bluuebunny/crossref_metadata_2025_split huggingface dataset:
Nonesterified fatty acid accumulation and release during heart muscle‐cell (myocyte) injury: Modulation by extracellular “acceptor”
Authors: Janero David R. , Burghardt Charles 
Published: February 2005

AbstractLong‐chain nonesterified fatty acid (NEFA) accumulation in the heart muscle cell (myocyte) and NEFA release to the extracellular milieu are considered contributors to the pathogenesis of myocardial injury in a number of cardiovascular disease states. Reported here is a study of the factors which influence and control the interactions among NEFA formation, intracellular NEFA accumulation, and NEFA release to the extracellular compartment by the irreversibly injured myocyte. Under conditions of metabolic inhibition, neonatal rat myocytes in primary monolayer culture became virtually depleted of ATP within 8 h. The metabolically inhibited myocytes evidenced membrane phospholipid degradation and a resultant net accumulation of NEFA produced thereby in the extracellular medium. However, under conditions of nutrient deprivation, the injured myocytes retained the NEFA produced from phospholipid catabolism intracellularly and did not release it to the culture medium, although the extent of myocyte ATP depletion was the same as it had been from metabolic inhibition. Serum could elicit, in a concentration‐dependent fashion, the quantitative release of NEFA from metabolically inhibited myocytes to the culture medium but did not influence the net production of NEFA by the injured cells. Similarly, NEFA release from nutrient‐deprived myocytes incubated in serum‐free, substrate‐free medium or in physiological buffer could be induced by supplementing the medium or buffer with bovine serum albumin (BSA), and the extent of NEFA release, but not NEFA formation, was dependent upon the extracellular BSA concentration. No manipulations to media other than changing their serum content or supplementing them with BSA were found to influence the disposition of NEFA produced during phospholipid catabolism in the irreversibly injured, ATP‐depleted myocyte. Therefore, although progressive metabolic compromise in the myocyte was correlated with increasing, net NEFA formation, the distribution of the NEFA between the intracellular and the extracellular compartments was not determined by the magnitude of ATP loss or by the nature or duration of at least two injury stimuli, metabolic inhibition and nutrient deprivation. Rather, the net release of NEFA from the ATP‐depleted myocyte to the culture medium and the consequent reduction of intracellular myocyte NEFA overload were critically and causally dependent upon the presence and concentration of extracellular NEFA “acceptor.” The influence of acceptor on the mobilization of NEFA from the injured myocyte has implications regarding the use of NEFA release as an index of myocyte pathology and could serve to modify the progression and extent of myocardial injury in vivo.
Heparin stimulation of plasminogen activator secretion by macrophage‐like cell line raw264.7: Role of the scavenger receptor
Authors: Falcone Domenick J. 
Published: February 2005

AbstractSecretion of urokinase‐type plasminogen activator (uPA) by RAW264.7 cells was stimulated by heparin in a dose‐ and time‐dependent manner. Secretion of uPA was not detected when cells were exposed to heparin at 4°C, indicating that heparin was not simply releasing receptor‐bound uPA. Furthermore, prior removal of membrane‐associated uPA did not influence heparin's ability to stimulate the release of uPA from the macrophage‐like line. Low molecular weight and weakly anticoagulant heparins stimulated uPA secretion but less effectively than other heparin fractions. The observed stimulation in macrophage uPA secretion by heparin is similar to that previously reported for polyanions recognized by the scavenger receptor including fucoidan, polyinosinic acid, dextran sulfate, and acetyl‐LDL (Falcone and Ferenc: J. Cell. Physiol. 135: 387–396, 1988). Evidence that heparin's binding to RAW264.7 cells is mediated by the scavenger receptor is derived from experiments in which fucoidan blocked the specific binding of [3H]‐heparin to RAW264.7 cells. However, heparin partially inhibited the stimulation of cholesteryl [3H]‐oleate synthesis observed in these cells upon incubation with acetyl‐LDL and weakly inhibited cellular binding of 125l‐acetyl‐LDL at 4°C. These data indicate that heparin's binding to RAW264.7 cells is mediated, only in part, by the scavenger receptor. Nonetheless, neither heparin nor fucoidan was able to stimulate the release of plasminogen activator activity from monocyte‐like U937 cells which are devoid of scavenger receptor activity.
Characterization of the epidermal growth factor receptor associated with cytoskeletons of A431 cells
Authors: Roy Linda M. , Gittinger Cynthia K. , Landreth Gary E. 
Published: February 2005

