bluuebunny/crossref_metadata_2025 huggingface dataset:
Epidermal growth factor: Modulator of murine embryonic palate mesenchymal cell proliferation, polyamine biosynthesis, and polyamine transport
Authors: Gawel‐Thompson Kathleen J. , Greene Robert M.
Published: February 2005

Polyamines (putrescine, spermidine, and spermine) are normal cellular constituents able to modulate cellular proliferation and differentiation in a number of tissues and cell types. This investigation explores the response of murine embryonic palate mesenchymal (MEPM) cells to epidermal growth factor (EGF) in terms of biosynthesis of putrescine and its transport across the plasma membrane and tests the hypothesis that polyamine transport can serve as an alternative mechanism (other than biosynthesis) for elevating intracellular polyamines during stimulation of MEPM cellular proliferation. MEPM cells treated with EGF were stimulated to proliferate and showed a dose‐ and time‐dependent stimulation of ornithine decarboxylase (ODC) which was maximal at 4‐6 hours. EGF also stimulated the initial rate of putrescine transport in dose‐ and time‐dependent manner. This stimulation was found to be maximal 3 hours after treatment and specific for the putrescine transport system. The kinetic parameters of putrescine transport shifted from 2.52 μM (Km) and 23.6 nmol/mg protein/15 minutes (Vmax) in nonstimulated cells to 4.48 μM (Km) and 39.8 nmol/mg protein/15 minutes (Vmax) in EGF‐treated cells. This kinetic shift did not require de novo protein or RNA synthesis, as cycloheximide (10 μg/ml) and actinomycin D (50 μg/ml) had little effect on the ability of EGF to stimulate the initial rate of putrescine uptake. The rate of transport, however, was found to be inversely related to cell density. The addition of exogenous putrescine concomitantly with EGF blocked the induction of ODC, while in the presence of difluoromethylornithine (DFMO) (irreversible inhibitor of ODC) the initial rate of putrescine transport remained elevated throughout the time course studied. This stimulation of putrescine uptake caused by polyamine deprivation was reversed by exogenous putrescine and Ca+ + while α‐aminoisobutryic acid (AIB) further stimulated the rate of uptake. EGF's ability to stimulate cellular DNA synthesis was inhibited by DFMO. If DFMO‐treated cells were stimulated with EGF in the presence of exogenous putrescine, this stimulatory effect was preserved. These studies indicate that the rate of polyamine transportation is highly responsive to a signal which initiates biosynthesis of polyamines. Further, this transportation system provides a compensatory mechanism allowing the cell to increase intracellular levels of polyamines when environmental conditions inhibit biosynthesis or when polyamines are abundant.
Protein kinase C activation and down‐regulation in relation to phorbol ester‐induced differentiation of SH‐SY5Y human neuroblastoma cells
Authors: Heikkilä Jari E. , Åkerlind Göran , Åkerman Karl E. O.
Published: February 2005

The role of protein kinase C activation in changes in muscarinic receptor functions and in the appearance of biochemical properties characteristic of neuronal cells was studied in SH‐SY5Y human neuroblastoma cells induced to differentiate with the phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA). A decrease in muscarinic receptor sensitivity with respect to agonist induced Ca2+ mobilization and receptor number parallelled the increase in membrane‐associated protein kinase C (PK‐C) activity. These changes occurred during the first 6 h of culture, and they were associated with rounding‐up of cells. A subsequent decrease in particulate PK‐C activity was followed by an increase in noradrenaline content, the appearance of an electrically excitable membrane, and an increase in the level of neuron‐specific enolase. These changes were accompanied by a pronounced neurite outgrowth. 1‐(5‐lsoquinolinesulphonyl)‐2‐methylpiperazine (H‐7), an inhibitor of PK‐C and cyclic nucleotide‐dependent protein kinases, enhanced the morphological differentiation induced by TPA, whereas N‐(2‐guanidinoethyl)‐5‐isoquinolinesulphonamide (HA‐1004), which primarily inhibits cyclic nucleotide‐dependent protein kinases, had no effect on the TPA‐induced phenotypic differentiation. H‐7 inhibited the decrease in muscarinic receptor sensitivity and receptor number, but had no effect on the appearance of the electrically excitable membrane or on the increase in the neuron‐specific enolase level. Both H‐7 and HA‐1004 inhibited the TPA‐induced increase in noradrenaline content.
Transforming growth factor‐beta and fibroblast growth factor act synergistically to inhibit collagen II synthesis through a mechanism involving regulatory DNA sequences
Authors: Horton Walter E. , Higginbotham Jill D. , Chandrasekhar Srinivasan
Published: February 2005

Transforming growth factor‐beta (TGF‐β) and fibroblast growth factor (FGF) are two growth factors that will modulate chondrocyte growth and matrix synthesis. Here we report that these two growth factors act in a synergistic fashion to suppress the synthesis of type II collagen by embryonic chicken sternal chondrocytes. Treatment of chondrocytes with 20 ng/ml TGF‐β or 100 ng/ml FGF (acidic or basic) results in a 60–70% suppression of expression of the pro α1 chain of type II collagen. By comparison, when chondrocytes are exposed to a combination of 1 ng/ml TGF‐β and 10 ng/ml FGF, a complete suppression of type II collagen synthesis was observed. Epidermal growth factor (EGF), platelet‐derived growth factor (PDGF), or insulin‐like growth factor‐1 (IGF‐1) produce no suppression of synthesis either individually or in combination with TGF‐β. The decreased expression of the protein results from a decrease in the steady‐state level of the mRNA transcript coding for type II procollagen, as indicated by a northern analysis. Finally, chondrocytes transfected with a plasmid carrying the CAT gene driven by the collagen II promoter/enhancer sequence displayed high levels of CAT activity when cultured in control media, but treatment of the cells with a combination of the two growth factors resulted in a dramatic reduction of CAT activity, indicating diminished promoter activity. These results suggest that both TGF‐β and FGF can down‐regulate transcription of the collagen II gene through regulatory DNA sequences in the promoter and/or enhancer region. In addition, the finding of synergy suggests that these two growth factors may act through different pathways.
Modulation of growth of human carcinoma SW‐13 cells by heparin and growth factors
Authors: Halper Jaroslava , Carter Bobbie J.
Published: February 2005

