the repeats at the center. In contrast to simian virus 40 (SV40), where the repeats are nonoverlapping and all four repeats can be simultaneously occupied, the crystal structure of the four central murine polyomavirus sequence repeats in complex with the polyomavirus origin-binding domain reveals that only three of the four repeats (P1, P2, and P4) are occupied. Isothermal titration calorimetry confirms that the stoichiometry is the same in solution as in the crystal structure. Consistent with these results, mutation of the third repeat has little effect on DNA replication in vivo. Thus, the apparent 2-fold symmetry within the DNA repeats is not carried over to the protein-DNA complex. Flanking sequences, such as the AT-rich region, are known to be important for DNA replication. When the orientation of the central region was reversed with respect to these flanking regions, the origin was still able to replicate and the P3 sequence (now located at the P2 position with respect to the flanking regions) was again dispensable. This highlights the critical importance of the precise sequence of the region containing the pentamers in replication.     

A Second CRM1-Dependent Nuclear Export Signal in the Influenza A Virus NS2 Protein Contributes to the Nuclear Export of Viral Ribonucleoproteins

Influenza A virus NS2 protein, also called nuclear export protein (NEP), is crucial for the nuclear export of viral ribonucleoproteins. However, the molecular mechanisms of NEP mediation in this process remain incompletely understood. A leucine-rich nuclear export signal (NES2) in NEP, located at the predicted N2 helix of the N-terminal domain, was identified in the present study. NES2 was demonstrated to be a transferable NES, with its nuclear export activity depending on the nuclear export receptor chromosome region maintenance 1 (CRM1)-mediated pathway. The interaction between NEP and CRM1 is coordinately regulated by both the previously reported NES (NES1) and now the new NES2. Deletion of the NES1 enhances the interaction between NEP and CRM1, and deletion of the NES1 and NES2 motifs completely abolishes this interaction. Moreover, NES2 interacts with CRM1 in the mammalian two-hybrid system. Mutant viruses containing NES2 alterations generated by reversed genetics exhibit reduced viral growth and delay in the nuclear export of viral ribonucleoproteins (vRNPs). The NES2 motif is highly conserved in the influenza A and B viruses. The results demonstrate that leucine-rich NES2 is involved in the nuclear export of vRNPs and contributes to the understanding of nucleocytoplasmic transport of influenza virus vRNPs.