Time-dependent effects of electroacupuncture at the Ren channel on extracellular signal-regulated kinases 1/2 expression in focal cerebral ischemia rats

BACKGROUND： The onset of focal cerebral ischemia activates extracellular signal-regulated kinases 1 and 2, regulates cell cycle, promotes cell proliferation and differentiation, and affects the normal stage and function of brain cells. OBJECTIVE： To observe the effects of electroacupuncture at the Ren channel on extracellular signal-regulated kinases 1/2 expression in the lateral cerebral ventricle wall of rats with focal cerebral ischemia. The effects were analyzed at different time points after intervention. DESIGN： Randomized controlled study. SETTING： Department of Anatomy, Sun Yat-Sen University. MATERIALS： A total of 60 healthy adult male Wistar rats weighing （250±10） g were provided by the Experimental Animal Center, Medical College of Sun Yat-Sen University. The animal experiment was conducted with confirmed consent by the local ethics committee. The GB6805-Ⅱ electric acupuncture apparatus was provided by Shanghai Medical Equipment High-techno Company. METHODS： The experiment was performed at the Laboratory of Anatomy, Sun Yat-Sen University, from February to July 2007. All experimental animals were randomly divided into the following groups： normal group （n = 6）, sham operation group （n = 18）, model group （n = 18）, and electroacupuncture group （n = 18）. Middle cerebral artery occlusion （MCAO） was performed in the model group and electroacupuncture group. Zea Longa＇s grading standard was used to assess neurological impairment after reperfusion; animals whose grades were between l and 4 were included in this study. The normal control group was not exposed to MCAO. In sham operation animals, the right common carotid artery （CCA） was isolated, and the external carotid artery （ECA） was damaged, but no embolism was induced. The electroacupuncture group was given acupuncture on the second day after surgery. The acupoint locations were chosen according to Experimental Acupuncture （People＇s Publishing House; 1997; First Edition）. The Chengjiang, Qihai, and Guanyuan acupoints were labeled and connected to a G6805 electroacupuncture apparatus with sparse-dense waves （sparse waves were 30 Hz, dense waves were 100 Hz）, with a frequency of 6-15 V. The duration was 20 minutes. Two days after surgery, the model and sham operation groups were placed with their backs on the operating table, but they received no acupuncture. However, the normal group received acupuncture. The experimental animals under anesthesia were sacrificed on days 7, 14, and 28 post-surgery. Western blot analysis was used to measure expression of extracellular signal-regulated kinases 1/2 in the inferior region of the lateral cerebral ventricle wall. Expression was measured in the normal group at time points corresponding to the sham operation group. MAIN OUTCOME MEASURES： Expression of extracellular signal-regulated kinases 1/2 in the inferior region of the lateral cerebral ventricle wall at different time points after intervention. RESULTS： All 60 rats were included in the final analysis, without any loss. Seven days after MCAO, there was no significant difference in extracellular signal-regulated kinases 1/2 expression in the electroacupuncture group compared to the model group （P 〉 0.05）. However, extracellular signal-regulated kinases 1/2 expression significantly increased in the model group at 14 and 28 days after treatment （P 〈 0.05）. CONCLUSION： Electroacupuncture at the Ren channel can enhance extracellular signal-regulated kinasesl/2 expression in the inferior region of the lateral cerebral ventricle wall of rats with focal cerebral ischemia. However, this effect is not apparent until 14 days after electroacupuncture intervention.

Season, Environment Stress and Refrigerated Storage Affect Genomic DNA Isolation of Tung Tree

Many metabolites in leaf tissue disturbed plant genomic DNA isolation and always varied when leaves was harvested from different environments. Objective of this study was to investigate whether season, environment stress and refrigerated storage affect genomic DNA isolation of tung tree leaves. Five types of young leaves and two DNA isolation protocols, the recycling CTAB protocol I and II, were adopted to carry out the experiment. Our results showed that both leaf type and protocol affected DNA isolation of tung tree. Using the recycling CTAB protocol II, though little DNA were obtained from three types of young leaves, the other two have satisfying results. Whereas the recycling CTAB protocol I could produce high yield genomic DNA from all the five types of young leaves. All the detectable DNA samples in agarose gel electrophoresis were good templates for PCR reaction. Season, environment stress and refrigerated storage had a big effect on genomic DNA isolation of tung tree. The recycling CTAB protocol I was proved to be an effective and universal protocol for DNA isolation of tung tree. Five types of young leaves could all act as the tissue for isolation of genomic DNA, but the summer healthy young leaves without long-time refrigerated storage are the best. The optimal leaf tissue will benefit DNA isolation of plant species.

