bluuebunny/crossref_metadata_2025 huggingface dataset:
Alterations in cellular calcium handling as a result of systemic calcium deficiency in the developing chick embryo: I. Erythrocytes
Authors: Koide Masafumi , Smith Carol A. , Miyahara Takashi , Tuan Rocky S.
Published: February 2005

Chick embryos rendered calcium (Ca) deficient by shell‐less (SL) culture develop hypertension and tachycardia. Since hypocalcemia is accompanied by hypernatremia systemically but not by lower cellular Ca (Koide and Tuan, 1989), we speculate that cellular Ca handling may be altered in the SL embryo, perhaps involving Na transport. Using erythrocytes (RBC) from day‐14 SL and normal (NL) embryos as the experimental cell, cellular Ca handling was studied under varying extracellular osmotic and ionic conditions by analyzing 45Ca uptake and cell volume regulation. Two agents, p‐chloromercuriphenylsulfonate (PCM), and inosine/iodoacetamide (INI) were used to treat the RBCs to modify plasma membrane ion permeability and to deplete cellular ATP, respectively. Other cellular functions and activities related to Ca homeostasis, including ATP content and Ca2+‐ATPase activity, were also analyzed. These analyses showed: (1) in NaCl, Ca uptake was similar in NL and SL cells, except after INI treatment, which resulted in slower Ca uptake by the SL cells, (2) in choline and sucrose, Ca uptake by SL RBCs was higher, (3) Ca uptake by RBCs of both embryos changed depending on the osmotic agent (Na < K ≤ choline < sucrose), (4) Ca2+‐ATPase activity was higher in SL RBC, although there was no change in the size or charge of the enzyme, and (5) in any osmotic agent, cellular Na was significantly lower, whereas cellular K was higher, in SL RBC. Based on these results, three features of RBC Ca handling were apparent: (1) Na‐Ca exchange was functional and was more active in SL RBCs, (2) Ca uptake was dependent on the total ionic electrochemical gradient but not on bulk H2O movement, and (3) Ca pumping out capacity was directly correlated with Ca2+‐ATPase activity. Elevated Ca uptake in sucrose‐treated SL RBC is therefore indicative of its greater ion permeability. Taken together, these findings indicate that cellular Ca handling of the RBCs of SL chick embryos is characterized by a more active Na‐Ca exchange system, greater ion permeability, and higher Ca pumping out capacity, thereby suggesting an up‐regulated Ca handling function in the SL RBCs. The abnormal cellular Ca handling may be a direct result of the systemic Ca deficiency of the SL chick embryo and may be functionally related to its hypertension and tachycardia. © 1992 Wiley‐Liss, Inc.
Different relationships between cellular adenosine or 3′‐phosphorylation and cellular adenine ribonucleotide catabolism may be obtained
Authors: Overgaard‐Hansen Kay , Klenow Hans
Published: February 2005

Treatment of BALB/c‐3T3 mouse fibroblasts with 3′‐led to a rapid accumulation of 3′‐phosphates and the kinetics of this process has been determined. Concomitant with accumulation of these compounds, the adenine ribonucleotide pool was reduced. The kinetics of the two processes suggested that they were tightly coupled. The inhibitory effect of relatively high concentrations of coformycin indicated that IMP was an intermediate in the catabolic pathway. Similar experiments with Ehrlich ascites tumor cells were performed in Ringer‐Hepes solution at pH 6.5 or 7.5 and with varying concentrations of orthophosphate. The experiments were performed with cells where ATP was [3H]‐. This allowed the determination of the catabolism of adenine ribonucleotides to labeled nucleosides under conditions where added adenosine was phosphorylated. The results showed that at low phosphate concentration (5.8 mM) at pH 6.5 adenosine may be phosphorylated at a rate that was completely balanced to the concomitant catabolism of adenine ribonucleotides; that is, there was apparently a tight kinetic coupling between anabolism of adenosine and catabolism of adenine ribonucleotides. With 3′‐a corresponding effect was obtained although the apparent coupling between phosphorylation of 3′‐and catabolism of adenine ribonucleotides was not complete. When experiments were performed at the same pH but at high concentration of phosphate (45 mM) there was in contrast no coupling between the two processes; that is, ATP was present in constant amounts while 3′‐phosphates accumulated at a high rate. In experiments with adenosine under these conditions there was still some although a relatively limited degree of apparent coupling between phosphorylation of adenosine and catabolism of adenine ribonucleotides. In both lines of cells used and with both adenosine and 3′‐, the main products of the catabolism of adenine ribonucleotides were inosine and hypoxanthine. With 3′‐there was in addition (about 20%) formation of xanthosine, suggesting that IMP dehydrogenase had also been activated. These results lead to the suggestion that adenosine (or 3′‐) may be phosphorylated in two ways. 1) Phosphorylation may depend on an adenosine kinase unrelated to catabolism of adenine ribonucleotides. 2) Phosphorylation may be tightly coupled to catabolism of adenine ribonucleotides. A nucleoside phosphotransferase may catalyze the transfer of a phosphoryl group from IMP to adenosine (or 3′‐) to form AMP (or 3′‐) and inosine, a process that may be tightly coupled to an AMP deaminase reaction. The IMP formed in the latter reaction may not be released but transferred to the phosphotransferase. In contrast, the AMP formed in the phosphotransferase reaction should be in equilibrium with soluble AMP. It is assumed that a physical complex may exist, possibly in a membrane bound form, between AMP deaminase and the nucleoside phosphotransferase. © 1993 Wiley‐Liss, Inc.
GM‐CSF triggers a rapid, glucose dependent extracellular acidification by TF‐1 cells: Evidence for sodium/proton antiporter and PKC mediated activation of acid production
Authors: Wada H. Garrett , Indelicato Stephen R. , Meyer Lorraine , Kitamura Toshio , Miyajima Atsushi , Kirk Gregory , Muir Victoria C. , Parce J. Wallace
Published: February 2005

