Microsatellite genetic variation in the Chinese endemic Eucommia ulmoides (Eucommiaceae): implications for conservation

Eucommia ulmoides, a traditional Chinese medicinal plant, is endangered as a consequence of long‐term and widespread harvest in the late 20th century. It has been widely cultivated as a source of herbal medicine and for use in the organic chemical industry in China. In this study, eight microsatellite markers were applied to investigate genetic diversity in E. ulmoides. Three hundred individuals from one semi‐wild population and nine cultivated populations across its main production area were collected. A high level of genetic diversity at population levels (H_E = 0.716) was observed. The highly outcrossed mating system, high longevity of E. ulmoides and seed admixture may be responsible for high genetic variation within populations. A genetic bottleneck was observed in one population. Populations were only slightly differentiated from one another (F_ST = 0.063); this was also supported by AMOVA, which revealed that 94.05% of the total variation resided within populations. This is probably attributable to long‐distance gene flow mediated by the exchange of seeds by local farmers. Implications of these results for the conservation of genetic resources of E. ulmoides are discussed. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173 , 775–785.     

FOXO1 promotes wound healing through the up-regulation of TGF-β1 and prevention of oxidative stress

Keratinocyte mobilization is a critical aspect of wound re-epithelialization, but the mechanisms that control its precise regulation remain poorly understood. We set out to test the hypothesis that forkhead box O1 (FOXO1) has a negative effect on healing because of its capacity to inhibit proliferation and promote apoptosis. Contrary to expectations, FOXO1 is required for keratinocyte transition to a wound-healing phenotype that involves increased migration and up-regulation of transforming growth factor β1 (TGF-β1) and its downstream targets, integrin-α3 and -β6 and MMP-3 and -9. Furthermore, we show that FOXO1 functions in keratinocytes to reduce oxidative stress, which is necessary to maintain cell migration and prevent cell death in a TGF-β1–independent manner. Thus, our studies identify a novel function for FOXO1 in coordinating the response of keratinocytes to wounding through up-regulation of TGF-β1 and other factors needed for keratinocyte migration and protection against oxidative stress, which together promote migration and decrease apoptosis.     

Acquisition of Contextual Discrimination Involves the Appearance of a RAS-GRF1/p38 Mitogen-activated Protein (MAP) Kinase-mediated Signaling Pathway That Promotes Long Term Potentiation (LTP)

RAS-GRF1 is a guanine nucleotide exchange factor with the ability to activate RAS and RAC GTPases in response to elevated calcium levels. We previously showed that beginning at 1 month of age, RAS-GRF1 mediates NMDA-type glutamate receptor (NMDAR)-induction of long term depression in the CA1 region of the hippocampus of mice. Here we show that beginning at 2 months of age, when mice first acquire the ability to discriminate between closely related contexts, RAS-GRF1 begins to contribute to the induction of long term potentiation (LTP) in the CA1 hippocampus by mediating the action of calcium-permeable, AMPA-type glutamate receptors (CP-AMPARs). Surprisingly, LTP induction by CP-AMPARs through RAS-GRF1 occurs via activation of p38 MAP kinase rather than ERK MAP kinase, which has more frequently been linked to LTP. Moreover, contextual discrimination is blocked by knockdown of Ras-Grf1 expression specifically in the CA1 hippocampus, infusion of a p38 MAP kinase inhibitor into the CA1 hippocampus, or the injection of an inhibitor of CP-AMPARs. These findings implicate the CA1 hippocampus in the developmentally dependent capacity to distinguish closely related contexts through the appearance of a novel LTP-supporting signaling pathway.     

The Interaction of the Cellular Export Adaptor Protein Aly/REF with ICP27 Contributes to the Efficiency of Herpes Simplex Virus 1 mRNA Export

Herpes simplex virus 1 (HSV-1) protein ICP27 enables viral mRNA export by accessing the cellular mRNA export receptor TAP/NXF, which guides mRNA through the nuclear pore complex. ICP27 binds viral mRNAs and interacts with TAP/NXF, providing a link to the cellular mRNA export pathway. ICP27 also interacts with the mRNA export adaptor protein Aly/REF, which binds cellular mRNAs and also interacts with TAP/NXF. Studies using small interfering RNA (siRNA) knockdown indicated that Aly/REF is not required for cellular mRNA export, and similar knockdown studies during HSV-1 infection led us to conclude that Aly/REF may be dispensable for viral RNA export. Recently, the structural basis of the interaction of ICP27 with Aly/REF was elucidated at atomic resolution, and it was shown that three ICP27 residues, W105, R107, and L108, interface with the RNA recognition motif (RRM) domain of Aly/REF. Here, to determine the role the interaction of ICP27 and Aly/REF plays during infection, these residues were mutated to alanine, and a recombinant virus, WRL-A, was constructed. Virus production was reduced about 10-fold during WRL-A infection, and export of ICP27 protein and most viral mRNAs was less efficient. We conclude that interaction of ICP27 with Aly/REF contributes to efficient viral mRNA export.     

