bluuebunny/crossref_metadata_2025 huggingface dataset:
Human blood group A‐like determinants as marker of the intracellular pools of glycoproteins in secretory and absorbing of A+ rabbit jejunum
Authors: Bernadac A. , Gorvel J. P. , Feracci H. , Maroux S.
Published: February 2012

Human blood group A antigenicity of glycoproteins is retained on epon‐embedded jejunum sections after glutaraldehyde fixation and osmium treatment. The intracellular location of molecules bearing these determinants was visualized in the four types of epithelial cells of A+ rabbit jejunum sections with immuno‐colloidal gold labeling. The brush border membrane and in particular the glycocalyx of absorbing cells as well as the secretory granules of goblet and Paneth cells were heavily labeled. In enteroendocrine cells, the membrane of secretory granules and not their content was lightly labeled. The differential labeling of secretory or membrane bound glycoproteins is accompanied by different labels of the Golgi complex as expected if labeling of the Golgi saccules was due to the presence of glycoproteins in transit. In all cases the label is primarily concentrated in only half the cisternae on the trans side of the Golgi stacks. In absorbing cells, structures have been revealed in the terminal web that could be related to the brush border membrane and consequently implicated in its biogenesis. The fibrillar material of the glycocalyx appears as highly labeled tangled structures which apparently proceed from densely stained “carrier” vesicles arising from the Golgi apparatus. Vesicles fusing at the lower part of microvilli could result of integration of this material into the lightly labeled vesicles strictly found in the terminal web. These last vesicles could also contain newly synthesized brush border hydrolases.
Distribution of fibrillar centers and silver‐stained components in the nucleolus of human Sertoli cells
Authors: Devictor M. , Hartung M. , Stahl A.
Published: February 2012

The nucleolus of the human Sertoli cell consistently showed three distinct, spontaneously segregated parts: 1. one or two large, silver‐positive fibrillar centers; 2. strands of dense fibrillar component continuous with the dense cords surrounding the fibrillar center. These components were also silver‐positive; 3. a granular, silver‐negative mass. These observations show that in the human Sertoli cell the number of fibrillar centers is far lower than the diploid number of NORs. They also suggest that the fibrillar center might contain several NORs in this cell type.
Isolation of Kurloff cells by Percoll density gradient centrifugation. Protein labeling with 35S‐methionine of these cells
Authors: Landemore G. , Debout C. , Quillec M. , Izard J.
Published: February 2012

Large populations of splenic Kurloff (150–200 × 10(6) Kurloff cells) were obtained from estrogenized guinea pigs by isopycnic centrifugation in a Percoll solution of 1.085 g/ml starting density. The Kurloff cells settled at a buoyant density of about 1.100 g/ml. The purity of these cell suspensions reached 95%, as assessed by phase contrast microscopy and by specific staining. The viability assessed by Trypan blue exclusion test was also about 95%. Moreover, the good transmission electron microscopic appearance of these Kurloff cells and their ability to take up 35S‐methionine in culture confirmed their physiological integrity. By autohistoradiography, this protein labeling was localized between the nucleus and the Kurloff body, and also on the Kurloff body itself. This data reinforces the hypothesis of de novo synthesis of the Kurloff body.
Polyploidy and nuclear phenotypes in salivary glands of the rat
Authors: Vidal B. C.
Published: February 2012

This work describes polyploidy characteristics and nuclear phenotypes with advancing age in salivary glands of the rat. Feulgen‐stained nuclei from squashed salivary gland fragments and nuclei isolated from homogenized salivary glands were analyzed with automatic scanning cytophotometry using Zeiss equipment and a Microdata computer. Polyploidy was demonstrated even in the submaxillary glands of young animals which exhibited 2C and 4C Feulgen‐DNA classes. Glands from 1.5 year‐old rats displayed diploid, tetraploid, and octaploid nuclei. Parotid glands from young animals exhibited Feulgen‐DNA values pertaining to 2C, 4C, and 8C classes, whereas those from aged animals exhibited values pertaining to these classes and to the 16C one. The polyploid nuclei of both glands showed mean absorbances higher than those of diploid nuclei, which is assumed to be due to an increase in the chromatin packing state accompanied by redistribution of the heterochromatin areas. Feulgen‐DNA values ‐nuclear surface and average absorption ratio‐ surface covered by condensed chromatin correlations support these assumptions.
In vitro development of seminiferous tubules from immature rat testis: ultrastructural and morphometric studies
Authors: Gelly J. L. , Delongeas J. L. , Barrat E. , Hatier R. , Grignon G.
Published: February 2012

Seminiferous tubules from 5 day‐old rats maintained in a semi‐solid culture system were examined for 4, 8 or 12 days. Morphometric and ultrastructural studies show a significant increase in the seminiferous tubule diameter and normal Sertoli cell differentiation. All the germinal cells degenerate except the spermatogonia.
Analysis of pore‐rich areas on the nuclear envelope of spermatocytes
Authors: Couve E.
Published: February 2012

Serial sections analysis of spermatocytes of the rodent Phyllotis darwini, reveal a persistent positional relationship between nucleolar material and centrioles. Pore rich areas interposed between these nucleocytoplasmic components, are represented graphically. Centriole replication takes place during the pachytene stage. Pore rich areas could be the result of a preferential aggregational process which insures the exchange of information between nuclear and cytoplasmic regions.
Ag‐NOR" proteins are present when transcription is impaired
Authors: Sanchez‐Pina M. A. , Medina F. J. , Fernandez‐Gomez M. M. , Risueño M. C.
Published: February 2012

