Biomedical subjects

N Gas

Publications and source records attributed to N Gas.

At least 37 records · Page 2Linked to original sources

PubMed1991

Distribution of nucleolar proteins B23 and nucleolin during mouse spermatogenesis.

The intracellular distribution of nucleolar phosphoproteins B23 and nucleolin was studied during mouse spermatogenesis, a process that is characterized by a progressive reduction of nucleolar activity. Biochemical analyses of isolated germ cell fractions were performed in parallel with the in situ ultrastructural immunolocalization of these two proteins by means of specific antibodies and colloidal gold markers, and by silver staining. RNA blot experiments showed that mRNA for nucleolin progressively decreased during spermatogenesis whereas mRNA for B23 increased in amount during early spermatogenic stages. Immunoblotting confirmed that both proteins were present during early spermatogenesis up to the round spermatid stage and absent from mature sperm. Immunoelectron microscopy revealed that in spermatogonia, leptotene and pachtyene spermatocytes, and in Golgi phase spermatids, B23 and nucleolin were localized in the dense fibrillar component and granular component of the nucleolus but not in the fibrillar centers. In the dense fibrillar residue of the cap phase spermatids, labeling with anti-nucleolin but not with anti-B23 was observed. During nucleolar inactivation, neither of the two polypeptides was dispersed to the nucleoplasm. Silver salts stained the fibrillar centers and dense fibrillar component but not the granular component of the nucleolus. Our results suggest that there is no direct relationship between nucleolar activity and the occurrence of B23 and nucleolin or silver staining. Moreover, we confirm that silver staining and the presence of B23 or nucleolin are not directly related to each other.

Animals↗

PubMed1991

Location of ribosomal genes in CHO cells; in situ hybridization with a non-isotopic DNA probe on G-banded chromosomes.

A novel in situ hybridization technique using sulfonated probes is described. This non-radioactive approach, which employs chemically modified DNA and immunocytochemical procedures, is compatible with pre-G-banding and allows a rapid localization of the hybridized sequences on chromosomal spreads with a high spatial resolution. Using this technique we have localised the Chinese hamster ribosomal genes in the telomeric region of ten chromosomes, and among them in the subtelomeric q region of the Z5 chromosome. These results are discussed, the genetic markers confirming and locating the origin of Z group chromosomes by rearrangements of Chinese hamster chromosomes.

Animals↗

PubMed1990

Structural and immunological properties of the phenolic glycolipids from Mycobacterium gastri and Mycobacterium kansasii.

Mycobacterial species-specific antigens belong to the three following classes: phenolic glycolipids (Phe Gl), acyltrehalose-containing lipooligosaccharides and polar glycopeptidolipids. These antigens have been chemically defined and alkali-labile epitopes were found to characterize the lipooligosaccharide antigen type. In the present study the major Mycobacterium kansasii phenolic glycolipid epitope namely Phe Gl K-I was delineated as the distal monoacetylated disaccharidic residue: 2,6-dideoxy-4-O-methyl-alpha-D-arabino-hexopyranosyl-(1----3)-2-O-methyl -4-O- acetyl-alpha-L-fucopyranose. This acetoxy group is required for K-I epitope recognition demonstrating that alkali-labile epitopes also occur in the phenolic glycolipid antigen class. Using immunoelectron microscopy, the Phe Gl K-I epitope was localized around the electron-transparent layer on the M. kansasii cell-wall surface. Furthermore, two new phenolic glycolipids namely Phe Gl K-III and Phe Gl K-IV were discovered in minute amounts. They were purified and characterized by their retention time in direct-phase column HPLC. These molecules are also M. kansasii antigens, whose epitopes differ from that of Phe Gl K-I. The complete family of phenolic glycolipids Phe Gl K-I, K-II, K-III and K-IV was found in both rough and smooth variants of both M. kansasii and Mycobacterium gastri species.

Antibodies↗

PubMed1990

Effects of differentiation-inducing agents on maturation of human MCF-7 breast cancer cells.

