bluuebunny/crossref_metadata_2025_split huggingface dataset:
Clonal growth of lymphoid cells in serum‐free media requires elimination of H2O2 toxicity
Authors: Darfler Frederick J. , Insel Paul A. 
Published: February 2005

AbstractWe have recently described the development of a serum‐free medium that contains casein, insulin, testosterone, transferrin, and linoleic acid and that supports the long‐term growth of a wide variety of lymphoid cells. A problem of culturing cells in this medium is the difficulty of cloning cells or growing cells at low density. We now describe the formulation of a chemically defined medium that supports the clonal growth of the murine S49 T lymphoma cell line. This medium contains catalase, insulin, transferrin, testosterone, Na2SeO3, and dilinoleoyl phosphatidylcholine and contains less than 50 μg/ml total protein. The two novel additions in this medium are catalase, which replaces casein and dilinoleoyl phosphatidylcholine, which substitutes for linoleic acid in this defined medium. In addition to S49 cells, the medium described above supports the long‐term growth of other lymphoid cells, including human and murine hybridomas. We propose that catalase functions to degrade H2O2 that is present in the cultures and that casein, bovine serum albumin, and other proteins commonly included in media for cultured cells may also scavenge H2O2. Na2SeO3 also partially protects against the death of cells at clonal density and this protection may, like catalase, be due to removal of H2O2. Our results suggest that H2O2 is an important cytotoxic agent that prevents growth of lymphoid cells during culture in serum‐free media and perhaps in serum‐containing media as well.
Segregation of mouse hemopoietic progenitor cells using the monoclonal antibody, YBM/42
Authors: Watt Suzanne M. , Gilmore David J. , Metcalf Donald , Cobbold Stephen P. , Hoang Trang K. , Waldmann Herman 
Published: February 2005

AbstractA rat monoclonal antibody, YBM/42, directed against mouse leukocyte common antigen, was used for the analysis and separation of hemopoietic progenitor cells from mouse bone marrow and fetal liver. Cells were fractionated on a FACS‐II cell sorter and the resulting subpopulations examined for their morphology and ability to form colonies in agar (for day 7 colonies) and methylcellulose (for day 2 erythroid clones). The antibody bound to all leukocytes, including blast cells and day 7 hemopoietic progenitor cells (day 7 colony forming cells, CFC), but not to erythrocytes or nucleated erythroid cells. This antibody can be used to advantage to enrich for early progenitor cells from mouse fetal liver, in which the majority of cells (70%) are nucleated erythroid cells. In day 12 fetal liver, approximately 10% of all cells bind this antibody strongly and, of these approximately 70% are blast cells. Contained within this positive population are 95% of all day 7 CFC. In the most enriched fraction about 20% of the cells formed day 7 colonies. This represents a 25‐fold enrichment over unsorted fetal liver. The negative fractions contain 94% of all cells forming erythroid clones (≥8 cells) on day 2 of culture (day 2 CFU‐E). In the most enriched fraction, 20% of the cells are day 2 CFU‐E. Day 7 CFC can therefore be well separated from day 2 CFU‐E, with good recovery of both cell types, by use of a single label. Day 7 colony forming cells were classified as granulocyte (G‐CFC), macrophage (M‐CFC), mixed granulocyte/macrophage (GM‐CFC), pure erythroid (E), or mixed erythroid (Emix). A high enrichment for multipotential cells is achieved and constitues 3–5% of cells in the most enriched fraction. Most types of day 7 CFC could not be separated with YMB/42, but GM‐CFC and M‐CFC exhibit a broader distribution than the other CFC with regard to fluorescence intensity. This implicit heterogeneity in GM‐CFC and M‐CFC is further substantiated by the finding that myeloid progenitors in the different FACS fractions also share a differential reactivity to different sources of growth factors.
Glucocorticoids and prostaglandins inhibit the induction of macrophage DNA synthesis by macrophage growth factor and phorbol ester
Authors: Hamilton John A. 
Published: February 2005

