bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quantitative ultrastructural analysis of receptor‐mediated insulin uptake into adipocytes
Authors: Smith Robert M. , Jarett Leonard 
Published: February 2005

AbstractMonomeric 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

AbstractAdult 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.
Recovery of HeLa cell population growth after treatment with 5,6‐dichloro‐1‐β‐D‐ribofuranosylbenzimidazole (DRB)
Authors: Tamm Igor 
Published: February 2005

AbstractDose‐response curves for the inhibition of heterogeneous nuclear RNA (hnRNA) synthesis in HeLa cells by 5,6‐dichloro‐1‐β‐D‐ribofuranosylbenzimidazole (DRB; 5–100 μM; 30 min) are biphasic and indicate the existence of two subpopulations of hnRNA molecules, one highly sensitive and the other completely resistant, as previously reported for molecules >1,000 nucleoides long (Tamm et al., 1976; Sehgal et al., 1976a). In the short‐term experiments, the drug‐sensitive synthesis of hnRNA was inhibited 50% at a DRB concentration of ∼7 μM, and 70% at 20 μM, whereas drug‐resistant synthesis, which comprises ∼20% of total, continued at DRB concentrations as high as 100 μM. After 24 hr of DRB treatment in medium containing 5% fetal calf serum, the increase in cell number in the exponentially growing population was inhibited by only 42% at 20 μM DRB, and the formation of colonies of ≥ ten cells was not decreased. DRB at 40 μM concentration decreased population growth by 76% and colony formation by 63%. Treatment with 60 μM DRB was sufficient to prevent a net increase in cell number and to reduce colony formation by 78%. After termination of treatment, the time required for the surviving population to begin rapid proliferation was directly related to the concentration of DRB used to treat cells and to the duration of treatment. After 24‐hr treatment with 40 μM DRB, cultures recovered within 1 day, whereas after 60 μM DRB, 3–4 days were required. After 40‐hr treatment with 60 μM DRB, 5–6 days were required for recovery, and after 80 μM DRB, 9–11 days. During the “dormant” period the cell number ranged from 15 to 60% of the initial number and was fairly stable for given conditions. After the “dormant” period, recovery was rapid. The population growth rate in cultures undergoing treatment with DRB is directly related to serum concentration; however, the recovery rate during the post‐treatment period is unaffeced by serum concentration.
Regulation of prostaglandin production and ectoenzyme activities in cultured aortic endothelial cells
Authors: Chesterman Colin N. , Ager Ann , Gordon John L. 
Published: February 2005

AbstractProstaglandin production, angiotensin‐converting enzyme, and 5′‐nucleotidase were measured in porcine aortic endothelial cells in situ (with a multiwell template on an opened aorta), in primary culture and in subcultures. Changes during culture were monitored and the effects of culture conditions were investigated by growing cells on a biological matrix or on plastic, by adding different sera to the growth medium, and by harvesting cells enzymically or mechanically. Prostacyclin production by endothelium in primary culture is highest immediately after cell isolation and subsequently declines; this pattern is repeated each time the cells are subcultured. The level at which production stabilises is ∼ 200 pgċ106 cells−1ċh−1. Detaching cells by physical means stimulates production much more than enzymic dispersion; the type of serum or the presence of a biological matrix does not alter prostaglandin production. The relative amount of prostaglandin E produced increases with time, from ∼20% of the prostacyclin production shortly after isolation to > 100% in subcultured cells. None of the culture conditions that we tested altered this trend. Angiotensin‐converting enzyme activity decreases during primary culture, but activity can be sustained by including homologous serum (from whole blood or from platelet‐free plasma) in the culture medium. The method of harvesting cells, or the presence of a matrix, did not affect enzyme activity. 5′‐Nucleotidase also declines during culture, with a progressive decrease in both Km and Vmax from template to primary culture to subcultures. None of the variations in culture conditions prevented this change. Ectoadenosine‐deaminase activity, not detectable in cultured cells, can be measured in the template. Part of this activity was released by the vascular wall and could be due to plasma diffusing from the interstitial space.
Growth of the mitochondrial inner membrane in synchronous cultures of Tetrahymena pyriformis: An examination of phospholipid accumulation
Authors: Hemmingsen Sean M. , Young Paul G. 
Published: February 2005

