摘要:

AbstractHuman bone cells grown in culture, representative of a preosteoblastic stage of maturation, produce an extracellular matrix composed of collagen several noncollagenous glycoproteins, hyaluronan, and four distinct proteoglycans (PGs). The influence of donor age on the levels of expression of these molecules in vitro has not been well characterized. In this study, human bone cells derived from sources ranging from fetal to 60‐year‐old donors were grown in culture, radiolabeled for 24 h, and the amount of incorporation of [35S]sulfate into PGs, [3H]glucosamine into hyaluronan, [3H]leucine/proline into osteonectin, and [3H]proline into collagen was determined. Cell proliferation was most rapid in fetal‐derived bone cells and decreased with increasing age. Total protein and PG synthesis also decreased with increasing age, falling to 1/3 and 1/4, respectively, of fetal levels after age 30. A large chondroitin sulfate PG (Mr∼ 600,000 Da) was the major fetal PG and its levels were highly correlated with cellular proliferation. [3H]Collagen and [35S]decorin levels increased with the increasing age of the donor, reached a maximum in puberty‐derived cells, and decreased to 1/3 maximal levels after age 20. The heparan sulfate PG (Mr∼ 400,000 Da) exhibited steadystate levels regardless of donor age. [3H]Osteonectin and [35S]biglycan levels were high in fetal‐derived cells and in cells derived from pubescent donors. The percentage of collagen and four proteoglycans associated with the cell layer pool changed with donor age. All fetal‐derived PG core proteins possessed more N‐ and O‐linked oligosaccharides than newborn or adult derived PGs. ©

ISSN:0021-9541    

DOI:10.1002/jcp.1041510202

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractThe effect of hypoxia on subsequent susceptibility of porcine pulmonary artery endothelial cells (PAEC) to hydrogen peroxide (H2O2) injury was studied. Preexposure of PAEC to hypoxia for 3 or more h significantly increased susceptibility to subsequent H2O2challenge. Analysis of the activities of antioxidant enzymes and xanthine oxidase/dehydrogenase suggested that changes in these enzymes in hypoxic PAEC were not responsible for the increased susceptibility. However, hypoxia resulted in significant time‐dependent decreases in total glutathione at 12 h or more. The rate of glutathione regeneration in diethylmaleate‐treated PAEC and the rate of uptake of cystine and glycine were significantly lower during hypoxia. Hypoxia also caused depletion of ATP and NADPH levels in PAEC, but these did not occur until well after hypoxia‐enhanced susceptibility to H2O2injury was demonstrable. Alterations in glutathione levels and enhanced susceptibility were reversible when hypoxic PAEC were returned to normoxia. These results indicate that hypoxia increased the susceptibility to H2O2injury by decreasing the ability of PAEC to maintain and regenerate cellular glutathione content in response to H2O2challenge. © 1992 Wiley‐L

ISSN:0021-9541    

DOI:10.1002/jcp.1041510203

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractThe enzyme alkaline phosphatase (AP) has been shown to be lost or inappropriately expressed during carcinogenesis in some tissues. Because retinoic acid (RA) appears to play a role in the normal regulation of the enzyme (RA up‐regulates AP in a variety of cell types) we have suggested that altered AP expression in some cancers may be caused by a defect in the ability of the cells to respond normally to retinoid. We have begun to use the chemically transformable mouse embryo fibroblast cell, C3H10T1/2, to investigate this possibility. In this initial study we characterized AP regulation in normal C3H10T1/2 cells and show that: (1) 10−7M RA increases AP activity within 3–4 h in serum‐free medium; (2) serum inhibits short‐term induction (0–8 h) in a concentration‐dependent manner (10% serum causes complete inhibition); (3) during long‐term RA exposure (24 h and 48 h), induction can be detected in serum‐containing medium; (4) AP induction is dose related at RA concentrations from 10−10M to 10−6M in serum‐free medium; (5) 10−5M RA is ineffective at inducing AP in serum‐free medium during 8 h but is the most effective concentration in serum‐containing medium during 24 h and 48 h exposures; (6) AP inducibility by RA requires near‐confluent cell densities; and (7) when cultures become confluent, cells become constitutive for AP and no longer require RA for enzyme expression. The effects of serum and cell density on AP inducibility by RA and implications of the RA up‐regulation of AP for teratogenesis are

