bluuebunny/crossref_metadata_2025 huggingface dataset:
Exosome formation during maturation of mammalian and avian reticulocytes: Evidence that exosome release is a major route for externalization of obsolete membrane proteins
Authors: Johnstone R. M. , Mathew A. , Mason A. B. , Teng K.
Published: February 2005

We have assessed whether exosome formation is a significant route for loss of plasma membrane functions during sheep reticulocyte maturation in vitro.

Although the recovery of transferrin binding activity in exosomes is at best ∼25–30% of the lost activity, recoveries of over 50% of the lost receptor can be obtained if 125I‐labelled transferrin receptor is measured using an immunological approach. Degradation products of the transferrin receptor in the medium suggest that receptor instability may contribute to the less than quantitative recovery of the transferrin receptor. Significantly higher (75–80%) levels of the nucleoside transporter can be recovered in exosomes during red cell maturation using 3H‐nitrobenzylthioinosine binding to measure the nucleoside transporter.

These data suggest that exosome formation is a major route for removal of plasma membrane proteins during reticulocyte maturation and plasma membrane remodelling.

We have also shown that both in vivo and in vitro, embryonic chicken reticulocytes form exosomes which contain the transferrin receptor. Thus, exosome formation is not restricted to mammalian red cells, but also occurs in red cells, which retain organelles, such as nuclei and mitochondria, into the mature red cell stage.
Insulin‐induced mitogenesis associated with transformation by the SV40 large T antigen
Authors: Wang Hanlin , Scott Robert E.
Published: February 2005

Simian virus 40 (SV40) large T antigen‐transformed cells typically show a markedly reduced serum requirement for growth and the inability to growth arrest and differentiate. An SV40 large T antigen‐transformed 3T3 T cell line, CSV3‐1, that can growth arrest and differentiate into adipocytes with high efficiency has, however, recently been described (Scott et al: Proc. Natl. Acad. Sci. U.S.A. 86: 1652–1656, 1989; Estervig et al: J. Virol. 63: 2718–2725, 1989; J. Cell. Physiol. 142:552–558, 1990). The results of the current studies using these cells show that whereas quiescent 3T3 T cells show no mitogenic response to insulin, quiescent CSV3‐1 cells show a highly significant insulin‐induced mitogenic responsiveness in the absence of other added growth factors. Maximum mito‐genesis was observed at an insulin concentration of 1 μg/ml, which induced 40–70% of the cells to undergo DNA synthesis within 48 hours. The half maximum response was achieved with 1–10 ng/ml of insulin. Insulin's mitogenic effect on CSV3‐1 cells was evident under several different culture conditions that induce quiescence and was not mediated by any detectable autocrine growth factors that might make CSV3‐1 cells competent to respond to insulin. In CSV3‐1 cells insulin appears to act on its own receptor rather than on the IGF‐1 receptor, because at comparable dosages IGF‐1 is 10‐to 100‐fold less effective than insulin. Insulin also is shown to be a mitogen for another SV40‐transformed cell line, CSV3–35, which can be growth arrested; in contrast insulin has no mitogenic effect on two control cell lines that are stably transfected with pSV2neo, a plasmid containing SV40 early promoter/enhancer but lacking large T antigen gene. These results suggest a significant relationship between SV40 T antigen‐associated transformation and the expression of mitogenic responsiveness to insulin.
Regulatory mechanisms for thrombomodulin expression in human umbilical vein endothelial cells in vitro
Authors: Hirokawa Kazunori , Aoki Nobuo
Published: February 2005

It has been reported that thrombomodulin (TM) expression in endothelial cells is modulated by various agents. We investigated cellular regulatory mechanisms for TM expression in human umbilical vein endothelial cells (HUVECs), incubated with agents, by measuring the time course changes in surface TM activity, total TM antigen in cell lysates, and TM mRNA levels. While dibutyryl cAMP (3 mM) increased TM mRNA levels in HUVECs and was followed by increased TM activity, dibutyryl cGMP had no effect on TM activity. Phorbol myristate acetate (PMA) induced rapid loss of surface TM activity (∼8 h) and later increased TM mRNA levels between 4 h and 40 h (maximum at 24 h), resulting in biphasic effects on TM activity. Tumor necrosis factor or interleukin‐1β suppressed surface TM activity and TM mRNA levels. Internalization/degradation of TM in HUVECs incubated with PMA or cytokines was suggested by co‐culture with chloroquine. The decrease in surface TM activity observed was not caused by the release of TM molecules from the cells into the conditioned media. These results suggest that TM activity in HUVECs is modulated by independent mechanisms involving cytoplasmic TM mRNA levels and internalization/degradation of TM molecules. These regulatory mechanisms may involve protein kinase A and protein kinase C‐dependent mechanisms but are independent of protein kinase G.
Altered cellular responses to serum mitogens, including platelet‐derived growth factor, in cultured smooth muscle cells derived from arteries of patients with moyamoya disease
Authors: Aoyagi Masaru , Fukai Naomi , Sakamoto Hiroshi , Shinkai Tadashi , Matsushima Yoshiharu , Yamamoto Mari , Yamamoto Kiyotaka
Published: February 2005

