RNA transport in isolated myeloma nuclei. Transport from membrane- denuded nuclei

Nuclei prepared from MOPC-21 cells were treated with the nonionic detergents Triton X-100 or Nonidet P-40. Chemical analysis revealed that nearly 90% of the nuclear phospholipid was removed by detergent treatment. The membrane-denuded nuclei remained intact with preservation of nuclear pore complexes as demonstrated by electron microscopy. Ribonucleic acid transport from detergent-treated nuclei proceeded at the same rate and to the same extent as in control nuclei. Normal nuclear restriction of nucleic acids was unaltered by removal of the nuclear membranes. The effect of temperature on transport of RNA from freshly isolated myeloma nuclei with intact nuclear envelopes was studied. No temperature transition was associated with the transport process. These data indicate that the transport of macromolecules from isolated myeloma nuclei is independent of the nuclear membrane.     

Ultrastructure of chick erythrocyte nuclei undergoing reactivation in heterokaryons and enucleated cells

The reactivation of chick erythrocyte nuclei after Sendai virus induced fusion of chick erythrocytes with intact or anucleate rat myoblasts or rat epithelial cells was studied by electron microscopy. Both in heterokaryons and in reconstituted cells formed by the fusion of chick red cells with anucleate rat L6 myoblasts the amount of highly condensed chromatin in the chick nuclei decreased with time after fusion at the same time as the proportion of dispersed chromatin increased. Nuclear organelles, typical of active nuclei but absent in the nuclei of unfused erythrocytes, appeared during reactivation. The percentage of chick nuclei containing a nucleolus was low 24 h after fusion but increased so that almost all nuclei contained one or more nucleoli 120 h after fusion. In reconstituted cells the frequency of nucleoli was much lower than in heterokaryons. In other respects, the erythrocyte nuclei introduced into anucleate rat cells underwent a normal reactivation and appeared to be well integrated with the cytoplasm. Thus, the nuclear envelope consisted of two normal leaflets in direct contact with the cytoplasm. Nuclear pores were observed in front of interchromatin channels. A normal cytoplasmic geometry appeared to be re-established since the Golgi apparatus occupied a position close to the poles of the chick nucleus.     

Selective localization of neptunium-237 in nuclei of mammalian cells]

After injection in the rat of soluble neptunium salt, the distribution of this element was studied at the subcellular level by electron microscopy and electron probe microanalysis. Abnormal structures have been observed by electron microscopy in the nuclei of hepatocytes, and the same structures have also been observed in the nuclei of the proximal tubules cells of the kidney. These structures are formed of clusters of very small and dense particles, several nanometers in diameter. The clusters are localized in the central part of the nuclei and they are separate from nucleoli and heterochromatin. Electron probe X-ray analysis of this cluster have shown that they contain neptunium associated with phosphorus. In the cell containing neptunium inclusions, other non specific lesions are also observed (nuclear pycnosis, mitochondrial depletion).     

Cytologic characteristics and origin of naked nuclei in breast aspirate smears

To elucidate the origin of "naked nuclei" in breast aspiration smears, 17 cases of fibroadenoma were studied by light and electron microscopy. The ATPase reaction was also studied at both levels. The aspirates contained two types of naked nuclei: denuded degenerated nuclei and oval to spindle-shaped nuclei with very scanty cytoplasm. The cytoplasm of the latter was rich in free ribosomes and rough-surfaced endoplasmic reticulum but was devoid of cytoplasmic filaments and dense bodies. These cells were negative for ATPase activity. Stromal cells, not myoepithelial cells, characteristically demonstrated such cellular features in the aspirates and tissue sections studied. We conclude that most naked nuclei are derived from stromal cells.     

The effect of sodium tetrathionate stabilization on the distribution of three nuclear matrix proteins in human K562 erythroleukemia cells

Using both conventional fluorescence and confocal laser scanning microscopy we have investigated wheter or not stabilization of isolated human erythroleukemic nuclei with sodium tetrathionate can maintain in the nuclear matrix the same spatial distribution of three polypeptides (M_r 160 kDa and 125 kDa, previously shown to be components of the internal nuclear matrix plus the 180-kDa nucleolar isoform of DNA topoisomerase II) as seen in permeabilized cells. The incubation of isolated nuclei in the presence of 2 mM sodium tetrathionate was performed at 0° C or 37° C. The matrix fraction retained 20–40% of nuclear protein, depending on the temperature at which the chemical stabilization was executed. Western blot analysis revealed that the proteins studied were completely retained in the high-salt resistant matrix. Indirect immunofluorescence experiments showed that the distribution of the three antigens in the final matrix closely resembled that detected in permeabilized cells, particularly when the stabilization was performed at 37° C. This conclusion was also strengthened by analysis of cells, isolated nuclei and the nuclear matrix by means of confocal laser scanning microscopy. We conclude that sodium tetrathionate stabilization of isolated nuclei does not alter the spatial distribution of some nuclear matrix proteins.     