AbstractBased on morphological evidence, mitochondrial inner membrane growth has been reported to be discontinuous in heat shock‐synchronized Tetrahymena pyriformis. As a biochemical measure of membrane growth under these conditions, we have examined phospholipid accumulation in the cell. No marked modulation of the accumulation of any of the major phospholipids could be detected through the cell cycle. At least 89% of the cardiolipin in the cells is restricted to the mitochondria, and we have used it as a marker for the growth of the mitochondrial inner membrane. During the heat shock synchrony, cardiolipin accumulates uniformly in parallel with the exponential rate of increase of total cellular phospholipids. These results suggest that at least the phospholipid component of all membrane systems in the cell grow continuously and uniformly. Additionally, we have shown that the total phospholipid content of Tetrahymena increases by a factor of 2.4 per generation following a series of heat shocks. No such net overaccumulation is observed for protein content.
The regulation of macrophage protein turnover by a colony stimulating factor (CSF‐1)
Authors: Tushinski R. J. , Stanley E. R. 
Published: February 2005

AbstractCSF‐1 is a hemopoietic growth factor that specifically regulates the survival, proliferation, and differentiation of mononuclear phagocytic cells. A homogeneous population of mononuclear phagocytes, bone marrow derived macrophages (BMM), were used to study the regulation of protein turnover by CSF‐1. Removal of CSF‐1 (∼0.4 nM) from exponentially growing BMM cultured in 15% fetal calf serum containing medium decreases the rate of DNA synthesis by more than 100‐fold. Addition of CSF‐1 to these cells causes them to resume DNA synthesis within 12 h. More immediate effects of CSF‐1 were observed on BMM protein metabolism. BMM cultured for 24 h in the absence of CSF‐1 reduce their protein synthetic rate by 50–60%. The protein synthetic rate commences to decrease at 2–3 h after CSF‐1 removal. Readdition of CSF‐1 to BMM previously incubated in its absence causes a return to the protein synthetic rate of exponentially growing cells within 2 h. In the presence of CSF‐1, BMM synthesize protein at a rate of ∼8.7%/h and degrade it at a rate of ∼0.9%/h. Removal of CSF‐1 results in a decrease in the protein synthetic rate to ∼3.4%/h and an increase in the rate of protein degradation to ∼3.4%/h. The rate of protein synthesis by BMM increases linearly with CSF‐1 concentration over the range of concentrations stimulating both survival and proliferation, while the rate of protein degradation decreases exponentially over the range of concentrations stimulating survival without proliferation. Therefore, it appears that the stimulation of the rate of protein synthesis and inhibition of the rate of protein degradation are two distinct effects of CSF‐1, both part of the pleiotropic response to this growth factor. The inhibition of the rate of protein degradation by CSF‐1 may be most significant for its survival inducing effect.
Degradation of microinjected proteins: Effects of lysosomotropic agents and inhibitors of autophagy
Authors: Rote Kevin V. , Rechsteiner Martin 
Published: February 2005

AbstractHeLa cells, injected with radioiodinated proteins by fusion with RBC ghosts, were exposed to inhibitors of lysosomal proteolysis and autophagy. The degradation of injected [125I]bovine serum albumin (BSA) was unaffected by chloroquine, NH4Cl, nocodazole, colcemid, puromycin, cycloheximide, or enucleation. Although degradation of [125I]lactate dehydrogenase (LDH) and [125I]pyruvate kinase (PK) was inhibited one‐third by chloroquine or ammonia, their degradation was unaffected by the other compounds. In contrast, enhanced degradation of 125I‐PK resulting from depriving injected HeLa cells of amino acids and serum was inhibited 70% by colcemid and abolished by chloroquine or ammonia. Similarly, degradation of [14C]sucrose‐labeled BSA‐polylysine conjugates that entered HeLa cells by endocytosis was inhibited as much as 80% by chloroquine and ammonia.Sensitivity of both enhanced proteolysis and degradation of exogenous proteins to ammonia or chloroquine indicates they are effective inhibitors of lysosomal proteolysis in HeLa cells. Failure of ammonia or chloroquine to inhibit degradation of injected 125I‐BSA and the modest inhibition of degradation of injected 125I‐LDH or 125I‐PK indicates that virtually all BSA molecules and most PK or LDH molecules are degraded by a nonlysosomal proteolytic system. Components of this degradative system are present in vast excess or are long lived, since inhibition of protein synthesis for 20 hr had no effect on the degradation of injected proteins.
Susceptibility to merocyanine 540‐mediated photosensitization: A differentiation marker on murine hematopoietic progenitor cells
Authors: Meagher Richard C. , Sieber Fritz , Spivak Jerry L. 
Published: February 2005