Progressive stenosis or occlusion of bi ateral internal carotid arteries by fibrocellular intimal thickening results in cerebral ischemia in moyamoya disease. The etiology is unknown. We examined cultured arterial smooth muscle cells (SMC) from scalp arteries of five patients with moyamoya disease. In this study we investigated the responsiveness of the cells in culture to serum mitogens including platelet‐derived growth factor (PDGF), a major mitogen of SMC, and compared the response to that of cells derived from age‐matched control patients. SMC from patients with moyamoya disease proliferated less rapidly in a medium with 15% serum than did control SMC and responded poorly to the addition of PDGF to 5% serum. PDGF alone did not stimulate SMC in a quiescent state to initiate DNA synthesis in moyamoya disease, without serum factors other than bovine serum albumin, though it significantly stimulated the controls. Simultaneous additions of epidermal growth factor, insulin‐like growth factor‐I, and PDGF stimulated initiation of DNA synthesis in cells from moyamoya disease, but not as much as PDGF alone did in the controls. Although direct correlations with the pathogenesis of the disease remain to be clarified, the results indicate altered interrelations between serum factors and the cellular responses in vessels of moyamoya disease.
Genetic characterization of adenovirus transformed cell revertants resistant to methylglyoxal bis(guanylhydrazone): Evidence for the involvement of three genetic loci
Authors: Alami Nezha , Chopra Arvind , Thirion Jean‐Paul
Published: February 2005

Five new independent rat somatic cell mutants resistant to the antileukemic drug methylglyoxal bis(guanylhydrazone) (MGBG) were isolated after mutagen treatment. The mutants were 7‐ to 10‐fold more resistant to MGBG than were the parental wild‐type cells. When the MGBG‐resistant (MGR) mutants were exposed to the drug in the presence of Tween‐80, a nonionic detergent, they became as sensitive (MGS) to MGBG as the wild‐type cells, indicating that they were probably permeability mutants. Genetic analysis of hybrids between MGR mutants and wild‐type cells showed the MGR and the nontransformed alleles to be recessive to the MGS (wild‐type) and transformed phenotype, respectively. Complementation analysis of the seven mutants revealed three functional genetic units or loci responsible not only for the MGR phenotype but also for tumorigenicity as determined in nude mice. Only the MGS hybrids produced tumors in the nude mice, whereas the MGR hybrids and mutants did not. Our results suggest the existence of cellular membrane components that are responsible both for cellular tumorigenicity and resistance to MGBG.
Altered levels of phosphoinositide metabolites and activation of guanine‐nucleotide dependent phospholipase C in rat hepatic tumors
Authors: Lalwani Narendra D. , Hylemon Philip B. , Strom Stephen C.
Published: February 2005

The metabolism of phosphatidylinositol was studied in normal quiescent hepa‐tocytes, hepatocellular carcinomas induced by single dose of diethylnitrosamine, followed by 2‐acetylaminofluorene and partial hepatectomy (Solt‐Farber model), and in an established hepatoma cell line, JB1. The JB1 hepatoma cell line and hepatocellular carcinomas demonstrated a 4‐ to 5‐fold higher rate of turnover of [3H]‐inositol and [3H]‐glycerol than the control hepatocytes. Significantly, elevated levels of second messengers inositol 1,4,5‐trisphosphate and sn‐1,2‐diacylglycerol were noted in hepatic tumor cells within 4 hr of labeling with precursor molecules, whereas no detectable level of 3H‐labelled inositol trisphos‐phate was noted in quiescent hepatocytes, even after incubation with 10 mM LiCl for 30 min. Approximately 2.5‐fold higher specific activities of a guanine nucleotide and Ca+2 dependent phosphatidylinositol 4,5‐bisphosphate specific phospholipase C were detected in the hepatocellular carcinoma cells. The cellular location of the phospholipase C activity was also different, being membrane bound in hepatocytes and equally distributed between cytosolic and membrane factions in the hepatomas. These data are consistent with the hypothesis that the enhanced production of diacylglycerol and inositol 1,4,5‐trisphosphate in hepa‐tocellular carcinomas may be due to the activation of a guanine nucleotide dependent phosphatidylinositol 4,5‐bisphosphate specific phospholipase C. These data are the first to compare phosphoinositide turnover in normal liver and hepatic tumor cells and suggest that the sustained levels of second messengers is closely associated with the transformation and enhanced growth rate in hepatic tumor cells.
Insulin and other regulatory factors modulate the growth and the phosphoenolpyruvate carboxykinase (PEPCK) activity of primary rabbit kidney proximal tubule cells in serum free medium
Authors: Wang Yue , Taub Mary
Published: February 2005

Insulin was observed to modulate the growth and the phosphoenolpyruvate carboxykinase(PEPCK) activity of primary cultures of rabbit renal proximal tubule cells in serum free medium. Insulin was stimulatory to primary proximal tubule cell growth at a concentration of 10−8 M. In contrast, insulin was inhibitory to a proximal tubule function, PEPCK activity, following a 5‐minute incubation period. An insulin dosage as low as 10−10 M was inhibitory to PEPCK activity, suggesting the involvement of insulin receptors. Although insulin was required at a significantly higher dosage to stimulate the growth of the primary renal proximal, tubule cells than to inhibit PEPCK activity, the elevated dosage required in order to observe a growth effect may be explained by the degradation of insulin by the primary renal proximal tubule cells. However the possible involvement of receptors for Insulin‐like Growth Factor I (IGF‐I) and Insulin‐like Growth Factor II (IGF‐II) in mediating the effects of insulin cannot be excluded. Other effector molecules were also examined with respect to their effects on PEPCK activity. The possible involvement of cyclic AMP in the control of the PEPCK activity of the primary renal cells was indicated by the stimulatory effects of 8 bromocyclic AMP, isobutyl methylxanthine (a cyclic AMP phosphodiesterase inhibitor), and forskolin (an activator of adenylate cyclase). Phorbol 12‐myristate 13‐acetate (TPA), which activates protein kinase C, was inhibitory. The actions of these effector molecules and insulin on the PEPCK activity of the primary renal cultures are remarkably similar to their effects on hepatic PEPCK. Several growth factors, fibroblast growth factor (FGF), and transforming growth factor beta (TGF beta) were also examined. FGF was observed to be stimulatory, whereas TCF beta was inhibitory to the PEPCK activity of the primary renal proximal tubule cells.
In vitro kinetics of basic fibroblast growth factor diffusion across a reconstituted corneal endothelium
Authors: Dabin Isabelle , Courtois Yves
Published: February 2005

