bluuebunny/crossref_metadata_2025 huggingface dataset:
Metabolic effects of heparin on rat cervical epithelial cells
Authors: Wright Thomas C. , Karnovsky Morris J.
Published: February 2005

The glycosaminoglycan heparin inhibits the growth of a number of different cell types in vitro including smooth muscle cells, mesangial cells, fibroblasts, and rat cervical epithelial cells (RCEC). Studies investigating the antiproliferative effects of heparin on smooth muscle cells have demonstrated the site of the cell cycle block and revealed several metabolic alterations that could be causally associated with growth inhibition. We have investigated these metabolic parameters in RCEC to determine whether they are also associated with the antiproliferative effects of heparin in epithelial cells. Heparin acts rapidly to inhibit RCEC growth with inhibition detectable by autoradiography 7 h after the addition of heparin. Heparin treated RCEC begin to enter S‐phase 12 h after the removal of heparin. These findings suggest that heparin blocks RCEC in the early‐to‐mid G1 phase of the cell cycle rather than late in G1 or early in S‐phase as has previously been demonstrated for smooth muscle cells. Unlike smooth muscle cells, the uptake of thymidine and uridine is not inhibited by heparin in RCEC. Treatment of medium with heparin‐Sepharose does not reduce the subsequent growth of RCEC; heparin inhibits the growth of RCEC in heparin‐Sepharose treated medium in a manner identical to that in nontreated medium. Therefore the growth inhibitory effects of heparin cannot be explained by the inactivation of mitogens present in serum. In contrast to its effects on smooth muscle cells, heparin treatment of RCEC does not result in a reduction in the binding of epidermal growth factor (EGF) to the cells. These results indicate that although heparin inhibits the growth of a variety of cell types, significant differences exist in the responses of the different cells to heparin.
Casein secretion by mammary gland epithelia from collagen gel cultures and lactating glands
Authors: Rocha Victor , Hwang Soo‐In , Ortiz C. Leo
Published: February 2005

Amino acid incorporation experiments show that epithelial cells from lactating mouse mammary glands and from collagen gel culture both synthesize and secrete four principal phosphocaseins (p45, p40, p27, and p23 KD). In both cases, however, the casein production is largely dominated by the p27 species. The average percentage distribution of the above casein species in medium from cultured epithelia is approximately 13%, 6%, 68%, and 14%, respectively; for milk the distribution is approximately 23%, 7%, 54%, and 16%. The predominance of the p27 species is not a consequence of extensive extracellular differential degradation of the secreted caseins since no significant casein degradation was observed in culture medium, either in contact or isolated from epithelial cell monolayers. Synthesis and secretion of all the caseins by cultured epithelia is dependent upon insulin, prolactin, and hydrocortisone. Presumably some intracellular events result in the secretion of p27 as the principal casein in mouse milk. Apparently, some selection factor(s) operate to make p27 a major nitrogenous nutritional component for a newborn mouse. In addition, on a quantitative basis, the relative levels of various caseins secreted by epithelia from lactating mammary glands is essentially duplicated by epithelia in collagen gel culture.
Thrombin‐induced thromboxane generation by neutrophils and lymphocytes: Dependence on enzymic site
Authors: Bizios R. , Lai L. , Fenton J. W. , Malik A. B.
Published: February 2005

We examined the effects of thrombin on thromboxane generation by sheep neutrophils and lymphocytes in vitro. Physiological concentration of thrombin (50 nM) resulted in thromboxane B2 generation from both neutrophils and lymphocytes, which was comparable to that obtained with zymosan activated serum challenge of the cells. Thromboxane B2 generation was dependent on the enzymic region of the thrombin molecule responsible for clotting activity because the complexing of thrombin with hirudin (1:1 U:U mixture of thrombin and hirudin) abolished thromboxane generation from both cell types. Further studies with modifed forms of α‐thrombin (which were produced by irreversible conjugation at the catalytic site and lacked enzymic activity) also showed no generation of thromboxane B2 from neutrophils or lymphocytes. The results indicate that thrombin stimulates thromboxane generation from neutrophils and lymphocytes and that this response is dependent on the proteolytic activity of thrombin.
Effects of dissolved oxygen concentration on hybridoma growth and metabolism in continuous culture
Authors: Miller William M. , Wilke Charles R. , Blanch Harvey W.
Published: February 2005

Oxygen transport is a major limitation in large‐scale mammalian cell culture. The effects of the dissolved oxygen concentration (DO; from 0.1 to 100% saturation with air) on Sp2/0‐derived mouse hybridomas were investigated using continuous culture. The steady‐state concentration of viable cells increased with decreasing DO until a critical dissolved oxygen concentration of 0.5% of air saturation was reached. The cell concentration declined at lower DO because of incomplete glutamine oxidation, and the specific lactate production from glucose increased to offset the reduced energy production from glutamine. Cell viability increased as the DO was decreased; the viability continued to increase even when the DO was reduced below 0.5%. The specific oxygen uptake rate was essentially constant for DO ≥ 10% of air saturation and then decreased with decreasing DO. The P/O ratio (ATP molecules produced per O atom consumed) appears to change from 2 to 3 between 10 and 0.5% DO. The specific ATP production rate calculated using this assumption decreases only slightly with decreasing DO. The optimum DO of 50% for antibody production is different than the optimum (∼ 0.5% DO) for cell growth.
Characterization of murine monoclonal antibodies directed against basic fibroblast growth factor
Authors: Massoglia S. L. , Kenney J. S. , Gospodarowicz D. J.
Published: February 2005

