川百合花粉管的生殖细胞分裂过程中微管骨架的分布变化

应用透射电镜辅以免疫荧光定位技术研究了川百合 (Lilium davidii Duch.)花粉管中生殖细胞分裂过程中染色体动态和微管分布的关系。在生殖细胞分裂前和有丝分裂前期 ,电镜观察一直未见微管结构 ,但免疫荧光图象显示生殖细胞中有微管蛋白存在。直到分裂的前中期—中期 ,染色体出现 ,它们沿花粉管的长轴前后排列 ,横向的着丝点对相应地一对对地纵向排列。这时 ,生殖细胞中才出现大量微管 ,它们分布于细胞周质区和染色体之间 ,并跨越染色体的整个长度。前中期—中期开始时 ,只有 1～ 2对着丝点从横向转为纵向 ,微管垂直插入着丝点形成着丝点微管 ,而非前人用免疫荧光方法观察到的微管与着丝点侧向联接的图象。随着横向的着丝点对逐渐转变成纵向的过程 ,着丝点微管数量逐渐增多 ,但不形成典型的纺锤体。分裂后期 ,染色体交错分离 ,微管的分布与前中期—中期的基本相同。晚后期 ,染色体呈明显的两群 ,除极区和细胞中央区有微管残余外 ,大部分微管消失。通过染色体长度的测量 ,间接证明了分裂后期 B的存在。分裂末期的晚期 ,核膜形成后 ,在两精核之间的区域 ,微管数量开始增多。此区可能代表用免疫荧光所观察到的微管重叠区。细胞板出现后 ,微管消失     

The role of gap junction membrane channels in development

In most developmental systems, gap junction-mediated cell-cell communication (GJC) can be detected from very early stages of embryogenesis. This usually results in the entire embryo becoming linked as a syncytium. However, as development progresses, GJC becomes restricted at discrete boundaries, leading to the subdivision of the embryo into communication compartment domains. Analysis of gap junction gene expression suggests that this functional subdivision of GJC may be mediated by the differential expression of the connexin gene family. The temporal-spatial pattern of connexin gene expression during mouse embryogenesis is highly suggestive of a role for gap junctions in inductive interactions, being regionally restricted in distinct developmentally significant domains. Using reverse genetic approaches to manipulate connexin gene function, direct evidence has been obtained for the connexin 43 (Cx43) gap junction gene playing a role in mammalian development. The challenges in the future are the identification of the target cell populations and the cell signaling processes in which Cx43-mediated cell-cell interactions are critically required in mammalian development. Our preliminary observations suggest that neural crest cells may be one such cell population.     

Electron transfer in reaction centers of Rhodobacter sphaeroides and Rhodobacter capsulatus monitored by fluorescence of the bacteriochlorophyll dimer

Spectral and kinetic characteristics of fluorescence from isolated reaction centers of photosynthetic purple bacteria Rhodobacter sphaeroides and Rhodobacter capsulatus were measured at room temperature under rectangular shape of excitation at 810 nm. The kinetics of fluorescence at 915 nm reflected redox changes due to light and dark reactions in the donor and acceptor quinone complex of the reaction center as identified by absorption changes at 865 nm (bacteriochlorophyll dimer) and 450 nm (quinones) measured simultaneously with the fluorescence. Based on redox titration and gradual bleaching of the dimer, the yield of fluorescence from reaction centers could be separated into a time-dependent (originating from the dimer) and a constant part (coming from contaminating pigment (detached bacteriochlorin)). The origin was also confirmed by the corresponding excitation spectra of the 915 nm fluorescence. The ratio of yields of constant fluorescence over variable fluorescence was much smaller in Rhodobacter sphaeroides (0.15±0.1) than in Rhodobacter capsulatus (1.2±0.3). It was shown that the changes in fluorescence yield reflected the disappearance of the dimer and the quenching by the oxidized primary quinone. The redox changes of the secondary quinone did not have any influence on the yield but excess quinone in the solution quenched the (constant part of) fluorescence. The relative yields of fluorescence in different redox states of the reaction center were tabulated. The fluorescence of the dimer can be used as an effective tool in studies of redox reactions in reaction centers, an alternative to the measurements of absorption kinetics.Abbreviations Bchl bacteriochlorophyll - Bpheo bacteriopheophytin - D electron donor to P⁺ - P bacteriochlorophyll dimer - Q quinone acceptor - Q_A primary quinone acceptor - Q_B secondary quinone acceptor - RC reaction center protein - UQ₆ ubiquinone-30     