AbstractThe tumor‐promoting phorbol ester, 12‐0‐tetradecanoyl‐phorbol‐13‐acetate (TPA), stimulates starch‐elicited mouse peritoneal macrophages to undergo DNA synthesis in vitro, apparently without the generation of an endogenous macrophage growth factor (MGF). No evidence was found for any synergistic interaction between TPA and exogenous colony stimulating factors (CSFs) for macrophage DNA synthesis. Low concentrations of glucocorticoids and also prostaglandins E1 and E2 suppress both the CSF‐1‐stimulated and the TPA‐stimulated macrophage DNA synthesis; these same drugs inhibit the CSF‐1‐mediated and TPA‐mediated enhancement of macrophage plasminogen activator (PA) activity. Thus glucocorticoids and prostaglandins E1 and E2 oppose the action of growth factors and the tumor promoter on macrophage and precursor cell function.
Culture of quiescent arterial smooth muscle cells in a defined serum‐free medium
Authors: Libby Peter , O'Brien Kathleen V. 
Published: February 2005

AbstractAn ideal medium for metabolic studies would maintain cultured vascular smooth muscle cells in a quiescent, viable state, as they are in normal arteries in vivo, and would be chemically defined so that the concentrations of hormones and nutrients could be manipulated precisely. In unsupplemented serum‐free media these cultures lose protein and DNA, indicating impaired viability. Addition of maximally effective concentrations of insulin (10−6 M) and transferrin (5 m̈g/ml) prevents loss of DNA and produces near neutral protein balance. Further addition of ascorbic acid (10−4 M) actually promotes net gain of protein with little or no increase in DNA. Ascorbate consistently increased noncollagen protein synthesis by cultured aortic smooth muscle cells. This novel action of the vitamin did not require insulin but was additive to the effect of this hormone, and was produced by isoascorbate, but not by a variety of other reducing agents. Thus, vascular smooth muscle cells can be maintained in a quiescent but noncatabolic state in simple chemically defined culture media. This finding should facilitate studies of the effects of nutrients and hormones on the metabolism of these cells under conditions that resemble those in the normal artery in vivo. Such an approach may also prove valuable for culture of other differentiated cell types that do not usually divide in the intact organism.
Stimulation of tyrosinase activity of cultured melanoma cells by lysosomotropic agents
Authors: Saeki Hisaaki , Oikawa Atsushi 
Published: February 2005

AbstractThe tyrosinase (EC 1.14.18.1) activity of cell‐free extracts (TyH) of B16 melanoma cells cultured in the presence of 5 to 10 mM ammonium chloride was considerably higher than that of cells from control cultures. This increase in TyH in the presence of ammonium chloride seemed to be due to de novo synthesis of the enzyme, because it was inhibited by 1 μg/ml of cycloheximide. In the presence of the latter, however, ammonium chloride did increase the tyrosinase activity of living cells in culture (TyC) resulting in about threefold increase in the TyC/TyH ratio, a measure of the extent of tyrosinase reaction exerted by the enzyme present in living cells. This higher TyC/TyH ratio induced by ammonium chloride was also observed in the absence of cycloheximide. Similar increases in TyH, TyC, and TyC/TyH occurred in the presence of methylamine or ethylamine instead of ammonium chloride, but not in the presence of tetraethylammonium chloride, and also in culture medium of higher pH. The apparently similar effects of lysosomotropic bases and medium of higher pH on the TyC/TyH ratio suggest that there are some mechanisms that control the intramelanosomal pH lower than the cytoplasmic pH.
Microtubules and lymphocyte responses: Effect of colchicine and taxol on mitogen‐induced human lymphocyte activation and proliferation
Authors: Cuthbert Jennifer A. , Shay Jerry W. 
Published: February 2005

AbstractThe role of microtubules in mitogen‐induced human lymphocyte activation and proliferation was examined. The effect of colchicine, a microtubule‐disrupting agent, was compared with taxol, a microtubule‐stabilizing drug, and with isaxonine (N‐isopropyl‐amino‐2‐pyrimidine orthophosphate), a proposed microtubular‐active drug. Lymphocyte proliferation, assessed by measuring the increase in the number of cells in mitogen‐stimulated cultures, was completely suppressed by both colchicine and taxol (100 nM) whereas significant inhibition by isaxonine required much higher concentrations (5 mM). In order to characterize the inhibition, initial lymphocyte blast transformation and subsequent DNA synthesis were investigated. Neither colchicine nor taxol inhibited lymphocyte blast transformation assessed by quantitating the change in volume of the stimulated cells after a 24‐hour incubation. In contrast, isaxonine (2–5 mM) suppressed blast transformation. Initial DNA synthesis, evaluated by measuring the cumulative incorporation of [3H]thymidine between 30 and 48 hours of culture, was inhibited in a concentration‐dependent manner by both isaxonine and colchicine but not by taxol. Electron microscopic studies confirmed that both taxol and colchicine (10 nM) arrested the responding lymphocytes in mitosis, and that isaxonine inhibited initial activation. These results suggest that normal microtubule function is only necessary for cell division and that drug effects on blast transformation and initial DNA synthesis are unrelated to microtubules.
Factors influencing calcium‐induced terminal differentiation in cultured mouse epidermal cells
Authors: Hennings Henry , Holbrook Karen A. , Yuspa Stuart H. 
Published: February 2005

AbstractMouse epidermal cells can be grown as a proliferating monolayer in medium containing 0.02‐0.1 mM calcium. Terminal differentiation of these cells with formation of cornified cells and cell death is induced by elevating calcium in the medium to >0.1 mM. A variety of agents were studied as potential modifiers of this calcium‐induced terminal differentiation. Other than calcium, no cation tested was active in inducing or preventing epidermal maturation. Modifiers of calcium or sodium fluxes, local anesthetics and protease inhibitors were also without effect. Modulators or analogues of cyclic nucleo‐tides did not influence epidermal differentiation, and cyclic nucleotide levels did not change significantly in the first 10 min after increasing calcium.Effective inhibition of calcium‐induced differentiation, as estimated by morphology, ultrastructure and cornified envelope formation, was seen with the divalent cation ionophore A23187 and the Na+K+ATPase inhibitor ouabain. The well‐known effects of ouabain on intracellular sodium and potassium suggested the possible involvement of these ions in the program of calcium‐induced epidermal maturation. The increase in medium calcium produced an elevation of both intracellular sodium and potassium within 12–24 hours. The calcium‐induced increase in intracellular potassium appears to be the more relevant of these changes since the increase was blocked by both ouabain and A23187. Other inhibitors of calcium‐induced differentiation, including harmaline, 8(diethylamino) octyl 3,4,5‐trimethoxybenzoate hydrochloride (TMB‐8) and low potassium medium, also blocked the rise of intracellular potassium. The five inhibitors had no consistent effect on intracellular sodium. Thus, elevated intracellular potassium may be necessary for the later stages of epidermal differentiation. However, neither ouabain nor A23187 affected the assembly of desmosomes, the earliest ultrastructural change noted after increasing medium calcium. This rapid change in cell‐cell contact, beginning within minutes after calcium elevation, appears to be independent of changes in sodium and potassium, but may instead be modulated by increased calcium at the cell surface.
Deposition of fibronectin on material surfaces exposed to plasma: Quantitative and biological studies
Authors: Grinnell Frederick , Phan Trung V. 
Published: February 2005

AbstractExperiments were carried out to characterize plasma fibronectin deposition onto material surfaces exposed to plasma solutions. Under nonclotting conditions, the amount of fibronectin adsorption on the surfaces, determined by an indirect radioactive antibody assay, was maximal at low plasma concentrations (0.1%). At higher concentrations of plasma, other plasma proteins appeared to compete with and inhibit adsorption of fibronectin. Biological activity (fibronectin‐promoted cell spreading) was also greatest at low plasma concentrations and decreased as the plasma concentration was raised. When surfaces were exposed to plasma under clotting conditions (i.e., addition of Ca2+ and thrombin), fibronectin deposition on the surfaces and biological activity remained constant or increased as the plasma concentration was raised. Based on indirect immunofluorescent antibody assays, the fibronectin deposited from clotting plasma appeared to be in a punctate distribution over the entire material surface and occasionally was associated with discrete fibrillar structures. The increased deposition of fibronectin from clotting plasma compared to nonclotting plasma (approximately a 10‐fold difference with 10% plasma) was partially a result of covalent crosslinking of fibronectin to fibrin based upon studies with putrescine added to inhibit crosslinking during clotting. On the other hand, the increase in biological activity that occurred if the surfaces were exposed to clotting plasma was completely inhibited by putrescine, indicating that fibronectin had to be crosslinked to fibrin to have biological activity under these conditions. Finally, fibronectin deposition also occurred on surfaces exposed to whole blood and was markedly enhanced when clotting occurred.
Modulation of cyclic 3′5′‐adenosine monophosphate in cultured renal (MDCK) cells by endogenous prostaglandins
Authors: Hassid Aviv 
Published: February 2005

