bluuebunny/crossref_metadata_2025_split huggingface dataset:
Potentiation of oncogenic N‐ras‐induced neurite outgrowth and ornithine decarboxylase activity by phorbol dibutyrate and protein kinase inhibitor H‐8
Authors: Trotta Robert J. , Thomson Timothy M. , Lacal Juan‐Carlos , Pellicer Angel , Burstein David E. 
Published: February 2005

AbstractA recombinant N‐ras oncogene, under the transcriptional control of a corticosteroid‐inducible mouse mammary tumor virus (MMTV) promoter, has been stably transfected into a PC12 rat pheochromocytoma subline. This cell line, designated UR61, undergoes N‐ras‐induced neurite outgrowth and cessation of division when treated with dexamethasone (Guerrero et al.: Biochemical and Biophysical Research Communications 150:1185–1192, 1988). We have employed the UR61 cell line as a model for ras oncogene‐induced neuronal differentiation. In UR61 cells, dexamethasone‐induced expression of the recombinant N‐ras gene resulted in time‐dependent expression of ornithine decarboxylase enzyme (ODC) activity. Prompted by recent reports of possible functional (Lacal et al.: Molecular and Cellular Biology 7:4146–4149, 1987; Wolfman and Macara: Nature 325: 359–361, 1987) and direct (Jeng et al.: Biochemical and Biophysical Research Communications 145:782–788, 1987) interactions between oncogene ras‐coded p21 and protein kinase C (PK‐C; Ca+ +/phospholipid‐dependent protein kinase), we employed the protein kinase inhibitor H‐8 (N‐[2‐(methylamino)ethyl]‐5‐isoquinoline sulfonamide dihydrochloride) and phorbol 12, 13‐dibutyrate (PDBu) to investigate this putative interaction in the UR61 cells, where ODC activity and neurite outgrowth were used as indicators of oncogenic N‐ras action. Treatment of UR61 cells with PDBu depleted cells of PK‐C and failed to promote neurite outgrowth but enhanced N‐ras‐induced neurite outgrowth and ODC activity. H‐8, which suppressed ODC induction by forskolin and phorbol myristate acetate, enhanced both N‐ras‐induced ODC activity and neurite outgrowth. Inhibition of ODC activity by difluoromethylornithine (DFMO) did not suppress oncogenic ras‐induced neurite outgrowth, suggesting that these two ras‐triggered events are mechanistically independent. These findings suggest that certain actions of N‐ras can occur in cells depleted of PK‐C, and thus, the role of PK‐C in ras‐induced differentiation differs from its role in ras‐induced mitogenesis and transformation.
Role of Na+/H+ exchange in the granulocyte‐macrophage colony‐stimulating factor‐dependent growth of a leukemic cell line
Authors: Caracciolo Daniele , Pannocchia Antonella , Treves Silvia , Ghigo Dario , Gallo Eugenio , Tarella Corrado , Bussolino Federico , Turrini Franco , Tamponi Giacomo , Bosia Amalia 
Published: February 2005

AbstractThe growth of the human leukemia cell line AML‐193 in a serum‐free medium is strictly dependent on the presence of the cytokine granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), which is one of the major regulators of the myelomonocytic lineage. At present, little is known about the mechanisms by which this growth factor transduces the signal intracellularly. The results of this study demonstrate that GM‐CSF needs the operation of a Na+/H+ exchanger, which is located in the plasma membrane of almost every vertebrate cell. In fact, the GM‐CSF‐dependent proliferation of AML‐193 cells is strongly reduced in the presence of the amiloride analog EIPA, a specific inhibitor of the Na+/H+ exchanger. When acidified, AML‐193 cells are able to recover the original pH, in a Na+‐dependent and EIPA‐inhibitable way; this demonstrates for the first time the presence of the Na+/H+ exchanger in these cells. Finally, GM‐CSF, at doses superimposable to those needed for triggering proliferation, induces in AML‐193 cells a sustained alkalinization, which is dependent on a operating Na+/H+ exchange, as it is inhibited by EIPA. These results suggest that GM‐CSF, like other growth factors in other cell systems, exerts its mitogenic activity in AML‐193 cells by inducing a Na+/H+ exchanger‐mediated rise in pHi.
DNA synthesis inhibition and reduced functional differentiation of midpregnant mouse mammary epithelia on collagen gels
Authors: Trumpbour Virginia , Rocha Victor 
Published: February 2005

