bluuebunny/crossref_metadata_2025 huggingface dataset:
Fibroblast contraction of collagen gels requires activation of protein kinase C
Authors: Guidry Clyde
Published: February 2005

Fibroblasts stimulated to contract collagen gels with serum were completely inhibited by staurosporine, a broad spectrum kinase inhibitor. Further analysis demonstrated that staurosporine is potent (IC50 17 nM), rapid in onset, and completely reversible. Complete inhibition of gel contraction was also observed with calphostin C (IC50 48 nM), an inhibitor specific for protein kinase C (PKC). Similar effects were not observed with KT5926 or KT5720, inhibitors for myosin light chain kinase and cAMP‐dependent kinase, respectively. These data suggested that serum‐stimulated fibroblast contraction is dependent upon activation of PKC. This was also observed with fibroblast contraction stimulated with endothelin‐1, platelet‐derived growth factor, and transforming growth factor beta. PKC activated directly with low concentrations of phorbol ester was observed to stimulate fibroblast contraction of collagen gels, in some cases within 30 minutes of exposure. © 1993 Wiley‐Liss, Inc.
Pericyte growth and contractile phenotype: Modulation by endothelial‐synthesized matrix and comparison with aortic smooth muscle
Authors: Newcomb Patricia M. , Herman Ira M.
Published: February 2005

We compared the effects of endothelial‐synthesized matrix and purified matrix molecules on pericyte (PC) and aortic smooth muscle cell (SMC) growth, heparin sensitivity, and contractile phenotype in vitro. When PC are plated on endothelial‐synthesized (EC) matrix, cell number is, on average, 3.1‐fold higher than identical populations grown on plastic. Under the same conditions, SMC proliferation is stimulated 1.6‐fold. Purified matrix molecules, such as collagen type IV (COLL) or fibronectin (FN), both major components of the EC matrix, stimulate PC/SMC growth 1.2–1.7‐fold. Heparin (100 μg/ml), which inhibits the growth of early passage SMC by 60%, inhibits PC growth ∼50%, when cells were plated on plastic. However, PC plated on EC matrix in the presence of heparin (100 μg/ml) grow as well as parallel cultures grown on plastic (in the absence of heparin). Concomitant with matrix‐stimulated proliferation, we observed a marked reduction in PC containing alpha vascular smooth muscle actin (αVSMA), as seen by immunofluorescence using affinity‐purified antibodies (173/615 positive pericytes on DOC matrix (28%) vs. 221/285 (77%) positive on glass). SMC respond similarly. Whereas αVSMA protein is markedly altered when PC and SMC are cultured on EC matrix, similar reductions in mRNA are not observed. However, Northern blotting does reveal that PC contain 17–30 times the steady‐state levels of αVSMA mRNA compared to SMC. When SMC and PC cultures on plastic are treated with heparin, the steady‐state levels of vascular smooth muscle actin mRNA increase 5 and 1.5 fold, respectively. Similarly, heparin treatment of PC grown on plastic induces a 1.8 fold increase in nonmuscle actin mRNA. These heparin‐induced alterations in isoactin mRNA levels are not seen when PC are cultured on EC matrix. We also observed reductions in αVSMA and β actin mRNA levels when PC are plated on FN, where they maintain a ratio of 13:1 (α:β). Similar ratios are found in SMC present in rat and bovine aortae in vivo. These steady‐state isoactin mRNA ratios are slightly different from those seen in cultured PC (8–10:1; α:β). These results suggest that selective synthesis and remodelling of the endothelial basal lamina may signal alterations in pericyte growth and contractile phenotype during normal vascular morphogenesis, angiogenesis, or during the microvascular remodelling that accompanies hypertensive onset. © 1993 Wiley‐Liss, Inc.
Effect of phospholipid acyl chain modulation on vitamin E incorporation into pulmonary artery endothelial cell membranes
Authors: Patel Jawaharlal M. , Abeles Alan J. , Block Edward R.
Published: February 2005

Incorporation of vitamin E (α‐tocopherol) was measured in total membranes of pulmonary artery endothelial cells (PAEC) following treatment with eight synthetic phosphatidylethanolamines (PE) (Palmitoyloleoyl, 16:0–18:1 PE1; distearoyl, 18:0–18:0 PE2; dioleoyl, 18:1–18:1 PE3; stearoyl‐ linoleoyl, 18:0–18:2 PE4; dilinoleoyl, 18:2–18:2 PE5; stearoyl‐arachiod‐nyl, 18:0–20:4 PE6; diarachidonyl, 20:4–20:4 PE7; and stearoyl‐docosahexenoyl, 18:0–22:6 PE8). Endogenous PE content of native membranes was 0.88 ± 0.01 nmol/mg protein. Incorporation of PE irrespective of fatty acid content significantly (P < 0.02) increased the PE content of total membranes. Vitamin E incorporation in control membranes was 63 ± 9 nmol/mg protein. Incorporations of vitamin E in PE1‐ to PE7‐treated cells were significantly (P < 0.05) increased compared to controls and were comparable to each other. Vitamin E incorporation into PE8‐treated cells was threefold greater (P < 0.001) thatn controls and twofold greater (P < 0.001) than PE1‐ to PE7‐treated cells. Increased PE content results in increased vitamin E incorporation into PAEC membranes irrespective of the fatty acids present on the acyl chain, and maximal incorporation of vitamin E in PE8‐treated cells may relate to the increased carbon chain length rather than to the degree of unsaturation at the sn2 position. © 1993 Wiley‐Liss, Inc.
Deficiencies in collagen phagocytosis by human fibroblasts in vitro: A mechanism for fibrosis?
Authors: McCulloch Christopher A. G. , Knowles Gisele C.
Published: February 2005

