bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthesis and apical and basolateral secretion of thyroglobulin by thyroid cell monolayers on permeable substrate: Modulation by thyrotropin
Authors: Chambard Marianne , Mauchamp Jean , Chabaud Odile
Published: February 2005

In the thyroid glands, thyroglobulin (Tg) is specifically synthesized by follicular cells and then secreted into the apical lumen where it is concentrated and used as a substrate for thyroid hormone synthesis. The presence of Tg in the circulation has been reported in normal and pathological situations. To determine the domains of the plasma membrane, apical and/or basolateral, involved in Tg secretion, porcine thyroid epithelial cells were cultured as a monolayer on the porous bottom of a culture chamber in which both apical and basal media are independently accessible. Control experiments using labeled Tg ascertained the tightness of the monolayer and showed that within 48 h only 0.2–0.5% of the Tg introduced in the apical medium was transferred through the cell layer into the basal compartment. For kinetic studies of Tg synthesis and secretion, monolayers were cultured for up to 72 h in the presence of 35S‐methionine and with or without 100 μU/ml thyrotropin (TSH) in the basal medium. Labeled Tg was measured by double immunoprecipitation and by fluorography of polyacrylamide gel electrophoresis. We showed that 80–95% of total secreted Tg was recovered in the apical medium. The remainder was secreted through the basolateral membranes in the basal medium. The amount of Tg secreted into the apical compartment was stimulated two‐ to threefold by TSH whereas no TSH effect was observed on secretion in the basal compartment. Moreover, measuring apical and basal volumes, we observed a net water flow from the apical to the basal side. It was stimulated threefold by TSH, increasing the Tg concentration in the apical compartment of the stimulated cell layer. During the culture time, the amount of Tg synthesized and secreted was increased by TSH, as was the Tg mRNA content, as determined by the dot‐blot hybridization method.
Cyclic activity of the receptors of cobalamin bound to transcobalamin II
Authors: Hall Charles A. , Colligan Pamela D. , Begley James A.
Published: February 2005

The activity of receptors specific for human transcobalamin II‐Cobalamin (TC II‐Cbl) were measured in virus‐transformed lymphoblasts, hepatocytes (hepatoma) and diploid fibrolasts. In all three types of human cells the receptor activity increased as cells went from a resting phase to the most actively dividing phase. Receptor activity declined as cell division slowed. The changes in activity of lymphoblasts and hepatocytes were produced by changes in receptor numbe and not by changes in affinity between receptors and TC II‐Cbl. The basis of the change in fibroblasts was not clear. The Cbl‐dependent methionine synthetase activity of fibroblasts, in contrast, tended to be greatest when the cultures were confluent and replication had slowed. As the fibroblasts became senescent the receptor activity for TC II‐Cbl declined and the fluctuations with the phase of the cell were blunted. However, the release of apo TC II from the cells was maintained. These observations must be taken into consideration when the respective cells are used as models. Even more important are the implications of the observations of the changes in receptor activity for TC II‐Cbl for the regulation of the entry of Cbl into cells.
Neutrophil C3bi receptors: Formation of membrane clusters during cell triggering requires intracellular granules
Authors: Petty Howard R. , Francis Joseph W. , Todd Robert F. , Petrequin Pamela , Boxer Laurence A.
Published: February 2005

Video‐intensification fluorescence microscopy has been used to study the cell surface distribution of the complement receptor (CR) for C3bi (CR3) on human neutrophils. Fluorescin‐ or rhodamine‐labeled monoclonal IgG or Fab fragments of antireceptor antibody were used as probes of receptor localization. C3bi receptors are uniformly distributed on untreated cells. Glass coverslips were coated with lipopolysaccharide (LPS) and serum was added; the serum deposits complement components, including C3bi, on the surface. When neutrophils were adherent to these coverslips, receptors were found in large clusters, and a fraction of the fluorescence remained uniform. Double‐labeling studies were conducted by first labeling with anti‐CR3 followed by attachment to LPS/serum‐treated slides. This, in turn, was followed by labeling with the antibody conjugated to a second fluorophore. These studies revealed that the CR3 clusters were predominantly new antigenic sites exposed after attachment to the LPS/serum‐treated slides. To determine the contribution of granule‐associated CR3, we have studied neutrophils defective in receptor up‐regulation, neutrophil cytoplasts, and a stimulator of granule release, A23187. Neutrophils from a patient with specific granule deficiency were found to be defective in granular CR3 and did not form clusters on C3‐modified surfaces. The patient's neutrophils were defective in CR3 up‐regulation and enzyme release as shown by fluorescence flow cytometry and gelatinase release, respectively. Cytoplasts also failed to show CR3 clusters on LPS/serum‐treated coverslips. Furthermore, neutrophils treated with A23187 demonstrated numerous CR3 clusters. We suggest that formation of CR3 membrane domains during immune recognition requires the participation of intracellular granules. We speculate that these domains are formed by fusion of CR3‐bearing granules at local sites of adhesion.
Regulation of glutathione levels in mouse spleen lymphocytes by transport of cysteine
Authors: Ishii Tetsuro , Sugita Yoshiki , Bannai Shiro
Published: February 2005