The use of nucleolar silver staining in plant cells in conditions of impaired transcription, either natural (quiescent cells) or induced (actinomycin D, ethidium bromide and cordycepin treatments), shows that nucleoli with very low transcription rates, compared with controls, contain similar amounts of argyrophilic proteins, as well as the same structural location. This demonstrates the absence of correlation between silver staining and present transcriptional activity. The effects of conditions used on the structure of nucleolar chromatin support our previous suggestion on the involvement of “Ag‐NOR” proteins in the process of decondensation of NOR chromatin.
Dynamics of antibody transfer from mother to fetus through the yolk‐sac cells in the rat
Authors: Laliberté F. , Mucchielli A. , Laliberté M. F.
Published: February 2012

In rodents, maternal immunoglobulins are transported intact by the yolk‐sac visceral epithelium from mother to fetus. The main purpose of the present paper is to study the dynamics of the uptake and transport of immunoglobulins by the rat yolk‐sac using a new experimental design. The results show the rapid binding of IgG to the cell membrane microvilli since only 30 sec were sufficient for this attachment to occur. The endocytic process also appears to be very fast as localization of IgG in clusters, pits and microvesicles were observed after 5 min of contact between the yolk‐sac and the IgG solution. Moreover, the antibodies were detected in the intracellular spaces within 15 min of incubation.
Quantitative aspects of membrane shifts in rat parathyroid cells initiated by decrease in serum calcium
Authors: Wild P. , Schraner E. M. , Eggenberger E.
Published: February 2012

The secretory activity of parathyroid glands in rats was stimulated by decreasing the serum Ca++ concentration through constant intravenous infusion of EGTA. The morphometric analysis of the nuclear and cytoplasmic volume and of the surface area of the rough endoplasmic reticulum, Golgi complex, secretory granules and plasma membrane revealed a membrane shift from secretory granules and Golgi complex to the plasma membrane within 1 hr of calcium depression. Subsequently, between 1 and 3 hr of calcium depression, the membrane shift was from the plasma membrane to the Golgi complex. It is considered likely that these membrane shifts are related to a rise in release of parathyroid hormone by exocytosis and a subsequent increase in retrieval of plasma membrane by endocytosis—probably through the compartment of coated pits and coated and uncoated vesicles.
Polymorphism of acetylcholinesterase and identification of new molecular forms after sedimentation analysis
Authors: Dreyfus P. A. , Friboulet A. , Tran L. H. , Rieger F.
Published: February 2012

Acetylcholinesterase (AChE) is composed of several distinct molecular forms, which are identified and partly resolved by velocity sedimentation analysis on sucrose gradients. We made the assumption that each AChE form sediments as a peak of activity with a gaussian shape in the continuous sucrose gradient. We experimentally demonstrate that the complex AChE profiles can be decomposed in gaussian distributions of separate molecular entities. We performed a high salt‐detergent extraction of AChE from mouse skeletal muscle and isolated fractions enriched in each particular from. These fractions were then submitted to a second sedimentation, to assess the stability and to further characterize each AChE form. Then, we calculated the statistical significance level of each AChE form and identified up to 9 separate molecular specifies in mouse adult muscle. These forms are the major “4 S”, “6.5 S”, “10 S”, “12 S” and “16 S” and minor molecular active components of AChE. These results suggest complex structural interactions between catalytic and non catalytic subunits of AChE and do not simply fit the tailed asymmetric globular model of AChE with six molecular species.
Endosomes transfer yolk proteins to lysosomes in the vitellogenic oocyte of the trout
Authors: Busson‐Mabillot S.
Published: February 2012

The internalization of the yolk proteins has been investigated by electron microscopy and cytochemistry in the oocyte of the trout which stores up large quantities of yolk. The oocyte evolution has been followed for 18 months in a homogeneous group of animals. Anionic ferritin has been injected during vitellogenesis. The results indicate that as in other oocytes the yolk proteins are absorbed by coated vesicles during vitellogenesis. But unlike most other oocytes the yolk proteins are then transferred via typical endosomes to a conspicuous lysosomal compartment built up very early at the onset of the cytoplasmic differentiation of the oocyte e.i. 10 months earlier. During vitellogenesis yolk progressively accumulates in this lysosomal compartment. Injected anionic ferritin follows the same pathway of internalization. These findings indicate that in this oocyte, the whole yolk cycle presumably represents an adaptation of a general cellular activity, the receptor‐mediated endocytosis, largely amplified, sequenced and spread over several months.
Biochemical and immunological characterization of the microfibrillar ecto‐endoplasmic boundary in the ciliate Isotricha prostoma
Authors: Vigues B. , Metenier G. , Groliere C. A.
Published: February 2012

