bluuebunny/crossref_metadata_2025 huggingface dataset:
Continuously applied compressive pressure induces bone resorption by a mechanism involving prostaglandin E2 synthesis
Authors: Imamura Kazunobu , Ozawa Hiroyuki , Hiraide Takatoshi , Shibasaki Yoshinobu , Fukuhara Tatsuo , Takahashi Naoyuki , Suda Tatsuo
Published: February 2005

In previous research, we devised a specific culture chamber to examine the effect of continuously applied compressive pressure (CCP) on bone formation and resorption. The chamber was infused with compressed mixed gases with different O2 and CO2 composition to maintain the pO2, pCO2, and pH in the culture medium under pressures of +0.5 atm (1.5 atm total) to +2.0 atm (3.0 atm total) at the same levels as those at the ordinary pressure (1 atm). Using the specific culture chamber, we demonstrated that CCP greatly suppressed the differentiation of mouse osteoblast‐like MC3T3‐E1 cells. The inhibition by CCP appeared to be mediated by prostaglandin E2 (PGE2). In the present study, we examined the effect of CCP on osteoclastic bone resorption. CCP treatment of mouse bone marrow culture markedly increased both the PGE2 production and the number of tartrateresistant acid phosphatase (TRACP)‐positive mononuclear cells (possibly precursors of multinucleated osteoclasts). An autoradiographic study using [125l]‐salmon calcitonin showed clearly that those TRACP‐positive cells had calcitonin receptors. The CCP effect was the greatest at + 1.0 atm (2.0 atm total). Isobutylmethylxanthine potentiated the production of TRACP‐positive cells induced by CCP. Adding indomethacin completely inhibited both the TRACP‐positive cell formation and the PGE2 production induced by CCP. CCP also increased the release of 45Ca from prelabeled mouse calvaria during later stages (2–6 days) of the 6‐day culture period. CCP markedly increased PGE2 but not interleukin 1 in the culture media of mouse calvaria. These results indicate that, besides inhibiting osteoblast differentiation, CCP stimulates bone resorption by generating new osteoclasts through a mechanism involving PGE2 production.
Retinyl acetate inhibits platelet‐derived growth factor‐induced Ca2+ signals in C3H 10T1/2 fibroblasts
Authors: Hill Timothy D. , Boynton Alton L. , Dean Nicholas M. , Mordan Lawrence J.
Published: February 2005

Mitogenic stimulation of density‐arrested C3H 10T1/2 mouse fibroblasts by serum or purified platelet‐derived growth factor (PDGF) was potently inhibited by retinyl acetate (RAc; IC50 = 0.1 μg/ml, 0.3 × 10−6 M) when administered during the frist 2 hours of mitogen exposure. This inhibitory effect of RAc coincided with a period early in the cell growth‐division cycle when density‐arrested C3H 10T1/2 cells stimulated by PDGF were found to require Physiological levels of extracellular Ca2+ for the transition from G0 to G1 of the cell cycle. To determine if the inhibitory effect of RAc was mediated through alterations in the Ca2+ signaling pathway induced by mitogens, we examined Fura‐2‐loaded fibroblasts for changes in the Ca2+ response elicited by PDGF. Addition of PDGF (5 ng/ml) induced a transient increase in the [Ca2+]i that was not significantly effected by the extracellular Ca2+ concentration. Treatment of cells with RAc caused a concentration‐ and time‐dependent inhibition of this PDGF‐stimulated Ca2+ flux (IC50 = 0.45 μg/ml or 1.5 × 10−6 M; t1/2 = 15 min), whereas release of intracellularly stored Ca2+ by thrombin was unaffected by RAc (1.2 μg/ml, 4 × 10−6 M). Treatment with RAc did not significantly affect PDGF binding to cell surface receptors or the generation of inositol phosphates. These results suggest that the mechanism by which RAc inhibits PDGF‐ or serum‐induced mitogenesis is through modulation of the Ca2+ signal stimulated by PDGF, and thereby depriving the cell of a rise in intracellular Ca2+ necessary for progression through the cell cycle.
Patterns of spectrin transcripts in erythroid and non‐erythroid cells
Authors: Prchal Josef T. , Yoon Sa‐Hyun , Papayannopoulou Thalia
Published: February 2005

Spectrin is the major protein of the membrane erythrocyte skeleton. More recently, homologous but non‐identical spectrins (fodrins) were also found in various non‐erythroid tissues. Spectrin mRNA in erythroid and various non‐erythroid cells was examined by direct hybridization with human α‐spectrin, β‐spectrin (erythroid spectrins), and α‐fodrin (non‐erythroid spectrin) cDNA probes. Northern blot analysis of poly (A)+ RNA revealed a distinct pattern of expression in erythroid vs. non‐erythroid cells. Erythroid cells from early erythroblasts to reticulocyte stage expressed two mRNA species of β‐spectrin, whereas they expressed only a single speeies of α‐spectrin, and no α‐fodrin mRNA. In contrast, non‐erythroid cells (platelets, myeloid cells, liver, muscle, heart, cerebellum, and eye lens) expressed either no α‐spectrin mRNA or a different molecular weight transcript(s) of this gene, and a single species of β‐fodrin mRNA. Additionally, they also expressed from none to multiple species of β‐spectrin, and these were of different molecular size(s) from that found in erythroid cells (with the exception of platelets). Transcripts of non‐erythroid spectrin, α‐fodrin, were found as a single copy only in non‐erythroid tissues. Human and murine erythroleukemia cells expressed both erythroid spectrin transcripts in addition to α‐fodrin and raise the possibility that erythroid progenitors may have the potential to express both erythroid and non‐erythroid species. These data indicated that several mRNA species of β‐spectrin could be detected in both erythroid and some non‐erythroid cells. Whether multiple spectrin peptides could also be found with functional heterogneity is unclear. However, in each case, the pattern combination observed appeared to be tissue‐specific.
Evidence for probenecid‐sensitive organic anion transporters on polarized thyroid cells in culture
Authors: Géarard Corinne , Boudier Jeanne‐Andrée , Mauchamp Jean , Verrier Bernard
Published: February 2005

