THE FINE STRUCTURE OF THE NUCLEAR MATERIAL OF A BLUE-GREEN ALGA,ANABAENA CYLINDRICA LEMM

The chromatinic material of the blue-green alga Anabaena cylindrica has complex configurations in the central regions of the cells. The distribution of the chromatin within the cells varies in different filaments, probably in response to variations in the disposition of other cellular components. In electron micrographs of thin sections of organisms fixed by the method of Kellenberger, Ryter, and Séchaud (1958) the centroplasm contains fibrillar and possibly granular components which can be identified as the nuclear material by comparison with stained preparations viewed in the light microscope. The fibrils in the nuclear regions have diameters in the range of 5 to 7 mµ and are embedded in a matrix of lower density. The nuclear regions are not greatly different from the cytoplasm in their electron density. Reducing the calcium content of the fixative results in coagulation of the fibrils to form coarser structures. The significance of the observations is discussed in relation to observations on the fine structure of other classes of algae and of bacteria.     

Spatial distribution of lamin A and B1 in the K562 cell nuclear matrix stabilized with metal ions.

When the nucleus is stripped of most DNA, RNA, and soluble proteins, a structure remains that has been referred to as the nuclear matrix, which acts as a framework to determine the higher order of chromatin organization. However, there is always uncertainty as to whether or not the nuclear matrix, isolated in vitro, could really represent a skeleton of the nucleus in vivo. In fact, the only nuclear framework of which the existence is universally accepted is the nuclear lamina, a continuous thin layer that underlies the inner nuclear membrane and is mainly composed of three related proteins: lamins A, B, and C. Nevertheless, a number of recent investigations performed on different cell types have suggested that nuclear lamins are also present within the nucleoplasm and could be important constituents of the nuclear matrix. In most cell types investigated, the nuclear matrix does not spontaneously resist the extraction steps, but must rather be stabilized before the application of extracting agents. In this investigation, by immunochemical and morphological analysis, we studied the effect of stabilization with different divalent cations (Zn(2+), Cu(2+), Cd(2+)) on the distribution of lamin A and B1 in the nuclear matrix obtained from K562 human erythroleukemia cells. In intact cells, antibodies to both lamin A and B1 mainly stained the nuclear periphery, although some immunoreactivity was detected in the nuclear interior. The fluorescent lamin A pattern detected in Cu(2+)- and Cd(2+)-stabilized nuclei was markedly modified, whereas Zn(2+)-incubated nuclei showed an unaltered pattern of lamin A distribution. By contrast, the distribution of lamin B1 in isolated nuclei was not modified by the stabilizing cations. When chromatin was removed by nuclease digestion and extraction with solutions of high ionic strength, a previously masked immunoreactivity for lamin A, but not for lamin B1, became evident in the internal part of the residual structures representing the nuclear matrix. Our results indicate that when metal ions are used as stabilizing agents for the recovery of the nuclear matrix, the distribution of both lamin A and lamin B1 in the final structures, corresponds to the pattern we have very recently reported using different extraction procedures. This observation strengthen the concept that intranuclear lamins may act as structural components of the nuclear matrix.     

Analysis of Borna Disease Virus Trafficking in Live Infected Cells by Using a Virus Encoding a Tetracysteine-Tagged P Protein

Borna disease virus (BDV) is a nonsegmented, negative-stranded RNA virus characterized by noncytolytic persistent infection and replication in the nuclei of infected cells. To gain further insight on the intracellular trafficking of BDV components during infection, we sought to generate recombinant BDV (rBDV) encoding fluorescent fusion viral proteins. We successfully rescued a virus bearing a tetracysteine tag fused to BDV-P protein, which allowed assessment of the intracellular distribution and dynamics of BDV using real-time live imaging. In persistently infected cells, viral nuclear inclusions, representing viral factories tethered to chromatin, appeared to be extremely static and stable, contrasting with a very rapid and active trafficking of BDV components in the cytoplasm. Photobleaching (fluorescence recovery after photobleaching [FRAP] and fluorescence loss in photobleaching [FLIP]) imaging approaches revealed that BDV components were permanently and actively exchanged between cellular compartments, including within viral inclusions, albeit with a fraction of BDV-P protein not mobile in these structures, presumably due to its association with viral and/or cellular proteins. We also obtained evidence for transfer of viral material between persistently infected cells, with routing of the transferred components toward the cell nucleus. Finally, coculture experiments with noninfected cells allowed visualization of cell-to-cell BDV transmission and movement of the incoming viral material toward the nucleus. Our data demonstrate the potential of tetracysteine-tagged recombinant BDV for virus tracking during infection, which may provide novel information on the BDV life cycle and on the modalities of its interaction with the nuclear environment during viral persistence.     

Melting fine structure of filamentous fungus nuclear DNA.