We have studied the ability of bFGF to traverse and be trapped within basement membranes. An extract of EHS tumor (matrigel) coated on culture chamber filters was used as an in vitro model of basement membranes. Our results showed a slow diffusion of bPGF dependent on the amount of low affinity binding sites present within the matrigel. High amounts 01 bFGF and heparin increased the initial rate of diffusion by displacement of bFGF bound to matrigel. An in vitro corneal endothelium model (endothelial cells overlying matrigel) was also developed. This monolayer, with a weak permeability, decreased the kinetic rate of bFGF diffusion compared with matrigel alone. These results indicate that modulation of bFGF distribution in a tissue by a basement membrane is dependent on bFGF concentration, basement membrane composition, permeability of associated cells, and local presence of heparin. This selective control may be a regulating step in bFGF action.
Effects of a putative prostaglandlin E2 antagonist, AH6809, on chondrogenesis in serum‐free cultures of chick limb mesenchyme
Authors: Capehart Anthony A. , Biddulph David M.
Published: February 2005

In the present study, we have examined the effects of a putative antagonist of prostaglandin E2 (PGE2), AH6809, on chondrogenesis in serum‐free cultures of mesenchyme from distal tips of stage 25 chick limb buds in order to test the hypothesis that endogenous PGE2, through receptor‐linked adenylate cyclase (AC), initiates differentiation of cartilage in limb mesenchyme. Daily addition of 10−4 M concentrations of AH6809 produced marked inhibition of chondrogen‐esis over a 5‐day period of cell culture as evaluated by Alcian green binding to cartilage matrix components. Inhibition of chondrogenesis by this compound was further shown to be reversible and treatment of cells with the antagonist limited to periods when chondrocytes had differentiated and were actively secreting cartilage‐specific matrix components had little effect. Preincubation of control cells in 1094 M concentrations of AH6809 inhibited PGE induced activation of AC by greater than 80% without significant (P>.05) inhibition of basal activity by the antagonist. Responses to parathyroid hormone, which increased AC activity by 7‐fold, and forskolin which increased AC activity by 23‐fold in control cells, were also uninhibited by preincubation in AH6809. The results demonstrate that blockade of PGE2‐AC linked receptors in prechondrogenic limb mesenchyme inhibits chondrogenesis supporting the hypothesis that endogenous PGE2 concentrations in undifferertiated limb mesenchyme play aninitiating role in the differentiation of cartilage.
Replacement of media in cell culture alters oxygen toxicity: Possible role of lipid aldehydes and glutathione transferases in oxygen toxicity
Authors: Sullivan S. J. , Roberts R. J. , Spitz D. R.
Published: February 2005

Replacement of media in cell cultures during exposure to hyperoxia was found to alter oxygen toxicity. Following 100 hr of exposure to 95% or 80% O2, the surviving fraction (SF) of Chinese hamster fibroblasts, as assayed by clonogenicity, was less than 1 × 10−3 when the culture media was replaced only at the onset of the O2 exposure. Media replacement every 24 hr throughout the hyperoxic exposure resulted in SFs of 1.7 × 10−1 (95% O2) and 1.9 × 10−1 (80% O2) at 95 hr. Cellular resistance to and metabolism of 4‐hydroxy‐2‐nonenal (4HNE), a cytotoxic byproduct of lipid peroxidation, was examined in cells 24 hr following exposure to 80% O2 for 144 hr with media replacement. These O2‐exposed cells were resistant to 4HNE, requiring 2.6 times as long in 80 μM 4HNE to reach 30% survival as compared to density‐matched normoxia control. Furthermore, during 40 and 60 min of exposure to 4HNE, the O2‐preexposed cells metabolized greater quantities of 4HNE (fmole/cell) relative to control. The activity of glutathione S‐transferase (GST), an enzyme believed to be involved with the detoxification of 4HNE, was significantly increased in the O2‐preexposed cells compared with controls. Catalase activity was significantly increased, but no change was found in total glutathione content, glutathione peroxidase, manganese superoxide dismutase, and copper‐zinc superoxide dismutase activities at the time of 4HNE treatment in the O2‐preexposed cells relative to density‐matched control. The results demonstrate that in vitro tolerance to the cytotoxic effects of hyperoxia can be achieved through media replacement during O2 exposure. Tolerance to oxygen toxicity conferred resistance to the cytotoxic effects of 4HNE, possibly through GST‐catalyzed detoxification. These results provide further support for the hypothesis that toxic aldehydic byproducts of lipid peroxidation contribute to hyperoxic injury.
Amplification of the metallothionein‐1 and metallothionein‐2 genes in copper‐resistant hepatoma cells
Authors: Czaja Mark J. , Weiner Francis R. , Freedman Jonathan H.
Published: February 2005