The cytoplasm of the ciliated protozoan Isotricha prostoma is compartmented by a continuous fibrillar system made up of a double layer of 4 nm‐diameter filaments: the microfibrillar ecto‐endoplasmic boundary (EEB). Isolation of this structure after treatment of the cells in a buffer of low ionic strength in the presence of the detergent Triton X‐100 evidenced connections linking the two filamentous layers. One dimensional electrophoresis on SDS‐polyacrylamide gel of EEB fractions revealed several major proteins with apparent molecular weights between 11 and 23 K. Of these, two neighboring bands of MW22 and 23 K were removed from gels and used as antigens to obtain rabbit antibodies. The antiserum obtained reacted specifically with injected proteins as shown by the technique of immunological detection on nitrocellulose sheets using the peroxidase reaction product. Electron microscopy localization of the antigens with anti‐IgG coupled with colloidal gold showed significant labeling of the EEB within the cortex of Isotricha permeabilized with Triton X‐100. We hope that the 22‐23 K antiserum will prove to be a useful tool for the comparative study of other non‐actin filament systems in Protozoa.
Spatial conformation of glycans and glycoproteins
Authors: Montreuil J.
Published: February 2012

Ten years ago, we anticipated future results by building the Y‐shaped molecular model of a biantennary glycan. Progressively, this structure has been refined and modified thanks to experimental data obtained by using physical methods: X‐ray diffraction, electron spin resonance (EPR), nuclear magnetic resonance (NMR) including two‐dimensional NMR and one‐dimensional 1H‐nuclear Overhauser effect (NOE) experiments, neutron scattering and hard‐sphere exo‐anomeric (HSEA) calculations. So, the concept evolved successively from the Y‐, to the T‐, the bird‐ and the “broken wing”‐conformation, until the demonstration, that these conformers are interconvertible. The bird‐conformation as well as the concept of the mobility of antennae are in a good agreement with the reactivity of lectins, including membrane lectins, by rendering accessible any specific sugar structure, and with the activity of glycosyltransferases by making reachable the substitutable hydroxyl groups even in the case of pentaantennary structures. Along this line, we know now that the tetraantennary glycans adopt an “umbrella conformation” in which the four antennae are disposed parallely to the protein surface and act as protective shields. So could be explained the resistance towards proteases and the weak antigenicity of numerous glycoproteins as well as the peculiar behaviour and resistance of metastatic cancerous cells since it has been recently demonstrated that membrane glycoproteins and fibronectin of this kind of cells are significantly enriched in tri‐ and tetraantennary glycans.
The cytoplasmic matrix of the human spermatozoon: cross‐filaments link the various cell components
Authors: Escalier D.
Published: February 2012

This transmission electron microscopic study demonstrated a periodic arrangement of short cross‐filaments in all the cytoplasmic layers of the human spermatozoon. These filaments were connected with adjacent cellular components (of the same type or not) thus appearing to link the sperm structures to one another. The filaments of the peripheral cytoplasm, those of the perinuclear space and those between the cytoskeletal structures of the flagellum were 3 to 5 nm, 7 to 9 nm and 2 to 4 nm wide respectively. These cross‐links displayed a 14 to 20 nm periodicity and measured 6 to 35 nm in length, depending upon their location. They were associated with electron dense patches on the outer acrosomal membrane. Plasma membrane swelling was associated with a disruption of the cortical filaments on the inside surface of the membrane. This suggested a relation between the normal morphology of the plasmalemma and the cross‐filaments. In altered sperm heads, a particular modification of the perinuclear space was found consisting of an aggregation of the cross‐filaments into repeated bundles. Many of the morphological characteristics of these cross‐filaments could be compared to similar cytoskeletal structures as known in somatic cells. The data of this study suggest that this filamentous network may play an essential role in the maintenance of the topographical relations between the various organelles which may be especially necessary due to the kinematics of this cell.
Micrometric in situ marking apparatus for an object on a microscope grid. Application of observation of serial sections
Authors: Jésior J. C. , Bois J. M.
Published: February 2012

A device for the precise localization (better than 0.5 micron) of an object on a grid has been developed and connected to the translation system of an electron microscope. Applied to biological thin sections, this device enables to easily find and to observe a selected microcrystal projection obtained by the grid sectioning technique. Moreover, low dose observations on ribbons of ultrathin sections can be made because the information is repeated in the successive sections of the ribbon: With the device the position of the selected object is determined at high dose in one section and low dose measurement is made on the following section.
Testosterone‐induced nuclear changes in the rat uterine luminal epithelium. A stereological study
Authors: Cidadão A. J. , David‐Ferreira J. F.
Published: February 2012

Testosterone administration to the spayed immature female rat produces significant changes in the nuclear ultrastructure of the uterine epithelial cells. In general, these changes increase when a higher dose is used. The most intense changes occur in the nucleolus: volume density and volume increase; volume increases in all nucleolar compartments; granular and vacuolar volume density increase; fibrillar center number increase, but a reduction of their individual volume is observed. Condensed chromatin volume density and volume decrease. Nuclear volume, nuclear envelope surface, and nuclear body volume density and frequency remain almost the same. The surface density of the interface between condensed and dispersed chromatin, fibrillar centers and fibrillar component, and condensed chromatin and fibrillar component, show significant variations even with a small dose of testosterone (0.5 mg). Such a dose is unable to change the volume density of condensed chromatin or fibrillar centers. Correlations between our results and biochemical or other morphological data are discussed.
Independent variation in the number of coated pits and of coated vesicles in cultured fibroblasts
Authors: Lubinski J. , Huet C.
Published: February 2012

