bluuebunny/crossref_metadata_2025 huggingface dataset:
Relationships between cytokeratin filaments and centriolar derivatives during ciliogenesis in the quail oviduct
Authors: Lemullois M. , Gounon P. , Sandoz D.
Published: February 2012

In the quail oviduct, the mature ciliated cells contain a well developed and polarized cytokeratin network which is bound to desmosomes and in close contact with the striated rootlets associated with basal bodies. In ovariectomized quail, the immature epithelial cells of oviduct present a rudimentary cytokeratin network associated with the centrioles of the diplosome (one of them forming a primary cilium) and with the short striated rootlets. The development of the cytokeratin network which occurs simultaneously with the ciliogenesis was observed by electron microscopy and immunocytochemistry (immunofluorescence and immunogold staining) using a prekeratin antiserum. During estrogen‐induced ciliogenesis, cytokeratin intermediate filaments are always found associated with the different ciliogenic structures i.e. [dense granules, deuterosomes, procentrioles and centrioles]. In ciliogenic cells, the procentrioles and centrioles seem to be associated with the intermediate filaments by their pericentriolar material. These direct contacts decrease once the centrioles/basal bodies are anchored to the plasma membrane. Simultaneously the striated rootlets develop and associate with cytokeratin. The ciliogenic cells appear as a suitable system for studying in vivo, the possible association between centrioles and intermediate filaments and its functional meaning.
The histologist Sigmund Freud and the biology of intracellular motility
Authors: Triarhou L. C. , Del Cerro M.
Published: February 2012

Sigmund Freud, the acclaimed founder of psychoanalysis, invested nine years of his early scientific effort investigating animal histology, cell biology and basic neuroscience prior to concentrating on human nervous and mental disorders. Through his histological studies Freud provided coherent evidence suggesting that the protoplasm consists of a contractile fibrillary network, the present‐day cytoskeleton; he was one of the original founders of the fibrillary theory on the structure of the protoplasm. Concerning the biology of the cell nucleus, Freud appears to have been the first author who documented movements of nucleoli in nerve cells, a phenomenon presently referred to as nuclear rotation. In certain instances, Freud's observations antedate later views by more than half a century and are important to our current understanding of cell structure and basic processes of intracellular motility.
Biochemical and ultrastructural features of human fibroblasts cultured from a new variant of type 3 lipid storage myopathy
Authors: Radom Jean , Salvayre Robert , Mussini Jean‐Marie , De Lisle Bernadette , Negre Anne , Maret Arlette , Billaudel Sylviane , Douste‐Blazy Louis
Published: February 2012

A new variant of multisystemic lipid storage myopathy (type 3) has been identified. Human cultured fibroblasts present a major triacylglycerol storage whereas other neutral lipids and phospholipids are in the normal range. When feeding the cells in the presence of radiolabelled oleic acid we observed an accumulation of radiolabelled triacylglycerols demonstrating the endogenous biosynthesis of the stored triacylglycerols. After a 72‐hr chase period, no degradation of radiolabelled triacylglycerols was observed. Histochemical examination of multisystemic lipid storage myopathy skin fibroblasts showed a massive accumulation of neutral lipids (stained by the fluorescent probe Nile Red) in cells grown in medium supplemented with 10% fetal calf serum. These cytoplasmic vacuoles were not obviously membrane‐surrounded as shown by electron microscopy.
Replication bands and nucleoli in the macronucleus of Euplotes eurystomus: An ultrastructural and cytochemical study
Authors: Olins Ada L. , Olins Donald E. , Derenzini Massimo , Hernandez‐Verdun Danièle , Gounon Pierre , Robert‐Nicoud Michel , Jovin Thomas M.
Published: February 2012

The principal structural compartments of the macronucleus of Euplotes eurystomus were examined by ultrastructural and cytochemical procedures. Interphase chromatin is condensed in highly compact granules that stain intensely with the DNA‐specific osmium‐amine procedure. Nucleoli react strongly with silver and with thiol‐specific reagents, but are almost completely unstained by osmium‐amine. The organelle of DNA synthesis, the replication band, is composed of 2 zones. The forward zone consists of highly ordered chromatin fibers, stains strongly with osmium‐amine, with silver, and with thiol‐specific reagents. The rear zone, which is the site of DNA synthesis, is impoverished in DNA, and is very sensitive to collapse induced by in vivo heat shock, or during nuclear isolation.
G1 and G2 arrest achieved in root meristems by black light irradiation under anoxia, after DNA bromosubstitution
Authors: Sans Jorge , Mergudich Darinka , Galanti Norbel , de la Torre Consuelo
Published: February 2012

Irradiation under anoxic conditions with near‐UV light (300–400 nm walength) of Allium cepa L. root meristems whose cycling cells have their DNA bromosubstituted brings about accumulation of cells in both G1 and G2 stages, in frequencies similar to those found in dormant roots. On the other hand, cells in mitosis nearly disappear from the 12th hour onwards after light irradiation. Since fidelity of transcription is lost after such treatment, the data suggest that altering the pattern of gene expression in the cell cycle is a way to achieve dormancy in these previously proliferating meristems.
Changes in iodine mapping in rat thyroid during the course of iodine deficiency: imaging and relative quantitation by analytical ion microscope
Authors: Fragu Philippe , Briançon Colette , Halpern Sylvain , Larras‐Regard Eliane
Published: February 2012