The effects of the differentiation inducing agents (DIAS), sodium butyrate (NaBu), retinoic acid (RA), dimethylformamide (DMF), hexamethylene bisacetamide (HMBA), forskolin, and 12-O-tetradecanoylphorbol-13-acetate (TPA), on the growth, morphology, and estrogen receptor (ER) content and epithelial membrane antigen (EMA) expression on a serumless human breast cancer cell line (MCF-7) were compared. All these agents reversibly caused a concentration-dependent growth inhibition in monolayers and markedly reduced colony-forming efficiency in soft agar. A twofold increase in doubling time was obtained with RA (1 microM), but cell replication ceased with NaBu (1 mM), forskolin (50 microM), DMF (1%), HMBA (5 mM), and TPA (8 nM). Total growth arrest induced by these last compounds was preceded by an accumulation of cells in G0/G1 phase observed at 24 h by flow cytometry and accompanied by a change in cell morphology as seen by light and electronic microscopy. An increase in cell volume and the presence of lipid droplets was noted in treated cells that were spread out, as compared with controls. The acquisition of a more mature phenotype was confirmed by an increased expression of EMA monitored by flow cytometry. A specific reduction in the number of ER without any constant dissociation (Kd) modification was also observed after treatment with the 5 DIAs. No modification of morphological or biochemical characteristics, including EMA expression and ER binding, were observed for RA (1 microM)-treated cells. All these results suggest that induction of a more differentiated phenotype is associated with a block in G1 cell cycle phase, resulting in total growth arrest. Apparently, RA (1 microM)-treated cells did not fulfill these criteria, since only a slight accumulation in G1 and a slowed growth rate were evaluated.

Acetamides↗

PubMed1990

Nucleolar distribution of proteins B23 and nucleolin in mouse preimplantation embryos as visualized by immunoelectron microscopy.

The ultrastructural distribution of proteins B23 and nucleolin in the nucleolus of mouse embryos from the zygote to the early blastocyst has been analyzed by means of specific antibodies and immunocytochemistry using colloidal gold complexes as markers. In parallel, silver staining of nucleoli was carried out on ultrathin sections. Our results show that the compact prenucleolar bodies at 1- and 2-cell stage as well as the compact residual fibrillar masses observed up to the morula stage, are labelled with the two antibodies. These masses, however, are not stained with silver up to the 4-cell stage. In well-developed nucleoli, the two antibodies co-localize in the dense fibrillar component (DFC) and the granular component (GC) while fibrillar centers (FCs) are devoid of label. On the contrary, silver staining occurs in the FCs and DFC but not in the GC. Our observations suggest that there is no direct relationship between the occurrence of silver staining and the distribution of protein B23 or nucleolin. Moreover, neither the localization of the two above proteins nor silver staining are unequivocally related to the nucleolar activity.

Animals↗

PubMed1989

Aspartate aminotransferase isoenzymes in Leptosphaeria michotii. Properties and intracellular location.

Two forms of aspartate aminotransferase were obtained from the fungus Leptosphaeria michotii and purified to a state of apparent homogeneity by a five-step purification procedure ending with blue Ultrogel chromatography. Holoenzyme specific activities were 13430 and 9110 nkat oxalacetate/mg protein-1 and isoelectric points were 7.1 and 7.0 for forms A and B, respectively. Both isoenzymes were isologous dimers of Mr 92,000. They differed mainly in their Km for 2-oxoglutarate and aspartate, their ability to use cysteine sulfinate as a substrate and their ability in vitro to be specifically tightly associated as follows: form A with a malate dehydrogenase monomer of Mr 25,000; form B with an unidentified protein of Mr 40,000-44,000. Rabbit antiserum raised against the form A holoenzyme was not reactive against the form B holoenzyme and vice versa. Association of the holoenzyme with the complex essentially provoked a shift of the isoelectric point to 5.8 for form B [corrected] and to 5.2 for form B, without affecting kinetic parameters. In order to localize in situ the two transaminase forms, ultrastructural detection was carried out by immunogold staining of thin sections of Lowicryl-K4M-embedded colonies. Antiserum against form A essentially labelled cytoplasm and cell wall and, to some extent, mitochondria, while antiserum against form B heavily labelled mitochondria and cell wall and to a lesser extent cytoplasm. Moreover, mitochondria were isolated and purified by Percoll-density-gradient centrifugation. Only form A was identified in this subcellular fraction using ELISA.

Ascomycota↗

PubMed1989

The lateral motion of lipid molecules in the apical plasma membrane of endothelial cells is reversibly affected by the presence of cell junctions.

