bluuebunny/crossref_metadata_2025 huggingface dataset:
Localization of thymulin (FTS‐Zn) in mouse thymus. Comparative data using monoclonal antibodies following different plastic embedding procedures
Authors: Auger C. , Monier J. C. , Savino W. , Dardenne M.
Published: February 2012

The distribution of thymulin (FTS‐Zn) was studied in thymuses from normal mice (OF1) or autoimmune mice (NZB). Thymulin localization was investigated using immunocytochemical techniques on sections of GMA and epon‐embedded mouse thymuses. Two monoclonal antibodies were used: anti‐synthetic thymulin and anti‐intracellular thymulin. In the immunofluorescence assay, GMA sections allowed a more subtle localization of thymulin in the cytoplasm of epithelial cells (with a vacuolar pattern) compared to the epon sections (with a homogeneous fluorescence in the cytoplasm). In both cases, the number of labeled cells was greater in the medullary region than in the cortex of the thymus. At the electron microscopic level, immunolabeling of epon ultrathin sections showed ferritin distributed in some of the epithelial cell vacuoles. The two monoclonal antibodies revealed similar distributions of thymulin in the thymus. The results obtained in this study confirm that the amount of thymulin is greater in the epithelial cells of normal compared to autoimmune thymuses.
Changes in protein synthesis and in RNA poly A+ population after treatment of Dictyostelium amoebae by 5‐bromo‐2′‐deoxyuridine
Authors: Scrive‐Menahem M. , Ayala J. , Jacquet M. , Felenbok B.
Published: February 2012

Incorporation of 5‐bromodeoxyuridine (5‐BUdR) into nuclear DNA severely interrupts the life cycle of Dictyostelium discoideum after the first generation of growth. Loose cellular aggregates are then formed, but no spore or stalk cells are detectable and no other morphological transformations are observed. The perturbation of gene expression in the life cycle has been studied at the protein level by two‐dimensional gel electrophoresis after pulse labelling with 35S‐methionine and also by changes in the patterns of polysomal messenger RNA population. The latter was monitored by hybridisation studies using specific cDNA probes for “vegetative” and “18 hr” messenger RNAs. In the presence of 5‐BUdR major anomalies in polypeptide synthesis were observed after the loose aggregation stage. Some vegetative polypeptides, including actin, which are normally abundant only during growth to the aggregation stage, are oversynthesised during the period 12‐24 hr after starvation. In this same interval the normal decline in the abundance of vegetative mRNA species was not observed. In marked contrast virtually half the normal “18 hr‐specific polypeptides” were poorly synthesised. Likewise, the normal increase in abundance of the corresponding “18 hr‐specific” poly A + RNA species in the polysomes did not occur. No major alteration in the timing of the appearance of new macromolecules during the cell cycle was observed in spite of extensive modification of gene expression by the incorporation of 5‐BUdR into genomic DNA.
Modulation of CALLA and HLA‐DR on a non T non B leukemic cell line by lipid treatment
Authors: Martyre M. C. , Vaigot P. , Kayibanda M. , Rosenfeld C.
Published: February 2012

The relationship between membrane lipid fluidity and expression of HLA‐DR and cALL (CALLA) antigens was studied in a human non T non B acute lymphoblastic leukemia cell line (Reh). The membrane fluidity was modulated by treatment with cholesteryl hemisuccinate or phospholipids (e.g. egg lecithin) and monitored by fluorescence polarization. HLA‐DR and CALLA expression was measured in an indirect immunofluorescence test with a Fluorescence Activated Cell Sorter (FACS 440), on 24, 48, 72 and 96 hour‐cultured cells. Significant antigenic modulation was obtained with cholesteryl hemisuccinate treatment on 48 hour‐cells where a slight increase in HLA‐DR and a marked decrease in CALLA were observed. In contrast no antigenic modification was observed on lecithin‐treated cells.
Intercellular junctions in the hepatopancreas of the lobster Nephrops norvegicus
Authors: Lane N. J. , Harrison J. B. , Lee W. M.
Published: February 2012

The hepatopancreas of the lobster has recently been found to be a rich source of material from which to isolate arthopod gap junctions biochemically (Finbow et al., 1983a; 1984). It has therefore been studied here to assess the features of these intercellular junctions and any others that may be present, in vivo. The tissue consists of columnar epithelial cells which possess apical microvilli and basal infoldings. In thin sections the lateral borders of these cells are characterized by desmosomes and smooth septate junctions as well as by gap junctions. The desmosomes exhibit no apparent freeze fracture profile but the septate junctions display parallel rows of ridges or aligned intramembranous particles (IMPs) with complementary grooves on the other membrane half; these IMPs shift in their preferential fracturing plane depending on whether the tissue has first been fixed, always remaining on the EF if unfixed. The IMPs or connexons, of which the gap junctions are composed, fracture onto the E face, leaving complementary pits on the P face, regardless of whether the tissue is fixed or not. At the base of the pancreatic cells, the lateral borders are thrown into interdigitating folds which display endocytotic profiles and possible internalization of junction‐bearing membranes. This phenomenon, which is readily visualized both after tracer incubation and in replicas, may represent junctional degradation relating to membrane turnover.
In situ hybridization: a routine method for parallel localization of DNA sequences and of their transcripts in consecutive paraffin sections with the use of 3H‐labelled nick translated cloned DNA probes
Authors: Guelin M. M. , Kejzlarovà‐Lepesant J. J. , Lepesant J A JA.
Published: February 2012

