bluuebunny/crossref_metadata_2025 huggingface dataset:
Lymphocyte cell-cycle analysis by flow cytometry. Evidence for a specific postmitotic phase before return to G0.
Authors: Richman D P
Published: May 2004

We studied the cell cycle of lectin-stimulated human lymphocytes, making use of a flow cytometer. The RNA and DNA content of large numbers of individual cells was determined by supravital staining with acridine orange. The present study confirmed previous observations by others of a progression from G0 through G1 and S phase to G2/mitosis during the first 3 d in culture. It was also found that on subsequent days stimulated cells, before their return to G0, remained stationary in a state in which they contained the G0 complement of DNA and approximately twice the G0 complement of RNA. Cell-cycle manipulation with vinblastine and 5-bromo-2-deoxyuridine (BUdR) revealed that previous passage through both S phase and mitosis was required for entry into this newly observed late phase. In addition, there was high correlation (r = 0.973, P less than 0.001) between the number of cells in the late phase and measured [3H]thymidine uptake. It therefore appears that, in this system, stimulated cells remain in a distinct cell-cycle phase for a number of hours before their return to the resting state.
Localization of calmodulin in rat cerebellum by immunoelectron microscopy
Authors: Lin C , Dedman J , Means A
Published: May 2004

Calmodulin, a multifunctional Ca(++)-binding protein, is present in all eucaryotic cells. We have investigated the distribution of this protein in the rat cerebellum by immunoelectron microscopy using a Fab-peroxidase conjugate technique. In Purkinje and granular cell bodies, calmodulin reaction product was found localized both on free ribosomes and on those attached to rough endoplasmic reticulum (RER) and the nuclear envelope. No calmoduline was observed in the cisternae of RER or the Golgi apparactus. Calmodulin did not appear to be concentrated in the soluble fraction of the cell under the conditions used. Rather, peroxidase reaction product could be seen associated with membranes of the Golgi apparatus the smooth endoplasmic reticulum (SER), and the plasma membrane of both cell bodies and neuronal processes. In the neuronal dendrites, calmodulin appeared to be concentrated on membranes of the SER, small vesicles, and mitochondria. Also, granular calmodulin was observed in the amorphous material. In the synaptic junction, a large amount of calmodulin was seen attached to the inner surface of the postsynaptic membrane, whereas very little was observed in the presynaptic membrane or vesicles. These observations suggest that calmodulin is synthesized on ribosomes and discharged into the cytosol, and that it then becomes associated with a variety of intracellular membranes. Calmodulin also seems to be transported via neuronal processes to the postsynaptic membrane. Calmodulin localization at the postsynaptic membrane suggests that this protein may mediate calcium effects at the synaptic junction and, thus, may play a role in the regulation of neurotransmission.
Cell-substratum attachment and cell surface hyaluronate of Rous sarcoma virus-transformed chondrocytes.
Authors: Mikuni-Takagaki Y , Toole B P
Published: May 2004

Hyaluronate is associated with the cell surface of cultured Rous sarcoma virus-transformed chondrocytes. Detachment of these cells from their substratum by a variety of reagents is accompanied by release of 75-100% of this hyaluronate into solution. Treatment of the cells with 200 U/ml protease-free Streptomyces hyaluronidase at 37 degrees C cause release of greater than 90% of the cell surface hyaluronate and complete cell detachment. Treatment with a lower concentration of Streptomyces hyaluronidase (30 U/ml) at 25 degrees C or a corresponding activity of testicular hyaluronidase gives similar results, but only in the presence of mM EGTA. Treatment with the lower activities of either hyaluronidase or with 1 mM EGTA alone release only approximately 45% of the cell surface hyaluronate and does not cause significant cell detachment. It is concluded that there are two populations of cell surface hyaluronate differing in their accessibility or their resistance to dissociation from other components of the cell surface. It is proposed that the less readily released fraction is located between the transformed chondrocyte surface and substratum and is necessary for their interaction.
Structural comparison of several actin-binding macromolecules.
Authors: Tyler J M , Anderson J M , Branton D
Published: May 2004

The cytoskeletal components, macrophage actin-binding protein and filamin, were dried from glycerol and examined by low-angle rotary shadowing electron microscopy. Both are elongate, flexible molecules whose general morphologi is similar to that of erythrocyte spectrin. Neither actin-binding protein nor filamin binds to spectrin-depleted erythrocyte membranes.
Direct association of fibronectin and actin molecules in vitro.
Authors: Keski-Oja J , Sen A , Todaro G J
Published: May 2004

Affinity chromatography with actin-Sepharose conjugates of purified human fibronectin, normal human plasma, or serum-free culture fluid from human fibroblasts showed that fibronectin molecules can directly bind to actin. A quantitative recovery of soluble human fibronectin was accomplished by chromatography on actin immobilized on Sepharose beads. Human fibronectin molecules bound to actin-Sepharose were eluted with 0.25--0.35 M potassium bromide, and these molecules competed in a species-specific radioimmunoassay for human fibronectin. The subunits of fibronectin isolated by actin-Sepharose chromatography comigrated in SDS polyacrylamide gel electrophoresis with those of electrophoretically homogeneous fibronectin purified by conventional procedures. The efficient direct binding of fibronectin to actin suggests that interactions between these proteins might also take place in vivo but further studies are needed to elucidate the biological significance of this affinity.
Dissociation of opsonized particle phagocytosis and respiratory burst activity in an Epstein-Barr virus-infected myeloid cell line.
Authors: Newburger P E , Pagano J S , Greenberger J S , Karpas A , Cohen H J
Published: May 2004

