bluuebunny/crossref_metadata_2025 huggingface dataset:
Attenuation of LPS‐induced neutrophil thromboxane B2 release and chemiluminescence
Authors: Zheng Hui , Crowley Jeffrey J. , Chan Jane C. , Raffin Thomas A.
Published: February 2005

Polymorphonuclear leukocytes (PMN) may play a key role in acute lung injury and ARDS. The mechanisms of PMN‐mediated lung injury include the release of inflammatory mediators, such as oxygen free radicals which cause direct tissue injury, and arachidonic acid metabolites which cause pulmonary vasoconstriction and increased vascular permeability. The goals of this in vitro study were (1) to assess the effects of PMN‐activating agents (lipopolysaccharide, LPS; phorbol myristate acetate, PMA; tumor necrosis factor, TNF) on PMN thromboxane B2 (TXB2) release and oxygen free radical production and (2) to determine the effects of agents purported to suppress PMN activity (pentoxifylline, PTX; adenosine; dibutyryl cyclic AMP, DBcAMP; and terbutaline, TBN) on activator‐induced PMN TXB2 release and oxygen free radical production. PMN TXB2 release was determined by radioimmunoassay and oxygen free radical production was monitored by Chemiluminescence. Our results show that (1) LPS and PMA significantly increase PMN TXB2 release, whereas tumor necrosis factor (TNF) has no effect; (2) LPS and PMA significantly increase PMN Chemiluminescence; (3) DBcAMP and TBN significantly reduce LPS‐induced PMN TXB2 release whereas PTX and adenosine do not; (4) TBN significantly reduces PMA‐induced PMN TXB2 release whereas other agents do not; (5)All agents(PTX, adenosine, DBcAMP, and TBN) significantly reduce LPS‐induced PMN Chemiluminescence but none attenuate PMA‐induced PMN Chemiluminescence. We conclude that:LPS and PMA activate PMN manifested by TXB2 release and Chemiluminescence. Additionally, all the PMN suppressing agents do attenuate some PMN functions. Of interest, PTX, adenosine, DBcAMP, and TBN have different effects depending upon functional assay and activating agent. It will be important to investigate the mechanisms by which PMN suppressing agents alter signal transduction resulting in differential effects on PMN function.
Developmental patterns in the antioxidant defenses of the housefly, Musca domestica
Authors: Allen R. G. , Oberley L. W. , Elwell J. H. , Sohal R. S.
Published: February 2005

The activities of superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione S‐transferases, GSSG reductase, thiol transferases, gamma glutamyl‐cysteine synthetase, and glucose‐6‐phosphate dehydrogenase, and the concentrations of H2O2 and reduced and oxidized glutathione were determined in the various developmental stages of houseflies. Housefly development was correlated with a progressive increase of cellular oxidizing equivalents and a loss of cellular reducing capacity. The loss of reducing equivalents appeared to result from a decrease in the activity of enzymes involved in glutathione and NADPH synthesis and a concomitant increase in glutathione‐oxidizing enzymes. Relatively little change was observed in SOD activity during housefly development; however, the electrophoretic pattern of MnSOD varied in a manner specific to developmental stage. A striking increase in H2O2 concentration occurred prior to pupation possibly due to changes in substrate catabolism. These results support the hypothesis that the cellular environment becomes progressively more oxidizing during development.
WS‐1 human fibroblasts contain distinct calcium and protein kinase C‐mediated pathways for activation of Na+/H+ exchange: Contrasting effects of thrombin and PMA
Authors: Hendey Bill , Mamrack Mark D.
Published: February 2005

PMA and thrombin were examined for their ability to activate Na+/H+ exchange in growth‐arrested WS‐1 human fibroblasts. PMA or thrombin caused a cytoplasmic alkalinization that required extracellular sodium and was sensitive to 1 mM amiloride, suggesting that the rise in pH was mediated by the Na+/H+ exchanger. However, PMA and thrombin activated Na+/H+exchange by distinctly different mechanisms. The rate of cytoplasmic alkalinization caused by 30 nM PMA was slower than 10 nM thrombin. The PMA‐induced pH change was sensitive to the protein kinase inhibitors staurosporine (50 nM) and H‐7 (100 μM) No increase in intracellular calcium was observed after PMA treatment and the cytoplasmic alkalinization caused by PMA was not sensitive to the drug TMB8 (200 μM) or the intracellular calcium‐chelator BAPTA. In contrast, the thrombin‐induced rise in cytoplasmic pH was insensitive to 50 nM staurosporine and only partially reduced with 100 μM H‐7. The thrombin‐induced activation of Na+/H+ exchange was inhibited by 200 μM TMB8 or pretreatment with BAPTA. PMA caused translo‐cation of PKC activity from a cytoplasmic to membrane fraction whereas thrombin did not. Pretreatment with 50 nM staurosporine significantly reduced measurable PKC activity with or without PMA treatment. PMA and thrombin were also examined for their ability to induce DNA synthesis in growth‐arrested WS‐1 human fibroblasts. Unlike thrombin, PMA did not stimulate [3H]‐thymidine incorporation in cells serum‐deprived for 48 hours. In addition, PMA inhibited thrombin‐induced DNA synthesis when added at the same time or as late as 10 hours after thrombin addition. Therefore, thrombin and PMA activate Na+/H+ exchange by distinct pathways, but only the thrombin‐induced pathway correlates with a mitogenic response.
Membrane potential and amino acid transport in a mutant chinese hamster ovary cell line
Authors: Rotoli Bianca Maria , Bussolati Ovidio , Dall'asta Valeria , Gazzola Gian Carlo
Published: February 2005

