bluuebunny/crossref_metadata_2025 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

The 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

The 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

The 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

Sodium 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

Lysosomotropic 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

To 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

The 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

Incubation 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

The 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

TGF‐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

Chondrogenesis 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

When 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

Normal 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

Several 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.
Identification of insulin receptors on the insulin‐independent variant 1246‐3A cell line
Authors: Gazzano Helene , Serrero Ginette
Published: February 2005

1246‐3A cell line is an insulin‐independent variant isolated from the adipogenic cell line 1246 which can proliferate in the absence of insulin, has lost the ability to differentiate, and secretes an insulin‐related factor called IRF similar to pancreatic insulin and different from IGFs. In contrast, the parent adipogenic cell line 1246 is dependent on the presence of insulin to proliferate and differentiate in defined medium. In the present paper, we examined if the loss of response to insulin observed for 1246‐3A cells was accompanied by alterations in the insulin receptor properties. Insulin binding and tyrosine kinase activity of insulin receptors isolated from 1246‐3A cells and from the parent cell line 1246 were measured; 125I‐insulin binding to intact cells was 75% lower for the 1246‐3A cells than for the 1246 cells. This was due to a decrease in receptor number without major change in receptor affinity. However, when the cells were solubilized in 1% Triton X‐100 and the insulin receptor was partially purified by chromatography on wheat germ agglutinin‐agarose, a similar pattern of binding was observed for both cell lines. Down regulation of insulin receptors by insulin occurred in a dose‐dependent fashion, which was similar for both cell lines. Phosphorylation experiments were performed by incubation of the partially purified insulin receptor with insulin and [γ‐32P]ATP. They indicated that insulin stimulated phosphorylation of the 95‐kDa molecular weight β subunit of the receptor, in a similar fashion for both cell types. These data suggest that the insulin‐independent cell line 1246‐3A does not possess a specific defect in the insulin receptor which alters both its binding and autophosphorylation properties and that the loss of response to insulin can be attributed to the fact the 1246‐3A cells secrete IRF which bind to cell surface receptors and stimulate their proliferation.
Similarities in glycosylation and transport between the secreted and plasma membrane forms of the epidermal growth factor receptor in A‐431 cells
Authors: Soderquist Ann Mangelsdorf , Stoscheck Christa , Carpenter Graham
Published: February 2005

We have studied the synthesis and oligosaccharide processing of the 110,000 dalton form of the epidermal growth factor (EGF) receptor that is secreted into the medium of A‐431 cells. Its 90,000 dalton precursor is soluble within the lumen of intracellular membrane vesicles shortly after synthesis, indicating that it lacks a membrane anchor. Analysis of labeled glycopeptides reveals that the glycosylation of the 110,000 dalton, secreted receptor is very similar to that of the 170,000 dalton, plasma membrane receptor. Based on Concanavalin A‐Sepharose elution profiles of its glycopeptides, the secreted receptor has both complex and high‐mannose N‐linked oligosaccharides. Also, like the plasma membrane receptor, the secreted receptor contains N‐acetylgalactosamine residues in its complex chains. Not only are major features of oligosaccharide processing of the soluble and membrane‐bound forms of the receptors similar, but the kinetics of transport to the cell exterior is the same for each. These data indicate that the glycosylation pattern and kinetics of cellular transport of the EGF receptor are determined by factors other than the sequence of its cytoplasmic and transmembrane domains.
Inhibition of macrophage activation by isoquinolinesulfonamides, phenothiazines, and a napthalenesulfonamide
Authors: Holian Andrij , Jordan Mark K. , Nguyen Hanh V. , Devenyi Zoltan J.
Published: February 2005

