Adrenalectomy Potentiates Immediate Early Gene Expression in Rat Brain

Administration of kainate or pentylenetetrazole increased c-fos, c-jun, junB, and junD mRNA levels in rat brain in a dose-dependent manner. Kainate increased these mRNA levels predominantly in the hippocampus, and pentylenetetrazole was more effective in the cortex. Adrenalectomy (3 days) was used to eliminate endogenous glucocorticoid hormones. Adrenalectomy significantly potentiated kainate-induced increases, compared with increases caused by kainate (4 mg/kg) alone, in the hippocampal mRNA levels of c-fos and junB by 6.5-fold and of junD by twofold and tended to augment c-jun mRNA. Corticosterone administration blocked the potentiated stimulation of these mRNA levels caused by adrenalectomy. Adrenalectomy also significantly increased pentylenetetrazole-induced levels of c-fos mRNA in the cortex. These results demonstrate that glucocorticoids modulate immediate early gene expression in the brain, raising the possibility that this interaction contributes to interneuronal and interindividual differences in responses to stimuli and to the effects of stress- or disease-induced changes in glucocorticoid concentrations.     

Temporal Control of Immediate Early Gene Induction by Light

Background

The light-gated cation channel channelrhodopsin-2 (ChR2) is a powerful tool for the optical induction of action potentials in neurons. Mutations of the cysteine 128 (C128) residue have been shown to greatly extend the lifetime of the conducting state of ChR2. However, until now, only subthreshold depolarizations have been reported from C128 mutants.

Methods and Findings

Here we report the induction of long high-frequency spike trains by brief light pulses in ChR2(C128A)-transfected pyramidal cells in hippocampal slice culture. ChR2(C128A)-mediated spike bursts triggered expression of the immediate early gene c-fos in pyramidal neurons. Robust and cell-specific expression of c-Fos protein was detected after a single blue light pulse and depended on action potential firing, but not on synaptic activity. However, photocurrents diminished upon repeated stimulation and limited the number of action potential bursts that could be elicited.

Conclusions

We conclude that the C128A mutant is not suitable for chronic stimulation of neurons, but very useful for light-controlled induction of immediate early genes. This property of ChR2(C128A) could be harnessed to control the expression of proteins under control of the c-fos promoter with precise timing and single cell specificity.     

Using Evolutionary Conserved Modules in Gene Networks as a Strategy to Leverage High Throughput Gene Expression Queries

Background

Large-scale gene expression studies have not yielded the expected insight into genetic networks that control complex processes. These anticipated discoveries have been limited not by technology, but by a lack of effective strategies to investigate the data in a manageable and meaningful way. Previous work suggests that using a pre-determined seed-network of gene relationships to query large-scale expression datasets is an effective way to generate candidate genes for further study and network expansion or enrichment. Based on the evolutionary conservation of gene relationships, we test the hypothesis that a seed network derived from studies of retinal cell determination in the fly, Drosophila melanogaster, will be an effective way to identify novel candidate genes for their role in mouse retinal development.

Methodology/Principal Findings

Our results demonstrate that a number of gene relationships regulating retinal cell differentiation in the fly are identifiable as pairwise correlations between genes from developing mouse retina. In addition, we demonstrate that our extracted seed-network of correlated mouse genes is an effective tool for querying datasets and provides a context to generate hypotheses. Our query identified 46 genes correlated with our extracted seed-network members. Approximately 54% of these candidates had been previously linked to the developing brain and 33% had been previously linked to the developing retina. Five of six candidate genes investigated further were validated by experiments examining spatial and temporal protein expression in the developing retina.

Conclusions/Significance

We present an effective strategy for pursuing a systems biology approach that utilizes an evolutionary comparative framework between two model organisms, fly and mouse. Future implementation of this strategy will be useful to determine the extent of network conservation, not just gene conservation, between species and will facilitate the use of prior biological knowledge to develop rational systems-based hypotheses.     