The analytical ion microscope (AIM) makes possible imaging and relative quantitation of multiple stable or labeled elements on an even tissue section, according to their mass. The purpose of this work was to follow at the rat thyroid follicle level the changes in 127I mapping during low iodine diet (LID) in relation to the ability of thyroid to pick up radioiodine (129I) and to synthesize Tg from its precursor, 2H‐labeled leucine. The overall picture of images and countings of 127I shows a progressive decrease of the luminal iodine concentration which on day 80 was 10‐fold lower than that of control value. In control rat thyroid cell, concentration was 10‐fold lower than that of follicular lumina and was unchanged until 35 days, but the size of the cytoplasmic compartment increased, suggesting a redistribution of iodine stores between thyroid cells and follicular lumina. 129I was always found in colloid as well as in cells at all stages. After 35 days of LID, cytoplasmic and luminal radioiodine concentrations decreased. In control rats, [2H]leucine was found mainly in the cells. During LID its localization was evidenced progressively in most of the lumina. The most striking fact was the presence up to 35 days of some large residual follicles with high 127I concentration and low 129I and 2H incorporation. These data demonstrate the follicular heterogeneity of thyroid response to progressive chronic TSH stimulation induced by LID.
Blastema cell proliferation in vitro: Effects of limb amputation on the mitogenic activity of spinal cord extracts
Authors: Boilly Bénoni , Albert Philippe
Published: February 2012

Primary cultures of mesenchymal cells of axolotl limb blastemas provide a very sensitive in vitro bioassay for studying nerve dependence of newt regeneration. These cells can be stimulated by crude spinal cord extracts on non‐amputated animals in a dose‐dependent manner up to 60 μg protein/ml of culture medium; at this concentration the mitotic index is increased 4‐fold. Spinal cord extracts of axolotls 14 days after forelimb amputation (i.e., late bud stage) are more efficient in stimulating blastema cell proliferation (+50%) than extracts of axolotls 7 days after forelimb amputation (i.e., early bud stage) or of axolotls without amputation. In a similar manner, spinal cord extracts of young axolotls 14 days after forelimb amputation, are more stimulatory than older axolotls 14 d after forelimb amputation which regenerate only a very small blastema during the same time. It appears that spinal cord mitogenic activity is enhanced after limb amputation, probably in correlation with blastema cell requirements for limb regeneration.
Morphological and functional differentiation of human thyroid cells in collagen gel culture
Authors: Thomas‐Morvan Colette , Caillou Bernard , Schlumberger Martin , Fragu Philippe
Published: February 2012

In order to study the expression of the morphological and functional characteristics of human thyroid cells, 3‐dimensional cultures were carried out in collagen gel. This substrate allows the cells to retain their organization in follicles with a normal polarity. Cellular polarities appeared normal at the time of collagen embedding, but there was a delay of 4–5 days in culture before the maximal TSH stimulation of 125I− uptake and of cAMP accumulation occurred. In normal and adenoma‐derived cells, 125I− uptake, which could be increased by TSH, was demonstrated. cAMP accumulated in the culture medium and thyroglobulin was secreted into the follicle lumen. Of the 4 differentiated carcinomas for which the 72‐hr uptake of 125I− was measured, only 2 displayed slight 125I− uptake and response to TSH. Thus, human thyroid cells exhibit better morphological and functional differentiation in collagen gel culture than in monolayer culture. Furthermore, in a variety of pathological cases studied, the expression of specific characteristics in culture varied in a fashion similar to differences observed in vivo.
In vivo binding of auxin to the plasmalemma and tonoplast of parenchymal cells in the wheat coleoptile
Authors: Perbal Gérald , Driss‐Ecole Dominique
Published: February 2012

Tritiated auxin applied by an agar block on the wheat coleoptile tip for 2 hr was covalently fixed to adjacent protein by treatment with 1‐(3‐dimethylaminopropyl)‐3‐ethylcarbodiimide hydrochloride (DCC). The density of labelled auxin in the nucleus, the cell wall, the cytoplasm, and the vacuole was determined by autoradiography. Localization of tritiated auxin was studied at high resolution at the tonoplast and the plasmalemma lining the transverse (distal and proximal) and the longitudinal walls. The radioactivity along the tonoplast was always less than along the plasma membrane. The distribution of 3H‐auxin was different across the longitudinal and transverse regions of the plasmalemma. The labelling was distributed asymmetrically on the longitudinal plasma membrane with a peak observed on the external surface. Tritiated auxin was distributed more symmetrically on the distal and the proximal plasma membranes. Our results are in agreement with the hypothesis that there are 2 different specific binding sites on the plasmalemma. The ratio of auxin present at the proximal and distal regions of the plasmalemma was 1.28.
Selective intracellular beryllium localization in rat tissue by mass‐resolved ion microprobe imaging
Authors: Levi‐Setti Riccardo , Berry Jean‐Pierre , Chabala Jan M. , Galle Pierre
Published: February 2012

Beryllium absorption sites in the kidney and liver of rats have been located and imaged at approximately 70 nm lateral resolution with a scanning ion microprobe utilizing secondary ion mass spectrometry. Embedded sections and lyophilized cryosections of these organs were prepared after in vivo administration of beryllium in soluble form. Beryllium distribution images were correlated with the histological microstructure revealed by CN− images. In the kidney, beryllium concentrates selectively within the nuclei of proximal tubule cells and occasionally within modified podocytes or mesangial cells in the glomerulus. In the liver, beryllium is seen to localize within severely altered lysosomal structures as well as within hepatocyte nuclei. These observations are relevant to understanding aspects of the toxic and carcinogenic properties of absorbed beryllium compounds.
Anionin sites of the basement membrane of rat seminiferous tubules during ontogenesis
Authors: Leheup Bruno P. , Gelly Jean‐Louis , Delongeas Jean‐Luc , Grignon Georges
Published: February 2012

