bluuebunny/crossref_metadata_2025 huggingface dataset:
Gangliosides stimulate astroglial cell proliferation in the absence of serum
Authors: Katoh‐Semba Ritsuko , Facci Laura , Skaper Stephen D. , Varon Silvio
Published: February 2005

Previous studies with microcultures of astroglial (AG) cells from newborn rat cerebrum had shown an ability of gangliosides to interact with AG cells cultured under defined conditions. We have now investigated the capability of gangliosides to stimulate DNA synthesis and cell number increases in similar secondary microcultures of newborn rat cerebrum AG cells. At a concentration of 6 × 10−5M, GM1 ganglioside stimulated DNA synthesis and increased cell numbers, with DNA synthesis leading cell increases by 12–24 hr. The ganglioside‐induced AG cell proliferative response occurred with GD1a, GD1b and GT1b, GT1b being the most potent at 10−5M—while asialo GM1 and sialic acid were without effect. In the standard test cultures, DNA synthesis declined very steeply after the first day, with cell numbers stabilizing at the level reached after 2 days. Ganglioside was not itself responsible for the restricted proliferative response, as serum produced the same behaviors.
Effects of hypoxia and hyperoxia on proteoglycan production by bovine pulmonary artery endothelial cells
Authors: Humphries Donald E. , Lee Sheu‐Ling , Fanburg Barry L. , Silbert Jeremiah E.
Published: February 2005

Bovine pulmonary artery endothelial cells in culture were used to assess the influence of oxygen tension on proteoglycan synthesis. Cells exposed to 3% O2 (hypoxia) for 72 h and then labeled with [35S]sulfate for 5 h accumulated significantly less [35S]proteoglycan in medium than cells exposed to 20% O2 (control). This decrease was due primarily to a reduction in heparan sulfate. Cells exposed to 80% O2 (hyperoxia) for 72 h secreted slightly more [35S]proteoglycan into medium than controls. Greater accumulation of chondroitin sulfate was responsible for the increase. The amount of cell‐associated proteoglycan did not change significantly in cells cultured in 3% or 80% O2 as compared with control cells cultured in 20% O2. Proteoglycans produced by hypoxia‐ or hyperoxia‐treated cells were found to be similar in size to proteoglycans produced by cells cultured at 20% O2. Glycosaminoglycan sulfation, as measured by ion‐exchange chromatography, did not appear to change with varying oxygen tensions. Our results demonstrate that production of proteoglycans secreted by endothelial cells in culture is sensitive to oxygen tension.
Markedly altered membrane transport and intracellular binding of vincristine in multidrug‐resistant Chinese hamster cells selected for resistance to vinca alkaloids
Authors: Sirotnak F. M. , Yang C.‐H. , Mines L. S. , Oribé E. , Biedler J. L.
Published: February 2005

Studies of a multidrug‐resistant variant (DC‐3F/VCRd‐5L) of Chinese hamster lung cells selected for resistance to vinca alkaloids revealed marked alterations in transport and intracellular binding of [3H]vincristine compared to parental DC‐3F cells. Influx of [3H]vincristine in DC‐3F cells appears to be an equilibrating, but mediated, process. Although saturation kinetics for [3H]vincristine influx were not demonstrated, an extremely high temperature‐dependence (Q1027–37°C = 5–6) and trans‐inhibition of influx following preloading of cells with nonradioactive vincristine argue in favor of a carrier‐mediated process. Efflux of [3H]vincristine from parental cells conformed to first‐order kinetics (t1/2 37° = 3.6 ± 0.4) and exhibited a lower temperature‐dependence (Q10 27–37°C = 3–3.5) than influx. In variant vs. parental cells, influx of [3H]vincristine was reduced 24‐fold and efflux was increased twofold, accounting for the large (approximately 48‐fold) reduction in steady‐state level of exchangeable drug accumulating in variant cells. Otherwise, transport of [3H] vincristine in these cells showed characteristics similar to parental DC 3F cells. Also, the rate and amount of intracellular binding of [3H]vincristine in variant cells was almost 40‐fold lower than in parental cells. These alterations in influx and efflux of [3H]vincristine and its intracellular binding appear to account, at least to a major extent, for the high level of resistance (2,750‐fold) of this variant to vinca alkaloids. In contrast, cross‐resistance of this variant to daunomycin (178‐fold) could be explained only minimally by a transport alteration. Only a two‐fold increase in efflux of [3H]daunomycin was demonstrated in variant vs. parental cells along with some decrease in intra‐cellular binding. Influx of [3 H]daunomycin was unaltered. In view of these results, we conclude that these two agents most likely do not share the same route for entry in these cells but might share the same efflux route.
Alterations in plasma membrane lipid organization during lymphocyte differentiation
Authors: Del Buono Brian J. , Williamson Patrick L. , Schlegel Robert A.
Published: February 2005

The fluorescent probe merocyanine 540, which binds preferentially to bilayers in which the lipids are loosely packed, was used to investigate changes in the organization of the lipids of the lymphocyte plasma membrane during primary and secondary lymphopoiesis. When mouse thymocytes were incubated with the dye, most immature cells stained, while most mature cells, about to enter the peripheral circulation, did not. Similarly, mature lymphocytes from both mouse and human peripheral blood did not stain, but these same cells did when activated by in vitro mitogenic stimulation. Freshly isolated splenic lymphocytes, presumably activated in vivo by antigen, also bound merocyanine 540, but after 48 hours of culture in the absence of stimulus they displayed only a low affinity for the dye, a phenotype that reverted to a high affinity upon mitogenic stimulation. These results suggest that changes in the organization of the lipids of the plasma membrane take place during lymphocyte differentiation: viz., immature cells possess a disordered membrane that becomes increasingly ordered as the cells mature and enter the peripheral circulation; then, upon antigen‐induced differentiation, the plasma membrane again becomes disordered. These lipid organization changes are discussed in the context of their possible role in the regulation of lymphocyte circulation via intercellular interactions between lymphocytes and cells of the reticuloendothelial system.
Norepinephrine decreases EGF binding in primary rat hepatocyte cultures
Authors: Cruise Jennifer L. , Cotecchia Susanna , Michalopoulos George
Published: February 2005