When tissue culture cells were maintained at 37 degrees C in a serum‐free medium for 4 hr no change in the number of coated pits could be detected using ultrastructural techniques. However, the number of coated vesicles was highly significantly increased, being 179% more than in the control cultures. If the cells were put back into a medium supplemented with 5% calf serum, the number of coated pits was unchanged, but the number of coated vesicles decreased and returned to the control level within a few minutes. The same results were obtained when using ligands such as Low Density Lipoprotein or alpha‐2‐macroglobulin which are known to be internalized via coated structures. It is concluded that coated pits appear and disappear at equal rates and that coated vesicles can accumulate independently. It is suggested that this could be due to the presence of a large reserve of soluble clathrin. This pool would have a low turnover rate because cycloheximide did not block coated vesicle accumulation over the period studied.
Changes in organization and microtubule assembly activity of the centrosome during lymphocyte stimulation
Authors: Schweitzer I. , Brown D. L.
Published: February 2012

Changes in the organization of centrosomes in mouse splenic T lymphocytes stimulated by concanavalin A (con A) were examined by electron microscopy of serial sections. In both resting and stimulated lymphocytes the single centrosome consists of a pair of centrioles, satellite bodies, and pericentriolar material. In resting cell centrosomes the satellite bodies are preferentially associated with, and appear to be attached by short stalks to, one of the centrioles. The satellite bodies are the primary sites of microtubule termination in the resting cell centrosome. During stimulation by con A there is a several‐fold increase in microtubule content. This is correlated with an overall increase in centrosome size, an apparent increase in the size and in the number of satellite bodies, and a redistribution of satellite bodies to occupy a position between the two centrioles. Increased numbers of microtubules are detected terminating on the satellite bodies and in the pericentriolar material of the stimulated cell centrosome. Microtubule assembly from centrosomes in vitro was assessed by electron microscopy using detergent‐permeabilized lymphocytes that had been pretreated to remove endogenous microtubules and supplied with purified bovine brain tubulin. These studies indicate that satellite bodies are major sites of microtubule assembly in both resting and stimulated cell centrosomes and show that the centrosomes of stimulated cells assemble more microtubules in vitro than resting cell centrosomes. This parallels the increase in microtubule content in intact lymphocytes stimulated by con A and suggests that the changes in centrosome organization and microtubule assembly capacity that occur during stimulation are causally related.
Ultrastructure of a temperature‐induced Balbiani ring in Chironomus thummi
Authors: Santa‐Cruz M. C. , Morcillo G. , Diez J. L.
Published: February 2012

A Balbiani ring‐like structure (T‐BR III) is induced at the right telomere on chromosome III by a 35 degrees C heat‐shock. The location of T‐BR III was identified in 3 micron‐semithin sections that were afterwards resectioned to obtain ultrathin sections. These were stained either by uranyl acetate‐lead citrate or PTA. The puff appeared composed of different structures: small compact chromatin bodies, loose chromatin with an apparently fibrillar organization, and granules. The granules, 200‐250 A in diameter, appeared either in linear arrays or in a clustered form. The three components described above were interspersed within the T‐BR without a compartmentalized organization. EDTA preferential ribonucleoprotein staining technique evidenced an EDTA‐positive material within the T‐BR that corresponded to 200‐250 A granules as well as apparently fibrillar structures. However, EDTA did not completely stain some clustered granules. Neither free nor clustered granules were found in T‐BRs formed in the presence of actinomycin D. The significance of the different T‐BR structures in relation to the transcriptional activity of the puff is discussed.
Intra‐individual length heterogeneity of Rana esculenta mitochondrial DNA
Authors: Monnerot M. , Mounolou J. C. , Solignac M.
Published: February 2012

Mitochondrial DNA extracted from Rana esculenta oocytes appears heterogeneous in size. The length of these molecules varies continuously from 18,700 bp to 19,700 bp. Each animal is heteroplasmic and can be characterized by the range of the variation (400‐700 bp) and the extreme sizes of the various molecules it carries. The variable region of the genome has been localized between the coding region and the replication origin area.
Role of carbohydrates in rat leukemia cell‐liver macrophage cell contacts
Authors: Schlepper‐Schäfer J. , Holl N. , Kolb‐Bachofen V. , Friedrich E. , Kolb H.
Published: February 2012

The mechanism by which macrophages recognize tumor cells is still unknown. We have studied interactions between rat liver macrophages and rat L 5222 leukemia cells. These tumor cells, but not normal leukocytes or erythrocytes, adhere to freshly isolated macrophages in vitro. Binding of tumor cells by macrophages can be inhibited by N‐acetyl‐D‐galactosamine, D‐galactose and more potently by glycoproteins with terminal N‐acetyl‐D‐galactosamine or D‐galactose residues. Tumor cell adhesion is calcium‐dependent. The relevant leukemia cell membrane structures which bear terminal beta‐D‐galactosyl or related residues have been determined as trypsin‐ and pronase‐sensitive, and hence may presumably be glycoproteins. The tumor cell receptor on liver macrophages appears to be a lectin with the carbohydrate specificity N‐acetyl‐D‐galactosamine greater than D‐galactose greater than L‐fucose.
Fluid phase and mannose receptor‐mediated uptake of horseradish peroxidase in mouse bone marrow‐derived macrophages. Biochemical and ultrastructural study
Authors: Lang T. , de Chastellier C.
Published: February 2012