Epithelial thyroid cells in primary cultures loaded with BCECF/AM rapidly released the impermeant fluorescent dye BCECF (bis(carboxyethyl)carboxyfluorescein) in the incubation medium. Cells organized into follicles rapidly cleared BCECF (80% within 10 min) whereas fluorescence microscopy did not show any fluorescence in the follicular cavity. Cells organized into monolayers on plastic exported BCECF into the medium (70% within 40 min) whereas fluorescence microscopy showed intense fluorescence under the domes. BCECF efflux was blocked by probenecid, one of the known inhibitors of organic anion transporters, with similar efficiency in both structures. Maximal and half‐maximal effects were respectively observed for 5 mM and 0.4 mM probenecid. The polarity of BCECF efflux was studied by using monolayers on collagen‐coated Nuclepore filters: 85% of BCECF released was found in the basal compartment and 15% in the apical compartment. These findings suggested that thyroid cells in culture expressed a transport mechanism for the anionic form of BCECF. Furthermore, the observed activation of the Na+/H+ exchanger by probenecid suggested that the presence of this blocker did not overcome problems arising in the use of BCECF as intracellular pH indicator for thyroid cells.
Effect of histidine on histidinol‐induced heat protection in chinese hamster ovary cells
Authors: Lee Yong J. , Kim Dooha , Corry Peter M.
Published: February 2005

The possible mechanism for heat protection by the protein synthesis inhibitor histidinol was investigated in CHO cells. Histidinol (HST, 5 mM), an analogue of the essential amino acid L‐histidine, added for 2 hr before and during heating at 43°C, protected cells from killing at 43°C. Treatment with HST produced a 600‐fold increase in survival from 3 × 10−4 to 1.8 × 10−1 after 2.5 hr at 43°C. Although the cells were washed after HST treatment, substantial protective effect was still observed during heating at 43°C. This protective effect gradually decreased with increased incubation time after the drug treatment. However, the protective effect was immediately reduced by treatment with histidine (HIS, 0.25‐5 mM) during heating. The amount of reduction was dependent upon HIS concentration: five millimolar HIS completely inhibited HST‐induced heat protection. Furthermore, protein synthesis which was inhibited by 95% by 5 mM HST, resumed immediately with 5 mM HIS treatment. In addition, when cells were labeled during or after HST treatment, neither preferential accumulation of heat shock protein families nor phosphorylation of 28 kDa protein was observed. Therefore, these results suggest that the cessation of protein synthesis itself is one of the events involved in protection.
Nuclear localization of 68 kDa calmodulin‐binding protein is associated with the onset of DNA replication
Authors: Subramanyam C. , Honn Susan C. , Reed William C. , Reddy G. Prem Veer
Published: February 2005

In Chinese hamster embryo fibroblast cells, an increase in intracellular calmodulin levels coincided with the nuclear localization of a calmodulin‐binding protein of about 68 kDa as the cells progressed from G1 to S phase. When cells were limited from entering into S phase, by omitting insulin a defined medium, intracellular CaM levels did not increase and the 68 kDa calmodulin‐binding protein was completely absent from the nuclei. Corresponding to the nuclear localization of calmodulin and the 68 kDa calmodulin‐binding protein in S phase cells, there was a dramatic increase in DNA polymerase and thymidine kinase activities in the nuclei of S phase cells as compared to G1 phase cells. In addition, the 68 kDa calmodulin‐binding protein along with calmodulin, is observed to be an integral component of replitase complex responsible for nuclear DNA replication in S phase cells. These observations point to the association of calmodulin and calmodulin‐binding protein(s) with the replication machinery responsible for nuclear DNA replication during S phase. A possible regulatory role of these proteins in the onset of DNA replication and cell proliferation is discussed.
Epoxyeicosatrienoic acids activate Na+/H+ exchange and are mitogenic in cultured rat glomerular mesangial cells
Authors: Harris R. C. , Jacobson H. R. , Capdevila J. , Homma T.
Published: February 2005

The present study examined responses of cultured rat glomerular mesangial cells to exogenous exposure of epoxyeicosatrienoic acids (EET's), products of cytochrome P450 epoxygenase. One day after administration of 8, 9‐ or 14, 15‐EET, cultured rat mesangial cells demonstrated significant increases in [3H]thymidine incorporation (10−7 M 14, 15‐EET: 120 ± 7% of control; n = 6; P < 0.025; 10−6 M 14, 15‐EET: 145 ± 10%; n = 20; P < 0.0005; 10−6 M 8,9‐EET: 167 ± 31%; n = 9; P < 0.05), which was not affected by addition of the cyclooxygenase inhibitor indon ethacin. In addition to stimulation of [3H]thymidine incorporation, the epoxides stimulated mesangial cell proliferation. 14, 15‐EET administration induced intracellular alkalinization of 0.2–0.3 pH units, which was prevented by extracellular Na+ removal and blunted by amiloride (0.5 mM). Following intracellular acidification with NH4Cl addition and removal, > 85% of 3 mM 22Na uptake into mesangial cells was inhibited by I mM amiloride, indicating Na+ /H+ exchange. Under these conditions, 14, 15‐EET stimulated Na+ /H+ exchange by 42% and 8, 9‐EET stimulated Na+ /H+ exchange by 59%. Neither protein kinase C depletion nor addition of the protein kinase C inhibitor, staurosporine, affected this stimulation. In [3H]myo‐inositol loaded mesangial cells, no significant stimulation of phosphoinositide hydrolysis was detected in response to administration of 14, 15‐EET.

