bluuebunny/crossref_metadata_2025 huggingface dataset:
Low tumorigenicity of canine cells transformed by the human cytomegalovirus
Authors: Yelle Jocelyn , Lussier Gilles , Pramatarova Albena , Hamelin Claude
Published: August 2004

Summary—

Dog embryo kidney cells transformed by the human cytomegalovirus (HCMV) were obtained after non‐permissive infection or transfection with viral DNA digested by restriction endonuclease EcoR I. The transformed cells, growing rapidly and showing an unlimited division potential, could use medium with only 2% serum for growth, contained nuclear virus antigens, and formed small colonies (< 0.2 mm) in agarose. From 40 mice inoculated with transformed canine cells, only one eventually developed a tumor. Results indicate that dog cells are immortalized but not tumorigenically transformed by the human cytomegalovirus.
Intermitochondrial junctions in a subpopulation of peripheral blood lymphocytes from healthy subjects*
Authors: Richard Stéphane , Masse Jean‐Marc , Guichard Josette , Breton‐Gorius Jeannine
Published: August 2004

Summary—

This paper reports the observation of intermitochondrial junctions (IMJ) with a periodicity of 15.4 ± 0.65 nm in peripheral blood lymphocytes of healthy subjects. The percentage of IMJ‐containing cells appears constant (about 10% of examined lymphocytes) and is independent of the delay of fixation. Immunogold staining reveals that lymphocytes with IMJ exhibit a T‐phenotype. IMJ have been reported in other types of tissues but, to our knowledge, have not been previously described in the blood cell. The signification of these structures is discussed.
An efficient isolation procedure of Ca‐tolerant ventricular myocytes from ferret heart for applications in electrophysiological studies
Authors: Bouron Alexandre , Potreau Daniel , Besse Colette , Raymond Guy
Published: August 2004

Summary— A simple procedure which provides a large yield of isolated ferret ventricular myocytes is described. The enzymatic dissociation was performed by perfusion of the whole heart with the “Langendorff method” at 37°C, without an incubation period. Special attention was given to the period of perfusion with Ca‐free or low‐calcium containing solutions and to the proportion of both collagenase and elastase used. The viability and calcium tolerance of the isolated cells were tested by ultrastructural and electrophysiological studies. Photo‐microscopy showed that 60 to 80% of the isolated cells had an elongated shape (18 μm in diameter, 150 μm in length) and did not beat spontaneously in normal Tyrode solution. The morphological and ultrastructural integrity of these cells was shown in SEM by their smooth surface with regularly spaced T‐tubule openings and in TEM by the regular distribution of the transverse tubular system, mitochondrium and sarcomeres. Using the whole‐cell patch‐clamp technique, they had a resting membrane potential of −72 mV, two types (“Purkinje like” and “ventricular like”) of action potentials could be elicited and they were correctly affected by well‐known modulators of calcium channels. This technique was successfully applied to the rat heart and could be used for heart dissociation of small mammals. It can simultaneously provide isolated cells of different regions of the heart and can be easily and routinely used by any investigator.
Effects of a series of homologous α,ω‐dimethylaminoalkanes on cell proliferation: binding and uptake of putrescine by a human glioblastoma cell line (U251) in culture
Authors: Quemener Véronique , Moulinoux Jacques‐Philippe , Khan Naim Aktar , Seiler Nikolaus
Published: August 2004

Summary— The first step of polyamine uptake is the binding of polyamines to the cell membrane. In order to characterize the specificity of the putrescine binding sites at the surface of the glioblastoma cells (U251), we have carried out competition experiments between putrescine bound to latex microspheres and vizualized by scanning electron microscopy and a series of N,N'‐tetramethyl‐α, ω‐diaminoalkanes. N,N'‐tetramethyl‐1,4‐butanediamine (N,N'‐tetramethylputrescine) and higher homologs inhibit the latex putrescine binding to the cell surface and concomitantly cell proliferation. [14C] putrescine uptake was mainly inhibited by the lower homologs, which were devoid of antiproliferative effects. Our results suggest that putrescine uptake by the human glioblastoma cell line U251, and putrescine binding to the surface of these cells are independent processes. The potential relationship between antitumor effect of N,N'‐tetramethyl‐α,ω‐diaminoalkanes and its binding to a specific putrescine acceptor site is discussed.
Microcalorimetric investigations on human leukemia cells — Molt 4
Authors: Nittinger Jürgen , Tejmar‐Kolar Liana , Fürst Peter
Published: August 2004

Summary— Heat production by leukemia cells Molt 4, growing in suspensions with 1 and 3 × 105 cells/ml for 4 days, was studied by microcalorimetry. Heat production rates were related to cell growth, glucose consumption, lactate production and cellular ATP‐content. The results show that the time course of heat dissipation is dependent on initial cell number. However, observed thermal power maxima were fairly identical in all experiments. Heat production rates per cell were similar during the initial phase of the study independently of initial cell number, while higher cell densities resulted in significantly lower rate of heat production. Glycolytic conversion of glucose into lactate is nearly stoichiometric. Our results indicate a relationship between heat production and amounts of glucose and lactate in the medium. ATP concentration in cells decreased after 24 hours of culture.
Ultrastructural localization of fibronectin mRNA in chick embryo by in situ hybridization using 35S or biotin labeled cDNA probes
Authors: le Guellec Dominique , Frappart Lucien , Willems Réjane
Published: August 2004

