bluuebunny/crossref_metadata_2025_split huggingface dataset:
Amino acid pools in CHL V79 cells during induction of thermotolerance: Reduction in free intracellular glutamine
Authors: Goh Chin Joo , Dumbroff Erwin B. , Lepock James R. 
Published: February 2005

AbstractThe amino acid pools in Chinese hamster lung V79 cells were measured as a function of time during hyperthermic exposure at 40.5° and 45.0°C. Sixteen of the 20 protein amino acids were present in sufficient quantity to measure accurately. The total amino acid pool and all individual amino acids, except glutamine, remained relatively constant for at least 90 min at 40.5°C and for 30 min at 45°C. The glutamine pool decreased rapidly to 20% of its control value within 30 min at 40.5°C with a T½ = 15 min. At 45°C, the decrease was 36%. Thermotolerance developed at 40.5°C with a T½ =30 min; thus, glutamine depletion preceeds the development of thermotolerance. The depletion of glutamine is probably due to increased metabolism and oxidation of glutamine through the TCA cycle at hyperthermic temperatures. Glutamine, as is true for other amino acids, was shown to protect proteins from thermal inactivation and V79 cells from hyperthermic killing when added in excess (4–10 mM) to the medium during heat stress. However, the stability of the total amino acid pool during the development of thermotolerance indicates that resistance to heat does not result from the accumulation of amino acids which then protect against thermal damage. The effects of the large decrease in the glutamine pool are unknown, although glutamine depletion may act as a signal for part of the heat shock response.
Evidence for independent metabolism and cell surface localization of cellular proteoglycans and glycosaminoglycan free chains
Authors: Piepkorn Michael , Hovingh Peter , Linker Alfred 
Published: February 2005

AbstractThe synthesis and turnover of metabolically labeled proteoglycans from medium, cell layer, and substratum‐associated compartments were characterized in four cell lines of fibroblastic origin, including a fibrosarcoma line, and in the murine melanoma cell type, B16.F10. Substantial differences were apparent between the various cell types with regard to quantities, hydrodynamic sizes, and compartmentalization of labeled product. Such variations were greater between the different cell lines than between separately labeled cultures of the same cell type. Greater than 85% of cell‐associated proteoglycans were accessible to glycosaminoglycan‐degrading enzymes added to the medium of monolayer cultures, demonstrating their principal location to be external to the cell membrane. Apparent glycosaminoglycan free chains, determined by a lack of change in hydrodynamic size following alkaline elimination, were among the products from each cell line and were similarly found to be in a principally pericellular location. Results from label‐chase studies demonstrated apparent independent kinetics for proteoglycans and glycosaminoglycan free chains, with little conclusive evidence for precursor‐product relationships. Also, their processing by the cells was different, since the proteoglycans were shed largely unchanged into the medium for the three cell lines evaluated, whereas the free chains were not recoverable from the medium in significant amounts. The latter observation suggests the internalization of cell surface‐associated free chains and their depolymerization at an intracellular site. The results, which indicate that the content, cellular disposition, and turnover of proteoglycans are quite variable between the cell lines studied, may reflect fundamental cell type‐specific specialization in the metabolism of these complex substances. Furthermore, the data raise the interesting possibility that glycosaminoglycan free chains may have biological functions at the cellular level, independent of proteoglycans.
Prostacyclin and prostaglandin E2 secretions by bovine pulmonary microvessel endothelial cells are altered by changes in culture conditions
Authors: Chung‐Welch Nancy , Shepro David , Dunham Bernadette , Hechtman Herbert B. 
Published: February 2005

