bluuebunny/crossref_metadata_2025 huggingface dataset:
Temporal behavior of abrin in the intoxication of chinese hamster cells (line CHO)
Authors: Tsuzuki Junji , Wu Henry C.
Published: February 2005

Abrin, a potent cytotoxin, was utilized as a probe to elucidate the mechanism by which external proteins are delivered to the cytoplasm of mammalian cells. Abrin bound rapidly to the surface receptors of the Chinese hamster cells (line CHO) and appeared to be internalized immediately without any significant lag. The maximum level of abrin internalization was achieved within eight minutes, based on both biochemical and electron microscopic autoradiographic studies with [125|] abrin. About 10% of the silver grains of internalized [125|] abrin were associated with vesicular structures, irrespective of the incubation time. Inhibition of protein synthesis began 30 minutes postincubation, and this latent period was not dependent on extracellular toxin concentration. SDS‐polyacrylamide gel electrophoresis of the internalized [125|] abrin indicated that internalized abrin molecules remained intact even after two hours of incubation.
A survey of intracellular Na+ and K+ of various normal, transformed, and tumor cells
Authors: Moyer Mary Pat , Mover Rex C. , Waite Marilynn R. F.
Published: February 2005

Intracellular concentrations of Na+ and K+ of various normal, transformed, and tumor cell cultures were analyzed by atomic absorption spectrophotometry. In all of the cultures analyzed there were markedly different concentrations in the transformed and tumor cells when compared to their normal counterparts. Although increased Na+ was often observed, there were no definitive correlations between absolute ion concentrations, or Na+:K+ ratios, and cell transformation.
Dissociation of hemoglobin accumulation and commitment during murine erythroleukemia cell differentiation by treatment with imidazole
Authors: Gusella James F. , Tsiftsoglou Asterios S. , Volloch Vladimir , Weil Susan C. , Neumann Jeffrey , Housman David E.
Published: February 2005

The effect of imidazole on DMSO‐induced murine erythroleukemia (MEL) cell differentiation has been examined. While imidazole does inhibit heme, globin mRNA, and hemoglobin accumulation in DMSO‐induced MEL cells, it does not affect the commitment of MEL cells to the specific limitation of proliferative capacity associated with the in vitro differentiation program. Furthermore, imidazole treatment does not affect DMSO‐induced changes in cell volume, in the relative proportion of nuclear protein IP25, and in the specific activity of the enzyme cytidine deaminase. A clonal analysis in the presence of imidazole indicated that the drug prevents heme accumulation even in MEL cells already committed to terminal differentiation. These observations suggest that imidazole effectively dissociates two aspects of the erythroid differentiation program of MEL cells: globin gene expression and commitment to loss of proliferative capacity.
Kinetics of chick embryo cell types in culture
Authors: La Rocca Paul J. , Rafferty Keen A.
Published: February 2005

The growth kinetics and population doubling limits of chick embryonic fibroblasts, chondroblasts, and retinal pigment cells were compared. Chondroblasts were found to have a cumulative population doubling level (37 ± 3 PDL) similar (p = 0.05) to that of control fibroblasts (42 ± 2 PDL), in individual and pooled clones. While both cell types have similar doubling potential, the proportion of tritium‐labeled nuclei decreases, and differs significantly as doubling level increases. This age‐associated decline is due to an extension in the population doubling time. Direct cell‐cycle analysis shows this increase to occur in the G1 phase. Furthermore, cartilage colonies maintain their phenotypic expression (metachromasia) throughout their lifespan under conditions of subcloning at sparse density.

When fibroblasts derived from 15 day chick embryos are compared with fibroblasts from 10 day embryos (41 ± 2 PDL) there is no significant difference (p = 0.05) in cumulative PDL or percent labeled nuclei, indicating that fibroblasts of different embryonic age have similar potential. The addition of hydrocortisone and insulin to the medium significantly shortens (25 ± 2 PDL) the lifespan of 10 day chick fibroblasts. Kinetics of retinal pigment cells show a population doubling potential (29 ± 1 PDL) different from fibroblasts and chondroblasts, suggesting that different cell types may not have similar limits on doubling potential when first determined in embryogenesis. Hydrocortisone and insulin have no effect on the growth kinetics or lifespan of retinal pigment cells in culture.
Platelet‐derived growth factor is a chemoattractant for vascular smooth muscle cells
Authors: Grotendorst G. R. , Chang T. , Seppä H. E. J. , Kleinman H. K. , Martin G. R.
Published: February 2005

In previous experiments (Grotendorst et al, 1981), we showed that platelet‐derived growth factor promotes the migration of smooth muscle cells in vitro. Using a “checkerboard” analysis, we now establish that platelet‐derived growth factor (PDGF) acts as a true chemoattractant for cultured aortic smooth muscle cells. Other growth factors such as epidermal growth factor, fibroblast growth factor, and insulin are not chemoattractants. The chemotactic response occurs before the initiation of DNA synthesis and is not affected by inhibition of DNA synthesis. Chemotaxis occurs at levels of PDGF lower than required for mitogenesis. RNA and protein synthesis are required for the chemotactic response. As found previously in bacteria and leucocytes, we find that methylation reactions are required for the chemotactic response. The possibility is discussed that PDGF acts in vivo at sites of vascular injury to attract smooth muscle cells from the medial layer to the luminal surface, and is involved in the early stages of the formation of atherosclerotic plaques.
An early mitosis‐determining event in regenerating rat liver and its possible mediation by prostaglandins or thromboxane
Authors: Rixon R. H. , Whitfield J. F.
Published: February 2005