Cysteine and cystine transport activities of resting and activated mouse spleen lymphocytes were characterized in order to examine the contributions of cysteine and cystine to intracellular glutathione contents. Following stimulation with lipopolysaccharide, the lymphocytes markedly increased their capacity to transport cysteine. The uptake of cysteine was mediated mainly by the ASC system (Na+‐dependent neutral amino acid transport system especially reactive with alanine, serine, and cysteine). On the other hand, both the resting and the activated lymphocytes had extremely low cystine transport activities. Because of the instability of cysteine, the culture media usually contained cystine but not cysteine. Therefore, both the resting and the activated lymphocytes rapidly decreased their glutathione contents owing to their poor capacities to take up cystine. The effects of freshly added cysteine on the cellular glutathione contents were examined in the presence of bath‐ocuproinedisulfonate, a nontoxic copper‐specific chelator that inhibits autoxidation of cysteine. Cysteine added at 25–400 μM only partially prevented the rapid decrease of the glutathione contents in fresh resting lymphocytes. In the lipopolysaccharide‐activated cells, however, cysteine enhanced the cellular glutathione contents in a dose‐dependent manner. These results indicate that the enhanced activity of the ASC system increases the level of intracellular glutathione in the presence of cysteine.
Heparin potentiates the action of plasma membrane‐associated growth stimulatory activity
Authors: Nagasaki Takayuki , Lieberman Michael A.
Published: February 2005

Plasma membranes prepared from mouse liver have been previously shown to contain growth stimulatory activity as determined with cultured mouse fibroblasts. This growth stimulatory activity, termed plasma membrane‐associated growth stimulatory activity (PMGA), is highly mitogenic in the presence of platelet‐poor plasma. We now demonstrate that the growth stimulatory action of PMGA is dramatically enhanced by the addition of heparin. The half‐maximal effect of heparin was observed at 1–3 μg/ml. The synergistic effect was seen in two distinct assays; the stimulation of DNA synthesis in quiescent cells, and an increase of cell number over a 3‐day culture period. Heparin, by itself, does not have any measurable influence on the growth of fibroblasts. The action of heparin is not unique to this glycosaminoglycan, as several other highly sulfated polysaccharides, including dextran sulfate, pentosan polysulfate, and fucoidan, also exhibited the highly synergistic effect. Among other glycosaminoglycans examined, chondroitin sulfate B and heparan sulfate had a small, but significant, effect on enhancing the growth stimulatory action of PMGA. Chondroitin sulfate A, chondroitin sulfate C, hyaluronic acid dextran, and poly‐L‐glutamic acid, however, had no detectable effect. Further experiments suggested that the effect of heparin is twofold, namely, both a potentiation of growth stimulatory activity and a protection of PMGA activity. The data presented here suggest that the association of various cell surface components, such as PMGA and specific proteoglycans, can modulate the growth potential of a cell.
Quantitation and tissue localization of the cellular retinoic acid‐binding protein
Authors: Eriksson Ulf , Hansson Eva , Nordlinder Hans , Busch Christer , Sundelin Johan , Peterson Per A.
Published: February 2005

The distribution of the cellular retinoic acid‐binding protein (CRABP) in some rat tissues has been determined, and the protein has been localized by immunocytochemical techniques in sections from rat testis. In the testis CRABP was found in the seminiferous tubuli with Sertoli cells and the spermatogonia most intensely stained. All other cells of the germinal epithelium appeared largely devoid of CRABP. By use of an enzyme‐linked immunosorbent assay CRABP was quantitatively estimated in several tissues and the highest levels were found in testis and eye. Comparisons of the tissue levels of CRABP and of the cellular retinol‐binding protein (CRBP) did not reveal any apparent correlation.
Effects of polyamine depletion on serum stimulation of quiescent 3T3 murine fibroblast cells
Authors: Schaefer Eugene L. , Seidenfeld Jerome
Published: February 2005

Numerous reports have shown that polyamines are required for cell proliferation. A current model for regulating commitment to DNA replication in cultured fibroblasts stimulated from quiescence by serum addition postulates sequential action by specific growth factors. To temporally localize polyamine‐dependent steps within this defined sequence, mouse Balb/c‐3T3 fibroblasts were partially depleted of polyamines by treatment with DL‐α‐difluoromethylornithine (DFMO), next rendered quiescent by serum deprivation, then stimulated by 10% serum with or without exogenous putrescine (Pu). Depletion of polyamines was verified by HPLC, and entry of cells into S phase was monitored by autoradiography. After 24 h of incubation with [3H]‐thymidine, polyamine‐depleted cells had labeling indices similar to quiescent cells if they were serum‐stimulated without Pu, but progressed to S phase to the same degree as control cultures if polyamines were restored by adding Pu at the time of serum stimulation. These observations suggested that commitment of quiescent cells to DNA replication may require polyamines. To determine if polyamine‐dependent steps occur during the pre‐commitment period (up to 12 h after serum stimulation) or only in traverse of G1 (12 h to 24 h, post‐commitment), polyamine‐depleted quiescent cells were serum‐stimulated for 12 h without Pu, then returned to low serum with Pu. Labeling indices of these cultures remained nearly as low as those of unstimulated cells. Reducing serum concentration from 10% to 0.5% at 12 h after stimulation did not effect labeling indices of control cells not depleted of polyamines by DFMO. These results supported the postulated requirement for polyamines during pre‐commitment events. However, polyamine‐deficient quiescent cells serumstimulated without Pu for periods longer than 24 h had labeling indices at 36 and 48 h significantly greater than at 24 h. This suggested that polyamine depletion may decrease the rate at which quiescent cells commit to DNA replication, rather than producing an absolute blockade during the precommitment period.
Messenger RNAs coding for enzymes of polyamine biosynthesis are induced during the G0‐G1 transition but not during traverse of the normal G1 phase
Authors: Stimac Eva , Morris David R.
Published: February 2005