A routine in situ hybridization method is described and discussed, which allows parallel detection of repeated DNA sequences and of their abundant transcripts in consecutive tissue sections of a same biological sample with a unique probe. The protocol, based on the use of classical 3H‐labelled nick translated cloned DNA probes and of conventional paraffin sections of ethanol‐acetic acid‐fixed tissues, consists of a simple combination of procedures in current use for separate detection of either RNA or DNA. Different treatments recommended in other methods are omitted or simplified, making the protocol suitable for routine use. The method is successfully applied here to a test‐system where ribosomal sequences are sought in the ovarian follicles of the Lepidopteran Ephestia kühniella, by using a recombinant plasmid containing a Drosophila melanogaster rDNA repeating unit as a probe. Specific and reproducible results are obtained. Sensitivity is sufficient though moderate specific activities are used. Background level is very low. The regionalized distribution of sequences of both types in the chosen model allows to demonstrate that specific detection of RNA requires the systematic removal of DNA from the tissue sections prior to hybridization.
Reutilization of insulin receptor and hormonal response in cultured foetal hepatocytes: the effects of chloroquine and vinblastine
Authors: Pringault E. , Plas C. , Desbuquois B. , Clauser H.
Published: February 2012

The effects of chloroquine and vinblastine (10‐100 microM) on insulin degradation and biological action were studied in cultured foetal rat hepatocytes. Insulin degradation, as measured by the release of trichloroacetic acid‐soluble radioactivity from 125I‐insulin into the medium, was strictly cell‐associated, saturable with respect to insulin concentrations and linearly related to the amount of cell‐associated hormone. The maximal rate of insulin degradation was 4,700 molecules/min per cell, and its KM about 5 nM. Thus, insulin receptors (30,000 sites/cell; half‐life close to 13 hr) must be reutilized 450‐fold before being degraded with an average time of reutilization inferior to 10 min. In the presence of 70 microM chloroquine or 100 microM vinblastine, insulin degradation was inhibited by 80% and the amount of cell‐associated hormone enhanced 2‐3‐fold. Nearly total inhibition of insulin‐stimulated glycogenesis was obtained with 70 microM chloroquine and 45 microM vinblastine. When hepatocytes were preincubated with chloroquine or vinblastine, insulin binding remained high for up to 4 hr, then progressively decreased thereafter. The addition of 10 nM native insulin during preincubation with the drugs resulted in an earlier and more pronounced decrease in insulin binding, whereas native insulin alone did not induce any change. Both the inhibition of insulin degradation and onset of receptor down‐regulation suggest a drug‐induced impairment in the receptor reutilization. This defect is correlated to a loss of the glycogenic effect of insulin in cultured foetal rat hepatocytes.
Relationship between growth inhibition and mitochondrial function in petite‐negative yeasts. I. Effects of antibiotics and dyes upon pathogenic and non‐pathogenic Candida species
Authors: Marmiroli N. , Tedeschi F. , Truzzi G. , Ferrari C. , Puglisi P. P.
Published: February 2012

Antibiotics and dyes which preclude growth of Saccharomyces cerevisiae in media containing oxidizable carbon sources arrested the growth of Candida albicans, Candida tropicalis and Candida utilis even in glucose medium. The growth in the presence of sub‐inhibitory concentrations of the various antibiotics and dyes determined a reduction in the cell survival but with no accumulation of respiratory deficient mutants. Under these culture conditions, the total respiration declined leaving a residual antimycin A‐resistant—hydroxamate‐sensitive O2 uptake, and the amount of the respiratory cytochromes aa3 and b synthesized was reduced. SDS gel electrophoresis of soluble proteins prepared from the antibiotic‐treated cells showed some bands in the MW range 92‐100 K, which became faint after the cells were grown in the presence of some mitochondrial inhibitors. The ultrastructural analysis of these cells evidenced disappearance of the mitochondrial cristae and their replacement by unfolded membranes. The data obtained suggest that the petite negative trait of Candida could depend on the non‐viability or on the very low viability of those cells which have lost their mitochondrial function.
Some biochemical observations on gluconeogenesis from propionate in hepatocytes isolated from normal and biotin‐deficients rats
Authors: Djabal A. , Mangeot M. , Cherruau B. , Lemonnier A.
Published: February 2012

Propionate and pyruvate added to isolated normal and biotin‐deficient adult rat hepatocytes increase the production of glucose. This production decreases about 30% on biotin deficiency. Malonate inhibits gluconeogenesis from propionate showing the metabolic transformation of propionyl‐CoA via the Krebs cycle. Neither glucagon nor dibutyryl‐cyclic AMP significantly stimulate gluconeogenesis.
Chromosomal banding by the action of two compounds: sodium glycocholate and arginine
Authors: Truchini J. P. , Geneix A. , Perissel B. , Talvard T. , Turchini M. F.
Published: February 2012

In human lymphocytic metaphasis chromosomes sodium glycocholate induces a banding of chromatids and an intense apparently perichromatic labelling, reproducible on the same points of various chromosomal pairs. Arginine makes centromeric heterochromatin visible.
Effects of 2‐mercapto‐1‐(beta‐4‐pyridethyl)‐benzimidazole (MPB) on Ehrlich cell nucleoli: stereological analysis
Authors: Lepoint A. , Goessens G.
Published: February 2012