The bioenergetics of amino acid transport system A was studied in two Chinese hamster ovary (CHO) cell lines, the parent line CHO‐PEOT/1 and CHY‐1, a mutant of the former exhibiting a low activity of the same transport system. The steady‐state transmembrane distribution ratio of the cationic amino acid L‐arginine (RARG) was employed as an indicator of membrane potential (Δψ). Evidence for the reliability of RARG to measure Δψ can be summarized as follows: (1) L‐arginine transmembrane distribution increased under conditions of cell hyperpolarization and decreased under conditions of cell depolarization; (2) L‐arginine distribution conformed closely to that expected for a probe of Δψ in conditions in which Δψ depends largely on the transmembrane potassium gradient; and (3) the value of Δψ obtained through a valinomycin null point experiment (−72.7 mV) was very similar to the value calculated from L‐arginine distribution using the Nernst equation (−73.4 mV). The transmembrane gradient of sodium electrochemical potential (Δ\documentclass{article}\pagestyle{empty}\begin{document}$ \tilde\mu $\end{document}Na), the driving force for the operation of system A, was slightly higher in the mutant cell line CHY‐1. In the same line, the intracellular level of the specific system A substrate MeAlB at steady state was also higher. Studies of the rheogenicity of system A in the two lines indicated that the depolarization associated with the entry of substrates of system A was proportional to the amount of amino acid taken up by the cells. Kinetic analysis showed that the low activity of system A in the mutant cell line was referrable to a decrease in transport Vmax. It is concluded that neither a decrease in energy available for the operation of system A nor a decreased efficiency of coupling of the system to Δψ is responsible for the defect observed in the mutant line.
Phorbol ester TPA inhibits the stimulation of bumetanide‐sensitive Na+/K+/Cl– transporter by different mitogens in quiescent BALB/c 3T3 mouse fibroblasts
Authors: Snyder David , Markus Miriam , Atlan Henri , Panet Rivka
Published: February 2005

In this study we examined the effect of the phorbol ester 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA) on the bumetanide‐sensitive Na+/K+/Cl– transporter in quiescent BALB/c 3T3 cells. We have shown that exposure of quiescent BALB/c 3T3 cultures to phorbol ester did not inhibit the basal bumetanide‐sensitive Rb+ influx or efflux. In fact, at high concentration (100 ng/ml), TPA slightly stimulated the bumetanide‐sensitive Rb+ influx and efflux. However, when the quiescent cultures were stimulated by serum or by defined growth factors, the stimulated fraction of the bumetanide‐sensitive Rb+ influx was drastically inhibited by exposure of the cells to the phorbol ester TPA. Based on the above findings, we propose that activation of protein kinase C by the phorbol ester TPA does not inhibit the Na+/K+/Cl– cotransport activity; however it does suppress only the growth‐factors‐stimulated fraction of the cotransport in quiescent BALB/c 3T3 cells. These data propose that activation of kinase C has a regulatory feedback effect on the stimulation of the Na+/K+/Cl– cotransport activity by growth factors.
Multiple signal transduction pathways lead to extracellular ATP‐stimulated mitogenesis in mammalian cells: I. Involvement of protein kinase C–dependent and –independent pathways
Authors: Wang Ding‐Ji , Huang Ning‐Na , Gonzalez Fernando A. , Heppel Leon A.
Published: February 2005

We recently reported that extracellular ATP was mitogenic for Swiss 3T3, 3T6, and A431 cells (Huang et al.: Proc. Natl. Acad. Sci. USA, 86:7904–7908, 1989). Here we examined the possible involvement of activation of the protein kinase C (PKC) signal transduction pathway in the mechanism of action of extracellular ATP. A potent synergistic stimulation of DNA synthesis in quiescent cultures of 3T3 and 3T6 cells was observed when ATP was presented in combination with growth factors that activate PKC, such as bombesin, vasopressin, or tumor‐promoting phorbol esters. This finding suggests that ATP and these mitogens do not act through a common mechanism. In contrast, ATP was unable to show synergism with phorbol esters in A431 cells. We discovered striking differences when we examined the kinetics of formation of diacylglycerol (DAG) stimulated by ATP among these cell lines. Thus, ATP stimulated a sustained biphasic increase of DAG in A431 cells, but only a rapid transient increase of DAG formation was observed in 3T3 and 3T6 cells. The breakdown of phosphatidylcholine was stimulated by ATP in A431 cells; however, a significantly reduced effect was displayed in 3T6 cells. Furthermore, we found that the diacylglycerol‐kinase inhibitor, 1‐monooleoylglycerol, greatly potentiated ATP‐stimulated DNA synthesis in A431 cells. Finally, down‐regulation of PKC by long‐term exposure to phorbol dibutyrate (PDBu) prevented stimulation of DNA synthesis induced by bombesin, vasopressin, or phorbol esters in 3T3 or 3T6 cells, while it had no such effect on ATP‐stimulated mitogenesis in the presence of insulin or epidermal growth factor. On the other hand, PDBu‐mediated down‐regulation of PKC partially inhibited [3H]thymidine incorporation stimulated by ATP in A431 cells. Taken together, we conclude that a protein kinase C‐dependent pathway is partially involved in ATP‐stimulated DNA synthesis in A431 cells, but a protein kinase C‐independent pathway exists in 3T3 and 3T6 cells. Pertussis toxin (PTX) inhibited the sustained phase of DAG formation and the breakdown of phosphatidylcholine stimulated by ATP in A431 cells. This suggests involvement of a PTX‐sensitive G protein.
Regulation of myosin and overall protein degradation in mouse C2 skeletal myotubes
Authors: Gulve Eric A. , Mabuchi Katsuhide , Dice J. Fred
Published: February 2005