The events occurring during emergence of cells from quiescence ( “G0”) are not necessarily identical to those in the G1 phase of continously dividing cells. Cellular levels of the mRNAs coding for ornithine decarboxylase (ODC) and S‐adenosyl‐methionine decarboxylase (SDC), key enzymes in polyamine synthesis, increased maximally within 5 h after addition of serum to resting 3T3 cells, following a kinetic course similar to that of c‐myc mRNA. In a pure early G1 population of cells, prepared by centrifugal elutriation of growing fibroblasts, the levels of ODC and SDC mRNAs were not significantly lower than in other phases of the cell cycle and approximated serum‐induced levels rater than the reduced values found in serum‐starved cells. Thus, we conclude that the mRNAs coding for the polyamine biosynthetic enzymes, like c‐myc, are growth controlled, but not regulated during traverse of a normal cell cycle.
Human platelet alpha granules contain a nonspecific inhibitor of megakaryocyte colony formation: Its relationship to type β transforming growth factor (TGF‐β)
Authors: Mitjavila M. T. , Vinci G. , Villeval J. L. , Kieffer N. , Henri A. , Testa U. , Breton‐Gorius J. , Vainchenker W.
Published: February 2005

Whole blood serum (WBS) and platelet‐poor plasma‐derived serum (PDS) from the same normal subject were compared for their abilities to support human megakaryocyte (MK) colony formation. In all cases, PDS promoted the growth of a higher number (20–50%) of MK colonies than did WBS. Increasing amounts of WBS decreased the number of colonies, whereas increasing concentration of PDS had no marked effects. Crude platelet extracts or platelet secretory products from thrombin‐activated platelets also elicited an inhibition of MK colony formation in a dose‐dependent manner. A complete inhibition was found for a dose equivalent to 1.109 platelets/ml and a 50% inhibition in a range of 1.107–1.108 platelets/ml. These platelet products were also inhibitory for erythroid progenitor growth. Platelets from two patients with gray platelet syndrome elicited only a minor inhibition of MK growth, suggesting that the platelet alpha granule is the origin of this inhibition. When platelet extracts were acid‐treated, the biological activity of the inhibitor on CFU‐MK and CFU‐E growth was 20–50‐fold higher. In addition, a potent stimulatory activity on the growth of day 7 CFU‐GM was observed. The enhancement of biological activities by acid treatment suggests that type β transforming growth factor (TGF‐β) could be involved in this platelet inhibitory activity. The homogeneous native TGF‐β (from 1 pg to 1 ng/ml) produced the same effects previously induced by platelet products. It totally inhibited CFU‐MK growth (at a 500 pg/ml), it inhibited CFU‐E growth, and it stimulated growth of day 7 CFU‐GM in the presence of a colony‐stimulating factor. The inhibition of CFU‐MK growth was also observed on purified progenitors. In conclusion, these results suggest that TGF‐β may be implicated in negative autocrine regulation of megakaryopoiesis. However, since this molecule has ubiquitous biological activities, its physiologic relevance as a normal regulator of megakaryopoiesis requires further investigation.
Plasma membrane fluidity measurements in intact endothelial cells: Effect of hyperoxia on fluorescence anisotropies of 1‐[4‐(trimethylamino)phenyl]‐6‐phenyl hexa‐1,3,5‐triene
Authors: Sheridan N. P. , Block E. R.
Published: February 2005

Fluorescence anisotropy measurements are widely used as sensitive indicators of cell membrane fluidity. 1‐[4‐(trimethylamino)phenyl]‐6‐phenyl hexa‐1,3,5‐triene (TMA‐DPH) is a cationic fluorescent aromatic hydrocarbon that anchors at the lipid‐water interface of membrane lipid bilayers. Its uptake into porcine pulmonary artery and aortic endothelial cells was monitored and the probe remained specifically localized on the cell surface for at least 4 h. It can therefore be recommended for use for specific plasma membrane lipid fluidity measurements in these cells. The effect of hyperoxia on plasma membrane fluidity was measured by using TMA‐DPH. In both cell types, hyperoxic damage resulted in decreases in plasma membrane fluidity. Recovery was achieved 48 h after a 42‐h hyperoxic exposure. These results indicate that TMA‐DPH is a sensitive probe of plasma membrane lipid domains of pulmonary artery and aortic endothelial cells and that hyperoxia causes reversible changes in the physical state of superficial lipid domains of the plasma membrane of these cells.
Joint oxygen‐glucose deprivation as the cause of necrosis in a tumor analog
Authors: Hlatky Lynn , Sachs Rainer K. , Alpen Edward L.
Published: February 2005

The sandwich system was recently developed as an in vitro tumor analog. Like spheroids, sandwiches are organized, multicellular systems in which the interplay between diffusion and consumption leads to the formation of spatial gradients; a necrotic center and a viable cell border subsequently develop. Using sandwiches of the 9L and V79 cell lines, the effects of oxygen and glucose deprivation on the onset and formation of necrosis were investigated. The data indicate that in sandwiches necrosis is a result of a shortage of both substances. Complementary cell monolayer experiments to determine a number of consumption parameters were performed. On the basis of the data, we propose a joint oxygen‐glucose deprivation model for V79 cell necrosis. It is assumed a cell dies when oxygen deprivation in conjunction with glucose deprivation lowers the cell's ATP production rate below a critical value. Interactions of the concentrations and consumptions of oxygen and glucose are analyzed theoretically; concentration profiles are obtained by numerically solving coupled non‐linear integral equations arising from the diffusion equation. The predicted viable border widths are in good agreement with the observed values.
Localization of protease nexin‐1 on the fibroblast extracellular matrix
Authors: Farrell David H. , Wagner Steven L. , Yuan Robert H. , Cunningham Dennis D.
Published: February 2005

