bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional studies of newly synthesized benzoic acid derivatives: Identification of highly potent retinoid‐like activity
Authors: Sato Mayumi , Shudo Koichi , Hiragun Akiyoshi
Published: February 2005

Three newly synthesized benzoic acid derivatives (terephthalic acid anilides, chalcone carboxylic acid, and azobenzene carboxylic acid), with a certain structural similarity to retinoic acid, were examined for their retinoid‐like bioactivity and their capacity to bind to cellular retinoid binding proteins. Two in vitro systems were used to evaluate their retinoid‐like bioactivity: inhibition of adipose conversion of ST 13 murine preadipose cells and growth promotion of murine sarcoma virus (MSV)‐transformed 3T3 cells in serum‐free culture. All three compounds tested inhibited ST 13 adipose conversion at nanomolar concentrations in a manner similar to classical retinoids such as retinoic acid. The growth‐stimulating activity of these compounds on MSV‐transformed 3T3 cells was one to two orders of magnitude greater than that of retinoic acid. Simultaneous treatment with these compounds and retinoic acid produced only a barely detectable additive effect, suggesting a common mechanism of action, whereas unrelated mitogens, thrombin, and insulin worked synergistically in combination with retinoic acid. None of the compounds competed with retinol for binding to cellular retinol binding protein. However, two of the three competed with retinoic acid for binding to cellular retinoic acid binding protein. This study provides evidence that the newly synthesized compounds should be included among the retinoids and that their strong biological activity will undoubtedly contribute to the biological and medical application of retinoids.
Selective purine release from P388D1 macrophages injured by silica
Authors: Dobson Michael E. , Stern Richard O. , Kane Agnes B.
Published: February 2005

Silica particles are toxic to primary cultures of macrophages or the P388D1 cell line in vitro. Loss of viability in these model systems is accompanied by depletion of ATP content within 3 to 6 hours. The mechanisms responsible for ATP depletion will be explored in this paper. After prelabeling for 1 hour with 3H‐adenine, silica‐treated cells released 60–80% of their labeled acid‐soluble pool into the culture medium. This release did not occur after phagocytosis of nontoxic titanium dioxide particles and was specific for purines. ATP depletion was accompanied by purine catabolism: inosine, hypoxanthine, xanthine, and uric acid were detected in the culture medium using thin layer or high‐performance liquid chromatography. The final xanthine oxidase step in purine catabolism generates reactive oxygen metabolites. Silica toxicity was not prevented by the xanthine oxidase inhibitor allopurinol nor exogenous purines. It is concluded that adenine nucleotide depletion and purine catabolism are not solely responsible for irreversible injury in silica toxicity. It is hypothesized that purine catabolism and release from injured macrophages may lead to generation of reactive oxygen species, injury to surrounding tissue, and fibrosis.
Growth characteristics of human epidermal melanocytes in pure culture with special reference to genetic differences
Authors: Hirobe Tomohisa , Flynn Evelyn , Szabo George , Vrabel Michael , Garcia Raul I.
Published: February 2005

Human melanocyte cultures were established using disaggregated epidermal cell suspensions derived from foreskins and plated onto culture dishes in medium containing 2% fetal bovine serum, growth factors, hormones, and melanocyte growth factor (MGF) extracted from bovine hypothalamus (Wilkins et al., J. Cell. Physiol., 122: 350, 1985). After 2 days in culture the cells were transferred to serum‐free medium to eliminate keratinocyte and fibroblast growth. Melanocytes grew preferentially and pure melanocyte populations could be harvested after 12–16 days in vitro. Melanocytes were later subcultured in the presence of 1% FBS. Pure melanocyte cultures were characterized by light and electron microscopic criteria, as well as by cytochemical demonstration of the melanocytes specific enzyme, tyrosinase. At the ultrastructural level, cultured melanocytes derived from black (negroid) neonatal skin (B‐M) had numerous mature rodshaped stage IV melanosomes, while white (caucasoid) skin‐derived melanocytes (W‐M) in culture contained no mature melanosomes. Growth rate, cell yield, and in vitro lifespan for B‐M were more than twice that for W‐M in pure melanocyte cultures in the presence of MGF. Our results suggest that MGF‐dependent growth of B‐M differs from that of W‐M.
Mitogen‐stimulated tyrosine phosphorylation of a 42‐kD cellular protein: Evidence for a protein kinase‐C requirement
Authors: Vila Jordi , Weber Michael J.
Published: February 2005

Tyrosine phosphorylation of a 42‐kD, cytosolic protein is a rapid consequence when quiescent cells are stimulated with any one of a diverse group of mitogenic agents. Among the inducers of this tyrosine phosphorylation are activators of protein kinase C, raising the possibility that this serine/threonine‐specific protein kinase plays a role in mitogen‐induced tyrosine phosphorylation. Using fibroblastic cells depleted of protein kinase C by chronic treatment with the tumor promoter tetradecanoyl phorbol acetate (TPA), we now show that protein kinase C is required for the tyrosine phosphorylation of the 42‐kD protein, even when epidermal growth factor (EGF), whose receptor is a tyrosine‐specific protein kinase, provides the initial stimulus. EGF is able to induce other cellular phosphorylations independent of protein kinase C, whereas thrombin appears to require the protein kinase C‐dependent pathway. These findings suggest that phosphorylation of the 42‐kD protein is part of a protein kinase C‐dependent kinase cascade involved in intracellular signalling.
Possible cyclic AMP‐dependence of the prereplicative surge of cytosolic calmodulin in proliferatively activated rat liver cells
Authors: Soriano M. , Pujol M. J. , Bachs O.
Published: February 2005