We compared the breakdown of total cellular protein with that of the contractile protein, myosin, in cultured C2 mouse skeletal myotubes. The degradation of long‐lived cellular proteins (which comprise the vast majority of myotube proteins) was inhibited by serum, insulin, and rat insulin‐like growth factor‐2. A physiological concentration of insulin was effective, but most of the effect of insulin occurred at concentrations well above the physiological range. IGF‐2 inhibited protein breakdown at concentrations well within the range of total IGF‐2 known to be present in the serum of fetal and neonatal rats. The breakdown of short‐lived proteins was not altered by insulin or serum. We measured myosin degradation using a monoclonal antibody directed against myosin heavy chain. The half‐life of myosin was 27 hours, and myosin breakdown was not altered by withdrawal of serum. Therefore, the enhanced protein degradation in response to serum withdrawal applies to certain proteins, but not to others.
Macrophage membrane glycoproteins that bind Griffonia simplicifolia I‐B4: Effect on cytotoxicity and protein secretion
Authors: Warfel Alwin H. , Zucker‐Franklin Dorothea , Zheng Zhaoyao Y.
Published: February 2005

Thioglycollate elicited peritoneal (TG‐Mϕs) but not resident peritoneal Mϕs (R‐Mϕs) were found to bind the lectin Griffonia simplicifolia isotype I‐B4 (GSI‐B4). This was demonstrated by ultrastructural studies and FACS analyses. Membranes from TG‐Mϕs were isolated, separated on SDS‐PAGE, electrotransferred onto nitrocellulose, and exposed to peroxidase‐labeled GSI‐B4. These procedures revealed two major membrane glycoproteins of molecular weights 180,000 and 94,000 daltons that bound the lectin GSI‐B4 which has a specificity for recognizing terminal α‐galactosyl residues. The presence of these epitopes on the two membrane glycoproteins was further substantiated by the fact that treatment of the membranes with α‐galactosidase destroyed their capacity to bind GSI‐B4 and that α‐D‐galactopyranoside but not N‐acetyl‐D‐glucosamine competitively inhibited GSI‐B4 from binding to the glycoproteins. Treatment of TG‐Mϕs with GSI‐B4 reduced the capacity of interferon (IFN) and lipopolysaccharide (LPS), or IFN alone, to induce Mϕ mediated cytotoxicity towards tumor cells by as much as 40%. GSI‐B4 also caused alterations in the pattern of biosynthetically 35S‐methionine labeled secreted proteins as early as 2 hours after contact with TG‐Mϕs. Out of 35 discernible proteins on fluorograms of SDS‐PAGE separated proteins, 5 were down‐regulated and 9 were enhanced. It is suggested that the two novel Mϕ membrane proteins may play a role in regulating the response of Mϕ subpopulations to their humoral and cellular environments.
A serum factor promotes collagenase synthesis by an osteoblastic cell line
Authors: Puccinelli Joseph M. , Omura Terry H. , Strege David W. , Jeffrey John J. , Partridge Nicola C.
Published: February 2005

Regulation of the synthesis of collagenase was investigated in the osteoblastic cell line, UMR 106‐01. The cells were stained by the avidin‐biotin‐complex technique for the presence of the enzyme. By this method, it was possible to identify cells producing collagenase. Synthesis, but not secretion, was found to be constitutive in these cells with the enzyme located intracellularly in cytoplasmic vesicles and the Golgi apparatus. The amount of collagenase contained within UMR cells and the number of cells synthesizing the enzyme were proportional to the concentration of fetal bovine serum in the incubating medium. When serum was withdrawn from the osteosarcoma cells, the content of collagenase decreased with time and the enzyme became undetectable by 48 h of serum depletion. The decrease in collagenase content could be completely reversed by resupplying serum to the cells. The collagenase promoting activity of serum could not be eliminated by adsorption on activated charcoal but was retained by a dialysis membrane with a 12,000 mol wt cutoff. A range of bone‐seeking hormones or agents known to affect collagenase secretion was added to the medium in an attempt to mimic the effect of serum on collagenase accumulation. None of these agonists, including parathyroid hormone, could reproduce the effect of serum on these cells, although parathyroid hormone could act as a collagenase secreta‐gogue in the presence or absence of serum. It is concluded that fetal bovine serum contains a yet unidentified factor or factors greater than 12,000 mol wt responsible for the continued synthesis of collagenase by UMR 106‐01 cells.
Characterization of calcium transport by basal plasma membranes from human placental syncytiotrophoblast
Authors: LaFond J. , LeClerc M. , Brunette M. G.
Published: February 2005

We have studied the mechanisms involved in calcium (Ca2+) transport through the basal plasma membranes (BPM) of the syncytiotrophoblast cells from full‐term human placenta. These purified membranes were enriched 25‐fold in Na+/ K+‐adenosine triphosphatase (ATPase), 37‐fold in [3H]dihydroalprenolol binding sites, and fivefold in alkaline phosphatase activity compared with the placenta homogenates. In the absence of ATP and Mg2+, a basal Ca2+ uptake was observed, which followed Michaelis‐Menten kinetics, with a Km Ca2+ of 0.18 ± 0.05 μM and Vmax of 0.93 ± 0.11 nmol/mg/min. The addition of Mg2+ to the incubation medium significantly decreased this uptake in a concentration‐dependent manner, with a maximal inhibition at 3 mM Mg2+ and above. The Lineweaver‐Burk plots of Ca2+ uptake in the absence and in the presence of 1 mM Mg2+ suggest a noncompetitive type of inhibition. Preloading the BPM vesicles with 5 mM Mg2+ had no significant effect on Ca2+ uptake, eliminating the hypothesis of a Ca2+/Mg2+ exchange mechanism. This ATP‐independent Ca2+ uptake was not sensitive to 10−6 M nitrendipine nor to 10−4 M verapamil. An ATP‐dependent Ca2+ transport was also detected in these BPM, whose Km Ca2+ was 0.09 ± 0.02 μM and Vmax 3.4 ± 0.2 nmoles/mg/3 min. This Ca2+ transport requires Mg2+, the optimal concentration of Mg2+ being approximately 1 mM. Preincubation of the membrane with 10−6 M calmodulin strongly enhanced the initial ATP‐dependent Ca2+ uptake. Finally, no Na+ /Ca2+ exchange process could be demonstrated.
Activation of signal transduction pathways protects quiescent Balb/c‐3T3 fibroblasts against death due to serum deprivation
Authors: Tamm Igor , Kikuchi Toyoko
Published: February 2005