The molecular basis for increased metallothionein concentrations in copperresistant hepatoma cells was examined. The copper‐resistant cell line HAC600, which is maintained in 600 μm copper, had increased steady‐state mRNA levels for both the metallothionein‐1 (MT‐1) and the metallothionein‐2 (MT‐2) genes. Levels of mRNA were increased 11‐fold for MT‐1 and 15‐fold for MT‐2, with no significant change in α‐tubulin mRNA content. HAC600NM cells, which are copper‐resistant cells kept in a normal copper concentration for over 1 year, also had eight‐ and tenfold increases in MT‐1 and MT‐2 mRNA levels. Nuclear run‐on assays showed that MT‐1 and MT‐2 gene transcription was increased nine‐ and eightfold in HAC600 cells and seven‐ and tenfold in HAC600NM cells, respectively. Southern blot analysis showed amplification of both metallothionein genes in HAC600 and HAC600NM cells. Thus the molecular basis of increased metallothionein in these hepatoma cells involved a stable gene amplification of both MT genes. The greater increase in metallothionein mRNA levels in HAC600 cells relative to the changes in transcription suggests that posttranscriptional mechanisms of gene regulation may also be acting in these cells.
Transforming growth factor type beta 1 (TGF‐β1) down‐regulates interleukin‐2 production and up‐regulates interleukin‐2 receptor expression in a thymoma cell line
Authors: D'Angeac Arnaud Dupuy , Dornand Jacques , Emonds‐Alt Xavier , Jullien Pierre , Garcia‐Sanz Jose A. , Erard François
Published: February 2005

Transforming growth factor type β1 (TGB‐β1) belongs to a family of polypeptides with regulatory effects on growth and differentiation of a variety of cell types. TGB‐β1 plays an important role in regulation of immune response by acting as a negative control signal for T cell proliferation through still unknown mechanisms. In this study we have analysed the effects of TGB‐β1 on EL 4–6.1, a variant of the murine EL 4 thymoma, which can be induced by phorbol 12‐myristate 13‐acetate (PMA) and/or interleukin 1 (IL‐1) to secrete interleukin 2 (IL‐2) and express IL‐2 receptors (IL‐2R). Using this defined model system, we show that TGB‐β1 simultaneously down‐regulates IL‐2 expression and up‐regulates the number of both high and low affinity IL‐2R. These changes correlate with changes at the mRNA level, suggesting an effect at the pre‐translational level. The specificity of both TGF‐β1 effects was demonstrated using a neutralizing antiserum to TGF‐β1. Our data also suggest that TGF‐β1 does not interfere with early activation signals of PMA and/or IL‐1. This model might be useful for elucidating the complex role of TGF‐β1 in the regulation of T cell responses.
Increased proteoglycan synthesis by the cardiovascular system of coarctation hypertensive rats
Authors: Lipke David W. , Couchman John R.
Published: February 2005

Proteoglycan (PG) synthesis in the cardiovascular system of coarctation hypertensive rats was examined by in vivo and in vitro labeling of glycosaminoglycans with 35SO4 in rats made hypertensive for short (4 days) and longer (14 days) durations. With in vivo labeling, only tissues directly exposed to elevated pressure (left ventricle, LV and aorta above the clip, AOR ↑) exhibited elevated PG synthesis after 4 days of hypertension. By 14 days, tissues both exposed to (LV and AOR ↑) and protected from elevated pressure (right ventricle and kidney) exhibited elevated PG synthetic rates. Slight elevations in the proportion of galactosamnoglycans were observed with a concurrent proportional decrease in heparan sulfate PGs. Using the in vitro labeling procedure, no significant increases in PG synthesis were observed in any tissue at either 4 days or 14 days of hypertension. These data indicate that: (1) coarctation hypertension stimulates PG production that is dependent initially on increased pressure and later, on additional non‐pressure related factors, (2) these other factors are responsible for enhanced PG production in tissues not directly exposed to pressure overload, (3) pressure and/or these other factors are essential for enhanced PG production in coarctation hypertension, and (4) synthesis of all GAG types appears to be affected.
Serum factor induces selective increase in Na‐channel expression in cultured skeletal muscle
Authors: Brodie Chaya , Sampson S. R.
Published: February 2005

We have examined effects of horse serum (HS) and various fractions (1 million‐1M, 300K, 100K, and 30K nominal molecular weight limit) obtained by ultrafiltration on expression of TTX‐sensitive Na‐channels and on activities of the Na‐K pump and glucose transport systems in cultured myotubes obtained from 1‐2‐day‐old neonatal rat pups. Five‐day‐old cells were transferred to serum‐free medium with no hormone or growth factor supplements (DMEM) for 24 hr and then treated with the various serum fractions for 48 hr. Measurements were made of specific [3H]‐saxitoxin (STX) binding, action potential properties, 86Rb‐uptake and 2‐deoxyglucose (2‐DG) uptake. HS significantly increased all parameters compared to DMEM (increases in STX‐binding, 69%; Rb‐uptake, 65%; 2‐DG uptake, 93%). Results of treatment with the separate fractions showed that the 300K fraction caused a significantly greater increase in STX‐binding than either HS or the other fractions. In contrast, the increases in Rb and 2‐DG uptakes induced by the different fractions were not different from that obtained with HS. We conclude that serum contains a factor that selectively increases expression of TTX‐sensitive Na‐channels in skeletal muscle.
Kidney‐specific enzyme expression by human kidney cell lines generated through oncogene transfection
Authors: Robinson Polly S. , Goochee Charles F.
Published: February 2005