The effects of MPB, a strong inhibitor of RNA synthesis, have been analysed at the ultrastructural level by means of stereological methods. After treatment, an increase in the nucleolar volume is observed. This enlargement is due to vacuolization of the nucleoli. However, the relative volumes of the nucleolar components are modified in various directions: the volume of the granular component decreases whereas the fibrillar centres increase in size. These results are discussed in terms of relations between morphology and function of the nucleolus.
Cytochalasin D suppresses beta‐adrenergic induced protein discharge without inhibiting membrane fusion
Authors: Busson‐Mabillot S. , Chambaut‐Guérin A. M. , Huleux‐Maurs C. , Ovtracht L. , Rossignol B.
Published: February 2012

Biochemical studies have shown that low doses of Cytochalasin D (CD) inhibit protein discharge evoked by isoproterenol, a beta‐adrenergic inducer, in isolated lobules of Rat parotid gland. Ultrastructural and radioautographic studies reported here indicate that, when lobules are treated by CD before isoproterenol stimulation, the connection of secretory granules with the acinar lumen is indeed prevented, but fusion of membrane granules evoked by isoproterenol still occurs. This fusion takes place with vacuoles induced by CD and possibly with other granules.
Rapid changes in levels of individual molecular forms of acetylcholinesterase after denervation of mouse sternocleidomastoid muscle
Authors: Goudou D. , Blondet B. , Friboulet A. , Rieger F.
Published: February 2012

Experimental denervation of adult mouse sternocleidomastoid muscle results in a decrease in total AChE. The most rapid change essentially affects the tailed, asymmetric 16 S AChE, since one day after nerve section, “16S” AChE is already significantly decreased to about 70% of its control value. We found that both background and junctional “16S” AChE are affected by this rapid decrease. Later, a sharp fall in “10S” and “4S” AChE occurs about seven days after denervation when muscle atrophy develops with loss of weight and proteins. A gaussian analysis of the sedimentation profiles of AChE extracted from denervated muscle shows that there is not only an early rapid decrease in 16 S AChE but also a decrease in the monomeric 3.3S AChE. This result suggests that there is a very rapid turn‐over of two molecular forms of AChE, the supposedly monomeric precursor and the complex asymmetric 16S AChE.
Histochemical and biochemical studies of cholinesterases in the carotid labyrinth of amphibians
Authors: Poullet M. , Verdiere M.
Published: February 2012

Acetylcholinesterase activity was detected in the carotid labyrinth of amphibians by both biochemical and histochemical methods. The histochemical tests showed enzyme activity in surfaces of afferent and efferent nerve terminals in contact with type I cells, but none within the type I cells. Acetylcholinesterase activity occurred on some, though not all, nerve fibers in the extra‐cellular spaces. These fibers might be parasympathetic cholinergic fibers innervating blood vessels.
Localization by immunoelectron microscopy of somatostatin‐containing cells in the gastric mucosa of a teleost fish, Perca fluviatilis
Authors: Noaillac‐Depeyre J.
Published: February 2012

Somatostatin‐immunoreactive cells were localized on semithin and ultrathin sections of Epon‐embedded samples of perch gastric mucosa, classically fixed with aldehydes and osmium tetroxide. On semithin sections, somatostatin cells were identified by using the immunoperoxidase method. The ultrastructural localization of somatostatin immunoreactivity was achieved using the colloidal gold method. Cells showing somatostatin immunoreactivity are found to be scattered among the surface mucous cells and the mucous neck cells. Somatostatin appears to be localized in cytoplasmic granules. Somatostatin‐containing cells are identified as the type I cells which were described in a previous ultrastructural study. The present report also points out that tissue samples which have been classically processed for ultrastructural study could be in some cases suitable for immunocytochemical investigations.
The expression of chick alpha A2‐crystallin RNA during lens development and transdifferentiation
Authors: Errington L. H. , Bower J. , Cuthbert J. , Clayton R. M.
Published: February 2012

During development of the vertebrate lens, the lens epithelium undergoes a final stage of differentiation into lens fibre cells, during which the major lens proteins, the crystallins, are synthesised. Lentoids, comprising clusters of lens fibre cells can also be produced by transdifferentiation from certain non‐lens tissues, including neural retina and pigmented retinal epithelium. We have isolated an alpha A2‐crystallin genomic probe and used it to study the transcription and processing of alpha A2‐crystallin mRNA both during lens development and transdifferentiation. We relate these results to earlier measurements in this laboratory of delta‐crystallin transcription and alpha‐ and delta‐crystallin protein synthesis, to compare the expression of these two sets of genes. Tissue specific differences in gene expression were found. delta‐crystallin mRNA is transcribed before alpha A2‐crystallin RNA in the lens, but after it in transdifferentiating neural retina and pigmented epithelia.
Ultrastructural changes in the rat thyroid gland during iodine deficiency
Authors: Beaumont A. , Fragu P.
Published: February 2012

Thyroid ultrastructure changes were studied during the course of a low iodine diet in rats. At day 20, follicles were normal, but a number of them contained cells of higher density and with greatly elongated microvilli. Endoplasmic reticulum cisternae were frequently dilated. From day 20 until day 80, the most characteristic changes in the thyroid cells were the progressive accumulation of subapical peroxidase‐positive exocytotic vesicles. After 80 days of the low iodine treatment, Golgi apparatuses were very active. Cell division could be observed. At this stage, exocytotic vesicles were generally very abundant. These data suggest that the remarkable accumulation of subapical exocytotic vesicles between day 20 and day 120 might represent an adaptation to the moderate and gradual increase in TSH stimulation that occurs in the conditions of low iodine diet.
Mitochondria alterations in Cd2+‐treated rats: general regression of inner membrane cristae and electron transport impairment
Authors: Toury R. , Boissonneau E. , Stelly N. , Dupuis Y. , Berville A. , Perasso R.
Published: February 2012

