bluuebunny/crossref_metadata_2025 huggingface dataset:
Bumetanide‐sensitive Na+ /K+ /Cl− transporter is stimulated by phorbol ester and different mitogens in quiescent human skin fibroblasts
Authors: Panet Rivka , Atlan Henri
Published: February 2005

In this study we investigated the correlation between the mitogenic effect and stimulation of Rb + (K + ) fluxes in human skin fibroblasts treated by purified growth factors. Both K+ transporters, bumetanide‐sensitive and ouabain‐sensitive, are stimulated 2‐3‐fold after addition of either fetal calf serum or purified recombinant growth factors to quiescent G0/G1 human skin fibroblasts. Three groups of mitogens were compared: (i) the phorbol ester 2‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA); (ii) growth factors that stimulate inositol phosphate hydrolysis and subsequently activate protein kinase C—fibroblast growth factor (FGF), platelet derived growth factor (PDGF), and α‐thrombin; and (iii) growth factors that do not activate kinase C—insulin‐like growth factor‐1 (IGF‐1), and transforming like growth‐factor‐α (TGF‐α). The three groups of mitogens stimulated human skin fibroblasts proliferation and Rb+ influxes in a similar dose‐dependent fashion. The results indicate that both the bumetanide‐sensitive and the ouabain‐sensitive Rb+ fluxes are stimulated by protein kinase C‐dependent and by the protein kinase C‐independent pathways of the mitogenic signal.
Stimulation of fibronectin production by TGF‐β1 is independent of effects on cell proliferation: The example of bovine adrenocortical cells
Authors: Shi De Li , Savona Catherine , Chambaz Edmond M. , Feige Jean‐Jacques
Published: February 2005

We reported previously that transforming growth factor β (TGF‐β) does not influence the proliferation of bovine adrenocortical cells but is a very potent inhibitor of their steroidogenic functions (Feige et al.:Journal of Biological Chemistry 262:13491‐13495, 1987). In the present study, we addressed the question of whether these cells modify the synthesis of their extracellular matrix (in particular of fibronectin) in response to TGF‐β1, similarly to the changes observed in cell types whose growth is modified by this factor (e.g., fibroblasts). Immunofluorescence studies using anti‐fibronectin antibodies revealed that TGF‐β1, treatment in serum‐free medium induced the formation of fibronectin‐containing fibrils associated with adrenocortical cells. Metabolic labeling of adrenocortical cells with [35S]‐methionine showed that fibronectin synthesis and secretion were highly stimulated by low concentrations of TGF‐β1. Half‐maximal stimulation was observed for TGF‐β1 concentrations in the range of 0.1 to 0.5 ng/ml and maximal stimulation reached 35‐fold over control at the concentration of 2 ng/ml. The earlier detectable effect was observed after 8 h of treatment (6‐fold stimulation) and the maximal increase was reached after 24 h of treatment. Stimulation of adrenocortical fibronectin synthesis by TGF‐β1 appeared to imply a transcriptional event since it was no longer observed in the presence of DRB, a potent inhibitor of RNA polymerases, and because the level of fibronectin mRNA was stimulated under TGF‐β1 treatment. Taken together, these results indicate that the increased expression of fibronectin is not closely related to growth‐regulatory effects of TGF‐β1 since it is also observed in adrenocortical cells, whose proliferation is unaffected by TGF‐β1.
Osteoblasts display receptors for and responses to leukemia‐inhibitory factor
Authors: Allan E. H. , Hilton D. J. , Brown M. A. , Evely R. S. , Yumita S. , Metcalf D. , Gough N. M. , Ng K. W. , Nicola N. A. , Martin T. J.
Published: February 2005

Specific binding of leukemia‐inhibitory factor (LIF) to osteoblasts, but not multinucleated osteoclasts, was demonstrated by receptor autoradiography by *using cells isolated from newborn rat long bones. The clonal rat osteogenic sarcoma cells, UMR 106‐06, which have several phenotypic properties of osteoblasts, expressed 300 LIF receptors per cell, with an apparent KD of 60 pM. Treatment of calvarial osteoblasts or UMR 106‐01 cells with LIF resulted in a dose‐dependent inhibition of plasminogen activator (PA) activity. Both calvarial osteoblasts and osteogenic sarcoma cells were shown by Western blotting and reverse fibrin autography to produce plasminogen activator inhibitor‐1 (PAI‐1), the production of which was increased by LIF treatment. Northern blot analysis revealed that LIF treatment resulted in a rapid (peak 1 hour), dose‐dependent increase in mRNA for PAI‐1. LIF treatment of the preosteoblast cell line, UMR 201, enhanced the alkaline phosphatase response of these cells to retinoic acid. Each of the osteoblast‐like cell types (calvarial osteoblasts, UMR 106‐06, and UMR 201) was shown to produce LIF by bioassay and, by using the polymerase chain reaction (PCR), was shown to express low levels of mRNA for LIF. These data establish that cells of the osteoblast lineage are targets for LIF action. The reported anabolic effects of this cytokine on bone formation in vivo could be related to inhibition of protease activity. LIF may be an important paracrine modulator in bone, or perhaps an autocrine one, based on the evidence for its production by osteoblasts and osteoblast‐like cells.
Regulation of tubulin synthesis during the cell cycle in the synchronous plasmodia of Physarum polycephalum
Authors: Ducommun B. , Cance J. , Wright M.
Published: February 2005