A continuous tissue culture cell line (Karpas line 120), derived from a patient with acute myeloblastic leukemia, not only demonstrates myeloblastic morphology and in vitro expression of several myeloid-specific biochemical markers but also contains Epstein-Barr virus (EBV) nuclear antigen. The present studies demonstrate EBV-genome-specific DNA within the total cellular DNA by molecular hybridization, thus establishing the presence of stable viral genome integration. The cells demonstrate complex coordinated myeloid functions including ingestion, degranulation, and respiratory burst activity. Line 120 cells show a respiratory burst (superoxide and hydrogen peroxide generation and hexosemonophosphate shunt activity) in response to soluble (phorbol myristate acetate) and particulate (latex beads) stimuli, as do normal granulocytes. They ingest complement-opsonized particles (lipopolysaccharide-oil droplets, zymosan, and bacteria), and degranulate in response to them. However, unlike normal granulocytes, the line 120 cells do not demonstrate respiratory burst activity in response to these complementopsonized particles. The dissociation between ingestion of complement-opsonized particles and activation of oxygen-dependent bactericidal activity severely impairs bacterial killing as compared with normal polymorphonuclear phagocytes.
Cytoskeletal network underlying the human erythrocyte membrane. Thin-section electron microscopy.
Authors: Tsukita S , Tsukita S , Ishikawa H
Published: May 2004

A filamentous network underlying the human erythrocyte membranes can be clearly visualized in situ by electron microscopy of thin sections of specimens fixed with tannic acid-glutaraldehyde. The network is composed of two layers: the first, a layer of vertical components with granular appearance, which are seen to be directly associated with the membrane proper, and the second, a horizontally disposed, anastomosing meshwork of filamentous components, approximately 9 nm in thickness, which are attached to the vertical components. The diameter and appearance of the filamentous components are similar to those of purified spectrin. EDTA treatment (0.1 mM, pH 8.0), which was used to extract spectrin and actin, resulted in the disappearance of the filamentous meshwork, leaving only the granular components.
Integrated stereological and biochemical studies on hepatocytic membranes. I.V. Heterogeneous distribution of marker enzymes on endoplasmic reticulum membranes in fractions.
Authors: Bolender R P , Paumgartner D , Muellener D , Losa G , Weibel E R
Published: May 2004

The purpose of the study was to consider quantitatively the relationships between the surface area of the endoplasmic reticulum (ER) and constituent marker enzyme activities, as they occur in fractions collected from rat liver homogenates. The ER surface area was estimated in five membrane-containing fractions by use of a combined cytochemical-stereological technique (5), while, at the same time, ER marker enzymes were assayed biochemically. Fraction/homogenate recoveries for the ER enzymes averaged 100%, total membrane surface area 98%, and ER surface area 96%. Relative specific activities, which compare the relative amounts of ER marker enzyme activities to the relative ER surface area in the membrane-containing fractions, indicate variable distributions for glucose-6-phosphatase and NADPH cytochrome c reductase, but not for esterase.
Transformation of isolated rat hepatocytes with simian virus 40.
Authors: Isom H C , Tevethia M J , Taylor J M
Published: May 2004

Rat hepatocytes were transformed by simian virus 40 (SV40). Hepatocytes from two different strains of rats and a temperature-sensitive mutant (SV40tsA 1609), as well as wild-type virus were used. In all cases, transformed cells arose from approximately 50% of the cultures containing hepatocytes on collagen gels or a collagen gel-nylon mesh substratum. Cells did not proliferate in mock-infected cultures. SV40-transformed hepatocytes were epithelial in morphology, retained large numbers of mitochondria, acquired an increased nucleus to cytoplasm ratio, and contained cytoplasmic vacuoles. Evidence that these cells were transformed by SV40 came from the findings that transformants were 100% positive for SV40 tumor antigen expression, and that SV40 was rescued when transformed hepatocytes were fused with monkey cells. All SV40-transformed cell lines tested formed clones in soft agarose. Several cell lines transformed by SV40tsA 1609 were temperature dependent for colony formation on plastic dishes. Transformants were diverse in the expression of characteristic liver gene functions. Of eight cell lines tested, one secreted 24% of total protein as albumin, which was comparable to albumin production by freshly plated hepatocytes; two other cell lines produced 4.2 and 5.7%, respectively. Tyrosine aminotransferase activity was present in five cell lines tested but was inducible by dexamethasone treatment in only two. We conclude from these studies that adult, nonproliferating rat hepatocytes are competent for virus transformation.
Phagosome-lysosome fusion. Characterization of intracellular membrane fusion in mouse macrophages.
Authors: Kielian M C , Cohn Z A
Published: May 2004