利用基因芯片对一高保守基因进行肿瘤相关分析

本文对大规模人类cDNA 测序过程中获得的一条高保守基因进行了初步功能研究,生物信息学研究发现该基因在人类、小鼠、果蝇、拟南芥和裂殖酵母中都有很高的保守性,其他分析预测该基因可能具有肿瘤相关性。RT-PCR分析表明,该基因在成人和胎儿组织中广谱表达。利用基因芯片分析该基因在7例肝癌、5例胰腺癌、2例喉癌和2例肺癌中表达情况,结果证实了该基因的肿瘤相关性,并且提示该基因在不同肿瘤类型中可能处于不同的地位。 Abstract:Preliminary function research of a highly conserved human gene,which was cloned from human fetal cDNA library during large-scale cDNA sequencing,is illustrated in this article.Bioinformatics analysis indicates that this gene is highly conserved in human、mouse、fruit fly、thaliana and fission yeast.Other bioinformatics analysis implies its relevance with tumors.RT-PCR analysis shows its wide-ranging expression patterns.Its expression in 16 cancer cases(including 7 liver cancer cases、5 pancreas cancer cases、2 larynx cancer cases and 2 lung cancer cases) is studied by using gene microarray analysis.The result shows its relevance with tumors and implies it may have different status in different classification of tumors.     

利用基因芯片对一高保守基因进行肿瘤相关分析

本对大规模人类cDNA测序过程中获得的一条高保守基因进行了初步功能研究,生物信息学研究发现该基因在人类、小鼠、果蝇、拟南芥和裂殖酶母中都有很高的保守性,其他分析预测该基因可能具有肿瘤相关性。RT－PCR分析表明,该基因在成人和胎儿组织中广泛谱表达。利用基因芯片分析该基因在7例肝癌、5例胰腺癌、2例喉癌和2例肺癌中表达情况,结果证实了该基因的肿瘤相关性,并且提示该基因在不同类型中可能处于不同的地位。     

Functional Organization of Brain Pathways Subserving the Baroreceptor Reflex: Studies in Conscious Animals Using Immediate Early Gene Expression

1. This paper reviews studies carried out in our laboratory in which we have used the c-fos functional mapping method, in combination with other methods, to determine the functional organization of central baroreceptor pathways as they operate in the conscious rabbit.2. First, we showed that periods of induced hypertension or hypotension each result in a specific and reproducible pattern of activation of neurons in the brainstem and forebrain. In particular, hypotension (but not hypertension) results in the activation of catecholamine neurons in the medulla and pons and vasopressin-synthesizing neurons in the hypothalamus.3. The activation of medullary cell groups in response to induced hypertension or hypotension in the conscious rabbit is almost entirely dependent on inputs from arterial baroreceptors, while the activation of hypothalamic vasopressin-synthesising neurons in response to hypotension is largely dependent on baroreceptors, although an increase in circulating angiotensin also appears to contribute.4. Discrete groups of neurons in the rostral ventrolateral medulla (RVLM) and A5 area in the pons are the major groups of spinally projecting neurons activated by baroreceptor unloading. In contrast, spinally projecting neurons in the paraventricular nucleus in the hypothalamus appear to be largely unaffected by baroreceptor signals.5. Direct afferent inputs to RVLM neurons in response to increases or decreases in arterial pressure originate primarily from other medullary nuclei, particularly neurons located in the caudal and intermediate levels of the ventrolateral medulla (CVLM and IVLM), as well as in the nucleus tractus solitarius (NTS).6. There is also a direct projection from barosensory neurons in the NTS to the CVLM/IVLM region, which is activated by baroreceptor inputs.7. Collectively, the results of our studies in conscious animals indicate that baroreceptor signals reach all levels of the brain. With regard to the baroreceptor reflex control of sympathetic activity, our studies are consistent with previous studies in anesthetized animals, but in addition reveal other previously unrecognized pathways that also contribute to this reflex regulation.     

用杆状病毒为载体在昆虫细胞中表达马立克病病毒pp38基因

鸡马立克病病毒(MDV)38kd磷蛋白(pp38)基因中包括起始密码子和终止密码子的完整编码序列被整合进杆状病毒AcNPV的转移载体质粒pVL1392,用所得的含pp38基因的重组转移载体质粒pVLpp38I与野生型杆状病毒AcNPV的DNA共转染昆虫传代细胞系Sf9细胞后,用荧光抗体法以抗MDV单克隆抗体H_(19)筛选到能表达MDVpp38的重组杆状病毒克隆BP38 I。免疫印迹试验表明,在重组病毒BP38 I感染的Sf9细胞溶解物中,可表现一条分子量约为35—36kd的为单克隆抗体H_(19)识别的MDV特异性蛋白带。     

