Imaging activity of neuronal populations with new long-wavelength voltage-sensitive dyes

We have assessed the utility of five new long-wavelength fluorescent voltage-sensitive dyes (VSD) for imaging the activity of populations of neurons in mouse brain slices. Although all the five were capable of detecting activity resulting from activation of the Schaffer collateral-CA1 pyramidal cell synapse, they differed significantly in their properties, most notably in the signal-to-noise ratio of the changes in dye fluorescence associated with neuronal activity. Two of these dyes, Di-2-ANBDQPQ and Di-1-APEFEQPQ, should prove particularly useful for imaging activity in brain tissue and for combining VSD imaging with the control of neuronal activity via light-activated proteins such as channelrhodopsin-2 and halorhodopsin.     

Dissolved organic carbon and fluorescence in Lake Hovsgol: factors reducing humic content of the lake water

 Lake Hovsgol is a large tectonic lake located in northern Mongolia, which has extremely transparent lake water. In our survey, the dissolved organic carbon of the lake water was 80–100 μM-C, and the fluorescence intensity in an excitation and emission matrix was very low. The brown color and high content of humic substances in river water flowing from a watershed consisting of grassland and forests rapidly declined in the coastal area of the lake. The decrease in humic content may be due not only to dilution by the lake water but also to flocculation and photobleaching. Among tectonic lakes in Asia, Lake Hovsgol would appear to have unique biological and hydrological features that reduce humic content and help to maintain water transparency. Received: June 25, 2002 / Accepted: January 10, 2003 Acknowledgments The authors acknowledge helpful discussion with Dr. J. Urabe. We thank Dr. T. Galbaatar, Mongolian Academy of Science, Mongolia, for his arrangements on the expeditions in 1999. We are also indebted to Mr. D. Hadbaatar, B. Ganbat, and the cruise staff of the R/V Suchbaatar for their assistance in the course of the study. This study was supported by Grant-in-Aid No. 09041159 and 13575034 for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Correspondence to:K. Hayakawa     

Coevolution of Interacting Fertilization Proteins

Reproductive proteins are among the fastest evolving in the proteome, often due to the consequences of positive selection, and their rapid evolution is frequently attributed to a coevolutionary process between interacting female and male proteins. Such a process could leave characteristic signatures at coevolving genes. One signature of coevolution, predicted by sexual selection theory, is an association of alleles between the two genes. Another predicted signature is a correlation of evolutionary rates during divergence due to compensatory evolution. We studied female–male coevolution in the abalone by resequencing sperm lysin and its interacting egg coat protein, VERL, in populations of two species. As predicted, we found intergenic linkage disequilibrium between lysin and VERL, despite our demonstration that they are not physically linked. This finding supports a central prediction of sexual selection using actual genotypes, that of an association between a male trait and its female preference locus. We also created a novel likelihood method to show that lysin and VERL have experienced correlated rates of evolution. These two signatures of coevolution can provide statistical rigor to hypotheses of coevolution and could be exploited for identifying coevolving proteins a priori. We also present polymorphism-based evidence for positive selection and implicate recent selective events at the specific structural regions of lysin and VERL responsible for their species-specific interaction. Finally, we observed deep subdivision between VERL alleles in one species, which matches a theoretical prediction of sexual conflict. Thus, abalone fertilization proteins illustrate how coevolution can lead to reproductive barriers and potentially drive speciation.     

Calpastatin, an endogenous calpain-inhibitor protein, regulates the cleavage of the Cdk5 activator p35 to p25

Cyclin-dependent kinase 5 (Cdk5) is a Ser/Thr kinase that is activated by binding to its regulatory subunit, p35. The calpain-mediated cleavage of p35 to p25 and the resulting aberrant activity and neurotoxicity of Cdk5 have been implicated in neurological disorders, such as Alzheimer's disease. To gain further insight into the molecular mechanisms underlying the pathological function of Cdk5, we investigated the role of the calpain inhibitor protein calpastatin (CAST), in controlling the aberrant production of p25. For this purpose, brain tissue from wild-type, CAST-over-expressing (transgenic), and CAST knockout mice were analyzed. Cleavage of p35 to p25 was increased in extracts from CAST knockout mice, compared with wild-type. Conversely, generation of p25 was not detected in brain lysates from CAST-over-expressing mice. CAST expression was 5-fold higher in mouse cerebellum than cerebral cortex. Accordingly, p25 production was lower in the cerebellum than the cerebral cortex. Furthermore, the Ca(2+) -dependent degradation of p35 by proteasome was evident when calpain was inhibited. Taken together, these results suggest that CAST is a crucial regulator of calpain activity, the production of p25, and, hence, the deregulation of Cdk5. Therefore, impairment of CAST expression and its associated mechanisms may contribute to the pathogenesis of neurodegenerative disorders.     