Several approaches have been used to study the determinants of phagosome-lysosome fusion in intact mouse macrophages. Lysosomes were labeled with the fluorescent vital dye acridine orange and the rate and extent of their fusion with yeast-containing phagosomes was monitored by fluorescence microscopy. Fusion was also assayed by electron microscopy, using horseradish peroxidase or thorium dioxide as a marker for secondary lysosomes. Good agreemen samples with an enzymatic marker, and thorium dioxide-labeled samples evaluated by stereology. The rate of usion as assayed by fluorescence was not affected by the number of particles ingested, serum concentration, or prior uptake of digestible or nondigestible substances. With this assay it was possible to observe the rate of fusion separate from and uninfluenced by the phagocytic rate. Both the rate and extent of fusion were dramatically increased after several days in culture and similar changes were found by use of the EM assays. Fusion was strongly affected by incubation temperature, having a Q10 of 2.5 No detectable fusion occurred below 15 degrees C, and this inhibition was rapidly reversed when cells were returned to 37 degrees C.
Inhibition of DNA chain elongation in a purine-auxotrophic mutant of Chinese hamster.
Authors: Zannis-Hadjopoulos M , Taylor M W , Hand R
Published: May 2004

DNA synthesis has been examined in a purine-auxotrophic mutant cell line of Chinese hamster (V79 pur 1) under conditions of purine deprivation. At 6 h after the removal of purines from the growth medium, there is a decrease in semiconservative DNA replication. Alkaline velocity centrifugation of the DNA synthesized during a 1-min pulse under conditions of purine deprivation shows that approximately 50% of the newly replicated DNA is the size of Okazaki pieces. These are not incorporated into bulk DNA during a 1-h chase. If the purine supply is restored to the growth medium, these short DNA pieces are jointed to full-sized DNA within 1 h. DNA fiber autoradiolgraphy reveals a retardation in the rate of DNA replication fork movement but no apparent inhibition of initiation of synthesis on replication units within clusters actively engaged in replication. Our results indicate that purine deprivation specifically inhibits elongation of nascent dna chains.
Evidence for a pronounced secretion of cyclic AMP by Tetrahymena.
Authors: Nandini-Kishore S G , Thompson G A
Published: May 2004

The unicellular eukaryote Tetrahymena pyriformis secretes significant amounts of cyclic AMP into its external medium. Cells transferred from growth medium into any of the following three different non-nutrient media: (a) 5 mM phosphate buffer containing 47 mM NaCl and 1 mM MgSO4, (b) 10 mM Tris, or (c) 1.3 mM Tris containing 1 mM citrate and 1 mM Ca(OH)2, released to the outside almost 60--80% of the total cyclic AMP produced during 2--5 h of incubation. Tris-citrate-Ca+2 medium was chosen for further experiments because of its minimal nonspecific interference in the cyclic AMP radioimmunoassay. The identity of the secreted material recognized as cyclic AMP by radioimmunoassay was confirmed by demonstrating its almost complete hydrolysis with commerical beef heart phosphodiesterase. Furthermore, the radioimmunoassay-active material in the concentrated medium co-chromatographed on paper with [3H]cyclic AMP, as judged by assay of the eluted material. After resuspending cells in Tris-citrate-Ca2+ medium, the extracellular concentration of cyclic AMP rose steadily over a 5-h period, reaching a level equvalent to approximately 35--50 pmol cyclic AMP/10(6) cells vs. an internal cyclic AMP quantity at 5 h of 8--10 pmol/10(6) cells. After 5 h, the level of extracellular cyclic AMP reached a plateau. There was no degradation or uptake of external cyclic AMP by the cells during this period.
Filament organization revealed in platinum replicas of freeze-dried cytoskeletons.
Authors: Heuser J E , Kirschner M W
Published: May 2004

This report presents the appearance of rapidly frozen, freeze-dried cytoskeletons that have been rotary replicated with platinum and viewed in the transmission electron microscope. The resolution of this method is sufficient to visualize individual filaments in the cytoskeleton and to discriminate among actin, microtubules, and intermediate filaments solely by their surface substructure. This identification has been confirmed by specific decoration with antibodies and selective extraction of individual filament types, and correlated with light microscope immunocytochemistry and gel electrophoresis patterns. The freeze-drying preserves a remarkable degree of three-dimensionality in the organization of these cytoskeletons. They look strikingly similar to the meshwork of strands or "microtrabeculae" seen in the cytoplasm of whole cells by high voltage electron microscopy, in that the filaments form a lattice of the same configutation and with the same proportions of open area as the microtrabeculae seen in whole cells. The major differences between these two views of the structural elements of the cytoplasmic matrix can be attributed to the effects of aldehyde fixation and dehydration. Freeze-dried cytoskeletons thus provide an opportunity to study--at high resolution and in the absence of problems caused by chemical fixation--the detailed organization of filaments in different regions of the cytoplasm and at different stages of cell development. In this report the pattern of actin and intermediate filament organization in various regions of fully spread mouse fibroblasts is described.
Association of centrioles with the marginal band of a molluscan erythrocyte.
Authors: Cohen W D , Nemhauser I
Published: May 2004

Continuous circumferential bundles of microtubules, or marginal bands (MBs), are best known as a prominent structural feature of all nonmammalian vertebrate erythrocytes and mammalian blood platelets. Since their discovery in the late 19th century, MBs have been thought to play a cellular morphogenetic role, but no cytological clues to the mechanism of MB biogenesis have been reported. In previous work we have established the presence of MBs in serveral invertebrate blood cell types, including amebocytes and coelomocytes of certain Arthropod species and erythrocytes of a Sipunculan. We report here the occurrence of MBs in erythrocytes of the ark Anadara transversa (Mollusca) and four closely related species. The MBs of these arks have a striking structural feature; each is physically associated with a pair of centrioles. The centrioles are identified as such on the basis of morphological criteria: size, cylindrical shape, right-angle orientation, pairing, and 9-triplet ultrastructure. This intimate association between centrioles and MBs suggests that centrioles may be MB-organizing centers and invites comparative investigation of their possible role in vertebrate erythrocyte and platelet morphogenesis.
The membrane proteins of the vacuolar system I. Analysis of a novel method of intralysosomal iodination.
Authors: Muller W A , Steinman R M , Cohn Z A
Published: May 2004

A method has been developed to deliver an idoinating system into the confines of the phagolysosome, allowing us to study the nature of the phagolysosomal membrane. Lactoperoxidase (LPO) is covalently coupled to carboxylated latex spheres (LPO-latex) in a stable, enzymatically active form. The addition of LPO-latex to cultured macrophages leads to their rapid attachment, ingestion, and enclosure in a plasma membrane-derived phagocytic vacuole. These organelles rapidly fuse with preexisting lysosomes and are converted to phagolysosomes (PL) that demonstrates both acid phosphatase and lactoperoxidase activities. The exposure of LPO-latex containing cells to 125I- and an extracellular peroxide-generating system, glucose oxidase-glucose, at 4 degrees C leads to incorporation of label into TCA-precipitable material. The incorporated cel-associated label was present as monoiodotyrosine, and negligible amounts were found in lipids. Cell viability remained > 99%. Autoradiography at both the light and EM level revealed that > 97% of the cells were labeled, and quantitative analysis demonstrated the localization of grains to LPO-latex containing PL. PL were separated on sucrose gradients, and their radiolabel was confined almost exclusively to the membrane rather than soluble contents. SDS-polyacrylamide gel electrophoretic analysis of the peptides iodinated from within PL demonstrated at least 24 species with molecular weights ranging from 12,000 to 250,000. A very similar group of proteins was identified on the plasma membrane (PM) after surface iodination, and on latex phagosomes derived from iodinated PM. No novel proteins were detected in PL, either immediately after phagosome-lysosome fusion or after 1 h of intracytoplasmic residence. We conclude that the membrane proteins accessible to LPO-catalyzed iodination on the luminal surface of the PL and on the external face of the PM are similar, if not identical.
Three-dimensional crystals of an integral membrane protein: an initial x-ray analysis.
Authors: Garavito R M , Rosenbusch J P
Published: May 2004

Matrix protein, a pore-forming transmembrane protein spanning the outer membrane of Escherichia coli, has been obtained in a variety of three-dimensional crystal forms amenable to both electron microscope and x-ray analyses. Successful association into large crystals depended on the use of alpha-octyl glucoside, a detergent with relatively low affinity for the protein. Electron micrographs of thin-sectioned crystals show a high degree of order. Preliminary crystallographic data suggest that the crystals, which exhibit diffraction to 3.8 A, have a cubic space group.
The adherence of human neutrophils and eosinophils to schistosomula: evidence for membrane fusion between cells and parasites.
Authors: Caulfield J P , Korman G , Butterworth A E , Hogan M , David J R
Published: May 2004

Human neutrophils and eosinophils adhere to the surface of schistosomula of Schistosoma mansoni that have been preincubated with antischistosomular sera with or without complement. Neutrophils are seen to form small (< 0.5 micrometer), heptalaminar and large (5-8 micrometer), pentalaminar fusions with the normal pentalaminar parasite surface membrane. By freeze-fracture techniques, attachment areas 5-8 micrometer in diameter are seen to form between neutrophils and schistosomula. These areas have three zones--an edge and two centrally located areas, one of which is rich and one of which is poor in intramembrane particles (IMPs). The edge zone is continuous around the attachment areas and is usually composed of a skip-fracture that passes out of the schistosomular outer membrane into the inner membrane. In some cases, the edge zone is made up of a string of IMPs. The IMP-rich central areas have an IMP concentration similar to that of unattached neutrophil membranes, are raised off of the surface of the schistosomulum, and have two normal schistosomular membranes underneath indicating that they are indeed unattached. the IMP-poor central areas are composed of a fused or hybrid membrane that is continuous with the neutrophil plasma membrane but that bears the same spatial relationship to the schistosomular inner membrane that the normal outer membrane does. Similar changes are seen in samples prepared with glycerination. Eosinophils generally do not fuse with the schistosomular outer membrane but, instead, discharge their granular contents onto the surface of the schistosomula and appear to adhere to the parasite through this discharged material. It is suggested that schistosomula have a capability to fuse with mammalian cells and that this fusion proceeds from a fusion of the outer leaflets to a fusion of the bilayers, as appears also to be the case in other systems.
Development of cell surface saccharides on embryonic pancreatic cells
Authors: Maylie-Pfenninger MF , Jamieson JD
Published: May 2004

Using a battery of seven lectin-ferritin conjugates as probes for cell surface glycoconjugates, we have studied the pattern of plasmalemmal differentiation of cells in the embryonic rat pancreas from day 15 in utero to the early postpartum stage. Our results indicate that differentiation of plasmalemmal glycoconjugates on acinar, endocrine, and centroacinar cells is temporally correlated with development and is unique for each cell type, as indicated by lectin-ferritin binding. Specifically, (a) expression of adult cell surface saccharide phenotype can be detected on presumptive acinar cells as early as 15 d in utero, as indicated by soybean agglutinin binding, and precedes development of intracellular organelles characteristic of mature acinar cells; (b) maturation of the plasmalemma of acinar cells is reached after intracellular cytodifferentiation is completed, as indicated by appearance of Con A and fucoselectin binding sites only at day 19 of development; conversely, maturation of the endocrine cell plasmalemma is accompanied by "loss" (masking) of ricinus communis II agglutinin receptors; and (c) binding sites for fucose lectins and for soybean agglutinin are absent on endocrine and centroacinar cells at all stages examined. We conclude that acinar, centroacinar, and endocrine cells develop from a common progenitor cell(s) whose plasmalemmal carbohydrate composition resembles most closely that of the adult centroacinar cell.