Monoclonal antibodies (McAbs) were developed that identify the complete (1–146 aa) and the NH2‐terminal truncated (des 1–15) form of bovine basic fibroblast growth factor (bFGF). Four McAbs, designated McAbs 6, 8, 38, and 42, bind the complete form of bFGF found in bovine pituitary, brain, and adrenal gland. One of these McAbs, McAbs 42, also binds to the des 1–15 aa form of bFGF found in bovine adrenal gland, kidney, and corpus luteum. None of the McAbs binds bovine‐brain‐derived acidic FGF (aFGF). McAbs 6, 8, and 38 recognized the same epitope located within the first ten residues of the NH2‐terminal of complete bFGF. McAb 42 recognizes a “core” epitope found on both the complete and des 1–15 aa bFGFs. The McAbs are murine lgGs with affinity constants of 107‐108 liter/M for bovine‐pituitary‐derived bFGF. McAbs 8 and 42 have been used in a two‐site ELISA to detect the complete form of bFGF. The ELISA is sensitive to 38.5 fmole/well of bFGF and is not affected by the presence of calf serum or bovine‐brain‐derived aFGF. These McAbs should be useful in distinguishing the native and des 1–15 aa forms of bFGF from each other, and from aFGF and other growth factors.
Production of tumor growth‐inhibiting protein by the neonatal mouse brain: Dependence on intracellular glutamine metabolism
Authors: Miwa Nobuhiko , Matsuno Tetsuya , Mizuno Satoshi
Published: February 2005

We previously reported that the mouse brain at the neonatal stage but not at the adult stage secreted a carcinostatic factor of 62,000 Da, termed NBCF, which inhibited clonal growth and DNA synthesis of malignant cells preferentially over those of normal cells. In the present study, NBCF production by the neonatal mouse brain in culture was investigated. Addition of L‐glutamine to the culture medium markedly promoted NBCF production in a concentration‐dependent manner. The production seemed to be specific to glutamine, since no promotive effect was exerted by L‐glutamic acid, its analogue DL‐α‐aminoadipic acid, L‐aspargine, or L‐aspartic acid or by other amino acids or vitamins. NBCF production was markedly reduced in culture medium either devoid of L‐glutamine or containing 6‐diazo‐5‐oxo‐L‐norleucine, a glutamine antagonist, or L‐metionine sulfoximine, an inhibitor of glutamine synthetase. Thus NBCF production was promoted by extracellular supply, intracellular synthesis, and utilization of L‐glutamine but was not affected by its deamidated form or homologue amino acids. On the other hand, NBCF production was completly repressed by addition of cycloheximide to the culture medium. The repressive effect was also exerted by actinomycin D although not completely, whereas cytosine β‐D‐arabinofuranoside did not repress NBCF production. These results indicated that NBCF production by cultivation was independent of DNA replication but dependend mostly on a transcription stage and its following stages and partly on a translation stage from the preexisting mRNA to the protein.
Perturbation of cultured human vascular endothelial cells by phorbol ester or thrombin alters the cellular von Willebrand factor distribution
Authors: Reinders Jan Hendrik , Vervoorn Richard C. , Verweij Cornelis L. , Van Mourik Jan A. , De Groot Philip G.
Published: February 2005

We have studied the influence of perturbation of cultured human umbilical vein endothelial cells on the distribution of the von Willebrand factor. As shown previously, short‐term ( ≤ 1 hr) treatment of endothelial cells with the phorbol ester 4β‐phorbol 12‐myristate 13‐acetate (PMA) or thrombin resulted in the release of cellular stored von Willebrand factor. Long‐term treatment with PMA or thrombin evoked a distinct change in the endothelial cell distribution of von Willebrand factor, evident 24 to 48 hrs after exposure. Whereas the contents of the von Willebrand factor storage sites in the cells were gradually restored within 48 hrs, enhanced amounts of von Willebrand factor were secreted into the medium. However, PMA did not increase the endothelial cell contents of mRNA encoding for von Willebrand factor. The number as well as the size of von Willebrand factor storage granules in the endothelial cells increased after exposure to the phorbol ester, as determined by immunofluorescence microscopy. A second treatment with PMA or thrombin, 48 hrs after cells had been stimulated with these agents, resulted again in the instantaneous release of von Willebrand factor. PMA and thrombin caused a decrease in the von Willebrand factor contents of the extracellular matrix. Pulse‐chase experiments revelaed that PMA blocked the deposition of von Willebrand factor in the subendothelium, whereas PMA did not affect the degradation of matrix von Willebrand factor. Thus, perturbation of endothelial cells changes the cellular distribution of von Willebrand factor.
Iron uptake from transferrin and transferrin endocytic cycle in friend erythroleukemia cells
Authors: Hradilek A. , Neuwirt J.
Published: February 2005

Several aspects of iron metabolism were studied in cultured Friend erythroleukemia cells before and after induction of hemoglobin synthesis by dimethyl sulfoxide. The maximal rate of iron uptake from 59Fe‐labeled transferrin, 1.5 × 106 atoms of Fe/cell per 30 min in uninduced cells, increased to 3 × 106 atoms/cell after 5 days of induction. The increase in iron uptake was not accompanied by a proportional increase in the number of transferrin receptors detected by 125I‐labeled transferrin binding, suggesting a more efficient iron uptake by transferrin receptors in induced cells, with the rate of about 26 iron atoms per receptor per hour, compared to 15 atoms in uninduced cells. In agreement with this conclusion are results of the study of cellular 125I or 59Fe labeled transferrin kinetics. In the induced cells transferrin endocytosis and release proceeded with identical rates and all the endocytosed iron was retained inside the cell. On the other hand, transferrin release by uninduced cells was significantly slower and a substantial part of internalized 59Fe was released. On the basis of these results, different efficiency of iron release from internalized transferrin, accompanied by changes in cellular transferrin kinetics, is proposed as one of the factors determining the rate of iron uptake by developing erythroid cells.
Monensin inhibits ligand dissociation only transiently and partially and distinguishes two galactosyl receptor pathways in isolated rat hepatocytes
Authors: Oka Janet A. , Weigel Paul H.
Published: February 2005

