bluuebunny/crossref_metadata_2025 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

Long‐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

Secretion 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

Epidermal 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

We 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

The 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

Polymorphonuclear 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

Osteoclasts 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

Two 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

Human 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

The 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

In 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

A 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

We 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.
Casein accumulation in distended rough endoplasmic reticulum of collagen gel‐cultivated mouse mammary epithelia
Authors: Hurley David , Hwang Soo‐In , Rocha Victor
Published: February 2005

Mouse mammary epithelial cells cultivated on collagen gels synthesize and secrete casein in a hormone‐dependent manner. Fine‐structure electron microscopy of secretory cultures revealed numerous cytoplasmic structures surrounded by membrane that is studded with ribosomes. The structures appear to be distended rough endoplasmic reticulum (RER). Electron microscope protein A‐colloidal gold immunolocalization showed casein antiserum‐specific deposition of gold particles over the RER cytoplasmic vesicles in cells provided insulin, prolactin, and hydrocortisone (IPF). Nonimmune antiserum showed no gold particle deposition over these cytoplasmic structures. Epithelia provided only insulin showed no such cytoplasmic vesicles nor any specific deposition of gold particles. Immunoblot analysis of cell lysate and culture medium showed casein only in IPF‐treated cultures. It appears that the casein secretory pathway in collagen gel cultured mammary epithelia is blocked at the step that fuses RER vesicles to Golgi membrane. The data raise questions regarding the processing and maturation of casein and the mechanism of casein secretion in these cultures.
Membrane‐associated hyaluronate‐binding activity of chondrosarcoma chondrocytes
Authors: McCarthy Mary T. , Toole Bryan P.
Published: February 2005

The association of hyaluronate with the surface of chondrocytes was examined by several approaches using primary cultures of chondrocytes derived from the Swarm rat chondrosarcoma. In culture, chondrosarcoma chondrocytes produced large pericellular coats, which can be visualized by particle exclusion, and which can be removed by Streptomyceshyaluronidase. Exposure of chondrocytes, which had been metabolically labelled with 3H‐acetate, to exogenous hyaluro‐nate or to Streptomyceshyaluronidase resulted in the release of 36–38% of the endogenous, labelled chondroitin sulfate from the cell layer into the incubation solution. These results imply that at least 37% of the cell layer chondroitin sulfate proteoglycan is retained there by an interaction with hyaluronate. Thus membranes were prepared from cultured chondrocytes and examined for sites which bind3H‐hyaluronate. Binding was observed and found to be saturable, specific for hyaluronate, of high affinity (Kd =∼10−10M),and destroyed by treating the membranes with trypsin. The 3 H‐hyaluronate‐binding activity was inhibited competitively by hyaluronate decasaccharides but not by hexasaccharides or octasaccharides, indicating that the binding sites recognize a sequence of hyalu‐ronate composed of five disaccharide repeats. The binding activity was partially purified from a detergent extract of chondrocyte membranes by ion exchange chromatography on DEAE‐cellulose, followed by affinity chromatography on wheat germ agglutinin‐agarose. Analysis of the partially purified binding activity by SDS‐PAGE revealed five protein bands of 48,000–66,000 daltons in silver‐stained gels. SDS‐PAGE followed by Western blotting and exposure to mono‐clonal antibodies which recognize epitopes present in link protein and in the hyaluronate‐binding region of cartilage proteoglycan revealed no immunoreac‐tive protein bands in the partially purified material. We conclude that one mechanism by which hyaluronate associates with the chondrocyte surface may be via interaction with a membrane‐bound hyaluronate‐binding protein which is distinct from link protein and proteoglycan.
Possible role of prostaglandins as negative regulators in growth stimulation by tumor necrosis factor and epidermal growth factor in human fibroblasts
Authors: Hori Takamitsu , Kashiyama Satoshi , Hayakawa Makio , Shibamoto Sayumi , Tsujimoto Masafumi , Oku Naoto , Ito Fumiaki
Published: February 2005