AbstractCyclic AMP plays an important regulatory role in transport activity and proliferation of renal MDCK cells. This observation and the ability of MDCK cells to synthesize prostaglandins provided the impetus to assess the hypothesis that endogenous prostaglandins modulate cyclic AMP concentrations in MDCK cells. Three dissimilar cyclo‐oxygenase inhibitors: acetylsalicylate, 5, 8, 11, 14‐eicosatetraynoate, and meclofenamate significantly decreased cellular cyclic AMP levels and inhibited basal prostaglandin E2 synthesis. On the other hand, three dissimilar stimulators of prostaglandin synthesis: bradykinin, Ca2+‐ionophore A23187 and arachidonate, increased cellular cyclic AMP levels, and stimulated prostaglandin E2 synthesis. Acetylsalicylate inhibited the bradykinin‐ and A23187‐evoked increases of cyclic AMP as well as that of prostaglandin E2 synthesis. Prostaglandin E2, the major prostaglandin synthesized by MDCK cells, dose‐dependently increased cAMP levels when added exogenously. Acetylsalicylate did not significantly affect increases of cyclic AMP evoked by exogenous prostaglandin E2, documenting that acetylsalicylate inhibited cellular cyclic AMP levels by decreasing endogenous prostaglandin synthesis, rather than by a direct effect on cyclic AMP metabolism. Other prostaglandins synthesized by MDCK cells, i.e., prostaglandins I2, 6‐keto‐F1α, and F2α added exogenously did not significantly affect MDCK cyclic AMP levels, suggesting that they were probably ineffective as endogenous modulators of cyclic AMP. Moreover, endogenous prostaglandin E2 appeared four‐ to eightfold more potent as a stimulator of cyclic AMP levels than exogenous prostaglandin E2. The results support the concept that prostaglandin E2 is an endogenous cellular mediator that acts between an extracellular effector such as bradykinin and a second endogenous mediator of hormone action: cyclic AMP.
The centriolar antigen expression in TC7 cells is dependent on growth conditions and occurs at a particular time point in G1
Authors: Segarini P. R. , Shyamala M. , Atcheson C. L. , Kasamatsu H. 
Published: February 2005

AbstractThe correlation between growth conditions and centriolar antigen (Cag) expression in TC7 cells, a subline of African green monkey kidney cells, was studied. TC7 cells became quiescent when their number reached a high cell density, or when serum factors were depleted from media containing a low concentration of fetal bovine serum (FBS). There was a stoichiometric relationship between the concentration of serum present and the number of new cells produced. During proliferation, the projected cell area decreased as a function of cell density with two abrupt transitions. The first transition appeared to be independent of cell‐cell contact. However, the second transition seemed to occur mainly as a result of the limitation of the available substratum surface on which the cells could grow. The appearance of Cag in TC7 cells was found to be associated with the cells' growth conditions as well as with the particular phase in cell cycle. In an exponentially growing culture of cells with 10% FBS and in cells that were growth restricted due to a high density (above 2–3 × 105 cells/cm2), the incidence of cells with Cag‐positive staining was about 10–20%. It increased, however, to about 40–60% at cell densities between 2 × 104 cells/cm2 and 1 × 105 cells/cm2. The frequency of cells with positive Cag staining was as high as 80% in TC7 cells that were growth restricted by depletion of serum factor(s). Thus, the quiescent states attained by the two different growth restrictions seem to be different in their ability to express Cag. The frequency of Cag could be further increased by stimulating the quiescent cell population by FBS. In mitotically selected TC7 cells, Cag staining appeared about 4 hr after mitosis and about 2–4 hr before the onset of DNA synthesis. Thus, expression of Cag in TC7 cells is related to their growth conditions, and is characteristic of a part of the G1 phase of the cell cycle.
Regulation of cyclic adenosine 3′:5′‐monophosphate phosphodiesterases: Altered pattern in transformed myoblasts
Authors: Seth Prem K. , Rogers Jackilynn , Narindrasorasak Suree , Sanwal Bishnu D. 
Published: February 2005