Monensin has been shown to inhibit the dissociation of internalized asialoorosomucoid (ASOR) from galactosyl (Gal) receptors in hepatocytes (Harford et al., J. Cell. Biol., 96: 1824, 1983). Examination of the long‐term kinetics of dissociation of a single round of surface‐bound 125I‐ASOR in the presence of monensin revealed, however, that dissociation resumed after a lag of 30–40 min. Dissociation proceeded slowly with apparent first order kinetics (k = 0.006–0.022 min−1) and reached a plateau after 4 h, both in freshly isolated cells in suspension and in cells cultured for 24 h. Only a portion of the ligand bound to surface Gal receptors was capable of dissociating. The degree of dissociation was correlated with the expression of a subpopulation of receptors we have recently designated as state 1 Gal receptors (Weigel et al., Biochem. Biophys. Res. Commun. 140: 43, 1986). The recovery and dissociation of a portion of 125I‐ASOR‐receptor complexes after the lag period is not due to a depletion of monensin, since a second addition of the drug has no affect once dissociation resumes. Furthermore, as assessed by the accumulation of the fluorescent dye acridine orange, cells have not recovered the ability to acidify intracellular compartments during the time that dissociation occurs. The results support a model for the hepatic Gal receptor system, in which there are two functionally different receptor populations, recycling pathways, and ligand processing pathways. Monensin blocks dissociation of 125I‐ASOR from receptors in the major pathway completely. In the minor pathway dissociation proceeds to completion only after a lag. In this minor pathway monensin appears to temporarily delay a maturation or translocation process that must occur prior to dissociation. We conclude that the observed dissociation in the presence of monensin cannot be mediated by low pH, or by pH or pNa gradients.
Effects of inhibition of RNA or protein synthesis on CHO cell cycle progression
Authors: Traganos Frank , Kimmel Marek , Bueti Connie , Darzynkiewicz Zbigniew
Published: February 2005

Chinese hamster ovary (CHO) cells, synchronized by selective detachment at mitosis, were treated with various concentrations of actinomycin D (AMD) or cycloheximide (CHX) either immediately, or 1, 2, or 3 hr after mitosis. Since the minimum duration of G1 phase in these cultures was 3.4 hr, the addition of RNA or protein synthesis inhibitors took place at the beginning, first third, second third, or end (G1–S boundary) of G1 phase. The kinetics of exit from G1 phase, the rate and extent of traverse of S phase, and the reaccumulation of RNA were estimated under each set of growth conditions by flow cytometry of acridine orange‐stained cells. A mathematical model was constructed to describe the trajectories of the cell populations with respect to their increase in RNA and DNA content in the absence or presence of the inhibitor. The chronologic synchrony imposed on the CHO cell population began to decay within 3 hr, resulting in stochastic entrance of cells into S phase in the absence of inhibitor. Addition of AMD or CHX at 0, 1, 2, or 3 hr after mitosis, regardless of the inhibitor concentration, did not provide evidence of a critical restriction point in G1 beyond which cells were committed to enter S phase and were no longer sensitive to moderate suppression of RNA or protein synthesis. The observed kinetics of cell entrance into and traverse of S phase were consistent with an inherently heterogenous response to serum stimulation occurring at or just after cell division.
Megakaryocyte colony growth‐supporting activities in human plasma: Modification by platelets and platelet membranes
Authors: Yamasaki Kazuo , Jamal Nazir , Mann Kenneth G. , Messner Hans A.
Published: February 2005

Human megakaryocyte colonies are grown in methylcellulose with platelet‐poor plasma and medium conditioned by phytohemagglutinin‐stimulated leukocytes (PHA‐LCM) as a source of megakaryocyte colony stimulating factor (MEG‐CSF). The megakaryocyte colony growth‐supporting activity in human plasma can be absorbed by intact platelets or degranulated platelet membranes. It was possible to recover the activity by solubilized platelet membrane with cholic acid. Filtration of the solubilized platelet membrane preparations through a Sephadex G‐100 column yielded at least two activity peaks. The molecular weight of these two activities differs from that of the growth‐promoting activity in PHA‐LCM.
Effect of 5′‐difluoromethylthioadenosine, an inhibitor of methylthioadenosine phosphorylase, on proliferation of cultured cells
Authors: Nishikawa Seiji , Ueno Akemichi , Inoue Hideo , Takeda Yoshiro
Published: February 2005

5′‐Methylthioadenosine (MTA) is formed from decarboxylated S‐adenosylme‐thionine during biosynthesis of polyamines. This nucleoside is cleaved by methylthioadenosine phosphorylase (MTA Pase) to adenine and 5‐methyl‐thioribose‐l‐phosphate in mammalian cells. 5′‐Difluoromethylthioadenosine (DFMTA), a synthetic analog of MTA, was not a substrate for MTA Pase, but was a strong competitive inhibitor of the enzyme (Ki = 0.48 μM). DFMTA caused marked accumulation of labeled MTA formed from [35S]methionine in Raji cells, which contain MTA Pase, but not in CCRF‐CEM cells, which do not contain this enzyme, suggesting that it also inhibits the enzyme in intact cells. DFMTA inhibited the growth of a variety of cultured cells and its cytostatic effect was roughly proportional to the MTA Pase activity of the cells. MTA also depressed the growth of cultured cells but, in contrast with DFMTA, its inhibitory effect was greater in MTA Pase‐deficient cells (CCRF‐CEM) than MTA Pase‐containing cells (Raji). Inhibition of growth of Raji cells by DFMTA was partially reversed by exogenous adenine, a reaction product of MTA Pase. These results suggest that the utilization of adenine formed from MTA was important for proliferation of cells containing MTA Pase under the culture conditions employed, and that DFMTA inhibited cell growth by inhibiting MTA Pase activity.
Cell cycle and growth factor‐dependent phosphoprotein of 78kD differently regulated in normal and transformed mouse fibroblasts
Authors: Yang Henry C. , Pardee Arthur B.
Published: February 2005