Recombinant tumor necrosis factor (TNF), epidermal growth factor (EGF), and transforming growth factor β (TGF‐β) stimulated growth of confluent human diploid fibroblasts (FS‐4 cells) in the presence of fetal calf serum. TGF‐β synergistically enhanced both the TNF‐ and EGF‐stimulated cell growth, whereas synergism between the mitogenic action of EGF and that of TNF was not observed. When indomethacin or acetylsalicylic acid, an inhibitor of prostaglandin production, was added to FS‐4 cells, cell growth stimulated by EGF or TNF was increased, suggesting that prostaglandins induced by these mitogens antagonize their growth stimulatory actions. In contrast, neither indomethacin nor acetylsalicylic acid had a significant effect on the TGF‐β‐induced growth of FS‐4 cells. Mitogenic responses of indomethacin‐treated cells to EGF, TNF, and TGF‐β were similarly suppressed by the addition of exogenous prostaglandin D2 (PGD2). Other prostaglandins such as PGE2 and PGF2α produced less inhibition of the cell growth.
Enucleation of the rat pheochromocytoma clonal cell line, PC12: Effect on neurite outgrowth
Authors: Nichols Robert A. , Chandler Charles E. , Shooter Eric M.
Published: February 2005

The effect of removal of PC12 cell nuclei on neurite outgrowth was studied. Enucleation (80–90%) was accomplished in the presence of cytochalasin B using a centrifugation technique that exploited the very tight adhesivity of PC12 cells for a substratum composed of an extracellular matrix secreted by bovine corneal endothelial cells in response to epidermal growth factor treatment. Neither nucleated nor enucleated PC 12 cells showed significant neurite outgrowth on this particular matrix in the absence of nerve growth factor. In the presence of nerve growth factor both PC12 cell types initiated neurite outgrowth, but whereas neurites from nucleated cells grew continuously for two days, those from enucleated cells reached a maximum length after one day. The results suggest that neurite initiation but not continued neurite growth or stabilization can occur in the absence of transcription.
Role of protein kinase c in interleukin 1, anti‐T3, and mitogenic lectin‐induced interleukin 2 secretion
Authors: Mills Gordon B. , May Christopher , Hill Mary , Gelfand Erwin W.
Published: February 2005

Activation of T‐lymphocytes by antigen, mitogenic lectins, or antibodies against the T‐cell receptor complex, particularly in the presence of IL1, induces the secretion of the T‐cell growth factor IL2. IL2 then has a major role in regulating the duration and magnitude of the immune response. Interaction of antigen, antibodies against the T‐cell receptor complex, or mitogenic lectins with T‐lymphocytes also induces hydrolysis of membrane phospholipids, leading to the production of diacylglycerol, an activator of the Ca2+‐ and phospholipid‐dependent phosphotransferase, protein kinase C (PKC). Phorbol esters, potent activators of PKC, augment secretion of the T‐cell growth factor, interleukin 2 (IL2). Activation of PKC may therefore serve as an important early event in the production and secretion of IL2. We have determined whether IL2 secretion can be induced in the murine cell T‐lymphocyte line LBRM 331A5, where PKC is inhibited by staurosporine or sphingosine or in cells where PKC is depleted by prolonged incubation with high concentrations of phorbol esters. In cells in which PKC was either inhibited or depleted, antibodies against the T3 portion of the T‐cell receptor complex and the mitogenic lectin phytohemagglutinin (PHA) still triggered IL2 secretion. In addition, the monokine Ill augmented this IL2 secretion irrespective of whether PKC was inhibited or depleted. These data indicate that activation of PKC is not an obligatory step for IL2 secretion in LBRM 331A5 murine T‐lymphocytes.
Analysis of effect of age on synthesis of specific proteins by hepatocytes
Authors: Butler James A. , Heydari Ahmad R. , Richardson Arlan
Published: February 2005