Twenty‐four hours after addition of [14C] 14, 15‐EET, > 90% was preferentially esterified to cellular lipids, with predominant incorporation into phosphatidylinositol, phosphatidylethanolamine, and diacylglycerol.

Thus, these results demonstrate epoxyeicosatrienoic acids stimulate Na+ /H+ exchange and mitogenesis in mesangial cells. These effects do not appear to be mediated via phospholipase C activation. In addition, 14, 15‐EET was selectively incorporated into cellular lipids known to mediate signal transduction. These observations extend the potential biologic roles of c‐P450 arachidonate metabolites to include stimulation of cell proliferation and suggest a role for these compounds in vascular and renal injury.
Nerve growth factor and fibroblast growth factor influence post‐fusion expression of Na‐channels in cultured rat skeletal muscle
Authors: Brodie Chaya , Sampson S. R.
Published: February 2005

We have examined effects of nerve growth factor (NGF) and fibroblast growth factor (FGF) on the density of tetrodotoxin (TTX)‐ sensitive Na‐channels in cultured rat skeletal muscle. Measurements were made of specific binding of [3H] saxitoxin (STX) and the frequency and rate of rise of spontaneously occurring action potentials, the Physiological expression of Na‐channel density. Cells were transferred to various growth conditions at 6 days in vitro, and measurements were made beginning 24 hr later. Both growth factors (GF) caused dose‐related in creases in Na‐channels compared with myotubes maintained in normal, serum‐supplemented growth medium. Maximum effects occurred with a concentration of NGF of 50 ng/ml and FGF of 15 ng/ml. Scatchard analysis of specific STX binding showed an increase in Bmax with no significant change in Kd. Similar increases occurred on rate of rise and frequency spontaneous action potentials. Treatment of cultures with cycloheximide or actinomycin D, inhibitors of protein and RNA synthesis, completely prevented the increase in STX‐binding induced by GF treatment, The results indicate that NGF and FGF have important effects on regulation of excitable cell gene products after differentiation.
DNA polymerase‐primase complex in wild‐type and ts A1S9 mouse L‐cells, temperature‐sensitive for dna replication during cell cycle progression
Authors: Munsch Nicole , Sheinin Rose
Published: February 2005

Ts A1S9 mutant cells, derived from wild type WT‐4 mouse L‐cells, are temperature‐sensitive (ts) for DNA synthesis and cell division. We try to determine the cause of the arrest of DNA replication in ts A1S9 cells at the nonpermissive temperature by comparing the modifications induced by the shift of temperature on the activity and the synthesis of DNA polymerase‐alpha and DNA primase as a function of time. Forty‐seven hours after temperature upshift DNA polymerase‐alpha activity of ts A1S9 cells was inhibited by 90% while primase activity was barely detectable. By contrast, the activities of both enzymes increased to a plateau level in WT‐4 cultured at either temperature and in ts A1S9 cells grown at the low permissive temperature. Study of the synthesis of DNA polymerase‐alpha primase and of the structure of the enzyme complex during cell cycle progression was approached by immunoprecipitation of [35S]‐labelled cells, with a specific monoclonal antibody directed against DNA polymerase‐alpha. We have found that, irrespective of temperature of cultivation of WT‐4 or ts A1S9 cells, this antibody precipited polypeptides of 220, 186, 150, 110, 68–70, 60, and 48 kDa from cell extracts. With ts A1S9 cells cultivated at 38.5°C for 48 hr the polypeptides of 220 and 186 kDa, associated with alpha‐polymerase activity, were considerably more abundant than in the control cells, with a concomitant decline in the polypeptides of 60 and 48 kDa, implicated in primase activity. Thus the inhibition of DNA polymerase‐alpha cannot be due to a decreased synthesis of the 186 kDa subunit but to its temperature inactivation. Consistent with a recent asymmetric dimeric model where polymerase‐alpha complex and polymerase delta complex synthesize co‐ordinately at the replication fork lagging and leading DNA strands, the observed alterations of polymerase‐alpha and primase content explain the inhibition of DNA synthesis and the cell cycle arrest of the ts A1S9 cells at the nonpermissive temperature.
Modulation of growth and differentiation in normal human keratinocytes by transforming growth factor‐β
Authors: Matsumoto Kunio , Hashimoto Koji , Hashiro Makoto , Yoshimasa Hidenobu , Yoshikawa Kunihiko
Published: February 2005

The effect of transforming growth factor‐type β 1(TGF‐β) on the growth and differentiation of normal human skin keratinocytes cultured in serum‐free medium was investigated. TGF‐β markedly inhibited the growth of keratinocytes at the concentrations >2 ng/ml under low Ca2+ conditions (0.1 mM). Growth inhibition was accompanied by changes in cell functions related to proliferation. Remarkable inhibition of DNA synthesis was demonstrated by the decrease of [3H]thymidine incorporation. The decrease of [3H]thymidine incorporation was observed as early as 3 hr after addition of TGF‐β. TGF‐β also decreased c‐myc messenger RNA (mRNA) expression 30 min after addition of TGF‐β. This rapid reduction of c‐myc mRNA expression by TGF‐β treatment is possibly one of the main factors in the process of TGF‐β‐induced growth inhibition of human keratinocytes. Since growth inhibition and induction of differentiation are closely related in human keratinocytes, the growth‐inhibitory effect of TGF‐β under high Ca2+ conditions (1.8 mM Ca2+ differentiation‐promoting culture environment) was examined. TGF‐β inhibited the growth of keratinocytes under high Ca2+ conditions in the same manner as under low Ca2+ conditions, suggesting that it is a strong growth inhibitor in both low and high Ca2+ environments. The induction of keratinocyte differentiation was evaluated by measuring involucrin expression and cornified envelope formation: TGF‐β at 20 ng/ml increased in‐volucrin expression from 9.3% to 18.8% under high Ca2+ conditions, while it decreased involucrin expression from 7.0% to 3.3% under low Ca2+ conditions. Cornified envelope formation was modulated in a similar way by addition of TGF‐β: TGF‐β at 20 ng/ml decreased cornified envelope formation by 53% under low Ca2+ conditions, while it enhanced cornified envelope formation by 30.7% under high Ca2+ conditions. Thus, the effect of TGF‐β on keratinocyte differentiation is Ca2+ dependent. It enhances differentiation of human keratinocytes under high Ca2+ conditions, but inhibits differentiation under low Ca2+ conditions. Taken together, there is a clear discrepancy between TGF‐β effects on growth inhibition and induction of differentiation in human keratinocytes. These data indicate that growth inhibition of human keratinocytes by TGF‐β is direct and not induced by differentiation.
Evidence for a role of the Na+/H+ exchanger in the colony‐stimulating‐factor‐induced ornithine decarboxylase activity and proliferation of the human cell line M‐07e
Authors: Ghigo Dario , Brizzi Maria F. , Avanzi G. Carlo , Bussolino Federico , Garbarino Giovanni , Costamagna Costanzo , Pegoraro Luigi , Bosia Amalia
Published: February 2005

