bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of hexose transport by adenosine derivatives in human erythrocytes
Authors: May James M.
Published: February 2005

The human erythrocyte membrane carriers for hexoses and nucleosides have several structural features in common. In order to assess functional similarities, the effects of adenosine derivatives on hexose transport and cytochalasin B binding sites were studied. Adenosine inhibited zero‐trans uptake of 3‐0‐methylglucose half‐maximally at 5 mM, while more hydrophobic adenosine deaminase‐resistant derivatives were ten‐ to 20‐fold more potent transport inhibitors. However, degradation of adenosine accounted for very little of this difference in potency. Hexose transport was rapidly inhibited by N6‐(L‐2‐phenylisopropyl)adenosine at 5°C in a dose‐dependent fashion (EC50 = 240 μM), to lower the transport Vmax without affecting the Km. A direct interaction with the carrier protein was further indicated by the finding that N6‐(L‐2‐phenylisopropyl)adenosine competitively inhibited [3H]cytochalasin B binding to erythrocytes (Ki = 143 μM) and decreased [3H]cytochalasin B photolabeling of hexose carriers in erythocyte ghosts. The cross‐reactivity of adenosine and several of its derivatives with the hexose carrier suggests further homologies between the carriers for hexoses and nucleosides, possibly related to their ability to transport hydrophilic molecules through the lipid core of the plasma membrane.
Differential expression of c‐myc and the transferrin receptor in G1 synchronized M1 myeloid leukemia cells
Authors: Neckers Leonard M. , Tsuda Hiroyuki , Weiss Ervin , Pluznik Dov H.
Published: February 2005

We have studied the transcriptional activity, steady‐state mRNA levels, and steady‐state protein levels of the c‐myc and transferrin receptor (TfR) genes in murine M1 myeloid leukemia cells arrested in G1 phase of the cell cycle by different methods. When cells are growth‐arrested by density inhibition, a technique that places the majority of cells in early G1, c‐myc protein, as detected by Western analysis, is expressed at 80% of the level seen in proliferating cells. Steady‐state mRNA levels and, to a lesser extent, transcriptional activity of the c‐myc gene, parallel the protein findings. Under these conditions, TfR gene expression is much lower than in normally cycling cells. We have previously demonstrated that density‐inhibited M1 cells, released from density inhibition and treated with the DNA polymerase alpha inhibitor aphidicolin, remain in G1, but at a point temporally closer to S phase. Cells treated in this manner demonstrate reduced transcriptional activity and expression of the c‐myc gene, but TfR gene expression approximates the level found in proliferating cells. These data suggest that neither c‐myc nor TfR gene expression is constant throughout the G1 phase of the cell cycle in M1 cells. c‐myc gene expression is highest in early G1 and falls to low levels by late G1, while the reverse is true for TfR gene expression.
Effects of human neutrophil chemotaxis across human endothelial cell monolayers on the permeability of these monolayers to ions and macromolecules
Authors: Huang Ada J. , Furie Martha B. , Nicholson Susan C. , Fischbarg Jorge , Liebovitch Larry S. , Silverstein Samuel C.
Published: February 2005

We have developed a method for studying the permeability properties of human endothelia in vitro. Human umbilical vein endothelial cells (HUVEC) were cultured on a substrate of human amnion. Confluent monolayers of these cells demonstrated 6–12 ωcm2 of electrical resistance (a measure of their permeability to ions) and restricted the transendothelial passage of albumin from their apical to their basal surface. To determine whether leukocyte emigration alters endothelial permeability in this model, we examined the effects of migrating human polymorphonuclear leukocytes (PMN) on these two parameters. Few PMN migrated across the HUVEC monolayers in the absence of chemoattractants. In response to chemoattractants, PMN migration through HUVEC monolayers was virtually complete within 10 minutes and occurred at random locations throughout the monolayer. PMN migrated across the monolayer via the paracellular pathway. Although one PMN migrated across the monolayer for each HUVEC, PMN migration induced no change in electrical resistance or albumin perme‐ability of these monolayers. At this PMN:HUVEC ratio, these permeability findings were correlated morphologically to measurements that HUVEC paracellular pathway size increases by less than 0.22% with PMN migration. This increase is insufficient to effect a measurable change in the electrical resistance of the endothelial cell monolayer. These findings demonstrate that increased permeability of cultured endothelial cell monolayers is not a necessary consequence of PMN emigration.
Acetyl‐LDL stimulates macrophage‐dependent plasminogen activation and degradation of extracellular matrix
Authors: Falcone Domenick J. , Ferenc Michael J.
Published: February 2005