Degradation of collagen by fibroblast phagocytosis is an important pathway for physiological remodelling of soft connective tissues. Perturbations of this pathway may provide a mechanism for the development of fibrotic lesions. As collagen phagocytosis may be regulated by either a change of the proportions or the activity of phagocytic cells, we quantified phagocytosis with an in vitro model system. Collagen‐coated fluorescent latex beads were incubated with human gingival fibroblasts and the fluorescence associated with internalized beads was measured by flow cytometry. Cells from normal tissues that had been incubated with beads for 3 hours contained a mean of 64% phagocytic cells; however, a small subpopulation (10% of phagocytic cells) contained more than threefold higher numbers of beads per cell than the mean. In contrast, cells from fibrotic lesions exhibited a large reduction of the proportions of phagocytic cells (mean = 13.8%) and there were no cells with high numbers of beads. On the basis of 3H‐Tdr labeling, cells from fibrotic lesions that had internalized beads failed to proliferate, in contrast to phagocytic cells from normal tissues, which underwent repeated cell divisions. This result was not due to variations of cell cycle phase as there was no preferential internalization of beads during different phases of the cell cycle. The low phagocytic rate of cells from fibrotic lesions was also not due to asymmetric partitioning of phagosomes at mitosis as videocinemicrography of bead‐labeled phagosomes in single, pre‐mitotic cells demonstrated that > 90% of phagocytic cells equally partitioned beads to daughter cells. To investigate if inhibition of phagocytosis could be replicated in vitro, cells were incubated with the fibrosis‐inducing drugs nifedipine or dilantin. These cultures exhibited marked (15–75%), dose‐dependent reductions in the proportions of phagocytic cells, but there was no reduction in bead number per cell. Fibrotic lesions appear to contain fibroblasts with marked deficiencies in phagocytosis and the reduced phagocytic activity of these cells may contribute to unbalanced degradation and fibrosis. © 1993 Wiley‐Liss, Inc.
Myocyte DNA synthesis with aging: Correlation with ventricular loading in rats
Authors: Capasso Joseph M. , Bruno Silvia , Li Peng , Zhang Xun , Darzynkiewicz Zbigniew , Anversa Piero
Published: February 2005

To determine whether the detrimental mechanical and anatomical changes that occur biventricularly with aging are associated with activation of DNA synthesis, flow cytometric analysis was performed on myocyte nuclei prepared from the left and right ventricles of rats at 4, 12, 20, and 29 months of age. Heart weight increased significantly with age, and this growth adaptation was associated with the development of left ventricular failure and right ventricular dysfunction. These phenomena were coupled with marked elevations in diastolic wall stress and increases in the percentage of myocyte nuclei in S+G2M in both ventricles. Linear regression analyses revealed a direct correlation between the fraction of myocytes that entered the cell cycle and diastolic pressure and wall stress. An inverse relation was found between the percentage of myocyte nuclei in S+G2M and +dP/dt and systolic wall stress. Thus the depression of hemodynamic performance coupled with alterations in the loading conditions contributes, at least in part, to increased DNA synthesis in cardiac myocytes with age. © 1993 Wiley‐Liss, Inc.
Oleic acid supplementation reduces oxidant‐mediated dysfunction of cultured porcine pulmonary artery endothelial cells
Authors: Hart C. Michael , Andreoli Sharon P. , Patterson Carolyn E. , Garcia Joe G. N.
Published: February 2005

We have previously shown that supplementing cultured porcine pulmonary artery endothelial cells (PAEC) with exogenous oleic acid (18:1ω9) alters the fatty acid composition of the cells and reduces oxidant‐mediated cytotoxicity. Because the mechanisms by which lipid alterations modulate oxidant susceptibility have not been defined, the ability of 18:1 to reduce hydrogen peroxide (H2O2)‐mediated PAEC dysfunction was evaluated. PAEC monolayers on polycarbonate filters were incubated for 3 h in maintenance medium supplemented with either 0.1 mM 18.1 in ethanol vehicle (ETOH) or with an equivalent volume of vehicle alone. Twenty‐four hours later monolayers were treated for 30 min with 50 or 100 μM H2O2 in Hanks' balanced salt solution (HBSS) or with HBSS alone (nonoxidant control). As a functional index of PAEC monolayer integrity, the permeability of monolayers to albumin was then measured for 3 h. Treatment with 100 μM H2O2 caused cytotoxicity and progressive increases in PAEC monolayer permeability that were attenuated by 18:1 supplementation, whereas 50 μM H2O2 caused only a transient increase in permeability without cytotoxicity. Supplementation with 18:1 also attenuated H2O2‐induced reductions in PAEC adenosine triphosphate (ATP) content and disruption of PAEC microfilament architecture. The ATP content of PAEC monolayers was reversibly reduced in the absence of oxidant stress by incubation with glucose‐depleted medium containing deoxyglucose and antimycin A. Metabolic inhibitor‐induced ATP depletion increased monolayer permeability and altered cytoskeletal architecture, alterations that resolved during recovery of PAEC ATP content. These results demonstrate that ATP depletion plays a critical role in barrier dysfunction and suggests that the ability of 18:1 to reduce oxidant‐mediated PAEC dysfunction and injury may relate directly to its ability to preserve PAEC ATP content. © 1993 Wiley‐Liss, Inc.
Regulation of RNA degradation in cultured rat hepatocytes: Effects of specific amino acids and insulin
Authors: Balavoine Sophie , Feldmann Gérard , Lardeux Bernard
Published: February 2005