Regulation of α‐ and β‐tubulin isotype synthesis during the cell cycle has been studied in the myxomycete Physarum polycephalum, by subjecting synchronous plasmodia to temperature shifts and pharmacological perturbations. Temperature *shifts interfered with the regulation of tubulin synthesis. Inhibition of DNA synthesis prevents tubulin degradation after completion of the cell cycle (Ducommun and Wright, Eur. J. Cell Biol., 50:48–55, 1989) but did not perturb the initiation of tubulin synthesis. The constant increase of tubulin synthesis in the presence of tubulin‐sequestering drugs and the decrease of tubulin synthesis during a treatment with aphidicolin in late G2 phase suggest the existence of an autoregulatory mechanism of tubulin synthesis. Moreover, the microtubule poison methyl benzimidazole carbamate dissociated synthesis of the α1‐tubulin isotype from the generally strictly coordinated synthesis of all tubulin isotypes during the transient interruption of mitosis. These observations show that a microtubular poison can perturb regulation of the synthesis of specific isotubulins.
Effects of differentiation‐inducing agents on maturation of human MCF‐7 breast cancer cells
Authors: Guilbaud Nicolas F. , Gas Nicole , Dupont Marie Ange , Valette Annie
Published: February 2005

The effects of the differentiation inducing agents (DIAs), sodium butyrate (NaBu), retinoic acid (RA), dimethylformamide (DMF), hexamethylene bisacetamide (HMBA), forskolin, and 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), on the growth, morphology, and estrogen receptor (ER) content and epithelial membrane antigen (EMA) expression on a serumless human breast cancer cell line (MCF‐7) were compared. All these agents reversibly caused a concentration‐dependent growth inhibition in monolayers and markedly reduced colony‐forming efficiency in soft agar. A twofold increase in doubling time was obtained with RA (1 μM), but cell replication ceased with NaBu (1 mM), forskolin (50 μM), DMF (1%), HMBA (5 mM), and TPA (8 nM). Total growth arrest induced by these last compounds was preceded by an accumulation of cells in GO/G1 phase observed at 24 h by flow cytometry and accompanied by a change in cell morphology as seen by light and electronic microscopy. An increase in cell volume and the presence of lipid droplets was noted in treated cells that were spread out, as compared with controls. The acquisition of a more mature phenotype was confirmed by an increased expression of EMA monitored by flow cytometry. A specific reduction in the number of ER without any constant dissociation (Kd) modification was also observed after treatment with the 5 DIAs. No modification of morphological or biochemical characteristics, including EMA expression and ER binding, were observed for RA (1 μM)‐treated cells. All these results suggest that induction of a more differentiated phenotype is associated with a block in G1 cell cycle phase, resulting in total growth arrest. Apparently, RA (1 μM)‐treated cells did not fulfill these criteria, since only a slight accumulation in G1 and a slowed growth rate were evaluated.
Reversal of lovastatin‐mediated inhibition of natural killer cell cytotoxicity by interleukin 2
Authors: Cutts J. Lynn , Bankhurst Arthur D.
Published: February 2005

The activation of human natural killer (NK) cell cytotoxicity by interleukin 2 (IL‐2) is well established, although the biochemical mechanisms ofthis stimulation have not yet been fully delineated. Earlier, we reported that treatment of NK cells with an inhibitor of 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG CoA) reductase such as compactin or lovastatin significantly abrogates the in vitro killing of a susceptible human erythroleukemic cell line and that this inhibition can be completely reversed by 2 hr of exposure to mevalonate (J. Cell. Physiology 139:550–557, 1989). We report here that 24 hr of treatment with IL‐2 also reverses lovastatin inhibition of NK cell function. In addition to natural cytotoxicity, IL‐2 also restores chemotactic and antibody dependent cellular cytotoxicity functions to lovastatin‐treated cells. IL‐2 does not stimulate proliferation of these cells during this time period, nor does it affect the phenotypic composition of the NK cell preparations. Although IL‐2 was able to reverse the lovastatin‐mediated inhibition of every cell function we examined, it had no effect on the inhibition of cholesterol biosynthesis as measured by [3H]acetate incorporation into non‐saponifiable lipids, nor did it stimulate HMG CoA reductase activity. These findings support the hypothesis that there is a non‐sterol isoprenoid product which is required for NK cell cytotoxicity and chemotaxis. In addition, the data suggest that IL‐2 stimulation of NK cells proceeds by an isoprenoid‐independent pathway.
Growth factor‐induced DNA synthesis in cells that overproduce protein kinase C
Authors: Hoshina Sadayori , Ueffing Marius , Weinstein I. Bernard
Published: February 2005

In previous studies (Housey et al.: Cell 52: 343–354, 1988), our laboratory demonstrated that a cell line R6‐PKC3 that stably overproduces high levels of the βisoform of PKC displayed several abnormalities in growth control, and these phenotypic changes were also markedly enhanced when the cells were exposed to TPA. The present studies indicate that these cells also display marked changes in their response to certain growth factors. A striking finding was that several agents when tested alone in serum‐free medium, including EGF, PDGF, TPA, teleocidin, and OAG, stimulated DNA synthesis in quiescent R6‐PKC3 cells but had a negligible effect in quiescent R6‐C1 cells, a vector control cell line with jiormal levels of PKC. R6‐PKC3 cells also show an exaggerated response to very low concentrations of serum, when compared to R6‐C1 control cells. These studies provide direct genetic evidence that alterations in ce lular levels of PKC can markedly influence the responses of cells to specific growth factors.
Establishment of an osseous cell line from fetal rat calvaria using an immunocytolytic method of cell selection: Characterization of the cell line and of derived clones
Authors: Bernier Suzanne M. , Desjardins Johanne , Sullivan Arthur K. , Goltzman David
Published: February 2005