Inhibitors of prostaglandin and thromboxane production such as mefenamic acid, hydrocortisone, and dexamethasone prevented a large proportion of the parenchymal cells of rat liver, proliferatively activated by a two‐thirds partial hepatectomy, from entering mitosis without preventing them from initiating or completing DNA synthesis. This specific mitosis‐inhibiting action was maximum when the drugs were present during the first few hours after partial hepatectomy. In contrast, indomethacin, another inhibitor of prostaglandin and thromboxane production, maximally inhibited both DNA synthetic and mitotic activities when present during the same early period of prereplicative development, which showed that it had an action not shared by the other inhibitors. Arachidonic acid completely reversed the hydrocortisone‐ or dexamethasone‐induced inhibition of mitotic activity, but it did so only when it was injected between 2 and 3 hours after partial hepatectomy and glucocorticoid injection. Arachidonate's reversal of the hydro‐cortisone‐induced inhibition did not occur in the presence of mefenamic acid. These observations indicate that there is an early, prostaglandin‐ or thromboxane‐mediated, prereplicative process in proliferatively activated hepatocytes which determines their later entry into mitosis and which is separate from the early events leading to DNA synthesis.
A clonal analysis of the differentiation of 3T3‐L1 preadipose cells: Role of insulin
Authors: Steinberg Marcia M. , Brownstein Barbara L.
Published: February 2005

Cells of the established preadipose line, 3T3‐L1, appear to be undifferentiated fibroblasts during exponential growth. When cells become quiescent, a small percentage of them accumulate triglyceride and become morphologically indistinguishable from mature adipocytes. When insulin is added to quiescent cultures, up to 50% of the cells differentiate into adipocytes. The distribution of lipid‐containing cells which appear in clusters of varying sizes was analyzed to determine whether commitment to differentiation occurred after quiescence or during exponential growth and whether insulin was required as an inducer of commitment. The spatial arrangement of 3T3‐L1 cells at quiescence on some culture dishes was destroyed by replating. This resulted in random distribution of these cells. The distribution of adipocytes among replated and nonreplated cells in these experiments was compared to a computer generated random distribution of differentiated among undifferentiated cells. Dispersal of cells at confluence resulted in a distribution of fat among nonfat cells not significantly different from the computer generated random distribution. In undisturbed cultures, the distribution of fat cells is not random and is consistent with a commitment event in single cells at any cell division during exponential growth followed by divisions of both committed and uncommitted cells. Since insulin affected the number of mature adipocytes only when added after cessation of exponential growth, insulin is not the inducer of commitment but merely enhances lipid production in previously committed cells.
Factors involved in supporting the growth and steroidogenic functions of bovine adrenal cortical cells maintained on extracellular matrix and exposed to a serum‐free medium
Authors: Ill C. R. , Gospodarowicz D.
Published: February 2005

Bovine adrenal cortex cells maintained on extracellular matrix (ECM)‐coated dishes will proliferate actively when serum is replaced by HDL (25 μg protein/ml), insulin (10 ng/ml), and FGF (100 ng/ml). The cells have an absolute requirement for HDL in order to survive and grow. The omission of insulin, FGF, or both results in a slower growth rate and lower final cell density of the cultures. A requirement for transferrin (1 μg/ml) becomes apparent only when cells have been grown for at least four generations in the absence of serum.

Early passage (P1–P3) bovine adrenal cortex cells cultured in serum‐free medium responded to ACTH (10−8M) with increased 11‐deoxycortisol production; this effect was not observed in later passage cells (P7–P15). The cells' ability to utilize LDL‐derived cholesterol and to respond to db cAMP (1mM) by increased steroid release was preserved in cells cultured for over 60 generations in the serum‐free medium. HDL, although also able to increase steroid production in early‐passage cultures exposed to ACTH or to ACTH and dibutyryl cyclic AMP (db cAMP), was 10 fold less potent than LDL. It did not support steroidogenesis in cultures not exposed to these trophic agents.

The life span of bovine adrenal cortex cells grown in the serum‐free medium on fibronectin (FN)‐ versus ECM‐coated dishes was compared. Cells seeded in serum‐containing medium and grown in serum‐free medium had a life span of 34 versus 60 generations when maintained on fibronectin‐ or ECM‐coated dishes, respectively. Cells seeded in the complete absence of serum in the serum‐free medium on ECM‐ or fibronectin‐coated dishes could be passaged for 26 or 13 generations, respectively. While FGF was an absolute requirement for cells cultured on fibronectin‐coated dishes, it was not required when cells were maintained on ECM. These observations demonstrate the influence of the ECM not only in promoting cell growth and differentiation but also on the life span of cultured cells.
Formyl peptide stimulation of superoxide anion release from lung macrophages: Sodium and potassium involvement
Authors: Holian Andrij , Daniele Ronald P.
Published: February 2005