The anionic sites of the basement membrane of rat seminiferous tubules were demonstrated ultrastructurally in the lamina densa by using cationic polyethyleneimine (PEI). The sites were largely digested out after incubation with heparitinase, indicating a large proportion of heparan sulfates. The anionic sites were present as early as day 16 of gestation on the interstitial side of the lamina densa, and after gestation day 20 they were symmetrically organized on both sides of the lamina densa. The number of sites is not modified postnatally. They appear more irregular in density with advancing age. Experimental conditions as crytorchidism, fetal irradiation, and ligation of the ductuli efferentes lead to unspecific alterations in the distribution of the anionic sites that are parallel to the modifications in the basement membrane.
Ultrastructural localization of N‐acetylglucosamine residues in the cell wall of Gigaspora margarita throughout its life‐cycle
Authors: Grandmaison Jacques , Benhamou Nicole , Furlan Valentin , Visser Simon A.
Published: February 2012

The cell ultrastructure of the VAM fungus Gigaspora margarita was examined, and its N‐acetylglucosamine content and distribution were assessed by means of WGA/ovomucoid‐gold labelling. The various ontogenic stages of the fungus were studied with particular reference to cell walls. The results provide new information on chitin incorporation during spore wall development. A decrease of chitin was observed which was correlated to the structural simplification of the fungus wall throughout its life‐cycle. This suggests an involvement of chitin in specific biological functions such as mechanical resistance and plasticity.
Development and advantages of serum‐free, chemically defined nutrient media for culturing of nerve tissue
Authors: Romijn Herms J.
Published: February 2012

This paper describes in a historical perspective the development of serum‐free nutrient media suitable for long‐term culturing of nerve tissue. Several disadvantages of the use of serum are discussed, coupled with an acknowledgement that it is not always advisable to replace a routinely used serum‐supplemented medium by a chemically defined medium with the expectation of immediate success. Therefore a strategy is given on how to develop a chemically defined medium that is thoroughly tuned to the specific needs of the cell type to be cultured. It is argued that such a medium has several substantial advantages over the use of serum.
Electron microscope analysis of the E polytene chromosome of Drosophila subobscura: region 70B to 72D
Authors: Pérez‐Alonso Manuel , Cuenca Juan Bautista , De Frutos Rosa
Published: February 2012

Revision of the reference map of the polytene chromosomes of Drosophila subobscura was started by means of the electron microscope. We present a map of regions 70B to 72D of the E chromosome obtained from squashed and thin‐sectioned salivary glands. It was observed that the total number of bands in divisions 70B to 72D is considerably higher than those depicted in the reference map of Kunze‐Mühl and Müller. Functional considerations are made of some regions that show puff structures.
Enucleation of phagocytic cells with adenine, guanine, and their nucleosides in combination with centrifugation
Authors: Saito Takuya , Yamaguchi Junji
Published: February 2012

Several purine compounds, such as adenine, guanine, adenosine, guanosine, and their related compounds, exhibited enucleation activity on adherent mouse peritoneal exudate cells (macrophages) during centrifugation at 25,000 and 35,000 g for 60 min at 34°–36°C in medium containing one of these compounds. Enucleation activity, however, did not occur in cells treated with adenine nucleotides, inosine, xanthine, or any of the tested pyrimidines. The purine compounds also had enucleation activity on mouse macrophage‐like cell lines (P388D1 and RAW 264) and mouse polymorphonuclear leukocytes, but not on other typical cell lines such as a human epithelial cell line (HeLa S‐3) or a mouse fibroblast cell line (L929). Cytochalasin B (CB) treatment, however, resulted in the enucleation of all cell types tested, even at a centrifugal force as low as 5,000 g. The process of macrophage enucleation was observed by both light microscopy and scanning electron microscopy. In enucleated macrophages that had been treated with purine compounds, but not with CB, a newly formed cytoplasmic crater‐like structure (about 3–9 μm in diameter) was observed at the original site of the nucleus. Surface structures, such as microvilli and membrane ruffles, remained relatively intact in macrophages that had been enucleated by treatment with purine compounds. By contrast, these surface structures were markedly changed in CB‐treated macrophages. Purine compounds may affect cytoskeletal elements in ways similar to the well characterized effects of CB, and thus result in the enucleation of phagocytes. However, the characteristic differences in the enucleation activity exhibited by purine compounds and CB may indicate that purines have a mechanism of action different from that of CB.
Hyaluronic acid‐degrading enzymes in rat alveolar macrophages and in alveolar fluid: stimulation of enzyme activity after oral treatment with the immunomodulator RU 41740
Authors: Fiszer‐Szafarz Berta , Rommain Michèle , Brossard Claudine , Smets Pierre
Published: February 2012

RU 41740 (Biostim®) is an immunomodulator clinically used for the treatment of chronic bronchitis and recurrent pulmonary infections. In these diseases large amounts of mucus are produced which congest the bronchi. A major glycosaminoglycan constituent of this mucus is hyaluronic acid, one of the largest molecules in nature; its metabolic degradation is carried out by 3 acid hydrolases: hyaluronidase, β‐N‐acetylglucosaminidase, and β‐glucuronidase.