AbstractRat skeletal myoblasts, L6 and L8, have two major forms of phosphodiesterases, PDE II and PDE III. Only the former is activated by treatment with proteases. When the myoblasts are exposed to cAMP for 10–16 h, the activity of PDE III increases considerably. This increase is accompanied by a loss of activatability of PDE II by proteases. Leupeptin prevents the increase in the levels of PDE III suggesting that a protease in vivo may be responsible for the formation of PDE III from PDE II. Spontaneously or Rous sarcoma virus‐transformed myoblasts, however, show altered regulation of the two forms of PDE. In the presence of cAMP in the medium, unlike the nontransformed cells, the levels of PDE III do not increase but the activity of PDE II rises. Simultaneously, PDE II becomes refractory to activation by proteases. The altered mode of PDE regulation in transformed cells is dominant in hybrids between normal and transformed myoblasts, which suggests that altered regulation is due to an “acquisition” of some new property by transformed cells.
Regulation of self‐renewal of human T lymphocyte colony‐forming units (TL‐CFUs)
Authors: Wu A. M. 
Published: February 2005

AbstractFormation of human T lymphocyte colonies in semisolid medium from T lymphocyte colony‐forming units (TL‐CFUs) under stimulation of phytohemagglutinin (PHA) has been reported by several authors. These TL‐CFUs were present in unsensitized lymphocyte populations. We report here that such TL‐CFUs are capable of renewing themselves. This was observed when colony cells from primary T cell colonies that developed in the presence of PHA were replated in methylcellulose medium containing irradiated autologous leukocytes and PHA. We have also been able to demonstrate serial transfer of TL‐CFU for up to six passages. At each passage, colony‐forming frequency was determined from the proportional relationship between the number of new colonies obtained and the number of colony cells plated. Examination of the number of new colonies derived from each individual T cell colony (“burst size of TL‐CFU”) showed that most colonies contained very few new TL‐CFU and only a very small number of colonies contained many new TL‐CFU. The distribution of burst sizes could be well fitted to a gamma distribution, in agreement with prediction from a stochastic model. We have identified an activity that enhanced the mean TL‐CFU burst size three to ten times. This work provides the first evidence in vitro that self‐renewal of human T lymphocyte progenitor cells can be stimulated by specific regulatory proteins.
Separate intracellular pathways for insulin receptor recycling and insulin degradation in isolated rat adipocytes
Authors: Marshall Stephen , Olefsky Jerrold M. 
Published: February 2005

AbstractTo gain insight into the sequence of events that follow endocytotic uptake of insulin receptor complexes, we have examined the interrelationship between the degradative pathway of the insulin ligand and the recycling pathway of the insulin receptor. Tris(hydroxymethyl)aminomethane and other nonamphoteric amines were found to selectively impair insulin receptor recycling while leaving the insulin‐degradative pathway intact. In contrast, low concentrations of the lysosomotropic agent chloroquine markedly inhibited intracellular insulin degradation but had little or no affect on the recycling of internalized receptors. Thus, we conclude: (1) that insulin dissociates from its receptor after endocytotic uptake and both receptor and ligand follow a separate intracellular pathway; and (2) that receptor recycling and insulin degradation can be selectively inhibited by Tris and chloroquine, respectively, highlighting the potential usefulness of these agents as intracellular probes in the study of receptor‐ligand metabolism.
Occurrence of a monocyte/macrophage colony‐stimulating factor in the continuous ambulatory peritoneal dialysis fluids and its chromatographic behaviors and antigenicity compared with human urinary colony‐stimulating factor
Authors: Sakai N. , Tsuneoka K. , Shikita M. 
Published: February 2005

AbstractContinuous ambulatory peritoneal dialysis (CAPD) fluid from three patients with chronic renal failure exhibited the activity of colony‐stimulating factor (CSF) in amounts varying from 5 to 40 units per ml. Like the CSF obtained from normal human urine, the peritoneal CSF predominantly produced monocyte/macrophage colonies in soft‐agar culture of mouse bone marrow cells. Semipurified peritoneal CSF showed its isoelectric point at pH 3.6 and 4.9 before and after the treatment with neuraminidase. Under the same conditions, the urinary CSF was focused at pH 3.1 and 4.6. The position of elution of the peritoneal and urinary CSF in ordinary gel‐filtration chromatography corresponded to a molecular weight of 62,000 and 117,000, whereas both CSFs exhibited a molecular weight of 28,000 upon gel‐filtration in the presence of 6 M guanidine HCI. Furthermore, the two CSFs from the human sources were neutralized by antimouse L cell CSF serum in the same manner. We conclude that the peritoneal CSF is a sialoglycoprotein which is nearly identical with the urinary CSF despite processing of the latter through kidneys.