Young adult rats absorbed 50 p.p.m. Cd2+ added to drinking water. After 6 weeks, 3, 6 and 9 months of treatment, the ultrastructural condition of liver, kidney and muscle was observed by electron microscopy. The choice of these tissues was determined by their differences in the capacity to accumulate Cd2+: the liver is able to concentrate a considerable amount of metal, but redistributes it throughout the entire organism, while the kidney collects it in view of its elimination. Muscle contains the least Cd2+. A general regression in mitochondria cristae accompanied by a vesiculation and a fragmentation of endoplasmic reticulum appeared simultaneously in the three tissues, at as early as 6 weeks of treatment, and extended progressively with its continuation supporting evidence of a general attack of the intracellular membrane systems. Cd2+ stimulation of membrane‐degrading enzymes such as phospholipases and proteases was suggested. A concomitant diminution in glycogen stores was noted. Active synthesis of neutral lipids, especially cholesterol esters, took place in liver mitochondria of treated rats in collaboration with rough endoplasmic reticulum, and progressively generated a multiplication of electron‐transparent inclusions in cytoplasm. Isolated mitochondria from liver, kidney and muscle of Cd2+‐treated rats maintained partial energy coupling, but displayed a rapid early fall in cytochrome oxidase followed by a partial restoration after 6 months of treatment, and a progressively slackening of succinate dehydrogenase. Isolated vesicles of liver mitochondria inner membrane of treated rats behaved as intact mitochondria, indicating changes inside the membrane itself. Addition in vitro of the metal ion to mitochondria and also to inner membrane vesicles isolated from control rats revealed that Cd2+ was able to stop completely succinate dehydrogenase, but was totally ineffective on cytochrome oxidase. Membrane fixation of Cd2+ on the flavoprotein or SH associated with succinate dehydrogenase is proposed. Considering the close parallelism of the extensive depression of microsomal NADPH cytochrome c reductase and the rapid fall in mitochondrial cytochrome oxidase, it is suggested that an indirect inhibition process occurs, through Cd2+‐induced diminution of a constituent common to all cytochromes in the cell.
Biochemical and ultrastructural findings in a lymphoid cell line from Niemann‐Pick disease type A
Authors: Levade T. , Salvayre R. , Bes J. C. , Maret A. , Douste‐Blazy L.
Published: February 2012

A lymphoid cell line (LCL) established by Epstein‐Barr Virus (EBV)‐transformation of blood B‐lymphocytes from a patient affected with Niemann‐Pick disease (NPD) Type A exhibited a severe deficiency of sphingomyelinase activity (less than 10% residual activity). Ultrastructural investigation showed in LCL from NPD type A, the presence of numerous osmiophilic, electron‐dense inclusions with myelin‐like figures characteristic of the accumulation of sphingomyelin (and other amphiphilic lipids) similar to those observed in tissues of patients affected with NPD.
Structural and cytochemical differentiation of membrane elements of the apical membrane of amphibian urinary bladder epithelial cells. A label fracture study
Authors: Chevalier J. , da Silva P. Pinto , Ripoche P. , Gobin R. , Wang X. Y. , Grossetete J. , Bourguet J.
Published: February 2012

It is now generally accepted that ADH‐induced increase in water permeability in responsive epithelia is associated with the insertion of specific structures in the apical membrane of epithelial cells. Up to now, these structures have only been recognized in freeze‐fractured preparations and their chemical nature is still unknown. In this study, we used the label‐fracture method (Pinto da Silva and Kan, J. Cell Biol., 99, 1156‐1161, 1984) to investigate the distribution of wheat germ agglutinin (WGA) on the luminal plasma membrane of freeze‐fractured frog urinary bladder epithelial cells. With label‐fracture, the cytochemical markers are seen superimposed with the conventional high resolution image of the E face. Label‐fracture of tissue treated for 15 min with WGA and subsequently labeled with colloidal gold coated with ovomucoid showed uniform distribution of gold particles along the exoplasmic fracture face. Stereomicrographs show that the gold label is under the fracture face as it is attached to the outer surface of the membrane. Preincubation of the bladder with WGA for 3 hr induced a segregation of the intramembranous particles of the apical plasma membrane. In this condition, we observed a co‐distribution of WGA‐gold complexes with the segregated particles on the E face. This indicates that WGA‐binding sites are located on glycoproteins which probably comprise the large intramembranous particles dispersed on the exoplasmic faces of freeze‐fractured luminal membranes. In contrast, the numerous small intramembrane particles observed on P faces remained evenly distributed even after exposure to WGA and are, therefore, unrelated to WGA receptor sites. After WGA treatment, ADH still induced the formation of aggregates inside the smooth domains. A few WGA‐binding sites appeared to be associated to these aggregates.
Hydrosmotic response to vasopressin in frog skin. Control by endogenous factors
Authors: Svelto M. , Casavola V.
Published: February 2012

