bluuebunny/crossref_metadata_2025 huggingface dataset:
Fura 2 analysis of cytosolic calcium regulation in elutriated rat gastric parietal cells
Authors: Black E. W. , Cornwell T. L. , Lincoln T. M. , Strada S. J. , Thompson W. J.
Published: February 2005

The calcium probe, Fura 2, is used to establish and partially characterize histamine‐, carbachol‐ and forskolin‐induced calcium transients in enriched parietal cell populations prepared by centrifugal elutriation of dispersed rat fundic mucosa cell isolates. The magnitude of the maximal carbachol response, which is blocked by atropine but not cimetidine, is nearly five times that of histamine or forskolin. Time to peak responses for carbachol, forskolin, and histamine are approximately 7, 17, and 28 sec, respectively. Carbachol‐, histamine‐, and forskolin‐induced increases in Fura 2 fluorescence appear dependent upon extracellular calcium, since these responses are attentuated in low calcium media and blocked by EGTA in low‐calcium media or by lanthanum in high‐ or low‐calcium medium. Trifluoperazine and fenoctimine, at concentrations that inhibit secretion, have no effect on either carbachol‐ or histamine‐induced increases in cytosolic calcium. Seven major calcium/EGTA‐sensitive phosphopro‐teins are identified by SDS‐PAGE electrophoresis of ATP 32P‐labeled cell sonicates. We conclude that cytosolic calcium in enriched rat gastric parietal cell populations is regulated by secretagogue receptor‐controlled calcium channels. We postulate that these channels may be controlled by cyclic AMP‐dependent phosphorylation, since neither changes in cyclic AMP nor calcium alone mediate the effects of secretagogues entirely, but the interplay between these two second‐messenger systems potentiates the actions of these agents. The role of cytosolic calcium as a second messenger in secretagogue action appears similar to that of cyclic AMP in that a specific cellular concentration must be reached to initiate acid secretion.
Stochastic model for multipotent hemopoietic progenitor differentiation
Authors: Tsuji Kohichiro , Nakahata Tatsutoshi
Published: February 2005

In this study, the authors propose a stochastic model for multipotent hemopoietic progenitor differentiation, which assumes that there is a fixed probability (P) that a progenitor with a potential for differentiation along a particular lineage maintains the potential in each cell division in each daughter cell, and this differentiation process of each lineage proceeds independently. To examine the applicability of this model, a sequential micromanipulation of paired progenitors was carried out and followed by cytological examination of the cells contained in the colonies derived from these progenitors; then calculation was made of the ratio of the number of paired colonies containing cell(s) with a particular lineage to the number of paired colonies in which only one colony contained cell(s) with the lineage at the first and second cell division. The ratios were similar at the first and second cell division within each lineage. Furthermore, the frequences of each lineage in multilineage hemopoietic colonies were calculated using the P values obtained from these micromanipulation experiments. The expected frequencies were similar to those in the actual experiments. These results suggested that the stochastic model was applicable to multipotent hemopoietic progenitor differentiation.
Novel action of carnitine: Inhibition of aggregation of dispersed cells elicited by clusterin in vitro
Authors: Fritz Irving B. , Burdzy Krystyna
Published: February 2005

A novel effect of carnitine and O‐acylcarnitine derivatives has been described. The presence of these compounds has been shown to inhibit the aggregation of erythrocytes otherwise elicited by the addition of clusterin or fetuin. The specificity of carnitine action has been investigated by comparing influences of chemically related compounds. The concentrations required for inhibition by approximately 50% of aggregation of erythrocytes by clusterin under in vitro conditions defined were determined to be 1.5 mM for L(‐) or D(+) enantiomers of carnitine; 0.5 mM for decanoyl(−)‐ or (+)‐carnitine; 0.13 mM for lauroyl(−) ‐or (+)‐carnitine, and 0.05 mM for myristoyl(−)‐ or (+)‐carnitine. In contrast, concentrations up to 12.5 mM of dimethylcarnitine, deoxycarnitine, acetylcho‐line, acetyl‐β‐methylcholine, or inositol had no detectable inhibitory effect on aggregation elicited by clusterin. Clusterin addition also resulted in the aggregation of three other cell types examined (guinea pig spermatozoa, a cell line derived from testes of neonatal mice called TM4 cells, and Sertoli cells from testes of 20 day‐old rats). As in the case with erythrocytes, the presence of carnitine inhibited aggregation of spermatozoa, TM4 cells, and Sertoli cells in suspension. We consider possible mechanisms by which carnitine inhibits aggregation of erythrocytes and other populations of dispersed cells incubated in the presence of clusterin.
Rapidly reversible enzyme inhibition in a temperature‐sensitive mammalian cell mutant lacks thermotolerance
Authors: Vidair Charles A. , Dewey William C.
Published: February 2005

The temperature‐sensitive (ts) Chinese hamster ovary (CHO) cell mutant tsH1 contains a thermolabile leucyl‐tRNA synthetase. Upon incubation at the nonpermissive temperature of 39.5°C, the enzyme became reversibly inhibited over a period of minutes, and the cells lost viability over a period of many hours. However, killing of tsH1 by acute heating at 45°C was identical to that of wild‐type (SC) cells. In addition, the heat‐induced inhibition of protein synthesis was similar for both cell types, as measured after acute heating at 45°C. Furthermore, both killing and inhibition of protein synthesis showed thermotolerance in both cell types. In contrast to the effects at 45°C, at 39.5°C, neither the inhibition of leucyltRNA synthetase activity nor the killing of tsH1 expressed thermotolerance. Also, treatment of tsH1 at 39.5°C did not induce thermotolerance to killing at 45°C. The inhibition of leucyl‐tRNA synthetase activity in tsH1 at 39.5°C was further distinguished from the 45°C‐induced inhibition of protein synthesis in SC cells by a much more rapid reversal of the inhibition of leucyl‐tRNA synthetase activity. Also, the rate of reversal of the inhibition of protein synthesis by 45°C in SC cells was decreased by increased heat dose. Such was not true for the 39.5°C inhibition of leucyl‐tRNA synthetase activity in tsH1. The data indicate that there exist two distinct types of thermal inhibition‐one slowly reversible type which was observed during and after heating at 45°C and both induced and expressed thermotolerance, and a second, rapidly reversible type, which was evident only during heating of tsH1 at 39.5°C and neither induced nor expressed thermotolerance.
Cell enlargement: One possible mechanism underlying cellular senescence
Authors: Angello John C. , Pendergrass William R. , Norwood Thomas H. , Prothero John
Published: February 2005