The influence of isoquinolinesulfonamides (H‐7 and H‐8), phenothiazines(trifluoperazine and fluphenazine), and a naphthalenesulfonamide (N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide (W‐7)) on stimulated superoxide anion production and phosphatidyl inositol (PI) cycle activity was investigated in the guinea pig alveolar macrophage. All five drugs were able to inhibit superoxide anion production stimulated by n‐formyl‐nel‐leu‐phe (FNLP), leukotriene B4 (LTB4), and phorbol‐12,13‐dibutyrate (PDB). The order of potency was trifluoperazine ≥ fluphenzine > H‐7 = W‐7 > H‐8. The dose response curves could be shifted to less efficacy by increasing extracellular calcium. By itself, W‐7 markedly stimulated 45Ca+2 efflux, efflux, fluphenazine and trifluoperazine slightly stimulated 45Ca+2 efflux from macrophages preloaded with 45Ca+2. Consistent with these results, W‐7 markedly stimulated PI cycle activity, fluphenazine and trifluoperazine slightly stimulated PI cycle activity, while H‐7 and H‐8 had no significant effects on PI cycle activity. In addition, W‐7 by itself was able to stimulate a weak and short‐lived “burst” of superoxide anion production. In order to evaluate whether a site of action of the inhibitors was at protein kinase C and whether protein kinase C was involved in terminating the normally short‐lived FNLP‐ and LTB4‐stimulated macrophage activation, fluphenazine and H‐7 were used to evaluate the duration of FNLP‐ and LTB4‐stimulated PI cycle activity, at concentrations of the inhibitors that significantly blocked stimulated superoxide anion production. In all cases, FNLP and LTB4 still stimulated PI cycle activity, which still terminated even though protein kinase C was inhibited. These results suggest that all five drugs block protein kinase C, but H‐7 was the most specific in its action at protein kinase C, while the phenothiazines and W‐7 have multiple sites of action. In addition, these results suggest that protein kinase C may not function to cause the termination of FNLP‐ and LTB4‐stimulated PI cycle activity and subsequent superoxide anion production.
Stimulation by thyroid hormone of phosphate transport in primary cultured renal cells
Authors: Noronha‐Blob Lalita , Lowe Valerie , Sacktor Bertram
Published: February 2005

The regulation by thyroid hormone of phosphate transport in primary cultured chick renal cells was examined. The more physiologically active L‐analogs of triiodothyronine and thyroxine, but not the D‐analogs of the hormones, stimulated the Na+ ‐dependent phosphate uptake system. Na+ ‐independent phosphate uptake and Na+ ‐dependent uptakes of α‐methylglucoside and L‐proline were unaffected. The increase in Na+ ‐dependent phosphate uptake was concentration dependent, exhibited an induction period, and was blocked by inhibitors of RNA and protein synthesis. The stimulation of phosphate uptake by triiodothyronine was due to an increased Vmax rather than to an altered affinity for phosphate. These findings demonstrate that thyroid hormone acts directly on renal cells to modulate phosphate transport and suggest that the renal cell system may serve as a model to examine the mechanism by which thyroid hormone controls gene expression and regulates plasma membrane transport function.
Intercellular adhesion molecule‐1 (ICAM‐1) is involved in the cytolytic T lymphocyte interaction with a human synovial cell line
Authors: Mentzer Steven J. , Rothlein Robert , Springer Timothy A. , Faller Douglas V.
Published: February 2005

