bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of the nuclear antigen p105 as a function of cell‐cycle progression
Authors: Clevenger Charles V. , Epstein Alan L. , Bauer Kenneth D.
Published: February 2005

The characterization of the proliferation‐associated nuclear antigen designated p105 in quiescent and proliferating lymphocytes is described. Through the use of novel flow cytometric and cell‐sorting strategies the intracellular content of p105 was assessed in situ on a per cell basis. These analyses demonstrated the presence of multiple cellular subpopulations within the cell cycle differing significantly in p105 content. The data revealed that the flow cytometric quantitation of p105 levels may effectively discriminate cycling from noncycling cells. Immunogold electron microscopy revealed that the modulation of this interchromatin‐associated antigen was correlated with a significant degree of nuclear restructuring. In conjunction with cell sorting, immunogold electron microscopy and immunoblot controls demonstrated that the cell‐cycle‐related modulation in p105 cannot be accounted for by increased cellular mass or antigen sequestration. The significance of these controls and of the potential role of p105 in cellular proliferation is discussed.
Growth‐promoting activity in serum‐free medium of kallikreinlike arginylesteropeptidases from rat submaxillary gland
Authors: Catalioto R. M. , Négrel R. , Gaillard D. , Ailhaud G.
Published: February 2005

The characterization and purification of the growth‐promoting activity present in rat submaxillary gland extracts, known to be required for the proliferation of adipose precursor cells in serum‐free medium, have been undertaken. Fractionation of the extracts by ion‐exchange chromatography, gel filtration, and affinity chromatography on immobilized benzamidine allowed the copurification of the mitogenic activity with two distinct arginylesteropeptidases of apparent molecular weight 25,000; one of these enzymes has been purified to homogeneity and shown to be immunologically related to tonin, a well‐characterized kallikreinlike protease from submaxillary gland. The specificity of both enzymes was similar to that of plasma and glandular kallikreins, as indicated by the relative rates of hydrolysis of peptide p‐nitroanilide substrates. Prior treatment of the kallikreinlike proteases with phenylmethylsulfonylfluoride or aprotinin abolished completely both mitogenic and arginylesteropeptidase activities, indicating that enzymatic activity was essential for the manifestation of their growth‐promoting ability. The kallikreinlike proteases from rat submaxillary gland were able to replace thrombin to support the proliferation of Chinese hamster lung fibroblasts in serum‐free medium. These results underline the role of proteases in controlling cell growth and are discussed in light of adipose tissue development.
Effect of potassium depletion of Hep 2 cells on intracellular pH and on chloride uptake by anion antiport
Authors: Madshus Inger Helene , Tønnessen Tor Inge , Olsnes Sjur , Sandvig Kirsten
Published: February 2005

The effect of K+ depletion of Hep 2 cells on ion fluxes, internal pH, cell volume, and membrane potential was studied. The cells were depleted of K+ by incubation in K+‐free buffer with or without a preceding exposure to hypotonic medium. Efflux of K+ in cells not exposed to hypotonic medium was inhibited by furosemide or by incubation in Na+‐free medium, indicating that in this case at least part of the K+ efflux occurs by Na+/K+/Cl− cotransport. After exposure to hypotonic medium, K+ efflux was not inhibited by furosemide, whereas it was partly inhibited by 4,4′‐diisothiocyano‐2,2′‐stilbene‐disulfonic acid (DIDS). Exposure to hypotonic medium induced acidification of the cytosol, apparently because of efflux of protons from intracellular acidic vesicles. When isotonicity was restored, a rebound alkalinization of the cytosol was induced, because of activation of the Na+/H+ antiporter. While hypotonic shock and a subsequent incubation in K+‐free buffer rapidly depolarized the cells, depolarization occurred much more slowly when the K+ depletion was carried out by incubation in K+‐free buffer alone. The cell volume was reduced in both cases. K+ depletion by either method strongly reduced the ability of the cells to accumulate 36Cl− by anion antiport, and K+‐depleted cells were unable to increase the rate of 36Cl− uptake in response to alkalinization of the cytoscl.
Effect of reduced endocytosis induced by hypotonic shock and potassium depletion on the infection of Hep 2 cells by picornaviruses
Authors: Madshus Inger Helene , Sandvig Kirsten , Olsnes Sjur , van Deurs Bo
Published: February 2005

Potassium depletion after a brief exposure of the cells to hypotonic medium was used to inhibit endocytosis from coated pits in Hep 2 cells. After such treatment the endocytic uptake of transferrin was arrested, and electron microscopy revealed that virtually no coated pits were present at the cell surface, while smooth (uncoated) pits were abundant. Under the same conditions the cells were strongly protected against poliovirus, while the cytopathogenic effect of human rhinovirus type 2, HRV 2, was increased. The cytopathogenic effect of encephalomyocarditis (EMC) virus was only slightly affected.

