bluuebunny/crossref_metadata_2025 huggingface dataset:
Growth‐inhibitory effect of TGF‐B on human fetal adrenal cells in primary monolayer culture
Authors: Riopel Lise , Branchaud Charlotte L. , Goodyer Cynthia G. , Adkar Vinita , Lefebvre Yves
Published: February 2005

We examined the effects of transforming‐growth factor‐B (TGF‐B) on growth ([3H]‐thymidine uptake) and function (dehydroepiandrosterone sulfate [DHAS] and cortisol production) of human fetal zone adrenal cells. Results indicate that TGF‐B significantly inhibits, in a dose‐related manner, both basal and epidermal growth factor (EGF)‐stimulated cell growth: IC50= 0.1–;0.25 ng/ml. EGF is ineffective in overcoming the inhibitory effect of TGF‐B, suggesting a noncompetitive antagonism between the two factors. Also, the inhibitory effect of TGF‐B is additive to that of adrenocorticotropic hormone (ACTH). On the other hand, TGF‐B (1 ng/ml) does not significantly change basal or ACTH‐stimulated DHAS or cortisol secretion. We conclude that, unlike its effect on other steroid‐producing cells, TGF‐B inhibits growth of fetal zone cells and does not appear to have a significant inhibitory effect on steroidogenesis.
Induction of glucose‐regulated proteins in Xenopus laevis A6 Cells
Authors: Winning R. S. , Heikkila J. J. , Bols N. C.
Published: February 2005

We have characterized the induction of glucose‐regulated proteins (GRPs) in Xenopus laevis A6 cells, a kidney epithelial cell line. Exposure of A6 cells to medium in which 2‐deoxyglucose replaced galactose resulted in enhanced synthesis of two proteins at 78 and 98 kd. The 78 kd protein was determined by two‐dimensional PAGE to consist of two isoelectric variants with pls of 5.3 and 5.2 whereas the 98 kd protein resolved into a single spot with a pl of 5.1. The 78 kd protein cross‐reacted with antiserum against chicken GRP78 (glucose‐regulated protein), suggesting that the Xenopus protein shares homology with a previously characterized GRP. This was supported by the finding that a rat GRP78 probe hybridized with a 2‐deoxyglucose‐inducible mRNA. Synthesis of the two proteins was also induced by tunicamycin, 2‐deoxygalactose, and dithiothreitol. However, the GRPs were not induced by glucosamine or calcium ionophore A23187 at concentrations and exposure periods that have previously been shown to elicit a GRP response in mammalian and avian cells. Enhanced synthesis of the two GRPs by 2‐deoxyglucose was transient, reaching maximal levels by 12‐24 h and decreasing to near control levels by 48 h. Removal of the stress at the point of peak synthesis resulted in decreased synthesis of both proteins within 6 h and a return to control levels within 24 h of recovery. These data suggest that Xenopus cells have a GRP response that is similar, but not identical, to that found in mammalian cells.
Effects of transforming growth factor β and epidermal growth factor on cell proliferation and the formation of bone nodules in isolated fetal rat calvaria cells
Authors: Antosz Mark E. , Bellows Carlton G. , Aubin Jane E.
Published: February 2005

When cells enzymatically isolated from fetal rat calvaria (RC cells) are cultured in vitro in the presence of ascorbic acid and Na β‐glycerophosphate, discrete three‐dimensional nodules form with the histologic, immunohistochemical, and ultra‐structural characteristics of bone (Bellows et al; Calcified Tissue International 38:143–154, 1986; Bhargava et al., Bone, 9:155–163, 1988). Quantitation of the number of bone nodules that forms provides a colony assay for osteoprogenitor cells present in the RC population (Bellows and Aubin, Develop. Biol., 133:8–13, 1989). Continuous culture with either epidermal growth factor (EGF) or transforming growth factor beta (TGF‐β) results in dose‐dependent inhibition of bone nodule formation; however, the former causes increased proliferation and saturation density, while the latter reduces both parameters. Addition of EGF (48 h pulse, 2‐200 ng/ml) to RC cells at day 1 after plating results in increased proliferation and population saturation density and an increased number of bone nodules formed. Similar pulses at confluence and in postconfluent multilayered cultures when nodules first begin forming (approx. day 11) inhibited bone nodule formation and resulted in a smaller stimulation of cell proliferation. Forty‐eight hour pulses of TGF‐β (0.01‐1 ng/ml) reduced bone nodule formation and proliferation at all times examined, with pulses on day 1 causing maximum inhibition. The effects of pulses with TGF‐β and EGF on inhibition of nodule formation are independent of the presence of serum in the culture medium during the pulse. The data suggest that whereas EGF can either stimulate or inhibit the formation of bone nodules depending upon the time and duration of exposure, TGF‐B inhibits bone nodule formation under all conditions tested. Moreover, these effects on osteoprogenitor cell differentiation do not always correlate with the effects of the growth factors on RC cell proliferation.
Inositol 1,3,4,5‐tetrakisphosphate stimulates the initiation of DNA synthesis in Ca2+‐deprived rat liver cells
Authors: Hill Timothy D. , Zwiller Jean , Boynton Alton L.
Published: February 2005

