bluuebunny/crossref_metadata_2025_split huggingface dataset:
Loss of three major auto phosphorylation sites in the EGF receptor does not block the mitogenic action of EGF
Authors: Clark Stella , Cheng Dorothy J. , Hsuan J. Justin , Haley John D. , Waterfield Michael D. 
Published: February 2005

AbstractThe EGF receptor cDNA has been transfected into receptor‐negative Chinese hamster ovary (CHO) cells. A mutant cell line (CHO 11) was isolated that expresses a receptor of lower molecular weight than the EGF receptor from A431 cells (150,000 daltons compared to 170,000 daltons) and which appeared as a doublet on SDS‐PAGE. By digestion of the receptor with endoglycosidase F it was shown that an altered pattern of glycosylation could not account for the smaller size of the protein, although it could explain the appearance of the CHO 11 receptor as a doublet protein. A deletion was located to the transfected cDNA and shown to involve the removal of coding sequences for the most C‐terminal 20,000 daltons of the EGF receptor, which contains the three major autophosphoryation sites. Despite the loss of these sites the EGF receptor from CHO 11 cells binds EGF, demonstrates protein tyrosine kinase activity in response to EGF, and transduces a mitogenic signal. The CHO 11 receptor protein is still autophosphorylated on alternative tyrosine residues. We conclude that phosphorylation of the three tyrosines (P1, P2, and P3) in the C‐terminal domain of the receptor is not required for signal transduction by the EGF receptor in these cells.
Potassium inhibition of transforming protein P85gag‐mos and reversal of the transformed phenotype in 6m2 cells
Authors: Lai Chiu‐Nan , Gallick Gary E. , Maxwell Steve A. , Brinkley Bill R. , Becker Frederick F. 
Published: February 2005

AbstractK+ at high concentrations (52–72 mM hypertonic KCI) has been reported to induce reverse transformation in the 6m2 cell, which is a clone of normal rat kidney cells (NRK) infected with a temperature‐sensitive transformation virus. When exposed to high K+, 6m2 cells grown at the permissive temperature (33°C) exhibit normal morphology and reduced soft agar growth, characteristics of cells grown at nonpermissive temperature (39°C). In the current study, flattening of cells and rearrangement of surface microvilli were demonstrated by scanning electron microscopy to occur within 6 hr of exposure to high K+, similar to the effect of temperature shift to 39°C. Exposure to K+ resulted in a 90% inhibition of P85gag‐mos‐associated serine kinase activity within 5 min, with a subsequent reduction of up to 75% of the synthesis of this protein. These alterations in the putative transforming protein were similar to those induced by temperature shift and were considered to be the basis for retrotransformation. The cell microtubular system and F‐actin cables were affected more slowly by K+ than by a temperature shift to 39°C. The former did not achieve the fine reticulum network seen in NRK cells until 72 hr later, but the latter remained aberrant. The effect on the enzyme might be mediated by alteration in phosphorylation, but the mechanism by which kinase inactivation induces retrotransformation is not yet known.
Rapid expression of ornithine decarboxylase mRNA in a macrophage‐like cell line: cAMP repression of the requirement for prior protein synthesis
Authors: Shurtleff Sheila A. , McElwain Chris M. , Taffet Steven M. 
Published: February 2005

AbstractOrnithine decarboxylase (ODC) activity was rapidly induced in the RAW264 marcophage‐like cell line after treatment with bacterial lipopolysaccharide (LPS). ODC mRNA leveis were determined by isolating cellular RNA, followed by Northern blot and dot blot analysis using a 32P‐labeled cDNA probe. ODC mRNA levels increased within 1 hour of stimulation of RAW264 cells with 1.0 μg/ml LPS. Transcription rate analysis on isolated nuclei indicated that an increase in transcription rate contributed to this increase in ODC mRNA. ODC mRNA levels continued to rise for 4 hours, peaking at eight times the basal level. ODC mRNA appeared as a single 2.2‐kb band prior to stimulation. After stimulation, the 2.2‐kb band intensified, and a second (2.7 kb) band was seen by Northern gel analysis. Similar induction was demonstrated when 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA) was used as the stimulus. The induction of ODC mRNA by either LPS or TPA was blocked by the addition of cycloheximide (25 μg/ml) or anisomycin (0.1 mM) to the cellular incubation mixture. This indicated that protein synthesis was required as a prerequisite to LPS or TPA induction of ODC mRNA. Experiments in which cycloheximide addition was delayed after LPS treatment indicated that some of the required protein synthesis occurred within the first 30 minutes and that complete expression of ODC mRNA was possible if protein synthesis continued for at least 2 hours before cycloheximide was added. Stimulation with 8‐bromo‐cAMP in addition to LPS has been shown to enhance the induction of ODC over that induced by LPS or TPA alone. It was not possible to block ODC mRNA induction with cycloheximide or anisomycin after treatment with the combined stimulus of LPS and cAMP or TPA and cAMP, indicating that protein synthesis was not required when cAMP was used as a coinducer. Thus, we have shown that in the same cell, ODC mRNA can be induced by two different pathways, one requiring protein synthesis and one not requiring protein synthesis.
Trans‐stimulation of L‐system amino acid transport in normal and chronic leukemic human lymphocytes: Phorbol ester restores function in CLL
Authors: Segel George B. , Woodlock Timothy J. , Lichtman Marshall A. 
Published: February 2005