Protease nexin‐1 (PN‐1) is a protease inhibitor that is secreted by fibroblasts and several other cultured cells. PN‐1 forms complexes with certain serine proteases in the extracellular environment including thrombin, urokinase, and plasmin. The complexes then bind to the cells and are rapidly internalized and degraded. This report demonstrates that PN‐1 is present on the surface of fibroblasts, bound to the extracellular matrix. Immunofluorescent studies showed that PN‐1 colocalized with fibronectin on both intact cells and in preparations of extracellular matrix made from these cells. In contrast, PN‐1 did not colocalize with the epidermal growth factor receptor, a plasma membrane marker. An enzyme‐lined immunosorbent assay was developed which showed that the extracellular matrix contained at least 60–80% of the cellular immunoreactive PN‐1. Extraction of the matrix with 2 M NaCl removed PN‐1 in a form which reacted with 125l‐thrombin to form complexes which were immunoprecipitated by anti‐PN‐1 lgG and were of identical size as complexes made from soluble PN‐1 and 125l‐thrombin. These data indicate that in addition to its role as a soluble protease inhibitor, PN‐1 is also a component of the extracellular matrix and might control its proteolysis.
Thermal adaptation and heat shock response of Tilapia ovary cells
Authors: Chen J. Don , Yew F. H. , Li Gloria C.
Published: February 2005

The growth of tissue culture TO‐2 cells derived from the warm water fish Tilapia, the induction of thermotolerance, and protein synthesis profiles of these cells in response to temperature changes were examined. TO‐2 cells can grow between 15 to 34°, with an optimal growth temperature of 31°. There is no apparent killing of the cells when the temperature is lowered to 4° for up to 3 days. Survival of TO‐2 cells at 43° was studied after various preheat treatments: (1) acute heating at 40° for 15 min followed by 31° incubation, (2) chronic exposure at 37° for several hr, or (3) long‐term thermal adaptation at 34°. The cells acquire thermotolerance from pre‐exposure to 37° for as short as 6 hr. Preheating at 40° followed by incubation at 31° also induces thermotolerance against a subsequent 43° heat challenge. In addition, 34° thermal adapted cells are resistant to 43° heating. One‐ and two‐dimensional gel electrophoresis of proteins after heat treatments show that three major heat shock proteins with molecular weights around 87, 70, and 27 kD are preferentially synthesized. The synthesis of two additional proteins with an isoelectric point of 6.9 and molecular weights of 60 and 44 kD are significantly enhanced in 34° thermal‐adapted and 37° chronic heated cells, but not in cells subjected to an acute heat shock at either 40° or 43°. On the other hand, the 27 kD heat shock proteins are mainly present in the 43°, 40°, and 37° heat‐shocked cells, but not in the 34° thermal‐adapted cells.
Characterization of plasminogen activator produced by an established cell line from human ovary
Authors: Matsuo Chikami , Fukao Hideharu , Matsuo Osamu
Published: February 2005

An established cell line (OC‐1) was obtained from human ovarian tissue, which yielded a high concentration of plasminogen activator (PA) in the culture medium. The PA (OC‐1‐PA) produced by the cell line was purified and compared with urokinase (UK), proform of UK (pro‐UK), and tissue‐type PA (t‐PA) purified from human melanoma cells (Bowes). OC‐1‐PA was purified by Zn chelate‐Sepharose affinity chromatography followed by high‐performance liquid chromatography with a Zn chelate‐5PW column and with a p‐amino‐benzamidine‐5PW column, giving a yield of 58.3% and a purification factor of 15,439. This purified material revealed a single band of Mr 55,000 on sodium dodecylsulfate polyacrylamide gel electrophoresis in the presence or absence of reducing agents. Electrophoresis enzymography demonstrated that the Mr 55,000 protein band had a plasminogen‐dependent fibrinolytic activity. Treatment with plasmin did not change the Mr even in the presence of reducing agents. These results suggest that OC‐1‐PA has a single‐chain structure protected from protease degradation, which is completely different from UK. The activator had higher affinities for lysine and fibrin than those of UK or pro‐UK. An immunological study demonstrated that OC‐1‐PA cross reacted with anti‐UK lgG but not with anti‐t‐PA lgG. All these findings indicate that OC‐1‐PA belongs immunologically to the UK type, but its structure differs from that of UK.
Decreased membrane permeability to potassium is responsible for the cell volume increase that drives lens fiber cell elongation
Authors: Parmelee Judith T. , Beebe David C.
Published: February 2005