The infusion of a solution containing triiodothyronine, amino acids, glucagon, and heparin (TAGH solution) triggered rat liver cell proliferation. It also induced a transient prereplicative surge of cytosolic calmodulin (between 6 and 20 hr postinfusion) similar to that observed in liver cells proliferatively activated by partial hepatectomy. The injection of the β‐adrenergic blocker dl‐propanolol (20 mg/kg of body weight) at the time of the infusion prevented this transient rise of cytosolic calmodulin and also inhibited the early prereplicative surge of total liver cyclic AMP, which usually occured between 1 and 4 hr after infusion. Propanolol also inhibited the early prereplicative surge of cyclic AMP and the increase of calmodulin in liver cells proliferatively activated by partial hepatectomy. The infusion of a solution containing cyclic AMP (5 μmoles) and theophylline (10 mg) into normal rats produced an increase of cytosolic calmodulin similar to that observed after infusion of TAGH solution or after partial hepatectomy. Thus it seems that the prereplicative rise of cytosolic calmodulin observed in proliferatively activated liver cells may be regulated by the early prereplicative surge of cyclic AMP.
Mammary gland Ca2+‐binding (‐dependent) proteins: Identification as calelectrins and calpactin I/p36
Authors: Hom Yun Kit , Sudhof Thomas C. , Lozano Joseph J. , Haindl Alfred H. , Rocha Victor
Published: February 2005

Calcium‐binding (‐dependent) proteins (CBPs) associated with the spreading of mammary epithelial cell cultures have been identified as various calelectrins and calpactins (p36). In immunoblot analysis, the CBPs of 30–36 kD and 68–70 KD variously react with different calelectrin and calpactin I monomer/p36 antisera. The same immunoreactive proteins were shown to be present in virgin mammary glands and collagen gel mouse mammary epithelial cell cultures. The mammary CBPs show extensive immunochemical relatedness; however, they fail to show cross‐reaction with antiserum to calpactin II (lipocortin) antiserum. These immunoreactive CBPs comigrate in electrophoresis with 35S‐methionine‐labeled CBPs isolated from mammary epithelial cell cultures. Unlike calmodulin, the mammary CBPs that correspond to calelectrins and calpactin I monomer/p36 are not stable to thermal denaturation. The mammary CBPs bind to epithelial cell membranes in a Ca2+‐dependent manner and are differentially released from ruptured cells, compared with calmodulin, suggesting subcellular localization. Phenothiazineagarose and phenylagarose are equivalent in their ability to bind the mammary CBPs. Thus, mammary gland CBPs of 30–36 kD and 68–70 kD have been shown to be related or equivalent to the calelectrins and to calpactin I monomer/p36. Since these proteins are known to bind Ca2+, we conclude that the mammary gland CBPs are also Ca2+‐binding proteins. The mammary gland CBPs are immunologically related and probably represent members of a larger family of related proteins.
Extracellular matrix assembly of cell‐derived and plasma‐derived fibronectins by substrate‐attached fibroblasts
Authors: Allio Andrea E. , McKeown‐Longo Paula J.
Published: February 2005

Using a previously described model system for the incorporation of plasma fibronectin into the extracellular matrix (McKeown‐Longo, P.J. and Mosher, D.F., 1985. J. Cell Biol., 100: 364–374), we compared the binding of cell‐derived and plasma‐derived fibronectins to human fibroblast cell layers. Binding was measured in time course experiments using metabolically labeled cell‐derived, iodinated cell‐derived, and iodinated plasma‐derived fibronectins. The kinetics of matrix assembly of cell‐ and plasma‐derived fibronectins were the same. Competitive binding curves using intact fibronectin or the 70‐kD amino‐terminal fragment of fibronectin suggested that cell surface binding sites have equal affinity for cell‐ and plasma‐derived fibronectins. Iodinated fibronectins did not bind to isolated matrices containing collagen type I, fibronectin, and thrombospondin. These results suggest that fibroblasts do not distinguish between cell‐derived and plasma‐derived fibronectins when assembling exogenous fibronectin into extracellular matrix.
Growth stimulation by PGE2 and EGF activates cyclic AMP‐dependent and ‐independent pathways in primary cultures of mouse mammary epithelial cells
Authors: Imagawa Walter , Bandyopadhyay Gautam K. , Wallace Daiana , Nandi Satyabrata
Published: February 2005

Mammary epithelial cells from virgin Balb/c mice were isolated by collagenase digestion and cultured within collagen gels in serum‐free basal medium containing insulin (10 μg/ml). Previous work has shown that linoleate or its metabolite, prostglandin E2 (PGE2), stimulate the growth of these cells only in the presence of a growth stimulant such as epidermal growth factor (EGF). Since PGE2 can stimulate cyclic AMP (cAMP) production, the role of cAMP in linoleate and EGF‐stimulated growth was examined. The cAMP phosphodiesterase inhibitor, IBMX (0.1 mM), was found to augment growth when cells were cultured in the presence of both EGF and linoleate or PGE2, but not either factor alone. These results indicated that EGF does not stimulate proliferation via cyclic AMP mediated events but could synergize with cAMP events if cAMP levels were elevated by PGE2. When assayed in cells plated on top of collagen‐coated culture dishes, cellular cyclic AMP levels were stimulated by PGE2, but only marginally by EGF. Although the stimulation of endogenous cAMP by PGE2 and IBMX was insufficient to stimulate growth in the absence of EGF, exogenous dibutyryl‐cAMP (>100 μg/ml) was able to do so showing that a sustained, and high level of cAMP (>100 μg/ml) could stimulate growth in insulin‐containing basal medium. EGF was capable of enhancing the cellular sensitivity to dibutyryl‐cAMP but the converse was not observed. cAMP stimulation of growth was dependent upon a superphysiological concentration of insulin (10 μg/ml) or a physiological concentration of somatomedin‐C. These results indicate that the proliferation of mouse mammary epithelial cells can be stimulated separately or in synergism by cAMP‐dependent or ‐independent events.
Genetic characterization of human KB cell lines resistant to epidermal growth factor: Pseudomonas exotoxin conjugates
Authors: Amano Fumio , Pastan Ira , Gottesman Michael M.
Published: February 2005