As with other epithelia, the question arises of whether the endothelial cell junctions participate in cell polarization, restrict the localization of lipid molecules, and lead to differences in their lateral motion between the apical and basolateral plasma membranes. We found that in bovine aortic endothelium in culture, the localization of the lipophilic probe 5N-(hexadecanoyl)-aminofluorescein (HEDAF) was markedly affected by the presence of cell junctions. At confluence, the probe was observed to be restricted to the exoplasmic leaflet of the apical plasmalemma. It was detected in the basal membrane only after disruption of the cell junctions, whereas the cells remained firmly bound to the underlying extracellular matrix. Fluorescence recovery after photobleaching (FRAP) experiments indicated that the endothelial confluent cell monolayer forms a mosaic of closed diffusion areas in which the probe molecules are free to diffuse. For the first time, and using a new mathematical approach, it was possible to estimate the diameter of these diffusion areas. Values in the range 14-33 microns were found which correlate well with the actual measured diameters of 14 to 26 microns for the apical pole of individual cells. Moreover, cell junctions were found to affect the dynamics of the probe. When the cell junctions were disrupted, the lateral diffusion coefficient D of HEDAF was found to be the same in both apical and basal membranes. It regained its initial higher value in the apical poles when cell contacts were restored. This strongly suggests that in vascular endothelium, cell junctions affect overall plasma membrane organization in a reversible manner.

Animals↗

PubMed1989

Nucleolin from Xenopus laevis: cDNA cloning and expression during development.

Nucleolin is a key nucleolar protein in higher eukaryotic cells and is involved directly in ribosome biogenesis. Using an antiserum raised against hamster nucleolin, the homologous protein was detected in nucleoli of Xenopus laevis hepatocytes as well as in the amplified nucleoli of oocytes. A cDNA encoding Xenopus nucleolin has been isolated and sequenced. The deduced protein sequence reveals similar domains in Xenopus and in mammals, but they have undergone separate evolutions. In particular, each of the four RNA-binding domains has evolved differently--the carboxy-proximal domain is twice as conserved (87%) as the amino-proximal domain (42%). These data shed some light on the possible roles of each domain. The expression of nucleolin has been followed throughout oogenesis and embryogenesis. The appearance of nucleolin during early development precedes the transcription of rDNA and the synthesis of ribosomal proteins. The maximal accumulation of nucleolin at gastrulation coincides with nucleolar reformation. Furthermore, when ribosomal synthesis is activated during oogenesis and embryogenesis, peptides immunorelated to nucleolin appear and accumulate. The results suggest that nucleolin plays a role not only in ribosome assembly but also in nucleologenesis.

Amino Acid Sequence↗

PubMed1989

The effect of adenosine analogue (DRB) on a major nucleolar phosphoprotein nucleolin.

Nucleolin, a phosphorylated nucleolar protein, of 100 kDa selectively stained with bismuth tartrate and silver nitrate, is implicated in the transcription and maturation of pre-ribosomal RNA. Nucleolin also fulfills a structural function in nucleolar organization. Using immunocytochemistry the action of 5-6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), an inhibitor of hn/RNA synthesis known to modify the organization of the nucleolus, was studied for its effects on the distribution and the amount of nucleolin present. After DRB treatment, the morphology of the nucleolus was rapidly disturbed, but the distribution of the nucleolin remained unchanged: the dense fibrillar and the granular components were always positively immunostained. Thirty min after incubation with the drug, a strong increase of the amount of nucleolin occurred. Prolonged treatment led to a marked loss of label. Silver and bismuth staining showed that DRB does not seem to significantly affect the phosphorylation of nucleolin.

Animals↗

PubMed1988

Reversible plasma membrane ultrastructural changes correlated with electropermeabilization in Chinese hamster ovary cells.