The ability of acetyl‐LDL to stimulate macrophage‐dependent plasminogen activation and degradation of extracellular matrix was examined. We have found that expression of plasminogen activator activity in response to the scavenger receptor ligand varied among cell populations. Exposure to acetyl‐LDL stimulated plasminogen activator expression by cells which constitutively released low levels of activator. These include a virally transformed macrophage‐like cell line (RAW246.7), concanavalin A and C. parvum‐activated macrophages. The stimulation of plasminogen activator activity was independent of cellular lipid accumulation since nonlipoprotein inhibitors of acetyl‐LDL binding to the scavenger receptor stimulated activator expression in great excess to that observed with acetyl‐LDL. In contrast, acetyl‐LDL was unable to induce soluble plasminogen activator activity in cells which normally do not express it. These include a macrophage‐like cell line (J774A.1) and resident peritoneal macrophages. Furthermore, acetyl‐LDL was unable to modulate the copious secretion of activator by inflammatory macrophages elicited with thioglycolate. When macrophages were tested for their ability to degrade smooth muscle cell derived matrix, solubilization by resident, elicited, and activated cells was variously increased in the presence of plasminogen. Furthermore, exposure to acetyl‐LDL enhanced plasmin‐dependent degradation by resident cells and activated cells, whereas matrix degradation by elicited cells was unaffected.
Binding of diphtheria toxin to CHO‐K1 and vero cells is dependent on cell density
Authors: Schaefer Erik M. , Moehring Joan M. , Moehring Thomas J.
Published: February 2005

We studied the binding of 125I‐labeled diphtheria toxin (DTX) to receptors on monolayer cultures of Chinese hamster ovary cells (CHO‐K1) and Vero cells. The number of DTX receptors detected on the cell surface was shown to be dependent on the cell density (number of cells per unit area). Cells at low density (≤23,000 cells per cm2 for CHO‐K1 cells; ≤80,000 cells per cm2 for Vero cells) had more receptors for DTX than cells at higher densities. The difference in receptor number between low‐ and high‐density cells was 33‐fold for CHO‐K1 cells and 19‐fold for Vero cells. We estimated the maximum number of DTX receptors on low‐density CHO‐K1 and Vero cells to be 50,000 and 370,000 per cell, respectively. The cell density at which the binding of DTX was reduced to 50% of maximum was considerably lower for CHO‐K1 cells than for Vero cells (33,000 vs. 220,000 cells per cm2, respectively). Vero cells grown on a surface that had been conditioned by high‐density cells bound less DTX, suggesting that interaction of these cells with the underlying extracellular matrix might regulate the number of cell surface receptors for DTX. Low‐density cells were more sensitive to DTX than high‐density cells, suggesting that low‐density cells possessed an increased number of functional receptors that actively transported DTX to the cytosol. CHO‐K1 and Vero cells were equally protected by SITS (4‐Acetamido‐4prime;‐Isothiocyano‐Stilbene‐2,2′‐disulfonic Acid), a compound that has been shown to inhibit the binding and entry of DTX in Vero cells, suggesting that intoxication of CHO‐K1 and Vero cells is mediated by a similar mechanism. The data illustrate the importance of taking into account the cell density when measuring the number of DTX receptors on adherent cells.
Plasminogen activation and lysis of blood clots induced by cells in vitro
Authors: Knox Peter , Crooks Sheila
Published: February 2005

