bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of extracellular signaling on endothelial cell proliferation and protein N‐glycosylation
Authors: Oliveira Caroline M. , Banerjee Dipak K.
Published: February 2005

In vitro studies of angiogenic phenomenon have been limited due to nonavailability of a simple and biologically relevant model of the capillary wall. Recent development of a capillary endothelial cell line from the vascular bed of bovine adrenal medulla made us to study the effect of heparin, thrombin, thyroxine, glucagon, insulin, and phorbol myristate acetate (PMA) on the proliferative and metabolic activities such as glycosylation of asparagine‐linked glycoproteins of these cells in culture. Out of six different agents studied here, only heparin, thrombin, and thyroxine reduced the doubling time of these cells by 24 hr with no observed morphological abnormality. Glucagon, showed marginal reduction in the cell doubling time. By contrast, insulin and PMA enhanced the doubling time. Insulin treatment though induced the S phase of cell cycle but it blocked the cells entry into the G2 + M phase. PMA arrested the cells in Go/G1 phase. The cellular response to protein N‐glycosylation is increased in the presence of thyroxine, insulin, and thrombin and the effect is dose dependent. Further analysis on SDS‐PAGE indicated that glycosylation of 80–120 kDa and 43 kDa glycoprotein species are enhanced when these cells are treated with insulin and thrombin. Glycopeptide generated from these glycoproteins suggested that they all carry “high mannose” and “complex” type oligosaccharide chains attached to their protein core.
Comparison of the function of the tight junctions of endothelial cells and epithelial cells in regulating the movement of electrolytes and macromolecules across the cell monolayer
Authors: Milton Shirlette G. , Knutson Victoria P.
Published: February 2005

In cell culture, both endothelial and epithelial cell monolayers have been found to generate structurally similar tight junctional complexes, as assessed by thin section electron microscopy. Although structurally similar, the tight junctional complexes of the two cell types are, at least in part, responsible for the very different permeability characteristics of native endothelial and epithelial cell monolayers. The purpose of this work was to compare cultured endothelial and epithelial cells with respect to the function of their tight junctional complexes in regulating the movement of macromolecules and ions across the cell monolayers, and define functional parameters to characterize the tight junctional complexes. Bovine aorta endothelial cells and T84 colonic carcinoma epithelial cells were cultured on a microporous membrane support. The permeability coefficients of inulin, albumin, and insulin were determined with the cell monolayers and compared with the permeability coefficients obtained with 3T3‐C2 fibroblasts, a cell line that does not generate tight junctions. Electrical resistance measurements across the monolayer‐filter systems were also compared. The permeability coefficient of albumin across the endotnelial cell monolayer compared favorably with other reported values. Likewise, the electrical resistance across the T84 cell monolayer was in good agreement with published values. Utilizing permeability coefficients for macromolecules as an index of tight junction function, we found that a distinction between a lack of tight junctions (fibroblasts), the presence of en‐dothelial tight junctions, and the presence of epithelial tight junctions was readily made. However, when utilizing electrical resistance as an index of tight junction function, identical measurements were obtained with fibroblasts and endothelial cells. This indicates that more than one index of tight junction function is necessary to characterize the junctional complexes. Although structurally similar, epithelial cell and endothelial cell tight junctions perform very different functions, and, from our data, we conclude that the demonstration of tight junctional structures by electron microscopy is not relevant to the functional nature of the junction: structure does not imply function. A minimal assessment of tight junction function should rely on both the determination of the electrical resistance across the cell monolayer, and the determination of the permeability coefficients of selected macromolecules.
Dual effects of glucagon and cyclic amp on dna synthesis in cultured rat hepatocytes: Stimulatory regulation in early G1 and inhibition shortly before the s phase entry
Authors: Thoresen G. Hege , Sand Tor‐Erik , Refsnes Magne , Dajani Olav F. , Guren Tormod K. , Gladhaug Ivar P. , Killi Anne , Christoffersen Thoralf
Published: February 2005

Although several lines of evidence implicate cyclic AMP in the humoral control of liver growth, its precise role is still not clear. To explore further the role of cyclic AMP in hepatocyte proliferation, we have examined the effects of glucagon and other cyclic AMP‐elevating agents on the DNA synthesis in primary cultures of adult rat hepatocytes, with particular focus on the temporal aspects. The cells were cultured in a serum‐free, defined medium and treated with epidermal growth factor (EGF), insulin, and dexamethasone. Exposure of the hepatocytes to low concentrations (10 pM‐1 nM) of glucagon in the early stages of culturing (usually within 6 h from plating) enhanced the initial rate of S phase entry without affecting the lag time from the plating to the onset of DNA synthesis, whereas higher concentrations inhibited it. In contrast, glucagon addition at later stages (24–45 h after plating) produced only the inhibition. Thus, if glucagon was added at a time when there was a continuous EGF/insulin‐induced recruitment of cells to S phase, the rate of G1‐S transition was markedly decreased within 1–3 h. This inhibitory effect occurred at low glucagon concentrations (ID50 < 1 nM) and was mimicked by cholera toxin, forskolin, isobutyl methylxanthine, and 8‐bromo cyclic AMP. The results indicate that cyclic AMP has dual effects on hepatocyte proliferation with a stimulatory modulation early in the prereplicative period (Go or early G1), and a marked inhibition exerted immediately before the transition from G1 to S phase.
Comparison of bone and parathyroid hormone as stimulators of osteoclast development and activity in calvarial cell cultures from normal and osteopetrotic (mi/mi) mice
Authors: Graves Leland , Jilka Robert L.
Published: February 2005