Chinese hamster ovary cells (CHO) grown in monolayers were permeabilized to molecules with molecular weight up to 1000 by high intensity 100 mus square wave electric field pulses. This permeability was transient and the cell viability was not affected. It was not possible for molecules with molecular weight larger than 1500 to penetrate inside the cytoplasm if lytic pulsing conditions were not used. In order to investigate the ultrastructural changes associated with this transient and limited permeabilization, cells were chemically fixed a few seconds after their pulsation and observed by electron microscopy. By scanning electron microscopy, numerous microvilli and blebs were observed almost immediately after application of the field. No other membrane changes were observed. Permeabilization of the membrane was visualized at the electron microscopic level by penetration of Ruthenium red. The appearance of osmotic pressure-dependent 'blebs' was indicative of local weakening of the plasma membrane. Most of these effects were fully reversible and disappeared within 30 min at 37 degrees C with the formation of huge polykaryons when cells were in contact before pulsing.

Animals↗

PubMed1987

Influence of 12-O-tetradecanoylphorbol-13-acetate on proliferation and maturation of human breast carcinoma cells (MCF-7): relationship to cell cycle events.

Exposure of MCF-7 human mammary carcinoma cells to 12-O-tetradecanoylphorbol-13-acetate (TPA) results in changes in cell morphology and arrest of cell growth. The inhibition of cell proliferation and the increase in cell volume are concentration dependent; these effects are reversible upon removal of the tumor promoting agent. Electron microscopic studies reveal that TPA increases endoplasmic reticulum and induces the appearance of secretory granules. MCF-7 cells treated by TPA therefore present morphological characteristics of secretory cells. These effects of TPA on MCF-7 cells are accompanied by specific disruption of cell cycle events, a block of cells in G1 at the expense of S base, and a delayed passage through G2. Studies in which a cell cycle lock in G1 is produced by tamoxifen show that exposure of such cells to PA produces cell morphological changes and an inability to progress through the cell cycle when estradiol is added.

Breast Neoplasms↗

PubMed1987

Basic fibroblast growth factor enters the nucleolus and stimulates the transcription of ribosomal genes in ABAE cells undergoing G0----G1 transition.

The cellular action of growth factors, among them basic fibroblast growth factor (bFGF), is mediated by their interaction with a cell surface receptor, but the mechanism of transfer of mitogenic (or other) signals to the nucleus has not been identified. In this work, we show that bFGF is translocated to and accumulated in the nucleolus. Furthermore, the nucleolar localization of bFGF is correlated with a stimulation of transcription of ribosomal genes during G0----G1 transition induced by bFGF alone in adult bovine aortic endothelial cells (ABAE cells). Stimulation of ribosomal gene transcription is preceded by a significant increase of the major nonhistone nucleolar protein, nucleolin. In vitro, the growth factor has a direct effect on the enhancement of RNA polymerase I activity in isolated nuclei from quiescent sparse (G0) ABAE cells. The direct action of bFGF on the level of ribosomal gene transcription could correspond to an additional growth-signaling pathway, mediated by this growth factor.

Animals↗

PubMed1987

Distribution of nuclear matrix proteins in interphase CHO cells and rearrangements during the cell cycle. An ultrastructural study.

The nuclear matrix contains a group of residual non-histone proteins which remain structurally organized after extensive extraction of isolated nuclei with a high salt buffer, nucleases and a non-ionic detergent. Electron microscopic examination shows that the nuclear matrix is composed of a pore-complex lamina, an intranuclear network and residual nucleoli. In CHO cells biochemical analyses performed by one-dimensional SDS-PAGE show three major nuclear matrix polypeptides with molecular weights between 60 and 70 kDa. Polyclonal antibodies produced against these polypeptides were used to determine their nuclear distribution. Using immunoblotting, these proteins were found in whole nuclei, nuclear matrix, and in the intranuclear network but not in the pore-complex lamina. In order to determine the relationship between these structural proteins and the organization of the nucleus, the proteins were localized in situ. Ultrastructural detection was carried out by immunogold staining of thin sections of Lowicryl K4M-embedded cells. In interphase nuclei all condensed chromatin clumps were labelled. The nucleolus and the interchromatin granules were never immunogold-stained. During mitosis, the label was found to be associated with the chromosomes. This study shows that unlike the lamins, these 60-70 kDa nuclear matrix proteins are associated with the condensed chromatin throughout the cell cycle.

Animals↗

PubMed1985

Correlation between rDNA transcription and distribution of a 100 kD nucleolar protein in CHO cells.