Early replication signals in nuclei of Chinese hamster ovary cells

Summary DNA replication sites generally known as replicon domains were resolved as individual replication signals in interphase nuclei of permeabilized Chinese hamster ovary cells by immunofluorescent microscopy. Biotin-11-dUTP was utilized as a tool to label newly replicated DNA in permeable cells and to study the distribution of nascent DNA in pulselabel and in pulsechase experiments. Active sites of DNA replication were visualized in exponentially growing cells and in synchronized cultures throughout the S phase. Fluorescent images of replication sites were analyzed by standard fluorescense microscopy and in three dimensions by confocal laser scanning microscopy. The rapid increase in number of discrete foci of newly replicated DNA is an indication that DNA synthesis starts at limited number of sites in mammalian nuclei rather than at thousands of foci at the same time.     

ENTRY OF BASIC MACROMOLECULES INTO BARLEY ROOTS

The entry of labelled calf-thymus histone, lysozyme, and poly-L-lysine into barley root tips was studied at concentrations which strongly inhibit root elongation. The macromolecules were suitably labelled and at these concentrations it was found, by autoradiography and fluorescence microscopy, that histone and lysozyme readily entered the roots and appeared to bind mainly to cell walls of the epidermis and cortex and to penetrate the cytoplasm occasionally. Except in cap cells, nuclei were rarely penetrated. Poly-L-lysine readily permeated cell walls and invaded cytoplasm and nuclei throughout the root tip. Some cells were damaged by contact with basic macromolecules, as evidenced by a change in appearance of protoplasts under phase contrast and by the inability of these same protoplasts to exclude labelled β-lactoglobulin. Such damage was restricted to cells in contact with the outer solution. Interior to the epidermis, development of many cells was inhibited without visible signs of damage. Evidence supports the conclusion that in the presence of polybasic polymers the integrity of cell membranes is altered, thereby allowing leakage of some cell constituents essential for normal development.     

The nuclei of <Emphasis Type="Italic">Giardia lamblia</Emphasis>—new ultrastructural observations

Giardia lamblia is a parasite possessing a complex cytoskeleton and an unusual morphology of bearing two nuclei. Here, the interphasic nuclei of trophozoites, using field emission scanning electron microscopy, routine scanning and transmission electron microscopy, immunocytochemistry, and 3D reconstruction, are presented. An approach using plasma-membrane extraction allowed the observation of the two nuclei still attached in their original positions. The observations are as follows: (1) Giardia nuclei and cytoskeleton were studied in demembranated cells by routine scanning electron microscopy and field emission; (2) both nuclei are anchored to basal bodies of the anterior flagella and to the descending posterior-lateral and ventral flagella, at the right and left nuclei, respectively, in cells attached by its ventral disc; (3) this attachment occurs by proteinaceous links, which were labeled by anti-actin and anti-centrin but not by anti-dynein or anti-tubulin antibodies; (4) fibrilar connections between the nuclei and the disc were also observed; and (5) nuclei exhibited a pendular movement when living cells were treated with cytochalasin, although the nuclei were still connected by their anterior region. Our analysis indicated that the nuclei have a defined position, and fibrils perform an anchoring system. This raises the possibility of a mechanism for nuclei-fidelity migration during mitosis.     

The polypeptide composition and ultrastructure of nuclear ghosts isolated from mammalian cells

The polypeptide species of non-membranous nuclear ghosts from purified cell nuclei are conserved among a variety of human, hamster and mouse cell types studied, including HeLa, BHK, 3T6 and Hep-2 cell lines. The polypeptide species present in nuclear ghosts from HeLa cells synchronized in various stages of the cell cycle are largely the same with minor variations. The isolated nuclear ghosts are similar, in terms of polypeptide composition, to other residual nuclear structures isolated by independent techniques. The nuclear ghosts appear as flattened sac-like structures when viewed by scanning electron microscopy. Transmission electron microscopy of the nuclear ghosts reveals ring-like structures which may represent the nuclear pores. Also observed are novel rod-shaped structures approximately 260 nm in length and 50 nm in diameter. The latter images either arise by a rearrangement during isolation of the nuclear ghost macromolecules or are a heretofore undescribed structure of intact nuclei.     