We previously demonstrated an inverse relationship between the G1 volume of human diploid fibroblast‐like (HDFL) cells obtained from foreskin tissue and clonal replicative potential. On the basis of these results, we suggested that one process underlying in vitro senescence is a progressive increase in the mean cell volume of successive progeny within clonal lineages. We now report that the size of HDFL cells, as well as of chick embryo fibroblasts, can be increased in the virtual absence of cell division by culturing at low density and at low serum concentration (0.1‐1.0%). Consequent to an increase in cell size, the replicative potential of the cells is reduced to the level of later‐passage cells of similar size. By clonal analysis, the populations of enlarged cells contain up to three times as many nondividing cells as do controls. In the enlarged populations, the proportion of cells producing attenuated clones (four or fewer progeny) increases by about 30%, whereas the proportion of cells yielding >32 cells declines by a similar percentage. These observations lead us to propose that replicative potential may be limited by cell size, which in turn may be regulated by a kinetic relationship between cellular growth and cell division cycles.
Heparin stimulates the synthesis and modifies the sulfation pattern of heparan sulfate proteoglycan from endothelial cells
Authors: Nader Helena B. , Buonassisi Vincenzo , Colburn Patricia , Dietrich Carl P.
Published: February 2005

Heparin stimulates 2‐3‐fold, in a concentration‐dependent manner, the synthesis of heparan sulfate secreted by cultured endothelial cells. The increase in synthetic rate takes place immediately after exposure of the cells to heparin, affects only heparan sulfate, and is specific for the endothelial cell. No stimulation by other glycosaminoglycans was observed. Analysis of the disaccharide products formed by the action of heparitinases reveals a higher degree of sulfation of the uronic acid residues in the heparan sulfate of cells exposed to heparin.
Biphasic regulation of macrophage attachment by activators of cyclic adenosine monophosphate‐dependent kinase and protein kinase C
Authors: Issaad C. , Ventura M. A. , Thomopoulos P.
Published: February 2005

A method is described that enabled us to study the adhesiveness of J‐774 murine macrophages. Cell attachment was stimulated by activators of kinase C (i.e., phorbol esters) as well as kinase A (cyclic adenosine monophosphate; cAMP). This novel effect of cAMP was observed when its levels were increased via receptor triggering (prostaglandin E1, β‐adrenergic agonists), activation of Ns (cholera toxin), or inhibition of phosphodiesterase (Ro 20‐1724) or when the kinase was directly activated by Br8‐cAMP. The simultaneous treatment with kinase A and kinase C activators at the time of attachment resulted in a partially additive response. On the other hand, preincubation of the cells in suspension with one of the activators rendered them refractory to subsequent stimulation at the onset of the adhesion assay, whatever agent was used. Such a refractoriness was also observed in cells preincubated with oleoyl‐acetyl‐glycerol (OAG). On the other hand, when added at the time of attachment, this near‐physiological activator of kinase C evoked a biphasic response: the early stimulation of cell attachment was followed by an accelerated rate of “detachment.” In conclusion, kinase C and kinase A play a role in the sequence of events leading to cell adhesion. The cross desensitization observed is distal and takes place at or beyond the kinase step.
Autoregulation of MeWo metastatic melanoma cell growth: Characterization of intracellular (FGF, MGSA) and secreted (PDGF) growth factors
Authors: Pichon Frederic , Lagarde Alain E.
Published: February 2005

MeWo melanoma cells (clone LC1) secrete a potent mitogenic activity susceptible to reinitiate DNA replication in quiescent rodent fibroblasts (CCL39, NRK‐49F, NIH‐3T3) but not in BHK‐21 kidney cells. This activity appears to be closely related to platelet‐derived growth factor (PDGF) based on (1) its cationic nature, heat and acid resistance, but sensitivity to reducing agents; (2) its apparent molecular weight (33 kDaltons) as estimated by Biogel filtration, once dissociated from binding proteins by mild acidic treatment; (3) its weak affinity for heparin; and (4) its ability to compete with 125I‐PDGF for binding to human and rodent fibroblasts, and to be recognized by anti‐PDGF antibodies. Although MeWo cells coexpress the PDGF‐A and PDGF‐B (c‐sis) chain gene transcripts, the secreted product shows reactivity on CCL39 fibroblasts more compatible with the PDGF‐BB than with the PDGF‐AB isoform. MeWo cell lysates contain activities that bind moderately and strongly to heparin‐Sepharose, being eluted with 1.0 and 2.0 M NaCI, respectively. The latter may correspond to basic fibroblast growth factor (basic FGF), consistent with the expression of basic FGF gene mRNAs. The former has not been fully characterized and is probably not the product of the acidic FGF gene. In addition, MeWo cells react positively with the FB2 AH7 antibody, thus indicating that they elaborate material similar to melanoma growth‐stimulating activity (MGSA). MeWo cells proliferate in serum‐free medium in a cell‐density‐dependent fashion, both in liquid and semisolid cultures. Their division is modestly enhanced by basic FGF and by human and porcine PDGF but not by the factors that they release. However, the absence of demonstrable 125I‐PDGF binding sites on MeWo cells, in conjunction with their lack of sensitivity to suramin growth inhibition, suggests that the secreted PDGF does not act as an autocrine factor. Instead, the autonomous proliferation of MeWo melanoma cells may result from the concerted action of basic FGF and MGSA, which are mostly cell‐associated.
Abnormal fatty acid utilization by cultured cardiac cells from 7‐day‐old obese zucker rats
Authors: Quignard‐Boulange Annie , Freyss‐Beguin Marianne , Brigant Lucienne , Millanvoye‐Van Brussel Elisabeth
Published: February 2005