AbstractEpidermal growth factor receptors (EGF‐R) have been shown to be associated with the detergent‐insoluble cytoskeleton of A431 cells, where they retained both a functional ligand‐binding domain and tyrosine kinase activity. In the present study we have characterized the tyrosine kinase and ligand binding activities of this cytoskeletally associated EGF‐R. The tyrosine kinase activity of the cytoskeletally associated EGF‐R was stimulated by EGF treatment of intact cells as evidenced by increased autophosphorylation and phosphorylation of the exogenous substrate angiotensin II (AII). The kinetic behavior of the EGF‐R associated with cytoskeletons of EGF‐treated cells was similar to that of purified receptors. The stimulation of the receptor kinase activity required EGF treatment of intact cells prior to Triton extraction. If cytoskeletons were prepared from untreated cells and then incubated with EGF, there was no stimulation of the detergent‐insoluble receptor kinase activity, indicating that the immobilized receptor was unable to undergo EGF‐stimulated activation. Comparison of peptide maps from soluble and cytoskeletally associated EGF‐R revealed qualitatively similar patterns; however, they are distinguished by a prominent 46 kD band in digests of the cytoskeletal EGF‐R. Saturable binding of 125I‐EGF to A431 cytoskeletons prepared from adherent and suspended cells demonstrated the presence of specific receptors on the cytoskeleton. High‐affinity EGF‐R were preferentially retained upon detergent extraction of adherent cells, whereas both low‐ and high‐affinity receptors were solubilized from the cytoskeletons of suspended cells. Suspension of cells resulted in the solubilization of an additional 15% of the EGF‐R to that solubilized in adherent cells, indicating that EGF‐R can reversibly associate with the structural elements of the cell.
Biological oxidation and the mobilization of mitochondrial calcium during the differentiation of Physarum polycephalum
Authors: Nations C. , Allison V. F. , Aldrich H. C. , Allen R. G. 
Published: February 2005

AbstractWe have previously reported that calcium is required for the starvation‐induced differentiation of the slime mold, Physarum polycephalum. With the exception of calcium, each component of the complex starvation medium may be withheld and the organism will still differentiate into spherules. The results of the present study reveal that spherulation will proceed normally when the microplasmoidal cells are transferred from nutrient medium to a citrate buffer containing only 8 mM Ca CI2. Electron microscopy and X‐ray microprobe analysis reveal that there is an initial increase in the population of calcium‐containing mitochondrial granules when the microplasmodia are induced to differentiate. However, as differentiation proceeds, these granules decrease in number and are virtually absent from the mitochondria of mature spherules. The accumulation and depletion of calcium‐containing granules is not observed in a nondifferentiating strain of Physarum cultured under standard conditions, but is observed when this strain is first treated with a calcium‐enriched nutrient medium that conditions it for spherulation. Changes in the cellular concentrations of NADH and lipid peroxides, and in the activity of superoxide dismutase, correspond temporally to the pattern of increase and depletion of the calcium‐containing inclusions. The oxidative stress associated with starvation‐induced spherulation may be a consequence of the active accumulation of calcium; the mobilization of this calcium may then be the event that initiates differentiation.
Protein kinase C and a viral K‐RAS protein cooperatively enhance the response of adenylate cyclase to stimulators
Authors: Franks Douglas J. , Durkin Jon P. , Whitfield James F. 
Published: February 2005