Cryo X-ray nano-tomography of vaccinia virus infected cells

We have performed full-field cryo X-ray microscopy in the water window photon energy range on vaccinia virus (VACV) infected cells to produce tomographic reconstructions. PtK2 cells were infected with a GFP-expressing VACV strain and frozen by plunge fast freezing. The infected cells were selected by light fluorescence microscopy of the GFP marker and subsequently imaged in the X-ray microscope under cryogenic conditions. Tomographic tilt series of X-ray images were used to yield three-dimensional reconstructions showing different cell organelles (nuclei, mitochondria, filaments), together with other structures derived from the virus infection. Among them, it was possible to detect viral factories and two types of viral particles related to different maturation steps of VACV (immature and mature particles), which were compared to images obtained by standard electron microscopy of the same type of cells. In addition, the effect of radiation damage during X-ray tomographic acquisition was analyzed. Thin sections studied by electron microscopy revealed that the morphological features of the cells do not present noticeable changes after irradiation. Our findings show that cryo X-ray nano-tomography is a powerful tool for collecting three-dimensional structural information from frozen, unfixed, unstained whole cells with sufficient resolution to detect different virus particles exhibiting distinct maturation levels.     

Ultrastructural and autoradiographic analysis of the cellular reactivity of the exo- and endocrine epithelium of the frog pancreas under the action of cobalt chloride]

The frog pancreatic epithelium was studied using morphometry, autoradiography and electron microscopy during the treatment with cobalt chloride (10 mg/100 g body wieght). In response to this treatment, generative changes in some exocrine cells were revealed. At the same time, those of cells, whose nuclei were in the state of DNA synthesis, increased in number. Cobalt chloride resulted in endocrine A-cells damage. A compensation of the broken endocrine A-cell function was maintained in a transformation of the exocrine epithelium into endocrine A-cells and in the appearance of intermediate acinar-islet cells, as well as in islet A-cell hyperplasia.     

犬传染性肝炎病毒在体外细胞质内的发生

通过对犬传染性肝炎病毒（ＩＣＨＶ）在犬肾传代细胞内形态发生及其抗原定位的电镜和免疫胶体金电镜研究,发现ＩＣＨＶ除了在宿主细胞核内发生外,还有一条细胞质内的发生途径。在细胞质内病毒核壳体的装配是以均质致密包涵体和副晶格包涵体为“基地”,这与人们熟知的细胞核内形态发生方式相似。免疫胶体金标记显示,细胞质包涵体中含有大量的ＩＣＨＶ抗原成分,显核壳体在细胞质内装配病毒的结构蛋白来源。此外,在感染的细胞质内还观察到与核内相同的病毒核心样结构。     

甲状腺浆细胞瘤1 例及文献复习

目的:探讨甲状腺浆细胞瘤的病理学特点及临床表现。方法:对1例甲状腺浆细胞瘤进行组织学表现、免疫组化染色观察及文献复习。结果:组织学特点:肿瘤细胞成分单一,大多数为分化较为成熟的肿瘤性浆细胞,胞核圆形或卵圆形,大小较一致,常偏位,染色质呈车辐状或钟面状。免疫组化特点:肿瘤细胞表达CD20(++)、CD79a(+)、CD38(++)、CD138(+)、κ链(++)。结论:甲状腺浆细胞瘤是甲状腺一种少见的肿瘤,应同炎症反应的浆细胞增生及低分化癌、淋巴瘤、髓样癌等相鉴别,可以通过免疫组化染色及形态学观察进行鉴别诊断。     