AbstractMerocyanine 540 (MC 540) is an impermeant fluorescent dye that binds preferentially to fluidlike domains of the cell membrane. Photoexcitation of membrane‐bound dye causes a breakdown of the normal permeability properties of the membrane and, eventually, cell death. We have used in vitro and in vivo clonal assays to determine the relative sensitivities of different classes of normal murine hematopoietic progenitor cells to MC 540‐mediated photo‐sensitization. Late erythroid progenitors (CFU‐E) were the most sensitive cells, followed in order of decreasing sensitivity by early erythroid progenitors (BFU‐E), megakaryocyte progenitors (CFU‐Meg), day 7‐spleen colony forming cells (day 7‐CFU‐S), granulocyte/macrophage progenitors (CFU‐GM), and day 11‐spleen colony forming cells (day 11‐CFU‐S). Bipotent progenitors of the granulocyte/macrophage lineage were more sensitive than unipotent macrophage progenitors but less sensitive than unipotent granulocyte progenitors. Progenitors giving rise to large granulocyte/macrophage colonies were more sensitive than progenitors giving rise to small colonies (“clusters”). We conclude that sensitivity to MC 540‐mediated photosensitization is developmentally regulated and that differences occur even between the most closely related classes of progenitor cells. Our findings indicate the usefulness of MC 540 as a plasma membrane probe. They also support the contention that early and late‐appearing spleen colonies are the progeny of two distinct classes of progenitor cells.
The respiration‐linked limiting step of tumor cell transition from the non‐cycling to the cycling state: Its inhibition by oxidizable substrates and its relationships to purine metabolism
Authors: Olivotto M. , Caldini R. , Chevanne M. , Cipolleschi M. G. 
Published: February 2005

AbstractThe recruitment into the cycling state of resting Yoshida AH 130 hepatoma cells was studied with respect to its dependence on respiration in an experimental system wherein the overall energy requirement for this recruitment can be supplied by the glycolytic ATP. The G1‐S transition of these cells, unaffected by 2,4‐dinitrophenol (DNP) at concentrations which uncouple the respiratory phosphorylation, is impaired either by blocking the electron flow to oxygen by antimycin A or by adding an excess of some oxidizable substrates, chiefly pyruvate and oxalacetate. An experimental analysis, focused on pyruvate activity, showed that the inhibition of cell recruitment into S is not related to the depressing effects of this substrate on aerobic glycolysis of tumor cells, nor is it modified by forcing, in the presence of DNP, pyruvate oxidation through the tricarboxylic acid cycle as well as the overall oxygen consumption. Addition of suitable concentrations of preformed purine bases (mainly adenine), completely removes the block of the G1‐S transition produced either by the excess of oxidizable substrates or by antimycin A. These findings indicate the existence of a respiration‐linked step in purine metabolism, which restricts the above transition and is equally impaired by blocking the respiratory chain or by saturating it with an excess of reducing equivalents derived from unrelated oxidations. The inhibitory effects of pyruvate and antimycin A can be largely removed by the addition of folate and tetrahydrofolate, suggesting that the respiration‐linked restriction point of tumor cell cycling involves the folate metabolism and its connections to purine synthesis.
Growth‐stimulating effect of catecholamines on rat aortic smooth muscle cells in culture
Authors: Blaes Nelly , Boissel Jean‐Pierre 
Published: February 2005

AbstractThe effect of epinephrine was tested on the proliferation of rat arterial smooth muscle cells (SMC) in secondary cultures. Epinephrine added daily to the culture medium caused a striking stimulation of growth. The effect increased with time and was dose‐dependent. Maximal stimulation was observed at a concentration of 1010−5 M and after 72 hours. At higher concentrations (10−3 M) epinephrine exhibited toxic effects on SMC. When SMC were maintained quiescent by deprivation of serum, the subsequent addition of epinephrine required serum to significantly enhance growth. This growth stimulation increased with serum concentration (from 0.1 % to 10%). All the adrenergic agonists tested were found to stimulate SMC growth, with an activity classified by decreasing order as follows: norepinephrine > epinephrine > isoproterenol. Finally, this mitogenic response of SMC to catecholamines was specific since it could be blocked by adrenergic blocking agents, phentolamine being more efficient than propranolol in that connection. The results suggest that epinephrine and other catecholamines may act as growth factors for aortic SMC, at least in rat, mostly through adrenoreceptors.
Effect of 5‐azacytidine treatment on mouse embryonal carcinoma cells
Authors: Cremisi Chantal 
Published: February 2005