In the lung these enzymes are especially synthesized and active in alveolar macrophages. It was thus interesting to study the effect of RU 41740 administration on the hyaluronic acid‐degrading activity of these cells.

This compound was given by gastric gavage to rats and the activities of lung alveolar macrophage and alveolar fluid hyaluronidase, β‐N‐acetylglucosaminidase, β‐glucuronidase, and acid phosphatase as a lysosomal marker were determined. The effect on macrophage proliferation was also examined.

The results obtained showed that: (1) unstimulated alveolar macrophages display the remarkable property, compared with other cell types, that hyaluronidase activity is about equally distributed between the inside and the outside of the cell; (2) RU 41740 administration increases the total activity of the 4 enzymes studied in the alveolar macrophages without inducing any increase in the number of macrophages; (3) the intracellular activities of β‐N‐acetylglucosaminidase and β‐glucuronidase are markedly increased, whereas intracellular hyaluronidase activity is not changed. However, in the extracellular fluid only hyaluronidase activity is highly increased; (4) even the lysosomal marker enzyme acid phosphatase has only its intracellular activity is increased. This would suggest the possibility that other lysosomal enzymes may also be increased by this immunomodulator.

Most probably, the activation of the hyaluronic acid‐degrading enzymes by RU 41740 has the following consequences: extracellular hyaluronidase degrades hyaluronic acid to smaller molecules, which are then phagocytized by the macrophages; the intracellular hydrolytic enzymes, also strongly stimulated, complete the hydrolysis. Moreover, it was observed that the molecular form of rat macrophage hyaluronidase is the same as that of serum and other somatic cell hyaluronidase.
Actin cytoskeletal lesions in differentiated human colon carcinoma Caco‐2 cells after exposure to soybean agglutinin
Authors: Draaijer Marjan , Koninkx Jos , Hendriks Henno , Kik Marja , Van Dijk Jaap , Mouwen Johan
Published: February 2012

We have investigated the effects of soybean agglutinin on the cytoskeletal element actin in differentiated Caco‐2 cells. The actin cytoskeleton of the cells was visualized by fluorescence microscopy using 7‐nitrobenz‐2‐oxa‐1, 3‐diazole phallacidin as a specific marker for F‐actin. Compared with control Caco‐2 cells no changes in the fluorescence pattern were observed after incubation with soybean agglutinin. However, using the deoxyribonuclease‐I inhibition assay a dose‐related response was noted in the increase of intracellular G‐actin after a 2‐hour incubation period with soybean agglutinin. Already after exposure for 15 min to soybean agglutinin a decrease in intracellular F‐actin was demonstrable. This apparent depolymerization could be prevented by incubating the Caco‐2 cells with soybean agglutinin and the appropriate monosaccharide simultaneously. The increase in the amount of G‐actin appeared to be correlated with a shortening of microvilli on the Caco‐2 cells.
High resolution scanning electron microscopy of the cell
Authors: Tánaka Keiichi
Published: February 2012

The scanning electron microscope (SEM) has become a powerful tool for ultrastructural research with improvement of the instrument's resolution and progress in specimen preparation techniques. With regard to resolution, it has been improved step‐by‐step in this decade and, in 1985, an ultra‐high resolution SEM (UHS‐T1) was developed, with a resolution of 0.5 nm. Concerning specimen preparation, the osmium‐DMSO‐osmium method, which is effective for revealing intracellular structures, has come to be widely used. Techniques for observing smaller objects, such as bacteriophages, viruses, and biological macromolecules, have also been devised in recent years. As a result of these preparation techniques and the availability of the ultra‐high resolution SEM, the application of SEM in biology is expanding rapidly. In this paper, an outline of the ultra‐high resolution SEM, techniques for specimen preparation, findings of some biological materials by these techniques, and guidelines to making the specimens, are described.
Translocation and proteolytic processing of nascent secretory polypeptide chains: two functions associated with the ribosomal domain of the endoplasmic reticulum
Authors: Amar‐Costesec Alain , Dublet Bernard , Beaufay Henri
Published: February 2012

Rat liver microsomes were subfractionated by isopycnic centrifugation in sucrose gradient. The subfractions were assayed for translocation and proteolytic processing of nascent polypeptides in a rabbit reticulocyte lysate programmed with total RNA from human term placenta. The distribution of the translocation and processing of prelactogen through the gradient correlated with that of the microsomal RNA (ribosomes). Microsomes became inactive upon incubation with elastase, but the proteolyzed membranes recovered their activity by recombination with the soluble and active fragment of the docking protein (SRP‐receptor) from dog pancreas. When this fragment was combined with the gradient subfractions, or with the subfractions inactivated by incubation with elastase, the density profile of the translocation activity remained similar to that of RNA. Thus, its distribution cannot be accounted for merely by that of the docking protein; another membrane constituent, still unidentified, is both necessary for translocation of polypeptides and restricted to the rough portions of the endosplamic reticulum. Signal peptidase was assayed in the absence of protein synthesis, by use of preformed prelactogen and detergent‐disrupted microsomes. Its density distribution was also similar to that of RNA. Several components of the endosplamic reticulum now appear to be segregated within restricted areas on either side of the membrane, and to make up a biochemically distinct domain. We propose to call it the ribosomal domain in consideration of its contribution to protein biosynthesis by bound ribosomes. This domain probably accounts for a greater part of the membrane area at the cytoplasmic than at the luminal surface, as postulated earlier to explain how enzymes of the cytoplasmic surface are relatively less abundant in the rough microsomes than those of the luminal surface [Amar‐Costesec A. & Beaufay H. (1981) J. Theor. Biol. 89, 217–230].
Kurloff cell proteoglycans: presence of two main size‐populations of intracellular protease‐resistant proteochondroitin sulphate. Effect of D‐xyloside
Authors: Letaíef Slim‐Eric , Landemore Gérard , Bocquet Jean , Izard Jacques
Published: February 2012