Finally, appearance of acinar lumina beginning at approximately 17 d in utero is accompanied by differenetiation of apical and basolateral plasmalemmal domains of epithelial cells, as indicated by enhanced binding of several lectin-ferritin conjugates to the apical plasmalemmal, a pattern that persists from this stage through adult life.
Calcium-labile mitotic spindles isolated from sea urchin eggs (Lytechinus variegatus).
Authors: Salmon E D , Segall R R
Published: May 2004

We isolated calcium-labile mitotic spindles from eggs of the sea urchin Lytechinus variegatus, using a low ionic strength, EGTA lysis buffer that contined 5.0 mM EGTA, 0.5 mM MgCl2, 10-50 mM PIPES, pH 6.8, with 1% Nonidet P-40 (detergent) and 20-25% glycerol. Isolated spindles were stored in EGTA buffer with 50% glycerol for 5-6 wk without deterioration. The isolated spindles were composed primarily of microtubules with the chromosomes attached. No membranes were seen. Isolated spindles, perfused with EGTA buffer to remove the detergent and glycerol, had essentially the same birefringent retardation (BR) as spindles in vivo at the same mitotic stage. Even in the absence of glycerol and exogenous tubulin, the isolated spindles were relatively stable in the EGTA buffer: BR decayed slowly to about half the initial value within 30-45 min. However, both the rate and extent of BR decay increased with concentrations of Ca2+ above 0.2-0.5 muM as assayed using Ca-EGTA buffers (0.2 mM EGTA, 0.5 mM MgCl2, 50 mM PIPES, pH 6.8, plus various amounts of CaCl2). Microtubules depolymerized almost completely in < 6 min at Ca2+ concentrations of 2 muM and within several seconds at 10 muM Ca2+. Of several divalent cations tested, only Sr2+ caused comparable changes in BR. The absence of membranes in the isolated spindles appeared to be associated with a lack of calcium-sequestering ability. Our results suggest that calcium ions play an important role in the depolymerization of spindle microtubules and that membrane components may function within the mitotic apparatus of living cells to sequester and release calcium ions during mitosis.
Hypomethylation of DNA during differentiation of friend erythroleukemia cells
Authors: Christman JK , Weich N , Schoenbrun B , Schneiderman N , Acs G
Published: May 2004

DNA from mammalian cells has been shown to contain significant amounts of 5-methyl cytosine resulting from enzymatic transfer of methyl groups from s-adenosylmethionine to cytosine residues in the DNA polymer. The function of this modification is not known. We have found that DNA synthesized during chemically induced differentiation of friend erythroleukemia cells is hypomethylated, as measured by its ability to accept methyl groups transferred by homologous DNA methyltransferases in vitro. The extent of hypomethylation detected by this sensitive method is small, a decrease of less than 1.6 percent in 5-methylcytosine content.

Hypomethylated DNA can be isolated from friend erythroleukemia cells grown in the presence of dimethyl sulfoxide, butyrate, hexamethylene-bis- acetamide, pentamethylene-bis acetamide, and ethionine. However, hypomethylated DNA is found only under conditions where differentiation is actually induced. DNA isolated from cells of a dimethyl sulfoxide- resistant subclone grown in the presence of that agent is not hypomethylated, although DNA of these cells becomes hypomethylated after growth in the presence of inducers that can trigger their differentiation. We also find that the DNA of friend erythroleukemia cells does not become hypomethylated when the cells are exposed to inducing agents in the presence of substances that inhibit differentiation. These results suggest a close link between genome modification by methylation and differentiation of friend erythroleukemia cells.
Changes of cell morphology and prolactin secretion induced by 2-Br-alpha-ergocryptine, estradiol, and thyrotropin-releasing hormone in rat anterior pituitary cells in culture.
Authors: Antakly T , Pelletier G , Zeytinoglu F , Labrie F
Published: May 2004

The secretion of prolactin in cultured pituitary cells was studied in correlation with the cellular changes induced by stimulatory or inhibitory agents. The techniques used in this study were: radioimmunoassay, immunocytochemistry, scanning (SEM) as well as transmission (TEM) electron microscopy. Prolactin secretion was stimulated by 17 beta-estradiol (10 nM) as well as thyrotropin-releasing hormone (TRH) (3 nM) and inhibited by 2-Br-alpha-ergocryptine (CB-154) (1 muM). The total prolactin (release and cell content) increased between 2 and 8 d of estradiol treatment, indicating an increase of both synthesis and release of prolactin. This finding was in agreement with TEM observations because, in estradiol-treated prolactin cells, the Golgi saccules were distended and Golgi elements were increased, thus indicating increased synthetic activity of these cells. The addition of TRH over a 4-h period resulted in a significant degranulation of prolactin cells. In contrast, prolactin secretory granules became accumulated in the cells after CB-154 treatment for a period ranging from 4 to 24 h. In agreement, light microscope immunocytochemistry showed an increased reaction for prolactin after short-term (< 24 h) incubation with CB-154. Because prolactin cells represent approximately 70% of the glandular cell population as revealed by immunocytochemistry, it was then possible to observe the changes of cell surface by SEM. In most cells, estradiol and TRH led to an increase in the number and prominence of microvilli and blebs, whereas CB-154 treatment resulted in a slightly decreased number of microvilli and an increased occurrence of membrane foldings. This report thus provides morphological evidence for the stimulatory effects of estradiol and TRH, and the inhibitory effects of CB-154 on prolactin secretion in pituitary cells in primary culture. These data, moreover, show that acute changes in secretory activity of prolactin-secreting cells are accompanied by marked changes of their morphological characteristics.
Cell division in two large pennate diatoms Hantzschia and Nitzschia III. A new proposal for kinetochore function during prometaphase.
Authors: Tippit D H , Pickett-Heaps J D , Leslie R
Published: May 2004