Osteoclast development was studied in cell cultures prepared from calvaria of neonatal osteopetrotic (mi/mi) mice or their normal littermates, using tartrate‐resistant acid phosphatase (TRAPase), as an osteoclast marker. In cultures from normal mice, treatment with 10 nM PTH for 4–5 days stimulated the formation of osteoclasts. However in cultures from mi/mi mice, this response was only 7% ± 5% that of normal mice and they were significantly smaller than osteoclasts of normal mice. Mineralized bone particles elicited osteoclast development in cultures from both normal and mi/mi mice, and osteoclast size was identical for both genotypes. Seventy‐eight to 96% of the TRAPase‐positive cells bound 125 I‐CT, as demonstrated by autoradiography. 125I‐CT binding characteristics were identical in cultures from both genotypes treated with bone particles, exhibiting a Kd of 3.3–3.6 × 10−10 M. Addition of PTH stimulated 45Ca release from the added bone particles only in the case of cultures prepared from normal mice, and CT inhibited this response. Cells from normal mice were capable of excavating bone from the surface of smooth cortical bone wafers, but such excavations were rarely seen in the case of calvarial cells from mi/mi mice. Thus, PTH‐driven differentiation of osteoclasts is arrested in calvarial cell cultures from mi/mi mice, but mi/mi preosteoclasts retain the ability to express certain osteoclast markers in response to bone derived signals. We hypothesize that the lack of activity of mi/mi osteoclasts is due to the failure of mi/mi preosteoclasts to respond appropriately to resorptive agents, or to cytokines elicited by these agents.
Sarcolectin and interferon in the regulation of cell growth
Authors: Chany‐fournier Françoise , Jiang Pan Hong , Chany Charles
Published: February 2005

Sarcolectin is an endolectin present in a great variety of conjunctival tissues (muscles, cartilage, sarcomas), but also in brain or placental extracts of vertebrates, including primates. When purified to electrophoretical homogeneity as a 65‐kd protein, it agglutinates cells and has an affinity for simple sugars. In addition, it is able to inhibit the synthesis of interferon (IFN)‐dependent secondary proteins and to restore cells to their status ad primum. The biological effect of Poly(I). Poly(C)‐induced feedback interferon is inhibited by the addition of sarcolectins, which also abolishes cellular refractoriness to repeated IFN induction. Similarly, sequential association of, first, Poly(I). Poly(C); 4–5 h later, sarcolectin restores the full capacity of both to promote cell growth, unrestrained by IFN. Indeed, the secondary proteins which are in the process of being synthesized are inhibited. In a great variety of animal cells, sarcolectin can also initiate growth after it has been blocked by IFN. This is not an all‐or‐none effect, but a balance may be struck by IFN and sarcolectin, depending on their respective concentrations and specific activity. We propose that the coordination of these cellular functions of Poly(I).Poly(C), IFN, and sarcolectin takes place in the form of a triangular growth‐regulatory cycle and postulate that they thus maintain a balance during differentiated normal tissue development.
Interaction of Epidermal growth factor receptors with the cytoskeleton is related to receptor clustering
Authors: van Belzen Nico , Spaargaren Marcel , Verkleij Arie J. , Boonstra Johannes
Published: February 2005

Recently it has been established that cytoskeleton‐associated epidermal growth factor (EGF) receptors are predominantly of the high‐affinity class and that EGF induces a recruitment of low‐affinity receptors to the cytoskeleton. The nature of this EGF‐induced receptor‐cytoskeleton interaction, however, is still unknown. Therefore, we have studied the association of mutated EGF receptors with the cytoskeleton. Receptor deletion mutants lacking almost all intracellular amino acid residues displayed no interaction with the cytoskeleton, demonstrating that the cytoplasmic receptor domain is involved in this interaction. Further analysis revealed that receptor‐cytoskeleton interaction is independent of receptor kinase activity and the C‐terminal 126 amino acid residues, which include the autophosphorylation sites. Furthermore, it is shown that the high‐affinity receptor subclass is not essential for association of low‐affinity receptors to the cytoskeleton. EGF receptor‐cytoskeleton interaction was increased, however, by treatment with sphingomyelinase, an enzyme known to induce membrane protein clustering, indicating that EGF receptor clustering may cause the association to the cytoskeleton.
TGF‐β inhibits the platelet‐derived growth factor‐induced formation of inositol trisphosphate in MG‐63 human osteosarcoma cells
Authors: Pontbriant Candace M. , Chen Jan‐Kan , Orlando Joseph A.
Published: February 2005

Epidermal growth factor (EGF), platelet‐derived growth factor (PDGF), and heparin‐binding growth factor‐1 (HBGF‐1) stimulated the proliferation of a variant of the human osteosarcoma cell line, MG‐63‐LS = low serum. Transforming growth factor beta (TGF‐β) completely inhibited cell growth in basal medium supplemented with 2% fetal calf serum (FCS), blocked PDGF‐ and EGF‐stimulated cell proliferation, and modulated that of HBGF‐1. PDGF, but not EGF or HBGF‐1, activated the inositol trisphosphate/diacylglycerol (IP3/DAG) second message system in a dose‐dependent manner. EGF inhibited phosphoinositol lipid turnover and HBGF‐1 and TGF‐β stimulated phosphatidylinositol hydrolysis to produce inositol phosphate (IP) but not IP3. Preincubation of quiescent cells with TGF‐β for 30–40 minutes prior to the addition of PDGF resulted in an inhibition of PDGF‐induced production of IP3. This suggested that TGF‐β was an indirect inhibitor and blocked PDGF‐stimulated cell growth in part by interfering with the generation of the second messenger, IP3.
Tumor necrosis factor enhances the neutrophil‐dependent increase in endothelial permeability
Authors: Gibbs Lester S. , Lai Linda , Malik Asrar B.
Published: February 2005

