bluuebunny/crossref_metadata_2025 huggingface dataset:
Update on the concept of the cell cycle: the contribution of flow cytometry
Authors: Ronot X. , Adolphe M.
Published: February 2012

Flow cytometry has been extensively used to provide accurate estimates of the relative amounts of various cellular constituents (DNA, RNA, proteins) for cell kinetic studies. Multiparametric analysis also supports the recent concept that cell growth and the DNA division cycle may be under distinct regulatory mechanisms. Moreover, metabolic subcompartments of the cell cycle, distinguished by flow cytometry, have offered a highly sensitive cell classification in comparison with the conventional distinction of the four main phases of the cell cycle. Finally, a new sensitive and powerful technology, BrdU/DNA analysis, represents a remarkable maturing of a very useful alternative for the study of DNA synthesis and cell cycle traverse.
Applications of flow cytometry in cellular pharmacology
Authors: Adolphe M. , Ronot X.
Published: February 2012

Cellular pharmacology is defined as the study of drug effects on various cell functions. Flow cytometry enriches cellular pharmacology by the following possibilities for efficient analysis. Firstly, the determination of toxic concentrations can be approached by the assessment of cell viability. However, due to the existence of many fluorescent DNA probes, most studies are devoted to the investigation of products acting on cell division, particularly in the area of antineoplastic drugs. The effects of drugs on respiration can be approached by analysis of mitochondrial activities. On the other hand, the studies of drug actions on cell differentiation functions have been started using antisera or monoclonal antibodies to cell‐specific proteins such as collagen and keratin. Flow cytometry appears to be more and more important in the progress of cellular toxicology and pharmacology.
Flow cytometry for immunology
Authors: Cordier G.
Published: February 2012

Improvement in our knowledge in cellular biology is largely related to the use of new tools in quantitative cytology. Among them, flow cytometry was developed with numerous applications in the field of immunology including fundamental and applied research. Since its early beginning it has been associated with monoclonal antibodies to identify immuno‐competent cells, to quantify changes in expression of surface determinants, to separate cells subsets prior to the test of their functional properties. Major advances gained using either single or dual‐laser systems, multicolour fluorescence and computer facilities for multi‐parametric analysis. Using this methodology it was possible to correlate analysis of cell cycle phases and membrane antigens expression. Applications have been developed for the analysis of new drugs in vitro, the evaluation of immunomodulating treatment and for clinical investigations.
Nuclear accumulation of HMG1 protein is correlated to DNA synthesis
Authors: Bonne‐Andrea C. , Harper F. , Puvion E. , Delpech M. , De Recondo A. M.
Published: February 2012

The subcellular localization of HMG1 protein was studied by immunoelectron microscopy during growth of CV1 cells in culture and in confluent CV1 cells subsequently lytically infected with SV40. HMG1 was always detected in the cytoplasm of both non‐infected and infected cells. On the other hand, this protein displayed a nuclear localization only in those cells active in cellular and/or viral DNA replication, that is, in actively dividing non‐infected cells and in confluent cells following SV40 infection. The combination of electron microscope immunocytochemistry and autoradiography revealed that during SV40 lytic infection, HMG1 accumulates at sites of active viral DNA replication. Since HMG1 is a single‐stranded DNA binding protein and acts in vitro as a physiological nucleosome assembly factor, we suggest that its presence in the nucleus is related to its requirement in the DNA replication process.
Expression of a subclass of human blood group A antigenicity in A+ rabbit jejunum: specific glycosylation of the glycocalyx and a 140 K brush border glycoprotein
Authors: Gorvel J. P. , Bourges F. , Ferrero A. , Bernadac A. , Rasongles P. , Maroux S.
Published: February 2012

A monoclonal antibody (14 A4) raised against human A erythrocytes has been produced. It specifically reacts with a subclass of human blood group A determinants. Whereas all the major secreted and membrane‐bound glycoproteins of A+ rabbit jejunum epithelium bear human blood group A‐like determinants recognized by anti‐A polyclonal serum or a monoclonal antibody with broad specificity (Cl 3.3), expression of the A‐subclass recognized by 14 A4 is very restricted. The contents of secretory granules of Paneth cells but not the mucins of goblet cells were labeled by 14 A4. In the enterocytes, glycans recognized by 14 A4 were present in the glycocalyx, on an early expressed 140 K glycoprotein of brush border membranes and also on a glycoconjugate of the basolateral membrane of immature crypt cells. In the jejunal brush border membrane of blood group A secretor humans, only one glycoprotein of molecular weight 140 K bears the A‐subclass recognized by 14 A4.
Establishment, identification and characterization of a new sheep sinus tumor cell line
Authors: Shrivastava R. , Sowala H. , Nicolas A. , Julien R.
Published: February 2012

A cell line designated SRT was established from a sheep sinus tumor. Following primary culture, the cells were serially passaged 40 times. SRT cells maintained an epithelioid fibroblast‐like appearance and had a population doubling time of approximately 18 hr. Karyotype analysis of 14th passage cells showed the modal 2n chromosome number to be between 46 to 60, due to a large variation in acrocentric chromosome number. The electrophoretic mobilities of enzymes extracted from SRT cells were identical with those from normal sheep sinus cells. It propagated a number of ovine, bovine and canine viruses. Some virus‐like particles (80‐120 nm) were observed under the electron microscope. The tumor origin, good growth and wide range of virus susceptibility make SRT a highly suitable cell line for in vitro cancer research and for comparative virology studies.
Connections of intermediate filaments with the nuclear lamina and the cell periphery
Authors: Katsuma Y. , Swierenga S. H. , Marceau N. , French S. W.
Published: February 2012

