bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of the cellular mechanisms responsible for platelet‐derived growth factor induced alterations in cytoplasmic vinculin distribution
Authors: Herman B. , Harrington M. A. , Olashaw N. E. , Pledger W. J. 
Published: February 2005

AbstractExposure of quiescent density arrested BALB/c‐3T3 cells (clone A31) to platelet‐derived growth factor (PDGF;6–12 ng/ml) results in a rapid, reversible, time‐and dose‐dependent removal of vinculin from adhesion plaques (Herman and Pledger, 1985). Potential cellular mechanisms involved in PDGF‐induced removal of vinculin from adhesion plaques were examined. Removal of vinculin from adhesion plaques following exposure of cells to PDGF was temperature dependent, occurred in many fibroblast cell lines, and could be mimicked by 12‐tetradecanoyl phorbol‐13‐acetate (TPA; 5‐125 nM) or melittin (0.35 μM). Unlike the effect of PDGF, TPA‐or melittin‐induced vinculin disruption was not reversible. The removal of vinculin from adhesion plaques was inhibited by trifluoroperazine (TFP; 2.5 μM). 8‐(N,N‐diethylamino) octyl‐3,4,5‐trimethoxy benzoate (TMB‐8; 1.0 μM), mepacrine (220 μM), n‐α‐p‐tosyl‐L‐lysine chloromethylketone (TLCK; 100 μM), phenylmethoxysulphonylfluoride (PMSF; 500 μM), and e‐aminocaproic acid (e‐ACA; 100 μM); however, amiloride (100 μM), A23187 (20 μM), and chloroquine (1 mM) were unable to inhibit this effect. Melittin disruption of vinculin was inhibited by (in order of decreasing effectiveness) mepacrine > TMB‐8 > TFP > leupeptin > PMSF, whereas A23187 and amiloride had no effect. The return of vinculin to adhesion plaques following PDGF treatment required de novo mRNA transcription and protein synthesis and was associated with PDGF‐stimulated synthesis of vinculin.The observation that both PDGF‐ and melittin‐induced removal of vinculin from adhesion plaques is inhibited by mepacrine suggests that phospholipase activation may be an early and important step in PDGF‐induced disruption of vinculin from adhesion plaques. In addition, TFP, TMB‐8 and protease inhibitor inhibition of both the PDGF and melittin effects on vinculin distribution, coupled with the finding that TPA can mimic the PDGF or melittin response, suggests that Ca2+, calmodulin, protein kinase C, and /or proteolysis may play an important role(s) in the removal of vinculin from adhesion plaques following PDGF addition. The lack of effect of A23187 addition on vinculin distribution suggests that alterations in cellular Ca2+ is necessary but not sufficient for vinculin removal from adhesion plaques.
Class specificity of transferrin as a muscle trophic factor
Authors: Shimo‐Oka Tadashi , Hagiwara Yasuko , Ozawa Eijiro 
Published: February 2005

AbstractThe specificity of transferrin (Tf) in its exertion of a growth‐promoting effect on myogenic cells was examined using serum Tfs from chick, dove, goose, turkey, bovine, horse, rabbit, rat, and swine and primary myogenic cells from chick, duck, quail, rabbit, and rat, and rat L6 cells. Avian Tfs were effective on avian cells but not on mammalian cells, while mammalian Tfs were effective on mammalian cells but not on avian cells. Dove and bovine Tfs were exceptional in that they were effective on some class‐heterologous cells at higher concentrations and less so or completely ineffective on some class‐homologous cells. Despite these exceptions, however, the relationship between Tfs and cells can be summarized as a class specificity.To exert the growth‐promoting effect, it is prerequisite for Tf to bind its specific receptor on the cell surface. Using quail and L6 cells, we found that the binding of 125I‐labeled chick and rat Tfs to the respective receptors of quail and L6 myoblasts was competitively inhibited by other kinds of effective Tfs, but not by ineffective ones.We conclude that the class specificity in myotrophic activity of Tf is due to the affinity between Tf and Tf receptor.
Influence of retinoic acid on the ultrastructure and hyaluronic acid synthesis of adult human epidermis in whole skin organ culture
Authors: Tammi Raija , Tammi Markku 
Published: February 2005