A detailed study of horseradish peroxidase (HRP) uptake by in vitro cultured bone marrow‐derived macrophages was undertaken. Biochemical quantitations performed over a wide range of HRP concentrations, in the absence or presence of yeast mannan, showed that these macrophages pinocytose HRP by both fluid phase and mannose receptor‐mediated uptake. The relative contribution of these two types of endocytosis varied with the concentration of enzyme in the extracellular medium. A morphological study at the light and electron microscope levels conducted in parallel confirmed the biochemical data.
Secretory cell activity in the hamster seminal vesicle following castration. A morphometric ultrastructural study
Authors: Mata L. R. , David‐Ferreira J. F.
Published: February 2012

The ultrastructure of hamster seminal vesicle epithelium was studied 7, 14, 21 and 28 days after castration using a stereological approach. The results show that castration promotes epithelial reorganization, mainly characterized by reduced epithelial cell size and number, decreased rough endoplasmic reticulum and Golgi complex, increased lysosomes and lipid droplets, increased apical secretory granule size and number, and increased intracellular secretory products per average epithelial cell. It is concluded that after testosterone withdrawal the secretory activity of hamster seminal vesicle epithelial cells, although reduced, is not abolished, and that exocytosis is relatively more reduced than secretory protein production. We suggest that an extracellular androgen source is responsible for secretory activity not being lost in the epithelial cells of castrated hamster seminal vesicle.
Sialyl glycoprotein distribution on the plasma membrane of ejaculated ram spermatozoa
Authors: Malik Z. , Bartoov B.
Published: February 2012

Localization of sialyl residues on unfixed ejaculated ram sperm membrane using the direct covalent probes of either ferritin hydrazide or latex hydrazide revealed a unique regional distribution on the plasmalemma covering the sperm head only. Three different labelling zones were identified based on the intensity and the nature of the sialyl glycoconjugates: a patchy‐like zone which included the plasma membrane overlaying the post‐nuclear cap and the convex side of the apical body of the acrosome; highly ordered heavily labelled zones including the plasmalemma adjacent to the concave apical body of the acrosome and to the posterior part of the equatorial acrosomal segment; a paucity‐labelling zone which included the plasma membrane underlying the principal acrosomal region and the anterior part of the equatorial acrosomal segment. The possible physiological role of the highly ordered labelled zones is discussed.
Twisted architectures in cell‐free assembled collagen gels: study of collagen substrates used for cultures
Authors: Bouligand Y. , Denefle J. P. , Lechaire J. P. , Maillard M.
Published: February 2012

Reprecipitated fibrils from collagen solutions assemble into aggregates often showing a remarkable twisted structure. We first observed these aggregates in collagen gels prepared to facilitate culture of epithelial cells. We verified that these structures form in the absence of cells and correspond to a process of self‐assembly. Studies on reconstructed fibrils of collagen are generally based on the examination of thin specimens mounted onto coated grids prepared for electron microscopy. We rather applied the classical methods of fixation, embedding and ultramicrotomy, which allowed us to analyze the structure of these aggregates, several microns in diameter. Our gels were prepared from 2.5 mg/ml tropocollagen solutions usually chosen for cell and organ cultures. The time required to obtain twisted architectures, in these aggregates, depends on temperature and the presence of factors such as fetal calf serum proteins. Twist is observed at two different levels of organization. Microfibrils are gathered into twisted bundles which condense into cross‐striated fibrils. These fibrils themselves aggregate and show a mutual twist whose orientation is left‐handed as is the twist observed within each microfibril bundle. Several models of these architectures are presented. Planar twist, cylindrical twist and toroidal twist are described and their relation to the structure of certain liquid crystals is considered. Examples of orthogonal packing also have been observed. These structures obtained in vitro are very close to patterns already described in vivo in numerous collagen matrices.
Renewal of Leydig cells in the neonatal and adult monkey: a radioautographic study
Authors: Fouquet J. P. , Raynaud F.
Published: February 2012

The populations of interstitial cells in the testis of three 2‐3 month old monkeys and of three 5‐6 years old adults (Macaca fascicularis) were analyzed: percentage, mitotic index and labeling index after 3H‐thymidine injection. In the young monkey and the adult well differentiated Leydig cells incorporate the precursor as fibroblast‐like cells. This demonstrates their ability of renewal.
Influence of embedding media on DNA structure in herpes simplex virus type 1
Authors: Puvion‐Dutilleul F. , Pichard E. , Leduc E. H.
Published: February 2012

The organization of encapsidated herpes simplex viral DNA in situ was examined by use of the osmium‐amine stain specific for DNA. After either formaldehyde or glutaraldehyde fixation the DNA is packaged in a compact toroid without inner structure with Epon or GMA embedment but revealed a complex inner structure with Lowicryl K4M embedment. In the latter there was an inner cylindrical core, 50 × 80 nm, around which were apposed one or more thick filaments of 5‐8 nm diameter. Thinner DNA filaments of 3‐4 nm diameter form a cage of loose coils around the core with an intervening space of approximately 15 nm. Lowicryl embedding may be considered as a tool to investigate the packaging of viral DNA in virions.
Subunit arrangement of Escherichia coli F1‐ATPase studied by scanning transmission electron microscopy
Authors: Curgy J. J. , Bonnet N. , Colliex C. , Iftode F. , Issartel J. P. , Tence M. , Satre M. , Vignais P. V.
Published: February 2012

