bluuebunny/crossref_metadata_2025_split huggingface dataset:
The 73 kilodalton heat shock cognate protein purified from rat brain contains nonesterified palmitic and stearic acids
Authors: Guidon Peter T. , Hightower Lawrence E. 
Published: February 2005

AbstractA protein related to the 71 kilodalton inducible rat heat shock protein was purified to electrophoretic homogeneity in milligram amounts from brain tissue of nonheat‐stressed rats. The protein has been designated as a stress cognate protein based on previous studies and data presented herein that this protein cross‐reacted with a monoclonal antibody originally raised against the Drosophila 70 kilodalton heat shock protein. The purified protein had an apparent molecular mass of 73 kilodaltons when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and an apparent mass of 150 kilodaltons as determined by nondissociative gel chromatography, suggesting that the purified protein is a homodimer. The purified protein had isoelectric points of 5.0 under nondissociative conditions and 5.6 when exposed to protein denaturants, suggesting loss of bound anionic molecules and/or net exposure of basic residues upon denaturation. Chloroform/methanol extraction of the purified protein and subsequent analyses by thin layer and gas‐liquid chromatography resulted in the identification of palmitic and stearic acids noncovalently bound to the protein. Approximately four molecules of fatty acids were bound per dimer with palmitic and stearic acids present in a one‐to‐one ratio. The purified protein did not bind exogenously added radio‐active palmitate, indicating that the fatty acid‐binding sites of the cognate protein were fully occupied and that the associated fatty acids were too tightly bound to exchange readily. The possible significance of the fatty acids associated with the 73 kilodalton stress cognate protein is discussed.
Comparative studies of the binding and growth‐supportive ability of mammalian transferrins in human cells
Authors: Penhallow Robert C. , Brown‐Mason Anne , Woodworth Robert C. 
Published: February 2005

AbstractThe ability of human‐derived cells in culture to bind, remove iron from, and grow in the presence of transferrins (Tf) isolated from the sera of species commonly included in tissue culture medium was investigated. Kinetic studies on HeLa cells reveal apparent first‐order association rate constants of 0.43 min−1 for human Tf and 0.15 min−1 for equine Tf. Labeled chicken ovo‐Tf and fetal bovine Tf were not recognized by the HeLa cells. Competition experiments with HeLa cells that use either isolated Tf or parent serum confirm these findings. Equilibrium binding experiments performed on HeLa cells at 37°C in the presence of 2,4‐dinitrophenol to prevent iron removal indicate 1 × 106 Tf bound/cell with a dissociation constant (K'D) of 28 nM for human Tf and 182 nM for equine Tf. Equilibrium binding performed at 0°C to prevent endocytosis reveals 4.1–6.7 × 105 Tf binding sites/cell with a K'D of 8.3 nM for human Tf and 41.5 nM for equine Tf. Parallel experiments in normal human diploid fibroblast‐like MRC‐5 cells indicate expression of 0.82–2.78 × 105 Tf binding sites/cell with a K'D of 8.2 nM for human and 39.1 nM for equine Tf. Thus, the results of equilibrium binding studies of a more differentiated cell type are consistent with those found for HeLa cells. Fetal bovine Tf was found to compete weakly with labeled human Tf for human receptor on HeLa cells in a soluble receptor assay, with an approximately 500‐fold excess needed to reduce binding to half maximal. Iron uptake experiments show an iron donating hierarchy where human > horse > calf, suggesting that the rate of iron uptake depends on the affinity of receptor for transferrin. Growth experiments involving HeLa cells in chemically defined serum‐free medium demonstrate that bovine Tf will support growth as well as human Tf, but at concentrations much higher than are required of human Tf.
Alpha1‐adrenergic stimulation and cytoplasmic free calcium concentration in cultured renal proximal tubular cells: Evidence for compartmentalization of quin‐2 and fura‐2
Authors: Goligorsky Michael S. , Hruska Keith A. , Loftus David J. , Elson Elliot L. 
Published: February 2005

AbstractThis study was designed to examine the role of changes in cytoplasmic free calcium concentration ([Ca2+]i) during the response to α1‐adrenergic agonists in cultured renal proximal tubular cells. Experiments were carried out on primary cultures of canine proximal tubular cells grown in defined culture medium on a solid support, on collagen‐coated polycarbonate membranes, or on collagen‐coated glass coverslips. Quin‐2 and fura‐2 were used to monitor [Ca2+]i. The basal level of [Ca2+]i was 101 nM, as measured with quin‐2, and 122 nM, as determined using fura‐2. Fluorescence flow cytometry revealed that about 85% of the population of proximal tubular cells responded to phenylephrine with an increase in [Ca2+]i. Phenylephrine (10−5 M) caused an immediate actual increase in [Ca2+]i by 18 and 24%, as determined with quin‐2 and fura‐2, respectively, with the peak increase in [Ca2+]i averaging 22% and 44% over the basal level (180–300 sec). This effect did not require extracellular calcium. The effect of phenylephrine was abolished by prazosin and verapamil. Fluorescence microscopy of quin‐2 or fura‐2 loaded cells revealed punctate areas of fluorescence within the cytoplasm suggesting vesicular uptake of the dyes. Pinocytotic entrapment of the dyes was demonstrated by the transfer of cell‐impermeant fura‐2 across tubular cell monolayers mounted in Ussing chambers. The transfer of the dye was similar to that of a marker of fluid‐phase pinocytosis, Lucifer Yellow (LY). This pinocytotic entrapment of Ca2+‐indicators would lead to underestimation of the actual calcium transients. Microfluorometric study of single proximal tubular cells “scrape‐loaded” with fura‐2 revealed a four‐fold increase in [Ca2+]i concentration following stimulation with phenylephrine.
Stimulation of sulfated‐proteoglycan synthesis by forskolin in monolayer cultures of rabbit articular chondrocytes
Authors: Malemud Charles J. , Mills Teresa M. , Shuckett Rhonda , Papay Robert S. 
Published: February 2005

AbstractForskolin, a plant cardiotonic diterpene, stimulated proteoglycan biosynthesis by chondrocytes in monolayer culture. The quantitative increase in proteoglycans was dependent on the concentration of forskolin, but was relatively independent of the presence of serum. At forskolin concentrations that stimulated proteoglycan synthesis, a significant stimulation of adenylate cyclase and cAMP was also measured. The quantitative increase in proteoglycans was characterized, qualitatively, by an increased deposition of newly synthesized proteoglycan in the cell‐associated fraction. An analysis of the most dense proteoglycans (fraction dA1) in the cell‐associated fraction showed that more of the proteoglycans eluted in the void volume of a Sepharose CL‐2B column, indicating that an increased amount of proteoglycan aggregate was synthesized in forskolin‐treated cultures. The proteoglycan monomer dA1D1 secreted into the culture medium of forskolin‐stimulated cultures overlapped in hydrodynamic size with that of control cultures, although cultures stimulated with forskolin and phosphodiesterase inhibitors produced even larger proteoglycans. The hydrodynamic size of 35SO4 and 3H‐glucosamine‐labelled glycosaminoglycans isolated from the dA1D1 fraction of the culture medium was greater in forskolin‐treated chondrocytes, especially from those in which phosphodiesterase inhibitors had been added. These results indicated that forskolin, a direct activator of chondrocyte adenylate cyclase mimicked the effects of cAMP analogues on chondrocyte proteoglycan synthesis previously reported. These results implicate activation of adenylate cyclase as a regulatory event in the biosynthesis of cartilage proteoglycans, and more specifically in the production of hydrodynamically larger glycosaminoglycans.
α‐Thrombin‐induced inositol phosphate formation in go‐arrested and cycling hamster lung fibroblasts: Evidence for a protein kinase C‐mediated desensitization response
Authors: L'Allemain Gilles , Paris Sonia , Magnaldo Isabelle , Pouysségur Jacques 
Published: February 2005

AbstractIn resting Chinese hamster fibroblasts (CCL39) α‐thrombin rapidly induces the breakdown, of phosphoinositides. Accumulation of inositol phosphates (IP), measured in the presence of Li+, is detectable within 5s (seconds) of thrombin stimulation. Formation of inositol tris‐ and bisphosphates slightly precedes that of inositol monophosphate, indicating that thrombin activates primarily the phospholipase C‐mediated generation of inositol trisphosphate from phosphatidylinositol 4,5‐bisphosphate. Initial rates of IP production increase with thrombin concentration, with no apparent saturability over the range 10−4‐10 U/ml. Thrombin‐induced phosphoinositide hydrolysis rapidly desensitizes (t1/2 < 5 min), but a residual activity, corresponding to about 10% of the initial stimulation is sustained for at least 9 h, in contrast with the undetectable activity of G0‐arrested cells. This apparent desensitization may be due to a feedback regulation by protein kinase C, since pretreatment with the phorbol ester 12‐O‐tetradecanoyl phorbol 13‐acetate (TPA) markedly inhibits (by up to 70%) subsequent thrombin‐induced inositol phosphate formation. Conversely, growth factor deprivation of CCL39 cells results in a progressive increase of thrombin‐induced phosphoinositide hydrolysis, from the very low level of exponentially growing cells to the maximal level of G0‐arrested cells. This “up regulation” was found maximal in A51, a very well growth‐arrested CCL39 derivative, and reduced or virtually abolished in two tumoral and growth factor‐relaxed derivatives of CCL39. Although preliminary, this observation suggests that a persistent activation of phosphatidyl inositol breakdown might operate in variants selected for autonomous growth.
Role of lipoproteins in growth of human adult arterial endothelial and smooth muscle cells in low lipoprotein‐deficient serum
Authors: Chen Jan‐Kan , Hoshi Hiroyoshi , McClure Don B. , McKeehan Wallace L. 
Published: February 2005