The G1‐S boundary of non‐neoplastic cells requires extracellular Ca2+ for successful transition. Inositol 1,3,4,5‐tetrakisphosphate but not inositol 1,4,5‐trisphosphate can partially replace Ca2+ and stimulate the initiation of DNA synthesis of Ca2+‐deprived T51B rat liver cells but only if sufficient extracellular Ca2+ (i.e., 0.075 mM) is present. The potent tumor promoter and protein kinase C activator 12‐O‐tetradecanoylphorbol acetate is also capable of replacing ex‐tracellular Ca2+ and partially stimulating the initiation of DNA synthesis. In addition, both inositol‐1,3,4,5‐tetrakisphosphate and 12‐O‐tetradecanoylphorbol acetate added together elicit a full DNA synthetic response.
Asymmetric distribution of phospholipids in the membrane of vesicles released during in vitro maturation of guinea pig reticulocytes: Evidence precluding a role for “aminophospholipid translocase”
Authors: Vidal Michel , Sainte‐Marie Josette , Philippot Jean R. , Bienvenue Alain
Published: February 2005

Guinea pig reticulocytes lose their transferrin (Tf) binding activity during maturation, in the form of vesicles (exosomes) released into the extracellular medium. Vesicles were prepared from cultures of reticulocytes to study the possible externalization of a particular membrane‐associated activity, i.e., that of “aminophospholipid translocase.” Analysis of the peptide composition of these vesicles revealed that the major proteins are the Tf receptor and another peptide (70kDa), which is probably the “clathrin‐uncoating ATPase” described by johnstone et al. (1987). The exosome had a lipid composition similar to erythrocyte membrane, although with a lightly but significantly lower phosphatidylethanolamine content. The aminophospholipid distribution in the vesicle membrane was determined by fluorescamine labeling. The exosomes showed an asymmetric aminophospho‐lipid distribution similar to that of erythrocytes. “Aminophospholipid translocase” activity was absent, as no transverse diffusion of spin‐labeled phospholipids occurred over more than 2 hours at 37°C.
Ultrastructural analysis of acute immune thrombocytopenia in mice: Dissociation between alterations in megakaryocytes and platelets
Authors: Stenberg Paula E. , Levin Jack
Published: February 2005

Thrombopoiesis was studied in mice after the induction of acute immune throm‐bocytopenia with platelet antiserum (PAS). Utilizing electron microscopy, we examined platelets and megakaryocytes (MK) obtained 4, 8, 12, 24, 48, 72, and 120 hr after administration of PAS. Four to 24 hr after injection of PAS, the majority of bone marrow MK were normal in size and organelle distribution. The demarcation membrane system (DMS) extended normally throughout the mature cell cytoplasm at these times. However, approximately 50% of MK observed 48 hr and 72 hr after injection of PAS were significantly larger than normal, and often had demarcation membranes confined to an area between a peripheral organelle‐deficient zone and a central nuclear zone. The median values for sectional areas of platelets obtained 8–72 hr after administration of PAS were significantly greater than the median value for sectional areas of platelets in a pooled control sample. The proportion of cytoplasm to surface‐connected canalicular system appeared greater than normal in most large platelets from the PAS samples; and increased numbers of profiles of Golgi complex and endoplasmic reticulum were observed. By 48 hr post‐injection of PAS (at which time the modal ploidy class of MK has shifted from 16N to 32N; Corash et al.,Blood 70:177, 1987), most platelets were normal in size and cytoplasmic appearance. At 120 hr post‐injection of PAS, virtually all platelets exhibited a normal size and complement of organelles, and MK also had returned to normal. Our data indicate that in response to acute thrombocytopenia, MK prematurely release platelets which differ from normal platelets in size and cytoplasmic appearance. There was a marked dissociation between alterations in platelets and MK, since a statistically significant increase in platelet sectional area occurred 40 hr before the shift in modal ploidy class of MK, and platelet size subsequently decreased toward normal during the period that has been shown to be associated with the maximum shift in MK ploidy. These results strongly suggest that the characteristics of platelet release do not depend on the ploidy or cytoplasmic characteristics of MK.
Modulation of TGF‐β type 1 receptor: Flow cytometric detection with biotinylated TGF‐β
Authors: Newman Walter , Beall L. Dawson , Bertolini Donald R. , Cone James L.
Published: February 2005

Transforming growth factor β‐ type 1 (TGF‐β) was reacted with NHS‐biotin to yield a derivative of TGF‐β1 which was biotinylated on lysine residues. The biotinylated form of TGF‐β1 was separated from the unreacted material by reverse phase chromatography. In three separate bioassays, the derivatized peptide was as active as the starting material. The use of FITC‐avidin in conjunction with flow cytometry demonstrated that the binding of biotinylated TGF‐β to its receptor is saturable, competable, and specific. A 100‐fold molar excess of unde‐rivatized TGF‐β1 gave 85% inhibition of binding of the biotinylated peptide to the mink lung cell line CCL‐64, while TGF‐β2 showed no inhibition of binding, nor did insulin, calcitonin, or TGF‐α. Both CCL‐64 cells and human umbilical vein endothelial cells showed a density‐dependent down‐regulation of receptor expression in culture. Several factors were examined that might mediate this effect. The down‐regulation was shown not to be due to the secretion of an active form of TGF‐β1. The extracellular matrix from high‐density cells did not decrease expression of the receptor. Fibronectin, collagen, and gelatin were also unable to signal changes in receptor expression, even though in other systems such matrix components can regulate the responsiveness of cells to TGF‐β1. Lastly, staining simultaneously for DNA content and TGF‐β1receptor expression showed that there was no correlation between cell cycle and receptor levels.
Different pattern of differentiation in two LLC‐PK1 Clones
Authors: Van Den Bosch Ludo , De Smedt Humbert , Borghgraef Roger
Published: February 2005