This paper reports that the Kurloff cell sulphated and chondroitinase AC sensitive material previously described filtered on Sepharose CL4B columns as 2 main populations with Kav of 0.25 and 0.44. Its alkaline treatment resulted in the elution of 2 peaks with Kav of 0.52 and 0.78. Their reduction in size observed after alkaline treatment and the 6‐fold increase in the (35S) sulphate incorporation after addition of 0.1 mM xyloside to the incubation medium indicate that these intracellular sulphated glycosaminoglycans exist in the form of proteoglycans. They were characterized by their resistance to degradation by pronase, papain or cathepsin D, as assessed by gel filtration chromatography on Sepharose CL6B or CL4B. After the glycosaminoglycans were digested with chondroitinase AC, thin‐layer chromatography analysis indicated the presence of Δ di‐4S and Δ di‐6S in a ratio of 7:1. The presence of such protease‐resistant proteochondroitin sulphate in intracytoplasmic granules of both Kurloff cells and other natural killer cell types is emphasized.
Contribution of the vasopressin V1 receptor to its hydrosmotic response
Authors: Yorio Thomas , Satumtira Nimman
Published: February 2012

Toad urinary bladder epithelial cells grown in culture (primary) show a significant increase in water‐soluble inositol phosphates when treated with 10−8 M vasopressin (AVP), but not with (3‐deamino‐8‐d‐arginine)vasopressin (ddAVP), a V2‐agonist. The increase in inositol phosphates was blocked by the V1‐antagonist, d(CH2)5Tyr(Me)AVP, suggesting a V1‐coupled phosphoinositide breakdown. The V1‐antagonist had no effect on basal adenylate cyclase activity nor on that stimulated by AVP. However, the V1‐antagonist was found to attenuate the hydrosmotic response of AVP, suggesting some role of the V1‐receptor cascade in the water flow response. Mezerein (MZ), a non‐phorbol activator of protein kinase C (PKC) increased osmotic water flow response. Mezerein (MZ), a non‐phorbol activator of magnitude and occurred over a longer period (90 min) than that observed with AVP. In an attempt to emulate the V1‐response, activation of PKC, and an increase in intracellular calcium, toad bladders were incubated with MZ and the calcium ionophore A23187 (IP). It was found that IP enhanced the water flow response to MZ at all times measured. Mz and IP were also found to enhance cAMP‐mediated water flow, suggesting that apical membrane permeability may be regulated in part through V1‐receptor stimulation and its respective second messengers. Collectively, these observations suggest that the V1 receptor may play a role not only as part of a negative feedback system, but also as an integral component of the enhanced water permeability that occurs at the apical membrane.
Microtubules and actin microfilaments in the amphibian bladder granular cells
Authors: Hugon J.S. , Ibarra C. , Valenti G. , Bourguet J.
Published: February 2012

Microtubules and microfilaments were localized by an immunocytochemical method in the granular cells of the frog bladder after fixation and isolation.

An extensive array of microtubules was observed in the granular cells with an orientation towards the luminal plasma membrane in the supranuclear zone. Actin filaments formed a continuous bundle that underlined the cellular membrane.

After incubation in the presence of colchicine, nocodazole, or tubulozole, the microtubular network appeared fragmented but did not disappear completely.

These observations are related to the role of the cytoskeleton in the permeability response of the frog bladder epithelium to vasopressin.
Subcellular fractionation of epithelial cells from toad urinary bladder. 1. Assay of marker enzymes and differential centrifugation
Authors: Ripoche Véronique , Beauwens Renaud , Bouisset Michèle , Amar‐Costesec Alain , Beaufay Henri
Published: February 2012

We have undertaken the analytical fractionation of epithelial cells from toad urinary bladder, a tissue extensively used to study epithelial transport of ions and water. In an attempt to establish markers for the main subcellular organelles, a number of enzymes were assayed in cell homogenates. The nearly ubiquitous plasma membrane marker 5′‐nucleotidase, and the transferases that donate N‐acetylglucosaminyl, galactosyl, and sialyl residues to glycoproteins and glycolipids in the Golgi complex were not detectable. Glucose‐6‐phosphatase activity was low in relation to that of nonspecific phosphatases and, therefore, not suitable for identifying the endoplasmic reticulum. Like the cytosolic enzyme lactate dehydrogenase, catalase was essentially found in the high‐speed supernatant, with a noteworthy part of aminopeptidase (substrate, leucyl‐ß‐naphthylamide) and NAD glycohydrolase. Other enzymes, including cytochrome c oxidase, acid phosphatase, acid N‐acetyl‐ß‐glucosaminidase, alkaline phosphatase, alkaline phosphodiesterase I, nucleoside diphosphatase (substrate ADP), oligomycin‐resistant Mg++‐ATPase, and mannosyltransferase (acceptor, dolichylphosphate) were fairly active and largely sedimentable. After differential centrifugation, cytochrome oxidase, acid phosphatase, and acid N‐acetyl‐ß‐glucosaminidase were typically associated with the large granule fraction, whereas the other sedimentable enzymes exhibited a broad distribution profile overlapping the nuclear, large granule, and microsome fractions. Their behavior in density equilibrium centrifugation is examined in a companion paper.
Subcellular fractionation of epithelial cells from toad urinary bladder. 2. Isopycnic centrifugation and effect of density perturbants
Authors: Ripoche Véronique , Amar‐Costesec Alain , Beauwens Renaud , Beaufay Henri
Published: February 2012