AbstractMammary epithelial cells were examined for a link between DNA synthesis and subsequent synthesis and secretion of casein. Cells isolated from mice midway through pregnancy and cultured on collagen gels spread to form monolayers (spreading phase). Release of monolayer/gels to float in surrounding culture medium induces synthesis and secretion of casein (secretory phase). DNA synthesis was blocked during the spreading phase with cytosine arabinofuranoside (ARAC). Culture medium was assayed for casein by direct quantification of protein from SDS‐PAGE fluorographs, and by immunoblotting. When induced to become secretory, cells exposed to ARA C during the spreading phase showed a marked reduction of secretion of casein as compared to control cultures (72% reduction). In contrast, cells exposed to ARA C during the secretory phase (after monolayer formation was complete) showed no significant reduction in secretion of casein. Measurement of intracellular casein in secretory phase cells showed that reduced secretion of casein by cultures blocked during the spreading phase occurs as a consequence of reduced levels of casein synthesized, and not because of an inability to secrete intracellular accumulations. The inhibitor effect was specific; there was no significant reduction in levels of total intracellular protein synthesis, and neither cell spreading nor monolayer formation was impaired by treatments. These data support the notion that DNA synthesis is a prerequisite to functional differentiation of midpregnant mouse mammary epithelia maintained on floating collagen gels.
Mycoplasmas produce double‐stranded ribonuclease
Authors: Marcus Philip I. , Yoshida Itsuro 
Published: February 2005

AbstractMycoplasmas (Mollicutes) constitute a constant threat as insidious contaminants of animal cell cultures. They are responsible for myriad biochemical reactions associated with the cells they infect, and undoubtedly have been the source of metabolic and physiological activities attributed to their hosts. In an attempt to demonstrate a dsRNA‐inducible double‐stranded ribonuclease (dsRNase) in mammalian cells, comparable to that reported in avian cells, we discovered high levels of dsRNase “induced” by a particular stock of vesicular stomatitis virus. We now report that the double‐stranded ribonuclease resulted from the activity of a contaminant in that stock–;a “noncultivable” Mycoplasma hyorhinis. This report demonstrates the ubiquitous distribution of dsRNase among mycoplasmas, presents some characteristics of the enzyme and its production, and implicates once again mycoplasmas as contaminants of cell culture and potential perturbers of cellular physiology.
HSP 70 gene expression in Trypanosoma cruzi is regulated at different levels
Authors: de Carvalho Elizeu F. , de Castro F. T. , Rondinelli E. , Soares Celia M. A. , Carvalho Jose Francisco 
Published: February 2005

AbstractThe level of HSP 70 mRNA is altered in Trypanosoma cruzi cells incubated at supra‐optimal temperatures: the total amount of this RNA per cell is increased at 37°C, and slightly decreased at 40°C relative to its level at 29°C. However, its amount is greater in the polysomes at either temperature. The relative increase of this RNA is larger in the polysomes fraction than it is in the total RNA. In addition the level of HSP 70 protein in heat‐shocked cells is greater than would be expected from the recruitment of HSP 70 mRNA in the polysomal fraction. Taken together the data are interpreted as indicating that at 37°C and 40°C the HSP 70 gene regulation in T. cruzi involves both the selective accumulation of the HSP 70 mRNA in the polysomes and its preferential translation. At 37°C, in addition, an increase in the total amount of this template is observed in the cells.
Novel ATP‐binding heat‐inducible protein of Mr = 37,000 that is sensitive to transformation in BALB/3T3 cells
Authors: Nakai Akira , Hirayama Chisato , Ohtsuka Kenzo , Hirayoshi Kazunori , Nagata Kazuhiro 
Published: February 2005