AbstractThe protein kinase C stimulator TPA (12‐0‐tetradecanoyl phorbol‐13‐acetate) enhanced the responsiveness of adenylate cyclase to IPR (isoproterenol) and PGE1 (prostaglandin E1) in quiescent tsKSV‐NRK cells at the nonpermissive 41°C Reactivating the thermolabile mitogenic/oncogenic K‐ras protein in tsKSV‐NRK cells by dropping the temperature to 36°C also enhanced the responsiveness of adenylate cyclase to IPR and PGE1. The enhancement was transient and peaked at 6 hours after the temperature shift. This enhanced the responsiveness was specifically due to the reactivated viral K‐ras protein rather than the temperature shift because the same temperature shift did not affect adenylate cyclase responsiveness in uninfected NRK cells, nor was it a result of the mitogenic stimulus since reactivating the mitogenic pp60v‐src protein in tsASV‐NRK cells did not affect adenylate cyclase responsiveness. The increased responsiveness of adenylate cyclase at 6 hours after the temperature shift was not a result of elevated membrane‐associated PKC activity. However, the reactivated viral K‐ras protein strongly increased the stimulability of membrane‐associated PKC by TPA and it further increased TPA's ability to enhance the responsiveness of adenylate cyclase to IPR and PGE1. Thus, a viral K‐ras protein and membrane‐associated protein kinase C can cooperate to increase the responsiveness of adenylate cyclase to agonists.
Effect of pentoxifylline on developmental changes in neutrophil cell surface mobility and membrane fluidity
Authors: Newton J. Allen , Ashwood Edward R. , Yang Kuender D. , Augustine Nancy H. , Hill Harry R. 
Published: February 2005

AbstractPolymorphonuclear leukocytes (PMNs) from human neonates respond less efficiently to chemotactic factor stimulation than do PMNs from adults. The biologic mechanisms underlying this developmental process are poorly understood. In previous studies, we have found that pentoxifylline, an agent reported to enhance membrane deformability, increased the chemotactic response of neonatal PMNs. In the present studies, we have examined the effect of pentoxifylline on cell surface mobility and membrane fluidity by assessing fluorescent concanavalin A (Con A) capping and fluorescent polarization (FP). Baseline Con A capping was lower in the PMNs of neonates when compared to PMNs from adult controls. Colchicine, which increases capping by disrupting microtubules, exaggerated the differences between the adult and neonatal PMNs. Following exposure of neonatal PMNs to pentoxifylline, colchicine enhanced Con A capping to levels equivalent to those of colchicine‐treated PMNs from adults. Employing a fluorescence polarization (FP) assay, we found the fluid state of the membrane of PMNs from neonates was significantly less than that of adult controls. Pentoxifylline alone significantly increased the fluidity of the cell membranes of neonatal PMNs while decreasing elevated basal levels of F‐actin in the cell. These data suggest an intrinsic cytoskeletal difference in the PMNs of neonates that may be responsive to pharmacologic manipulation.
Generation of osteoclasts from hemopoietic cells and a multipotential cell line in vitro
Authors: Hattersley G. , Chambers T. J. 
Published: February 2005

AbstractOsteoclasts are the cells that resorb bone. It is generally presumed, on the basis of indirect experiments, that they are derived from the hemopoietic stem cell. However, this origin has never been established. We have developed an assay for osteoclastic differentiation in which bone marrow cells are incubated in liquid culture on slices of cortical bone. The bone slices are inspected in the scanning electron microscope after incubation for the presence of excavations, which are characteristic of osteoclastic activity. We have now incubated bone marrow cells at low density, or a factor‐dependent mouse hemopoietic cell line (FDCP‐mix A4) with 1,25 dihydroxyvitamin D3 (a hormone which we have previously found induces osteoclastic differentiation) with and without murine bone marrow stromal cells, or with and without 3T3 cells, on bone slices. Neither the bone marrow cells nor the bone marrow stromal cells alone developed osteoclastic function even in the presence of 1,25 dihydroxyvitamin D3. However, extensive excavation of the bone surface was observed, only in the presence of 1,25 dihydroxy‐vitamin D3, on bone slices on which bone marrow stromal cells were cocultured with low‐density bone marrow cells or the hemopoietic cell line. Similar results were obtained when the bone marrow stromal cells were killed by glutaraldehyde fixation; 3T3 cells were unable to substitute for stromal cells. These results are strong evidence that osteoclasts derive from the hemopoietic stem cell and suggest that although mature osteoclasts possess neither receptors for nor responsiveness to 1,25 dihydroxyvitamin D3, the hormone induces osteoclastic function through a direct effect on hemopoietic cells rather than through some accessory cell in the bone marrow stroma. The failure of 3T3 cells, which enable differentiation of other hemopoietic progeny from this cell line, to induce osteoclastic differentiation suggests that bone marrow stroma possesses additional characteristics distinct from those that induce differentiation of other hemopoietic cells that are specifically required for osteoclastic differentiation.
Cultures of fibroblasts in fibrin lattices: Models for the study of metabolic activities of the cells in physiological conditions
Authors: Gillery Philippe , Bellon George , Coustry Françoise , Borel Jacques‐Paul 
Published: February 2005