The effect of age on the synthesis of specific proteins by hepatocytes was studied in Fischer F344 rats using two‐dimensional polyacrylamide gel electrophoresis. Almost all proteins synthesized by hepatocytes from young rats were synthesized by hepatocytes isolated from old rats. Of over 500 proteins visually compared by two‐dimensional polyacrylamide gel electrophoresis, only 11 proteins were observed to disappear and/or appear consistently with increasing age. The rates of synthesis of 36 randomly chosen proteins were quantified. Interestingly, the synthesis of 35 of the 36 proteins decreased between 5 and 30 months of age. The decrease in protein synthesis varied (15% to 70%) from one protein to another; i.e., a heterogeneity was observed in the age‐related decrease in the synthesis of proteins. The age‐related decrease in protein synthesis was statistically significant for 53% of the proteins studied. The total decrease in the rate of synthesis of all 36 proteins studied was 40% between 5 and 30 months of age, which is essentially the same as the decrease in total protein synthesis by suspension of hepatocytes isolated from 5‐ and 30‐month‐old rats. The results of this study demonstrate that the mechanism underlaying aging is different from development, which is characterized by a major change in the species of proteins synthesized by a cell.
Contribution of endosomes to intrahepatic distribution of apolipoprotein B and apolipoprotein E
Authors: Wong Laurence
Published: February 2005

The endosomal contribution of apolipoprotein B and apolipoprotein E to the intrahepatic distribution of these 2 apolipoproteins was studied. The endosomal contribution of the apolipoproteins was estimated by comparing the specific radioactivities of the injected apolipoproteins with that of apolipoproteins in the liver. The endosomal contribution is further validated by using a least‐squares linear combination method that has been described previously (Wong and Pino: European Journal of Biochemistry 164: 357–367, 1987). The results using these two independent analyses were similar. There was no difference in endosomal contribution of the apolipoproteins between the fasted and fed rats. In both cases, total endosomal contribution in the microsomal fraction of apolipoprotein B was estimated at 7–10% and that for apolipoprotein E was estimated at 62–66%. The molecular weights of the two apolipoproteins in the endosomes closely resembles those of the injected apolipoproteins in the very low density lipoproteins, indicating little or no apolipoprotein loss during receptor‐mediated endocytosis. It is concluded that while there was no substantial contribution of apolipoprotein B to the intrahepatic distribution of that protein, the contribution of extrahepatic apolipoprotein E to intrahepatic apolipoprotein E distribution is substantial.
Calcium‐independent growth of human ovarian carcinoma cells
Authors: Chan Thomas C. K.
Published: February 2005

Evidence in the literature suggests that cancer cell growth in vitro is generally not sensitive to external calcium. A human ovarian carcinoma cell line (SKOV3) retained 60% of its normal growth in Dulbecco modified Eagle's medium (DME) when the calcium concentration was reduced from 3 mM to 10 μM. Chinese hamster ovary cells (CHO) were growth‐arrested in media containing less than 500 μM calcium. In low‐calcium (10 μM) DME, 10 μM of a calmodulin antagonist W7 inhibited the growth of SKOV3 cells by more than 90%, while 100 μM of its inactive analog W5 was mildly inhibitory (20%). The growth inhibition by W7 was antagonized by increasing calcium concentrations in the culture media, while the inhibition by W5 was calcium‐independent. The phorbol ester TPA was also effective in antagonizing W7's growth inhibition in low‐calcium DME, suggesting that the W7 effect is mediated via protein kinase C inhibition. SKOV3 total cellular protein kinase C activity was 1.6 times higher than CHO cells when incubated in normal DME. When incubated in low‐calcium DME, a large drop in protein kinase C activity in the CHO cells was observed while the enzyme activity was unchanged in the SKOV3 cells. Our data suggest that these human ovarian tumor cells have altered cellular calcium regulatory processes associated with the defective down‐regulation of protein kinase C. This defect may confer these cells the ability to proliferate independently of the external calcium concentration. Targeting the cellular signal transduction components may be useful in cancer chemotherapy.
Heat protectors and heat‐induced preferential redistribution of 26 and 70 kDa proteins in chinese hamster ovary cells
Authors: Lee Yong J. , Armour Elwood P. , Borrelli Michael J. , Corry Peter M.
Published: February 2005