The extracellular acidification rate of the human bone marrow cell line, TF‐1, increases rapidly in response to a bolus of recombinant granulocyte‐macrophage colony stimulating factor (GM‐CSF). Extracellular acidification rates were measured using a silicon microphysiometer. This instrument contains micro‐flow chambers equipped with potentiometric sensors to monitor pH. The cells are immobilized in a fibrin clot sandwiched between two porous polycarbonate membranes. The membranes are part of a disposable plastic “cell capsule” that fits into the microphysiometer flow chamber. The GM‐CSF activated acidification burst is dose dependent and can be neutralized by pretreating the cytokine with anti‐GM‐CSF antibody. The acidification burst can be resolved kinetically into at least two components. A rapid component of the burst is due to activation of the sodium/proton antiporter as evidenced by its elimination in sodium‐free medium and in the presence of amiloride. A slower component of the GM‐CSF response is a consequence of increased glycolytic metabolism as demonstrated by its dependence on D‐glucose as a medium nutrient. Okadaic acid (a phospho‐serine/threonine phosphatase inhibitor), phorbol 12‐myristate 13‐acetate (PMA, a protein kinase C (PKC) activator), and ionmycin (a calcium ionophore) all produce metabolic bursts in TF‐1 cells similar to the GM‐CSF response. Pretreatment of TF‐1 cells with PMA for 18 h resulted in loss of the GM‐CSF acidification response. Although this treatment is reported to destroy protein kinase activity, we demonstrate here that it also down‐regulates expression of high‐affinity GM‐CSF receptors on the surface of TF‐1 cells. In addition, GM‐CSF driven TF‐1 cell proliferation was decreased after the 18 h PMA treatment. Short‐term treatment with PMA (1–2h) again resulted in loss of the GM‐CSF acidification response, but without a decrease in expression of high‐affinity GM‐CSF receptors. Evidence for involvement of PKC in GM‐CSF signal transduction was obtained using calphostin C, a specific inhibitor of PKC, which inhibited the GM‐CSF metabolic burst at a subtoxic concentration. Genistein and herbimycin A, tyrosine kinase inhibitors, both inhibited the GM‐CSF response of TF‐1 cells, but only at levels high enough to also inhibit stimulation by PMA. These results indicate that GM‐CSF activated extracellular acidification of TF‐1 cells is caused by increases in sodium/proton antiporter activity and glycolysis, through protein kinase signalling pathways which can be both activated and down‐regulated by PMA. © 1993 Wiley‐Liss, Inc.
Polarized secretion of IGF‐I and IGF‐I binding protein activity by cultured aortic endothelial cells
Authors: Taylor W. Robert , Nerem Robert M. , Alexander R. Wayne
Published: February 2005

Insulin‐like growth factor‐I (IGF‐I) secretion by the vascular endothelium has been proposed to play a role in the regulation of vascular smooth muscle cell proliferation. Because vascular smooth muscle cells are adjacent to the abluminal surface of the endothelium, we tested the hypothesis that secretion of IGF‐I by endothelial cells is polarized. Porcine aortic endothelial cells were cultured on permeable membranes and IGF‐I measured by radioimmunoassay. Basal secretion exceeded apical secretion by a ratio of 2.3 ± 0.2:1.0 (P < 0.05). We also identified 35 kDa IGF‐I binding protein activity that is preferentially secreted on the basal surface of endothelial cells. We conclude that both IGF‐I and IGF‐I binding protein activity secretion by endothelial cells is polarized towards the basal surface of the endothelium. A polarized secretion mechanism for IGF‐I may be of importance in the normal growth and differentiation of the vasculature as well as in the development of vascular pathology. © 1993 Wiley‐Liss, Inc.
Cell cycle‐dependent gene expression in V point‐arrested BALB/c‐3T3 cells
Authors: Olson James E. , Winston Jeffrey T. , Whitlock James A. , Pledger W. J.
Published: February 2005

Density‐arrested BALB/c‐3T3 cells stimulated to proliferate in an amino acid‐deficient medium arrest in mid‐G1 at a point termed the V point. Cells released from V point arrest require 6 hr to traverse late G1 and enter S phase. As data presented here show that mRNA synthesis is needed for 2–3 hr after release of cells from the V point, after which inhibition of mRNA synthesis does not prevent entry into S phase, we used this mid‐G1 arrest protocol to analyze gene expression in late G1. We found that although stimulation of cells in amino acid‐deficient medium did not inhibit the induction of genes expressed in early G1, genes normally expressed in late G1 were expressed only after release from the V point. The expression of late G1 genes in cells released from the V point was temporally similar, in respect to G1 location, as was seen in stimulation of quiescent Go cells. As this protocol effectively divides gene expression into early (pre‐V point) and late (post‐V point) categories, it should be useful in studies of growth factor‐modulated events that regulate traverse of late G1 and commitment to DNA synthesis. In addition, we used c‐myb antisense oligonucleotides to show that c‐myb expression, which occurs in late G1, is required for BALB/c‐3T3 fibroblasts to traverse late G1 and initiate DNA synthesis. © 1993 Wiley‐Liss, Inc.
Orthovanadate both mimics and antagonizes the transforming growth factor β action on normal rat kidney cells
Authors: Rijksen Gert , Völler Maureen C. W. , Van Zoelen Everardus J. J.
Published: February 2005