AbstractRecently improved culture conditions for human adult arterial endothelial and smooth muscle cells from a wide variety of donors have been used to study the effects of lipoproteins on proliferation of both cell types in low serum culture medium. Optimal growth of endothelial and smooth muscle cells in an optimal nutrient medium (MCDB 107) containing epidermal growth factor, a partially purified fraction from bovine brain, and 1% (v/v) lipoprotein‐deficient serum was dependent on either high‐ or low‐density lipoprotein. High‐ and low‐density lipoprotein stimulated cell growth by three‐ and five‐fold, respectively, over a 6‐day period. Optimal stimulation of both endothelial and smooth muscle cell growth occurred between 20 and 60 μg/ml of high‐ and low‐density lipoproteins, respectively. No correlation between the activation of 3‐hydroxyl‐3‐methylglutaryl coenzyme A reductase activity and lipoprotein‐stimulated cell proliferation was observed. Lipid‐free total apolipoproteins or apolipoprotein C peptides from high‐density lipoprotein were partially effective and together with oleic acid effectively replaced native high‐density lipoprotein for the support of endothelial cell growth. In contrast, apolipoproteins or apolipoprotein C peptides from high‐density lipoprotein alone or with oleic acid had no effect on smooth muscle cell proliferation. The results suggest a functional role of high‐ and low‐density lipoproteins and apolipoproteins in the proliferation of human adult endothelial and smooth muscle cells.
The role of cyclic GMP in cells with the properties of smooth muscle cultured from the rat myometrium
Authors: Krall J. Frederick , Morin Anne 
Published: February 2005

AbstractCells growing in culture with previously described properties of rat uterine smooth muscle accumulated 45Ca2+ from the medium. Ca2+ uptake by these cells was stimulated by the addition to the medium of 8‐bromo‐cGMP but not by 8‐bromo‐cAMP. Ca2+ uptake was also stimulated by carbachol and by the nitro‐vasodilator nitroprusside. Although cholinergic agonists have been shown previously to stimulate contraction but not cGMP synthesis in the rat myometrium, both carbachol and nitroprusside stimulated cGMP production by the cultured cells. These results suggested the cells had cholinergic receptor‐mediated functions that reflected some neurotransmitter‐sensitive properties of uterine smooth muscle in situ. When determined by a specific radioligand binding assay, subcellular fractions of the cultured cells bound muscarinic cholinergic agonists and antagonists with affinities expected of the muscarinic receptor.The cells were also sensitive to the β‐adrenergic catecholamine agonist isoproterenol, which stimulated cAMP production but not Ca2+ uptake. Carbachol failed to inhibit isoproterenol‐dependent cAMP production, which is an important property of the cholinergic receptor in uterine smooth muscle in situ. These results suggest some but not all acetylcholine‐sensitive properties of uterine smooth muscle may be retained in cell culture.
System a transport activity in normal rat hepatocytes and transformed liver cells: Substrate protection from inactivation by sulfhydryl‐modifying reagents
Authors: Chiles Thomas C. , Kilberg Michael S. 
Published: February 2005