The subclone M‐07e, derived from the interleukin‐3 (IL‐3)‐responsive human myeloid cell line M‐07, is strictly dependent on either IL‐3 or granulocyte‐macrophage‐colony‐stimulating factor (GM‐CSF) for its growth and survival. This cell line may be regarded as a candidate model to investigate the poorly understood events triggered by growth factors binding to human hemopoietic cells. Both IL‐3 and GM‐CSF induce in M‐07e cells an increase of ornithine decarboxylase (ODC) activity, which reaches its maximum at 24–30 h and fully depends on de novo protein synthesis. The growth factors do not elicit translocation of protein kinase C to the membrane; thus a role of the kinase in ODC induction is ruled out. An amiloride‐inhibitable Na+/H+ exchanger is present in the membrane of M‐07e cells; its apparent Km for extracellular Na+ is 47.77 mM; and its activity is greatly enhanced when the cytoplasm is acidified. Growth‐factor‐evoked ODC activation and DNA synthesis are blocked in a dose‐ and time‐dependent manner when M‐07e cells are incubated with ethylispropyl‐amiloride, a specific inhibitor of Na+/H+ exchanger. The exchanger does not appear to be directly activated by IL‐3 or GM‐CSF, but its operation is strictly required for the biological effects of these growth factors on M‐07e cell line.
Characterization of glutamine transport into resting and concanavalin A‐stimulated peripheral human lymphocytes
Authors: Schröder Marie‐Theres , Schäfer Gertrud , Schauder Peter
Published: February 2005

Characteristics of glutamine transport, its substrate specificity, and its pattern of competitive and non‐competitive inhibition in response to amino acid analogues were determined in peripheral human lymphocytes, incubated with or without concanavalin A (Con A). Maximum capacity of transport (Vmax) at 37°C and 136.9 mM Na+ was 30 pmol/106 cells/30 seconds, while the apparent Km was 142 μM. In cells exposed to 10 mM histidine, asparagine, serine, or leucine transport of glutamine declined to 28%, 15%, 17%, and 21%, respectively, of the rates in controls. Inhibition by histidine (Ki = 0.58 mM) and serine (Ki = 0.25 mM) was competitive, by leucine was non‐competitive (Ki = 0.64), while α‐methylamino‐isobutyric acid and 2‐amino carboxy‐bicyclo (2.2.1)‐heptane had no effect. In cells cultured for 24 hours with or without 10 μg/ml Con A, the apparent Km was 70 μM vs. 89 μM and Vmax 73 vs. 26 pmol/106 cells/30 seconds. Sodium depletion (9.0 mM NaCl) greatly diminished glutamine transport in resting and stimulated cells. Inhibition of glutamine transport by serine was sodium sensitive, while inhibition by histdine and asparagine was not. Serine had no competitive effect in sodium‐depleted media. The data demonstrate what appear to be two carrier systems for glutamine, sodium sensitive and sodium insensitive. It is suggested that glutamine transport into lymphocytes occurs via processes similar to System N and System ASC described in other cells, with System ASC as the sodium‐sensitive component. Con A augments the capacity rather than the affinity of glutamine transporting systems.
Establishment of a cyclic adenosine monophosphate‐dependent growing human T‐cell line derived from an interleukin‐2‐dependent cell line
Authors: Takeshita Toshikazu , Ohbo Kazuyuki , Nakamura Masataka , Goto Yuso , Sugamura Kazuo
Published: February 2005

An adenosine 3′,5′‐cyclic monophosphate (cAMP)‐dependent growing cell line called CT‐Mat was established by the long‐term cultivation of an interleukin‐2 (IL‐2)‐dependent human T‐cell line, ILT‐Mat, in the presence of cholera toxin instead of IL‐2. CT‐Mat cells can grow in the medium containing either cholera toxin or forskolin or cAMP derivatives. Although the CT‐Mat cell line can still grow dependent on IL‐2, the forskolin‐induced growth of CT‐Mat cells was demonstrated not to be mediated by an autocrine mechanism of IL‐2 or any other growth factor. The intracellular cAMP level was elevated by treatment with the chemical agents but little by treatment with IL‐2. These suggest that cAMP transduces intracellular growth signals different from those through the IL‐2 receptor in an IL‐2‐dependent T‐cell line CT‐Mat.
Characterization of the D‐allose‐mediated regulation of sugar transport in Chinese hamster fibroblasts
Authors: Germinario R. J. , Kristof A. , Chang Z. , Manuel S.
Published: February 2005