Using selective media and complement‐mediated lysis of primary cultures of a fetal rat calvarial cell population, we have developed a cell line (OBCK6) that exhibits osteoblastic characteristics. OBCK6 cells demonstrated enhanced para‐thyroid hormone (PTH)‐stimulated adenylate cyclase activity relative to the primary, calvarial population, production of alkaline phosphatase activity and type I collagen, and the capacity to form mineralized nodules in unsupplemented medium after prolonged (22–26 day) culture. Two sublines, CFK1 and CFK2, which were isolated by dilution cloning, differed morphologically and with respect to growth rate. CFK1 cells demonstrated high PTH and prostaglandin E2‐stimulated adenylate cyclase activity, whereas only low PTH‐stimulated activity was observed in CFK2 cells. Retinoic acid and 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3] each reduced PTH‐stimulated adenylate cyclase activity in both the cell types. Retinoic acid and dexamethasone reduced and 1,25(OH)2D3 enhanced alkaline phosphatase activity in these cells. PTH significantly augmented alkaline phosphatase activity to a much greater extent in CFK1 than in CFK2 cells. Both CFK1 and CFK2 cells expressed type I but not type III collagen, and neither expressed osteocalcin. Strong Alcian blue staining of CFK2 cells was suggestive of a cartilaginous phenotype. These three cell lines, therefore, demonstrated discrete characteristics of skeletal cell function and should provide important models for evaluation of mechanisms of mineralization and for control of skeletal cell growth and mesenchymal differentiation in vitro.
Signal transduction in N‐formyl‐methionyl‐leucyl‐phenylalanine and concanavalin A stimulated human neutrophils: Superoxide production without a rise in intracellular free calcium
Authors: Liang Songlin , Woodlock Timothy J. , Whitin John C. , Lichtman Marshall A. , Segel George B.
Published: February 2005

Changes in intracellular ionized free calcium ([Ca]i), inositol triphosphate (IP3), and‐sn‐1,2‐diacylglycerol (DAG) were determined in relation to agonist‐induced human neutrophil superoxide (O2−) production. With 0.1 μM N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP) stimulation, generation of IP3 and a peak rise in [Cai] occurred at 30 sec, preceding maximal O2− production (1.5 min) and the maximal rise in DAG mass (4 min). FMLP‐induced O2 production was inhibited by pertussis toxin. In cytochalasin B‐primed, concanavalin A (Con A) stimulated neutrophils, a peak rise in [Ca], but not IP3 proceeded O2 production, and pertussis toxin did not inhibit O2− production. EGTA inhibited the cytochalasin B/fMLP‐induced increment in [Ca]i and O2− production by 75% and 50% respectively, and completely ablated the response to cytochalasin B/Con A, suggesting a role for extracellular as well as intracellular calcium in the respiratory burst. However, three types of experiments indicate that an increase in [Ca]i is neither sufficient nor always required for O2 − production. First, treatment with ionomycin resulted in a marked increase in [Ca]i but did not cause O2− production. Second, pertussis toxin inhibited both fMLP‐induced IP3 generation and O2− production but did not inhibit the rise in [Ca]i Third, following neutrophil priming with dioctanoylglycerol (diC8), maximal O2− production occurred in response to 0.015 μM fMLP or Con A without a rise in [Ca]i, and diC8/fMLP‐induced O2− production was not inhibited by EGTA. Taken together, these data suggest that (1) an increment in [Ca]i is not strictly essential for neutrophil O2− production, (2) unlike fMLP, Con A‐induced O2− production does not proceed through a pathway involving the pertussis toxin‐sensitive G protein, and (3) regulation of neutrophil [Ca]i involves mechanisms independent of IP3 concentration.
Involvement of Hexose transport in myogenic differentiation
Authors: Kudo P. A. , Lo T. C. Y.
Published: February 2005

A high (HAHT) and a low (LAHT) affinity hexose transport system are present in undifferentiated rat L6 myoblasts; however, only the latter can be detected in multinucleated myotubes. This suggests that HAHT is either down‐regulated or modified as a result of myogenesis. The present investigation examined the relationship between HAHT and myogenic differentiation. While myogenesis could be inhibited by the potent hexose transport inhibitor phloretin, it was not affected by phlorizin which had no effect on hexose transport. This relationship was further explored using six different HAHT‐defective mutants. All six mutants, altered in either the HAHT transport affinity (Type I mutants) or capacity (Type II mutants), were impaired in myogenesis. Since these mutants were selected from both mutagenized and non‐mutagenized cells with different reagents, or with different concentrations of the same reagent, the deficiency in myogenesis was likely due to changes in HAHT properties. This notion was confirmed by the observation that growth of Type I mutants in high D‐glucose concentrations could rectify the defect in myogenesis. D‐glucose was unlikely to rectify the defect in myogenesis, if this defect was due to a second unrelated mutation that may have arisen during isolation of the mutants. Since both types of mutants were not altered in LAHT, D‐glucose should still be taken up into the cells. The fact that the glucose‐mediated increase in fusion could not be observed in Type II mutants (deficient in the HAHT transporter) suggested that myogenesis was dependent on the presence of D‐glucose or its metabolites in specific HAHT‐accessible compartments. It is tempting to speculate that trans‐acting regulators involved in myogenesis may be synthesized from the glucose metabolites in these specialized HAHT‐accassible compartments.
Platelet‐derived growth factor‐induced destabilization of smooth muscle alpha‐actin mRNA
Authors: Corjay Martha H. , Blank Randal S. , Owens Gary K.
Published: February 2005