AbstractThe transport of amino acids by normal rat hepatocytes and several hepatoma cell lines has been examined for inactivation by various protein‐modifying reagents, including the sulfhydryl‐preferring reagents N‐ethylmaleimide (NEM) and p‐chloromercuribenzene sulfonate (PCMBS). Uptake of 2‐aminoisobutyric acid (AIB), a specific probe for hepatic System A‐mediated transport, was equally sensitive to inhibition by the organic mercurial PCMBS in each of the cell types tested. In contrast, the sensitivity of System A to inactivation by NEM was substantially different among the five cell types. Normal hepatocytes showed the greatest sensitivity, while the hepatoma cells varied in their responsiveness from moderate to no inhibition. PCMBS inactivated greater than 85% of the System A activity in rat H4 hepatoma cells within 10 min (t1/2 = 3 min). The inhibition by PCMBS was rapidly reversed by treatment of the cells with dithiothreitol. Amino acids showing a high affinity for System A protected the transport system from inactivation, whereas non‐substrates produced little or no protection. Amino acid‐dependent protection was stereospecific and system‐specific. L‐norleucine competitively inhibited AIB uptake (Ki = 1.9 ± 0.1 mM) in H4 cells and also protected System A from PCMBS‐dependent inactivation (half‐maximal protection occurred at an amino acid concentration of 0.6 + 0.1 mM). N‐bromosuccinimide was completely ineffective as an inhibitor of System A activity in hepatocytes, whereas treatment of H4 rat hepatoma cells with this reagent resulted in greater than 95% inhibition.
Platelet‐derived growth factor stimulation of [3H]‐glucosamine incorporation in density‐arrested BALB/c‐3T3 cells
Authors: Harrington Maureen A. , Wharton Walker , Pledger W. J. 
Published: February 2005

AbstractG0/G1 traverse in density‐arrested BALB/c‐3T3 cells is controlled by multiple serum‐derived growth factors. Platelet‐derived growth factor (PDGF) initiates a proliferative response, whereas factors present in plasma facilitate progression through G0/G1. In the absence of competence formation, progression factors are unable to stimulate cell cycle traverse. We have identified the stimulation of a biochemical process specific to competence formation in BALB/c‐3T3 cells. PDGF treated BALB/c‐3T3 cells incorporated 5–10‐fold more [3H]‐glucosamine (GlcN) into acid‐insoluble material as compared to plateletpoor plasma (PPP) treated cultures. Increased GlcN incorporation occurred in density‐arrested BALB/c‐3T3 cells in response to treatment with other competence factors, fibroblast growth factor, and Ca3 (PO4)2 and was not due to cellcycle traverse. Stimulation of [3H]‐GlcN incorporation by PDGF was time dependent, and increased incorporation of [3H]‐GlcN into protein required de novo protein synthesis. Several mechanisms through which PDGF could increase GlcN incorporation into cellular material were examined. Results of these studies suggest an increase in the cellular capacity to glycosylate proteins is a response to or a part of competence formation.
Phosphorylation of regulatory subunit of type I cyclic AMP‐dependent protein kinase: Biphasic effects of cyclic AMP in intact S49 mouse lymphoma cells
Authors: Russell Joanne L. , Steinberg Robert A. 
Published: February 2005

AbstractIntact S49 mouse lymphoma cells were used as a model system to study the effects of cyclic AMP (cAMP) and its analogs on the phosphorylation of regulatory (R) subunit of type I cAMP‐dependent protein kinase. Phosphorylation of R subunit was negligible in mutants deficient in adenylate cyclase; low levels of cAMP analogs, however, stimulated R subunit phosphorylation in these cells to rates comparable to those in wild‐type cells. In both wild‐type and adenylate cyclase‐deficient cells, R subunit phosphorylation was inhibited by a variety of N6‐substituted derivatives of cAMP; C‐8‐substituted derivatives were generally poor inhibitors. Two derivatives that were inactive as kinase activators (N6‐carbamoylmethyl‐5′‐AMP and 2′‐deoxy‐N6‐monobutyryl‐cAMP) were also ineffective as inhibitors of R subunit phosphorylation. Preferential inhibition by N6‐modified cAMP analogs could not be ascribed simply to selectivity for the more aminoterminal (site I) of the two cAMP‐binding sites in R subunit: Analog concentrations required for inhibition of R subunit phosphorylation were always higher than those required for activation of endogenous kinase; 8‐piperidino‐cAMP, a C‐8‐substituted derivative that is selective for cAMP‐binding site I, was relatively ineffective as an inhibitor; and, although thresholds for activation of endogenous kinase by site I‐selective analogs could be reduced markedly by coincubation with low levels of site II‐selective analogs, no such synergism was observed for the inhibitory effect. The uncoupling of cyclic nucleotide effects on R subunit phosphorylation from activation of endogenous protein kinase suggests that, in intact cells, activation of cAMP‐dependent protein kinase requires more than one and fewer than four molecules of cyclic nucleotide.
Internalisation and recycling of the granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) receptor on a murine myelomonocytic leukemia
Authors: Walker Francesca , Burgess Antony W. 
Published: February 2005