Fatty acid utilization by muscle and nonmuscle heart cells in culture has been investigated in the 7‐day‐old Zucker rat to determine if this tissue could contribute to the lower energy expenditure reported in obese rats at the onset of obesity. The partitioning of oleate to oxidation and esterification products and the effect of genotype on this partitioning according to cell types were studied. Results showed that the fatty acid β‐oxidation and its esterification in neutral lipid was decreased by 30% in beating muscle cells from obese animals when compared with those from lean animals. In contrast, nonmuscle cells exhibited a decreased β‐oxidation alone. A similar fatty acid composition of the phospholipids was found in non‐muscle cells of obese animals and their lean litter mates. In muscle cultures, palmitic and oleic acids are lower in cells of obese rats than in those of lean rats. The present study indicates that a defect in energy metabolism could be found in heart cells at the onset of obesity, suggesting that this defect is determined by intrinisic factor(s).
Comparison of uptake kinetics in freshly isolated suspensions and short‐term primary cultures of rat hepatocytes
Authors: Kukongviriyapan Veerapol , Stacey Neill H.
Published: February 2005

The apparent kinetics of uptake of various model substrates were examined for hepatocytes in suspension and primary culture up to 72 h. The ability of hepatocytes to take up taurocholate and ouabain was decreased in culture. Vmax for uptake of both substrates diminished rapidly with increasing time in culture. An increase in Km was observed in cultures 6 h after plating, but there was no further change with prolongation of culture time. The decrease of uptake of taurocholate and ouabain during culture may be due to the reduction in the number of transport carriers plus a decrease of affinity of the carrier to substrates. The nonsaturable component of cadmium uptake was much reduced in cultured cells compared with the suspensions. The saturable process was lower in 6 h culture but increased to a level comparable with the fresh cells at longer culture time. No significant change was found in the Km between suspensions and cultures. Uptake of alpha‐aminoisobutyric acid was greater in culture while that of 3‐O‐methyl‐D‐glucose was relatively stable but about one‐half that found in cell suspension. Thus, uptake of two substrates, taurocholate and ouabain, is clearly compromised with increasing time in primary culture, while uptake of the other substrates does not reflect such a dramatic decrease. It is therefore apparent that the cell preparation of choice in uptake studies depends on the substrate and the nature of the experiments.
Rapid deformation of “passive” polymorphonuclear leukocytes: The effects of pentoxifylline
Authors: Needham D. , Armstrong M. , Hatchell D. L. , Nunn R. S.
Published: February 2005

Entry times for spherical (no pseudopods) polymorphonuclear leukocytes (PMNs) into a 4 μm micropipet have been measured as a function of pipet suction pressure (2,500‐20,000 dyn/cm2) and concentration of the drug pentoxifylline (PTX, 0.1‐10.0 mM). For control cells (0 mM PTX), entry rates (reciprocal entry times) increased almost linearly with increasing suction pressure, indicating a Newtonian‐like behavior. With incubation in PTX solutions, entry rate vs. suction pressure became increasingly non‐linear, suggesting a shear‐thinning effect for the dissipative structure. At a given suction pressure the rate of entry showed a dose‐dependent increase with increasing PTX concentration, the effect being most pronounced at high suction pressures (20,000 dyn/cm2). Also, with increasing PTX concentration two other effects were observed: (i) there was a decreased incidence of cells that displayed pseudopodia, and (ii) there was an increased incidence of cells forming hernias and an increased streaming of cell cytoplasm during aspiration. The first observation points to a down‐regulation of the cell's functional ability to “activate” in response to surface/chemical stimuli, and the second indicates that both the cortical and cytoskeletal networks are weakened either by disruption and/or reduction in density of the protein polymers. These observations are in line with other recently published experiments which suggest that the rheological effects of pentoxifylline on PMNs may be associated with the state of actin.
Serum‐free culture of normal human melanocytes: Growth kinetics and growth factor requirements
Authors: Pittelkow Mark R. , Shipley Gary D.
Published: February 2005