ISSN:0021-9541    

DOI:10.1002/jcp.1041510204

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractThe correspondence between K+uptake in platelets to their responsiveness was studied using86Rb+as an analogue of K+. An average86Rb+uptake rate of 0.73 (± 0.140) × 10−15mole Rb+/min‐plt (n = 20) was observed. By the use of K+‐influx inhibitors, we were able to distinguish three distinct86Rb+uptake pathways: an ouabain‐sensitive (61% ± 2% inhibitable) pump and two equivalent channels, only one of which is sensitive to furosemide. Other platelet parameters were also examined in conjunction with K+‐uptake. Platelets incubated with ouabain exhibited an overall rise in their cell volume (MPV) with incubation time (ΔMPV = 7.4 × 10−17L/min1plt−1). Concomitantly, over 24 hours, a steady decrease in platelet number was recorded by blood cell coulter, which correlated inversely with the counts of particles, which by their size resemble white blood cells (r = 0.89). On a cellular level. incubation with ouabain induced greater expression of surface fibrinogen‐receptor (GPIIb), increased binding of FITC‐labelled fibrinogen, and increased responsiveness to ADP. Our observations suggest the following sequence of events: Ouabain turns off the Na+/K+‐ATPase pump, which leads to water accumulation in platelets and concomitant increased MPV. Greater expression of fibrinogen receptors on the distended platelet surface corresponds to spontaneous microaggregate formation as well as greater respon‐siveness to agonists. Our model links volume regulation, the expression of fibrinogen receptors, and the sensitivity of platelets to agonists to the activity of the Na+K+‐ATPase

ISSN:0021-9541    

DOI:10.1002/jcp.1041510205

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractWe established that plasmin (10−10M to 10−6M) caused neutrophils (PMN) to aggregate using an in vitro assay. Plasminogen had no PMN aggregatory activity even at a concentration of 2 μM. However, plasminogen caused PMN to aggregate when incubated with plasminogen activators [tissue plasminogen activator (25–200 U/ml) or urokinase (5–500 U/ml)]. Tissue plasminogen activator and urokinase alone had no PMN aggregatory activity. Analysis of these incubation mixtures indicated that plasmin was generated in the process and that the time course of plasmin generation correlated with the aggregation response. Active‐site‐inhibited plasmin did not induce PMN aggregation, indicating that a functional catalytic site was required for the response. Pretreatment of PMN with either active‐site‐inhibited plasmin or tranexamic acid prevented PMN aggregation by plasmin, indicating that both binding of plasmin to the cell surface via the lysine binding sites and catalysis were required for the response. The generation of plasmin during activation of fibrinolysis may play a pro‐inflammatory role by mediating aggregation of PMN. © 1