The A23187 calcium ionophore strongly inhibits the hydrosmotic response to vasopressin. On the contrary neither exogenous cAMP nor theophylline‐induced hydrosmotic response are affected by Ca++‐ionophore. The effects obtained with A23187 suggest that calcium ions modulate vasopressin‐induced osmotic water flow by interfering with a pre‐cAMP step. The increase in intracellular calcium concentration induced by A23187, in fact, may inhibit the rate of cAMP formation by interfering with the vasopressin‐stimulated adenylate cyclase system. This inhibition seems to be restricted to a locus proximal to the catalytic subunit of adenylate cyclase, whereas the hydrosmotic response to forskolin, a non‐hormonal activator, is not affected by calcium ionophore addition. In order to clarify whether calcium ions act directly on the adenylate cyclase system or activate a more complex pathway, we studied the interaction between calcium and prostaglandins in modulating the hydrosmotic response to vasopressin. Direct measurements by radioimmunoassay of PGE2 release in the serosal medium show that A23187 notably increases PGE2 release. Furthermore, the inhibition of prostaglandin biosynthesis by hydrocortisone (a phospholipase inhibitor) or by indomethacin and naproxen (agents that inhibit arachidonic acid oxygenase) results in augmented vasopressin‐stimulated water flow and prevents the inhibitory effect of the ionophore. Collectively these results strongly suggest that the effects obtained with A23187 on hydrosmotic response to ADH, are closely linked to calcium‐stimulated PGE2 biosynthesis.
Evidence for cycling of aggregate‐containing tubules in toad urinary bladder
Authors: Ding G. , Franki N. , Hays R. M.
Published: February 2012

Antidiuretic hormone (ADH) promotes the fusion of cytoplasmic tubular structures with the luminal membrane of receptor tissues such as toad urinary bladder. To determine whether fusion is a continuous cyclic process, bladders were stimulated with ADH with colloidal gold in the luminal bathing medium. After as little as 15 min of stimulation, gold‐filled tubules were seen in the cytoplasm, evidence that cycling was indeed taking place. Serial sections confirmed that these tubules had no connection with the luminal membrane, and had returned to the cytoplasm. Cessation of ADH stimulation, followed by a second stimulation, greatly reduced the number of gold‐filled cytoplasmic tubules, suggesting that many tubules were capable of refusion. Mean fusion event diameter underwent significant changes, enlarging at 15 min, and contracting at 60 min. Thus, ADH initiates a process of continuous cycling of cytoplasmic tubules between cytoplasm and luminal membrane.
Regulation of luminal membrane water permeability by water flow in toad urinary bladder
Authors: Muller J. , Kachadorian W. A.
Published: February 2012

Intramembranous particle aggregates (presumed sites for water flow) which appear in the luminal membrane consequent to ADH treatment are derived from cytoplasmic membrane structures (now termed “aggrephores”) which fuse with the luminal membrane. We have previously shown that bladders stimulated in the absence of an osmotic gradient have about twice as many aggregates and about three times as many sites of aggrephore fusion as bladders stimulated with ADH in the presence of a 175 milliosmolal gradient. The present studies show that the frequency of fused aggrephores and luminal membrane aggregates can be modified as a consequence of alterations in transmembrane water flow initiated by changing the transbladder osmotic gradient during hormone stimulation. Bladders treated with ADH for 1 hr without a gradient and then for 1 hr with a gradient had approximately 1/3 as many aggregates and fusion sites as paired bladders treated for 2 hr without a gradient. Conversely, bladders treated with ADH for 1 hr with a gradient and then for 1 hr without a gradient had approximately 2x as many aggregates and fusion sites as bladders treated for 2 hr with a gradient. In other experiments we demonstrate that the time course of hormone washout is greatly accelerated if carried out in the presence of an osmotic gradient. In paired bladders that were first stimulated with ADH for 30 min in the absence of a gradient, aggregates and fusion sites as well as osmotic water permeability determined in fixed bladders, persisted at near maximum levels for 15 min of washout in the absence of a gradient.(ABSTRACT TRUNCATED AT 250 WORDS)
Regulation of ADH‐stimulated water flow at a post‐luminal barrier in toad bladder
Authors: Kachadorian W. A.
Published: February 2012

Recent studies show that ADH‐stimulated water flow across toad bladder may be regulated at a site other than the luminal membrane. In these studies luminal membrane particle aggregate frequency has been used as a measure of luminal membrane water permeability. In fully stretched bladders the relationship between total tissue permeability and aggregate frequency is curvilinear, rather than linear. This implies a resistance in series with the luminal membrane that can become rate‐limiting for water flow during ADH stimulation. The possibility that transtissue water movement is actually regulated at such a post‐luminal membrane resistance is suggested by the finding that within 30 min following exposure to hormone, water flow becomes attenuated without any change in aggregate frequency. Supporting this possibility, recent data from follow‐up studies suggest that the apparent water permeability per luminal membrane aggregate is not reduced with time. Finally, for bladders in which prostaglandin synthesis is inhibited (by naproxen), increases in both base‐line water flow and water flow consequent to treatment with a submaximal dose of ADH (0.125 mU/ml), are much less than expected from simultaneously observed changes in luminal membrane aggregate frequency. In parallel experiments to these, moreover, direct measurements of luminal membrane water permeability from the rate of change of cell volume consequent to a transluminal membrane osmotic challenge, confirm that luminal membrane water permeability increases to the extent expected from changes in aggregate frequency. All of the data taken together argue for a post‐luminal membrane barrier in toad bladder which regulates tissue permeability during ADH stimulation.
The role of mitochondria‐rich cells in the chloride current conductance across toad skin
Authors: Katz U. , van Driessche W. , Scheffey C.
Published: February 2012