We have previously shown that treatment of postconfluent, quiescent rat vascular smooth muscle cells (SMC) with platelet‐derived growth factor (PDGF) dramatically reduced smooth muscle (SM) alpha‐actin synthesis and SM alpha‐actin mRNA abundance, suggesting a role for this mitogen in the control of SMC differentiation. In the present studies, we explored the molecular mechanisms whereby PDGF decreases SM alpha‐actin mRNA levels. Treatment of postcon‐fluent SMC with both platelet PDGF and recombinant PDGF‐BB resulted in a dramatic and concentration‐dependent decrease in SM alpha‐actin mRNA levels. We observed no differences in efficacy between platelet PDGF and PDGF‐BB, indicating that the PDGF‐A chain is not required for the effect. The rate of decrease in SM alpha‐actin mRNA abundance in PDGF‐treated SMC was greater than that observed in cells treated with the transcriptional inhibitor, actinomycin D, with or without PDGF, indicating that PDGF induced a transcriptionally dependent destabilization of the cytosolic SM alpha‐actin mRNA pool. This effect appeared selective for SM alpha‐actin, in that there was no evidence of a similar change in non‐muscle (NM) beta‐actin mRNA stability following PDGF treatment. Results of nuclear run‐on analyses showed no differences in SM alpha‐actin transcription between PDGF‐ and vehicle‐treated SMC at either 4 or 24 hours following treatment, demonstrating that decreases in transcription of the SM alpha‐acti gene did not contribute to PDGF‐induced changes in SM alpha‐actin mRNA abundance, Results of these studies support a possible role for PDGF in regulation of SMC differentiation via a post‐transcriptional control mechanism.
Significance of extracellular zinc‐binding ligands in the uptake of zinc by human fibroblasts
Authors: Ackland M. Leigh , McArdle Harry J.
Published: February 2005

Extracellular zinc (Zn)‐binding ligands were investigated as vehicles for uptake of Zn by human fibroblasts. The uptake of α‐macroglobulin, a major serum Zn‐binding protein proposed to have a function in Zn transport, was less than 1/200 that of the Zn uptake rate. The fibroblast growth medium, BME with 10% FBS, contains several Zn‐binding ligands. These were separated into components of MW greater than 30,000 and components of MW less than 30,000 using an Amicon microconcentrator. Cells accumulated Zn from both fractions; however, there was more uptake from the filtrate (MW 30,000), containing ligands with low affinity for Zn, hence with greater free Zn concentration. Zn uptake from a number of ligands with a range of affinities for Zn was examined and found to be inversely proportional to the Ka value for the ligands and therefore proportional to the free Zn concentration. When histidine and desferrioxamine, two structurally different Zn‐binding ligands were compared, analysis of the concentration curves of calculated free Zn against Zn uptake gave similar Vmax and Km values (S.E.M.) (±S.E.M.) of 373 ± 6 pmol/μg DNA'h and 0.08 ± 0.004 μM for histidine, and 349 ± 10 pmol/μg DNA/h and 0.06 ± 0.008 μM for DFO, suggesting that the same transport mechanism was operating in both systems. We conclude that no specific ligands are essential for transport of Zn into fibroblasts, but that ‘free’ Zn is acquired by the cell.
Cell‐cell interactions in the process of differentiation of thyroid epithelial cells into follicles: A study by microinjection and fluorescence microscopy on in vitro reconstituted thyroid follicles
Authors: Munari‐Silem Yvonne , Mesnil Marc , Seimi Samia , Bernier‐Valentin Françoise , Rabilloud Rachida , Rousset Bernard
Published: February 2005

Thyroid cells, cultured in the presence of thyroid stimulating hormone, reorganized within 36–48 hr into follicular structures, the in vitro reconstituted thyroid follicles or RTF. By microinjection of fluorescent probes either into the neoformed intrafollicular lumen (IL) or into cells forming the follicles, we have studied the development and some functional properties of cell‐cell contacts involved in (a) the formation of the thyroid follicular lumen and (b) the communication between thyrocytes within the follicle. The probes were compounds of either low (Lucifer Yellow: LY) or high molecular weight (Dextran labeled with fluorescein: FITC‐Dextran and Cascade Blue conjugated to bovine serum albumin: CB‐BSA). LY microinjected into IL of 2–9‐day‐old RTF was seen to label circular spaces with a diameter ranging from 10 to 100 μm. The cells delimiting the IL remained unlabeled. The fluorescent dye remained concentrated in IL for up to 24 hr. FITC‐Dextran or CB‐BSA microinjected into IL behaved as LY; the probes were restrained into the iumen. A 2 hr incubation of RTF with iodide induced alterations of the structure of IL; an effect mediated by an organic form of actively trapped iodide. A 15–30 min incubation of RTF in a low CA2+ medium caused the opening of IL visualized by the progressive decrease of the fluorescence of probes preinjected into the lumenal space. The same but more rapid effect was obtained by microinjection of EGTA into the IL. The low Ca2+‐dependent opening of IL was also demonstrated by the release into the medium of thyroglobulin present in IL. Microinjection of LY in a cell involved in the follicle structure led to the rapid labeling of the other cells forming the follicle but LY did not penetrate the IL. Unlike LY, the distribution of FiTC‐Dextran or CB‐BSA injected into cells delimiting the lumen was restricted to the microinjected cells. Alterations of medium or intralumenal Ca2+ concentration which caused the opening of IL did not affect the cell‐to‐cell transfer of LY. By using fluorescent probe microinjection, we show that the in vitro thyrocyte histiotypic differentiation leads to the reconstitution of functional intercellular junctions: tight junctions insuring the tightness of the neoformed lumen and gap junctions mediating the cell‐to‐cell exchange of small molecules. The structure of the thyroid follicles appears to be under the control of both extracellular and intralumenal Ca2+ concentrations.
Evidence that the adenylate cyclase secreted from Bordetella pertussis does not enter animal cells by receptor‐mediated endocytosis
Authors: Donovan Maura G. , Storm Daniel R.
Published: February 2005