Prometaphase in two large species of diatoms is examined, using the following techniques: (a) time-lapse cinematography of chromosome movements in vivo; (b) electron microscopy of corresponding stages: (c) reconstruction of the microtubules (MTs) in the kinetochore fiber of chromosomes attached to the spindle. In vivo, the chromosomes independently commence oscillations back and forth to one pole. The kinetochore is usually at the leading edge of such chromosome movements; a variable time later both kinetochores undergo such oscillations but toward opposite poles and soon stretch poleward to establish stable bipolar attachment. Electron microscopy of early prometaphase shows that the kinetochores usually laterally associate with MTs that have one end attached to the spindle pole. At late prometaphase, most chromosomes are fully attached to the spindle, but the kinetochores on unattached chromosomes are bare of MTs. Reconstruction of the kinetochore fiber demonstrates that most of its MTs (96%) extend past the kinetochore and are thus apparently not nucleated there. At least one MT terminates at each kinetochore analyzed. Our interpretation is that the conventional view of kinetochore function cannot apply to diatoms. The kinetochore fiber in diatoms appears to be primarily composed of MTs from the poles, in contrast to the conventional view that many MTs of the kinetochore fiber are nucleated by the kinetochore. Similarly, chromosomes appear to initially orient their kinetochores to opposite poles by moving along MTs attached to the poles, instead of orientation effected by kinetochore MTs laterally associating with other MTs in the spindle. The function of the kinetochore in diatoms and other cell types is discussed.
The localization of protein carboxyl-methylase in sperm tails.
Authors: Bouchard P , Gagnon C , Phillips D M , Bardin C W
Published: May 2004

Protein carboxyl-methylase (PCM), an enzyme known to be involved in exocytotic secretion and chemotaxis, has been studied in rat and rabbit spermatozoa. PCM activity and its substrate methyl acceptor protein(s) (MAP) were demonstrated in the supernate after solubilization of the sperm cell membrane by detergent (Triton X-100). A protein methylesterase that hydrolyzes methyl ester bonds created by PCM was demonstrated in rabbit but not in rat spermatozoa. This enzyme was not solubilized by nonionic detergent. The specific activities of PCM in rat spermatozoa from caput and cauda epididymis were similar and lower than that found in testis. By contrast, MAP substrates were low in testis and increased in parallel with sperm maturation in the epididymis. Multiple MAP were demonstrated in spermatozoa by polyacrylamide gel electrophoresis. The pattern of these proteins was similar in spermatozoa from different portions of the reproductive tract. Fractionation of heads and tails of rat spermatozoa on sucrose gradients indicated that PCM was found exclusively in the tail fraction, whereas MAP was detected both in head and tail fractions. The presence of all the components of the protein carboxyl-methylation system in spermatozoa and the localization of PCM and some of its substrates in the sperm tail are consistent with their involvement in sperm cell motility.
Mechanism of phagocytosis in dictyostelium discoideum: phagocytosis is mediated by different recognition sites as disclosed by mutants with altered phagocytotic properties
Authors: Vogel G , Thilo L , Schwarz H , Steinhart R
Published: May 2004

The recognition step in the phagocytotic process of the unicellular amoeba dictyostelium discoideum was examined by analysis of mutants defective in phagocytosis, Reliable and simple assays were developed to measure endocytotic uptake. For pinocytosis, FITC-dextran was found to be a suitable fluid-phase marker; FITC-bacteria, latex beads, and erythrocytes were used as phagocytotic substrates. Ingested material was isolated in one step by centrifuging through highly viscous poly(ethyleneglycol) solutions and was analyzed optically.

A selection procedure for isolating mutants defective in phagocytosis was devised using tungsten beads as particulate prey. Nonphagocytosing cells were isolated on the basis of their lower density. Three mutant strains were found exhibiting a clear-cut phenotype directly related to the phagocytotic event.

In contrast to the situation in wild-type cells, uptake of E. coli B/r by mutant cells is specifically and competitively inhibited by glucose. Mutant amoeba phagocytose latex beads normally but not protein-coated latex, nonglucosylated bacteria, or erythrocytes. Cohesive properties of mutant cells are altered: they do not form EDTA-sensitive aggregates, and adhesiveness to glass or plastic surfaces is greatly reduced.