Recycling Isolation of Plant DNA,A Novel Method

DNA is one of the most basic and essential genetic materials in the field of molecular biology. To date, isolation of sufficient and good- quality DNA is still a challenge for many plant species, though various DNA extraction methods have been published. In the present paper, a recycling DNA extraction method was proposed. The key step of this method was that a single plant tissue sample was recycled for DNA extraction for up to four times, and correspondingly four DNA precipitations （termed as the 1st, 2nd, 3rd and 4th DNA sample, respectively） were conducted. This recycling step was integrated into the conventional CTAB DNA extraction method to establish a recycling CTAB method. This modified CTAB method was tested in eight plant species, wheat, sorghum, barley, corn, rice, Brachy- podium distachyon, Miscanthus sinensis and tung tree. The results showed that high-yield and good-quality DNA samples could be obtained by using this new method in all the eight plant species. The DNA samples were good templates for PCR amplification of both ISSR and SSR markers. The recycling method can be used in multiple plant species and can be integrated with multiple conventional DNA isolation methods, and thus is an effective and universal DNA isolation method.

Coordination of light, circadian clock with temperature: The potential mechanisms regulating chilling tolerance in rice

Rice(Oryza sativa L.)is a major staple food crop for over half of the world’s population.As a crop species originated from the subtropics,rice production is hampered by chilling stress.The genetic mechanisms of rice responses to chilling stress have attracted much attention,focusing on chilling-related gene mining and functional analyses.Plants have evolved sophisticated regulatory systems to respond to chilling stress in coordination with light signaling pathway and internal circadian clock.However,in rice,information about light-signaling pathways and circadian clock regulation and their roles in chilling tolerance remains elusive.Further investigation into the regulatory network of chilling tolerance in rice is needed,as knowledge of the interaction between temperature,light,and circadian clock dynamics is limited.Here,based on phenotypic analysis of transgenic and mutant rice lines,we delineate the relevant genes with important regulatory roles in chilling tolerance.In addition,we discuss the potential coordination mechanism among temperature,light,and circadian clock in regulating chilling response and tolerance of rice,and provide perspectives for the ongoing chilling signaling network research in rice.

Transferability of Microsatellite Markers from Brachypodium distachyon to Miscanthus sinensis,a Potential Biomass Crop

Miscanthus sinensis has high biomass yield and contributed two of the three genomes in M. x giganteus, a bioenergy crop widely studied in Europe and North America, and thus is a potential biomass crop and an important germplasm for Miscanthus breeding. Molecular markers are essential for germplasm evaluation, genetic analyses and new cultivar development in M. sinensis. In the present study, we reported transferability of simple sequence repeat （SSR） markers from Brachypodium distachyon to M. sinensis. A set of 57 SSR markers evenly distributed across the B. distachyon genome were deliberately designed. Out of these B. distachyon SSR markers, 86.0% are transferable to M. sinensis. The SSR loci amplified in M. sinensis were validated by re-sequencing the amplicons. The polymorphism information content （PIC） of the transferable SSR markers varied from 0.073 to 0.375 with a mean of 0.263, assessed based on 21 M. sinensis genotypes. Phylogenetic tree based on 162 alleles detected by 49 SSR markers could unambiguously distinguish B. distachyon from M. sinensis, and cluster 21 M. sinensis genotypes into three groups that are basically in coincidence with their geographical distribution and ecotype classifications. The markers developed by the comparative genomic approach could be useful for germplasm evaluation, genetic analysis, and marker-assisted breeding in Miscanthus.