We examined the effect of tumor necrosis factor α (TNFα) on the increase in pulmonary microvascular endothelial monolayer permeability induced by activated neutrophils (PMN). Layering of PMN onto endothelial monolayers followed by activation of PMN with phorbol 12‐myristate 13‐acetate (PMA) increased125I‐albumin clearance rate across the monolayers. Pretreatment of endothelial monolayers for 6 hr with TNFα(200 U/ml) potentiated the PMN‐dependent increase in endothelial permeability, whereas 1 hr or 6 hr pretreatment of endothelial monolayers with 200 U/ml and 100 U/ml, respectively, TNFαdid not enhance the reponse. Adherence of PMN to the endothelial cells was increased at 1 and 6 hr after TNFα(200 U/ml) treatment, but the adherence reponse was markedly greater following 6 hr of TNFα. The TNFαtreatment of endothelial cells did not enhance neutrophil activation responses to PMA. Pretmeatment of PMN with IB4, a MAb to the CD18 integrin, the common β subunit of the adhesion proteins LFA‐1, Mac‐1, and p150,95 of PMN, reduced the increases in PMN adherence and the endothelial monolayer permeability induced by the 6 hr TNFα treatment. In contrast, pretreatment of PMN with OKM‐1, a MAb to the CD11b epitope (‐subunit), had no effect on the adherence and the potentiation of the increase in permeability. The potentiation of the PMN‐dependent permeability increase and enhanced endothelial adhesivity at 6 hr after TNFαpriming of endothelial cells was dependent on protein synthesis. The results indicate that protein synthesis‐dependent expression of an endothelial ligand for CD18 and resultant endothelial hyperadhesiveness potentiates the PMN‐mediated increase in endothelial permeability after TNFαactivation of endothelial cells. The priming of endothelial cells by TNFαmay be a critical step in the mediation of endothelial injury.
Effects of hypertonic and sodium‐free medium on transport of a membrane glycoprotein along the secretory pathway in cultured mammalian cells
Authors: Docherty Pamela A. , Snider Martin D.
Published: February 2005

Incubation of cultured cells in hypertonic medium and sodium‐free medium have been shown to block transport at two different stages along the endocytic pathway. To determine the effects of these treatments on the exocytic pathway, we studied the transport of the membrane glycoprotein of vesicular stomatitis virus (VSV‐G) in cells infected with tsO45 mutant virus. This mutant synthesizes a VSV‐G that accumulates in the endoplasmic reticulum (ER) when cells are incubated at 39.5°C. In addition, VSV‐G accumulates in the post‐ER pre‐Golgi compartment when cells are incubated at 15°C and in the trans‐Golgi network (TGN) when cells are incubated at 18°C. Upon transfer of cells to 32°C in control medium, VSV‐G exits each of these compartments and is transported to the cell surface. Incubation in sodium‐free medium at 32°C did not block transport from any of these three compartments. In contrast, incubation in hypertonic medium blocked export from the ER, transport from the pre‐Golgi compartment to the Golgi complex, and transport from the TGN to the cell surface. Our results, in combination with previous studies, suggest that hypertonic medium blocks at least five distinct transport steps: the three exocytic steps described here, endocytosis from the cell surface, and transport of cell surface proteins into the Golgi complex. This raises the possibility that vesicular transport in different parts of the cell shares common elements that are inhibited by this treatment.
Homocysteine export from cells cultured in the presence of physiological or superfluous levels of methionine: Methionine loading of non‐transformed, transformed, proliferating, and quiescent cells in culture
Authors: Christensen Benedicte , Refsum Helga , Vintermyr Olav , Ueland Per Magne
Published: February 2005

Determination of the transient increase in plasma homocysteine following administration of excess methionine is an established procedure for the diagnosis of defects in homocysteine metabolism in patients. This so‐called methionine loading test has been used for 25 years, but the knowledge of the response of various cell types to excess methionine is limited. In the present paper we investigated homocysteine export from various cell types cultured in the presence of increasing concentrations (15–1,000 μM) of methionine. For comparison of homocysteine export, the export rates per million cells were plotted versus cell density for proliferating cells, and versus time for quiescent cells. The homocysteine export from growing cells was greatest during early to mid‐exponential growth phase, and then decreased as a function of cell density. The export rate was higher from phytohemagglutinin‐stimulated than non‐stimulated lymphocytes, and higher from proliferating than from quiescent fibroblasts. The hepatocytes showed highest export rate among the cell types investigated. The enhancement of homocysteine export by excess methionine ranged from no stimulation to marked enhancement, depending on cell type investigated, and three different response patterns could be distinguished: (1) quiescent fibroblasts and growing murine lymphoma cell showed no significant increase in homocysteine export following methionine loading; export from human lymphocytes was only slightly enhanced in the presence of excess methionine; (2) the homocysteine export from proliferating hepatoma cells and benign and transformed fibroblasts was stimulated three to eightfold by increasing the methionine concentration in the medium from 15 to 1,000 μM; and (3) the response to methionine loading was particularly increased (about 15‐fold) in non‐transformed primary hepatocytes in stationary culture. The results outline a potentially useful procedure for the comparison of homocysteine export during cell growth in the presence of various concentrations of methionine. The results are discussed in relation to the special feature of homocysteine metabolism in various cell types and tissues including liver, and to the possible source of plasma homocysteine following methionine loading in vivo.
Epidermal growth factor treatment of A431 cells alters the binding capacity and electrophoretic mobility of the cytoskeletally associated epidermal growth factor receptor
Authors: Roy Linda M. , Gittinger Cynthia K. , Landreth Gary E.
Published: February 2005