In early studies of salt transport across frog and toad skin, it was assumed that chloride movement is extracellular. However, later studies suggested that chloride movement is largely transcellular. Chloride transport across toad skin is greatly diminished in skins of salt‐acclimated toads (Bufo viridis) and was correlated with the number of mitochondria‐rich (m.r.) cells in the epithelium. The activated chloride conductance could be recovered upon in vitro incubation with theophylline. It was found that the short‐circuit current (Isc) and the chloride conductance (Gcl) in toad skin could be separated experimentally by selective use of synthetic oxytocin (Syntocinon) or theophylline, and by substituting impermeable anions for chloride. With the use of the vibrating probe we demonstrated directly that chloride‐dependent peak currents are localized only over m.r. cells, under hyperpolarized (V = −100 mV) conditions. It is concluded that the m.r. cells form the principal site for passive chloride movement across amphibian skin. This cellular pathway is regulated through a cyclic AMP‐mediated process. It is suggested that the spatial separation of the sodium and chloride channels is essential to maintain the granulosum cells which are engaged in sodium transport hyperpolarized, and thus providing the driving force for the sodium entry into the cells.
The intracellular lumen: origin, role and implications of a cytoplasmic neostructure
Authors: Remy L.
Published: February 2012

Ultrastructurally, the intracellular lumen appears as a more or less spherical cavity with osmiophilic substance, bordered with microvilli and surrounded by a filamentous network. Epithelial cancer cells, both healthy cultured and hormonally stimulated in vivo, often present such structures, which probably result from a dysfunction of the Golgi apparatus and cytoskeleton. This characterizes an abortive secretory process, which may be the consequence of a great hormonal sensitivity of the cells or of a loss of differentiation criteria when they are cancerous or isolated for culture.
Correspondence of two nuclear networks observed in situ with the nuclear matrix
Authors: Pouchelet M. , Anteunis A. , Gansmuller A.
Published: February 2012

In resting lymphocytes, three well defined networks are observed and attempts were made to superimpose them upon the networks described in isolated nuclear matrices. These three nuclear structures are seen after DNase and RNase treatment. They are digested by pepsin but their sensitivity to this enzyme is different. The more resistant network corresponds to the outer lamina of the isolated nuclear matrix. The second network is located in the inter‐chromatin area. It is more sensitive to pepsin than the lamina and this sensitivity is increased ten‐fold when digestion with pepsin is preceded by RNase digestion. This network corresponds to the internal network of isolated nuclear matrices. The third network is located in the intrachromatin area and is the most sensitive to pepsin action.
Distribution of microtubules during the breakdown of the nuclear envelope of the Xenopus oocyte: an immunocytochemical study
Authors: Jessus C. , Huchon D. , Ozon R.
Published: February 2012

Xenopus oocytes were stained by anti‐tubulin and anti‐MAP1 antibodies during the first meiotic cell division. In the prophase‐blocked oocytes, only few microtubules are present around the upper part of the nuclear envelope. These microtubules are resistant to cold, calcium and antimitotic drug treatments. At this stage, monoclonal anti‐MAP1 antibody and polyclonal anti‐centrosome antibody reveal punctate staining of the nucleus and nucleoli. During the progesterone‐induced maturation, a microtubular network appears at the basal part of the disrupting nucleus. Anti‐MAP1 and anti‐centrosome antibodies stain a dense layer at the basal part of this microtubular array. Microtubules present in this array are cold, calcium‐ and antimitotic drug sensitive. Anti‐MAP1 and anti‐tubulin antibodies stain the whole metaphase II spindle, whereas only the poles of the metaphase II spindle are stained by the anti‐centrosome antibody.
Identification of an early expressed marker of the luminal membrane of rabbit small intestinal columnar cells. Presence of a homologous antigen in kidney proximal tubules and glomeruli
Authors: Gorvel J. P. , Rigal A. , Olive D. , Mawas C. , Maroux S.
Published: February 2012

Four monoclonal antibodies have been produced that specifically react with a rabbit intestinal brush‐border glycoprotein which migrates in SDS‐polyacrylamide gel electrophoresis as a protein of molecular weight 140,000. Contrarily to brush‐border hydrolases, it is expressed in poorly differentiated crypt cells of the small intestine. Absent from colon columnar cells, it can be considered to be an early marker of differentiation of absorbing cells of the small intestine. Immunofluorescence technique showed the presence of a homologous antigen in kidney proximal tubule brush‐border and podocytes of kidney glomeruli.
Ultrastructural analysis of the in vitro differentiation of female rat preadipocytes
Authors: Gregoire F. , Hauser‐Gunsbourg N. , Remacle C.
Published: February 2012