Rapid reactions of spruce cells to elicitors released from the ectomycorrhizal fungus Hebeloma crustuliniforme, and inactivation of these elicitors by extracellular spruce cell enzymes

Elicitors released from hyphae or cell walls of the ectomycorrhizal fungus Hebeloma crustuliniforme (Bull. ex Fries.) Quél. induced in suspension-cultured cells of Picea abies (L.) Karst. a set of fast reactions: (i) an immediate efflux of Cl^– into the medium, followed by a K⁺ efflux; (ii) an influx of Ca²⁺ (measured as accumulation of ⁴⁵Ca²⁺ in the cells); (iii) a phosphorylation of a 63-kDa protein and dephosphorylation of a 65-kDa protein (detectable by 4 min after elicitor application); (iv) an alkalinization of the medium, and (v) a transient synthesis of H₂O₂. The removal of extracellular Ca²⁺ by EGTA delayed the elicitor-induced alkalinization. A further reduction of this response could be achieved by TMB-8 an inhibitor of Ca²⁺ release from intracellular stores. Moreover, the inhibition of protein kinase activity by staurosporine prevented the extracellular alkalinization completely. However, the effectiveness of the elicitors in inducing the extracellular alkalinization was strongly impaired by constitutively secreted enzymes of spruce cells which cleaved the elicitors to inactive fragments. It is suggested that in ectomycorrhizae the efficacy of elicitors released from fungal cell walls is controlled by apoplastic enzymes of the host; the plant itself is able to reduce the activity of fungal elicitors on their way through the plant cell wall. But those elicitors which finally reach the plasma membrane of host cells induce reactions that are similar to the early defense reactions in plant-pathogen interactions.Abbreviations DW dry weight - FW fresh weight - TMB-8 3,4,5 trimethoxybenzoic acid 8-(diethylamino)-octyl ester We thank Prof. M. Zenk (Universität München, Germany) for providing spruce cell cultures, and Dr. I. Kottke (Universität Tübingen, Germany) for isolates of Hebeloma crustuliniforme Tü 704. We are also thankful to Dr. W. Mayer (Universität Tübingen) for valuble discussions. This work was supported by Deutsche Forschungsgemeinschaft. B. Zitterell-Haid was financed by Graduiertenkolleg Interaktion in Waldökosystemen (supported by Deutsche Forschungsgemeinschaft) and G. Hebe by a scholarship of the Landesgraduiertenförderungsgesetz.     

Immunocytochemical localization of callose in the germinated pollen ofCamellia japonica

Summary A polyclonal antibody against -1,3-glucan, callose, extracted from the pollen tube wall ofCamellia japonica was raised in mice and, using it as a probe, the localization of callose in the germinated pollen was studied. By confocal laser scanning microscopy, callose was found in the tip region of the pollen tube and the tube wall; the immuno-fluorescence in the tube wall was less toward the base of the tube. In contrast, the tip region did not fluoresce although the whole of the tube wall did strongly with aniline blue, the specific dye for callose. Immuno-electron microscopy showed that callose was also found in Golgi vesicles which concentrated in the tip region of the pollen tube, the inner layer of the tube wall, callose plugs, and Golgi vesicles in the pollen grain. Immuno-gold labeling was often detected on the fibrous structures in Golgi vesicles and callose plugs. Based on these results, the participation of Golgi vesicles in the formation of the tube wall and callose plugs was discussed.Abbreviation TBS Tris-buffered saline - Tris Tris(hydroxy-methyl)-aminomethane - PBS phosphate-buffered saline - BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay - CLSM confocal laser scanning microscopy - DP degree of polymerization     

Distribution and frequency of plasmodesmata in relation to photoassimilate pathways and phloem loading in the barley leaf