We investigated the relationship between intermediate filaments (IFs) and other detergent‐ and nuclease‐resistant filamentous structures of cultured liver epithelial cells (T51B cell line) using whole mount unembedded preparations which were sequentially extracted with Triton X‐100 and nucleases. Immunogold labelling and stereoscopic observation facilitated the examination of each filamentous structure and their three‐dimensional relationships to each other. After solubilizing phospholipid, nucleic acid and soluble cellular protein, the resulting cytoskeleton preparation consisted of a network of cytokeratin and vimentin IFs linked by 3 nm filaments. The IFs were anchored to and determined the position of the nuclear lamina filaments (NLF) network and the centrioles. The NLF was composed of the nuclear lamina filaments measuring 3‐6 nm in diameter which radiated from and anchored to the skeleton of the nuclear pores. The IFs located in the nuclear region appeared to be interwoven with the NLF. At the cell surface, the IFs seemed to be attached to the putative actin filament network. They formed a focally interrupted plexus‐like structure at the cell periphery. Fragments of vimentin filaments were found among the filamentous network located at the cell surface, and some filaments terminated blindly there.
Homologous desensitization of rat Sertoli cells by non‐stimulating concentrations of follicle‐stimulating hormone
Authors: Guillou F. , Martinat N. , Combarnous Y.
Published: February 2012

Pre‐incubation of rat Sertoli cells with concentrations of follicle‐stimulating hormone (FSH) too low to stimulate plasminogen activator (PA) secretion, provoked an inhibition of its subsequent stimulation by an effective dose of the hormone. A kinetic study of this desensitization was performed using equine FSH (which exhibits prolonged stimulation of PA secretion) and porcine FSH (which like all other FSH tested, provokes a transient response). Low non‐stimulating concentrations of both hormones were shown to inhibit the subsequent PA response to each of them. Desensitization of rat Sertoli cells by low (non‐stimulating) concentrations of FSH did not modify the typical time course (transient or prolonged) of PA secretion under subsequent stimulation by porcine or equine FSH, respectively. Only the intensity of the response to each hormone was dramatically reduced. Besides, the induction of desensitization by these non‐stimulating concentrations of FSH was shown to be very rapid (10‐15 min). The precise mechanism of this desensitization is not yet clear but its abolishment by the cyclic nucleotide phosphodiesterase (PDE) inhibitor MIX is consistent with the hypothesis that activation of PDE occurs at lower FSH concentration than adenylate cyclase activation.
Quantitative variations in nucleolar components within pineal gland cells during the rat estrous cycle
Authors: López‐Iglesias C. , Arias J. L.
Published: February 2012

Quantitative changes in the size of pinealocyte nucleoli have been reported in various studies on this cell type. However, the significance of quantitative changes in the nucleolar components is unknown. The present study is an attempt to analyze ultrastructural and morphometric modifications occurring in the pinealocyte nucleolar components during the estrous cycle in female rats. The fibrillar centers showed an increase during estrus consistent with a decrease in pinealocyte nucleolar activity and melatonin pineal levels. The fibrillar components and granular components tended to display a reciprocal relationship. An increase in the dense fibrillar component took place at metaestrus and diestrus when melatonin synthesis increased in pinealocytes. Maximum values of granular and interstitial components were found at the proestrus phase before the day of ovulation.
In situ structural organization of encapsidated and unencapsidated Ectromelia virus DNA
Authors: Derenzini M. , Farabegoli F. , Puvion‐Dutilleul F.
Published: February 2012

The structural organization of Ectromelia virus DNA in infected mouse liver cells has been studied by using thin sections stained with the Feulgen‐like osmium‐ammine reaction. We found that in the cytoplasmic factories, free viral DNA was structured into completely extended filaments 2‐3 nm thick. Viral DNA in immature virions, however, appeared to have a structural organization that superimposed that of eukaryotic chromatin. This was constituted by roundish subunits, with a diameter of 11‐13 nm, composed of a DNA ring encircling an unstained inner core. The mature virion was composed of the same type of subunits, which were arranged in threads twisted into a figure 8 configuration. The distribution of basic proteins was also investigated with the acrolein silver‐methenamine technique. In the viral particles only nucleoids were stained; a uniformly distributed positive reaction was observed in the cytoplasmic factories.
Aggregate cultures of foetal rat liver cells: development and maintenance of liver gene expression
Authors: Guigoz Y. , Werffeli P. , Favre D. , Juillerat M. , Wellinger R. , Honegger P.
Published: February 2012