ISSN:0021-9541    

DOI:10.1002/jcp.1041510206

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractHuman keratinocytes isolated from a skin biopsy and cultured in vitro on a feeder‐layer of irradiated fibroblasts reconstitute a stratified squamous epithelium suitable for grafting onto patients suffering from large burn wounds. Since conditioned medium from 3T3‐J2 cells can partially substitute for the intact feederlayer, we studied the possible involvement of insulin‐like growth factors acting in a paracrine fashion. IGFs were measured (after Sephadex G‐50 gel‐chromatography in acid conditions) in media conditioned by a feeder‐layer of lethally irradiated 3T3‐J2 fibroblasts on which keratinocytes were grown. Immunoreactive (IR) IGF‐I, IGF‐II, and IGF binding activity were present in the medium conditioned by the feeder‐layer. The medium conditioned by keratinocytes showed nearly undetectable amounts of IR IGF‐I and IGF‐II, suggesting that keratinocytes are unable to synthesize IGFs peptides. Recombinant IGF‐I and IGF‐II, and conditioned medium from 3T3‐J2 cells, caused a dose‐dependent increase of3H‐thymydine incorporation in cultured keratinocytes. The stimulatory effect of IGF and of 3T3‐J2 conditioned medium was inhibited by the MoAb Sm 1.2, which recognizes both IGF‐I and IGF‐II but not insulin, and by the MoAb αIR‐3, which is a specific antagonist of type‐1 IGF receptor. Fetal mouse‐derived 3T3‐J2 cells and adult human skin fibroblasts were equally able to sustain keratinocyte growth and in both cases addition of Sm 1.2 MoAb causes a 50% decrease in the keratinocyte number. When the non‐IGF‐producing BALB/c 3T3 cells were used as a feederlayer, the keratinocytes number was similar to that observed with 3T3‐J2 and with human fibroblasts plus the Sm 1.2 MoAb. IGF‐I and IGF‐II restored the BALB/c 3T3 growth promoting activity to the level of 3T3‐J2 and of normal human fibroblasts. Our results suggest that fetal mouse 3T3‐J2 and human fibroblasts synthesize IGF peptides, while keratinocytes do not. Fibroblast‐derived IGFs stimulate keratinocyte growth in a paracrine fashion, suggesting their role in the regulation of keratinocyte prolifera

ISSN:0021-9541    

DOI:10.1002/jcp.1041510207

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractWhole‐cell recordings were made from rat basophilic leukemia cells. The properties of the cells in the potential range negative to −20 mV could be accounted for by two potassium conductances, a leakage conductance (0.54 ± 0.01 nS, mean ± s.e.m., n − 6) and an inward rectifier conductance. The inward rectifier conductance activated at a potential close to Ekand had a maximal value of 2.7 ± 0.22 nS (n = 6 ). Cesium (2 mM, n = 6) decreased the inward current at every potential. Strontium (10 mM, n = 7) and rubidium (2 mM, n = 3) had a similar effect, but they also increased the slope conductance between −63 and − 103 mV. Barium (1–100 pM) blocked selectively the inward rectifying current without affecting the leakage current; the effect was use‐, voltage‐, and temperature‐dependent. TEA decreased the current; the concentration giving 50% inhibition was 37.5 mM. The current was unaffected by phorbol esters as well as several hormones and transmitters that werg tested. ©

ISSN:0021-9541    

DOI:10.1002/jcp.1041510208

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractWe have previously reported that the addition of lithium chloride (LiCl) to murine Dexter cultures results in increased numbers of progenitor and mature hematopoietic cells of the granulocyte, macrophage, and megakaryocyte lineages. We now report the effect of various levels of LiCl on the high proliferative potential colony‐forming cell (HPP‐CFC) in Dexter culture and on the induction of growth factors from Dexter stromal cells. LiCl (4 mEq/L) stimulated supernatant HPP‐CFC for the first 4 weeks of culture (150–275%), and stimulated stromal HPP‐CFC at week 3 (170–222%). Higher levels of lithium (8 and 12 mEq/L) selectively stimulated supernatant HPP‐CFC, macrophage, and eosinophil production, whereas granulocytes and granulocyte‐macrophage colony‐forming cells (CFU‐C) were inhibited. mRNA expression was evaluated from week 4 Dexter cultures that received a pulse or continuous exposure to lithium and had received either 0 or 1,100 cGy irradiation. Four mEq/L LiCl stimulated increased expression of G‐CSF, GM‐CSF, IL‐6, and, in the nonirradiated stroma continuously exposed to lithium, CSF‐1 mRNA. In general, the higher levels of lithium stimulated increased mRNA expression for these same growth factors mRNA for the recently described Steel factor was decreased with increasing levels of lithium added to either normal or irradiated stroma. Bioassays of conditioned medium (cm) from irradiated cultures against the FDC‐P1 and T1165 cell lines indicated cytokine activity, which was blocked by antibodies to GM‐CSF and IL‐6, respectively. Altogether these data show that lithium stimulates Dexter HPP‐CFC, and this stimulation appears to be mediated by multiple growth factors that are induced from st