We examined the role of the monovalent cations Na+ and K+ in the events encompassing the release of O by alveolar macrophages after stimulation with formyl methionyl phenylalanine (FMP). This was accomplished by determining the effect of changing the extracellular [Na+] and/or [K+] on FMP‐stimulated O production; and measuring 22Na+ 42K+ and 86Rb+ influx and efflux and intracellular [K+] for control and FMP‐stimulated alveolar macrophages. Stimulated O production was relatively insensitive to changes in extracellular K+ or Na+ concentrations until the [Na+] was decreased below 35 mM. At 4 mM [Na+], the rate of O production remained at 75% of the maximal rate observed at physiological concentrations of [Na+]. Both influx and efflux of 22Na+ were stimulated above control rates by FMP. The increased rates of fluxes lasted for a few minutes suggesting a transient increase in membrane permeability to Na+. Ouabain partially inhibited 22Na+ efflux but had no effect on O release. The influx of 86Rb+ and 42K+ was not altered by the addition of FMP but was virtually abolished in the presence of 10 μM ouabain or 1 mM quinine. In the presence of extracellular calcium, FMP‐stimulated a prolonged (> 20 minutes) increase in 86Rb+ or 42K+ efflux which was inhibitable by 1 mM quinine. In the absence of extracellular calcium, FMP stimulation of K+ efflux was greatly diminished and was not affected by quinine, although quinine still inhibited O production under these conditions. It was also observed that there was a loss of intracellular K+ when cells were stimulated by FMP in the presence of Ca+2, but not in the absence of Ca+2. Taken together, these results suggest a minimal direct role, if any, for K+ in the events that lead to FMP‐stimulated O release by alveolar macrophages.
Genetic evidence that a phorbol ester tumor promoter stimulates ornithine decarboxylase activity by a pathway that is independent of cyclic AMP‐dependent protein kinases in CHO cells
Authors: Lichti Ulrike , Gottesman Michael M.
Published: February 2005

Ornithine decarboxylase (ODC) inductions by cholera toxin and by the phorbol ester tumor promoter, TPA, were compared in wild‐type Chinese hamster ovary (CHO) cells and in mutant cells having altered cyclic AMP‐dependent protein kinase activity. The aim of these studies was to determine whether cyclic AMP‐dependent protein kinase is involved in these inductions. The time course and the magnitude of ODC inductions by either 100 ng/ml cholera toxin or 100 ng/ml TPA were similar in wild‐type cells with a maximum at 3–4 hours after treatment and a return to unstimulated levels by 8 hours. Induction of ODC by cholera toxin was suppressed more than 80% in the four protein kinase mutants studied (10215, 10248, 10260, and 10265), strongly implicating a cyclic AMP‐dependent kinase step in the mechanism of induction. Similar results were found with the cyclic AMP analog 8‐Br‐cyclic AMP and the phosphodiesterase inhibitor, methyl‐isobutylxan‐thine. The induction of ODC by TPA, on the other hand, was only partially inhibited (approximately 50%) in three of four mutants. Lower ODC activity in two mutants stimulated by cholera toxin or TPA whose kinetics were studied in more detail could not be ascribed to a reduced affinity (Km) of ornithine for the enzyme, but appeared to be due to reduced catalytic activity (Vmax) in the extracts. These results suggest that the induction of ODC by TPA proceeds by a mechanism which is only partially dependent on an intact cyclic AMP‐dependent protein kinase activity.
A stochastic model of self‐renewal and commitment to differentiation of the primitive hemopoietic stem cells in culture
Authors: Nakahata Tatsutoshi , Gross Alan J. , Ogawa Makio
Published: February 2005

We recently identified a murine hemopoietic stem cell colony which consists of undifferentiated (blast) cells and appears to be more primitive than CFU‐GEMM in the stem cell hierarchy. The progenitors for the colony which we termed “stem cell colony” possess an extensive self‐renewal capacity and the ability to generate many secondary multipotential hemopoietic colonies in culture. We replated a total of 68 stem cell colonies from cultures of murine spleen cells and analyzed the number of stem cell–and granulocyte(neutrophil)‐erythrocyte‐macrophage‐megakaryocyte (GEMM) colony‐forming cells in individual stem cell colonies. Of the 68 stem cell colonies, 35 contained progenitors (abbreviated as “S”‐cells) for stem cell colonies. The distributions of S‐cells and CFU‐GEMM in individual stem cell colonies were extremely heterogeneous. Neither the frequency distributions of S‐cells nor CFU‐GEMM in stem cell colonies could be fitted well by Poisson distribution. Rather, the frequency distribution of the s‐cells could be approximated by a geometric distribution and that of CFU‐GEMM by an exponential distribution, both of which are variates of the gamma distribution. Our observations are in agreement with those on the distributions of CFU‐S in individual spleen colonies and provided support for a stochastic model for stem cell self‐renewal and commitment in culture. Application of the theory of the branching process to the distribution of S‐cells revealed a distributional parameter “p” of 0.589 which is also in agreement with the earlier report on the p value for reproduction of CFU‐S.
Proliferative variability of endothelial clones derived from adult bovine aorta: Influence of fibroblast growth factor and smooth muscle cell extracellular matrix
Authors: Loncenecker J. P. , Kilty L. A. , Ridge J. A. , Miller D. C. , Johnson L. K.
Published: February 2005