The cell surface molecules involved in the human cytolytic T lymphocyte (CTL)‐synovial cell interaction may play an important role in T cell interactions with connective tissue mesenchymal cells. To examine the molecular basis for the CTL‐synovial cell interaction, we immortalized synovial cell explants to establish the cell line SYN.SPP. The SYN.SPP cell line was compared to the established B lymphoblastoid cell line JY. Cell surface immunofluorescence demonstrated significantly different levels of the immunologically relevant cell surface molecules ICAM‐1 and LFA‐3. Both cell lines were used to stimulate CTL precursors. After several months in culture, CTL lines stimulated by the SYN.SPP and JY cell lines demonstrated HLA class I‐directed cytolytic activity. The cell surface molecules utilized by the anti‐SYN.SPP and anti‐JY CTL lines were identified by monoclonal antibody (MAb) inhibition. MAb recognizing the CTL cell surface molecules CD3, CD8 and LFA‐1 (CD11a) significantly inhibited CTL‐mediated lysis of both target cells. An interesting observation was that the anti‐SYN.SPP CTL line appeared to utilize the ICAM‐1 and not the LFA‐3 target cell molecule. In contrast, the anti‐Y CTL line utilized the LFA‐3 and not the ICAM‐1 membrane molecule. These results indicate that CTL interactions with connective tissue mesenchymal cells may be regulated by a unique pattern of antigen nonspecific cell‐cell interaction molecules.
Elevation of angiotensin converting enzyme by 3‐isobutyl‐1‐methylxanthine in cultured endothelial cells: A possible role for calmodulin
Authors: Dasarathy Yamuna , Fanburg Barry L.
Published: February 2005

Incubation of cultured bovine pulmonary artery endothelial cells with 200 μM of 3‐isobutyl‐1‐methylxanthine (IBMX) for 24 hr produced a five‐to tenfold increase in cellular angiotensin converting enzyme activity (ACE) above that of untreated control cells. A lesser increase was observed in medium ACE. Other methylxanthines produced a similar, but less marked, effect. The elevation of ACE seemed to require de novo protein synthesis since it was reduced by 0.1 μ/ml cycloheximide. Elevation of cellular cAMP was detected at 30 min after introduction of IBMX, then rapidly returned to control levels at 1 hour, while elevation in cellular ACE at 24 hr required contact with IBMX for at least 2 hr. Hence, the transient elevation in cAMP is unlikely to be the cause of the elevation of ACE. Phorbol ester and synthetic diacyl glycerol OAG, activtors of protein kinase C, did not elevate ACE. Indomethacin, at a concentration known to inhibit cycloxygenase activity, had no effect on the elevation of ACE. The elevation of ACE by IBMX was not affected by the calcium channel blocker verapamil or the calcium chelator EGTA. In contrast, the effect of IBMX was totally abolished by the calmodulin inhibitors trifluoperazine and calmidazolium. The data show that IBMX elevates endothelial cell ACE and suggest that the elevation is mediated by a calcium‐calmodulin complex. The studies demonstrate a novel effect of methylxanthines on endothelial cells in culture.
Influence of the extracellular matrix on the proliferative response of human skin fibroblasts to serum and purified platelet‐derived growth factor
Authors: Rhudy Roberta W. , McPherson John M.
Published: February 2005

The culture of adult human skin fibroblasts on reconstituted bovine type I fibrillar collagen gels, ranging in concentration from 2.5–35.0 mg/ml, results in a reduction in proliferation rate by 40%–60% as measured by (3H) thymidine incorporation. The suppressive effect was noted when cells were cultured in both human and bovine serum. Drying the gels into thin films abolishes the suppressive effect of the fibrillar collagen on cell proliferation. Cell attachment studies showed that differences in the proliferation rate of cells on the various substrata were not simply due to differences in initial attachment. Studies with purified platelet‐derived growth factor (PDGF) demonstrated that the reduced responsiveness of cells to this factor, when cultured on collagen gels as compared to plastic, was largely responsible for the reduced proliferative activity of the cells when cultured in the presence of serum. The reduced proliferative activity of fibroblasts in response to PDGF, when cultured on collagen gels, was confirmed by total DNA determination. It was shown that the reduced responsiveness of cells to PDGF was not simply because the factor bound to the fibrillar collagen gel or was inaccessible to the cells. The data indicate that the reduced proliferation rate of fibroblasts cultured on collagen gels is a direct result of the influence of the extracellular matrix on the cells' ability to respond to a soluble mitogenic mediator.
Effect of hypo‐osmotic incubation on membrane recycling
Authors: Novak Jeanne M. , Ward Diane McVey , Buys Saundra S. , Kaplan Jerry
Published: February 2005