AbstractRadioiodinated granulocyte‐macrophage colony‐stimulating factor (125I‐GM‐CSF) binds to specific receptors (molecular weight approximately 50,000 daltons) on the murine myeiomonocytic leukemia, WEHI‐38D+. At 4°C 125I‐GM‐CSF remains on the surface of the cells and can be eluted by washing the cells with acidified isotonic buffer. When the cells are warmed to 37°C, the 125I‐GM‐CSF is internalized rapidly (t1/2: 7 min). The internalisation appears to be entirely receptor mediated and is independent of energy sources inhibited by sodium azide. This GM‐CSF‐mediated internalisation is not due to a general increase in the turnover of cell surface molecules as the specific binding of 25I‐transferrin is not affected by incubation of WEHI‐3BD+ cells with GM‐CSF. The initial 125I released when the cells are warmed to 37°C appears to be intact 125I‐GM‐CSF; however, after 2 h 80% of the 125I released was not precipitable with trichloroacetic acid and presumably represented degraded 125I‐GM‐CSF. Ammonium chloride or monensin reduced the release of 125I‐GM‐CSF from the cells, suggesting that the receptor‐bound ligand was processed through the lysosomes. A cor siderable proportion of the internalised GM‐CSF receptors were recycled to the surface and were available for ligand binding. Synthesis of new GM‐CSF receptors contributed to the re‐expression of GM‐CSF receptors after down‐regulation and it is possible that the GM‐CSF enhances the synthesis of its own receptors.
Enucleation of normal and transformed cells
Authors: Degaetano Douglas , Schindler Melvin 
Published: February 2005

AbstractA quantitative analysis based on cenrifugal force requirements for enucleation was developed to examine the response of a number of untransformed and transformed cell lines to cytochalasin mediated enucleation. Examination of the extent of cell enucleation as a function of centrifugal force resulted in a series of response curves demonstrating that enucleation g force requirements varied between Balb/c 3T3, Swiss 3T3, and Kirsten sarcoma virus transformed Balb/c 3T3 (3T3‐K). A four times greater centrifugal force was required to reach 50% enucleation for transformed Balb/c 3T3‐K when compared to Swiss 3T3. A qualitative correlation could be observed between ease of enucleation and the existence of a well‐formed stress fiber network. A comparison of cytochalasin B and D suggested that cytochalasin D was far more effective in the enucleation of transformed cells. Experiments with 2‐deoxyglucose and monensin provided evidence that decreasing cellular ATP levels, either directdly or potentially by uncoupling ion transport from ATP generation, can decrease the efficiency of enucleation It is suggested that the organization of the cytoskeleton is affected by the altered cellular ATP levels which can affect the centrifugal requirements of enucleation.
Effect of recombinant tumor necrosis factor on HL‐60 cells: Cell‐cycle specificity and synergism with actinomycin D
Authors: Darzynkiewicz Zbigniew , Carter Sally P. , Old Lloyd J. 
Published: February 2005

AbstractThe tumor necrosis factor (TNF) exhibits a multitude of activities depending on the type of target cells. We characterized the cytostatic and cytotoxic effects of recombinant TNF, alone and in combination with actinomycin D (AMD), on the human leukemic cell line HL‐60. Because HL‐60 cells, when triggered to monocytic differentiation by phorbol esters, are known to produce and secrete TNF, their sensitivity to the factor could indicate an autocrine function of TNF in this cell system. Indeed, HL‐60 cells were affected by TNF; their doubling time was increased by about 50% and progression through the cell cycle was perturbed. Initially, (up to 8 h) TNF induced a temporary arrest in G2 while later (24–48 h) it delayed progression through the G1 phase. Also, a transient increase in RNA content peaking at 6–8 h was apparent. The cytotoxicity of TNF alone was low. Thus, TNF may be involved in the regulation of the cell cycle of HL‐60 cells during early stages of their differentiation.The cytotoxicity of TNF was markedly potentiated in the presence of AMD; the effect was AMD but not TNF concentration‐dependent. Whereas at 20 and 50 ng/ml of AMD alone nonviable cells did not exceed 20% during the first 24 h of treatment, their proportion increased to 80 and 90%, respectively, in the presence of TNF. The most sensitive were cells in the S phase of the cell cycle. The observed synergistic effect of TNF and AMD does not appear to be caused by the action of TNF increasing the permeability of the cell membrane to AMD. The results indicate that HL‐60 cells, ordinarily resistant to the cytotoxic action of TNF, can be rendered sensitive by treatment with AMD. This implies that a combination of TNF and AMD may be considered in oncology for treatment of tumors otherwise nonresponding to TNF alone.