Based upon these findings, a model for recognition in phagocytosis is proposed: (a) A lectin-type receptor specifically mediates binding of particles containing terminal glucose (E. coli B/r). (b) A second class of "nonspecific" receptors mediate binding of a variety of particles by hydrophobic interaction. Nonspecific binding is affected by mutation in such a way that only strongly hydrophobic (latex) but not more hydrophilic particles (e.g., protein-coated latex, bacteria, erythrocytes) can be phagocytosed by mutant amoebae.
Brush-border alpha-actinin? Comparison of two proteins of the microvillus core with alpha-actinin by two-dimensional peptide mapping.
Authors: Mooseker M S , Stephens R E
Published: May 2004

The bundle of filaments within the intestinal microvillus contains four major polypeptides in addition to actin calmodulin, a 70-kdalton subunit and two polypeptides with molecular masses similar to that of the Z-line component alpha-actinin (95 and 105 kdaltons). Two-dimensional mapping of tryptic peptides indicates that (a) alpha-actinins from chicken skeletal, cardiac, and smooth muscle are similar but not identical proteins and that skeletal alpha-actinin in more similar to the cardiac subunit than to the alpha-actinin from gizzard; (b) the brush-border 95- and 105-kdalton subunits are closely related to each other, but the smaller subunit is not a proteolytic fragment of the 105-kdalton subunit; and (c) although there is considerable peptide overlap between the brush-border subunits and the three alpha-actinins, the peptide maps of the 95- and 105-kdalton proteins are substantially distinct from the various alpha-actinin maps, suggesting that neither brush-border subunit is a bona fide alpha-actinin. Nevertheless, on the basis of peptide mapping criteria alone, one cannot exclude the possibility that the brush-border subunits are "alpha-actinin-like." However, there is no immunological cross-reactivity between the brush-border subunits and alpha-actinins, using antibodies prepared against gizzard alpha actinin.
Membranes in the miotic apparatus of barley cells
Authors: Hepler PK
Published: May 2004

Membranes in the mitotic apparatus have been investigated ultrastructually in dividing cells of barley (Hordeum vulgare). After osmium tetroxide- potassium ferricyanide or ferrocyanide postfixation (OsFeCN) of material that had been fixed in glutaraldehyde in the presence of Ca(++), the nuclear envolope (NE)-endoplasmic reticulum (ER) complex is selectively stained, permitting observations on the cellular pattern and structural ramifications of this membrane system that have not been previously recognized. Specifically, it is observed that membrane system that have not been previously recognized. Specifically, it is observed that during mitosis the NE-ER forms a continuous membrane system that ensheathes and isolates the mitotic apparatus (MA). Elements of ER progressively accumulate in the region of the spindle pole, becoming most concentrated by early anaphase. Within the MA itself, there are striking spindle- membrane associations; in particular, tubular elements of predominantly smooth NE-ER invade the spindle interior selectively along kinetochore microtubules. The membrane elements at the pole and surrounding the MA consist of tubular reticulum and fenestrated lamellae. Membranes of the MA thus resemble in considerable detail the tubular network and fenestrated elements of the sarcoplasmic reticulum of muscle. It is suggested that the NE-ER of the dividing barley cell may function in one or both of the following ways: (a) to control the concentration of free Ca(++) in the MA and (b) to serve as an anchor to chromosome motion.
Analysis of the structure of intramembrane particles of the mammalian urinary bladder.
Authors: Robertson J D , Vergara J
Published: May 2004

The luminal and discoid vacuole membranes of the superficial cell layer of the transitional epithelium of the mammalian urinary bladder have been studied by thin-sectioning and freeze-fracture-etch (FFE) electron microscope methods. For the FFE studies membranes were deposited on a cationized glass surface, covered by a thin copper disc, and fractured under liquid N2. Specimens were etched at -100 degrees C and replicated at -190 degrees C. A model of the lattice membrane derived from thin sections was used to predict the heights of the fracture faces above the glass surface. A hexagonal pattern of globular intramembrane particles spaced 160 A apart was seen in the external fracture (EF) face plaques as previously described and regarded as the dominant structure. However, very extensive areas of another pattern, seen before in only limited areas, have beeen found in the EF faces. The pattern consists of a smooth hexagonal lattice with the same space constant as the globular one but a different structure. By image analysis it consists of overlapping domains bordered by shared but incomplete metal rims. Each domain has a central spot of metal encircled by a shadow. The surface of the smooth lattice is partly complementary to the corresponding protoplasmic fracture (PF) face which shows a similar hexagonal lattice with the same space constant. The height of the smooth EF lattice above the glass substrate is the same as the plane of the center of the lipid bilayer predicted by the model. The mean heights of the particles of the globular EF lattice are greater than the total thickness of the membrane as predicted by the model and confirmed by measurements. The globular EF lattice is not complementary and it is concluded that the globular particles do not exist in the native membrane but arise artifactually during the preparatory procedures.
A collagen-binding protein on the surface of ejaculated rabbit spermatozoa.
Authors: Koehler J K , Nudelman E D , Hakomori S
Published: May 2004

