De novo DNA methylation independent establishment of maternal imprint on X chromosome in mouse oocytes

In female mouse embryos, the paternal X chromosome (Xp) is preferentially inactivated during preimplantation development and trophoblast differentiation. This imprinted X-chromosome inactivation (XCI) is partly due to an activating imprint on the maternal X chromosome (Xm), which is set during oocyte growth. However, the nature of this imprint is unknown. DNA methylation is one candidate, and therefore we examined whether disruptions of the two de novo DNA methyltransferases in growing oocytes affect imprinted XCI. We found that accumulation of histone H3 lysine-27 trimethylation, a hallmark of XCI, occurs normally on the Xp, and not on the Xm, in female blastocysts developed from the mutant oocytes. Furthermore, the allelic expression patterns of X-linked genes including Xist and Tsix were unchanged in preimplantation embryos and also in the trophoblast. These results show that a maternal disruption of the DNA methyltransferases has no effect on imprinted XCI and argue that de novo DNA methylation is dispensable for Xm imprinting. This underscores the difference between imprinted XCI and autosomal imprinting.     

Nicotine Acts on Growth Plate Chondrocytes to Delay Skeletal Growth through the α7 Neuronal Nicotinic Acetylcholine Receptor

Background

Cigarette smoking adversely affects endochondral ossification during the course of skeletal growth. Among a plethora of cigarette chemicals, nicotine is one of the primary candidate compounds responsible for the cause of smoking-induced delayed skeletal growth. However, the possible mechanism of delayed skeletal growth caused by nicotine remains unclarified. In the last decade, localization of neuronal nicotinic acetylcholine receptor (nAChR), a specific receptor of nicotine, has been widely detected in non-excitable cells. Therefore, we hypothesized that nicotine affect growth plate chondrocytes directly and specifically through nAChR to delay skeletal growth.

Methodology/Principal Findings

We investigated the effect of nicotine on human growth plate chondrocytes, a major component of endochondral ossification. The chondrocytes were derived from extra human fingers. Nicotine inhibited matrix synthesis and hypertrophic differentiation in human growth plate chondrocytes in suspension culture in a concentration-dependent manner. Both human and murine growth plate chondrocytes expressed alpha7 nAChR, which constitutes functional homopentameric receptors. Methyllycaconitine (MLA), a specific antagonist of alpha7 nAChR, reversed the inhibition of matrix synthesis and functional calcium signal by nicotine in human growth plate chondrocytes in vitro. To study the effect of nicotine on growth plate in vivo, ovulation-controlled pregnant alpha7 nAChR +/− mice were given drinking water with or without nicotine during pregnancy, and skeletal growth of their fetuses was observed. Maternal nicotine exposure resulted in delayed skeletal growth of alpha7 nAChR +/+ fetuses but not in alpha7 nAChR −/− fetuses, implying that skeletal growth retardation by nicotine is specifically mediated via fetal alpha7 nAChR.

Conclusions/Significance

These results suggest that nicotine, from cigarette smoking, acts directly on growth plate chondrocytes to decrease matrix synthesis, suppress hypertrophic differentiation via alpha7 nAChR, leading to delayed skeletal growth.     

Patterns of Plant Invasions: A Case Example in Native Species Hotspots and Rare Habitats

Land managers require landscape-scale information on where exotic plant species have successfully established, to better guide research, control, and restoration efforts. We evaluated the vulnerability of various habitats to invasion by exotic plant species in a 100,000 ha area in the southeast corner of Grand Staircase-Escalante National Monument, Utah. For the 97 0.1-ha plots in 11 vegetation types, exotic species richness (log₁₀) was strongly negatively correlated to the cover of cryptobiotic soil crusts (r = −0.47, P < 0.001), and positively correlated to native species richness (r = 0.22, P < 0.03), native species cover (r = 0.23, P < 0.05), and total nitrogen in the soil (r = 0.40, P < 0.001). Exotic species cover was strongly positively correlated to exotic species richness (r = 0.68, P < 0.001). Only 6 of 97 plots did not contain at least one exotic species. Exotic species richness was particularly high in locally rare, mesic vegetation types and nitrogen rich soils. Dry, upland plots (n = 51) had less than half of the exotic species richness and cover compared to plots (n = 45) in washes and lowland depressions that collect water intermittently. Plots dominated by trees had significantly greater native and exotic species richness compared to plots dominated by shrubs. For the 97 plots combined, 33% of the variance in exotic species richness could be explained by a positive relationship with total plant cover, and negative relationships with the cover of cryptobiotic crusts and bare ground. There are several reasons for concern: (1) Exotic plant species are invading hot spots of native plant diversity and rare/unique habitats. (2) The foliar cover of exotic species was greatest in habitats that had been invaded by several exotic species.(3) Continued disturbance of fragile cryptobiotic crusts by livestock, people, and vehicles may facilitate the further invasion of exotic plant species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Systemic arterial compliance, systemic vascular resistance, and effective arterial elastance during exercise in endurance-trained men