Two clones (LD3 and LC3) were isolated from the established renal cell line LLC‐PK1. They differed with respect to the development of the Na+‐dependent α‐methyl‐D‐glucoside (AMG) uptake. The higher uptake capacity in LD3 as compared to LC3 was owing to the expression of a higher number of carrier molecules as was shown by the specific phlorizin binding. The intracellular cyclic AMP level, the D‐glucose concentration in the growth medium and the cell density could be excluded as the causes of the difference between both clones. We found that the faster development of the Na+‐dependent hexose carrier in LD3 was parallelled by a faster expression of trehalase in this clone. This suggests that the expression of both apical proteins is correlated. From the growth curves it was concluded that renewal of the undifferentiated population was roughly the same in both clones. The recruitment of cells from the undifferentiated to the growth arrested state seems also very similar as was deduced from the development of tight junctions and from the down‐regulation of the α‐methylaminoisobutyric acid (meAIB) uptake. The start of differentiation was identical as was shown by the similar rate of expression found for γ‐glutamyl transferase. The difference between both clones is most likely situated at the traverse to a fully differentiated cell. This process takes more time in LC3 than in LD3. Also the fully differentiated state seemed to be different in both clones. We conclude that the pattern of differentiated characteristics found in both clones is different and a model incorporating these differences is presented.
Cytochemical and molecular properties of simian virus 40 transformed Kaposi's sarcoma‐derived cells: Evidence for the secretion of a member of the fibroblast growth factor family
Authors: Werner Sabine , Hofschneider Peter Hans , Stürzl Michael , Dick Irene , Roth Willi Kurt
Published: February 2005

Contact‐inhibited Kaposi's sarcoma‐derived cells (KS cells) were transfected with Simian Virus 40 (SV40) DNA. Transformed cells (SV‐KSC) were selected for their capacity to form foci on monolayers of the low‐malignant KS cells. Isolated SV‐KSC foci were found to contain integrated SV40 DNA sequences and to express SV40 large T‐antigen. Several differentiation properties of KS cells are retained in the SV40 transformants, e.g., expression of vimentin and the endothelial cell marker BMA 120. In contrast to the maternal KS cells, SV‐KSC are capable of growing in platelet‐derived growth factor (PDGF)‐depleted plateletpoor‐plasma serum (PPPS) and in soft agar. However, they are not tumorigenic in nude mice. Expression of the oncogenes c‐myc, c‐N‐ras, c‐Ha‐ras, and p53 is significantly elevated in SV‐KSC, whereas c‐fos and c‐erb B expression is comparable to that of KS cells and fibroblasts. Conditioned medium from SV‐KSC can substitute for PDGF when PDGF‐dependent, nontransformed KS cells are grown in PPPS. Biochemical analysis of the SV‐KSC supernatant and PDGF A and B mRNA expression analysis provide evidence that the mitogenic activity is not due to a PDGF‐like growth factor. On the other hand, there is evidence to indicate that the SV‐KSC mitogen is a member of the fibroblast growth factor family. SV‐KSC represent an interesting model system for the study of different degrees of malignancy of cultured mesenchymal cells and especially provide an important source for the isolation of a potent growth factor for KS cells and other mesenchymal cells in vitro.
Internal binding sites for MSH: Analyses in wild‐type and variant Cloudman melanoma cells
Authors: Orlow Seth J. , Hotchkiss Sara , Pawelek John M.
Published: February 2005

Cloudman S91 mouse melanoma cells express both external (plasma membrane) and internal binding sites for MSH. Using 125I‐beta melanotropin (β‐MSH) as a probe, we report here an extensive series of studies on the biological relevance of these internal sites. Cells were swollen in a hypotonic buffer and lysed, and a particulate fraction was prepared by high‐speed centrifugation. This fraction was incubated with 125 l‐β‐MSH with or without excess nonradioactive β‐MSH in the cold for 2 hours. The material was then layered onto a step‐wise sucrose gradient (8–80%) and centrifuged (156,000g, 60 min); fractions were collected and counted in a gamma counter or assayed for various enzymatic activities. The following points were established: (1) Specific binding sites for MSH were observed sedimenting at an average density of 50% sucrose in amelanotic cells and at higher densities in melanotic cells. (2) These sites were similar in density to those observed when intact cells were labeled externally with 125I‐β‐MSH and then warmed to promote internalization of the hormone. (3) Most of the internal binding sites were not as dense as fully melanized melanosomes. (4) In control experiments, the MSH binding sites were not found in cultured hepatoma cells. (5) Variant melanoma cells, which differed from the wild‐type in their responses to MSH, had reduced expression of internal binding sites even though their ability to bind MSH to the outer cell surface appeared normal. (MSH‐induced responses included changes in tyrosinase, dopa oxidase, and dopachrome conversion factor activities, melanization, proliferation, and morphology.) (6) Isobutylmethylxanthine, which enhanced cellular responsiveness to MSH, also enhanced expression of internal binding sites. The results indicate that expression of internal binding sites for MSH is an important criterion for cellular responsiveness to the hormone.
Neurite outgrowth activity of protease nexin‐1 on neuroblastoma cells requires thrombin inhibition
Authors: Gurwitz David , Cunningham Dennis D.
Published: February 2005