A rabbit antibody to mouse 3T3 cell fibronectin was used in conjunction with a fluorescein-tagged second antibody to detect fibronectin-like activity on the surface of rabbit spermatozoa. Only ejaculated sperm displayed an intense and highly localized fluorescence over the acrosomal region. Cauda epididymal sperm of the rabbit as well as several other species did not exhibit any reaction. The fluorescent activity could be eliminated by trypsin treatment but was re-established by incubation in cell-free seminal fluid. Sperm recovered from females 10-12 h after mating showed a reduction or absence of antifibronectin fluorescence, suggesting that this component's loss could be a factor in sperm capacitation. Because fibronectins show strong binding to collagen, mixtures of ejaculated sperm and collagen were examined in the light and electron microscope. Living sperm appear to have a strong affinity for collagen and quickly adhere to the filaments by their heads, while continuing vigorous flagellations. Surface labeling of sperm with the galactose-oxidase-NaB[3H]4 technique, extraction with urea-detergent mixtures and affinity chromatography of extracts on gelatin-Sepharose revealed a single radioactive band of mot wt approximately 40,000 after SDS polyacrylamide gel electrophoresis and fluorography.
Cyclic 3', 5'-AMP relay dictyostelium discoideum. V. Adaptation of the cAMP signaling response during cAMP stimulation
Authors: Dinauer M , Steck TL , Devreotes P
Published: May 2004

In dictyostelium discoideum, extracellular cAMP activates adenylate cyclase, which leads to an increase in intracellular cAMP and the rate of cAMP secretion. The signaling response to a constant cAMP stimulus is terminated after several minutes by an adaptation mechanism. The time- course of adaptation stimuli of 10(-6) or 10(-7) M cAMP was assessed. We used a perfusion technique to deliver defined cAMP stimuli to [(3)H]adenosine-labeled amoebae and monitored their secretion of [(3)H]cAMP. Amoebae were pretreated with 10(-6) or 10(-7) M cAMP to periods of 0.33-12 minutes, and then immediately given test stimuli of 10(-8) M to 2.5 x 10(-7) M cAMP. The response to a given test stimulus was progressively attenuated and finally extinguished as the duration of the pretreatment stimulus increased. During concentration of the test stimulus. The responses to test stimuli of 10(-8), 5 x 10(-8), 10(-7), or 2.5 x 10(-7) M cAMP were extinguished after approximately 1, 2.25,2.5, and 10 min, respectively. 1.5 min of stimulation with 10(-7) M cAMP was necessary to extinguish the response of a test stimulus of 10(-8) M cAMP. Our data suggest that adaptation begins within 20 s of stimulation, rises rapidly for approximately 2.5 min, and reaches a plateau after approximately 10 min. The absolute rate of rise was faster during pretreatment with 10(-6) than with 10(-7) M cAMP. These results support a working hypothesis in which the occupancy of surface cAMP receptors leads to changes in two opposing cellular processes, excitation and adaptation, that control the activity of D. discoideum adenylate cyclase.
Detection of apolipoprotein C in human and rat enterocytes.
Authors: Schonfeld G , Grimme N , Alpers D
Published: May 2004

Apoproteins have important physiologic functions in lipoprotein metabolism. Several apoproteins are produced in the intestine including ApoA-I, ApoA-IV, and ApoB. Each appears to participate in intestinal lipid transport. The liver also produces several apoproteins, including ApoC-II and ApoC-III, but the data demonstrating the ability of the intestine to produce ApoC is incomplete. Our aim was to ascertain whether ApoC-II and ApoC-III were present in human and rat jejunum, and if so, whether their presence was altered by fat feeding. The technique of immunolocalization and a newly developed double antibody radioimmunoassay for rat ApoC-III3 were used. ApoC-III3 was found in the supranuclear regions of enterocytes along the entire lengths of villi in the jejuna of 12-h-fasted rats. 1 hour after the gastric ingestion of corn oil. ApoC-III3 was found primarily in between cells and in the lamina propria. Similar results were obtained in human jejunal biopsies with ApoC-II and ApoC-III. ApoC-III3 was also detected by radioimmunoassay in enterocytes isolated from jejuna of neonatal and adult rats. Thus, ApoC-II and ApoC-III are clearly present in the intestine as well as in the liver. In addition, because their localization is altered after fat feeding, they are also likely to be produced in the enterocyte.
Distribution of flourescently labeled α-actinin in living and fixed fibroblasts
Authors: JR Feramisco , Blose SH
Published: May 2004

The distribution of flourescently labeled α-actinin after microinjection into fibroblasts has been determined in both living and fixed cells. We have found that the distribution of the injected tetramethylrhodamine isthiocyanate-labeled protein (TMRITC-α-actinin) in living cells, which is in ruffling membranes, actin microfilament bundles, and polygonal microfilament networks (Feramisco, 1979, Proc. Natl. Acad. Sci. U. S. A. 76:3967-3971), was virtually unaffected by the fixation (3.5 percent formaldehyde) and extraction (absolute acetone) used for the preparation of the cells for immunoflourescence. Also, these patterns were found to coincide with the α-actinin revealed by immunoflourescence. Also, these patterns were found to coincide with the α-actinin revealed by immunoflourescence. These findings offer, for the first time, evidence indicating the validity of the immunoflourescence technique in the localization of α-actinin in cultured cells. With the combination of the injection procedure and the immunoflourescence localization of endogenous structural proteins, it was determined that nearly all of the actin stress fibers were decorated in a periodic manner with the injected α-actinin. Endogenous tropomyosin in the injected cells was found to be distributed with a periodic pattern along the stress fibers that was antiperiodic to the pattern observed for the microinjected α-actinin. The tropomyosin antibody stained the polygonal microfilament networks and was excluded from the foci, whereas the microinjected α-actinin was incorporated into the foci of the networks. Thus, the microinjected fluorescent derivative of α-actinin appears to be incorporated into the functional pools of α-actinin within the living cell and to be utilized by the cell with fidelity.