AbstractThe isolation and culture of pulmonary microvascular endothelial (MVE) cells from bovine lungs were established. Primary and early passaged cultures grew best in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% equine plasma‐derived serum, bovine retinal growth extract (1%), and heparin (90 μ/ml) on gelatin coated plates. A second tissue culture procedure was prepared in which the isolation technique was the same except the culture medium consisted of DMEM supplemented with 10% plasma‐derived serum. Either growth medium produced homogeneous, long term, serial cultures for up to 16 passages. MVE cells were characterized in part based on their morphology by light and electron microscopy and positive reaction to Factor VIII‐related antigen and uptake of 1,1′‐dioctacecyl‐1,3,3,3′3‐tetramethyl‐indocarbocyanine perchlorate acetylated low density lipoprotein (Dil‐Ac‐LDL). MVE cells were also positive for angiotensin‐converting enzyme (ACE) activity and the presence of ACE was localized on the cells by indirect immunofluorescence. MVE cells maintained in the presence of heparin and growth factor principally synthesized prostaglandin (PG) E2 (1512 ± 159 pg/mg protein at 15 min) and smaller amounts of prostacyclin (PGl2) and thromboxane (Tx) A2 (316 ± 43 and 588 ± 105 pg/mg protein/15 min respectively) as measured by radioimmunoassay. However, prostanoid release was not elevated from basal levels upon incubation with arachidonic acid, bradykinin, or ionophore A23187. In contrast, MVE cells cultured without heparin and growth factor secreted more PGl2 than PGE2 (862 ± 84 and 89 ± 12 respectively). Incubation with arachidonic acid, bradykinin, or ionophore A23187 induced significant increases in PGl2 and PGE2 production (P ≤ 0.01). Pulmonary artery endothelial (PAE) cell cultures used as a control for comparison predominantly synthesized PGl2. These findings suggest that in vitro the vessel source and culture conditions may qualitatively and quantitatively affect the pattern and levels of prostanoid synthesized and secreted.
Sodium transport in rat renal papillary collecting tubule cells in culture
Authors: Konieczkowski M. , Dunn M. J. 
Published: February 2005

AbstractSodium transport in the papillary collecting duct (PCD) is poorly understood because of the inaccessibility of the distal nephron to micropuncture. Cultured rat renal papillary collecting tubule (RPCT) cells were investigated as a model for the PCD. RPCT cells have the morphologic appearance and hormonal responsiveness of the papillary collecting tubule. Sodium transport was studied using 22Na+ uptake measurements. Sodium uptake, measured at 23°C in the absence of K+ and in the presence of 0.5 mM ouabain, was saturable at 100 mM extracellular NaCI, and half‐maximal uptake occurred at 40 mM NaCI. The accumulation of 22Na+ appeared to be intracellular and was regulated by (Na+, K+)‐ATPase activity, since activation of the Na+/K+ pump with K+ reduced 22Na+ accumulation by 90%. The time course for uptake was linear, showed only a single component, and followed first order kinetics with a t½ of 16 min. Amiloride and lithium inhibited 22Na+ influx, and a Dixon plot was linear, with a Ki of 16 μM amiloride. Chloride replacement or 1 mM furosemide, with or without K+, reduced uptake by only 20%. Sodium efflux from RPCT cells in the presence of ouabain showed a similar time course (t½, 15 min) and was also inhibited by amiloride (IC50 =20 μM). Increased extracellular pH stimulated 22Na+ uptake and inhibited 22Na+ efflux. Addition of permeable organic acids, acetate, and bicarbonate, enhanced 22Na+ uptake. These results are consistent with Na+/H+ and Na+/Na+ exchange as mechanisms of 22Na+ uptake in the RPCT cell. This exchanger may be important in regulation of transepithelial sodium flux, maintenance of intracellular pH and cell volume, and hormonal stimulation of the papillary collecting duct.
Lysosomotropic amines modulate neutrophil calcium homeostasis
Authors: Styrt Barbara , Klempner Mark S. 
Published: February 2005

AbstractLysosomotropic amines can raise the acidic internal pH of the neutrophil lysosome and inhibit neutrophil function. Because pH and calcium regulation are intimately connected in various types of excitable cells, we studied the effects of several lysosomotropic weak bases on neutrophil calcium homeostasis. Base‐treated cells had normal to minimally elevated resting cytosol free calcium, but weak bases produced significant release of calcium from organelles when this release was directly measured in permeabilized cells, even after depletion of inositol‐triphosphate‐sensitive stores. Collapse of transmembrane pH gradients with monensin similarly released organelle calcium. The initial cytosol calcium response to f‐met‐leu‐phe was enhanced by some of the lysosomotropic amines but the calcium rise was more transient in base‐treated cells than in control samples. These findings suggest that existence of an acidic intracellular compartment, such as the lysosome, is important to normal calcium homeostasis in the neutrophil and that pH sensitivity and inositol triphosphate sensitivity may define two pools of releasable organelle calcium. The effect of pH perturbation on calcium homeostasis may partially account for the inhibition of neutrophil function by lysosomotropic amines.
Modulation of nuclear proto‐oncogene expression and cellular growth in myeloid leukemic cells by human interferon alpha
Authors: Marshall Adele H. , Alper Deborah , Hiscott John 
Published: February 2005