Platelet‐derived growth factor (PDGF), epidermal growth factor (EGF), insulin‐like growth factor‐1 (IGF‐1), and insulin protect density‐inhibited murine Balb/c‐3T3 fibroblasts against death by distinctive mechanisms. Determination of the cell survival‐enhancing activity of growth factors by cell enumeration and neutral red uptake measurement gives equivalent results. PDGF displays a steep doseresponse relationship in the 1−5 ng/ml range. The other factors display shallow log‐linear relationships in the following ranges: EGF: 0.2−5 ng/ml; IGF‐1: 2−80 ng/ml; and insulin: 57−4,500 ng/ml. Agonists that lead to the activation of protein kinase A, including forskolin, 8‐bromoadenosine 3′:5′‐cyclic monophosphate (Br‐cAMP) and N6,2′‐O‐dibutyryladenosine 3′:5′‐cyclic monophosphate (db‐cAMP), markedly increase both short‐term (5‐h) and long‐term (20‐h) survival of cells. 2‐lsobutyl‐1‐methylxanthine (IBMX) markedly enhances short‐term survival, but its effect decays with time. The protein kinase C agonist 12‐O‐tetradecanoyl phorbol‐13‐acetate (TPA) has a moderate protective effect at concentrations of 16‐32 nM, and 64 nM TPA is highly effective. The synthetic diacylglycerols 1,2‐dioctanoylglycerol (DiC8) and 1‐oleoyl‐2‐acetylglycerol (OAG) and the calcium ionophore ionomycin show low activity. Supplemation of EGF with a protein kinase A or C agonist results in a varying additive increase in short‐term (5‐h) cell survival and supplementation of EGF+insulin or PDGF+EGF+insulin increases further the already high level of protection given by the growth factor combinations. Combining a protein kinase A and a protein kinase C agonist in the absence of growth factors gives an approximately additive increase in cell survival. Results obtained with kinase, RNA, and protein synthesis inhibitors suggest that: (1) activated protein kinase C catalyzes one or more phosphorylation events in quiescent Balb/c‐3T3 cells that lead to gene expression with the protein product(s) mediating protection of quiescent cells against death, and (2) phosphorylation events Catalyzed by protein kinase A largely serve to protect cells by a mechanism not requiring de novo RNA and protein biosynthesis.
Xyloside effects on in vitro hematopoiesis: Functional and biochemical studies
Authors: Kirby Suzanne L. , Bentley Stuart A.
Published: February 2005

Xyloside supplementation of long‐term bone marrow cultures (LTBMCs) has been reported to result in greatly enhanced proliferation of hematopoietic stem cells. This was presumed to be the result of xyloside‐mediated perturbation of proteoglycan synthesis by marrow‐derived stromal cells. To investigate this phenomenon, we first studied the effects of xyloside supplementation on proteoglycan synthesis by D2XRadll bone marrow stromal cells, which support hematopoietic stem cell proliferation in vitro. D2XRadll cells were precursor labelled with 35S‐sulfate, and proteoglycans separated by ion exchange chromatography, isopyknic CsCI gradient centrifugation, and gel filtration HPLC. Xylo‐side‐supplemented cultures showed an approximately fourfold increase in total 35S incorporation, mainly as free chondroitin‐dermatan sulfate (CS/DS) gly‐cosaminoglycan chains in the culture media. Both xyloside supplemented and nonsupplemented cultures synthesized DS1, DS2, and DS3 CS/DS proteoglycans as previously described. In contrast to previous reports, xyloside was found to inhibit hematopoietic cell growth in LTBMC. Inhibitory effects were observed both in cocultures of lL‐3‐dependent hematopoietic cell lines with supportive stromal cell lines and in primary murine LTBMCs. Xyloside was found to have a marked inhibitory effect on the growth of murine hematopoietic stem cells and IL‐3‐dependent hematopoietic cell lines in clonal assay systems and in suspension cultures. In contrast, dialyzed concentrated conditioned media from LTBMCs had no such inhibitory effects. These findings suggest that xyloside‐mediated inhibition of hematopoietic cell growth in LTBMC resulted from a direct effect of xyloside on proteoglycan synthesis by hematopoietic cells.
Generation of superoxide anion by brain endothelial cell xanthine oxidase
Authors: Terada Lance S. , Willingham Irene R. , Rosandich Mary E. , Leff Jonathan A. , Kindt Glenn W. , Repine John E.
Published: February 2005

Bovine brain endothelial cells (EC) that were isolated and propagated in pure culture had increased ( 20‐fold) levels of xanthine oxidase and xanthine dehydrogenase activity compared to whole brain homogenate. Brain EC also released superoxide anion (O) in to the extracellular medium. Treatment of EC with tungsten decreased (P < 0.05), both XO activity and O release. XO appears to be highly concentrated in cerebral vascular endothelium and may be an important source o; O.
Induction of plasminogen activator inhibitor 1 biosynthesis by hyperthermia
Authors: Bergonzelli Gabriela E. , Kruithof Egbert K. O.
Published: February 2005