Systemic arterial compliance (C) and vascular resistance (R) regulate effective arterial elastance (Ea), an index of artery load. Increases in Ea during exercise are due primarily to reductions of C and maintain optimal ventricular-arterial coupling. Because C at rest and left ventricular functional reserve are greater in endurance-trained (ET) compared with sedentary control (SC) humans, we hypothesized that reductions of C and increases in Ea are greater in ET than SC individuals. The aim of this study was to investigate C, R, and Ea during exercise in ET and SC humans. C, R, Ea, and cardiac cycle length (T) were measured at rest and during exercise of 40, 60, and 80% maximal oxygen uptake using Doppler ultrasonography in 12 SC and 13 ET men. C decreased in an exercise intensity-dependent manner in both groups, but its reductions were greater in the ET than SC subjects. Consequently, although C at rest was greater in the ET than SC group, the intergroup difference in C disappeared during exercise. Exercise-related changes in R/T were relatively slight and R/T was lower in the ET than the SC group, both at rest and during exercise. Although Ea at rest was lower in the ET than SC group, there were no intergroup differences in Ea at 40, 60, or 80% maximal oxygen uptake. We conclude that the reductions of C from rest to exercise are more marked in ET than SC humans. This may be related to the exercise-associated disappearance of the difference in Ea between ET and SC humans.     

Acylated anthocyanins from red radish (Raphanus sativus L.)

Twelve acylated anthocyanins were isolated from the red radish (Raphanus sativus L.) and their structures were determined by spectroscopic analyses. Six of these were identified as pelargonidin 3-O-[6-O-(E)-feruloyl-2-O-beta-D-glucopyranosyl]-(1-->2)-beta-D-glucopyranoside]-5-O-(beta-D-glucopyranoside), pelargonidin 3-O-[6-O-(E)-caffeoyl-2-O-(6-(E)-feruloyl-beta-D-glucopyranosyl)-(1-->2)-beta-D-glucopyranoside]-5-O-(beta-D-glucopyranoside), pelargonidin 3-O-[6-O-(E)-p-coumaroyl-2-O-(6-(E)-caffeoyl-beta-D-glucopyranosyl)-(1-->2)-beta-D-glucopyranoside]-5-O-(beta-D-glucopyranoside), pelargonidin 3-O-[6-O-(E)-feruloyl-2-O-(6-(E)-caffeoyl-beta-D-glucopyranosyl)-(1-->2)-beta-D-glucopyranoside]-5-O-(beta-D-glucopyranoside), pelargonidin 3-O-[6-O-(E)-p-coumaroyl-2-O-(6-(E)-feruloyl-beta-D-glucopyranosyl)-(1-->2)-beta-D-glucopyranoside]-5-O-(beta-D-glucopyranoside), and pelargonidin 3-O-[6-O-(E)-feruloyl-2-O-(2-(E)-feruloyl-beta-D-glucopyranosyl)-(1-->2)-beta-D-glucopyranoside]-5-O-(beta-D-glucopyranoside).     

A fast, simple method for screening radiation susceptibility genes by RNA interference

Radiotherapy can cause unacceptable levels of damage to normal tissues in some cancer patients. To understand the molecular mechanisms underlying radiation-induced physiological responses, and to be able to predict the radiation susceptibility of normal tissues in individual patients, it is important to identify a comprehensive set of genes responsible for radiation susceptibility. We have developed a simple and rapid 96-well screening protocol using cell proliferation assays and RNA interference to identify genes associated with radiation susceptibility. We evaluated the performance of alamarBlue-, BrdU-, and sulforhodamine B-based cell proliferation assays using the 96-well format. Each proliferation assay detected the known radiation susceptibility gene, PRKDC. In a trial screen using 28 shRNA vectors, another known gene, CDKN1A, and one new radiation susceptibility gene, ATP5G3, were identified. Our results indicate that this method may be useful for large-scale screens designed to identify novel radiation susceptibility genes.     

Establishment of stable hFis1 knockdown cells with an siRNA expression vector

Yeast Fis1p participates in mitochondrial fission, together with Dnm1p and Mdv1p. Recently, human Fis1 (hFis1) was reported to be involved in mitochondrial fission, together with Drp1. We established stable transformants with an hFis1 siRNA expression vector. In the stable hFis1 knockdown cells, hFis1 expression was suppressed to approximately 10%, and mitochondrial fission, induced by cisplatin treatment, was delayed. In addition, mouse Fis1 (mFis1) expression promoted mitochondrial fission and cell death in the hFis1 knockdown cells, suggesting that mFis1 complements the function of hFis1. These hFis1 siRNA expression vectors may be useful for studying the molecular function of mammalian Fis1.     

Generation of a highly pathogenic avian influenza A virus from an avirulent field isolate by passaging in chickens

Highly virulent avian influenza viruses can arise from avirulent strains maintained in poultry, but evidence to support their generation from viruses in wild birds is lacking. The most likely mechanism for the acquisition of virulence by benign avian viruses is the introduction of mutations by error-prone RNA polymerase, followed by the selection of virulent viruses. To investigate whether this mechanism could apply to wild waterfowl, we studied an avirulent wild-swan virus that replicates poorly in chickens. After 24 consecutive passages by air sac inoculation, followed by five passages in chicken brain, the avirulent virus became highly pathogenic in chickens, producing a 100% mortality rate. Sequence analysis at the hemmaglutinin cleavage site of the original isolate revealed a typical avirulence type of sequence, R-E-T-R, which progressed incrementally to a typical virulence type of sequence, R-R-K-K-R, during repeated passages in chickens. These results demonstrate that avirulent viruses maintained in wild waterfowl in nature and bearing the consensus avirulence type sequence R-E-T-R have the potential to become highly pathogenic while circulating in chickens.