(Z)-4-Bromo-5-(bromomethylene)-3-methylfuran-2(5H)-one sensitizes Escherichia coli persister cells to antibiotics

Persisters are a small subpopulation of bacterial cells that are dormant and extremely tolerant to antibiotics. The intrinsic antibiotic tolerance of persisters also facilitates the development of multidrug resistance through acquired mechanisms based on drug resistance genes. In this study, we demonstrate that (Z)-4-bromo-5-(bromomethylene)-3-methylfuran-2(5H)-one (BF8) can reduce persistence during Escherichia coli growth and revert the antibiotic tolerance of its persister cells. The effects of BF8 were more profound when the pH was increased from 6 to 8.5. Although BF8 is a quorum sensing (QS) inhibitor, similar effects were observed for the wild-type E. coli RP437 and its ΔluxS mutant, suggesting that these effects did not occur solely through inhibition of AI-2-mediated QS. In addition to its effects on planktonic persisters, BF8 was also found to disperse RP437 biofilms and to render associated cells more sensitive to ofloxacin. At the doses that are effective against E. coli persister cells, BF8 appeared to be safe to the tested normal mammalian cells in vitro and exhibited no long-term cytotoxicity to normal mouse tissues in vivo. These findings broadened the activities of brominated furanones and shed new light on persister control.     

A novel melatonin agonist Neu-P11 facilitates memory performance and improves cognitive impairment in a rat model of Alzheimer' disease

Previous studies have shown that melatonin is implicated in modulating learning and memory processing. Melatonin also exerts neuroprotective activities against Aβ-induced injury in vitro and in vivo. Neu-P11 (piromelatine, N-(2-(5-methoxy-1H-indol-3-yl)ethyl)-4-oxo-4H-pyran-2-carboxamide) is a novel melatonin (MT1/MT2) receptor agonist and a serotonin 5-HT1A/1D receptor agonist recently developed for the treatment of insomnia. In the present study we firstly investigated whether Neu-P11 and melatonin enhance memory performance in the novel object recognition (NOR) task in rats, and then assessed whether Neu-P11 and melatonin improve neuronal and cognitive impairment in a rat model of Alzheimer' disease (AD) induced by intrahippocampal Aβ_(1–42) injection. The results showed that a single morning or afternoon administration of Neu-P11 enhanced object recognition memory measured at 4 or 24 h after training. Melatonin was effective in the memory facilitating effects only when administered in the afternoon. Further results showed that intrahippocampal Aβ_(1–42) injection resulted in hippocampal cellular loss, as well as decreased learning ability and memory in the Y maze and NOR tasks in rats. Neu-P11 but not melatonin attenuated cellular loss and cognitive impairment in the rat AD model. The current data suggest that Neu-P11 may serve as a novel agent for the treatment of AD.     

The Arabidopsis oligopeptidases TOP1 and TOP2 are salicylic acid targets that modulate SA‐mediated signaling and the immune response

Salicylic acid (SA) is a small phenolic molecule with hormonal properties, and is an essential component of the immune response. SA exerts its functions by interacting with protein targets; however, the specific cellular components modulated by SA and critical for immune signal transduction are largely unknown. To uncover cellular activities targeted by SA, we probed Arabidopsis protein microarrays with a functional analog of SA. We demonstrate that thimet oligopeptidases (TOPs) constitute a class of SA‐binding enzymes. Biochemical evidence demonstrated that SA interacts with TOPs and inhibits their peptidase activities to various degrees both in vitro and in plant extracts. Functional characterization of mutants with altered TOP expression indicated that TOP1 and TOP2 mediate SA‐dependent signaling and are necessary for the immune response to avirulent pathogens. Our results support a model whereby TOP1 and TOP2 act in separate pathways to modulate SA‐mediated cellular processes.     

Agrobacterium tumefaciens-mediated transformation of Penicillium expansum PE-12 and its application in molecular breeding

Lipase produced by Penicillium expansum is widely used in laundry detergent and leather industry; however, the absence of an efficient transformation technology sets a major obstacle for further enhancement of its lipase productivity through advanced gene engineering. In this work, Agrobacterium tumefaciens-mediated transformation (ATMT) was investigated for P. expansum PE-12 transformation, using hygromycin phosphotransferase (hph) as a selectable marker gene. As a result, we revealed that the frequency of transformation surpassed 100 transformants/10⁵ condida, most of the integrated T-DNA appeared as a single copy at a random position in chromosomal DNA, and all the transformants showed mitotic stability. Facilitated by this newly established method, for the first time, P. expansum PE-12 was genetically engineered to improve the lipase yield, through a homologous expression vector carrying the endogenous lipase gene (PEL) driven by the strong constitutive promoter of the glyceraldehydes-3-phosphate dehydrogenase gene (gpdA) from Aspergillus nidulans. The highest expression level of the engineered strain reached up to 1700 U/mL, nearly 2-fold of the original industrial strain (900 U/mL). Our reproducible ATMT system has not only revealed the great potential of homologous expression-directed genetic engineering, which is more efficient and specific compared to traditional mutagenesis, but also provided new possibilities and perspectives for any other practical applications of P. expansum-related genetic engineering in the future.