Fluorescent in situ hybridization technique for cell type identification and characterization in the central nervous system

Central nervous system consists of a myriad of cell types. In particular, many subtypes of neuronal cells, which are interconnected with each other, form the basis of functional circuits. With the advent of genomic era, there have been systematic efforts to map gene expression profiles by in situ hybridization (ISH) and enhancer-trapping strategy. To make full use of such information, it is important to correlate “cell types” to gene expression. Toward this end, we have developed highly sensitive method of fluorescent dual-probe ISH, which is essential to distinguish two cell types expressing distinct marker genes. Importantly, we were able to combine ISH with retrograde tracing and antibody staining including BrdU staining that enables birthdating. These techniques should prove useful in identifying and characterizing the cell types of the neural tissues. In this article, we describe the methodology of these techniques, taking examples from our analyses of the mammalian cerebral cortex.     

The production and elimination of supernumerary blast cells in the leech embryo

 Different species of leech vary greatly in body size but all have 32 body segments. It is unclear how the development of this precise number of segments is regulated, although it is known that the teloblasts of the early leech embryo initially produce more than the required numbers of segment founder cells (blast cells). We used fluorescent dextrans to show that the M teloblast of the Helobdella robusta embryo produces a variable number of additional (supernumerary) cells. These cells fail to enter the germinal band (which contains cells of all lineages and gives rise to the adult leech), but detach from its posterior end and disappear. Our observations suggest that some suffer an increase in membrane permeability while others fuse with the M teloblasts, but that they do not undergo apoptosis. The supernumerary cells of different lineages detach from the germinal band at different times, suggesting that detachment is not triggered by a global signal acting simultaneously on all lineages. We tested the hypothesis that the elimination of the supernumerary m blast cells results from a requirement of m blast cells for close interactions with cells of the other lineages for their survival, a condition that would not be achieved by the last-born m blast cells that fail to enter the germinal band. We cultured isolated M teloblasts and found that they do produce blast cells that themselves divide, indicating that cells of the M lineage can survive in the absence of any interactions with cells of the other lineages. Received: 17 August 1998 / Accepted: 20 November 1998     

8-Hydroxy-(+)-delta-cadinene is a precursor to hemigossypol in Gossypium hirsutum

[(3)H](+)-delta-Cadinene and its 8-hydroxy derivative, prepared from (1RS)-[1-(3)H]FPP by the action of one and two recombinant enzymes, respectively, were infiltrated into cotyledons of bacterial blight-resistant cotton plants as they biosynthesized sesquiterpene phytoalexins in response to infection by Xanthomonas campestris pv. malvacearum. Following both treatments, tritium appeared in the HPLC fraction that contained hemigossypol. Hemigossypol was isolated from the cotyledons that had been treated with [(3)H](+)-8-hydroxy-delta-cadinene and was trimethylsilylated and purified. In two experiments, specific radioactivity of the hemigossypol derivative indicated that 5% and 10%, respectively, of the [(3)H](+)-8-hydroxy-delta-cadinene had been converted to hemigossypol.     

Eat and run? The hunger/satiation hypothesis in vertical migration: history,evidence and consequences

The study of vertical migrations in aquatic organisms has a long and colourful history, much of it to do with the effects of changing sampling technology on our understanding of the phenomenon. However, the overwhelming majority of such studies carried out today still depend on detecting differences in vertical distribution profiles during some course of time, or acoustic echoes of migrating bands of organisms. These can not distinguish migratory activity of individual organisms, but can only assess net results of mass transfers of populations, which may integrate many individual migrations. This is an important distinction, for without knowing the actual movements of individuals it seems unlikely that we will be able to understand their causes, nor the effects of vertical migrations on the environment or on the migrators themselves. This review examines evidence for individual vertical movements gathered from 'tracers', mainly gut contents, and reviews the evidence for the hypothesis that such movements are in fact driven by hunger and satiation. The more recently appreciated vertical migrations of phytoplankters and their similarities in form and driving forces to those of zooplankton and nekton are also discussed. Finally, the role of vertical migrators in vertical fluxes of materials is discussed, along with the consequences of satiation-driven descent for such estimates.     