Pleiotropic human KB cell mutants, selected for resistance to a conjugate of epidermal growth factor with Pseudomonas exotoxin (PE‐EGF), were characterized genetically. These mutants have a pleiotropic phenotype, which includes reduced number of EGF receptors and reduced growth rate. Hybrid cells between HeLa D98 and four out of five of these resistant cell lines were more resistant to PE‐EGF than hybrids formed between HeLa D98 and parental KB cells. This result indicates that the phenotype of PE‐EGF resistance is incompletely dominant in four out of five cases and recessive in one out of five variants. In three separate experiments, transfection of DNA from two of the dominant resistant cell lines resulted in transformation of wild‐type KB cells to PE‐EGF resistance, confirming the dominant nature of these mutations, which affect levels of EGF receptor in KB cells.
Density‐related changes of potassium (86Rb) uptake by amphibian endothelial cells
Authors: Fava P. , Geck P. , Brändle K. , Bereiter‐Hahn J.
Published: February 2005

Potassium influx has been investigated in XTH‐2 cells, a line derived from tadpole heart endothelia. In this line, the density at which the cultures become confluent is clearly separated from the density at which growth arrest takes place. Densityrelated changes in K+ influx were monitored by determining the uptake of 86Rb into well adhering cells kept in culture medium. The main observations were (1) 86Rb uptake is highest in single cells, and on confluency it reaches a low level, which is kept constant at higher cell density regardless of whether the cultures are stationary or still in logarithmic growth phase; (2) the relative amount of 86Rb taken up via the Na+−K+−2Cl− cotransport pathway and via the Na+/K+ pump changes from low cell density to confluent cultures; 86Rb uptake of single cells is nearly insensitive to ouabain, a maximum of ouabain sensitivity is reached around confluency, whereas piretanide‐sensitive 86Rb uptake is highest in single cells and seems to reach a minimum at the onset of confluency; (3) the variations in Na+/K+ pumping rate reflect neither differences in the amount of enzyme present nor changes in enzyme repartition between apical and basolateral plasma membranes; they seem to result from either “masking” or “unmasking” of the enzyme; (4) no alterations in K+ uptake occur that would be characteristic of the “stationary growth phase.” The only changes that seem to be related to arrest of proliferation are concerned with the Na+/K+ ‐ATPase, which achieves an extraordinary susceptibility to stimulation by monensin and exhibits an increase in PNPPase activity.
Laminin inhibits human keratinocyte migration
Authors: Woodley David T. , Bachmann Peter M. , O'Keefe Edward J.
Published: February 2005

A quantitative migration assay for human keratinocytes was developed to assess the influence of extracellular matrix molecules on cell motility independently from their effect on cell proliferation. Fibronectin and collagen types I and IV markedly promoted keratinocyte migration, but albumin, type V collagen, and heparan sulfate proteoglycan had little effect. In contrast, laminin inhibited keratinocyte motility and dramatically reduced type IV collagen‐induced migration in a concentration‐dependent manner. Laminin was not toxic, since it had no apparent effect on morphology, growth, or ability of cells to be passaged. Laminin, a major component of the lamina lucida, may inhibit motility of keratinocytes in vivo. Absence of contact with laminin, which accompanies wounding, may facilitate motility and healing in the epidermis.
Effect of serum on the intracellular pH of BALB/c‐3T3 cells: Serum deprivation causes changes in sensitivity of cells to serum
Authors: Martinez R. , Gillies R. J. , Giuliano K. A.
Published: February 2005

One of the earliest responses of quiescent mammalian cells to the addition of serum is an increase in intracellular pH (pHin). This pHin change is generally believed to be due to an increased activity of Na+/H+ exchange. A number of investigators have observed steady‐state differences in pHin between cells in the presence and absence of serum. However, no one has examined differences in pHin regulation that may exist between cells chronically exposed to, or deprived of serum. In this study, we investigated the effects of serum deprivation to identify those components of pHin regulation that were associated with quiescence. To do this, we examined pHin in cells growing chronically in 10% serum as well as in cells that were either acutely (1.5–2 hr) or chronically (48 hr) deprived of serum. Intracellular pH was monitored using the fluorescence of intracellularly loaded pyranine dye. Our results indicate that the resting pHin values of chronically or acutely serum‐deprived cells were not significantly different from each other yet, in both cases, were lower than those observed in cells exposed to 10% serum. Furthermore, we observed significant increases in pHin of both acutely or chronically serum‐deprived cells in response to the addition of serum at various concentrations, in the presence of 24 mM bicarbonate. Chronically serum‐deprived cells had slightly smaller responses and were more sensitive to lower concentrations of serum than were acutely deprived cells. Therefore, our data suggest that long‐term serum deprivation affects the magnitude and sensitivity of pHin to serum stimulation and causes the loss of some form of pHin regulatory mechanism(s).
Enhancement of transformed cell growth in agar by serine protease inhibitors
Authors: Cook Jeffry R. , Chen Jan‐Kan
Published: February 2005