Norepinephrine (NE) produced a dose‐dependent inhibition of 125I‐epidermal growth factor (EGF) binding to adult rat hepatocytes in primary culture. This effect was maximal after 1 hr of incubation with NE and could be blocked by the presence of an α1‐specific adrenergic receptor antagonist. The inhibition of binding correlates with the ability of NE to enhance hepatocyte DNA synthesis in the presence of EGF and appears to be mediated by a reduction in EGF receptor number, without a significant change in receptor affinity.
Transforming growth factor type β regulation of actin mRNA
Authors: Leof Edward B. , Proper Jacqueline A. , Getz Michael J. , Moses Harold L.
Published: February 2005

Stimulation of quiescent cultures of AKR‐2B cells with transforming growth factor type β (TGFβ) resulted in a transitory increase in actin cytoplasmic poly(A) + RNA. Levels of actin mRNA peaked approximately 4–8 hours subsequent to TGFβ addition and approached basal levels by 24 hours. The accumulation of actin transcripts was dose dependent and insensitive to inhibitors of protein synthesis; 1–3 ng/ml TGFβ induced maximal actin gene expression. Actin isotype‐specific probes demonstrated that both β‐ and γ‐cytoplasmic actins are induced by TGFβ.
Altered polyamine metabolism in chinese hamster cells growing in a defined medium
Authors: Sertich Gary J. , Glass James R. , Fuller David J. M. , Gerner Eugene W.
Published: February 2005

Chinese hamster cells (line CHO) maintained in McCoy's 5A medium (modified) supplemented with insulin (10 μg/ml), transferrin (5 μg/ml), and ferrous sulfate (1.1 μg/ml) proliferate at rates similar to cultures growing in the McCoy's medium supplemented with 10% fetal bovine serum. Colony‐forming ability is similar in cultures supplemented with either serum or the combination of growth factors. By 6 hours after replacement of serum with growth factors, ornithine decarboxylase (ODCase) activity increases, reaching a maximum value by 24 hours after serum replacement. This maximum is cell density dependent and can exceed a 30‐fold increase over enzyme activity in cultures supplemented with serum. The increased enzyme activity is due to a decrease in the turnover rate of the enzyme, based on protein synthesis inhibition studies, and an accumulation of active enzyme molecules rather than an activation of existing molecules, since the catalytic activity of ODCase, determined using the radiolabeled form of α‐difluoromethylornithine (an enzyme‐activated, irreversible inhibitor of ODCase) in concert with immuno‐chemical techniques, is unchanged by the different growth medium supplements. Intracellular putrescine and spermidine levels are substantially decreased when cultures are maintained in medium supplemented with insulin, transferrin, and ferrous sulfate, rather than serum, which is the sole source of exogenous ornithine. Titration of cultures growing in the defined medium with ornithine leads to a decrease in ODCase activity and an increase in intracellular putrescine and spermidine levels. Putrescine‐ and spermidine‐dependent S‐adenosyl‐L‐methionine decarboxylase activities are similar in cultures maintained in either medium. These data demonstrate that some, but not all, aspects of polyamine biosynthesis are affected by the availability of ornithine, the first substrate in the pathway.
Isolation of a human stromal cell strain secreting hemopoietic growth factors
Authors: Tsai Schickwann , Emerson Stephen G. , Sieff Colin A. , Nathan David G.
Published: February 2005

A diploid fibroblastoid cell strain, termed “ST‐1,” has been established from a long‐term liquid culture of human fetal liver cells. ST‐1 cells are nonphagocytic, nonspecific esterase negative and do not possess factor VIII‐related antigen but stain with antibodies specific for fibronectin and type I collagen. The ST‐1 cells produce nondialyzable hemopoietic growth factors capable of stimulating the development of erythroid bursts, mixed granulocyte‐macrophage colonies, pure granulocyte colonies, and pure macrophage colonies. These factors are active on both human fetal liver and human adult bone marrow progenitors. When liquid cultures of human fetal liver hemopoietic progenitors are established with a preformed monolayer of ST‐1 cells, the yields of nonadherent cells, erythroid progenitors, and myeloid progenitors are greatly increased. These studies demonstrate that the fibroblastoid ST‐1 cells support hemopoiesis in vitro and may be a critical element in the stromal microenvironment in vivo.
A cartilage‐derived growth factor enhances hyaluronate synthesis and diminishes sulfated glycosaminoglycan synthesis in chondrocytes
Authors: Hamerman David , Sasse Joachim , Klagsbrun Michael
Published: February 2005

Cartilage‐derived growth factor purified to homogeneity by affinity chromatography on columns of heparin‐Sepharose was mitogenic for early passage bovine fetal chondrocytes. Hyaluronate and sulfated glycosaminoglycan synthesis in these cells was analyzed by differential enzymatic digestion of the glycosaminoglycans labeled with [14C] glucosamine or [35S]. It was found that chondrocyte proliferation was accompanied by about a four‐fold increase in hyaluronate synthesis over a two‐day period, while the synthesis of sulfated glycosaminoglycans decreased by about 2‐fold. Chromatographic analysis of the sulfated glycosaminoglycans showed decreases in chondroitin 4 and 6 sulfates. It was concluded from these results that cartilage‐derived growth factor was a proliferative factor for chondrocytes and differed from the somatomedins.
In vitro expression of a 38,000 dalton heparin‐binding glycoprotein by morphologically differentiated smooth muscle cells
Authors: Millis Albert J. T. , Hoyle Marian , Kent Lorraine
Published: February 2005