AbstractChron c lymphocytic leukemia (CLL) B‐lymphocytes have a unique and specific diminution of L‐system (leucine favoring) amino acid uptake; the maximal velocity is approximately 10% of normal B‐lymphocytes. Treatment of CLL B‐cells with the maturational agent, tetradecanoyl phorbol acetate, results in restoration of L‐system amino acid uptake to normal velocity. To further characterize the effect of phorbol ester on the L‐system of CLL B‐cells, we have examined the ability of normal and CLL lymphocytes to exchange intracellular for extracellular amino acids by the L‐system (trans‐stimulation). A 60% increase in L‐system uptake was noted in normal B‐ and T‐lymphocytes in the presence of a high intracellular concentration of 2‐amino‐2‐carboxy‐bicycloheptane (BCH), a largely L‐system‐specific substrate. L‐system transport was not trans‐stimulated in CLL B‐lymphocytes. Phorbol ester treatment restored L‐system uptake in CLL to a normal Vmax of 900 umol/liter cell water per minute in the absence of BCH loading. The Vmax could be increased further to 2,400 if phorbol ester‐treated CLL cells were loaded with BCH. Hence, phorbol esters result not only in a normalization of L‐system uptake in CLL B‐cells but the transport system demonstrates exchange rates comparable to normal lymphocytes.
Effects of confluence on phosphate transport capacity in cultured renal cell lines
Authors: Scheinman Steven J. 
Published: February 2005

AbstractThe LLC‐PK1 cell line transports phosphate (Pi), glucose, and amino acids using carriers similar to those in proximal tubular cells. Others have reported that when monolayers reach confluence, hexose transport increases and activity of the A‐amino acid transporter falls. The present study evaluates Pi uptake by two continuous cell lines derived from renal proximal tubule, and demonstrates that phosphate uptake falls sharply upon reaching confluence in LLC‐PK1 cells but not in cultured opossum kidney (OK) cells. The fall in Pi uptake in LLC‐PK1 cells at confluence represents a halving in Vmax for Na‐dependent phosphate uptake (2.33 vs. 5.00 nmol/mg protein/5 min) without a change in Km (82 vs. 94 μM). Suppression of phosphate transport in confluent monolayers of LLC‐PK1 cells is completely reversed by bringing the cells into suspension. As has been shown for the phorbol ester 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA), exposure of monolayers to serum stimulates phosphate uptake, but unlike phorbol ester, serum does so without stimulating alanine uptake. OK cells differ from LLC‐PK1 in that no change occurs in Pi uptake at confluence, although they resemble LLC‐PK1 cells in that sugar uptake rises and alanine uptake falls at confluence. The different temporal patterns for Pi uptake in the two cell lines indicates that developmental change in the uptake of Pi is not linked to that of glucose or alanine.
Energetic mechanism of system a amino acid transport in normal and transformed mouse fibroblasts
Authors: Leister Kirk J. , Schenerman Mark A. , Racker Efraim 
Published: February 2005

AbstractOuabain treatment (0.4 mM) of normal and transformed C3H‐10T1/2 cells caused a progressive increase in 2‐aminoisobutyrate (AIB) transport reaching a maximum after 16 to 18 h exposure. There was a virtually complete blockage of this stimulated rate when 3 μM cycloheximide (CHX) was added together with ouabain at T = 0. In the transformed cell, addition of CHX after 14 h had no effect; in the normal cell, it inhibited (ca. 50%) the final AIB transport rate achieved after 24 h. The t½ for reaching maximal activity (insensitive to CHX exposure) was thus shifted from 8 h in the transformed cell to 15 h in the normal cell. Since the rate of achieving maximal activity in the absence of CHX was about the same in the two cells, the shift in t½ in the presence of CHX suggests that the rate of degradation is more rapid in the normal cell. Following ouabain treatment, the apparent Km for Na+ was decreased in both cells. The Km returned to the basal level 1 h after ouabain removal in the normal cell, but remained low in the transformed cell during this time period. The stimulation of AIB transport following ouabain removal was largely abolished by a proton ionophore (1799), a lipophilic cation (tetraphenyl‐phosphonium), or ouabain. These results suggest that, under the conditions of ouabain stress, there is a switch in the bioenergetic mechanism. The Na+/K+ pump and System A transporter appear to be linked and the membrane potential generated by the Na+/K+ pump activity becomes a major driving force for AIB uptake.
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

AbstractThree 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

AbstractSilica 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

AbstractHuman 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

AbstractTyrosine 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

AbstractThe 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

AbstractCalcium‐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

AbstractUsing 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

AbstractMammary 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

AbstractPleiotropic 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.