Hyperthermia is a clinical sign of inflammation and constitutes in itself an adaptive defense mechanism. The fibrinolytic system, a highly regulated proteolytic system, a involved in inflammatory processes. Plasminogen activator inhibitor 1 (PAI‐1) is the principal inhibitor of the two activators of the fibrinolytic system: tissue‐ and urokinase‐type PAs (t‐PA and u‐PA). Our present paper provides the first evidence that hyperthermia can directly induce PAI‐1. A moderate heat stress, sufficient to induce heat shock protein 70 mRNA approximately 100‐fold, resulted in a two‐to three‐fold increase in functionally active PAI‐1 in the conditioned medium of human HT‐1080 fibrosarcoma and Hep G2 hepatoma cells. Exposure of these cells to 42°C led to a similar two‐fold and two‐to five‐fold induction of PAI‐1 mRNA expression in HT‐1080 and Hep G2 cells, respectively, as has been determined by using both oligo d(T) selected and total RNA preparations. These results suggest that the observed increase in PAI‐1 accumulation is due to an induction of PAI‐1 biosynthesis. Run‐on transcription analysis indicates that the induction of PAI‐1 biosynthesis by hyperthermia is mediated by a stimulation of PAI‐1 gene transcription. No significant effect of hyperthermia was found on t‐PA or u‐PA at the level of antigen accumulation, mRNA, and gene transcription in human HT‐1080 fibrosarcoma cells. These results point to an additional regulatory mechanism of fibrinolysis in the context of inflammation.
Growth regulated expression of B‐MYB in fibroblasts and hematopoietic cells
Authors: Reiss Krzysztof , Travali Salvatore , Calabretta Bruno , Baserga Renato
Published: February 2005

The B‐myb cDNA has extensive sequence similarities to the c‐myb protooncogene, but, at variance with c‐myb, it is expressed in cells other than hematopoietic cells. In this paper, we show that (1) B‐myb is expressed in mouse, human, and hamster fibroblasts; (2) B‐myb mRNA levels are growth‐regulated in both fibroblasts and peripheral blood mononuclear cells; (3) by its mode of growth regulation (peak of expression, behavior in G1‐specific temperature sensitive (ts) mutants and in the presence of cycloheximide), B‐myb can be classified, like c‐myb, thymidine kinase, PCNA, and others, as a late growth‐regulated gene; (4) B‐myb mRNA levels decrease when HL‐60 cells are induced to differentiate; and (5) the increase in mRNA levels in serum‐stimulated cells is only partially explained by an increase in rate of transcription. The possibility that the B‐myb gene may be the equivalent in fibroblasts and epithelial cells of the c‐myb proto‐oncogene of hematopoietic cells is discussed.
Expansion of myelopoietic precursors and inhibition of B‐cell precursors in mice that express a T‐cell receptor gamma (Vγ 1.1JγM4Cγ4) transgene
Authors: Ferrick David A , Cumano Ana , Furlonger Caren , Min Xia , Iscove Norman N. , Paige Christopher J. , Mak Tak W.
Published: February 2005

Knowledge of the genetic determinants that can affect renewal of multipotential stem cells and their commitment to specific cell lineages is essential to our understanding of multicellular development. However, despite the vast amount of accumulated knowledge in this area, genetic determinants that affect renewal and commitment of precursor cells are unknown. In this study, we demonstrate that three independently derived founder mouse strains, transgenic for the TcR Vγ1.1Jγ4Cγ4 (TcRγ4) chain gene, differed significantly from normal mice in their development of T and B cells as well as myelopoietic precursor cells. Ontogenic programs consistent with an acceleration of T‐cell development and a delayed appearance and suppressed levels of pre‐B‐ and B‐cell precusors were evident in these transgenic mice. In addition, TcRγ4 transgenic mice possessed a significantly elevated level of myelopoietic pluripotent precursors. 3H‐thymidine cell suicide studies suggest that higher percentages of pluipotential precursors from the bone marrow of the TcRγ4 transgenic mice were in the S phase of the cell cycle. These modulations of the lymphoid and myelopoietic compartments, however, were not found in other T‐cell receptor transgenic mice (e.g., TcR Vγ1.2Jγ2Cγ2, TcRγ2; or Vβ8.1Dβjβ2.4Cβ2, TcRβ) constructed with the same or similar cDNA expression vector. The results suggest that the expression of a specific T‐cell receptor γ chain gene, and/or an elevated level of particular subset of TcRγδL cells, may affect the proliferation and relative proportions of haemopoietic and lymphoid precursors.
DNA‐mediated transfer of a human gene that confers resistance to mitomycin C
Authors: Buchwald Manuel , Clarke Carol
Published: February 2005

Attempts to complement the defect in the mitomycin C (MMC)‐sensitive Chinese hamster ovary (CHO) mutant MMC3 led to the isolation of hybrids with high resistance to the cytotoxic action of the drug. Hybrid cells selected with MMC after fusion of MMC3 cells to human diploid fibroblasts were approximately five times more resistant to MMC than wild‐type CHO cells but retained the original MMC3 sensitivity to another DNA cross‐linking agent, diepoxybutane. To confirm that the MMC resistance was genetically determined and was of human origin, DNA from the resistant hybrids was introduced into MMC3 cells, and transfectants were selected in MMC. These cells had the same level of MMC resistance as the hybrids. Thus we have identified a human gene that can confer MMC resistance to CHO cells. Identification of the gene should help understand the mechanisms of MMC resistance in mammalian cells.
GM‐CSF in association with IL‐1 triggers day‐8 CFU‐S into cell cycle: Role of histamine
Authors: Piquet‐Pellorce Claire , Schneider Elke , Dy Michel
Published: February 2005

