bluuebunny/crossref_metadata_2025 huggingface dataset:
Correlations between heparan sulfate metabolism and hepatoma growth
Authors: Ishihara Masayuki , Conrad H. Edward
Published: February 2005

A rat hepatoma cell line (Gershenson et al., Science, 170:859–861, 1970) contains a dynamic steady‐state pool of free heparan sulfate (HS) chains in the nucleus that increases in amount when growing cells reach confluence (Fedarko and Conrad, j. Cell Dioi, 102:587–599, 1986). In logarithmically growing cells labeled with 35SO42− steady‐state levels of [35SO4]HS in the nucleus are altered by a variety of culture conditions. Rapidly dividing cells (doubling time = 18–22 h) growing under optimized conditions had steady‐state levels of nuclear HS within the range of 40‐50 pmol 35SO4 in nuclear HS/106 cells. The steady‐state levels of nuclear HS were lowered by several changes in culture conditions, including 1) additions of 1 mM p‐nitrophenyl‐β‐D‐xyloside, 0.25–0.5 mM (+)‐catechin, 0.5 ng/ml transforming growth factor β, 20 ng/ml phorbol‐12‐myristate‐13‐acetate, 1 mM dibutyryl cAMP, or 1 mM inositol‐2‐PO4; 2) decreased levels of D‐glucose; or 3) deletions of serum, insulin, or inositol. In all cases lowering of the nuclear HS level was accompanied by an increase in the cell doubling times, suggesting a correlation in which nuclear HS levels must be optimized for maximal growth rates. When cells cultured under optimal growth conditions reached confluence, the level of nuclear HS increased threefold and the cells stopped dividing. The same culture conditions that lowered the steady‐state levels of HS in the logarithmically growing cells prevented this rise in the nuclear HS as the cells reached confluence and resulted in loss of contact inhibition and overgrowth of the confluent cultures. These observations suggest a second correlation in which elevated nuclear HS levels are found when cell growth is inhibited at confluence; prevention of this rise results in continued growth. Consistent with this correlation between elevated nuclear HS and reduced growth rates, it was observed that addition of either 0.5 μg/ml hydrocortisone or 0.05 μg/ml retinoic acid to the culture medium of logarithmically growing cultures resulted in.increases in steady‐state levels of nuclear HS that were accompanied by increased cell doubling times. The two agents that increased the levels of nuclear HS in logarithmically growing cultures had little effect on levels of nuclear HS in confluent cells or on contact inhibition. These results show that cells growing at maximal rates maintain nuclear HS levels within a range of 40–50 pmol SO4 in HS/106 cells and that when the steady‐state levels of nuclear HS either rise above or fall below this range, the growth rates in subconfluent cultures are decreased. As log‐phase cells reach confluence and stop dividing, there is a rise in the level of nuclear HS. Under growth conditions which prevent this rise, cells lose contact inhibition.
Comparison of phorbol‐12‐myristate‐13‐acetate and dioctanoyl‐sn‐glycerol in the activation of EL4/6.1 thymoma cells
Authors: Stoeck Michael , Lees Rosemary , Szamel Marta , Pantaleo Giuseppe , MacDonald H. Robson
Published: February 2005

The present study compared the role of two protein kinase C (PK‐C) activating agents, the phorbol ester phorbol‐12‐acetate‐13‐myristate (PMA) and the membrane‐permeating diacylglycerol dioctanoyl‐sn‐glycerol (Dic8) in the activation of EL4/6.1 thymoma cells. These cells have been shown to express interleukin‐2 receptors (IL‐2R) upon stimulation with optimal amounts of PMA (10 ng/ml); also, suboptimal amounts of PMA (1 ng/ml) synergized with the Ca2+ ionophore ionomycin and recombinant interleukin‐1 (rlL‐1) (Lowenthal et al., 1986). Comparing PMA and DiC8 led to the following results: PMA at 10 ng/ml induced IL‐2R; in contrast, DiC8 (30‐3 μg/ml) alone was unable to induce IL‐2R, although it did synergize with ionomycin (0.5 μg/ml) and rlL‐1. Bihourly additions of DiC8 did not change this pattern. The addition of DiC8 together with rlL‐2 also resulted in no IL‐2R expression. Furthermore, DiC8 (10 μg/ml) effectively translocated PK‐C. Therefore, the differences observed between PMA and DiC8 do not seem to be due to differences in metabolism or to an inability to translocate PK‐C. Analysis of messenger (m) RNA produced in stimulated EL4/6.1 cells revealed that DiC8 was also unable to induce mRNA for IL‐2R.

Our data suggest that PMA, especially at “optimal” concentrations, might have effects that cannot be mimicked by diacylglycerol. Furthermore, it seems that the deficient activity of diacylglycerols can be compensated for by a Ca2+ ionophore and, depending on the cellular system, by further signals such as IL‐1.
Effects of temperature on the degradation and biliary secretion of asialoorosomucoid by the perfused rat liver: Evidence for two intracellular pathways
Authors: Kloppel Thomas M.
Published: February 2005