Four endothelial cell clones derived from adult bovine aorta were examined with respect to their proliferative characteristics In vitro. Three of these clones, derived in the absence of fibroblast growth factor (FGF), displayed variable basal proliferative rates. One of these non‐FGF derived clones grew at a maximal rate which could not be further enhanced with FGF. The other two clones grew at a suboptimal rate which was stimulated by low doses of FGF (10–50 ng/ml) and inhibited by higher doses (100–250 ng/ml). The fourth clone, derived in the presence of FGF, was stimulated by FGF in a dose‐dependent manner (10–250 ng/ml) and was not growth inhibited at high FGF concentrations (250–1,000 ng/ml). Growth of all four clones on extracellular matrix (ECM) derived from bovine aortic smooth muscle (BASM) cells was optimal in the absence of FGF. ECM‐coated dishes also significantly increased the sensitivity of all clones by at least fivefold to mitogenic stimulation by serum. The proliferative lifespans of the clones ranged between 60 and 120 generations with the most actively proliferating clones attaining the greatest lifespan. Continuous subculture of two of the endothelial clones in the presence of FGF or on ECM‐coated dishes did not induce a dependence of the cells on either factor for subsequent growth in its absence. The results indicate that aortic endothelial cells display considerable clonal variability in ther basal proliferative rate and in their response to FGF. This clonal variability is not observed when the cells are maintained on ECM‐coated dishes derived from vascular smooth muscle cells.
Age dependent production of a competence factor by human fibroblasts
Authors: Clemmons David R.
Published: February 2005

Several cell types such as Balb/c 3T3 have been shown to require platelet‐derived growth factor (PDGF); however, strains of human fibroblasts from fetal donors have been shown to divide in medium containing plasma free of PDGF. Since human fibroblasts have been demonstrated to secrete other peptide growth factors such as somatomedin‐C, we have undertaken a study to determine if fibroblasts derived from fetal donors are capable of producing a mitogen(s) which will substitute for PDGF and support growth in plasma alone. Quiescent human fibroblasts from donors ages 12‐wk embryo, newborn, and 3‐yr‐old were exposed to serum‐free minimum essential medium (MEM) for 24 hr. The conditioned media collected from embryonic and newborn fibroblast donors were demonstrated to stimulate growth in the 3‐yr‐old cells with the addition of plasma alone, whereas conditioned medium from the 3‐yr‐old donor cells was without effect. The increases in growth and DNA synthesis were dependent upon concentration of media used. Conditioned medium derived from newborn fibroblasts also supported 3‐yr‐old cell growth but embryonic conditioned medium was more potent. The embryonic conditioned medium factor was heat and acid stable but destroyed by trypsin and excluded by a 5,000 (MW) molecular weight filter. The factor(s) had full competence factor activity since transient exposure to fibroblasts (3‐yr‐old donor) stimulated 78% nuclear labeling vs. 81% with continuous exposure. These results support the concept that there is an age‐dependent production of a competence factor by human fibroblasts which may partially account for their capacity to grow in medium devoid of PDGF and supplemented with plasma alone.
Altered aminoacyl‐tRNA synthetase complexes in CHO cell mutants
Authors: Pahuski Eddie , Klekamp Mark , Condon Tom , Hampel A. E.
Published: February 2005

The Chinese hamster ovary (CHO) aminoacyl‐tRNA synthetase mutants Gln‐2, His‐1, and Lys‐101 were analyzed for alterations in respective particulate enzyme forms. The mutant Gln‐2 showed a preferential loss of the lower molecular weight enzyme form for glutamine. His‐1 showed alterations of the enzyme complexes for several other aminoacyl‐tRNA activities but only decreased activity for itself. The mutant Lys‐101 only showed an altered Lysyl‐tRNA synthetase. These results provide evidence for a model of the intracellular role of the aminoacyl‐tRNA synthetase complexes wherein the high molecular weight forms utilize amino acids directly from the extracellular pool while the low molecular weight forms utilize intracellular pools.
Influence of physical exercise after long‐lasting hypodynamia on the morphological parameters of muscle fibers
Authors: Marciniak Marianna , Barańska Wanda , Rózycka Monika
Published: February 2005

The purpose of this research was morphometric ultrastructure evaluation of the fibers in muscles taking part in the flight of pigeons forced to exercise after a long period of hypodynamia. It was found that following physical exercise, after 12 and 18 months of mobility limitation, there appeared marked qualitative and quantitative changes: a diminution of the volume fraction and number of mitochondria, increase of smooth sarcoplasmic reticulum and sarcoplasm, and a significant decrease of the number of glycogen granules as compared with those after 18 months hypodynamia. The above described changes were more pronounced in the supracoracoideus than in the pectoralis muscle.
Complementation for the growth response to insulin and MSA occurs at a step distal to hormone‐receptor interaction in mouse embryo fibroblast × melanoma cell hybrids
Authors: Coppock Donald L. , Straus Daniel S.
Published: February 2005

The PG19 mouse melanoma cell line does not respond to the mitogenic effects of insulin either in serum‐deprived quiescent cultures or when growing in hormone‐supplemented serum‐free medium, while melanoma × mouse embryo fibroblast hybrids respond under both conditions. It has been proposed that some growth effects of insulin are mediated by its binding to receptors for insulin‐like growth factors. To determine whether the PG19 cells do not respond to insulin because they lack receptors for MSA (multiplication‐stimulating activity, an insulin‐like growth factor produced by BRL‐3A cultured rat liver cells), we have examined growth effects and receptor binding of MSA in the melanoma and hybrid cells. MSA stimulates [3H]thymidine incorporation in three melanoma × mouse embryo fibroblast hybrid clones but not in the parental melanoma cells. Thus the pattern of response of the melanoma cells and hybrids to MSA directly parallels their response to insulin. The melanoma cells and hybrids all have high‐affinity specific MSA binding sites (KD = 10–27 nM), indicating that the inability of the melanoma cells to respond to MSA is not attributable to a lack of MSA receptors. Insulin competes very poorly for binding of MSA to hybrid clone 7 but is nevertheless able to stimulate [3H]thymidine incorporation in this clone. This strongly suggests that the effects of insulin on [3H]thymidine incorporation in this clone are not mediated by its binding to MSA receptors. Insulin and MSA stimulate protein synthesis and inhibit protein degradation in the hybrids but not in the parental melanoma cells. These results suggest that both insulin and MSA action are blocked in the melanoma cells at a step distal to the binding of either hormone to its cellular receptors but prior to stimulation of protein synthesis.
Intracellular pH and the cell cycle of mitogen‐stimulated murine lymphocytes
Authors: Gerson Donald F. , Kiefer Hansruedi
Published: February 2005