Differentiation of lens epithelial cells into fiber cells involves an increase in cell volume which previously was proposed to be the direct cause of the extensive cell elongation which accompanies fiber formation. In this study we have continued to investigate the mechanism underlying cell elongation by defining the minimum nutrient and ion requirements of elongating cells, measuring potassium and sodium fluxes in stimulated and unstimulated lens epithelia, and determining the effects of several pharmacological agents on elongation and ion transport. We have shown that elongation will occur in a basic salt/glucose solution with Insulin‐like growth factor I/somatomedin‐C stimulation. Neither sodium nor any metabolite appears to be the osmotically active species which drives the increase in cell volume. However, potassium efflux rate coefficent was 47% lower in differentiating cells than in unstimulated cells, whereas potassium uptake rates and ouabain effects were similar. Cells did not elongate in potassium‐free medium nor in the presence of several drugs which prevent the accumulation of intracellular potassium or hinder osmotic water flux. Unstimulated cells elongated, however, with the application of quinidine, a drug known to block potassium channels. We propose that stimulation of lens epithelial cells with an insulin‐like growth factor signals the closure of a certain population of potassium channels. As a result, potassium efflux from the differentiating cells slows while active potassium uptake continues at a constant rate. Potassium then accumulates within the cell causing water influx, an increase in cell volume, and cell elongation.
Optical microscopy of antibody‐dependent phagocytosis and lysis of erythrocytes by living normal and chronic granulomatous disease neutrophils: A role of superoxide anions in extra‐ and intra‐cellular lysis
Authors: Francis Joseph W. , Boxer Laurence A. , Petty Howard R.
Published: February 2005

Novel optical microscopic techniques have been developed to observe neutrophil‐mediated effector functions at the level of individual cells. Conventional absorption spectrophotometry has shown that exposure of hemoglobin to superoxide anions decreases the intensity of the Soret band and shifts it to lower wavelengths. This oxidative event can be visualized within intact erythrocytes using bright‐field microscopy in conjunction with violet illumination at 430 nm. The sequential oxidation of lgG‐opsonized sheep erythrocytes bound to normal human neutrophils can be observed. Chronic granulomatous disease (CGD) neutrophils which do not generate superoxide anions were not capable of influencing target absorption at 430 nm. Cytolytic events were visualized by fluorescence microscopy. Cytosolic or membrane compartments of sheep erythrocytes were labeled with eosin Y or fluorescein isothiocyanate, respectively. Time‐dependent studies of erythrolysis show that targets are lysed extra‐ and intra‐cellularly. The fluorescent diffusion gradient generated at the site of membrane rupture suggests that a pore of approximately 30 nm in diameter is formed in the target membrane. The site of pore formation is not found at the target‐effector cell interface. CGD neutrophils did not display these cytolytic phenomena. Furthermore, the cytosolic label eosin Y could be followed into an associated granule compartment; we suggest that the phenomenon of piranhalysis may participate in antibody‐dependent effector mechanisms. Phagocytosis can also be observed using fluorescently‐labeled erythrocytes. Determinations of phagocytic index are more reliable with this approach. These microscopical methods are both simple and efficient. To our knowledge, these are the first direct microscopic studies of effector cell‐mediated target cell oxidation and cytolysis. These experiments provide a fresh approach to the study of phagocyte effector functions at the cellular level and illuminate the importance of superoxide anions in antibody‐dependent erythrolysis.
A monoclonal antibody that recognizes a basolateral membrane protein in A6 epithelial cells
Authors: Moberly James B. , Fanestil Darrell D.
Published: February 2005

The A6 cell line is a model for tight epithelia and studies of epithelial polarity. Monoclonal antibodies (MAbs) were produced by immunization of mice with intact A6 cells and fusion of spleen cells to generate hybridomas. Hybridoma supernatants were screened by ELISA to select MAbs binding to the apical membrane of confluent A6 cells. Localization of MAb binding was examined by indirect immunofluorescence using cross sections of A6 monolayers grown on collagen coated filters. One MAb, designated 13F12, was positive by apical surface ELISA but localized specifically to the basolateral membrane of cross sections of A6 monolayers on filters. Immunofluorescence labeling of confluent A6 cells grown on glass cover slips revealed that MAb 13F12 does not bind to the apical membrane, but binds to basolateral determinants in the regions of domes, where it appears able to penetrate cellular junctions. Subconfluent A6 cells express the antigen all over the cell surface. Cells approaching confluency express the antigen on the apical membrane of some cells but not others, and as the cells reach confluency, the antigen disappears from the apical surface, and the cells become fully polarized. A6 cells at confluency on glass cover slips are equally polarized as cells grown on filters with respect to this antigen. The antigen has been identified by immunoprecipitation as a 22 kDa protein. High concentrations of MAb 13F12 did not inhibit cell plating, indicating that the antigenic site is not directly involved in cell adhesion to the substrate. MAb 13F12 should prove to be a useful tool to study many aspects of epithelial polarity, including the signals involved in sorting of proteins to specific membrane domains.
Sodium butyrate preserves aspects of the differentiated phenotype of normal adult rat hepatocytes in culture
Authors: Staecker Jeffrey L. , Sattler Carol A. , Pitot Henry C.
Published: February 2005