Normal human epidermal melanocytes were selectively propagated from mixed (keratinocyte‐melanocyte) cultures and primary epidermal cell suspensions in serum‐free medium, MCDB 153 containing insulin, bovine pituitary extract (BPE), phorbol‐12‐myristate‐13‐acetate (PMA), ethanolamine, phosphoethanolamine, and hydrocortisone. Neonatal foreskin melanocytes (NFMs) replicated more readily than adult melanocytes in culture. Early passage NFMs grown in serum‐free medium exhibited a population generation time of 24‐48 hours. NFMs assumed a less dendritic appearance and were less pigmented than adult melanocytes. PMA or other protein kinase C‐activating phorbol esters significantly enhanced mitogenesis of NFMs; however, cAMP‐elevating agents were not required for efficient replication of NFMs. Basic fibroblast growth factor (bFGF) was a potent mitogen for NFMs and replaced the requirement for BPE in the culture medium. NFMs expressed a single class of specific, high‐affinity receptors for bFGF, exhibiting a Kd = 3 × 10−11 M and approximately 76,500 receptors/cell. Neither EGF nor TGF‐α were mitogenic for NFMs, and TGF‐β reversibly inhibited NFM growth. Rapidly growing, early passage NFMs were shown to have cell cycle times of 19.5, 7.5, and 9 hours for G1, S, and G2 /M phases of the cell cycle, respectively. Culture of NFMs to confluence or depletion of growth factors from the culture medium caused reversible, G1 phase‐specific, cell cycle growth arrest. Senescence of NFMs was associated with irreversible growth arrest in the G1 phase after 40‐45 population doublings in culture. Our data demonstrate that basal medium MCDB 153 can be supplemented with defined factors to cultivate selectively two major constituent cell types of the epidermis, the melanocyte and the keratinocyte.
Extracellular matrix heparan sulfate proteoglycans modulate the mitogenic capacity of acidic fibroblast growth factor
Authors: Gordon Portia B. , Choi Haing U. , Conn Greg , Ahmed Asra , Ehrmann Bettina , Rosenberg Lawrence , Hatcher Victor B.
Published: February 2005

Confluent cultures of human endothelial cells deposit into extracellular matrix (ECM) distinct heparan sulfate proteoglycans (HSPG) which modulate acidic fibroblast growth factor's (aFGF) ability to stimulate human endothelial cell mitogenic capacity. Extracellular matrix 35S‐HSPG were isolated from cultures metabolically labelled with Na235SO4 by DEAE‐Sepharose, Sepharose CL‐4B, and aFGF‐Affi‐Gel 15 column chromatography and identified by resistance to chon‐droitinase ABC and sensitivity to nitrous acid. Fifty to sixty percent of the 35S‐HSPG deposited into ECM do not bind aFGF. The bound 35 S‐HSGP (40‐50% of the total counts applied) eluted from the aFGF‐Affi‐Gel column after the addition of buffer containing 2 M NaCI. aFGF‐binding and aFGF‐nonbinding 35S‐HSPG were individually pooled and further purified by Sepharose CL‐4B column chromatography. 35S‐HSPG which bind aFGF, designated HSPGp, were 100‐fold superior to heparin in augmenting the mitogenic efficacy of aFGF in sparse proliferating cultures. In contrast, however, 35S‐HSPG, which did not bind aFGF, designated HSPG1, inhibited aFGF‐stimulated proliferation in both sparse and subconfluent endothelial cell cultures. The majority of the biological activity of both aFGF‐potentiating HSPGP and aFGF‐inhibitory HSPG1 was contained in the glycosaminoglycan chains released by alkaline borohydride treatment of intact HSPGP or HSPG1, respectively. 3H‐Core protein derived from HSPGP or HSPG1 contained only minor biological activity. The ability of heparitinase or hepnrinase (Flavobacterium heparinum) to abolish biological activity differed, depending upon the HSPG tested, also suggested that these are two distinct HSPGs.
Differentiation‐inducing and cytotoxic effects of tumor necrosis factor and interferon‐gamma in myeloblastic ML‐1 cells
Authors: Craig Ruth W. , Buchan Heather L.
Published: February 2005

The effects of the tumor necrosis factor (TNF), and a second pleiotropic cytokine interferon‐gamma (IFN), were examined in a line of human myeloblastic leukemia cells (ML‐1). By itself, TNF causes ML‐1 to differentiate along the monocytic pathway. The cells exhibit an increase in Fc receptors and acquire the morphological characteristics of maturing phenotype. They remain viable and continue to proliferate (at ≥ 50% of the control growth rate) even with 102–104 units/ml TNF. IFN alone has similar effects, causing an increase in Fc receptors but little cytotoxicity. In contrast to either cytokine alone, the combination of TNF plus IFN causes a cessation of proliferation and extensive cell death. Cytotoxicity occurs in a synergistic fashion; it requires the simultaneous presence of both cytokines, occurring with concurrent but not sequential exposure. These different responses, differentiation (TNF alone) and cytotoxicity (TNF + IFN), occur with a similar range of doses (∼ 102–104 units/ml) and in a similar time frame (beginning on day 2). In other cell types, IFN can augment either the differentiation‐inducing or the cytotoxic effect of TNF. In ML‐1, the combined application of TNF plus IFN results in a shift from differentiation to cytotoxicity.
Protein kinase C inhibitor H‐7 alters the actin cytoskeleton of cultured cells
Authors: Birrell G. B. , Hedberg K. K. , Habliston D. L. , Griffith O. H.
Published: February 2005

The effects of the protein kinase C inhibitor H‐7 on the actin cytoskeleton of cultured cells (Swiss 3T3 and PTK2) are described. As documented by fluorescence microscopy and the higher‐resolution technique of photoelectron microscopy, the effects are rapid and dramatic; exposure to 30 μM H‐7 in culture medium for less than 6 min is sufficient to induce a significant reduction in the numbers and thickness of actin microfilament bundles and alterations in the morphology of cell‐cell boundaries in PTK2 cells. One‐hour exposure to 30 μM H‐7 results in nearly complete depletion of normal actin microfilament bundles from all of the cell types examined, without dramatic changes in overall cell shape. The intermediate filament and microtubule cytoskeletal networks did not appear to be affected to any extent over the times and doses examined. Forty‐five minutes of exposure of Swiss 3T3 cells to 200 μM of either HA1004 (which is comparable to H‐7 with respect to inhibition of cyclic nucleotide dependent kinases) or to the protein kinase C inhibitor sangivamycin did not induce the actin alterations characteristic of H‐7. In addition, depletion of protein kinase C from Swiss 3T3 cells by means of phorbol ester‐induced down‐regulation did not prevent the effects of H‐7 on the actin cytoskeleton. These results demonstrate that the protein kinase C inhibitor H‐7 has a specific and rapid effect on the actin cytoskeleton, and furthermore H‐7 may have biochemical effects beyond those mediated by inhibition of protein kinase C or the cyclic nucleotide dependent kinases.
Tumor necrosis factor causes amplification of arachidonic acid metabolism in response to interleukin 1, bradykinin, and other agonists
Authors: Burch Ronald M. , Tiffany Carol W.
Published: February 2005