Incubation of alveolar macrophage in hypo‐osmotic media causes a time‐and temperature‐dependent increase in the number of surface receptors for three different ligands. Exposure of cell to solutions of 210 mOsM or less, at 37°C but not at 0°C, resulted in an increase in the number of surface receptors for diferric transferrin, alpha‐macroglobulin‐protease complexes, and mannose‐terminated glycoproteins. Upon media dilution at 37°C, surface receptor number reached a maximum within 5 min and returned to near‐normal values by 30 min. The increase in surface receptor number was the result of a decrease in the rate of internalization of receptors, either occupied or unoccupied. The rate of receptor exteriorization was unaltered by hypo‐osmotic incubation of cells. The rate of fluid‐phase pinocytosis was also inhibited upon incubation in hypo‐osmotic solution. In experiments in which both receptor‐mediated endocytosis and fluid phase pinocytosis were measured on the same samples, inhibition of both processes occurred with the same kinetics and to a similar extent. The rate of receptor‐mediated endocytosis recovered to normal rates after 60 min in hypo‐osmotic solutions, whereas the rate of fluid phase pinocytosis did not recover to the same extent.
Identification of neutrophil alkaline phosphatase‐inducing factor in cystic fluid of a human squamous cell carcinoma as granulocyte colony‐stimulating factor
Authors: Sato Noriharu , Asano Shigetaka , Koeffler H. Phillip , Yoshida Satoru , Takaku Fumimaro , Takatani Osamu
Published: February 2005

We have previously shown that a factor termed neutrophil alkaline phosphatase‐inducing factor (NAP‐IF) has the capacity to induce neutrophil alkaline phosphatase (NAP) in postmitotic granulocytes (PMGs). This factor has characteristics similar to those of granulocyte colony‐stimulating factor (G‐CSF), suggesting that the two factors assayed by different methods may be attributable to an identical macromolecule. In a preliminary experiment, we showed that purified natural G‐CSF (nG‐CSF) could induce NAP in vitro in the presence of 10% (v/v) fetal calf serum (FCS). In this study, purified human nG‐CSF and recombinant G‐CSF (rG‐CSF) induced NAP in granulocytes from both normal individuals and patients with chronic myelogenous leukemia in a dose‐dependent fashion in serum‐free and serum‐containing culture conditions. The induction of NAP by G‐CSF was detectable at 0.4 ng/ml and became maximal between 10 and 20 ng/ml. Anti‐G‐CSF serum incubated with either NAP‐IF or rG‐CSF inhibited induction of NAP. Morphological examinations revealed that granulocytes cultured with G‐CSF were more mature than those cultured without G‐CSF, indicating that G‐CSF promoted maturation of granulocytes in parallel with NAP induction. These results indicate that NAP‐IF in the cystic fluid of a human squamous cell carcinoma is identical to G‐CSF and that induction of NAP by G‐CSF is really a reflection of cell maturation promoted by G‐CSF.
Pyrimido‐pyrimidine modulation of EGF growth‐promoting activity and p21ras expression in rat mammary adenocarcinoma cells
Authors: Lichtner Rosemarie B. , Gallick Gary E. , Nicolson Garth L.
Published: February 2005