AbstractNormal human skin was maintained in organ culture under chemically defined conditions. All‐trans retinoic acid was added to the culture medium at the final concentration of 5 μmol/l. After 5 days in culture samples were either harvested for electron microscopy or labeled with 3H‐glucosamine for 24 h. After labeling, epidermis was separated from dermis and both tissue compartments were analyzed for the content of 3H‐labeled glycosaminoglycans (GAGs) using CPC‐precipitation and thin layer chromatography after enzymatic degradation into specific disaccharides. Retinoic acid caused a marked change in the epidermal tissue architecture. The epidermal cells were flattened and contained fewer desmosomes and tonofilaments than control explants. Retinoic acid induced accumulation of fine granular material in the intercellular spaces in the upper, and less dense, flocculent material in the lower epidermis. The analysis of 3H‐glycosaminoglycans showed that in the epidermis retinoic acid elevated the amount of labeled hyaluronate by 70%, whereas sulfated GAGs were not significantly increased. In dermis the incorporation of 3H‐glucosamine into neither hyaluronate nor sulfated GAGs was stimulated by the retinoic acid. It is concluded that retinoic acid significantly modifies the differentiation of normal adult human epidermis by decreasing cytoskeleton components and by inducing the synthesis of new intercellular material, at least a part of which is hyaluronic acid. As a consequence, the cohesion between the epidermal cells was apparently weakened.
Effect of amino acid levels on matrix vesicle formation by epiphyseal growth plate chondrocytes in primary culture
Authors: Ishikawa Yoshinori , Chin Jia E. , Schalk Elaine M. , Wuthier Roy E. 
Published: February 2005

AbstractThe effect of varying the amino acid concentrations of the culture medium on matrix vesicle formation was studied in primary cultures of chicken epiphyseal growth plate chondrocytes grown in Dulbecco's modified Eagle's medium (DME) supplemented with 10% fetal bovine serum (FBS). Decreasing the levels of free amino acids in the culture medium to levels of one‐half, one quarter, and one eighth of the values normally present in DME caused a progressive decline in matrix vesicle (MV) formation. Increasing the level in the culture medium of those amino acids that are enriched in extracellular fluid (ECF) of growth plate cartilage significantly increased formation of matrix vesicles (MV), as assayed by the alkaline phosphatase (AP) activities present in high‐speed sediments from spent culture media. However, adjusting the levels of all amino acids to match those of the ECF produced the greatest stimulation of MV formation. Of the amino acids that are notably enriched in ECF, glutamate (GLU), alanine (ALA), serine (SER), asparagine (ASN), and taurine (TAU) individually enhanced MV production, whereas proline (PRO), glycine (GLY), and aspartate (ASP) had essentially no effect. The simple combination of ECF levels of ALA and GLU resulted in a stimulation of MV formation equal to that observed when the eight aforementioned amino acids were elevated to ECF levels. Other combinations of ASP and GLY, or of TAU, SER, and ASN showed some stimulation, but at a lower level. Increasing the amino acid concentrations, alone or in combination, also increased the levels of cellular AP, and to a lesser extent cellular protein. While increases in cellular AP were generally correlated with increased formation of AP‐rich MV, this was not uniformly true. These results indicate that in addition to hormones and growth factors, nutritional factors such as the levels of amino acids are also critical for normal phenotypic expression, growth, and matrix formation by epiphyseal chondrocytes.
Increased free calcium in endothelial cells under stimulation with adenine nucleotides
Authors: Lückhoff Andreas , Busse Rudi 
Published: February 2005