In vitro, high density monolayer cultures of vascular smooth muscle cells can be induced to form multicellular nodules. The nodular cells appear to be morphologically differentiated smooth muscle cells. Sodium dodecyl sulfate (NaDodSO4)‐polyacrylamide gel electrophoresis was used to compare the proteins synthesized and secreted by monolayer and nodular cultures of smooth muscle cells. Although most proteins appeared to be similar, the nodular cultures contained a unique heparin binding protein of Mr = 38,000 (38kD protein) (Millis, A.J.T., Hoyle, M., Reich, E., and Mann, D.M., 1985, J. Biol. Chem., 260: 3754–3761). The 38kD protein was glycosylated and its apparent molecular weight was shifted to Mr = 32,500 after synthesis in the presence of tunicamycin or digestion with endoglycosidase F. The production of 38kD protein by nodular cell cultures did not appear to result from the degradation of a high molecular weight precursor in nodular conditioned medium. Further, it was not detected in monolayer cell conditioned medium that had been incubated with nodular cells. Finally, its synthesis was not induced in monolayer cell cultures that had been labeled in nodular cell conditioned medium. The 38kD protein appears to be uniquely associated with nodular cultures of smooth muscle cells.
Mechanisms of cytoskeletal regulation: Modulation of aortic endothelial cell protein band 4.1 by the extracellular matrix
Authors: Leto Thomas L. , Pratt Bruce M. , Madri Joseph A.
Published: February 2005

The bovine aortic endothelial cell (BAEC) cytoskeleton is a complex structure modulated by many stimuli including release from contact inhibition and various components of the extracellular matrix (ECM). Transduction of information from the ECM to the cell nucleus proceeds via several complex pathways including the cytoskeleton. We have demonstrated the presence of an immunoreactive isoform of the human erythrocyte cytoskeletal protein band 4.1 (4.1) in BAEC. BAEC 4.1 is similar in molecular weight to the erythroid protein by immunoblot analyses and produces a similar pattern of cysteine specific cleavage products consistent with a cluster of cysteine residues previously described in the erythroid molecule. We have also examined the effects of defined ECM proteins on the distributions of cultured BAEC 4.1 and actin filaments (AF) at confluency and following release from contact inhibition. The distribution of 4.1 in BAEC on a plasma fibronectin substrate is complex, having partial codistribution with cytoplasmic AF and a unique perinuclear staining. In contrast, on a collagen type I/III substrate, 4.1 is localized, in part, to peripheral areas of cell‐cell contact distinct from the dense peripheral band staining of AF. During migration on this substrate, 4.1 had a filamentous distribution having partial codistribution with AF. Indirect immunofluorescence staining of cross‐sections of bovine calf aortae revealed a cortical staining pattern in the aortic endothelial cells with staining noted on the luminal and basolateral aspects of the cells. These data suggest that, in endothelial cells, protein 4.1 is a cortical membrane protein which may function to link actin filaments to other skeletal proteins such as spectrin. These findings also suggest an active role for protein 4.1 in cytoskeletal reorganization events which can occur in response to external stimuli, such as the extracellular matrix or contact with other cells.
Alternative interpretation for the osmotic response of human erythrocytes
Authors: Massaldi H. A. , Richieri G. V. , Mel H. C.
Published: February 2005

In a recent publication, Heubusch et al. (J Cell. Physiol, 122:266–272, 1985) reported changes of erythrocyte volume measured by the Coulter counter technique over a wide range of osmolalities (160 to 3000 mOsm). Their results showed a partially hindered, nonlinear response, in contrast to classical observations made over more restricted osmolality ranges, using other methods. The authors suggested the underlying cause of this behavior to be a mechanical resistance of the membrane cytoskeleton. In this paper, we wish to offer a different interpretation of their results on erythrocyte osmotic behavior, based on similar experiments carried out in our laboratory, and supported by previous analyses from the literature. In particular, it is shown that the shape‐factor correction to the electronic sizing measurement can correctly account for the observed deviations from linearity in the hypotonic range. In contrast, increased chemical nonideality and eventual hemolysis are the likely factors responsible for the behavior in the hypertonic range.
Electrophysiological responses of osteoclasts to hormones
Authors: Ferrier Jack , Ward Angela , Kanehisa Junya , Heersche Johan N. M.
Published: February 2005

Electrophysiological measurements were carried out on osteoclasts in vitro. Such isolated osteoclasts are able to resorb bone in vitro and contract in response to calcitonin (CT). Our measurements show that individual osteoclasts respond to CT with a significant transient hyperpolarization of membrane potential. Application of parathyroid hormone (PTH) and dibutyryl cAMP produced a transient hyperpolarization in some osteoclasts. Measurements on an osteoblastlike line (ROS 17/2.8) showed a sustained hyperpolarizing response to CT, which is similar to but smaller than the hyperpolarizing response to PTH and dibutyryl cAMP in this and some other osteoblastlike lines. In contrast to osteoblastlike cells, the osteoclasts have no long term membrane potential response to CT, to PTH, or to dibutyryl cAMP. These results show that there are distinct differences between osteoclasts and osteoblasts in their ion transport responses to hormones.
Regulation of inositol phosphate levels by prostaglandins in cultured endometrial cells
Authors: Orlicky D. J. , Silio M. , Williams C. , Gordon J. , Gerschenson L. E.
Published: February 2005

Stimulation of cultured rabbit endometrial cells by one of the rabbit endometrial cell culture proliferation factors, prostaglandin F2α (PGF2α), resulted in a very rapid increase in the intracellular levels of [3H]‐inositol triphosphate (IP3), [3H]‐inositol biphosphate (IP2), and [3H]‐inositol monophosphate (IP1) in cells prelabeled with [3H]‐inositol. These increases in inositol phosphate levels were detected in periods of stimulation as short as 30 seconds, reached a maximum by 1 1/2−2 min and declined to control levels by 6–10 min. The stimulation was dose‐dependent with maximal increases observed near 10−6 M PGF2α. The cholinergic agent, carbachol, also led to time and dose‐independent increases in IP3. Lithium, cadmium, silver, copper, and zinc ions had no effect either on the breakdown of IP3 or on the accumulation of IP1. In contrast, vanadate at 10−6 or 10−5 M did lead to a decrease in the breakdown of IP1 and a concomitant increase in IP1, IP2, and IP3. PGF2α was found previously to induce an increase in rabbit endometrial cell DNA synthesis which was inhibited by concomitant or prior addition of prostaglandin E1 (PGE1). PGE1, in a dose‐dependent manner, was found to inhibit the observed IP3 increase by PGF2α at 1 1/2 min of stimulation. PGF2α treated and control cultures did not differ in cAMP or cGMP levels, cellular 45Ca uptake, nor cellular 22Na uptake. We propose that IP3 may be one of the intracellular messenger(s) synthesized following the treatment of rabbit endometrial cell cultures with the proliferation agent PGF2α and that it may play a crucial role with cAMP in growth regulation.
Energy‐dependent volume regulation in primary cultured cerebral astrocytes
Authors: Olson James E. , Sankar Raman , Holtzman David , James Adrian , Fleischhacker Deborah
Published: February 2005

Cell volume regulation and energy metabolism were studied in primary cultured cerebral astrocytes during exposure to media of altered osmolarity. Cells suspended in medium containing 1/2 the normal concentration of NaCl (hypoosmotic) swell immediately to a volume 40–50% larger than cells suspended in isoosmotic medium. The cell volume in hypoosmotic medium then decreases over 30 min to a volume approximately 25% larger than cells in isoosmotic medium. In hyperosmotic medium (containing twice the normal concentration of NaCl), astrocytes shrink by 29%. Little volume change occurs following this initial shrinkage. Cells resuspended in isoosmotic medium after a 30 min incubation in hypoosmotic medium shrink immediately to a volume 10% less than the volume of cells incubated continuously in isoosmotic medium. Thus, the regulatory volume decrease (RVD) in hypoosmotic medium involves a net reduction of intracellular osmoles. The RVD is partially blocked by inhibitors of mitochondrial electron transport but is unaffected by an inhibitor of glycolysis or by an uncoupler of oxidative phosphorylation. Inhibition of RVD by these metabolic agents is correlated with decreased cellular ATP levels. Ouabain, added immediately after hypoosmotic induced swelling, completely inhibits RVD, but does not alter cell volume if added after RVD has taken place. Ouabain also inhibits cell respiration 27% more in hypoosmotic medium than in isoosmotic medium indicating that the (Na, K)‐ATPase‐coupled ion pump is more active in the hypoosmotic medium. These data suggest that the cell volume response of astrocytes in hypoosmotic medium involves the net movement of osmoles by a mechanism dependent on cellular energy and tightly coupled to the (Na, K)‐ATPase ion pump. This process may be important in the energy‐dependent osmoregulation in the brain, a critical role attributed to the astrocyte in vivo.
Development of a neural phenotype in differentiating ganglion cell‐derived human neuroblastoma cells
Authors: Scott Ian G. , Åkerman Karl E. O. , Heikkilä Jari E. , Kaila Kai , Andersson Leif C.
Published: February 2005

Human neuroblastoma cells (clone SHSY‐5Y) induced to differentiate by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) are shown to possess properties characteristic of mature ganglion cells. Elevation of the external K+ concentration, exposure to Ca2+ ionophore A23187, and acetylcholine all stimulate the release of preloaded 3H‐noradrenaline in the presence but not in the absence of added Ca2+. Acetylcholine causes a fall in the 86Rb+ or 14C‐TPMP equilibrium potential across the plasma membrane and stimulates 86Rb+ efflux. These responses are prevented by atropine. Acetylcholine and muscarine but not nicotine stimulate an increase in 45Ca2+ influx, an effect blocked by atropine. None of these responses have been observed in nondifferentiating cells. Muscarinic receptors, however, as measured by the binding of tritiated quinuclidinyl benzilate (3H‐QNB), were present to a similar extent in control and differentiated cells. Both cell types also exhibit an accelerated release of Ca2+ in response to acetylcholine, but the control cells were at least 1 order of magnitude more sensitive to the agonist.
Human skeletal growth factor stimulates collagen synthesis and inhibits proliferation in a clonal osteoblast cell line (MC3T3‐E1)
Authors: Linkhart Susan , Mohan Subburaman , Linkhart Thomas A. , Kumegawa Masayoshi , Baylink David J.
Published: February 2005