When human diploid fibroblasts were seeded onto the surface of blood clots, lysis of the clot occurred as a result of the release of cellular plasminogen activator. A number of aspects of this lysis were studied. 1. There was no significant difference in rates of lysis of whole blood clots, platelet‐rich plasma clots, and platelet‐poor plasma clots brought about by the same number of fibroblasts. 2. Clot lysis was promoted by nondividing cells and by proliferating cells. 3. Using cycloheximide to block protein synthesis it was found that the plasminogen activator released by fibroblasts had an active half‐life of less than an hour. 4. When clots were washed prior to the addition of cells then lysis occurred at an increased rate. This was probably due to the removal of α2‐antiplasmin from the clots, since when antisera to α2‐antiplasmin was added to clots, lysis also proceeded at an increased rate. 5. Medium conditioned by fibroblasts did not promote clot lysis even when antiplasmin was removed by washing or by addition of antisera. 6. Cells had to be in direct contact with the clot in order to bring about lysis; when cells were separated from clots by permeable membranes there was no lysis. 7. When cross‐linking of fibrin was reduced by the inhibition of transglutaminase, the rate of clot lysis was increased.
DNA synthesis in U‐2 OS human osteosarcoma cells is independent of PDGF binding to functional cell surface receptors
Authors: Richter Michael R. , Graves Dana T.
Published: February 2005

Previous studies have shown that suramin reveals specific PDGF binding sites on U‐2 OS human osteosarcoma cells. Stud es presented here indicate that U‐2 OS cells pretreated with suramin internalize and degrade 125l‐PDGF and respond to PDGF by increased tyrosine kinase activity and amino acid transport. However, DNA synthesis in these cells is not reduced by incubation with the PDGF blocking agent suramin and is not stimulated by exogenous PDGF. These data indicate that U‐2 OS cells possess functional PDGF receptors but that high levels of DNA synthesis in these cells is unrelated to the binding of secreted PDGF to these cell surface receptors. Thus, it is unlikely that the PDGF mitogen produced by U‐2 OS cells stimulates proliferation through an autocrine mechanism involving secretion and subsequent binding to PDGF receptors.
Regulation of murine plasmacytoma transferrin receptor expression and G1 traversal by plasmacytoma cell growthfactor
Authors: Neckers L. M. , Nordan R. P.
Published: February 2005

Many plasmacytomas arising in BALB/c mice require a specific, macrophage‐derived growth factor in order to proliferate in vitro. Since transferin receptor expression is normally regulated by tissue‐specific growth factors and because expression of these receptors is required for cell proliferation, we examined the interaction of plasmacytoma growth factor (PCT‐GF) on transferrin receptor expression and cell cycle progression in several PCT‐GF‐dependent and independent plasmacytoma cell lines maintained in vitro. We found that removal of PCT‐GF results in a rapid and specific loss of transferrin receptor expression with concomitant G1 arrest in early G1. The time required for G1 arrest to become maximal correlates closely to the initial level of surface transferrin receptor expression and the rate of decay following removal of PCT‐GF. The calcium channel blocker diltiazem interferes with the ability of PCT‐GF to maintain transferrin receptor expression in PCT‐GF‐dependent cell lines and causes a G1 arrest of the cell population. When added to a PCT‐GF‐independent cell line, diltiazem also inhibited transferrin receptor expression and caused G1 arrest. Thus, both PCT‐GF‐dependent and ‐independent plasmacytoma cell lines require transferrin receptor expression for growth. In factor dependent cell lines, transferrin receptor expression requires exogenous PCT‐GF, while in factor‐independent cells, transferrin receptor expression is constitutive. In both cell types, intracellular calcium levels may play a role in receptor expression.
Epidermal growth factor‐dependent growth of human KB cells in a defined medium and altered growth factor requirements of KB mutants resistant to EGF‐Pseudomonas exotoxin conjugates
Authors: Amano Fumio , Gottesman Michael M. , Pastan Ira
Published: February 2005