Our recent evidence for the requirement of endogeneous histamine in IL‐3‐induced proliferation of day‐8 CFU‐S has prompted us to investigate whether or not GM‐CSF, which shares with IL‐3 the ability to stimulate bone marrow histamine synthesis, could also affect the cell cycle status of CFU‐S via this mediator. We show herein that recombinant GM‐CSF alone fails to trigger day‐8 CFU‐S into S phase, but supports their survival. However, in the same experimental conditions, GM‐CSF in combination with IL‐1 induces a CFU‐S proliferation similar to that obtained in response to IL‐3, while IL‐1 by itself has no effect on this biological activity. We further provide evidence that this phenomenon is completely abolished: (i) by preventing GM‐CSF‐induced histamine synthesis by α‐FMH, the specific inhibitor of histidine decarboxylase (HDC), or (ii) by blocking the binding sites of H2 histamine receptors with their specific antagonist oxmetidine. Similar results are obtained when progenitor‐enriched bone marrow cells are used instead of the unfractionated population. In addition, we provide an argument in support of a histamine receptor modulation by GM‐CSF that could explain the lack of effect of factor‐induced histamine on day‐8 CFU‐S cell cycling. Indeed, the entry of these progenitors into S phase that is normally promoted by dimaprit, a specific histamine H2 receptor agonist, is abolished by a preincubation with GM‐CSF. Taken together, our data support the conclusion that IL‐1 makes CFU‐S sensitive to GM‐CSF‐induced endogeneous histamine that will trigger them into cell cycle, while GM‐CSF alone has no such effect on this biological activity.
Isolation of variants of chinese hamster ovary cells with abnormally low levels of GSH: Decreased ability to cleave endocytosed disulfide bonds
Authors: Mandel Richard , Ryser Hugues J.‐P. , Niaki Bijan , Ghani Farooq , Shen Wei‐Chiang
Published: February 2005

Mutants of Chinese hamster ovary cells were selected for resistance to a 3 hour exposure to 4 μ/ml N‐methyl‐N'‐nitro‐N‐nitrosoguanidine and tested for glutathione (GSH) levels. Six of eight clones that survived the initial treatment had reduced GSH levels ranging from 26 to 61% of wild‐type values. These eight cell lines were tested for their susceptibility to a drug conjugate in which methotrexate (MTX) is disulfide‐linked to poly(D‐lysine) (MTX‐SS‐PDL) to test their capacity to cleave the endocytosed disulfide bond and release free MTX from this otherwise undegradable drug conjugate. We had shown that wild‐type cells were killed by ∼1 × 10−7 M MTX given as free drug, as MTX‐poly(L‐lysine) or as MTX‐SS‐PDL, but were not affected by MTX‐poly(D‐lysine). All six lines with abnormally low levels of GSH were resistant to MTX‐SS‐PDL. The variants with the lowest levels of GSH (MNR‐5) and MNR‐10 were tested further and showed near‐normal sensitivity to MTX and MTX poly(L‐lysine). As expected, both lines were hypersensitive to melphalan. They were, however, normally sensitive to diphtheria toxin and ricin, indicating that some cleavage opf the interchain disulfides in these protein toxins occurs even when cellular GSH is abnormally low. The lesser GSH requirement for toxin activation may be due to their extraordinary potency.
Interleukin‐6 and transforming growth factor‐β synergistically stimulate chondrosarcoma cell proliferation
Authors: Guerne Pierre‐André , Lotz Martin
Published: February 2005

This study examines the regulation of Swarm rat chondrosarcoma (SRC) cell proliferation in vitro. In serum‐free cultures, SRC cells showed only transient DNA synthesis and this was increased by serum. Transforming growth factor‐β (TGF‐β) was identified as an essential serum component, since the mitogenic effect of sera was related to their TGF‐β content and neutralized by antibody to TGF‐β.

Among a large panel of agents tested, TGF‐β was the only factor that stimulated proliferation in serum‐free media. The TGF‐β isoforms TGF‐β1 and TGF‐β2 induced similar dose‐dependent increases with maximal 62.5‐fold stimulation at 10 ng/ml.

Interleukin‐6 (IL‐6) was identified as a new factor that stimulated SRC proliferation. IL‐6 effects were serum‐dependent and their magnitude correlated with the TGF‐β content in different serum preparations. In serum‐free cultures where IL‐6 by itself had no detectable effect it caused up to 7.6‐fold increased proliferation in the presence of small doses of TGF‐β (0.01–0.1 ng/ml). This synergy was unique, since no other factor tested synergized with IL‐6 or TGF‐β. In examining potential mechanisms for this synergy it was found that TGF‐β increased IL‐6 receptor expression.

In summary, these results identify IL‐6 as a new and TGF‐β as the most potent growth factor for chondrosarcoma cells and describe novel interactions between these factors in the regulation of cell growth.
Maintenance and mobility of hemoglobin and water within the human erythrocyte after detergent disruption of the plasma membrane
Authors: Cameron Ivan L. , Cox Laura A. , Liu Xi Rong , Fullerton Gary D.
Published: February 2005

Is an intact plasma membrane responsible for keeping hemoglobin and water within the human erythrocyte? If not, what is responsible? How free is Hb to move about within the erythrocyte? To answer these questions erythrocytes were taken for phase contrast microscopy, transmission electron microscopy (TEM), determination of water‐holding capacity, and proton NMR studies both before and after membrane disruption with a nonionic detergent (Brij 58). Addition of 0.2% Brij to a D2O saline solution of hemoglobin (Hb) caused particles of Hb to appear and to aggregate. This aggregation of Hb caused the amplitude of the Hb proton NMR spectra to decrease. Thus, the less mobile the Hb the lower the Hb proton spectra amplitude. Erythrocytes washed in D2O saline showed proton NMR spectra of relatively low amplitude. Addition of Brij (0.2%) to these erythrocytes caused increased Hb mobility within these erythrocytes. The TEM of fixed and thin‐sectioned erythrocytes treated with Brij showed disruption of the plasma membrane of all erythrocytes regardless of whether or not they had lost Hb. Brij‐permeabilized erythrocytes washed in D2O saline or in a D2O K buffer maintained a higher heavy water‐holding capacity upon centrifugation as compared to nonpermeabilized erythrocytes. The TEM of Brij‐treated and washed erythrocyte “shells” revealed a continuous submembrane lamina but no other evidence of cytoskeletal elements. The water‐holding capacity of the erythrocyte can be accounted for by the water‐holding capacity of hemoglobin. The evidence favors a relatively immobile state of Hb and of water in the erythrocyte that is not immediately dependent on an intact plasma membrane but is attributed to interactions between Hb molecules and the submembrane lamina.
The increase of calmodulin in PC12 cells induced by NGF is caused by differential expression of multiple mRNAs for calmodulin
Authors: Bai Guang , Weiss Benjamin
Published: February 2005