AbstractThe release of vasodilating substances from the vascular endothelium has been postulated to depend on a rise in the level of intracellular free calcium (Cai++). We measured Cai++ in intact monolayers of calf endothelial cells, grown in culture, that were loaded with the fluorescent calcium indicator quin 2. Fluorescence (excitation wavelength 340 nm, emission wavelength 492 nm) was calibrated by raising Cai++ to a maximum with the calcium ionophore ionomycin (0.1 μM) and by lowering it to a minimum with ionomycin plus manganese (0.4 mM), which quenches quin 2 fluorescence completely. Loss of fluorescent dye from the cells was calculated from fluorescence at the isosbestic excitation wavelength (365 nm). Resting Cai++ was 71 ± 3 (SEM) nM. ATP (adenosine ‐5′‐triphosphate) raised Cai++ dose‐dependently and reversibly to 458 ± 60 nM at a concentration of 10 μM, and at 0.1 mM to values close to those that occurred under ionomycin. ADP (A‐5′‐PP) and AMP (A‐5′‐P) had smaller effects with a maximal Cai++ of 287 ± 72 nM at 30 μM ADP and 176 ± 17 nM at 0.1 mM AMP. At these concentrations, ADP and AMP attenuated significantly the increase of Cai++ under ATP (10 μM). Adenosine (0.1 or 0.3 mM) and acetylcholine (0.1 to 30 μM) enhanced Cai++ inconsistently, by a maximum of 50 nM. These effects were abolished by theophylline and atropine, respectively. In the absence of extracellular calcium, ATP still raised Cai++, although endothelial responsiveness declined after repetitive stimulations. We conclude that activation of purinergic receptors increases intracellular free calcium in endothelial cells, and that this increase is probably an essential trigger for synthesis of prostacyclin and the labile endothelium‐derived relaxant factor.
Characterization of plasminogen activator from two human renal carcinoma cell lines
Authors: Nelson Norman F. , Cieplak Witold , Dacus Stephen C. , Prager Morton D. 
Published: February 2005

AbstractPlasminogen activator (PA) activity was identified in the conditioned medium of two human renal carcinoma cell lines, Cur and Caki‐1, PA activity of medium, following chromatography on Con A‐Sepharose, was divided into effluent and eluate fractions, the latter obtained after elution with methyl mannoside. The ratio of PA activity in effluent:eluate was 90:10 for Caki‐1 and 60:40 for Cur. The PA of both effluent fractions and the Caki‐1 eluate fraction was of the urokinase (UK) type. Identification rested on molecular weight determination by zymography (major component with Mr 52,000 and a less prominent component of 93,000), lack of binding to fibrin, inhibition by anti‐UK antibodies, and lack of inhibitory effect of anti‐tissue type PA (TPA) antibodies or the Erythrina trypsin inhibitor, which inhibits TPA but not UK. PA of the Cur eluate fraction gave a more complex pattern in that it bound significantly to fibrin (like TPA), was completely inhibited by both anti‐UK and anti‐TPA antibodies, but was unaffected by Erythrina trypsin inhibitor. These results raise the possibility of an unusual PA‐like enzyme that immunologically cross reacts with anti‐UK and anti‐TPA. Most of the PA of both cell lines was secreted in a latent form that could be activated by trypsin treatment. The latency appears to result largely from secretion of urokinase proenzyme, which is consistent with the Mr 52,000 of the major PA species and the insensitivity to diisopropyl fluorophosphate inhibition prior to trypsin activation. However, in addition, a UK binding component was found in the conditioned medium, which produced an Mr 93,000 component by reaction with UK.
Modifications of the adenylate cyclase complex during differentiation of cultured myoblasts
Authors: Morris Stephen A. , Bilezikian John P. 
Published: February 2005

