电刺激听神经诱发小鼠脑干神经元活动的光信号特征(英文)

背景:光学记录技术是以电压敏感染料为介质,以硅光电二极管转换技术为特点的新型电生理检测方法,有助于分析复杂的神经结构中膜电位变化的时间-空间分布。目的:使用光学记录的方法观察小鼠脑干听神经电刺激诱发冲动的时间-空间分布,并分析抑制性神经递质γ-氨基丁酸和γ-氨基丁酸A受体拮抗剂荷包牡丹碱对听觉诱发冲动的影响。设计:随机对照实验。单位:解放军总医院老年医学研究所,日本关西医科大学耳鼻喉科。材料:实验于2002-05/11在日本关西医科大学耳鼻喉科实验室完成。选取出生后0～5d的ddy/ddy小鼠100只,清洁级,雌雄不拘。方法:将全部动物麻醉后断头处死,在冷冻条件下制作有活性的脑干切片。脑片一侧与无损伤的听神经根残端连接。将脑片置于硅胶铺底的有机玻璃平皿中,用直径30μm钨丝固定。用钨丝电极刺激脑片一侧的听神经根残端,在耳蜗核、前庭核记录诱发反应。对照情况下使用正常的人工脑脊液作为灌流液;灌流液中加入50μmol/Lγ-氨基丁酸的人工脑脊液代替正常人工脑脊液孵育脑片20min后,记录γ-氨基丁酸对脑干听觉通路诱发信号的影响;灌流液中加入50μmol/Lγ-氨基丁酸和200μmol/L荷包牡丹碱的人工脑脊液代替含50μmol/Lγ-氨基丁酸的人工脑脊液孵育脑片20min后,记录荷包牡丹碱对脑干听觉通路诱发信号的影响。刺激条件为正相矩形波脉冲,电流强度5mA,持续时间5ms,频率0.1Hz,电刺激起始时间被设置为89.9ms。脑片染色采用电压敏感染料NK3041,使用16×16像素的硅光电二极管阵列设备记录刺激听神经所诱发的光学信号。主要观察指标:①主要结局;电刺激听神经诱发的脑干光学信号及其特点。②次要结局:γ-氨基丁酸和荷包牡丹碱光学反应记录结果。结果:实验纳入ddy/ddy小鼠100只,中途死亡56只,共44只进入结果分析。①电刺激听神经诱发的脑干光学信号及其特征:刺激听神经诱发的光学信号以时间-空间分布的形式被记录。在同侧耳蜗核,光学信号的潜伏期为(4.63±1.01)ms,前庭核的峰潜伏期为(6.00±0.89)ms。每一个光学信号分为两个成分:快的峰电位样反应及慢的长时程反应。快电位的起始相具有突触前性质,晚期相具有突触后性质;慢的长时程反应可能与多突触传递有关。②γ-氨基丁酸和荷包牡丹碱光学反应记录结果:灌流液中加入50μmol/Lγ-氨基丁酸可最大限度地降低听神经诱发的脑干神经元信号的幅度,快反应起始相的潜伏期没有延长,但幅度有所降低,晚期相以及慢反应的幅度被明显抑制;而灌流液中加入50μmol/Lγ-氨基丁酸、200μmol/L荷包牡丹碱后则可部分逆转γ-氨基丁酸对此信号的作用,快的峰电位样反应和慢反应的幅度有部分恢复。结论:多部位的光学记录系统可以收集电刺激听神经的诱发反应,光学信号显示了时间-空间分布的类型。γ-氨基丁酸能够使电刺激听神经诱发的脑干神经元信号的幅度明显降低,而γ-氨基丁酸A受体拮抗剂荷包牡丹碱可以竞争性地对抗部分而非全部的γ-氨基丁酸的抑制作用,提示γ-氨基丁酸能神经元对听神经诱发冲动的抑制作用除部分通过γ-氨基丁酸A受体实现外,还涉及其他亚型的γ-氨基丁酸受体。

Effects of Light and Temperature on the Expression of the Lhcb2 Gene in Pea

An approximately 800 bp cDNA ( Lhcb 2) encoding light_harvesting chlorophyll a/b_binding protein complex (type Ⅱ) was cloned from the seedling of pea ( Pisum sativum L.) with RT_PCR method. Southern blotting using special probe demonstrated that there existed one copy of Lhcb 2 in pea genome. RT_PCR and Northern blotting revealed the expression of Lhcb 2 which was regulated by light in a time_dependent expression manner. The Lhcb 2 gene didn't express untill 2 h after irradiated with white light. Low temperature (4 ℃) also affected the Lhcb 2 gene by decreasing half of its expression under 25 ℃.

