bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ultraviolet light‐induced inhibition of small nuclear RNA synthesis
Authors: Eliceiri Brian P. , Choudhury Kanakendu , Scott Quincy O. , Eliceiri George L. 
Published: February 2005

AbstractTwo 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

AbstractErythroid 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

AbstractPlatelet‐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

AbstractA 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

AbstractThe 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

AbstractIn 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

AbstractCyclic 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

AbstractPlatelet‐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

AbstractWe 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

AbstractThe 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

AbstractFibroblasts 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.