Human skeletal growth factor (hSGF), an 11‐kD polypeptide purified from human bone, has been proposed to be a local regulator of bone formation. To investigate the underlying cellular mechanisms in an in vitro model system, we examined the effects of hSGF on proliferation and collagen synthesis in cells of the clonal osteoblast cell line MC3T3‐E1. This line was isolated from newborn mouse calvarial cells and retains many characteristics of mature osteoblasts (Sudo, H., et al., (1984) J. Cell Biol. 96:191). A 14‐hr treatment with hSGF increased noncollagenous protein synthesis to 215% of unstimulated controls and increased collagen synthesis to 630% of controls as determined by [3H]proline incorporation and high‐pressure liquid chromatographic separation of [3H]proline and [3H]hydroxyproline in acid hydrolysates of trichloroacetic acid‐insoluble protein. HSGF did not increase cell number over a 48‐hr period and caused a reversible inhibition of DNA synthesis. Half‐maximal hSGF concentration for stimulation of [3H]proline incorporation and inhibition of [3H]thymidine incorporation was 100 ng/ml. HSGF also inhibited DNA synthesis in cells stimulated by serum. In contrast, hSGF stimulated both collagen synthesis and DNA synthesis in primary cultures of chick embryo bone cells, which may be developmentally less mature than MC3T3‐E1 cells. The results suggest that hSGF directly stimulated mature osteoblast matrix synthetic activity and that hSGF has differential effects on proliferation of osteoblast progenitor cells and mature osteoblasts.
Reversible inactivation of the Ah receptor associated with changes in intracellular ATP levels
Authors: Gudas Jean M. , Hankinson Oliver
Published: February 2005

We have previously demonstrated that incubation of Hepa‐1 cells in the presence of cytochalasin B (CB) results in a time‐ and temperature‐dependent loss of 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) binding activity (Gudas et al., 1986). We show here that this loss of binding activity is probably attributable to a CB induced inhibition of glucose transport, as incubation of cells in the presence of glycolytic inhibitors or in glucose free medium caused a similar effect. All conditions leading to loss of binding also caused a marked reduction in cellular ATP concentration, suggesting that ATP (or perhaps another energy‐dependent molecule) is required for maintaining the receptor in the active state. Inactivation of the Ah receptor occurred in the cytosol but not when it had translocated to the nucleus. Reactivation of receptor binding activity occurred readily in vivo and did not require de novo protein synthesis. However, attempts to restore receptor binding activity in vitro were not successful. To our knowledge this is the first reported evidence indicating that the TCDD binding Ah receptor can exist in both an inactive and an active form, with the amount present in the active ligand binding form being coupled to the energy state of the cells.
Mc3T3‐G2/PA6 preadipoctyes support in vitro proliferation of hemopoietic stem cells through a mechanism different from that of interleukin 3
Authors: Kodama Hiro‐Aki , Hagiwara Hajime , Sudo Hiroko , Amagai Yuji , Yokota Takashi , Arai Naoko , Kitamura Yukihiko
Published: February 2005

Both MC3T3‐G2/PA6 preadipocytes and interleukin 3 (IL 3) can support in vitro proliferation of mouse hemopoietic stem cells (CFU‐S). We examined whether MC3T3‐G2/PA6 cells produce IL 3 and whether a common mechanism might underlie the action of both of these agents. We used cultured mast cells, DA‐1 cells, and FDC‐P2 cells as the targets of IL 3 and conditioned medium (CM) of WEHI‐3 cells as a source of IL 3. MC3T3‐C2/PA6 CM did not support the growth of the above cells. IL 3 mRNA was not detected in the preadipocytes. Since CM obtained from the cocultures of bone marrow cells and MC3T3‐G2/ PA6 cells did not have a significant effect on the growth of the IL 3‐dependent cells, none of the bone marrow cells seem to produce IL 3 under the influence of the preadipocytes. When the factor‐dependent cells were cocultured with MC3T3‐G2/PA6 cells, the former did not survive, whereas mast cells and DA‐1 cells intimately associated with the preadipocytes. Even when bone marrow cells, mast cells, and MC3T3‐G2/PA6 cells were cocultured, the number of CFU‐S increased, but not that of mast cells. These results seem to exclude the possibility of the action of IL 3 in the microenvironment provided by MC3T3‐G2/PA6 preadipocytes.
Modulation of WI‐38 cell proliferation by elevated levels of CaCl2
Authors: Praeger Frank C. , Cristofalo Vincent J.
Published: February 2005

Elevating the level of extracellular calcium (CaEx2+) increases the saturation density achieved by the normal human diploid cell line, WI‐38, but does not change the growth rate. Day 7 cell yields remain unchanged when [CaEx2+] is between 0.5 mM and 3.0 mM, decrease when [CaEx2+] < 0.5 mM, and increase when [CaEx2+] > 3.0 mM. Combining hydrocortisone with additional CaCl2 results in an additive effect on the saturation density relative to that obtained with each treatment separately. The stimulatory effect of elevated [CaCl2] is independent of serum concentration but is lost when WI‐38 cells are grown in conditioned medium. Stimulation is recovered when conditioned medium is diluted with serum‐free medium. In the case of young cultures grown in conditioned medium, stimulation can also be recovered when higher than usual levels of additional CaCl2 are used (2–3 mM). A glutamine supplementation to the conditioned medium potentiates cell response to elevated [CaCl2]. These results indicate that the loss of an enhanced saturation density when cells are grown in conditioned medium is not due to serum depletion but is more likely the effect of metabolites and/or nutrient depletion. When older or less vigorously growing cultures are grown in conditioned medium, additions of up to 3 mM CaCl2 only lead to inhibition, suggesting an age‐related change in proliferative regulation. Elevated levels of CaEx2+ also enhance the proliferative response of quiescent monolayers to serum stimulation. This finding, along with the increase in saturation density of Ca2+‐treated cultures, suggests that an elevated level of CaEx2+ affects cell entry into and exit from quiescence brought on by density‐dependent inhibition.
Na+‐dependent amino acid transport is a major factor determining the rate of (Na+, K+)‐ATPase mediated cation transport in intact HeLa cells
Authors: Zibirre Reinhard , Poronnik Philip , Koch Gebhard
Published: February 2005