Exposure to D‐allose has been demonstrated to lead to decreased 2‐deoxyD‐glucose (2‐DG) and 3‐0‐methyl‐D‐glucose transport in the V79 Chinese hamster lung fibroblast cell line. The effect of D‐allose (1) was maximal after 4 hours exposure to the cells; (2) was optimal between 2.77 and 5.55 mM D‐allose; and (3) led to a decreased Vmax for 2‐DG transport with no change in the transport Km value. The decrease in 2‐DG transport induced by D‐allose was reversible and the reversal was differentially affected by cycloheximide, being blocked by a low concentration of cycloheximide (0.05 μg/ml) but not a high concentration of the inhibitor (5 μg/ml). D‐allose did not competitively inhibit the transport of 2‐DG while D‐glucose under similar conditions yielded a K1 for 2‐DG transport inhibition of 1.7 mM. Additionally, D‐allose did not affect the phosphorylation of 2‐DG by hexokinase in cell‐free cytosol. The data indicate that D‐allose has significant lowering effects on sugar transport activity. Additionally, while the sugar itself may be the active component in sugar transport regulation, the effect is not blocked by inhibition of protein synthesis but the synthesis of a regulatory protein(s) may be involved in the return of sugar transport following D‐allose removal.
Estrogen regulates peptidylarginine deiminase levels in a rat pituitary cell line in culture
Authors: Nagata Saburo , Yamagiwa Miho , Inoue Kinji , Senshu Tatsuo
Published: February 2005

A Nonidet P‐40 extract of growth hormone‐producing rat pituitary MtT/S cells was found to contain peptidylarginine deiminase (EC 3.5.3.15), which was indistinguishable from an enzyme preparation from rat muscle in Western immunoblotting and immunoprecipitation. This enzyme was immunocytochemically detected in the cytoplasm but was not secreted into the medium during the cultivation. When the cells were cultured for 2 days with various concentrations of 17 β‐estradiol (E2), the enzyme activity increased in a dose‐dependent manner, reaching a maximum level (four‐ to five old higher than control) at about 10−9 M. This increase in the enzyme activity was evident by 14 hr of culture and became relatively stable after 24 hr. It correlated well with the increase in the amount of the muscle type enzyme per cell as analyzed by Western immunoblotting. Estriol and a synthetic estrogen, diethylstilbestrol, also increased the enzyme activity, whereas testosterone, progesterone, and corticosterone were without effect. An antiestrogen, tamoxifen, which by itself was inactive, partially suppressed the effect of E2. Exposure of MtT/S cells for 14 hr to E2 increased incorporation of 35S‐labeled amino acids into the immunoprecipitable peptidylarginine deiminase. This increase was dependent on the concentration of E2, attaining a maximum level (about tenfold higher than the control) at about 10−9 M. These results indicate that estrogen effects the increase in peptidylarginine deiminase content in the pituitary cells by stimulating enzyme synthesis.
Accessory cells induce a polyphosphatidylinositol response when cultured with mitogen‐activated T lymphocytes
Authors: Akeson Ann L. , Mitani Kiminobu , McCarthy Becky M. , Harmony Judith A. K.
Published: February 2005

Monocytes (MO) influenced phosphoinositide metabolism when human T lymphocytes, isolated from peripheral blood, were activated by polyclonal mitogens. In the 3 hr immediately following mitogenic challenge, the synthesis of phosphatidylinositol (PI) was augmented and the synthesis of PI‐4‐phosphate (PIP) and PI‐4,5‐bisphosphate (PIP2) was induced in cultures of T lymphocytes and MO. In addition, MO induced a rapid and transient degradation of PIP and PIP2 in T cells prelabeled with [32P]PL and subsequently activated by mitogen. Induction of a PIP/PIP2 response correlated well with induction of DNA replication by MO when T cells were activated by phytohemagglutinin or by neuraminidase plus galactose oxidase. MO did not influence polyphosphoinositide metabolism when T cells were stimulated by the nonmitogenic lectin wheat germ agglutinin. Interleukin 1 could not substitute for monocytes in inducing a polyphosphoinositide response. By causing a rapid and transient release of the second messengers diacylglycerol and inositol phosphates and by subsequently increasing their cellular precursors, MO may induce the interleukin 2 responsive state in T lymphocytes.
Partial down‐regulation of protein kinase C reverses the growth inhibitory effect of phorbol esters on HepG2 cells
Authors: Duronio Vincent , Huber Brian E. , Jacobs Steven
Published: February 2005

Phorbol ester treatment of HepG2, a human tumorigenic cell line, caused rapid morphological changes characterized by a flattening and spreading of the cells that coincided with a rapid inhibition of thymidine incorporation. Within 24 h, cell division was completely inhibited, suggesting the cells had entered a quiescent state. Continued incubation n the presence of phorbol esters resulted in the resumption of thymidine incorporation and cell division, but this coincided with only a partial down‐regulation of PKC activity. Seventy two hours of treatment was required to obtain down‐regulation of greater than 80% of the PKC activity, but reversal of the inhib tory effects occurred between 24 and 48 h after the addition of phorbol esters, when a large proportion of the PKC activity was still present. Northern blot analysis of a number of transcripts showed that the steady‐state levels of c‐myc and transforming growth factor β1 (TGF‐β1) messages increased only after 3 h of phorbol ester treatment and returned to normal levels after 24 h. C‐fos, albumin, and alphafetoprotein messages were not affected, suggesting the differentiation state of the cells was not altered. Therefore, phorbol ester activation of PKC causes an inhibition of HepG2 cell growth initially, but this is unlike the promotion of differentiation seen in other systems. Partial downregulation of PKC activity causes a reversal of the growth inhibition and the cells return to a normal growth rate. This effect is also clearly different from systems in which phorbol esters have been shown to have a mitogenic effect on cells.
Sex‐dependent and independent expression of the P‐glycoprotein isoforms in Chinese hamster
Authors: Bradley Gracf , Georges Eijas , Ling Victor
Published: February 2005