Rotation‐mediated aggregate cultures of foetal rat liver cells were prepared and grown in a chemically defined medium. Their capacity for cellular organisation and maturation was studied over a culture period of 3 wk by using both morphologic and biochemical criteria. It was found that within each aggregate, distinct liver cell types were present and attained their normal, differentiated phenotype. Parenchymal cells formed small acini with a central lumen. Within the first 2 wk in culture, albumin and ferritin mRNA levels were maintained, while the alpha‐fetoprotein mRNA levels decreased, and tyrosine aminotransferase (TAT) gene expression increased. No significant response to glucocorticoids was observed in early cultures, whereas after 3 wk a marked increase in TAT mRNA levels was elicited by dexamethasone and glucagon (additive stimulatory effects). The results show that foetal rat liver cells cultured in a chemically defined medium are able to rearrange themselves into histotypic structures, and display a developmental pattern of gene expression comparable to that of perinatal rat liver in vivo. This culture system offers therefore a useful model to study the development and function of liver cells.
Effect of cetiedil on acetylcholine release and intramembrane particles in cholinergic synaptosomes
Authors: Israel M. , Manaranche R. , Gaudry‐Talarmain Y. Morot , Lesbats B. , Gulik‐Krzywicki T. , Dedieu J. C.
Published: February 2012

The release of acetylcholine (ACh) from instantly frozen Torpedo electric organ synaptosomes in the course of stimulation is systematically associated with an increase in the number of large intramembrane particles counted on freeze‐fracture replicas. The drug cetiedil, which is a potent inhibitor of ACh release, also blocks the increase in the number of large particles. The blockage was studied either after ionophore A 23187 or Glycera neurotoxin action in the presence of calcium.
Characterization of cell types in human breast tumor primary cultures
Authors: Muller D. , Fricker J. P. , Millon‐Collard R. , Abecassis J. , Pusel J. , Eber M. , Methlin G.
Published: February 2012

Primary cultures of cells from breast carcinomas were attempted in 74 cases. Growth was observed in 46 cases. Using immunochemical demonstration of keratin proteins (KER), epithelial membrane antigen (EMA) and carcinoembryonic antigen (CEA), three morphologically distinct cell populations were characterized and described. Two cell types (E‐ and E'‐cells) were identified as epithelial by their positive staining for KER and EMA. The third type (F cells) displayed a negative staining for these two epithelial markers; they were considered as stromal cells (fibroblasts). More than 50% of the cultures consisted of pure epithelial cells. Positive CEA staining was observed only in KER‐ and EMA‐positive cells. Out of the 30 cultures, 15 displayed positive staining for CEA. In 7 of these, 30‐50% of cells displayed positive, diffuse staining for CEA. The other 8 cultures consisted of more than 50% CEA‐positive cells. Strong and homogeneous positivity was restricted to the E‐cells, while in the same cultures, E'‐cells displayed heterogeneous and diffuse staining. Efficiency and value of this cell culture system are discussed, in comparison with other human breast tumor cell (HBTC) culture techniques. Identification of growing cells and cellular composition of primary cultures are emphasized.
Localization of highly phosphorylated proteins in cells altered by Herpes simplex virus infection
Authors: Puvion‐Dutilleul F. , Laithier M.
Published: February 2012

Highly phosphorylated proteins detectable by their ability to bind bismuth ions were localized in rabbit fibroblasts before and during infection with Herpes simplex viruses type 1 and type 2. The bismuth tartrate procedure of Locke and Huie applied to glutaraldehyde‐fixed cells revealed a low level of bismuth binding in a restricted portion of the normal nucleolus in non‐infected cells. From 2.5‐17 hr post‐infection during virus development and maturation, the phosphorylated proteins were more widespread and the intensity of reaction was augmented. Bismuth deposits were then associated with virus‐modified pre‐existing structures including all of the nucleolar fibrils, the more abundant interchromatin granules, reduplications of some areas of the inner nuclear membrane and the Golgi apparatus. Virus‐induced structures which were stained included nuclear dense bodies, the teguments of enveloped virions and the contents of extranuclear enveloped structures devoid of capsids. Following detergent‐induced destruction of membranes, staining was lost from the nuclear envelope and cytoplasmic virions, which demonstrated that the highly phosphorylated proteins were tightly bound to nuclear and viral membranes. Bismuth staining of nitrocellulose sheets containing proteins extracted from whole cells revealed no reaction in normal cells but three positive bands were found in infected cells.
Lectin‐like components in the macronuclear replication bands of Euplotes eurystomus
Authors: Olins Donald E. , Olins Ada L. , Sève Annie‐Pierre , Bourgeois Claire A. , Hubert Jean , Monsigny Michel
Published: February 2012

Upon incubation with fluoresceinylated neoglycoproteins, isolated macronuclei from the ciliated protozoan Euplotes eurystomus display different labelling patterns depending on the nature of the sugar bound to the neoglycoproteins. Specific sugar‐binding components (i.e., lectin‐like molecules) are associated with presumed nucleoli and with the macronuclear replication bands. This is the first demonstration that DNA synthesis and sugar‐binding components are co‐localized in an eukaryotic cell.
The somatic function of the micronucleus in asexual reproduction of Paramecium: thermosensitivity of amicronucleates
Authors: Chau Miu Fun , Ng Stephen F.
Published: February 2012