A rat pheochromocytoma cell line (PC12 cells) was used as a model to investigate the role of calmodulin and its multiple mRNAs in NGF‐induced neuronal differentiation. The effect of NGF on the degree of differentiation was assayed using a simple differentiation scoring system. Significant increases in the differentiation score were seen by one day, and the scores increased about 10‐fold by 8 days of treatment. NGF also increased calmodulin in the PC12 cells; significant increases were seen by 2 days of treatment, and a maximum increase of 3‐fold was seen by 4 days. Northern blot analysis using a calmodulin riboprobe revealed that all five calmodulin mRNAs found in rat tissue were present in PC12 cells. The relative abundance of the calmodulin mRNAs was 1.7 ≥ 1.4 ≥ 2.3 ≥ 4.1 ≥ 0.9 kb. NGF treatment caused a differential increase in these mRNAs. The 1.4 kb transcript (from Gene II) was increased earlier (at 1 day) and to a greater extent (3‐fold) than any of the other mRNAs. Studies of the half‐lives (t1/2) of these mRNAs suggested that the t1/2 varied with the mRNA; the smaller the mRNA, the shorter the t1/2. However, there were no significant effects of NGF on the t1/2 of any of the mRNAs. These studies indicate that NGF elevates calmodulin in PC12 cells by causing a differential increase in the multiple mRNAs for calmodulin and that the increase in calmodulin may play some part in NGF‐induced neuronal differentiation in PC12 cells.
Sheep amniotic fluid has a protein factor which stimulates human fibroblast populated collagen lattice contraction
Authors: Rittenberg T. , Longaker M. T. , Adzick N. S. , Ehrlich H. P.
Published: February 2005

Sutured incisional wounds made in fetal sheep and rabbits heal without scarring. Fetal sheep excisional wounds can close by contraction, but those in fetal rabbits do not. In vivo and in vitro evidence suggests that rabbit amniotic fluid inhibits wound contraction. The question arises: does sheep amniotic fluid promote wound contraction because their fetal wounds close by contraction? Sheep amniotic fluid (SAF) from 100 and 125 days gestation was tested in fibroblast populated collagen lattice (FPCL) system, an in vitro model of wound contraction. SAF stimulated FPCL contraction in a dose responsive manner. SAF from a 100 day fetus was more stimulating than a 125 day SAF. SAF enhanced FPCL contraction in the presence or absence of serum. SAF was fractionated by size, using column chromatography. It yielded a fraction with an estimated molecular weigh near 40,000 daltons, which stimulated FPCL contraction. The factor was inactivated by proteolytic digestion and heat denaturation. This protein fraction which stimulates FPCL contraction is not related to (1) actin‐myosin filaments enhanced contraction by ATP‐induced cell contraction, (2) promotion of fibroblast elongation on glass surface or in collagen, or (3) increased cell number by enhanced fibroblast duplication in a collagen matrix. A mechanism for SAF promotion of FPCL contraction was investigated but not identified.
Glutamine synthetase expression in rat oligodendrocytes in culture: Regulation by hormones and growth factors
Authors: Fressinaud Catherine , Weinrauder Halina , Delaunoy Jean‐Pierre , Tholey Georges , Labourdette Géarard , Sarliève Louis Léon
Published: February 2005

Glutamine synthetase (GS, EC 6.3.1.2.) has long been considered as a protein specific for astrocytes in the brain, but recently GS immunoreactivity has been reported in oligodendrocytes both in mixed primary glial cell cultures and in vivo. We have investigated its expression and regulation in “pure” cligodendrocyte cultures. “Pure” oligodendrocyte secondary cultures were derived from newborn rat brain primary cultures enriched in oligodendrocytes as described by Besnard et al. (1987) and were grown in chemically defined medium. These cultures contain more than 90% galactocerebroside‐positive oligodendrocytes and produce “myelin” membranes (Fressinaud et al., 1990) after 6–10 days in subcultures (30–35 days, total time in culture). The presence of GS in oligodendrocytes from both primary glial cell cultures and “pure” oligodendrocyte cultures was confirmed by double immunostaining with a rabbit antisheep GS and guinea pig antirat brain myelin 2′, 3′‐cyclic nucleotide 3′‐phosphodiesterase. In “pure” oligodendrocyte cultures, about half of cells were labeled with anti‐GS antibody. Furthermore, on the immunoblot performed with a rabbit antisheep GS, the GS protein in “pure” oligodendrocyte secondary cultures was visualized as a single band with an apparent molecular mass of about 43 KDa. In contrast, two protein bands for GS were observed in cultured astrocytes. On the immunoblot performed with a rabbit antichick GS, two immunopositive protein bands were observed: a major one migrating as the purified adult chick brain GS and a minor one with a lower molecular mass. Two similar immunoreactive bands were also observed in pure rat astrocyte cultures. Compared to pure rat astrocyte cultures, “pure” oligodendrocyte cultures of the same age displayed an unexpectedly high GS specific activity that could not be explained by astrocytic contamination of the cultures (less than 5%). As for cultured astrocytes, treatment of oligodendrocyte cultures with dibutyryl‐adenosine 3′: 5′‐cyclic monophosphate, triiodothyronine, or hydrocortisone increased significantly GS specific activity. Interestingly, epidermal growth factor, basic fibroblast growth factor, and platelet‐derived growth factor that increase the GS activity in astrocytes do not affect this activity in oligodendrocytes. Thus we confirm the finding of Warringa et al. (1988) that GS is also expressed in oligodendrocytes. We show that its activity is regulated similarly in astrocytes and oligodendrocytes by hormones, but that it is regulated differently by growth factors in these two cell types.
5‐Azacytidine‐induced demethyelation of DNA to senescent level does not block proliferation of human fibroblasts
Authors: Gray Matthew D. , Jesch Stephen A. , Stein Gretchen H.
Published: February 2005