AbstractAlterations in receptor‐independent activation of adenylate cyclase during proliferation and differentiation of L6E9 myoblasts were studied using Mn2+, forskolin, and Gpp(NH)p. Analyses were performed 3,6, and 10 days following subculture, corresponding to onset of proliferation, end of proliferation with start of differentiation, and completion of differentiation, respectively. The apparent activation constant for Mn2+ decreases with the age of the culture; the apparent activation constant for Mg2+ does not. Bimodal activation by Mn2+, i.e., at concentrations greater than 10 mM, results in total adenylate cyclase activity less than the Vmax and occurs exclusively in differentiated cultures. Independent of the presence of Mg2+, forskolin activation occurs with low‐ and high‐affinity constants in differentiated cultures and with a low affinity constant in youngest cultures; intermediate cultures (day 6) demonstrate low‐ and high‐affinity activation only in the presence of high Mg2+. In contrast, the Vmax for forskolin increases with increasing Mg2+ in all culture ages. Although Gpp(NH)p‐dependent adenylate cyclase activation occurs with an apparent activation constant independent of culture age and Mg2+, low Mg2+ fosters bimodal activation by Gpp(NH)p, i.e., above 100 μM nucleotide, total adenylate cyclase activity is less than the Vmax. The loss of stimulatory capacity by high Gpp(NH)p is greatest in differentiated cultures. Additional experiments are presented to substantiate that bimodal activation by Gpp(NH)p is specific. Cholera‐ and pertussis toxin‐dependent ADP ribosylation patterns demonstrate a marked decrease in both Ns and Ni in differentiated cultures. The data suggest that alterations in postreceptor activation of adenylate cyclase during the course of differentiation and proliferation are mediated by guanine nucleotide binding proteins as well as by allosteric cation regulatory units.
Characterization of reticulofibroblastoid colonies (CFU‐RF) derived from bone marrow and long‐term marrow culture monolayers
Authors: Lim B. , Izaguirre C. A. , Aye M. T. , Huebsch L. , Drouin J. , Richardson C. , Minden M. D. , Messner H. A. 
Published: February 2005

AbstractThe maintenance of hemopoietic precursors in long‐term liquid bone marrow cultures (LTBMC) is associated with the presence of an adherent stromal layer composed of heterogeneous cell populations. We have used a culture assay to promote the growth of one of its cellular components and characterize its properties. Freshly obtained bone marrow cells and cells derived from the adherent layer of LTBMC were grown in methylcellulose‐clotted plasma in the presence of phytohemagglutinin‐stimulated leukocyte‐conditioned medium (PHA‐LCM), hydrocortisone (HC), and citrated normal human plasma. Both sources contained cells (CFU‐RF) that gave rise to colonies of cells with a reticulofibroblastoid appearance. In the presence of HC, most colonies contained lipid‐laden cells. Colonies could be further propagated as adherent layers when transferred into liquid cultures. These cells produced laminin, fibronectin, and collagen types I, III, IV, and V. They were negative for Von Willebrand factor VIII. The ability to synthesize laminin and collagen type IV distinguished these cells from a population of previously described bone marrow fibroblasts (CFU‐F). The relationship of CFU‐RF to hemopoietic precursors was investigated using patients with chronic myeloid leukemia and bone marrow transplant recipients. Cells within CFU‐RF‐derived colonies were uniformly negative for the Philadelphia chromosome, thus making it unlikely that they belonged to the malignant hemopoietic clone. CFU‐RF‐derived colonies in bone marrow transplant recipients were found to be exclusively of host origin. Both observations support the view that CFU‐RF is not part of the repertoire of hemopoietic stem cells.
Prostaglandin F2α and E1 regulation of proliferation in primary cultures of rabbit endometrial cells
Authors: Orlicky David J. , Lieberman Rita , Gerschenson L. E. 
Published: February 2005

AbstractThe study of growth of endometrial cells is of importance in reproductive biology. Several factors and hormones are thought to play important roles in the control of growth. Prostaglandin F2α (PGF2α) causes an increase in both tritiated thymidine ([3H]Tdr) incorporation into DNA and in the cell number of primary cultures of rabbit endometrial cells cultured in a serum‐free, chemically defined media. Prostaglandins F1α, E1, E2, A2, and B2 and arachidonic acid (all tested at 10−7 M) do not affect [3H]Tdr incorporation as compared to control cultures. The increase in [3H]Tdr incorporation into DNA in response to PGF2α stimulation is concentration‐dependent (optimal ∼3 × 10−7 M) and is seen starting ∼9 hr poststimulation. Both prostaglandin E1 (PGE1) and prostaglandin E2 (PGE2), but not PGs F1α, I2, A2, B2, their parent molecules, or related molecules, antagonize and can completely block the PGF2α‐induced increase in [3H]Tdr incorporation into DNA. This antagonism is seen both when the cells are pretreated with PGE1 prior to the PGF2α stimulation and when the cells are exposed to both PGE1 and PGF2α simultaneously. Exogenously added 8‐Br‐cAMP mimics the PGE1 antagonism of PGF2α. The PGF2α‐induced increase in [3H]Tdr incorporation is not synergistic, antagonistic, or additive with the [3H]Tdr incorporation increase in response to either estradiol‐17β or epidermal growth factor. The specific effect of PGF2α on primary culture endometrial cell growth and its antagonism by PGE1, PGE2, and 8‐Br‐cAMP are new findings.
Hexose transport in L6 rat myoblasts. I. Rate‐limiting step, kinetic properties, and evidence for two systems
Authors: D'amore Tony , Lo Theodore C. Y. 
Published: February 2005

AbstractThe hexose transport system of undifferentiated L6 rat myoblasts was investigated. 2‐Deoxy‐D‐glucose (2‐DOG) and 2‐deoxy‐2‐fluoro‐D‐glucose (2FG) were used as analogues to investigate the rate‐limiting step of hexose uptake into the cell. Virtually all of the 2‐DOG or 2FG taken up into the cell was found to be in the phosphyorylated form. No significant pool of intracellular free sugar could be detected. This demonstrates that hexose transport, not phosphorylation, is the rate‐limiting step. The inhibitory effect of various glucose analogues on 2‐DOG and 3‐O‐methyl‐D‐glucose (3‐OMG) uptake revealed that these two sugars may be taken up into the cell by different carriers. In addition, kinetics analysis of the transport of both sugars also indicates that two hexose transport systems may be present in L6 cells. 2‐DOG is transported by high and low affinity transport systems (Km 0.6 mM and 2.9 mM, respectively), whereas 3‐OMG is transported by a low affinity system (Km 3.5 mM). Treatment of cells with ionophores or energy uncouplers results in inactivation of the high affinity system, but not the low affinity system.
Further studies on the enhancing factor and its possible mechanism of action
Authors: Mulherkar Rita , Deo M. G. 
Published: February 2005

AbstractIn this study the nature of binding of enhancing factor (EF) and its mode of action are examined. EF binds to A431 cells through its own receptor, which is distinct from the receptor for epidermal growth factor (EGF). EF binds to the cell membrane and in turn provides a binding site for EGF. Data analyzed from Scatchard plots show that prior treatment of formalin‐fixed A431 cells with EF for 30 minutes results in an increase in the number of binding sites for 125I‐EGF. 3H‐Thymidine incorporation studies, using the EGF‐receptorless cell line NR‐6, indicate that neither EF nor EGF alone stimulates the cells to synthesise DNA, but when both are added together the cells show 3H‐thymidine incorporation. The role of EF may be to trap EGF and make it available to the cells through its own receptors even in the absence of EGF receptors. EF also induces anchorage‐independent growth of normal fibroblasts in soft agar only in the presence of EGF.
Retinoids stimulate the release of superoxide by neutrophils and change their morphology
Authors: Badwey John A. , Robinson John M. , Curnutte John T. , Karnovsky Morris J. , Karnovsky Manfred L. 
Published: February 2005