A major nucleolar protein with a molecular weight of 100 kD is directly implicated in the transcription of pre-ribosomal RNA (pre-rRNA) and appears to be cleaved into specific maturation products during pre-ribosome biogenesis. Polyclonal antibodies which recognize the 100 kD protein and its products were used to determine the correlation between rDNA transcription and these proteins. Actinomycin D (AMD) was used to block selectively rDNA transcription (AMD 0.1 microgram/ml). Immunoperoxidase and immunogold staining were carried out in untreated and treated cells. Digitalization allowed the quantification of label according to the nucleolar components and the cellular areas. In exponentially growing cells, the dense fibrillar component was shown to contain more 100 kD protein than the granular RNP component but both nucleolar components were positively immunostained. The distribution of the 100 kD protein was rapidly modified by AMD: loss of label occurred first in the dense fibrillar zone of the nucleolus, demonstrating the correlation between rDNA transcription and the presence of this protein. However, one part of the protein remains in the segregated nucleolus after 1 h of AMD treatment, thus supporting the structural function of this protein.

Animals↗

PubMed1985

Immunolocalization of the 100 K nucleolar protein in CHO cells.

The localization of a major nucleolar protein with a molecular weight of 100,000 has been followed in Chinese hamster ovary cells using specific antibodies to this protein and immunocytochemical techniques. By immunofluorescence and immunocytochemistry at ultrastructural level in situ, the 100 K protein was detected abundantly in the nucleolus of interphase cells. In exponentially growing cells, the dense fibrillar component was shown to contain more 100 K protein than the granular RNP component but both the nucleolar components were positively immunostained. Fibrillar centers consistently showed weaker or no staining. The 100 K association with the preribosomal RNP components of the interphase nucleolus supports evidence for a role of this protein in pre-rRNA transcription and preribosomal processing.

Animals↗

PubMed1985

Immunolocalization of the 100 kDa nucleolar protein during the mitotic cycle in CHO cells.

The localization of a major nucleolar protein with a molecular weight of 100,000 has been followed during mitosis in Chinese hamster ovary CHO cells using specific antibodies to this protein and immunocytochemical techniques. The 100 kDa protein was visualized at discrete sites on metaphase chromosomes, corresponding to nucleolus organizer regions, and in large, immunostained nucleolar remnants that are discarded in the cytoplasm after nucleolar disintegration. After mitosis, the 100 kDa protein was shown to play an early role in nucleolar reformation. It was first detected in small deposits around the anaphase chromosomes. In telophase, the protein accumulated simultaneously in prenucleolar bodies and in the reforming nucleoli. The early presence of the 100 kDa protein in the telophase nucleus suggests that it is essential for the reestablishment of nucleolar function after mitosis. Thus this protein is present throughout the CHO cell cycle, an observation which supports the hypothesis that it plays a fundamental role in cell organization.

Animals↗

PubMed1984

Induction of new proteins in the nuclear matrix of CHO cells by a heat shock: detection of a specific set in the nucleolar matrix.

An early response to an incubation of CHO cells for one hour at 43 degrees C is the appearance in the nuclear matrix of new proteins: 2 proteins (48 and 46 kDa) were associated with the extranucleolar part of the nuclear matrix, 6 other proteins (130, 100, 95, 85, 76 and 70 kDa) were present in matrices prepared from isolated nucleoli. In addition, a large increase in the amount of associated minor components is observed. It is suggested that these early modifications in composition of the matrix are related to the inhibition of rRNA and hnRNA synthesis and maturation.

Animals↗

PubMed1984

Bismuth staining of a nucleolar protein.

A major nucleolar protein in Chinese hamster ovary cells with a molecular weight (MW) of 100 kD has been found to stain selectively with the bismuth tartrate technique of Locke & Huie [19]. After glutaraldehyde fixation and bismuth staining of electrophoretic transfers of total nucleolar proteins separated by SDS-PAGE, a single band corresponding to the 100 kD protein is revealed. When the technique is applied to whole cells, small punctate regions of the nucleoli are strongly stained. At the ultrastructural level, bismuth selectively contrasts the fibrillar centers and the adjoining cords of the dense fibrillar component. The remainder of the dense fibrillar component is not stained. It is proposed that the high phosphorylation level of the 100 kD protein is responsible for its glutaraldehyde-insensitive bismuth staining. The concentration of this protein in certain localized regions of the nucleolus suggests that it plays a metabolic rather than a structural role.

Animals↗