Cloning of Brassica napus EIN3 Gene and Its Expression Induced by Sclerotinia sclerotiorum

[Objective] The study was to investigate roles of Brassica napus EINB in （ BnEIN3 ） resistance to Sclerotinia sclerotiorum. [ Methods] Genomic PCR and RT-PCR were carded out to isolate genomic DNA and cDNA sequences of BnEIN3 from oilseed rape, based on the highly conserved region of EIN3 gene from Arabidopsis thaliana and the homologous sequences of oilseed rape ESTs. Expression levels of BnEIN3 were detected in three varieties of oilseed rape inoculated with S. sclerotiorum by real-time quantitative PCR.[Results] A 1 947 bp DNA fragment was obtained from oilseed rape. The fragment shared 82% identity to A. thaliana EIIV3, encoded 614 amino acids containing an EIN3 domain, and was named as BnEIN3. Real-time PCR results showed that expression patterns of BnEIN3 were drastically different in different varieties. In highly resistant oilseed rape variety D083, BnEIN3 expression level was significantly increased 72 h after S. sclerotiorum inoculation whereas in middle resistant and susceptible varieties Zhongshuang 9 and 84039, BnEIN3 expression was suppressed. [ Conclusion ] BnEIIV3 may play an important role in oilseed rape resistance to S. sclerotiorum.

Aression of TLR_9 in human pulmonary adenocarcinoma cell line A549

Objective:Being considered as a bridge between the innate immunity and acquired immunity,Toll-like receptors(TRLs) are very important innate immunity moleculars.Recent researchs on the innate immunity have focused on the relationship between TLRs and human tumor.This paper investgated the expression and significance of TLR9 in human pulmonary adenocarcinoma cell(A549 cell) and human bronchial epithelial cell(HBE cell).Methods:After culturing A549 cell and HBE cell in vitro,the expression of TLR9 mRNA and protein in both cells were detected by immunocytochemistry,Real-time Quantitative Reverse Transcriptase-Polymerase Chain Reaction(Real-time Quantitative PCR) and Western blot,respectively.Results:By immunocytochemistry staining,TLR9 was mainly expressed in both cells' cell membrane and endochylema as brown-yellow material.It showed that the expressions of TLR9 mRNA and protein in A549 cell were stronger than those in HBE cell(P < 0.01).Conclusion:The results suggest TLR9 might cause the progression of human pulmonary adenocarcinoma,and the mechanism needs to be further investgatied.

TaqMan real-time fluorescent quantitative RT-PCR in detection of macrophage inflammatory protein-2γ mRNA in myocarditis murine

Objective: To study the role of macrophage inflammatory protein (MIP)-2γ in myocarditis pathogenesis in BALB/c mice. Methods: The relationship between the progression of Coxsarckie virus B3(CVB3) viral myocarditis and experimental autoimmune myocarditis and MIP-2γ mRNA expression in mouse was studied by TaqMan real-time fluorescent quantitative RT-PCR. Results: MIP-2γ mRNA expression rose on 3 to 5 d after CVB3 infection, reached peak on 7 d, and returned to normal level until 14 d, which corresponded well with the disease course. The MIP-2γ mRNA expression level rose significantly on the day 18 d after immunization with porcine cardiac myosin, which was consistent with pathological examination. Conclusion: MIP-2γ may be involved in the pathogenesis of myocarditis.

