bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of idle ribosomes in the response of Chinese hamster ovary cells to depletion of histidyl‐tRNA
Authors: Scornik Oscar A. 
Published: February 2005

AbstractIn Chinese hamster ovary cells, histidine starvation and inactivation of histidyl‐tRNA synthetase by mutations or histidinol result in stimulation of protein breakdown. We have previously shown that the regulatory mechanism recognizes the level of aminoacylation of tRNAHis. We now report that it is also sensitive to the functional state of the ribosomes. Cycloheximide, an inhibitor of peptidyl‐tRNA translocation, decreases the sensitivity of the regulation. In the presence of 1.5 μg cycloheximide/ml, protein synthesis is inhibited to 6% of control; a full response can still be elicited by appropriate concentrations of histidinol, but it requires a more extensive depletion of histidyl‐tRNA than in the absence of cycloheximide. The response is attained only when the depletion is sufficient to inhibit protein synthesis further and to increase the number of ribosomes idling in the histidine codon with an empty aminoacyl site, measured by their reactivity in vivo to low concentrations of puromycin. The results indicate that a simple depletion of his‐tRNA is not sufficient to elicit the response and suggest that idle ribosomes are required for regulation.
Methylglyoxal‐bis(guanylhydrazone)‐resistant Chinese hamster ovary cells: Genetic evidence that more than a single locus controls uptake
Authors: Heaton Mary Anne , Flintoff Wayne F. 
Published: February 2005

AbstractChinese hamster ovary cells spontaneously resistant to the cytotoxic action of methylglyoxal‐bis(guanylhydrazone) have been isolated in a multistep selection scheme. A low‐level resistant isolate has been shown to be defective in the ability to accumulate the drug intracellularly. This was reflected in a 10‐fold lower Vmax than wild‐type cells for drug uptake as well as a slight enhancement of drug efflux. More highly resistant isolates selected from this low‐level resistant isolate were totally deficient in the ability to take up the drug. A partial revertant, selected from this low‐level resistant isolate, retained some change in the Vmax for uptake but lost the accelerated rate of efflux characteristic of the low‐level resistant line. Genetic analysis by somatic cell hybridization indicated that the low‐level resistant phenotype was recessive to the wild‐type phenotype. In addition, the low‐level resistant phenotype could be complemented by a previously isolated highly resistant cell also defective in drug uptake (Mandel and Flintoff 1978) J. Cell. Physiol., 97: 335–344). Taken together, these data suggest that more than one locus controls drug uptake in Chinese hamster ovary cells.
Heparin and heparan sulfate binding sites on B‐16 melanoma cells
Authors: Biswas Chitra 
Published: February 2005

AbstractWe have reported previously that the production of a tumor cell factor that stimulates synthesis of fibroblast collagenase is influenced by a fibroblast‐deposited matrix component, possibly heparan sulfate‐proteoglycan. In this study, binding sites for heparin and heparan sulfate on mouse B‐16 melanoma cells have been demonstrated. Binding of 3H‐heparin and 35S‐heparan sulfate has been shown to occur to whole cells, isolated membranes, and to a component(s) of detergent extracts of the membranes. Scatchard analysis of binding of 3H‐heparin yielded a Kd of 2–5 × 10−8 M and a Bmax of 0.5 × 107 heparin molecules bound per cell. Binding of 35S‐heparan sulfate was of at least an order of magnitude lower affinity than heparin, but the Bmax was similar to that for heparin. Competition studies showed that 35S‐heparan sulfate binding was inhibited totally by heparin and heparan sulfate and partially by dermatan sulfate, but no inhibition was obtained with hyaluronate or chondroitin sulfate. Binding of 3H‐heparin was inhibited totally by heparin but to different extents by preparations of heparan sulfate from different tissue sources. The heparin/heparan sulfate binding activity is a protein(s) because it is destroyed by treatment with trypsin. Binding of 3H‐heparin to transblots of the detergent extract of the B‐16 cell membranes indicated that at least part of the binding activity is a 14,000‐dalton protein.
Intracellular acidification inhibits the proliferative response in BALB/c‐3T3 cells
Authors: Lucas C. A. , Gillies R. J. , Olson J. E. , Giuliano K. A. , Martinez R. , Sneider J. M. 
Published: February 2005

AbstractOne of the earliest events to occur upon the addition of serum to quiescent cells is an increase in the intracellular pH (pHin). The relationship between this pH change and proliferation is not known. In the present study, we investigate the consequences of acidifying the cytosol using the weak acid, 5′, 5″‐dimethyl oxazolidine 2,4‐dione (DMO). At a concentration of 50 mM, DMO inhibits the serum‐induced increases in pHin, DNA synthesis, and cell number. This concentration of DMO is shown not to inhibit the steady‐state rate of mitochondrial respiration and not to inhibit DNA synthesis in a pH‐independent fashion. The effects of DMO treatments are also shown to be reversible, indicating that this compound is not cytotoxic. These observations indicate that DMO inhibits cell proliferation by lowering intracellular pH. One important event that must occur prior to the initiation of DNA synthesis is an elevated rate of protein synthesis. The rate of protein synthesis in situ is extremely pH sensitive. Addition of 50 mM DMO to serum‐stimulated cultures reduces the rate of leucine incorporation to unstimulated levels. These observations suggest that cytoplasmic acidification may inhibit proliferation through its effects on protein synthesis.
Fetal type 2 pneumocytes form alveolar‐like structures and maintain long‐term differentiation on extracellular matrix
Authors: Blau Hannah , Guzowski Dorothy E. , Siddiqi Zafar A. , Scarpelli Emile M. , Bienkowski Robert S. 
Published: February 2005

AbstractWe investigated the effects of reconstituted basement membrane (a crude extract of the Engelbreth‐Holm‐Swarm tumor) on type 2 pneumocyte differentiation during long‐term culture. Cells were derived from mature 29 d fetal rabbits. Morphology was studied by light and electron microscopy. On thin gel, the cells initially segregated into clumps; they were cuboidal with apical microvilli and contained lamellar bodies, but dedifferentiated by 8 d. On thick gel, epithelial cells associated into spherical clusters surrounding a central lumen. These alveolarlike structures persisted at least 22 d. The cells were cuboidal and had lamellar bodies and intercellular tight junctions; they exhibited polarity, with apical microvilli facing the lumen, basally located nuclei, and gel matrix abutting the basal surface. In contrast, cells cultured on plastic formed colonies, then a monolayer, but dedifferentiated 5–7 d after plating. [14C]Acetate was used to label newly synthesized phospholipids. The amount of disaturated phosphatidylcholine (DSPC), expressed as a percentage of total phosphatidylcholine (PC), was used as an indicator of surfactant lipid production; percentage DSPC synthesized by cells cultured on thick gel did not change significantly, from 55 ± 3 at 3 d, to 63 ± 2 at 22 d in culture. DSPC synthesized by cells cultured on plastic decreased from 57 ± 1% at 3 d to 45 ± 2% at 22 d (p < 0.001), which is consistent with the morphologic evidence of dedifferentiation. Synthesis of total PC compared with total phospholipid did not vary with either time in culture or substrate. This study emphasizes the importance of a complex extracellular matrix for maintenance of type 2 pneumocyte differentiation. The system should prove useful for studying the interaction of these cells with basement membrane, including the role of events occurring at the cell surface in modulating expression of a differentiated phenotype.
Regulation of gene expression of myeloperoxidase during myeloid differentiation
Authors: Tobler Andreas , Miller Carl W. , Johnson Keith R. , Selsted Michael E. , Rovera Giovanni , Koeffler H. Phillip 
Published: February 2005

AbstractMyeloperoxidase (MPO) is a major heme enzyme involved in inflammatory responses of polymorphonuclear leukocytes. Using cDNA and intron specific probes for MPO we studied the regulation of MPO expression during myeloid differentiation of the promyelocytic HL‐60 leukemia cell line. Mature MPO mRNA species of 3.3, 2.8 and 1.6 kb and heterogenous nuclear (hn)RNA of > 8 and ∼4 kb were observed in wildtype HL‐60 cells. Induction of differentiation of the cells towards either granulocytes or macrophages resulted in a profound decrease (> 95%) in the concentration of MPO mRNA levels, showing that gene expression of MPO mRNA is closely linked to the stage of development of myeloid cells. Studies using normal and leukemic hematopoietic cells confirmed these findings and showed that myeloblasts and promyelocytes contain MPO mRNA. Rate of transcription of MPO was measured by a nuclear run‐on assay in wild‐type and day 3– and day –4 differentiated HL‐60 cells and was nearly the same in all three. In contrast, rate of transcription of c‐myc in the same nuclei became almost undetectable with induction of differentiation. Overall transcription decreased by 60% and 80% on day 3 and 4 of differentiation, respectively, compared to wild‐type cells. Stability of mature MPO mRNA was also measured and found to be the same in wild‐type and differentiated HL‐60. Half‐life of MPO hnRNA was ≤ 30 min in wild‐type HL‐60; nevertheless, this hnRNA was easily detectable 3 days after induction of differentiation of these cells. Taken together, the results show that decreased expression of MPO mRNA with differentiation occurs in part post‐transcriptionally, possibly due to a failure in RNA processing. In addition, as overall transcription decreases during differentiation, MPO transcription is concomitantly reduced. This indicates that transcriptional and post‐transcriptional mechanisms cooperate in the control of MPO gene expression.
Hormonal effects of vitamin D3 on epidermal melanocytes
Authors: Abdel‐Malek Zalfa A. , Ross Richard , Trinkle Linda , Swope Viki , Pike John W. , Nordlund James J. 
Published: February 2005

AbstractThe role of cholecalciferol, 25(OH) D3, and 1,25(OH)2 D3, as modulators of melanocyte function and proliferation has been examined. Topical application of 100 μg cholecalciferol to the pinnal epidermis of DBA/2J mice for 5 or 10 days increased the number of L‐dihydroxyphenylalanine‐positive (DOPA‐positive) melanocytes and had a synergistic effect with a low dose of ultraviolet B light (UVB). Application of 1 μg 1,25(OH)2 D3 had a transient effect on epidermal melanocytes. Addition of cholecalciferol to pure cultures of human melanocytes did not alter their tyrosinase activity (therefore, melanin synthesis) or growth rate even after 72 hours of treatment. However, treatment of similar cultures with 1,25(OH)2 D3 at a concentration equal to or greater than 10−8 M suppressed tyrosinase activity but did not affect proliferation. The effect of 25(OH) D3 was similar to, but lower in magnitude than, that of 1,25(OH)2 D3. We attempted to demonstrate the presence of specific receptors for 1,25(OH)2 D3 in normal human melanocytes using the monoclonal antibody (Mo Ab) 9A7γ and to a secondary biotinylated Ab and analyzed by the fluorescence activated cell sorter (FACS). An increase in the specific fluorescent signal was constantly observed. By using the immunoblotting technique, we observed a major immunoreactive species that migrated in the 53‐kD region in normal melanocytes. The size of this major immunoreactive species was smaller in melanoma cells than in normal melanocytes. This correlates with the finding that the former cells were unresponsive to cholecalciferol, 25(OH) D3, or 1,25(OH)2 D3 treatment. These results predict a direct role for 1,25(OH)2 D3 as an effector of normal melanocyte function.
Phorbol esters can persistently replace interleukin‐2 (IL‐2) for the growth of a human IL‐2‐dependent T‐cell line
Authors: Takeshita Toshikazu , Goto Yuso , Nakamura Masataka , Fujii Masahiro , Iwami Masafumi , Hinuma Yorio , Sugamura Kazuo 
Published: February 2005

AbstractA selected clone from an IL‐2‐dependent human T‐cell line was persistently propagated in the presence of phorbol esters with the ability to activate protein kinase C (PKC), such as 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) or phorbol‐12,13‐dibutylate (PDBu). Thus, a TPA(PDBu)‐dependent T‐cell line, designated TPA‐Mat, was established from IL‐2‐dependent T cells. The TPA‐dependency of TPA‐Mat was not lost during cultivation for more than a year in the presence of TPA, and TPA‐Mat cells still showed IL‐2‐dependent growth. However, the TPA (PDBu)‐dependent growth of TPA‐Mat did not seem to be mediated by an autocrine mechanism of IL‐2 or by any other growth factor production, because these factors were not detected in TPA‐Mat cell supernatants. Therefore, the phorbol esters substituted for IL‐2 and may be directly involved in transduction of growth signals in TPA‐Mat cells. Although activity of PKC was down‐regulated, messenger ribonucleic acid (mRNA) of the PKC β‐gene was detected in TPA‐Mat cells was stimulated not only by phorbol esters but also by nonphorbol ester tumor promoters with the ability to activate PKC. These observations suggest that the sustained activation of PKC by the phorbol esters could induce continuous growth of the IL‐2‐dependent TPA‐Mat cells.
Altered molecular properties of tubulin in a multidrug‐resistant variant of Chinese hamster cells selected for resistance to vinca alkaloids
Authors: Pain J. , Sirotnak F. M. , Barrueco J. R. , Yang C‐H. , Biedler J. L. 
Published: February 2005