The regulation of RNA degradation by specific amino acids and insulin was investigated in cultured rat hepatocytes from fed rats previously injected in vivo with [6‐14C]orotic acid. The effects of three groups of amino acids were compared to those of a complete amino acid mixture. The first one consisted of the eight amino acids (leucine, proline, glutamine, histidine, phenylalanine, tyrosine, methionine, tryptophan) previously found to be particularly effective in the control of proteolysis. The two other groups were defined from our study with single additions of amino acids, one consisting of proline, asparagine, glutamine, alanine, phenylalanine, and leucine and the other including the latter group with serine, histidine, and tyrosine. The results showed that these three groups were able to strongly inhibit deprivation‐induced RNA breakdown at one and ten times normal plasma concentrations but to a lower extent than the complete amino acid mixture. Six amino acids (proline, asparagine, glutamine, alanine, phenylalanine, leucine) inhibited individually RNA degradation by more than 20%. However, the deletions of proline, asparagine, glutamine, or alanine from the group of these six amino acids were not followed by a loss of inhibitory effect. On the contrary, an important loss of inhibition was observed when leucine and phenylalanine were deleted. Furthermore, only these two amino acids exhibited an additive inhibitory effect. Thus leucine and phenylalanine could be considered as important inhibitors of RNA breakdown in cultured rat hepatocytes. Finally, insulin which had no significant effect on RNA degradation in the absence of amino acids, was able to potentiate the inhibitory effect of different amino acid groups. © 1993 Wiley‐Liss, Inc.
Pretreatment with vitamin A inhibits transforming growth factor alpha stimulation of human mammary carcinoma cells
Authors: Halter Susan A. , Winnier Angela R. , Arteaga Carlos L.
Published: February 2005

Vitamin A or retinol is an important agent in the normal differentiation and growth of cells. Retinol is an effective inhibitor of the growth of many transformed cells in vitro and in vivo but its mechanism of action is unclear. Transforming growth factor alpha (TGFα) is a known mitogen. We examined the effect of retinol treatment on TGFα stimulation of two human mammary carcinoma cell lines, one which is growth inhibited by retinol and one which is not. Pretreatment of both cell lines for 48 hours with retinol resulted in inhibition of TGFα stimulation of growth. In the T47D cell line the mechanism was not related to an effect on the cellular content of TGFα, epidermal growth factor (EGF) receptor protein, EGF receptor mRNA, or on the binding of TGFα to the EGF receptor. However, TGFα‐induced stimulation of the EGF receptor substrate, phospholipase C‐γ1, was abrogated in the T47D cell line with retinol pretreatment. In the MDA‐MB‐468 cell line, pretreatment with retinol resulted in a decrease in tyrosine phosphorylation of the EGF receptor. These results suggest that pretreatment with retinol decreases cellular proliferation seen with TGFα treatment by altering phospholipase C‐γ1 response and/or EGF receptor tyrosine kinase activity. Alteration of phospholipase C‐γ1 activity does not appear to be responsible for the inhibition of cell growth seen in the absence of TGFα stimulation. © 1993 Wiley‐Liss, Inc.
Characterization of selective glucocorticoid‐dependent responses in a glucocorticoid‐resistant smooth muscle tumor cell line
Authors: Fan Weimin , Cooper Tina M. , Norris James S.
Published: February 2005

The DDT1 MF2 smooth muscle cell line was derived from an estrogen/androgeninduced leiomyosarcoma arising in the hamster ductus deferens. Growth of this cell line is arrested in Go/G1 by treatment with glucocorticoids. To facilitate the study of the mechanism of glucocorticoid‐induced cell growth arrest, a glucocorticoid‐resistant variant cell line, DDT1 MF2 GR1 (GR1), was developed by genetic selection. Growth of this mutant cell line is completely resistant to the inhibitory action of glucocorticoids. However, we now demonstrate that both primary and secondary glucocorticoid‐induced events still exist in the GR1 cell line. By analyzing the expression and genetic pattern of glucocorticoid receptor, no detectable rearrangement of the glucocorticoid receptor gene was found although the expression of both mRNA and protein levels of the receptor were lower in the variant compared to wild‐type cells. In addition, we found that the expression of two growth‐associated genes, Ha‐ras and transforming growth factor β1 (TGF‐β1) are down‐regulated by glucocorticoids in wild‐type DDT1 MF2 cells but not in GR1 cells. These results indicated that the function or activity of glucocorticoid receptor in the GR1 cells is not qualitatively altered. Our data suggest that a lower glucocorticoid receptor level is not the real cause or at least not the single cause for the GR1 cell's loss of sensitivity to the inhibitory action of glucocorticoid. Instead, we postulate the existence of a defect downstream of the primary site of action of glucocorticoid receptor complexes in GR1 cells. © 1993 Wiley‐Liss, Inc.
Comparative analysis of mitogenic and morphogenic effects of HGF and EGF on rat and human hepatocytes maintained in collagen gels
Authors: Michalopoulos George K. , Bowen William , Nussler Andreas K. , Becich Michael J. , Howard Tamara A.
Published: February 2005

Hepatocytes maintained in collagen gels remain differentiated for prolonged periods of time compared to cells maintained on conventional cultures. Previous studies with other culture systems in which chemical supplements or substratum modifications enhanced hepatocyte differentiation showed that in all of these systems hepatocytes do not respond to mitogens. In this study it is shown that hepatocytes maintained between two layers of collagen gels respond to mitogens HGF (also known as scatter factor (HGF/SF)) and epidermal growth factor (EGF). Cell density did not affect the responsiveness to mitogens as in conventional cultures. In addition both mitogens (HGF more pronounced) induce characteristic morphogenic changes in which hepatocytes form processes and join in formation of cords. Hepatocytes respond to mitogens for up to 6 days in culture at which point they become refractory to further mitogenic stimulation. This occurs despite electron microscopic evidence that these cells are fully viable when they become refractory to mitogenesis. The refractory state is not modified by substitution of one growth factor for the other or by addition of growth factors at different times. Hepatocytes in the refractory state become again responsive to mitogens when the collagen gels are dispersed by collagenase and the cells are replated on conventional substrates. © 1993 Wiley‐Liss, Inc.
Contributions of K+, Na+, and Cl− to the membrane potential of intact hamster vascular endothelial cells
Authors: Miao Kai , Wondergem Robert , Hossler Fred E. , Joyner William L.
Published: February 2005