P‐glycoprotein (Pgp) is a small family of membrane proteins which belongs to a superfamily of energy‐dependent membrane transport proteins identified in phylogenetically distant species, from bacteria to man. Among mammalian species, some of the Pgp isoforms can mediate multidrug resistance by acting as an energy‐dependent drug efflux pump. However, the physiologic functions of the Pgp isoforms have not been defined. In this study we examined the expression of the three hamster Pgp isoforms in normal hamster tissues, by using isoform‐specific monoclonal antibodies in a competitive immunohistochemical assay. We showed that each Pgp isoform is predominantly expressed in a small, distinct group of differentiated cells, where it is likely to function in specific secretory pathways. The expression of the Pgp isoforms appears to be tightly regulated and, at least in some cells, under complex hormonal control. Furthermore, there is a striking sex difference in Pgp content of the adrenal cortex. These findings are important for the ultimate understanding of the normal physiologic roles of the Pgp gene family members.
Adenylate cyclase and protein kinase C mediate opposite actions on endothelial junctions
Authors: Oliver Juan A.
Published: February 2005

To determine whether the endothelial paracellular pathway is regulated, the effect of intracellular messengers on the transendothelial flux of inert radiolabeled molecules of diverse molecular size was examined in bovine aortic endothelial cells grown on collagen‐coated filters. The endothelial monolayers showed a modest electrical resistance (21 ± 10 Ω. cm2; m ± SD) and restricted the passage to 14C‐sucrose, 3H‐inulin, 14C‐dextran (70 kDa), and 125l‐polyvinyl pyrrolidone (125l‐PVP, 360 kDa) according to their molecular mass. 8‐Bromoadenosine 3′‐5′ cyclic monophosphate (8‐Br‐cAMP) reduced by more than 30% the permeability coefficients of 14C‐sucrose and 3H‐inulin but had no effect on the permeability of 125l‐PVP. The permeabilities of 14C‐sucrose and of 14C‐inulin were strikingly increased by activating protein kinase C (PKC) by phorbol 12‐myristate 13‐acetate or sn‐1,2‐dioctanoly‐glycerol whereas the latter compound had no effect on the permeability of 125l‐PVP. In addition, the permeability of 14C‐sucrose was unchanged by a phorbol ester that does not activate PKC. Increasing intracellular calcium with ionomycin had no effect on the permeability of 14C‐sucrose. None of these maneuvers significantly affected the protein content of the endothelial monolayers. The results indicate that 8‐Br‐cAMP and PKC activators modulate a pathway across the endothelial monolayer that excludes 125l‐PVP (360 kDa) but readily accepts 14C‐sucrose and 3H‐inulin, suggesting that this pathway is the paracellular pathway. Hence, low molecular weight molecules such as sucrose and inulin can be used to probe the behavior of the paracellular pathway of endothelial monolayers grown in vitro. The results also indicate that the paracellular pathway in endothelium is regulated and suggest that endothelial junctions can be closed by stimulating adenylate cyclase and opened by stimulating protein kinase C.
Binding of epidermal growth factor to its receptor is affected by membrane phospholipid environment
Authors: Kano‐Sueoka Tamiko , King David M. , Fisk Harold A. , Klug Stefanie J.
Published: February 2005

Cells of epithelial origin generally require ethanolamine to grow in culture; when these cells are grown without ethanolamine, the phosphatidylethanolamine content of their membrane phospholipid becomes 1/2 to 1/3 of the normal amount, and growth stops. We have hypothesized that growth ceases because the phospholipid environment becomes unsuitable for membrane‐associated function. Using ethanolamine‐requiring rat mammary cells, we have investigated the possible effect of phosphatidylethanolamine deficiency on the binding characteristics of epidermal growth factor. Apparent dissociation constant for the high‐affinity sites in cells having normal membrane phospholipid was 1.7 × 10−10M, whereas that of phosphatidylethanolamine‐deficient cells was 2.7 × 10−10M: the difference was small, but significant. Pretreatment with phorbol ester caused the loss of high‐affinity sites in cells having normal membrane, whereas binding characteristics of epidermal growth factor became refractory to the pretreatment in phosphatidylethanolamine‐deficient cells. In addition, the rate of internalization of bound epidermal growth factor in phosphatidylethanolamine‐deficient cells was about 1/4 of normal cells. Further, whether cells had normal or phosphatidylethanolamine‐deficient membranes seemed to affect the phosphorylation patterns of membrane proteins in response to epidermal growth factor or phorbol ester. These results suggest that membrane phospholipid environment affects the activity of the epidermal growth factor receptor.
Effect of polyunsaturated fatty acids and phospholipids on “3H”‐vitamin E incorporation into pulmonary artery endothelial cell membranes
Authors: Sekharam K. Madhavi , Patel Jawaharlal M. , Block Edward R.
Published: February 2005

