bluuebunny/crossref_metadata_2025_split huggingface dataset:
Thermal adaptation and heat shock response of Tilapia ovary cells
Authors: Chen J. Don , Yew F. H. , Li Gloria C. 
Published: February 2005

AbstractThe 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

AbstractAn 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

AbstractDifferentiation 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

AbstractNovel 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

AbstractThe 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

AbstractWe 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

AbstractA 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

AbstractThe 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

AbstractThe 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

AbstractComparative 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

AbstractIt 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

AbstractA 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

AbstractAddition 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

AbstractA 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.