The human kidney cell line 293 was generated by transfection of adenovirus DNA into normal human embryonic kidney (HEK) cells (Graham et al., 1977), whereas the human kidney cell lines ST‐1i and STt‐4i were generated by transfection of HEK cells with plasmids encoding SV40 viral oncogenes (Abcouwer et al., 1989). In this study, we examined kidney‐specific enzyme activity levels in 293, ST‐1i, and STt‐4i cells to determine their ability to exhibit kidney‐specific gene expression. Enzymes examined were leucine aminopeptidase (LAP), γ‐glutamyl transpeptidase (γ‐GTP), and the disaccharidases trehalase and maltase. Enzymatic activity levels were compared to three other kidney cell lines (MDCK, OK, and LLC‐PK1) as well as to normal human embryonic kidney (HEK) cells and the human hepatoma cell line, Hep G2. Modulation of kidney‐specific enzyme activities was assessed in response to several differentiation‐inducing agents (adenosine, n‐butyric acid, hexamethylene bisacetamide (HMBA), dimethyl sulfoxide (DMSO), N, N′‐dimethylformamide (DMF), isobutyl methyl xanthine (IBMX), di butyryl cAMP, and retinoic acid). ST‐1i and STt‐4i exhibit elevated levels of LAP, γ‐GTP, trehalase, and maltase, consistent with their kidney cell origin, whereas 293 cells exhibit elevated levels of just γ‐GTP and maltase. Maltase and γ‐GTP enzyme activities in ST‐1i and STt‐4i cells were very responsive to the various inducing agents; 293 cells were less responsive at the inducer concentrations examined. None of the three human cell lines formed domes under any of the experimental conditions. In summary, ST‐1i and STt‐4i are comparable to normal HEK cells in expression of kidney‐specific enzymes and in responsiveness to differentiation‐inducing agents, in spite of continued expression of SV40 oncogenes.
Analysis of the c‐myc P2 promoter
Authors: Moberg Kenneth H. , Andrew Tyndall W. , Pyrc Janina , Hall David J.
Published: February 2005

The cis‐acting elements governing transcription from the murine c‐myc P2 promoter have not been well defined. To gain a better understanding of the nature of the protein‐DNA interactions that take place on the P2 promoter, protein binding assays were performed. The ME1a2 and E2F factors appear to be the predominant proteins bound to a region spanning positions −140 to −24 relative to the P2 transcription start site. By a number of criteria, these factors appear to be distinct. When c‐myc promoter sequences were coupled to the chloramphenicol acetyltransferase gene (CAT) and transiently transfected into tissue culture cells it was found that optimal transcription from P2 was heavily dependent on the ME1a2 element.
Characterization of sarcoplasmic reticulum in skinned heart muscle cultures
Authors: Weinstein Leah , Brik Hadassa , Rotmensch Heschi H. , Shainberg Asher
Published: February 2005

The plasma membranes of rat heart muscle, grown in cell culture, were made permeable with saponin in a Ca‐free solution. The cells were then supplied with a medium resembling the cytosol, and the adenosine triphosphate (ATP)‐dependent Ca2+ sequestration was measured in the presence of oxalate. The nonmitochondrial component accounts for about 50% of the total Ca2+ uptake. The nonmitochondrial accumulation of Ca2+ within myocardial cells was found to be reversible by addition of the Ca2+ ionophore A23187. On the other hand, the Ca2+ antagonist D‐600 (50 μM) had almost no effect on Ca2+ accumulation. Caffeine reduced Ca2+ accumulation in the skinned cardiomyocytes in a concentration‐dependent manner. In addition, the anticalmodulin drug trifluo‐perazine (TFP) reduced Ca2+ accumulation in the skinned cells. Because of the analogy between nonmitochondrial ATP‐dependent Ca2+ accumulation and the sarcoplasmic reticulum (SR) function with regard to the influence of various agents, it is assumed that we actually measure Ca2+ accumulation in the SR. The rate of Ca2+ accumulation into the SR measured during the development of the cardiomyocytes in culture shows an almost linear increase as a function of culture age. Amiodarone, a potent antiarrhythmic agent, and its metabolite, desethyl‐amiodarone, inhibited Ca2+ accumulation into SR, which may explain their therapeutic effect.
Relationship of IgE receptor topography to secretion in RBL‐2H3 mast cells
Authors: Seagrave JeanClare , Pfeiffer Janet R. , Wofsy Carla , Oliver Janet M.
Published: February 2005

In RBL‐2H3 rat leukemic mast cells, cross‐linking IgE‐receptor complexes with anti‐lgE antibody leads to degranulation. Receptor cross‐linking also stimulates the redistribution of receptors on the cell surface, a process observed here by labeling the anti‐lgE with 15 nm protein A‐gold particles that are visible by back‐scattered electron imaging in the scanning electron microscope. We report that anti‐lgE binding stimulates the redistribution of IgE‐receptor complexes at 37°C from a dispersed topography to distributions dominated sequentially by short chains, small clusters, and large aggregates of cross‐linked receptors. Cells incubated with 1 μg/ml anti‐IgE, a concentration that stimulates maximum net secretion, redistribute receptors into chains and small clusters during a 15 min incubation period. At 3 and 10 μg/ml anti‐IgE, net secretion is reduced and the majority of receptors redistribute rapidly into clusters and large aggregates. The addition of Fab fragments with the high anti‐IgE concentrations, to reduce cross‐linking, delays receptor aggregation and enhances secretion. The progression of receptors from small clusters to large aggregates is prevented in cells treated with dihydrocytochalasin B to prevent F‐actin assembly. These results establish that characteristic patterns of receptor topography are correlated with receptor activity. In particular, they link the formation of large receptor aggregates to reduced signalling activity. Cytoskeleton‐membrane interaction is implicated in the formation or stabilization of the large receptor clusters.
Retinoic acid inhibits basement membrane protein biosynthesis while stimulating dome formation by madin darby canine kidney cells in hormonally defined serum‐free medium
Authors: Taub Mary
Published: February 2005