AbstractTwo techniques for fibroblast culture in three‐dimensional fibrin matrices (fibrin lattices) were used to study the behavior and metabolism of cells in this physiological support. When the fibrin lattices were prepared in silicone‐coated glass petri dishes, fibroblasts induced retraction of lattices to an extent dependent on both cell density and serum concentration, the cells stopped dividing, and their protein and collagen syntheses proceeded at a lower rate, like that in collagen lattices. When fibrin lattices were prepared in plastic petri dishes, the matrix attached to the walls, there was no retraction, and the protein synthesis was very active even as regards collagen. The optimal concentration of ascorbic acid in nonretracting fibrin lattices was lower (10 μg/ml) than in monolayers (50 μg/ml). The comparison of collagen and fibrin lattices showed that the collagenic nature of the lattice was not compulsory for supporting the phenomenon of retraction and that fibroblasts, when exposed to a stress in a fibrin matrix prevented from retracting, secreted far more collagen.
Fibronectin and fibrinolysis are not required for fibrin gel contraction by human skin fibroblasts
Authors: Tuan Tai‐Lan , Grinnell Fred 
Published: February 2005

AbstractHuman skin fibroblasts contracted fibrin gels in a time‐ and cell‐dependent manner. Under optimal conditions, gel contraction amounted to more than 50% in 2 hr. Fibronectin did not promote contraction, and fibrinolysis was not required for contraction, although gels contracted without serum or aprotinin were lysed. Before contraction, fibrin was present in loosely packed, randomly organized fibrils. After contraction, the fibrils were more densely packed and aligned in the plane of cell spreading. Cycloheximide treatment of fibroblasts inhibited gel contraction in serum‐free medium but not in serum‐containing medium. Fibronectin could not substitute for serum in overcoming the cycloheximide effect. Binding sites for fibrin were distributed randomly over the cells' surfaces based on electron microscopic observations. Often small groups of fibrils were localized in indentations at the cell surface. Finally, peptides containing the arg‐gly‐asp‐ser sequence inhibited gel contraction.
Temperature‐dependent oligomerization of hsp85 in vitro
Authors: Lanks Karl W. 
Published: February 2005

AbstractThe failure of conventional subcellular fractionation methods to identify interactions between the bulk of hsp85 and other cellular structures suggested that critical stress protein interactions might be detectable only at elevated temperatures. This was confirmed by showing that incorporation of hsp85 and grp95 into sedimentanble complexes in Triton X‐100 extracts of L929 cells increased progressively over the 30°C‐43°C temperature range. Whereas several other proteins, including hsp 110 and hsp69, became sedimentable under these conditions, this effect required temperatures of ∼43°C and was only partially detergent‐dependent. In contrast, hsp85 became sedimentable at temperatures as low as 33°C, and this effect was highly detergent‐dependent. Temperature‐dependent conversion of purified hsp85 to a sedimentable form was shown to result from limited oligomerization of the protein, which occurred in the presence of detergent. Since the detergent requirement could be met by a variety of compounds, including sphingosine, these findings suggest that hsp85 oligomerization may occur when intact cells are exposed to elevated temperature.
RNA synthesis and stability in UV‐irradiated and nonirradiated mouse L cells
Authors: Jones Robert W. , Eliceiri Brian P. , Eliceiri George L. 
Published: February 2005