Melting fine structure of the nuclear DNA isolated from the filamentous fungus Fusarium graminearum Schwabe is presented. Optical melting profiles of nuclear DNA were analyzed by using a combination of curve fitting and derivative techniques. The "melting components" were obtained from the derivative curve by a simple decomposition technique. Differential optical melting curves of unsheared nuclear DNA indicate the presence of 15 "melting components" in filamentous fungus nuclear genome. It should be emphasized that the "melting components" observed here are different from the "thermalites" which can be observed in bacteriophage DNA. The "melting components" reported here represent the separately melting of large "blocks" of fungus nuclear DNA.     

Intracellular localization of adeno-associated viral proteins expressed in insect cells

Production of vectors derived from adeno-associated virus (AAVv) in insect cells represents a feasible option for large-scale applications. However, transducing particles yields obtained in this system are low compared with total capsid yields, suggesting the presence of genome encapsidation bottlenecks. Three components are required for AAVv production: viral capsid proteins (VP), the recombinant AAV genome, and Rep proteins for AAV genome replication and encapsidation. Little is known about the interaction between the three components in insect cells, which have intracellular conditions different to those in mammalian cells. In this work, the localization of AAV proteins in insect cells was assessed for the first time with the purpose of finding potential limiting factors. Unassembled VP were located either in the cytoplasm or in the nucleus. Their transport into the nucleus was dependent on protein concentration. Empty capsids were located in defined subnuclear compartments. Rep proteins expressed individually were efficiently translocated into the nucleus. Their intranuclear distribution was not uniform and differed from VP distribution. While Rep52 distribution and expression levels were not affected by AAV genomes or VP, Rep78 distribution and stability changed during coexpression. Expression of all AAV components modified capsid intranuclear distribution, and assembled VP were found in vesicles located in the nuclear periphery. Such vesicles were related to baculovirus infection, highlighting its role in AAVv production in insect cells. The results obtained in this work suggest that the intracellular distribution of AAV proteins allows their interaction and does not limit vector production in insect cells.     

Tritium nuclear magnetic resonance spectroscopy. Distribution patterns and nuclear overhauser enhancements in some tritiated steroids

The distribution of tritium atoms in some labeled steroids has been determined by tritium nuclear magnetic resonance spectroscopy. In addition, the distribution of molecular types (mono vs. polytritiated) and nuclear Overhauser enhancements have been determined. As near maximal NOE's were observed, quantitative distribution information was derived from NOE suppressed spectra.     

Atomic resolution structures in nuclear transport

There are currently at least 53 structures of components of nuclear transport in the Protein Databank. In addition to providing critical insights into molecular mechanisms of nuclear transport, these atomic resolution structures provide a large body of information that could guide biochemical and cell biological analyses involving nuclear transport proteins. This paper catalogs 53 crystal and NMR structures of nuclear transport proteins, with the emphasis on providing information useful for mutagenesis and overexpression of recombinant proteins.     

植物细胞中NuMA类似蛋白的分布及其在细胞周期中的变化

免疫荧光染色结果说明植物细胞核内含有与抗动物ＮｕＭＡ多抗呈阳性交叉反应的多肽。选择性抽提并结合免疫荧光染色结果说明这种多肽位于核基质纤维蛋白网络上。免疫印迹反应显示胡萝卜（ＤａｕｃｕｓｃａｒｏｔａＬ．）悬浮培养细胞核基质蛋白与抗动物ＮｕＭＡ蛋白多抗的阳性反应条带为７４ｋＤ和７６ｋＤ。有丝分裂各期免疫荧光染色的结果表明植物细胞中的ＮｕＭＡ类似蛋白在有丝分裂过程中呈现有规律的变化。结合选择性抽提的有丝分裂各期的免疫荧光染色的结果表明核基质在此过程中也发生明显变化。应用选择性抽提并结合ＤＧＤ包埋去包埋电镜技术对植物细胞间期及有丝分裂期核基质的形态结构进行了观察。结果显示胡萝卜悬浮培养细胞间期核内存在一个非染色质性的纤维蛋白网络体系,而在正处于分裂的细胞中则未观察到。以上结果说明ＮｕＭＡ类似蛋白是核基质的组分之一并与有丝分裂密切相关。     

Morphometric analysis and topological organization of nuclear matrix in freeze-fractured electron microscopy

The ultrastructural organization of nuclear matrix, purified from intact or membrane-denuded rat liver nuclei, has been analysed by means of freeze-fracturing technique. This method avoids dehydration and embedding which, in conventional thin sectioning, partly distort or mask the matrix ultrastructure. The various matrix components, and mainly the peripheral lamina and the inner network revealed complex arrangements undetectable with conventional techniques. Morphometric analyses performed with a Texture Analysis System (TAS) Leitz, allowed to obtain precise information on the matrix constituents, based on the histograms of their size distribution. These textural characteristics have been utilized in order to identify, by means of a particular computer programme, the putative matrix localization within intact freeze-fractured nuclei.     