The effect of retinoic acid on basement membrane protein biosynthesis and dome formation by Madin Darby canine kidney cells was examined. Retinoic acid inhibited the biosynthesis of laminin, collagen IV, and heparan sulfate proteo‐glycan by confluent MDCK monolayers in a hormonally defined serum‐free medium. Retinoic acid inhibited laminin biosynthesis by as much as 70% after an 8 day incubation period. The inhibitory effect of retinoic acid on laminin biosynthesis preceded temporally the stimulatory effect of retinoic acid on dome formation. This observation is consistent with the existence of a causal relationship between these two phenomena. Not only did retinoic acid inhibit the biosynthesis of laminin, but in addition the biosynthesis of the cellular 67 kd laminin binding protein was inhibited.
Mechanism of control of trophoblast invasion in situ
Authors: Graham Charles H. , Lala Peeyush K.
Published: February 2005

We have previously shown that first trimester human trophoblast cells share in vitro invasive properties with malignant cells. In this study we show that the in situ control of trophoblast invasion is provided by the uterine microenvironment. Trophoblast cells were labeled with 125l‐deoxyuridine and examined for their ability to invade an epithelium‐free human amniotic membrane in vitro under various conditions. The degree of invasion was determined as the percentage of the radioactivity retained within the membrane. Conditioned media from first trimester human decidual cells (DCM) suppressed invasion of trophoblast cells in the amnion invasion assay. This suppression was prevented by addition of neutralizing anti‐TGF β antibody or neutralizing antibody to tissue inhibitor of metalloproteinases (TIMP‐1) to the DCM, and mimicked by TGFβ,. These antibodies also augmented invasion beyond control levels, suggesting that trophoblast cells may also produce these factors. A bioassay for TGFβ activity, measured by antiproliferative effect on the mink lung epithelial cell line Mv 1 Lu, revealed that decidual cells produced this factor only in the latent form, whereas the active form was produced by the trophoblast. A decrease in collagenase type IV activity in the conditioned media of trophoblast cultures was observed when TGFβ was added to these cultures. Removal of endogenous TGFβ in trophoblast cultures by addition of anti‐TGFβ antibody resulted in down‐regulation of TIMP message as determined by Northern analysis. These results indicate that (a) decidua‐derived (and to a minor extent trophoblast‐derived) TGFβ is the prime mediator in the control of invasion by first trimester trophoblast, the latent form of TGFβlikely being activated by trophoblast‐derived proteinases; (b) induction of TIMPby TGFβ in both trophoblast and decidua is the final pathway in this control.
Heat shock induces two distinct S6 protein kinase activities in quiescent mammalian fibroblasts
Authors: Jurivich Donald A. , Chung Jongkyeong , Blenis John
Published: February 2005

The regulation of S6 kinase activity was used to monitor perturbations of intracellular signaling activity during heat shock of quiescent murine and human fibroblasts. Previous reports on exponentially growing insect and plant cells had indicated that 40S ribosomal protein S6 is dephosphorylated during heat shock; thus inhibition of S6 kinase activity by heat shock was anticipated in NIH 3T3 fibroblasts and human cells (HeLa, diploid embryonic fibroblasts MRC‐5, and skin‐derived fibroblasts). Unexpectedly, two distinct S6 protein kinases were activated in quiescent fibroblasts after heat exposure. One of the enzymes was partially purified by sequential column chromatography and was determined to be equivalent to the enzyme activated by serum and other growth factors, referred to here as pp70‐S6 protein kinase. The other protein S6 kinase, pp90rsk, was identified by a specific immunoprecipitation assay. Monitoring both enzymatic activities during heat shock revealed a temporal pattern of activation that was reversed when compared to non‐stressed, mitogen‐stimulated cells. Finally, heat shock stimulated protein S6 phosphorylation in cultured, quiescent mammalian cells. These data demonstrate that specific protein kinases can be activated during heat shock, and that some early mitogenic signals may also participate in the response of cells to physiologic stress.
Apoptosis of murine BW 5147 thymoma cells induced by dexamethasone and γ‐irradiation
Authors: Kruman Inna I. , Matylevich Natalia P. , Beletsky Igor P. , Afanasyev Vladimir N. , Umansky Samuil R.
Published: February 2005

The mode and the kinetics of the death of T‐thymoma cells upon dexamethasone treatment and γ‐irradiation (10Gy) have been studied using flow cytometry and biochemical analysis. It has been shown that the hormone and γ‐irradiation induce cell death by apoptosis. In both cases the cells are initially blocked in G2/M and die only after overcoming the blockage and cytokinesis. A short exposure to dexamethasone results in a cytostatic effect, whereas a cytotoxic effect is absent. Reducing serum concentration to 2% causes more rapid death both following γ‐irradiation and dexamethasone. These results are discussed in relation to cell death and proliferation.
Secretion and biological actions of insulin‐like growth factor binding proteins in two human tumor‐derived cell lines in vitro
Authors: Camacho‐Hubner Cecilia , McCusker Robert H. , Clemmons David R.
Published: February 2005

The insulin‐like growth factors (IGFs) I and II are present in extracellular fluids associated with specific binding proteins (IGFBPs) that can modify their biologic actions. These studies were undertaken to determine which forms of IGFBP are secreted by endometrial carcinoma (HEC‐1B) and breast carcinoma (MDA‐231) cells, to characterize variables that control IGFBP secretion, and to study the effect of IGFBP‐1 and IGFBP‐2 on IGF‐I stimulated cell proliferation. Secreted IGFBPs were identified by ligand blotting and IGFBP‐1 was quantified using a specific radioimmunoassay (RIA). MDA‐231 cell conditioned media (CM) contained four (43,000, 39,000, 30,000 and 24,000 Mr) forms of IGFBP, and HEC‐1B cell CM contained three forms (39,000, 34,000 and 30,000 Mr). Immunoblotting showed that the 30,000 Mr form secreted by both cell types was IGFBP‐1. Likewise the 34,000 Mr band in HEC‐1B media reacted with IGFBP‐2 antiserum and the 39,000 and 43,000 Mr bands reacted with IGFBP‐3 antiserum.