Little is known concerning the effects of Na+‐coupled solute transport on (Na+, K+)‐ATPase mediated cation pumping in the intact cell. We investigated the effect of amino acid transport and growth factor adition on the short term regulation of (Na+, K+)‐ATPase cation transport in HeLa cells. The level of pump activity in the presence of amino acids or growth factors was compared to the level measured in phosphate buffered saline. These rates were further related to the maximal pump capacity, operationally defined as ouabain inhibitable 86Rb+ influx in the presence of 15 μM monensin. Of the growth factors tested, only insulin was found to moderately (22%) increase (Na+, K+)‐ATPase cation transport. The major determinant of pump activity was found to be the transport of amino acids. Minimal essential medium (MEM) amino acids increased ouabain inhibitable 86Rb+ influx to a level close to that obtained with monensin, indicating that the(Na+, K+ )‐ATPase is operating near maximal capacity during amino acid transport. This situation may apply to tissue culture conditions and consequently measurements of (Na+, K+)‐ATPase activity in buffer solutions alone may yield little information about cation pumping under culture conditions. This finding applies especially to cells having high rates of amino acid transport. Furthermore, rates of amino acid transport may be directly or indirectly involved in the long‐term regulation of the number of (Na+, K+)‐ATPase molecules in the plasma membrane.
Control of heme synthesis during Friend cell differentiation: Role of iron and transferrin
Authors: Laskey Jennifer D. , Ponka Prem , Schulman Herbert M.
Published: February 2005

In many types of cells the synthesis of δ‐aminolevulinic acid (ALA) limits the rate of heme formation. However, results from our laboratory with reticulocytes suggest that the rate of iron uptake from transferrin (Tf), rather than ALA synthase activity, limits the rate of heme synthesis in erythroid cells. To determine whether changes occur in iron metabolism and the control of heme synthesis durihg. Erythroid cell development Friend erythroleukemia cells induced to erythroid differentiation by dimethylsulfoxide (DMSO) were studied. While added ALA stimulated heme synthesis in uninduced Friend cells (suggesting ALA synthase is limiting) it did not do so in induced cells. Therefore the possibility was investigated that, in induced cells, iron uptake from Tf limits and controls heme synthesis. Several aspects of iron metabolism were investigated using the synthetic iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH). Both induced and uninduced Friend cells take up and utilize Fe for heme synthesis directly from Fe‐SIH without the involvement of transferrin and transferrin receptors and to a much greater extent than from saturating levels of Fe‐Tf (20 μM). Furthermore, in induced Friend cells 100 μM Fe‐SIH stimulated 2‐l4C‐glycine incorporation into heme up to 3.6‐fold as compared to the incorporation observed with saturating concentrations of Fe‐Tf. In contrast, Fe‐SIH, even when added in high concentrations, did not stimulate heme synthesis in uninduced Friend cells but was able to do so as early as 24 to 48 h following induction. In addition, contrary to previous results with rabbit reticulocytes, Fe‐SIH also stimulated globin synthesis in induced Friend cells above the level seen with saturating concentrations of transferrin. These results indicate that some step(s) in the pathway of iron from extracellular Tf to protoporphyrin, rather than the activity of ALA synthase, limits and controls the overall rate of heme and possibly hemoglobin synthesis in differentiating Friend erythroleukemia cells.
Elevation of intracellular glutathione content associated with mitogenic stimulation of quiescent fibroblasts
Authors: Shaw Jill P. , Chou Iih‐Nan
Published: February 2005

The relationship between total glutathione (GSH) content and cell growth was examined in 3T3 fibroblasts. The intracellular GSH level of actively growing cultures gradually decreases as these cells become quiescent by either serum deprivation or high cell density. Upon mitogenic stimulation of sparse, quiescent (G0/G1) cultures with serum, there is a rapid 2.3‐fold elevation in intracellular GSH levels which, is maximal by 1 h and returns to baseline by 2 h. This is followed by a more gradual increase in GSH content as cells enter the S phase. In addition, the elevation in GSH content is required for maximum induction of DNA synthesis. Treatments that prevent the early increase in intracellular GSH levels do not affect protein synthesis but result in a reversible dose‐dependent decrease in the percent of cells capable of entering S phase. These results indicate that GSH may be important in the regulation of cellular proliferation.
Purification of melanoma growth stimulatory activity
Authors: Richmond Ann , Thomas H. Greg
Published: February 2005

The Hs0294 human malignant melanoma cell line produces a monolayer mitogen that stimulates the serum free growth of low‐density cultures of Hs0294 cells. This report describes the purification of that mitogen, termed MGSA for melanoma growth stimulatory activity, from serum‐free conditioned medium from the Hs0294 cultures. MGSA has been purified from acetic acid extracts of lyophilized conditioned medium by gel filtration, reverse‐phase high‐pressure liquid chromatography (RP‐HPLC), and preparative electrophoresis, resulting in a >400,000‐fold purification. MGSA bioactivity resides in acid‐ and heat‐stable polypeptides of high and low molecular weight (24–28 kd and < 14–16 kd). However, the majority of the activity is reproducibly associated with the ˜16‐kd moiety eluting from RP‐HPLC at ∼35% acetonitrile. Reduction with dithiothreitol or B‐mercaptoethanol results in a loss of biological activity but does not convert the 24–28‐kd moieties to the<14–16‐kd forms of MGSA. 125l‐MGSA that has been purified by preparative electrophoresis (16 kd) specifically binds to Hs0294 melanoma cells and retains 100% of the growth‐stimulatory activity. The 16‐kd MGSA stimulates the proliferation of Hs0294 cells at concentrations of 0.3–30 pM. The electrophoretic mobility of MGSA is also unaltered by the preparative electrophoresis procedure, further demonstrating that this procedure does not alter the biochemical integrity of the growth factor. Purified MGSA does not enable anchorage‐independent growth of normal rat kidney (NRK) cells and is therefore different from the previously described transforming growth factors. The amino acid composition of MGSA differs from that of other previously described growth factors. These data demonstrate that MGSA represents a separate class of growth factors with biological and biochemical properties different from other growth factors.
Studies on the mechanism by which glutamine and heat shock increase lactate synthesis by l929 cells in the presence of insulin
Authors: Lanks Karl W.
Published: February 2005