AbstractTo address the mechanisms that regulate expression of specific growth‐related nuclear proto‐oncogenes, the transcript levels of the c‐fos, c‐myc, (2′5′)‐oligoadenylate synthetase, IFN‐α1, and IFN‐β1 genes have been measured in the human leukemic cell lines KG‐1, U937, and HL‐60 following growth stimulation by serum, induction of differentiation by tumor‐promoting agents, and/or treatment of cells with exogenously supplied alpha interferon (rIFN‐α2). Production of fos and myc RNA was measured by S1 mapping, using fos DNA probes which identified either primary unspliced transcripts or steady‐state‐spliced mRNA levels, and using a myc probe which spanned the two major c‐myc start sites, P1 and P2. Pretreatment of a quiescent KG‐1 cell population with IFN for 18 hours before serum addition decreased the stimulation of both fos and myc RNA production. In HL‐60 and U937 cells, IFN pretreatment had no inhibitory effect on serum‐induced fos or myc transcription; however, in U937, rIFN‐α2 treatment alone stimulated fos mRNA 11‐fold. Expression of 2′5′ oligoadenylate synthetase was induced in IFN‐treated cultures but not in cells stimulated with serum alone. No serum‐induced IFN‐α1 or IFN‐β1 gene expression was observed in KG‐1 or U937 cells. These results demonstrate that exogenous rIFN‐α2 treatment of quiescent KG‐1 cells can antagonize the effect of growth factors by altering expression of nuclear proto‐oncogenes, but in general growth inhibition is not obligatorily coupled to inhibition of proto‐oncogene transcription.
Interferon induces cell fusion and the formation of multinuclear cells in a culture of ehrlich ascites tumor cells
Authors: Tominaga Shin‐Ichi 
Published: February 2005

AbstractThe increase in number of Ehrlich ascites tumor (EAT) cells was diminished significantly when the cell culture was treated with 1,000 lU/ml of recombinant mouse alpha or beta interferon (IFN). Microscopical observation revealed that almost all the cells showed bi‐or multinuclear morphology 3 to 5 days after IFN treatment. Furthermore, a videorecording showed that each multinuclear cell arose by fusion after mitotic division of one parental cell.
Effect of heat shock on RNA metabolism in HeLa cells
Authors: Sadis Seth , Hickey Eileen , Weber Lee A. 
Published: February 2005

AbstractIncubation of HeLa cells at 42°C results in pronounced inhibition of the accumulation of 18S and 28S ribosomal RNA (rRNA) and non‐heat shock polyadenylated messenger RNA (mRNA) in the cytoplasm. Accumulation of transfer RNA and 5S ribosomal RNA is not affected. Transcription of rRNA precursor is reduced to approximately 50% of the 37°C rate after 10 min of hyperthermia and declines to 30 of the control rate after 1 hr. In contrast, the accumulation of mature rRNA in the cytoplasm is inhibited more than 95%. Quantitative hybridization experiments and Northern blot analysis detect little accumulation of rRNA precursor sequences in nuclei, suggesting that the majority of the rRNA that is synthesized is degraded. Heat stress at 42°C was found to have little effect on transcription of most non‐heat shock mRNAs. However, accumulation of individual non‐heat shock mRNAs in the cytoplasm proceeds at reduced rates. These results indicate that the primary effect of elevated temperature on RNA metabolism in mammalian cells is inhibition of processing and/or transport. Despite this, steady‐state levels of abundant and rapidly turning over mRNA species remain unchanged during prolonged heat stress. We find that the half‐life of c‐myc mRNA increases greater than twofold at 42°C. Thus, 42°C heat stress appears to inhibit both accumulation and turnover of non‐heat shock mRNA.
Preparation of basolateral membranes that transport p‐aminohippurate from primary cultures of rabbit kidney proximal tubule cells
Authors: Yang Il Suk , Goldinger James M. , Hong Suk Ki , Taub Mary 
Published: February 2005