Large, intermediate, and small bundles and contiguous tissues of the leaf blade of Hordeum tvulgare L. ‘Morex’ were examined with the transmission electron microscope to determine their cellular composition and the distribution and frequency of the plasmodesmata between the various cell combinations. Plasmodesmata are abundant at the mesophyll/parenchymatous bundle sheath, parenchymatous bundle sheath/mestome sheath, and mestome sheath/vascular parenchyma cell interfaces. Within the bundles, plasmodesmata are also abundant between vascular parenchyma cells, which occupy most of the interface between the sieve tube-companion cell complexes and the mestome sheath. Other vascular parenchyma cells commonly separate the thick-walled sieve tubes from the sieve tube-companion cell complexes. Plasmodesmatal frequencies between all remaining cell combinations of the vascular tissues are very low, even between the thin-walled sieve tubes and their associated companion cells. Both the sieve tube-companion cell complexes and the thick-walled sieve tubes, which lack companion cells, are virtually isolated symplastically from the rest of the leaf. Data on plamodesmatal frequency between protophloem sieve tubes and other cell types in intermediate and large bundles indicate that they (and their associated companion cells, when present) are also isolated symplastically from the rest of the leaf. Collectively, these data indicate that both phloem loading and unloading in the barley leaf involve apoplastic mechanisms.     

Application of DNA amplification fingerprinting (DAF) to mixed culture bioreactors

Summary The use of DNA amplification fingerprinting (DAF) as a tool for monitoring mixed microbial populations in bioreactors was evaluated. Short (8-mer or 10-mer) oligonucleotides were used to prime DNA extracts from various biological reactors during polymerase chain reaction (PCR) amplification. The reactors examined in this study included two sets of anaerobic stirred tank continuous flow bioreactors. One set of anaerobic reactors was operated under methanogenic conditions and one set was operated under sulfate-reducing conditions. The anaerobic reactor communities in the methanol-fed reactors showed extensive DAF homology. DAF was also applied to a fixed-film azo dye degrading reactor to examine the degree of uniformity of colonization of the substratum in representative regions of the reactor. This method is a quick and relatively inexpensive means of monitoring microbial community structure during biological processes. Since no cultivation of the sample is involved, the genetic profile of the community is not biased by outgrowth conditions. DAF profiles may be useful for comparisons of population changes over time or of bench-scale vs pilot-scale reactors but not adequate for assessing community diversity.     

二核苷酸重复多态性的非同位素检测及其在基因诊断中的应用

本文报道了一种检测二核苷酸重复多态性的简便的非同位素法,利用重复序列两侧的特异引物进行ＰＣＲ扩增,产生的等位片段在薄层变性聚丙烯酰胺凝胶电泳上分离,再用灵敏的银染法显色。该方法不需要标记ＰＣＲ产物,简便、快速,分辨率可达１ｂｐ,并可用多对引物同时进行多重ＰＣＲ分析。用此方法对ＤＭＤ家系成员ｄｙｓｔｒｏｐｈｉｎ基因的５＇－脑型外显子止游区和３＇－非翻译区的两个（ＣＡ）。位点进行了扩增片段长度多态性分     

抗原捕获聚合酶链式反应（AC－PCR）直接分型检测单纯疱疹病毒

用抗单纯疱疹病毒（ＨＳＶ）型共同性ｇＣ和ｇＤ羊克隆抗体（ＭｃＡｂ）,包被即Ｅｐｐｅｎｄｏｒｆ管,捕捉ＨＳＶ,同时加入３个引物：一个是ＨＳＶ─１／ＨＳＶ─２型共同性上游引物,另两个分别是ＨＳＶ─１和ＨＳＶ─２型特异性下游引物。借此建立了能直接分型检测ＨＳＶ的抗原捕获聚合酶链式反应（ＡＣ─ＰＣＲ）。ＨＳＶ─１的扩增产物为４７７ｂｐ,ＨＳＶ─２的为３９９ｂｐ两型病毒经ＡＣ─ＰＣＲ扩增后产生分子量不同的ＤＮＡ片段,致使ＡＣ─ＰＣＲ能直接分型检测ＨＳＶ。ＨＳＶ─１和ＨＳＶ─２扩增产物的克隆和序列分析表明,本方法特异性好。用本法检测Ｂａｌｂ／ｃ幼鼠中枢神经系统ＨＳＶ感染的脑标本,进一步证实本方法不仅敏感、特异,而且分型准确。     

应用PCR技术检测细小病毒H－1DNA在人肝癌与裸鼠正常组织中复制的差异

应用ＰＣＲ技术检测细小病毒Ｈ－１ＤＮＡ在人肝癌与裸鼠正常组织中复制的差异黄青山,马承武,郭兰萍,陈献华,罗祖玉（上海复旦大学生理与生物物理学系,上海２００４３３）关键词：自主性细小病毒Ｈ－１及ＭＶＭ,聚合酶链式反应（ＰＣＲ）,人肝癌模型,抑瘤作用,肿...