We investigated the effects of three serine protease inhibitors (leupeptin, soybean trypsin inhibitor, and aprotinin) on the serum‐free growth of two transformed cell lines in soft agar. Aprotinin markedly enhanced the growth of rat embryo fibroblasts that had been transformed by polyoma middle T antigen (PyMLV‐REF52), while having only a slight effect on the colonial growth of SV40 transformed Balb/c 3T3 cells (SV3T3‐Aga). Leupeptin and soybean trypsin inhibitor, on the other hand, significantly enhanced the growth of SV3T3‐Aga cells while having little effect on PyMLV‐REF52 growth. We observed no stimulatory effect of any of the protease inhibitors on serum‐free monolayer growth. Under conditions of excess aprotinin, PyMLV‐REF52 cells were found to be unresponsive to epidermal growth factor (EGF) at a concentration that would normally stimulate agar colony growth. However, aprotinin was not capable of supporting colony formation with transforming growth factor‐beta. These results indicate that aprotinin acts primarily as a protease inhibitor in spite of its structural homology to EGF and that EGF may promote the soft agar growth of these cell lines either by inhibiting proteolysis directly or by enhancing the synthesis of a serine protease inhibitor.
Growth‐dependent subcellular redistribution of protein kinase C in cultured porcine aortic endothelial cells
Authors: Uratsuji Yuko , Dicorleto Paul E.
Published: February 2005

We have previously observed major differences in the phosphorylation of membrane proteins in sparse, proliferating versus confluent, quiescent pig aortic endothelial cells (EC) (Kazlauskas and DiCorleto, 1987). In the present study we examined whether EC growth state can influence the activity of a specific phosphorylating enzyme, protein kinase C (PKC) in cytosolic and membrane fractions of pig aortic EC. Levels of PKC were measured using two methods: (1) Ca2+ and phospholipid‐dependent phosphorylation of exogenous histones using gamma‐labeled [32P]ATP, and (2) [3H]phorbol‐12, 13‐dibutyrate (PDBu) binding activity. The total amount of PKC activity in the quiescent versus proliferating cells was similar but the percentage of PKC activity in the membrane fraction correlated with the proliferative index of the cells: confluent, quiescent cultures exhibited a majority of PKC activity in the cytosolic fraction (67%), whereas sparse, proliferating cultures contained principally membrane‐bound PKC (70%). We also examined the role of PKC in the mitogenic response of pig aortic EC to fetal calf serum. Following serum stimulation of sparse, serum‐deprived pig aortic EC, PKC activity redistributed from the cytosolic to the membrane fraction in a rapid process that correlated with subsequent DNA synthesis. A potent activator of PKC, 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), induced a minimal mitogenic response in pig aortic EC when added alone but acted synergistically with low concentrations of fetal calf serum to greatly stimulte DNA synthesis. Furthermore, pig aortic EC treated with TPA for 24 h to down‐regulate PKC exhibited only 25% of the serum‐stimulated mitogenic activity of control cultures. These results suggest a role for PKC activation and translocation in the proliferation of pig aortic EC.
Recombinant gibbon interleukin‐3 stimulates megakaryocyte colony growth in vitro from human peripheral blood progenitor cells
Authors: Mazur Eric M. , Cohen Janet L. , Bogart Lee , Allan Mufson R. , Gesner Thomas G. , Yang Yu‐Chung , Clark Steven C.
Published: February 2005

Gibbon interleukin‐3 (rlL‐3) has recently been cloned and found to have a high degree of homology with the human IL‐3 molecule. In this investigation, we evaluated the effects of gibbon rlL‐3 on normal human peripheral blood megakaryocyte progenitor cell growth in vitro. Gibbon rlL‐3 exhibited substantial megakaryocyte colony stimulatory activity (Meg‐CSA), supporting peak colony numbers at a concentration of 1 U/ml. Megakaryocyte colony growth induced by rlL‐3 reached 58% of the maximum achieved with the active, Meg‐CSA‐containing protein fraction of aplastic canine serum. Increasing gibbon rlL‐3 concentrations also stimulated a 4–5‐fold increase in megakaryocyte colony size and resulted in a decrease in geometric mean megakaryocyte ploidy. Ploidy values fell from 8.5N ± 1.4 (±SEM) at an rlL‐3 concentration of 0.1 U/ml to a minimum of 2.9N ± 0.3 at 10 U/ml. In the presence of rlL‐3 at 1.0 U/ml, megakaryocyte colony growth was linear with cell plating density and the regression line passed approximately through the origin. The effects of rlL‐3 on megakaryocyte colony growth were independent of the presence of T‐lymphocytes in the cultures. Cross‐species evaluation of murine and gibbon lL‐3 indicated that its bioactivity is species restricted. Murine lL‐3 did not support colony growth from human megakaryocyte progenitors and gibbon rlL‐3 showed no activity in stimulating acetylcholinesterase production by murine bone marrow cells. Gibbon rlL‐3 is a potent stimulator of the early events of human megakaryocyte progenitor cell development promoting predominantly mitosis and early megakaryocytic differentiation.
Cells with marrow and spleen repopulating ability and forming spleen colonies on day 16, 12, and 8 are sequentially ordered on the basis of increasing rhodamine 123 retention
Authors: Ploemacher R. E. , Brons N. H. C.
Published: February 2005