Potassium depletion without hypotonic shock reduced the endocytic uptake of transferrin 2–3‐fold and the number of coated pits at the cell surface about 3‐fold. Furthermore, the cells were not protected against poliovirus after such treatment. The data indicate that the productive uptake of poliovirus occurs by receptor‐mediated endocytosis from coated pits, while the productive uptake of the other two picornaviruses may occur by another endocytic pathway. In order to efficiently arrest endocytosis from coated pits in these cells, hypotonic shock seems to be a critical component of the potassium depletion protocol.
Establishment and some mutational characteristics of 3T3‐like near‐diploid mouse cell line
Authors: Sasaki M. S. , Kodama S.
Published: February 2005

A stable and nonmalignant near‐diploid cell line, designated m5S, has been established by serial in vitro transfers of embryonic skin cells of ICR mouse. The cells are stable in karyotype and growth, display high sensitivity to contact inhibition of cell division, are nontumorigenic in nude mice, and the transformation and mutation may be reproducibly tested. Unlike most of the mouse cell lines, the m5S underwent a unique karyotypic change during establishment and showed high stability in a diploid range, making amenable to the study of mutagenesis. The m5S is nearly unique; it represents the second near‐diploid cell line sensitive to postconfluence inhibition of cell division derived from mouse embryo cells to be reported and characterized. These cells are being successfully used for qualitative and quantitative studies of oncogenic transformation as well as mutation to drug resistance by radiation and chemicals.
Antiproliferative effects of heparin on vascular smooth muscle cells are reversed by epidermal growth factor
Authors: Reilly Christopher F. , Fritze Linda M. S. , Rosenberg Robert D.
Published: February 2005

Heparin and related glycosaminoglycans are potent inhibitors of both in vivo and in vitro smooth muscle cell (SMC) proliferation. We have found that epidermal growth factor (EGF) reverses the antiproliferative effects of heparin. Other known SMC mitogens, including platelet‐derived growth factor (PDGF), insulin‐like growth factor‐1 (IGF‐1), and thrombin, were unable to prevent heparin action. The EGF specificity was further demonstrated by developing a biological growth assay in which EGF or PDGF, at concentrations as low as 1 ng/ml, stimulated SMC growth in the absence of other serum components. Under these conditions, EGF, but not PDGF, suppressed heparin inhibition as well. The ability of EGF to reverse heparin inhibition was only observed when mitogen and glycosaminoglycan were added to SMC at similar times. If SMC were pretreated with heparin for 48 hours prior to EGF addition, the protective effects of EGF were lost. Heparin did not directly prevent 125I‐EGF or platelet‐derived EGF‐like peptides from binding to the EGF receptor on SMC. However, cultures that were pretreated with heparin for 48 hours bound 49% less 125I‐EGF than cultures that had been pretreated with the mucopolysac‐charide for only 2 hours or that had not been preexposed to heparin. In previous studies, we have established that heparin exerts its maximal inhibitory activity after a 48‐hour treatment of SMC (Reilly et al. 1986). Taken together, these data suggest that heparin may exert its antiproliferative potential by slowly and specifically altering SMC response to EGF‐like mitogens of platelet origin.
Irreversible reduction in potassium fluxes accompanies terminal differentiation of human myoblasts to myotubes
Authors: Panet Rivka , Digregorio Debra M. , Brown Robert H.
Published: February 2005

Potassium and sodium fluxes believed to be important in the cellular response to serum and growth factors have not been widely investigated in cells which have undergone terminal differentiation. In this study we have analyzed two main K+ transport systems—the ouabain‐sensitive Na+/K+ pump and the bumetanide‐sensitive transporter—in human muscle in vitro at two developmental stages: proliferating myoblasts and differentiated myotubes. Myoblast differentiation to myotubes was accompanied by a marked decrease in both the ouabain‐sensitive and the bumetanide‐sensitive K+ (Rb+) influxes. The addition of serum to the terminally differentiated myotubes had no effect on these K+ transporters. However, serum addition to serum‐deprived, undifferentiated myoblasts produced a marked stimulation of these K+ fluxes.

The bumetanide‐sensitive K+ transporter in human myoblasts and myotubes has the following properties: (1) It carries 30% and 40% of the total K+ influx in myoblasts and myotubes, respectively. (2) It performs net efflux of K+ in the undifferentiated myoblasts and zero net flux (self‐exchange) in terminally differentiated myotubes. (3) It is dependent on extracellular Na+ and Cl− in addition to K+. (4) In myoblasts, the Km value for K+ is 1.36 mM, similar to the Km for K+ of the Na+/K+ pump. (5) It is resistant to ouabain (up to 2 mM) and sensitive to furosemide (K0.5 = 5 × 10−6 M) and bumetanide (K0.5 = 10−7 M). These data indicate that following terminal differentiation of proliferating myoblasts to mitotically inactive myotubes there is an irreversible reduction of K+ fluxes with a change in the net flux of K+ carried by the bumetanide‐sensitive transporter.
Parallel changes in amino acid transport and protein kinase C localization in LLC‐PK1 cells treated with TPA or diradylglycerols
Authors: Dawson W. David , Cook John S.
Published: February 2005

Protein kinase C is considered to be a major target for tumor promoting phorbol esters such as 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA). We have analyzed the correlation between A‐system amino acid transport and the distribution of protein kinase C (PKC) between a membrane‐rich fraction (100,000 g pellet) and cytosol (supernatant) from homogenized LLC‐PK1 cells, a pig kidney epithelial cell line grown in culture. During log growth 1 day after seeding the cells onto culture plates, PKC activity is high in the membrane fraction and low in the cytosol. As the cells become confluent the PKC distribution shifts to a cytosolic pool. Concomitantly, A‐system amino acid transport, as measured by methylaminoisobutyric acid [14C]MeAIB uptake, decreases. TPA (0.01–1.0 μM) induces a shift of PKC activity from the cytosol back to the membrane‐rich fraction in post‐confluent cells with a concomitant 2–3 fold stimulation of MeAIB uptake. The same responses can be achieved by treating cells with certain diradylglycerols, either diacylglycerols such as 1‐oleyl‐2‐acetyl‐sn‐glycerol (OAG) or alkylacylglycerols such as 1‐hexadecenyl‐2‐oleyl‐sn‐glycerol. Both responses to TPA are blocked by cytochalasin B, but cycloheximide inhibits the transport response without affecting PKC redistribution. It is suggested that the redistribution may be a necessary but not sufficient concomitant to the transport activation.
Novel regulatory actions of 1α,25‐dihydroxyvitamin D3 on the metabolism of polyphosphoinositides in murine epidermal keratinocytes
Authors: Tang Wilson , Ziboh Vincent A. , Isseroff R. Rivkah , Martinez D.
Published: February 2005