It has been established that following removal of the micronucleus in Paramecium tetraurelia, the amicronucleate cell line enters a depression period, characterized by slow growth rate and oral abnormalities, at normal growth temperature (27°C). Such cell lines gradually recover in growth rate and stomatogenesis. In the present study, 4 recovered amicronucleate cell lines were challenged with high temperatures (35°C, 36°C, and 36.5°C). They exhibited growth rate reduction and abortive cytokinesis at 35°C and 36°C, and died at 36.5°C. In addition, they demonstrated oral defects similar to those observed in the depression period: disruption of the regular oral membranellar pattern, reduction in the length of the oral apparatus, and impaired phagocytosis (food vacuole formation). These high temperature‐induced abnormalities were largely restricted to amicronucleates, and were rare or seen to a much lesser extent in sister micronucleate cell lines. This study demonstrates the participation of the micronucleus in conferring thermotolerance on the cells. It is hypothesized that the micronucleus specifies heat‐shock proteins to maintain the integrity of oral and somatic cytoskeletal elements at high temperature.
A membrane preparation that contains proteins characteristic of the rough endoplasmic reticulum
Authors: Amar‐Costesec Alain , Hortsch Michael , Turu Christine
Published: February 2012

We describe a procedure for disassembling rat liver rough microsomes, which allows the purification of the rough endoplasmic reticulum (ER) membrane. Membrane‐bound ribosomes and adsorbed proteins are first detached by washing rough microsomes with 5 mM Na‐pyrophosphate. In a second step, the vesicle membrane is opened by digitonin, with concomitant release of the luminal content. The purification is monitored at each step by electron microscopy, and by assaying chemical constituents (protein, phospholipid, RNA) and marker enzymes for the main subcellular organelles. The final membrane preparation is representative of the ER, since it contains 24.1% of the liver glucose 6‐phosphate with a relative specific activity of 14.2. Contaminants represent less than 5% of its protein content. SDS‐polyacrylamide gel electrophoresis, followed by immunoblot analysis, reveals that the ribophorins I and II, two established markers of the rough (d) domain are still present in the final membrane preparation. It also containes the docking protein (or signal recognition particle receptor) and protein disulfide isomerase, and has conserved the functional capacity to remove co‐ and post‐translationally the signal peptide of pre‐secretory proteins. The membrane preparation is suitable for studies on the polypeptide composition of the d domain.
Chromatin and nuclear envelope of freeze‐fractured, neuronal interphase nuclei, resolved by scanning electron microscopy
Authors: de Boni Umberto
Published: February 2012

High‐resolution scanning electron microscopy (SEM) has previously been used to study intracellular detail, including chromatin. It has, however, been commonly carried out either on cellular subfractions or following extraction methods to visualize detail. In the work presented here, intracellular detail of neurons of the dorsal root was visualized in situ by viewing freeze‐fracture faces obtained after hypotonic expansion. This procedure permits the detailed resolution, by SEM, of juxtanuclear and intranuclear detail to a degree impossible without hypotonic dispersal. In agreement with work previously reported, nuclear chromatin of these interphase cells presents largely as 30‐nm fibers, with a next higher hierarchical structure imparted by swelling in magnesium chloride. Detailed analyses showed that particles as small as 10‐nm nucleosomes comprising the 30‐nm chromatin fiber could be resolved, with ≪end‐on≫ views of such fibers showing 5 nucleosomes per helical turn of the fiber. Chromatin fibers positioned subjacent to nuclear pores, or associated with ≪ nuclear spaces ≫ communicating with nuclear pores, were frequently found to be resolved as clusters, in an apparently more decondensed conformation, rather than tightly coiled into the 30‐nm fiber. In addition, details of the nuclear envelope, including nuclear pores and perinuclear filaments as well as membranes of the endoplasmic reticulum, decorated with ribosomes, were clearly resolved.
Modulation of lymphocyte nuclear matrix organization in vivo by 5,6‐dichloro‐1‐β‐D‐ribofuranosyl benzimidazole: an autoradiographic and immunofluorescence study
Authors: Chaly Nathalie , Cadrin Monique , Kaplan J. Gordin , Brown David L.
Published: February 2012

Assembly of active nuclei in lymphocytes stimulated by mitogen is paralleled by the elaboration of a structurally and biochemically complex nuclear matrix (NM). To examine the dynamics of individual NM polypeptide components during blastogenesis, we have applied immunofluorescence labelling with anti‐NM antibodies to concanavalin A‐stimulated mouse splenocytes. Whereas peripherin and PI2 antigens did not reorganize during stimulation, labelling of PI1 and small nuclear ribonucleoprotein (snRNP) antigens increased markedly in intensity and redistributed in concert with the previously reported NM restructuring. Double‐labelling showed, furthermore, that snRNPs and the internal staining component of P11 were largely co‐localized. As an approach to studying the role of RNA and RNA synthesis in NM organization, we have further examined the effects of the inhibitor of RNA synthesis, 5,6‐dichloro‐1‐β‐d‐ribofuranosyl benzimidazole (DRB), on NM antigen distribution. The rapid inhibition of 3H‐uridine incorporation by DRB was accompanied by coordinate aggregation of snRNPs and of the internal PI1 component into large, brightly stained patches. Both 3H‐uridine incorporation levels and antigen localization were readily reversed upon removal of DRB. We conclude that NM antigens behave independently during nuclear and NM assembly and that NM organization, as reflected by NM antigen distribution, is modulated by con A‐ and DRB‐induced alterations in RNA synthesis. We propose, furthermore, that the PI1 antigen plays a role in RNA metabolism, and is possibly involved in RNA transport to the nuclear periphery.
Localization of DNA within Ehrlich tumour cell nucleoli by immunoelectron microscopy
Authors: Thiry Marc , Scheer Ulrich , Goessens Guy
Published: February 2012

