bluuebunny/crossref_metadata_2025_split huggingface dataset:
A clonal derivative of tunicamycin‐resistant Chinese hamster ovary cells with increased N‐acetylglucosamine‐phosphate transferase activity has altered asparagine‐linked glycosylation
Authors: Waldman Barbara Criscuolo , Oliver Constance , Krag Sharon S. 
Published: February 2005

AbstractA population of Chinese hamster ovary (CHO) cells resistant to the antibiotic tunicamycin (TM) had previously been isolated (Criscuolo, B.A., and Krag, S.S. (1982) J. Cell Biol. 94:586–591) by a stepwise selection procedure using progressive increments of TM added to the medium. TM inhibits asparaginelinked glycoprotein biosynthesis by blocking the transfer of N‐acetylglucosamine‐1‐phosphate from the sugar nucleotide UDP‐N‐acetylglucosamine to the isoprenoid lipid carrier, dolichyl phosphate. Four clonal derivatives were isolated from the TM‐resistant population in the presence of 27 μg TM/ml and were found to overproduce the N‐acetylglucosamine‐phosphate transferase activity to the same extent (approximately 15‐fold compared to wild‐type cells). One of these clones, 3E11, was > 550‐fold more resistant to TM than wild‐type cells. The resistance phenotype remained during at least 2.5 months of growth in the absence of TM. 3E11 cells exhibited chromosomal translocations, but no homogeneously staining regions (HSR) or double minute chromosomes. The N‐acetylglucosamine‐phosphate transferase activity in 3E11 cells was membrane‐associated and was inhibited by TM. A 140,000‐dalton membrane protein and at least four other membrane proteins were enriched in 3E11 cells. Mannosylphosphoryldolichol synthase and glucosylphosphoryldolichol synthase activities were not elevated in membranes prepared from 3E11 cells. Asparagine‐linked glycosylation was altered such that 3E11 cells synthesized primarily a truncated oligosaccharide, Man5GlcNAc2, perhaps due to the reduced amount of mannosylphosphoryldolichol relative to wild‐type cells.
Apical trehalase expression associated with cell patterning after inducer treatment of LLC‐PK1 monolayers
Authors: Yoneyama Yoshitaka , Lever Julia E. 
Published: February 2005

AbstractTrehalase, a differentiation‐specific marker of renal proximal tubule brush border membrane, is expressed in confluent long‐term cultures of the renal epithelial cell line LLC‐PK1. The level of trehalase is greatly increased after treatment of cultures with differentiation inducers such as hexamethylene bisacetamide (HMBA), accompanied by increases in other apical membraneassociated differentiated functions (Yoneyama and Lever: J. Cell. Physiol. 121: 64–73, 1984). In the present study, we utilize a polyclonal antibody specific for renal trehalase to demonstrate that trehalase expression induced in LLC‐PK1 cultures after HMBA treatment is localized in cells forming a three‐dimensional network of strands across the confluent monolayer. The antitrehalase antibody recognized an apical membrane antigen of apparent molecular weight 100–110 kD both in LLC‐PK1 cultures and in the corresponding pig renal brush border membranes. Strand formation and total trehalase activity increased in parallel as a function of inducer concentration and duration of exposure. Strand formation and trehalase expression were also greatly enhanced in monolayers grown on a Nuclepore filter support even in the absence of inducer. Strand formation was not a prerequisite for induced trehalase expression in culture, since strands did not develop in cultures treated with N, N'‐dimethylformamide (DMF) an equally potent inducer of trehalase expression. In this case, cells which expressed increased levels of trehalase were dispersed at random over the monolayer. Induction of strand formation and trehalase expression by HMBA required a minimum exposure period of 48 hr and persisted up to a week after removal of inducer. By contrast, the response to DMF required continuous presence of inducer. Levels of trehalase declined even in the continuous presence of inducer in local regions of low cell density created by wound‐repair of the monolayer. In addition to the membrane‐bound form, trehalase activity was also recoverable from the culture medium, but release of trehalase was not affected by inducers.These observations are consistent with the view that a cell type committed to express a program of differentiation after HMBA treatment or growth on a permeable support is organized in specific cell patterns visible as strands over the confluent cell monolayer.
Toxicity of ligand and antibody‐directed ricin A‐chain conjugates recognizing the epidermal growth factor receptor
Authors: Vollmar Angelika M. , Banker Deborah E. , Mendelsohn John , Herschman Harvey R. 
Published: February 2005