The epidermal growth factor (EGF) receptor interacts with structural elements of A431 cells and remains associated with the cytoskeleton following extraction with nonionic detergents. Extraction of cells with 0.15% Triton X‐100 resulted in detection of only approximately 40% of the EGF binding sites on the cytoskeleton. If the cells were exposed to EGF prior to extraction, approximately twofold higher levels of low‐affinity EGF binding sites were detected. The difference in number of EGF binding sites was not a consequence of differences in numbers of EGF receptors associated with the cytoskeleton; equal amounts of 35S‐labeled receptor were immunoprecipitated from the cytoskeletons of both control and EGF‐treated cells. The effect of EGF pretreatment on binding activity was coincident with a change in the mobility of the receptor from a doublet of Mr ∼ 160–180 kDa to a single sharp band at 180 kDa. The alteration in receptor mobility was not a simple consequence of receptor phosphorylation in that the alteration was not reversed by alkaline phosphatase treatment, nor was the shift produced by treatment of the cells with phorbol ester. The two EGF receptor species demonstrated differential susceptibility to V8 proteinase digestion. The EGF‐induced 180 kDa species was preferentially digested by the proteinase relative to the 160 kDa species, indicating that EGF binding results in a conformational change in the receptor. The EGF‐mediated preservation of binding activity and altered conformation may be related to receptor oligomerization.
Programmed cell death (apoptosis) is induced rapidly and with positive cooperativity by activation of cyclic adenosine monophosphate‐kinase I in a myeloid leukemia cell line
Authors: Lanotte M. , Riviere J. B. , Hermouet S. , Houge G. , Vintermyr O. K. , Gjertsen B. T. , Døskeland S. O.
Published: February 2005

Programmed death (apoptosis) of the rat myelocytic leukemic cell line IPC‐81 was triggered by cyclic adenosine monophosphate (cAMP) analogs or by agents (cholera toxin, prostaglandins) increasing the endogenous cAMP level. The induction of cell death by cholera toxin was preceded by increased activation of cAMP‐kinase. Cell lysis started already 5 hr after cAMP challenge and was preceded by internucleosomal DNA fragmentation and morphological changes characteristic of apoptosis. The cell suicide could be prevented by inhibitors of macromolecular synthesis. cAMP analogs induced cell death in a positively cooperative manner (apparent Hill coefficient of 2.9), indicating that triggering of the apoptotic process was under stringent control. There was a strong synergism between cAMP analogs complementing each other in the activation of cAMP‐dependent protein kinase I (cAKI). No such synergism was noted for analogs complementing each other in the activation of cAKII. It is concluded that apoptosis can be induced solely by activation of cAKI. The IPC‐81 cells expressed about four times more cAKI than cAKII. The expression of cAK subunits, on the protein and mRNA levels, was only minimally affected by cholera toxin treatment.
Role of intracellular‐free calcium in the cornified envelope formation of keratinocytes: Differences in the mode of action of extracellular calcium and 1,25 dihydroxyvitamin D3
Authors: Pillai Sreekumar , Bikle Daniel D.
Published: February 2005

Extracellular calcium (Cao) and the steroid hormone 1,25(OH)2D, induce the differentiation of human epidermal cells in culture. Recent studies suggest that increases in intracellular free calcium (Cai) levels may be an initial signal that triggers keratinocyte differentiation. In the present study, we evaluated cornified envelope formation, the terminal event during keratinocyte differentiation, and correlated it with changes in the Cai levels during differentiation of keratinocytes in culture induced by Cao or 1,25(OH)2D, Keratinocytes were grown in different Cao concentrations (0.1 or 1.2 mM) or in the presence of 1,25(OH)2D OCT'1 to 10−7 M, and the Cai levels were measured using the fluorescent probe lndo‐1. Our results suggest that the induction of cornified envelope formation is associated with an increase in Cai level during calcium‐induced differentiation. Cao and the calcium ionophore ionomycin acutely increased Cai and cornified envelope formation. In contrast, the effect of 1,25(OH)2D on increasing Cai levels and stimulating cornified envelope formation was long‐term, requiring days of treatment with 1,25(OH)2D. Our data are consistent with other recent studies and support the hypothesis that Cao regulates keratinocyte differentiation primarily by acutely increasingtheirCai levels. The roleofcalcium in the mechanism ofaction of 1,25(OH)2D keratinocyte differentiation is less clear. The increase in Cai of keratinocytes during 1,25(OH)2D induced differentiation may be essential for or subsequent to its prodifferentiation effects.
Role of growth factors in the contraction and maintenance of collagen lattices made with arterial smooth muscle cells
Authors: Chen Jan‐Kan , Haimes Howard B. , Weinberg Crispin B.
Published: February 2005