We have determined that sodium butyrate and, to a lesser extent, dimethylsulfoxide (DMSO) and 3‐aminobenzamide (3‐AB) preserve aspects of the differentiated phenotype of primary cultures of adult rat hepatocytes. The histone deacetylase inhibitor, butyrate, inhibits the increase in y‐glutamyltranspeptidase (GGT) activity and the decrease in basal tyrosine aminotransferase (TAT) activity normally observed when hepatocytes are cultured under appropriate conditions. The effects of butyrate on GGT and TAT activities are accompanied by parallel changes in GGT and TAT mRNA levels. The poly(ADP)ribose‐synthetase inhibitor, 3‐aminobenzamide, has effects similar to butyrate on GGT activity and mRNA levels, while both 3‐AB and DMSO increase basal TAT activity in cultured hepatocytes. Under appropriate conditions all three agents—butyrate, 3‐AB, and DMSO‐extend the length of time cultured hepatocytes can be maintained as confluent monolayers. However, under all the conditions studied, butyrate extended the length of time hepatocytes could be maintained as monolayers more than any other treatment used. Butyrate‐treated hepatocytes maintained ultrastructural features that were more similar to those of hepatocytes in vivo than hepatocytes treated with any other of the agents tested. Histone acetylation levels of primary cultures of adult rat hepatocytes declined concomitant with the loss of the differentiated phenotype of the cells. These results suggest that histone acetylation may play a role in the changes in gene expression observed when hepatocytes are placed in culture.
A function that relates protein synthetic rates to potassium activity in vivo
Authors: Horowitz Samuel B. , Lau Ying‐Tung
Published: February 2005

A newly developed experimental system allows the controlled alteration of intracellular K+ activity (aK) and the measurement of amino acid incorporation rates in a single cell, the Xenopus oocyte. We found that as aK is increased by microinjecting a K+ salt, [3H]leucine incorporation (R) varies over a 100‐fold range, first stimulated and then inhibited as it passes through four response regions (A–D). In region A (aK ∼ 60–100 mM), R is at a nongrowth or maintenance level and is stimulated weakly by increasing aK. In region B (aK ∼ 100–130 mM), R is stimulated intensely by increasing aK, roughly tripling with every 10 mM increase. In region C (aK ∼ 130–160 mM), R is inhibited intensely by increasing aK. Finally, in region D (aK > 160 mM), R is inhibited weakly as aK increases. Collectively, the four response regions constitute the oocyte's R/aK response function. The function provides a comprehensive description of how K+ activity influences the rate of protein synthesis in an intact cell. In the subsequent discussion, we compared the oocyte response function with the K+ response determined in cell‐free translational systems. While in vivo and in vitro functions are similar, differences exist that may be important in a cellular control system. We then considered the relevance of the oocyte R/aK response function to “normal” processes in the oocyte and in somatic cells, i.e., those in which aK is varied by physiological changes in the plasma membrane. We concluded that the intensely stimulatory region B is importantly involved in hormonal action and other growth‐activating processes and that the entire R/aK response function may play a role in control of protein synthesis during the cell cycle.
Requirement for extracellular calcium or magnesium in mitogen‐induced activation of human peripheral blood lymphocytes
Authors: Modiano Jaime F. , Kelepouris Ellie , Kern Jeffrey A. , Nowell Peter C.
Published: February 2005

The importance of calcium in lymphocyte activation is well recognized, but the levels of extracellular ionized free calcium (Ca++) necessary for lymphocyte proliferation via various pathways have not been investigated in detail. We studied the ability of a lectin mitogen (PHA) and a calcium ionophore (ionomycin) to induce interleukin 2 receptors, interleukin 2 (IL2) production, and proliferation over various concentrations of extracellular Ca++. Reducing the Ca++ levels from the normal 200 μM to 10 μM in PHA‐stimulated cultures partially inhibited IL2 receptor expression, IL2 production, and subsequent proliferation. At 1 μM Ca++, both IL2 activity and proliferation were eliminated, but partial IL2 receptor expression was still observed. Ionomycin did not induce any of these events in cultures where the extracellular Ca++ concentration was below 100 μM. Restoring calcium in the medium resulted in normal levels of IL2 receptor expression, IL2 activity, and proliferation when PBL were stimulated with either mitogen. Exogenous magnesium partially restored these events in PHA‐stimulated cultures, but had no effect when ionomycin was used as the mitogen. These data indicate that stimulation by ionomycin is much more dependent upon the levels of extracellular Ca++ than is PHA. Extracellular calcium also appears to be necessary subsequent to IL2 receptor acquisition, since the latter was seen without IL2 activity or proliferation at very low extracellular Ca++, and IL2 failed to restore the proliferative response under these conditions. The data also suggest that PHA, but not ionomycin, can activate lymphocytes via a magnesium dependent pathway, or that PHA has a lower specificity for divalent cation cofactors.
Studies on the mechanism of zinc uptake by human fibroblasts
Authors: Ackland M. Leigh , Danks David M. , McArdle Harry J.
Published: February 2005

The mechanisms of zinc uptake from a complete culture medium by human fibroblasts have been studied. The metal is accumulated in a biphasic pattern; an initial rapid phase followed by a slower linear phase. We suggest that the former represents binding to carriers or receptors on the cell surface followed by uptake to within the cell, or at least to a compartment inaccessible to proteolytic digestion. The uptake correlates well with estimates of the zinc requirement of a growing fibroblast. The process of uptake is saturable, with an apparent association constant of 1.1 × 107 M−1. Interestingly, there appears to be a very large number of binding sites, 2 × 107 per cell. No explanation for this observation is immediately apparent. The mechanism of uptake is not dependent on metabolic energy, or at least on ATP levels within the cell, but N‐ethyl maleimide does block uptake in a dose‐dependent manner. Weak bases and ionophores, apart from nigericin, do not affect uptake. The results suggest that zinc is not taken up by a receptor‐mediated endocytic pathway as has been described for transferrin and iron.
Interaction of rat asialotransferrin with adult rat hepatocytes: Its relevance for iron uptake and protein degradation
Authors: Rudolph John R. , Regoeczi Erwin
Published: February 2005