Bordetella pertussis, the pathogen responsible for whooping cough, produces a calmodulin‐sensitive adenylate cyclase. Several investigators have shown that the partially purified adenylate cyclase is capable of entering animal cells and elevating intracellular cAMP levels (Confer and Eaton:Science 217:948–950, 1982; Shattuck and Storm: Biochemistry 24:6323–6328, 1985). However, the mechanism for entry of the catalytic subunit of this adenylate cyclase into animal cells is unknown. It has been reported that the B. pertussis adenylate cyclase extracted from bacterial cells with urea does not enter animal cells by receptor‐mediated endocytosis. There is, in addition to the cell associated form of the B. pertussis adenylate cyclase, a cell‐invasive form of the enzyme secreted into the bacterial culture media. The properties of the cell‐associated and secreted enzymes are significantly different (Masure and Storm: Biochemistry 28:438–442, 1989). In this study, we report evidence that the secreted form of the B. pertussis adenylate cyclase enters animal cells by a mechanism distinct from receptor‐mediated endocytosis.
M‐07e human leukemic factor‐dependent cell line provides a rapid and sensitive bioassay for the human cytokines GM‐CSF and IL‐3
Authors: Avanzi Gian Carlo , Brizzi Maria Felice , Giannotti Joann , Ciarletta Agnes , Yang Yu‐Chung , Pegoraro Luigi , Clark Steven C.
Published: February 2005

We have isolated a subline of the M‐07 human megakaryoblastic leukemia cell line, designated M‐07e, that requires either interleukin‐3 (IL‐3) or granulocyte macrophage colony‐stimulating factor (GM‐CSF) for growth, even in the presence of fetal calf serum. This cell line will not grow long term in any other cytokine although it responds slightly to IL‐2, IL‐4, IL‐6, IL‐9, and interferon‐γ. We have used the M‐07e subline to develop a quantitative bioassay for the measurement of levels of either GM‐CSF or IL‐3. This assay is as sensitive to either factor as the human bone marrow colony assay (CFU‐GM) or the chronic myelogeneous leukemic (CML) blast cell proliferation assay for these factors and is much more convenient and reliable than either. With this assay, as little as 25–50 pg/ml of either IL‐3 or GM‐CSF can be detected, a level that should render the assay useful for analysis of these molecules in samples from patients undergoing colony‐stimulating factor therapy and from conditioned media from natural sources of the factors. In these cases, neutralizing antisera to each cytokine are required to demonstrate the specificity of the assay. This assay, in combination with quantitative immunoassays should greatly facilitate the analysis of the roles of IL‐3 and GM‐CSF in regulating hematopoiesis both in patients and in natural sources of the cytokines.
Epithelioid macrophages secrete a deactivating factor for superoxide release
Authors: Camarero Valeria C. P. C. , Junqueira Virginia B. , Colepicolo Pio , Karnovsky Manfred L. , Mariano Mario
Published: February 2005

The release of superoxide anion (O) by inflammatory macrophages, multinucleated giant cells, and epithelioid cells, obtained by the insertion of round glass coverslips into the subcutaneous tissue of mice, was investigated. O was shown to be spontaneously released by cells on the surface of glass coverslips implanted up to 7 days but not by cells obtained 14 or 21 days after coverslip implantation. The formel showed increased O release when stimulated by phorbol myristate acetate, whereas cells harvested after 14 or 21 days implantation did not. The induction of delayed type hypersensitivity around coverslips implanted for 5 days increased spontaneous O release by these cells by 40%. On the other hand, when the same protocol was used with coverslips implanted for 14 days, O release was not detected. These results were viewed in regard to the composition of the cell population at each time point. When coverslips were removed after 14 days of implantation and the cells incubated for 30 minutes in vitro, the medium so conditioned inhibited O release by cells of 5 day old preparations. This indicates the release by cells on the longer term coverslips of a substance that inhibits O production by cells of coverslips implanted for 5 days only. This inhibitory activity could be suppressed by treating the conditioned medium with proteases. The factor was, however, heat stable and exerted its effects even when the test cells were exposed to phorbol myristate acetate.
Evidence for c‐myc in the signaling pathway for TGF‐β in well‐differentiated human colon carcinoma cells
Authors: Mulder Kathleen M. , Humphrey Lisa E. , Choi Ho Gene , Childress‐Fields Karla E. , Brattain Michael G.
Published: February 2005

Previously, we described a model culture system for comparing responsiveness of poorly differentiated and well‐differentiated human colon carcinoma cells to exogenous growth factors. While polypeptide growth stimulators elicited an up‐regulation of c‐myc, as well as a mitogenic response in the well‐differentiated cells, the poorly differentiated cells were insensitive to exogenous growth stimulators. We now show, by thymidine incorporation experiments and autoradiographic analysis, that transforming growth factors, β1 (TGF‐β) abrogated the mitogenic responses to the growth factors epidermal growth factor + insulin + transferrin (lC,50 = 0.8 ng/ml), as well as to nutrients (basal medium; lC50 = 0.2 ng/ml) in the well‐differentiated cells. The poorly differentiated cells did not respond to TGF‐β. Moreover, TGF‐β (10 ng/ml) completely abrogated the growth factor‐stimulated up‐regulation of c‐myc in the TGF‐β responsive, well‐differentiated colon carcinoma cells. Addition of TGF‐β to the TGF‐β‐responsive, well‐differentiated cells, at a time after c‐myc had been transiently up‐regulated in response to growth stimulatory factors, resulted in a loss of responsiveness to TGF‐β. Addition of TGF‐β to these cells at increasing time periods after EIT stimulation also resulted in a loss of the TGF‐β‐induced repression of c‐myc. The results suggest an important role for c‐myc in the mechanism of action of TGF‐β in well‐differentiated human colon carcinoma cells.
Phagocytosis in Acanthamoeba: I. A mannose receptor is responsible for the binding and phagocytosis of yeast
Authors: Allen Philip G. , Dawidowicz Eliezar A.
Published: February 2005