Tumor necrosis factor stimulated prostaglandin E2 synthesis in Swiss 3T3 fibroblasts. Interleukin 1 also stimulated prostaglandin synthesis. Simultaneous addition of tumor necrosis factor and interleukin 1 synergistically stimulated prostaglandin synthesis, even when both growth factors were added at what would be supramaximal concentrations by themselves. Several small peptides and nonpeptides rapidly stimulate prostaglandin synthesis in these cells. Pretreatment with tumor necrosis factor synergistically enhanced prostaglandin synthesis in response to bradykinin, bombesin, thrombin, norepinephrine, and platelet‐activating factor. Thus, tumor necrosis factor stimulates prostaglandin synthesis and greatly amplifies prostaglandin synthesis in response to other agonists. This finding may have significance in chronic inflammatory diseases such as rheumatoid arthritis in which several hormones and growth factors may synergistically augment eicosanoid synthesis.
Histones synthesized at different stages of myogenesis are differentially degraded in myotube cells
Authors: Wunsch Ann M. , Lough John
Published: February 2005

We recently reported that cultures of terminally differentiating myotube cells synthesize histones in reduced but significant amounts in comparison with proliferating myoblasts (Wunsch et al., 1987, Dev. Biol., 119: 85–93). In this study, the stability of myotube histone has been determined, comparing the degradation of de novo–synthesized histones in nascent (day 3) and maturing (day 4) myotubes with histones in the same cells that had been previously made during myoblast proliferation (day 1). Histones synthesized in proliferating myoblasts and myotubes were pulse‐labeled with 3H‐lysine and chased up to seven days, followed by determinations of radioactivity remaining in histone bands using fluorography of one‐ and two‐dimensional polyacrylamide gels. Considered in aggregate, core histones synthesized de novo in nascent (day 3) myotubes were degraded most rapidly, followed by myotube histones that had been previously made during the proliferative phase (day 1) of myogenesis. De novo–synthesized histones in maturing (day 4) myotubes were relatively stable. Individual histone classes were degraded in the following order of increasing half‐life, regardless of the differentiative stage at which they were synthesized: H2A.Z, H2A, H2B, H3(.2, day 1;.3, days 3 and 4), H4.
Modulation of fibronectin adhesive functions for fibroblasts and neural cells by chemically derivatized substrata
Authors: Lewandowska Kristine , Balachander Natarajan , Sukenik Chaim N. , Culp Lloyd A.
Published: February 2005

Adhesion responses of fibroblasts (Balb/c 3T3 cells) and human neuron‐derived (Platt neuroblastoma) cells have been examined with plasma fibronectin (pFN) adsorbed to glass surfaces derivatizeci with an alkyl chain and six chemical end groups interfacing with the bound pFN to test regulation of pFN function. Using new derivatization protocols, the following surfaces have been tested in order of increasing polarity: [CH3], [CC], [Br], [CN], [Diol], [COOH], and underivatized glass ([SiOH]). For all substrata, pFN bound equivalently using either a supersaturating amount of pFN or a subsaturating amount in competition with bovine albumin. Attachment of both cell types was also equivalent on all substrata. However, spreading/differentiation responses varied considerably. F‐actin reorganization was tested in 3T3 cells with rhodamine‐phalloidin staining. While stress fibers formed effectively on pFN‐coated [SiOH] and [Br] substrata, only small linear bundles of F‐actin and a few thin stress fibers were observed on the [COOH], [Diol], and [CN] substrata; the hydrophobic substrata ([CH3]) and [CC] gave an intermediate response. When a synthetic peptide containing the Arg‐Gly‐Asp‐Ser sequence required for integrin binding to FNs was included in the medium as an inhibitor, additional differences were noted: Stress fiber formation was completely inhibited on [SiOH] but not on [Br] and stress fiber formation was very sensitive to inhibition on the hydrophobic substrata while the F‐actin patterns on the [CN] and [COOH] substrata were unaffected. Evaluation of neurite outgrowth by neuroblastoma cells on these substrata revealed both qualitative and quantitative differences as follows: [Diol]  [COOH] > [SiOH] ≫ [CN]  [Br] > [CH3]  [CC]. While there was poor cytoplasmic spreading and virtually no neurites formed on the hydrophobic surfaces when pFN alone was adsorbed, neurite formation could be “rescued” if a mixture of pFN with an excess of bovine albumin was adsorbed, demonstrating complex conformational interactions between substratum‐bound pFN and adhesion‐inert neighboring molecules. In summary, these studies demonstrate that different chemical end groups on the substratum modulate pFN functions for cell adhesion, principally by affecting the conformation of these molecules rather than the amounts bound. Furthermore, these studies confirm multiple‐receptor interactions with the FN molecules in cell type‐specific adhesion patterns.
Membrane pinch‐off and reinsertion observed in living cells of Drosophila
Authors: Narita Kazuhiko , Tsuruhara Takashi , Koenig J. H. , Ikeda Kazuo
Published: February 2005