Vitamin E, a dietary antioxidant, is presumed to be incorporated into the lipid bilayer of biological membranes to an extent proportional to the amount of polyunsaturated fatty acids or phospholipids in the membrane. In the present study we evaluated the distribution of incorporated polyunsaturated fatty acids (PUFA) and phosphatidylethanolamine (PE) in various membranes of pulmonary artery endothelial cells. We also studied whether incorporation of PUFA or PE is responsible for increased incorporation of 3 H)‐vitamin E into the membranes of these cells. Following a 24‐hr incubation with linoleic acid (18:2), 18:2 was increased by 6.9‐, 9.2‐, and 13.2‐fold in plasma, mitochondrial, and microsomal membranes, respectively. Incorporation of 18:2 caused significant increases in the unsaturation indexes of mitochondrial and microsomal polyunsaturated fatty acyl chains (P <.01 versus control in both membranes). Incubation with arachidonic acid (20:4) for 24 hr resulted in 1.5‐, 2.3‐, and 2.4‐fold increases in 20:4 in plasma, mitochondrial, and microsomal membranes, respectively. The unsaturation indexes of polyunsaturated fatty acyl chains of mitochondrial and microsomal membranes also increased (P.01 versus control in both membranes). Although incubations with 18:2 or 20:4 resulted in several‐fold increases in membrane 18:2 or 20:4 fatty acids, incorporation of “3 H”‐vitamin E into these membranes was similar to that in controls. Following a 24‐hr incubation with PE, membrane PE content was significantly increased, and “3H”‐vitamin E incorporation was also increased to a comparable degree, i.e., plasma membrane > mitochondria > microsomes. Endogenous vitamin E content of the cells was not altered because of increased incorporation of PE and “3H”‐vitamin E. When “3H”‐vitamin E was incorporated into lipid vesicles prepared from the total lipid extracts of endothelial cells and varying amounts of exogenous PE, vitamin E content was directly related to PE content. These results demonstrate that PUFA and PE distribute in all pulmonary artery endothelial cell membranes. However, only increases in PE were associated with increased incorporation of “3H”‐vitamin E in membranes of these cells.
Improved coupling between proliferation‐arrest and differentiation‐induction in ML‐1 human myeloblastic leukemia cells
Authors: Kozopas Karen M. , Buchan Heather L. , Craig Ruth W.
Published: February 2005

Proliferation and differentiation are coupled in normal cells and are aberrant in leukemia cells. The studies reported here were aimed at more effectively coupling proliferation‐arrest and differentiation‐incuction in a human myeloblastic leukemia cell line (ML‐1). This was accomplished by using reduced serum conditions in conjunction with a differentiation‐inducing agent: cells were first inncubated in reduced serum “0.3% fetal bovine serum (FBS)” instead of standard conditions (7.5% FBS) and, second, exposed to 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA). The effects of this protocol were as follows: first, cell proliferation was slowed and cells accumulated in Go/G1, phase of the cell cycle; this occurred with only a minimal decrease in viability “to∼88–92% (0.3% FBS) from ≥ 96% (7.5% FBS)”. Second, the induction of differentiation was accelerated; this allowed the time of exposure to TPA to be decreased. Acceleration of induction was very pronounced when cells were maintained in 0.3% FBS both before and during exposure to TPA, with TPA at concentrations above the minimum sufficient for induction but below those causing significant cytotoxicity; as little as 1 hour of TPA exposure resulted in near‐maximal induction (∼80%) with this protocol, compared to the ≥ 1 day required with previous standard protocols. In sum, conditions that slow ML‐1 cell proliferation (0.3% FBS) enhance TPA‐induced differentiation, substantially narrowing the time frame of induction; these conditions should be useful for studying the molecular mechanisms that underlie the induction process.
Activation of cell membrane potassium conductance by mercury in cultured renal epitheloid (MDCK) cells
Authors: Jungwirth A. , Ritter M. , Paulmichl M. , Lang F.
Published: February 2005

To elucidate mechanisms of mercury toxicity, the cell membrane potential has been determined continuously in cultured kidney (MDCK)‐cells during reversible application of mercury ions to extracellular perfusate. Exposure of the cells to 1μM mercury ions is followed by rapid, sustained, and slowly reversible hyperpolarization of the cell membrane, increase of cell membrane potassium selectivity, and decrease of cell membrane resistance. Thus, mercury ions enhance the potassium conductance of the cell membrane. Half maximal hyperpolarizing effect is elicited by ≈0.2 μM. Higher concentrations of mercury ions (> 10 μM) eventually depolarize the cell membrane. At extracellular calcium activity reduced to less than 0.1 μM, 1 μM mercury ions still leads to a sustained hyperpolarization and increase of potassium selectivity of the cell membrane. As evident from fluorescence measurements, 10 μM, but not 1 μM mercury ions leads to a rapid increase of intracellular calcium activity. Pretreatment of the cells with either pertussis toxin or cholera toxin does not blunt the hyperpolarizing effect of mercury ions. In conclusion, mercury ions activate the potassium conductance by a mechanism independent of increase of intracellular calcium activity and of cholera toxin‐or pertussis toxin‐sensitive G‐proteins. This activation of potassium conductance may account for early effects of mercury intoxication, such as kaliuresis.
Modulation of milk protein synthesis through alteration of the cytoskeleton in mouse mammary epithelial cells cultured on a reconstituted basement membrane
Authors: Seely Keith A. , Aggeler Judith
Published: February 2005

Recent studies indicate that the cytoskeleton may be involved in modulating tissue‐specific gene expression in mammalian cells. We have studied the role of the cytoskeleton in regulating milk protein synthesis and secretion by primary mouse mammary epithelial cells cultured on a reconstituted basement membrane that promotes differentiation. After 8 days in culture, cells were treated with cytochalasin D (CD) (0.5–1 μg/ml) to alter actin filaments or acrylamide (Ac) (5 mM) to alter intermediate filaments (cytokeratins). CD inhibited synthesis of most proteins in a concentration‐dependent manner, with β‐casein being the first affected. In contrast, Ac increased protein synthesis and secretion by 17–31% after a 12 hr treatment. Polyacrylamide gel electrophoresis of total secreted proteins indicates that synthetic rates of most proteins were increased equally by Ac treatment. This increase is apparently controlled at the level of translation, because control and Ac‐treated cells contained the same amount of poly‐A+ RNA, and neither CD nor Ac altered mRNA levels for β‐casein. There was also no indication that either CD or Ac can induce the expression of milk proteins in quiescent cells cultured on a plastic substratum. In conjunction with the biochemical studies, changes in cytoskeletal morphology caused by the drug treatments were analyzed by immunofluorescence microscopy. As has been observed in other cell types, low concentrations of CD caused cells to round up by disrupting actin filaments. Ac treatment slightly decreased the intensity of actin staining, but no changes in microfilament organization were observed. Ac‐treated cells showed slight disorganization of the cytokeratin filaments, with some peripheral interfibrillar bundling, but the cytokeratin network did not collapse and no retraction of cell extensions or breakdown of cell‐cell contacts was observed. These results confirm previous reports that the actin cytoskeleton may play a role in regulating tissue‐specific protein synthesis. How Ac stimulates protein synthesis is unknown, but it is unlikely that this effect is directly mediated through intermediate filaments.
Thrombin increases proliferation and decreases fibrinolytic activity of kidney glomerular epithelial cells
Authors: He Ci‐Jiang , Rondeau Eric , Medcalf Robert L. , Lacave Roger , Schleuning Wolf‐Dieter , Sraer Jean‐Daniel
Published: February 2005

