bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ca++‐ionophore‐induced dissipation of intracellular proton gradients in rat thymocytes
Authors: Dell'Antone Paolo 
Published: February 2005

AbstractRat thymocytes incorporate large amounts of acridine orange at concentrations of approximately 10 μM in about 10 minutes at 37oC. The addition of (NH4)2SO4 (at a final concentration of several mMolars) releases about 30% of the incorporated dye into the medium. The NH4+‐releasable dye uptake is almost completely abolished by 20 minutes' incubation with 4 μM of the Ca++‐ionophore A23187. Dye uptake is associated with an absorbance change at 492 mm and thus may be followed spectrophotometrically. However, NH4+ at the above concentrations or nigericin (0.5 mg/ml) completely annul this change in absorbance, indicating that it reflects only the accumulation of the dye within the acidic cellular compartments. In a Ca++‐containing physiological saline, the addition of A23187 (at a final concentration of 8 μM) at the end of the dye uptake phase initiated a reversal in the absorbance change; in the absence of Ca++, reversal occurred at a much lower rate. Incubation of cells for 30 minutes with 2 μM A23187 in Ca++‐containing saline completely abolished NH4+‐sensitive dye uptake; less A23187 (0.5 μM) and like or a longer incubation period brought about a striking decrease in NH4+‐sensitive dye uptake. Similar results were obtained with cells suspended in RPLM 1640 medium. In the absence of external Ca++, A23187 impaired cell capacity to incorporate dye in a ΔpH‐dependent manner, but a longer incubation time or higher concentrations of the ionophore were required to obtain a comparable effect. It is thus concluded that the ionophore dissipates intracellular pH gradients by an intracellular divalent cation (Ca++ or also Mg++)‐H+ exchange.
Evidence for two pathways of protein kinase C induction of 2AR expression: Correlation with mitogenesis
Authors: Smith James H. , Denhardt David T. 
Published: February 2005

Abstract2ar is a cDNA clone of an mRNA that is inducible by 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA) in mouse JB6 epidermal cells. This mRNA is stably induced in confluent cells but only transiently induced in subconfluent cells; the elevated level of 2ar mRNA in TPA‐treated cells appears to be the result of enhanced transcription (Smith and Denhardt: Journal of Cellular Biochemistry 34:13–22, 1987). Phorbol dibutyrate, teleocidin, and aplysiatoxin, which activate protein kinase C and are also tumor promoters, are shown here to induce 2ar to the same extent as TPA at both cell densities. The increased expression by TPA was prevented by cycloheximide and by H7, a specific inhibitor of protein kinase C. Epidermal growth factor, platelet‐derived growth factor, and the nonphorbol promoter diethylhexylphthalate were more effective inducers in confluent than in subconfluent cultures. All‐trans retinoic acid, dexamethasone and fluocinolone acetonide, inhibitors of tumor promotion, diminished 2ar induction in both confluent and subconfluent cells. In TPA‐treated subconfluent cultures indomethacin produced a slight inhibition, whereas difluoromethyl ornithine potentiated the induction. In TPA‐treated confluent cultures, in contrast, indomethacin enhanced 2ar mRNA levels and difluoromethyl ornithine was inhibitory. We conclude that the protein kinase C‐mediated induction of 2a‐ expression is controlled by different pathways in subconfluent cultures and confluent cultures, indicative of apparent changes in the regulation of gene expression as proliferating cells become quiescent.
Inhibition of heat shock (stress) protein induction by deuterium oxide and glycerol: Additional support for the abnormal protein hypothesis of induction
Authors: Edington Brent V. , Whelan Sandra A. , Hightower Lawrence E. 
Published: February 2005