This study reports on the effects of heparin, basic and acidic fibroblast growth factors (bFGF and aFGF, respectively), and transforming growth factor type‐e (TGFe) on the growth of a human adrenocortical carcinoma cell line, SW‐13. Heparin has previously been shown to inhibit growth in several cell types, including smooth muscle cells, certain fibroblasts, and epithelial cells, and to modulate the effects of fibroblast growth factors. Whereas bFGF and aFGF bind tightly to heparin and elute from a heparin‐Sepharose column with 2 M NaCl and 1.6 M NaCI, respectively, TGFe binds to heparin with lower affinity and can be eluted from heparin‐Sepharose column with 0.5 M NaCI. TGFe is a polypeptide unrelated to FGF, is present in neoplastic and nonneoplastic tissues, and stimulates the growth of certain epithelial cells and fibroblasts in soft agar and monolayer. Since the growth of SW‐13 cells is stimulated by TGFe and by bFGF, we hypothesized that heparin would inhibit the growth of SW‐13 cells by binding to these growth factors and that the effects of heparin could be overcome with the addition of either growth factor. Our experiments confirmed that heparin inhibits the growth of SW‐13 cells. A dose‐dependent growth inhibition was observed in both monolayer and soft agar. The inhibition in monolayer was partially reversed upon heparin withdrawal. The effects of heparin in both monolayer and soft agar were at least partially overcome by TGFe and by basic or acidic FGF. Overall protein synthesis does not appear to be affected by heparin as measured by [35S]methionine uptake. In contrast, epidermal growth factor (EGF) and insulin‐like growth factor I (IGF‐I) were unable to overcome heparin‐induced inhibition both in monolayer and in soft agar. Heparin also inhibited [3H]thymidine incorporation in AKR‐2B and partially inhibited AKR‐2B cell stimulation by TGFe; however, it further potentiated the already potent stimulation by bFGF. We propose that heparin, TGFe, bFGF, and aFGF modulate the growth of SW‐13 cells and possibly of other epithelial cells in complex ways and that heparin‐like substances present in the extracellular matrix play an important role in the control of epithelial growth.
ATP hydrolysis by ischemic Mitochondria
Authors: Classen J. Barthelow , Mergner Wolfgang J. , Costa M.
Published: February 2005

Cellular ATP levels are determined by the rates of ATP production and ATP hydrolysis. Both phenomena are affected by ischemia. Mitochondrial enzymes are damaged, inhibiting this organelle's ability to make ATP. Mitochondria are also uncoupled by ischemia and have the ability to hydrolyze ATP. We designed a series of experiments to determine whether decreased production or increased hydrolysis of ATP was the primary effect of mitochondrial damage. Rat hearts were subjected to 45 min of warm ischemia in order to induce irreversible cell damage. ATP or ADP was injected into cuvettes containing mitochondria isolated from normal myocardium or myocardium damaged by ischemia. Luciferin‐luciferase, which fluoresces in the presence of ATP, was also added to the tubes as an indicator of ATP levels. Mixtures of uncoupled and coupled mitochondria were made and compared with the mitochondria damaged by ischemia. The results showed that mitochondria damaged by prolonged ischemia hydrolyze ATP more rapidly than normal mitochondria; however, normal mitochondria can easily compensate for increased ATP hydrolysis when in mixture with equal amounts of uncoupled mitochondria. These data suggests that the low cellular levels of ATP following irreversible ischemia are primarily due to decreased ATP synthesis and not to increased hydrolysis.
Heat shock stimulates the release of arachidonic acid and the synthesis of prostaglandins and leukotriene B4 in mammalian cells
Authors: Calderwood Stuart K. , Bornstein Bruce , Farnum Elizabeth K. , Stevenson Mary Ann
Published: February 2005

Heat shock has a profound influence on the metabolism and behavior of eukaryotic cells. We have examined the effects of heat shock on the release from cells of arachidonic acid and its bioactive eicosanoid metabolites, the prostaglandins and leukotrienes. Heat shock (42–45°) increased the rate of arachidonic acid release from human, rat, murine, and hamster cells. Arachidonate accumulation appeared to be due, at least partially, to stimulation of a phospholipase A2 activity by heat shock and was accompanied by the accumulation of lysophosphatidyl‐inositol and lysophosphatidylcholine in membranes. Induction of arachidonate release by heat did not appear to be mediated by an increase in cell Ca+ +. Stimulation of arachidonate release by heat shock in hamster fibroblasts was quantitatively similar to the receptor‐mediated effects of β thrombin and bradykinin. The effects of heat shock and β thrombin on arachidonate release were inhibited by glucocorticoids. Increased arachidonate release in heat‐shocked cells was accompanied by the accelerated accumulation of cyclooxygenase products prostaglandin E2 and prostaglandin F2α and by 5‐lipoxygenase metabolite leukotriene B4. Elevated concentrations of arachidonic acid and metabolites may be involved in the cytotoxic effects of hyperthermia, in homeostatic responses to heat shock, and in vascular and inflammatory reactions to stress.
Heterogeneity of the changes in cytoplasmic pH upon serum stimulation of quiescent fibroblasts
Authors: Bright Gary R. , Whitaker James E. , Haugland Richard P. , Taylor D. Lansing
Published: February 2005