A novel 78kD phosphoprotein (pp78) of BALB/c 3T3 mouse fibroblasts is reported. Its properties of appearance in late G1 phase, dependence on specific growth factors, and altered constitutive kinetics in benzo(a)pyrene‐transformed (BPA31) cells suggest its role in growth and transformation. Pp78 phosphorylation is in a dynamic state and is stabilized by inhibitors of protein and mRNA synthesis, possibly because of inhibition of a labile phosphatase or protease. Its disappearance in S phase and its low level in exponential cells also indicate a dynamic control that is dependent on growth conditions. Enhancement by phorbol myristate acetate indicates that phosphorylation of pp78 is a consequence of protein kinase C activation, but it appears much later than does an 80kD phosphoprotein (pp80), which is a recognized substrate of kinase C. No simple relation between the appearance of pp78 and mitogenesis was found. Two other phosphoproteins varied with growth conditions. One is the pp80 kinase C substrate which was found in the untransformed (A31) 3T3 cells early after stimulation but was absent in BPA31 cells. The other is an 18kD phosphoprotein that appeared shortly after quiescent 3T3 cells were stimulated.
Regulation of thyroidal inducibility of Na,K‐ATPase and binding of epidermal growth factor in wild‐type and cold‐sensitive E1a mutant type 5 adenovirus‐transformed CREF cells
Authors: Guernsey Duane L. , Duigou Gregory J. , Babiss Lee E. , Fisher Paul B.
Published: February 2005

We have analyzed the relationship between expression of the transformed phenotype and thyroid hormone (triiodothyronine, T3) inducibility of Na,K‐ATPase and binding of 125I‐epidermal growth factor (EGF) to cell membrane receptors in wild‐type (wt) and mutant type 5 adenovirus (Ad5)‐transformed CREF cells displaying a cold‐sensitive (cs) expression of the transformed phenotype. CREF cells respond to thyroid hormone treatment with increased Na,K‐ATPase activity and bind similar levels of 125I‐EGF at 32°C, 37°C and 39.5°C. In contrast, CREF cells transformed by wt Ad5 or the E1A plus E1b‐transforming genes of wt Ad5 are refractile to T3 treatment and bind lower levels of 125I‐EGF than CREF cells at all three temperatures. By employing a series of cloned CREF cell lines transformed by a host‐range cold‐sensitive mutant virus, H5hr1 or H5dl101, or the E1a or E1a plus E1b genes from these viruses, we have investigated expression of the transformed state and its relationship with hormone inducibility and EGF binding. When cs virus‐, cs E1a‐ or cs E1a plus E1b‐transformed CREF clones were grown at 32°C, a nonpermissive transforming temperature in which cs‐transformed cells exhibit properties similar to untransformed CREF cells, T3 induced Na,K‐ATPase activity and these cells bound similar levels of 125I‐EGF as CREF cells. However, when cs virus‐and cs Ela plus Elb‐transformed CREF clones were incubated at 37°C or 39.5°C, temperatures at which cs‐transformed cells exhibit properties similar to wt Ad5‐transformed CREF cells, they did not respond to T3 and bound lower levels of 125I‐EGF than CREF cells. In the case of cs E1a‐transformed CREF clones, thyroid hormone responsiveness was observed at both 32°C and 37°C, but not at 39.5°C. By performing temperature shift experiments‐i.e. 32°C to 37°C, 32°C to 39.5°C, 37°C to 32°C, and 39.5°C to 32°C, it was demonstrated that after a shift from lower to higher temperature a 24‐hr lag period was required for cs‐transformed CREF cells to lose T3 inducibility and exhibit reduced EGF binding, whereas 96 hr after a shift from higher to lower temperature a 96‐hr lag period was required for cs‐transformed cells to regain T3 inducibility and increased 125I‐EGF binding. Flow cytometric analysis indicated that the ability of T3 to induce Na,K‐ATPase in CREF cells was not a consequence of alterations in cell‐cycle distribution. These results indicate that the loss of thyroid hormone inducibility of Na,K‐ATPase and a decreased binding of 125I‐EGF are properties which correlate with expression of the transformed phenotype in Ad5‐transformed CREF cells.
Recombinant human interleukin‐1 inhibits the induction by dexamethasone of alkaline phosphatase activity in murine capillary endothelial cells
Authors: Mulkins Mary A. , Allison Anthony C.
Published: February 2005