Both glutamine and heat shock increase lactate production in the L929 cell system, Glutamine is now shown to increase hexose uptake in the presence of insulin, to inhibit pyruvate oxidation, and to provide reducing equivalents to the cytosolic compartment. The relative contribution of these processes to lactate production depends on the availability of pyruvate. When ample pyruvate is available from the culture medium, stimulation of lactate synthesis by glutamine and heat shock is transaminase dependent, suggesting that shuttling of reducing equivalents from mitochondria to cytoplasm is involved. In the absence of medium pyruvate, stimulation of glycolysis by both glutamine and heat shock is largely responsible for increased lactate synthesis. None of the observed effects of glutamine appears to be sufficient to explain the observed stimulation of glycolysis.
Effects of nutritional and hormonal factors on the metabolism of retinol‐binding protein by primary cultures of rat hepatocytes
Authors: Dixon Joseph L. , Goodman DeWitt S.
Published: February 2005

Studies were conducted to explore hormonal and nutritional factors that might be involved in the regulation of retinol‐binding protein (RBP) synthesis and secretion by the liver. The studies employed primary cultures of hepatocytes from normal rats. When cells were cultured in Dulbecco's modified Eagle's medium alone, a high rate of RBP secretion was observed initially, which declined and became quite low by 24 hr. Supplementing the medium with amino acids maintained RBP and albumin secretion at moderate (but less than initial) rates for at least 3 days. Further addition of dexamethasone maintained the production and secretion rates of RBP, transthyretin, and albumin close to the initial rates for up to 3–5 days in culture. The effects of dexamethasone were not rapid and were not specific for RBP; half‐maximal effects were seen at 10−9 to 10−8 M levels. Hormonally treated hepatocytes produced and secreted RBP, transthyretin, and albumin at both absolute and relative rates similar to physiological values, as estimated from rates reported by others from studies in vivo (with both rats and humans) and with perfused livers. Glucagon addition partially maintained the secretion rates of these 3 proteins, but less effectively than did dexamethasone. A number of other hormones, added singly or in combination, did not affect RBP production or secretion. Addition of retinol to the cultured normal hepatocytes was without effect upon RBP secretion. These studies show that supplementing the culture medium of hepatocytes with amino acids and dexamethasone maintains RBP production and secretion for several days. In normal hepatocytes, with ample supply of retinol available within the cell, addition of exogenous retinol does not appear to influence RBP metabolism or secretion by the cells.
Calcium mobilization in permeabilized fibroblasts: Effects of inositol trisphosphate, orthovanadate, mitogens, phorbol ester, and guanosine triphosphate
Authors: Muldoon Leslie L. , Jamieson Gordon A. , Villereal Mitchel L.
Published: February 2005

Utilizing a digitonin‐permeabilized cell system, we have studied the release of calcium from a non‐mitochondrial intracellular compartment in cultured human fibroblasts (HSWP cells). Addition of 1 mM MgATP to a monolayer of permeabilized cells in a cytosolic media buffered to 150 nM Ca with EGTA rapidly stimulates 45Ca uptake, and the subsequent addition of the putative intracellular messenger inositol trisphosphate (InsP3) induces rapid release of 85% (±6% n = 6) of the 45Ca taken up in response to ATP. Mitogenic peptides (bradykinin, vasopressin, epidermal growth factor [EGF], and insulin) and orthovanadate, which are effective in mobilizing intracellular Ca in intact cells, have little or no effect when added alone to permeabilized cells. However, in the presence of GTP these agents stimulate accumulation of inositol phosphates and release Ca from the InsP3‐sensitive pool. These data suggest that a GTP binding protein is involved in receptor mediated activation of phospholipase C, which leads to release of inositol phosphates. The GTP‐dependent release of InsP3 and the mobilization of 45Ca from the intracellular compartment are inhibited by pretreatment of cells, prior to permeabilization, with the protrein kinase C activator 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA). TPA pretreatment does not affect the InsP3 stimulated Ca release. These results suggest that protein kinase C is involved in down‐regulation or inhibition of phospholipase C, or the GTP binding protein responsible for relaying the mitogenic signal from the cell surface receptor to the phospholipase C activity.
Mitogenic effect of double‐stranded RNA in human fibroblasts: Role of autogenous interferon
Authors: Vilček Jan , Kohase Masayoshi , Henriksen‐Destefano Dorothy
Published: February 2005