Protease nexin‐1 (PN‐1) is a protein proteinase inhibitor recently shown to be identical with the glial‐derived neurite‐promoting factor or glial‐derived nexin. It has been shown to promote neurite outgrowth in neuroblastoma cells and in sympathetic neurons. The present experiments were designed to further test the hypothesis that this activity on neuroblastoma cells is due to its ability to complex and inhibit thrombin. It has been suggested that PN‐1 :thrombin complexes might mediate the neurite outgrowth activity of PN‐1. However, the present studies showed that such complexes, unlike free PN‐1, did not promote neurite outgrowth. The neurite outgrowth activity of PN‐1 was only detected in the presence of thrombin or serum (which contains thrombin). PN‐1 did not affect the rate or extent of neurite outgrowth that occurred when neuroblastoma cells were placed in serum‐free medium. Retraction of neurites by thrombin was indistinguishable in cells whose neurites had been extended in the presence or absence of PN‐1. The neurite‐promoting activity of PN‐1 was inhibited by an anti‐PN‐1 monoclonal antibody, which blocks its capacity to complex serine proteinases. The plasma thrombin inhibitor, antithrombin III, stimulated neurite outgrowth but only when its thrombin inhibitory activity was accelerated by heparin. The neurite outgrowth activity of both antithrombin III and PN‐1 corresponded to their inhibition of thrombin. Together, these observations show that PN‐1 promotes neurite outgrowth from neuroblastoma cells by inhibiting thrombin and suggest that this depends on the ability of thrombin to retract neurites.
Adenine nucleotides modulate phosphatidylcholine metabolism in aortic endothelial cells
Authors: Pirotton Sabine , Robaye Bernard , Lagneau Christian , Boeynaems Jean‐Marie
Published: February 2005

ATP and ADP, in concentrations ranging from 1—100 μM, increased the release of [3H]choline and [3H]phosphorylcholine (P‐choline) from bovine aortic endothelial cells (BAEC) prelabelled with [3H]choline. This action was detectable within 5 minutes and was maintained for at least 40 minutes. ATP and ADP were equiactive, and their action was mimicked by their phosphorothioate analogs (ATP‐γS and ADPβS) and adenosine 5′‐(β,γ imido) triphosphate (APPNP), but not by AMP, adenosine, and adenosine 5‐(α,β methylene)triphosphate (APCPP): these results are consistent with the involvement of P2Y receptors. ATP also induced an intracellular accumulation of [3H]choline: the intracellular level of [3H]choline was increased 30 seconds after ATP addition and remained elevated for a least 20 minutes. The action of ATP on the release of choline metabolites was reproduced by bradykinin (1 μM), the tumor promoter phorbol 12‐myristate 13‐acetate (PMA, 50 nM), and the calcium ionophore A23187 (0.5 μM). Down‐regulation of protein kinase C, following a 24‐hour exposure of endothelial cells to PMA, abolished the effects of PMA and ATP on the release of choline and P‐choline, whereas the response to A23187 was maintained. These results suggest that in aortic endothelial cells, ATP produces a sustained activation of a phospholipase D hydrolyzing phosphatidylcholine. The resulting accumulation of phosphatidic acid might have an important role in the modulation of endothelial cell function by adenine nucleotides. Stimulation of phospholipase D appears to involve protein kinase C, activated following the release of diacylglycerol from phosphatidylinositol bisphosphate by a phospholipase C coupled to the P2Y receptors (Pirotton et al., 1987a).
Comparison between transport and degradation of leucine and glutamine by peripheral human lymphocytes exposed to concanavalin A
Authors: Koch Brigitte , Schröder Marie‐Theres , Schäfer Gertrud , Schauder Peter
Published: February 2005

Transport and pathways of leucine and glutamine degradation were evaluated in resting human peripheral lymphocytes and compared with the changes induced by concanavalin A (ConA). Cells were incubated with [1‐14C]leucine (0.15 mM), [U‐14C]leucine (0.15 mM), or [U‐14C]glutamine (0.4 mM) after culture with or without 2, 5, 7, or 10 μg/ml ConA for 2, 18, or 24 hours, respectively. Initial rates of transport of leucine and glutamine were augmented 2.7‐fold and threefold by the mitogen. Leucine transamination, irreversible oxidation, and catabolism beyond isovaleryl‐CoA were increased by 90%, 20%, and 60%, respectively. Glu‐tamine utilization increased threefold; accumulation of glutamate, aspartate, and ammonia increased by 700%, 50%, and 100%, respectively, and 14CO2 production by about 400% in response to ConA. The results indicate that ConA stimulates to about the same extent transport of leucine and glutamine into lymphocytes. Glutamine is mainly channeled into catabolic pathways, while leucine remains largely preserved. It is suggested that these metabolic changes provide more leucine for incorporation into protein and more N‐ and C‐atoms required for the synthesis of macromolecules and energy from glutamine.
Propagation of fetal human RPE cells: Preservation of original culture morphology after serial passage
Authors: Song Mi‐Kyoung , Lui Ge Ming
Published: February 2005

The permissive effects of extracellular matrix (ECM) on in vitro growth and differentiation of fetal human retinal pigment epithelial (RPE) cells have been studied. Factors which enhanced the effect of ECM to support cell division were also examined, including growth factors, culture media, and serum requirement. Under the specific culture conditions we have defined, it is possible to propagate these RPE cells at low density (less than 20 cells/mm2) with excellent growth properties for greater than 72 doublings (fourteen passages) in serial culture. Later‐passaged cells maintained the morphological appearance of early‐passaged cultures. ECM produced by bovine corneal endothelial cells was by far the most predominant factor in promoting rapid cell proliferation and viability over repeated passaging. Basic fibroblast growth factor (bFGF) exerted a substantial effect on the rate of cell division at different serum concentrations on plastic dishes. In addition, this factor showed profound synergistic effect when RPE cells were maintained on ECM, both in the preservation of cell morphology and also in long term viability. Other growth factors, such as epidermal growth factor (EGF) and transforming growth factor‐beta (TGF‐B), were also tested, but EGF effects were less prominent than those observed with bFGF, and TGF‐B had an inhibitory effect at high concentrations. The ability to obtain a relatively large number of human RPE cells in vitro which preserve the appearance of early passage cells may provide useful opportunities to study the physiological properties and pathological alterations involving this important cell type.
Cell size and RNA content correlate with cell differentiation and proliferative capacity of rat keratinocytes
Authors: Poot Martin , Rizk‐Rabin Marthe , Hoehn Holger , Pavlovitch Jana Henriette
Published: February 2005