AbstractThe patterns of radioactively labeled proteins from cultured chicken embryo cells stressed in the presence of either D2O or glycerol were analyzed by using one‐dimensional polyacrylamide gel electrophoresis. These hyperthermic protectors blocked the induction of stress proteins during a 1‐hour heat shock at 44°C. The inhibitory effect of glycerol but not D2O on the induction of heat shock proteins could be overcome by increased temperature. By using transcriptional run‐on assays of isolated nuclei and cDNA probes to detect hsp70‐ and hsp88‐specific RNA transcripts, it was shown that the D2O and glycerol blocks occurred at or before transcriptional activation of the hsp70 and hsp88 genes. After heat‐stressed cells were returned to 37°C and the protectors were removed, heat shock proteins were inducible by a second heating. This result and the fact that the chemical stressor sodium arsenite induced stress proteins in glycerol medium indicated that the treatments did not irreversibly inhibit the induction pathways and that the stress response could be triggered even in the presence of glycerol by a stressor other than heat. In principle then, cells incurring thermal damage during a 1‐hour heat shock at 44°C in D2O or glycerol medium should be competent to respond by inducing heat shock proteins during a subsequent recovery period at 37°C in normal medium. We found that heat shock proteins were not induced in recovering cells, suggesting that glycerol and D2O protected heat‐sensitive targets from thermal damage. Evidence that the heat‐sensitive target(s) is likely to be a protein(s) is summarized. During heat shocks of up to 3 hours duration, neither D2O nor glycerol significantly altered hsp23 gene activity, a constitutively expressed chicken heat shock gene whose RNA transcripts and protein products are induced by stabilization (increased half‐life). During a 2‐hour heat shock, glycerol treatment blocked the heat‐induced stabilization of hsp23 RNA and proteins; however, D2O treatment only blocked RNA transcript stabilization, effectively uncoupling the hsp23 protein stabilization pathway from hsp23 RNA stabilization and transcriptional activation of hsp70 and hsp88 genes.
Elevated c‐fos expression inhibits differentiation of L6 rat myoblasts
Authors: Rahm Magnus , Jin Pei , Sümegi Janos , Sejersen Thomas 
Published: February 2005

AbstractExpression of c‐fos is induced by a number of signals in several cell systems. Although the exact function of the c‐fos product is unknown, it has been implicated to be of importance for both cell growth and differentiation (Verma and Sassone‐Corsi, 1987). To analyze how c‐fos expression relates to in vitro myogenic differentiation, the kinetics of c‐fos mRNA expression during spontaneous in vitro differentiation of L6J1 myoblasts was examined; c‐fos transcripts were most abundant at day 4 of the differentiation process. Multinucleated myotubes and expression of α‐actin and myosin heavy chain (MHC) mRNA appeared later, at day 6 or 7, and increased to maximal levels after 10 days in culture. To analyze further the relation between c‐fos expression and L6J1 myogenic differentiation, L6J1 myoblasts were transfected with expression vectors containing the murine c‐fos gene driven by a metallothionein promoter. The growth rate of c‐fos‐transfected L6J1 cells did not differ from that of control cells. However, formation of myotubes was significantly reduced in c‐fos‐transfected L6J1 cultures compared with neo‐transfected controls. Myotube formation and expression of the myogenic markers α‐actin and MHC were reduced in subclones expressing high levels of c‐fos, but not in subclones with lower levels of c‐fos expression. These results indicate that a marked elevation of c‐fos expression at least partially inhibits L6J1 myogenic differentiation.
Human microvascular endothelial cells express integrin‐related complexes that mediate adhesion to the extracellular matrix
Authors: Cheng Yao‐Fen , Kramer Randall H. 
Published: February 2005

AbstractMicrovascular endothelial cells (MEC) must use a set of surface receptors to adhere not only to the vascular basement membrane but, during angiogenic stimulation, to the interstitium. We examined how cultured MEC Isolated from human foreskin interact with their subendothelial matrix. MEC were able to attach to diverse extracellular matrix proteins, including fibronectin (Fn), vitronectin (Vn), laminin (Ln), type I and IV collagen, as well as to fibrinogen and gelatin. Adhesion to Fn, but not to laminin or collagens, was specifically blocked in the presence of Arg‐Gly‐Asp (RGD)‐containing peptides. When surface radioiodinated MEC were solubilized and subjected to affinity chromatography on Fn‐Sepharose columns, two polypeptides of 150 and 125 kD, corresponding to the integrin heterodimer α5β1, were identified. MEC also express a complex of 150 (α) and 95 kD (β3) that is related to the Vn receptor. Immunofluorescent staining of MEC cultures with antibodies to the integrin β1 subunit demonstrated receptors on the basolateral surface at focal adhesion plaques that co‐localized with vinculin and with Fn‐positive matrix fibers. Occasionally, antibodies to the Vn receptor stained the vinculin‐positive focaladhesion plaques that frequently co‐localized with the β1 complex. However, in cultures of MEC that were attached to substrates coated with alternating strips of Fn and Vn, the β1 complex was preferentially localized to the Fn substrate, while the Vn receptor was concentrated on the Vn substrate. The results indicate that MEC express at least two different heterodimer adhesion receptors that belong to the integrin superfamily and appear to have distinct ligand specificities: the Fn receptor and the Vn receptor. These receptors mediate cell adhesion to the extracellular matrix and presumably have an important role in hemostasis and neovascularization.
Control of cell division in hepatoma cells by exogenous heparan sulfate proteoglycan
Authors: Fedarko Neal S. , Ishihara Masayuki , Conrad H. Edward 
Published: February 2005