AbstractApproximately equal amounts of 125I‐mAb 225 (a monoclonal antibody recognizing the human epidermal growth factor receptor) and 125I‐labeled epidermal growth factor (125I‐EGF) were bound by HeLa cells. However, these two EGF receptor binding moieties had different fates after binding. Sixty percent of cell‐associated 125I‐EGF was internalized. The majority of internalized 125I was released from the cell within 2 hr. In contrast, whereas only 30% of bound 125I‐mAb 225 was internalized by HeLa cells, the internalized radioactivity remained cell‐associated. EGF and mAb 225 were used to construct ricin A‐chain (RTA) conjugates. The two chimeric molecules, EGF‐RTA and mAb 225‐RTA, were equally toxic to human HeLa cells. EGF‐RTA was also toxic to murine 3T3 cells. In contrast, mAb 225‐RTA was not toxic to 3T3 cells, consistent with the human EGF‐receptor specificity of mAb 225. Neither conjugate was cytotoxic to EGF‐receptor‐deficient 3T3‐NR6 cells. Rapidity and potency of protein synthesis inhibition of HeLa cells were equivalent for the two chimeric conjugates, as was the degree to which colony‐forming ability was reduced. However, ammonium chloride enhanced the toxicity of EGF‐RTA but not mAb 225‐RTA, suggesting that the two toxic chimeric toxins‐‐like the unconjugated receptor‐binding moieties‐‐are processed differently by HeLa cells.
PDGF induces c‐myc mRNA expression in MG‐63 human osteosarcoma cells but does not stimulate cell replication
Authors: Womer Richard B. , Frick Kevin , Mitchell Christopher D. , Ross Alonzo H. , Bishayee Subal , Scher Charles D. 
Published: February 2005

AbstractThe platelet‐derived growth factor (PDGF) modulated growth response of the MG‐63 human osteosarcoma cell line, which neither expresses c‐sis mRNA nor secretes a PDGF analogue, was characterized. Scatchard analysis demonstrated that the MG‐63 cells have 23,000 receptors per cell with a kd of 5 × 10−11 M. The receptor became phosphorylated, in a PDGF concentration‐dependent manner, when 32P‐orthophosphate‐labeled cells were treated with PDGF for 3 h at 4°C. The phosphorylated receptor was identified by autora‐diography and gel electrophoresis after isolation of the 32P‐labeled receptor using a solid‐phase monoclonal antibody directed against phosphotyrosine. Binding of the receptor to the antibody was inhibited by 5 mM phenyl phosphate, further suggesting that PDGF stimulated tyrosine‐specific receptor autophosphorylation. In addition, treatment of MG‐63 cells with PDGF for 3 h at 37°C induced a 7.5‐fold increase in c‐myc mRNA accumulation as analyzed on Northern gels. However, MG‐63 cells grew equally well in either serum‐(which contains PDGF) or plasma‐(which does not) supplemented medium. Furthermore, PDGF did not stimulate DNA synthesis in growth arrested MG‐63 cells, nor did it potentiate DNA synthesis modulated by somatomedin C. Thus MG‐63 cells are a naturally occurring cell variant in which PDGF stimulates c‐myc expression but does not modulate mitogenesis.
Hormonal regulation of acid phosphatase release by osteoclasts disaggregated from neonatal rat bone
Authors: Chambers T. J. , Fuller K. , Darby J. A. 
Published: February 2005

AbstractOsteoclasts disaggregated from neonatal rat long bones and incubated on plastic or glass substrates were found to release a considerable proportion of tartrate‐resistant acid phosphatase into culture supernatants. Enzyme release was detectable in the supernatant medium of cultures containing as few as ten cells after 1 hr of incubation and proceeded in a linear manner for the ensuing 6 hr. Calcitonin (1 pg/ml) and cytochalasin B (5 μ/ml) inhibited release into the supernatant, suggesting that release represents enzyme secretion. Prostaglandin E1 induced transient inhibition followed by recovery; parathyroid hormone and 1,25(OH)2 vitamin D3 were without influence. Acid phosphatase release in these cultures shows a pattern of hormone responsiveness that coincides with the effects of these hormones on bone resorption by isolated osteoclasts. The extent of acid phosphatase release and its regulation by calciotropic hormones imply a central role for acid hydrolase secretion in osteoclastic bone resorption. The experimental system described in this study may facilitate analysis of the pharmacological hormonal and cellular regulation of osteoclastic function.
Metabolic effects of heparin on rat cervical epithelial cells
Authors: Wright Thomas C. , Karnovsky Morris J. 
Published: February 2005