We have examined the initial events in phagocytosis by Acanthamoebe castellanii in order to understand this process at the molecular level and have determined that phagocytosis in this organism is mediated by a receptor which recognizes mannose‐ricn elements in the particle to be phagocytosed. We demonstrate that the binding and internalization of yeast particles can be inhibited by the sugars D(+)‐mannose and D(−)‐fructose in a stereospecific, concentration‐dependent manner. This inhibition is specific; these sugars did not inhibit the uptake of latex beads by this organism. Using mannosylated neoglycoproteins, which are much more potent inhibitors of particle binding as compared with the free sugar, we demonstrate the presence of a receptor on the amoeba cell surface which is necessary for the binding of yeast as the initial event of phagocytosis. The Acanthamoeba mannose receptor also appears to be able to mediate the delivery of soluble mannose‐rich molecules to a degradative compartment such as the lysosome. Knowledge of this receptor will allow a better understanding of the molecular events of phagocytosis.
Differential modes of action of fibronectin and epidermal growth factor on rabbit corneal epithelial migration
Authors: Nishida Teruo , Nakamura Masatsugu , Mishima Hiroshi , Otori Toshifumi
Published: February 2005

In order to clarify the roles of fibronectin (FN) and epidermal growth factor (EGF) in corneal wound healing, we cultured blocks of excised rabbit cornea for 24 hours in media containing one of these agents, then measured the length of the path of the epithelial layer that had migrated down the side of the block. Both FN and EGF stimulated epithelial migration significantly in a dose‐dependent fashion. Responses to EGF involved a time ag of at least 12 hours before stimulation could be observed, but there was no lag‐time for FN‐stimulated migration. FN was maximally effective only if it was continuously present. In contrast, exposure to EGF for 6 hours did not stimulate epithelial migration, but exposure for 9 hours resulted in the same stimulatory effects as were observed after 24 hours' continuous exposure. Anti‐FN antibody inhibited the FN‐ and EGF‐stimulated migration of corneal epithelium. But anti‐EGF antibody inhibited only EGF‐stimulated migration and had no effect on FN‐stimulated migration. These results indicate that, unlike FN, EGF need not be present, once the epithelial cells have recognized its signal. Furthermore, the stimulatory effect of EGF depended on FN, while that of FN was independent of EGF. The effects of EGF on migration of corneal epithelium may, therefore, be mediated by FN.
Demonstration of a direct role for myosin light chain kinase in fibroblast‐populated collagen lattice contraction
Authors: Ehrlich H. Paul , Rockwell W. Bradford , Cornwell Trudy L. , Rajaratnam Joseph B. M.
Published: February 2005

Mixing feed fibroblasts with soluble collagen and serum‐supplemented culture medium at 37°C results in the entrapment of cells within the polymerizing collagen matrix. This cellular‐collagen complex is referred to as a fibroblast‐populated collagen lattice (FPCL). In time, this FPCL undergoes a reduction in size called lattice contraction. The proposed mechanism for lattice contraction is cellular force produced by cytoplasmic microfilaments which organize collagen fibrils compacting the matrix. When the regulatory subunits of myosin, myosin light chains, are phosphorylated by myosin light chain kinase (MLCK), myosin ATPase activity is increased and actin‐myosin dynamic filament sliding occurs. Elevated levels of myosin ATPase are required for maximal lattice contraction. Cholera toxin inhibits lattice contraction by increasing intracellular levels of cAMP. It is proposed that increased cytoplasmic concentrations of cAMP promote phosphorylation of MLCK, the enzyme important for maximizing myosin ATPase activity. Phosphorylating MLCK in vitro inhibits activity by decreasing its sensitivity to calcium‐calmodulin complex. A decrease in MLCK activity would result in lower levels of myosin ATPase activity. MLCK, purified from turkey gizzard, was subjected to limited proteolytic digestion to produce calmodulin‐independent‐MLCK. The partially digested kinase does not require calcium‐calmodulin for activation. Independent‐MLCK is not subject to inhibition by phosphorylation. The electroporetic inoculation of independent‐MLCK into fibroblasts before FPCL manufacture produced enhanced lattice contraction. Lattice contraction, in the presence of cholera toxin, was restored to normal levels by the prior electroporetic introduction of independent‐MLCK. These findings support the hypothesis that increases in cAMP hinder lattice contraction by a mechanism involving inhibition of MLCK and myosin ATPase.
Macrovascular and microvascular endothelium during long‐term hypoxia: Alterations in cell growth, monolayer permeability, and cell surface coagulant properties
Authors: Shreeniwas R. , Ogawa S. , Cozzolino F. , Torcia G. , Braunstein N. , Butura C. , Brett J. , Lieberman H. B. , Furie M. B. , Joseph‐Silverstein J. , Stern D.
Published: February 2005