The in vitro incubation of murine keratinocytes in the presence of 1α,25‐dihydroxyvitamin D3 enhanced the rapid hydrolysis of the prelabeled keratinocyte polyphosphoinositides (polyPtdIns) when compared to untreated cells. The rapid hydrolysis of the polyPtdIns and the release of the inositol phosphosphates (particularly InsP3 and InsP2) precede the onset of differentiation of these cells. These data therefore suggest that 1α,25‐didydroxyvitamin D3 functions in vitro to initiate the rapid generation of InsP3 from cellular polyPtdIns; this in turn may mobilize intracellular Ca2+, thus providing the signal which program the murine keratinocytes from a proliferating mode into a differentiating mode.
Dexamethasone inhibits feedback regulation of the mitogenic activity of tumor necrosis factor, interleukin‐1, and epidermal growth factor in human fibroblasts
Authors: Kohase Masayoshi , Henriksen‐Destefano Dorothy , Sehgal Pravinkumar B. , Vilĉek Jan
Published: February 2005

Tumor necrosis factor (TNF), interleukin‐1 (IL‐1), and epidermal growth factor (EGF) were mitogenic for human diploid FS‐4 fibroblasts. Dexamethasone amplified the growth‐stimulating action of all three agents. Amplification of the growth‐stimulating action was maximal when dexamethasone was added along with TNF or EGF; no amplification was seen if the addition of dexamethasone was delayed for more than 3 hr. Prolonged simultaneous treatment with TNF and EGF resulted in less growth stimulation than treatment with EGF alone. Dexamethasone abolished this apparent antagonistic interaction between TNF and EGF. Dexamethasone also inhibited the antiviral action of TNF against encephalomyocarditis (EMC) virus in FS‐4 cells. TNF and IL‐1 increased the steady state level of interferon (IFN)‐β2 mRNA but failed to induce detectable levels of IFN‐β1 mRNA in FS‐4 cells. Dexamethasone inhibited the increase of IFN‐β2 mRNA levels by IL‐1 or TNF. Inhibition of IFN‐β synthesis is likely to be responsible for the inhibition of the TNF‐induced antiviral state by dexamethasone. Since IFNs suppress cell growth, inhibition of endogenous IFN‐β synthesis may also be responsible for the amplification by dexamethasone of the growth‐stimulating action of TNF and IL‐1. Amplification of the mitogenic action of EGF by dexamethasone appears to be mediated by a different mechanism.
Effects of EGF and calcium on adult parenchymal hepatocyte proliferation
Authors: Eckl Peter M. , Whitcomb Wendi R. , Michalopoulos George , Jirtle Randy L.
Published: February 2005

Adult rat hepatocytes were grown in serum‐free medium containing 0.05‐4 mM Ca+ + and 40 ng/ml EGF. After 48 hours of cultivation the mitotic index and the percentage of second division metaphases were determined. The results demonstrated a maximum proliferation response to EGF at a Ca+ + concentration of 0.4 mM. With lower and higher external Ca+ + concentrations the fraction of cells undergoing more than one cell division decreased. At lower Ca+ + concentrations this decrease appears to result from a reduced viability. In contrast, the low response to EGF at higher Ca+ + concentrations—especially in the physiological range—may reflect the influence of Ca+ + on the state of hepatocyte differentiation.
Free sterol metabolism and low density lipoprotein receptor expression as differentiation markers of cultured human keratinocytes
Authors: Williams Mary L. , Mommaas‐Kienhuis Anne‐Marieke , Rutherford Suzanne L. , Grayson Stephen , Vermeer Bert Jan , Elias Peter M.
Published: February 2005

In contrast to most tissues, epidermis and its derivatives appear to lack low density lipoprotein (LDL) receptors and exhibit sterologenesis rates unaffected by circulating lipoprotein (LP) cholesterol content. Since LDL receptors have been demonstrated in both cultured squamous cell carcinoma cells and human foreskin keratinocytes, when maintained in low‐calcium media, LDL receptor expression may be related to keratinocyte differentiation. We compared receptor binding and internalization of LDL‐gold in normal keratinocytes at different stages of growth at physiological calcium concentrations (early, 3–5 days; preconfluent, 6–10 days; postconfluent, 12–17 days), and correlated receptor expression with sterologenesis in LP‐replete vs.‐depleted media. Whereas in early cultures about 60% of sterologenesis was LP dependent, this fraction declined in preconfluent and confluent cultures despite continued culture growth and little decline in total sterologenesis. Accordingly, LDL receptors were most evident in early cultures, declining in preconfluent cultures in parallel with the decrease in LP‐dependent sterol synthesis. In contrast, sterologenesis in human foreskin fibroblasts was profoundly influenced by exogenous LP at all stages of confluence; total and LP‐dependent sterologenesis declined in parallel with growth cessation. These studies represent the first demonstration that normal keratinocytes express functional LDL receptors at physiologic calcium concentrations. Moreover, they demonstrate that LDL receptor expression in keratinocytes, in contrast to fibroblasts, can only in part be attributed to growth requirements. Instead, loss of LDL receptor expression serves as a distinctive marker of keratinocyte differentiation and may reflect the specific functional requirements of the epidermis in vivo.
Effects of epidermal growth factor on transferrin receptor phosphorylation and surface expression in malignant epithelial cells
Authors: Castagnola Jan , MacLeod Carol , Sunada Hironobu , Mendelsohn John , Taetle Raymond
Published: February 2005