We have utilized the in situ perfused rat liver under nonrecirculating conditions to examine the effect of temperature on the metabolism and biliary secretion of [125I]‐asialoorosomucid (ASOR). In this manner we were able to follow the fate of a single round of internalized ligand. In control livers perfused at 37°C, approximately 50% of [125I]‐ASOR injected into the portal vein was extracted on first pass. Five minutes after the injection, radioactivity, which had been extracted initially, began to appear in the hepatic venous effluent. Within 25 min, 50% of the initially extracted radioactivity was released into the perfusion medium; the bulk of this radioactivity (>95%) was soluble in trichloroacetic acid. In livers perfused at temperatures slightly less than 37°C (30–35°C), first‐pass extraction of [125I]‐ASOR was similar to that observed at 37°C. However, a severalfold decrease in the rate of release of radioactivity from the liver into the perfusion medium was noted at the lower perfusion temperatures; whereas greater than 50% of the initially extracted radioactivity was released within 30 min from livers perfused at 37°C, only 5% was released at 30°C. At the lower perfusion temperature, a larger proportion of the released radioactivity was acid precipit‐able (24% vs. 5%). Some radioactivity also was recovered in the bile; of the total amount of radioactivity released from the liver in 30 min at 37°C, approximately 5% was directed into the bile. At lower temperatures of perfusion, a greater fraction of the radioactivity that was released from the liver was directed into the bile (20% at 30°C vs. 5% at 37°C). The data imply that the endosomal pathway to the lysosome is highly sensitive to slight reductions in temperature while the transcytotic route into bile is less sensitive. Lower temperatures might prolong the residence time of ASOR in the prelysosomal endosomal compartments, and thereby increase the likelihood that undegraded ligand will be returned to the blood or be missorted into bile.
Ultraviolet light‐induced inhibition of small nuclear RNA synthesis
Authors: Eliceiri Brian P. , Choudhury Kanakendu , Scott Quincy O. , Eliceiri George L.
Published: February 2005

Two apparently distinct types of inhibition of the synthesis of U1, U2, U3, U4, and U5 small nuclear RNA, induced by ultraviolet (UV) radiation, have been described before: immediate and delayed. Our present observations can be summarized as follows: a) neither the immediate nor the delayed inhibition appear to be mediated by the formation of cyclobutane pyrimidine dimers, since they were not prevented by photoreactivating light, in ICR 2A frog cells; b) the inhibition of U1 RNA synthesis, monitored in HeLa cells within the first few minutes after irradiation, extrapolated to a substantial suppression at time zero of postirradiation cell incubation, providing further support for the proposal that the immediate inhibition is a reaction separate from the delayed UV light‐induced inhibition of U1 RNA synthesis; c) the transition from the pattern of the immediate inhibition to that of the delayed inhibition (disappearance of the UV‐resistant fraction of U1 RNA synthesis and increased rate of inhibition) occurred gradually, without an apparent threshold, within the first 2 hr of incubation after irradiation; and d) the incident UV dose that resulted in a 37% level of residual U1 RNA synthesis (D37) during the delayed inhibition was about 7 J/m2, with an apparent UV dose threshold, and was about 60 J/m2 for the immediate inhibition.
Effects of five recombinant hematopoietic growth factors on enriched human erythroid progenitors in serum‐replaced cultures
Authors: Mitjavila Maria Teresa , Natazawa Masaki , Brignaschi Pascale , Debili Najet , Breton‐Gorius Janine , Vainchenker William
Published: February 2005

Erythroid progenitors from normal human marrow were purified by a two‐step immune panning method permitting both the enrichment of erythroid progenitors (plating efficency up to 10%) and the separation of CFU‐E from BFU‐E. The purified erythroid progenitors were grown in serum‐replaced conditions; in some experiments at an average of one cell per well. Human recombinant granulocytemacrophage colony stimulating factor (GM‐CSF), interleukin 3 (IL3), erythroid potentiating activity (EPA), and human erythropoietin (Epo) either recombinant or homogenous native were tested for their effect on CFU‐E growth. Epo was an absolute requirement for CFU‐E growth and was sufficient to obtain colony formation at the unicellular level whereas GM‐CSF and IL3 did not further increase the plating efficiency. EPA potentiated the effect of Epo on this progenitor only in experiments performed at unicellular level. Human recombinant GM‐CSF, IL3, Interleukin 1 α (IL 1α), and Epo were subsequently tested for their ability to promote BFU‐E growth. GM‐CSF and IL3 supported the growth of erythroid bursts in the presence of Epo, even at the unicellular level. However, IL3 promoted a higher number of bursts than GM‐CSF under all conditions tested. These two growth factors have no or very small additive effects when tested in combination. IL1α added to Epo alone had no effect on the growth of BFU‐E whereas it potentiated the combined action of IL3 and GM‐CSF on the primitive BFU‐E. In conclusion, this study confirms at the unicellular level and under serum‐free conditions that erythroid progenitors are regulated by multipotential growth factors in early phases of erythropoiesis and become sensitive only to Epo in later phases of differentiation.
Platelet‐derived growth factor and angiotensin II cause increases in cytosolic free calcium by different mechanisms in vascular smooth muscle cells
Authors: Roe Michael W. , Hepler John R. , Harden T. Kendall , Herman Brian
Published: February 2005

Platelet‐derived growth factor (PDGF) and angiotensin II (All) are thought to mediate their biological effects in vascular smooth muscle cells (VSMCs) by causing alterations in cytosolic free calcium ([Ca2+]i). In this study we examine the pathways by which PDGF and All alter [Ca2 +]i in VSMCs. Addition of PDGF resulted in a rapid, transient, concentration‐dependent increase in [Ca2+]i; this rise in [Ca2 +]i was blocked completely by preincubation of cells with ethylene glycol‐bis (β‐aminoethyl ether) N,N,N′,N′‐tetraacetic acid (EGTA) or CoCl2, by the voltage‐sensitive Ca2 +‐channel antagonists verapamil or nifedipine, by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), or by pertussis toxin. All also caused an increase in [Ca2 +]i; however, All‐stimulated alterations in [Ca2+]i displayed different kinetics compared with those caused by PDGF. Pretreatment of cells with 8‐(diethylamine)‐octyl‐3,4,5‐trimethyoxybenzoate hydrochloride (TMB‐8), almost totally inhibited All‐induced increases in [Ca2+]i. EGTA or CoCl2 only slightly diminished All‐stimulated increases in [Ca2+]i. Nifedipine, verapamil, TPA, and pertussis toxin pretreatment were without effect on All‐induced increases in [Ca2+]i. PDGF and All both stimulated increases in total inositol phosphate accumulation, although the one‐half maximal concentration (ED50) for alterations in [Ca2+]i and phosphoinisitide hydrolysis differed by a factor of 10 for PDGF (3 × 10−10 M for Ca2+ vs. 2.5 × 10−9 M for phosphoinositide hydrolysis), but they were essentially identical for All (7.5 × 10−9 M for Ca2+ vs. 5.0 × 10−9 M for phosphoinositide hydrolysis). PDGF stimulated mitogenesis (as measured by [3H]‐thymidine incorporation into DNA) in VSMCs with an ED50 similar to that for PDGF‐induced alterations in phosphoinositide hydrolysis. PDGF‐stimulated mitogenesis was blocked by pretreatment of cells with voltage‐sensitive Ca2 + channel blockers, TPA, or pertussis toxin. These results suggest that PDGF and All cause alterations in [Ca2+]i in VSMCs by at least quantitatively distinct mechanisms. PDGF binding activates a pertussis‐toxin‐sensitive Ca2+ influx into cells via voltage‐sensitive Ca2+ channels (blocked by EGTA, verapamil, and nifedipine), as well as stimulating phosphoinositide hydrolysis leading to release of Ca2+ from intracellular stores. All‐induced alterations in [Ca2+]i are mainly the result of phosphoinositide hydrolysis and consequent entry of Ca2+ into the cytoplasm from intracellular stores. Our data also suggest that changes in [Ca2 +]i caused by PDGF are required for PDGF‐stimulated mitogenesis.
Permeabilization of transformed mouse fibroblasts by 3′‐O‐(4‐benzoyl)benzoyl adenosine 5′‐triphosphate and the desensitization of the process
Authors: Gonzalez Fernando A. , Ahmed Ahmed H. , Lustig Kevin D. , Erb Laurie , Weisman Gary A.
Published: February 2005

A photoreactive analogue of ATP, 3′‐O‐(4‐benzoyl)benzoyl adenosine 5′‐triphosphate (BzATP) altered the plasma membrane permeability of transformed 3T6 mouse fibroblasts to normally impermeant molecules as previously reported for ATP, but at lower concentrations. BzATP‐induced permeabilization was modulated by pH, temperature, and the ionic composition of the medium similar to the permeabilizing effects of ATP. Conditions known to enhance ATP‐induced permeabilization, such as treatment with the mitochondrial uncoupler carbonyl cyanide‐m‐chlorophenylhydrazone (CCCP) or the Ca2+‐calmodulin antagonist trifluoperazine also enhanced BzATP‐induced permeabilization. Conditions inhibitory to ATP‐induced permeabilization, including chloride replacement or treatment with furosemide or dithiothreitol (DTT), inhibited permeabilization induced by BzATP. The ionic strength of the medium modulated the responsiveness of the cells to ATP and BzATP; a decrease in the ionic strength below isotonicity increased the sensitivity of the cells to the nucleotides, whereas an increase in ionic strength above isotonicity inhibited permeabilization. Prolonged exposure to ATP under non‐permeabilizing conditions caused the cells to become insensitive to ATP and BzATP. The densensitization phenomenon provides support for the theory that the permeabilization process is mediated by a receptor for ATP.
Effect of donor age and prior sun exposure on growth inhibition of cultured human dermal fibroblasts by all trans‐retinoic acid
Authors: Stanulis‐Praeger Betzabe M. , Gilchrest Barbara A.
Published: February 2005

The effect of retinoic acid on human fibroblasts was studied in a cell culture model of chronologic aging and photoaging. During early exponential phase, all trans‐retinoic acid significantly stimulated growth rate of adult arm‐derived dermal fibroblasts but not of newborn or adult foreskin‐derived fibroblasts. Retinoic acid also significantly reduced saturation density in most young adult arm‐derived lines and all 24 lines derived from old adult arm and foreskin. However, four of ten young adult outer arm (relatively sun‐exposed) and one of ten young adult inner arm (relatively sun‐protected) fibroblast lines increased their saturation density in response to retinoic acid. These data suggest that prior sun exposure and/or donor age may influence cellular responsiveness to retinoic acid. Neither the stimulatory nor the inhibitory effect of retinoic acid could be attributed to cell density, to breakdown of retinoic acid in culture, to nutrient depletion orto serum dependency. However, stationary phase fibroblasts from all sites (foreskin, inner and outer arm) showed an increase in filopodia and in intracellular actin after treatment with retinoic acid that was roughly proportional to the degree of growth inhibition, irrespective of donor age. We suggest that retinoic acid induces premature density dependent growth inhibition at least in part by increasing filopodia‐mediated cell contact that is in turn directly related to an increase in fibrillar actin.
Endothelial adherence under shear stress is dependent upon microfilament reorganization
Authors: Wechezak Arlene R. , Wight Thomas N. , Viggers Robert F. , Sauvage Lester R.
Published: February 2005

In response to externally applied shear stress, Cultured endothelial monolayers develop prominent, axially‐aligned, microfilamentous bundles, termed “stress fibers” (Dewey: Journal of Biomechanical Engineering 106:31–35, 1984; Franke et al.: Nature 81:570–580, 1984; Franke et al.: Klin. Wochenschr 64:989–992, 1986; Wechezak et al.: Laboratory Investigation 53:639–647, 1985). It is unclear, however, whether similar stress fibers develop in noncontiguous endo‐thelial cells and whether these structures are necessary for adherence of individual cells under shear stress. It also is unknown what alterations occur in microtubules, intermediate filaments, and focal contacts as a consequence of shear stress. In this study, endothelial cells, free of intercellular contact, were exposed to 93 dynes/cm2 for 2 hr. With the aid of specific labeling probes and interference reflection microscopy, the distributional patterns of microfilaments, microtubules, intermediate filaments, and focal contacts were examined. Following shear stress, microfilament bundles and their associated focal contacts were concentrated in the proximal (relative to flow direction) cell regions. In contrast, microtubules were distributed uniformly within cell contours. Intermediate filaments displayed only an occasional tendency for accumulation at proximal edges. When cells were shear‐tested in the presence of cytochalasin B to inhibit microfilament assemly, considerable cell loss occurred. Following inhibition of tubulin polymerization, no increase was observed in the percentage of cells lost due to shear over nontreated controls. Nocodazole‐treated cells, however were characterized by prominent stress fibers throughout the cell. These results indicate that stress fiber and focal contact reorganization represent major responses in isolated endothelial cells exposed to shear stress and that these cytoskeletal structures are necessary for adherence.
Role of cyclic adenosine monophosphate in the induction of endothelial barrier properties
Authors: Stelzner Thomas J. , Weil J. V. , O'Brien R. F.
Published: February 2005

Cyclic adenosine monophosphate (AMP) has numerous important effects on cell structure and function, but its role in endothelial cells is unclear. Since cyclic AMP has been shown to affect transmembrane transport, cell growth and morphology, cellular adhesion, and cytoskeletal organization, it may be an important determinant of endotelial barrier properties. To test this we exposed bovine pulmonary artery endothelial cell monolayers to substances known to increase cyclic AMP and measured their effect on endothelial permeability to albumin and endothelial cell cyclic AMP concentrations. Cholera toxin (CT), a stimulant of the guanine nucleotide binding subunit of adenylate cyclase, led to a concentration‐dependent 2‐6‐told increase in cyclic AMP which was associated with a 3–10‐fold reduction in albumin transfer across endothelial monolayers. The effect was not specific to albumin as similar barrier‐enhancing effects were also noted with an unrelated macromolecule, fluorescein isothiocyanate (FITC)‐dextran (MW 70,000). Barrier enhancement with cyclic AMP elevation was also observed with forskolin, a stimulant of the catalytic subunit of adenylate cyclase. The temporal pattern of barrier enhancement seen with these agents paralleled their effects on increasing cyclic AMP, and the barrier enhancement could be reproduced by incubation with either dibutyryl cyclic AMP or Sp‐cAMPS, cyclic AMP‐dependent protein kinase agonists. Furthermore, the forsko‐lin effect on barrier enhancement was partially reversed with Rp‐cAMPS, an antagonist of cyclic AMP‐dependent protein kinase. Since endothelial actin polymerization may be an important determinant of endothelial barrier function, we sought to determine whether the cyclic AMP‐induced effects were associated with increases in the polymerized actin pool (F‐actin). Both cholera toxin and forskolin led to apparent endothelial cell spreading and quantitative increases in endothelial cell F‐actin fluorescence. In conclusion, increased endothelial cell cyclic adenine nucleotide activity was an important determinant of endothelial barrier function in vitro. The barrier enhancement was associated with increased endothelial apposition and increases in F‐actin, suggesting that influences on cytoskeletal assembly may be involved in this process.
Relationship of cytosolic ion fluxes and protein kinase C activation to platelet‐derived growth factor induced competence and growth in BALB/c‐3T3 cells
Authors: Zagari Martin , Stephens Marcy , Earp H. Shelton , Herman Brian
Published: February 2005

Platelet‐derived growth factor (PDGF) and other agents that activate protein kinase C (PKC) rapidly alter cytosolic pH (pHi) and intracellular free calcium ([Ca++]i) in BALB/c‐3T3 fibroblasts. To define whether changes in pHi or [Ca++]i are linked to PDGF‐stimulated mitogenesis, these parameters were assessed in control and PKC depleted fibroblasts. PDGF addition to BALB/c‐3T3 fibroblasts resulted in transient acidification of the cytoplasm followed by prolonged cytosolic alkalinization. Exposure of cells to 12‐tetradecanoylphorbol‐13‐acetate (TPA), a phorbol ester that activates PKC, resulted in cytosolic alkalinization without prior acidification. Overnight incubation with 600 nM TPA decreased the total cell PKC histone phosphorylating activity in BALB/c‐3T3 fibroblasts by >90%. In PKC‐deficient fibroblasts, TPA, and PDGF‐induced alkalinization was abolished. In addition, the transient drop in pHi seen initially in control cells treated with PDGF is sustained to the point where pHi is fully 0.6–0.7 pH units below control cell values for up to 30 minutes.

PDGF increased [Ca+ +]i threefold; this transient rise in [Ca++]i was only minimally affected (<15%) by lowering of the extracellular calcium level with ethylene glycol bis(b‐aminoethyl ether)O N,N,N′ tetraacetic acid (EGTA) or blocking calcium influx with CoCI2. In contrast, 8‐(diethylamine)‐octyl‐3,4,5‐trimethoxybenzoate (TMB‐8), an agent thought to inhibit calcium release from intracellular stores, substantially inhibited the rise in [Ca++]i caused by PDGF. TPA and 1‐oleoyl‐2‐acetylglycerol (OAG) increased [Ca++]i but in contrast to PDGF this effect was blocked by pretreatment of cells with EGTA or CoCI2. In PKC‐deficient fibroblasts, PDGF still increased [Ca++]i and stimulated DNA synthesis as effectively as in controls. TPA and OAG however, no longer increased [Ca++]i. The continued ability of PDGF to stimulate DNA synthesis in the face of sustained acidification and the absence of PKC activity suggests that cytosolic alkalinization and PKC activation are not essential for PDGF‐induced competence in BALB/c‐3T3 fibroblasts.
Characterization of the increased biological potency in BALB/C 3T3 cells of two analogs of human insulinlike growth factor I which have reduced affinity for the 28 K cell‐derived binding protein
Authors: Cascieri Margaret A. , Hayes Nancy S. , Bayne Marvin L.
Published: February 2005

We have characterized the biological activity of two analogs of insulinlike growth factor I (IGF I) which have significantly reduced affinity for the soluble 28 K binding proteins which are secreted by various cell types. The analogs, which were made by site‐directed mutagenesis of a synthetic gene encoding for IGF I, are [Gln 3, Ala 4, Tyr 15, Leu 16] IGF I and an analog in which the first 16 amino acids of IGF I were replaced with the first 17 amino acids of insulin (B‐chain mutant). These two peptides have 100‐fold and >1,000‐fold lower affinity, respectively, than IGF I for the 28 K binding protein present in the conditioned medium of two cell types, the clonal rat vascular smooth muscle line A10, and BALB/C 3T3 cells. The 28 K protein secreted by BALB/C 3T3 cells has fivefold‐lower apparent affinity for both IGF I and [Gln 3, Ala 4, Tyr 15, Leu 16] IGF I than does the 28 K protein secreted by A10 cells. Conditioned medium from these two cells types has similar amounts of unoccupied 28 K protein as evidenced by the ability of 125 I‐IGF I to specifically bind to and be covalently bound to the protein after treatment with the bifunctional cross‐linking reagent disuccinimidyl suberate.

In the presence of 0.1% calf serum, IGF I and [Gln 3, Ala 4, Tyr 15, Leu 16] IGF I stimulate DNA synthesis in A10 cells with ED50 = 0.4 nM, and in BALB/C 3T3 cells with ED50 = 10 nM and 1.3 nM, respectively. Thus, these peptides are equipotent in A10 cells, but the mutant peptide is ten times more active than IGF I in BALB/C 3T3 cells. A10 cells can be made ten times less sensitive to IGF I by performing the incubation in the presence of conditioned media from BALB/C 3T3 cells but not from A10 cell?. The activity of [Gln 3, Ala 4, Tyr 15, Leu 16] IGF I is not altered under these conditions. Thus, the conditioned media, which contain 28 K proteins secreted by A10 cells and BALB/C 3T3 cells, have different effects on the biological action of IGF I. These data suggest that the 28 K binding proteins can have important effects on the sensitivity of tissues to IGF I and that the B‐chain mutant and [Gln 3, Ala 4, Tyr 15, Leu 16] IGF I will be useful in assessing the biological role of these proteins.
Control of DNA polymerase‐α activity in regenerating rat liver by calcium and 1α,25(OH)2D3
Authors: Rixon R. H. , Isaacs R. J. , Whitfield J. F.
Published: February 2005

The late G1 surge of DNA polymerase‐α activity and the initiation of DNA replication in the hepatocytes of partial hepatectomy‐induced regenerating liver were severely reduced when the mitogenic partial hepatectomy was carried out in the hypocalcemic and 1,25(OH)2D3 (1α,25‐dihydroxycholecalciferol)‐deficient environment of parathyroidectomized (PTX) or thyroparathyroidectomized (TPTX) rats. These inhibitions were prevented in TPTX rats by a postpartial hepatectomy injection of 1,25(OH)2D3, which also restored blood calcium to normocalcemic levels. Inhibition of active DNA polymerase‐α accumulation and initiation of DNA synthesis in TPTX rats were also completely prevented by prefeeding the rats a low phosphorus diet, which stopped the lowering of the blood levels of calcium and 1,25(OH)2D3 following parathyroid removal. These studies indicate that the rise of DNA polymerase‐α activity and the initiation of DNA replication in regenerating liver are controlled by cellular processes that rely on normal blood levels of calcium and 1,25(OH)2D3. Because DNA polymerase‐α is the third DNA replication enzyme (the others are ribonucleotide reductase and thymidylate synthase) that has been shown to depend on parathyroid hormone and/or the circulating levels of calcium and 1,25(OH)2D3 that it controls, the authors concluded that the processes dependent on calcium and 1,25(OH)2D3 are parts of a mechanism that coordinately activates the DNA‐replicating enzymes. The possibility that cyclic adenosine monophosphate (cAMP)‐dependent protein kinases are involved in this replication mechanism is considered.
Collagen lattice effects on fibroblast arachidonic acid metabolism
Authors: Pentland Alice P.
Published: February 2005

Fibroblasts are routinely maintained in vitro on tissue culture plastic, in an environment which i s devoid of collagen, the most abundant extracellular protein in dermis. Recent work has shown that by seeding fibroblasts into a collagen matrix, many aspects of their metabolism change dramatically: they stop proliferation, organize and contract the collagen matrix, and secrete much larger quantities of the usual extracellular matrix components. Because so many fibroblast functions are dramatically altered by the presence of the collagen matrix, matrix effects on fibroblast metabolism of arachidonic acid were examined. The studies presented here show that during the period of matrix contraction, metabolism of arachidonate to prostaglandins by fibroblasts is increased sixfold compared to cells plated on plastic, and that this increase i s correlated with contraction but does not regulate it. The increase in prostaglandin synthesis is due in part to an increased new synthesis of the rate‐limiting enzyme in prostaglandin synthesis, cyclooxygenase. No change in the profile of products the fibroblasts synthesize from arachidonate is induced by the presence of the matrix. After the lattice contraction is complete, the basal arachidonate metabolism of matrix‐embedded fibroblasts is the same as controls grown on plastic. In addition, the matrix‐embedded cells have the same capacity to synthesize PCE2 in response to IL‐1 as do cells grown on plastic. However, the response to the hormone agonist bradykinin by the matrix‐embedded cells is present on day 1 but not on day 3, the time when cells grown on plastic are most responsive. These data indicate that while basal prostaglandin metabolism is unaffected in quiescent fibroblasts which have been embedded in a collagen matrix, response to hormone agonists may be greatly attenuated. The changes in the metabolism of arachidonate which occur during the process of matrix contraction and organization may play a part in the regulation of wound repair.
Analysis of the growth factor requirements for stimulation of WI‐38 cells after extended periods of density‐dependent growth arrest
Authors: Owen Thomas A. , Soprano Dianne Robert , Soprano Kenneth J
Published: February 2005

When cultures of WI‐38 human diploid fibroblasts reach high cell densities, they cease to proliferate and enter a viable state of quiescence. WI‐38 cells can remain in this quiescent state for long periods of time; however, the longer the cells remain growth arrested, the more time they require to leave G0, progress through G1, and enter S after stimulation with fresh serum. The experiments presented here compare the response of long‐term quiescent WI‐38 cells (stimulated 26 days after plating) and short‐term quiescent WI‐38 cells (stimulated 12 days after plating) to treatment with a variety of individual purified growth factors instead of whole serum. Our results show that the qualitative and quantitative growth factor requirements necessary to stimulate G1 progression and entry into S were the same for both short‐ and long‐term quiescent WI‐38 cells, in that the same defined medium supplemented with epidermal growth factor [EGF], recombinant human insulin‐like growth factor 1 [IGF‐1], and dexamethasone [DEX] stimulated both populations of cells to proliferate with the same kinetics and to the same extent as serum. However, the long‐term quiescent WI‐38 cells were found to exhibit a difference in the time during which either serum or these individual growth factors were required to be present during the prereplicative period. We believe that this difference may be the cause of the prolongation of the prereplicative phase after stimulation of long‐term density‐arrested WI‐38 cells.
Binding modes of IgG from Pemphigus autoimmune sera onto guinea pig keratinocytes and the fate of bound IgGs
Authors: Milner Yoram , Sagi Ephraim , Timberg Rina , Michel Beno , Me'te'zeau Phlllppe , Goldberg Michel
Published: February 2005

Pemphigus is an intraepidermal autoimmune blistering disease of humans caused by circulating IgGs. We have investigated the binding mode and the fate of bound antibodies from Pemphigus sera (P‐IgG) on guinea pig keratinocytes in suspension in order to find clues to the loss of cell adhesion in vivo (acantholysis). Flow cytometry, following indirect immunofluorescent labeling of the keratinocytes, and dead cells' staining with ethidium bromide, demonstrated the specific surface binding of P‐IgG onto living keratinocytes only. This was shown with several Pernphigus sera or purified P‐IgG. This technique, used with various Pemphigus sera, showed that the specific binding is increased when the serum titer is higher, and “Km” values for P‐IgG were roughly and inversely correlated to the titers. Upon saturation the same average number of Pemphigus IgG sites per cell were found for the sera of different patients. Analysis of the specific binding of [125I]‐P‐lgC onto Percollseparated (living) keratinocytes showed the existence of two classes of sites: 2 × l06 sites/cell high‐affinity sites (Kd = 1.5 × 10 −6 M total IgG) and 25 × l06 sites/cell low‐affinity sites (Kd = 6 × 10−5 M total IgG). Cell sorting and flow cytometry of individual cells allowed us to correlate the light‐scattering signal, the RNA content, the size and morphology, and the P‐IgG binding to the cells. The results indicated that P‐IgG binding is homogeneous within the living keratinocytes and increases with cell size (cell maturity). Cell‐sorter analysis of cells with membrane‐bound P‐IgC, coupled to direct determination of P‐IgC released in the medium, revealed the fate of bound P‐IgG: 40–60% of the P‐IgGs were released in the medium within 30 minutes at 37°C. This was accompanied and followed by a much slower, metabolic energydependent, internalization process of the membrane‐bound P‐IgG. The internalization has been confirmed by electron microscopy of bound P‐lgC labeled with protein A‐gold. Internalized IgGs were seen in the cells in coated membranous vesicles and other endocytic compartments. Similar behavior was also observed with two other membrane ligands: i.e., concanavalin A and multispecific rabbit “antisurface” antibodies.
Actions of insulin‐like growth factor‐I on the B104 neuronal cell line: Effects on cell replication, receptor characteristics, and influence of secreted binding protein on ligand binding
Authors: Orlowski Craig C. , Chernausek Steven D. , Akeson Richard
Published: February 2005

Several peptide growth factors influence the growth and differentiation of neural cells. To investigate further the growth‐promoting effects of the somatomedins on cells of neural origin, the authors characterized the binding and mitogenic effects of insulin‐like growth factor‐1 (IGF‐1) on a functionally differentiated rat neuronal cell line (B104). Specific, high‐affinity (Kd ≅ 10−9 M) receptors for IGF‐1 were abundant (approximately 124,000 binding sites/B104 cell). These IGF‐1 receptors were similar to those of non‐neural tissue in that they contained 135,000 dalton binding subunits (demonstrated by affinity labeling and autoradiography) and recognized insulin at high concentrations. IGF‐1 was more potent than insulin at stimulating B 104 cell replication in serum‐free medium and, at an initial concentration of 100 ng/ml, was the only exogenous growth factor needed to maintain growth through several cell divisions. Furthermore, cells of later passage were found to secrete specific IGF binding proteins that produced an unusual, biphasic binding curve in radioligand displacement studies. These binding proteins apparently sequester IGF‐1, limiting its access to the cell. Experiments with B 104 cells may provide useful information about the role of IGFs and their binding proteins as potential regulators of growth and differentiation of the primitive neuroblast.
Differential inhibitory effects of TGF‐β on EGF‐, PDGF‐, and HBGF‐1‐stimulated MG63 human osteosarcoma cell growth: Possible involvement of growth factor interactions at the receptor and postreceptor levels
Authors: Mioh Hiroyuki , Chen Jan‐Kan
Published: February 2005

The growth of MG63 human osteosarcoma cell line in 5% serum is stimulated by epidermal growth factor (EGF), platelet‐derived growth factor (PDGF), or heparin‐binding growth factor‐1 (HBGF‐1). The mitogenic effect of EGF and PDGF is completely blocked by TFG‐β at 1 ng per ml and the effect of HBGF‐1 is attenuated by 75–80%. Treatment of MG63 cells with TGF‐β reduces HBGF‐1 receptor binding affinity from 1.24 × 10–11 M to 3.51 × 10–11 M with no change on the receptor number (1.1. × 103 per cell). The receptor‐binding affinity of EGF and PDGF is not altered by TGF‐β treatment; however, the number of EGF receptor is increased by 25%. Both EGF and PDGF stimulate MG63 cellular tyrosine kinase activity, and such stimulation is inhibited by TGF‐β pretreatment. No change in the cellular protein tyrosine phosphorylation pattern can be detected in HBGF‐1‐stimulated cells with and without TGF‐β pretreatment. These data suggest that TGF‐β inhibits EGF and PDGF mitogenicity by blocking EGF‐ and PDGF‐stimulated tyrosine kinase activity and attenuates HBGF‐1 mitogenicity by decreasing its receptor affinity.
Electrophysiological properties of isolated rat liver cells
Authors: Sawanobori Tohru , Takanashi Hideo , Hiraoka Masayasu , Iida Yoshitaka , Kamisaka Kazuaki , Maezawa Hidenori
Published: February 2005

The electrophysiological properties of isolated rat liver cells were studied using the patch clamp method in whole‐cell configuration. The membrane potential in isolated hepatocytes was −42 ± 17 mV (n = 20). The input resistance (Rin) and the time constant (τm) were 51 ± 17 M (the range of 34 to 180 MΩ) (n = 20) and 4.2 ± 1.0 msec (the range of 3 to 16.5 ms) (n = 20). Assuming that the specific membrane capacitance is 1 μF/cm2, the membrane resistance and membrane capacitance were 42. ± 9.0 KΩ cm2 and 87 ± 27 pF. These values indicate that isolated rat hepatocytes are not abnormally permeable or leaky. The current‐voltage relationship was linear with no rectification. The depolarizing pulse from the resting potential did not induce fast or slow inward currents even when norepinephrine or high Ca2 (3.6 mM) were applied. This indicates that there is no voltage‐sensitive Ca2+ channel in the isolated hepatocytes.
Regulation of beta‐adrenergic receptor expression in rat liver
Authors: Schleifer Leonard S. , Black Ira B. , Reid Lola M.
Published: February 2005

To begin defining the factors regulating neurotransmitter receptor expression, we examined beta‐adrenergic receptors in rat liver in vivo and in primary liver cultures under defined hormonal conditions. β‐receptors described a remarkable developmental profile in vivo, increasing fivefold between embryonic days 16 and 20, and decreasing tenfold by early adulthood. The developmental decrease reflected reduced receptor number without a change in receptor properties. The ontogenetic decrease appeared to be specific for β‐receptors α‐receptors developed in a hyperbolic fashion, reaching high plateau values by the third postnatal week.

Adult rat liver cells plated into culture re‐expressed high β‐receptor levels, exhibiting a 4‐8‐fold increase. A similar pattern of expression of the β‐receptors, having similar pharmacological properties, was observed in primary liver cultures maintained in serum‐free medium, in a serum‐supplemented medium or in several variations of a serum‐free, hormonally defined medium designed for primary liver cultures. Thus, the degree of expression of the β‐receptors was not found affected by various hormones, by serum, or by any medium condition. By contrast, the degree of expression of the β‐receptors was markedly sensitive to cell density. High expression of the β‐receptors was observed at low cell densities (1‐3 × 106 cells/150 mm dish), and low expression or no expression was observed in confluent cultures (10‐20 × 106 cells/150 mm dish). Our experiments suggest that β‐receptor expression does not follow an immutable program, but may be regulated by density‐dependent cell‐cell interactions.
Platelet‐derived growth factor, epidermal growth factor, and insulin‐like growth factor I regulate specific cell‐cycle parameters of human diploid fibroblasts in serum‐free culture
Authors: Chen Yuhchyau , Rabinovitch Peter S.
Published: February 2005

The growth regulation of human diploid fibroblasts by platelet‐derived growth factor (PDGF), epidermal growth factor (EGF), insulin‐like growth factor I (IGF‐I) (somatomedin C), dexamethasone, and transferrin was investigated in a serumfree, chemically defined culture system. Cell‐cycle kinetic parameters were determined using 5′‐bromodeoxyuridine (BrdU) incorporation and flow cytometric analysis with the DNA‐specific dye Hoechst 33258. We found that PDGF and EGF regulate the proportion of cells capable of entering the cell cycle from the quiescent state, with smaller effects upon the rate of cell transition from G1 into S phase. IGF‐I, on the other hand, regulates the rate of cell exit from G1 without affecting the cycling fraction. Transferrin and dexamethasone showed less effect upon the cell‐cycle kinetics under these culture conditions. The data provide functional evidence that PDGF and EGF regulate similar cell‐kinetic parameters in human fibroblast cultures. IGF‐I is functionally distinct from both PDGF and EGF in its role of regulating G1 exit rate without affecting the cycling fraction. These observations made by BrdU‐Hoechst flow cytometric techniques provide a novel perspective on the regulatory effects exerted by different classes of growth factors, and suggest a mode of interdependence of these mitogens in regulating the net growth rate which could be a feature of growth regulation in vivo. These data also provide a different perspective on the regulation of the growth of fibroblast‐like cells than that of the “competence/progression” cell‐cycle model.
Effect of Na+ flux inhibitors on induction of c‐fos, c‐myc, and ODC genes during cell cycle
Authors: Panet Rivka , Amir Iris , Snyder David , Zonenshein Leora , Atlan Henri , Laskov Reuven , Panet Amos
Published: February 2005

The role of Na + transport systems in the mitogenic signal induced by growth factors was studied, and it was shown that two Na + transport systems contribute to the early increase in cytoplasmic Na + in response to serum growth factors, namely the amiloride‐sensitive Na+/H + antiport and the bumetanide‐sensitive Na + /K + /CI‐ cotransport. Bumetanide or amiloride, when added separately, inhibited part of the increase in cytoplasmic Na +, as a response to the addition of serum to quiescent BALB/c mouse 3T3 fibroblasts. Each drug also suppressed part of the stimulation of the ouabain‐sensitive Rb + influx, which was controlled by intracellular Na +. However, when both drugs were added together with serum growth factors, a complete inhibition of the early increase in [Na +], and subsequently a complete blockage of Na + /K + pump stimulation was obtained.