In bovine aortic or capillary endothelial cells (ECs) incubated under hypoxic conditions, cell growth was slowed in a dose‐dependent manner at lower oxygen concentrations, as progression into S phase from G1 was inhibited, concomitant with decreased thymidine kinase activity. Monolayers grown to confluence in ambient air, wounded, and then transferred to hypoxia showed decreased ability to repair the wound, as a result of both decreased motility and cell division. Hypoxic ECs demonstrated a ≈3‐fold increase in the total number of high‐affinity fibroblast growth factor receptors, and levels of endogenous FGF were suppressed. Consistent with the presence of functional FGF receptors, addition of basic FGF overcame, at least in part, hypoxia‐mediated suppression of EC growth, and enhanced wound repair in hypoxia, stimulating both motility and cell division. Despite slower growth in hypoxia, ECs could achieve confluence, and the monolayers consisted of larger cells with altered assembly of the actin‐based cytoskeleton and small gaps between contiguous cells. The permeability of these hypoxic EC monolayers to macromolecules and lower molecular weight solutes was increased. Cell surface coagulant properties were also perturbed: the anticoagulant cofactor thrombomodulin was suppressed, and a novel Factor X activator appeared on the EC surface. These data indicate that micro‐ and macrovascular ECs can grow and be maintained at low oxygen tensions, but hypoxic endothelium exhibits a range of altered functional properties which can potentially contribute to the pathogenesis of vascular lesions.
Evidence for extralysosomal hydrolysis of high‐density lipoprotein cholesteryl esters in rat hepatoma cells (Fu5AH): A model for delivery of high‐density lipoprotein cholesterol
Authors: DeLamatre John G. , Carter Robert M. , Hornick Conrad A.
Published: February 2005

Rat hepatoma cells (Fu5AH) were studied as a model for the net delivery of apoE‐free high‐density lipoprotein (HDL) cholesterol to a cell. Incubating cells with HDL results in (1) a decrease in both media‐free cholesterol and cholesteryl ester concentration; (2) decreased cell sterol synthesis; and (3) increased cell cholesteryl ester synthesis. HDL cholesteryl ester uptake is increased when cells are incubated for 18 hr in cholesterol poor media. Coincubation of 3H‐cholesteryl ester‐labeled low‐density lipoprotein (LDL) with 50 μM chloroquine or 25 μM monensin results in a decrease in the cellular free cholesterol/cholesteryl ester (FC/CE) isotope ratio, indicating an inhibition in the conversion of cholesteryl ester to free cholesterol. In contrast, chloroquine and monensin do not alter the cellular FC/CE isotope ratio for 3H‐CE HDL. This evidence indicates that acidic lysosomal cholesteryl ester hydrolase does not account for the hydrolysis of HDL‐CE. Free cholesterol generated from 3H‐cholesteryl ester of both LDL and HDL is reesterified intracellularly. At higher HDL concentrations (above 50 μg/ml) HDL cholesteryl ester hydrolysis is sensitive to chloroquine. We propose that an extralysosomal pathway is operating in the metabolism of HDL cholesterol and that at higher HDL concentrations a lysosomal pathway may be functioning in addition to an extralysosomal pathway.
Characterization of atrial natriuretic peptide receptors in brain microvessel endothelial cells
Authors: Whitson Peggy A. , Huls M. Helen , Sams Clarence F.
Published: February 2005

Atrial natriuretic peptide (ANP) binding and ANP‐induced increases in cyclic guanosine monophosphate (cGMP) levels have been observed in brain microvessels (Chabrier et al., 1987; Steardo and Nathanson, 1987), suggesting that this fluid‐regulating hormone may play a role in the fluid homeostasis of the brain. This study was initiated to characterize the ANP receptors in primary cultures of brain microvessel endothelial cells (BMECs). The apparent equilibrium dissociation constant, Kd, for ANP increased from 0.25 nM to 2.5 nM, and the number of ANP binding sites as determined by Scatchard analysis increased from 7,100 to 170,000 sites/cell between 2 and 10 days of culture following monolayer formation. Time‐ and concentration‐dependent studies on the stimulation of cGMP levels by ANP indicated that guanylate cyclase‐linked ANP receptors were present in BMECs. The relative abilities of ANP, brain natriuretic peptide (BNP), and a truncated analog of ANP containing amino acids 5–27 (ANP 5–27) to modulate the accumulation of cGMP was found to be ANP >BNP ≫ ANP 5–27. Affinity cross‐linking with disuccinimidyl suberate and radiolabeled ANP followed by gel electrophoresis under reducing conditions demonstrated a single band corresponding to the 60–70 kD receptor, indicating the presence of the nonguanylate cyclase‐linked ANP receptor. Radiolabeled ANP binding was examined in the presence of various concentrations of either ANP, BNP, or ANP 5–27 and suggested that a large proportion of the ANP receptors present in blood‐brain barrier endothelial cells bind all of these ligands similarly. These data indicate both guanylate cyclase linked and nonguanylate cyclase linked receptors are present on BMECs and that a higher proportion of the nonguanylate cyclase linked receptors is expressed. This in vitro culture system may provide a valuable tool for the examination of ANP receptor expression and function in blood‐brain barrier endothelial cells.
Extinction of the human insulin gene expression in insulinoma X fibroblast somatic cell hybrids involves cis‐acting DNA elements
Authors: Besnard Corinne , Monthioux Eliane , Loràs Patrick , Jami Jacques , Daegelen Dominique
Published: February 2005

Insulin gene expression in rat insulinoma (RIN) cells is extinct in RIN × fibroblast hybrids and can reappear upon loss of DNA contributed by the fibroblast parent. (Besnard et al., Exp. Cell Res. 185:101–108, 1989). In the present study, we looked for the role of 5′‐flanking sequences of the human insulin gene in the negative control observed in the hybrids. RIN cells were transformed with composite genes which consisted of the coding sequence of the gpt gene placed under the control of 5′‐flanking regions of the human insulin gene (Ins. gpt gene). Upon hybridization of these cells with mouse fibroblasts, the expression of both Ins. gpt and endogenous rat insulin genes were suppressed together. The results obtained indicate that cis‐acting DNA elements are involved in the negative control of the gene. These elements are located in a fragment spread from ‐258 to +241 of the transcription origin of the human insulin gene.
Transforming growth factor‐β and platelet‐derived growth factor synergistically stimulate contraction by testicular peritubular cells in culture in serum‐free medium
Authors: Tung Pierre S. , Fritz Irving B.
Published: February 2005