Keratinocytes from rat skin were separated according to their size in a specially designed unit‐gravity sedimentation chamber. The fractions obtained with this technique showed clear morphological differences, and analysis of size distribution confirmed that size was the criterion for separation. Simultaneous DNA and RNA staining of the fractions with acridine orange and subsequent flow cytometric analysis enabled one to classify cells into resting, proliferating, and differentiating stages. Cell size was not directly correlated with proliferation in situ as determined with acridine orange flow cytometry, nor with proliferative capacity in culture as assayed by BrdU/Hoechst flow cytometry. The smallest cells, exhibiting low DNA and RNA content, which do not proliferate in vivo, required a prolonged period of serum stimulation in vitro to initiate RNA and DNA synthesis. Cells of intermediate size exhibited early RNA synthesis and maximal proliferative capacity, whereas the largest cell population displayed no RNA synthesis in culture and the least proliferative capacity. In conclusion, these results suggest that RNA synthesis early after serum stimulation, in addition to a specific, optimal cell size, correlates with the proliferative capacity of keratinocytes in cell culture.
New role for histamine in interleukin‐3‐induced proliferation of hematopoietic stem cells
Authors: Schneider Elke , Piquet‐Pellorce Claire , Dy Michel
Published: February 2005

This study investigated the effect of histamine generated by murine bone marrow cells in response to IL‐3 on one particular biological activity of this growth factor, i.e., triggering of cells forming colonies in spleen (CFU‐S) into S phase. Evidence is provided that i) IL‐3‐induced day‐8 CFU‐S cell cycling, evaluated by hydroxyurea suicide, is completely abrogated when the binding of histamine to its H2 receptors is blocked by the specific antagonist oxmetidine, whereas cetirizine, a H1 receptor antagonist, is ineffective; and ii) the entry of day‐8 CFU‐S intoS phase in response to IL‐3 is likevise abolished when the histamine synthesis promoted by the growth factor is prevented by α‐fluoromethylhistidine, a specific inhibitor of the histamine‐forming enzyme, histidine decarboxylase. Similar results are obtained with both drugs, when a progenitor‐enriched bone marrow population is used instead of total cells. Furthermore, i.v. injection of recombinant (r)IL‐3 results within 2 hr in a substantial increase in bone marrow cell histamine synthesis together with triggering of day‐8 CFU‐S into cycle, the latter being completely abolished by a simultaneous injection of the H2 histamine receptor antagonist oxmetidine. Thus, our findings support the notion that both in vitro and in vivo the proliferation of early CFU‐S in response to IL‐3 is modulated by histamine via its H2 receptors. This conclusion is also consistent with the observation that dimaprit, a specific agonist of these receptors not only enhances the sensitivity of day‐8 CFU‐S to HU after a 2 hr incubation with bone marrow cells but also increases, to the same extent as IL‐3, the number of colonies formed in irradiated spleens after a 5 hr pretreatment.
Relationship of c‐myc gene copy number and gene expression: Cellular effects of elevated c‐myc protein
Authors: Pallavicini M. G. , Rosette C. , Reitsma M. , Deteresa P. S. , Gray J. W.
Published: February 2005

The relationship between gene copy number and expression and cellular consequences of elevated levels of c‐myc protein has been investigated using recombinant Chinese hamster ovary cell lines transfected with DNA coding for the murine c‐myc gene. HC‐8 and LC‐5 recombinant cells carry approximately 800 and 50 copies of c‐myc sequences, respectively, under control of an inducible heat shock promoter. Multivariate flow cytometric analysis and clonogenic assays were used to measure the relationship among c‐myc expression, rate of DNA synthesis, and cell survival. Following heat exposure, maximally induced HC‐8 cells produced approximately tenfold more c‐myc protein than heated LC‐5 cells, suggesting a close relationship between gene copy number and level of expression. However, considerable heterogeneity in the level and time of c‐myc expression was observed following heat induction, even though the amounts of genomic c‐myc were reatively constant. Heterogeneity in gene expression was not attributable to variation in heat induction methodologies and/or cell cycle phase distributions. The presence of high levels of recombinant c‐myc protein was associated with a decreased rate of bromodeoxyuridine incorporation into DNA. High levels of c‐myc protein in HC‐8 cells were inversely correlated with cell survival postheating, suggesting that high levels of c‐myc protein are incompatible with cell survival.
Fibroblasts from wounds of different stages of repair vary in their ability to contract a collagen gel in response to growth factors
Authors: Finesmith Tina H. , Broadley Kenneth N. , Davidson Jeffrey M.
Published: February 2005

Wound contraction is one function of granulation tissue which is critical to repair. This study compares the ability of fibroblast‐like cells derived from granulation tissue of various ages to contract a tissue equivalent, or a collagen gel, and examines the influence of growth factors implicated in wound repair on collagen gel contraction by these different cell populations. Cells from older granulation tissue (21 and 28 days) have an enhanced ability to contract a tissue equivalent when compared to cells from younger granulation tissue (7 and 14 days) or normal rat skin fibroblasts. Transforming growth factor‐b̃1 (TGF‐b̃1) enhanced contractility most in those cells which had a greater basal contractile ability. While basic fibroblast growth factor (bFGF) alone had moderately stimulatory effects at low doses (0.1‐1.0 ng/ml), higher doses (≥ 10 ng/ml) inhibited basal contraction. Pretreatment with bFGF followed by exposure to TGF‐b̃1, with or without the continued presence of bFGF, delayed gel contraction by cells from skin and early granulation tissue, but bFGF enhanced TGF‐b̃1 activity in highly contractile cells. Transforming growth factor‐α moderately enhanced contraction by cells from older granulation tissue. While both TGF‐b̃1 and bFGF enhanced wound repair, their differential effects on the fibroblast‐like cell derived from granulation tissue of different ages suggest that phenotypic differences exist between these cell populations. In addition, our results predict significant interactions between polypeptide cytokines at the site of repair.
Basic fibroblast growth factor expression in human omental microvascular endothelial cells and the effect of phorbol ester
Authors: Bikfalvi A. , Alterio J. , Inyang A. L. , Dupuy E. , Laurent M. , Hartmann M. P. , Vigny L. , Raulais D. , Courtois Y. , Tobelem G.
Published: February 2005