The shape and the arrangement of subunits in Escherichia coli F1‐ATPase (ECF1) lacking the delta subunit have been explored with a high performance scanning transmission electron microscope. In tilting experiments, the ECF1 molecule appeared as a flat cylinder whose width (approx. 120 A) was about twice its height. The symmetry of front view projections of ECF1 has been investigated by computer analysis. In a population taken at random from the data bank, one third of the particles showed five‐fold radial symmetry components, one third six‐fold radial symmetry components and the last third no typical symmetry. The six‐fold radial symmetry was consistent with a hexagonal arrangement of six large peripheric masses, which probably correspond to the three alpha and the three beta subunits of ECF1. The five‐fold radial symmetry was tentatively explained by a fusion of two juxtaposed peripheric subunits. Lateral projections showed a zig‐zag organization of the large masses, suggesting that the large alpha and beta subunits are located on two levels, with some degree of intercalation between the subunits of the two levels.
Enzymatic response to glucocorticoids of the chick intestinal endoderm associated with various mesenchymal cell types
Authors: Lacroix B. , Kedinger M. , Simon‐Assmann P. , Haffen K.
Published: February 2012

The aim of the present study was to test the morphological and functional maturation of recombinants composed of chick intestinal endoderms associated to different mesenchymal supports and their enzymatic response to glucocorticoids. For this purpose 5.5‐day chick embryonic intestinal endoderm has been associated to 14‐day fetal rat gut mesenchyme, to rat intestinal fibroblasts (6‐day neonatal rat intramucosal fibroblasts) or to rat control fibroblasts, originating from 20‐day fetal rat skin and lung and from 6‐day neonatal rat intestinal muscle. The recombinants were grown as intracoelomic grafts either for 12 days or for 10 days plus 2 days in organ culture in the presence of dexamethasone. The data show that heterospecific recombinants achieve subnormal morphogenesis and enzymatic maturation. The organ culture experiments further reveal that sucrase activity is insensitive to dexamethasone in all types of recombinants whereas, alkaline phosphatase is highly stimulated over the levels present in the intestine developed in situ whatever the stromal support, except when this support is provided by rat gut mesenchyme. These results support the view that in the intestine the hormonal response is mediated by epithelial‐mesenchymal interactions.
Comparative aspects of circulating erythrocytes in the trophic and reproductive phases of European eel. Ultrastructure and cytochemistry
Authors: Barni S. , Gerzeli G.
Published: February 2012

Different cytochemical and morphological parameters were used to compare the functional patterns of the erythrocytes in the transition from the trophic (yellow eel) to the reproductive (silver eel) phases of European eel (Anguilla anguilla L.). Data on the nucleus were obtained by microdensitometric (Feulgen reaction) and cytofluoremetric (propidium iodide and Hoechst 33342 fluorochromization) evaluations of DNA staining intensity. In the silver eel, chromatin condensation was more heterogeneous and chromatin also appeared to be looser. The fluorochromes provided information on the different degrees of organization of the chromatin filament, with regard to both primary and higher order DNA structure. In the silver eel, the fine structure showed a higher number of erythrocytes with large euchromatinic areas and the heterochromatinic component was occasionally present in scattered clumps. As for the cytoplasm, in the silver eel a positive correlation could be established between the presence of intact cytoplasmic organelles (mitochondria, Golgi complexes, ribosomes, etc.) and the high fluorescence intensity. This aspect is revealed through the detection of definite membrane components such as glycoconjugates and primary amino groups (PAS reaction and fluorescamine staining, respectively). All these findings allowed us to establish that the transition from the trophic to the premigratory reproductive phase is marked by a higher heterogeneity of the erythrocytes, due to different cell maturation levels. This fact can be ascribed to different metabolic requirements as well as to a more intense erythropoiesis, typical of the most active phase of the eel's life.
In situ ultrastructural localization of sugar‐binding sites in lizard granulosa cell nuclei
Authors: Hubert J. , Seve A. P. , Bouvier D. , Masson C. , Bouteille M. , Monsigny M.
Published: February 2012

Mannose‐binding sites were detected at the ultrastructural level in nuclei of lizard granulosa cells in situ by means of mannosylated ferritin. When ultrathin sections of material embedded in glycol methacrylate were incubated with mannosylated ferritin, a strong labelling was observed over nucleoli, chromatin and the external leaflet of the nuclear envelope, but no labelling was detected in the perinuclear space except for nuclear pores. This labelling was clearly inhibited when sections were incubated in a solution containing both mannosylated ferritin and a sugar‐related neoglycoprotein (mannosylated serum albumin). These ultrastructural data supporting the existence of nuclear lectin‐like components in reptilian cells are in agreement with our previous findings about such components in nuclei isolated from mammalian cells. Owing to the unique organization of the granular component of the nucleoli in specialized granulosa cells, we are able to show that some of the mannose‐binding sites are associated with ribosomal precursors.
ATP‐dependent calcium transport in rat parotid microsomes. I. Localization, properties, Ca2+‐ATPase activity and phosphoenzyme formation
Authors: Bonis D. , Chambaut‐Guérin A. M. , Rossignol B.
Published: February 2012

ATP‐dependent Ca2+ transport was studied in rat parotid microsomes; the activity appears to be associated with rough endoplasmic reticulum vesicles, as indicated by marker distribution in subcellular fractions and by electron microscopic observations. Purified rough microsomes exhibit an ATP‐dependent Ca2+ accumulation and a Ca2+‐dependent ATPase activity; these activities are similarly stimulated by K+ and display an apparent Km for free calcium of 0.6‐0.7 microM. A phosphoprotein, with a molecular weight of about 110,000, was detected after short incubation with [gamma 32P] ATP and CaCl2; it is suggested that this compound represents a phosphorylated intermediate form of the Ca2+‐ATPase.
Chondrogenesis in mouse limb bud in vitro. I. Effect of the ectoderm
Authors: Desbiens X. , Carette F. , Meunier L. , Bart A.
Published: February 2012