The contraction of collagen lattices made with arterial smooth muscle cells was studied in medium MCDB 107 without serum or supplemented with 1% fetal bovine serum, plus insulin, transferrin, and low‐density lipoprotein. Under these conditions, smooth muscle cell mitogens including HBGF‐1 (aFGF), PDGF, and EGF stimulated contraction. Stimulation by HBGF‐1 was more profound than with other factors tested. HBGF‐1 stimulation of lattice contraction was blocked by protein synthesis inhibitors, but not by inhibitors of DNA synthesis. Histological observations indicated that HBGF‐1 also enhanced the maintenance of healthy cells in the lattice. Taken together, these observations suggest that HBGF‐1 stimulates lattice contraction, not by a mitogenic effect, but by stimulating synthesis of specific cellular proteins. Since the greatest effects of HBGF‐1 on lattice contraction were seen during the first 72 h following casting, the effects on maintenance of cell viability are probably less important in promoting lattice contraction.
Effects of prostaglandin E2 and F2α on cytoplasmic pH in a clonal osteoblast‐like cell line, MOB 3–4
Authors: Kawase Tomoyuki , Orikasa Michiaki , Suzuki Akitoshi
Published: February 2005

Prostaglandin E2 (PGE2, 5 ng/ml to 5 μg/ml) induced a dose‐dependent increase in cAMP accumulation, inositol phosphates (IPs) accumulation, and cytoplasmic free Ca2+ ([Ca2+]i) in a clonal osteoblast‐like cell line, MOB 3–4. In contrast, prostaglandin F2α (PGF2α, 5 ng/ml to 5 μg/ml) stimulated increases in IPs accumulation and [Ca2+]i without stimulating an increase in cAMP accumulation. Both PGE2 (>0.5 μg/ml) and PGF2α (≥0.5 μg/ml) increased cytoplasmic pH (pHi) from approximately 7.15 to 7.35 in BCECF‐loaded cells. A tumor promotor, phorbol 12‐myristate 13‐acetate (PMA, 0.1–100 nM) also increased pHi without effect on phosphoinositide hydrolysis. Both PGE2‐(5 μg/ml) and PMA‐ (100 nM) induced cytoplasmic alkalinization was inhibited by removal of extracellular Na+, or by pretreatment of the cells with amiloride (0.5 mM), an inhibitor of Na+/H+ exchange, or H‐7 (100 μM), a nonspecific inhibitor of protein kinase C. Thus, MOB 3–4 cells appeared to possess PGE2 receptors and PGF2α receptors: the former are coupled to adenylate cyclase and phospholipase C, and the latter are predominantly coupled to phospholipase C. Also the cells appeared to possess an amiloride‐sensitive Na+/H+ exchange activity, which increases pHi in response to PGE2 and PGF2α, as well as to PMA. Long‐term (48 hr) exposure of the cells to PGE2 at a high concentration (5 μg/ml), but not to PGF2α and PMA, decreased DNA synthesis in the serum‐deficient medium. Thus, cytoplasmic alkalinization appeared insufficient for cell replication. At least in MOB 3–4 cells, the inhibitory effect of PGE2 on DNA synthesis may be due to the cAMP messenger system.
Nature of enhanced mitochondrial oxidative metabolism by a calf blood extract
Authors: Kuninaka Termichi , Senga Yoshinori , Senga Hirobumi , Weiner Murray
Published: February 2005

The effect of calf blood extract (Solcoseryl, SS) on mitochondrial oxidative function in various states was studied polarographically in vitro. (1) Mitochondrial respiration in all 4 conventional study states (Estabrook, 1967) was enhanced by the addition of SS, including states 1 and 2 (endogenous substrates only). (2) The effect of SS on mitochondrial oxygen consumption was concentration dependent, while ADP/O ratio remained constant. The effect of added respiratory substrates varied with the particular substrate at optimally active concentrations. With suboptimal substrate levels, ADP/O ratios were concentration dependent, in contrast to the SS effect. Under oligomycin ATPase inhibition, SS was no longer active, in contrast to DNP, which remained active. (3) In states 3 (added ADP) and 4 (ADP exhausted), oxygen consumption and oxidative phosphorylation were enhanced by SS in the presence or absence of citrate, glutamate, pyruvate, lactate, or ascorbate. However, in the presence of succinate, SS had no effect. (4) ADP/O ratio was decreased by SS in the presence of added substrate, suggesting that SS activation of H+‐ATPase enhances ATP hydrolysis as well as oxidative phosphorylation and ATP synthesis. (5) The enhancing effect of SS on mitochondrial function is due to hydrophilic components of SS. The lipidic components obtained by Folch fraction of SS have no effect. It is concluded that the effects of SS respiratory substrates and uncouplers on mitochondrial function are essentially different. SS enhances both ATP synthesis and oxygen consumption by mitochondria.
Differential effects of interferon gamma and alpha on in vitro model of angiogenesis
Authors: Maheshwari R. K. , Srikantan V. , Bhartiya D. , Kleinman H. K. , Grant D. S.
Published: February 2005

The formation of blood vessels in vivo (angiogenesis) is an important process and is usually initiated in response to injury, tumor growth, or normal tissue development. We have studied the effect of human interferon (IFN) alpha (α) and gamma (γ) on the capillary‐like network formation in an in vitro model of angiogenesis using human umbilical vein endothelial cells (HUVEC). When HUVEC cells are plated on Matrigel (reconstituted basement membrane matrix enriched in laminin), a network of capillary like structures (endotubes) rapidly forms. IFN‐α enhanced the tube formation in a dose‐dependent manner, whereas IFN‐γ significantly inhibited the tube formation. In addition, both the enhancement and inhibition of angiogenesis by IFN‐α and γ was found to be greater if the cells were pretreated with IFN for 12 hr before plating on the Matrigel. These results suggest that IFN may play an important role in several vascular processes including early stages of wound healing, recanalization of thrombi, tumor growth, metastasis, normal growth, and development.
Glutamine is a powerful effector of heat shock protein expression in Drosophila Kc cells
Authors: Sanders Marilyn M. , Kon Christine
Published: February 2005