RA 233, a pyrimido‐pyrimidine analogue developed originally as an antiplatelet agent, has reduced the incidence of tumor metastases in clinical trials. However, in animal tumor models antimetastatic therapy using RA 233 has been inconsistent. We therefore tested RA 233 for additional effects, such as its direct action on tumor cells. Using the rat 13726NF mammary adenocarcinoma tumor system, low, nontoxic concentrations of RA 233 had pleiotropic and differential effects on two 13762NF tumor cell clones. The growth of MTC cells (low spontaneous metastatic potential) was not affected by low concentrations of RA 233 (50 m̈M) or epidermal growth factor (EGF) (up to 10 ng/ml) for 3 days in 0.5–10% fetal bovine serum. In contrast, MTLn3 (high spontaneous metastatic potential) cell cultures maintained for 3 days in low (0.5–1%) serum in the presence of 1.25–10 ng/ml EGF doubled in cell numbers compared with control cultures, and addition of 50 m̈M RA 233 abrogated the growth‐stimulatory effect of EGF. The inhibitory effect of RA 233 on MTLn3 cells was dose dependent and not due to cell toxicity as determined by cell viability, cell growth, and colony formation properties after drug removal. In addition, incubation of MTLn3 cell with 50 m̈M RA 233 resulted in an increase of p21ras protein expression, whereas there was no effect on the level of p21ras in identically treated MTC cells or when either clone was treated with 10 ng/ml EGF. The results suggest that among the heterogeneous effects of RA 233 on tumor cells, modulation of growth factor responses and regulatory molecules may be important.
Relative contribution of the leukocyte molecules MO1, LFA‐1, and p150,95 (LeuM5) in adhesion of granulocytes and monocytes to vascular endothelium is tissue‐ and stimulus‐specific
Authors: Arnaout M. Amin , Lanier Lewis L. , Faller Douglas V.
Published: February 2005

Adhesion of human monocytes and granulocytes to vascular endothelium plays an important role in migration of these cells to inflammatory sites in tissues. A family of three human leukocyte heterodimeric surface molecules named Mo1, LFA‐1, and p150,95 (LeuM5) has been shown to mediate leukocyte adhesion to confluent monolayers of human umbilical vein endothelial cells (HUVE). The relative contribution of each of the three molecules in leukocyte endothelial adhesion was studied using a variety of stimuli. Purified human granulocytes and monocytes were radiolabelled and incubated with HUVE for 45 minutes in a 37°C humidified 5% CO2 incubator in the presence or absence of subunit‐specific monoclonal antibodies (MAbs). Adhesion was assessed by quantitation of endothelial cell‐associated radioactivity and confirmed by microscopic evaluation. MAbs directed against the alpha subunit of LFA‐1 as well as to the beta subunit common to all three antigens significantly inhibited unstimulated monocyte adhesion to HUVE. Small but significant inhibition was also observed using MAbs directed against Mo1a and p150. Phorbol myristate acetate (PMA)‐induced grranulocyte adhesion to HUVE was significantly inhibited by anti‐Mo1a and anti‐beta, but not by anti‐LFA‐1a or anti‐p150. When HUVE were prestimulated by recombinant IL‐1, a different pattern of antigen utilization by granulocytes was observed. MAbs directed against each of the three alpha subunits as well as the common beta subunit all inhibited granulocyte adhesion to HUVE. Furthermore the effect of the three anti‐alpha subunit MAbs on granulocyte‐HUVE adhesion was additive. These studies show that relative contribution of Mo1, LFA‐1, and p150,95 to leukocyte endothelial adhesion varies depending on the cell type and the stimulus used. These studies also reveal a novel role for p150,95 in promoting monocyte and granulocyte adhesion to HUVE.
Cell‐to‐cell heterogeneity in the expression of carbohydrate‐based epitopes of a mucin‐type glycoprotein on the surface of human mammary carcinoma cells
Authors: Moss Lenny , Greenwalt Dale , Cullen Betsey , Dinh Nina , Ranken Ray , Parry Gordon
Published: February 2005