The transferrin (Tf) receptor is a major transmembrane protein which provides iron for normal and malignant cell growth. Epidermal growth factor (EGF) has been reported to rapidly and transiently alter the number of surface Tf receptors in normal and transformed epithelial cells. To investigate mechanisms of EGF‐induced changes in surface Tf display, EGF effects on surface Tf receptors were compared in two cell lines which differ in their number of EGF receptors and growth responses to EGF. In cloned A431 cells with high receptor numbers which are growth‐inhibited by EGF, EGF caused a 50% decrease in Tf receptor expression after 30 min. In contrast, EGF induced a rapid, transitory increase (within 5 min) in the number of surface Tf receptors on KB carcinoma cells which returned to basal levels by 15 min. The observed changes in Tf receptor display were due to altered receptor distribution and not changes in ligand affinity or total cellular transferrin receptor pools. Anti‐EGF receptor monoclonal antibody blocked effects of EGF on transferrin receptor expression. Since the antibody is internalized and causes EGF receptor down‐regulation, effects on transferrin receptor expression were independent of these events. EGF‐induced alterations in Tf receptor display occurred even when cells were pretreated with colchicine, suggesting that changes in surface Tf binding were not mediated by cytoskeletal components. Na orthovanadate, which mimics some early cellular effects of EGF, duplicated EGF's effects on A431 Tf receptors, but had no effect on KB cells, suggesting these responses occur by differing mechanisms. To determine whether EGF caused changes in Tf receptor phosphorylation, 32P‐labelled Tf receptors were immunoprecipitated after EGF treatment. After exposure to EGF, A431 cells showed no change in Tf phosphorylation, but KB cells showed a transient, 6‐fold increase in transferrin receptor phosphorylation on serine residues. In both A431 and KB cells, phorbol ester (PMA) also increased phosphorylation on transferrin receptors, but had little effect on surface Tf receptor expression. In malignant cell lines, EGF induces rapid, variable changes in transferrin receptor expression and phosphorylation which differ from the effects of PMA. These early responses to EGF appear to differ with the cell type and correlate poorly with alterations in Tf receptor phosphorylation. These results suggest Tf receptor phosphorylation does not regulate Tf receptor display in all cells.
Effect of dexamethasone on parathyroid hormone stimulation of cyclic AMP in an opossum kidney cell line
Authors: Rizzoli R. , Bonjour J.‐Ph.
Published: February 2005

The influence of the glucocorticoid dexamethasone on the cAMP response to parathyroid hormone (PTH) and various agonists was studied in epithelial monolayers of opossum kidney (OK) cells. The incubation with dexamethasone for 72 hours led to a dose‐dependent higher cAMP response to PTH or forskolin in intact cells as well as in digitonin‐permeabilized cells. This effect did not appear to result from changes in phosphodiesterase (PDE) activity nor from alterations in cAMP efflux from the cells. Moreover, dexamethasone increased the formation of domes by OK cell epithelium. Thus, dexamethasone seems to promote a more differentiated renal epithelial phenotype as suggested by enhanced hormonal response.
Comparison of oxidative metabolism in vitro in endothelial cells from different species and vessels
Authors: Kjellström B. Thomas , Örtenwall Per , Risberg Bo
Published: February 2005

Oxygen consumption was compared in confluent cultures of endothelial cells from human umbilical cord veins, rat pulmonary arteries, and bovine aortas. A microrespirometric method utilizing oxygenated hemoglobin as oxygen supply and indicator of respiration was used. Respiratory rate was equal in human and murine cells (2.0 × 10−6 and 2.2 × 10−6 μl O2/cell/hour respectively), compared on a cell‐to‐cell basis, while respiratory rate of the bovine endothelium was significantly lower (0.4 × 10−6 μl O2/cell/hour) (P < .001).
Lipid transfer between endothelial and smooth muscle cells in coculture
Authors: Stoll Lynn L. , Spector Arthur A.
Published: February 2005

A coculture system was employed to study the interactions between endothelium and vascular smooth muscle cells in arachidonic acid metabolism. Bovine aortic endothelial cells grown on micropore filters impregnated with gelatin and coated with fibronectin are mounted on polystyrene chambers and suspended over confluent smooth muscle cultures. The endothelial basal laminae are oriented toward the underlying smooth muscle, and the two layers are separated by only 1 mm. Each cell layer was assayed individually: apical and basolateral fluid also was collected separately for assay. Fatty acids, including arachidonic acid, are readily transferred between the endothelial and smooth muscle cells in this system. Distribution of the incorporated fatty acids among the lipids of each cell is the same as when the fatty acid is added directly to the culture medium. Arachidonic acid released from endothelial cells is available as a substrate for prostaglandin production by smooth muscle. In addition, fatty acids released from the smooth muscle cells can pass through the endothelium and accumulate in the fluid bathing the endothelial apical surface. These fatty acid interchanges may be involved in cell‐cell signaling within the vascular wall, the clearance of lipids from the vascular wall, or the redistribution of arachidonic acid and other polyunsaturated fatty acids between adjacent cell types. Furthermore, the findings suggest that prostaglandin production by smooth muscle cells can occur in response to stimuli that cause arachidonic acid release from endothelial cells.
Insulin‐like growth factor‐I: Specific binding to high and low affinity sites and mitogenic action throughout the life span of WI‐38 cells
Authors: Phillips Paul D. , Pignolo Robert J. , Cristofalo Vincent J.
Published: February 2005