IGF‐I stimulated the secretion of IGFBP‐3 from both cell types and IGFBP‐2 from HEC‐1B cells but either decreased or caused no change in secretion of IGFBP‐1 and a 24,000 Mr form. In contrast, insulin inhibited the secretion of IGFBP‐1 but increased the secretion of the 24,000 Mr form. Compounds that elevate intracellular cAMP levels increased the secretion of IGFBP‐3, IGFBP‐1, and the 24,000 Mr form from both MDA‐231 and HEC‐1B cells.

When sparse cultures of MDA‐231 cells were used, addition of IGF‐I caused a 24% increase in cell number after 48 hr. This mitogenic response was enhanced by the presence of recombinant human IGFBP‐1 (45% increase in cell number, P < 0.001). Bovine IGFBP‐2 did not potentiate IGF‐I stimulated cell proliferation.

These findings show that two tumor cell lines secrete distinct forms of IGFBPs and that there is differential regulation of IGFBP secretion. At least one form secreted by both tumors may act as a positive autocrine modulator of IGF‐l's growth stimulating actions.
Effect of trifluoperazine on renal epitheloid madin‐darby canine kidney cells
Authors: Paulmichl M. , Wöll E. , Weiss H. , Waldegger S. , Lang F.
Published: February 2005

Following exposure to a number of hormones, the cell membrane in Madin‐Darby Canine Kidney (MDCK) cells is hyperpolarized by increase of intracellular calcium activity. The present study has been performed to elucidate the possible role of calmodulin in the regulation of intracellular calcium activity and cell membrane potential. To this end trifluoperazine has been added during continuous recording of cell membrane potential or intracellular calcium. Trifluoperazine leads to a transient increase of intracellular calcium as well as a sustained hyperpolarization of the cell membrane by activation of calcium sensitive K+ channels. Half‐maximal effects are observed between 1 and 10 μmol/L trifluoperazine. A further calmodulin antagonist, chlorpromazine, (50 μmol/L), similarly hyperpolarizes the cell membrane. The effects of trifluoperazine are virtually abolished in the absence of extracellular calcium. Pretreatment of the cells with either pertussis toxin or phorbolester TPA does not interfere with the hyperpolarizing effect of trifluoperazine. In conclusion, calmodulin is apparently involved in the regulation of calcium transfer across the cell membrane but not in the stimulation of K+ channels by intracellular calcium.
Enhanced inhibition of tissue factor by the extended form of an endothelial cell glycoprotein (an extrinsic pathway inhibitor)
Authors: Colburn P. , Crabb J. W. , Buonassisi V.
Published: February 2005

We have identified, in the supernatant medium of cultured endothelial cells, an additional inhibitor of tissue factor that is eluted at higher salt concentrations during heparin‐Sepharose chromatography and is a much more potent inhibitor of the activation of the coagulation cascade than the species we studied earlier (Colburn and Buonassisi: In Vitro Cellular and Developmental Biology 24:1133‐1136, 1988). The inhibitor we described earlier has been shown to be functionally and immunologically identical to a rabbit plasma extrinsic pathway inhibitor, EPI (Warn‐Cramer et al.: Thrombosis Research 61:515–527, 1991). The new species (molecular mass, 47 kDa) is susceptible to proteolytic cleavage which results in a sharp reduction of its ability to inhibit tissue factor and an increase in electrophoretic mobility compatible with a molecular mass of 45 kDa, the same as that of the inhibitor reported earlier. Based on these data, we suggest that the new inhibitor yields, through proteolytic cleavage of an amino acid sequence of about 25 residues, the N‐glycansulfated compound previously described.
Differential expression of two classes of lck transcripts upon phorbol ester treatment of human leukemic T cells
Authors: Leung Stewart , Miyamoto Neil G.
Published: February 2005

The human lymphocyte‐specific tyrosine kinase gene, Ick, is transcribed from two distinct promoters, resulting in two classes of transcripts (type I and II) differing in their 5′ untranslated regions. The steady‐state levels of the type I and II Ick transcripts were measured in a variety of lymphoid and non‐lymphoid human tumor cell lines by S1 nuclease mapping and by a sensitive assay system using the polymerase chain reaction. Human thymocytes and all the leukemic T cell lines tested express both type I and II Ick transcripts, albeit at different relative levels. Peripheral blood T cells express mainly type II Ick transcripts, whereas two colonic carcinoma lines, COLO 201 and COLO 205, express exclusively type I Ick transcripts. Treatment of the leukemic T cell lines, P30/OKUBO and Jurkat, by the phorbol esters tetradecanoylphorbol acetate (TPA) or phorbol dibutyrate (PDB) results in the down‐regulation of the type I, and the up‐regulation of the type II, Ick transcript levels. The effect of PDB on the in vitro differentiation of Jurkat cells, and the expression of Ick transcripts, is reversible. The modulation of Ick transcript levels in TPA‐treated Jurkat cells is not due to differential RNA stability, suggesting that the two Ick promoters are utilized differentially during T cell differentiation. The leukemic T cell line, Jurkat, may thus serve as a model for the elucidation of molecular mechanisms that regulate Ick transcription and T cell differentiation.
Murine mast cell colony formation supported by IL‐3, IL‐4, and recombinant rat stem cell factor, ligand for c‐kit
Authors: Tsuji Kohichiro , Zsebo Krisztina M. , Ogawa Makio
Published: February 2005