The distribution of DNA in Ehrlich tumour cell nucleoli was investigated by means of an immunocytochemical approach involving a monoclonal antibody directed against double‐ and single‐stranded DNA. Immunolabelling was performed either before or after the embedding process. The postembedding labelling method allows better ultrastructural preservation than the preembedding labelling method. In particular, the various nucleolar components are well preserved and identifiable. In the nucleolus, labelling is particularly concentrated over the perinucleolar chromatin and over its intranucleolar invaginations, which penetrate the nucleolar body and often terminate at the fibrillar centres. In addition, aggregates of gold particles are found in the fibrillar centres, preferentially towards the peripheral regions. By contrast, the dense fibrillar component is completely devoid of labelling. The result seem to indicate that DNA containing the rDNA genes is located in the fibrillar centres, with a preference for the peripheral regions. This finding suggests that transcription of the rDNA genes should occur within the confines of the fibrillar centre, probably close to the boundary region of the surrounding dense fibrillar component. The results are discussed in the light of present knowledge of the functional organization of the nucleolus.
Differentiation of a clone isolated from the HT29 cell line: polarized distribution of histocompatibility antigens (HLA) and of transferrin receptors
Authors: Godefroy Odile , Huet Christian , Blair Leslie A.C. , Sahuquillo‐Merino Christian , Louvard Daniel
Published: February 2012

The HT29 cell line, derived from a human colon adenocarcinoma, is able to differentiate if galactose replaces glucose in the culture medium. We have isolated a clone (HT29‐18) from this cell which displays differentiated properties of the parent cell line. HT29‐18 cells grown in glucose‐containing medium form multiple layers of round cells without specific cell‐cell adhesion. In contrast, when grown in galactose‐containing medium, they form a monolayer with tight junctions and exhibit a well differentiated brush border at their apical membrane, which faces the culture medium. The polarized properties of HT29‐18 cells grown galactose‐containing medium were demonstrated by immunofluorescent techniques with antibodies against 2 plasma membrane proteins. Class I histocompatibility antigens (HLA) and transferrin receptors, 2 well characterized integral membrane proteins, are uniformly distributed on the cell surface of undifferentiated HT29‐18 cells, but acquire a polarized distribution during differentiation, localized on the basolateral membranes and absent from the apical surface. Binding of 125I‐labeled transferrin was used to determine transferrin receptor distribution on apical and basolateral membranes. Functional tight junctions in the differentiated cultures were demonstrated, as the monolayer was impermeable to a permeation dye (ruthenium red) as well as to antibodies. The sealing of these tight junctions is, as in vivo, Ca++‐dependent as they could be opened by a short incubation in Ca++‐free medium.
Macrophages express glial markers
Authors: Alliot Françoise , Pessac Bernard
Published: February 2012

We have investigated the relationships between the glial cells of the central nervous system and the resident macrophages. The data reported here show that mouse peritoneal, bone marrow and spleen macrophages are immunoreactive to antibodies specific for astrocytes and oligodendrocytes.
The aminoterminal domain of alpha‐tubulin probed by monoclonal antibodies. Recognition of a rarely exposed epitope by the monoclonal antibody 111 B52 C2**
Authors: Wolff Annie , de Nechaud Béatrice , Chillet Denise , Gros François
Published: February 2012

The production and identification of a monoclonal antibody, 111 B52 C2, raised against fragments obtained after limited proteolysis of purified tubulin is described. The recognized epitope is located on the aminoterminal domain of the α‐tubulin subunit and differs from the antigenic sites reacting with the presently existing panel of available monoclonal antibodies. This monoclonal antibody thus constitutes a potentially useful tool to explore interaction between tubulin and other specific ligands.
Monotonic versus oscillating microtubule assembly: a cryo‐electron microscope study
Authors: Wade R.H. , Pirollet Fabienne , Margolis Robert L. , Garel Jean‐Renaud , Job Didier
Published: February 2012

Depending on the free GTP concentration, microtubules can assemble following either a monotonic or an oscillatory mode. We have used cryoelectron microscopy to compare the tubulin assemblies characteristic of each polymerization pathway. We focus on the first assembly peak. At this particular time point, despite their strikingly different subsequent evolution, both systems are similar with regard to the extent of tubulin polymerization and to the microtubule length distribution. The present study shows that whilst the observed microtubule structures are the same in both systems, the oscillatory system shows quantities of closed ring‐like tubulin oligomers, far in excess of those observed in the monotonic system. Furthermore, the conversion of the oscillating system to a monotonic one by GTP addition during the first oscillation is accompanied by a marked decrease in the number of rings. Based on these results we propose that the GTP dependent step which governs microtubule oscillations is the opening of inactive tubulin oligomers.
Ultrastructural and cytochemical studies on extranucleolar bodies in rat oocytes at the preovulatory follicle stage
Authors: Antoine Nadine , Thiry Marc , Goessens Guy
Published: February 2012