AbstractThe effects of cell surface heparan sulfate proteoglycan (HSPG) prepared from log and confluent monolayers of a rat hepatoma cell line on hepatoma cell growth were studied. When HSPG isolated from confluent cells was added exogenously to log phase cells, it was internalized and free heparan sulfate (HS) chains appeared transiently in the nucleus. Concurrently, the growth of the treated cells was inhibited, but the cells resumed logarithmic growth as the level of nuclear HS fell, and the cells grew to confluence and became contact inhibited. When HSPG prepared from log‐phase hepatoma cells was added exogenously to log phase cells, it was internalized but very little of the internalized HS appeared in the nucleus, and there was no change in the rate of cell growth. However, when the rate of cell growth was reduced by culture of the cells in serum‐ and insulin‐deficient medium, HSPG prepared from log‐phase cells stimulated the growth rate of these slow‐growing cells. The cell cycle dependency of HSPG uptake and growth inhibition was studied in cultures synchronized by a thymidine/aphidicolin double block. When [35SO4]HSPG from confluent cells was added to synchronized cells just as they were released from the second block, a portion of the [35SO4]HSPG was internalized and [35SO4]HS appeared in the nucleus. However, at mitosis the [35SO4]HS disappeared almost completely from all of the cellular pools, and after mitosis, more of the [35SO4]HSPG was taken up and [35SO4]HS reappeared in the nucleus and remained in the nucleus until the cells divided again. When cultures were released from the aphidicolin block, both control and HSPG‐treated cells progressed through the S, the G2, and the M phases of the cell cycle. However, the length of the G1 phase of the cycle was increased in the HSPG‐treated cells. The treated cultures then progressed through the second S, G2, and M phases. Thus, the inhibition of cell division occurred in the G1, phase of the cell cycle, prior to the G1,/S boundary. Addition of the HSPG to the synchronized cultures just after the first mitosis resulted in an immediate arrest of the cell cycle in G1, These results support the earlier suggestion (M. Ishihara, N.S. Fedarko, and H.E. Conrad 1987 J. Biol. Chem. 262:4708–4716; M. Ishihara and H.E. Conrad (1989) J. Cell Physiol., in press) that the HSPG formed by confluent hepatocytes plays a role in the prevention of cell division, whereas the HSPG formed by exponentially growing cells plays a role in the stimulation of cell division. The inhibition of cell growth results from a block in the G1, phase of the cell cycle prior to the G1,/S boundary.
Differentiation of germ cells in seminiferous tubules transplanted to testes of germ cell‐deficient mice of W/Wv and SI/SId genotypes
Authors: Kuroda Hideya , Nakayama Hiroki , Namiki Mikio , Matsumoto Keishi , Nishimune Yoshitake , Kitamura Yukihiko 
Published: February 2005

Abstract(WB × C57BL/6)F1‐W/W (hereafter, WCB6F1‐SI/SId) mice are sterile due to the deficient spermatogenesis in the testes. The cause of deficient spermatogenesis in WBB6F1‐W/W mice is considered to be a defect in germ cells themselves, whereas that in WCB6F1‐SI/SId mice is considered to be a defect in tissue environment necessary for differentiation of germ cells. Seminiferous tubules isolated from cryptorchid testes of C57BL/6‐+/+ mice were transplanted into the testes of WBB6F1‐W/W and WCB6F1‐SI/SId mice to clarify that the extratubular environment of these mice was intact or not. Type A spermatogonia in the transplanted tubules normally differentiated into spermatids, suggesting that the extratubular environment is intact in both WBB6F1‐W/W and WCB6F1‐SI/SId mice.
Cyclic AMP can partially restore platelet‐derived growth factor‐stimulated prostaglandin E2 biosynthesis, and calcium mobilization in EJ‐ras‐transformed NIH‐3T3 cells
Authors: Olinger P. L. , Benjamin C. W. , Gormacn R. R. , Connor J. A. 
Published: February 2005