The human omentum contains a potent, not yet identified angiogenic activity. The omentum is very vascularized. Therefore, we investigated whether human omental microvascular endothelial cells (HOME cells) express the angiogenic peptide basic fibroblast growth factor (bFGF). Cytosol prepared from HOME cells stimulated DNA synthesis in bovine epithelial lens cells (BEL cells). The mitogenic activity could be neutralized by an anti‐bFGF antibody. Basic FGF‐like material from the HOME cell cytosol was bound onto a heparin‐Sepharose column at 0.6 M and was eluted at 3 M NaCl. The 3 M NaCl eluted material reacted with the specific anti‐bFGF antibody in an ELISA and stimulated DNA synthesis. It did not react with a specific anti‐acidic fibroblast growth factor (aFGF) antibody. Western blotting experiments using the same bFGF antibody showed the presence of a major band of 17 Kd and a doublet of 20‐22 Kd. Northern blotting of nonstimulated HOME cells using a specific 1.4 kb bFGF probe showed the presence of 5 molecular species of 6.6, 3.7, 2.2, 2.0, and 1.0kb. No aFGF mRNA was detected with a specific previously characterized 4.04 kb probe. 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) did not influence significantly the expression of bFGF at the protein and mRNA level in HOME cells. Thus, protein kinase C activation by TPA did not appear to modulate significantly the expression of bFGF for that cell type. Contrastingly, human umbilical vein endothelial cells (HUVE cells), which expressed no bFGF and aFGF mRNA at a basal level, were induced to express bFGF but not aFGF mRNA when stimulated by TPA. These results suggest that the described angiogenic activity could be the bFGF‐like mitogen contained in HOME cells and that these cells are different from endothelial cells derived from large vessels (HUVE cells) regarding the expression of bFGF.
Cell cycle regulation of an exogenous human poly(ADP‐ribose) polymerase cDNA introduced into murine cells
Authors: Bhatia Kishor , Kang Veronica H. , Notario Vicente , Haque Saikh J. , Cherney Barry W. , Smulson Mark E. , Stein Gary S. , Bustin Michael , Huppi Konrad
Published: February 2005

We have evaluated the regulation of expression of the poly(ADP‐ribose) polymerase gene during cell growth and replication. In a synchronized population of HeLa cells or in serum‐stimulated WI‐38 cells, steady‐state levels of the polymerase mRNA were highest at late S and S‐G2 phases and negligible in early S phase. Transcription did not solely account for the significant increase in the mRNA levels observed in late S phase by Northern analysis. The stability of the mRNA was dependent upon the percent proliferating cells in the culture. Accordingly, polymerase mRNA from cells in early exponential phase was significantly more stable than from cells in stationary phase of asynchronous growth. To clarify these observations, we utilized a novel heterologous expression system that involved murine 3T3 cells transfected with a human poly(ADP‐ribose) polymerase cDNA under the control of a non‐cell cycle‐specific promoter. Cells were synchronized, and a comparison was made of the endogenous (murine) and exogenous (human) polymerase mRNA levels. Both the endogenous and the exogenous mRNA were specifically stabilized by the same mechanisms and only during late S phase; therefore, we concluded that mRNA pools for the polymerase are regulated at the post‐transcriptional level. The heterologous expression system confirmed that the post‐transcriptional regulation system in the mouse cells can recognize and faithfully regulate the human cDNA in response to the murine cell cycle signals. More importantly, the presence of extra copies (human) of the polymerase gene did not provide an increased amount of the total polymerase mRNA or protein and, in fact, the sum of the endogenous and exogenous mRNA in the transfected cells was approximately the same as the level of endogenous transcript in the control cells. This suggested that there might be a limit to the amount of polymerase protein accumulating in the cellular pool and thus levels of poly(ADP‐ribose) polymerase may be autoregulated.
Plasminogen activator inhibitor type‐2 Is a major protein induced in human fibroblasts and SK‐MEL‐109 melanoma cells by tumor necrosis factor
Authors: Pytel Barbara A. , Peppel Karsten , Baglioni Corrado
Published: February 2005

Tumor necrosis factor (TNF) induces the synthesis of two proteins of Mr 42 and 36 kDa in human fibroblasts and SK‐MEL‐109 melanoma cells. To identify these proteins, a λgt10 cDNA library was prepared from the mRNA of TNF‐treated SK‐MEL‐109 cells. By screening this library, we found a cDNA that preferentially hybridized to TNF‐induced RNA. Hybrid‐selected mRNA was translated into a protein of 42 kDa; cDNA sequence analysis followed by a comparison with other known protein sequences identified this protein with plasminogen activator inhibitor, type‐2 (PAI‐2). After removal of TNF, PAI‐2 mRNA turned over rapidly, with an apparent half‐life of ∼ 2.5 h. Addition of dexamethasone increased the turnover of this mRNA, suggesting that the level of PAI‐2 mRNA could be regulated post‐transcriptionally by glucocorticoids. PAI‐2 was not secreted, but accumulated in fibroblasts continuously treated with TNF.
Pertussis toxin catalyzed ADP‐ribosylation of a 41 kDa G‐protein impairs insulin‐stimulated glucose metabolism in Bc3H‐1 myocytes
Authors: Moises Regina S. , Heidenreich Kim A.
Published: February 2005