Insulin‐like growth factor‐I (IGF‐I) (13 nM) can replace insulin (0.8 μM) in a serum‐free medium containing epidermal growth factor (EGF) (16 nM) and dexamethasone (DEX) (140 nM) and stimulate DNA synthesis in young cultures of WI‐38 cells, similar to the stimulation of serum‐supplemented medium. By contrast, senescent cells become unresponsive to all of these hormones. The effect of IGF‐I, EGF, and DEX is synergistic in stimulating multiple rounds of low density cell division. Total specific binding of [125]IGF‐I per cell in monolayer culture does not change with age, which indicates, in light of increased cell size with age, an actual decrease in specific binding per μM2 of cell surface area. Binding can be traced to two separate cell proteins. Binding to the α subunit of the IGF‐I transmembrane receptor may increase slightly with age while the 50% displacement remains unchanged. The remainder of the IGF‐I specific binding (five‐ to thirty‐fold more) is to a low molecular weight, cell‐associated binding protein whose 50% displacement is 10 times higher, but also remains unchanged with age. Specific binding to the lower affinity sites decreases slightly with age at equal cell densities. IGF‐I binding to the α subunit of the transmembrane receptor is independent of cell density, while binding to the low molecular weight binding protein is inversely proportional to cell density and may vary by as much as tenfold.
Contact‐inhibition‐induced quiescent state is marked by intense nuclear expression of Statin
Authors: Wang Eugenia
Published: February 2005

Statin, a nuclear protein of 57,000 daltons, is found in in vitro aged, nonproliferating human fibroblasts but not in their young, replicating counterparts or transformed derivatives; it is also found in the nuclei of young fibroblasts when their growth is arrested but rapidly disappears from the cells once the restriction to growth is removed. We reported earlier that as cells leave the quiescent state, the loss of Statin from the nucleus precedes the initiation of DNA synthesis; here we report that in a confluent culture, as cells leave the traverse of the replicative cycle and assume the quiescent phenotype, Statin is not expressed maximally until total contact inhibition of growth is achieved. This full manifestation of Statin occurs in monolayer culture with cells forming the typical swirling pattern and fibronectin organized into large intercellular cables. The late expression of Statin in cells approaching the quiescent state is also verified biochemically by immunoblotting assays. The present results, taken together with those reported earlier, indicate that the nuclear appearance of Statin occurs only after the complete cessation of DNA synthesis and that the full manifestation of this protein can be used as a marker for the G0 quiescent state.
Superoxide dismutase activity and glutathione concentration during the calcium‐induced differentiation of Physarum polycephalum microplasmodia
Authors: Nations C. , Allen R. G. , Balin A. K. , Reimer R. J. , Sohal R. S.
Published: February 2005

Microplasmodia of Physarum polycephalum differentiate into spherules when the CaCl2 concentration of their nutrient medium is increased to 54mM (high‐calcium). The salts starvation medium routinely used to induce differentiation contains 8mM CaC12. This medium will not induce spherulation in the absence of a calcium salt; no other metal is essential. High‐calcium also induces the spherulation of a strain of Physarum that had not been previously observed to spherulate. The striking increase in superoxide dismutase activity (SOD) and the decrease in glutathione concentration (GSH) that are characteristic of salts‐induced spherulation do not occur in salts media containing high‐calcium. In the absence of calcium, no significant change in SOD is observed and very little change in GSH occurs. The immediate effect of the oxidative stress associated with spherulation may be the release of calcium stores into the cytosol. The parameters modulating this stress are, in turn, sensitive to exogenous calcium concentrations.
Differentiation of human keratinocytes: Changes in lipid synthesis, plasma membrane lipid composition, and 125I‐EGF binding upon administration of 25‐hydroxycholesterol and mevinolin
Authors: Ponec Maria , Kempenaar Johanna , Weerheim Ari , Boonstra Johannes
Published: February 2005

We have studied the relationship between differentiation capacity, plasma membrane composition, and epidermal growth factor (EGF) receptor expression of normal keratinocytes in vitro. The plasma membrane composition of the cells was modulated experimentally by cholesterol depletion, using specific inhibitors of cholesterol synthesis, such as 25‐hydroxycholesterol and mevinolin. Exposure of the cells towards these inhibitors resulted in a drastic decrease of cholesterol biosynthesis, as determined from 14C‐acetate incorporation into the various lipid fractions. This effect on cholesterol biosynthesis was reflected by changes in plasma membrane composition, as determined by lipid analysis of isolated plasma membrane fractions, these resulting in a decreased cholesterol‐phospholipid ratio. The experimental modulation of plasma membrane composition by 25‐hydroxycholesterol or mevinolin were accompanied by a decreased cornified envelope formation and by high expression of EGF binding sites. These phenomena were more pronounced in cells induced to differentiate by exposure of cells grown under low Ca2+ to normal Ca2+ concentrations, as compared to cells grown persistantly under low Ca2+ concentrations. These results suggest a close correlation between plasma membrane composition, differentiation capacity, and EGF receptor expression.
Insulin‐like growth factor‐II receptors in cultured rat hepatocytes: Regulation by cell density
Authors: Scott Carolyn D. , Baxter Robert C.
Published: February 2005