We have investigated the effects of extracellular anions on the regulation of expression of the heat shock response in DrosophilaKc cells incubated in defined balanced salt solutions. Widely varying chloride concentrations had no effect on normal or heat shock protein (hsp) expression. Increasing glutamate concentrations from zero to 15 mM increased hsp expression more than 100‐fold while affecting expression of non‐heat‐shock proteins minimally. Glutamine was 20–100‐fold more potent than glutamate in supporting hsp expression, while other amino acids were less effective or supported no detectable hsp synthesis in heat shock. Inhibition of glutamine synthetase with methionine‐sulfoximine resulted in very low hsp expression with glutamate and normal high level expression with glutamine, confirming the importance of glutamine. The absence of glucose and treatment with 2‐deoxyglucose did not change the requirement for adequate glutamine for hsp expression. Cells heat shocked under conditions which gave very low hsp expression resumed growth when returned to normal medium as well as cells which expressed normal levels of hsps. Measurements of free amino acid levels in cells heat shocked in the presence and absence of glutamine showed a correlation between glutamine levels and amount of hsp expression. We conclude that a physiological process regulated by glutamine or a glutamine metabolite is important for normal hsp expression in heat shock conditions in Drosophila.
Voltage‐dependent calcium channels regulate GH4 pituitary cell proliferation at two stages of the cell cycle
Authors: Ramsdell John S.
Published: February 2005

Calcium is an intracellular signal implicated in the regulation of cell proliferation. We have examined the growth regulatory role of voltage‐dependent calcium channels (VDCC) in a rat pituitary cell line (GH4C1) that expresses two well‐characterized VDCC subtypes (L and T) and is growth‐inhibited by several agents known to enhance calcium entry. Thyrotropin‐releasing hormone (TRH), tetrade‐canoylphorbol acetate (TPA), and epidermal growth factor (EGF), each known to enhance calcium entry in GH4 cells, decrease GH4 cell number and incorporation of [3H]‐thymidine. The growth inhibitory action of these agents is cytostatic with a predominant effect to block G1 cells from entering S‐phase. We next examined the growth regulatory action of pharmacologic agents that interact directly and specifically with type L VDCC. Activation of type L VDCC with the dihydropyridine BAY K8644 inhibits GH4 proliferation as measured by cell number and [3H]‐thymidine incorporation. This action of BAY K8644 is enhanced by a submaximal K+‐maintained depolarization, and the growth inhibitory action of these agents is also cytostatic as evident by the block of G1 cells from entering S‐phase. Nimodipine, an antagonist specific for type L VDCC blocks (IC50 = 30 nM) BAY K8644‐inhibited cell proliferation by substantially reducing the S‐phase block. Taken together these findings indicate that calcium entry through type L VDCC inhibits GH4 cell proliferation by blocking entry into S‐phase. By contrast, nimodipine caused only a small reversal of the TRH‐induced S‐phase block, suggesting that TRH inhibits proliferation by a mechanism that differs at least in part from L‐channel activation. Unexpectedly, nimodipine, given alone, caused a substantial inhibition of GH4 cell proliferation. This action of nimodipine was cytostatic, yet differed from calcium channel activators in that the percentage of S‐phase cells was unchanged whereas G2‐M‐phase cells increased with a parallel decrease in G1‐phase cells. Similar effects were also observed with other classes of calcium channel blockers. Taken together these results indicate that calcium entry through VDCC regulates GH4 cell proliferation differently cepending on the stage of the cell cycle. In G1‐phase cells, sustained entry of calcium through type L VDCC blocks entry into S‐phase. In G2‐M‐phase cells entry of calcium promotes progression through mitosis.
Regulation of glucose transport as well as glucose transporter and immediate early gene expression in 3T3‐L1 preadipocytes by 8‐bromo‐cAMP
Authors: Cornelius Peter , Marlowe Melissa , Call Kenneth , Pekala Phillip H.
Published: February 2005

In the present study we have examined the ability of 8‐bromoadenosine cyclic 3′, 5′‐phosphate (8‐bromo‐cAMP; the membrane permeant analog of cAMP which can activate protein kinase A) to mimic hormone action and stimulate glucose transport and glucose transporter (GLUT‐1) gene expression as well as the expression of several growth‐related protooncogenes in quiescent 3T3‐L1 fibroblasts. 8‐Bromo‐cAMP induced a rapid and prolonged increase in the rate of hexose transport. Early activation of hexose transport (within 30 min) was associated with increased plasma membrane immunoreactive glucose transporters, which corresponded to a doubling in the number of D‐glucose‐displaceable, plasma membrane cytochalasin B binding sites. The time course for 8‐bromo‐cAMP‐induced hexose transport preceded the accumulation of GLUT‐1 mRNA, which peaked between 4 and 8 h after exposure to the agent, and subsequently declined to approach basal (control) levels. Expression of the immediate‐early genes c‐fos and jun‐B was induced by 8‐bromo‐cAMP on a rapid, but sustained time course, whereas induction of c‐jun expression was delayed. Alterations in specific mRNAs following exposure to 8‐bromo‐cAMP were due to increased gene transcription (as judged by nuclear transcription run‐on assays), although with respect to GLUT‐1, an increase in mRNA stability was also observed. Treatment of the cells with forskolin resulted in the induction of GLUT‐1 expression as well as expression of the immediate early genes. Exposure of quiescent 3T3‐L1 fibroblasts to 8‐bromo‐cAMP resulted in a substantial increase in rates of total protein and RNA synthesis, but had little effect on DNA synthesis. The results demonstrate that 8‐bromo‐cAMP initiated a G0/G1 transition, but did not permit progression into S‐phase. The results further suggest that increased cytosolic cAMP results in the stimulation of glucose transport by three distinct mechanisms to include translocation of pre‐existing transporters, increased transcription of the GLUT‐1 gene and increased stability of GLUT‐1 mRNA.
Regulation of motility in bovine brain endothelial cells
Authors: Rosen Eliot M. , Jaken Susan , Carley William , Luckett Peter M. , Setter Eva , Bhargava Madhu , Goldberg Itzhak D.
Published: February 2005