The transmembrane potential (Vm) of vascular endothelial cells (EC) is an important property that may be involved in intra‐ and intercellular signal transduction for various vascular functions. In this study, Vm of intact aortic and vena caval EC from hamsters were measured using conventional microelectrodes. Vascular strips with the luminal surface upwards were suffused in a tissue chamber with krebs solution in physiological conditions. The resting Vm of aortic and vena caval EC was found to be −40± 1 mV (n = 55) and −43± 1 mV (n = 15), respectively. The Vm recordings were confirmed to have originated from EC by scanning and transmission electron microscopy combined with the comparison of electrical recordings between normal and endothelium‐denuded aortic strips. The input resistance varied from 10–240 MΩ, which implied the presence of electrical coupling between vascular EC. Elevating the K+ level in the suffusate from 4.7 mM to 50 and 100 mM depolarized aortic EC by 19% and 29% and vena caval EC by 18% and 29%, respectively. These low percentages indicated a relatively small contribution of [K+] to the resting Vm of vascular EC. A positive correlation (r> 0.69) between the resting Vm and the magnitude of depolarization by the high [K+]0 may be related to the involvement of voltage‐dependent K+ channels. The hyperpolarization caused by lowering both [Na+]0 and [CI−]0 suggested the disengagement of some electrogenic transport systems in the membrane, such as a Na+ ‐K+ ‐CI− cotransporter. The transference number (tion), as an index of membrane conductance for specific ions, was calculated for K+ (15‐20%), Na+ (16%), and Cl− (9‐15%), demonstrating that both Na+ and Cl− as well as K+ contribute to the overall resting Vm. Our study documented some basic electrophysiology of the vascular EC when both structural and functional properties of the cell were maintained, thus furthering the understanding of the essential role of endothelial cells in mediating vascular functions. © 1993 Wiley‐Liss, Inc.
An N‐cadherin–like protein contributes to solute barrier maintenance in cultured endothelium
Authors: Alexander J. S. , Blaschuk O. W. , Haselton F. R.
Published: February 2005

We investigated the role of cadherins in the solute barrier maintained by endothelial cells in vitro. Cell‐column chromatographic measurement of endothelial barrier showed that reducing normal extracellular calcium from 1.2 to 0.12 mM increased endothelial permeability to 250% of baseline after 20 min. Restoring normal calcium restored the barrier within 15 min which remained stable for at least 60 min. We used sulfo‐NHS‐biotin and anti‐cadherin antibodies to characterize endothelial proteins with possible roles in the maintenance of endothelial barrier. The non‐specific probe sulfo‐NHS‐biotin identified at least ten endothelial cell surface proteins, with greatest labelling occurring at molecular weights of 125 and 145 kD. Six proteins, including the 125 and 145 kD proteins, associated with the cytoskeleton. Western blotting for the presence of classical cadherins containing the conserved cytoplasmic sequence CDPTAPPYDSLLVFDYEG detected two bands at 145 and 125 kD which associated with the cytoskeleton. Western blotting with an antibody, which recognizes FHLRAHAVDINGNQV, an extracellular homotypic binding region of N‐cadherin, detects three bands. Of these three, one protein had a molecular weight of 125 kD and was associated with the cytoskeleton. Immunofluorescence with both N‐cadherin and anti‐peptide 1 antibodies found staining at endothelial cell borders. The utility of a newly developed cell‐column calcium switch assay was tested by verifying the functional role of the previously described epithelial cadherin, uvomorulin, in epithelial barrier. We then applied this method to endothelial cell columns and found the N‐cadherin antibody interfered with the reforming of interendothelial junctions. These results suggest that, as in epithelial cells, cadherins in bovine endothelial cells have a functional role in forming the calcium sensitive endothelial junction and may play an important role in the formation of normal barrier. © 1993 Wiley‐Liss, Inc.
Leukocyte‐deactivating factor from macrophages: Partial purification and biochemical characterization. A novel cytokine
Authors: Camarero Valéria C. P. C. , Colepicolo Pio , Ribeiro José M. C. , Karnovsky Manfred L.
Published: February 2005

A deactivating factor (MDF) is released from granuloma‐like lesions of mice (giant and epithelioid macrophages) to the surrounding medium. Test cells incubated in the presence of MDF display dramatic inhibition of superoxide anion (O2−) release when stimulated. This failure to manifest O2 release is observed whether PMA, all‐transretinal, or fMet‐Leu‐Phe is the stimulating agent. MDF acts on different cell types from different species; mouse macrophages as well as guinea pig, human, and mouse neutrophils. Such results suggest that it is a universal regulatory cytokine with high affinity for phagocytic lineages. The factor was subjected to various purification methods: ultrafiltration, gel chromatography, and reversed phase HPLC. A crude preparation that resulted from conditioning of medium by old macrophages (MCM) shows two peaks of activity when subjected to gel filtration. These correspond to molecular weights for the active principle of 3 and 11 kD. When the factor was obtained by extraction of the same cells after washing and sonication. Only the former peak was seen. Fractions corresponding to a MW of 3 kD from several preparations were combined and subjected to HPLC. MDF activity then appeared in a single fraction. MDF is thus putatively a modulator of the cidal activity of phagocytic cells that utilize release of reactive oxygen species for cytocidal activity. © 1993 Wiley‐Liss, Inc.
Effects of ultraviolet irradiation on human skin‐derived epidermal cells in vitro
Authors: Dissanayake N. S. , Greenoak G. E. , Mason R. S.
Published: February 2005