Cytoplasmic granules obtained from toad urinary bladder epithelial cells were brought to buoyancy in a linear sucrose gradient. The gradient was loaded either with untreated cytoplasmic granules, or with granules treated with Na pyrophosphate (PPi), with digitonin, or with PPi and digitonin in succession. The following enzymes were assayed in the gradient subfractions: oligomycin‐insensitive Mg++‐ATPase, alkaline phosphodiesterase I, alkaline phosphatase, acid N‐acetyl‐β‐glucosaminidase, cytochrome oxidase, nucleoside diphosphatase (substrate, ADP), aminopeptidase (substrate, leucyl‐β‐naphthylamide), and mannosyltransferase (acceptor, dolichylphosphate). Comparison of the density distributions of enzymes in untreated and treated preparations led to the characterization of 4 distinct subcellular entities. In agreement with the properties of mitochondria from other cell types, cytochrome oxidase buoys at 1.18 within a narrow density range and its behavior is not significantly altered by PPi or digitonin. Under all conditions, acid N‐acetyl‐β‐glucosaminidase is recovered over a broad density range in the lower part of the gradient and appears as a qualified lysosomal marker. Mg++‐ATPase, alkaline phosphodiesterase I, and alkaline phosphatase belong to a group with the distinguishing features of a low equilibrium density in native cytoplasmic granules and a marked shift (+0.03 density units) after digitonin treatment. Such properties are typical of the plasma membranes. Part of the aminopeptidase activity probably also belongs to plasma membrane‐derived elements. Minor differences between alkaline phosphatase and the other 2 members of that group make it possible that their distribution domains in the membrane do not overlap or coincide. Finally, mannosyltransferase is identified as an endoplasmic reticulum marker: its distribution is shifted to lower densities after PPi. This treatment releases ribosomes from rough microsomes and accordingly decreases the density of these vesicles. The subcellular localization of nucleoside diphosphatase, which displays distinct properties, is still unclear.
Water permeability in different epithelial barriers
Authors: Capurro C. , Escobar E. , Ibarra C. , Porta M. , Parisi M.
Published: February 2012

The water permeability properties of a series of epithelial barriers (the toad urinary bladder [TUB], the rat caecum [RC], the distal human colon [DHC], and the human amnion [HA] were studied in different experimental conditions. Three parameters were simultaneously determined: the water permeability coefficient in the presence of a transepithelial hydrostatic gradient (Phydr); the water permeability coefficient in the presence of an osmotic gradient (Posm); and the transepithelial potential difference (dV). All experiments were performed with the same experimental device, allowing comparison of the permeability properties of the barriers tested. The results obtained were: (1) TUB (N=8): Phydr = 0.079±0.008 cm/s; Posm = 0.0004±0.0002 cm/s; dV=31±5 mV; (2) TUB after ADH (N=8): Phydr = 0.093±0.012 cm/s; Posm = 0.0065±0.0011 cm/s; dV=52±8; (3) RC (N=10): Phydr = 0.18±0.02 cm/s; Posm = 0.0019±0.0004 cm/s; dV=3.9±0.1 mV; (4) RC adapted to a high K diet (N=10): Phydr = 0.21±0.02 cm/s; Posm = 0.0018±0.0006 cm/s; dV=4.5±0.5 mV; (5) DHC (N=6): Phydr = 0.22±0.03 cm/s; Posm = 0.002±0.05 cm/s; dV=15±3 mV; (6) HA (N=10): Phydr = 0.32±0.05 cm/s; Posm = 0.0154±0.0015; dV=0. The results show a good correlation between Phydr and dV, but not between dV and Posm or between Posm and Phydr.
(Na,K)‐pump: cellular role and regulation in nonexcitable cells
Authors: Cohen Brian J. , Lechene Claude
Published: February 2012

The (Na,K)‐pump develops and maintains ionic gradients that are of fundamental importance for proper function of most animal cells. These gradients are utilized in the form of ionic leak pathways by a number of special and general cell processes (e.g., nerve conduction, nutrient transport, pH regulation). As the sodium gradient in particular energizes many vital cell processes, alterations in cell activity will often be manifest as changes in sodium entry. The (Na,K)‐pump rate varies accordingly, in order to maintain balance between Na entry and exit thereby maintaining the potential energy of the cell.

Acute changes in sodium influx are balanced by increases in activity of existing pump units, with only a small change in intracellular sodium concentration. This is posible because intracellular is normally poised on the steep limb of the concentration versus activity curve for the (Na,K)‐pump, at a point well below maximal activity, allowing large increases in (Na,K)‐pump rate with only small changes in sodium concentration. If the increase in sodium influx is prolonged, it appears that the cell responds by synthesizing new pumps, allowing intracellular sodium concentration to return to its original values.