An overall increase of 40% in nuclear‐associated protein has been shown to be one of the sequellae of exposure of eukaryotic cells to elevated temperatures. Several investigators have shown that the increased protein/DNA ratios correlated well with the degree of cytotoxicity. In previous investigations, we have shown that cycloheximide, which protects the cell from the killing effects of heat, produces a dramatic reduction of the bulk nuclear‐associated proteins after heating. In this investigation, we studied a previously unobserved efflux of a 26 kDa protein after heat shock and the preferential accumulation of the 70 kDa protein. The 26 kDa protein was shown not to be a member of previously described heat shock protein families. Preferential reduction of a 26 kDa protein and accumulation of a 70 kDa protein was observed in nuclei isolated from Chinese hamster ovary cells after heating at 43°C. After heat treatment, the 26 kDa protein in the nucleus was decreased to a level 0.1–0.3 times the original amount in unheated cells, and the 70 kDa protein in the nucleus increased by a factor of 1.6–1.8. The normal levels of these two proteins were restored when cells were incubated at 37°C following heat shock. Cells treated with heat protectors, cycloheximide and histidinol, demonstrated approximately the same redistribution in nuclear 26 and 70 kDa proteins immediately after heating as those not exposed to these drugs. On the other hand, restoration to control levels was much faster in the protector‐treated cells, suggesting that “repair” of heat‐induced damage is an important factor in the cells ability to survive this insult. Return to normal protein levels did not require new protein synthesis.
Effects of D‐threo‐PDMP, an inhibitor of glucosylceramide synthetase, on expression of cell surface glycolipid antigen and binding to adhesive proteins by B16 melanoma cells
Authors: Inokuchi Jin‐Ichi , Momosaki Keiko , Shimeno Hiroshi , Nagamatsu Atsuo , Radin Norman S.
Published: February 2005

Incubating B16 melanoma cells with an inhibitor of glucosylceramide (GlcCer) synthetase, D‐threo‐1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol (D‐threo‐PDMP), led to a considerable decrease in the levels of GlcCer and lactosylceramide (LacCer). The content of ganglioside GM3 was little affected, but the ability to bind a monoclonal antibody against the ganglioside (M2590) was greatly reduced, suggesting that the reduction in the simple glycolipids led to encryption of the membrane antigen. This interpretation is supported by the observation that permeabilization of the treated cells with Triton X‐100 restored immunological reactivity. Specificity of the PDMP effect was shown by its lack of effect on the reactivity of two other surface antigens to anti‐melanoma monoclonal antibodies M562 and M622, and of the major histocompatibility antigens to anti‐H‐2KbDb monoclonal antibody. The ability of the treated cells to attach to laminin or type IV collagen was lost but that to fibronectin was not. The effects of the enzyme inhibitor were counteracted by including GlcCer in the culture medium. This indicates that the lipid was absorbed by the cells and utilized like endogenously‐formed GlcCer. Cells preattached to laminin or collagen could be induced to round up by addition of inhibitor. In contrast, L‐threo‐PDMP (which does not block the synthesis of GlcCer) had no effect on the immunologic reactivity of GM3 or the adhesion properties of the cells. However, it did produce considerable accumulation of LacCer. These data suggest that the simple glycolipid, GlcCer, is an essential factor for antigenic expression of the more complex glycolipids on cell surfaces and that there is a close association and interaction between glycolipids and adhesive receptors on the cell surface.
Hemin enhances differentiation and maturation of cultured regenerated skeletal myotubes
Authors: Funanage Vicky L. , Schroedl Nancy A. , Moses Priscilla A. , Smith Susan M. , Kirwin James J. , Hartzell Charles R.
Published: February 2005