Rapidly proliferating, polyclonally stimulated mouse spleen lymphocytes were separated by density‐gradient unit‐gravity sedimentation. The following measurements were made on each fraction: the average intracellular water volume, the distribution of DNA content by flow microfluorometry, the rate of 3H‐thymidine incorporation, and the intracellular pH. Fractions of cells with a small average intracellular volume were predominately in G0 or G1 phase of the cell cycle, while fractions of larger cells had higher proportions of cells in S or G2. Multiple regression analysis of the data for both T and B lymphocytes indicated that the intracellular pH of cells in G0, G1, or G2 is around pH 7.2, and that the intracellular pH of cells in S phase of the cell cycle is around pH 7.4.
The biochemical and ultrastructural effects of tunicamycin and D‐glucosamine in L1210 leukemic cells
Authors: Morin Michael J. , Porter Carl W. , McKernan Patricia , Bernacki Ralph J.
Published: February 2005

Tunicamycin was found to specifically inhibit the incorporation of a number of sugars into L1210 leukemia cell glycoproteins. This inhibition of glyco‐protein biosynthesis led to a cessation of cell growth which was reversible in a dose‐dependent and time‐dependent manner. After removal of the antibiotic from L1210 cell cultures resumption of sugar incorporation preceded that of thymidine incorporation and the recovery of cell growth. The treatment of cells with tunicamycin resulted in a significant increase in the intracellular pool of UDP‐N‐acetylglucosamine which occurred concurrently with alterations in cell ultrastructure including distentions of the endoplasmic reticulum and nuclear membranes. Similar ultrastructural changes and increases in the intracellular pools of UDP‐sugars were observed in L1210 cells exposed to 5 mM D‐glucosamine, which suggested that the antiproliferative effects of tunicamycin may be related to the accumulation in the endoplasmic reticulum of one or more nucleotide sugar precursors of asparagine‐linked glycoprotein biosynthesis. However, the biological effects of tunicamycin could be distinguished from those caused by D‐glucosamine. Exposure of L1210 cells to tunicamycin resulted in specific alterations in the biochemical composition of the plasma membrane and in the inhibition of cellular agglutination by wheat germ agglutinin which were not apparent following exposure to equitoxic concentrations of the aminosugar. These studies, together with those which demonstrated that recovery of the cellular capacity to synthesize glycoproteins was obligatory for the recovery of cellular proliferation in tunicamycin‐treated cells, suggested that inhibition of the synthesis of glycoproteins was the major factor limiting L1210 leukemic cell proliferation.
Growth‐dependent AIB and meAIB uptake in LLC‐PK1 cells: Effects of differentiation inducers and of TPA
Authors: Amsler Kurt , Shaffer Carolyn , Cook John S.
Published: February 2005

Cultured pig kidney cells designated LLC‐PK1, previously shown to acquire Na + ‐dependent concentrative transport of hexoses as the cells become growth arrested, also show Na + ‐dependent concentrative uptake of the amino acid analogs α‐aminoisobutyric acid (AIB) and (methyl) meAIB. This A system‐like transport is most active in sparse, growing cultures and becomes stepped down at confluence. The cell/medium equilibrium distribution ratio of the lipophilic cation tetraphenylphosphonium ion (TPP+) decreases in parallel fashion, suggesting that a decrease in membrane potential may be a major factor in the stepdown. Differentiation inducers (hexamethylene bisacetamide) and phosphodiesterase inhibitors (theophylline, methylisobutyl xanthine) accelerate the stepdown, but even in the presence of these compounds addition of the tumor promoter 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA) results in the maintenance of a high level of AIB and meAIB uptake. In all these respects the changes in A system‐like amino acid transport are the reciprocal of those seen for concentrative hexose transport, although the driving force appears to be the same for both systems. The TPA analogs phorbol and 4‐0‐methyl TPA which are inactive in tumor promotion are inactive in this system as well. In confluent, already stepped‐down cultures, addition of TPA leads to a rapid (2–6 hour) stimulation of AIB and meAIB uptake. The enhancement is sensitive to cycloheximide and actinomycin D. The ouabain‐sensitive fraction of meAIB uptake is not markedly changed in the TPA‐enhanced uptake, nor is the TPP+ distribution ratio elevated in TPA‐treated cells, making it unlikely that the TPA effect is through an alteration in the membrane potential.
Receptor‐mediated endocytosis and exocytosis of transferrin in concanavalin A‐stimulated rat lymphoblasts
Authors: Hamilton Thomas A.
Published: February 2005