AbstractAll‐trans‐retinal stimulated the release of superoxide by human and guinea pig neutrophils 63 ± 14 SD and 53 ± 5 SD nmol of O2−/min/107 cells, respectively. Superoxide release by unstimulated cells was negligible. All‐trans‐retinal also induced morphological changes (i.e., evaginations) in these cells. Other retinoids were effective in instigating these phenomena. The similarities of these effects to those instigated by cis‐unsaturated fatty acids (Badwey, J.A., et al., 1984, J. Biol. Chem., 259:7870–7877) are discussed in light of possible mechanisms.
Glucose metabolism in human spermatozoa: Lack of insulin effects and dissociation from alloxan handling
Authors: Gorus Frans K. , Pipeleers Daniel G. 
Published: February 2005

AbstractThe role of glucose metabolism in sperm cell motility was examined in purified human spermatozoa from the perspective of elucidating its possible significance in spontaneous and experimental diabetes. After a 4‐h incubation in the absence of D‐glucose, the mean progressive velocity of human spermatozoa was 40% lower than that of control cells kept in the presence of D‐glucose. The decline was rapidly overcome by the addition of D‐glucose or D‐fructose, the amplitude of this stimulatory effect being independent of the ambient hexose concentration. Between 1.4 and 16.7 mM glucose, spermatozoal glucose oxidation also proceeded independently of the extracellular glucose levels, whereas both insulin (100nM) and glucagon (100nM) failed to significantly affect the rate of glucose metabolism or cellular motility. It is speculated from these results that an alteration in seminal hexose concentrations or pancreatic hormone levels may be an unlikely cause for the reduced sperm motility that is characteristically observed in diabetic patients. Human spermatozoa rapidly incorporated D‐glucose and 3‐0‐methyl‐D‐glucose but excluded the glucose‐analogue alloxan, which may explain their resistance against the toxic effects of this diabetogenic drug, in spite of their intrinsic sensitivity to organic peroxides such as tert‐butyl hydroperoxide.
N‐myc and c‐src genes are differentially regulated in PCC7 embryonal carcinoma cells undergoing neuronal differentiation
Authors: Sejersen Thomas , Björklund Håkan , Sümegi Janos , Ringertz Nils R. 
Published: February 2005

AbstractWe examined the expression of N‐myc, c‐myc, and c‐src in four embryonic carcinoma (EC) cell lines during different states of cell growth and following induction of in vitro differentiation. N‐myc mRNA was detected in undifferentiated cells of four EC cell lines (PCC7, PCC3, PCC4, F9) neither of which showed N‐myc gene amplification. No N‐myc transcripts could be detected in mRNA prepared from a murine neuroblastoma cell line and from a murine fibroblast line. The level of N‐myc mRNA decreased by 85% when PCC7 EC cells were induced by retinoic acid and cAMP treatment to form nerve‐like cells. Six days after induction, the PCC7 cells changed into aggregates of neurofilament positive cells with massive neurite outgrowths. At this stage DNA replication had been reduced by more than 95%. The decreased N‐myc expression in induced PCC7 cells was parallelled by 300–500% increase in c‐src expression. Slowing of cell multiplication by serum starvation, on the other hand, did not affect the level of N‐myc or c‐src mRNA levels in PCC7 cells. C‐myc was expressed in all EC lines except PCC7, which surprisingly did not express c‐myc even at an exponential rate of proliferation. Chemical induction of F9 EC cells to form visceral endoderm or parietal endoderm resulted in markedly reduced (85%) levels of N‐myc transcripts. A similar decline in c‐myc expression was found in differentiated F9 cells. No c‐src transcripts were detected in proliferating or differentiated F9 cells. These results suggest that N‐myc may be expressed not only in neural development, but also in very early, undetermined embryonic cells. The activation of c‐src expression when PCC7 EC cells differentiate into nerve‐like cells shows that the pattern of proto‐oncogene expression may change during a differentiation process, some proto‐oncogenes increasing, others decreasing their representation in the mRNA pool.