Summary— We have studied the expression of the fibronectin gene in 7 day‐old chick embryo (stage 32) by in situ hybridization at the light and electron microscope levels, using a 397 base‐pairs chicken cDNA, labeled by radioisotope or biotin‐11dUTP. Cryostat sections of whole chick embryos displayed a selective label on the upper layer of the dermis, fibrous sclera and mesenchymal cells but not on cartilagenous sclera cells. These results show that the expression of the fibronectin gene varies in relation to the morphogenetic events. Hybridization at the ultrastructural level on thin sections of sclera embedded in Lowicryl K4M showed a selective labeling on various cell compartments. Biotin‐11dUTP and radiolabeled probes were compared. The labeling was found precisely on the membrane of the rough endoplasmic reticulum and on the nuclear envelope. A few silver grains were located on the nucleus and in the perinucleolar region. This study shows that the postembedding in situ hybridization is a powerful procedure to study the expression of the extracellular protein genes and gives further information on the localization of mRNA.
The role of pH in the intracellular acetylcholine receptor ligand processing
Authors: Park J , Bursztajn S
Published: August 2004

Summary— Previous studies have shown that the internalized AChRs are transported through many vesicular compartments: Golgi associated vesicles, coated vesicles, smooth vesicles, endosome‐like structures and lysosomes. These compartments have an acidic pH ranging from 4.5 to 6.5. The pH differences between organelles suggests that these differences may influence the sorting and final expression of AChRs. To test this hypothesis, we measured the number of counts of 125I‐αBTX or 125I‐Mab35 dissociated from myotube membranes containing AChRs as a function of pH. Neither the 125I‐αBTX nor 125I‐Mab35 showed an enhanced dissociation in the pH range 4.0–7.0, whereas lowering the pH to 6.0 or below enhanced the dissociation of 125I‐α 2‐macroglobulin from myotubes. In other experiments using Torpedo membrane we showed that neither 125I‐αBTX nor 125I‐Mab35 appreciably dissociated from the AChR unless the pH was less than 4 or above 11. Double‐label studies using a novel membrane permeable acidotropic molecule DAMP (3‐(2,4 nitroanilino) 3′amino‐N‐methyl‐dipropylamine), facilitated mapping the pH of the intracellular compartments containing internalized AChRs. This molecule accumulates inside acidic compartments in the cell and has a dinitrophenol (DNP) group recognized by DNP specific antibodies. Cells were treated with 30 μg DAMP for 30 min and allowed to internalize Mab35‐gold (15 nm) for various periods (0−15 h). At each time point we fixed and washed the cells, and incubated with anti‐DNP monoclonal antibodies followed by incubation with anti‐mouse IgG and protein A colloidal gold (5 nm). Different sized gold particles allowed us to simultaneously identify the AChR compartments and estimate their pH. Sister cultures were exposed to acidotropic drugs to destroy pH gradients. Under those conditions, AChR delivery to lysosomes was blocked. Our studies show that AChRs are transported through acidic compartments ranging from pH 4.5 to 6.5 and in contrast to other ligands they do not dissociate from the intracellular membranes at low pH.
Sample preparation of animal tissues and cell cultures for secondary ion mass spectrometry (SIMS) microscopy
Authors: Chandra Subhash , Morrison George H.
Published: August 2004

Summary—

Sample preparation is a critical step in the elemental analysis of animal tissues and cell cultures with ion microscopy. Since live cells cannot be analyzed with ion microscopy, a careful sample fixation is necessary which preserves the native structural and chemical integrity of a specimen. The evaluation of morphological and chemical integrity of a fixed specimen is necessary before any physiological explanation of ion fluxes is interpreted based on ion microscopy. For diffusible ion localization studies, strict cryogenic procedures are recommended. Examples are shown for diffusible ion microanalysis in frozen‐freeze‐dried tissues and cell cultures. Ion microscopy studies of tightly bound elements/molecules may be conducted in chemically fixed and/or plastic embedded specimens. Since it is not generally known which elements/molecules are tightly bound to the tissue matrix, a confirmation of elemental distribution with cryogenic procedures is desirable. A recent approach of combining laser scanning confocal fluorescence microscopy and ion microscopy on the same frozen freeze‐dried cell is also discussed for recognizing smaller cytoplasmic structures in ion microscopy images.
Scanning secondary ion analytical microscopy with parallel detection
Authors: Slodzian Georges , Daigne Bernard , Girard François , Boust Fabrice , Hillion François
Published: August 2004

Summary—

The secondary ion microscope described here allows to obtain the simultaneous registration of chemical and isotopic distribution maps of several elements composing the sample. The instrument has been specially designed to optimize both sensitivity and selectivity: bombardment with primary Cs+ ions to increase the ionization yields of negative secondary ions, efficient collection of secondary ions at the target surface, matching of the secondary ion beam etendue with the acceptance of the mass spectrometer working at high mass resolution, spectrometer with parallel detection capabilities. The probe diameter can be made as low as 30 nm and ion induced electron images registered at the same time as ion images. Presently, four ion micrographs are obtained simultaneously over a field of view up to 20 × 20 μm2 containing up to 512 × 512 pixels. Examples are shown with an ion probe diameter of 0.1 μm.
SIMS microscopy: a tool to measure the intracellular concentration of carbon 14‐labelled molecules
Authors: Hindie Elif , Coulomb Bernard , Galle Pierre
Published: August 2004