In this study, we examined the effects of pertussis toxin (PT) on the ADP‐ribosylation of guanine nucleotide binding proteins (G‐proteins) and various insulinstimulated processes in cultured BC3H‐1 myocytes. Treatment of intact myocytes with 0.1 μg/ml PT for 24 hours resulted in the complete ribosylation of a 41 kDa protein. The 41 kDa PT substrate was immunoprecipitated with antibodies directed against a synthetic peptide corresponding to a unique sequence in the alpha subunit of Gi‐proteins. PT treatment of intact cells had no effect on insulin receptor binding or internalization. However, PT inhibited insulin‐stimulated glucose transport at all insulin‐concentrations tested (1–100ng/ml). Maximally stimulated glucose transport was reduced by 50% ± 15%. Insulin‐stimulated glucose oxidation was also decreased by 31% ± 8%. The toxin had no significant effect on the basal rates of glucose transport and glucose oxidation. The time course of PT‐induced inhibition on glucose transport correlated with the time course of the “in vivo” ADP‐ribosylation of the 41 kDa protein. The results suggest that a 41 kDa PT‐sensitive G‐protein, identical or very similar to Gi, is involved in the regulation of glucose metabolism by insulin in BC3H‐1 cells.
Butyrate blocks the accumulation of CDC2 mRNA in late G1 phase but inhibits both the early and late G1 progression in chemically transformed mouse fibroblasts BP‐A31
Authors: Charollais Renée‐HéLène , Buquet Christine , Mešter Jan
Published: February 2005

Sodium butyrate (6 mM) blocks the resumption of the cell division cycle in serum‐deprived chemically transformed Balb/c‐3T3 mouse fibroblasts (BP‐A31). The inhibition of G1 progression by sodium butyrate is not restricted to a specific mitogenic signaling pathway and is equally effective when tetradecanoyl phorbol acetate (TPA), insulin, or fetal calf serum (FCS) is used as inducer. The inhibitor acts in early as well as late G1 phase as indicated by experiments in which inhibitor was added and withdrawn at different times after restimulation of quiescent cells by FCS. At the gene expression level, sodium butyrate does not affect the inducibility of early cell cycle‐related genes (c‐myc, c‐jun) while blocking the induction of cdc 2 mRNA, a late G1 marker. We conclude that sodium butyrate does not interfere with the growth factor signaling pathways regulating the (early) cell cycle‐related gene expression. However, the presence of sodium butyrate early in G1 phase inhibits the cascade of events leading eventually to the expression of late G1‐characteristic genes such as cdc2. The antimitogenic activity of sodium butyrate may be related to its interference with an (unknown) process involved in the “mitogenic” cascade.
Biochemical and functional characterization of proteoglycans produced by SI/SId murine bone marrow stromal cell lines
Authors: Bentley Stuart A. , Kirby Suzanne L. , Anklesaria Pervin , Greenberger Joel S.
Published: February 2005

The Steel anemia of mice results from an inherited defect in the hematopoietic microenvironment. Proteoglycans synthesized by bone marrow stromal cells are an important functional component of the hematopoietic microenvironment in normal animals. It is thus possible that Steel anemia results from a molecular abnormality involving bone marrow stromal proteoglycans. To investigate this possibility, we studied proteoglycan synthesis in three stromal cell lines from Steel anemic (SI/SId) animals and two control stromal cell lines, one (+/+2.4) from a non‐anemic littermate, and one (GBI/6) from a normal mouse. Proteoglycans were precursor labelled with 35S sulfate and separated by ion exchange HPLC, CsCI density gradient centrifugation, and molecular sieve HPLC. Glycosaminoglycan (GAG) moieties were characterized by molecular sieve HPLC and enzyme sensitivity. There were no consistent differences in total proteoglycan synthesis, proteoglycan heterogeneity, GAG hydrodynamic size, or enzyme sensitivity among the cell lines studied. Growth factor binding to stromal extracellular matrix (ECM) was studied by co‐culture of an IL‐3‐dependent cell line (FDC‐P1) with cell‐free ECM preparations from an SI/SId and a control (GBI/6) stromal cell line, with and without pre‐incubation with IL‐3. Cell‐free ECM preparations from SI/SId and control cell lines supported FDC‐P1 growth to an approximately equal extent after pre‐incubation with II‐3. FDC‐P1 growth support by ECM preparations from both cell lines was also observed without IL‐3 pre‐incubation, although to a lesser extent, suggesting ECM binding of endogenous growth factors synthesized by the stromal cells.
Basic fibroblast growth factor accumulates in the nuclei of various bFGF‐producing cell types
Authors: Tessler Shoshanna , Neufeld Gera
Published: February 2005

The intracellular localization of basic fibroblast growth factor (bFGF) was studied in BHK‐21 cells transfected with an expression vector containing the complementary DNA (cDNA) of the human bFGF gene (pbFGF). The intracellular location of bFGF was determined using indirect immunofluorescence. The antibodies used were polyclonal antibodies directed against either recombinant human bFGF or recombinant Xenopus: bFGF. The nuclei of transfected cells that produce bFGF, but not the nuclei ofuntransfeted cells, were labeled strongly by the antibodies. The nuclear staining was totally abolished when anti‐bFGF antibodies preadsorbed with bFGF were used. Several types of endothelial cells known to produce bFGF were also stained in their nuclei by the antibodies. Nuclear extracts prepared from transfected cells were found to contain bFGF as determined using heparin‐sepharose affinity chromatography, followed by Western blot analysis of fractions, which stimulated the proliferation BHK‐21 cells. The mitogenic cactivity associated with the nuclei was not destroyed when isolated cell nuclei were digested by trypsin. It is therefore likely that the nucleus associated bFGF is intranuclear, these findings suggest that some biologicaL activities of bFGF may be mediated by nuclear bFGF binding proteins or by the direct binding of bFGF‐ to DNA.
Transforming growth factor β1 enhances expression of 50 kDa protein related to 2′‐5′ oligoadenylate synthetase in human sperm cells
Authors: Naz Rajesh K. , Kumar Rakesh
Published: February 2005