Mouse bone marrow cells (BMC) were subjected to countercurrent centrifugal elutriation and subsequently separated on the basis of light scatter and fluorescence intensity after being labeled with the supravital dye Rhodamine 123 (Rh‐123). The sorted cells were then assayed for their in vivo spleen colony‐forming ability (day −8, −12, and −16 CFU‐S) and their ability to repopulate the bone marrow or spleen over a 13‐day period with CFU‐S‐12, CFU‐GM, or nucleated cells. Cells with marrow repopulating ability (MRA), as measured by the ability of the sorted cells to repopulate the marrow with secondary CFU‐S‐12 or CFU‐GM, had low affinity for Rh‐123. These cells showed minimal spleen colony‐forming ability, and the ratio of MRA to CFU‐S‐12 in this preparation was 309. Cells with spleen repopulating ability (SRA), CFU‐S‐16, CFU‐S‐12, and CFU‐S‐8 retained increasing amounts of Rh‐123, respectively, and CFU‐S‐8 were almost exclusively found among cells with high Rh‐123 affinity. These cells also included about half of all day‐12 CFU‐S, and the ratio of MRA to day‐12 CFU‐S was 0. The results show that MRA cells, SRA cells, CFU‐S‐16, CFU‐S‐12, and CFU‐S‐8 can be sequentially ordered on the basis of increasing mitochondrial activity. The data also demonstrate for the first time, and without the application of negative selection by the use of cytostatic agents, that MRA cells are a separate class of primitive hemopoietic stem cells that fully meet the criteria of pre‐CFU‐S.
Extent and properties of nonbulk “bound” water in crystalline lens cells
Authors: Cameron I. L. , Contreras E. , Fullerton G. D. , Kellermayer M. , Ludány A. , Miseta A.
Published: February 2005

Crystalline lenses provided good material to study and measure the properties of cellular water. Different methods were used to establish the extent and properties of nonbulk water in mammalian lenses. These methods include: NMR titration analysis, a test of the osmotic properties, a test of dye exclusion In lenses with intact cell membranes and in lenses with disrupted cell membranes, and the water‐holding capacity of lenses subjected to 40,000 × g for 1 hour with intact cell membranes and in lenses with disrupted cell membranes. The data from these methods, as well as other data from the literature, lead to the conclusion that most, if not all, of the water in lens cells (up to 2.2 g water/g dry mass) has motional and osmotic properties that distinguish it from bulk water. These findings call into question the common and convenient assumption that all but a small proportion of cellular water is like that in dilute solution.
Characterization of a high‐molecular‐weight protein immunoprecipitated by platelet‐derived growth factor antisera
Authors: Graves Dana T. , Antoniades Harry N.
Published: February 2005

We previously demonstrated that a high‐molecular‐weight glycoprotein could be immunoprecipitated from metabolically labeled U‐2 OS cells with platelet‐derived growth factor (PDGF) antiserum and that it appears to be derived from a different precursor than is the 30 kD PDGF‐like mitogen produced by these cells. These findings were unexpected, since the molecular weight of this glycoprotein is too large to be encoded by the PDGF structural genes. From experiments with metabolically labeled U‐2 OS human osteosarcoma, fibroblasts, and NRK cells, we report here that a 185 kD protein immunoprecipitated with PDGF antiserum has the following characteristics. (1) It is a PDGF binding protein that is unrelated to α2‐macroglobulin. (2) It is phosphorylated in response to PDGF stimulation. (3) It is immunoprecipitated by phosphoryrosine antibodies. (4) It is not a substrate of epidermal growth factor‐induced tyrosine kinase activity. These studies indicate that high‐molecular‐weight proteins immunoprecipitated by antiserum to PDGF represent a complex between PDGF and a binding protein capable of being phosphorylated by a PDGF‐induced tyrosine kinase. These characteristics are identical to those of the PDGF receptor.
DNA replication fork progression rate and temporal organization of S phase in normal epidermis and in basal cell carcinoma
Authors: Galand Paul , Heenen Michel
Published: February 2005

The double‐pulse labeling technique for DNA fiber autoradiography was applied to epidermal cells from normal human skin and from human basal cell carcinoma (BCC). We aimed to measure the size and replication rate of the replication unit (RU) for both types of cell and to account, from these results, for our previous observation of a near doubling of S‐phase duration in BCC, compared with normal skin. The mean RU size was 76 ± 4 m̈m in BCC, not significantly different from the 68 ± 6 μm value found in normal skin, so the mean of those two values (i.e., 72 μm), was used in further calculations. The rate of replication fork progression was 0.59 ± 0.005 μmlm/min in the normal epidermis and 0.33 ± 0.03 μm/min in BCC, corresponding to a replication time of the average RU equal to 61 min and 109 min, respectively. Thus, with an unchanged RU size in BCC, the observed 1.8‐fold decrease in the rate of fork progression in the tumor can account entirely for our previous observation of a 1.8‐fold increase in S‐phase duration in this tumor, without requiring the assumption of any change in the temporal organization of DNA synthesis in the malignant cells. Considering S phase as an ordered process in which a major part, if not all, of the genome replicates at genetically determined times, we suggest that the clusters of replication units are, in turn, organized into temporally defined “sets.” These sets are composed of all the clusters (whatever their chromosomal location) that are programmed to initiate replication during the same fraction of the S period. This hypothesis implies that DNA synthesis in a given set is triggered by some event coupled to progression of replication in the immediately preceding set. Based on a S‐phase duration of 10.2 hours in normal skin and of 19.2 hours in BCC (our previous data), and assuming perfect synchrony and homogeneity of the clusters within each set and of each cluster's constitutive RUs, the minimum number of sequentially replicating sets, in both instances, can be estimated as roughly equal to 10.
Dual effect of dihydropyridines on 45Ca uptake induced by the K+ —channel blocker 4‐aminopyridin on mast cells
Authors: Eleno N. , Botana L. , Espinosa J.
Published: February 2005