Participation of the adhesive disc during karyokinesis in Giardia lamblia

Evidence is presented for a potential involvement of the adhesive disc on the nucleus division in Giardia lamblia. The trophozoite mitotic nucleus was studied by transmission electron microscopy, freeze-fracture, freeze-substitution and also by immunofluorescence microscopy using anti-tubulin antibodies specific to spindle microtubules and Panotic staining. Prior to cell division the nucleus elongated and a displaced disc fragment, established contact with the nucleus. A progressive nucleus indentation was coincident with the concomitant presence of a disc fragment at the constricted region. One nucleus each time progressively divided until the karyokinesis was finished and two daughter-nuclei were observed. After the first karyokinesis a second karyokinesis takes place following the same procedure. When Giardia gets the four nuclei, cytokinesis occurs. Duplicated basal bodies were seen in between the first and the second karyokinesis. Immunofluorescence microscopy, using a panel of anti-tubulin antibodies, and electron microscopy of cells processed using microtubule stabilizer buffers, or cells fast-frozen and freeze-substituted, did not reveal the presence of a typical spindle. We propose that Giardia lamblia presents an uncommon mitotic behavior where the adhesive disc, a microtubular structure, seems to participate in the karyokinesis process.     

The chemical composition of nuclei and chromosomes isolated by the Langmuir trough technique

The pattern of staining for DNA, histone, and nonhistone protein has been studied in whole cells and in nuclei and chromosomes isolated by surface spreading. In whole interphase cells from bovine kidney tissue culture, nuclear staining for DNA and histones reveals numerous small, intensely stained clumps, surrounded by more diffusely stained material. Nuclei in whole cells stained for nonhistone proteins also contain intensely stained regions surrounded by diffuse stain. These intensely stained regions also stain for RNA, indicating that the regions contain nucleolar material. Electron microscopy of kidney cells confirms that multiple nucleoli are present. Kidney nuclei isolated by surface spreading show an even distribution of stain for DNA, histones, and nonhistone proteins, indicating that the surface forces disperse both condensed chromatin and nucleoli. DNA and protein staining was also studied in metaphase chromosomes from testes of the milkweed bug, Oncopeltus fasciatus. Staining for DNA and histones in metaphase chromosomes is essentially the same in sections of fixed and embedded testes as in preparations isolated by surface spreading. However, striking differences are noted in the distribution of nonhistone proteins. In sections, nonhistone stain is concentrated in extrachromosomal areas; metaphase chromosomes do not stain for nonhistone proteins. Chromosomes isolated by surface spreading, however, stain intensely for nonhistone proteins. This suggests that nonhistone proteins are bound to the chromosomes as a contaminant during the isolation procedure. The relationship of these findings to current work with chromosomes isolated for electron microscopy is discussed.     

The chemical composition of nuclei and chromosomes isolated by the Langmuir trough technique

The pattern of staining for DNA, histone, and nonhistone protein has been studied in whole cells and in nuclei and chromosomes isolated by surface spreading. In whole interphase cells from bovine kidney tissue culture, nuclear staining for DNA and histones reveals numerous small, intensely stained clumps, surrounded by more diffusely stained material. Nuclei in whole cells stained for nonhistone proteins also contain intensely stained regions surrounded by diffuse stain. These intensely stained regions also stain for RNA, indicating that the regions contain nucleolar material. Electron microscopy of kidney cells confirms that multiple nucleoli are present. Kidney nuclei isolated by surface spreading show an even distribution of stain for DNA, histones, and nonhistone proteins, indicating that the surface forces disperse both condensed chromatin and nucleoli. DNA and protein staining was also studied in metaphase chromosomes from testes of the milkweed bug, Oncopeltus fasciatus. Staining for DNA and histones in metaphase chromosomes is essentially the same in sections of fixed and embedded testes as in preparations isolated by surface spreading. However, striking differences are noted in the distribution of nonhistone proteins. In sections, nonhistone stain is concentrated in extrachromosomal areas; metaphase chromosomes do not stain for nonhistone proteins. Chromosomes isolated by surface spreading, however, stain intensely for nonhistone proteins. This suggests that nonhistone proteins are bound to the chromosomes as a contaminant during the isolation procedure. The relationship of these findings to current work with chromosomes isolated for electron microscopy is discussed.     

