bluuebunny/crossref_metadata_2025 huggingface dataset:
A marker of acid‐secreting membrane movement in rat gastric parietal cells
Authors: Mercier Frédéric , Reggio Hubert , DeVilliers Ginette , Bataille Dominique , Mangeat Paul
Published: February 2012

A monoclonal antibody (mab 146.14) marker of the movement of acid‐secreting membranes in rat gastric partial cells has been produced and characterized. Mab 146.14 recognized a 95‐kD major component of a purified membrane fraction of rat gastric mucosa, the protein composition of which was similar to that of well characterized porcine H+‐K+ ATPase‐enriched membranes, and that presented the characteristic shift of density depending on whether it was purified from resting or stimulated tissues. Further biochemical analysis characterized the antigen as a membranous protein that might be in its native form, part of a higher multimolecular complex.

Immunocytochemical localization of the antigen demonstrated that only membranes related to acid secretion in parietal cells expressed the 95‐kD antigen. In resting conditions, the 95‐kD antigen was diffusely distributed in the cell cytoplasm associated with inactive tubulovesicles. In stimulated cells, by contrast, all the antigen was recovered associated with secretory active microvilli formed by the apical insertion of the previously resting internal tubulovesicles. In conclusion, the 95‐kD antigen, presumably a part of the rat gastric proton pump, is a marker of acid‐secreting membranes in rat parietal cells. The translocation of antigen and membranes, observed by both light and electron microscopy supports the fusion model of membrane insertion from a cytoplasmic storage pool to the apical surface upon stimulation of acid secretion.
Heterogeneous distribution of phospholipids in membranes along the secretory pathway in pancreatic B‐cells
Authors: Coulombe Pierre A. , Bendayan Moise
Published: February 2012

The membrane content in phospholipids along the secretory pathway in rat pancreatic B‐cells was studied in situ by high‐resolution cytochemistry, applying the recently introduced phospholipase A2‐gold technique. The gold particles were mostly associated with cell membranes, and the various types of membranes were labeled to a different extent. Quantitation of the labeling over these membranes revealed a heterogenous distribution of the labeling across the secretory pathway. This heretogeneity occured mainly as a progressive, decreasing gradient in the first half of this pathway, between the rough endoplasmic reticulum and the mi‐cisternae of the Golgi apparatus. The labeling density remained at a lower level in the trans‐most Golgi cisternae and immature secretory granule membranes, to increase in the mature secretory granule membrane, where it reached the value found in the plasma membrane. These results provide evidence that the functional heterogeneity existing across the membrane forming the secretory pathway is parallelled by substantial changes in their phospholipid content.
Posttranslational modifications of tubulin in cultured mouse brain neurons and astroglia
Authors: Eddé Bernard , Denoulet Philippe , de Néchaud Béatrice , Koulakoff Annette , Berwald‐Netter Yoheved , Gros François
Published: February 2012

Posttranslational modifications of tubulin were analyzed in mouse brain neurons and glia developing in culture. Purified tubulin was resolved by isoelectric focusing. After 3 weeks of culture neurons were shown to express a high degree of tubulin heterogeneity (8 α and 10 β isoforms), similar to that found in the brain at the same developmental stage. Astroglial tubulin exhibits a less complex pattern consisting of 4 α and 4 β isoforms.

After incubation of neuronal and glial cells with 3H‐acetate in the presence of cycloheximide, a major posttranslational label was found associated with α‐tubulin and a minor one with β‐tubulin. The acetate‐labeled isotubulins of neurons were resolved by isoelectric focusing into as many as 6 α and 7 β isoforms, while those of astroglia were resolved into only 2 α and 2 β isoforms. The same α isoforms were also shown to react with a monoclonal antibody recognizing selectively the acetylated forms(s) of α‐tubulin [38]. Whether acetate‐labeling of α‐tubulin in these cells corresponds to the acetylation of Lys40, as reported for Chlamydomonas reinhardtii [30, 33, 34], is discussed according to very recent data obtained by protein sequence analysis.

Tubulin phosphorylation was analyzed by incubation of cell cultures with 32PO4. No phophorylation of α‐tubulin isoforms was detected. A single β‐tubulin isoform (β′2), expressed only in neurons, was found to be phosphorylated. This isoform is similar to that previously identified in differentiated mouse neuroblastoma cells [16, 18, 21].
Development of the large nucleolus in the oocytes of the copepod Acanthocyclops vernalis: an electron microscope study
Authors: Standiford David M. , Gregg Thomas G.
Published: February 2012

A central feature of oogenesis in the copepod crustacean, Acanthocyclops vernalis, is the development of a very large nucleolus in the oocytes. This nucleolus appears to be the only source of rRNA for the oocyte, as no helper cells are present. Previous work has suggested that ribosomal DNA sequences other than those found at the morphological nucleolar organizers are participating in the elaboration of this nucleolus. It has been hypothesized that chromatin diminution, which occurs during early embryonic development, may involve the loss of these rDNA sequences, which are needed only for the production of ribosomes during oogenesis. The present study examines the development of the large oocyte nucleolus at the electron microscopic level. Nucleologenesis in A. vernalis was found to proceed through 5 stages. During the first 3 stages nucleolar morphology resembled that described in other organisms. In the last 2, however, nucleolar morphology changed radically and the nucleolus was seen to increase greatly in size while breaking up into multiple subunits. The subunits initially resemble active nucleoli, although in the last stage, synthesis appears to stop, as the nucleolus was found to consist only of dense areas containing ribosome‐like particles. These observations are consistent with the hypothesis that diminuted DNA contains ribosomal RNA genes.
Induction of rat jejunal epithelial cell expression of sucrase‐isomaltase by glucocorticoids in priimary cell culture and in vivo
Authors: Yeh Kwo‐Yih , Yeh Mary , Holt Peter R.
Published: February 2012

The cell cycle phase that mediates the induction of intestinal sucrase‐isomaltase (SI) expression by glucocorticoids was investigated by measuring migration rates of 3H‐DNA‐labeled and of SI‐containing epithelial cells by autoradiography and indirect immunofluorescent staining after simultaneous administration of [3H]thymidine and cortisone to 12‐d‐old rat pups. By 24 and 48 h, lead 3H‐DNA‐labeled cells had migrated 7.8 and 12.4 cell positions higher on the villus than lead cells expressing SI. Cell migration rates from 12 to 24 h and 24 to 48 h were 0.68 and 0.97 cell position/h. Thus, commitment to SI expression occurred in cells 11.5–12.8 h after the S phase, which is calculated to be in the G1 phase.

To determine whether committed cells need to replicate to express SI, cell differentiation was examined in primary cultures of crypt cells originating from corticosterone‐treated rats. About two‐thirds of cultured cells were retarded in the S phase after plating, as judged by no increase of DNA labeling indices, no change in epithelial cell number, and the absence of mitosis (<0.01%). The proportion of cells expressing SI increased from 0 to 6–8% between 12 and 24 h, and reached 48% 48 h after plating on collagen‐coated dishes. SI expression did not occur in cells plated on glass or plastic surfaces. Pulse labeling with [35SH]methionine confirmed that de novo synthesis of SI occurred in cell cultures. Thus, additional cell cycling of committed cells occuring in vivo is not obligatory for the expression of SI.
Ultrastructural immunocytochemical localization of fibronectin deposition during corneal endothelial wound repair. Evidence for cytoskeletal involvement
Authors: Sabet Minu D. , Gordon Sheldon R.
Published: February 2012

The distribution of the extracellular matrix (ECM) protein, fibronectin (FN), has been examined ultrastructurally in noninjured and injured rat corneal endothelium in vivo and in vitro by immunoperoxidase cytochemistry. In noninjured endothelia, FN was observed within the rough endoplasmic reticulum (RER) cisternae but not along the cell‐Descemet's membrane (DM) interface. Twenty‐four and 48 h after a circular freeze injury, immunoperoxidase reaction product was detected at the cell‐DM interface as well as within cytoplasmic vesicles and intercellular spaces. By 1 and 2 wk post‐injury, a line of reaction product could still be demonstrated at the cell‐DM interface and evidence for newly deposited basement membrane material was observed in this region. In order to understand whether fibronectin deposition during wound repair was dependent on cytoskeletal influences, organ culture experiments were performed in which the media was supplemented with either 10−8M colchicine or 2.5 × 10−3M cytochalasin B. Without inhibitors, injured corneas cultured for 24 h had FN deposition at the cell‐DM interface similar to the in vivo results. Corneas cultured in the presence of cytochalasin B also showed FN deposition at the cell‐DM interface.

However, when injured endothelia were cultured in the presence of colchicine, no reaction product was observed at the cell‐DM interface, although it could be detected intracellularly within RER. Incubating the tissues in the presence of puromycin abolished all extracellular and intracellular staining. These results indicate that during wound repair, corneal endothelial cells produce fibronectin and deposit it upon Descemet's membrane by a mechanism that may be mediated by microtubules.
Nucleologenesis: use of non‐isotopic in situ hybridization and immunocytochemistry to compare the localization of rDNA and nucleolar proteins during mitosis
Authors: Jiménez‐García Luis F. , Rothblum Lawrence I. , Busch Harris , Ochs Robert L.
Published: February 2012

Using in situ hybridization and immunocytochemistry during interphase and mitosis, we have compared the distribution of ribosomal DNA (rDNA) to that of the nucleolar proteins fibrillarin and RNA polymerase I. During interphase, nucleolar proteins were localized at sites throughout the nucleolus while the bulk of rDNA was localized in a single restricted nucleolar area. During metaphase and anaphase, all six NORs were detected by in situ hybridization, Ag‐staining, or by the immunolocalization of RNA polymerase I. During telophase, rDNA and RNA polymerase I were found in a distinct subset of the prenucleolar bodies (PNBs) which obviously must contain the nucleolar organizers. Other numerous PNBs are smaller in size and do not contain detectable amounts of rDNA or RNA polymerase I. Therefore, reconstruction of the nucleolus originates in telophase‐specific domains which contain both rDNA and RNA polymerase I.
Keratinization of rat vaginal epithelium. III. Effect of estradiol on keratinization
Authors: Gupta P.D. , Vijayasaradhi S. , Reddy A.G.
Published: February 2012

Effect of estradiol‐17β on rat vaginal epithelial cells (VEC) was studied by transmission electron microscopy. During the normal estrous cycle in adult rats keratinization and exfoliation of VEC was observed only in the estrus phase, though tonofilament bundles were seen distributed in the cytoplasm of the VEC during the proestrus and, to a lesser extent, in the diestrus phase. Electron microscope data on adult ovariectomised, immature and neonatally estradiol‐primed rats demonstrated keratinization of the VEC within 24 h of injection. Proliferative activity was observed in the basal cells in primed animals. The basal cells remained cuboidal whereas cells of intermediate and luminal layers became flattened. These cells showed irregular profiles and, therefore, enhanced numbers of cellular junctions (desmosomes) were observed compared to control groups. Many tonofilament bundles were seen in the cytoplasm of cells belonging to intermediate and luminal layers in primed animals.
In vivo auxin binding sites in the pith meristem and its derivatives in tomato apical bud
Authors: Driss‐Ecole Dominique , Perbal Gérald
Published: February 2012

Tritiated auxin applied to the apical bud of tomato plantlets was covalently fixed to adjacent protein after treatment by glutaraldehyde. Localization of labelling was studied by high resolution autoradiography at the cellular membrane level in the cells of the pith meristem and in its derivatives (parenchymal cells of the pith). The quantity of binding sites was very low in the meristematic cells. There was only one small peak of affinity for auxin at the plasmalemma lining the longitudinal walls of these cells. During the following cell differentiation an increase occurred in the number of binding sites. Along the longitudinal and proximal walls of parenchymal cells, two peaks of affinity were obtained. The first peak was located on the plasmalemma and the other on the endoplasmic reticulum. For the distal wall only one peak was located on the internal surface of the plasmalemma. The high affinity of tritiated auxin across the plasmalemma of the distal region of the pith cells could be related to polar auxin transport. Auxin affinity was also observed for the external surface of the tonoplast in pith cells.
Traction fibres in chromosome movement: the pros and cons
Authors: Fuge Harald
Published: February 2012

The current discussion on the mechanism of chromosome movement in anaphase is dominated by a new hypothesis assuming active movement of the chromosome along stationary kinetochore microtubules. The aim of this article is to call attention to several older as well as to more recent observations on spindle structural dynamics and microtubule rearrangements which, in the author's opinion, cannot be explained by the new model. In fact, the observations seem to be more consistent with the classical concept of a traction fibre. For this reason, these observations should not be disregarded.
Three‐dimensional reconstructions of nucleolus‐organizing regions in PHA‐stimulated human lymphocytes
Authors: Hozák Pavel , Novák Jiřít. , Smetana Karel
Published: February 2012

Ultrastructure and three‐dimensional distribution of nucleolus‐organizing regions have been studied on ultrathin serial sections of PHA‐stimulated human lymphocytes. During the 48 hr of activation the size of fibrillar centers (FCs) decreased from 0.6–0.9 μm to 0.2–0.3 μm and the number of FCs increased rapidly from one to 75–107 per cell. The number of fibrillar complexes (i.e. associations of a different number of FCs connected by the dense fibrillar component) also increased but did not reach the maximum number of nucleolar organizers presented here. Three‐dimensional computer reconstructions of fibrillar complexes showed that lymphocyte activation was accompanied by early (2–4 hr) changes in the shape of the primary fibrillar center. Invagination of the dense fibrillar component on its surface occured and division into two or more smaller FCs followed. Gradually, the typical structure of the nucleolus with several fibrillar complexes and many FCs was formed. These results confirm the hypothesis of fibrillar complex‐nucleolar organizer correlation published recently.
Growth and characterization of isolated bovine tracheal gland cells in culture. Influence of a reconstituted basement membrane matrix
Authors: Benali Rachid , Dupuit Florence , Jacquot Jacky , Fuchey Claudette , Hinnrasky Jocelyne , Ploton Dominique , Puchelle Edith
Published: February 2012

We describe a method for establishing the culture of bovine tracheal submucosal gland (BTG) cells, in which we have also examined the influence of a reconstituted basement membrane matrix derived from the Engelbreth‐Holm‐Swarm tumor (EHS) on the growth and morphological differentiation of these cells. BTG cells have been isolated by tissue enzymatic digestion using trypsin, deoxyribonuclease I, elastase, hyaluronidase and EGTA for 1 hr at 37°C. Afterwards, cells and tissue were collected by centrifugation and were incubated for 15 min with 15% newborn calf serum to inactivate the proteolytic enzymes. Enzymatic digestion using only trypsin, centrifugation and inactivation steps were repeated three times. Using this protocol, we obtained 15±4 (×106) cells per g of tracheal submucosa with 72 ± 2% (n = 5) cell viability. On microscopic observation, isolated cells were mainly composed of serous type glandular cells. Cells were cultured in a 1: 1 medium of Dulbecco's Modified Eagle's/Ham's F12 supplemented with 10% fetal calf serum and subcultured in either plastic flasks or flasks coated with EHS matrix. On the plastic, the BTG cells exhibited at confluency an epithelloid appearance. They stained positively with the immunofluorescent anticytokeratin antibody and contained PAS‐staining granules. By electron microscopy, lactoferrin, a protein marker specific to the serous cells, was demonstrated immunocytochemically in small secretory vesicles. BTG cells cultured on EHS matrix revealed a significantly increased growth in comparison to those cultured on plastic. In post‐confluent culture of BTG cells on EHS matrix, we observed numerous dome‐like structures formed by differentiated cells which were joined together around luminal spaces.
Dynamics of pancreatic tissue cells in the rat exposed to long‐term caerulein treatment. 2. Comparative analysis of the various cell types and their growth
Authors: Morisset Jean , Grondin Gilles
Published: February 2012

This study was undertaken to evaluate the effects of caerulein, a CCK analog, on the different cell populations of the pancreatic tissue and their respective turnover. Rats received saline or caerulein subcutaneously and 3H‐thymidine intraperitoneally three times a day for 4 days. They were sacrificed immediately after termination of treatment and 2,15 and 50 days later. With age, the proportion of acinar cells decreased significantly whereas those of the ductal and interstitial cells increased. Although caerulein induced preferential acinar cell growth, it did not modify the proportion of this cell population with regard to the other cells. However, at specifc times after termination of treatment, caerulein induced modifications in the ductal, endothelial and interstitial cell populations. The growth promoting effect of caerulein was evident from the specific increases in total DNA content and DNA synthesis. The labeling indices indicate that all cell populations except the endocrine system were stimulated to grow in response to caerulein. Furthermore, all new cells remained for at least 50 days after termination of treatment. These data indicate that caerulein induced uniform growth of the pancreatic tissue during intensive treatment. The normal growth rate of these stimulated cells was, however, arrested for the following 50 days while that of the control group cell population proceeded normally.
Dual internalization pathway for lutropin and choriogonadotropin in porcine Leydig cells in primary culture
Authors: Salesse Roland , Dacheux Françoise , Genty Nicole , Garnier Jean
Published: February 2012

The role of the high affinity receptor in the internalization of porcine lutropin (pLH) and human choriogonadotropin (hCG) by porcine Leydig cells in primary culture during short‐term stimulation by the two hormones was investigated. The fate of the hormones was followed either by electron microscopy (with colloidal gold‐labeled hormones) or by measurement of the cellular distribution of [125I]pLH and [125I]hCG.

With both techniques, the internalization of pLH was found to be one order of magnitude greater than hCG, though the recycling rate of the high affinity receptors was the same with both hormones. However, when the cell surface was progressively depleted of its high affinity receptors by preincubation with increasing doses of hCG or pLH, the internalization of [125I]pLH remained high and largely independent of the number of high affinity receptors still available on the cell surface, while that of [125I]hCG was found to be proportional to this number. The endocytosis of [125I]pLH could only be inhibited by the simultaneous presence of micromolar concentrations of unlabeled pLH, hCG or α or β subunits of ovine LH (oLH). The intact α‐hCG subunit and the deglycosylated α‐oLH subunit were less potent, while β‐hCG and deglycosylated β‐oLH had no significant effect.

These results could be explained by the existence of a “carrier” or “scavenger” receptor for LH, but with a low affinity (≌3·106 M−1) and present in excess on the cell surface as compared to the high affinity receptor. The possible physiological significance of this receptor is discussed.
Modification of cell evagination and cell differentiation in quail oviduct hyperstimulated by progesterone
Authors: Perche Olivier , Lainé Marie‐Christine , Pageaux Jean‐François , Laugier Christian , Sandoz Daniel
Published: February 2012

Quail oviduct development is controlled by sex steroid hormones. Estrogens (E) induce cell proliferation, formation of tubular glands by epithelial cell evagination and cell differentiation. Progesterone (P) strongly increases the secretory process in E‐treated quails, but inhibits cell proliferation, cell evagination and differentiation of ciliated cells. The balance between E and P is critical for harmonious development of the oviduct.

After 6 daily injections of two doses of estradiol benzoate (10 or 20 μg/d) and high doses of P (4 mg/d), tubular gland formation by epithelial cell evagination was inhibited, while epithelial cell proliferation occurred, as shown by the height of the villi and the increase in DNA.

Secretory processes were strongly stimulated. Ovalbumin, a tubular gland cell marker and avidin, a mucous cell marker, were localized by immunofluorescence and immunogold labeling. Ovalbumin was localized only in the rudimentary tubular glands, whereas avidin was dispersed throughout the secretory cells.

High doses of progesterone inhibited tubular gland cell proliferation, disturbed the distribution of avidin and inhibited differentiation of ciliated cells. Ovalbumin synthesis occured only in epithelial cells which were evaginated despite the hyperstimulation. Ovalbumin gene expression appeared highly dependent upon the cell position.
Cytochemical study of abnormal intranuclear structures rich in beryllium
Authors: Berry Jean‐Pierre , Mentre Pascale , Hallegot Philippe , Levi‐Setti Ricardo , Galle Pierre
Published: February 2012

During prolonged intoxication with beryllium sulphate, intranuclear beryllium‐rich structures (IBRS) develop mainly in the cells of the convoluted tubules of the kidney. These structures are constituted by the accumulation of dense granules approximately 20 nm in diameter. The present work shows: 1) by electron probe microanalysis that IBRS are rich in phosphorus and calcium, and 2) by high resolution ion microanalysis that the granules are rich in beryllium and proteins. Staining with thallium alcoholate and regressive staining with ethylenediaminetetraacetate (EDTA) seem to demonstrate the presence of ribonucleoproteins in the granules. But the richness in calcium and phosphorus makes it difficult to interprete cytochemical reactions based on thallium and lead because complexes can be formed between calcium and thallium or lead, and between phosphorus and lead. Extraction with EDTA and digestion with RNase carried out on floating slices fixed with glutaraldehyde and embedded in glycol methacrylate show that: 1) the positive response of IBRS to cytochemical techniques used seems due solely to calcium; 2) the RNase forms a stable complex with a constituent of the granules that could be the highly phosphorylated acidic protein that binds preferentially to beryllium described by Parker and Stevens.
Immotile, aflagellate spermatozoa in Ptiliidae coleopterans
Authors: Baccetti Baccio , De Coninck Eliane
Published: February 2012

The first aflagellate and immotile Coleopteran spermatozoon is described in a group of species belonging to the family Ptiliidae. The mature spermatozoon is devoid of flagellum, centrioles and mitochondria, includes a three‐layered acrosomal complex (extraacrosomal layer, acrosome and perforatorium) and a long nucleus made up of two regions of different densities. A compact submembranary capsule and a thick glycocalyx are also present. Motility organelles are absent during the whole spermiogenesis, which is not regressive. The new structures peculiar for this type of sperma are designed for protection. No other sperm models with these characteristics have been described so far.
The effects of blue and red light on Acetabularia mediterranea after long exposure to darkness
Authors: Dazy Anne‐Catherine , Puiseux‐Dao Simone , Borghi Hélène
Published: February 2012

After Acetabularia mediterranea cells were kept in darkness for 2–8 weeks, all the cellular processes were arrested and the algae did not grow. In particular, the transcellular electrical potential (VAB) decreased to almost zero and cytoplasmic streaming was arrested. Upon illumination with continous blue light (BL), the first events were (as with white light (WL)), immediate increase in VAB and movements of water, followed, after a lag period of 1–3 min, by transient recovery of cytoplasmic streaming which lasted about 16 min. After 10 min (earlier than in WL), the frequency of the spontaneous action potentials increased much more than in WL. Then, after 1.5–4 hr during which VAB often decreased to zero while the cytoplasmic movements stopped, both activities resumed with diurnal oscillations. BL stimulated (as WL) rRNA synthesis, migration of rRNA from nucleus towards apex and cell growth. Upon illumination with red light (RL), VAB also increased, but water movements were much less pronounced than in BL. The transient streaming phase was shorter. The spontaneous action potentials increased in frequency much later (several hr) and much less than in BL or WL. VAB did not decreased at any time and was maintained at particularly high values. Cytoplasmic streaming resumed, but showed very attenuated or no rhythm. rRNA synthesis and migration remained low. Cell growth did not resume during the experiments.

By comparing our results with those of other studies relating to growth, morphogenesis and photosynthesis, we suggest that BL and RL could affect all these processes by differentially modifying the cytoplasmic concentrations of ions which may influence the functions of the cytoskeleton.
Chromosomal localization of the mouse gene coding for the 68 kDa neurofilament subunit
Authors: Matte Marie‐Geneviève , Duprey Philippe , Li Zhen Lin , Mattei Jean‐François , Paulin Denise
Published: February 2012

The chromosomal localization of the mouse gene coding for the 68 kDa intermediate filament subunit of neurones (NF‐L) was determined by in situ hybridization using specific 3H‐labelled DNA probes. There is only one copy of the NF‐L gene. The gene encoding NF‐L is located on chromosome 14 region (D1‐E1).
Solubilization and electrophoretic studies of cyst wall proteins of a hypotrichous ciliate
Authors: Rios Rosa Maria , Sarmiento Rafael , Torres Antonio , Fedriani Concepcion
Published: February 2012

A method for induction of synchronous encystment in a hypotrichous ciliate, Paraurostyla sp. is described. Cyst walls, isolated by shaking with glass beads, were analyzed by SDS‐polyacrylamide gel electrophoresis. To test optimal conditions of solubilization of cyst wall proteins, different treatments using Triton X‐100, EDTA, EGTA, urea, SDS and 2‐mercaptoethanol were carried out. At least, 15 different proteins were identified as specific to the cyst wall. Four low molecular weight polypeptides (40, 27–26, 20 and 18 kDa represented aproximately 70% of the cyst wall proteins. The 170, 135 and 40‐kDa bands exhibited a PAS‐positive reaction. Hydrogen and disulphide bonds were shown to be the most important interactions involving cyst wall proteins. Amino acid composition of cyst wall proteins was also investigated by HPLC. High amounts of glycine, cystine and proline were detected.
The acentriolar state of the Drosophila cell lines 1182
Authors: Debec Alain , Abbadie Corinne
Published: February 2012

A Drosophila melanogaster cell line devoid of centrioles has been recently described. In order to achieve an easier characterization of these acentriolar cells, we used the monoclonal antibody Bx 63 of M. Frusch which recognizes the Drosophila centrosome. Although centrosomes are detected at every mitotic pole in Drosophila cells with centrioles, no such structure has been observed in 1182‐4 acentriolar cells. The antigenic material is, however, present in these cells. Moreover, we noticed a certain proportion of acentriolar cells in 4 other 1182 lines. The lack of centrioles previously found only in the 1182‐4 cells seems therefore not accidental and should be linked to their particular origin.
Genetic evidence for linkage with the Z and W sex chromosomes of two distinct couples of alleles controlling larval and postmetamorphic skin pigmentation in salamander
Authors: Collenot Alain , Dournon Christian , Lauthier Michel
Published: February 2012

Summary— In Pleurodeles waltl, progeny resulting from a cross between 2 individuals of the Z/W sexual genotype include 25% of W/W individuals, while those issued from crossing a Z/W neomale with a W/W thelygenous female include 50% of W/W individuals. W/W individuals can be identified through the peptidase‐1 zymogram since, in P. waltl, this enzyme is controlled by codominant alleles which are linked to the sex chromosomes. In such progeny, we discovered 2 mutant phenotypes affecting larval and postmetamorphic skin pigmentation in W/W individuals. These phenotypes are described herein. The study of their inheritance in several offspring provides evidence that they are controlled by 2 distinct genes, the recessive mutant alleles of which are linked to the W sex chromosome; moreover, in thelygenous W/W females, the differential segment does not prevent the occurrence of meiotic recombinations between W sex chromosomes. Mutant skin pigmentary phenotypes are easily identified and constitute a tool for rapid, efficient selection of individuals of the W/W sexual genotype.
An analog of Xenopus N1N2 protein in Pleurodeles waltl
Authors: Moreau Nicole , Lautredou Nicole , Angelier Nicole
Published: February 2012

Summary— The oocyte nucleus of Pleurodeles waltl contains a major 185‐kDa protein analog of Xenopus N1N2. Rabbit antibodies were raised against the 185‐kDa protein. Affinity‐purified antibody directed against the acid part (pI 4.7) of the protein was prepared using antigens separated by two‐dimensional electrophoresis. Specificity of the antibody was controlled on two‐dimensional gels of nuclear proteins. It was shown that the 185‐kDa protein was separated into 2 forms: an acid form of pI 4.7–5.3, and a base form of pI 7. Peptide maps of the 2 spots revealed that they were closely related. The antibody was tested on: a) spread nuclear content, b) on sections of embryonic stages from stage 2 to stage 34, and c) the sections of adult tissies. The 185‐kDa protein appeared to be associated with the RNP matrix of a particular type of lampbrush chromosome loop, the granular loop. This protein was present in the nuclei of all embryonic cells. In adult tissues, it was present only in the nuclei of cells which presented high mitotic activity. These results confirm that, like N1N2, the 185‐kDa protein interacts with the constitution of chromatin; furthermore, they provide evidence for the role of this protein in transcriptional activity.
Anti‐fibronectin serum inhibits the disorganization of the dermal‐epidermal junction in cultured wounded skin
Authors: Denefle Jean‐Pierre , Zhu Quian‐Long , Lechaire Jean‐Pierre
Published: February 2012

Summary— During wound‐healing in cultured frog skin fragments, fibronectin (FN) was detected in the dermal‐epidermal junction. Intracellular fibronectin was stained using permeabilization and DAB immunoperoxidase. With electron microscopy intracytoplasmic FN granules were localized in the epidermal processes of the stratum germinativum cells protruding towards the dermis and in their marginal regions (membrane‐associated plaques). Faint staining was visible at the level of the lamina densa and inside some parts of the lamina lucida. In comparison, contrasted ultrathin sections revealed classical disorganization of the dermal‐epidermal junction. In the presence of anti‐fibronectin serum during the whole time of culture, fibronectin‐antifibronectin binding was visualized in the form of sparse cytoplasmic granules in the epidermal processes of the stratum germinativum cells. Contrasted ultrathin sections emphasized the continuity between the tonofilaments, the anchoring filaments and the anchoring fibrils. Briefly, anti‐fibronectin serum inhibits the disorganization of the dermal‐epidermal junction in cultured wounded skin.
Expression of liver peroxisomal proteins as compared to other organelle marker enzymes in rats treated with hypolipidemic agents
Authors: Malki Cherkaoui Mustapha , Bardot Olivier , Lhuguenot Jean‐Claude , Latruffe Norbert
Published: February 2012

Summary— Peroxisome proliferation induced by 2 hypolipidemic agents (clofibrate and ciprofibrate) was studied in rats by complementary approaches, ie cell fractionation, electron microscopy, marker enzyme activities, immunoblotting and nucleic acid hybridization techniques. Administration of clofibrates for 2 and 52 weeks in doses of 500 ppm and 50 ppm respectively, or ciprofibrate for 2,28 and 52 weeks in doses of 250, 25 and 25 ppm respectively, did not alter the behavior of the peroxisomes after induction as shown by ultracentrifugation profiles. The peroxisome mass was increased as shown by the purification procedure. Specific enzymes (catalase and mostly cyanide insensitive palmitoyl CoA oxidase) were induced. A mechanism of peroxisome biogenesis might have been initiated ie cytosolic factor, ligand‐receptor interaction and/or post‐translational modification of the import. Increase in marker enzyme activities showed that the peroxisomes are the most responsive organelles in comparison to lysosomes, mitochondria and smooth endoplasmic reticulum (except for cytochrome P‐450 LA ω‐hydroxylase). Peroxisomal integral membrane proteins appeared to be differently induced: some of them were virtually absent in untreated rat liver but were strongly expressed in treated liver. Induction was substained for 52 weeks, indicating that there was no compensatory mechanism.
Mass and molecular weight of isolated nuclear rings
Authors: Aréchaga Juan , Diaz Juan , Silió Margarita , Bahr Gunter F
Published: February 2012

Summary— Nuclear rings are cell structures found at the nuclear cortex wedged between the nuclear envelope and the chromatin fiber network. In previous publications we have dealt with their morphology, relationships with the nuclear membranes, chromatin fibers and cytoskeletal filaments; and more recently, with their measurements at high electron microscope resolution. In this article we have calculated the mass and molecular weight of 336 isolated nuclear rings from human circulating lymphocytes using a photometric procedure and polystyrene latex spheres as the standard for weight calibration. Our results show a range of mass of 0.4–35.5 × 10−16g (equivalent to 0.2–21.2 × 108 Da with a positively skewed distribution (median: 3.3 × 10−16g or 2.0 × 108 Da). Mass and volume of nuclear rings were highly correlated. In addition, it was possible to calculate the area, the whole mass and the mass per unit area of the nuclear envelope present in the center of the nuclear rings. The mass of this area also shows a lognormal distribution (median of mass/unit area: 37.3 × 10−8 pg/nm2 or 1.9 × 105 Da/nm2). We discuss the significance of this results as parameters for the characterization of the nuclear rings and their possible implications for a new interpretation of nuclear cortex architecture, nucleocytoplasmic traffic and macromolecule segregation between the two main cell compartments.
Fate of ecto‐NAD+ glycohydrolase during phagocytosis of normal and mannosylated latex beads by murine macrophages
Authors: Muller Christian D , Schuber Francis
Published: February 2012

Summary— In order to gain a better understanding of the role of ecto‐NAD+ glycohydrolase, an enzyme predominantly associated with phagocytic cells, we have studied its fate in murine macrophages (splenic, resident peritoneal and Kupffer cells) during phagocytosis of opsonized or mannosylated latex beads. In parallel, we have also monitored nucleotide pyrophosphatase, another ectoenzyme of macrophages. Phagosomes were isolated by flotation in a discontinuous sucrose gradient and the enzyme activities were determined with fluorometric methods. Low levels of NAD+ glycohydrolase and nucleotide pyrophosphatase could be measured associated with the phagosomal fractions, eg, respectively less than 4.5% and 10% in spleen macrophages. The phagosomal activities originate from the plasma membrane, ie they were latent and inactivation of ecto‐NAD+ glycohydrolase with the diazonium salt of sulfanilic acid resulted in a marked decrease of this enzyme activity in the phagosomal fractions. Pre‐labelling of the cell surface by [3H]‐galactosylation indicated that NAD+ glycohydrolase is internalized to a lesser extent than an average surface‐membrane unit. These results indicate that if ecto‐NAD+ glycohydrolase of macrophages can be internalized to a limited extent during phagocytosis of opsonized or mannosylated latex beads, this enzyme appears to be predominantly excluded from the surface area involved in the uptake of such particles.
An approach to the architecture of Scutigera coleoptrata hemocyanin by electron microscopy and image processing
Authors: Boisset Nicolas , Taveau Jean‐Christophe , Lamy Jean‐Noel
Published: February 2012

Summary— The 6 × 6meric hemocyanin of the centipede Scutigera coleoptrata, prepared by the single layer negative staining technique with uranyl acetate, has been investigated under the electron microscope. Isolated molecules were windowed from selected digitized micrographs. After alignment, they were submitted to correspondence analysis and hierarchical ascendant classification. Two main clusters of molecules were differentiated, and their average images were obtained. A 3D‐model constructed from these images is proposed.
In vivo decondensation of chromatin and nucleolar fibrillar component by Leucaena leucocephala ingredient
Authors: Vogt Günter
Published: February 2012

Summary— Feeding of the shrimp, Penaeus monodon, with diets containing leaf meal of the leguminous shrub, Leucaena leucocephala, resulted in complete chromatin decondensation of hepatopancreas cells. The fibrillar component of the nucleolus was decondensed in parallel, whereas the granular component remained intact. This unique combination of nuclear signs was accompanied by only moderate alterations of other cell organelles. Our findings therefore demonstrate an encouraging possibility to manipulate the chromatin organization in living cells. Furthermore, ultrastructural features obtained thus far only in isolated and chromatin‐depleted nuclei could be verified. These are, for instance, filament bundles which attach the nucleolus to the nuclear periphery, or a filamentous skeleton of the nuclear pores. In addition, the attachment of chromatin to the inner membrane of the nuclear envelope was observed. Decondensation was probably caused by the major Leucaena ingredient mimosine and is obviously related to its copper chelating properties.
Cytoskeletal control of the apical surface transformation of rat uterine epithelium
Authors: Luxford Karen A , Murphy Christopher R
Published: February 2012

Summary— During early pregnancy, in the lead up to blastocyst implantation, the apical cell surface of luminal epithelial cells of the rat uterus undergo a dramatic shape transformation. This study aims to investigate the role of the cytoskeleton in this apical transformation by considering the effects of the drugs cytochalasin D and colchicine on the uterine luminal cell surface. The results are determined using transmission and scanning electron microscopy. In vivo exposure to cytochalasin D during oestrus, as well as on day 1 of pregnancy, did not affect the long, regular surface microvilli. This drug, however, did disrupt the terminal web within the apical cytoplasm of these cells. Disruption of microfilament (MF) polymerization by cytochalasin D on day 4 of pregnancy induced a cell surface transformation, resulting in the appearance of numerous irregular projections normally present during blastocyst implantation on day 6 of pregnancy. Colchicine did not alter the uterine microvilli of oestrus or day 1 pregnant tissue. Unlike the effect of cytochalasin D, colchicine‐induced microtubule (MT) disruption on day 4 of pregnancy did not increase irregular projections and hence this treatment did not result in the cell surface appearance associated with blastocyst implantation. These results indicate that the disruption of MF, rather than MT, contributes to the transformation of the uterine luminal cell surface during the lead up to blastocyst attachment.
Raman Mapping of the Indentation‐Induced Densification of a Soda‐Lime‐Silicate Glass
Authors: Kassir‐Bodon Assia , Deschamps Thierry , Martinet Christine , Champagnon Bernard , Teisseire Jérémie , Kermouche Guillaume
Published: February 2012

Micro‐Raman spectroscopy was used to map the Vickers indentation‐induced densification on a window glass (soda‐lime silicate glass), from the correlation between Raman spectrum and density. Both top surface and cross‐section of the residual imprints were investigated. It is shown that the sample preparation needed for the investigation of the in‐depth densification does not alter the results. The maximum densification ratio measured is about 3.8%, which is 60% of the maximum densification measured after a high‐pressure hydrostatic loading. Within the experimental errors, densification maps are self‐similar for the two indentation loads used 10 N and 20 N. A comparison with finite element results based on a constitutive model developed for silica is discussed.
Comparison of the effects of marchantin C and fucoidan on sFlt-1 and angiogenesis in glioma microenvironment
Authors: Lv Yafeng , Song Qingxu , Shao Qianqian , Gao Wenjuan , Mao Haiting , Lou Hongxiang , Qu Xun , Li Xingang
Published: February 2012

Objectives

This study aimed to examine the effects of marchantin C and fucoidan on angiogenesis induced by glioma cells and monocytes, and to elucidate the role of sFlt-1 in this process.