AbstractIn mouse L cells, relatively low doses of UV light (e.g., about 35 J/m2) induced the rapid breakdown of the molecules of many RNA species transcribed shortly before irradiation. This included 28S, 18S, 5.8S, and 5S rRNA, U1, U2, U3, U4, and U5 small nuclear RNA, but not the main band of transfer RNAs or 7SL RNA. At higher UV doses, an RNA band that contains tRNAleu was also degraded rapidly after UV irradiation. RNA molecules synthesized long before irradiation (e.g., 22 h for small RNAs, 4 h for large rRNAs) were not affected. Our results suggest that the maturation and/or assembly into fully mature ribonucleoprotein particles of several small RNA species is not completed 4 h after transcription. The effect of UV radiation occurred in mouse L cells, but not in human HeLa or KB cells. In a previous report, L cells were transformed by DNA transfection with two mouse U1b RNA genes, named U1.1 and U1.2. We observed now that, in L cells transformed with the U1.2 gene, the ratio of radioactivity in the apparent U1b and U1a RNA precursors after 5 min of labeling was about 20 times higher than (a) this ratio in briefly labeled L cells that had been transformed with the U1.1 gene, and (b) the ratio of radioactive mature U1b and U1a RNA after 20 h of chase in L cells transformed with the U1.2 gene. These results suggest that very high levels of U1b RNA are transcribed from the exogenous U1.2 gene copies, followed by the rapid degradation of most of these transcripts.
Temporary complementation of temperature‐sensitive mutants of the cell cycle by transfection with a wild‐type or a mutant cDNA of ADP/ATP translocase
Authors: Alder Hansjuerg , Chang Chung‐Der , Chen Sing‐Tsung , Beck Ingrid , Chang Chen‐Yeh , Baserga Renato 
Published: February 2005

AbstractA number of cell‐cycle‐specific temperature‐sensitive (ts) mutants have been isolated from animal cells, especially Syrian hamster cells. These ts mutants, like cell cycle ts mutants of yeast, can be complemented by specific genes, some of which have been molecularly cloned. We have isolated a cDNA clone that complements TK− ts13 cells, but only temporarily. This clone, called B1, differs from a previously isolated clone (Sekiguchi et al.: EMBO Journal 7: 1683–1687, 1988) that specifically complements ts13 cells. In addition, B1 also complemented temporarily three other ts mutants of the cell cycle, tsAF8, ts694, and ts550C cells. These mutants have different mutations since, in cell fusion experiments, they complement each other. Sequencing of the B1 cDNA clone revealed that it was a mutant of human ADP/ATP translocase in which some human sequences at the 5′ end have been replaced by SV40 sequences. The wild‐type translocase was less effective but could still increase the survival time of cell cycle ts mutants at the restrictive temperature. Using the polymerase chain reaction, it was possible to demonstrate that the B1 plasmid is expressed in TK−ts13 cells undergoing temporary complementation.
Chemotactic peptide receptor‐cytoskeletal interactions and functional correlations in differentiated HL‐60 cells and human polymorphonuclear leukocytes
Authors: Rao K. Murali Krishna , Currie Mark S. , Cohen Harvey J. , Weinberg J. Brice 
Published: February 2005

AbstractWe studied the chemotactic peptide receptor/cytoskeletal interactions in HL‐60 cells induced to differentiate with different agents and attempted to correlate these observations with the acquisition of different functional responses. Dibutyryl cyclic AMP‐treated cells showed rapid superoxide anion production in response to N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP) and slow, sustained response to phorbol myristate acetate (PMA). Retinoic acid‐induced cells showed a slow, sustained response to both FMLP and PMA. Interferon‐γ‐treated cells produced no superoxide anion on stimulation with FMLP, whereas tumor necrosis factor (TNF)‐treated cells showed a slight response. Chemotactic peptide receptor association was the same in the HL‐60 cells treated with different agents, despite marked differences in the superoxide anion generation and actin polymerization responses to FMLP and PMA in these cells. In mature neutrophils chemotactic peptide receptor association with the cytoskeleton was not affected by either pertussis or cholera toxin. However, both toxins inhibited FMLP‐induced actin polymerization and superoxide anion generation. This suggested involvement of a G‐protein similar to Gt, rather than Gi or Gs. Neither toxin had any effect on PMA‐induced superoxide anion generation. These observations indicate that receptor association with the cytoskeleton may not have a significant role in affecting signal recognition and response. Among the several possible roles suggested, clearance of the occupied receptors may be the most important role of the cytoskeletal association. HL‐60 cells induced to differentiate with different agents (because of their varied functional responses) might prove very useful in dissecting the molecular mechanisms regulating stimulus‐induced activation of neutrophils.