The Pathogenic Properties of a Novel and Conserved Gene Product,KerV, in Proteobacteria

Identification of novel virulence factors is essential for understanding bacterial pathogenesis and designing antibacterial strategies. In this study, we uncover such a factor, termed KerV, in Proteobacteria. Experiments carried out in a variety of eukaryotic host infection models revealed that the virulence of a Pseudomonas aeruginosa kerV null mutant was compromised when it interacted with amoebae, plants, flies, and mice. Bioinformatics analyses indicated that KerV is a hypothetical methyltransferase and is well-conserved across numerous Proteobacteria, including both well-known and emerging pathogens (e.g., virulent Burkholderia, Escherichia, Shigella, Vibrio, Salmonella, Yersinia and Brucella species). Furthermore, among the 197 kerV orthologs analyzed in this study, about 89% reside in a defined genomic neighborhood, which also possesses essential DNA replication and repair genes and detoxification gene. Finally, infection of Drosophila melanogaster with null mutants demonstrated that KerV orthologs are also crucial in Vibrio cholerae and Yersinia pseudotuberculosis pathogenesis. Our findings suggested that KerV has a novel and broad significance as a virulence factor in pathogenic Proteobacteria and it might serve as a new target for antibiotic drug design.     

Fluorescence-Based Monitoring of In Vivo Neural Activity Using a Circuit-Tracing Pseudorabies Virus

The study of coordinated activity in neuronal circuits has been challenging without a method to simultaneously report activity and connectivity. Here we present the first use of pseudorabies virus (PRV), which spreads through synaptically connected neurons, to express a fluorescent calcium indicator protein and monitor neuronal activity in a living animal. Fluorescence signals were proportional to action potential number and could reliably detect single action potentials in vitro. With two-photon imaging in vivo, we observed both spontaneous and stimulated activity in neurons of infected murine peripheral autonomic submandibular ganglia (SMG). We optically recorded the SMG response in the salivary circuit to direct electrical stimulation of the presynaptic axons and to physiologically relevant sensory stimulation of the oral cavity. During a time window of 48 hours after inoculation, few spontaneous transients occurred. By 72 hours, we identified more frequent and prolonged spontaneous calcium transients, suggestive of neuronal or tissue responses to infection that influence calcium signaling. Our work establishes in vivo investigation of physiological neuronal circuit activity and subsequent effects of infection with single cell resolution.     

The BC200 RNA Gene and Its Neural Expression Are Conserved in Anthropoidea (Primates)

The gene encoding BC200 RNA arose from a monomeric Alu element. Subsequently, the RNA had been recruited or exapted into a function of the nervous system. Here we confirm the presence of the BC200 gene in several primate species among the Anthropoidea. The period following the divergence of New World monkeys and Old World monkeys from their common ancestor is characterized by a significantly higher substitution rate in the examined 5′ flanking region than in the BC200 RNA coding region itself. Furthermore, the conservation of CpG dimers in the RNA coding region (200 bp) is drastically increased compared to the 5′ flanking region (∼400 bp) over all 12 species examined. Finally, the brain-specific expression pattern of BC200 RNA and its presence as a ribonucleoprotein particle (RNP) are conserved in Old World and New World monkeys. Our studies indicate that the gene encoding BC200 RNA was created at least 35–55 million years ago and its presence, mode of expression, and association with protein(s) as an RNP are under selective pressure. Received: 1 December 1997 / Accepted: 3 June 1998     

Hermes, a Functional Non-Drosophilid Insect Gene Vector from Musca Domestica

Hermes is a short inverted repeat-type transposable element from the house fly, Musca domestica. Using an extra-chromosomal transpositional recombination assay, we show that Hermes elements can accurately transpose in M. domestica embryos. To test the ability of Hermes to function in species distantly related to M. domestica we used a nonautonomous Hermes element containing the Drosophila melanogaster white (w(+)) gene and created D. melanogaster germline transformants. Transgenic G(1) insects were recovered from 34.6% of the fertile G(0) adults developing from microinjected w(-) embryos. This transformation rate is comparable with that observed using P or hobo vectors in D. melanogaster, however, many instances of multiple-element insertions and large clusters were observed. Genetic mapping, Southern blotting, polytene chromosome in situ hybridization and DNA sequence analyses confirmed that Hermes elements were chromosomally integrated in transgenic insects. Our data demonstrate that Hermes elements transpose at high rates in D. melanogaster and may be an effective gene vector and gene-tagging agent in this species and distantly related species of medical and agricultural importance.     