Morpho-functional parameters of nucleoli in polyploid mucous and albumen cells of salivary gland in the snail Succinea lauta

Variation of some characteristics of nucleoli of polyploid mucous and albumen cells was examined in salivary glands of the snail Succinea lauta. The number, total area and Ag-protein content of nucleoli, and DNA content in each nucleus were estimated on squashed preparations incubated with AgNO3, decolorized and then Feulgen stained. The ultrastructure of nucleoli was studied by electron microscopy. Differentiated mucous cells had 4c-8c-16c-32c nuclei; albumen cells had 8c-16c-32c-64c-128c nuclei. The ultrastructure of nucleoli of the two cell types was essentially the same. Normally, a large fibrous to granular zone was observed in the nucleoli, without a clear distinction between fibrous and granular components. At the same time, aggregations of granular matter could be discerned at the periphery of nucleoli. No fibrous centers were observed. Occassionally, nucleolonema-like structures occurred. Normally each nucleolus contacted several chromosomes. On squashed preparations, the least size of nucleoli was 2-3 microm, and the largest size amounted to 14 microm in mucous cells, and to 50-80 microm in albumen cells. The number of nucleoli rose from 1-2 in tetraploid nuclei to 2-3 in 32c-nuclei, and to 5-7 in 128c-nuclei. The disparity between the ploidy levels of nuclei and the numbers of nucleoli may be due, presumably, to aggregation of chromosome NORs. The Ag-protein content in the nucleoli, and the total nucleolar area displayed a strong mutual correlation. Both parameters differed significantly by 1.5-2.2 times in mucous and albumen cells of the same ploidy level. Thus, in albumen and mucous cells the total Ag-protein content in octaploid nuclei was 3.3 and 2.2 relative units (r. u.), respectively. In 16c- and 32c-nuclei of albumen cells, it was 7.6 and 15.1 r. u.; and in the same nuclei of mucous cells--3.8 and 6.8 r. u., respectively. On the whole, in albumen cells, in the course of 4 endocycles (4c-128c), the total Ag-protein content increased by 17 times. Therefore, the mean multiplication factor for this parameter was found to be 2.05 per endocycle. In mucous cells, in the course of 3 endocycles (4c-32c), the total Ag-protein content increased by 5.2 times against 8 times expected, with the mean multiplication factor equal to 1.75 per endocycle. Thus, in the course of polyploidization of albumen and mucous cell nuclei, the gene dosage effect was fully pronounced in the former, and only partly in the latter. This differtence is due obviously to peculiarities of differentiation of the two cell types, in particular, to differences in the number of activated ribosomal genes.     

The development of branched silk gland nuclei

Nuclei in the giant polyploid silk gland cells of Calpodes ethlius grow by endomitosis and can develop hundreds of branches during larval life. The shape of the these nuclei is characteristic for each region of the gland. We have found shape to be correlated with arrangement of the nuclear matrix. Scanning electron microscopy showed nuclear matrices with shapes similar to those of feulgen stained nuclei. Profiles of isolated matrices seen by transmission electron microscopy had filaments aligned parallel to the long axis of nuclear branches. DNA stained by Hoechst had a similar parallel alignment within the branches. Nuclear shape may be maintained by a small number of components, since electrophoretic analysis showed only a few abundant polypeptides in the matrix fraction. Silk gland nuclei have some of the same nuclear matrix antigens found in smaller, more regularly shaped, eukaryote nuclei.     

Dry mass content in nuclei and cytoplasm during differentiation of the root cortex cells of Helianthus Annuus.

Dry mass content in nuclei and cytoplasm as well as its concentration during the growth and differentiation of the root cortex cells of Helianthus annuus were investigated by means of interference microscopy. Moreover, by incubation with radioactive thymidine, the endopolyploidization at the level of 4C was found to exist in this species. It was found that during the growth and differentiation of the root cortex cells, the average dry mass content in nuclei essentially does not increase, whereas its content in cytoplasm increases significantly. At the same time the progressive decrease of dry mass concentration takes place in both nuclei and cytoplasm. This drop is connected with their growth and results from the considerable hydratation of karyoplasm and cytoplasm.     

Ultrastructural changes in the male germ cells of Bulinus truncatus during spermatogenesis

The structure of the spermatogonia, spermatocytes, spermatids and Sertoli cells of the hermaphroditic snail Bulinus truncatus was studied by electron microscopy. The spermatogonia are small, with relatively large nuclei. The acrosome develops from a small proacrosomal granule which is probably derived from the Golgi apparatus in the spermatocyte stage. Condensation and elongation of the nuclei were found in the spermatids. The shape and components of the Sertoli cells did not change during the spermatogonium and spermatocyte stages. Before spermiation the Sertoli cells have the morphological features of steroid-producing cells. The study showed that the Sertoli cells are involved in the nutrition and transportation of the spermatogenic cells, in spermiation and in hormone production.