The role of the ectoderm in the chondrogenesis of mouse limb bud mesoderm was investigated in vitro at several developmental stages by analysis of the evolution of DNA content, the accumulation of sulfated glycosaminoglycans and histochemical procedures. Young limb buds or the undifferentiated apex of older buds (stages 17 and 19 of Theiler's table) from which the ectoderm had been removed with trypsin treatment initiated a large chondrogenesis but not morphogenesis. When the ectoderm was present, these limb buds showed a polarized proximal to distal outgrowth and differentiated skeletal primordia. Mesodermal cells of stage 20 limb bud apex were able to differentiate autopodial skeletons with or without the presence of the ectoderm: cartilaginous areas of the limb skeleton seem determined at this developmental stage. These results, which show the importance of the ectoderm in limb bud morphogenesis, are compared with results obtained using other methods with mouse or bird buds.
Cell determinants of vasopressin‐stimulated water flow
Authors: Carvounis C. P.
Published: February 2012

I present a technique that permits evaluation of the permeability to water of the luminal membrane of the toad urinary bladder, independently of constraints to water flow imposed by the remainder of the tissue. This technique essentially depends on fixation of the luminal membrane with 1% glutaraldehyde for 5 min, and subsequent elimination of cytosolic constraints by decreasing the tonicity of the serosal bath to 1/2 normal strength. The increased hydraulic conductivity found with serosal hypotonicity is readily reversible, as the bladder returns to an isotonic serosal bath. By evaluating water flow in luminally fixed bladders during bathing in normal and hypotonic bath, one may identify the relative contribution of the luminal membrane and the “cytosol” on water flow. Using this technique, I found that the effect of the prostaglandin inhibitor Naproxen to increase vasopressin‐stimulated water flow is due to increased luminal membrane permeability. The effect of histidine to increase vasopressin‐stimulated water flow, however, depends on increased permeability of both the luminal membrane as well as the underlying structures. The action of serosal hypertonicity to induce water flow is due to an increased luminal permeability. However, serosal hypertonicity decreases “cytosolic” permeability, so that its overall function is a composite effect of its action at the luminal membrane and the “cytosolic” level.
Action of photoreactive analogs of vasopressin in toad bladder
Authors: Eggena P. , Ma C. L. , Fahrenholz F. , Buku A. , Schwartz I. L.
Published: February 2012

A series of analogs of vasopressin with photoreactive groups in positions 1, 2, 3, 4, 8 or 9 of the nonapeptide sequence have been studied for their effects on water and urea permeability of the isolated toad urinary bladder. Compounds with photoreactive groups in positions 3 or 8 bound covalently to receptors as judged by a persistent increase in water and urea permeability following UV irradiation, prevention of photolabeling by incubation in the presence of vasopressin, and a persistent increase in membrane‐bound adenylate cyclase activity. Some analogs were inactive in the dark, but became active and bound covalently to receptors during photolysis. Other analogs were inhibitors or agonists in the dark, but did not bind to receptors following UV irradiation. Time course studies with photolabelled bladders showed a stable urea flux for 4 hr in the absence of osmotic water flow. However, in the presence of water flow urea flux was initially enhanced (solvent drag effect) and later retarded (diminished urea permeability). Binding of photoaffinity analogs to receptors was not diminished with acidification of the serosal bathing medium, lowering of the bath temperature from 21 degrees C to 4 degrees C or with addition of prostaglandin E1. However, the capacity of photoreactive analogs to effect an increase in transmural water flux, once the analog was bound covalently to receptors, was markedly diminished under these conditions.
Possible role of Ca2+‐binding sites in the regulation of Na+ transport in toad urinary bladder
Authors: Hardy M. A.
Published: February 2012

La3+ was used to assess the role of membrane‐bound Ca2+ in the regulation of basal and antidiuretic hormone (ADH)‐induced Na+ transport by the isolated toad urinary bladder. Na+ transport was monitored by means of a short‐circuit current (Isc) device. Mucosal La3+ (0.5‐5 mM) increased Isc, while serosal La3+ (5 mM) produced a biphasic response (stimulation followed by inhibition). The stimulatory effects of La3+ were additive when present on both sides and were suppressed by mucosal amiloride or serosal ouabain. The action of mucosal La+ was reversible but the inhibition produced by serosal La3+ was not. In the presence of serosal La3+ the natriferic effect of ADH was abolished, but Theophylline, dibutyryl‐cAMP, Amphotericin B, mucosal La3+, mucosal low pH, and phospho(enol) pyruvate, were able to increase Isc. These results suggest that Ca2+ binding sites in apical and basolateral membranes may play a key role in the modulation of both basal and ADH‐induced Na+ transport. Serosal La3+ apparently inactivates the hormone‐receptor interaction and/or the link between the ADH‐receptor complex and the activation of adenylate cyclase, but does not interfere with the operation of the Na+ “pump”, the basal activity of adenylate cyclase or any of the intracellular events that mediate the effect of ADH on Na+ transport.
Active Ca2+ transport in plasma membranes of branchial epithelium of the North‐American eel, Anguilla rostrata LeSueur
Authors: Flik G. , Bonga S. E. Wendelaar , Fenwick J. C.
Published: February 2012