Iron‐loaded transferrin has been shown to be necessary for the support of cell proliferation in culture. This function depends upon interaction of transferrin with a specific high‐affinity cell surface receptor. The present report is directed toward determining the consequences of the interaction of transferrin with this receptor on Concanavalin A‐stimulated rat lymphocytes. Three specific questions have been posed: (a) Is transferrin endocytosed following binding to its specific receptor in a temperature‐dependent fashion? (b) Following endocytosis, is the carrier protein released from the cell in a structurally and functionally intact form? and (c) Is the cell surface transferrin receptor also endocytosed following ligand binding? The results provide affirmative answers to all questions. Using two independent probes of the cell surface versus intracellular location of transferrin we observed that cell‐bound transferrin moved from the cell surface to the inside of the cell and subsequently back to the medium. This process occurred in a temperature‐dependent fashion. When cells containing only intracellular transferrin were further incubated at 37°C approximately 80% of cell‐bound transferrin was released to the medium. Nearly all of this material retained reactivity with antibody to transferrin. In addition, exocytosed transferrin exhibited qualitatively and quantitatively equivalent binding reactivity with the transferrin receptor and showed identical electophoretic mobility on SDS gel electrophoresis. Finally, using similar methodology to that employed with transferrin itself, we provide evidence that the specific receptor is also endocytosed.
Folate utilization in friend erythroleukemia cells
Authors: Steinberg Stephen E. , Fonda Susan , Campbell Caryl L. , Hillman Robert S.
Published: February 2005

In order to study the generation, factors controlling endogenous folate pools, and their functional importance, Friend erythroleukemia cells were grown in media containing 100; 1,000; and 10,000 ng/ml of tritiated pteroylglutamic acid (3H)PteGIu1 and then studied in unlabeled media with varying amounts of PteGlu1. The intracellular folate pool was directly proportional to the PteGlu1 in which the cells were incubated. At equilibrium, greater than 95% of the labeled intracellular folate pool chromatographed as polyglutamyl folate, regardless of the exogenous folate concentration. The functional importance of the intracellular folate pool was studied by varying the endogenous pool and the exogenous (media) supply. The ability of the cells to replicate in the absence of exogenous folate was directly proportional to the intracellular polyglutamyl folate pool. The maximal rate of replication, however, required exogenous PteGlu1 in addition. The cell doubling time was the most important determinant of intracellular folate turnover; changes in the intracellular pool size and the extracellular folate concentration had no effect on the turnover time. In a rapidly proliferating tissue, the onset of functional folate deficiency will be determined by dilution of intracellular polyglutamates among progeny until a critical level is reached.
Neovascular responses induced by cultured aortic endothelial cells
Authors: Harris‐Hooker Sandra A. , Gajdusek Corinne M. , Wight Thomas N. , Schwartz Stephen M.
Published: February 2005

Neovascularization was studied in the chorioallantoic memebrane of the chick embryo after implantation of bovine aortic endothelial and smooth muscule cells, Swiss and BALB/c 3T3 cells and human diploid fibroblasts cultured separately on microcarrier beads. Quantitative analysis of neovascularization indicated a 3 1/2‐fold increase in the number of blood vessels responding to endothelial cells while smooth muscle cells induced a twofold increase when compared to the response of beads without cells. Skin fibroblasts and Swiss 3T3 cells did not elicit a comparable response. The marked angiogenic response induced by endothelial cells was characterized by a 137% increase in total vessel length and a 35% increase in average vessel area when compared to controls. Two of the properties required for an angiogenesis factor—stimulation of cellular migration and proliferation—can also be demonstrated using endothelial cell‐conditioned medium in cell culture systems. Medium from cultured bovine aortic endothelium stimulates DNA synthesis, proliferation, and migration of smooth muscle cells. In adition, conditioned media from both endothelial cells and smooth muscle cells produced an angiogenic response in the chorioallantoic membrane assay, which was comparable to that produced by intact cells growing on microcarrier beads. Similar responses were not evident with medium conditioned by other cell types. These results indicate the potential importance of endothelial cells and endothelial cell products in regulating blood vessel growth.
Bovine endothelial cells transformed in vitro by benzo(a)pyrene
Authors: Grinspan Judith B. , Mueller Stephen N. , Levine Elliot M.
Published: February 2005

A cloned strain of bovine vascular endothelial cells with a finite in vitro lifespan was treated with benzo(a)pyrene (BP) after approximately 75% of its lifespan was completed. Untreated cultures of this strain senesced upon serial subcultivation and contained large, nondividing cells. In three out of seven trials, BP treatment produced transformed cells appeared in the cultures concomitant with the senescence of the parent cells. All transformed cell lines examined exhibited indefinite lifespans and altered karyotypes. Two of the lines retained most of the characteristics of normal endothelial cells, except that one became aneuploid and the other polyploid, Neither of these lines formed tumors when inoculated into nude mice. The remaining two lines retained mostly diploid kayotypes, but a high percentage of cells contained Robertsonian translocations. In one line cell volume was markedly reduced. In addition, these lines grew in multilayers, were anchorage independent, and proliferated in medium containing 0.5% serum. When 107 cells of these lines were injected into nude mice, tumors appeared within 1 week and were identified as malignant hemangioendotheliomas of bovine origin.
Lateral transmission of tension in frog myofibers: A myofibrillar network and transverse cytoskeletal connections are possible transmitters
Authors: Street Sibyl F.
Published: February 2005