AbstractThe organic anion p‐aminohippurate (PAH) is specifically secreted by the renal proximal tubule. The possibility was examined that the probenecid sensitive PAH transport system (which is involved in this secretory process in renal proximal tubule cells in vivo) is retained in primary cultures of rabbit kidney proximal tubule cells. Significant 3H‐PAH uptake into primary cultures of proximal tubule cells was observed. After 10 min, 150 pmole PAH/mg protein had accumulated intracellularly. Given an intracellular fluid volume of 10 μl/mg protein, the intracellular PAH concentration was estimated to be 15 μM. The initial rate of PAH uptake (when 50 μM PAH was in the uptake buffer) was inhibited 50% by 2 mM probenecid. Intact monolayers also exhibited Na+ ‐dependent alpha methyl‐D‐glucoside uptake (an apical marker). Basolateral membranes were purified from primary rabbit kidney proximal tubule cell cultures. Probenecid sensitive PAH uptake into the membrane vesicles derived from the primary cultures was observed. The rate of PAH uptake was equivalent to that obtained with vesicles obtained from the rabbit renal cortex. No significant Na+ ‐dependent D‐glucose uptake into the vesicles was observed, indicating that primarily basolateral membrane vesicles had indeed been obtained.
Norepinephrine modulates the growth‐inhibitory effect of transforming growth factor‐beta in primary rat hepatocyte cultures
Authors: Houck Keith A. , Cruise Jennifer L. , Michalopoulos George 
Published: February 2005

AbstractTGF‐beta is a potent inhibitor of EGF‐induced DNA synthesis in primary rat hepatocyte cultures. Norepinephrine (NE) was shown to modulate this inhibition of DNA synthesis. It produced a five‐fold increase, from 2.8 pM to 14.4 pM, in the ID50 for TGF beta. The effect was dose‐dependent and was significant at concentrations of 10−6M NE and greater. The modulation by NE was mediated by the alpha1‐adrenergic receptor as shown by the ability of the alpha1 antagonist prazosin to block the activity. This effect might be important during liver regeneration in allowing escape of hepatocytes from negative growth control exerted by TGF‐beta.
Chondrogenesis in chick limb mesenchyme in vitro derived from distal limb bud tips: Changes in cyclic AMP and in prostaglandin responsiveness
Authors: Biddulph David M. , Sawyer Linwood M. , Dozier Mandy M. 
Published: February 2005

AbstractChondrogenesis was monitored in micromass cultures of mesenchymal cells derived from the distal tip of stage‐25 chick limb buds over a 6‐day period. Alcian green staining and immunofluorescent localization of cartilage‐specific proteoglycans revealed the appearance of cartilage matrix by day 3 of cell culture. By day 6, cultures contained a uniform and homogeneous population of fully differentiated chondrocytes throughout the cell layer, with only a narrow rim of nonchondrogenic cells around the extreme periphery of the culture. Synthesis of sulfated glycosaminoglycans also progressively increased between days 3 and 6, being 8‐fold higher at day 6 than at day 1 of culture. Both adenylate cyclase (AC) activity and cAMP concentrations increased dramatically during the first 2 days of culture, reaching maximal levels by day 2, which remained elevated and stable throughout the remaining chondrogenic period (days 3–6). Responsiveness of both AC and cAMP concentrations of the cells to PGE2 was maximal by day 1 of culture and was increased over control cells by 12‐fold and 8‐fold respectively. Both responses, however, were dramatically reduced by day 3, at which time the initiation of cartilage formation was apparent. Responsiveness of cells during the prechondrogenic period to PGE2 was relatively specific in that no effects could be demonstrated with equivalent concentrations of PGF2α or 6‐keto‐PGF1α, although PGl2 did produce increases in cAMP concentrations of about 50% of those of PGE2. These results indicate that previously reported changes in the cAMP system in heterogeneous cell cultures derived from whole limb buds reflect changes occurring in the chondrogenic cell type and indicate further that peak responsiveness of the cAMP system of these cells to prostaglandins is restricted to prechondrogenic developmental periods.
Probability of transition into cell cycle does not depend on growth factor concentration
Authors: Böhmer Ralph M. , Beattie Lynette D. 
Published: February 2005