AbstractThe basis for the markedly altered intracellular binding of [3H]vincristine in a multidrug‐resistant variant (DC–3F/VCRd–5L) of Chinese hamster lung cells (DC–3F) was investigated. Binding of [3H]vincristine by protein in cytosol derived from each cell type exhibited a differing requirement for GTP in MgCl2 containing buffer of low‐ionic strength. Binding of [3H]vincristine occurred to cytosolic protein derived from both variant and parental DC–3F cells, but after removal of GTP, binding only occurred to cytosolic protein from parental cells regardless of the presence of added GTP. Although binding by cytosolic protein from parental DC–3F cells did not require GTP, the addition of 0.1 mM GTP increased by two‐fold the rate and extent of binding. When cytosol from variant and parental DC–3F cells was incubated with low concentrations of [3H]vincristine in high‐ionic strength buffer and analyzed by molecular‐sieve HPLC, most of the protein binding [3H]vincristine in parentally derived cytosol eluted as Mr 110,000–115,000 daltons, corresponding to that for dimeric tubulin. The same binding species was not detected in cytosol derived from variant cells. However, these same fractions derived with both parental and variant cytosols contained tubulin as shown by SDS‐PAGE and immunoblotting. A smaller peak of [3H]vincristine binding and an amount of tubulin equal to that found in later fractions were found in the void volume during the same HPLC elution runs with cytosol from both variant and parental DC–3F cells. Evidence was also obtained for differences between parental and variant DC–3F cells in β‐tubulin isoforms following isoelectric focusing and immunoblotting. Parental‐cell cytosol contains a single isoform of β‐tubulin. However, in variant cell cytosol the same isoform and, in addition, three more basic isoforms were found. These alterations in [3H]vincristine binding and in isoform compositions of β‐tubulin in variant versus parental DC–3F cells may have importance in regard to vincristine resistance in DC–3F cells.
Structure, biosynthesis, and function of the hexose transporter in Chinese hamster ovary cells deficient in N‐acetylglucosaminyltransferase 1 activity
Authors: Haspel Howard C. , Revillame Josefina , Rosen Ora M. 
Published: February 2005

AbstractWe have used a Chinese hamster ovary cell line deficient in N‐acetyl‐glucosaminyltransferase 1 activity (Lec1) to study the effects of altered asparaginelinked oligosaccharides on the structure, biosynthesis, and function of glucose transporter protein. Immunoblots of membranes of Lec1 cells show a glucose transporter protein of Mr 40,000, whereas membranes of wild‐type (WT) cells contain a broadly migrating Mr, 55,000 form similar to that observed in several other mammalian tissues. The total content of immunoreactive glucose transporters in Lec1 cells is 3.5‐fold greater than that of WT cells. Digestion with endoglycosidases, treatment with inhibitors of glycosylation, and interactions with agarose‐bound lectins demonstrate that glucose tranporters of both cell lines derive from a similar Mr 38,000 core polypeptide and that both contain asparagine‐linked oligosaccharide. Transporters in Lec1 cells contain primarily “undecorated” but “trimmed” mannose‐type asparagine‐linked oligosaccharides, while the protein in WT cells contains a mixture of “decorated” and “trimmed” asparagine‐linked oligosaccharides. Biosynthetic and turnover studies demonstrate that Lec1 cells, in contrast to WT cells, are unable fully to process the core asparagine‐linked oligosaccharides of maturing glucose transporters. When radiolabeled in methionine‐deficient medium both Lec1 and WT cells show similar rates of synthesis and turnover of glucose transporter proteins. It should be noted, however, that starvation for a critical amino acid may alter the ability of the cell to synthesize or degrade proteins. The abilities of Lec1 and WT cells to transport hexoses and to interact with the inhibitor cytochalasin B are very similar. The results indicate that, although altered asparagine‐linked glycosylation can affect the content and biogenesis of glucose transporters, these changes do not greatly modify cellular hexose uptake. The possibility that alterations in asparagine‐linked glycosylation may change the cell surface localization or acquisition of a “functional conformation” of the glucose transporter is also suggested.
Segregation of precursors with high proliferative potential from their differentiated progeny in a myeloma cell line
Authors: Wandl U. , Hoang T. , Minden M. , Messner H. A. 
Published: February 2005

AbstractHuman myeloma colonies were grown from the peripheral blood of a patient with multiple myeloma who was unresponsive to any further therapy. The majority of primary myeloma colonies contained cells that were able to form secondary colonies upon replating and facilitated the establishment of a myeloma cell line (OCI‐My5). The recloning procedure was repeated for colonies that contained the highest and lowest number of clonogenic progenitors. This approach allowed the selection of clonogenic cells with high self‐renewal potential. Using a cell separation procedure based on differences in sedimentation velocity, clonogenic myeloma cells with high self‐renewal were found to be smaller than the majority of clonogenic myeloma cells. Furthermore, cells with increasing size did not only display reduced self‐renewal ability but showed significant increases in the content of mRNA transcripts for lambda light chains. These data were consistent with the view that clonogenic myeloma cells are heterogeneous with respect to self‐replication and that they give rise to more differentiated progeny, as documented by increasing mRNA levels for the M‐protein.
Abnormal proteins as the trigger for the induction of stress responses: Heat, diamide, and sodium arsenite
Authors: Lee Kong‐Joo , Hahn George M. 
Published: February 2005