AbstractThe glycosaminoglycan heparin inhibits the growth of a number of different cell types in vitro including smooth muscle cells, mesangial cells, fibroblasts, and rat cervical epithelial cells (RCEC). Studies investigating the antiproliferative effects of heparin on smooth muscle cells have demonstrated the site of the cell cycle block and revealed several metabolic alterations that could be causally associated with growth inhibition. We have investigated these metabolic parameters in RCEC to determine whether they are also associated with the antiproliferative effects of heparin in epithelial cells. Heparin acts rapidly to inhibit RCEC growth with inhibition detectable by autoradiography 7 h after the addition of heparin. Heparin treated RCEC begin to enter S‐phase 12 h after the removal of heparin. These findings suggest that heparin blocks RCEC in the early‐to‐mid G1 phase of the cell cycle rather than late in G1 or early in S‐phase as has previously been demonstrated for smooth muscle cells. Unlike smooth muscle cells, the uptake of thymidine and uridine is not inhibited by heparin in RCEC. Treatment of medium with heparin‐Sepharose does not reduce the subsequent growth of RCEC; heparin inhibits the growth of RCEC in heparin‐Sepharose treated medium in a manner identical to that in nontreated medium. Therefore the growth inhibitory effects of heparin cannot be explained by the inactivation of mitogens present in serum. In contrast to its effects on smooth muscle cells, heparin treatment of RCEC does not result in a reduction in the binding of epidermal growth factor (EGF) to the cells. These results indicate that although heparin inhibits the growth of a variety of cell types, significant differences exist in the responses of the different cells to heparin.
Casein secretion by mammary gland epithelia from collagen gel cultures and lactating glands
Authors: Rocha Victor , Hwang Soo‐In , Ortiz C. Leo 
Published: February 2005

AbstractAmino acid incorporation experiments show that epithelial cells from lactating mouse mammary glands and from collagen gel culture both synthesize and secrete four principal phosphocaseins (p45, p40, p27, and p23 KD). In both cases, however, the casein production is largely dominated by the p27 species. The average percentage distribution of the above casein species in medium from cultured epithelia is approximately 13%, 6%, 68%, and 14%, respectively; for milk the distribution is approximately 23%, 7%, 54%, and 16%. The predominance of the p27 species is not a consequence of extensive extracellular differential degradation of the secreted caseins since no significant casein degradation was observed in culture medium, either in contact or isolated from epithelial cell monolayers. Synthesis and secretion of all the caseins by cultured epithelia is dependent upon insulin, prolactin, and hydrocortisone. Presumably some intracellular events result in the secretion of p27 as the principal casein in mouse milk. Apparently, some selection factor(s) operate to make p27 a major nitrogenous nutritional component for a newborn mouse. In addition, on a quantitative basis, the relative levels of various caseins secreted by epithelia from lactating mammary glands is essentially duplicated by epithelia in collagen gel culture.
Thrombin‐induced thromboxane generation by neutrophils and lymphocytes: Dependence on enzymic site
Authors: Bizios R. , Lai L. , Fenton J. W. , Malik A. B. 
Published: February 2005

AbstractWe examined the effects of thrombin on thromboxane generation by sheep neutrophils and lymphocytes in vitro. Physiological concentration of thrombin (50 nM) resulted in thromboxane B2 generation from both neutrophils and lymphocytes, which was comparable to that obtained with zymosan activated serum challenge of the cells. Thromboxane B2 generation was dependent on the enzymic region of the thrombin molecule responsible for clotting activity because the complexing of thrombin with hirudin (1:1 U:U mixture of thrombin and hirudin) abolished thromboxane generation from both cell types. Further studies with modifed forms of α‐thrombin (which were produced by irreversible conjugation at the catalytic site and lacked enzymic activity) also showed no generation of thromboxane B2 from neutrophils or lymphocytes. The results indicate that thrombin stimulates thromboxane generation from neutrophils and lymphocytes and that this response is dependent on the proteolytic activity of thrombin.
Effects of dissolved oxygen concentration on hybridoma growth and metabolism in continuous culture
Authors: Miller William M. , Wilke Charles R. , Blanch Harvey W. 
Published: February 2005