Human glomerular epithelial cells (GECs) in culture synthesize single‐chain, urokinase‐type plasminogen activator (SC‐uPA), tissue‐type plasminogen activator (t‐PA), and plasminogen activator inhibitor 1 (PAI‐1) and possess specific membrane‐binding sites for u‐PA. Using purified 125I‐alpha thrombin, we demonstrate here the presence of two populations of specific binding sites for thrombin on GECs (1.Kd = 4.3 ± 1.0 × 10−10 M, 5.4 ± 1.4 × 104 M sites per cell, 2. Kd = 1.6 ± 0.5 × 10−8 M, 7.9 ± 1.8 × 105 sites per cell). Purified human alpha thrombin promoted the proliferation of GECs and induced a time‐and dose‐dependent increase of SC‐uPA, t‐PA, and PAI‐1 antigens released by GECs. Thrombin‐mediated increase in antigen was paralleled by an increase in the levels of corresponding u‐PA and PAI‐1 messenger RNA. In contrast, thrombin decreased u‐PA activity in conditioned medium. This discrepancy between u‐PA antigen and u‐PA activity was explained by a limited proteolysis of SC‐uPA by thrombin, leading to a two‐chain form detected by immunoblotting and that could not be activated by plasmin. Thrombin also decreased the number of u‐PA binding sites on GECs (p < 0.05) without changing receptor affinity. Hirudin inhibited the binding and the cellular effects of thrombin, whereas thrombin inactivated by diisopropylfluorophosphate had no effect, indicating that both membrane binding and catalytic activity of thrombin were required. We conclude that thrombin, through specific membrane receptors, stimulates proliferation of GECs and decreases the fibrinolytic activity of GECs both at the cell surface and in the conditioned medium. These results suggest that thrombin could be involved in the pathogenesis of extracapillary proliferation and persistency of fibrin deposits in crescentic glomerulonephritis.
Bindings of the lectins Griffonia simplicifolia‐1 and pokeweed mitogen mark discrete stages of myoepithelial‐like differentiation of cell lines from the rat mammary gland
Authors: Rudland Philip S. , Hughes Christine M.
Published: February 2005

Individual single‐cell‐cloned cell lines of the different rat mammary (Rama) cell types have been tested for their ability to bind the lectins Griffonia simplicifolia‐1(GS‐1) and pokeweed mitogen (PWM) using fluorescent, histochemical, and radioactive assays. Myoepithelial‐like cell lines isolated from neonatal rat mammary glands and from nonmetastasizing tumors strongly bind GS‐1 and PWM, whereas the corresponding epithelial and fibroblastic cell lines do not. When the epithelial cell lines are grown on floating gels of polymerised rat tail collagen, the basally situated or peripheral cells are stained strongly with peroxidase‐conjugated lectins, whereas the apically or luminally situated cells are unstained. The capacity of cell lines intermediate in morphology between epithelial and myoepithelial‐like cells to bind to GS‐1 is as follows: Rama 25 epithelial < Rama 25–12 < Rama 25‐I1 < Rama 25–14 < Rama 29 myoepithelial‐like cells, the same order as for other markers of myoepithelial cells. Conjugated PWM, however, binds only to the myoepithelial‐like cell lines. Treatment of Rama 25 epithelial cells with agents that disrupt microtubules accelerates their conversion to elongated, myoepithelial‐like cells in culture. The binding of cells to GS‐1 is observed prior to, and that to PWM after, the major morphological change. It is suggested that the stepwise appearances of carbohydrate receptors for GS‐1 and PWM mark discrete stages in the differentiation of epithelial to myoepithelial‐like cells in culture, in the same way that they mark similar differentiation stages in ductal development in mammary glands of prepubertal rats.
IL‐3 specifically inhibits GM‐CSF binding to the higher affinity receptor
Authors: Taketazu Fumitoshi , Chiba Shigeru , Shibuya Kyoichi , Kuwaki Tomoaki , Tsumura Haruhiko , Miyazono Kohei , Miyagawa Kiyoshi , Takaku Fumimaro
Published: February 2005

The inhibition of binding between human granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and its receptor by human interleukin‐3 (IL‐3) was observed in myelogenous leukemia cell line KG‐1 which bore the receptors both for GM‐CSF and IL‐3. In contrast, this phenomenon was not observed in histiocytic lymphoma cell line U‐937 or in gastric carcinoma cell line KATO III, both of which have apparent GM‐CSF receptor but an undetectable IL‐3 receptor. In KG‐1 cells, the cross‐inhibition was preferentially observed when the binding of GM‐CSF was performed under the high‐affinity binding condition; i.e., a low concentration of 125I‐GM‐CSF was incubated. Scatchard analysis of 125I‐GM‐CSF binding to KG‐1 cells in the absence and in the presence of unlabeled IL‐3 demonstrated that IL‐3 inhibited GM‐CSF binding to the higher‐affinity component of GM‐CSF receptor on KG‐1 cells. Moreover, a chemical cross‐linking study has revealed that the cross‐inhibition of the GM‐CSF binding observed in KG‐1 cells is specific for the β‐chain, Mr 135,000 binding protein which has been identified as a component forming the high‐affinity GM‐CSF receptor existng specifically on hemopoietic cells.