The synthetic double‐stranded RNA polyinosinate‐polycytidylate [poly‐(I).poly(C)] was mitogenic in cultures in human foreskin fibroblasts, as demonstrated by a stimulation of 3H‐thymidine incorporation and an increase in cell density. Poly(I).poly(C) is a potent inducer of interferon (IFN)‐beta in human fibroblasts. Single‐stranded poly(I) or poly(C) were not mitogenic in human fibroblasts and did not stimulate IFN production. Antiserum to interferon (IFN)‐beta, added to poly(I).poly(C)‐stimulated cultures in order to neutralize endogenously generated IFN, markedly amplified the mitogenic action. Under similar experimental conditions, antiserum to IFN‐beta did not enhance the mitogenic action of epidermal growth factor (EGF). Dexamethasone enhanced the mitogenic action of poly(I).poly(C) in a manner similar to antiserum against IFN‐beta. This effect of dexamethasone correlated with its marked inhibitory action on poly(I).poly(C)‐stimulated IFN production. Together with the results of other related studies, these findings support the notion of an evolutionary link between the generation of a mitogenic signal and IFN induction. In addition, these results support the concept that autocrine secretion of IFN‐beta can exert negative feedback control of cell proliferation.
Quantitation of protein kinase C by immunoblot—expression in different cell lines and response to phorbol esters
Authors: Stabel S. , Rodriguez‐Pena A. , Young S. , Rozengurt E. , Parker P. J.
Published: February 2005

Antisera have been raised against human protein kinase C and also against a synthetic peptide based on the sequence of the bovine brain enzyme (LLNQEE‐GEYYNVPIPE). These antibodies react with protein kinase C from a number of species (human, murine, rat, rabbit, bovine), indicating substantial conservation of epitopes. These antisera have been used to quantitate directly protein kinase C by immunoblot analysis. We show here that there is a strict correlation between the leveis of immunoreactive polypeptide and extractable calcium‐ and phospholipid‐dependent kinase activity for various cell lines. Treatment of murine, rat, and human cells with phorbol dibutyrate was found to deplete levels of immunoreactive protein kinase C severely. A detailed study of the time course of this depletion in Swiss 3T3 cells shows that it follows precisely the loss of extractable activity. On exposure to 400 nM phorbol 12,13‐dibutyrate protein kinase C was essentially undetectable by 40 hours; the half‐life of this down‐regulation was 6.7 hours. This data thus demonstrate that the loss of immunoreactive protein kinase C and of extractable calcium‐ and phospholipid‐dependent kinase activity precisely parallels the phorbol ester induced down‐regulation of binding and responsiveness in Swiss 3T3 cells.
Retention of differentiated characteristics by cultures of defined rabbit kidney epithelia
Authors: Wilson Patricia D. , Anderson Robert J. , Breckon Ruth D. , Nathrath Walter , Schrier Robert W.
Published: February 2005

Rabbit nephron segments of proximal convoluted tubules (PCT); proximal straight tubules (PST); cortical and medullary thick ascending limbs of Henle's loop (CAL, MAL); and cortical, outer medullary, and inner medullary collecting tubules (CCT, OMCT, IMCT) were individually microdissected and grown in monolayer culture in hormone supplemented, defined media. Factors favoring a rapid onset of proliferation included young donor age, distal tubule origin, and the addition of 3% fetal calf serum to the medium. All primary cultures had polarized morphology with apical microvilli facing the medium and basement membrane‐like material adjacent to the dish. Differentiated properties characteristics of the tubular epithelium of origin retained in cultures included ultrastructural characteristics and cytochemically demonstrable marker enzyme proportions. PCT and PST were rich in alkaline phosphatase; CAL stained strongly for NaK‐ATPase; CCT contained two cell populations with regard to cytochrome oxidase reaction. A CCT‐specific antikeratin antibody (aLEA) was immunolocalized in CCT cultures, and a PST cytokeratin antibody stained PST cultures. The biochemical response of adenylate cyclase to putative stimulating agents was the same in primary cultures as in freshly isolated tubules. In PCT and PST adenylate cyclase activity was stimulated by parathyroid hormone (PTH) but not by arginine vasopressin (AVP); CAL and MAL adenylate cyclase was stimulated by neither PTH nor AVP; CCT, OMCT, and IMCT adenylate cyclase was stimulated by AVP but not by PTH. NaF stimulated adenylate cyclase activity in every cultured segment. It is concluded that primary cultures of individually microdissected rabbit PCT, PST, CAL, MAL, CCT, OMCT, and IMCT retain differentiated characteristics with regard to ultrastructure, marker enzymes, cytoskeletal proteins, and hormone response of adenylate cyclase and provide a new system for studying normal and abnormal functions of the heterogeneous tubular epithelia in the kidney.
Inhibition of fibroblast chemotaxis by recombinant human interferon γ and interferon α
Authors: Adelmann‐Grill Bernhard C. , Hein Rüdiger , Wach Franz , Krieg Thomas
Published: February 2005

Interferons have recently been recognized as potent mediators in inflammatory processes, exerting profound effects on fibroblasts. The influence of interferons γ and α on the chemotactic movement of fibroblasts toward various attractants was, therefore, investigated. Normal human adult and embryonal dermal fibroblasts, fibrosarcoma‐derived fibroblasts and SV40‐transformed fibroblasts were tested against conditioned medium from fibroblasts, the chemotactic peptide C‐140 of fibronectin, platelet‐derived growth factor, and leukotriene B4 as attractants in the presence or absence of the interferons. Interferons γ and α inhibited chemotaxis in a dose‐dependent manner and at concentrations at least as low as 10−2 ng/ml. Inhibition was noticeable when the cells were exposed to interferon for as short a period as 60 minutes, and the effect was not readily reversible. Inhibition occurred when the cells came from sparse or dense cultures, but when platelet‐derived growth factor was the attractant and the cells had been grown at low density there was no inhibition. It is concluded that this is a specific effect, not to be wholly explained by overall increase in membrane rigidity. Inhibition of fibroblast chemotaxis by interferons may be an important regulatory mechanism during wound healing or fibrosis and metastatic spread of tumor cells.