Summary—

Monolayer cultures of human fibroblasts were incubated for 24 h with 14C‐arginine and observed by means of SIMS microscopy (ion microscopy). Carbon 14 imaging showed the intracellular distribution of labelled arginine which featured high nuclear incorporation. The local concentration of this amino acid in different cells and intracellular structures was assessed through local isotopic 14C/12C ratio measurement. This relates the signal intensity of the labelling isotope carbon 14 to that of the corresponding natural isotope (carbon 12) of known tissular concentration. Using this method we were able to measure minor variations in the molecular concentration of arginine (expressed as μmol/g of tissue) between different fibroblasts. Results of this study indicate that SIMS microscopy is well adapted to carbon 14 detection and can provide quantitative maps of the cellular and subcellular distribution of 14C‐labelled molecules.
Mapping of intracellular halogenous molecules by low and high resolution SIMS microscopy
Authors: Berry Jean‐Pierre , Galle Pierre , Chassoux Danièle , Escaig Françoise , Linarez‐Cruz L. Gustavo , Lespinats Genevieve
Published: August 2004

Summary—

The subcellular distribution of halogenous molecules has been studied by SIMS microscopy in cultured cells of a human breast carcinoma (MCF‐7 cell line). Two instruments of microanalysis were used. A low lateral resolution ion microscope (SMI 300 CAMECA) and a prototype scanning ion microscope equipped with a cesium gun that gives high lateral resolution images. This apparatus has been developed by G Slodzian, in Onera Laboratories (Office National d'Etudes et de Recherches Aérospatiales). Molecules studied by low lateral resolution ion microscope were halogenous steroids: fluorometholone, triamcinolone, bromocriptine and bromoandrosterone. Analytical images show that the first two compounds are mainly localized in the nuclear structure of MCF‐7 cells whereas the last two molecules are localized in cytoplasm of these cells. Images were obtained with a resolution of 1 μm. With the scanning ion microscope, it is now possible to obtain images at the ultrastructural level. Four analytical images can be simultaneously obtained by a single scan of the imaged area, corresponding to a depth of erosion of the section of ten nm. The intranuclear distributions of three pyrimidine analogs, 5‐bromo‐2′‐deoxyuridine, 5‐iodo‐2′‐deoxyuridine and 5‐fluorouracil have been studied in phase S and M of MCF‐7 cells and these images have been compared to the distribution of sulfur, nitrogen and phosphorus. All these images have been obtained with a lateral resolution better that 100 nm.
Intracellular boron localization and uptake in cell cultures using imaging secondary ion mass spectrometry (ion microscopy) for neutron capture therapy for cancer
Authors: Bennett Brian D. , Zha Xiaohui , Gay Isabelle , Morrison George H.
Published: August 2004

Summary—

Quantitative ion microscopy of freeze‐fractured, freeze‐dried cultured cells is a technique for single cell and subcellular elemental analysis [1–3]. This review describes the technique and its usefulness in determining the uptake and subcellular distribution of the boron from boron neutron capture therapy drugs.
SIMS determination of the distribution of the main mineral cations in the depth of the cuticle and the pecto‐cellulosic wall of epidermal cells of flax stems: problems encountered with SIMS depth profiling
Authors: Ripoll Camille , Jauneau Alain , Lefèbvre Fabrice , Demarty Maurice , Thellier Michel
Published: August 2004

Summary—

Depth profiles of C, Na, Mg, Al, K and Ca were performed in the cuticle and wall of epidermal cells of flax hypocotyls, with current densities ranging from 0.2 to 1 pA μm−2. The crater bottoms were never flat, but exhibited fairly complex, filiform or alveolar structures. The profiles of K, Ca and Mg were reasonably parallel to one another. The Ca/Mg signal ratio was in the magnitude of 3.5 in the cuticle. The Na profile, except perhaps in the cuticle, did not parallel the K, Ca and Mg profiles, but rather paralleled the C profile. At the outset of the depth profiles, ie in the cuticle, the intensity of the Na signal, although fairly variable, was usually above that of K; then there was an abrupt decrease of the Na signal, possibly at the border of the cuticle and of the wall. The Al signal usually began to increase, thus revealing the occurrence of perforations through the epidermis sample, after 80 min sputtering at a current density of 1 pA μm−2; the mean sputtering rate was thus estimated to be in the order of 1 μm h−1.
The role of secondary ion mass spectrometry (SIMS) in biological microanalysis: technique comparisons and prospects
Authors: Linton Richard W. , Goldsmith Jack G.
Published: August 2004

Summary—

The virtues and limitations of SIMS ion microscopy are compared with other spectroscopic techniques applicable to biological microanalysis, with a special emphasis on techniques for elemental localization in biological tissue (electron, X‐ray, laser, nuclear, ion microprobes). Principal advantages of SIMS include high detection sensitivity, high depth resolution, isotope specificity, and possibilities for three‐dimensional imaging. Current limitations, especially in comparison to X‐ray microanalysis, center on lateral spatial resolution and quantification. Recent SIMS instrumentation advances involving field emission liquid metal ion sources and laser post‐ionization will help to minimize these limitations in the future. The molecular surface analysis capabilities of static SIMS, especially with the new developments in commercial time‐of‐flight spectrometers, are promising for application to biomimetic, biomaterials, and biological tissue or cell surfaces. However, the direct microchemical imaging of biomolecules in tissue samples using SIMS will be hindered by limited concentrations, small analytical volumes, and the inefficiencies of converting surface molecules to structurally significant gas phase ions. Indirect detection using elemental or isotopically tagged molecules, however, shows considerable promise for molecular imaging studies using SIMS ion microscopy.
Insulin, glucagon and somatostatin in fetal rat islets maintained free‐floating in culture: immunocytochemistry and radioimmunoassay
Authors: Leduque Patrick , Scharfmann Raphael , Chayvialle Jean‐Alain , Aratan‐Spire Sonia , Basmaciogullari Annie , Czernichow Paul , Dubois Paul‐M
Published: August 2004