Insulin‐like growth factor‐II (IGF‐II) receptors in primary cultures of adult rat hepatocytes were characterized and their regulation by cell density examined. In hepatocytes cultured at 5 × 105 cells per 3.8 cm2 plate, [125I]IGF‐II bound to specific, high affinity receptors (Ka = 4.4 ± 0.5 × 109 I/mol). Less than 1% cross‐reactivity by IGF‐I and no cross‐reactivity by insulin were observed. IGF‐II binding increased when cells were permeabilized with 0.01% digitonin, suggesting the presence of an intracellular receptor pool. Determined by Scatchard analysis and by polyacrylamide gel electrophoresis after affinity labeling, the higher binding was due solely to an increase in binding sites present on 220 kDa type II IGF receptors. In hepatocytes cultured at low densities, the number of cell surface receptors increased markedly, from 10–20,000 receptors per cell at a culture density of 6 × 105 cell/well to 70–80,000 receptors per cell at 0.38 × 105 cells/well. The increase was not due simply to the exposure of receptors from the intracellular pool, as a density‐related increase in receptors was also seen in cells permeabilized with digitonin. There was no evidence that IGF binding proteins, either secreted by hepatocytes or present in fetal calf serum, had any effect on the measurement of receptor concentration or affinity. We conclude that rat hepatocytes in primary culture contain specific IGF‐II receptors and that both cell surface and intracellular receptors are regulated by cell density.
Insulin‐like growth factor II binding and action in human fetal fibroblasts
Authors: Conover Cheryl A. , Rosenfeld Ron G. , Hintz Raymond L.
Published: February 2005

To investigate the role of insulin‐like growth factor II (IGF‐II) in human prenatal growth, IGF‐II binding and biological action were studied in four lines of fetal and three lines of postnatal human fibroblasts. Specific biniding of IGF‐II was similar in both groups: 15.7% and 14.9% for fetal and postnatal fibroblasts, respectively. This was 5–10 times the amount of IGF‐I binding found in these cells. IGF‐I and IGF‐II caused dose‐dependent increases in [14C]aminoisobutyric acid (AIB) uptake. IGF‐II was sevenfold less potent than IGF‐I in stimulating this metabolic response in both fetal and postnatal fibroblasts. The maximal effect of IGF‐II in stimulating [14C]AIB uptake approached that of IGF‐I. Similar results were obtained when IGF‐I and IGF‐II stimulation of [3H]thymidine incorporation was compared in fetal and postnatal fibroblasts. Incubation in the presence of alphalR‐3, a monoclonal antibody to the type I IGF receptor, inhibited the ability of both IGF‐I and IGF‐II to stimulate [14C]AIB uptake and [3H]thymidine incorporation in fetal and postnatal cells. A monoclonal antibody to the insulin receptor did not affect IGF action. These data indicate that IGF‐II is a potent metabolic and mitogenic stimulus for human fetal firoblasts. However, despite the presence of abundant type II IGF receptors on both fetal and postnatal human fibroblasts, IGF‐II stimulation of amino acid transport and DNA synthesis appears to be mediated through the type I rather than through its own type II IGF receptor.
Effect of osmolar and ionic composition of the extracellular fluid on transferrin endocytosis and exocytosis and iron uptake by reticulocytes
Authors: Bowen Barbara J. , Morgan Evan H.
Published: February 2005

The effects of osmolar and ionic factors on endocytosis and exocytosis were investigated using rabbit reticulocytes and 125I‐59Fe labelled transferrin. Endocytosis and exocytosis of transferrin and the uptake of iron were inhibited by increasing the osmolality or decreasing the ionic strength or pH of the cell incubation medium. However, elevation of the pH above 8.0 inhibited endocytosis but not exocytosis. Replacement of the NaCl in the incubation medium by Nal, NaF, NaSCN, NaCIO4, Na2SO4, Na phosphate, or Na Hepes inhibited endocytosis and iron uptake but only Nal, NaF, and NaSCN inhibited exocytosis. Transferrin exocytosis was insensitive to inhibitors of anion or cation transport, but endocytosis and iron uptake were inhibited by several anion transport inhibitors. Overall, transferrin endocytosis was more sensitive than exocytosis to most of the factors which were investigated, and the effects on the rates of endocytosis and iron uptake were quantitatively very similar. The results provide strong support for the concept that transferrin endocytosis is a necessary step in iron uptake by reticulocytes. They do not support the chemiosmotic models of exocytosis in their present formulations, out do not rule out the possible role of an osmotic event in exocytosis.
Activation and proliferation signals in murine macrophages: Stimulation of Na+, K+‐ATPase activity by hemopoietic growth factors and other agents
Authors: Vairo Gino , Hamilton John A.
Published: February 2005

Purified colony stimulating factor (CSF‐1) stimulates the Na+, K+‐ATPase activity of murine bone marrow‐derived macrophages (BMM) and resident peritoneal macrophages (RPM) measured as ouabain‐sensitive 86Rb+ uptake. Similar concentrations of CSF‐1 stimulate the Na+, K+‐ATPase activity and DNA synthesis in BMM whilst ouabain, a specific inhibitor of the Na+, K+‐ATPase, also inhibits this CSF‐1‐mediated DNA synthesis. Other purified hemopoietic growth factors, granulocyte‐macrophage CSF (GM‐CSF) and interleukin‐3 (IL‐3), and the tumor promoter, 12‐0‐tetradecanoyl‐phorbol‐13‐acetate (TPA), even though differing in their mitogenic capabilities, are also stimulators of the Na+,K+‐ATPase activity in BMM and RPM. The non‐mitogenic agents, lipopolysaccharide (LPS) and Concanavalin A (Con A), are also active. CSF‐1 stimulation of the Na+,K+‐ATPase was shown to be dependent on elevation of intracellular Na+ via an amiloride sensitive Na+‐channel, most likely representing Na+/H+ exchange activity. Such stimulation of Na+,K+‐ATPase activity via activation of the Na+/H+ exchange appears to be a necessary but insufficient early macrophage response for subsequent DNA synthesis.
Time dependence of transmembrane potential changes and intracellular calcium flux in stimulated human monocytes
Authors: Bernardo John , Brink Holly F. , Simons Elizabeth R.
Published: February 2005

