bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of glutamine transport into resting and concanavalin A‐stimulated peripheral human lymphocytes
Authors: Schröder Marie‐Theres , Schäfer Gertrud , Schauder Peter 
Published: February 2005

AbstractCharacteristics 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

AbstractAn 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

AbstractExposure 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

AbstractA 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

AbstractMonocytes (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

AbstractPhorbol 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

AbstractP‐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

AbstractTo 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

AbstractCells 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

AbstractVitamin 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

AbstractProliferation 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

AbstractTo 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

AbstractRecent 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.