AbstractNIH‐3T3 cells transformed by the EJ‐ras oncogene display reduced platelet‐derived growth factor (PDGF)‐stimulated phospholipase C activity as measured by inositol 1,4,5‐triphosphate (IP3) synthesis and Ca2 + mobilization. The lack of PDGF‐stimulated Ca2+ mobilization in EJ‐ras transformed cells is not due to a loss of IP3 sensitivity, because microinjected IP3 elevates intracellular Ca2+. Treatment of EJ‐ras transformed cells with cholera toxin or 8‐bromo‐cyclic AMP, but not pertussis toxin or the β‐subunit of cholera toxin, results in a slight recovery of PDGF‐stimulated IP3 synthesis, a marked increase in intracellular Ca2+ mobilization, and an almost complete recovery of prostaglandin E2 biosynthesis. These data suggest that EJ p21‐mediated inhibition of PDGF‐stimulated intracellular events can be partially and transiently reversed by cyclic AMP.
Regulation of mitosis onset and thymidine kinase activity during the cell cycle of Physarum polycephalum plasmodia: Effect of fluorodeoxyuridine
Authors: Wright M. , Tollon Y. 
Published: February 2005

AbstractThe effects of fluorodeoxyuridine were investigated during three events of the cell cycle: S‐phase, mitosis, and the cyclic synthesis of thymidine kinase in the synchronous plasmodium of the myxomycete Physarum. DNA synthesis was inhibited, and there was limited action on other macromolecular syntheses. When DNA synthesis was slowed down, onset of the following increase of thymidine kinase synthesis occurred at approximately the same time as in the control, but mitosis was blocked in a very early prophase stage and metaphase was never observed. These effects were suppressed when the action of fluorode‐oxyuridine was prevented by the addition of thymidine to the medium. In agreement with the action of aphidicolin and hydroxyurea, these observations show that: (1) perturbation of the S‐phase does not prevent the nuclei from entering a very early prophase stage, but it does prevent them from proceeding through metaphase; (2) blockage of DNA synthesis does not perturb the normal timing of the triggering of thymidine kinase synthesis; and (3) the signal that triggers the arrest of thymidine kinase synthesis is postmitotic and does not require extensive DNA synthesis. In contrast with hydroxyurea and aphidicolin, in the presence of fluorodeoxyuridine metaphase was not observed. Thus, the triggering of thymidine kinase synthesis is unambiguously dissociated from metaphase and postmitotic events. Because synthesis of thymidine kinase remains under the control of temperature shifts from 22 to 32°C, a simple model of the cell cycle involving two regulatory pathways could account for the triggering of thymidine kinase synthesis, early prophase stage, and metaphase.
Kinetics of α2‐macroglobulin endocytosis and degradation in mutant and wild‐type Chinese hamster ovary cells
Authors: Yamashiro Darrell J. , Borden Laurence A. , Maxfield Frederick R. 
Published: February 2005

AbstractThe production of Chinese hamster ovary (CHO) cell mutants which are defective in endocytosis has led to a greater understanding of the process by which cells sort ligands and their receptors. Robbins and coworkers have obtained CHO mutants which are resistant to diphtheria toxin, defective in the delivery of endocytosed lysosomal enzymes to lysosomes, and have a decreased uptake of iron from transferrin (Robbins et al.: 1. Cell Biol. 96:1064–1071, 1983). We have previously shown that these CHO mutants are markedly deficient in the acidification of early endocytic compartments (Yamashiro and Maxfield: J. Cell Biol. 105:2713–2721, 1987). In this study we examined the endocytosis of α2‐macroglobulin (α2M) to determine whether the defects in early endosome acidification would alter the processing of this ligand. We found that the CHO mutants DTG 1–5–4 and DTF 1–5–1 bind, internalize, and degrade 125l‐α2M in a manner similar to the wild‐type cells. We also found that the CHO mutants retain the ability to recycle the receptors for α2M. Since the binding of α2M is greatly reduced at mildly acidic pH (≈︁6.8), only slight acidification of the endosomal compartment should be sufficient to achieve sorting of a2M from its receptor. In contrast, lysosomal enzymes require more acidic conditions (pH < 6.0) for dissociation. The different behavior of the two ligands provides biochemical evidence for a partial (but not complete) defect in early endosome acidification in the mutants. The data also indicate that pH regulation in a relatively narrow range can achieve differential sorting of various ligands.
Murine B‐cell stimulatory factor‐1 (BSF‐1)/Interleukin‐4 (IL‐4) is a multilineage colony‐stimulating factor that acts directly on primitive hemopoietic progenitors
Authors: Kishi Kenji , Ihle James N. , Urdal David L. , Ogawa Makio 
Published: February 2005