The 1,4‐dihydropyridines (DHP) are calcium antagonists and represent a new class of drugs which act by a selective inhibition of Ca++ influx through voltage‐operated calcium channels. We report the effect of nifedipine (Bay A 1040), nisoldipine (Bay K 5552) ad nitrendipine (Bay E 5009) on the histamine release and on the 45Ca uptake promoted by 4‐aminopyridin in mast cells. These cells treated with DHP (10−12−10−3 M) activated the secretory response in a dose‐dependent manner in the range of concentrations 10−6−10−3 M did not significantly inhibit the secretion. 4‐Aminopridin, a known K+‐channel blocker, induced 45Ca uptake. Pretreatment of mast cells with DHP prior to 4‐aminopyridin stimulation inhibited or stimulated 45Ca uptake depending on concentration; thus, concentrations of DHP below 10−12 of nitrendipine and 10−9 for nisoldipine and nifedipine were inhibitory, while higher doses potentiated 45Ca uptake. These results demonstrate a diversity of pharmacological effects of DHP on mediator secretion and 45Ca uptake in mast cells and throw into question their only properties as Ca++ antagonists.
Effect of endothelial‐cell‐conditioned medium on proteoglycan synthesis in cultured smooth muscle cells from pig aorta
Authors: Berrou Eliane , Breton Monique , Deudon Elisabeth , Picard Jacques
Published: February 2005

The effect of porcine endothelial‐cell‐conditioned medium on proteoglycan synthesis by pig aorta smooth muscle cells was studied under serum‐free conditions. Maximal stimulation of [35S]‐sulfate incorporation (50%) into medium‐secreted and cell layer proteoglycans was observed after 20 min and 4 h incubation, respectively. This stimulation can be explained neither by increased secretion nor by oversulfation of medium‐secreted [35S]‐labeled proteoglycans. Those [35S]‐proteoglycans secreted (for 24 h) in the presence of endothelial cell‐conditioned medium were characterized by a higher hydrodynamic size than those secreted in the presence of control medium, without modification of glycosaminoglycan chain length. Agreement between the stimulation of incorporation of [35S]‐sulfate into glycanic chains (50.1%) and [14C]‐serine residues associated with glycosaminoglycans (49.9%) involved an increase in the number of glycanic chains linked to protein cores. The lesser stimulation of [14C]‐serine incorporation into secreted proteins (18%) suggested that stimulation of glycosaminoglycan synthesis was not the direct consequence of enhanced protein synthesis. Proteoglycan synthesis was studied in the presence of para‐nitrophenyl‐b̃‐D‐xyloside. Fractionation of medium‐secreted [35S]‐proteoglycans and xyloside‐initiated glycosaminoglycans revealed that stimulation of [35S]‐glycosaminoglycan synthesis by endothelial‐cell‐conditioned medium required a protein core acceptor for glycanic chain initiation. Our results suggest that the factor(s) secreted by endothelial cells are able to modify smooth muscle cell proteoglycan synthesis by stimulating the first step of protein core glycosylation. This stimulation was accompanied by an increase in proteoglycan hydrodynamic size.
Ca‐dependent slow action potentials in human skeletal muscle
Authors: Siri L. Nicola , Dubrovsky A. L. , Uchitel O. D.
Published: February 2005

Slow Ca‐action potentials (CaAP) were studied in normal human skeletal muscle fibers obtained during surgery (fibers with both ends cut). Control studies also were carried out with intact as well as cut rat skeletal muscle fibers. Experiments were performed in hypertonic Cl‐free saline with 10 or 84 mM Ca and K‐channel blockers; muscles were preincubated in a saline containing Cs and tetraethylammonium. A current‐clamp technique with two intracellular microelectrodes was used. In human muscle, 14.5% of the fibers showed fully developed CaAPs, 21% displayed nonregenerative Ca responses, and 64.5% showed only passive responses; CaAPs were never observed in 10 mM Ca. In rat muscle, nearly 90% of the fibers showed CaAPs, which were not affected by the cut‐end condition. Human and rat muscle fibers had similar membrane potential and conductance in the resting state. In human muscle (22–32± C, 84 mM Ca), the threshold and peak potential during a CaAP were + 26 ± 6 mV and + 70 ± 3 mV, respectively, and the duration measured at threshold level was 1.7 ± 0.5 sec. In rat muscle, the duration was four times longer During a CaAP, membrane conductance was assumed to be a leak conductance in parallel with a Ca and a K conductance. In human muscle (22–32° C, 84 mM Ca, 40 μS, fiber diameter), values were 0.4 ± 0.1 μS, 1.1 ± 0.7 μS, and 0.9 ± 0.4 μS, respectively. Rat muscle (22–24° C, 84 mM Ca) showed leak and K conductances similar to those found in human fibers. Ca‐conductance in rat muscle was double the values obtained in human muscle fibers.
Transferrin endocytosis and iron uptake in developing myogenic cells in culture: Effects of microtubular and metabolic inhibitors, sulphydryl reagents and lysosomotrophic agents
Authors: Sorokin L. M. , Morgan E. H. , Yeoh G. C. T.
Published: February 2005

The experiments described in this study were designed to investigate receptor‐mediated endocytosis of transferrin and its role in iron uptake by cultured chick presumptive myoblasts (dividing and non‐dividing) and myotubes. The effects of a variety of inhibitors on the internalization of transferrin and iron were investigated and three main effects were found: (i) sulphydryl reagents and microtubular inhibitors reduced the rate of transferrin and iron internalization to similar degrees, (ii) metabolic inhibitors reduced the rate of iron uptake more than that of transferrin endocytosis, and (iii) lysosomotrophic agents almost completely abolished iron accumulation by the cells without any effect on the rate of transferrin internalization. The results suggest that metabolic energy is required not only for the endocytosis of transferrin but also for subsequent steps in the iron uptake process, and that iron release from transferrin occurs in acidified endosomes. Overall, these experiments show that all or virtually all of the iron taken up by developing muscle cells from transferrin occurs as a consequence of receptor‐mediated endocytosis of the protein.
Stable variant of LM fibroblast defective in fluid‐phase but competent in receptor‐mediated endocytosis
Authors: Ryser Hugues J.‐P. , Li Wande , Mandel Richard , Shen Wei‐Chiang
Published: February 2005