A serum‐free culture system was established for human KB carcinoma (HeLa) cells that consisted of a chemically defined medium and several growth factors including epidermal growth factor (EGF), insulin, transferrin, hydrocortisone, and ethanolamine. EGF and insulin showed the greatest effects on the growth rate of KB cells. Insulin‐like growth factor l (IGF‐l) at the same concentration as insulin stimulated cell growth less than insulin. Transferrin, hydrocortisone, or ethanolamine had no growth‐stimulatory effects alone but were stimulatory when combined with EGF and/or insulin. Transforming growth factor‐beta inhibited growth and triiodothyronine stimulated growth. The growth factor requirements were established for several KB mutants with low EGF receptor levels that had been selected for resistance to a conjugate of EGF with Pseudomonas exotoxin (EGF‐PE). Three of five KB mutants did not respond to EGF; two other mutants responded to a lesser extent than the parental KB cells. Four mutants had a reduced response to insulin and responded to T3; one mutant (ET‐30) responded to neither. These results indicate that KB cells selected for EGF‐PE resistance have lost their growth response to EGF and illustrate the usefulness of serum‐free medium for studying the growth factor requirements of mutants with altered receptor levels.
Desensitization of adenylate cyclase and cyclic AMP flux during the early stages of liver regeneration
Authors: Mahler Stephen M. , Wilce Peter A.
Published: February 2005

Liver regeneration is controlled by a complex network of interactions between hormones, growth factors, and a variety of hepatotrophic factors. Transient increases in cAMP in the early stages of liver regeneration that are necessary for DNA synthesis and subsequent mitosis have been reported; however, studies on the mechanisms that control cellular cAMP levels during liver regeneration, namely adenylate cyclase activity, cAMP‐dependent phosphodiesterase activity, and cAMP efflux from the cell, have been generally incomplete. In this study we have shown that although there are three peaks in intracellular cAMP levels in the first 24 hours after partial hepatectomy, the adenylate cyclase activity stimulated by glucagon, prostaglandin E2, adrenaline, and fluoride in vitro decreases with time. Kd and BMAX of hepatocyte glucagon and β receptors were similar to the sham controls. Our results are consistent with a mixed homologous/heterologous desensitization of the adenylate cyclase system. There was also a loss of cAMP‐dependent phosphodiesterase activity after partial hepatectomy. We speculate that even though the hormone‐stimulated adenylate cyclase system has been desensitized, the system retains the ability to respond to the transient pulses of the variety of hormones secreted after partial hepatectomy and thus raise the intracellular concentration of cAMP. The decrease in cAMP‐dependent phosphodiesterase may be necessary to prevent rapid breakdown of cAMP.
Role of idle ribosomes in the response of Chinese hamster ovary cells to depletion of histidyl‐tRNA
Authors: Scornik Oscar A.
Published: February 2005

In 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

Chinese 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

We 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

One 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

We 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

Myeloperoxidase (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

The 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

A 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

The 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

We 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

Human 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

Thermotolerance 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

Drosophila 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

A 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

The 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.
Inductive effect of recombinant human interleukin‐1 α and β on differentiation of macrophage‐like tumor cell line P388D1
Authors: Hanazawa Shigemasa , Hanaizumi Chika , Amano Shigeru , Hirose Kimiharu , Ohmori Yoshihiro , Kumegawa Masayoshi , Yamaura Kohichi , Kitano Shigeo
Published: February 2005