The garland cell of Drosophila is a nephrocyte which takes up waste products from the haemolymph. Endocytosis is thought to occur by the pinch‐off of coated vesicles from deep invaginations of the plasma membrane called labyrinthine channels. Electron microscopic studies show that the length of these channels is variable, depending on the relative rates of membrane pinch‐off and reinsertion (recycling). Thus, in wild‐type garland cells, if the temperature is raised from 19°C to 30°C, the channels shorten, because at high temperature the pinch‐off rate exceeds the reinsertion rate. On the other hand, in garland cells of the temperature‐sensitive, single‐gene mutant shibirets1(shi), in which endocytosis is reversibly blocked at the pinch‐off stage at 30°C, the labyrinthine channels elongate considerably, as membrane insertion proceeds while pinch‐off is blocked. The rates of membrane pinch‐off and insertion were quantitated in living garland cells by observing the changes in the capacitance of the whole cell membrane which occur as a result of changes in the total area of the plasma membrane. In wildtype cells, the capacitance gradually decreased as the temperature was raised to 30°C, reflecting the shortening of the channels. In shi cells, the capacitance decreased between 19°C and 26°C but then began to increase at higher temperatures as the blockage of endocytosis caused by the shi gene took effect, causing the channels to elongate. The observations suggest that in shi cells the surface area of the cell more than doubles in 1 2 min by channel elongation. Estimates of the amount of membrane which is pinched off and reinserted were made.
Effect of phenylarsine oxide on fluid phase endocytosis: Further evidence for activation of the glucose transporter
Authors: Frost Susan C. , Lane M. Daniel , Gibbs E. Michael
Published: February 2005

We have shown previously that insulin stimulates fluid phase endocytosis in 3T3‐L1 adipocytes (Gibbs et al., 1986). Using [14C]sucrose as an endocytotic marker, we show here that phenylarsine oxide, a trivalent arsenical which binds neighboring dithiols, blocked not only insulin‐stimulated fluid phase endocytosis, but basal endocytosis as well. The K1 for this process was 6 μM in the presence or absence of insulin and the time required for inhibition was less than 2.5 min, the limit of detection in our assay system. These results can be compared with the inhibitory effect of phenylarsine oxide on insulin‐stimulated glucose transport. Although the K1 for insulin‐stimulated transport (7 μM) was similar to that for inhibition of endocytosis, basal glucose transport was not affected by the inhibitor. Further, when cells were prestimulated with insulin causing maximal stimulation of the glucose transport rate, phenylarsine oxide induced a time‐dependent reduction to the basal rate (t1/2 of 10 min), despite the fact that endocytosis was blocked immediately. This observation suggests that if the transporter is recycled by an exocytotic/endocytotic mechanism, it is distinct from fluid‐phase endocytosis/exocytosis, which is a vesicle‐mediated process, and provides further evidence that the transporter may undergo intrinsic activation/inactivation which does not require vesicle movement.
Differential effects of phorbol ester on the in vitro invasiveness of malignant and non‐malignant human fibroblast cells
Authors: Fridman Rafael , Lacal Juan Carlos , Reich Reuven , Bonfil Daniel R. , Ahn Chang‐Ho
Published: February 2005

The effect of the phorbol ester tumor promoter 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) on cell invasion was studied using an in vitro assay for cell invasion through a reconstituted basement membrane matrix (Matrigel). TPA inhibited the invasiveness of malignant human fibrosarcoma HT1080 cells. In contrast, WI‐38 lung fibroblasts, which show a very low invasive capacity, were stimulated (3‐fold) to invade Matrigel after exposure to TPA for 48 hours. The inhibitory or stimulatory effects of TPA on cell invasion were correlated with a decrease or an increase in cell motility and collagenase IV activity, respectively. Synthetic diacylglycerols partially mimicked the inhibitory action of TPA on HT1080 cells but failed to stimulate WI‐38 cell invasion. Immunoblots demonstrated that in both cell lines the α and β isoforms of protein kinase C were equally down‐regulated after a 5 hour exposure to TPA despite the basal low level of protein kinase C polypeptide in the malignant cells. Thus, whereas in WI‐38 cells induction of an invasive behavior could be observed in the absence of protein kinase C, in the malignant cells disappearance of the kinase was associated with a non‐invasive phenotype.
Role of lipocortin I in the glucocorticoid induction of the terminal differentiation of a human squamous carcinoma
Authors: Violette S. M. , King I. , Browning J. L. , Pepinsky R. B. , Wallner B. P. , Sartorelli A. C.
Published: February 2005

The human squamous cell carcinoma SqC/Y1 undergoes spontaneous terminal differentiation in the confluent state. The degree of maturation was markedly increased by glucocorticoids and by both human recombinant and placental lipocortin I. Western analyses demonstrated cellular secretion of lipocortin into the medium. Glucocorticoid‐induced maturation was antagonized by a lipocortin l‐specific monoclonal antibody, by phospholipase A2 (PLA2), and by arachidonic acid. Induction of the differentiation of SqCC/Y1 cells by lipocortin I was prevented by arachidonic acid. The PLA2 inhibitor, dibromoacetophenone, caused an increase in envelope‐competent cells indicating that inhibition of PLA2 results in induction of differentiation. Epidermal growth factor prevented the induction of differentiation by both lipocortin I and by glucocorticoids. The nonsteroidal lipoxygenase/cyclo‐oxygenase inhibitor, phenidone, also increased SqCC/Y1 differentiation, suggesting that leukotrienes, thromboxanes, and/or prostaglandins may be involved in lipocortin‐mediated regulation of SqCC/Y1 maturation. The findings support a role for lipocortin I in mediating the effects of glucocorticoids on epidermal cell differentiation.
Effect of a continuously applied compressive pressure on mouse osteoblast‐like cells (MC3T3‐E1) in vitro
Authors: Ozawa Hiroyuki , Imamura Kazunobu , Abe Etsuko , Takahashi Naoyuki , Hiraide Takatoshi , Shibasaki Yoshinobu , Fukuhara Tatsuo , Suda Tatsuo
Published: February 2005

Bone metabolism is often affected by a variety of mechanical forces, but the cytological basis of their action is not known. In this study, we examined the effect of a continuously applied compressive pressure (CCP) on the growth and differentiation of clonal mouse osteoblast‐like cells (MC3T3‐E1) cultured in a specifically devised culture chamber. The gas phase of the chamber was maintained at a pressure of 2 atmospheres (atm) above ambient (3 atm total, 3.1 kg/cm2; 3.0 × 105 Pa) by continuously infusing a compressed mixed gas (O2: N2:CO2 = 7.0%:91.3%:1.7%). The pO2, pCO2, and pH in the culture medium at 37°C under 3 atm were maintained at the same levels as those under 1 atm. MC3T3‐E1 cells were cultured in α minimal essential medium containing 10% fetal bovine serum under either 3 atm in the CCP culture chamber or 1 atm in an ordinary CO2 incubator. Alkaline phosphatase activity, a marker of osteoblasts, was greatly suppressed by the CCP treatment. The inhibition of alkaline phosphatase activity was rapidly restored when the cells were transferred to an ordinary CO2, incubator under 1 atm, indicating that the inhibition of alkaline phosphatase activity by CCP is reversible. Cell growth was not altered under CCP. The CCP treatment greatly increased the production and secretion of prostaglandin E2(PGE2). Adding either conditioned medium from the CCP culture or exogenous PGE2 to the control culture under 1 atm suppressed alkaline phosphatase activity dose‐dependently. The CCP treatment also suppressed collagen synthesis and calcification. These results suggest that CCP causes the cells to produce and secrete PGE2 which, in turn, inhibits differentiation of osteoblasts and the concomitant calcification.
Expression and amplification of cloned rat liver tyrosine aminotransferase in nonhepatic cells
Authors: Kohli Kulwant K. , Stellwagen Robert H.
Published: February 2005

A full‐length cDNA for the rat liver enzyme tyrosine aminotransferase has been used to construct mammalian expression vectors by recombinant DNA techniques. These vectors, which have employed either a simian virus 40 or a Rous sarcoma virus promoter, were transfected into a variety of nonhepatic mammalian cell lines in culture. Transient expression of tyrosine aminotransferase was readily observed after transfection into monkey COS cells and mouse L cells. Stable clones that express cloned tyrosine aminotransferase have been isolated from mouse L cells, hamster Wgla fibroblasts, and Chinese hamster ovary (CHO) cells. A vector capable of expressing both tyrosine aminotransferase and dihy‐drofolate reductase was stimulated to undergo amplification by treatment with methotrexate in a CHO cell line deficient in the latter enzyme. Levels of tyrosine aminotransferase as much as 50‐fold higher than typically seen in glucocorticoid‐induced hepatoma cells were achieved in some CHO clones by this technique. The tyrosine aminotransferase produced at these highly amplified levels appeared structurally normal and had no major harmful effects on the cells.
Glucocorticoid and thyroid hormones inhibit proliferation of serum‐free mouse embryo (SFME) cells
Authors: Loo Deryk , Rawson Cathleen , Schmitt Molly , Lindburg Katherine , Barnes David
Published: February 2005

Mouse embryo cells derived in a serum‐free medium formulation (SFME cells) do not exhibit growth crisis or chromosomal abnormalities and are nontumorigenic in vivo; these cells are also reversibly growth inhibited by serum or platelet‐free plasma (Loo et al.; Science, 236:200–202, 1987).A portion of the inhibitory activity of serum could be extracted by charcoal, a procedure that removes steroid and thyroid hormones. Both L‐3,5,3′‐triiodothyronine (T3) and hydrocortisone inhibited growth of SFME cells in a reversible manner. The inhibitory activity of serum also was partially removed by treatment with anion exchange resin in a procedure designed to deplete serum of thyroid hormone. However, the effect of serum on untransformed SFME cells could not be prevented by addition of the antiglucocorticoid RU38486, and ras‐transformed clones of SFME cells, which are capable of growing in serum‐containing medium, retained inhibitory responses to glucocorticoid and, with some clonal variability, to T3. These results suggest that glucocorticoid or thyroid hormones may contribute to the inhibitory activity of serum on SFME cells, but additional factors are also involved.
Alterations in electrophoretic mobility, diaphorase activity, and terminal differentiation induced in murine erythroleukemia lines by differentiating agents
Authors: Gascoyne Peter R. C. , Becker Frederick F.
Published: February 2005

The electrophoretic mobilities (EPMs) and semiquinone reductase activities of two clones of Friend murine erythroleukemia (MEL) cells were investigated as a function of treatment with the inducing agents dimethylsulfoxide (DMSO) and hexamethylene bisacetamide (HMBA) As reported previously by others, the inducible clone DS19 lost its ability to grow in soft agar and expressed hemoglobin as judged by benzidine/H2O2 staining after 96 hours of treatment with 1% DMSO or 4 mM HMBA. In addition, its EPM fell by 14%, its semiquinone reductase activity by 40%, and its mean diameter by 10%. The second clone, R1, retained its ability to grow in soft agar and lacked hemoglobin expression after treatment with HMBA and DMSO, characterizing it as noninducible. However, R1 did demonstrate alterations in EPM, semiquinone reductase activity, and cell diameter that closely paralleled those found in DS19. Such responses were not seen in three non‐MEL cell lines exposed to HMBA or DMSO, suggesting that clone R1 responded to these inducing agents in a cell‐line specific manner but that its ability to complete the sequences necessary for differentiation may be blocked at an unknown point distal to the block characteristic of untreated cells. The data show that while a reduction in EPM, semiquinone reductase activity, and cell diameter accompany induced differentiation in MEL cells, such changes can occur in the absence of a commitment to terminal differentiation.
c‐fos Reduces growth factor requirements for mitogenic stimulation of L6 rat myoblasts
Authors: Rahm Magnus , Sejersen Thomas
Published: February 2005

Addition of fetal calf serum (FCS) to serum‐deprived L6J1 rat myoblasts increases fos‐like immunoreactivity. The nuclear immunoreactivity reached a maximum 2 h after serum addition. Effects of the c‐fos protein on myoblast proliferation were analyzed in L6J1 rat myoblasts transfected with the murine c‐fos gene under control of a metallothionein promoter. L6J1 myoblasts with elevated expression of transfected c‐fos reached higher cell densities than neo transfected control myoblasts when approaching a stationary phase in normal culture conditions (5% FCS). The differences in cell densities were even more pronounced at low serum concentrations (0.5% FCS). c‐fos transfected cells also had a faster growth rate than did control cells in serum‐free medium supplemented with calcium chloride, lithium chloride, sodium selenite, hydrocortisone, and insulin. The cell morphology of c‐fos transfected L6J1 myoblasts was not affected compared to control myoblasts.

These results suggest that c‐fos protein expression in L6J1 myoblasts is activated by serum and that mitogenic stimulation of L6J1 myoblasts is facilitated by the presence of elevated amounts of c‐fos protein.
Na+/H+ antiporter is the primary proton transport system used by osteoclasts during bone resorption
Authors: Hall Tony J. , Chambers Timothy J.
Published: February 2005

We have examined the effects of inhibitors of proton transport systems on osteoclastic bone resorption using an in vitro bone slice assay, where osteoclasts (OCs) are free from the influence of other bone cells. Amiloride (AM) and dimethyl‐amiloride (DMA), inhibitors of the Na+ /H+ antiporter, were potent inhibitors of bone resorption (IC50 ∼ 9 and 0.7 μM for AM and DMA, respectively). Omeprazole (OM), a potent inhibitor of parietal cell K+ /H+ ‐ATPase, was a poor inhibitor of OC bone resorption (IC50 ∼ 100 μM). These results strongly suggest that the Na+ /H+ antiporter is the primary proton system used by OCs during bone resorption.
Epidermal growth factor and bombesin differ strikingly in the induction of early responses in Swiss 3T3 cells
Authors: Bierman Arjo J. , Koenderman Leo , Tool Anton J. , Siegfried W. De Laat
Published: February 2005

Swiss 3T3 cells express receptors for both the polypeptide epidermal growth factor (EGF) and the tetradecapeptide bombesin and respond mitogenically to these substances. These cells thus provide a system to analyze potential signal transduction pathways involved in mitogenic stimulation. Here we have determined and compared the early ionic responses elicited by EGF and bombesin and their relation to diacylglycerol (DG) and inositolphosphate (InsPn) production. Whereas EGF fails to cause any significant change in intracellular Ca2+ bombesin effectively induces prompt and transient Ca2+ mobilization from intracellular stores. Further support of the idea that these receptors utilize distinct signalling pathways comes from the measurements of cytoplasmic pH (pHi). As in most target cells, EGF induces a delayed (1 min) but sustained intracellular alkalinization that reaches a new steady state after ∼︁10 min. Bombesin, in contrast, elicits a biphasic response; within seconds, a rapid but transient rise in pHi is observed, followed by a further slower sustained alkalinization. Inhibition of the Na+/H+ exchanger prevents both EGF as well as bombesin‐induced alkalinization. However, under these conditions, bombesin evokes a rapid and sustained acidification related to the Ca2+ response. Apparently, bombesin initiates a Ca2+ ‐dependent acidifying process immediately after binding of the hormone to its receptor. Furthermore, we could demonstrate that the bombesin‐induced alkalinization depends on protein kinase C activation whereas the EGF response does not. Determination of the total DG and InsPn accumulation revealed that EGF is ineffective in stimulating phospholipase C‐mediated production of these second messengers. In contrast, bombesin causes a rapid DG and InsPn production coinciding with the Ca2+ response and the first phase of the rise in pHi followed by a slower DG accumulation coinciding with the second alkalinization phase. Our results show that in Swiss 3T3 cells the bombesin receptor activates the hydrolysis of inositol lipids as a mechanism of signal transduction, which consequently causes changes in Ca2+i and pHi. Clearly, the EGF receptor utilizes different pathways to evoke mitogenisis and stimulates Na+/H+ exchange independently of DG production and protein kinase C activation.
Heparin and acidic fibroblast growth factor interact to decrease prostacyclin synthesis in human endothelial cells by affecting both prostaglandin H synthase and prostacyclin synthase
Authors: Weksler Babette B.
Published: February 2005

Prostaglandin production by cultured human endothelial cells varies with growth conditions. We observed a marked diminution in both spontaneous and inducible production of prostacyclin (PGI2) by human umbilical vein and saphenous vein endothelial cells when they were cultured in the presence of the heparin‐binding growth factor, acidic fibroblast growth factor (aFGF) and heparin, compared with PGI2 production during culture in medium lacking these factors. Decreased PGI2 production was related to duration of exposure of the cells to aFGF and heparin and depended on the concentration of both substances. Heparin (1—100 μg/ml) strongly potentiated the effects of aFGF but had a limited and variable effect alone. The decrease in PGI2 production correlated with a reduction in the cellular content of immunoreactive prostaglandin H synthase and prostacyclin synthase. Arachidonate deacylation was not decreased. In addition, the eicosanoid profile of endothelial cells was changed by exposure to aFGF and heparin. These studies indicate that heparin acts as a modulator of prostaglandin synthesis in endothelial cells through its interaction with aFGF, mediated by alterations in two key enzymes in the arachidonate metabolic pathway.