The F‐40 cell line, a stable variant of LM fibroblasts selected for its resistance to polyethylene glycol (PEG)‐induced fusion (Roos and Davidson: Somatic Cell and Molecular Genetics 6:381‐‐391, 1980), has a decreased capacity to internalize fluid‐phase markers and nonspecifically surface‐bound macromolecules. It is not defective in exocytosis since, after a short sucrose pulse, it releases the same fraction of ingested sucrose into the medium as does the parental line. F40 cells have a normal capacity to carry out receptor‐mediated endocytosis, as tested with 125I‐alpha‐2 macroglobulin (alpha‐2 MG) and 125I‐transferrin (Tf), and to recycle Tf receptor to the cell surface. These data demonstrate that receptor‐mediated and non‐receptor mediated endocytosis are distinct processes that can be altered independently. Of the many membrane fusions occurring in the course of endocytosis, the only one that appears associated with the defect in cell fusion characteristic of F40 cells is the formation of primary endocytotic vesicles engaged in non‐receptor‐mediated internalizations.
Adenosine induction of rapid catabolism of adenine ribonucleotides and independent elevation of the ATP content in quiescent mouse fibroblasts
Authors: Klenow Hans , Østergaard Erik
Published: February 2005

The influence of adenosine on the ribonucleotide metabolism in quiescent BALB/c 3T3 cells was studied. The cellular adenine ribonucleotides were labelled by pretreating the cells with [2−3H]‐adenine. After addition of adenosine to the cell cultures, the amount and radioactivity of the cellular purine ribonucleotides and the radioactivity of the purine compounds in the medium were determined. It appeared that adenosine gave rise both to rapid catabolism of adenine ribonucleotides with inosine 5′‐monophosphate (IMP) as an intermediate and to expansion of the cellular adenosine 5′‐triphosphate (ATP) pool. The maximal rates and the apparent activation constants for the two processes have been determined. Experiments with varying concentrations of coformycin (an inhibitor of adenosine 5′‐monophosphate [AMP] deaminase and adenosine deaminase) and of 5′‐amino‐5′‐deoxyadenosine (an inhibitor of adenosine kinase), respectively, showed that each compound may almost completely inhibit the adenosine‐induced catabolism. This effect can be obtained under conditions where there was little or no effect by the two inhibitors on the rate of expansion of the cellular ATP pool. These results may best be explained by assuming that the process of expansion of the ATP pool is independent of the induced catabolism of adenine ribonucleotides, even though both processes seem to depend on the phosphorylation of adenosine to AMP. The total increase in the pool size of ATP and of guanosine 5′‐triphosphate (GTP), both caused by adenosine, seems not to have regulatory effect on adenine ribonucleotide catabolism.
Ethanolamine and choline transport in cultured bovine aortic endothelial cells
Authors: Lipton Beth A. , Yorek Mark A. , Ginsberg Barry H.
Published: February 2005

The transport of the polar head groups, ethanolamine and choline, was examined in cultured bovine aortic endothelial cells. Both ethanolamine and choline are taken up by high‐ and low‐affinity systems. The K'm and V'max for the Na+‐dependent, high‐affinity ethanolamine and choline transport system are 3.0 and 3.0 μM and 5.4 and 7.3 pmol/mg protein/min, respectively. Ethanolamine and choline competitively influence one another's transport as the presence of 50 μM ethanolamine increases the K'm but not the V'max of choline uptake. Likewise, 50 μM choline increases the K'm but not the V'max of ethanolamine transport. The concentration of ethanolamine that inhibits maximal velocity of 5 μM choline by 50% is 9.7 μM, while 12 μM choline inhibits 5 μM ethanolamine maximal velocity by 50%. Uptake of both head groups is only partially Na+‐dependent and is inhibited similarly by 2‐methylethanolamine and 2,2‐dimethyl‐ethanolamine at all concentrations examined. Hemicholinium‐3, a classic inhibitor of high‐affinity, Na+‐dependent choline transport, reduces both ethanolamine and choline accumulation in a concentration‐dependent fashion, but has a greater effect on choline transport at higher concentrations. The major portion of these data is consistent with our hypothesis that the uptake of physiological concentrations of ethanolamine and choline may occur through the same transport system. However, the results of the effect of hemicholinium‐3 and the extent of Na+‐dependency of choline and ethanolamine uptake could be interpreted as meaning that separate transport systems for choline and ethanolamine exist which cross react or that a single transport system exists which has separate active sites for the two compounds.
Cachectin/TNF kills or inhibits the differentiation of 3T3‐L1 cells according to developmental stage
Authors: Kawakami Masanobu , Watanabe Nobumasa , Ogawa Hiroko , Kato Arata , Sando Hiroyuki , Yamada Nobuhiro , Murase Toshio , Takaku Fumimaro , Shibata Seiichi , Oda Toshitsugu
Published: February 2005