Scatter factor (SF) is a fibroblast‐derived cytokine which stimulates motility of epithelial and vascular endothelial cells. We used a quantitative assay based on migration of cells from microcarrier beads to flat surfaces to study the regulation of motility in bovine brain endothelial cells (BBEC). Peptide growth factors (EGF, ECGF, basic FGF) did not stimulate migration. Tumor promoting phorbol esters (PMA, PDD) markedly stimulated migration, while inactive phorbol esters (4a‐PDD, phorbol‐13,20‐diacetate) did not affect migration. Both SF‐ and PMA‐stimulated migration were inhibited by (1) TGF‐beta; (2) protein kinase inhibitors (e.g., staurosporine, K‐252a); (3) activators of the adenylate cyclase signaling pathway (e.g., dibutyryl cyclic AMP, theophylline); (4) cycloheximide; and (5) anti‐cytoskeleton agents (e.g., cytochalasin B, colcemid). However, PMA and SF pathways were distinguishable: (1) PMA induced additional migration at saturating SF concentrations; (2) the onset of migration‐stimulation was immediate for PMA and delayed for SF; and (3) down‐modulation of protein kinase C (PKC) ablated PMA but not SF responsiveness. Assessment of PKC by (3H)‐phorbol ester (PDBu) binding and by immunoblot showed (1) scatter factor does not cause significant redistribution or down‐modulation of PDBu binding or alpha‐PKC; and (2) PDBu mediates redistribution and down‐modulation of both binding and alpha‐PKC. These findings suggest two pathways for BBEC motility: a PKC‐dependent pathway and an SF‐stimulated/PKC‐independent pathway.
Effect of transforming growth factor beta‐1 on ovine satellite cell proliferation and fusion
Authors: Hathaway Marcia R. , Hembree Joan R. , Pampusch Mary S. , Dayton William R.
Published: February 2005

We have evaluated the effect of transforming growth factor beta‐1 (TGF beta‐1) on proliferation and fusion of cultured ovine satellite cells isolated from 5‐month‐old wether lambs. The isolation and culture protocols were validated by clonal analysis of the original cell preparation and assessment of proliferation and fusion of control cultures. Approximately 85% of the original cells isolated were myogenic as assessed by clonal analysis. The ovine cells doubled approximately every 18 hours during their exponential growth period and achieved a maximum percent fusion of 39.5% after 144 hours in culture. TGF beta‐1 inhibited fusion of these cells in a dose‐dependent manner with half‐maximal inhibition occurring at.08 ng/ml. Maximal inhibition (95% suppression) occurred between.1 and.5 ng/ml. TGF Beta‐1 (.05–3.0 ng/ml) did not inhibit proliferation of cultured ovine satellite cells in serum‐containing medium or in serum‐free defined medium. In contrast, TGF beta‐1 did significantly suppress serum‐stimulated proliferation of either porcine or bovine satellite cells that were isolated by using a procedure identical to that used to isolate the ovine satellite cells. Thus, proliferation of ovine satellite cells appears to respond differently to TGF beta‐1 than does proliferation of either porcine or bovine satellite cells.
Multiple signal transduction pathways lead to extracellular ATP‐stimulated mitogenesis in mammalian cells: II. A pathway involving arachidonic acid release, prostaglandin synthesis, and cyclic AMP accumulation
Authors: Huang Ning‐Na , Wang Ding‐Ji , Gonzalez Fernando , Heppel Leon A.
Published: February 2005