We used the mouse monocyte/macrophage‐like tumor cell line P388D1 to test whether or not interleukin‐1 (IL‐1) stimulates differentiation of monocyte/macrophage progenitors. Incubation of these cells with recombinant human interleukin‐1 (rhIL‐1) α and β resulted in their increased adherence, stimulation of nonspecific esterase activity, and increased Fc rosette formation. rhIL‐1s inhibited cell growth and stimulated Fc rosette formation in a dose‐dependent fashion. The cell growth inhibition due to rhIL‐1s depended on the concentration of serum in culture medium. Synergism between rhIL‐1 and calcium ionophore A23187 was found for the cell growth inhibition and Fc rosette formation. The presence of ethylene glycol bis‐ (β‐aminoethyl ether) N,N,N,N‐tetraacetic acid(EGTA) in the medium abolished the stimulatory effect of rhIL‐1 on Fc rosette formation of the cell line. These results demonstrate that rhIL‐1s are a potent inducer of the differentiation of the macrophage‐like tumor cell line P388D1.
Transforming growth factor β responsiveness is modulated by the extracellular collagen matrix during hepatic ito cell culture
Authors: Davis Bernard H.
Published: February 2005

Hepatic sinusoidal Ito cells have the capacity to produce interstitial collagen types I and Ill as well as other matrix proteins and may be involved in hepatic fibrogenesis. Transforming growth factor β (TGF β) responsiveness was evaluated during in vitro cell culture, since increasing evidence suggests that this ubiquitous polypeptide can stimulate the production of collagenous proteins in a variety of cell types. TGF β induced marked inhibition of Ito cell proliferation for cells grown on either a type I or a type IV collagen matrix. In marked contrast, the collagen synthetic response was considerably different for cells grown on a type I versus a type IV collagen matrix. When cells were grown on a type I collagen matrix, TGF β caused a significant increase in the accumulation of collagen type I and III. When Ito cells were grown on a type IV collagen matrix, there was no stimulation of collagen production. TGF β respsonsiveness was also evaluated in the setting of altered vitamin A concentrations. Freshly isolated Ito cells are engorged with vitamin A, the usual physiologic storage site for hepatic vitamin A. During in vitro culture and during in vivo fibrogenesis, Ito cells lose their vitamin A stores coincident with a transformation to a collagen‐producing myofibroblast‐like cell. When cultured Ito cells were grown on a type I collagen matrix and re‐exposed to an increased concentration of vitamin A, the production of interstitial collagen was reduced. However, when the vitamin A‐enriched Ito cells were exposed to TGF β, the production of interstitial collagen was increased, similar to cells that had not received vitamin A.
Lysosomal enzymes in extracellular digestion in the unicellular eukaryote Tetrahymena
Authors: Tiedtke Arno , Rasmussen Leif
Published: February 2005

The ciliate Tetrahymena thermophila was starved for orthophosphate in a synthetic medium at pH 7.5. These cells did not utilize phosphorylcholine, final concentration 1 mM, as a phosphate source for cell growth and multiplication. If the phosphorylcholine solution, however, was incubated for 24 h at pH 5.5 with extracellular, “spent” medium from a culture in early stationary phase of growth, then it promoted culture growth readily at pH 7.5. It was shown that the spent medium in the same concentration did not stimulate growth in itself. It is concluded that extracellular digestion of phosphorylcholine enabled the cells to grow and multiply in a nutrient medium having organic phosphate compounds as the only phosphate source. It is argued that the phosphatases in the spent medium are of lysosomal origin.
Cytoarchitecture of kirsten sarcoma virus‐transformed rat kidney fibroblasts: Butyrate‐induced reorganization within the actin microfilament network
Authors: Ryan Michael P. , Higgins Paul J.
Published: February 2005

