bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

AbstractA 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

AbstractMeWo 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

AbstractFatty 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

AbstractThe 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

AbstractEntry 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

AbstractNormal 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

AbstractConfluent 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

AbstractThe 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

AbstractThe 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

AbstractTumor 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

AbstractWe 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

AbstractAdhesion 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

AbstractThe 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.