In an attempt to characterize the preadipocytes of the adipose tissue of female rat, we studied by electron microscopy the differentiation of the cells into mature adipocytes in in vitro cultures. The preadipocytes arose from the stroma‐vascular fraction of perirenal and perigenital adipose tissue. Culture of the preadipocytes in an enriched medium consisting of Dulbecco's medium supplemented with 10% fetal calf serum, antibiotics, rat triglycerides (0.5%), insulin (290 nM) and Tween 80 (0.1 mg/ml) induced their adipose conversion. The morphology of preadipocytes changed progressively. They accumulated fat granules, droplets and finally globules, which fused together. The cell organelles featured qualitative and quantitative modifications. The nucleus migrated with most mitochondria and a part of the Golgi system towards the cell periphery; the rough endoplasmic reticulum, dilated at the initial stage of differentiation became less and less conspicuous; the perinuclear Golgi system was dispersed between lipid droplets during fat accumulation; thick bundles of microfilaments, localized beneath the plasma membrane disappeared; large lipid droplets were surrounded by a network of microfilaments; many microvesicles and some “rosettes” typical of mature adipocytes could be observed. Nevertheless, the ultrastructural criteria did not allow to clearly discriminate the undifferentiated cells: early preadipocytes (without lipid droplets), adipoblasts and fibroblasts, all of these being probably present in the culture system.
Inhibitory effect of sodium arsenite and azide on asialoglycoprotein receptor mediated endocytosis in suspended rat hepatocytes
Authors: Scarmato P. , Durand G. , Agneray J. , Feger J.
Published: February 2012

The inhibitory effect of sodium arsenite and azide on asialoorosomucoid endocytosis was tested using isolated rat hepatocytes. Under either continuous flux conditions or a single synchronous wave of ligand endocytosis we confirm that azide inhibits the recycling of the receptors and we provide evidence for the involvement of thiol groups in the internalization step. In addition pretreatment of hepatocytes with azide allows us to demonstrate that receptor endocytosis proceeds independently of the presence of any specific ligand.
Mitochondrial analysis in living cells: the use of rhodamine 123 and flow cytometry
Authors: Ronot X. , Benel L. , Adolphe M. , Mounolou J. C.
Published: February 2012

Flow cytometry combines the advantages of microscopy and biochemical analysis in a single highly sensitive technique for a rapid examination of numerous individual living cells. It has become a potent and essential tool in the studies of the physiology of the whole cell and its organelles. Rhodamine 123 is a vital fluorescent dye used in flow cytometry. As it is specifically concentrated in mitochondria because of the transmembrane potential that these organelles maintain in living cells, rhodamine 123 is thus a useful probe for monitoring the abundance and activity of mitochondria. A critical survey of the routine use of rhodamine 123 together with flow cytometry in mitochondrial research is presented.
Human myotube differentiation in vitro in different culture conditions
Authors: Delaporte C. , Dautreaux B. , Fardeau M.
Published: February 2012

Human muscle cells derived from satellite cells, maintained in standard tissue culture conditions, do not differentiate as rapidly or as completely as myoblasts from other species (chicken, rat, mouse). In an attempt to improve myogenesis, we studied the effects of modifying the culture media and of coculturing muscle with nerve cells, using myoblasts grown in standard culture media as the basis for comparison. Myogenesis was measured by fusion index, creatine kinase (CK) activity; acetylcholinesterase (AChE) activity (total and molecular forms); and the number of acetylcholine receptors (AChR). Modification of culture media accelerated fusion of myoblasts, but the cell density decreased and myotubes were unable to survive for long periods. In contrast, coculturing muscle with nerve cells increased both cell density and the number of myotubes. CK, AChE and AChR increased in the presence of defined media. In the nerve‐muscle cocultures the increase was less marked. Manipulating culture conditions modified the molecular forms of AChE. Only a (4 + 6.5) S peak was present in control cultures, but a 10S peak appeared in defined media. The 16S form was detected only in nerve‐muscle cocultures. This study shows that fusion of human myoblasts and differentiation of myotubes in tissue culture can be accelerated by removal of serum macromolecules. Further differentiation of myotubes was achieved only in the nerve‐muscle cocultures.
Polarity reversal of inside‐out thyroid follicles cultured within collagen gel: an ultrastructural study
Authors: Barriere H. , Chambard M. , Mauchamp J. , Gabrion J.
Published: February 2012

Inside‐out porcine thyroid follicles in culture undergo polarity reversal after being embedded in collagen gel. The newly‐formed follicles reexpress some specific thyroid functions lost in inside‐out follicles (Chambard et al., 1984. We present here an ultrastructural study of the inversion of polarity in this model system. This process takes place within 24 to 48 hr, without any opening of the original tight junctions, as shown by fixation in the presence of ruthenium red. A general shrinkage of cellular aggregates was noted soon after embedding. At the apical pole, three different modifications were observed: structural changes appeared in the kinocilium, microvilli and underlying cytoskeleton as early as 10 min after embedding, mainly when the apical pole of the cells was in close contact with the collagen fibers; large cytoplasmic lamellipod‐ or pseudopod‐like extensions, covering the adjacent apical domain, protruded from outer apical regions; some other apical areas invaginated and formed channels inside the aggregates. The last two processes prevented close contact between apical cell surfaces and collagen fibers and allowed a persistence of the initial polarity in some of the cells. Newly‐formed lumens were closed 24 hr after embedding in gel and the outer surface of the cellular aggregates in close contact with collagen fibers looked like a basal membrane. These mechanisms proceeded at different rates and involved different numbers of cells, but they all appeared to be related to the transformation of inside‐out follicles into follicular structures.
Cytological and ultrastructural changes in the pituitary pars distalis of the goldfish kept in calcium‐free environments
Authors: Olivereau M. , Chambolle P. , Dubourg P. , Olivereau J.
Published: February 2012