AbstractUsing affinity chromatography on ATP‐agarose, we have identified a major ATP‐binding protein in Nonidet P‐40 extracts of avian and mammalian cells labeled with [35S]methionine. After washing ATP‐agarose beads with high‐ionic‐strength buffer (0.4 M NaCl), the 37‐kD protein was shown to be one of the major ATP‐binding proteins while p72 and grp78, which are members of the hsp70 family, also bound to ATP‐agarose. This protein consisted of several spots on two‐dimensional gel electrophoresis. The isoelectric point of the most basic spot was approximately 9.2 in chick embryo fibroblasts, whereas it was about 8.8 in mouse 3T3 cells. The identities of these proteins in mouse and chick cells were confirmed by peptide mapping. After heat‐shock treatment of BALB/3T3 cells, the major heat‐shock protein, hsp70, was shown to be induced very rapidly after heat shock and was recovered in the ATP‐binding fraction. Besides hsp70, a 37‐kD protein was also found to be induced by heat shock. This protein was drastically induced by treating the cells with α,α′‐dipyridyl, an iron chelating reagent, but not with sodium arsenite, calcium ionophore, or tunicamycin. The synthesis and the total amount of this ATP‐binding protein increased in mouse 3T3 cells transformed by simian virus 40, methylcholanthrene, or activated c‐Ha‐ras oncogene compared to their normal counterparts. The incorporation of [32P]orthophosphate was not detected in either normal or transformed cells. These studies established that a major ATP‐binding protein of Mr = 37,000 is a heat‐inducible protein and that the synthesis of this protein is regulated by malignant transformation.
Ca‐dependent slow action potentials in neuromuscular diseases
Authors: Siri L. Nicola , Dubrovsky A. L. , Uchitel O. D. 
Published: February 2005

AbstractSlow Ca‐dependent action potentials were studied in skeletal muscle fibers from different Neuromuscular Diseases (NMD). Byopsies were obtained from: 3 my‐opathies [Fascioscapulohumeral Dystrophy (FSH) and Polymyositis (PM)], 6 patients with other diseases (CD) [Amyotrophic Lateral Sclerosis (ALS), Central Core Disease, Mitochondrial Myopathy, Polyneuritis (PN), von Eulenberg's Paramyotonia], and 8 normal control muscles. Experiments were carried out in muscle fibers under current‐clamp conditions. Membrane currents other than Ca ones were abolished or greatly diminished. Muscle fibers produced any of 3 types of responses, when stimulated by depolarizing pulses: fully developed Ca‐action potentials (CaAP), abortive non‐regenerative Ca responses (NrR), or only capacitive passive responses (WR). The 3 types of responses were not dependent on the basal conditions of the fibers. The frequency of observation of CaAPs was significantly higher in myopathic disease. In myopathies, 46% of the muscle fibers had CaAPs, while only 22% of fibers from CD and 15% of the fibers from normal muscles showed CaAPs. No differences were observed in the resting constants as well as in the CaAPs parameters between normal and diseased muscle fibers.
Altered cell cycle responses to insulin‐like growth factor I, but not platelet‐derived growth factor and epidermal growth factor, in senescing human fibroblasts
Authors: Chen Yuchyau , Rabinovitch Peter S. 
Published: February 2005

AbstractHuman diploid fibroblasts (HDF) were used to study aging‐related changes in the proliferative response to platelet‐derived growth factor (PDGF), epidermal growth factor (EGF), and insulin‐like growth factor I (IGF‐I, somatomedin‐C) in serum‐free, chemically defined culture medium. Cell cycle kinetic parameters were determined by using 5‐bromodeoxyuridine incorporation and flow cytometric analysis with the DNA stain Hoechst 33258. This allowed analysis of the growth factor response tobe focussed exclusively upon of the cycling faction of cells within the culture, even in senescent cell cultures which contained predominantly nondividing cells. PDGF and EGF exert their primary effect upon regulation of the proportion of cycling cells in the culture. The doses of PDGF and EGF that produced a half‐maximal cycling fraction, analogous to Km, showed no large or consistent difference between young‐ and old‐passage cells. In contrast, IGF‐I primarily affects the rate of transition of cells from G1, into S phase, and the dose of IGF‐I which produced a half‐maximal rate of G1 exit increased up to 130‐fold in older‐passage cells. Unexpectedly, supraPhysiologic concentrations of IGF‐I were found to increase the G1, exit rate of the dividing subpopulation of cells in older‐passage cultures to rates higher than those seen in young cultures. In summary, among cells capable of cycling in aging cultures, there were few changes in the regulation of the growth fraction by PDGF and EGF, but there was a greatly increased dependence on IGF‐I for regulation of the rate of entry into S phase. The slower growth of the dividing population of cells in aging cultures may be related to a requirement for IGF‐I at levels which are greatly above those usually supplied.
Plasma membrane fluidity gradients of human peripheral blood leukocytes
Authors: Collins James M. , Grogan W. McLean , Scott Robert B. 
Published: February 2005