A novel, brain-specific mouse drebrin: cDNA cloning, chromosomal mapping, genomic structure, expression, and functional characterization

Drebrin A, a major neuronal actin-binding protein, regulates the dendritic spine shapes of neurons. Here, we have cloned and characterized a novel mouse cDNA clone encoding a truncated form of drebrin A, named s-drebrin A. Analysis of the genomic organization of the mouse drebrin gene (Dbn1), which mapped to the central portion of chromosome 13, revealed that isoforms including s-drebrin A are generated by alternative splicing from a single drebrin gene. The s-drebrin A mRNA was expressed in the brain, but not in non-neuronal tissues. The s-drebrin A expression was barely detected in the embryonic brain, but was upregulated during postnatal development of the brain. Overexpression of GFP-tagged s-drebrin A in fibroblasts showed it to be associated with actin filaments and with changes in actin cytoskeleton organization. These findings suggest that s-drebrin A has a role in spine morphogenesis, possibly by competing the actin-binding activity with drebrin A.     

Casein Kinase 2 dependent phosphorylation of eIF4B regulates BACE1 expression in Alzheimer’s disease

Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder. Increased Aβ production plays a fundamental role in the pathogenesis of the disease and BACE1, the protease that triggers the amyloidogenic processing of APP, is a key protein and a pharmacological target in AD. Changes in neuronal activity have been linked to BACE1 expression and Aβ generation, but the underlying mechanisms are still unclear. We provide clear evidence for the role of Casein Kinase 2 in the control of activity-driven BACE1 expression in cultured primary neurons, organotypic brain slices, and murine AD models. More specifically, we demonstrate that neuronal activity promotes Casein Kinase 2 dependent phosphorylation of the translation initiation factor eIF4B and this, in turn, controls BACE1 expression and APP processing. Finally, we show that eIF4B expression and phosphorylation are increased in the brain of APPPS1 and APP-KI mice, as well as in AD patients. Overall, we provide a definition of a mechanism linking brain activity with amyloid production and deposition, opening new perspectives from the therapeutic standpoint.Subject terms: Kinases, Alzheimer''s disease, Neuronal physiology, Pathogenesis     

人单核细胞系中HIV-1前病毒转录调节新型研究模型的建立

【目的】抗反转录病毒疗法(ART)能够有效控制人免疫缺陷病毒(Human immunodeficiency virus-1,HIV-1)的复制,但是不能将其完全清除。至2012年底,全球仍有3 500万HIV-1感染者,同年约160万人死于艾滋病(Acquired immune deficiency syndrome,AIDS)及其相关疾病。HIV-1感染难以根治的主要原因之一是机体内HIV-1潜伏储存库(Reservoir)的存在。HIV-1潜伏储存库主要由CD4+T细胞和单核巨噬细胞构成,与CD4+T细胞相比,目前研究者对单核巨噬系细胞中HIV-1病毒复制机制尚不明了,且缺乏适宜的研究体系。因此,为探讨单核细胞活化或分化信号对HIV-1复制的影响,我们建立了旨在研究HIV-1前病毒转录调控机制的人单核巨噬细胞系模型。【方法】构建env区域移码突变和nef区域携带EGFP或Nano Luc报告基因的HIV-1 NLn GFP-Kp或NLn Nano Luc-Kp重组病毒,分别感染2种人单核细胞系THP-1或U937细胞。通过有限稀释法制备单克隆细胞系,利用流式细胞术或Nano Luc荧光素酶活性分析检测报告基因的表达。筛选EGFP或Nano Luc阴性表达的细胞克隆,经激活剂佛波酯(Phorbol-12-myristate-13-acetate,PMA)刺激后鉴定潜伏感染的细胞克隆。【结果】研究中鉴定了4个HIV-1潜伏感染的细胞克隆。其中2个是表达EGFP的THP-1克隆,2个是以Nano Luc为报告基因的U937克隆。这些克隆在PMA刺激处理后皆有报告基因的表达,而在恒态条件下未检测到报告基因的表达。【结论】成功建立了4个HIV-1潜伏感染的人单核细胞系克隆,该模型有助于理解单核巨噬系细胞的HIV-1病毒复制机制,可能成为进一步研究HIV-1前病毒转录调控机制的有力工具。     

Evidence for the Involvement of Mitochondrial Respiration in Calcification in a Marine Coccolithophorid, Emiliania huxleyi

In the marine coccolithophorid, Emiliania huxleyi, CaCO₃ productionunder illumination showed a lag phase for about 3 h and thenincreased greatly. During the lag phase the rate of CaCO₃ productionin the light was similar to that in the dark. The productionof CaCO₃ in the dark was inhibited by the addition of 170 µMCCCP, 1 mM KCN and 1 mM SHAM. These results suggest that a littleproduction of CaCO₃ is supported by energy from mitochondrialrespiration, but that large amount of CaCO₃ production requiresphotosynthesis. ¹Present address: SDS Biotech K.K., Tsukuba Technology Center,Midorigahara 2-1, Tsukuba, Ibaraki, 300-26 Japan