A mutual antagonism exists between interleukin‐1s (IL‐1s) as pro‐inflammatory and glucocorticoids as anti‐inflammatory mediators. This report examines the effects of IL‐1 on the induction by dexamethasone of alkaline phosphatase in LEII murine endothelial cells. Dexamethasone increases the specific activity of alkaline phosphatase in a time‐ and dose‐dependent fashion (maximum 14‐fold induction at 10−6 M, IC50 = 10−8 M), and this induction can be completely inhibited by simultaneous incubation with picomolar concentrations of recombinant human IL‐1α or IL‐1β. This IL‐1‐mediated antagonism of dexamethasone activity is not due to a down‐regulation of glucocorticoid receptors in the cell line used, because the number of receptors and their affinity for dexamethasone is unchanged in IL‐1‐treated cells. However, induction of alkaline phosphatase by dexamethasone in LEII cells is receptor‐mediated, since it can also be inhibited by glucocorticoid‐receptor antagonists.
The cell cycle associated change of the Ki‐67 reactive nuclear antigen expression
Authors: Sasaki Kohsuke , Murakami Tomoyuki , Kawasaki Masahiro , Takahashi Manabu
Published: February 2005

The change of human nuclear antigen expression in proliferating cells recognized by a monoclonal antibody, Ki‐67, during the cell cycle was investigated in HeLa S3 cells using a bivariate‐flow‐cytometric analysis. The antigen was immunocytochemically stained with FITC, and DNA was stained with propidium iodide (PI). The expression of the antigen increased with cell‐cycle progression, especially in the latter half of S‐phase and reached a maximum at G2M‐phase, although its content varied greatly from cell to cell. The cells in which DNA synthesis was inhibited by treatment with hydroxyurea increased markedly in the antigen expression (as compared to untreated cells). Treatment with adriamycin also elevated the antigen content. After digestion with DNase I, but not after RNase treatment, FITC fluorescence from the antigen disappeared. These results suggest that the Ki‐67 antigen is bound to DNA and its expression does not depend on DNA replication. Although the biological implications of the antigen remain unresolved, the antigen may be considered to be essential for maintaining the proliferating state of cells.
Isolation of Thy‐1 caps and analysis of their phospholipid composition in mouse T‐lymphoma cells
Authors: Suchard Suzanne J. , Lo Hilda K. , Bourguignon Lilly Y. W.
Published: February 2005

In this study we have used a density perturbation method to isolate anti‐Thy‐1 antibody‐induced Thy‐1 caps from mouse T‐lymphoma cells in the absence of detergents, and then compared the phospholipid composit on of these capped membranes with that of uncapped membranes. Initial phospholipid analysis by two‐dimensional thin layer chromatography (2‐D TLC) reveals a significant increase in the amount of 32P‐labeled phosphatidylcholine in the Thy‐1 capped membrane. In contrast, no significant changes are observed in the labeling of phosphatidylserine, phosphatidylethanolamine, or the sphingomyelins. Therefore, it is suggested that phosphatidylcholine may be involved in the organization and/or regulation of Thy‐1 antigen redistribution. The composition of phosphoinositide in uncapped and capped membranes was analysed separately using one‐dimensional thin layer chromatography (1‐D TLC) to resolve phosphatidylinositol (PI), phosphatidylinositol 4‐phosphate (PIP), and phosphatidylinositol 4, 5‐bisphosphate (PIP2) from all other phospholipids. This analysis reveals a significant reduction in levels of PIP and PIP2, but not PI, in Thy‐1 caps. Through the use of ion exchange column chromatography, we have found an increased production of all three species of inositol phosphates during anti‐Thy‐1 antibody‐induced capping. Inositol 1, 4, 5 ‐triphosphate (IP3) shows the most significant increase, compared to the much smaller increases in inositol 4, 5‐bisphosphate (IP2) and inositol monophosphate (IP). These results suggest that the binding of anti‐Thy‐1 antibody to Thy‐1 antigen activates phospholipase C which, in turn, initiates polyphosphoinositide turnover and IP3 production. It is proposed that these observed effects are the result of early signal transducing events which are prerequisite steps in Thy‐1 receptor cap formation.
Synergistic antiproliferative effects of glucocorticoids and interferon‐α on some lymphoid cell lines
Authors: Elliott Keith R. F. , Princler Gerald L. , Urba Walter J. , Faltynek Connie R.
Published: February 2005

The Daudi B lymphoblastoid cell line was previously demonstrated to be highly sensitive to the antiproliferative effect of recombinant interferon‐αA (rlFN‐α). In the present study, glucocorticoid hormones were shown to act synergistically with rlFN‐α to further increase the sensitivity of Daudi cells to rlFN‐α.At 106M, dexamethasone, prednisolone, or hydrocortisone alone had little effect on Daudi cell growth, but they greatly potentiated the antiproliferative activity of rlFN‐α. The synergy between rlFN‐α and glucocorti‐coids on Daudi cells was not related to the inhibitory effects of glucocorticoids on prostaglandin or leukotriene synthesis, since no synergy was observed between rlFN‐α and indomethacin or nordihydroguaiaretic acid. Glucocorticoids and rlFN‐α also had appreciable synergistic antiproliferative effects on two out of five other IFN‐sensitive lymphoid cell lines. When Raji B lymphoblastoid cells, which were quite resistant to the antiproliferative effect of rlFN‐α, were treated with the combination of glucocorticoids and rlFN‐α, no significant synergistic effects were observed. The synergistic antiproliferative effects of glucocorticoids and rlFN‐α observed with some IFN‐sensitive lymphoid cell lines in this in vitro study may have clinical relevance in the treatment of certain lymphoid malignancies that are sensitive to rlFN‐α therapy.
Inhibition of growth and induction of enzyme activities in a clonal human hepatoma cell line (Li‐7A): Comparison of the effects of epidermal growth factor and an anti‐epidermal growth factor receptor antibody
Authors: Knowles Aileen F.
Published: February 2005