AbstractThe shape of the fluidity gradient of the outer hemileaflet of the plasma membrane of normal, living, human white blood cells was determined using a series of n‐(9‐anthroyloxy) fatty acid probes where n = 2, 3, 6, 7, 9, 11, 12, and 16, to establish a baseline for future studies on the consequences of various pathological states. Fluorescence uptake and steady‐state anisotropy values were obtained with a flow cytometer capable of continuous recording over time of vertical and horizontal emission intensities, with the output of these intensities as calculated anisotropy values. The fluorescence uptake of all of the membrane probes was rapid up to about 15 min. The magnitudes of the uptake of fluorescence was, for the n‐(9‐anthroyioxy) series, in the order 2 > 3 > 6 > 7 > 9 > 11 =12 = 16 for neutrophils, lymphocytes, and monocytes. Anisotropy values were constant from 5 to 30 min after addition of the various probes. The orders of the anisotropy magnitudes, indicative of the shapes of the fluidity gradient, were, for neutrophils, 6 > 7 > 9 > 2 = 3 = 11 = 12 > 16, and for lymphocytes, 7 > 6 > 9 > 11 > 2 = 3 > 11 = 12 > 16, and for monocytes, 9 > 7 > 6 > 11 > 2 = 3 > 12 > 16. The kinetics of anisotropy from 1 to 5 min after addition of the probes differed for each of the three cell types. Probes with an n‐value less than or equal to the maxima (n = 6, neutrophils; n = 7, lymphocytes; n = 9, monocytes) rapidly (1.2 min) reached equilibrium, whereas those probes with n‐values greater than the maxima took progressively longer times to equilibrate as n increased. This behavior is consistent with the existence of an energy barrier just below the approximate region sensed by the probes, which would correspond to just below 6AS for neutrophils, 7AS for lymphocytes, and 9AS for monocytes.
Ras and neu oncogenes reverse serum inhibition and epidermal growth factor dependence of serum‐free mouse embryo cells
Authors: Shirahata Sanetaka , Rawson Cathleen , Loo Deryk , Chang Yung‐Jin , Barnes David 
Published: February 2005

AbstractSerum‐free mouse embryo cells, cultured in basal nutrient medium supplemented with insulin, transferrin, epidermal growth factor, fibronectin, and high‐density lipoprotein, do not exhibit growth crisis, lack detectable chromosomal aberrations, are nontumorigenic in vivo, are dependent on epidermal growth factor for survival, and are growth inhibited by serum or platelet‐free plasma. These cells after transfection with the human Ha‐ras or rat neu oncogenes no longer required epidermal growth factor for survival, were tumorigenic in vivo, and also proliferated in serum‐containing medium. Autocrine activity capable of replacing epidermal growth factor was detected in conditioned medium from ras‐transformed cultures, but little such activity was detected in medium from neu‐transformed cultures. In addition, the capability of ras or neu‐transformed cells to grow in serum‐containing medium could not be mimicked in untransformed cells by the addition of growth factors or conditioned medium from transformed cells. These results suggest that the known structural similarity of the neu gene product to the EGF receptor is also reflected in a functional similarity by which the mutationally activated neu protein can replace the ligand‐activated EGF receptor. These results also suggest that the ability of ras‐and neutransformed cells to escape the effect of the inhibitory serum activity is a nonautocrine property distinct from the acquisition of EGF autonomy.
Role of messenger RNA subcellular localization in the posttranscriptional regulation of human histone gene expression
Authors: Zambetti G. , Stein J. , Stein G. 
Published: February 2005