An important characteristic of the functional differentiation of the blood monocyte is the development of its capacity to recognize and respond to stimuli. This ability is mediated to a large extent by specific receptor glyco‐proteins located on the cell surface. Stimulation of mononuclear phagocytes via these receptors results in a rapid rise in intracellular Ca+ + concentration, accompanied or followed by a change in membrane potential, generation of oxidative products, degranulation, and effector functions such as phagocytosis, aggregation, or locomotion. While the development of these characteristics is difficult to characterize in vivo, several investigators have demonstrated in vitro changes in these cells that correlate with the development of effector function. To examine the mechanisms of specific membrane‐stimulus interactions of monocytes as they differentiate into macrophage‐like cells, we studied the responses of human monocytes and of monocytes incubated in serum‐containing medium for up to 96 hr to the chemotactic peptide formyl‐methionyl‐leucyl‐phenylalanine (fMLP). Freshly isolated monocytes exhibited little change in transmembrane potential following stimulation with an optimal concentration of peptide and underwent a significant increase only after 48 hr in culture. While constant resting intracellular Ca+ + concentrations were maintained during the culture period, intracellular Ca+ + levels following fMLP stimulation increased with with incubation in serum, for up to 96 hr. In contrast, fMLP‐induced respiratory burst activity increased from 0 to 24 hr in culture; it remained elevated at 48 hr but declined again by 96 hr. Incubation of the cells for 24 hr increased their random (unstimulated) motility in modified Boyden chambers but did not alter the cells' directed (chemotactic) response to fMLP in comparison to the response of freshly isolated monocytes. Peptide binding to the cells did not increase during the incubation period, indicating that an increase in receptor number or in affinity for fMLP was not responsible for the enhanced responsiveness to fMLP as incubation time increased. These studies indicate that incubation of monocytes in serum‐containing medium leads to a complex, altered series of responses to fMLP that correlate with the differentiation of the original monocytes in vitro and may relate to the in vivo differentiation of monocytes to macrophages.
Roles of protein kinases in neurotransmitter responses in Xenopus oocytes injected with rat brain mRNA
Authors: Ito I. , Hirono C. , Yamagishi S. , Nomura Y. , Kaneko S. , Sugiyama H.
Published: February 2005

Microinjection of rat brain mRNA in Xenopus oocytes induced acetylcholine, neuroteisin, serotonin, and glutamate receptors in the cells. These receptors stimulate an intracellular reaction pathway, including G‐protein activation, inositol trisphosphate (IP3) formation, and Ca2+‐dependent CI− channels. In the present study, we examined the roles of several protein kinases in these responses by means of inhibitors and activators of these kinases. Isoquinolinesulfonamides, inhibitors of protein kinases, caused no current responses and affected no receptor‐mediated responses when injected into the oocytes at low doses (30–50 pmol), which inhibit cyclic nucleotide‐dependent kinases or kinase C specifically, but abolished the receptor‐mediated responses at a higher dose (300 pmol), which inhibit most protein kinases nonspecifically. Calmodulin inhibitors blocked the receptor‐mediated responses strongly. Activation of cyclic nucleotide‐dependent kinases or kinase C by injection of cAMP (or cGMP) or perfusion with phorbol esters caused no direct current responses but suppressed receptor‐mediated responses. Current responses triggered by IP3 injection were not suppressed by these treatments. These results suggest that cAMP‐ (or cGMP‐)dependent kinases or kinase C may not be involved in the pathway directly but may modulate it by inhibiting the initial part of the pathway (receptors, G‐proteins, and/or phospholipase C), and they suggest that calmodulin may most likely be involved in the activation of Ca2+‐dependent CI− channels.
Myogenic stem cell commitment probability remains constant as a function of organismal and mitotic age
Authors: Quinn Lebris Smith , Norwood Thomas H. , Nameroff Mark
Published: February 2005

Chicken myogenic stem cells can undergo symmetric and asymmetric cell divisions. Symmetric divisions produce two stem cells or two cells committed to terminal muscle differentiation. Asymmetric divisions produce one stem cell and one committed cell. Committed cells undergo four divisions, and their progeny differentiate into postmitotic, biochemically distinct muscle cells, which can be identified immunocytochemically. The control of stem cell commitment was investigated in vitro by means of cell cloning and subcloning experiments, and computer modeling. We found that stem cell commitment is a process which can be modeled as a stochastic event, with a central tendency or probability of 0.2 ± 0.1. This value is independent of organismal or mitotic age of the stem cells, cell density, or growth in a mitogen‐poor environment. Myogenic stem cells stop dividing after approximately 30 divisions in vitro. Since the probability of commitment to terminal differentiation remains below 0.5, clonal senescence and terminal differentiation are separate processes in this system.
Serial propagation of human ovarian surface epithelium in tissue culture
Authors: Siemens Craig H. , Auersperg Nelly
Published: February 2005