A cloned human hepatoma cell line (Li‐7A), possessing epidermal growth factor (EGF) receptors numbering in the range of 10‐20 pmol/106 cells, was inhibited in its growth by EGF as well as an antagonist monoclonal antibody (MoAb) to the EGF receptor. The mode of action of the two ligands of EGF receptors appeared to be different as indicated by the following results: (1) EGF induced marked alteration in cell morphology, whereas the antibody did not; (2) cellular protein accumulated in the EGF‐treated cells but not in the antibody treated cells; and (3) ectoATPase activities were greatly enhanced in Li‐7A cells treated with EGF and cholera toxin but were unaffected in cells treated with antibody and cholera toxin. The last result also suggests that expression of ectoATPase activities is under the regulation of both EGF and cholera toxin. Li‐7A cells provide an additional valuable experimental system for the study of EGF action, as well as the interactive effects of EGF and cholera toxin. The enrichment of the ATPase activities in the EGF‐cholera toxin‐treated cells can be exploited for the detailed study and isolation of these enzymes and elucidation of their physiological functions.
Interferon and phorbol esters down‐regulate slgM expression by independent pathways
Authors: Schaffer Frederick M. , Benedict Stephen H. , Petsche Dieter , Lau Alan , Williams Bryan R. G. , Mills Gordon B. , Gelfand Erwin W.
Published: February 2005

We studied the effects of recombinant, interferon, 12‐O‐tetradecanoylphorbol13‐acetate (TPA), and phorbol 12,13 dibutyrate (PDB) on surface immunoglobulin expression by Daudi cells. Incubation of cells with recombinant alpha2interferon (IFN‐α2) caused a 2.5‐fold (60%) decrease in slgM expression as measured by relative fluorescence index (RFI) using a flow cytometer. This decrease in slgM expression was independent of inhibitory effects on proliferation and cell cycle progression. TPA or PDB also caused a threefold (67%) decrease in slgM expression, while enhancing proliferation and cell cycle progression. Coincubation of cells with IFN‐α2 or TPA decreased slgM expression by more than fourfold (>75%), which was greater than the decrease induced by the optimal concentration of either agent alone. Molecular studies demonstrated that the treatment of cells with IFN‐α2 or TPA decreased the steady‐state levels of mRNA for the heavy chain of IgM (cμ), suggesting that down‐regulation of slgM occurred at a pretranslational level. Activation of the cell membrane sodium/proton antiport did not play an integral role in the IFN‐α2 or phorbol‐ester‐induced pathway of slgM down‐regulation. Whereas IFN‐α2 induced an increase in the activity of 2′,5′‐oligoadenylate (2–5A) synthetase, the addition of TPA to IFN‐α2 caused a significant decrease in the activity of this enzyme. Although IFN‐α2 and TPA exhibited additive effects on slgM expression, they had opposing effects on cell proliferation, cell cycle progression, and induction of 2–5A synthetase activity, suggesting that these agents down‐regulate slgM expression through independent pathways.
Evidence of intracellular and trans‐acting differentiation‐inducing activity in human promyelocytic leukemia HL‐60 cells: Its possible involvement in process of cell differentiation from a commitment step to a phenotype‐expression step
Authors: Okazaki Toshiro , Kato Yoshiki , Tashima Masaro , Sawada Hiroyoshi , Uchino Haruto
Published: February 2005

We previously reported that human promyelocytic leukemia HL‐60 cells, when treated with various inducers in magnesium‐deficient medium, became committed to differentiate but did not express the differentiation‐related phenotypes (Okazaki et al., J. Cell. Physiol., 131:50–57, 1987). In the present study we demonstrated the existence of an intracellular differentiation‐inducing activity (int‐DIA) in differentiation‐committed phenotype‐nonexpressing HL‐60 cells by using cybrid formation between untreated HL‐60 cells and cytoplasts from HL‐60 cells treated in magnesium‐deficient medium with 100 nM 1α,25‐dihydroxyvitamin D3 (1,25(OH)2D3). Cell extracts from similarly treated HL‐60 cells also showed int‐DIA, which when added (10 mg total protein/ml) to culture of untreated HL‐60 cells, could increase the percentages of nitroblue tetrazolium (NBT)‐ and nonspecific esterase (NSE)‐positive cells from 1% to 53%, and from 0 to 32%, respectively. They also induced differentiation of human monoblastic leukemia U‐937 cells and of human myeloblastic leukemia KG‐1 cells but not of erythroleukemia K‐562 cells. These results suggested that the int‐DIA had a common effect on differentiation induction in several human myeloid cell lines and may be involved in inducing cells to proceed from a commitment to a phenotype‐expression step during human myeloid cell differentiation.
Combinative ligand‐receptor interactions: Epinephrine depresses RAW264 macrophage antibody‐dependent phagocytosis in the absence and presence of met‐enkephalin
Authors: Petty Howard R. , Berg Kelly A.
Published: February 2005

Macrophages have been shown to possess cell surface receptors for opiates and catecholamines. The abilities of these ligands to affect RAW264 macrophage antibody‐dependent effector activity directed against sheep red blood cells were tested. Phagocytosis was measured by the uptake of 51Cr labeled erythrocytes and optical microscopy. Cytolysis was measured by 51Cr‐release assays. Met‐enkephalin increased specific antibody‐dependent phagocytosis in a dose‐dependent fashion; the optimal dose was found to be 10−8 M. Epinephrine diminished phagocytosis in a dose‐dependent manner exhibiting a maximal inhibition at 10−4–10−5 M. This inhibition can be blocked by propranolol. The combined effects of simultaneous treatment with met‐enkephalin and epinephrine were measured. At the several doses tested, the combined effects of these two ligands on the amount of phagocytosis were equivalent to or more inhibitory than epinephrine alone. Thioglycolate‐elicited murine peritoneal macrophages demonstrated similar responses to epinephrine, met‐enkephalin, and their combination. Therefore, in vitro models more closely approximating in vivo neuroregulation of macrophage function demonstrate phagocytic inhibition.
Characterization of the rebinding of 125I‐epidermal growth factor released from BALB/c‐3T3 cells following accumulation in the presence of chloroquine
Authors: Cooper Janet L. , Wakshull Eric , Wharton Walker
Published: February 2005