Satellite cells, isolated from marcaine‐damaged rat skeletal muscle, differentiate in culture to form contracting, cross‐striated myotubes. Addition of 20 μM hemin (ferriprotoporphyrin IX chloride) to the culture medium resulted in increases in the number, size, and alignment of myotubes; in the number of myotubes that exhibited cross‐striations; and in the strength and frequency of myotube contractions. Hemin increased satellite cell fusion by 27%, but decreased cell proliferative rate by 30%. Hemin increased the specific activity of creatine kinase (CK), a sensitive indicator of muscle differentiation, by 157%. Separation of CK isoenzymes by agarose gel electrophoresis showed that hemin increased only the muscle‐specific CK isoenzymes (MM‐CK and MB‐CK). Thus, hemin seems to duplicate some of the effects of innervation on cultured myotubes by increasing contraction frequency and strength, appearance of cross‐striations, and muscle‐specific isoenzymes. In contrast, 3‐amino‐1,2,4‐triazole, an inhibitor of heme biosynthesis, decreased the number of cross‐striated myotubes, the strength and frequency of myotube contractions, and CK activity. These inhibitory effects were reversed by hemin. Collectively, these results demonstrate a physiologically significant role for heme in myotube maturation.
Reversible forskolin‐induced impairment of sucrase‐isomaltase mRNA levels, biosynthesis, and transport to the brush border membrane in Caco‐2 cells
Authors: Rousset Monique , Chantret Isabelle , Darmoul Dalila , Trugnan Germain , Sapin Catherine , Green Fiona , Swallow Dallas , Zweibaum Alain
Published: February 2005

Hybridization analysis of mRNA with a cDNA probe for human sucrase‐isomaltase, pulse‐chase experiments with L‐[35S]‐methionine followed by SDS‐PAGE, and immunofluorescence detection of sucrase‐isomaltase were used to analyze the level(s) at which forskolin interferes with the expression of the enzyme in Caco‐2 cells in culture. Three effects are observed in Caco‐2 cells treated with forskolin: (1) a marked decrease in the level of sucrase‐isomaltase mRNA, (2) a marked decrease in the biosynthesis of the enzyme without any alteration of its stability, and (3) an almost total inhibition of its transport to the brush border membrane. All three effects are reversible when the drug is removed from the culture medium, though this reversibility is asynchronous: transport to the brush border membrane resumes after 24 h, sucrase‐isomaltase mRNA levels are back to the normal after 5 days, whereas the biosynthesis of the enzyme, although increasing progressively, remains lower than in control cells, even 10 days after removal of the drug. The possibility that some effects are directly dependent on cAMP and others a consequence of changes in glucose metabolism is discussed.
Pulmonary microvascular endothelial cell contractility on silicone rubber substrate
Authors: Morel Nicole M. L. , Dodge Andrea B. , Patton Wayne F. , Herman Ira M. , Hechtman Herbert B. , Shepro David
Published: February 2005

Endothelial cell (EC) motility may contribute to the regulation of microvascular perfusion and/or paracellular permeability. The experiments reported herein demonstrate that bovine pulmonary microvessel EC can reversibly deform a silicone substrate in response to agents known to contract and relax smooth muscle cells. Contracting pulmonary microvessel EC exerted a tension that created wrinkles in the underlying deformable substrate. Relaxation and loss of tension were revealed by the disappearance of these wrinkles without loss of cell adhesion to the substratum. Angiotensin II (Ang II) and bradykinin stimulated pulmonary microvessel EC to contract within 3 to 8 min in a Ca2+‐dependent fashion. The peak of contraction at 10 to 20 min was followed by relaxation. Forskolin and sodium nitroprusside (SNP) initiated relaxation of the microvessel EC within 3 to 10 min respectively. Relaxed EC contracted following the addition of Ang II, also within 3 min. Dibutyryl cAMP, dibutyryl cGMP, and the photoactivated internalized “caged” cAMP and cGMP promoted EC relaxation in a manner similar to forskolin and SNP. Increases in the intracellular concentration of inositol triphosphate (IP3) with the photoactivated IP3 complex promoted EC contraction in 2 min with a peak at 7 min. The contraction was followed by relaxation, which occurred at 20–25 min. Neither bovine pulmonary artery nor retinal microvessel EC, used as controls, contracted under these experimental conditions. One could speculate that this unique contractile property of pulmonary microvessel EC as observed in vitro may play a regulatory role in vivo, in local perfusion and/or in intercellular gap regulation.
Secretory character of a group of isoproterenol‐induced polypeptides in mouse parotid glands
Authors: Solís Remigio O. López , Miranda Dante , Alliende Cecilia , González Julieta , Díaz Nelson , Díaz Héctor , Ruiz Fernando
Published: February 2005