AbstractOxygen transport is a major limitation in large‐scale mammalian cell culture. The effects of the dissolved oxygen concentration (DO; from 0.1 to 100% saturation with air) on Sp2/0‐derived mouse hybridomas were investigated using continuous culture. The steady‐state concentration of viable cells increased with decreasing DO until a critical dissolved oxygen concentration of 0.5% of air saturation was reached. The cell concentration declined at lower DO because of incomplete glutamine oxidation, and the specific lactate production from glucose increased to offset the reduced energy production from glutamine. Cell viability increased as the DO was decreased; the viability continued to increase even when the DO was reduced below 0.5%. The specific oxygen uptake rate was essentially constant for DO ≥ 10% of air saturation and then decreased with decreasing DO. The P/O ratio (ATP molecules produced per O atom consumed) appears to change from 2 to 3 between 10 and 0.5% DO. The specific ATP production rate calculated using this assumption decreases only slightly with decreasing DO. The optimum DO of 50% for antibody production is different than the optimum (∼ 0.5% DO) for cell growth.
Characterization of murine monoclonal antibodies directed against basic fibroblast growth factor
Authors: Massoglia S. L. , Kenney J. S. , Gospodarowicz D. J. 
Published: February 2005

AbstractMonoclonal antibodies (McAbs) were developed that identify the complete (1–146 aa) and the NH2‐terminal truncated (des 1–15) form of bovine basic fibroblast growth factor (bFGF). Four McAbs, designated McAbs 6, 8, 38, and 42, bind the complete form of bFGF found in bovine pituitary, brain, and adrenal gland. One of these McAbs, McAbs 42, also binds to the des 1–15 aa form of bFGF found in bovine adrenal gland, kidney, and corpus luteum. None of the McAbs binds bovine‐brain‐derived acidic FGF (aFGF). McAbs 6, 8, and 38 recognized the same epitope located within the first ten residues of the NH2‐terminal of complete bFGF. McAb 42 recognizes a “core” epitope found on both the complete and des 1–15 aa bFGFs. The McAbs are murine lgGs with affinity constants of 107‐108 liter/M for bovine‐pituitary‐derived bFGF. McAbs 8 and 42 have been used in a two‐site ELISA to detect the complete form of bFGF. The ELISA is sensitive to 38.5 fmole/well of bFGF and is not affected by the presence of calf serum or bovine‐brain‐derived aFGF. These McAbs should be useful in distinguishing the native and des 1–15 aa forms of bFGF from each other, and from aFGF and other growth factors.
Production of tumor growth‐inhibiting protein by the neonatal mouse brain: Dependence on intracellular glutamine metabolism
Authors: Miwa Nobuhiko , Matsuno Tetsuya , Mizuno Satoshi 
Published: February 2005

AbstractWe previously reported that the mouse brain at the neonatal stage but not at the adult stage secreted a carcinostatic factor of 62,000 Da, termed NBCF, which inhibited clonal growth and DNA synthesis of malignant cells preferentially over those of normal cells. In the present study, NBCF production by the neonatal mouse brain in culture was investigated. Addition of L‐glutamine to the culture medium markedly promoted NBCF production in a concentration‐dependent manner. The production seemed to be specific to glutamine, since no promotive effect was exerted by L‐glutamic acid, its analogue DL‐α‐aminoadipic acid, L‐aspargine, or L‐aspartic acid or by other amino acids or vitamins. NBCF production was markedly reduced in culture medium either devoid of L‐glutamine or containing 6‐diazo‐5‐oxo‐L‐norleucine, a glutamine antagonist, or L‐metionine sulfoximine, an inhibitor of glutamine synthetase. Thus NBCF production was promoted by extracellular supply, intracellular synthesis, and utilization of L‐glutamine but was not affected by its deamidated form or homologue amino acids. On the other hand, NBCF production was completly repressed by addition of cycloheximide to the culture medium. The repressive effect was also exerted by actinomycin D although not completely, whereas cytosine β‐D‐arabinofuranoside did not repress NBCF production. These results indicated that NBCF production by cultivation was independent of DNA replication but dependend mostly on a transcription stage and its following stages and partly on a translation stage from the preexisting mRNA to the protein.
Perturbation of cultured human vascular endothelial cells by phorbol ester or thrombin alters the cellular von Willebrand factor distribution
Authors: Reinders Jan Hendrik , Vervoorn Richard C. , Verweij Cornelis L. , Van Mourik Jan A. , De Groot Philip G. 
Published: February 2005