Streptozotocin-induced expression of Ngn3 and Pax4 in neonatal rat pancreatic α-cells

AIM： To investigate the mechanism behind β-cell regeneration in neonatal rat pancreas treated with strep- tozotocin （STZ）. METHODS： Neonatal Sprague Dawley rats were intra- peritoneally injected with 70 mg/kg STZ. Body weight, pancreas weight and blood glucose were recorded every two days after the treatment. To identify the expression and location of transcription factors in the rat pancreas, double immunofluorescent staining was performed using antibodies to specific cell markers and transcription factors. RESULTS： Expression of Neurogenin 3 （Ngn3）, a marker for endocrine precursor cells, was observed by immunofluorescence in a few β-cells and many a-cells. The expression reached a peak 12 d after treatment. Pax4, a transcription factor that lies downstream of Ngn3 and plays an important role in β-cell differentiation, was also expressed in the α-cells of STZ-treated rats. We did not observe significant changes in Nkx6.1, which is essential for β-cell maturation in the treated rats. CONCLUSION： α-cells dedifferentiated into endocrine precursor cells and acquired the ability to dedifferentiate in the neonatal rat pancreas after STZ treatment.

Establishment of an orthotopic transplantation tumor model of hepatocellular carcinoma in mice

AIM:To improve the outcome of orthotopic transplantation in a mouse model,we used an absorbable gelatin sponge(AGS) in nude mice to establish an orthotopic implantation tumor model.METHODS:MHCC-97L hepatocellular carcinoma(HCC)cells stably expressing the luciferase gene were injected into the subcutaneous region of nude mice.One week later,the ectopic tumors were harvested and transplanted into the left liver lobe of nude mice.The AGS was used to establish the nude mouse orthotopic implantation tumor model.The tumor suppressor gene,paired box gene 5(PAX5),which is a tumor suppressor in HCC,was transfected into HCC cells to validate the model.Tumor growth was measured by bioluminescence imaging technology.Semi-quantitative reverse transcription polymerase chain reaction(RT-PCR) and histopathology were used to confirm the tumorigenicity of the implanted tumor from the MHCC-97L cell line.RESULTS:We successfully developed an orthotopic transplantation tumor model in nude mice with the use of an AGS.The success rate of tumor transplantation was improved from 60% in the control group to 100% in the experimental group using AGS.The detection of fluorescent signals showed that tumors grew in all live nude mice.The mice were divided into 3 groups:AGS-,AGS+/PAX5-and AGS+/PAX5 +.Tumor size was significantly smaller in PAX5 transfected nude mice compared to control mice(P < 0.0001).These fluorescent signal results were consistent with observations made during surgery.Pathologic examination further confirmed that the tissues from the ectopic tumor were HCC.Results from RT-PCR proved that the HCC originated from MHCC-97L cells.CONCLUSION:Using an AGS is a convenient and efficient way of establishing an indirect orthotopic liver transplantation tumor model with a high success rate.

QUANTIFICATION OF P4HA2 mRNA OF FIBROBLASTS WITH SYBR GREEN BASED RT-PCR FOR CORRECTING CMV INACTIVATION EFFICIENCY IN DONOR BLOOD

Objective To quantify proline 4-hydroxylase, alpha polypeptide ii （ P4HA2 ） mRNA of human embryo lung fibroblast （HELF） with SYBR green based reversed transcript PCR （RT-PCR） for correcting cytomegalovirus （CMV） inactivation or clearance efficiency in donor blood. Methods A pair of specific primers of exon 12a of P4HA2 was designed, and the related PCR-reaction system and condition were optimized. Then the recombinant plasmid containing the target fragment was constructed for making standard curve with SYBR green based real-time RT-PCR. Finally, the sensitivity, reproducibility, and specificity of this method were fully estimated. Results The sensitivity of the method was 1.5E ＋ 04 copies/mL of P4HA2 mRNA, corresponding to 10^3 fibroblasts. In addition, existence of 8. 67E ＋ 06 leukocytes could not interfere with the accurate quantification of HELF in the large dynamic range. The intra-assay variability and inter-assay variability both varied in different concentrations, being higher in low concentrations and lower in high concentrations. But all of them were below 13. 76% in variation, which showed acceptable stability of this method. Conclusion SYBR green and specific primer based real-time RT-PCR show up a good quality for quantifying HELF P4HA2 mRNA with good specificity, stability, and high sensitivity. Approximate 10 copies of P4HA2 mRNA per cell in average can be detected by the method. Therefore, this method can be used to deduct fibroblast-associated CMV for correcting CMV inactivation efficiency in leukocytes.