AbstractThermotolerance and synthesis of heat shock proteins are induced in cells in response to a variety of environmental stresses. We examined the suggestion of Hightower (1980) that modifications of intracellular proteins may be the triggering event that induces heat shock protein synthesis and thermotolerance. We did so by modifying cellular proteins, using diamide, a sulfhydryl oxicizing agent, and dithio‐bis (succinimidyl propionate), an agent that cross‐links bifunctional amino groups. Both of these agents induced heat shock proteins and thermotolerance in CHO (HA‐1) cells. Furthermore, we observed cross‐resistance and self‐tolerance with three seemingly unrelated stimuli (diamide, heat, and sodium arsenite). This observation suggests that the induction of protective responses to these stimuli is mediated by a common mechanism. The results support the hypothesis that production of abnormal proteins by various stresses induces the stress responses as well as tolerance.
Characterization of chloride uptake in Drosophila Kc cells
Authors: Sherwood Ann C. , John‐Alder Kathleen , Sanders Marilyn M. 
Published: February 2005

AbstractDrosophila Kc cells use at least two mechanisms for chloride uptake. These transport systems can be distinguished by their kinetic properties and by their differential sensitivity to various drugs. One transport system predominates at [Cl−]0 below 30 mM and is greater than fivefold more sensitive to disulfonic stilbenes than the second system. At [Cl−]0 above 30 mM, the predominant uptake mechanism is inhibited by vanadate and nitrate.
Mitochondrial DNA molecules and virtual number of mitochondria per cell in mammalian cells
Authors: Robin Eugene D. , Wong Ronald 
Published: February 2005

AbstractA new biochemical method for estimating the virtual number of mitochondria (mt) per cell was developed and used together with a plasmid probe to measure mt DNA/mitochondrion and mt DNA/cell. These methods were used in five cell types from four mammalian species. Mt DNA/mitochondrion was essentially constant in all cell types (mean 2.6 ± 0.30 SE mitochondrial DNA molecules/mt). Mt DNA molecules/cell encompassed an eight‐fold range between various cell types (low 220 ± 6.2; high 1,720 ± 162 mt DNA molecules/cell). Virtual mt number/cell ranged from 83 ± 17 to 677 ± 80 (SE) mt/cell in various cell types. All five mammalian virtual mitochondria contained the same genomic mass. The number of virtual mitochondria per cell and amount of mt DNA per cell appear to be closely regulated within a given cell type but differ widely from cell type to cell type.
Simultaneous addition of EGF prolongs the increase in cytosolic free calcium seen in response to bradykinin in NRK‐49F cells
Authors: Marks Peter W. , Kruskal Benjamin A. , Maxfield Frederick R. 
Published: February 2005

AbstractThe calcium‐sensitive fluorescent indicator fura‐2 and a microscope equipped for rapidly changing excitation wavelengths were used to look at the effects of growth factors on cytosolic free calcium ([Ca2+]i,) in NRK‐49F cells. In these cells bradykinin induced a rapid increase in [Ca2+]i, which generally decayed to near basal [Ca2+]i within 3 minutes. The initial rise in [Ca2+]i in response to bradykinin was relatively independent of extracellular calcium; however, the decay to basal [Ca2+]i was more rapid in the absence of extracellular calcium. Measurements made on individual cells showed a heterogeneity in the response to bradykinin. Epidermal growth factor (EGF) had no effect on [Ca2+]i in NRK‐49F cells when added alone in the presence of extracellular calcium. Simultaneous addition of bradykinin and EGF produced a more prolonged increase in [Ca2+]i than bradykinin alone. The prolongation was dependent on the presence of extracellular calcium and did not occur in its absence. Transient increases in [Ca2+]i occurring after the initial peak were occasionally seen in these cells. Our results indicate that there is rapid interaction between the signaling mechanisms for bradykinin and EGF. When this occurs, one effect is the transport of calcium into the cell from the extracellular environment, causing a more prolonged rise in [Ca2+]i. This effect occurs within 1 minute after combined addition of bradykinin and EGF.