The effects of cachectin/tumor necrosis factor (TNF) on growth and differentiation of 3T3‐L1 cells were examined. This fibroblastic cell line can be induced to differentiate into a mature cell type having the biochemical and morphological characteristics of normal adipocytes. At various stages of growth and differentiation, 3T3‐L1 cells were exposed to 2.5 × 10−16 to 2.5 × 10−8 M (4.2 fg/ml to 420 ng/ml = ca. 1.2 × 10−14 to 1.2 × 10−6 U/ml) recombinant human cachectin/TNF for 24 hr, after which cytotoxicity or differentiation was evaluated. During log‐phase cell growth, cachectin/TNF had no significant effect on cell viability, and the preadipocytic cells were also resistant to the cytotoxic effect of cachectin/TNF at the contact‐inhibited confluent stage. However, when cachectin/TNF was added to the cells during induced differentiation, only 20% of the cells survived. After differentiation into adipocytes, cells regained their resistance to cachectin/TNF‐induced cytotoxicity. Cachectin/TNF also markedly affected the differentiation of 3T3‐L1 cells into adipocytes. When cells in the confluent phase of growth were exposed to cachectin/TNF for 24 hr, their subsequent hormone‐induced differentiation to adipocytes was inhibited. Like cachectin/TNF, IL‐1 also induces suppression of lipoprotein lipase and enhances lipolysis in differentiated 3T3‐L1 adipocytes; however, in contrast to cachectin/TNF, IL‐1 had no effect on the viability or differentiation of pre‐adipocyte 3T3‐L1 cells. These results indicate that the cytotoxic action of cachectin/TNF varies in the same cell type depending on the stage of growth or differentiation. The results also imply that cachectin/TNF may play a normal role in controlling the differentiation of certain types of cells in vivo including adipocyte lineages.
Stochastic model for mast cell proliferation in culture of murine peritoneal cells
Authors: Kobayashi Toshimi , Nakahata Tatsutoshi
Published: February 2005

We recently identified two types of mast cell colonies derived from murine peritoneal cells: type 1 and type 2. Type 1 mast cell colonies consisted of berberine sulfate(+)‐ safranin(+) connective tissue‐type mast cells (CTMC) and were derived from mature CTMC in the heaviest fraction obtained by Percoll density gradient centrifugation. In contrast, type 2 mast cell colonies consisted of alcian blue(+)‐ berberine sulfate(−)‐ safranin(−) mucosal mast cells (MMC) and were derived from immature progenitors in low density fractions. We replated a total of 60 type 1 and 60 type 2 mast cell colonies and examined their capability for producing secondary colonies. Although all of the primary colonies yielded secondary colonies, the replating efficiencies of individual colonies varied over a wide range.

Cumulative distributions of secondary colonies from both type 1 and type 2 primary colonies could be fitted well by gamma distributions obtained by computer simulation. These findings are in agreement with the stochastic model for CTMC‐ and MMC proliferation. Cytological analyses of secondary colonies from primary type 1 colonies revealed heterogeneous distributions of alcian blue(+)‐ safranin(−)‐ berberine sulfate(−) mast cells, suggesting that transdifferentiation from mature CTMC to safranin(−)‐ berberine sulfate(−) mast cells is also governed by stochastic mechanisms.
Plasma membrane lipid organization and the adherence of differentiating lymphocytes to macrophages
Authors: Del Buono Brian J. , White Scott M. , Williamson Patrick L. , Schlegel Robert A.
Published: February 2005

Changes in the packing of phospholipids in the plasma membrane of lymphocytes occur during differentiation within primary and secondary lymphoid organs. As they differentiate, lymphocytes interact with a variety of reticuloendothelial cells, including macrophages. To investigate a possible relation between these two phenomena, the strength of the interactions between lymphocytes and macrophages was measured in vitro as a function of the tightness of packing of phospholipids on the lymphocyte surface. Strength of adherence was measured by the ability of lymphocytes to remain adherent to macrophages when subjected to increasing centrifugal forces. Phospholipid packing was assessed using the fluorescent lipophilic probe merocyanine 540 (MC540), which preferentially binds to bilayers in which the lipids are more loosely packed. Three subpopulations of murine thymocytes were resolved with respect to strength of adherence to peritoneal or thymic macrophages. To determine whether these subpopulations corresponded with the three classes of cells distinguishable by MC540 fluorescence, populations enriched for staining or non‐staining cells, and cells sorted on the basis of MC540 fluorescence intensity, were examined. The least fluorescent cells were the least strongly adherent; the most fluorescent cells were the most strongly adherent; and cells of intermediate fluorescence had intermediate adherence. When splenic lymphocytes were examined with respect to adherence to peritoneal or splenic macrophages, similar patterns of fluorescence and adherence were seen. These results suggest that the organization of the plasma membrane lipid bilayer of lymphocytes may be involved in their interactions with macrophages during primary and secondary differentiation. The adherence signal for lymphocytes thus may be similar to that proposed for other blood cells.
Modulation of growth factor induced fiber outgrowth in rat pheochromocytoma (PC12) cells by a fibronectin receptor antibody
Authors: Schwarz Martin A. , Brown Patricia J. , Eveleth David D. , Bradshaw Ralph A.
Published: February 2005

Rat pheochromocytoma PC12 cells respond to the binding of nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) by extending neurites in a manner resembling sympathetic neurons. This response requires cell attachment to an appropriate substratum (Fujii et al., J. Neurosci., 2:1157, 1982); attachment factors which function in this capacity include the adhesive proteins fibronectin and laminin. Incubating PC12 cells with a ployclonal antiserum directed against a putative 140‐kDa fibroblast cell surface fibronectin receptor (anti‐gp140) perturbed spreading but not attachment of the cells to fibronectin and laminin substrates. However, in the presence of anti‐gp 140 or its Fab fragments, NGF‐stimulated neurite outgrowth was dramatically reduced. The antibody also caused a retraction of previously extended neurites. SDS‐PAGE analysis of immunoprecipitates of PC12 cells surface labeled with 125I identified a prominent 120–140‐kDa band, suggesting that the site of anti‐gp 140 action in PC12 cells is also through a fibronectin receptor.