AbstractWhen quiescent cells in monolayer culture are stimulated to proliferate with growth factor, the entry into S‐phase or mitosis appears to follow first‐order kinetics, with a probability to enter the cell cycle that depends on growth factor concentration (Smith and Martin, 1973). Suboptimal growth factor concentrations also lead to a decreased fraction of the cell population that responds to the stimulation (Brooks et al., 1984). Using flow cytometry, we have re‐investigated this dual effect of growth factor concentration on cultures of quiescent normal human skin fibroblasts, stimulated with submaximal concentrations of fetal calf serum, epidermal growth factor, and platelet‐derived growth factor. The size of the responding population decreased with decreasing concentration of growth factor, but the time course of cell division within this responding population was identical for all growth factor concentrations. This is in conflict with previous concepts and indicates that the entry into the proliferative state is based on a decision mechanism that cannot be adequately described using transition probabilities determined by mitogen concentration.
Extracellular matrix modulates the function of human melanocytes but not melanoma cells
Authors: Ranson Marie , Posen Solomon , Mason Rebecca S. 
Published: February 2005

AbstractNormal human epidermal melanocytes are attached to a basement membrane, a specialized form of extracellular matrix (ECM), located between the epitheliumand underlying dermal tissues. To determine whether ECM influences pigmented cell behavior in vitro, human epidermal melanocytes and melanoma cells were cultured on uncoated or ECM‐coated plastic culture surfaces, and a comparison was made between growth and function in the presence or absence of ECM. Melanocytes cultured on ECM‐coated surfaces developed flatter and larger cell bodies and produced more melanin than melanocytes cultured on uncoated surfaces. In the presence of phorbol‐myristate‐acetate and cholera toxin, the rate of melanocyte replication was increased by ECM. In the absence of these mitogens, ECM significantly enhanced the adhesiveness of nonproliferating melanocytes. ECM had little or no effect on these parameters (morphology, tyrosinase activity, replication) in a pigmented human malignant melanoma cell line. These findings indicate that normal human epidermal pigment cells have the ability to recognize and respond to matrix signals, whereas this capacity appears to be absent in melanoma cells.
Protein synthesis inhibitors prevent the induction of laminin B1, collagen IV (α1), and other differentiation‐specific mRNAs by retinoic acid in F9 teratocarcinoma cells
Authors: Wang Sho‐Ya , Gudas Lorraine J. 
Published: February 2005

AbstractSeveral differentiation‐specific genes, including those for collagen IV and laminin, are induced by retinoic acid (RA) in mouse F9 teratocarcinoma cells. Dibutyryl cAMP can enhance the effect of RA in these cells, but dibutyryl cAMP alone does not induce these genes. Inhibition of RNA synthesis with 5‐6‐dichloro‐1‐B‐D‐ribofuranosylbenzimidazole prevents the induction of these genes by RA; inhibition of DNA synthesis with aphidicolin does not prevent the induction. In vitro transcription studies (Wang et al., Dev. Biol., 107:75–86, 1985) demonstrate that these differentiation‐specific genes are regulated by RA at least partially at the level of transcription. To determine whether the regulation of transcription of these differentiation‐specific genes is a primary effect of RA, we measured the sensitivity of the induction of mRNAs specific for these RA‐inducible genes to inhibitors of protein synthesis. RNA was isolated from F9 cells that had been treated for 20 hr with RA (with or without dibutyryl cAMP) in the presence or absence of either cycloheximide or puromycin. We then hybridized the 32P‐labeled recombinant plasmids collagen IV (α1) (pcl5), laminin B1 (pcl56), and pcJ6 to RNA from the treated cells. Both cycloheximide and puromycin inhibited the RA induction of the collagen IV (α1), laminin B1, and J6 mRNAs. In contrast, in a control experiment, a 20‐hr treatment with cycloheximide did not inhibit the accumulation of metallothionein l‐specific mRNA in response to zinc in F9 cells. Thus protein synthesis is required for the expression of the collagen IV (α1), laminin B1, and J6 genes, and this result suggests that the transcriptional regulation of these genes by RA is indirect.