Expression Level of a Transcription Factor Gene Mdhd-Zip Up-regulated during Apple Fruit Senescence

[Objective] This study aimed to explore the role of one apple transcription factor of homeodomain-leucine zipper(MdHD-Zip) during apple fruit senescence.[Method] Postharvest Red Fuji fruits(Malus domestica Borkh ‘Red Fuji') were stored at room temperature(18 ℃-20 ℃) and cold condition(0 ± 1 ℃) separately.Fruit firmness and ethylene production during storage process were analyzed.Transcript level of Md HD-Zip was detected by real-time quantitative PCR during apple fruit storage under room temperature and cold condition.[Result] Expression level of Md HD-Zip was found up-regulated in later stage of apple fruit senescence at room temperature,while it showed a peak level after one month of cold storage.[Conclusion] The results of the present study suggest that Md HD-Zip may play a role in regulating apple fruit senescence.

人牙髓细胞内Smad2、3在转化生长因子β_1信号转导中的作用

目的 探讨人牙髓细胞内Smad 2、3在转化生长因子 β1 (transforminggrowthfactor β1 ,TGF β1 )信号转导过程中的作用。方法 原代培养人牙髓细胞 ,用激光共聚焦显微镜观察TGF β1 刺激初期牙髓细胞内Smad 2、3由胞质向胞核转位的现象 ,同时采用Westernblot方法检测刺激后期Smad 2、3蛋白表达的变化。结果 TGF β1 刺激 2h内 ,Smad 2、3在胞质中表达渐弱 ,在胞核内表达渐强 ,呈现由胞质向胞核逐步转位的趋势。Smad 2蛋白总量在TGF β1 刺激前后几乎无变化 ,而Smad 3在刺激后 2 4h表达量明显下降 ,48h后表达十分微弱。结论 Smad 2、3可能是人牙髓细胞内TGF β1 的信号转导分子。TGF β1 刺激初期Smad 2、3通过发生转位参与转导TGF β1 信号至核内 ,刺激后期Smad 3表达水平的下调可能与TGF β1

Transcriptional and translational responses of rapeseed leaves to red and blue lights at the rosette stage

Under different red （R）：blue （B） photon flux ratios, the growth performance of rapeseed （Brassica napus L.） is significantly different. Rapeseed under high R ratios shows shade response, while under high B ratios it shows sun-type morphology. Rapeseed under monochromatic red or blue light is seriously stressed. Transcriptomic and proteomic methods were used to analyze the metabolic pathway change of rapeseed （cv. ＂Zhongshuang 11＂） leaves under different R：B photon flux ratios （including 100R：0B%, 75R：25B%, 25R：75B%, and 0R：100B%）, based on digital gene expression （DGE） and two-dimensional gel electrophoresis （2-DE）. For DGE analysis, 2054 differentially expressed transcripts （llog2（fold change）l〉1, q〈0.005） were detected among the treatments. High R ratios （100R：0B% and 75R：25B%） enhanced the expression of cellular structural components, mainly the cell wall and cell membrane. These components participated in plant epidermis development and anatomical structure morphogenesis. This might be related to the shade response induced by red light. High B ratios （25R：75B% and 0R：100B%） promoted the expression of chloroplast-related components, which might be involved in the formation of sun-type chloroplast induced by blue light. For 2-DE analysis, 37 protein spots showed more than a 2-fold difference in expression among the treatments. Monochromatic light （ML; 100R：0B% and OR： 100B%） stimulated accumulation of proteins associated with antioxidation, photosystem II （PSII）, DNA and ribosome repairs, while compound light （CL; 75R：25B% and 25R：75B%） accelerated accumulation of proteins associated with carbohydrate, nucleic acid, amino acid, vitamin, and xanthophyll metabolisms. These findings can be useful in understanding the response mechanisms of rapeseed leaves to different R：B photon flux ratios.

Homogeneous label-free fluorescent assay of small molecule-protein interactions using protein binding-inhibited transcription nanomachine

Quantitative analysis of interactions between small molecules and proteins is a central challenge in chemical genetics, molecular diagnostics and drug developments. Here, we developed a RNA transcription nanomachine by assembling T7 RNA polymerase on a small molecule-labeled DNA heteroduplex. The nanomachine, of which the RNA transcription activity can be quantitatively inhibited by protein binding, showed a great potential for small molecule-protein interaction assay. This finding enabled us to develop a novel homogeneous label-free strategy for assays of interactions between small molecules and their protein receptors. Three small molecule compounds and their protein receptors have been used to demonstrate the developed strategy. The results revealed that the protein-small molecule interaction assay strategy shows dynamic responses in the concentration range from 0.5 to 64 nM with a detection limit of 0.2 nM. Due to its label-free, homogeneous, and fluorescence-based detection format, besides its desirable sensitivity this technique could be greatly robust, cost-efficient and readily automated, implying that the developed small molecule-protein interaction assay strategy might create a new methodology for developing intrinsically robust, sensitive and selective platforms for homogeneous protein detection.