Lysosomotropic amines, such as chloroquine and methylamine, increase the intracellular accumulation of 125I‐EGF by inhibiting lysosomal degradation. It has been shown previously that BALB/c‐3T3 cells, prelabeled at 4°C with 125I‐EGF for 3 h and subsequently chased at 37°C in the presence of chloroquine, internalized the surface bound 125I‐EGF which was subsequently released into the extracellular medium in a high molecular weight form which co‐migrated with native 125I‐EGF. The secreted 125I‐EGF rebound to the cells from which it was released more efficiently than does peptide in the extracellular media. We now show that when the BALB/c‐3T3 cells were prelabeled at 37°C for 2 h in the presence of chloroquine, the internalized 125I‐EGF released into the medium was in a high molecular weight form which co‐migrated with native 125I‐EGF and did not rebind anymore efficiently than did peptide in the extracellular media. This lack of rebinding was not due to an alteration in the 125I‐EGF molecule since it was still capable of rebinding to naive A431 cells, nor was it due to the exhaustion of EGF receptors on the BALB/c‐3T3 cells. The inhibition of rebinding was observed only when the cells were treated with EGF in the presence of chloroquine, and was not due to a general down‐regulation of membrane receptors. The differences between the rebinding of 125I‐EGF at 4°C and 37°C suggest that EGF may be processed via different pathways in the cell.
Loss of three major auto phosphorylation sites in the EGF receptor does not block the mitogenic action of EGF
Authors: Clark Stella , Cheng Dorothy J. , Hsuan J. Justin , Haley John D. , Waterfield Michael D.
Published: February 2005

The EGF receptor cDNA has been transfected into receptor‐negative Chinese hamster ovary (CHO) cells. A mutant cell line (CHO 11) was isolated that expresses a receptor of lower molecular weight than the EGF receptor from A431 cells (150,000 daltons compared to 170,000 daltons) and which appeared as a doublet on SDS‐PAGE. By digestion of the receptor with endoglycosidase F it was shown that an altered pattern of glycosylation could not account for the smaller size of the protein, although it could explain the appearance of the CHO 11 receptor as a doublet protein. A deletion was located to the transfected cDNA and shown to involve the removal of coding sequences for the most C‐terminal 20,000 daltons of the EGF receptor, which contains the three major autophosphoryation sites. Despite the loss of these sites the EGF receptor from CHO 11 cells binds EGF, demonstrates protein tyrosine kinase activity in response to EGF, and transduces a mitogenic signal. The CHO 11 receptor protein is still autophosphorylated on alternative tyrosine residues. We conclude that phosphorylation of the three tyrosines (P1, P2, and P3) in the C‐terminal domain of the receptor is not required for signal transduction by the EGF receptor in these cells.
Potassium inhibition of transforming protein P85gag‐mos and reversal of the transformed phenotype in 6m2 cells
Authors: Lai Chiu‐Nan , Gallick Gary E. , Maxwell Steve A. , Brinkley Bill R. , Becker Frederick F.
Published: February 2005

K+ at high concentrations (52–72 mM hypertonic KCI) has been reported to induce reverse transformation in the 6m2 cell, which is a clone of normal rat kidney cells (NRK) infected with a temperature‐sensitive transformation virus. When exposed to high K+, 6m2 cells grown at the permissive temperature (33°C) exhibit normal morphology and reduced soft agar growth, characteristics of cells grown at nonpermissive temperature (39°C). In the current study, flattening of cells and rearrangement of surface microvilli were demonstrated by scanning electron microscopy to occur within 6 hr of exposure to high K+, similar to the effect of temperature shift to 39°C. Exposure to K+ resulted in a 90% inhibition of P85gag‐mos‐associated serine kinase activity within 5 min, with a subsequent reduction of up to 75% of the synthesis of this protein. These alterations in the putative transforming protein were similar to those induced by temperature shift and were considered to be the basis for retrotransformation. The cell microtubular system and F‐actin cables were affected more slowly by K+ than by a temperature shift to 39°C. The former did not achieve the fine reticulum network seen in NRK cells until 72 hr later, but the latter remained aberrant. The effect on the enzyme might be mediated by alteration in phosphorylation, but the mechanism by which kinase inactivation induces retrotransformation is not yet known.
Rapid expression of ornithine decarboxylase mRNA in a macrophage‐like cell line: cAMP repression of the requirement for prior protein synthesis
Authors: Shurtleff Sheila A. , McElwain Chris M. , Taffet Steven M.
Published: February 2005