Amiloride or bumetanide, when added separately, only partially inhibited DNA synthesis induced by serum, 24% and 8% respectively. However, when both drugs were added together, at the time of serum addition to the quiescent cells, cell entry into S‐phase was completely inhibited. To investigate the mode of cell‐cycle inhibition, analysis was done of the possible role of early Na + fluxes in the mitogenic signal transduced from cell membrane receptors to the nucleus. The effects of the two drugs amiloride and bumetanide on induction of three genes‐c‐fos, c‐myc, and ornithin decarboxylase (ODC)‐was measured during cell transition through the G1‐phase. Amiloride and bumetanide, when added separately or in combination, did not inhibit the induction of c‐fos, c‐myc, and ODC mRNAs. These results suggest that stimulation of Na+ fluxes by serum growth factors is essential for cell transition into the S‐phase of cell cycle, but it plays no apparent role in the growth factor signal transduced from the cell surface to the interior of the cell, as manifested by c‐fos, c‐myc, and ODC genes induction.
Regulation of hepatocyte growth: Alpha‐1 adrenergic receptor and ras p21 changes in liver regeneration
Authors: Cruise Jennifer L. , Muga Stephanie J. , Lee Yi‐Sheng , Michalopoulos George K.
Published: February 2005

Catecholamines, acting via the alpha‐1 adrenergic receptor, have been demonstrated to influence adult rat hepatocyte DNA synthesis in primary culture and in vivo during liver regeneration following partial hepatectomy (PHX). Earlier investigations have suggested that the alpha‐1 effect on DNA synthesis is significant only during the first day following PHX. We examined receptor binding at several early and late time points after surgery, and we observed a significant loss of specific [3H]‐prazosin binding to cells isolated from rat livers 48 and 72 hr after PHX. In contrast, the ability of norepinephrine to stimulate inositol phosphate production in isolated cells prelabeled with [3H]‐myo‐inositol was transiently reduced between 8 and 16 hr, when alpha‐1 binding capacity was virtually unchanged. This uncoupling of phosphoinositide turnover from binding was preceded by a drop in hepatic membrane ras p21 content, as assayed by liquid competition radioimmunoassay. The loss of immunoreactive p21 from membranes was significant by 2 hr after PHX. These findings suggest a role for alpha‐1 receptors and ras protein in the early events of liver regeneration.
Control of protein synthesis by extracellular Na+ in cultured fibroblasts
Authors: Petronini Pier Giorgio , Tramacere Mariarosaria , Mazzini Alberto , Kay John E. , Borghetti Angelo F.
Published: February 2005

In chick embryo fibroblasts (CEFs), a partial substitution of extracellular Na+ with other cations or carbohydrates decreased the intracellular Na+ content without altering the K+ level. Concomitantly, a significant decrease in the serum‐dependent rate of protein synthesis occurred. This phenomenon appeared to be quickly reversible upon reconstitution of the correct extracellular Na+ concentration in the culture medium. The presence of a transcriptional inhibitor such as actinomycin D during the treatment did not inhibit the reversibility of the phenomenon. The presence in the culture medium of K+ in such excess as to dissipate the membrane potential did not alter the observed relationship between the protein synthesis rate and the internal Na+ content. Analysis of the amino acid pool indicated that the observed inhibition of the rate of protein synthesis in CEFs incubated in low Na+ medium was not caused by an unbalanced availability of intracellular amino acids. In addition, intracellular pH, as estimated by the measurement of the equilibrium distribution of benzoic acid, did not show any significant alteration in cells incubated in the presence of bicarbonate buffer and in low extracellular Na+. Moreover, the relationship between the rate of protein synthesis and the internal Na+ content was still observed in CEFs cultured in bicarbonate‐containing media, but at lower or higher than physiological pH. Analysis by two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) of the proteins synthesized by CEFs cultured at a reduced extracellular Na+ concentration showed that specific alterations of gene expression occurred.
Sodium butyrate selectively antagonizes the inhibitory effect of retinoids on cornified envelope formation in cultured human keratinocytes
Authors: Schmidt Rainer , Cathelineau Christine , Cavey Marie T. , Dionisius Veronique , Michel Serge , Shroot Braham , Reichert Uwe
Published: February 2005

Sodium butyrate affects cell differentiation in confluent epidermal keratinocyte cultures by considerably increasing the spontaneous formation of cross‐linked envelopes in normal human keratinocytes (NHK). It also favors the development of envelope competence in the Simian virus‐40 (SV‐40)‐transformed human foreskin keratinocyte line SV‐K14. It completely abolishes the inhibitory effect of serum and retinoic acid on the expression of plasma membrane‐associated transglutaminase. However, other markers of epidermal differentiation that are also under the control of retinoids such as keratins or the enzyme cholesterol sulfotransferase are not affected by butyrate. The level of the cellular retinoic acid binding protein (CRABP) is considerably increased in its presence. Butyrate does not interfere with the binding of retinoids to their cellular binding proteins. Our observations suggest that sodium butyrate stimulates cornified envelope formation via the induction of the plasma membrane‐associated transglutaminase required for cornified envelope synthesis and, additionally, by abolishing the inhibitory effect of retinoids on the expression of this enzyme.