At the antral follicle stage, the nucleolus is entirely composed of a homogeneous proteinic compact mass. This nucleolar compaction during oogenesis seems to be a general feature in mammalian oocytes. However, when oocyte maturation is induced by gonadotropin hormone (LH), oocytes enter into preovulatory stage. All the nucleoli are vacuolated and extranucleolar bodies appear in the germinal vesicle near the nucleolar mass. Based on the results obtained by ultrastructural cytochemical stainings, we postulate that these extranucleolar bodies originate from the nucleolar mass itself. The presence of the extranucleolar bodies could reflect the extrusion of nucleolar material, essentially ribonucleoproteins, into the ooplasm. This material could persist after fertilization in the pronuclei until the resumption of transcription at the early stage of embryogenesis.
Electron microscope study of ribonucleoprotein polyparticles and their relation to perichromatin granules
Authors: Vazquez‐Nin Gerardo , Ortiz Alma , Lopez Aurora , Echeverria Olga
Published: February 2012

Nuclear ribonucleoprotein particles were studied in the central nervous system of the rat after fixation by perfusion with hypotonic‐detergent formaldehyde. Beads‐on‐a‐string structures (polyparticles or chains) formed of 14‐nm granules related by a 7‐nm thick filament were found. These polyparticles are positively contrasted by a preferential method for ribonucleoproteins and they are sensitive to RNase. Perichromatin granules are loosened by this fixation and their internal structure is clearly depicted. They are composed of a tangled mass of 3‐nm thick filaments, but lack 14‐nm granules. The internal filaments of the perichromatin granules display frequent continuities with polyparticles. These results show that polyparticles correspond to perichromatin fibrils visualized in nuclei by standard fixation procedures.
Internalization of atrial natriuretic peptide by adrenal glomerulosa cells
Authors: Morel Gérard , Mesguich Patrick , Chabot Jean‐Guy , Belles‐Isles Marthe , Jeandel Lydie , Heisler Seymour
Published: February 2012

Internalization of 125I‐labelled atrial natriuretic peptide ([125I]ANP) by rat adrenal glomerulosa cells in vivo was investigated by means of an ultrastructural autoradiographic approach. One to 30 min after IV injection of [125I]ANP, silver grains were found, at the light microscope level, over all glomerulosa cells; coinjection of 20 μg of unlabelled ANP inhibited this binding by 64%. At the electron microscope level, the time‐course study indicated maximal silver grain densities in plasma membranes 1 min after IV injection; grains were detected in mitochondria (external membranes and matrix) 2 min after injection, with maximal labelling at 15 min. The cytoplasmic matrix was labelled only 30 min after injection. During the time‐course, labelling of nuclei, Golgi apparatus, and lysosomes was minimal. The data suggest that after binding to plasma membranes ANP is rapidly internalized and distributed within glomerulosa cells. The association of radioactivity with mitochondria suggests that ANP may have intracellular sites of action complementary to those on plasma membranes.
Multiplication of the dictyosome during the formation of autospores in the green alga Chlorococcum infusionum
Authors: Chida Yukie , Noguchi Tetsuko
Published: February 2012

Numerical changes in dictyosomes during the formation of autospores in a green alga, Chlorococcum infusionum, were investigated by electron microscopy. Two dictyosomes were seen near the nucleus in young vegetative cells. Four dictyosomes were seen in large mononucleate cells which appeared to enter mitosis soon. Binucleate cells contained 4 or 8 dictyosomes, the latter number being found in the large binucleate cells. Large tetranucleate cells contained 16–25 dictyosomes in each cell. Dictyosomes consisted of about 4 cisternae with a diameter of 0.4–0.6 μm in mono‐ to tetranucleate cells. Cytokinesis began with the formation of the septa during the third nuclear division, and 16 cells were finally formed. Dictyosomes did not increase in number in 8‐ and 16‐nucleate cells. In septum‐forming cells, dictyosomes were 0.6–1.0 μm in diameter, with 6–9 cisternae. A single dictyosome was included in each of the 16 resultant cells. These observations suggest that the dictyosomes multiply in association with the multiplication of the nuclei without correlation with formation of the cell wall or cytokinesis.
F9 teratocarcinoma aggregates express villin upon differentiation into visceral endoderm‐like cells
Authors: Buc‐Caron Marie‐Hélène , Lamblin Danièle , Kellermann Odile
Published: February 2012

The visceral endoderm of the mouse embryo is a polarized epithelium which has recently been shown to express villin, a major actin binding component of absorptive epitheliums. We report here that villin is induced during differentiation of aggregates of the mouse embryonal carcinoma F9, an in vitro system widely used to study extraembryonic endoderm differentiation. Identical results were obtained with a variant of F9 which carries an immortalizing vector. Villin is coexpressed with F‐actin and with α‐foetoprotein, in most of the visceral endoderm‐like cells lining the aggregates. This system is potentially useful to study (i) the induction of villin expression and (ii) the establishment of polarity in the visceral endoderm epithelium.
Heat shock stress is deleterious to CNS cultured neurons microinjected with anti‐HSP70 antibodies
Authors: Khan Naim A. , Sotelo Julio
Published: February 2012

CNS cultured neurons microinjected with anti‐mouse immunoglobulin or anti‐hsp70 antibodies and cultured at 37°C remained healthy while those injected with anti‐hsp70 antibodies, exposed to 50°C for 15 min and returned to 37°C exhibited deleterious effects i.e. lesser interconnecting neurites and poor neuronal population. This shows that heat shock stress affects the survival of neurons in vitro.
Localization of replicated DNA‐containing sites in preimplantation bovine embryo in relation to the onset of RNA synthesis
Authors: Kopečný Václav , Fulka Josef , Pivko Juraj , Petr Jaroslav
Published: February 2012

The distribution of embryonic DNA in nuclei of blastomeres of early‐preimplantation cow embryos was studied by autoradiography. Two‐, 4‐, 8‐, and 16‐cell embryos were cultured for 18–20 h in the presence of [methyl‐3H]thymidine to ensure that all replicated embryonic DNA was labeled.