Temporal Decoding of MAP Kinase and CREB Phosphorylation by Selective Immediate Early Gene Expression

A wide range of growth factors encode information into specific temporal patterns of MAP kinase (MAPK) and CREB phosphorylation, which are further decoded by expression of immediate early gene products (IEGs) to exert biological functions. However, the IEG decoding system remain unknown. We built a data-driven based on time courses of MAPK and CREB phosphorylation and IEG expression in response to various growth factors to identify how signal is processed. We found that IEG expression uses common decoding systems regardless of growth factors and expression of each IEG differs in upstream dependency, switch-like response, and linear temporal filters. Pulsatile ERK phosphorylation was selectively decoded by expression of EGR1 rather than c-FOS. Conjunctive NGF and PACAP stimulation was selectively decoded by synergistic JUNB expression through switch-like response to c-FOS. Thus, specific temporal patterns and combinations of MAPKs and CREB phosphorylation can be decoded by selective IEG expression via distinct temporal filters and switch-like responses. The data-driven modeling is versatile for analysis of signal processing and does not require detailed prior knowledge of pathways.     

Chromatin Remodeling and Immediate Early Gene Activation by SLFN11 in Response to Replication Stress

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用改进的DDRT-PCR技术进行人胚差异基因筛选

介绍一种从不同类型细胞或不同生长状态细胞中分离差异表达基因的快速高效mRNA差异显示技术,其特点是利用Ready-To-Go RT-PCR反应珠和Ready-To-Go RAPD分析珠进行mRNA差异显示分析,使取样步骤降至最低程度,减少了潜在的取样误差和外源DNA污染,并确保每次反应的高度重复性.通过银染测序胶分析差异显示的cDNA带,便于DNA回收和进一步克隆.用此方法分析人胚发育早期不同阶段基因的差异表达,选用6条随机引物对3、4和5周龄人胚进行mRNA差异显示分析,从2 000多条带中共分离出14个差异产物,经二次扩增及反向RNA印迹确证其中6个片段为发育不同阶段差异表达基因.     

SSH法分离早期发育相关基因片段

目的:分离和鉴定与小鼠合子基因组激活(ZGA) 以及母源调控向胚胎调控(MZT) 过渡事件 相关的基因片段,为进一步认识ZGA 和MZT 发生的分子机制奠定基础。方法:利用单个植入前 胚胎抑制性消减杂交(SPE2SSH) 的方法,对单个小鼠MⅡ卵母细胞和22细胞期胚胎进行双向消减 杂交;利用cDNA array 对获得的基因片段作进一步的鉴定。结果:从构建的消减杂交库中随机挑 取得31 个克隆,经cDNA array 鉴定发现其中有5 个MⅡ期特异表达的基因片段和9 个22细胞期 特异表达的基因片段,Genbank 检索发现其中10 个片段是首次报道在植入前发育阶段表达。结 论:这些基因片段具有明显的期特异性表达的特点,可能在ZGA、MZT 和胚胎植入前发育过程中 起着重要的作用。     

Automated Visual Cognitive Tasks for Recording Neural Activity Using a Floor Projection Maze

Neuropsychological tasks used in primates to investigate mechanisms of learning and memory are typically visually guided cognitive tasks. We have developed visual cognitive tasks for rats using the Floor Projection Maze^1,2 that are optimized for visual abilities of rats permitting stronger comparisons of experimental findings with other species.In order to investigate neural correlates of learning and memory, we have integrated electrophysiological recordings into fully automated cognitive tasks on the Floor Projection Maze^1,2. Behavioral software interfaced with an animal tracking system allows monitoring of the animal''s behavior with precise control of image presentation and reward contingencies for better trained animals. Integration with an in vivo electrophysiological recording system enables examination of behavioral correlates of neural activity at selected epochs of a given cognitive task.We describe protocols for a model system that combines automated visual presentation of information to rodents and intracranial reward with electrophysiological approaches. Our model system offers a sophisticated set of tools as a framework for other cognitive tasks to better isolate and identify specific mechanisms contributing to particular cognitive processes.