A large, O‐linked glycoprotein, termed PAS‐O, is a major differentiation antigen on the surface of normal lactating breast epithelia and is also found on the surface of many mammary tumors and mammary carcinoma cell lines. A characteristic feature of populations of tumor cells that express PAS‐O is the cell‐to‐cell heterogeneity with respect to the presence or absence of the molecule. In this study, we used the human mammary carcinoma line 734B and a set of six monoclonal antibodies reactive with PAS‐O to study the basis of this heterogeneity. Extensive Western blot analysis of antibody binding to PAS‐O in milk fat globule membranes and in skim milk revealed that the antibodies all recognized different epitopes of PAS‐O. Moreover, the epitopes were destroyed by periodic acid oxidation, demonstrating their oligosaccharide basis. All six monoclonal antibodies stained the 734B cells heterogeneously. In addition, five clones derived from the parent 734B population also exhibited heterogeneity in the expression of each of the epitopes. An analysis of staining of the 734B clones revealed that, in some cases, certain cells within the cloned population stained with one monoclonal antibody but not with another antibody. Significantly, though, when the 734B cells were treated with neuraminidase prior to antibody staining, most of the heterogeneity was eliminated, and all but one of the monoclonal antibodies stained 90–100% of the cells. This increase in cell staining was matched by an increase in PAS‐O staining on Western blots. We conclude that heterogeneity in PAS‐O expression on 734B cells is due partly to masking of epitopes by sialic acid and a variation (on a cell‐to‐cell basis) in the extent of PAS‐O sialylation.
Effect of continuous heat stress on cell growth and protein synthesis in Aedes albopictus
Authors: da Gloria da Costa Carvalho Maria , Freitas Marcia Soares
Published: February 2005

Aedes albopictus (clone C6/36) cells, which normally grow at 28°C, were maintained at a supraoptimal temperature of 37°C. The effect of continuous heat stress (37°C) on cell growth was analyzed as were the modifications occurring with protein synthesis during short‐ and long‐term heat stress. We observed that cells in lag or exponential growth phase, present inhibition of cell growth, and cells in the lag phase showed more sensitivity to death than cells growing exponentially. During the first hour of exposing the cells to 37°C, they synthesized two heat shock proteins (hsps) of 82 kd and 70 kd, respectively, concomitant with inhibition of normally produced proteins at control temperature (28°C). However, for incubations longer than 2 hr at 37°C, a shift to the normal pattern of protein synthesis occurred. During these transitions, two other hsps of 76 kd and 90 kd were synthesized. Pulse chase experiments showed that the 70‐kd hsp is stable at least for 18 hr, when the cells are returned to 28°C. However, if cells were incubated at 37°C, the 70‐kd hsp is stable for at least 48 hr. The 70‐kd hsp was localized in the cytoplasmic and in the nuclear compartment. OUr results indicate a possible role of hsp 70‐kd protein in the regulation of cell proliferation.
Glucose elevates ornithine decarboxylase expression in vero cells
Authors: Lundgren David W. , Prokay Sandra L.
Published: February 2005

The addition to Earle's balanced salt solution (EBSS) of amino acids that are transported by a Na+‐dependent cotransport system was not required by Vero cells for ornithine decarboxylase (ODC:EC 4.1.1.17) amplification. Vero cell ODC activity was elevated tenfold above basal levels when confluent cells were incubated for 5 hr in EBSS alone. ODC activity increased as a function of the incubation time in EBSS and was not elevated above basal enzyme levels when cells were incubated in EBSS minus glucose. ODC expession increased as a function of the glucose concentration in EBSS, with 20 mM glucose producing a 90‐fold increase in ODC activity. ODC expression is more responsive to glucose in high‐density quiescent cultures than in low‐density growing cultures. Enhanced ODC expression by glucose depended on Na+ and K+ concentrations. The specific activity of ODC was also elevated above basal levels when mannose or fructose replaced glucose in EBSS. The addition of alanine or asparagine to EBSS enhanced ODC activity above levels obtained with EBSS containing standard (5.5 mM) glucose concentrations. In the absence of glucose, alanine was more effective than asparagine in enhancing ODC expression. These results suggest that the transport of amino acids is not an absolute requirement for Vero cell ODC expression and that ODC expression is linked to changes in cellular energetics and/or ion fluxes.