Though increases in (Na,K)‐pump activity must be accompanied by increases in potassium leak rates, in the experiments we have presented, there does not appear to be direct functional coupling between the (Na,K)‐pump and the K leak pathways. In these situations the matching of active influx and passive efflux of K short‐term appears to occur by mechanisms not directly related to (Na,K)‐pump activation.
Differential expression of 240 kDa ConA‐binding glycoprotein in vitro and in vivo detected by immunochemical methods
Authors: Barakat Ibtissam , Bezamahouta César , Zanetta Jean‐Pierre , Vincendon Guy
Published: February 2012

The expression of the 240 ConA‐binding glycoprotein (240 kDa), a marker of synaptic junctions isolated from the rat cerebellum, was studied by immunocytochemical techniques in forebrain and cerebellum from rat and chicken, and in chick dorsal root ganglia. Parallel studies were carried out either on tissue sections or in dissociated cell cultures. In all cases non neuronal cells were not immunostained.

The tissue sections of cerebellum from rat and chick exhibited 240 kDa glycoprotein immunoreactivity, especially in the molecular layer, while the forebrain sections from rat and chick did not show any significant immunostaining. In contrast, in dissociated forebrain cell cultures, all neuronal cells expressed 240 kDa glycoprotein immunoreactivity, while glial cells remained totally unlabelled. In tissue sections of dorsal root ganglion (DRG), sensory neurons expressed the 240 kDa only after the embryonic day (E 10). A large number of small neurons in the dorsomedial part of DRG were immunostained with 240 kDa glycoprotein antiserum, whereas only a small number of neurons in the ventrolateral part of the ganglia displayed 240 kDa immunoreactivity. In dissociated DRG cell cultures (mixed or neuron‐enriched DRG cell cultures) all the neuronal perikarya but not their processes were stained. These studies indicate that 240 kDa glycoprotein expression is completely modified in cultures of neurons of CNS or PNS since the antigen becomes synthetized in high amount by all cells independent of synapse formation. This demonstrates that the expression of 240 kDa is controlled by the cell environment.
Heterogeneity of progesterone receptor expression in epithelial cells of immature and differentiating quail oviduct
Authors: Pageaux Jean‐François , Dufrene Laurent , Laugier Christian , Perche Olivier , Sandoz Daniel
Published: February 2012

The localization of progesterone receptor (PR) in the quail oviduct was investigated before and after the onset of sexual maturation using an immunohistochemical technique. PR was revealed exclusively in nuclei of target cells whatever the hormonal state of the tissue (immature or not, pretreated or not with progesterone).

In the immature or ovariectomized quail oviduct, PR was principally localized in the undifferentiated epithelial cells; some mesothelial cells and a very few stromal cells expressed the PR. Only 40–45% of the epithelial cells were immunoreactive. These positive cells were mainly localized in the furrows of the villi where further evagination of the epithelium will occur to form the tubular glands.

The onset of sexual maturation was accompanied by an increase of the proportion of positive epithelial cells and stromal cells. In estradiol‐treated animals, more than 90% of the tubular gland cells were strongly stained while only 40% of the luminal epithelial cells were immunoreactive.

Our results show that there are two subpopulations of epithelial cells: those expressing the PR before the onset of sexual maturation even in ovariectomized quails (constitutive expression) and those expressing the PR during sexual maturation or after estrogen injection (inductive expression). These results, associated with previously published studies dealing with the cytodifferentiation of epithelial cells during natural development or after various hormonal treatments in ovarectomized animals, suggest that the first are the progenitors of tubular gland cells, and the second the progenitors of ciliated and goblet cells. In stromal cells, PR expression is also inducuible.
A technique for labelling the sample surface for quick‐freeze, deep‐etch, rotary replication electron microscopy: application to the study of geletin gel structure
Authors: Favard Pierre , Lechaire Jean‐Pierre , Maillard Maryvonne , Favard Nina , Djabourov Madeleine , Leblond Jacques
Published: February 2012

A method using magnesium oxide crystals to label the surface of physical gels, such as gelatin gel before quick‐freezing is described and discussed. The quick‐freeze, deep‐etch, rotary replication technique is most adapted to 3‐D visualization of physical gel structure. However, it is known that the depth which ultrarapid freezing may reach is limited by the growth of ice crystals as the distance from the surface of the specimen (rapidly cooled by smashing against a cooled metal plate) increases. Consequently, intact preservation of structures occurs only in superficial zones of the specimen.

The MgO surface labelling technique provides a simple means for surface recognition. It enables the estimation of a given replicated area depth, taking into account the angle of specimen scraping before etching and replicating. By comparison of views of the same replica at different depths, freezing artifacts may be recognized even when they cause only slight deformations in the structure. This is particularly necessary for interpretation of gel network geometry: interpretation can be made with certainty only if a reliable surface reference marker exists.

For gelatin gels, the depth of best freezing can be estimated to be around 5 μm from the frozen sample surface.
Attempted localization of a substrate for chitinases in plant cells reveals abundant N‐acetyl‐d‐glucosamine residues in secondary walls
Authors: Benhamou Nicole , Asselin Alain
Published: February 2012