Murine sarcoma virus‐transformed rat fibroblasts (KNRK cells) undergo marked cytoarchitectural reorganization during in vitro exposure to sodium‐n‐butyrate (NaB) resulting in restoration of (1) a more typical fibroblastoid morphology, (2) proper cell‐to‐cell orientation, and (3) substratum adherence. Augmented cell spreading, involving > 90% of the population was a function of culture density and time of exposure to NaB (2 mM final concentration). Induced cell spreading reflected a 2.5‐ to 3.0‐fold increase in both total cellular actin content and deposition of actin into the detergent‐resistent cytoskeleton. Cytoskeletal actin deposition in response to NaB was accompanied by the formation of occasionally dense, parallel alignments of F‐actin‐containing microfilaments and by a dramatic increase in the size and incidence of actin‐enriched membrane ruffles. Long‐term NaB‐treated cells exhibited parallel orientations of microfilaments similar to those found in untransformed fibroblasts. Increased cytoskeletal actin occurred within 24 hr of NaB exposure, correlating with the initial reorganization of actin‐containing microfilaments detected microscopically, and reflected concomitant 3‐fold increases in cellular alpha‐actinin and fibronectin content. In contrast, the amount of vimentin, tropomyosin, and tubulin in NaB‐treated cells was significantly decreased. NaB‐induced morphologic restructuring of sarcoma virus‐transformed fibroblast, thus, impacts on all three basic cytoskeletal systems. Selective increases, however, were evident in particular cytoskeletal proteins (actin, alpha‐actinin, fibronectin) implicated in microfilament networking and cell spreading.
Murine cerebral microvascular endothelium incorporate and metabolize 12‐hydroxyeicosatetraenoic acid
Authors: Moore Steven A. , Prokuski Laura J. , Figard Paul H. , Spector Arthur A. , Hart Michael N.
Published: February 2005

Cultured murine cerebromicrovascular endothelial cells were employed to study the metabolism of 12‐hydroxyeicosatetraenoic acid (12‐HETE) in an in vitro model of the blood‐brain barrier. These endothelial cells convert 12‐HETE to at least four, more polar compounds. Analysis of the least polar and predominant metabolite by gas chromatography combined with chemical ionization and electron impact mass spectrometry of reduced and nonreduced derivatives indicate that the compound is 8‐hydroxyhexadecatrienoic acid (8‐HHDTrE). The uptake of 12‐HETE into cell phospholipids peaks at 2 hr, and is not saturable up to the highest concentration tested, 5 μM. Seventy‐five to 92% of this 12‐HETE is incorporated into phosphatidylcholine, while the remainder is divided between the inositol and ethanolamine phospholipids. Incorporation into neutral lipids is slower, with radioactivity gradually accumulating in triglycerides over 24 hr. Saponification of cell lipids demonstrated that not only 12‐HETE, but also its major metabolite, 8‐HHDTrE, is incorporated into the cell lipids. Prostacyclin and prostaglandin E2 production by the cerebral endothelial cells is inhibited by up to 56% with 1 μM and 90% with 5 μM 12‐HETE. These data demonstrate that 12‐HETE is actively metabolized by cerebral endothelium and suggest at least two mechanisms through which 12‐HETE may alter cerebromi‐crovascular function: (1) incorporation into cerebral endothelial membranes and (2) inhibition of cerebral endothelial prostaglandin production. Conversion of 12‐HETE to more polar compounds, particularly 8‐HHDTrE, may be interpreted as either the inactivation of 12‐HETE or the production of additional, biological mediators.
Regulation of the heat‐shock response by interferon in mouse L cells
Authors: Dubois M. F. , Mezger V. , Morange M. , Ferrieux C. , Lebon P. , Bensaude O.
Published: February 2005

Interferon (IFN) is not able to induce heat‐shock protein (HSP) synthesis. However IFN pretreatment of mouse L cells has been shown to enhance the decrease of overall protein synthesis which follows a heat shock, and to stimulate the accumulation of HSPs. We show here that the synthesis of a protein (the hepatitis B virus surface antigen) under the control of a Drosophila HSP 70 promoter is also stimulated in IFN‐pretreated cells. The regulation by IFN takes place at two levels: first, the rate of HSP gene transcription is increased in nuclei isolated from IFN‐treated cells; second, the synthesis of HSPs is prolonged after pretreatment with IFN. Experiments performed in the presence of actinomycin D show that this effect is due to a stablization by IFN of mRNAs coding for HSPs.