AbstractHistone mRNAs are naturally localized on non‐membrane‐bound polysomes and selectively destabilized during inhibition of DNA replication. Targeting histone mRNA to membrane‐bound polysomes, by incorporating sequences coding for a signal peptide into the message, results in the stabilization of the histone fusion mRNA when DNA synthesis is interrupted (Zambetti et al.: Proceedings of the National Academy of Sciences of the United States of America 84:2683–2687, 1987). A single nucleotide substitution that abolishes the synthesis of the signal peptide results in the localization of the histone fusion mRNA on non‐membrane‐bound polysomes to the same extent as endogenous histone mRNA and fully restores the coupling of histone fusion mRNA stability to DNA replication. Signal peptide‐histone fusion mRNAs containing two point mutations that result in the incorporation of two positively charged amino acids into the hydrophobic domain of the signal peptide are partially retained on non‐membrane‐bound polysomes and are partially destabilized during inhibition of DNA synthesis. These data indicate that the degree to which the signal peptide‐histone fusion mRNAs are associated with non‐membrane‐bound polysomes is correlated with the extent to which the mRNAs are degraded during inhibition of DNA synthesis. These results suggest that the subcellular location of histone mRNA plays an important role in the posttranscriptional regulation of histone gene expression.
Effects of β‐all‐trans retinoic acid on growth, proliferation, and cell death in a multicellular tumor spheroid model for squamous carcinomas
Authors: Sacks Peter G. , Oke Victor , Vasey Tracey , Calkins Dennise P. , Terry Nicholas H. A. 
Published: February 2005

AbstractThe growth of multicellular tumor spheroids, MTSs, from squamous carcinoma line MDA 886Ln was inhibited by β‐all‐trans retinoic acid (RA). Inhibition occurred within 3 to 5 days of treatment, and MTS size then remained static for up to 2 weeks. Although their growth stopped, 10‐day‐treated MTSs incorporated [3H]thymidine into trichloroacetic acid‐precipitable material, and the [3H]‐thymidine labeling index, determined by autoradiography, was equivalent between control and RA‐treated MTSs. Bivariate flow cytometric analysis of bro‐modeoxyuridine‐labeled MTSs showed equivalent S phase progression of labeled cells over an 8‐hour chase. MTS growth stasis was not related to RA‐induced cell cycle effects. Monitoring of MTSs for cell sloughing showed no significant cell shedding that could account for stasis. Quantitation of cell number and DNA content per MTS showed an RA‐induced decrease. This was confirmed by histological analysis, which demonstrated the temporal appearance of accellular areas. MTS growth statis is thus related to an RA‐induced cell loss in this MTS model for squamous carcinomas.
Altered regulation of platelet‐derived growth factor A‐chain and c‐fos gene expression in senescent progeria fibroblasts
Authors: Winkles Jeffrey A. , O'Connor Mary L. , Friesel Robert 
Published: February 2005

AbstractThe study of human genetic disorders known as premature aging syndromes may provide insight into the mechanisms of cellular senescence. These diseases are clinically characterized by the premature onset and accelerated progression of numerous features normally associated with human aging. Previous studies have indicated that fibroblasts derived from premature aging syndrome patients have in vitro growth properties similar to senescent fibroblasts from normal individuals. As an initial approach to determine whether gene expression is altered in premature aging syndrome fibroblasts, RNA was prepared from various cell strains and used for gel blot hybridization experiments. Although normal fibroblasts only express platelet‐derived growth factor (PDGF) A‐chain mRNA for a brief period following mitogenic stimulation, one strain of Hutchinson‐Gilford (progeria) syndrome fibroblasts, AG3513, consdtutively expresses PDGF A‐chain mRNA and PDGF‐AA homodimers. The PDGF A‐chain gene does not appear to be amplified or rearranged in these fibroblasts. AG3513 progeria fibroblasts have properties characteristic of senescent cells, including an altered morphology and a diminished mitogenic response to growth promoters. The diminished response of AG3513 progeria fibroblasts to PDGF stimulation was examined in some detail. Studies using 125I‐PDGF‐BB, which binds with high affinity to both A‐ and B‐type PDGF receptors, indicate that normal and AG3513 progeria fibroblasts have a similar number of PDGF receptors. Although receptor autophosphorylation occurs normally in PDGF‐stimulated AG3513 progeria fibroblasts, c‐fos mRNA induction does not. The senescent phenotype of AG3513 fibroblasts is probably unrelated to their constitutive PDGF A‐chain gene expression; further studies are necessary in order to directly address this issue. Also, additional analysis of this progeria fibroblast strain may provide information on the control of mitogeninducible gene expression in normal cells.