The extensibility of the sarcolemma of single myofibers can be reduced locally by leaving a segment covered by a sleeve of surrounding tissue composed of cut myofibers, blood vessels, and connective tissue, hereafter referred to as “the splint.” Splinted fibers from frog semitendinosus muscle were used to study mechanical connections (transverse coupling) between myofibrillar components and sarcolemma. The transverse coupling is strong enough to insure a tight correlation between myofibril length and overlying sarcolemma length in both resting and activated fibers and to transmit nearly maximum isometric tension to the splint. Lateral transmission of active tension was demonstrated with a preparation which had the distal two‐thirds of an intact fiber covered by a splint and the proximal third dissected clean. When the outer end of the splint was pinned down and only the distal tendon was held, tension generated in the splinted fiber was transmitted to, and recorded from, the splint. Parameters of isometric tension transmitted laterally were not significantly different from those of tension transmitted longitudinally. Myofibrils branch profusely and form a network that may act as a unitary force generator and transmitter. In splinted fibers its output is possibly picked up circumferentially and transmitted across the sarcolemma by a microfilament network. A cap of relatively inextensible sarcolemma “splints” myofiber ends. Resting tension is transmitted to and from the myofibrils by transverse coupling beyond the cap and the region of short sarcomere spacing it covers. Transverse cytoskeletal connections at Z and M regions are described. Immobilization of the sarcolemma allows study of myofibril‐sarcolemma linkage in intact libers. Both active and resting tension were transmitted laterally.
Inhibition of carbohydrate incorporation in transformed cells by a cancer‐associated galactosyltransferase acceptor (CAGA)
Authors: Podolsky Daniel K. , Fournier Deborah , Isselbacher Kurt J.
Published: February 2005

The effect of cancer‐associated galactosyltransferase acceptor (CAGA) on incorporation of a variety of macromolecular precursors has been studied in transformed and nontransformed cells. Incorporation of [3H]‐mannose, [3H]‐galactose, and [3H]‐glucosamine into acid precipitable material after one‐hour pulse was inhibited more than 70% within four hours after exposure to CAGA in polyoma‐transformed BHK cells and within eight hours after exposure in chick embryo fibroblasts infected with a temperaturesensitive RSV mutant (Ts68) grown at the permissive temperature (CEF‐RSV 37°C). Initial short‐term rate of uptake (< one minute) and total long‐term uptake (one hour) of the labelled carbohydrates (acid‐soluble and acidinsoluble material) was inhibited less than 15% over this period. Incorporation of 14C‐leucine, 3H‐serine,3H‐uridine, and 3H‐thymidine into acid‐precipitable material was also inhibited > 85% in transformed cells, but more than 12‐hour exposure to CAGA was required before maximal inhibition was detected. Uptake of these labelled precursors was inhibition was detected. Uptake of these labelled precursors was inhibited less than 20% up to eight hours after exposure to CAGA. In nontransformed cells (BHK and CEF) incorporation of labelled monosaccharides as well as protein and nucleic acid precursors into acid‐precipitable material was reduced less than 25% up to 12 hours following exposure to CAGA. Infected CEF grown at the nonpermissive temperature (CEF‐RSV 14°C) were affected to an extent similar to other nontransformed cells. These data suggest that the specific action of CAGA on transformed cells may be due to inhibition of glycoconjugate synthesis.
Selective induction of enhanced complement receptor 3 activity on murine macrophages
Authors: Yen Shing‐Erh , Walker William S.
Published: February 2005

A high proportion of murine resident peritoneal macrophages bear complement receptors 1 and 3 (CR1, CR3) which bind C3b and iC3b components of complement, respectively. By contrast, macrophages derived from bone marrow, blood, and the elicited peritoneal exudate are predominantly CR1+3−. To determine if the microenvironment of the normal peritoneal cavity influences CR3 phenotype, we studied the effects of lavage from the cavity on cultures of primary peritoneal exudate macrophages, and on macrophages derived from progenitors in the bone marrow, blood, and peritoneal exudate. The cell‐free peritoneal lavage (CFPL), after 24 hr or culture, induced CR3 on primary and culture‐derived populations of peritoneal exudate macrophages but had no effect on the CR3‐ phenotype of macrophages derived from bone marrow or blood. The CR3‐inducing activity in CFPL was abolished by heating at 70°C for 30 min and by trypsin, and was not affected by adsorption with EA(IgM)iC3b indicator cells, demonstrating that it is not soluble CR3. Finally, exudate macrophages exposed to CFPL required at least 24 hr before they expressed CR3; such macrophages regenerated CR3 after the receptors were removed by trypsin. The selective effect of the activity in CFPL for peritoneal exudate macrophages indicates that the local microenvironment of the peritoneal cavity can influence the expression of CR3.
Cell repair after liver injury: An analysis of some metabolic conditions required for the stimulation of RNA synthesis in postischemic liver nuclei
Authors: Piccoletti Roberta , Bernelli‐Zazzera Aldo
Published: February 2005

Nuclei isolated from liver cells recovering from reversible (non‐necrogenic) ischemia show an increased RNA synthesis. The postischemic effect is not abolished by previous adrenalectomy and occurs in fasted as well as in fed animals. Treatment with cycloheximide, at a dose that severely inhibits protein synthesis without primary effects on RNA synthesis, suppresses the postischemic stimulation of RNA synthesis even if cycloheximide is administered when stimulation is already well developed. Postischemic liver nuclei respond only weakly to the additional stimulation of RNA synthesis caused by the presence of cytosol and albumin in the incubation medium.
Regulation of intracellular protein degradation in IMR‐90 human diploid fibroblasts
Authors: Auteri Joseph S. , Okada Annabelle , Bochaki Victor , Fred Dice J.
Published: February 2005