Comparative studies with rat transferrin (rTf) and asialotransferrin (asialo‐rTf) were performed on suspended adult rat hepatocytes with the aim of elucidating the mechanism of enhanced hepatic Fe acquisition from asialo‐rTf observed previously in vivo. At low ligand concentrations (0.05–20 μ/ml), the cells bound more asialo‐rTf thanrTf. However, the excess binding was abolished by incubation either in the presence of 1.55 mg/ml of diferric rTf or of 1 mg/ml of asialomucin. Following either treatment, asialo‐rTf and rTf were bound to comparable extents. These findings indicate that both transferrin receptors and the hepatic galactose recognition system (lectin) are essential for preferential binding of asialo‐rTf by hepatocytes. The possibility is considered that the lectin facilitates capture of asialo‐rTf by the same binding sites that are normally available for rTf rather than that it functions as an alternative pathway. In agreement with this view, asialo‐rTf could not be channeled into the lectin‐mediated degradative pathway by blocking Tf receptors with human Tf. Enhanced Fe uptake from asialo‐rTf was fully prevented by asialomucin and partially prevented by human Tf. The incomplete efficacy of human Tf in this regard supports reports in the literature about Fe uptake by the liver in a manner that is independent of Tf receptors. Rhesus asialo‐Tf was deployed to show that no recognition mechanism exists for heterologous asialo‐Tf in rat hepatocytes. The importance of using undenatured labeled proteins for studies with cells is demonstrated.
Antimitogenic actions of lectins in cultured human fibroblasts
Authors: Kaplowitz Paul B. , Haar Jack L.
Published: February 2005

It was previously reported that the lectins wheat germ agglutinin (WGA) and concanavalin A (Con A) inhibit the mitogenic actions of multiple peptide growth factors in human fibroblasts without having a significant effect on mitogen binding. The current studies were designed to further examine the mechanism of this antimitogenic action of lectins. Addition of WGA at progressively later times after stimulation of fibroblasts with peptide mitogens revealed significant inhibition of DNA synthesis even when the lectin was added 16–20 h after growth factors. This suggests an inhibitory effect on a pathway occurring late in G1, or close to the G1/S boundary. WGA also inhibited stimulation of DNA synthesis by non‐peptide agents such as colchicine and vanadate ion, indicating that the lectin inhibits a common distal step in the mitogenic response, rather than acting primarily on events occurring at the level of the growth factor‐receptor interaction. WGA had a rapid (within 30 min) inhibitory effect on insulin‐stimulated amino acid uptake, but Con A, which like WGA blocked mitogen‐stimulated 3H‐dT incorporation, had little effect on stimulation of amino acid uptake. Thus the inhibition of DNA synthesis and amino acid uptake by lectins appear to be mediated by distinct mechanisms. WGA binding to fibroblasts persisted even when the lectin was removed from the incubation medium, but unlike 125I‐EGF, which was rapidly internalized at 24deg;C, little 125I‐WGA was internalized. Incubation of fibroblasts for 20 h with WGA or Con A was not toxic to cells, since reversal of lectin binding by the appropriate saccharide allowed normal subsequent stimulation of DNA synthesis by EGF and insulin. However, the observation that cells exposed to antimitogenic lectins undergo a marked decrease in cell spreading suggests that changes in cell shape may be relevant to the mechanism by which lectin‐treated fibroblasts become unresponsive to mitogenic stimulation.
Temperature‐sensitive Chinese hamster cell mutant with a defect in glycoprotein synthesis: Accumulation of the EGF receptor in the endoplasmic reticulum and the role of the glucose‐regulated protein GRP78
Authors: Feige Jean‐Jacques , Keller Gilbert‐A. , Scheffler Immo E.
Published: February 2005

A temperature‐sensitive mutant of Chinese hamster fibroblasts with a defect in glycoprotein synthesis is investigated after transfection and amplification of the gene for the human EGF receptor. We demonstrate that at the nonpermissive temperature a partially glycosylated species of the receptor accumulates in the endoplasmic reticulum. The oligosaccharides present are the high mannose types, since they can be removed completely by treatment with endoglycosidase H. Pulse‐chase experiments show that the abnormal species of the receptor cannot be chased to a form that is either resistant to endoglycosidase H, or altered in its mobility on SDS polyacrylamide gels. The abnormal species of the receptor appears within the first hour of a shift to the nonpermissive temperature, and no further changes are observed upon prolonged incubation of cells at 40deg;C. However, after 3–4 hours immunoprecipitations of the receptor yield another protein, which has properties very similar, if not identical, to the glucose‐regulated protein GRP78. The induction of this protein at 40deg;C can be suppressed completely with an inhibitor of RNA synthesis, without any effect on the glycosylation defect, or on the accumulation of the EGF receptor in the endoplasmic reticulum.
Sodium butyrate induces differentiation in breast cancer cell lines expressing the estrogen receptor
Authors: Graham Kathryn A. , Buick Ronald N.
Published: February 2005