The cytology and ultrastructure of the pars distalis, mainly that of prolactin (PRL) cells, were investigated in goldfish adapted to fresh water (FW) or deionized water (DW) for 20 and 40 days, or gradually adapted to 1/3 artificial sea water (ASW) or 1/3 Ca‐free sea water. When compared to PRL cells of goldfish kept in FW, those of goldfish adapted to DW did not show signs of increased activity. The lack of exocytotic activity and the low development of various organelles suggested that cell activity was slightly reduced. In 1/3 ASW, PRL cells were smaller and less active. In 1/3 Ca‐free ASW, PRL cells appeared slightly stimulated compared with those of fish in 1/3 ASW. The Golgi area was more developed and a few lamellae of endoplasmic reticulum were observed in some cell islets. However, there was no significant difference between PRL cells of goldfish kept in 1/3 Ca‐free ASW and in FW. In 1/3 ASW, which is isosmotic to the blood, thyrotrophs (TSH cells) corticotrophs (ACTH cells) and somatotrophs (STH cells) were not clearly affected. In DW, these cells and their nuclei were significantly enlarged. Their stimulation was also evident in 1/3 Ca‐free ASW; values for cellular and nuclear areas were maximal in this environment and significantly higher than those of fish in FW and 1/3 ASW. These data suggest that in addition to the PAS‐positive cells of the pars intermedia, highly stimulated in Ca‐free environments, other cell types of the pars distalis may be involved in osmoregulation, and that the role of PRL cells is not primordial in the goldfish.
Redistribution of nuclear lamins in mitotic cells
Authors: Jost E. , Lepper K. , Högner D. , Zimmer A. , Boschek B.
Published: February 2012

The nuclear lamins are directed from the cytoplasm to chromosomes as part of the maturation pathway of the interphase nucleoskeleton. In mitosis, the three polypeptides lamin A, B and C were found in the cytoplasm from prophase until anaphase and shifted to chromosomal surfaces at telophase (Ely, D'Arcy and Jost, 1978; Gerace, Blum and Blobel, 1978). We show here that early events in nucleoskeleton formation could be regulated by extracellular pH. When exponentially growing tissue culture cells and cells arrested in mitosis were exposed to different extracellular pH values, three patterns of distribution of lamins were observed in mitotic cells: exclusively cytoplasmic distribution of mitotic lamins at low pH (6.8 to 7.3); a premature association of a lamin subfraction with metaphase chromosomes at intermediate pH 7.5; a more prominent relocation of lamins onto chromosomes in metaphase and in disorganized metaphase at pH 8.0. Reassembly of lamins occurred at telomeric ends of mitotic chromosomes followed by a lateral fusion to form a nuclear cage. Using immunogold localization, we show that pH‐induced, premature, partial deposition of lamins onto condensed chromosomes may occur prior to the formation of the bilamellar nuclear envelope. These results suggest that the pH‐induced redistribution of lamins acts to trigger early events of mitosis to interphase transition.
Lysosomal arylsulfatases A and B from horse blood leukocytes: purification and physico‐chemical properties
Authors: Wojczyk B.
Published: February 2012

Lysosomal arylsulfatases A and B (aryl‐sulfate sulfohydrolases, EC 3.1.6.1) from horse leukocytes were purified about 680‐fold and 70‐fold, respectively, starting from a crude extract of the azurophil and specific granules of leukocytes, by affinity, ion exchange, and gel filtration chromatography. Purified arylsulfatase A displayed anomalous kinetics, a pH optimum at 5.2, an isoelectric point at 4.3, and a Km value for p‐nitrocatechol sulfate (pNCS) of 0.37 mM. This enzyme was found to exist in two association states depending on pH: a high molecular weight form at pH 5.0 and a low molecular weight form at pH 7.5. Arylsulfatase B displayed normal kinetics, a pH optimum at 5.8, two isoelectric points at pH 8.6 and 8.9, and a Km value for pNCS of 3.38 mM. The thermostability of the two enzymes was different: arylsulfatase B was found to be more stable than arylsulfatase A. Arylsulfatase A was inhibited by sulfate, sulfite, silver, magnesium, manganese and calcium ions and arylsulfatase B by chloride, sulfate, sulfite and silver ions.
Fibronectin (FN) localizations in early wound healing of cultured frog skins
Authors: Zhu Q. L. , Denefle J. P. , Lechaire J. P.
Published: February 2012

The wound healing process of frog skin fragments in epibolic cultures has provided information on FN localizations during the migration of keratinocytes. Mainly two FN localizations were studied by indirect immunodetections: Epidermal localization around keratinocytes which have acquired a fibroblastic shape. Dermal localizations of the sectioned collagen of the stratum spongiosum and stratum compactum detected at the beginning of the culture. Both localizations were observed in this epibolic wound healing process during 6 hr and 24 hr in culture and showed a differential sensitivity to cycloheximide (CHX). It was worth noting that fibronectin was permanently detected in the subcutaneous tissue of non‐cultured or cultured skin fragments with or without CHX.
The use of Ultroser G as a serum substitute in the culture of human skin fibroblasts
Authors: Myara I. , Polini G. , Scotto J. , Lemonnier A. , Gautier M.
Published: February 2012

Growth of human skin fibroblasts was dramatically enhanced when serum in the culture medium was replaced by Ultroser G. Compared to the same cells cultured in the presence of serum, alterations in glucose and lipid metabolism and an increase in the activity of prolidase (EC 3.4.13.9) and prolinase (EC 3.4.13.8) were also observed. Consequently, we advise extreme caution in the use of Ultroser G in metabolic studies, especially for periods of culture exceeding 10 days. However, Ultroser G can help to produce a large number of cells and so facilitate purification of the proteins produced during the stationary growth phase.