The effects of UVA, mixed UVA + B, and solar‐simulated irradiation were examined in human keratinocytes and melanocytes cultured in vitro. Irradiation with UVA, UVA + 3, or the solar simulator caused a dose‐dependent decrease in keratinocyte cell numbers and thymidine incorporation at 24 hours, with recovery after 48 and 72 hours. Divided dose regimens reduced the inhibitory effect of ultraviolet (UV) irradiation on cell numbers measured 24 hours after the last irradiation. Exposure to both UVA and UVA + B increased formation of cornified envelopes. Similar irradiance doses of UVA 80 minutes (1.12 J/cm2) and UVA + B 40 minutes (1.04 J/cm2) caused 2.4‐ and 3.3‐fold increases in cornified envelope formation, respectively. With solar‐simulated irradiation, the cornified envelope formation was increased by 3.5‐fold after exposure of 8 minutes (2.6 J/cm2). Irradiation of melanocytes with UVA, UVA + B, or solar‐simulated irradiation resulted in a dose‐dependent decrease in melanocyte numbers after 24 hours compared with sham‐irradiated controls. As a result of UV irradiation, tyrosinase activity of melanocytes measured at 24 hours was stimulated. UVA + B irradiation (1.04 J/cm2) increased tyrosinase activity approximately twofold, while UVA alone (1.1 J/cm2) increased tyrosinase four to sixfold and solar‐simulated irradiation (1.3 J/cm2) increased tyrosinase approximately twofold compared to the control cells. Melanin content increased in cells after both UVA and mixed UVA + B irradiation. These results indicate that both UVA and mixed UVA + B irradiation had qualitatively similar effects on the proliferative and functional activity of skin‐derived cells but that the type of irradiation and the dosage regimen affect the dose‐response relationship. © 1993 Wiley‐Liss, Inc.
Insulin induces rapid accumulation of insulin receptors and increases tyrosine kinase activity in the nucleus of cultured adipocytes
Authors: Kim Sung‐Jin , Kahn C. Ronald
Published: February 2005

To better understand the mechanism by which insulin exerts effects on events at the cell nucleus, we have studied insulin receptors and tyrosine kinase activity in nuclei isolated by sucrose density gradient centrifugation following insulin treatment of differentiated 3T3‐F442A cells. Insulin stimulated nuclear accumulation of insulin receptors by approximately threefold at 5 min. The half‐maximal effect was observed with 1–10 nM insulin. Following insulin treatment, phosphotyrosine content associated with the nuclear insulin receptor was also increased by twofold at 5 min with a similar insulin concentration dependency. These nuclear insulin receptors differ from the membrane‐associated insulin receptors in that they were not efficiently solubilized with 1% Triton X–100. During the same period of time, insulin stimulaced nuclear tyrosine kinase activity toward the exogenous substrate poly Glu4: Tyr1 tenfold in a time‐dependent manner reaching a maximum at 30 min. The insulin receptor substrate protein 1 (IRS‐1) could not be detected in the nucleus by immunoblotting. However, a nuclear protein with Mr ≈ 220 kDa was tyrosine phosphorylated, and insulin further stimulated this process threefold >30 mins. Surface labeling was performed to determine if the nuclear insulin receptors would emerge from the plasma membrane fraction. Using 1251‐BPA‐insulin with intact cells, the intensity of nuclear insulin receptor labeling was negligible and not increased throughout 30 min incubation at 37°C. In contrast, there was an increase in labeled receptors in the microsomal fraction following insulin treatment. Taken together, these results indicate that insulin rapidly increases nuclear insulin receptor appearance and activates nuclear tyrosine kinase activity. The insulin‐induced accumulation of nuclear insulin receptors cannot be accounted for by internalization of surface membrane receptors. These effects of insulin may play an important role in action of the hormone at the nuclear level. © 1993 Wiley‐Liss, Inc.
In vitro expression of osteoblastic markers in cells isolated from normal fetal and postnatal human bone and from bone of patients with osteogenesis imperfecta
Authors: Morike M. , Schulz M. , Brenner R. E. , Bushart G. B. , Teller W. M. , Vetter U.
Published: February 2005

We studied the expression of osteoblastic markers in cultured cells isolated from the bone of 15 patients with different clinical forms of osteogenesis imperfecta (OI) and of seven fetal and postnatal controls. Cultured bone cells of ten OI patients produced abnormal collagen type I. Similar to controls, OI bone cells produced predominantly collagen type I with traces of collagen types III and V. The 1,25(OH)2 vitamin D3‐stimulated synthesis of osteocalcin, a specific osteoblastic marker protein, was similar in OI bone cells and age‐matched controls. Bone cells from fetal controls and from patients with the perinatal lethal OI type II produced less osteocalcin than bone cells from postnatal controls and surviving OI patients. OI bone cells responded to parath.yroid hormone (PTH) by increased production of cAMP similar to controls. Bone cells from fetal controls and from OI type II donors showed a decreased response to PTH. Activity of the bone‐liver‐kidney isoenzyme alkaline phosphatase (AP) was detected in all control and OI bone cells. The expression of all osteoblastic markers was similar in bone cells producing abnormal collagen type I. These observations show that OI bone cells in vitro express a pattern of osteoblastic markers similar to age‐matched control bone cells indicating that osteoblastic differentiation is not altered by the underlying defects of collagen type I metabolism in OI bone cells. © 1993 Wiley‐Liss, Inc.
Activation of the 92 kDa type IV collagenase by tissue kallikrein
Authors: Desriviéres Sylvane , Lu He , Peyri Nicole , Soria Claudine , Legrand Yves , Ménashi Suzanne
Published: February 2005