Addition of sodium butyrate (NaB) to 6 cultured human breast carcinoma cell lines results in a dose and time‐dependent growth inhibition. Kinetic evidence, related to the growth of a minority cell population which decreases in size with time of exposure, is presnted to indicate that the NaB effect is reversible. In those cell lines that express the estrogen receptor (ER), growth inhibition is accompanied by a more differentiated phenotype, which is characterized by increased accumulation of lipid and milk‐fat globule membrane glycoproteins. The poential for differentiation is not blocked by tamoxifen, indicating that the relationship to ER expression is likely secondary to the association of ER expression with a particular stage of secretory cell differentiation that is susceptible to NaB induction. Of the 3 lines shown to respond in this way (MCF‐7, ZR‐75‐1, and MDA‐134), ZR‐75‐1 is an extreme example that may serve as a model for studies of gene expression during human mammary epithelial cell differentiation.
A nuclear cAMP binding protein in retinoic acid‐treated HL‐60 cells
Authors: Briggs Robert C. , Casey Susan B.
Published: February 2005

A cAMP binding protein was detected in HL‐60 cells using photoaffinity labeling with 8‐azido [32P]cAMP. The binding protein was found in a 0.35 M NaCl nuclear protein extract from untreated HL‐60 cells and from the HL‐60 cells induced to mature with retinoic acid. While the quantity of the cAMP binding protein did not change following the induced differentiation, a second form of the subunit, altered in charge, was present at 3 and 5 days after retinoic acid treatment. The findings indicate that the regulatory subunit of the type II cAMP‐dependent protein kinase could be involved in nuclear functions associated with human myeloid cell differentiation.
Alteration of the Cloudman melanoma cell cycle by prostaglandins E1 and E2 determined by using a 5‐bromo‐2′‐deoxyuridine method of DNA analysis
Authors: Abdel‐Malek Zalfa A. , Swope Viki B. , Trinkle Linda S. , Ferroni Elisabeth N. , Boissy Raymond E. , Nordlund James J.
Published: February 2005

Prostaglandins (PGs) E1 and E2 stimulate tyrosinase activity and suppress the proliferation of Cloudman S91 melanoma cells by altering their progression through the cell cycle. Prostaglandin E1 and PGE2 have prolonged or residual effects on melanoma cells. Cells treated for 5 or 24 hours with 10 μg/ml PGE2 demonstrated decreased proliferation and increased tyrosinase activity for 48 hours after removal of the PGs. The effects of PGs on the cell cycle were investigated by determining total DNA content in cells stained with propidium iodide (Pl) and analyzed by a fluorescence activated cell sorter (FACS). Prostaglandin E1 blocked cells in G2 phase after 5 hours of treatment, corresponding to when inhibition of proliferation was first evident. Similarly, after 9 hours of treatment with PGE2, more cells were in late S, early G2 phase and less in G1 than their control counterparts. Also, melanoma cells were pulse‐labeled with 5‐bromo‐2′‐deoxyuridine (BrdUrd) prior to or at the end of PG treatment and then stained with a fluoresceinated monoclonal antibody to BrdUrd, and with Pl. This allows one to observe how BrdUrd‐labeled S‐phase cells cycle with time. Both PGE1 and PGE2 inhibit proliferation by blocking cells in G2 phase of the cell cycle. The PG‐induced block in G2 may be required by melanoma cells to synthesize mRNA and proteins that are essential for stimulation of tyrosinase activity. Ultrastructurally, only a subpopulation of the cells treated with PGE1 or PGE2 contained more mature melanosomes than control cells.
Uptake of copper by mouse hepatocytes
Authors: McArdle Harry J. , Gross Sharon M. , Danks David M.
Published: February 2005

This study has investigated the uptake of copper by mouse hepatocytes. The cells gave similar results whether they were used right after isolation or maintained overnight on collagen‐coated dishes. Uptake from cells in suspension followed two phases: an initial rapid binding followed by a linear uptake phase. The two phases were not so easily distinguishable in cells grown in culture where uptake was linear over the first hour. The uptake showed saturation but may not have followed simple kinetics. Histidine stimulated uptake in a concentration‐dependent manner, as did some other amino acids, but copper had very little effect on histidine uptake. The process was not dependent on intracellular adenosine triphosphate (ATP), since inhibitors that substantially reduced ATP levels inside the cell did not alter copper uptake. The inhibitors, however, blocked histidine uptake to varying degrees, suggesting that copper and histidine are taken up by different pathways. The uptake was reduced markedly by N‐ethyl maleimide, and preincubation of the cells with “Pronase” resulted in a decrease of uptake. A model for the uptake of copper by hepatocytes that incorporates the data presented in this paper with that produced by earlier workers is suggested.
Superoxide as a signal for increase in intracellular pH
Authors: Shibanuma Motoko , Kuroki Toshio , Nose Kiyoshi
Published: February 2005

We examined the role of superoxide in the increase in intracellular pH (pHi) of human histiocytic leukemia U937 cells treated with 4β‐phorbol‐12,13‐didecanoate (4β‐PDD) or serum. 4β‐PDD or serum induced a rapid increase in pHi, and antioxidants such as superoxide dismutase (SOD), vitamin E, and butylated hydroxyanisole (BHA) were found to inhibit the amiloride‐sensitive increase in pHi induced by 4β‐PDD. SOD inhibited the increase in pHi caused by serum, and essentially the same was found in concanavalin A‐stimulated mouse thymocytes. Also, a superoxide‐generating system, xanthine‐xanthine oxidase (X‐XOD), increased pHi of U937 cells as much as 4β‐PDD or serum. From these findings, it appears that superoxide is the basis for the modulation of pHi.