The secretory nature of the isoproterenol‐induced mouse parotid polypeptides C, D, E, F, and G (molecular weights 64,000, 61,000, 51,500, 38,000, and 37,000, respectively) is documented. Polypeptides C, D, E, F, and G, accumulated in response to successive daily stimulations with isoproterenol, were detected in a fraction enriched in hypertrophic parotid acinar cells. These cells, characterized by an increased content of cytoplasmic granules, maintain a secretory responsiveness to isoproterenol, which has been evidenced by light microscopy, enzymatic analysis, and unidimensional SDS‐polyacrylamide gel electrophoresis. Thus, a parallelism in the loss and recovery of both secretory granules, α‐amylase and polypeptides C, D, E, F, and G, was observed. Moreover, after secretion stimulation, polypeptides C, D, E, F, and G were detected in the fluid collected directly from parotid gland cannulation. Given the secretory character of polypeptides C, D, E, F, and G, mechanisms explaining both their progressive accumulation along the chronic administration of isoproterenol, as well as their progressive disappearance observed after suspending that treatment, are discussed.
Regulation of differentiation and keratin protein expression by vitamin A in primary cultures of hamster tracheal epithelial cells
Authors: Edmondson Susan W. , Wu Reen , Mossman Brooke T.
Published: February 2005

Hamster tracheal epithelial (HTE) cells maintained in primary culture show the induction of specific keratin species under vitamin A‐deficient conditions. A comparison was made between the morphology and the expression of keratins in HTE cells in vivo and in primary culture with and without vitamin A. HTE cells cultured in serum‐free, vitamin A‐supplemented medium formed a simple cuboidal, ciliated monolayer and produced four simple epithelial keratins (7, 8,18, and 19). In contrast, vitamin A‐deficient HTE cells, which were squamous‐like and stratified in culture, produced a more complex keratin pattern, with the induction of four additional keratin species (5,6, 14, and 17). A keratin pair whose expression serves as a marker of stratified epithelia was induced, as well as a single keratin species unique to lesions of squamous metaplasia in vitamin A‐deficient hamster tracheal organ cultures. Thus it appears that HTE cells retain the ability to respond to a deficiency in vitamin A through squamous differentiation and increased keratin production when removed from the intact organ and maintained in primary culture in a chemically defined medium. This system may be useful for the study of mechanisms underlying the squamous differentiation of respiratory epithelial cells in the development of bronchogenic tumors.
Distinct pathways regulate transforming growth factor β1‐stimulated proto‐oncogene and extracellular matrix gene expression
Authors: Howe Philip H. , Cunningham Muriel R. , Leof Edward B.
Published: February 2005

The effect of pertussis toxin (PT) on transforming growth factor β1 (TGFβl)‐induced proto‐oncogene expression was investigated in AKR‐2B fibroblasts. PT substantially abolished c‐sis and c‐myc mRNA expression following TGFβl stimulation. This inhibitory effect was specific for TGFβ1‐stimulated proto‐oncogene expression and associated with the ADP‐ribosylation of a 41‐kDa substrate. Actinomycin D decay and nuclear run‐on experiments demonstrated that the inhibitory effects of PT are a result of decreased transcriptional activation and not to an increased decay of proto‐oncogene message. PT did not, however, affect TGFβl‐stimulated fibronectin and collagen mRNA accumulation nor did it have any inhibitory effect on TGFβl‐induced morphological transformation. These data indicate that TGFβl‐stimulated gene expression is coupled to multiple pathways distinguished by their sensitivity to PT.