A branchial epithelial membrane fraction, more than 20‐fold enriched in Na+/K+‐ATPase activity when compared with the crude homogenate of the tissue, was obtained from adult freshwater American eels. In a membrane vesicle preparation that consisted of 33% inside‐out, 23% right‐side‐out and 44% leaky vesicles, the accumulation of 45Ca2+ was stimulated by ATP, but not by ADP. Accumulation of 45Ca2+ was prevented when vesicles were pretreated with detergent or the Ca2+ ionophore A23187; Ca2+ efflux was observed when the ionophore was added to actively 45Ca2+‐loading vesicles. Oxalate did not affect Ca2+ accumulation in these vesicles. Kinetic analysis of the Ca2+‐transport process by an Eadie‐Hofstee plot revealed that the process is homogeneous; its kinetic parameters are a K0.5 for Ca2+ of 0.053 microM and a Vmax of 2.25 nmol Ca2+/min.mg protein (at 37 degrees C). The calmodulin dependency of this Ca2+ transporting process was shown by the inhibitory action of calmodulin antagonists and by the stimulatory effect of calmodulin repletion after EGTA treatment of the membranes. We conclude that an ATP‐energized Ca2+ pump is present in the plasma membranes of branchial epithelium, that resembles the Ca2+ pumps of e.g. mammalian intestinal or renal plasma membranes, and propose its involvement in branchial Ca2+‐uptake from the water.
Attachment of DNA to nucleolar and nuclear skeletal structures as visualized by Kleinschmidt molecular spreading
Authors: Bureau J. , Hubert J. , Bouteille M.
Published: February 2012

Co‐isolated residual nuclear shells and residual nucleoli from membrane‐depleted rat liver nuclei were spread according to Kleinschmidt's method. Comparison of the spread residual structures isolated from nuclear shells and spread pore complex‐lamina isolated from nuclear envelopes showed that these residual structures are morphologically identical. Furthermore, our nuclear shell isolation procedure allowed visualization of DNA strands bound to a granular component of the lamina. The fragmentation of nuclear shells allowed us to obtain well‐spread nucleolar remnants, in which we observed DNA strands anchored on a residual nucleolar network attached to the lamina. The different molecular features revealed by the spreading of residual nucleolar structures suggest that both non‐transcribing nucleolar DNA and active ribosomal genes are linked to the nucleolar network. Although the exact nature of this network remains to be defined, the results of the present study strongly suggest that the DNA molecules of the chromosomes bearing ribosomal genes have many sites of attachment to a non‐chromatin nucleolar network which can be referred to as a nucleolar skeletal complex.
Formation and maturation of axo‐glandular synapses and concomitant changes in the target cells of the rat subcommissural organ
Authors: Marcinkiewicz M. , Bouchaud C.
Published: February 2012

Synapse formation and maturation in the subcommissural organ (SCO) of Wistar rats were studied from birth to the end of the first month. Modifications of the secretory ependyma were analyzed over the same period. On the 1st postnatal day, the large varicosities in contact with the SCO ependymocytes appeared immature (absence or low density of vesicular population, no synaptic membrane differentiation). The synaptic contacts were formed from the 3rd postnatal day, near the glandular cell nuclei (0.1 micron distance); progressively, the content of the axonal boutons and the pre‐ and post‐synaptic specializations became similar to those of adults. From the 21st day on, the axo‐glandular innervation was considered analogous to that in the adult. Using immunocytochemistry, it was found that the increase in the serotonin‐immunoreactive fiber density in the whole organ was time‐dependent. Light and electron microscopy demonstrated changes in the morphology of SCO ependymocytes during the first postnatal weeks, notably in the endoplasmic reticulum and content ot apical protrusions. On postnatal day 14, two types of ependymal cells, neonatal‐like and adult‐like, coexisted. The evolution of SCO ependymocytes coincided with the progressive onset and maturation of axo‐glandular innervation taking place after birth.
Ultrastructure of cell fusion and premature chromosome condensation (PCC) of thymocyte nuclei in metaphase II mouse oocytes
Authors: Szollosi D. , Czolowska R. , Soltynska M. S. , Tarkowski A. K.
Published: February 2012

Following PEG (polyethylene glycol) treatment of ovulated metaphase II mouse oocytes aggregated with thymocytes, fusion of cell membranes occurs. Prerequisites for cell fusion are: close apposition of lectin‐agglutinated (phytohemagglutinin‐treated) membranes of both cells, formation of firm punctual adhesion sites, and expansion of adhesion sites over a certain area. Establishment of the firm cell‐cell contact is associated with development of actin‐like filaments along both of the adhering plasma membranes. Membrane fusion occurs at single or multiple sites, and is followed by internalization of thymocyte‐oocyte membrane complexes decorated with actin filaments into the hybrid cell cytoplasm. A filamentous actin layer forms also along the inner surface of newly formed hybrid oocyte‐thymocyte plasma membrane. Thymocyte nuclei incorporated into oocyte cytoplasm undergo nuclear envelope breakdown and premature chromosome condensation (PCC) leading, eventually, to formation of single chromatids complete with kinetochores. Concomitantly with chromatin condensation an extensive polymerization of microtubules starts in the center of the chromatin mass which leads to the formation of an apparently non‐functional spindle‐like structure.