Methods

T98G glioma cells and THP1 monocytes were pretreated with marchantin C or fucoidan, respectively. Conditioned media were used for endothelial cell tube formation assay and detection of sFlt-1 by ELISA. Depletion of sFlt-1 was achieved by a neutralizing antibody to assess its role in the process.

Key findings

Marchantin C inhibited angiogenesis induced by T98G cells while fucoidan inhibited both T98G and THP1 cell-induced angiogenesis. In all three groups in which angiogenesis was inhibited, sFlt-1 level in the supernatants was elevated. Pretreatment of the conditioned media with sFlt-1 antibody restored the inhibited angiogenesis to a certain degree.

Conclusions

This study suggested for the first time that marchantin C and fucoidan could significantly inhibit angiogenesis induced by glioma cells or monocytes. Up-regulation of sFlt-1 played an important role in this process.
In-vitro dissolution methods for controlled release parenterals and their applicability to drug-eluting stent testing
Authors: Seidlitz Anne , Weitschies Werner
Published: February 2012

Objectives

Dissolution testing is a powerful tool for the characterization of dosage form performance in vitro under standardized conditions. In spite of the increasing number of parenterally administered medicinal products, currently there are no compendial dissolution test methods designed especially for these types of dosage forms. In addition to classical drug delivery systems, drug/device combination products, such as drug-eluting stents, are being used increasingly.

Key findings

This review describes the current methods that are used most often for in-vitro dissolution testing of parenteral dosage forms, i.e. the ‘sample and separate’ methods, the ‘dialysis’ methods, and the ‘flow-through’ methods, with a special emphasis on whether these methods can be used for drug-eluting stent testing. In the light of current regulatory requirements and with the exploding costs of preclinical and clinical development, test systems that include biorelevant parameters and are predictive of in-vivo performance are increasingly important. Published attempts to take biorelevant conditions into consideration in the design of dissolution test apparatus developed for parenteral dosage forms, including a method that was designed to emulate the embedding and flow-conditions at the site of stent implantation, have been outlined in this review.

In spite of the large quantity of highly potent controlled release parenteral products marketed today, there is still a lack of suitable methods for in vitro dissolution testing for these dosage forms especially with regard to biorelevant testing conditions. For dosage forms implanted into tissues it seems of major importance to reproduce the transport forces which are predominant in vivo (diffusive versus convective) in the in-vitro experimental setup.
Spectroscopic investigations of the molecular interaction of anticancer drug mitoxantrone with non-ionic surfactant micelles
Authors: Enache Mirela , Volanschi Elena
Published: February 2012

Objectives

The aim of this study was to investigate the interaction of the anticancer drug mitoxantrone with non-ionic micelles, as simple model systems of biological membranes.

Methods

UV-VIS absorption spectroscopy was used to quantify the drug–surfactant micelle interactions in terms of the binding constant and the micelle–water partition coefficient of the drug.

Key findings

Interaction of mitoxantrone with non-ionic micelles reduces the dimerization process of mitoxantrone, the drug molecules being encapsulated into micelles as monomer. The strength of the interaction between mitoxantrone and non-ionic micelles is higher at pH 10 than at pH 7.4, and depends on the surfactant in the order Tween 80 > Tween 20 > Triton X-100. The higher partition coefficient at pH 10 compared to pH 7.4 suggests that at basic pH the deprotonated mitoxantrone is incorporated more efficiently into the hydrophobic medium of non-ionic micelles compared to physiological pH, when the protonated drug is predominant.

Conclusions

These results on simple model systems miming the drug–membrane interactions contribute to the elucidation of the behaviour of the drug in vivo, as well as the possible utilization of surfactant micelles as drug carriers.
Serine-proteases as plasminogen activators in terms of fibrinolysis
Authors: Flemmig Martin , Melzig Matthias F
Published: February 2012

Objectives

This review should give an overview about the natural human plasminogen activators and their various modified variants as well as similar substances isolated from animals, microorganisms and plants. When a blood clot is formed in a blood vessel, it avoids the oxygen supply of the surrounding tissue. A fast fibrinolytic therapy should redissolve the blood vessel and reduce the degradation of the tissue. All proteases that are part of the human blood coagulation and fibrinolytic system belong to the serine protease family. t-PA (tissue plasminogen activator) and u-PA (urokinase plasminogen activator) are the naturally occurring fibrinolytic agents that are also used in therapy.

Key findings

Despite many years of research, t-PA is still the gold standard in fibrinolytic therapy. But it has to be given as an infusion, which needs time. Modified fibrinolytic substances are, were, or perhaps will be in the market. They have different advantages over t-PA, but often the disadvantages predominate.

Conclusion

Many substances have been developed but an optimal fibrinolytic agent combined with a simple administration is not in therapeutic use to date.