Addition of mitogens to quiescent cells results in rapid ionic changes in the cytoplasm, including pH. We studied the changes in cytoplasmic pH in single Swiss 3T3 cells upon serum stimulation using fluorescence ratio imaging microscopy. Quiescence was attained using two approaches, serum deprivation of subconfluent cells and confluence. All measurements were made in the presence of bicarbonate and the absence of other organic buffers. We also used BCECF coupled to dextran to avoid several artifacts associated with using BCECF‐AM, including leakage and phototoxicity. Analysis of the changes in cytoplasmic pH demonstrated a dramatic heterogeneity in the responses of single cells. There were six basic classes of responses, (1) a fast alkalinization, reaching a maximum pH in ∼2–5 min; (2) a slow alkalinization, reaching a maximum pH in 10‐20 min; (3) a very slow alkalinization, not reaching a plateau pH within the measurement time; (4) no apparent change in pH during the measurement time; (5) an early transient acidification, followed by either a fast or slow alkalinization; and (6) an acidification, followed by alkalinization and then by a decrease to some intermediate pH. Subconfluent cells exhibited greater heterogeneity in response than confluent cells, with no single dominant class of response. The dominant (55%) response for confluent cells was a gradual alkalinization of ∼0.01 pH units/min. A larger proportion (52%) of subconfluent cells exhibited an early transient acidification compared to confluent cells (7%). A significant proportion of both types of cells (23% subconfluent, 36% confluent) exhibited no change in cytoplasmic pH upon stimulation. In general, the kinetics of changes in cytoplasmic pH were significantly different from the published results with population averaging methods.
Cell‐type‐specific expression of mouse DNA polymerase β‐gene is regulated by silencer elements
Authors: Yamaguchi Masamitsu , Hayashi Yuko , Ajiro Kozo , Matsukage Akio
Published: February 2005

RNA blot hybridization analysis revealed that the steady‐state level of DNA polymerase β‐mRNA in mouse neuroblastoma N18TG2 cells was approximately fivefold higher than that in NIH/3T3 cells. In order to examine the function of DNA polymerase β‐gene silencers in these two cell lines, we employed a chloramphenicol acetyltransferase (CAT)‐transient expression assay using the CAT plasmids containing the silencers linked to various promoter‐enhancers. In NIH/3T3 cells, DNA polymerase β‐gene silencers effectively repressed the function of its own promoter and those of several other heterologous promoter‐enhancers. In contrast, the silencers only marginally affected the CAT expression directed by DNA polymerase β‐gene promoter and heterologous promoter‐enhancers in N18TG2 cells. The extent of the increase of CAT expression by removing silencer elements in NIH/3T3 cells was very similar to the ratio of DNA polymerase β‐mRNA content in N18TG2 cells to that in NIH/3T3 cells. These results indicate that cell‐type‐specific expression of DNA polymerase β‐gene is primarily controled by the function of its silencer elements.
Differential response among cells in the chick embryo myogenic lineage to photosensitization by merocyanine 540
Authors: Nameroff Mark , Rhodes Linda D.
Published: February 2005

Primary cultures of myogenic cells from progressively older embryonic and adult chickens were incubated in medium containing Merocyanine 540 (MC540) and were exposed to white light during the incubation period. After exposure, the cultures were followed to determine cell survival and differentiation. MC540 attached to the surface membranes of all cells. In cultures from 10‐day embryos (E10 cells), concentrations of MC540 ≥ 60 μg/ml resulted in death of nearly all myogenic cells upon exposure to light, but non‐myogenic cells survived and replicated. Below 60 μg/ml, there was a dose‐dependent reduction in muscle differentiation. At concentrations <40 μg/ml, there was no effect on myogenesis. Cultures of cells from 18‐day (E18) embryos (myogenic stem cells) and from adult muscle (satellite cells) were resistant to doses of MC540 that killed E10 cells. E14 myogenic cell populations contained both resistant and sensitive sub‐populations. Terminally differentiated muscle cells were more sensitive to MC540 than precursor cells from any age embryo. Progeny of E18 cells acquired sensitivity to MC540 as differentiation proceeded. In clonal cultures, cells that normally give rise to small muscle clones (committed cells) were selectively destroyed by exposure to the dye. These observations demonstrate that an MC540‐resistant myogenic population is present in low numbers in 10‐day embryonic pectoral muscle. As development proceeds, this population increases such that, by 18 days of gestation, most of the myogenic cells are resistant to MC540. The results also suggest that embryonic chick myogenic stem cells and adult satellite cells have surface membrane properties which differ from those of their committed progeny.
Preadipocyte stimulating factor in rat serum: Evidence for a discrete 63 kDa protein that promotes cell differentiation of rat preadipocytes in primary cultures
Authors: Li Zhen‐Hua , Lu Zhongding , Kirkland James L. , Gregerman Robert I.
Published: February 2005

In primary cultures of rat preadipocytes (PA) isolated from epididymal or perirenal depots, rat serum is more effective than other animal sera (fetal calf, newborn calf, human, horse, rabbit, cat, sheep, goat, dog, pig) in promoting adipogenic conversion, biochemical differentiation, and mitogenesis. Only mouse serum is comparable to rat serum. This activity is attributable to a specific growth factor (pre‐adipocyte stimulating factor, PSF). An assay for PSF in rat serum was devised using PA from perirenal fat of 3‐month‐old Fischer 344 rats grown first to confluence in FCS for 8 days and then for the next 3 days in test serum, followed by measurement of triglyceride (TG) and glycerol‐3‐phosphate dehydrogenase (GPDH). Rat serum induces dose‐dependent rapid cell division, which coincides with accumulation of TG and increase of GPDH; for routine quantitation, TG is assayed. The biochemical characteristics of PSF in serum are as follows: stable at 4°C for up to 1 year; inactivated at 100°C (80% loss, 30 min) but stable at 56°C for 1 hr; stable at pH 2–12; non‐dialyzable; completely resistant to pepsin, trypsin, and chymotrypsin but destroyed by pronase and subtilisin; stable to DTT and periodate; and m.w. between 68 kDa (Sephacryl‐300) and 58 kDa (Sephacryl‐300 in 5 M urea). PSF activity is greater in serum from Wistar than from Fischer 344 rats, while activity of serum from Zucker obese (fa/fa) rats is at least as great as that from Wistar rats and, like serum of rats made obese by feeding a high‐fat, high‐carbohydrate diet, is not suppressed. PSF activity is not due to insulin, insulin‐like growth factor‐1 (IGF‐1), growth hormone, glucocorticoids, or combinations of these hormones. PSF activity was not seen with a number of growth factors including colony‐stimulating factor (CSF‐1), GM‐CSF, interleukins 1, 2, and 3, neuroleukin, tumor necrosis factor, and others. PSF is distinct from the low molecular weight (4–8 kDa) differentiation factor present in rat serum, FCS, and human serum that promotes the adipogenic conversion and cellular differentiation of 3T3‐L1, 3T3‐F442A, and Ob17 cells. PSF appears to be a new differentiation factor for rat preadipocytes, has properties suggestive of a highly glycosy‐lated protein, and may be highly species specific.
GH4 pituitary cell variants selected as nonresponsive to thyrotropin‐releasing hormone‐enhanced substratum adhesion are nonresponsive to epidermal growth factor: Evidence for a common signaling defect
Authors: Ramsdell John S. , Tashjian Armen H.
Published: February 2005

Thyrotropin‐releasing hormone (TRH) and epidermal growth factor both enhance prolactin synthesis and substrate adhesion (a morphological change called stretching) of GH4 rat pituitary cells. We have examined TRH‐ and EGF‐induced cell stretching using genetic and pharmacologic approaches. We selected and isolated a series of GH4 cell variants nonresponsive to TRH‐induced cell stretching (str−). This selection yielded several variants that were nonresponsive to both TRH‐ and EGF‐induced stretching but were still responsive to stretching induced by several other agents (tetradecanoylphorbol acetate [TPA], butyrate, and Nepla‐nocin A). One of the str− variants (a14) was examined in detail. TRH, EGF, and TPA each enhanced prolactin synthesis in a14 cells, indicating that the a14 variant contained functional receptor binding sites for all 3 ligands as well as the capacity to generate those intracellular signals required for enhanced prolactin synthesis. Because the str− variants were isolated without selective pressure for EGF‐induced stretching and because the possibility of more than one selectable mutation in all the variants is unlikely, we suggest that TRH and EGF share a common mechanism to induce cell stretching. We next examined whether the str− variants had a defect in a signaling pathway or in the biochemical endpoint for TRH‐ and EGF‐induced cell stretching. A pharmacologic approach was utilized to investigate the biochemical basis for induced cell stretching. A synthetic Arg‐Gly‐Asp‐Ser tetrapeptide (RGDS), specific for fibronectin and vitronectin adhesion receptors, inhibited TRH‐, EGF‐, and TPA‐induced GH4 cell stretching and attachment to fibronectin‐ and vitronectin‐coated dishes. These results suggest that the interaction between fibronectin and/or vitronectin and their receptor(s) may be a biochemical endpoint by which several agonists induce stretching of GH4 cells. Because the str− variant has RGDS‐specific binding sites for fibronectin and vitronectin and responds to some agents that induce cell stretching via an RGDS receptor, we conclude that the a14 str− variant has a defect in an intracellular signaling pathway, shared by TRH and EGF, which induces cell stretching.
Characterization of neutral and cationic amino acid transport in Xenopus oocytes
Authors: Campa Michael J. , Kilberg Michael S.
Published: February 2005

Amino acid transport was characterized in stage 6 Xenopus laevis oocytes. Most amino acids were taken up by the oocytes by way of both Na+‐dependent and saturable Na+‐independent processes. Na+‐dependent transport of 2‐aminoisobutyric acid (AIB) was insensitive to cis‐ or trans‐inhibition by the System A‐defining substrate 2‐(methylamino)‐isobutyric acid (MeAIB), although threonine, leucine, and histidine were found to be effective inhibitors, eliminating greater than 80% of Na+‐dependent AIB uptake. Lack of inhibition by arginine eliminates possible mediation by System Bo,+ and suggests uptake by System ASC. The Na+‐dependent transport of characteristic System ASC substrates such as alanine, serine, cysteine, and threonine was also insensitive to excess MeAIB. Evidence to support the presence of System Bo, + was obtained through inhibition analysis of Na+‐dependent arginine transport as well arginine inhibition of Na+‐dependent threonine uptake. The Na+‐independent transport of leucine was subject to trans‐stimulation and was inhibited by the presence of excess phenylalanine, histidine, and, to a lesser extent, 2‐amino‐(2,2,1)‐bicycloheptane‐2‐carboxylic acid (BCH). These observations are consistent with mediation by System L. The characteristics of Na+‐independent uptake of threonine are not consistent with assignment to System L, and appear to be reflective of Systems asc and bo,+. In its charged state, histidine appears to be transported by a carrier similar in its specificity to System y+, but is taken up by System L when present as a zwitterion.
Agarose‐selected variants of two human tumor cell lines exhibit altered methionine auxotrophy
Authors: Liteplo Robert G. , Hipwell Sheridan E.
Published: February 2005

Our aim was to determine if the selection of human tumor cells with enhanced anchorage‐independent growth capacity was associated with alterations in methionine auxotrophy. Cells with an increased ability to form colonies on soft agarose were selected from human melanoma (MeWo) and neuroepithelioma (SK‐N‐MC) cell lines. In contrast to their respective parental lines, a high proportion of the agarose‐selected variants were completely unable to proliferate in methionine‐free medium containing its immediate precursor homocysteine. The variants exhibited no significant change in their total DNA 5‐methylcytosine content and showed no stimulation of either RNA or DNA synthesis upon the addition of homocysteine when the cells were cultured in methionine‐free medium. These variants were unable to synthesize [3H]S‐adenosylmethionine from [3H]adenine and homocysteine. The failure to detect the accumulation of [3H]S‐adenosylmethionine in these variant lines was not likely due to the enhanced turnover of S‐adenosylmethionine but rather to a reduced ability to synthesize methionine from homocysteine and 5‐methyltetrahydrofolic acid. These results support our hypothesis that alterations in the metabolism of methionine and/or intracellular transmethylating activities may contribute to, or be associated with, the autonomous growth of malignant human tumor cells.
Growth‐inhibiting effect of tumor necrosis factor on human umbilical vein endothelial cells is enhanced with advancing age in vitro
Authors: Shimada Yoshiya , Kaji Kazuhiko , Ito Hideki , Noda Kouichi , Matsuo Mitsuyoshi
Published: February 2005

We have examined the effects of in vitro aging on the growth capacity of human umbilical vein endothelial cells (HUVECs) under the influence of tumor necrosis factor (TNF) with or without interferon‐γ (IFN‐γ). The growth and colony‐forming abilities of control cells were impaired with advancing age in vitro, especially at later stages (more than 70–80% of life span completed). It was found that treatment with TNF inhibited growth and colony‐forming efficiency at any in vitro age. The effects of TNF were shown to increase with increasing in vitro age, as reflected by a more pronounced increase in doubling times, a decrease in saturation density, and a reduction in colony‐forming efficiency. However, the characteristics of TNF receptors, including the dissociation constant, and the number of TNF‐binding sites per cell‐surface area remained rather constant. The effect of TNF was augmented by IFN‐γ at a dose that alone affected growth and colony formation only slightly. The augmentation by IFN‐γ was also found to depend on in vitro age; the synergy with TNF in the deterioration of colony‐forming ability was observed only in “aged” cells. These results suggest that the intrinsic responsiveness of HUVECs to growth‐inhibiting factors, as well as to growth‐stimulating factors, changes during aging in vitro.
Cholinergic desensitization of pepsinogen secretion and calcium mobilization of dispersed guinea pig chief cells
Authors: Cherner Jay A. , Naik Latika , Singh Gurcharn
Published: February 2005

When dispersed chief cells from guinea pig stomach were first incubated with carbachol, washed, and then reincubated with carbachol in fresh incubation solution, the stimulation of pepsinogen secretion and the rise in intracellular calcium concentration during the second incubation were reduced. Carbachol did not cause residual enzyme secretion, but the same range of concentrations that causes enzyme secretion caused desensitization that was rapid, temperature dependent, and reversible with time. Preincubation with carbachol caused approximately a 65% reduction in enzyme secretion stimulated during a subsequent incubation with this agonist, but the potency of carbachol was unaffected. Prior exposure to carbachol also reduced subsequent stimulation caused by cholecystokinin (CCK‐8), gastrin I, ionophore A23187, or 12‐O‐tetradecanoylphorbol 13‐acetate but did not alter stimulation by any agonist that increases cellular cAMP. Carbachol pretreatment of Fura‐loaded chief cells caused a threefold increase in the EC50 for carbachol‐stimulated [Ca2+]i and approximately a 30% reduction in the maximal rise in [Ca2+]i in response to carbachol or CCK‐8. Inhibition of [N‐methyl‐3H] scopolamine binding by carbachol following carbachol pretreatment indicated that modulation of receptor affinity or number did not account for functional desensitization. These data indicate that carbachol causes heterologous desensitization of pepsinogen secretion stimulated by agonists that mobilize cellular Ca2+ or activate protein kinase C through a postreceptor action and suggest that an attenuated rise in chief cell calcium is one mechanism mediating the desensitization of enzyme secretion.
Thrombin‐induced prostacyclin biosynthesis in human endothelium: Role of guanine nucleotide regulatory proteins in stimulus/coupling responses
Authors: Garcia Joe G. N. , Painter Richard G. , Fenton John W. , English Denis , Callahan Karleen S.
Published: February 2005

The regulation of prostacyclin (PGl2) synthesis by cultured human umbilical vein endothelium (HUVEC) was investigated. HUVEC monolayer generation of PGl2 was monitored by RIA of 6‐keto PGF1α and dose‐dependent increases observed with human α‐and γ‐thrombins, histamine, or arachidonate. Alpha thrombin (10 nM) produced levels of 6‐keto PGF1α approximating responses with 1 μM γ‐thrombin, 5μM arachidonate, or 10 μM histamine. Diisopropyl phosphorofluo‐ridate‐inactivated α‐thrombin did not stimulate PGl2 release, demonstrating that catalytic activity was required for thrombin‐stimulated PGl2 release. Sodium fluoride (NaF), at concentrations known to activate guanine nucleotide regulatory proteins (G proteins), directly stimulated HUVEC PGl2 synthesis in a dose‐dependent and time‐dependent manner (20 mM NaF, 4.4 ± 0.5‐fold increase at 10 min, 11.9 ± 1.5‐fold increase at 30 min). Neither α‐thrombin nor NaF‐stimulated PGl2 release was dependent upon the availability of extracellular Ca+ +. The hypothesis that G proteins are involved in agonist‐stimulated PGl2 synthesis was further supported by studies using digitonin‐permeabilized HUVEC monolayers challenged with another G protein activator, guanosine 5′‐0‐3‐thiotrisphosphate (GTP γ S), which effected significant dose‐dependent increases in PGl2 synthesis compared with control levels of 6‐keto PGFα. In contrast, the G‐protein inhibitor GDP β S, (guanosine 5′‐0‐2‐thiodiphosphate), attenuated α‐thrombin‐mediated prostaglandin generation. Treatment of HUVEC monolayers with pertussis toxin (1 μg/ml) did not inhibit the PGl2 synthesis stimulated by either α‐thrombin, NaF, or histamine but catalyzed the ADP ribosylation of a 40 kDa membrane protein which cross‐reacted with antisera against a synthetic peptide corresponding to an amino acid sequence common to the α‐subunit of other G‐proteins. Preincubation of HUVEC microsomal membranes with α‐thrombin diminished pertussis toxin‐catalyzed ADP ribosylation in a time‐dependent manner. These data suggest that thrombin stimulation of PGl2 synthesis by HUVEC monolayers requires the catalytically functional enzyme and further suggests that the thrombin‐occupied receptor is coupled to phospholipase activities by a pertussis toxin‐insensitive guanine nucleotide regulatory protein in human endothelial cell membranes.
Purification of human blood burst‐forming units‐erythroid and demonstration of the evolution of erythropoietin receptors
Authors: Sawada K. , Krantz S. B. , Dai C‐H. , Koury S. T. , Horn S. T. , Glick A. D. , Civin C. I.
Published: February 2005

To facilitate the direct study of the molecular events that control the development of human burst‐forming units‐erythroid (BFU‐E), we have developed a method to purify BFU‐E from peripheral blood. Using density centrifugation, rosetting with a mixture of neuraminidase‐treated and IgG‐coated sheep erythrocytes, positive panning with anti‐My10 monoclonal antibody, overnight adherence to plastic dishes, negative panning with monoclonal antibodies, and density centrifugation, human blood BFU‐E were purified from 0.04% to 56.6%, a 1,400‐fold purification with a 13% yield. More than 90% of purified BFU‐E were recombinant interleukin‐3 (rlL‐3) dependent, which survived for 48 h with rlL‐3 in the absence of recombinant erythropoietin (rEP), and 80% gave rise to erythroid bursts of more than 500 hemoglobinized cells. rEP dependency was not evident until after 72 h of incubation in vitro. The purified cells (day 1) were incubated with rlL‐3 and rEP in liquid culture for 24 (day 2), 48 (day 3), and 72 (day 4) h and then were transferred into semisolid cultures and incubated until day 15. The size of the erythroid colonies observed in semisolid cultures decreased continuously in association with the incubation time of day 1 purified cells in liquid cultures. The first appearance of colony‐forming units‐erythoid (CFU‐E) that gave rise to colonies of 8 to 49 cells was observed after 72 h of incubation of day 1 cells in the liquid culture. 125I‐rEP was incubated for 5 h at 37°C with purified cells (day 1) or with the cells that had been incubated in liquid culture for an additional 24‐72 h, and the presence of erythropoietin (EP) receptors was investigated using auto‐radiography. Specific binding of 125I‐rEP was detected in 19 ± 7% of the initial day 1 BFU‐E. The percentage of 125I‐rEP‐binding to erythroid progenitor cells and the amount of binding continuously increased as day 1 BFU‐E matured. 125I‐rEP specific binding was observed with all of the erythroid progenitor cells that had been incubated in liquid culture for 72 h. These data demonstrate that primitive BFU‐E have a much lower number of EP receptors than CFU‐E and develop an increased concentration of EP receptors in association with their maturation and loss of proliferative capacity.
Comparison of the ability of basic and acidic fibroblast growth factor to stimulate the proliferation of an established keratinocyte cell line: Modulation of their biological effects by heparin, transforming growth factor β (TGFβ), and epidermal growth factor (EGF)
Authors: Gospodarowicz D. , Plouët J. , Malerstein B.
Published: February 2005

The bioactivity of both bFGF and aFGF in the BALB/MK‐1 cell line has been compared to that of EGF. Our results indicate that, for that cell type, aFGF was far more potent than bFGF in inducing cell proliferation. In the presence of heparin, aFGF was as potent as EGF. In addition, excess bFGF has an inhibitory effect on the proliferation of MK cells exposed to a saturating concentration of aFGF, therefore acting as a partial agonist of aFGF. Surprisingly, bFGF, although it had low biological activity, was capable of synergizing the effect of EGF. In its presence, culturesexposed to saturating concentration of EGF have a final cell density 3‐to 4‐fold higher than that of counterpart cultures exposed to EGF alone. TGFβ, which in previous studies has been shown to inhibit the growth of keratinocytes, also inhibited the growth of BALB/MK‐1 cells in response to either bFGF or aFGF. These studies suggest a role for FGF in regulating BALB/MK proliferation. aFGF provides positive growth signals which can be negatively modulated by excess bFGF or TGFβ, while bFGF, although a poor mitogen, could act by potentiating the effect of subsaturating concentrations of EGF.
Inhibition of replicon cluster ligation into chromosomal DNA at elevated temperatures
Authors: Warters Raymond L. , Lyons Bradley W.
Published: February 2005

The rate limiting enzymatic step for DNA replication in HeLa cells incubated at 43.5°C v/as the ligation of clusters of replicons into the cell's genome. At 43.5° the reciprocal slope for inhibition of DNA chain (replicon) initiation, or of the ligation of replicon clusters into the genome, was 18 or 7 min, respectively. The failure of replicon clusters to be ligated into chromosomal DNA was not a consequence of the failure of histone proteins to be deposited onto replicating DNA, or of chromatin replicated at 43.5°C to be organized into fully condensed chro‐matin. In addition it was not due to the failure of fully active topoisomerase II to be deposited at a normal frequency along replicating chromatin DNA. The failure of replicon clusters to be ligated into the genome resulted in the persistence of single, but not double, DNA strand breaks in the cell's genome 24 hours after cell heating.
Enhanced glycosyltransferase activity during thermotolerance development in mammalian cells
Authors: Henle Kurt J. , Monson Thomas P. , Stone Angie
Published: February 2005

The cellular heat shock response leads to the enhanced synthesis of a family of heat shock proteins and the development of thermotolerance. In CHO cells, however, heat shock also leads to enhanced synthesis of a 50 kD glycoprotein and elevated activity of N‐acetylgalactosaminyltransferase (GalNAcT). In this study we showed increased GalNAcT activity during thermotolerance expression in all of five mammalian cell lines included in the study. However, there was no simple correlation between cellular heat sensitivity of unheated control cells and basal levels of GalNAcT activity, measured toward the same exogenous acceptor apomucin. Although GalNAcT was elevated in thermotolerant cells, GalNAcT activity itself did not exhibit thermotolerance in terms of reduced sensitivity to heat inactivation. The increase in GalNAcT activity after heating was similar in exponentially growing and plateau‐phase cultures and was inhibited neither by cycloheximide nor actinomycin D. However, the inhibitors by themselves also increased GalNAcT activity in unheated control cells. Chemical inducers of ther‐motolerance (arsenite and diamide) increased GalNAcT activity, but the increase was modest when compared to that following hyperthermia. In addition to GalNAcT, two other glycosyltransferases with specificity for O‐glycans, α1,2‐fuco‐syltransferase and α2,6‐sialyltransferase, also showed increased activity after hyperthermia and during thermotolerance development. Together with previously published data, these results support the hypothesis that heat‐induced activation of O‐glycan‐specific glycosyltransferases plays a physiological role in the cellular heat shock response and in thermotolerance development.
Morphologic changes in human carcinoma cells (A‐431) stimulated by epidermal growth factor: Effect of cholesterol and low‐density lipoproteins on the ruffling response
Authors: Jackowski M. M. , Swift L. L. , Cohen S. , McKanna J. A.
Published: February 2005

Stimulation of A‐431 carcinoma cells with epidermal growth factor (EGF) causes dramatic morphologic responses including ruffling, rounding, and bulk‐phase pinocytosis. In attempts to explore the mechanisms responsible for changes in plasmalemma topography. we have investigated the effects of exogenous sterols thought to alter membrane fluidity.

Light and scanning electron microscopy revealed a time‐ and concentration‐dependent inhibition of ruffling (> 90%) by cholesterol. This effect could be duplicated by preincubation of the cells with comparable levels of low‐density lipoproteins (LDL). EGF‐stimulated bulk‐phase endocytosis also is inhibited by treatment with cholesterol. No alteration of EGF binding, kinase stimulation, or internalization was detected in cells incubated in cholesterol‐enriched medium (175 μg/ml in 0.5% ethanol), nor did cholesterol or LDL have any effect on EGF‐stimulated rounding.

Morphometry of electron micrographs from cholesterol‐treated cells revealed a selective depletion of interdigitating lateral surface membrane that normally appears to be recruited to generate apical ruffles. Thus, the sterol inhibition of ruffling may be due to redistribution of plasmalemma rather than to changes in membrane viscosity. Together with previous observations, these data suggest that EGF‐stimulated ruffling and bulk‐phase pinocytosis are related phenomena, whereas EGF‐stimulated cell rounding is an independent process.
Mechanism of human lymphotoxin and tumor necrosis factor induced destruction of cells in vitro: Phospholipase activation and deacylation of specific‐membrane phospholipids
Authors: Knauer Mary Fitzgerald , Longmuir Kenneth J. , Yamamoto Robert S. , Fitzgerald Thomas P. , Granger Gale A.
Published: February 2005

The role of phospholipase (PLase) activation and lipid metabolism in lymphotoxin (LT)‐ and tumor necrosis factor (TNF)‐mediated destruction of murine L929 cells was examined. At the levels of LT and TNF employed, cell destruction began at 4–6 h and was 99% complete by 30 h. Cell membrane phospholipids (PL), labelled in situ at the C2 position with 14C arachidonic acid, were analyzed by two‐dimensional thin‐layer chromatography and quantiated over a 30 h time course after LT or TNF treatment. The ratio of radiolabel incorporation relative to the actual amount of each PL present was determined by inorganic phosphate analysis. Radiolabelled arachidonic acid, eicosanoids, and neutral lipids were released into the medium prior to the onset of cell death (4–6 h) and continued to accumulate linearly throughout the destructive reaction. There was a quantitative relationship between the appearance of radiolabelled metabolites in the media and the loss of radiolabelled cellular PL. Cellular phosphatidylethanola‐mine was the primary PL deacylated by PLase action, showing a 75% reduction in radiolabel. The PLase inhibitors—quinacrine, hydrocortisone, dexamethasone, and indomethacin—were potent inhibitors of LT‐ and TNF‐mediated cell destruction, suggesting that selective deacylation of specific membrane PL by PLase activation is an important step in the events that lead to LT‐ and TNF‐mediated cellular destruction in vitro.
Protein kinase C levels and protein phosphorylation associated with inhibition of proliferation in a murine macrophage tumor
Authors: Goode Nigel T. , Hart Ian R.
Published: February 2005

Treatment of M5076 tumor cells with the phorbol esters 12‐O‐tetradecanoyl‐phorbol 13‐acetate (TPA) and phorbol 12, 13 dibutyrate (PdBu) inhibited cellular proliferation, whereas 1,2‐dioctanoyl‐glycerol (DiC8) and 1‐oleoyl2‐acetyl‐glycerol (OAG) did not affect cell growth. Inhibition of cellular proliferation in this cell line appears to be a consequence of protein kinase C (PKC) down‐regulation since phorbol esters, but not a single application of diacylglycerols (DGs) down‐regulated cellular PKC levels. By repeated application of DGs, PKC down‐regulation was achieved and correlated with inhibition of proliferation. Phorbol ester‐induced PKC down‐regulation was reversible, upon removal of the phorbol ester, and the reappearance of PKC was associated with resumption of proliferation. The mitogenic responsiveness of these cells to added serum depended upon cellular PKC levels. Phorbol esters also caused the phosphorylation of two proteins which were not phosphorylated in response to DG treatment. Inhibition of growth of M5076 cells appears to be associated with phosphorylation of two novel proteins and/or PKC down‐regulation.
Epidermal G1‐chalone and transforming growth factor‐β are two different endogenous inhibitors of epidermal cell proliferation
Authors: Richter K. Hartmut , Schnapke Ruben , Clauss Matthias , Fürstenberger Gerhard , Hinz Detlef , Marks Friedrich
Published: February 2005

Epidermal G1‐chalone and transforming growth factor‐β (TGFβ), two endogenous inhibitors of epidermal cell proliferation, were compared with regard to several effects on epidermis in vivo and in vitro. Both factors inhibited DNA labeling in a rat tongue epithelial cell line, with similar kinetics and half‐maximal effects at approximately 1 pg/ml (enriched chalone) and 1 ng/ml (TGFβ). For primary neonatal mouse keratinocytes, (TGFβ) was found to be a rather strong inhibitor of cell proliferation, whereas chalone showed only a weak effect on cells grown in medium containing 1.2 mM Ca2+ and no effect at all in the presence of 0.06 mM Ca2+. Vice versa, upon i.p. injection, only chalone was able to inhibit mouse epidermal DNA synthesis in vivo, whereas TGFβ had no effect at all. A moderate increase of transglutaminase activity in neonatal primary mouse keratinocytes was induced by both factors at concentrations of about 300 pg TGFβ/ml and 10 pg chalone fraction/ml. Chalone did not compete with [125]TGFβ for specific binding sites on primary murine keratinocytes. A polyclonal "chalone antiserum" did not interact with TGFβ, and a neutralizing TGFβ antibody that inhibited the effect of TGFβ on cell proliferation could not block the inhibitory effect of chalone on RTE2 cells. In contrast to TGFβ, epidermal G1‐chalone did not induce proliferation of NIH‐3T3 cells. These results indicate that epidermal G1‐chalone and TGFβ are two different inhibitors of epidermal cell proliferation.
Fibroblast growth factor‐dependent mitogenic signal transduction pathway in chemically transformed mouse fibroblasts is similar to but distinct from that initiated by phorbol esters
Authors: Buchou T. , Měster J.
Published: February 2005

Treatment of the quiescent, chemically transformed Balb/c mouse 3T3 cells (BP‐A31) with fibroblast growth factor (FGF) leads to reinitiation of the cell division cycle in a large proportion of the cells. The characteristics of the mitogenic action of FGF closely resemble those of phorbol esters (activators of protein kinases type C) and differ from those of insulin (mediated by insulin‐like growth factor 1 receptors). In particular, the effects of FGF as well as of phorbol‐2‐myristate‐13‐acetate (PMA), unlike the effects of insulin, are prevented by a low concentration (7.5 nM) of staurosporin (an efficient inhibitor of protein kinase C) as well as by 3‐isobutyl‐1‐methyl xanthin (IBMX). Both FGF and PMA are good inducers of the accumulation of c‐fos and c‐jun mRNAs, whereas insulin has little effect. However, FGF was fully active (both as a mitogen and as inducer of c‐fos mRNA accumulation) also in cells where the protein kinase C‐mediated pathway had been downregulated by a long exposure to phorbol dibutyrate. We propose that the mitogenic effect of FGF does not require activation of protein kinase C, but that the subsequent events in the transduction pathways initiated by FGF and PMA, respectively, are (in part) coincident.
Coordinate reciprocal trends in glycolytic and mitochondrial transcript accumulations during the in vitro differentiation of human myoblasts
Authors: Webster Keith A. , Gunning Peter , Hardeman Edna , Wallace Douglas C. , Kedes Larry
Published: February 2005

Changes in the mRNA levels during mammalian myogenesis were compared for seven polypeptides of mitochondrial respiration (the mitochondrial DNA‐encoded cytochrome oxidase subunit III, ATP synthase subunit 6, NADH dehydrogenase subunits 1 and 2, and 16S ribosomal RNA; the nuclear encoded ATP synthase β subunit and the adenine nucleotide translocase) and three polypeptides of glycolysis (glyceraldehyde‐3‐phosphate dehydrogenase, pyruvate kinase, and triose‐phosphate isomerase). Progressive changes during the conversion from myoblasts to myotubes were monitored under both atmospheric oxygen (nor‐moxic) and hypoxic environments. Northern analyses revealed coordinate, biphasic, and reciprocal expression of the respiratory and glycolytic mRNAs during myogenesis. In normoxic cells the mitochondrial respiratory enzymes were highest in myoblasts, declined 3‐ to 5‐fold during commitment and exit from the cell cycle, and increased progressively as the myotubes matured. By contrast, the glycolytic enzyme mRNAs rose 3‐ to 6‐fold on commitment and then progressively declined. When partially differentiated myotubes were switched to hypoxic conditions, the glycolytic enzyme mRNAs increased and the respiratory mRNAs declined. Hence, the developmental regulation of muscle bioenergetic metabolism appears to be regulated at the pretranslational level and is modulated by oxygen tension.