IMR‐90 human diploid fibroblasts (HDF) lose from 30–50% of their genomic 5‐methyldeoxycytidine (5mdC) during the cellular aging process. In contrast, immortal SV40‐transformed IMR‐90 maintain a constant level of 5mdC in culture. Precrisis SV40‐transformed HDF (AG3204) represent a stage in between normal cell aging and immortalization because these cells still have a finite proliferative lifespan, but it is longer than that of normal HDF and ends in cell death rather than in G1‐arrest. We find that AG3204 cells continue to lose from 12–33% of their 5mdC after a population has a become 99% positive for SV40 T‐antigen. Both IMR‐90 cells and AG3204 cells have similar levels of 5mdC (average of 2.25%) at the end of lifespan. We investigated whether this level of 5mdC is an absolute block to further proliferation by treating IMR‐90 and AG3204 cells with 5‐azacytidine (5azaC) to reduce their 5mdC levels below the terminal elvel normally achieved at end of lifespan. We find that both IMR‐90 and AG3204 cells undergo extensive proliferation with subterminal levels of 5mdC and that the lifespans of both cells types are shortened by 5azaC treatment. These studies indicate that random genomic DNA demethyelation to a specific level of 5mdC is not a direct cause of finite proliferative lifespan. However, the correlation between accelerated DNA demethylation and accelerated aging still suggests that these two phenomena are related. Two ways to explain this relationship are: (1) DNA demethylation during aging is not random, and/or (2) both DNA demethylation and other cases, accelerated random DNA demethylation could accelerate aging, but not necessarily in direct relationship to the final genomic level of 5mdC achieved during the normal aging process.
Lectin binding to gp60 decreases specific albumin binding and transport in pulmonary artery endothelial monolayers
Authors: Siflinger‐Birnboim Alma , Schnitzer Jan , Lum Hazel , Blumenstock Frank A. , Shen Chien‐Ping J. , Del Vecchio Peter J. , Malik Asrar B.
Published: February 2005

The effect of albumin binding to cultured bovine pulmonary artery endothelial cell (BPAEC) monolayers on the transendothelial flux of 125I‐labelled bovine serum albumin (BSA) was examined to determine its possible role on albumin transcy‐tosis. The transport of 125I‐BSA tracer across BPAEC grown on gelatin‐ and fibronectin‐coated filters (0.8 μm pore diam.) was affected by the presence of unlabelled BSA in the medium in that transendothelial 125I‐BSA permeability decreased, reaching a 40% reduction at BSA concentrations equal to or greater than 5 mg/ml. BSA binding to BPAEC monolayers was saturated at concentration of 10 mg/ml with an apparent binding affinity of 6 x 10−7 M. In contrast, gelatin added to the medium altered neither 125I‐BSA binding nor transport. Several lectins were tested for their ability to inhibit 125I‐BSA binding and transport. One lectin, Ricinus communis (RCA), reduced 125I‐BSA binding by 70% and transport by 40%. Other lectins, Ulex europaeus, Triticum vulgare, and Glycine max decreased neither 125I‐BSA binding nor transport. The reduction of 125I‐BSA transport by RCA was not observed in the presence of saturating levels of BSA, indicating that RCA influenced only the albumin‐dependent component of transport. RCA, but not other lectins, precipitated a 60 kDa plasmalemmal glycoprotein from cell lysates of surface radioiodinated BPAEC monolayers. This 60 kDa glycoprotein appears to be the equivalent of gp60 identified previously as an albumin binding glycoprotein in rat microvascular endothelium. In summary, approximately 40% of albumin transport across' BPAEC monolayers is dependent on albumin binding. This component of albumin transport is inhibited by 80% by the binding of RCA to gp60. These results suggest that binding of albumin to gp60 on pulmonary artery endothelial cell membrane is a critical determinant of transendothelial albumin flux involving mechanisms such as plasmalemmal vesicular transcytosis.
Receptor‐mediated autocrine growth‐stimulatory effect of 5‐hydroxytryptamine on cultured human pancreatic carcinoid cells
Authors: Ishizuka Jin , Beauchamp R. Daniel , Townsend Courtney M. , Greeley George H. , Thompson James C.
Published: February 2005

5‐hydroxytryptamine (5‐HT) is a mitogen for fibroblasts, vascular smooth muscle cells, renal mesangial cells, and jejunal crypt cells. The human carcinoid cell line (termed BON) that we established in our laboratory from a pancreatic carcinoid tumor produces and secretes 5‐HT. In this study, therefore, we examined the effect of 5‐HT on growth of BON cells. Furthermore, by use of selective 5‐HT receptor antagonists, we examined receptor and post‐receptor mechanisms by which 5‐HT‐induced responses were produced. 5‐HT stimulated growth of BON cells. 5‐HT stimulated phosphatidylinositol (PI) hydrolysis in a dose‐dependent fashion and inhibited cyclic AMP production in a dose‐dependent fashion. The 5‐HT1A/1B receptor antagonist, SDZ 21–009, prevented the reduction of cyclic AMP production evoked by 5‐HT and inhibited the mitogenic action of 5‐HT. The 5‐HT1C/2 receptor antagonist, mesulergine, competitively inhibited PI hydrolysis, but did not affect the mitogenic action of 5‐HT. The mitogenic action of 5‐HT and the reduction of cyclic AMP production evoked by 5‐HT were also inhibited by pertussis toxin. These results suggest that 5‐HT is an autocrine growth factor for BON cells and that mitogenic mechanism of 5‐HT involves receptor‐mediated toxin‐sensitive GTP binding protein. 8‐bromo‐cyclic AMP inhibited growth of BON cells whereas 8‐bromo‐cyclic GMP had no effect on cell growth. Involvement of protein kinase A in BON cell growth regulation was confirmed by the observation that a cAMP‐dependent protein kinase antagonist (Rp‐cAMPS) could stimulate BON cell growth.