AbstractWe have studied the influence of perturbation of cultured human umbilical vein endothelial cells on the distribution of the von Willebrand factor. As shown previously, short‐term ( ≤ 1 hr) treatment of endothelial cells with the phorbol ester 4β‐phorbol 12‐myristate 13‐acetate (PMA) or thrombin resulted in the release of cellular stored von Willebrand factor. Long‐term treatment with PMA or thrombin evoked a distinct change in the endothelial cell distribution of von Willebrand factor, evident 24 to 48 hrs after exposure. Whereas the contents of the von Willebrand factor storage sites in the cells were gradually restored within 48 hrs, enhanced amounts of von Willebrand factor were secreted into the medium. However, PMA did not increase the endothelial cell contents of mRNA encoding for von Willebrand factor. The number as well as the size of von Willebrand factor storage granules in the endothelial cells increased after exposure to the phorbol ester, as determined by immunofluorescence microscopy. A second treatment with PMA or thrombin, 48 hrs after cells had been stimulated with these agents, resulted again in the instantaneous release of von Willebrand factor. PMA and thrombin caused a decrease in the von Willebrand factor contents of the extracellular matrix. Pulse‐chase experiments revelaed that PMA blocked the deposition of von Willebrand factor in the subendothelium, whereas PMA did not affect the degradation of matrix von Willebrand factor. Thus, perturbation of endothelial cells changes the cellular distribution of von Willebrand factor.
Iron uptake from transferrin and transferrin endocytic cycle in friend erythroleukemia cells
Authors: Hradilek A. , Neuwirt J. 
Published: February 2005

AbstractSeveral aspects of iron metabolism were studied in cultured Friend erythroleukemia cells before and after induction of hemoglobin synthesis by dimethyl sulfoxide. The maximal rate of iron uptake from 59Fe‐labeled transferrin, 1.5 × 106 atoms of Fe/cell per 30 min in uninduced cells, increased to 3 × 106 atoms/cell after 5 days of induction. The increase in iron uptake was not accompanied by a proportional increase in the number of transferrin receptors detected by 125I‐labeled transferrin binding, suggesting a more efficient iron uptake by transferrin receptors in induced cells, with the rate of about 26 iron atoms per receptor per hour, compared to 15 atoms in uninduced cells. In agreement with this conclusion are results of the study of cellular 125I or 59Fe labeled transferrin kinetics. In the induced cells transferrin endocytosis and release proceeded with identical rates and all the endocytosed iron was retained inside the cell. On the other hand, transferrin release by uninduced cells was significantly slower and a substantial part of internalized 59Fe was released. On the basis of these results, different efficiency of iron release from internalized transferrin, accompanied by changes in cellular transferrin kinetics, is proposed as one of the factors determining the rate of iron uptake by developing erythroid cells.
Monensin inhibits ligand dissociation only transiently and partially and distinguishes two galactosyl receptor pathways in isolated rat hepatocytes
Authors: Oka Janet A. , Weigel Paul H. 
Published: February 2005

AbstractMonensin has been shown to inhibit the dissociation of internalized asialoorosomucoid (ASOR) from galactosyl (Gal) receptors in hepatocytes (Harford et al., J. Cell. Biol., 96: 1824, 1983). Examination of the long‐term kinetics of dissociation of a single round of surface‐bound 125I‐ASOR in the presence of monensin revealed, however, that dissociation resumed after a lag of 30–40 min. Dissociation proceeded slowly with apparent first order kinetics (k = 0.006–0.022 min−1) and reached a plateau after 4 h, both in freshly isolated cells in suspension and in cells cultured for 24 h. Only a portion of the ligand bound to surface Gal receptors was capable of dissociating. The degree of dissociation was correlated with the expression of a subpopulation of receptors we have recently designated as state 1 Gal receptors (Weigel et al., Biochem. Biophys. Res. Commun. 140: 43, 1986). The recovery and dissociation of a portion of 125I‐ASOR‐receptor complexes after the lag period is not due to a depletion of monensin, since a second addition of the drug has no affect once dissociation resumes. Furthermore, as assessed by the accumulation of the fluorescent dye acridine orange, cells have not recovered the ability to acidify intracellular compartments during the time that dissociation occurs. The results support a model for the hepatic Gal receptor system, in which there are two functionally different receptor populations, recycling pathways, and ligand processing pathways. Monensin blocks dissociation of 125I‐ASOR from receptors in the major pathway completely. In the minor pathway dissociation proceeds to completion only after a lag. In this minor pathway monensin appears to temporarily delay a maturation or translocation process that must occur prior to dissociation. We conclude that the observed dissociation in the presence of monensin cannot be mediated by low pH, or by pH or pNa gradients.