西南喀斯特地区典型林分土壤优先流特征及影响因素

研究喀斯特地区的优先流现象,明确影响优先流的主要因素,对当地植被恢复具有重要意义。通过野外染色示踪试验以及图像处理技术,获得染色溶液在垂直和水平方向上的分布情况,并提取总染色面积比、基质流深度、优先流比和长度指数作为优先流特征参数,运用灰色关联度分析法对影响优先流的14个因子进行分析。结果表明: 喀斯特地区典型林分土壤存在优先流现象,其优先流主要有漏斗状和树枝状两类,有横向水流运动现象发生。喀斯特地区林下的染色面积比平均为19.4%,基质流深度、优先流比和长度指数平均分别为4.96 cm、62.9%和385.5%。在影响优先流的14个环境因子中,对染色面积比影响最大的是土壤初始含水量,对基质流深度影响最大的是速效钾含量,对优先流比和长度指数影响最大的均为速效磷含量。喀斯特地区典型林分下优先流的发育程度高且空间变化剧烈,土壤初始含水量等物理性质对其有较大影响,土壤养分也是优先流的一个重要影响因素。     

The use of the fluorescent probe 8-anilino-1-naphthalene sulphonic acid (ANS) as a histochemical stain in plant tissues

Abstract. The fluorescent probe 8-anilino-l-naphthalene sulphonic acid (ANS) has been evaluated as a histochemical stain for plant tissues. The wide specificity of the compound for hydrophobic binding sites limits its analytical use, but renders it of considerable value as a general fluorescent stain for use in epi-illumination fluorescence microscopy. Used in this way it is analogous to the light-microscope stain toluidine blue. ANS has also been found to be a sensitive vascular tracer.     

应用^45Ca示踪技术研究烤烟对钙肥的吸收及分布规律

应用放射性核素^45Ca示踪方法,以烤烟品种“K326”为材料进行盆栽试验,研究在不同施钙水平下,烤烟对钙素营养的吸收分配规律。结果表明：烟株在旺长期体内积存Ca量最高。烤烟吸收的Ca肥量随施Ca水平的提高或生育期的进程而增加,有显著的正相关性。将烟吸收的钙肥在烟株的地上部分主要分配在叶中,其次是茎,花与侧芽较少。烟叶中吸收Ca肥量占地上部分的百分数随生育期进程下降,而烟茎的却增加。烟株地上部吸收的Ca肥量占施入量的百分数随施Ca水平的提高而减少,但随生育期进程而增加。     

Using isotopomer path tracing to quantify metabolic fluxes in pathway models containing reversible reactions

As a more complete picture of the genetic and enzymatic composition of cells becomes available, there is a growing need to describe how cellular regulatory elements interact with the cellular environment to affect cell physiology. One means for describing intracellular regulatory mechanisms is concurrent measurement of multiple metabolic pathways and their interactions by metabolic flux analysis. Flux of carbon through a metabolic pathway responds to all cellular regulatory systems, including changes in enzyme and substrate concentrations, enzyme activation or inhibition, and ultimately genetic control. The extent to which metabolic flux analysis can describe cellular physiology depends on the number of pathways in the model and the quality of the data. Intracellular information is obtainable from isotopic tracer experiments, the most extensive being the determination of the isotopomer distribution, or specific labeling pattern, of intracellular metabolites. We present a rapid and novel solution method that determines the flux of carbon through complex pathway models using isotopomer data. This time-consuming problem was solved with the introduction of isotopomer path tracing, which drastically reduces the number of isotopomer variables to the number of isotopomers observed experimentally. We propose a partitioned solution method that takes advantage of the nearly linear relationship between fluxes and isotopomers. Whereas the stoichiometric matrix and the isotopomer matrix are invertible, simulated annealing and the Newton-Raphson method are used for the nonlinear components. Reversible reactions are described by a new parameter, the association factor, which scales hyperbolically with the rate of metabolite exchange. Automating the solution method permits a variety of models to be compared, thus enhancing the accuracy of results. A simplified example that contains all of the complexities of a comprehensive pathway model is presented. Copyright John Wiley & Sons, Inc.     

Sites of lanthanum occlusion in the testis of the crayfish Procambarus paeninsulanus (Crustacea: Cambaridae)

The presence of stage-dependent occlusive junctions between adjacent Sertoli cells in the seminiferous epithelium of the crayfish testis was demonstrated by a lanthanum tracer study. The germinal epithelium did not appear to be compartmentalized, as evidenced by access of lanthanum to spermatogonia, spermatocytes, and spermatids. During late spermiogenesis, when encapsulated stage VI spermatids were concentrated in the center of an acinus, lanthanum was excluded apically, coincident with lumen formation. This is the first study examining occluding junctions using a barrier penetration method in the testis of a crustacean.