Type IV collagenases are secreted as latent 92 and 72 kDa proenzymes which are then activated extracellularly. The mechanisms by which they are activated in vivo are not clear. We have studied the activation of porcine endothelial cell type IV collagenases by tissue and plasma kallikrein, and found that tissue kallikrein was a very efficient activator of the 92 kDa type IV collagenase. Enzyme cleavage was observed at concentrations of tissue kallikrein as low as 0.1 μg/ml. Plasma kallikrein had no effect. By comparison, plasmin, which has been proposed to be the physiological activator of interstitial collagenase and stromelysin, and elastase were much less effective, and high concentrations (plasmin at 100–200 μg/ml and elastase at 20 μg/ml) were required to cause only a limited cleavage which was not associated with an increase in activity, as observed by the gelatin‐gel lysis assay. In addition tissue kallikrein was found by immunohistochemistry to be present in the extracellular matrix of the intima of porcine aortic vessel wall. These findings suggest that tissue kallikrein can be a potential activator of the 92 kDa type IV collagenase in vivo. © 1993 Wiley‐Liss, Inc.
Morphological and proliferative responses of endothelial cells to hydrostatic pressure: Role of fibroblast growth factor
Authors: Acevedo Arlene D. , Bowser Samuel S. , Gerritsen Mary E. , Bizios Rena
Published: February 2005

Subconfluent bovine pulmonary artery endothelial cells on rigid substrates were exposed to 1.5–15 cm H2O sustained hydrostatic pressure for up to 7 days and exhibited elongation, cytoskeletal rearrangement, increased cell proliferation, and bilayering. The role of basic fibroblast growth factor (bFGF) in the mechanism(s) of these endothelial cell responses to sustained hydrostatic pressure was investigated. Evidence that bFGF was released from endothelial cells exposed to sustained hydrostatic pressure or compression was provided by the following experimental results: (1) Cells exposed to control (3 mm H2O) pressure displayed intense nuclear and cytoplasmic bFGF staining by immunocytochemical techniques; this staining was absent in cells exposed to 10 cm H2O for 7 days. (2) Conditioned medium from endothelial cells exposed to 10 cm H2O for 7 days contained at ansferable, growth‐promoting activity exhibiting heparin‐Sepharose affinity, lability to both heat and freeze/thawing, and neutralization by anti‐bovine bFGF. (3) Suramin (0.1 mM), a growth‐factor receptor inhibitor, abrogated the proliferative and morphological responses of endothelial cells exposed to sustained hydrostatic pressure. Endothelial cells exposed to elevated hydrostatic pressure demonstrated no detectable decrement in cell viability as assessed by Trypan blue exclusion. The results of the present study indicate that hydrostatic pressure or compression can induce bFGF release from endothelial cells independent of cell injury or death; bFGF is subsequently responsible for the morphological, proliferative, and bilayering responses of endothelial cells to hydrostatic pressure. © 1993 Wiley‐Liss, Inc.
Isoproterenol‐ and insulin‐induced hyperpolarization in rat skeletal muscle
Authors: Li Kai‐Xun , Sperelakis Nicholas
Published: February 2005

Using conventional microelectrode techniques, we investigated the combined effects of isoproterenol (Iso) and insulin (Ins) on the resting membrane potential (RMP) of isolated rat skeletal muscles. In soleus muscle, Iso (1 μM) and Ins (4 units/L) separately induced a hyperpolarization of 9.2 mV and 4.8 mV, respectively. Combined administration of Iso and Ins induced a hyperpolarization of 12.4 mV, larger than either one separately. A similar observation was made in Na+‐loaded rewarming experiments. 8‐Br‐cAMP (1 mM and 3 mM) and forskolin (10 μM, an adenylate cyclase activator) induced a hyperpolarization of 5.3 mV, 8 mV, and 6.0 mV, respectively. This hyperpolarizing action was blocked by ouabain indicating that the Na‐K pump was involved in the hyperpolarization. 8‐BrcGMP (3 mM) had no effect on RMP; however, it blocked or reversed the hyperpolarization caused by 8‐Br‐cAMP (1 mM). In addition, 8‐Br‐cGMP partially inhibited the hyperpolarizing effect of Iso (1 μM) by 40% and completely prevented the effect of Ins. The phorbol ester, PMA, (1 μM, a PKC activator) induced a ouabain‐inhibitable hyperpolarization. These results suggest that cAMP and PKC are involved in the Iso‐ and Ins‐induced hyperpolarization and that Iso and Ins influence the RMP presumably through regulation of the electrogenic Na‐K pump via different mechanisms. © 1993 Wiley‐Liss, Inc.
Regulation of amino acid transport and protein metabolism in myotubes derived from chicken muscle satellite cells by insulin‐like growth factor‐I
Authors: Duclos M. J. , Chevalier B. , Goddard C. , Simon J.
Published: February 2005