Normal rat kidney [NRK] cells grown in the presence of epidermal growth factor (EGF) or platelet‐derived growth factor (PDGF) have a normal phenotype and undergo density‐dependent growth inhibition, whereas in the presence of multiple growth factors, density arrest is lost and the cells become phenotypically transformed. We studied the influence of the protein tyrosine phosphatease (PTPase) inhibitor sodium orthovanadate on the mitogenic stimulation of NRK cells by growth factors and on transformation‐linked properties as loss of density‐dependent growth inhibition and anchorage‐independent growth. The fraction of cells in serum‐deprived monolayer cultures that is induced to proliferate upon mitogenic stimulation by EGF or PDGF is only slightly enhanced upon addition of low concentrations (25–50 μM) of vanadate. Addition of vanadate per se induces proliferation of only a very limited amount of cells, but results in a shift of the dose‐response curves for other growth factors to lower concentrations. Vanadate added in combination with EGF or PDGF is able to mimic the effect of transforming growth factor β (TGFβ) in inducing phenotypic transformation. In monolayer cultures density‐dependent growth inhibition is lost and anchorage‐independent proliferation is observed on dishes coated with poly(2‐hydroxy‐ethyl methacrylate) (polyHEMA). The extent of these changes is similar to that induced by TGFβ. However, the morphology of the obtained colonies in polyHEMA‐coated dishes is quite different. Cells transformed by TGFβ in the presence of EGF form rather amorphous colonies, whereas in the presence of orthovanadate colonies are formed that tend to fall apart in loose cells. The effect of vanadate on cell transformation is dependent on the growth factor conditions in a bimodal way. When a suboptimal dose of growth factor(s) is used, 25 μM vanadate is very effective in preventing density‐induced growth inhibition and stimulating anchorage‐independent proliferation. However, the same concentration of vandate is inhibitory when cells are maximally stimulated and antagonizes the transforming effect of TGFβ added in combination with other growth factors. It is hypothesized that vanadate acts on a set of different protein tyrosine phosphatases. Some of these are positive and others negative regulators of growth. © 1993 Wiley‐Liss, Inc.
Intracellular free sodium concentrations in GH4C1 cells
Authors: Törnquist Kid , Ekokoski Elina
Published: February 2005

In the present investigation, intracellular sodium ([Na+]i) levels were determined in GH4C1 cells using the fluorescent probe SBFI. Fluorescence was determined by excitation at 340 nm and 385 nm, and emission was measured at 500 nm. Intracellular free sodium ([Na+]i) was determined by comparing the ratio 340/385 to a calibration curve. The ratio was linear between 10 and 60 mM Na+. Resting [Na+]i in GH4C1 cells was 26 ± 6.2 mM (mean ± SD). In cells incubated in Na+‐buffer [Na+]i decreased to 3 ± 3.6 mM. If Na+/K+ ATPase was inhibited by incubating the cells with 1 mM ouabain, [Na+]i increased to 47 ± 12.8 mM in 15 min. Stimulating the cells with TRH, phorbol myristyl acetete, or thapsigargin had no effect on [Na+]i. Incubating the cells in Ca2+‐buffer rapidly increased [Na+]i. The increase was not inhibited by tetrodotoxin. Addition of extracellular Ca2+, nimodipine, or Ni2+ to these cells immediately decreased [Na+]i, whereas Bay K 8644 enhanced the influx of Na+. In cells where [Na+]i was increased the TRH‐induced increase in intracellular free calcium ([Ca2+]i) was decreased compared with control cells. Our results suggest that Na+ enters the cells via Ca2+ channels, and [Na+]i may attenuate TRH‐induced changes in [Ca2+]i in GH4C1 cells. © 1993 Wiley‐Liss, Inc.
Parathyroid hormone‐induced changes in alkaline phosphatase expression in fetal calvarial osteoblasts: Differences between rat and mouse
Authors: Jongen J. W. J. M. , Bos M. P. , Van Der Meer J. M. , Herrmann‐Erlee M. P. M.
Published: February 2005

We studied the effects of parathyroid hormone (PTH) on two markers of the osteoblast phenotype: alkaline phosphatase (AP) (activity and mRNA) and cyclic adenosine monophosphate (cAMP) accumulation. Osteoblast‐like cells derived from fetal rat (ROB) and mouse (MOB) calvariae were isolated by collagenase treatment. Cells were cultured in α‐Minimal Essential Medium (MEM) with 2% fetal calf serum (FCS) for 4 days. In ROB and MOB bPTH(1‐34) induced a fast increase (up to 5 minutes) in cAMP accumulation. When equal amounts of cells were seeded, the cAMP accumulation was higher in MOB than in ROB. No difference in basal AP activity was observed between ROB and MOB. When bPTH(1‐34) was added to ROB for the last 24 or 48 hr, AP activity decreased dose dependently. However, MOB treated with bPTH(1‐34) for the last 24 or 48 hours showed an increase of AP activity. Basal AP activity was positively correlated with the seeding density of ROB and MOB cultures. Basal AP activity influenced the degree of inhibition (ROB) or stimulation (MOB) after incubation with bPTH(1‐34). © 1993 Wiley‐Liss, Inc.
Effects of growth factors on cell cycle arrest in dolichyl phosphate‐depleted cultures
Authors: Doyle J. W. , Ward‐Bailey P. F. , Kandutsch A. A.
Published: February 2005

Previously we showed that CHO cell growth is arrested in the G1 or G0 phase within 24 h after the biosynthesis of mevalonic acid is blocked. The growth‐limiting factor under these conditions appeared to be dolichyl phosphate or one of its glycosylated derivatives with consequent decrease in the synthesis of N‐linked glycoproteins (Doyle, J.W., and A.A. Kandutsch, 1988, J. Cell Physiol. 137:133–140; Kabakoff, B., J.W. Doyle, and A.A. Kandutsch, 1990, Arch. Biochem. Biophys. 276:382–389). We show herein that cell surface glycoproteins are depleted in the inhibited cultures and that growth arrest is delayed when supraphysiological concentrations of insulin, insulin‐like growth factor‐1 (IGF‐1) and bFGF are added to the culture medium. Apparently an elevated level of a growth factor increases the length of time during which a threshold level of occupied receptor is maintained as the number of glycosylated receptor molecules declines. The results support the idea that cellular levels of dolichyl phosphate and its derivatives may limit cell division by controlling the numbers of functional receptors for growth factors and of other glycoproteins on the cell surface. © 1993 Wiley‐Liss, Inc.
Mutant cell line demonstrating a block in insulin and insulin‐like growth factor type 1 (IGF‐1) induced mitogenesis
Authors: Leckett Blaine , Spurmanis Aleksandrs , Allen Cynthia , Germinario Ralph J.
Published: February 2005

Recently, we have isolated a Chinese hamster cell variant (IV‐A1‐j) resistant to an insulin‐diphtheria‐A chain toxic conjugate (Leckett and Germinario: Cytotechnology [in press]. This cell line exhibited a decreased level of insulin binding, but normal growth in serum‐containing medium when compared to the parental cell line (V‐79). In this paper we further demonstrate that while IV‐A1‐j cells are capable of growing in serum‐containing medium, they are insensitive to the mitogenic actions of either insulin or IGF‐1. In contrast, epidermal growth factor (EGF) and/or α‐thrombin (THR) generate a mitogenic effect in IV‐A1‐j cells comparable to that observed in the parental V‐79 cells. The combination of EGF and/or THR with either insulin or IGF‐1 results in an increase in V‐79 cell growth above EGF and/or THR alone. On the other hand, insulin or IGF‐1 in the presence of other mitogens did not stimulate further growth in IV‐A1‐j cells. While insulin binding was lower in IV‐A1‐j cells, internalization of 125I‐insulin was not different in the two cell types. Additionally, insulin‐stimulated glycogen synthesis and protein synthesis were not different in the two cell types. These observations are consistent with insulin and IGF‐1 sharing a mitogenic signalling pathway in Chinese hamster fibroblasts and that this pathway is distinct from other growth factor signalling pathways. The fact that this pathway is defective in the IV‐A1‐j cell line indicates the potential usefulness of these cells in identifying a key step(s) in the insulin (IGF‐1) mitogenic pathway. © 1993 Wiley‐Liss, Inc.
Position‐dependent nuclear accumulation of the retinoblastoma (RB) protein during in vitro myogenesis
Authors: Szekely Laszlo , Jin Pei , Jiang Wei‐Qin , Rosén Anders , Wiman Klas G. , Klein George , Ringertz Nils
Published: February 2005

The expression of the retinoblastoma (RB) protein has been studied during in vitro muscle differentiation by immunofluorescence staining with three different antibodies against RB protein. Proliferating mononucleate L6 rat myoblasts showed a low level of expression. As cells began to enter a nonreplicating G0 state, the cell population became heterogeneous. Some nonreplicating cells showed a high level of expression. Nuclei at the two ends of myotubes were strongly positive, whereas centrally located nuclei showed low RB expression. Overexpression of the human RB protein in rat L6 myotubes from a Semliki forest virus (SFV)‐based, transient expression vector produced a similar picture. Terminally located nuclei expressed human RB at a much higher level than did the centrally located nuclei. The results suggest that individual nuclei with a multinucleated syncytium may undergo position‐dependent specialization. © 1993 Wiley‐Liss, Inc.
Marrow stroma‐derived osteogenic clonal cell lines: Putative stages in osteoblastic differentiation
Authors: Fried A. , Benayahu D. , Wientroub S.
Published: February 2005

This report documents characterization of five osteogenic cell subpopulations of bone marrow stroma. The clonally derived cell lines were isolated from the parental line MBA‐15 known to express osteoblastic‐associated features in vitro and to form bone in vivo. The latter, presumably “arrested” at a particular stage along the osteogenic lineage, are useful models to study the processes involved in the differentiation of bone forming cells. The clones differ in their morphology, proliferation rate, quantities and distribution of extracellular matrix proteins, levels of alkaline phosphatase activity and activation of adenylate cyclase by parathyroid hormone and/or prostaglandin E. These properties have been retained during prolonged growth and subculturing through many passages. MBA‐15.4 is a presumptive preosteoblast with a fibroblast‐like appearance; it proliferates rapidly, synthesizes equal amounts of collagen and noncollagenous proteins, and produces constitutively low levels of alkaline phosphatase. This clone has PGE2‐stimulated adenylate cyclase activity and a very low constitutive response to PTH. On the other hand, MBA‐15.6 has a large polygonal morphology with limited proliferative potential, synthesizes twice as much noncollagenous proteins as collagen, has high alkaline phosphatase activity, and responds strongly to PTH. The characteristics of the other clones place them between these two categories. The effects of 10−7 M dexamethasone or 10−12–10−8 M 1,25 dihydroxyvitamin D3 on growth and differentiation further strengthen the variance between these clones. The different in vitro characteristics of the various clones were directly reflected in their bone formation ability in vivo. When transplanted under the renal capsule, MBA‐15.33 formed a thick fibrous tissue, MBA‐15.4 formed small foci of bone, and MBA‐15.6 formed massive woven bone at the same period of time. © 1993 Wiley‐Liss, Inc.
Interactions of sertoli cells with laminin are essential to maintain integrity of the cytoskeleton and barrier functions of cells in culture in the two‐chambered assembly
Authors: Tung Pierre S. , Fritz Irving B.
Published: February 2005

The addition of anti‐laminin IgG to the basal surfaces of rat Sertoli cells in culture in a two‐chambered assembly results in a perturbation of F‐actin arrangements, including disruption of the pericellular circumferal rings, impairments of the Sertoli cell permeability barrier, and subsequently focal defoliation, followed by cell reaggregation. The pentapeptide YIGSR, which competes with the laminin receptor for laminin (Kleinman and Weeks: Curr. Oph. Cell Biol., 1:964–967, 1989; Graf et al.: Biochemistry, 26:6896–6900, 1987) also elicited focal defoliation of Sertoli cells from the extracellular matrix‐coated filter in the two‐chambered assembly. Addition of YIGSR to Sertoli cell cultures resulted in cell detachment within 2 to 3 h. In contrast, the irrelevant peptide YIGSE had no detectable effects. The anti‐laminin IgG was effective only when added to the chamber in which access was readily available to the basal surfaces of Sertoli cells, but YIGSR was effective when added either to the outer chamber or to the inner chamber. These data were interpreted to indicate that the Sertoli cell barrier generated in the two‐chambered assembly allowed a relatively rapid diffusion of YIGSR between chambers, but prevented the rapid equilibration of anti‐laminin IgG between compartments. Addition of anti‐laminin IgG to the basal, but not to the apical surfaces of Sertoli cells, resulted in more rapid rates of equilibration of [3H]‐methoxyinulin and [86Rb]Cl across the Sertoli cell monolayer. This evidence of impairment to the integrity of the barrier was detected prior to the disruption of stress fibers and focal defoliation, but after evidence of dissolution of the circumferal F‐actin ring, which occurred within 1 h after addition of anti‐laminin IgG. We consider the possibility that a transmembrane link exists between extracellular laminin and cytoskeletal elements which modulates the circumferal F‐actin ring. We further postulate that this linkage can influence the nature of tight junctional complexes, and thereby the integrity of the Sertoli cell barrier. © 1993 Wiley‐Liss, Inc.
Induction of GTP‐cyclohydrolase I mRNA expression by lectin activation and interferon‐γ treatment in human cells associated with the immune response
Authors: Schott Karin , Gütlich Markus , Ziegler Irmgard
Published: February 2005

The development of tetrahydrobiopterin synthesis during lectin stimulation of resting human T lymphocytes (Kerler et al. [1989] FEBS Lett., 250:622–624), the interferon‐γ induced neopterin production by human monocytes/macrophages (Huber et al. [1984] J. Exp. Med., 160:310–16), and the control of tetrahydrobiopterin synthesis in activated T cells by the synergistic action of interferon‐γ and interleukin 2 (Ziegler et al. [1990] J. Biol. Chem., 265:17026–17030) were previously explained by modulation of the apparent GTP‐cyclohydrolase I activation. In this study we demonstrate that increases in GTP‐cyclohydrolase I activity which occur after lectin induction and after cytokine treatment correlate with increased steady state mRNA levels specific for this enzyme. The enhancement of interferon‐γ induced enzyme activity in primed T cells by interleukin 2 also corresponds to further increases in mRNA expression. The steady state GTP‐cyclohydrolase I mRNA levels in primed T cells, however, do not correlate with the steep decline which follows the culmination of enzyme activity 44 hours after treatment. This indicates that the down‐regulation of apparent GTP‐cyclohydrolase I activity is caused by posttranslational modification of the protein. © 1993 Wiley‐Liss, Inc.
Core proteins of soluble chondroitin sulfate proteoglycans purified from the rat brain block the cell cycle of PC12D cells
Authors: Katoh‐Semba Ritsuko , Oohira Atsuhiko
Published: February 2005

The effects of soluble chondroitin sulfate proteoglycans (CSPGs) purified from the rat brain on proliferation of and neurite outgrowth from PC12D cells (Katoh‐Semba et al., J Neurosci Res 17:36, 1987) were investigated. When PC12D cells are cultured under standard conditions, they proliferate with a doubling time of about 2 days, irrespective of the presence or absence of NGF. However, the addition of a mixture of several types of purified soluble brain CSPG (50 nmol uronic acid/ml) to the culture medium prevented the increase in the number of PC12D cells as well as the nerve growth factor (NGF)‐induced neurite extension. The dose for 50% inhibition (ID50) was 1.6 nmol/ml for cell proliferation and 2.7 nmol/ml for neurite elongation. The increase in cell number seemed to stop around 6 h after exposure to culture medium supplemented with brain‐derived CSPGs, and even substratum‐attached CSPGs were able to exert such inhibitory effects. Only brain‐type CSPGs, not a cartilage‐derived CSPG (PGH) or a hyaluronate‐binding PGH, had such inhibitory effects. Furthermore, these inhibitory activities were associated only with the core proteins of brain‐derived CSPGs, and not with polysaccharide chains from brain‐derived CSPGs. Incorporation of [3H]thymidine into DNA did not decrease for at least the first 12 h. Consequently, the amount of DNA per cell after 48 h of culture was about twofold higher in cells treated with brain CSPGs than in nontreated cells after exposure to the medium with CSPGs. Microspectrophotometry revealed that the population of cells with a high DNA content was greater in the culture treated with brain‐derived CSPGs than in the control culture. These findings indicate that purified soluble brain CSPGs block the cell cycle of PC12D cells at the G2 phase with resultant cessation of cell proliferation and the inhibition of neurite outgrowth. © 1993 Wiley‐Liss, Inc.
Assembly of the peripheral domain of the bovine vacuolar H+‐adenosine triphosphatase
Authors: Myers Melanie , Forgac Michael
Published: February 2005

The biosynthesis and assembly of the peripheral sector (V1) of the vacuolar protontranslocating adenosine triphosphatase (V‐ATPase) was studied in a bovine kidney epithelial cell line. Monolayer cultures of cells were metabolically radiolabeled with Tran 35S‐label and the V‐ATPase subsequently immunoprecipitated using a monoclonal antibody raised against the bovine brain‐coated vesicle proton pump. The V‐ATPase immunoprecipitated from the bovine kidney cell line has a subunit composition very similar to that of the bovine brain‐coated vesicle proton pump and the V‐ATPase prepared from other kidney tissues. Radiolabeling the cells for increasing times showed that the V1 or peripheral portion of the V‐ATPase is assembled within 10–15 min; the intact V1V0 complex is also detectable within 10–15 min. Fractionation of the cells into cytosolic and membrane components prior to immunoprecipitation revealed that there is a significant pool of V1 in the cytosol; a similar complex is also found in bovine brain cytosol. Pulse‐chase studies suggest that this cytosolic pool is not an obligate precursor for membranebound V1V0 and does not exchange with the membrane V1 population at later times. No qualitative differences in assembly were observed when pulse‐chase studies were performed at 15°C or in the presence of brefeldin A. This suggests that assembly of V1V0 is probably completed in the endoplasmic reticulum prior to distribution of the enzyme throughout the cell, with a cytosolic pool of V1 of unknown function existing in parallel with the fully assembled complex. © 1993 Wiley‐Liss, Inc.
Resolution of multiple AP‐1 complexes in HL‐60 cells induced to differentiate by 1,25‐dihydroxyvitamin D3
Authors: Kolla Sarah S. , Studzinski George P.
Published: February 2005

Activator protein‐1 (AP‐1) complex plays a central role in the regulation of both growth and differentiation in many cell types. Monocytic differentiation of HL‐60 cells by TPA (12‐0‐tetradecanoyl phorbol‐13‐acetate) has been reported to be paralleled by increased AP‐1 binding to DNA and by elevated c‐jun expression, suggesting transcriptional level of control. We show that two forms of AP‐1 complex, designated AP‐1/1 and AP‐1/2, can be demonstrated in logarithmically growing HL‐60 cells, that the exposure of these cells to 10−8 M 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3) results in increased binding of these complexes to the AP‐1 DNA element, and that the AP‐1 complex can be resolved into at least three forms in differentiated cells. Binding to, or competition with, a mutated form of the AP‐1 binding site shows that the most slowly migrating complex (AP‐1/3) binds to DNA with greater specificity than do complexes AP‐1/1 and AP‐1/2, while antibody inhibition and binding studies performed at 37°C indicate that jun proteins predominate in AP‐1/2 complexes. Exposure of extracts from differentiated, but not untreated, HL‐60 cells to 2 mM ATP increases the prominence of AP‐1/3 complexes, and reduces the DNA binding of AP‐1/1 complexes. Treatment of the extracts with phosphatases abolishes the binding of AP‐1/2 and AP‐1/3 to DNA, and increases the binding intensity of AP‐1/1. When extracts from differentiated cells are mixed with extracts from undifferentiated cells the AP‐1/3 complexes become less prominent, suggesting than an inhibitory activity in undifferentiated cells prevents the formation of AP‐1/3 complexes. These studies show the association of multiple forms of AP‐1 complex with the mature monocytic phenotype, and suggest several levels of control of monocytic differentiation. © 1993 Wiley‐Liss, Inc.
Induction of tolerance to hypothermia and hyperthermia by a common mechanism in mammalian cells
Authors: Glofcheski David J. , Borrelli Michael J. , Stafford Diane M. , Kruuv Jack
Published: February 2005

Pretreatment by hypothermic (25°C) cycling (PHC) of attached exponential‐phase V79 Chinese hamster cells by Method 4 (24 hr at 25°C + 1.5 hr at 37°C + 24 hr at 25°C + trypsin + 3 hr at 37°C) or by Method 3 (48 hr at 25°C + trypsin + 3 hr at 37°C) make mammalian V79 cells significantly more resistant to 43°C hyperthermia. There is no significant difference in the 43°C curves whether Method 3 or 4 is used for pre‐exposure. If pre‐exposure is at 15 or 10°C, the resistance to hyperthermia is significantly reduced. PHC by Method 4 significantly increases survival of cells exposed to 5°C and, to a lesser extent, to 10°C. The increase in hyper‐ and hypothermic survival after PHC cannot be accounted for by changes in cell cycle distribution. Heat‐shock protein synthesis is not induced by PHC; hence, protection does not result from newly synthesized proteins. When cells are made tolerant to hyperthermia by a pretreatment in 2% DMSO for 24 hr at 37°C (Method 8), the cells are not more resistant to subsequent exposures to hypothermia, either at 5 or 10°C. The results imply that there may be two mechanisms of inducing resistance to hyperthermia, only one of which also confers resistance to hypothermia. © 1993 Wiley‐Liss, Inc.
Physiological oxygen tension modulates the chemically induced mitogenic response of cultured rat hepatocytes
Authors: Maier Peter , Schawalder Hanspeter
Published: February 2005

Freshly isolated rat hepatocytes were cultured at periportal‐ (13% O2) or perivenous‐like (4% O2) oxygen tension and exposed to subtoxic exposure levels of cyproterone acetate (CPA: 10–330 μM), phenobarbital (PB: 0.75‐6 mM), and dimethylsulfoxide (DMSO: 0.1–3.3%) from 24–72 h after seeding. Induced alterations in ploidy, in the number of S‐phase cells, the degree of binuclearity, and cellular protein content were determined by twin parameter protein/DNA flow cytometry analysis of intact cells and isolated nuclei. CPA and PB increased whereas DMSO decreased dose dependently the total number of S‐phase cells. The changes differed within individual ploidy classes and were modulated by the oxygen tension. CPA increased and DMSO decreased the number of S‐phase cells preferentially among the diploid hepatocytes at periportal‐like oxygen tension. In contrast, PB increased binuclearity and S‐phase cells mainly among the tetraploid hepatocytes at perivenous‐like oxygen tension. Cellular protein content increased dose dependently after exposure to the hepatomitogens (CPA, PB) and decreased after exposure to DMSO at both oxygen tensions. Comparison with in vitro data proves that chemicals which interact with cells from the progenitor liver compartment (CPA, DMSO) exert their mitogenic activity best in cultures at periportal‐like oxygen tension preferentially in diploid hepatocytes, whereas chemicals which affect cells from the functional compartment show a higher activity at perivenous‐like oxygen tension. Physiological oxygen tension seems to be an effective modulator of the proliferative response of cultured rat hepatocytes similar to that expected for periportally or perivenously derived hepatocytes. © 1993 Wiley‐Liss, Inc.
Type I collagen substrate increases calcitonin and parathyroid hormone receptor‐mediated signal transduction in UMR 106–06 osteoblast‐like cells
Authors: Ikeda Kazuto , Michelangeli Valdo P. , Martin T. John , Findlay David M.
Published: February 2005

Components of the extracellular matrices (ECM) exert pleiotropic effects in many cell systems, but little is known of the effect of ECM on hormone signal transduction. We have investigated the effect of ECM substrates on cell growth and signal transduction by calcitonin (CT) and parathyroid hormone (PTH) using the rat osteosarcoma cell line, UMR 106–06. Type I collagen (collagen[I]) and Matrigel changed the morphology of the cells and significantly inhibited cell growth by 37% or 23%, respectively, compared with control. None of laminin, fibronectin, or type IV collagen affected cell shape or proliferation. Cells cultured on collagen (I)‐coated plates showed increased specific binding of labeled CT compared with cells on plastic plates. The effect was apparent by 24 h and persisted for at least 72 h. None of the other ECM affected CT binding. Scatchard analysis revealed that collagen(I) increased CT receptor numbers but not receptor affinity. Consistent with increased binding capacity, cells plated on collagen(I) had increased responses to each of CT and PTH in terms of cyclic adenosine monophosphate (cAMP) production compared to control cells. In addition, cAMP production by prostaglandin E2, cholera toxin, and forskolin was increased by 30–70% compared to control. These data suggest that collagen(I) had effects not only on membrane receptors but on guanosine triphosphate (GTP) binding proteins (G proteins). The effect of collagen(I) on CT binding was no longer present when the cells were freed from the plates by enzymatic dispersion and binding measured in cell suspensions. In UMR 106–01 cells transiently transfected with the porcine CT receptor cDNA, binding was similarly induced by collagen(I). These data are the first demonstration that collagen(I) may play an important role in signal transduction, affecting both receptors and G proteins in UMR 106–06 cells. These results draw attention to the potential role of the ECM of bone in hormone‐induced responses. © 1993 Wiley‐Liss, Inc.
Differences in transferrin response and numbers of transferrin receptors in rat and human mammary carcinoma lines of different metastatic potentials
Authors: Inoue Takatoshi , Cavanaugh Philip G. , Steck Peter A. , Brünner Nils , Nicolson Garth L.
Published: February 2005

We previously found that transferrin (Tf) differentially stimulated the growth of highly metastatic variant lines of murine melanoma and that these highly metastatic cells also had greater numbers of Tf receptors on their cell surfaces. In the present study we found that highly metastatic rat mammary adenocarcinoma cell lines also responded differentially to Tf in proliferation assays, and cell monolayers bound Tf in relation to their metastatic potential (MTPaB10 > MTPaB5 > MTLn3 > MTLn2 > MTC > MTF7 > MTPa). The brain‐colonizing lines PaB10 and PaB5 were the most responsive to Tf and had the highest numbers of Tf receptors. Different human breast cancer cell lines also responded differentially to Tf in proliferation assays and bound different amounts of Tf to their cell surface Tf receptors. Transferrin binding, but not growth response, correlated with metastatic and invasive properties of lines selected from the human MCF‐7 series (MCF7/LCC2 > MCF7/LCC1 > MCF7). In examining the transferrin binding and growth response of lines from the human MDA series, the Tf binding and growth response was MDA231 > MDA435 > MDA468. The lines MDA435 and MDA231 were metastatic in nude mouse assays, whereas the line MDA468 was not. Scatchard analysis indicated the presence of a single class of receptor for Tf on the rat and human mammary cell lines. The results suggest that neoplastic cells displaying various metastatic properties may express differing numbers of Tf receptors and respond differently to growth factors such as Tf. © 1993 Wiley‐Liss, Inc.
Secretion of transforming growth factor‐β isoforms by embryonic stem cells: Isoform and latency are dependent on direction of differentiation
Authors: Slager H. G. , Freund E. , Buiting A. M. J. , Feijen A. , Mummery C. L.
Published: February 2005

Murine embryonic stem (ES) cells are maintained in an undifferentiated state when cultured in medium conditioned by Buffalo rat liver (BRL) cells. BRL conditioned medium (CM) contains a differentiation inhibitory activity (DIA) that is synonymous with leukemia inhibitory factor (LIF). ES cells in monolayer culture can be induced to differentiate by addition of all‐trans retinoic acid (RA) to the BRL CM, when they mainly form cells resembling parietal endoderm, or by culture in medium not conditioned by BRL cells. ES cells thus deprived of LIF/DIA differentiate spontaneously to a cell type that expresses Brachyury (T), a marker of early mesoderm. Northern blot analyses have shown previously that transcripts for transforming growth factor beta 1 (TGF‐β1) are detected in undifferentiated cells while transcripts for TGF‐β2 and TGF‐β3 only become detectable after differentiation. We have now determined levels of TGF‐β protein in CM and in the extracellular matrix (ECM) and have used neutralizing antibodies specific for TGF‐β1 and TGF‐β2 that do not react with recombinant human TGF‐β3 to determine the isoform secreted. Using the growth inhibition of mink lung CCL64 cells as a bioassay for TGF‐β activity, we demonstrate that undifferentiated ES cells secrete latent TGF‐β1 into the medium but no activity is found in their ECM. Cells induced to differentiate with RA contain TGF‐β2 in both active and latent forms in their CM. Likewise their ECM contains TGF‐β2 as the sole isoform. ES cells deprived of LIF/DIA secrete both TGF‐β1 and TGF‐β2 isoforms in their CM but TGF‐β‐like activity remains after addition of neutralizing antibodies for TGF‐β1 and TGF‐β2. This active TGFβ is the major component of the TGF‐β activity in this CM. By contrast, ECM from LIF/DIA deprived cells contains only the TGF‐β1 and β2 isoforms. The remaining activity in CM correlates with high expression of TGF‐β3 by Northern blot analysis in these cells. We speculate that TGF‐β3 is secreted by these cells and may be activated more efficiently and/or in a different manner to TGF‐β1 and TGF‐β2, since it is present in CM only in its active form. © 1993 Wiley‐Liss, Inc.
Transferrin is made and bound by photoreceptor cells
Authors: Davis Alberta A. , Hunt Richard C.
Published: February 2005

Retinal pigment epithelial cells, which form one aspect of the blood‐retinal barrier, control the access of blood‐borne components such as diferric transferrin to the neural retina. It has recently been shown that RPE cells remove iron from diferric transferrin in a low pH compartment and subsequently release it in a low molecular weight form that can be chelated by apo‐transferrin (Hunt and Davis: J. Cell Physiol. 152:102–110, 1992). It is now shown that photoreceptor cells can bind diferric transferrin to receptors on their inner segments. Moreover, polymerase chain reaction and in situ hybridization show that cells of the neural retina, particularly photoreceptors, make apo‐transferrin. © 1993 Wiley‐Liss, Inc.
Interaction of facilitative glucose transporter with glucokinase and its modulation by ADP and glucose‐6‐phosphate
Authors: Lachaal Mohsen , Jung Chan Y.
Published: February 2005

Bacterial glucokinase (GK) binds to purified, human erythrocyte glucose transporter (GT) reconstituted in vesicles. The binding is largely abolished if GT is predigested with trypsin, indicating that GK binds to the cytoplasmic domain of GT. The binding is a saturable function of GK concentration showing two distinct affinities with apparent KD of 0.33 and 5.1 μM. The binding is stimulated by an increasing concentration of ADP with the 50% maximal effect at 5 mM. Glucose‐6‐phosphate (G6P) also stimulates the binding with a distinct optimum at 25 mM. The binding is stimulated only slightly by ATP. D‐glucose has no affect on the binding. KCl enhances the binding with the maximal effect at physiological intracellular concentrations. The binding is sensitive to changes in pH with an optimum at pH 4. The binding causes no detectable functional change in GT. However, the enzymatic activity of GK measured at nanomolar concentrations of GK is significantly greater in the presence of GT vesicles than in its absence or in the presence of protein‐free vesicles, indicating that GK interacts with GT at this low concentration range with an apparent KD of 10 mM. Although its physiological significance is not known, the GK‐GT interaction in vitro described here suggests that these two proteins may also interact in the cell and regulate carbohydrate metabolism. © 1993 Wiley‐Liss, Inc.
Adhesion molecules in skeletogenesis: II. Neural cell adhesion molecules mediate precartilaginous mesenchymal condensations and enhance chondrogenesis
Authors: Widelitz Randall B. , Jiang Ting‐Xin , Murray Ben A. , Chuong Cheng‐Ming
Published: February 2005

Neural cell adhesion molecules (NCAM) was expressed transiently by mesenchymal cells in precartilaginous condensations of the embryonic chicken limb but was lost upon differentiation into cartilage. Consequently, NCAM was present in the periphery of the limb anlagen but was absent in the cartilaginous center of the growing limb. To determine NCAM function in limb bud chondrogenesis we incubated dissociated stage 22/23 distal mesenchymal limb bud cells with Fab' fragments of antibodies to NCAM. Cell aggregation was inhibited by incubating the cells with anti‐NCAM Fab'. These results suggest that NCAM may mediate the formation of precartilaginous condensations. This hypothesis was further tested using micromass cultures. NCAM expression in micromass cultures in vitro recapitulated that in vivo. NCAM was enriched in condensations of 2 day cultures, but was diminished and concentrically distributed around cartilage nodules in 4 day cultures. Anti‐NCAM Fab' fragments reduced the area occupied by precartilaginous condensations and the degree of chondrogenic differentiation. Control antibody against chicken embryo fibroblasts had no effect. The effect of overexpressing NCAM was analyzed by electroporating expression vectors directing the synthesis of chicken NCAM. Limb bud cells cultured after electroporation with an NCAM expression vector displayed larger cartilage nodules and greater chondrogenic differentiation than cells electroporated with vector alone. The expression of NCAM in electroporated cells also increased. Control experiments using plasmids encoding β‐galactosidase indicated that approximately 10% of the limb bud cells were transfected under these conditions. The results suggest that NCAM is involved in the chondrogenesis pathway by mediating the formation of precartilaginous condensations. © 1993 Wiley‐Liss, Inc.