We report investigations on factors influencing contractility by testicular peritu‐bular cells (PC) maintained in culture in a three‐dimensional collagen gel system, and the behavior of PC in culture on a two‐dimensional system. At low and moderate cell densities, PC embedded in collagen gels in serum‐free Eagle's minimal essential medium (MEM) have a lesser degree of contractility than PC in culture in MEM containing calf serum. The contractility by PC, measured by determining changes in diameter of the collagen gel, was increased by addition of transforming growth factor‐β (TGF‐β) to serum‐free MEM, and this was further enhanced by supplementing the medium with platelet‐derived growth factor (PDGF). In the absence of TGF‐β, however, PDGF had no detectable effects on PC contractility. Other growth factors examined (epidermal growth factor, insulin, and fibroblast growth factor) did not influence the degree of contractility of PC in serum‐free MEM in the presence or absence of TGF‐β. PC maintained in MEM supplemented with platelet‐poor serum (PPS) have a lesser degree of contractility than their counterparts in MEM containing 2.5% calf serum. The addition of TGF‐β and PDGF to PPS‐supplemented MEM restored contractility by PC to a level comparable to that observed by PC in MEM containing complete serum. The addition of nonpurified bovine serum albumin (BSA) to MEM greatly increased PC contractility. By contrast, highly purified BSA had no such effect, suggesting that one or more components adsorbed to the impure BSA was implicated. Polyclonal antibody against fibronectin did not influence the contractility of PC in collagen gels in the presence or absence of serum. Antiserum against TGF‐β partially blocked the enhancement of contractility of PC in MEM containing non‐purified BSA. In PC plated on top of a collagen gel lattice, the attachment, spreading, and cell shape were greatly influenced by the presence of TGF‐β and PDGF, both singly and together. Data presented are interpreted to indicate that effects elicited by serum on the properties of PC in culture, and on the contractility of PC, can be attributed in part to the combined influences of TGF‐β and PDGF in serum.
Serotonin: An inducer of collagenase in myometrial smooth muscle cells
Authors: Jeffrey John J. , Ehlich Lynn S. , Roswit William T.
Published: February 2005

Rat myometrial smooth muscle cells in culture actively produce collagenase in medium containing fetal bovine serum, but not in medium containing newborn bovine serum or containing fetal serum adsorbed with dextran‐coated charcoal. A dialyzable molecule has been isolated from fetal bovine serum, which restores the ability of the smooth muscle cells to produce collagenase. The molecule has been purified and identified as serotonin (5‐hydroxytryptamine). Cells cultured in medium depleted of serotonin for 3 days fail to produce collagenase, as assessed both enzymatically and immunologically. Addition of serotonin promptly restores the ability of the cells to produce the enzyme. The EC50 for serotonin is approximately 2 μM; maximum stimulation of collagenase production is observed at 5 μM. The response is specific for serotonin: a wide variety of compounds tested, either related to serotonin or of potential eproductive significance, were without effect in the induction of collagenase production by the cells. No changes in DNA content, general protein synthesis, or cellular collagen production were observed as a consequence of serotonin depletion or restoration, suggesting a selective effect of the compound on collagenase production. The effect of serotonin was also selective to myometrial smooth muscle cells; collagenase‐producing fibroblasts from skin and cervix displayed no serotonin requirement for enzyme production. Studies using specific agonists or antagonists for a variety of serotonin receptor subtypes suggest that the 5‐HT‐2 receptor mediates the serotonin induction of collagenase in these cells. Preliminary evidence indicates that cultured human myometrial smooth muscle cells are also dependent upon serotonin for collagenase production. The evidence in this study suggests the possibility that serotonin serves as a signal to begin the massive collagen degradation that occurs in the postpartum uterus.
Chloride secretagogues stimulate inositol phosphate formation in shark rectal gland tubules cultured in suspension
Authors: Ecay Tom W. , Valentich John D.
Published: February 2005

Neuroendocrine activation of transepithalial chloride secretion by shark rectal gland cells is associated with increases in cellular cAMP, cGMP, and free calcium concentrations. We report here on the effects of several chloride secretagogues on inositol phosphate formation in cultured rectal gland tubules. Vasoactive intestinal peptide (VIP), atriopeptin (AP), and ionomycin increase the total inositol phosphate levels of cultured tubules, as measured by ion exchange chromatography. Forskolin, a potent chloride secretagogue, has no effect on inositol phosphate formation. The uptake of 3H‐myo‐inositol into phospholipids is very slow, preventing the detection of increased levels of inositol trisphosphate. However, significant increases in inositol monophosphate (IP1) and inositol biphosphate (IP2) were measured. The time course of VIP‐ and AP‐stimulated IP1 and IP2 formation is similar to the effects of these agents on the short‐circuit current responses of rectal gland monolayer cultures. In addition, aluminum fluoride, an artificial activator of guanine nucleotide‐binding proteins, stimulates IP1 and IP2 formation. We conclude that rectal gland cells contain VIP and AP receptors coupled to the activation of phospholipase C. Coupling may be mediated by G‐proteins. Receptor‐stimulated increases in inositol phospholipid metabolism is one mechanism leading to increased intracellular free calcium concentrations, an important regulatory event in the activation of transepithelial chloride secretion by shark rectal gland epithelial cells.