Is the "nonspecific" thalamus still "nonspecific"?

The classical concept of "nonspecific" thalamus, as distinguished from the principal thalamic nuclei (i.e. the primary sensory, motor and limbic relays) is here briefly revisited in the light of anatomical investigations performed in the last decades, and primarily those based on tract tracing techniques. Altogether these data pointed out that the so-called "nonspecific" thalamus is composed by a heterogeneous collection of nuclear masses, which display not only species differences, but also marked internuclear variations in their cytological and neurochemical features, connections, areal and laminar distribution upon the cortex, and functional properties. Thus, the "nonspecific" thalamus exerts a modulatory role on cortical activity, chiefly regulated at the intrathalamic level by the interplay between the thalamic reticular nucleus and the interneurons and projection neurons of the dorsal thalamus. However, each of the components that have been traditionally considered as "nonspecific" also subserves selective roles in the transfer of different kinds of information from the thalamus to the cerebral cortex and basal ganglia.     

A histological and NMR study of the melon of the striped dolphin (Stenella coeruleoalba)

The melon, the echolocation organ of the striped dolphin (Stenella coeruleoalba), was investigated by morphological and high-resolution (13)C nuclear magnetic resonance (NMR) methods, in order to characterize structure and composition gradient at the histological and molecular level. Morphological analysis showed that the lipidic components are organized in an adipose tissue; moreover, a clear muscular component was observed. Age-related structural differences also were noted. Furthermore, NMR yielded detailed information at a qualitative-quantitative level on the lipid components.     

Association of topoisomerase II with the hepatoma cell nuclear matrix: The role of intermolecular disulfide bond formation

Previous studies have resulted in conflicting data regarding the recovery of the nuclear enzymes topoisomerase (topo) II and topo I in the nuclear matrix fraction. In the present study we have assessed the effect of systematically altering a single extraction procedure on the distribution of these enzymes during the subfractionation of nuclei from HTC hepatoma tissue culture cells. When nuclear monolayers (prepared by treating attached cells in situ with the neutral detergent Nonidet-P40 at 4 degrees C) were isolated in the presence of the irreversible sulfhydryl blocking reagent iodoacetamide, subsequent treatment with DNase I and RNase A followed by 1.6 M NaCl resulted in structures which were extensively depleted of intranuclear components as assessed by phase contrast microscopy and conventional transmission electron microscopy. These structures contained 12 +/- 4% of the total protein present in the original nuclear monolayers. The lamins and polypeptides with molecular weights comparable to those of actin and vimentin were the predominant polypeptides present on SDS-polyacrylamide gels. Western blotting revealed that less than 5% of the total nuclear topo II molecules were present in these structures. In contrast, when the sulfhydryl cross-linking reagent sodium tetrathionate (NaTT) was substituted for iodoacetamide, the same extraction procedure yielded structures containing components of the nucleolus and an extensive intranuclear network. These structures contained a wide variety of nonlamin, nonhistone nuclear polypeptides including 23 +/- 4% of the total nuclear topo II. SDS-polyacrylamide gel electrophoresis performed under nonreducing conditions revealed that topo II in these nuclear matrices was present as part of a large disulfide cross-linked complex. Treatment of these structures with reducing agents in 1.6 M NaCl released the topo II. In contrast, topo I did not form disulfide cross-linked oligomers and was not detectable in any of these nuclease- and salt-resistant structures prepared at 4 degrees C. To assess the effect of in vitro heat treatment on the distribution of the topoisomerases, nuclear monolayers (isolated in the absence of iodoacetamide and NaTT) were heated to 37 degrees C for 1 h prior to treatment with nucleases and 1.6 M NaCl. The resulting structures (which retained 26 +/- 5% of the total nuclear protein) were morphologically similar to the NaTT-stabilized nuclear matrices and contained 15 +/- 4% of the total nuclear topo II. High-molecular-weight disulfide cross-linked oligomers of topo II were again demonstrated. Attempts to demonstrate these disulfide cross-linked oligomers in intact cells were unsuccessful.     

Differences in male and female macaque dispersal lead to contrasting distributions of nuclear and mitochondrial DNA variation

Male macaques typically leave their natal group before sexual maturity, while females remain for life. Thus genes flow between groups and populations almost solely through male transfer. This asymmetrical dispersal pattern, affects the distribution of variation in the nuclear and mitochondrial genomes differently Nuclear genetic variation, measured by allozyme polymorphisms, is relatively evenly distributed throughout the populations of a macaque species, provided there are no major geographical barriers. Conversely the distribution of maternally inherited mitochondrial DNA (mtDNA) diversity is characterized by local homogeneity and large interpopulational differences. Because of differences in inheritance, dispersal, and population structure, the information contained in nuclear and mitochondrial genomes is best used to address different types of behavioral, genetic, and conservation questions.