Recently, a novel cytokine designated stem cell factor (SCF) was isolated from medium conditioned by buffalo rat liver cells and proved to be the ligand for c‐kit. We have examined the effects of recombinant rat SCF alone and in various combinations with interleukin‐3 and interleukin‐4 on murine mast cell colony formation in methylcellulose culture. As a source of connective tissue‐type mast cells (CTMC), we used peritoneal mast cells. No individual factor supported colony formation by purified peritoneal mast cells. When cells were grown in combinations of two factors, significant mast cell colony growth was seen. When cells were grown in the presence of three factors, not only the number of colonies was increased but also the colonies were larger. Mast cells in these colonies contained safranin‐ and berberine sulfate‐positive cells, but the proportions of positive and negative cells varied depending on the factor combinations. We then examined the effects of these factors on proliferation of bone marrow‐derived mast cells (BMMC) by replating pooled mast cell colonies. As a single factor, only interleukin‐3 supported mast cell colony formation. Combinations of two of the three factors supported mast cell colony formation. However, the most impressive synergism was seen again with the combination of the three factors. Not only was the number of colonies increased, but there was a significant increase in size. These results indicate that SCF is an important factor for the proliferation of both CTMC and BMMC.
Enhancement by transforming growth factor‐β 1 (TGF‐β 1) of the proliferation of leukemic blast progenitors stimulated with IL‐3
Authors: Suzuki Toshiya , Bessho Masami , Hirashima Kunitake , Tohda Shuji , Nagata Kaoru , Morio Tomohiro , Imai Yasufumi , Nara Nobuo
Published: February 2005

We studied the effect of transforming growth factor‐beta 1(TGF‐β 1) on colony formation of leukemic blast progenitors from ten acute myeloblastic leukemia (AML) patients stimulated with granulocyte colony‐stimulating factor (G‐CSF), granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), interleukin‐3 (IL‐3), interleukin‐6 (IL‐6), or interleukin‐1β (IL‐1β). These CSFs and interleukins by themselves stimulated the proliferation of leukemic blast progenitors without adding TGF‐β 1. G‐CSF, GM‐CSF, and IL‐3 stimulated blast colony formation in nine patients, IL‐6 stimulated it in five, and IL‐1β stimulated in four. TGF‐β 1 significantly reduced blast colony formation stimulated by G‐CSF, GM‐CSF, or IL‐6 in all patients. In contrast, TGF‐β 1 enhanced the stimulatory effect of IL‐3 on blast progenitors from three cases, while in the other seven patients TGF‐β 1 reduced blast colony formation in the presence of IL‐3. To study the mechanism by which TGF‐β 1 enhanced the stimulatory effect of IL‐3 on blast progenitors, we carried out the following experiments in the three patients in which it occurred. First, the media conditioned by leukemic cells in the presence of TGF‐β 1 stimulated the growth of leukemic blast progenitors, but such effect was completely abolished by anti‐IL‐1 β antibody. Second, the addition of IL‐1 β in the culture significantly enhanced the growth of blast progenitors stimulated with IL‐3. Third, leukemic cells of the two patients studied were revealed to secrete IL‐1 β and tumor necrosis factor‐α (TNF‐α) constitutively; the production by leukemic cells of IL‐1 β and TNF‐α was significantly promoted by TGF‐β 1. Furthermore, the growth enhancing effect of TGF‐β 1 in the presence of IL‐3 was fully neutralized by anti‐IL‐1 β antibody. These findings suggest that TGF‐β 1 stimulated the growth of blast progenitors through the production and secretion of IL‐1 β by leukemic cells.
Differentiation induction of blast cells in two cases of childhood acute megakaryoblastic leukemia in vitro by interleukin‐3 and interleukin‐6: An ultrastructural cytochemical study
Authors: Miyauchi Jun , Sugita Kiyoko , Okui Masahito , Taguchi Nobuyuki , Clark Steven C. , Shimizu Koichi
Published: February 2005

Although hematopoietic growth factors influence renewal and differentiation of blast progenitors in acute myelogenous leukemia (AML), morphological maturation of leukemic blasts is thought a rare event, even when cultured in the presence of appropriate growth stimulants. However, light microscopic observation may not be sufficient to clarify precisely the effects of hematopoietic growth factors on the morphological differentiation of leukemic blasts. In this study, using cell culture techniques and electron microscopic cytochemistry for platelet peroxidase (PPO), we studied the effects of interleukin‐3 (IL‐3) and interleukin‐6 (IL‐6), both of which are considered to play an important role in normal megakaryocytopoie‐sis, on the growth and differentiation of blast cells from two patients with childhood acute megakaryoblastic leukemia (AMKL). In both of the two cases, IL‐3 stimulated leukemic colony formation in methylcellulose culture, whereas IL‐6 showed little such activity. However, in suspension culture, IL‐6 was active in promoting megakaryocytic differentiation, although incomplete, as detected by increase in the number of PPO‐positive cells, some having demarcation membrane‐like structure. This effect was evident in culture with IL‐6 alone in one patient, but it was detectable only when IL‐6 was used in combination with IL‐3 in the other patient. In contrast, IL‐3 alone stimulated differentiation towards myeloid but not megakaryocytic lineage. These results indicate that IL‐3 and IL‐6 have a distinct role in leukemic megakaryocytopoiesis (IL‐3 stimulates growth, whereas IL‐6 promotes morphological differentiation) and that cooperation between these two cytokines functions most effectively for megakaryocytic differentiation of AMKL cells in a fashion similar to that for normal megakaryo‐cytopoiesis.