Ultrathin sections of healthy and fungus‐infected plant tissue were treated with either wheat‐germ agglutinin (WGA) ovomucoid‐gold complex or microbial chitinase‐gold complexes for localizing putative chitin‐like macromolecules. Fungal cell walls, known to contain chitin, were labeled with both probes and were considered as positive controls. Plant secondary cell walls of both healthy and infected tissues were also intensely labeled whereas compound middle lamella‐primary walls and cell cytoplasm were free of labeling. Enzymatic digestion of plant tissues with chitinase from Streptomyces griseus abolished the fungal cell wall labeling but did not interfere with that of plant secondary cell walls. This suggests that polymers analogous to fungal chitin are absent in plant cell walls. Tissue digestions with either proteinase K or lipase led to surprising results as far as the possible nature of N‐acetylglucosamine‐containing molecules is concerned. The loss of labeling over plant secondary walls following lipase digestion suggests that N‐acetylglucosamine residues may be linked to lipids to form glycolipids. However, these results have to be viewed with caution since the possibility that peptides may be present but inacessible to proteinase K should be considered. The role of the detected N‐acetylglucosamine containing molecules as possible substrates for plant chitinases is discussed.
A possible role of actin in the mechanical control of the cell cycle
Authors: Tripathi Satish C.
Published: February 2012

Sail‐sheet Cultures (SSC) are those in which the cells are i) grown within the meshes of inert grids ii) exposed to nutrients from most sides iii) attached to one another only at the edges like sail of a yacht (hence, the name ‘sail‐sheet’) and iv) have the advantage of three‐dimensional structure similar to an in vivo situation. We grew fibroblasts from chicken heart explants as SSC and studied the effect of mechanical stretching on the F‐actin content of these cells. This study was designed to investigate the hypothesis that the effect of tension on the cell cycle may be channeled through the microfilaments. Data from this preliminary study suggested that short‐term mechanical stretching of sail‐sheets, using low frequency tension (1.0 Hz), diminishes F‐actin. Thus, it may be possible to relate the decrease in the F‐actin content of these cells to the slowing down of their locomotory activity, possible rounding up, and division. This study might contribute to the understanding of the mechanical control of the cell cycle and be of relevance in the phenomena such as healing of wounds and control of the cell division in tumors.
Restoration of dysgenic murine (mdg) myotube contraction after addition of Schwann cells from normal mice in vitro
Authors: Courbin Pierre , Do Thi Ahn , Ressouches Annie , Dussartre Christian , Powell Jeanne Anne , Koenig Jeanine
Published: February 2012

Muscular dysgenesis in mutant mice is characterised by failure of excitation‐contraction (E‐C) coupling and consequent loss of skeletal muscle contraction. Contractile activity is restored in vitro by the addition of normal mice cells (11) (18) (7). In the present study we show a new model: contraction and ultrastructural organization of dysgenic myotubes are restored by coculture with Schwann cells from normal mice.
Identification of the 16°C compartment of the endoplasmic reticulum in rat liver and cultured hamster kidney cells
Authors: Morré D. James , Minnifield Nita , Paulik Mark
Published: February 2012

Summary— In many systems transfer between the endoplasmic reticulum and the Golgi apparatus is blocked at temperatures below 16°C. In virus‐infected cells in culture, a special membrane compartment is seen to accumulate. Our studies with rat liver show a similar response to temperature both in situ with slices and in vitro with isolated transitional endoplasmic relticulum fractions. With isolated transitional endoplasmic reticulum fractions, when incubated in the presence of nucleoside triphosphate and a cytosol fraction, temperature dependent formation of vesicles occurred with a Q10 of −2 but was apparent only at temperatures greater than 12°C. A similar response was seen in situ at 12°C and 16°C where fusion of transition vesicles with cis Golgi apparatus, but not their formation, was blocked and transition vesicles accumulated in large numbers. At 18°C and below and especially at 8°C and 12°C, the cells responded by accumulating smooth tubular transitional membranes near the cis Golgi apparatus face. With cells and tissue slices at 20°C neither transition vesicles nor the smooth tubular elements accumulated. Those transition vesicles which formed at 37°C were of a greater diameter than those formed at 4°C both in situ and in vitro. The findings show parallel responses between the temperature dependency of transition vesicle formation in vitro and in situ and suggest that a subpopulation of the transitional endoplasmic reticulum may be morphologically and functionally homologous to the 16°C compartment observed in virally‐infected cell lines grown at low temperatures.
X‐ray microanalysis of freeze‐dried digestive mucus in Anguilla anguilla L: modifications of ion content during the early steps of secretion
Authors: Humbert Willy , Simonneaux Valérie , Kirsch Raymond
Published: February 2012

Summary— Digestive mucus of sea‐water adapted eels has been observed and analyzed by the scanning electron microscope (SEM) after rapid freezing at liquid nitrogen temperature followed by freeze‐drying. No chemical procedures were used in this technique. This allowed the maintenance of the mucous coating.

Preliminary X‐ray microanalysis carried out on freeze‐fractured and freeze‐dried samples of the oesophagus showed a decrease of K+ and an increase of Ca2+ and Cl− from the basal part of the mucous cell towards its the apical part. This technique has proven to be satisfactory for it prevents translocation and loss of diffusible elements in situ and allows X‐ray microanalysis in the SEM.
Partial reversion at the bobbed locus of Drosophila melanogaster
Authors: Terracol Régine , Iturbide Yvonne , Prud'Homme Nicole
Published: February 2012

Summary— In Drosophila melanogaster the tandemly arranged repetitive sequences coding for 18S and 28S rRNA are heterogenous at the level of the spacers between units and insertions that interrupt many 28S rRNA genes. This heterogeneity contrasts with the homogeneity of the regions transcribed into 18S and 28S rRNA. Homogenization and evolution of repetitive genes are usually explained by conversion, amplification events or unequal crossovers. In this paper we studied the change in rDNA patterns associated with partial reversion of bobbed mutations. In most cases, no increase in rDNA gene number, but a new repartition of gene types were found.