The effects of insulin and insulin‐like growth factor‐I (IGF‐I) on amino acid transport and protein metabolism were compared in myotubes derived from chicken breast muscle satellite cells. Protein synthesis was assessed by continuous labelling with [3H]‐tyrosine. Protein degradation was estimated by the release of trichloroacetic acid (TCA) soluble radioactivity by cells which had been previously labelled with [3H]‐tyrosine for 3 days. Amino acid transport was measured in myotubes incubated in Dulbecco's modified Eagle's medium (DMEM) 0.5% bovine serum albumin (BSA) with or without insulin or IGF‐I. Subsequent [3H]‐aminoisobutyric acid (AIB) uptake was then measured in amino acid‐free medium. IGF‐I was more efficient than insulin at equimolar concentration (3.2 nmol/l) in stimulating protein synthesis (127 and 113% of basal, respectively) and inhibiting protein degradation (32% and 13% inhibition of protein degradation following 4 h incubation). Half maximal effective concentrations for stimulation of AIB uptake were 0.27 ± 0.03 nmol/l and 34.8 ± 3.1 nmol/l for IGF‐I and insulin respectively, with maximal stimulation of about 340% of basal. Cycloheximide (3.6 μmol/l) diminished IGF‐I‐stimulated AIB uptake by 55%. Chicken growth hormone had no effect on basal AIB uptake in these cells and neither glucagon nor dexamethasone had an effect on basal or IGF‐I‐stimulated AIB uptake. This study demonstrates an anabolic effect for IGF‐I in myotubes derived from primary chicken satellite cells which is mediated by the type I IGF receptor, since the cation‐independent mannose 6‐phosphate receptor does not bind IGF‐II in chicken cells. © 1993 Wiley‐Liss, Inc.
Induction by vasoactive intestinal peptide of interferon α/β synthesis in glial cells but not in neurons
Authors: Chelbi‐Alix Mounira K. , Brouard Aline , Boissard Claudine , Pelaprat Didier , Rostene William , Thang Ming Nguy
Published: February 2005

Vasoactive intestinal peptide (VIP), a 28‐aminoacid peptide, plays a multifunctional neuromodulatory role in both peripheral and central nervous systems. We have recently reported that VIP induces interferon (IFN) α/β synthesis in human colon adenocarcinoma cell line HT‐29. It has been reported that VIP may counteract HIV‐induced neuronal cell death; therefore, we postulated that the action of VIP may be mediated by a cascade regulation, involving the production of some cytokines such as IFN. Here we demonstrate that primary cultures of rat mesencephalic neurons and glial cells respond differently to VIP. Thus VIP enhanced 2′5′ oligoadenylate (2′5′A) synthetase activity and inhibited vesicula stomatitis virus multiplication in glial cultures only. However, both cell cultures had functional adenylate cyclase coupled receptors for VIP. The increase in 2′5′A synthetase activity in glial cultures reached a maximum with 10−6M VIP and required cellular RNA and protein synthesis. Anti‐IFN α/β, but not anti‐IFN γ, antibodies abolished the induction of the antiviral and 2′5′A synthetase activities by VIP in rat glial‐enriched cultures, suggesting that these inductions were mediated through IFN α/β synthesis. Moreover, VIP or poly (i).poly (C12U) caused, in the glial cultures, the induction and secretion of an IFN of type α/β with a titer value of 16 and 32 units/ml respectively. In contrast, neither of these two substances was able to induce IFN synthesis in neurons, which were, however, sensitive to IFN α/β produced by VIP‐treated glial cells. IFN produced by VIP in glial cells may therefore play an important role in defending the brain against viruses. © 1994 Wiley‐Liss, Inc.
Age‐related changes in DNA synthesis stimulated by epinephrine and isoproterenol in primary cultured rat hepatocytes
Authors: Ishigami Akihito , Roth George S.
Published: February 2005

We examined epinephrine‐ and isoproterenol‐stimulated DNA synthesis in primary cultured hepatocytes from 6‐, 12‐, and 24‐month‐old rats. Epinephrine‐stimulated DNA synthesis in 6‐month‐old rat hepatocytes began after 20 h and reached a maximum at 50 h. Similarly, isoproterenol‐stimulated DNA synthesis in 6‐month‐old rat hepatocytes began after 10 h and reached a maximum at 45 h. In contrast, both epinephrine‐ and isoproterenol‐stimulated DNA synthesis in 12‐ and 24‐month‐old rat hepatocytes were reduced approximately 40–60% and 80%, respectively, as compared to that at 6 months. Both epinephrine‐ and isoproterenol‐stimulated DNA synthesis were strongly inhibited by the betaadrenergic antagonist, propranolol, but not by the alpha1‐adrenergic antagonist, prazosin, or the alpha2‐adrenergic antagonist, yohimbine. However, in the presence of EGF, epinephrine‐stimulated DNA synthesis activity was inhibited by prazosin but not by propranolol. These results indicate that stimulated DNA synthesis in rat hepatocytes declines with age and that there are two different pathways for epinephrine‐stimulated DNA synthesis in the presence or absence of EGF. © 1994 Wiley‐Liss, Inc.

This article is a US Government work and, as such, is in the Public domain in the United States of America.
Thrombomodulin lacking the cytoplasmic domain efficiently internalizes thrombin via nonclathrin‐coated, pit‐mediated endocytosis
Authors: Conway Edward M. , Nowakowski Barbara , Steiner‐Mosonyi Marta
Published: February 2005

Thrombomodulin (TM) is a transmembrane vascular endothelial cell receptor that is a cofactor in a major physiologically relevant natural anticoagulant system. We recently developed a cell model to examine one mechanism of regulation of TM cell surface expression and visually demonstrated that the receptor undergoes internalization predominantly via noncoated pits (Conway et al., 1992, J. Cell. Phys., 151:604–612). We have extended these studies to examine the role of the cytoplasmic domain of TM by deleting this region and expressing the truncated version of the molecule in COS cells (COS.Cyto.Del cells). Electron microscopy demonstrated internalization of gold‐labeled anti‐TM antibody or thrombin in a time‐ and temperature‐dependent manner, similar to that seen with the wild‐type transfected cells (COS.TM‐CR). Endocytosis was characterized by initial surface clustering of gold particles, followed by aggregation into noncoated pits, early endosome formation, and, finally, entry into multivesicular bodies and lysosomes. There was a notable absence of gold particles in clathrin‐coated pits and vesicles. The kinetics of binding and internalization of 125I‐labeled ligand in COS.Cyto.Del cells was compared with that of COS.TM‐CR cells and was not significantly different. These studies provide ultrastructural and quantitative data to indicate that TM efficiently undergoes endocytosis via nonclathrin‐coated pits when the receptor is lacking the cytoplasmic domain. This finding suggests that there may be alternative regions of the molecule that mediate those signals necessary for internalization. © 1993 Wiley‐Liss, Inc.
Regulation of in vitro glia‐induced microvessel morphogenesis by urokinase
Authors: Laterra John , Indurti Ravi R. , Goldstein Gary W.
Published: February 2005