We have previously shown that extracellular ATP acts as a mitogen via protein kinase C (PKC)‐dependent and independent pathways (Wang, D., Huang, N., Gonzalez, F.A., and Heppel, L.A. Multiple signal transduction pathways lead to extracellular ATP‐stimulated mitogenesis in mammalian cells. I. Involvement of protein kinase C‐dependent and independent pathways in the mitogenic response of mammalian cells to extracellular ATP. J. Cell. Physiol., 1991). The present aim was to determine if metabolism of arachidonic acid, resulting in prostaglandin E2 (PGE2) synthesis and elevation of cAMP levels, plays a role in mitogenesis mediated by extracellular ATP. Addition of ATP caused a marked enhancement of cyclic AMP accumulation in 3T3, 3T6, and A431 cells. Aminophylline, an antagonist of the adenosine A2 receptor, had no effect on the accumulation of cyclic AMP elicited by ATP, while it inhibited the action of adenosine. The accumulation of cyclic AMP was concentration dependent, which corresponds to the stimulation of DNA synthesis by ATP. The maximal accumulation was achieved after 45 min, with an initial delay period of about 15 min. That the activation of arachidonic acid metabolism contributed to cyclic AMP accumulation and mitogenesis stimulated by ATP in 3T3, 3T6, and A431 cells was supported by the following observations: (a) extracellular ATP stimulated the release of [3H]arachidonic acid and PGE2 into the medium; (b) inhibition of arachidonic acid release by inhibitors of phospholipase A2 blocked PGE2 production, cyclic AMP accumulation, and DNA synthesis activated by ATP, and this inhibition could be reversed by adding exogenous arachidonic acid; (c) cyclooxygenase inhibitors, such as indomethacin and aspirin, diminished the release of PGE2 and blocked cyclic AMP accumulation as well as [3H]thymidine incorporation in response to ATP; (d) PGE2 was able to restore [3H]thymidine incorporation when added together with ATP in the presence of cyclooxygenase inhibitors; (e) pertussis toxin inhibited ATP‐stimulated DNA synthesis in a time‐and dose‐dependent fashion as well as arachidonic acid release and PGE2 formation. Other evidence for involvement of a pertussis toxin‐sensitive G protein(s) in ATP‐stimulated DNA synthesis as well as in arachidonic acid release is presented. In A431 cells, the enhancement of arachidonic acid and cyclic AMP accumulation by ATP was partially blocked by PKC down‐regulation, implying that the activation of PKC may represent an additional pathway in ATP‐stimulated metabolism of arachidonic acid. In all of these studies, ADP and AMP‐PNP, but not adenosine, were as active as ATP. In summary, the data support a role for arachidonic acid metabolism in ATP‐dependent DNA synthesis in 3T3, 3T6, and A431 cells.
Phosphorylation of HSP27 during development and decay of thermotolerance in Chinese hamster cells
Authors: Landry Jacques , Chrétien Pierre , Laszlo Andrei , Lambert Herman
Published: February 2005

The small molecular weight heat shock protein HSP27 was recently shown to confer a stable thermoresistant phenotype when expressed constitutively in mammalian cells after structural gene transfection. These results suggested that HSP27 may also play an important role in the development of thermotolerance, the transient ability to survive otherwise lethal heat exposure after a mild heat shock. In Chinese hamster O23 cells increased thermoresistance is first detected at 2 h after a triggering treatment of 20 min at 44°C, attains a maximum at 5 hours, and decays thereafter with a half‐life of 10 h. We found that the development and decay of transient thermotolerance cannot be solely explained on the basis of changes in the cellular concentration of HSP27. The cellular HSP27 concentration is not increased appreciably at 2 h after heat shock and attains a maximum at 14 h. Similar results were obtained in the case of another heat shock protein, HSP70. HSP70 follows slightly faster kinetics of accumulation (peaks at 10 h) and decays much more rapidly (t1/2 = 4h) than HSP27 (t1/2 = 13h). HSP27 has 3 isoelectric variants A, B, and C of which B and C are phosphorylated. In cells maintained at normal temperature, HSP27A represents more than 90% of all HSP27. Shifting the cell culture temperature from 37 to 44°C induces the incorporation of 32P into the more acidic B and C forms, a process that occurs very rapidly since the reduction in the concentration of the A form and a corresponding increase in the level of B and C is detectable by immunoblot analysis within 2.5 min at 44°C. Analyses performed at various times during development and decay of transient thermo‐tolerance revealed a close relationship between the effect of heat shock on HSP27 phosphorylation and cell ability to survive. For example, fully thermotolerant cells (5 h post‐induction) are refractory to induction of HSP27 phosphorylation by a 20‐min heat shock. The induction of HSP27 phosphorylation was also studied in a family of clonal cell lines of O23 cells that are thermoresistant as a result of the constitutive expression of a transfected human HSP27 gene. In these thermore‐sistant cells, phosphorylation of the endogenous hamster HSP27 is induced to a level comparable to that found in the thermosensitive parental cells. However, phosphorylation of the exogencus human protein, which represents more than 80% of total HSP27 in these cells, was much less induced. Although different mechanisms are involved in the maintenance of a high level of unphosphorylated HSP27 in induced‐thermotolerant and thermoresistant transfectant O23 cells, our results suggest that the degree of HSP27 phosphorylation could be a determinant of the ability of cells to survive hyperthermia.
Histamine inhibits cell spreading and C3bi receptor clustering and diminishes hydrogen peroxide production by adherent human neutrophils
Authors: Francis Joseph W. , Todd Robert F. , Boxer Laurence A. , Petty Howard R.
Published: February 2005

Cell adherence plays a central role in many host defense mechanisms. Human peripheral blood neutrophils possess cell surface receptors that contribute to cell adherence or detachment. Receptors specific for the C3bi cleavage fragment of the third component of complement (CR3) promote adhesion, whereas histamine receptors promote detachment. In the present study, we tested the ability of histamine to down‐regulate the physiological effects of CR3 receptors. Histamine decreased the binding of 51Cr‐labeled neutrophils to complement‐coated surfaces (C3‐coated surfaces) in a dose‐dependent fashion. Scanning electron microscopic and optical microscopic observations of neutrophils on C3‐coated surfaces revealed polarized or spherical cell morphologies in the absence or presence of histamine, respectively. Histamine inhibited the ability of CR3 to cluster on plasma membranes of neutrophils adherent to C3‐coated surfaces as shown by fluorescence microscopy. In addition, histamine diminished but did not abolish the FMLP‐stimulated increase in plasma membrane CR3 expression as measured by fluorometry. Histamine did not inhibit the release of marker proteins from specific or gelatinase containing granules by neutrophils in suspension. Histamine also diminished the FMLP‐stimulated production of respiratory burst oxidants from cells in suspension or cells allowed to adhere to fibrinogen substrates. We suggest that histamine may modulate selective changes in neutrophil function by diminishing adherence and preventing changes in cell shape following cell activation.