Human cellular polypeptide factors, namely interferon‐α, interferon‐γ, transforming growth factor (TGF)‐α, and TGF‐β, were analyzed for their effect on motility of human sperm cells. Both interferons caused an inhibition of sperm cell motility due to direct cytotoxic effects without inducing 2′‐5′ oligoadenylate [2–5(A)]synthetase activity. TGF‐α affected neither motility nor the levels of 2–5(A) synthetase in sperm cells. TGF‐β, had no affect on sperm motility, yet it caused an induction of 2–5(A)synthetase activity. Western immunoblot analysis of TGF‐β1‐treated sperm indicated an enhancement of a 50 kDa protein. Metabolic labeling of sperm cells revealed biosynthesis of one major protein of 50 kDa and at least five minor proteins in the range of 30–92 kDa; the level of 50 kDa protein increased after treatment with TGF‐β1. The treatment of sperm cells with TGF‐β1 did not affect their penetration in zona‐free hamster eggs (SPA). These results indicate that TGF‐β1 enhances expression of a 50 kDa protein related to 2–5(A)synthetase in human sperm cells along with other minor proteins, and this increase does not affect sperm motility and SPA.
Decline of poly(ADP‐ribosyl)ation during in vitro senescence in human diploid fibroblasts
Authors: Dell'orco R. T. , Anderson L. E.
Published: February 2005

Poly(ADP‐ribose) polymerase activity was determined at various times during the in vitro life span of two human diploid fibroblast‐like cell lines of different donor ages. The cell lines differed in their ability to transfer ADP‐ribose, with cells from an embryonic donor exhibiting 2 to 3 times the activity found in cells obtained from a newborn donor. The activity in both cell lines decreased by 30–60% as the cells moved through their in vitro life spans. The decline could not be attributed to increases in glycohydrolase or the leakage of polymerase from older cell preparations. Enzyme activation with DNase I indicated that similar levels of enzyme were present in both cell lines at all in vitro ages. These results indicate that although poly(ADP‐ribosyl)ation is inversely related to donor age as well as in vitro age the decrease is in response to other factors which change with increasing age.
Growth factor‐independent proliferation of normal human neonatal keratinocytes: Production of autocrine‐ and paracrine‐acting mitogenic factors
Authors: Cook Paul W. , Pittelkow Mark R. , Shipley Gary D.
Published: February 2005

When normal human foreskin keratinocytes were cultured in the absence of polypeptide growth factors at densities above 5 × 103/cells cm2, the cells proliferated continuously and the addition of IGF‐I, EGF, TGFα, or aFGF did not significantly alter growth rate. Heparin sulfate, TGFβ, or suramin inhibited keratinocyte growth factor‐independent proliferation. The addition of EGF, TGFα, or aFGF reversed heparin‐induced growth inhibition, while bFGF partially negated this effect. RIA of keratinocyte‐derived conditioned medium (CM) indicated the presence of TGFα peptide at a concentration of approximately 235 pg/ml. In contrast, clonal growth of keratinocytes required the addition of growth factors to the basal medium. Keratinocyte‐derived CM replaced EGF in stimulating keratinocyte clonal growth, and an anti‐EGF receptor mAb inhibited CM‐induced keratinocyte clonal growth. In addition to its effect on keratinocytes, keratinocyte‐derived CM stimulated the incorporation of [3H]thymidine by quiescent cultures of human foreskin fibroblasts, mouse AKR‐2B cells, and EGF‐receptorless mouse NR6 cells. CM‐stimulated [3H] thymidine incorporation into quiescent normal human fibroblasts was partially reduced in the presence of anti‐EGF receptor mAb. Heparin sulfate partially inhibited CM‐induced keratinocyte clonal growth and [3H] thymidine incorporation into quiescent AKR‐2B cells. We hypothesize from these data that autocrine and paracrine‐acting factors produced by kerati‐nocytes modiated their effect through the activation of both EGF receptor‐dependent and EGF receptor‐independent mitogenic pathways and that some of these factors appear to be sensitive to inhibition by heparin.
Induction of cell cycle‐dependent genes during cell cycle progression of arterial smooth muscle cells in culture
Authors: Gadeau Alain‐Pierre , Campan Michel , Desgranges Claude
Published: February 2005

Serum stimulation of arterial smooth muscle cells in culture induces a progression through the cell cycle and cell proliferation. Most genes previously described as cell cycle‐dependent in various cell types also demonstrate a cell cycle‐dependent expression in arterial smooth muscle cells. As in other cell types, these genes can be classified into three groups according to their mode of expression: “immediate early” genes (c‐fos, c‐myc,…), “delayed early” genes (2F1,…), and “late‐G1” genes (proliferating cell nuclear antigen, thymidine kinase,…). In addition to these previously described genes, three genes isolated from a cDNA library of stimulated smooth muscle cells have been demonstrated to be cell cycledependent: A21, the rat JE gene, and L51 can be classified as “immediate early” genes, while M11 represents a new member of the “delayed early” gene family.