The sulfur metabolism regulator MetR is a global regulator controlling phytochrome-dependent light responses in Aspergillus nidulans

Phytochrome-dependent light signaling has been studied in several fungi.In Aspergillus nidulans lightstimulated phytochrome activates the high-osmolarity glycerol(HOG)signaling pathway and thereby controls the expression of a large number of genes,many of which are related to stress responses.In a genome-wide expression analysis in A.nidulans we found that phytochrome,fph A,is under strict expression control of the central regulator of the sulfur-starvation response,Met R.This transcriptional regulator is required for the expression of genes involved in inorganic sulfur assimilation.In the presence of organic sulfur,Met R is probably ubiquitinated and possibly degraded and the transcription of sulfur-assimilation genes,e.g.,sulfate permease,is turned off.The expression analysis described here revealed,however,that Met R additionally controls the expression of hundreds of genes,many of which are required for secondary metabolite production.We also show that met R mutation phenocopies fph A deletion,and five other histidine-hybrid kinases are down-regulated in the met R1 mutant.Furthermore,we found that light and phytochrome regulate the expression of at least three carbon–sulfur hydrolases.This work is a further step towards understanding the interplay between light sensing and metabolic pathways.

A flexible metal-organic framework with double interpenetration for highly selective CO_2 capture at room temperature

MicroRNAs （miRNAs） are small non-coding RNAs that regulate gene expression at the post-transcriptional level, and their aberrant expression occurs during the development of malignant diseases. Recently, miRNAs have been proposed as potential prognostic and predictive biomarkers for early diagnosis. However, a major obstacle in rapid miRNA analysis from real samples is the lack of ultrasensitive and quantitative techniques. In this regard, the use of chemiluminescence （CL） system offers a highly sensitive strategy for detecting miRNAs. In this article, an ultrasensitive approach has been established for the quantification ofmiRNAs, using magnetic beads （MBs） and alkaline phosphatase （AP）-based CL system. This technique depends on sandwich hybridization among MBs-labeled capture probes, target miRNAs and biotin-labeled reporter probes, conjugation of streptavidin-alkaline phosphatase （SA-AP） to biotin-labeled reporter probes, and CL detection of AP-linked targets. Detection of miR-21 with this technique demonstrated a high selectivity and an ultralow limit of detection （LOD） of 60 fM with an extraordinarily wide range of six orders of magnitudes. The quantitation could be achieved by direct detecting target miRNA in serum samples within a total time of 1.5 h and did not require reverse transcription and polymerase chain reaction （PCR） amplification. Therefore, this developed method shows great potential for early cancer diagnosis based on miRNAs as biomarkers.

Unanchored ubiquitin chain sustains RIG-I-induced interferon-I activation and controls selective gene expression

Ubiquitination plays a crucial role in retinoic acid-inducible gene I(RIG-I)-induced antiviral responses.However, the precise regulatory mechanisms of RIG-I activity mediated by conjugated and unanchored ubiquitin chains remain to be determined. In this study, we discovered that T55 of RIG-I was required for its binding ability for the unanchored ubiquitin chains. Experimental and mathematical analysis showed that unanchored ubiquitin chains associated with RIG-I were essential for sustained activation of type I interferon(IFN) signaling. Transcriptomics study revealed that the binding of RIG-I with unanchored ubiquitin chains additionally regulated the expression of a subset of metabolic and cell fate decision genes. Moreover, we found that ubiquitin-specific peptidase 21(USP21) and USP3 deubiquitinate conjugated and unanchored ubiquitin chains on RIG-I respectively. Taken together, characterization of the regulation mode and functions of conjugated ubiquitination and the unconjugated ubiquitin chainbinding of RIG-I may provide means to fine-tune RIG-I-mediated type I IFN signaling.