AbstractInterleukin‐4 (IL‐4), which was originally identified as a B‐cell growth factor, has been shown to produce diverse effects on hemopoietic progenitors. The present study investigated the effects of purified recombinant murine IL‐4 on early hemopoetic progenitors in methylcellulose culture. IL‐4 supported the formation of blast cell colonies and small granulocyte/macrophage (GM) colonies in cultures of marrow and spleen cells of normal mice as well as spleen cells of mice treated with 150 mg/kg 5‐fluorouracil (5‐FU) 4 days earlier. When the blast cell colonies were individually picked and replated in cultures containing WEHI‐3 conditioned medium and erythropoietin (Ep), a variety of colonies were seen, including mixed erythroid colonies, indicating the multipotent nature of the blast cell colonies supported by IL‐4. To test whether or not IL‐4 affects multipotent progenitors directly, we replated pooled blast cells in cultures under varying conditions. In the presence of Ep, both IL‐3 and IL‐4 supported a similar number of granulocyte/erythrocyte/macrophage/megakaryocyte (GEMM) colonies. However, the number of GM colonies supported by IL‐4 was significantly smaller than that supported by IL‐3. When colony‐supporting abilities of IL‐4 and IL‐3 were compared using day‐4 post‐5‐FU spleen and day‐2 post‐5‐FU marrow cells, IL‐4 supported the formation of fewer blast cell colonies than did IL‐3. IL‐4 and IL‐6 revealed synergy in support of colony formation from day 2 post‐5‐FU marrow cells. These results indicate that murine IL‐4 is another direct‐acting multilineage colonystimulating factor (multi‐CSF), similar to IL‐3, that acts on primitive hemopoietic progenitors.
Role of mevalonic acid in the regulation of natural killer cell cytotoxicity
Authors: Cutts J. Lynn , Scallen Terrence J. , Watson John , Bankhurst Arthur D. 
Published: February 2005

AbstractConsiderable evidence has accumulated for a role of a nonsteroidal mevalonate product in the regulation of DNA replication and cell division. We report here a similar requirement for mevalonate in a nonreplicative function, that of natural killer (NK) cell cytotoxicity. Treatment of NK cells with 10 μM compactin for 48 hr results in a significant inhibition of cytotoxicity which can be completely reversed by treatment with 1 mM mevalonate, but not cholesterol, dolichol, or isopentenyl adenine. Protein and RNA synthesis appear to be involved in this reversal. Treatment with compactin and reversal with mevalonate do not affect the phenotypic distribution of the effector cell population, and the cell type involved in the inhibition and reversal of cytotoxicity is a CD16 (Leu 11)‐, Leu 19‐positive, large granular lymphocyte. The conjugation of the target and effector cell early in the lytic pathway is inhibited by compactin treatment of the effector cell population, and this inhibition is reversed by mevalonate.
Injury‐induced release of basic fibroblast growth factor from bovine aortic endothelium
Authors: Gajdusek Corinne M. , Carbon Sheila 
Published: February 2005

AbstractAlthough the basic fibroblast growth factor (bFGF) gene lacks a traditional consensus signal peptide domain indicative for secretion, many cell types have receptors for bFGF. Since endothelium is a rich source of cell‐associated bFGF, we asked under what conditions could bFGF be released or secreted from confluent cultures of bovine aortic endothelial (BAE) cells. The level of bFGF in BAE cell lysates was compared with the level of heparin‐releasable bFGF in intact BAE cell monolayers, intact cells with exposed extracellular matrix (nonlytic matrices), and extracellular matrices prepared by cell lysis (lytic matrices). Less than 10% of total cell‐associated bFGF was released from intact cell monolayers and nonlytic matrices. In contrast, the levels of bFGF released from lytic matrices depended upon the conditions used to prepare the matrices. Cell lysis at neutral pH generated matrices that released the highest bFGF levels (approximately 50% of total cell‐associated bFGF). These matrices were heavily contaminated by histones, indicating the cellular release and adsorption of intracellular proteins to the matrix. Matrices prepared by BAE cell exposure to basic pH (100 mM NH4OH) contained low bFGF content and minor histone contamination. These latter matrices were chosen to study bFGF sequestration, under physiological conditions, into the extracellular matrix of confluent BAE cell cultures. Incubation with endotoxin, an agent acutely toxic to BAE cells, resulted in cellular release and adsorption of endogenous bFGF to cells and matrices, accompanied by histone deposition in the matrices. These results suggested that one mechanism for bFGF release from BAE cell monolayers was passive release induced by severe cell injury and/or cell lysis with secondary adsorption to the matrix.