Ornithine decarboxylase (ODC) activity was rapidly induced in the RAW264 marcophage‐like cell line after treatment with bacterial lipopolysaccharide (LPS). ODC mRNA leveis were determined by isolating cellular RNA, followed by Northern blot and dot blot analysis using a 32P‐labeled cDNA probe. ODC mRNA levels increased within 1 hour of stimulation of RAW264 cells with 1.0 μg/ml LPS. Transcription rate analysis on isolated nuclei indicated that an increase in transcription rate contributed to this increase in ODC mRNA. ODC mRNA levels continued to rise for 4 hours, peaking at eight times the basal level. ODC mRNA appeared as a single 2.2‐kb band prior to stimulation. After stimulation, the 2.2‐kb band intensified, and a second (2.7 kb) band was seen by Northern gel analysis. Similar induction was demonstrated when 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA) was used as the stimulus. The induction of ODC mRNA by either LPS or TPA was blocked by the addition of cycloheximide (25 μg/ml) or anisomycin (0.1 mM) to the cellular incubation mixture. This indicated that protein synthesis was required as a prerequisite to LPS or TPA induction of ODC mRNA. Experiments in which cycloheximide addition was delayed after LPS treatment indicated that some of the required protein synthesis occurred within the first 30 minutes and that complete expression of ODC mRNA was possible if protein synthesis continued for at least 2 hours before cycloheximide was added. Stimulation with 8‐bromo‐cAMP in addition to LPS has been shown to enhance the induction of ODC over that induced by LPS or TPA alone. It was not possible to block ODC mRNA induction with cycloheximide or anisomycin after treatment with the combined stimulus of LPS and cAMP or TPA and cAMP, indicating that protein synthesis was not required when cAMP was used as a coinducer. Thus, we have shown that in the same cell, ODC mRNA can be induced by two different pathways, one requiring protein synthesis and one not requiring protein synthesis.
Trans‐stimulation of L‐system amino acid transport in normal and chronic leukemic human lymphocytes: Phorbol ester restores function in CLL
Authors: Segel George B. , Woodlock Timothy J. , Lichtman Marshall A.
Published: February 2005

Chron c lymphocytic leukemia (CLL) B‐lymphocytes have a unique and specific diminution of L‐system (leucine favoring) amino acid uptake; the maximal velocity is approximately 10% of normal B‐lymphocytes. Treatment of CLL B‐cells with the maturational agent, tetradecanoyl phorbol acetate, results in restoration of L‐system amino acid uptake to normal velocity. To further characterize the effect of phorbol ester on the L‐system of CLL B‐cells, we have examined the ability of normal and CLL lymphocytes to exchange intracellular for extracellular amino acids by the L‐system (trans‐stimulation). A 60% increase in L‐system uptake was noted in normal B‐ and T‐lymphocytes in the presence of a high intracellular concentration of 2‐amino‐2‐carboxy‐bicycloheptane (BCH), a largely L‐system‐specific substrate. L‐system transport was not trans‐stimulated in CLL B‐lymphocytes. Phorbol ester treatment restored L‐system uptake in CLL to a normal Vmax of 900 umol/liter cell water per minute in the absence of BCH loading. The Vmax could be increased further to 2,400 if phorbol ester‐treated CLL cells were loaded with BCH. Hence, phorbol esters result not only in a normalization of L‐system uptake in CLL B‐cells but the transport system demonstrates exchange rates comparable to normal lymphocytes.
Effects of confluence on phosphate transport capacity in cultured renal cell lines
Authors: Scheinman Steven J.
Published: February 2005

The LLC‐PK1 cell line transports phosphate (Pi), glucose, and amino acids using carriers similar to those in proximal tubular cells. Others have reported that when monolayers reach confluence, hexose transport increases and activity of the A‐amino acid transporter falls. The present study evaluates Pi uptake by two continuous cell lines derived from renal proximal tubule, and demonstrates that phosphate uptake falls sharply upon reaching confluence in LLC‐PK1 cells but not in cultured opossum kidney (OK) cells. The fall in Pi uptake in LLC‐PK1 cells at confluence represents a halving in Vmax for Na‐dependent phosphate uptake (2.33 vs. 5.00 nmol/mg protein/5 min) without a change in Km (82 vs. 94 μM). Suppression of phosphate transport in confluent monolayers of LLC‐PK1 cells is completely reversed by bringing the cells into suspension. As has been shown for the phorbol ester 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA), exposure of monolayers to serum stimulates phosphate uptake, but unlike phorbol ester, serum does so without stimulating alanine uptake. OK cells differ from LLC‐PK1 in that no change occurs in Pi uptake at confluence, although they resemble LLC‐PK1 cells in that sugar uptake rises and alanine uptake falls at confluence. The different temporal patterns for Pi uptake in the two cell lines indicates that developmental change in the uptake of Pi is not linked to that of glucose or alanine.
Energetic mechanism of system a amino acid transport in normal and transformed mouse fibroblasts
Authors: Leister Kirk J. , Schenerman Mark A. , Racker Efraim
Published: February 2005

Ouabain treatment (0.4 mM) of normal and transformed C3H‐10T1/2 cells caused a progressive increase in 2‐aminoisobutyrate (AIB) transport reaching a maximum after 16 to 18 h exposure. There was a virtually complete blockage of this stimulated rate when 3 μM cycloheximide (CHX) was added together with ouabain at T = 0. In the transformed cell, addition of CHX after 14 h had no effect; in the normal cell, it inhibited (ca. 50%) the final AIB transport rate achieved after 24 h. The t½ for reaching maximal activity (insensitive to CHX exposure) was thus shifted from 8 h in the transformed cell to 15 h in the normal cell. Since the rate of achieving maximal activity in the absence of CHX was about the same in the two cells, the shift in t½ in the presence of CHX suggests that the rate of degradation is more rapid in the normal cell. Following ouabain treatment, the apparent Km for Na+ was decreased in both cells. The Km returned to the basal level 1 h after ouabain removal in the normal cell, but remained low in the transformed cell during this time period. The stimulation of AIB transport following ouabain removal was largely abolished by a proton ionophore (1799), a lipophilic cation (tetraphenyl‐phosphonium), or ouabain. These results suggest that, under the conditions of ouabain stress, there is a switch in the bioenergetic mechanism. The Na+/K+ pump and System A transporter appear to be linked and the membrane potential generated by the Na+/K+ pump activity becomes a major driving force for AIB uptake.