In nuclei of blastomeres before the onset of transcription, taking place in the progressed 8‐cell cleavage division of the cow embryo1, labeled DNA was distributed excentrically at the nuclear periphery. After transcription started, the distribution of the labeled DNA was uniform throughout the nucleus.

The progressive association of DNA with the nucleolus‐precursor body (NPB) also correlated with the expected onset of rRNA synthesis. The penetration of the labeled DNA into NPB was seen to start contemporaneously with the formation of a big central vacuole in NPB, but remained restricted, at this stage, to a few points of contact with the adjacent nucleolus‐associated chromatin. No labeled DNA was detected inside this big vacuole, still showing a uniform nucleoplasmic texture.

The inside of the nucleolus was penetrated by DNA in the next step of nucleologenesis when secondary small vacuoles were formed and when the first signs of nucleolar transcription were detected1. These small vacuoles contained a coarse fibrillar component which was a frequent site of labeled DNA detection.

This study of shifts of DNA‐containing sites during transition from maternal to embryonic genome expression correlates closely with previous evidence obtained by a study of genome reactivation which was situated in the late 8‐cell cleavage division of cattle early embryogenesis. It is during this stage, according to the present evidence, that the embryonic DNA occupies the whole of the nucleus, probably due to the extension of the DNA molecule at the onset of transcription. The association of rDNA with the nucleolus‐precursor body leading to the activation of rRNA synthesis is a morphologically well defined process which may reveal, in different experimental systems, the transcription capacity of cattle embryonic genome.
Synthesis and characterization of a long‐acting fluorescent analog of vasotocin
Authors: Eggena Patrick , Buku Angeliki
Published: February 2012

This study reports the synthesis of 1‐desamino‐7‐lysine‐ (fluorescein)‐8‐arginine‐vasotocin (7‐lys(flu) dAVT), and describes its biological activity in the isolated urinary bladder of the toad Bufo marinus. 7‐lys(flu)dAVT was fully active in increasing bladder permeability to water. A half‐maximal hydroosmotic response was obtained at a concentration of 3×10−8 M. A unique feature of this analog was that its reponse was not readily reversed after reoval of the analog from the serosal bathing solution. The residual response to 7‐lys(flu) dAVT was abolished (reversibly) by reducing serosal bath pH from 7.4 to 6.0, suggesting that acidification inhibits the response to analog at a step after the interaction of the ligand with its receptor. Although 8‐arginine‐vasopressin (AVP) was about 20 times more potent than 7‐lys(flu)dAVT in increasing membrane permeability to water, the response to AVP was readily reversed. Preincubation of bladders with 7‐lys(flu)dAVT in the presence of AVP blocked the residual response to 7‐lys(flu) dAVT. These studies suggest that 7‐lys(flu)dAVT forms a stable and physiologically active complex with hydrosmotic toad bladder receptors, and it may, therefore, serve as a useful fluorescent marker for receptors in tissues from this and other species that use vasotocin as an antidiuretic/pressor principle.
Evidence that monensin inhibits vasopressin‐stimulated water flow at an early step in the receptor‐adenylate cyclase sequence
Authors: Franki Nicholas , Mosenkis Bruce , Hays Richard M.
Published: February 2012

Monensin, a highly selective sodium ionophore, inhibits vasopressin‐stimulated water flow in toad urinary bladder pretreated with naproxen, an inhibitor of prostaglandin synthesis. Inhibition is partially dependent on the presence of sodium in the serosal medium, but not on serosal calcium. We have found that monensin does not inhibit water flow generated by forskolin, cyclic AMP, or isobutyl methyl xanthine (MIX); indeed, an enhancement of water flow was seen following cAMP and MIX, as well as following 0.2 μM forskolin. Our findings suggest that monensin uncouples the vasopressin‐receptor‐G protein‐adenylate cyclase sequence at some early step, by a mechanism that remains unknown, but that may directly or indirectlyinvolve intracellular sodium.
Role of an apical cation‐selective channel in function of tight epithelia
Authors: Van Driessche Willy , Erlij David , Simaels Jeannine
Published: February 2012

Some features of oxytocin stimulation of a cation‐selective channel of the apical membrane of amphibian tight epithelia were examined in an attempt to understand the channel's role in the regulation of epithelial transport. We first examined the ability of the channel to pass alkaline‐earth cations. We found that oxytocin can stimulate the movement of alkaline‐earth ions through the channel. This stimulation became greatly enhanced by treatment with Ag+. The stimulation of alkaline‐earth movements is discussed together with recently reported experiments which suggest that the channel may be involved in K+ secretion. In addition we carried out comparative studies of the effects of oxytocin on the channel in a variety of epithelia obtained from different amphibians to examine whether the stimulation of ionic currents through the channel and the enhancement of hydrosmotic permeability caused by the hormone are linked. The results of our experiments showed that oxytocin activates the channel in the urinary bladders of Bufo marinus, and Rana catesbeiana as well as in the skin of B. marinus. It is well known that in all these tissues the hormone increases water permeability of the apical membrane. On the other hand, in skins of Rana catesbeiana, Rana pipiens, and Rana temporaria, where oxytocin does not have a hydrosmotic effect, the hormone did not increase the currents through the cation‐selective channel.
Intramembrane particle structures in epithelial cells of the toad urinary bladder: a quantitative freeze‐fracture study
Authors: Shinowara Nancy L. , Palaia Thomas A. , Discala Vincent A.
Published: February 2012