Most human ovarian cancers are thought to arise in the ovarian surface epithelium (OSE). The precise role of OSE in carcinogenesis has not been defined because no appropriate animal models for the study of this tissue exist and culture of human OSE has been limited to primary outgrowths. In this report, we describe conditions for serial cultivation of normal human OSE. Premenopausal ovarian tissue was obtained at surgery. OSE growth was compared in media MCDB 202, 199 and Waymouth's 752/1 (WM) supplemented with 5, 15, or 25% fetal bovine serum (FBS), with/without 20 ng/ml epidermal growth factor (EGF) and 0.4 γg/ml hydrocortisone (HC). The rate and extent of OSE outgrowths from explants in primary culture were greatest in either WM or 199/202 (1:1), supplemented with 15% FBS/EGF/HC. In early passage cultures, cell proliferation was most rapid and extensive in 199/202 with 15% FBS, EGF, and HC. In this medium, OSE cells were subcultured up to 10 times and underwent 20–25 population doublings over 5 weeks. The population doubling time during rapid growth was approximately 48 h. Seeding efficiencies of up to 53% and cloning efficiencies of up to 13% were obtained. Early passage OSE cells reversibly modulated from a slow growing, epithelial, intensely keratin‐positive form in 199/202 medium lacking EGF/HC, to a rapidly proliferating, elongate, less keratin‐positive form in medium with EGF/HC. OSE cells grown in WM/5–15% FBS were epithelial and near‐stationary. Thus, culture conditions have been defined for ovarian carcinogen assays requiring either proliferating or stationary cell populations, and for further studies of the role of OSE in ovarian biology.
Effects of diacylglycerols on LLC‐PK1 renal epithelia: Similarity to phorbol ester tumor promoters
Authors: Mullin James M. , McGinn Mary T.
Published: February 2005

As previously shown using phorbol ester tumor promoters (see Mullin and O'Brien: Am. J. Physiol., 251:C597–C602, 1986), diacylglycerols induce leakiness in LLC‐PK1 renal epithelial tight junctions. The similarity between phorbol ester and diacylglycerol action includes effects on (1) cell morphology, (2) dome formation, (3) transepithelial resistance and potential difference, (4) transepithelial flux of D‐mannitol, and (5) mitogenesis. Four diacylglycerols have been tested: 1,2‐dioctanoylglycerol; 1,2‐dicaprylglycerol; 1,2‐dioleoylglycerol; and 1‐oleoyl‐2‐acetoyl‐sn‐3‐glycerol. Their relative effectiveness depended upon the phenomenon being observed. Unlike phorbol esters, diacylglycerol effects were reversible within hours at 37°C in the continued presence of diacylglycerol, and effects were more pronounced when cell sheets were exposed to diacylglycerols from the basolateral cell surface. Overall, these findings indicate that previous results with phorbol esters may be attributed to the protein kinase C signal transduction system, and this system may therefore exert a role in transepithelial permeability.
Direct visualization and quantitative analysis of epidermal growth factor‐induced receptor clustering
Authors: van Belzen Nico , Rijken Philip J. , Hage Willem J. , de Laat Siegfried W. , Verkleij Arie J. , Boonstra Johannes
Published: February 2005

Several observations have indicated that clustering of growth factor receptors plays an important role in the action of growth factors. In this investigation, we have used the label fracture method to study the effects of epidermal growth factor (EGF) on the lateral distribution of its receptors in A431 epidermoid carcinoma cells. This method allows a direct visualization of immunogold‐labeled plasma membrane receptors on ultrastructural level and in addition permits an quantitative analysis of their lateral distribution. EGF receptors were immunogold‐labeled according to standard procedures with the monoclonal anti‐EGF receptor antibody 2E9 (lgG1), which binds to the EGF receptor in a 1:1 ratio. In the absence of EGF, EGF receptors located on the surface of A431 cells were found to be clustered, as deduced from Poisson variance analysis (p < 0.001). Following treatment of A431 cells with EGF, receptor clustering increased rapidly, reaching the maximum within 10 min. Maximal clustering was maintained for 1 h, after which the lateral distribution of receptors returned to the control situation within another hour.
Role of the cytoskeleton in laminin induced mammary gene expression
Authors: Blum Joanne L. , Wicha Max S.
Published: February 2005

The differentiation of rat mammary epithelial cells is characterized both by morphologic changes and by the expression of a group of milk protein genes. We have previously shown that by culturing these cells on the basement membrane glycoprotein laminin, the synthesis of the milk proteins, transferrin, α‐casein, and α‐lactalbumin is induced. In order to determine if this effect is mediated through the cytoskeleton, we have treated these cells with cytochalasin D and colchicine. Treatment with cytochalasin D or colchicine for 24 h inhibits the accumulation of α‐casein, transferrin, and α‐lactalbumin without significant effect on general protein synthesis. Pulse chase studies show that cytochalasin D does not alter the intracellular turnover of α‐casein or transferrin. Additionally, treatment with cytochalasin D causes an early (within 1 h) increase in secretion of α‐casein and transferrin suggesting that the actin cytoskeleton provides a meshwork for secretory vesicles. The disruption of this network enhances the secretion of preformed proteins. However, long term (24 h) treatment with cytochalasin D inhibits synthesis of these milk proteins. Northern blot analysis indicates that treatment with cytochalasin D or colchicine inhibits the laminin induced increase in α‐casein, α‐lactalbumin, and transferrin mRNAs. These studies indicate that the major effect of the cytoskeleton on laminin induced milk protein gene expression occurs at the level of accumulation of mRNAs for these proteins. We conclude that the expression of laminin induced milk protein gene expression in primary rat mammary cultures depends on the integrity of the actin and microtubule cytoskeleton.