Plasminogen activators (PAs) regulate a variety of processes involved in tissue morphogenesis and differentiation. We used a coculture system in which microvascular endothelial cells are induced by glial cells to form capillary‐like structures in order to examine the role of urokinase‐type PA (uPA) during microvessel morphogenesis within the central nervous system (CNS). Endotheliaderived uPA activity decreased sevenfold within glial‐endothelial cocultures when capillary‐like structures were formed. Incubation of cocultures with concentrations of phorbol 12‐myristate 13‐acetate (0.1 and 1.0 nM) that induced endothelial uPA activity (45–210%) inhibited endothelial differentiation (25–70%). Furthermore, incubation of cocultures with proteolytically active low molecular weight uPA (5–500 IU/ml) inhibited endothelial differentiation (37–75%), whereas the amino terminal cell‐binding fragment of uPA had minimal effect. Inhibition of plasminogen activation in cocultures with the serine protease/plasmin inhibitors aprotinin and soybean trypsin inhibitor increased glia‐induced capillary‐like structure formation (96–98%). These findings establish a paracrine/autocrine function for urokinase and its inhibitors in regulating endothelial responses to perivascular glia and provide insight into mechanisms of microvascular reactions to CNS pathology. © 1994 Wiley‐Liss, Inc.
Culture of a prostatic cell line in basement membrane gels results in an enhancement of malignant properties and constitutive alterations in gene expression
Authors: Freeman Michael R. , Bagli Darius J. , Lamb Carolyn C. , Guthrie Paul D. , Uchida Toshi , Slavin Richard E. , Chung Leland W. K.
Published: February 2005

Interaction of a transformed rat prostate epithelial cell (NbMC‐2) with basement membrane gels (Matrigel) has been evaluated using a long‐term matrix culture system. NbMC‐2 cells, and single‐cell clonal derivatives, formed spheroidal multicellular structures (aggregates) on Matrigel surfaces and were weakly invasive or noninvasive during a 1 week period. During subsequent 2–4 week periods, invasive cells originating from the aggregates and exhibiting a fusiform morphology became evident and increased in number in the matrix cultures. This biphasic pattern of behavior did not occur on laminin, type I or type IV collagen, or fibronectin substrates, but it did occur on Matrigel in serum‐free medium. Characterization of sublines enriched in fusiform cells indicated that they maintained their distinct morphology with continuous culture. Further, they exhibited significantly greater invasive potential, saturation density, and random motility (chemokinesis) than the parent cell line. Steady‐state levels of fibronectin mRNA were highly elevated in the tusiform variants, demonstrating a constitutive alteration in patterns of gene expression coinciding with the altered morphology. These results indicate that clonal NbMC‐2 cells differentiate at a reproducible frequency into a more aggressive cell type in response to culture in the basement membrane–‐like matrix. The altered phenotypic properties appear to be stable since they can be inherited by daughter cells and because they are evident in the absence or matrix. These observations suggest a cell‐specific mechanism for promotion of malignant growth by matrix‐mediated induction of novel cell properties. © 1994 Wiley‐Liss, Inc.
Transition metal chelator TPEN counteracts phorbol ester–induced actin cytoskeletal disruption in C6 rat glioma cells without inhibiting activation or translocation of protein kinase C
Authors: Hedberg Karen K. , Birrell G. Bruce , Mobley Philip L. , Griffith O. Hayes
Published: February 2005

Phorbol ester–induced reorganization of the actin cytoskeleton was investigated in C6 rat glioma cells. Observations by fluorescence microscopy and photoelectron microscopy indicated that pretreatment with the transition metal chelator N,N,N',N'‐tetrakis(2‐pyridylmethyl)ethylenediamine (TPEN) for 1–2 h at 50 μM reduced the sensitivity of the actin cytoskeleton to disruption by the subsequent addition of 200 nM phorbol myristate acetate (PMA). The protective effect of TPEN was eliminated by adding back Zn2+ prior to PMA addition, implicating chelation of metal ions as the mechanism of action of TPEN. C6 cells exposed to PMA experience potent activation of protein kinase C (PKC) and substantial redistribution of the kinase from a soluble to a particulate cellular fraction (translocation). TPEN pretreatment did not block PKC translocation in PMA‐exposed cells. By two‐dimensional gel analysis, TPEN also did not reduce, but rather slightly increased, the PMA‐stimulated phosphorylation of the acidic 80 kDa endogenous PKC substrate, as well as two other proteins at 18 kDa and 50 kDa. In contrast, TPEN significantly suppressed phosphorylation of a 20 kDa protein, both in cells treated with TPEN only and in TPEN‐pretreated PMA‐exposed cells. The results indicate that the ability of TPEN to protect against PKC‐mediated actin cytoskeletal disruption is not due to either a block of PKC translocation or to general inhibition of PKC activity. Rather, the action of TPEN is more selective and probably involves chelation of Zn2+ at a critical Zn2+ ‐dependent phosphorylation step downstream from the initial tumor promoter–‐induced effects on PKC. © 1994 Wiley‐Liss, Inc.