Summary— Fetal rat islets maintained free‐floating in tissue culture represent a source of B‐cells. Because we recently noted the occurrence of other cell types during long‐term tissue culture, this in vitro model was used to examine the possible development of non B‐cells. The changes in the numbers and percentages of B, A and D‐cells in vitro were estimated by counting the hormone‐positive cells after immunocytochemical staining. Insulin, glucagon, and somatostatin contents were determined in extracts of the cultured tissue. The experiments described here showed that the cultured islets maintained their viability over a two‐week culture period, as evidenced by the increase of both the number of B‐cells per islet and the DNA content per islet. During the first few days of culture, immunocytochemically stained free‐floating islets indicated the presence of rare A‐ and D‐cells at the periphery of B‐cells; thereafter, numerous A‐ and D‐cells were seen interdigitating with B‐cells. Expressed per islet, the number of A‐ and D‐cells increased during the culture; within the endocrine cell population, the percentage of these cells increased with time, at the expense of the percentage of B‐cells. The glucagon and somatostatin contents of the free‐floating islets were also increased. These converging observations suggest that additional non B‐cells may have been produced by free‐floating islets during long‐term tissue culture.
Target cell deformability determines the type of phagocytic mechanism used by Entamoeba histolytica‐like, Laredo strain
Authors: Lejeune Agne´s , Gicquaud Claude
Published: August 2004

Summary— Morphological study of red blood cell phagocytosis by Entamoeba histolytica‐like (Laredo strain) has shown that this amoeba is able to ingest by two distinct mechanisms. One is classical phagocytosis and the other is by suction or microphagocytosis. Rigidification of red blood cells by treatment with glutaraldehyde shows that there is a correlation between the deformability of the ingested cell and the type of phagocytosis observed. Indeed, as the red cells become more rigid, less microphagocytosis is observed. To demonstrate that this shift in phagocytic mechanisms is not induced by the modification of a surface receptor by the glutaraldehyde treatment, the amoebas were fed with erythrocyte ghosts. Since these have lost most of their hemoglobin content, they are less rigid than the intact erythrocytes. The ghosts, even after glutaraldehyde treatment, are always ingested by microphagocytosis. These results have therefore led us to conclude that the type of erythrocyte phagocytosis used by E histolytica‐like (Laredo strain) is determined by the deformability of the targetted red blood cells.
Comparative studies of protein biosynthesis: the main experimental parameters in pulse and pulse‐chase experiments must be standardized
Authors: Appel Martine , Couderc Elisabeth , Feger Jeanne
Published: August 2004

Summary— The influence of experimental conditions was investigated in pulse and pulse‐chase experiments involving l‐[35s]methionine incorporation by isolated rat hepatocytes. The incorporation of the labelled amino acid must be linear as a function of dose and time, and similar hepatocyte densities should be used to compare radioactive uptake. High hepatocyte densities give greater precision. In pulse‐chase experiments, it was shown that a pool of free radioactivity moves progressively during the chase phase from the intracellular compartment. Protein‐associated radioactivity must therefore be expressed as a function of both total radioactive uptake and total labelled protein.
A radioautographic study of [3h]‐A 23187 (calcimycin) in components of the brain. Distribution in different cell types of the diencephalo‐mesencephalic roof
Authors: Meiniel Robert , Didier Robert , Meiniel Annie , Jeminet Georges , Prudhomme Michelle
Published: August 2004

Summary— The distribution of the ionophore [3h]‐A 23187 was examined by means of light and electron microscopy in elements of the central nervous system located in the diencephalo‐mesencephalic roof. A 23187 is not evenly distributed in the components studied (ependyma, secretory ependyma of the subcommissural organ and neurons of the mesencephalon). At the cellular level, A 23187 appears preferentially associated with the cytoplasmic membrane as well as with the internal membranous system.
Early and late replicating DNA involved in the G1 to S transition in Allium cepa L meristematic cells
Authors: Gonzalez‐Fernandez Aurora , Aller Patricio , Sans Jorge , de la Torre Consuelo
Published: August 2004

Summary— The involvement of portions of the genome replicated at different times of the S period in the regulation of the G1 to S transition was analyzed in Allium cepa L meristem cells. For this, DNA bromosubstitution confined to discrete portions of a previous S period followed by anoxic UVA irradiation (300–400 nm light) was performed in synchronous cells. Sequences replicated in late S appeared to be involved in the positive regulation of the initiation of replication. Hence, cells were prevented from initiating replication if irradiated at mid G1 only when the DNA sequences replicated in the last third of the previous S period were bromosubstituted. Cycloheximide‐induced inhibition of protein synthesis at late G1 also prevented the G1 to S transition. Sequences replicated in mid S appeared unrelated to any control of the initiation of replication. On the other hand, sequences replicated in the first third of the S period seemed to be involved in the negative regulation of the initiation of replication, since irradiation after previous bromosubstitution of early replicating DNA sequences advanced G1 cells into the next S phase and increased the proliferative fraction of the population. Finally, the simultaneous inactivation of DNA sequences involved in both positive and negative regulation of replication allowed the cells to enter into S.
Pattern of arborization of the motor nerve terminals in the fast and slow mammalian muscles
Authors: Tomas Josep , Santafe´ Manel , Fenoll Rosa , Mayayo Emilio , Batlle Jordi , Lanuza Angel , Piera Veronica
Published: August 2004

Summary— A silver impregnation method and a morphometric approach were used to define differences existing in the motor nerve terminal branching pattern between a fast‐twitch muscle (extensor digitorum longus) and a slow‐twitch one (soleus) of the normal adult rat. Because no single measure can describe precisely all geometrical properties (ie both topology and metrics) of the nerve terminals, we evaluated morphologic parameters defining length and angular characteristics in the different terminal segments classified according to their centrifugal order. The main results indicate that the distal free‐end segments in the extensor digitorum longus muscle are shorter and less divergent than in the soleus nerve terminals. The endings in the two muscles have different fractal dimensions. Findings are discussed in the context of the hypothetical mechanisms governing motor nerve terminal size and complexity.
Reversible dissociation of the plant golgi apparatus by brefeldin A
Authors: Satiat‐Jeunemaitre Be´atrice , Hawes Chris
Published: August 2004

Summary— The effects of the drug Brefeldin A, shown to block the translocation of proteins between the endoplasmic reticulum and the Golgi apparatus in animal cells, were studied on different plant cell systems. In suspension culture cells and root tissues, the Golgi aparatus was affected by Brefeldin A treatments resulting in distortion and dissociation of the Golgi stacks, coupled with appearance of numerous vesicles in the cytoplasm. This process was reversible. Therefore, Brefeldin A provides a powerful tool with which to study Golgi dynamics and function in plant as well as in animal cells.
Ultrastructural immunodetection of a Pichia anomala killer toxin: a preliminary study
Authors: Cailliez Jean‐Charles , Gerloni Mara , Morace Giulia , Conti Stefania , Cantelli Cristina , Polonelli Luciano
Published: August 2004

Summary— A monoclonal antibody (mAb KT4), produced against a Pichia anomala killer toxin, was used to study the secretion process of toxin producing cells. The indirect immunofluorescence assay, performed with large concentrations of mAb KT4, showed a homogeneous distribution of the epitope at the cell surface of the P anomala cells. When increasing dilutions of mAb KT4 were employed, a ‘punctuated’ labeling appeared on the yeast's cell wall which suggested a heterogeneous secretion of the killer toxin. Similar labeling was also observed by immunodetection on live yeast cells held in buffered suspension. These results confirmed that 'punctuated' labeling was not an artefact due to a distortion of the cell's shape by having been dried on glass slides. Indirect immunodetection was performed in electron microscopy on ultra‐thin sections of cells embedded in Araldite resin. The labeling thus obtained showed both the presence of the epitope in the cytoplasm and its sensitivity to strong glutaraldehyde fixation. Indirect immunodetection, performed on ultra‐thin frozen sections, showed a cytoplasmic and cell wall labelling. However, the amount of gold particles observed in the cell wall was too low to confirm the heterogeneous killer toxin secretion observed in immunofluorescence. In this case, killer cells were fixed with a low concentration of glutaraldehyde which preserved the structure of the epitope complementary with mAb KT4.
Effect of reduced temperatures and brefeldin A on prolactin secretion and on subcellular distribution of the secretory product and membrane antigens in GH3 pituitary cells*
Authors: Nasciutti Luiz‐Eurico , Picart Renée , Rosenbaum Etelka , Tixier‐Vidal Andrée , Tougard Claude
Published: August 2004

Summary— The effects of reduced temperatures (20, 15 or 10°C) and brefeldin A (BFA) on prolactin (PRL) secretion in the GH3 rat pituitary cell line have been compared. Both treatments inhibit PRL release to different extents. Ultrastructural immunocytochemistry reveals that, depending on the treatment, PRL is blocked at different steps during its intracellular transit. The temperatures of 20 and 15°C block the PRL transport at one face of the Golgi stacks whereas both the temperature of 10°C and BFA treatment induce an arrest of PRL at the level of the rough endoplasmic reticulum (RER) cisternae. Moreover, exposure to 10°C or BFA induces an accumulation of a specific Golgi membrane antigen in the dilated RER structures. However, although disorganized and no longer definable under BFA treatment, the Golgi apparatus remains visible at 10°C. These two last treatments cause also an increase in the number of partly rough, partly smooth tubular structures tentatively called ‘paired cisternae’.
Bombesin‐like immunoreactivity in eosinophils. A light and electron microscopic study: effect of fixatives and cross‐reactivity with various compounds
Authors: Soranzo Lydia , Ganem Yves , Chatelet François , Launay Jean‐Marie , Roland Jean
Published: August 2004

Summary— Eosinophils immunopositive for bombesin tetradecapeptide were detected by means of light and electron microscopy in human and rat gastrointestinal tract and pulmonary tissue. This immunoreaction was only evidenced after the use of acetic acid‐containing fixative such as Bouin's fluid. The dependence of this immunostaining on fixatives and time course were extensively studied. This immunoreaction promotes mainly one epitope probably associated with the C‐terminal sequence. This epitope seems also to be present in other neuropeptides such as substance P (SP) and, to a lesser extent in chemotactic factors like formyl peptide (fMLP) or eosinophil chemotactic factor of anaphylaxis (ECF‐A). At the electron microscopic level, the immunopositivity was associated with eosinophil membranes.
Differential temperature sensitivity of cultured cells from cartilaginous or bone origin
Authors: Flour Marie‐Pierre , Ronot Xavier , Vincent Françoise , Benoit Bénédicte , Adolphe Monique
Published: August 2004

Summary— The effects of long‐term exposure to hyperthermia were studied on several cell cultures of cartilaginous or bone origin after a 4‐day treatment at 40°C. Chondrocytes proliferation, as well as mitochondrial activity were not modified by these culture conditions (40°C) but protein content and cell volume were increased. In contrast, the proliferative capacity of osteoblasts, MC3T3.E1 and ROS 17/2.8 was decreased and their protein content, cell volume and mitochondrial activity were increased. Chondrocytes appeared to be thermoresistant, and osteoblastic cells thermosensitive. Furthermore, temperature sensitivity was greater for the continuous established osteoblastic cell line MC3T3. E1 and for the cancerous established osteoblastic cell line ROS 17/2.8 than for chondrocytes.
The ultrastructural study of the interphase cell nucleus of Lacandonia schismatica (Lacandoniaceae: Triuridales) reveals a non‐typical extranucleolar particle
Authors: Jiménez‐Garcia Luis Felipe , Agredano‐Moreno Lourdes Teresa , de Segura‐Valdez Maria Lourdes , Echeverria Olga Margarita , Martinez Esteban , Ramos Clara Hilda , Vázquez‐Nin Gerardo Hebert
Published: August 2004

Summary—

By light and electron microscope cytochemistry we characterized the interphase nucleus of Lacandonia schismatica, the only known species of the new plant family Lacandoniaceae, whose most peculiar feature is the inverted position of the sexual organs, an aspect never found before among flowering plants. Furthermore, we compare it to Triuris alata, a related species, to Voyria aphylla (a dicotyledon), to Gymnosiphon divaricatus (a monocotyledon) and also to saprophytes. The reticulated chromatin of L schismatica and T alata is similar to that of other monocotyledons. In addition, we describe a unique type of RNP granules in the interchromatin space which are about 32 ± 3 nm SD in diameter and occur as huge clusters. They are intermediate in size and spatial distribution between inter‐ and peri‐chromatin granules. We term them ‘Lacandonia granules’. The granules were also found in T alata. They are 31 ± 2 nm in diameter. No significant differences in size were observed between them (P > 0.05). Synaptonemallike complexes and ring‐shaped structures were seen in interphase nuclei of somatic cells of these species. Coiled and nucleolus‐associated bodies, as well as centromeres were also found in these two organisms. On the contrary, Vaphylla and G divaricatus display a chromocentric nuclear organization. The nuclear similarities between L schismatica and T alata suggest extremely close phylogenetic relationships between them.
The association of microtubules with the plasmalemma in epidermal tendon cells of the river crab
Authors: Nakazawa Eiko , Katoh Kazuo , Ishikawa Harunori
Published: August 2004

Summary—

The mode of association of microtubules (MTs) with the plasmalemma in epidermal tendon cells of the river crab, Potamon dehaani was studied by thin‐section electron microscopy. In the leg muscle, the tendon cells connect striated muscle cells with the cuticle, forming specialized junctions at both ends. At the muscle‐tendon cell junction, the apposed plasmalemmas are interdigitated in a zig‐zag pattern separated by a uniform space of about 50 nm, where the basal lamina is shared by two cells. At the tendon cell‐cuticle junction, the plasmalemma of the tendon cell forms many conical invaginations, into which dense fibrous material extends from the cuticle. Inside the tendon cell, numerous microtubules run parallel to the direction of tension transmission and are arranged into parallel bundles of various sizes. Within such bundles, fine filamentous structures cross‐link adjacent MTs. MTs span the entire length of the cell and attach at their both ends to the junctional domains of the plasmalemma. The junctional plasmalemma is characterized by formation of an electron‐dense undercoat, through which MTs are connected with the plasmalemma proper. The ultrastructural features of MT association with the plasmalemma are basically the same at both junctions. At the junctions, MTs usually terminate with free ends and are linked laterally to the plasmalemmal undercoat with fine filamentous structures. These observations emphasize the role of the plasmalemmal undercoat as a device of the attachment of MTs to the plasmalemma.
Alterations in erythrocyte membrane fluidity in children with trisomy 21: a fluorescence study
Authors: Kantar A. , Giorgi PL , Curatola G. , Fiorini R.
Published: August 2004

Summary—

Membrane fluidity of erythrocytes obtained from 15 children with trisomy 21 and 20 healthy controls were studied by measuring steady‐state fluorescence anisotropy and fluorescence lifetime of 1,6‐diphenyl‐1,3,5‐hexatriene (DPH) and 1‐(4‐trimethylammoniumphenyl)‐6‐phenyl‐1,3,5‐hexatriene (TMA‐DPH) incorporated in hemoglobin‐free erythrocyte membranes. Our results demonstrate a significant decrease in DPH fluorescence anisotropy and a significant increase in TMA‐DPH fluorescence anistropy in erythrocytes from subjects with trisomy 21. No significant differences between the two groups were observed in the fluorescence lifetime of DPH and TMA‐DPH. These data suggest an increase in membrane fluidity in the interior part of the membrane and a decrease in fluidity at the lipid‐water interface region. This could be in part attributed to an increased oxidative damage in trisomy 21.
Opposing effects of heparin with TGF‐β or aFGF during repair of a mechanical wound of human endothelium. Influence of cAMP on cell migration
Authors: Klein‐Soyer Claudine , Archipoff Gisèle , Beretz Alain , Cazenave Jean‐Pierre
Published: August 2004

Summary—

The effects on vascular wound repair in vitro of aFGF and TGF‐β, growth factors having opposite influences on endothelial cell growth and angiogenesis, were studied using as a model a mechanical lesion of confluent endothelium. Modulation by heparin of the activities of these growth factors during the repair process was also examined. Whereas heparin alone inhibited repair by lowering both cell proliferation and cell migration, TGF‐β alone mainly inhibited cell proliferation. When added together, TGF‐β and heparin exerted a combined inhibitory effect resulting in a residual lesion 50% larger than in controls. aFGF alone accelerated lesion coverage and this effect was enhanced by 40% over control values when heparin was added with aFGF. This acceleration was slightly (less than 10%) but consistently diminished by TGF‐β. Cell density in confluent unwounded areas was increased by 40% in the presence of aFGF, but TGF‐β diminished cell density by 20%. A small (30%) increase in intracellular cAMP was measured whenever aFGF was present during the repair process. In comparison, intracellular cAMP inducing agents (forskolin, dbcAMP) accelerated cell migration by 20% during lesion recovery without affecting cell proliferation or density. The present results show that the inhibitory effects of TGF‐β during vascular wound repair are opposed by aFGF. Furthermore, heparin (or heparan sulfates in vivo) modulates growth factors having activating or inhibiting functions and thus plays a regulatory role during the repair process. cAMP‐inducing substances other than growth factors are able to accelerate cell migration.
A hypothesis on p34cdc2 sequestration based on the existence of Ca2+‐coordinated changes in H+ and MPF activities during pus egg activation
Authors: Charbonneau Michel , Grandin Nathalie
Published: August 2004

Summary— The entry into, and exit from, mitosis are controlled by a universal M‐phase promoting factor (MPF) composed of at least p34cdc2 and a cyclin. Embryonic systems are convenient for studying the association and dissociation of the active MPF complex because oocytes and eggs are naturally arrested at a specific point of the cell cycle until progression to the next point is triggered by a hormonal signal or sperm. In amphibians, eggs prior to fertilization are arrested at metaphase 2 of meiosis due to the presence of a stabilized MPF complex. Fertilization (egg activation) produces a transient increase in intracellular free Ca2+, a propagating Ca2+ wave, that specifically triggers the destruction of cyclin, leading to MPF inactivation and entry into the first embryonic interphase. We have recently shown that intracellular pH (pHi) variations in amphibian eggs, a large increase at fertilization and small oscillations during the embryonic cell cycle, were temporally and functionally related to the corresponding changes in MPF activity. In addition, the recent finding that the pHi increase at fertilization in Xenopus eggs is a propagating, Ca2+‐dependent pH wave which closely follows the Ca2+ wave, together with the absence in the egg plasma membrane of pHi‐regulating systems responsible for that pHi increase, suggest the existence of cortical or subcortical vesicles acidifying in the wake of the Ca2+ wave, thus producing the pH wave. Given the known functions of intracellular acidic compartments in various systems and the existence of acidic vesicles in marine invertebrate eggs, we propose that these hypothetic acidic vesicles in Xenopus eggs might serve as a storage for the p34cdc2 released in the cytoplasm following egg activation, both the pHi increase and MPF inactivation being triggered and synchronized by the same signal, the Ca2+ wave.
The role of exocytosis in the apocrine secretion of milk lipid globules in mouse mammary gland during lactogenesis
Authors: Metka Kralj , Nada Pipan
Published: August 2004

Summary— Functional relations between exocytotic vesicle membranes, plasmalemma and milk fat globule membranes (MFGM) were studied during the final stages of mouse mammary gland differentiation, in the gland during full lactation and in the postpartum gland in which the synthesis of secretory products was partly inhibited by application of 2‐Br‐α‐ergokryptin. Analysis of ultrathin sections, freeze‐fracture replicas, scanning electron microscopy and application of a cytochemical marker filipin showed that the apocrine secretion of lipid globules was closely related to the exocytosis of milk proteins. During the last days of gestation the secretion of lipid globules resulted from many exocytotic events of the secretory vesicles that accumulated and fused around the cytoplasmic lipid droplets. Seldom the lipid droplet protruded partly into the gland lumen and a part of its surface became covered with the apical plasmalemma. Although apical plasmalemma became more important in the formation of MFGM in the postpartum period, we could still confirm a direct contribution of secretory vesicle membranes to the final detachment of the lipid globule. The application of 2‐Br‐α‐ergokryptin hindered the apocrine secretion of the lipid globules and a situation similar to the situation in the prepartum gland was observed.
Three‐dimensional architecture of the higher plant nucleolonema disclosed on serial ultrathin sections
Authors: Sato S
Published: August 2004

Summary— The three‐dimensional architecture of the nucleolonema of Vicia faba has been studied by applying a silver impregnation technique to serial ultrathin sections. This technique disclosed lateral and transverse segments of the nucleolonema which were heavily impregnated with silver. The lateral profiles of the nucleolonema segments were classified into three main categories; a segment made up of one to several rod‐like filaments (type I); a ladder‐like segment consisting of two parallel and of transverse filaments (type II); and a last type constructed from two parallel filaments (type III). Tracing of the lateral segments through serial sections has indicated that type I first appears, then either type II or III and finally type I reappears at the corresponding sites on sections. Types II and III remained constant in width, about 1.0 μm, along their longitudinal axes whereas the width of type I was significantly smaller than that of the two former. The lateral filaments of both types II and III showed heterogeneity in width on account of the presence of knobs intermittently distributed along them. The thickness of these knobs was about 0.35 μm. Combining the observations on serial ultrathin sections and the morphometrical data it is very probable that the elementary structure of the nucleolonema is a 0.35‐μm thick filament that tightly coils up into a solenoid structure with a thickness of approximately 1.0 μm. This model can explain the appearance of open‐ and closed‐argyrophilic rings in serial sections since transverse segments of the solenoid are expected to show the argyrophilic rings. The elementary filament of the nucleolonema solenoid was sometimes loosened. Judging from our cytochemical data at the electron microscope level, some argyrophilic proteins appear to reside in the axial space of the solenoid but both DNA and RNA were not detectable in this space.
Cyclical transformations of the actin cytoskeleton of hyacinth pollen subjected to recurrent vapour‐phase hydration and dehydration
Authors: Heslop‐Harrison John , Heslop‐Harrison Yolande
Published: August 2004

Summary— Transformations of the actin cytoskeleton of the pollen of Hyacinthus orientalis during cycles of vapour‐phase hydration and dehydration have been examined using a non‐fixation, DMSO permeabilisation method for TRITC‐phalloidin staining, coupled with microwave stabilisation. In freshly shed pollen actin appears: a) at the plasmalemma in the form of extended, thin fibrils co‐oriented with the cellulosic microfibrils of the contiguous intine; b) as a sheath investing the generative cell; c) as spicules around the vegetative nucleus; and d) in scattered spicules in the cytoplasm. During hydration in 85–95% relative humidity (RH), actin from all of these sites is progressively translated into a system of extended fibrils in the vegetative cell, concurrently with the onset of movement in the cytoplasm. Dehydration at 5–7% RH reverses this process, actin accumulating in rodlets, spicules or larger fusiform bodies in close association with the generative cell and vegetative nucleus, and also in the cytoplasm. The fibril system initially present at the plasmalemma is not restored. After ten cycles of hydration and dehydration 3.6% of the grains remained germinable. The ecological significance of the findings is noted, and the possibility that the observed transformations result from variation in the Ca2+ concentration in the cytosol as the water content of the cytosol changes is considered.
Subcellular localization of acid carboxypeptidase in rat liver and human fibroblasts
Authors: Gérard Nathalie , Thirion Jacqueline , Wattiaux‐De Coninck Simone , Wattiaux Robert
Published: August 2004

Summary— The subcellular distribution of acid carboxypeptidase was investigated in rat liver, normal human skin (CRL 1501) and lung (WI‐38) fibroblasts, galactosialidosis skin fibroblasts (GM 00806) and transformed lung fibroblasts (WI‐38 VA 13). Results of differential and isopycnic centrifugations and osmotic activation experiments clearly indicate that the enzyme is located in lysosomes, in agreement with observations suggesting that carboxypeptidase is the protective protein of the ‘Galjaard complex’ which is defective in galactosialidosis.
Assessment of fluorochromes for cellular structure and function studies by flow cytometry
Authors: Petit Jean‐Michel , Denis‐Gay Michelle , Ratinaud Marie‐Hélène
Published: August 2004

Summary— Because flow cytometry permits the analysis of individual whole cells, one of the key requirements in selecting a probe is its ability to target the site of interest into cells. In addition, dyes must possess ideal properties (ie extinction coefficient, Stoke's shift) rendering them appropriate for this methodology. Other characteristics, such as fluorescence quenching and energy transfer, inherent to the staining, provide numerous applications in flow cytometry. The available fluorophores used in flow cytometry are classified according to their cellular incorporation and binding. Thus, probes are presented and discussed as follows: 1) dyes of cellular components (DNA, RNA, proteins, lipids); 2) probes of membrane potential; 3) fluorophores that are sensitive to their microenvironment (pH, calcium, etc); and 4) those used for measurement of enzymatic activities into cells.
Flow cytometric analysis for reproductive biology
Authors: Spanò Marcello , Evenson Donald P.
Published: August 2004

Summary— Flow cytometric studies of spermatogenesis have been advanced by the need for: i) rapid, sensitive, objective and multiparameter measurements of reproductive effects due to environmental, occupational, and therapeutic exposure to toxicants; and ii) assessment of fertility potential of human and animal sperm. As a consequence, various flow cytometric techniques are already available to identify germ cell subpopulations undergoing both proliferative and maturative processes in normal and perturbed conditions. Significant improvements have been introduced in order to investigate the spermatogenic complex differentiation pathway and the apparent uniformity of mature sperm. Flow cytometry (FCM) has been applied to the measurement of both testis and sperm cells in a variety of species, including man. End points considered in toxicology studies are: altered testicular germ cell ratios, DNA and RNA content, increase of the coefficient of variation, induction of diploid elongated spermatids and diploid sperm, altered nuclear morphology, sperm cell viability, mitochondrial function and sperm chromatin structure. Precise DNA content measurements allow accurate analysis to determine the proportion of X‐ and Y‐chromosome bearing sperm and sorting of these subpopulations for gender preselection. FCM technology has reached a maturation level that allows its inclusion in the list of available and routine methods for reproductive studies in human and animal populations.