AbstractSeveral properties of embryonal carcinoma (EC) cell lines, such as multipotent PCC4‐aza‐1 cells and nullipolent F9 cells originating from murine teratocarcinoma cells, were examined after treatment with 5‐azacytidine, which produces undermethylated DNA. Drug‐treated PCC4‐aza‐1 cells exhibited morphological changes and differentiated, whereas azacytidine‐treated F9 cells displayed no detectable morphological change. After treatment with 5 azacytidine, PCC4‐aza‐1 cells, whether or not they differentiated, as well as F9 cells, became permissive for polyoma even though both cell types are usually resistant to polyoma. In contrast, only the differentiated azacytidine‐treated PCC4‐aza‐1 cells became sensitive to SV40 infection, i.e., synthesized T antigen, despite the resistance normally shown by such cells to this viral infection. In some PCC4‐aza‐1 and F9 cells, drug treatment induced expression of H2 antigen but did not derepress plasminogen activator synthesis. These results suggest that undermethylation of certain cellular genes in PCC4‐aza‐1 and F9 cells is correlated with the establishment of Py permissivity, SV40 sensitivity, H2 antigen expression, and the triggering of a differentiation process. The relationship between the expression of these characters and differentiation is discussed.
Proliferative effects of purified granulocyte colony‐stimulating factor (G‐CSF) on normal mouse hemopoietic cells
Authors: Metcalf D. , Nicola N. A. 
Published: February 2005

AbstractWhen granulocyte colony‐stimulating factor (G‐CSF), purified to homogeneity from mouse lung‐conditioned medium, was added to agar cultures of mouse bone marrcw cells, it stimulated the formation of small numbers of granulocytic colonies. At high concentrations of G‐CSF, a small proportion of macrophage and granulocyte‐macrophage colonies also developed. G‐CSF stimulated colony formation by highly enriched progenitor cell populations obtained by fractionation of mouse fetal liver cells using a fluorescence‐activated cell sorter, indicating that G‐CSF probably acts directly on target progenitor cells. Granulocytic colonies stimulated by G‐CSF were small and uniform in size, and at 7 days of culture were composed of highly differentiated cells. Studies using clonal transfer and the delayed addition of other regulators showed that G‐CSF could directly stimulate the initial proliferation of a large proportion of the granulocvte‐macrophage progenitors in adult marrow and also the survival and/or proliferation of some multipotential, erythroid, and eosinophil progenitors in fetal liver. However, G‐CSF was unable to sustain continued proliferation of these cells to result in colony formation. When G‐CSF was mixed with purified granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) or macrophage colony‐stimulating factor (M‐CSF), the combination stimulated the formation by adult marrow cells of more granulocyte‐macrophage colonies than either stimulus alone and an overall size increase in all colonies. G‐CSF behaves as a predominantly granulopoietic stimulating factor but has some capacity to stimulate the initial proliferation of the same wide range of progenitor cells as that stimulated by GM‐CSF.
Isozymes of creatine kinase in mammalian cell culures
Authors: Van Brussel Elizabeth , Yang Jing J. , Seraydarian Maria W. 
Published: February 2005

AbstractPrevious studies on the energy metabolism of rat myocardial cells in culture supported the hypothesis that the creatine‐phosphocreatine–creatine kinase system plays an important role in the intracellular transport of energy from the mitochondria to the myofibrils and in the regulation of energy production coupled to energy utilization in this model system. Effective functional compartmentation of ATP could result from the binding of creatine kinase to cellular organelles (e.g., myofibrils and mitochondria) such that high energy charge at the myofibrils is maintained by the reverse creatine kinase reaction, while phosphocreatine is synthesized mainly at the mitochondria in the forward creatine kinase reaction. It was, therefore, essential to demonstrate the presence of mitochondrial creatine kinase in the cultured myocardial cells to support this hypothesis, particularly since the mitochondrial creatine kinase was reportedly absent in fetal hearts. Using electrophoresis on cellulose acetate strips, the mitochondrial creatine kinase isozyme, as well as MM, MB, and BB isozymes, have now been demonstrated in myocardial cultures derived from neonatal rats. The mitochondrial creatine kinase increased with age in culture and with age of animal from which the culture is derived. Furthermore, the addition of creatine to culture media stimulates its synthesis. The mitochondrial creatine kinase isozyme was not detected in nonmuscle cells in culture derived from the neonatal rat hearts, nor in L6 muscle cell line. Phosphocreatine was present in all cells, but the regulation of energy metabolism and energy shuttle by creatine‐phosphocreatine–creatine kinase could be operative only in the cells where the mitochondrial creatine kinase is present. This regulatory mechanism provides for an efficient system concomitant with the continuous energy demand of the myocardium; it is not ubiquitous and its development in myocardial cells seems to be triggered postnatally.