Human diploid fibroblasts (IMR‐90) regulate their overall rates of proteolysis in response to the composition of the culture medium and the ambient temperature. The magnitude and, in some cases, the direction of the response depend on the half‐lives of the cellular proteins that are radioactively labeled and the time chosen for measurements of protein degradation. Fetal calf serum, insulin, fibroblast growth factor, epidermal growth factor, and amino acids selectively regulate catabolism of long‐lived proteins without affecting degradation of short‐lived proteins. Fetal calf serum reduces degradative rates of long‐lived proteins and is maximally effective at a concentration of 20%, but the effect of serum on proteolysis is evident only for the first 24 hr. Insulin inhibits degradation of long‐lived proteins in the presence or absence of glucose and amino acids in the medium, but is maximally effective only at high concentrations (10−5 M). Amino acid deprivation increases degradative rates of long‐lived proteins for the first 6 hr, but then decreases their catabolism for the subsequent 20 hr. Lowered temperature is the only condition tested that significantly alters degradative rates of short‐lived proteins. Although cells incubated at 27°C have reduced rates of degradation for both short‐lived and long‐lived proteins compared to cells at 37°C, lowered temperature reduces catabolism of long‐lived proteins to a greater extent.
Factors influencing survival and growth of mammalian cells exposed to hypothermia. I. Effects of temperature and membrane lipid perturbers
Authors: Kruuv J. , Glofcheski D. , Cheng K.‐H. , Campbell S. D. , Al‐Qysi H. M. A. , Nolan W. T. , Lepock J. R.
Published: February 2005

The Arrhenius plot of the rate of V79 Chinese hamster cell inactivation due to hypothermia has a “break” around 7–10°C with optimum storage temperature for unprotected cells being about 10°C. Addition of the membrane lipid perturber, butylated hydroxytoluene, improves survival of cells when compared to controls at temperatures below this break but not above. Arrhenius plots of growth rates of the cells show breaks at 30 and 40°C. Measurements of membrane fluidity by electron spin resonance or membrane polarization anisotropy by fluorescence spectrophotometry techniques as a function of temperature in these cells also reveal “breaks” centered around 8 and 30°C. Hence, the changes in the rate of cell inactivation and growth as a function of temperature may be related to membrane lipid phase changes.
Quantitative ultrastructural analysis of receptor‐mediated insulin uptake into adipocytes
Authors: Smith Robert M. , Jarett Leonard
Published: February 2005

Monomeric ferritin‐insulin was used as an ultrastructural marker to determine by quantitative electron microscopy the time course and route of insulin uptake in rat adipocytes. To approximate steady state membrane binding conditions prior to any internalization, adipocytes were prefixed with glutaraldehyde and incubated for 30 min with 70 nM monomeric ferritin‐insulin. Electron micrographs of these cells showed that the ferritin‐insulin particles were predominantly in small groups of receptor sites on the plasma membrane and in pinocytotic‐like invaginations of the plasma membrane. Significant amounts of ferritin‐insulin were observed in cytoplasmic vesicles of unfixed cells as early as 2 min and in multivesicular bodies and lysosome‐like structures within 5 to 10 min after the addition of the ligand. Ferritin‐insulin accumulation reached steady state levels in the cytoplasmic vesicles in 5 to 10 min and in the lysosome‐like structures in 15 min. Little ferritin‐insulin was bound to coated pits, and the relative paucity of coated pits found in adipocytes suggested that these specialized endocytotic structures have a relatively insignificant role in insulin uptake in fat cells. Quantitative analysis of the uptake process suggested that a proportion of the insulin internalized by the cell may not be transported to lysosomes, but may be recycled along with the insulin receptor to the plasma membrane.
Myeloproliferative sarcoma virus: Its effects on erythropoiesis in adult DBA/2J mice
Authors: Fagg Barbara , Ostertag Wolfram , Klein Bernard , le Bousse Caroline
Published: February 2005

Adult susceptible mice (DBA/2J) infected with MPSV (myeloproliferative sarcoma virus), a defective RNA tumour virus, develop splenomegaly and progressive disruption of the haematologic system culminating in death. The present study was specifically directed toward determining the effects of the virus on erythroid differentiation. Early and late precursor cells (erythroid burst‐forming units; BFU‐E and colony‐forming units; CFU‐E, respectively) were evaluated by the ability of bone marrow and spleen cells to form colonies of fully differentiated erythroid cells in vitro. MPSV caused substantial modification of both the BFU‐E and CFU‐E populations in the bone marrow and spleen of infected animals. Changes were detected in the CFU‐E population preceding any significant increase in spleen weight. In the bone marrow, the proportion of CFU‐E cells increased almost twofold by days 5–10 after virus infection but decreased by day 15. In the spleen, CFU‐E frequency rose 40‐fold by days 10–15 and then declined steadily prior to death. At the peak of CFU‐E expansion, a small proportion of the population appeared to be erythropoietin (Ep) independent, although there was no evidence of a complete switch to Ep‐independence which occurs in Friend virus‐induced erythroleukemia. Dose‐response curves showed that none of these data could be explained in terms of a changing responsiveness to Ep. However, evidence is presented that indicates that BFU‐E from MPSV‐infected animals lose or have a reduced requirement for burst‐promoting activity (BPA) relative to normal cells although their progeny still need Ep for terminal erythroid differentiation.