Freeze‐fracture electron microscopy reveals intramembrane particle arrays in basal membranes of granular epithelial cells as well as both upper and lower plasma membranes of the underlying basal cells in the toad urinary bladder. These particle arrays are morphologically indistinguishable from the luminal membrane aggregates which are known to be associated with antidiuretic hormone (ADH)‐stimulated water transport. In both granular and basal cells particle arrays are frequently located in and/or around the openings of vesicular and/or tubular structures fused to the plasma membranes, suggesting that they may be transferred from the cytoplasm by membrane fusion. Quantification of cytoplasmic aggrephores in control granular cells shows that they can be numerous and as close to the basolateral membrane as they are with the luminal membrane, to which they are known to fuse and deliver aggregates upon ADH stimulation. Aggrephore‐like tubules were also found in the basal cells. Particle array densities were quantified for 6 pairs of control and ADH‐stimulated hemibladders. At least 1440 μm2 area of plasma membrane for each membrane domain was examined. Results indicate that the presence of these particle arrays in granular and basal cell membranes is highly variable and that exposure to ADH does not cause a statistically significant increase in their frequency.
Isolation of large sheets of apical material from frog urinary bladder epithelial cells by freeze‐fracture
Authors: Verbavatz J.M. , Calamita G. , Hugon J.S. , Bourguet J.
Published: February 2012

In the amphibian urinary bladder, water permeability is correlated with the insertion of intramembrane particle aggregates (IMPAs) into the apical plasma membrane (AM) of the granular cells. These aggregates are believed to contain water channels. Characterization of the IMPAs by comparing AM fractions of antidiuretic hormone (ADH)‐treated and resting epithelia requires isolation and purification of AM‐rich material, free of other cytoplasmic aggregate‐containing organelles, in both cases. A technique derived from freeze‐fracture was chosen to isolate large sheets of apical membrane material from frog (Rana esculenta) urinary bladder epithelium. The apical side was plated on a polylysine‐coated glass slide, frozen with liquid nitrogen, and fractured. A nylon mesh was inserted between the glass slide and the bladder, in order to bring the fracture plane back to the AM periodically. Fluorescent markers were used to characterize the material having fractured with the glass slide. Samples were observed by fluorescence and phase contrast microscopy. We obtained evidence that numerous patches of fractured AM remained on the glass surface without nuclei. A phase contrast picture was obtained only at a high magnification, indicating a low thickness of the recovered material. Further characterization was made with SDS‐PAGE. Protein contents of samples were extracted under various experimental conditions and the patterns of ADH‐treated, resting AM samples, or whole epithelial cell crude homogenates, were compared. Staining of some bands increased under certain conditions, whereas many others disappeared. Both morphological and biochemical approaches demonstrate that the recovered material was apical in origin.
Neutral aminopeptidase: a potential marker enzyme of the amphibian urinary bladder epithelial cell apical membrane
Authors: Tacnet F. , Chevalier J. , Coudrier E. , Berthonaud V. , Ripoche P.
Published: February 2012

Antidiuretic hormone induces, in the apical plasma membrane of amphibian urinary bladder epithelial cells, the exocytotic insertion of intramembranous particle aggregates that probably contain water channels. Purification of the apical membrane is a way to characterize the aggregates. The isolation of such purified membranous fractions involves the use of specific exogenous or endogenous markers. One of them could be the neutral aminopeptidase (AP), whose activity was detected in urinary bladder. Enrichment in AP activity was observed in plasma membrane preparations compared to cell homogenates (×2.7). However, a large part of the enzyme activity was also recovered in the soluble fraction of the preparation, suggesting large proteolysis of the protein. The enzyme presents a low optimal pH (6.4) and a high specificity for proline‐p‐nitroanilide as compared to the AP present in kidneys and intestines. To localize the protein in the amphibian bladder epithelium, an immunological approach was necessary due to the low activity of the enzyme in this tissue. The low enzymatic activity also prevented the purification of sufficient amounts of the urinary bladder AP as antigen, and we prepared antibodies against purified AP from frog or toad kidneys where the activity is 60 tims higher than in the bladder. The serum specificity was verified by spot immunodetection, Western blot, inhibition capacity of antibodies, and immunoadsorption on a solid support with the renal enzyme. The sera were found to be able to react with native as well as denatured forms of the kidney enzyme. Antibodies cross‐reacted with several peptides of low molecular weight (40–60 kDa) from urinary bladder plasma membrane proteins (Western blot). Immunocytochemical localization of AP was observed on the apical side of toad bladder epithelial cells.

In conclusion, neutral aminopeptidase is a potential marker for amphibian urinary bladder apical membranes. However, due to the considerable proteolysis during the membrane preparation, rigorous precautions, such as the use of protease inhibitor to avoid its release in a soluble fraction, should be taken.