ISSN:0021-9541    

DOI:10.1002/jcp.1041510209

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractIn contrast to neurite outgrowth, pigment cell dendrite formation is relatively unstudied. Keratinocyte‐conditioned medium (KCM) induces a striking dendricity in human melanocytes and B16 melanoma cells that is detectable within 30 min, maximal in 24–48 hr, and quantifiable by computerized image analysis. Cyto‐chalasin B (CB), known to disrupt actin microfilaments, completely blocks dendrite formation if added to cultures before or with KCM. This effect is rapidly reversible, and dendrites appear within 1 hr after refeeding with KCM alone. In contrast, CB treatment fails to disrupt existing dendrites previously induced by KCM. Agents known to cause microtubule disassembly (colchicine, nocodazole, or vinblastine) do not inhibit dendrite formation if added before or with KCM. In contrast, these agents disrupt established dendrites Inhibition of protein synthesis with cycloheximide or actinomycin D completely blocks dendrite formation, but if cultures are provided fresh KCM lacking protein synthesis inhibitors, dendrites reappear within 24 hr. Actin microfilaments visualized with a monoclonal antibody or rhodamine‐phalloidin are poorly organized in untreated cells, but form numerous fibers localized along dendrites in KCM‐treated cells. Microtubules visualized with a monoclonal anti‐tubulin antibody are localized in the center of dendrites. These cytoskeletal changes occur without altering β actin or β tubulin mRNA levels. Taken together, these data implicate actin microfilaments in dendrite outgrowth, but not in maintenance, and conversely microtubules in dendrite maintenance but not in formation. These keratinocyte‐induced changes involving β actin and β tubulin polymerization appear to require both new protein synthesis and post‐translational regulation. The observed similarities between melanocytes and other neural crest‐derived cells suggest that cutaneous pigment cells might serve as an alternative model for studies of neurite outgrowth.

ISSN:0021-9541    

DOI:10.1002/jcp.1041510210

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractCytokines like tumor necrosis factor (TNF), interferon‐γ (IFN‐γ), and interleukin‐1 (IL‐1) are known to interfere with the differentiation of cultured cell lines of adipocyte precursors. In the present study, the effect of mouse and rat IFN‐γ, as well as human IL‐1β, was investigated on rodent preadipocytes in primary cultures, either in the presence of fetal bovine serum (FBS, 10%) or in serum‐free defined medium. IFN‐γ exerted an antiproliferative action that was more pronounced when cells reached confluency than during the growth phase of the culture. Morphological observation and quantifications of undifferentiated and differentiating cells revealed that IFN‐γ caused a decrease in the proportion of cells devoid of lipid droplets which would correspond to fibroblast‐like cells, whereas preadipocytes remained unaffected. IFN‐γ induced a marked retardation of adipoconversion, resulting in a partial inhibition of lipoprotein lipase (LPL) activity and a severe decrease in glycerol‐3‐phosphate dehydrogenase (GPDH) activity. The antiproliferative and anti‐LPL effects of IFN‐γ were neutralized by adding anti‐IFN‐γ antibodies, while these antibodies prevented only partially the depressing effect of IFN‐γ on GPDH activity. Contrary to IFN‐γ, IL‐1β slightly enhanced the proliferation in preadipocyte cultures. IL‐1β also depressed adipoconversion, inhibited markedly LPL activity, and partially reduced GPDH activity. These results show that the influence of cytokines on adipoconversion observed in preadipocyte cell lines can be tound in n

ISSN:0021-9541    

DOI:10.1002/jcp.1041510211

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY