bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sulfate transport in human lung fibroblasts (IMR‐90)
Authors: Elgavish Ada , Smith Jeffrey B. , Pillion Dennis J. , Meezan Elias 
Published: February 2005

AbstractSulfate transport in a fibroblast cell line derived from human lung (IMR‐90) occurred mainly via high‐ and low‐affinity, SITS‐sensitive pathways and to a lesser extent by an SITS‐insensitive mechanism. In low‐ionic‐strength media (sucrose substituted for salts) the apparent Km of the carrier‐mediated sulfate influx was 1 mM. At 0.3 mM, the sulfate concentration normally found in human serum, the contribution of the SITS‐insensitive pathway was negligible. In physiological salts solution, an SITS‐sensitive, high‐affinity (Km 34 ± 14 μM) sulfate influx system was observed at extracellular sulfate concentrations less than 100 μM. Between 100 and 500 μM sulfate, the range normally found in human serum, sulfate influx occurred via an SITS‐insitive, lowaffinity pathway and to a small extent by an SITS‐insensitive mechanism. Extracellular chloride inhibited the influx and stimulated the efflux of sulfate. Bicarbonate and thiosulfate inhibited sulfate influx but had no effect on sulfate efflux. Phosphate, arsenate, or Na+ did not affect sulfate uptake. These results indicate that in human lung fibroblast IMR‐90 cells sulfate is transported mainly via an SO42−/Cl‐ exchange system independent of the phosphate or Na+ transport. Since sulfate concentration as high as 50 mM only slightly increased sulfate efflux, SO42−/SO42− exchange is probably a minor component of sulfate uptake.
Expression of glucagon sensitivity by transformed MDCK cells normally unresponsive to glucagon: Early commitment to differentiation
Authors: Beckner Suzanne K. , Lin Michael C. 
Published: February 2005

AbstractA cloned line of canine kidney cells (MDCK) transformed with Harvey murine sarcoma virus, in contrast to the parental, untransformed line, expressed glucagon sensitivity only under controlled culture conditions. The glucagon sensitivity of transformed MDCK cells appeared after 10 days of culture if plated at less than 100,000 cells/dish or after 3 days if cells were plated at greater than 300,000 cells/dish. As there was no effect of conditioned medium from glucagon‐sensitive cells on insensitive cells, media components seemed not to be involved in this phenomenon. Glucagon sensitivity appeared more readily in defined as opposed to serum‐containing medium. In fact, as little as 2% fetal bovine serum inhibited the expression of glucagon sensitivity when included in defined medium over the course of the experiment. Furthermore, when transformed MDCK cells were exposed to serum for only the first 24 hr of culture, glucagon sensitivity on day 11 was identical to that of cells exposed to serum throughout the entire experiment. In contrast, exposure to serum later in culture (days 4–8) had no inhibitory effect on the expression of glucagon sensitivity on day 11. The data suggest that differntiation, or glucagon sensitivity, occurs when transformed, glucagon‐insensitive cells achieve a critical high density and that differentiation is sensitive to inhibition by serum only during the first 24 hr of culture.
Phosphorylation of rabbit skeletal muscle myosin in situ
Authors: Moore Russell L. , Houston Michael E. , Stull James T. , Iwamoto Gary A. 
Published: February 2005

AbstractMyosin light chain (P light chain) is phosphorylated by Ca2+. calmodulindependent myosin light chain kinase. Based on studies with rat skeletal muscles, it has been shown that P light chain phosphorylation correlated to the extent of potentiation of isometric twitch tension. It is not clear whether this correlation exists in rabbit skeletal muscle, which has been the primary source of contractile proteins for biochemical studies. Therefore, phosphorylation of myosin P light chain in rabbit slow–twitch soleus and fast‐twitch plantaris muscles in situ was examined. Electrical stimulation (5 Hz, 20 seconds) of plantaris muscle produced an increase in the phosphate content of P light chain from 0.17 to 0.45 mol phosphate/mol P light chain. This increase in phosphate content was accompanied by a 58% increase in maximal isometric twitch tension. Tetanic stimulation (100 Hz, 15 seconds) of rabbit soleus muscle resulted in only a small increase in P light chain phosphate content from 0.02 to 0.10 mol phosphate/mol P light chain, and posttetanic twitch tension did not increase significantly. The correlation between potentiated isometric twitch tension and P light chain phosphorylation in rabbit fast‐twitch muscle is similar to that observed in rat skeletal muscle. These results were consistent with the hypothesis that phosphorylation of rabbit skeletal muscle myosin, which results in an increase in actin‐activated ATPase activity, may be related to isometric twitch potentiation.
Subcellular localization of histone messenger RNAs on cytoskeleton‐associated free polysomes in HeLa S3 cells
Authors: Zambetti Gerard , Stein Janet , Stein Gary , Schmidt Warren 
Published: February 2005

AbstractWe have examined the subcellular distribution of histone mRNA‐containing polysomes in HeLa S3 cells to assess the possible relationship between localization of histone mRNAs and the regulation of cellular histone mRNA levels. The distribution of histone mRNAs on free and membrane bound polysomes was examined as well as the association of histone mRNA‐containing polysomes with the cytoskeleton. The subcellular localization of histone mRNAs was compared with that of HLA‐B7 mRNAs which encode a cell surface antigen. Histone mRNAs were localized predominantly on the free polysomes, whereas the HLA‐B7 mRNA was found almost exclusively on membrane bound polysomes. However, both species of mRNA were found associated with the cytoskeleton. Interruption of DNA synthesis by hydroxyurea treatment resulted in a rapid and selective destabilization of histone mRNAs in each subcellular fraction; in contrast, the stability of HLA‐B7 mRNA appeared unaffected. The results presented confirm that histone mRNAs are predominantly located on non‐membrane bound polysomes and suggest that these polysomes are associated with the cytoskeletal framework.
Rat fibroblasts expressing high levels of human c‐myc transcripts are anchorage‐independent and tumorigenic
Authors: Pellegrini Sandra , Basilico Claudio 
Published: February 2005

AbstractThe c‐myc oncogene has been implicated in the genesis of tumors as it has been found to undergo rearrangements and structural alterations in several types of neoplasms, However, the molecular mechanism of its involvement in transformation is still unclear as is the function of its protein product. We have studied the biological activity of the normal human c‐myc oncogene in a well‐characterized rat fibroblast line, F2408. The human gene, deleted of its 5′ non‐coding exon, was placed under the control of the Polyoma virus DNA regulatory region, linked to a dominant selectable marker (neomycin‐resistance gene) and transfected into F2408 cells. Several neoR colonies were assayed for ability to grow in agar suspension, and the steady‐state levels of mRNA transcripts of the exogenous c‐myc gene were analyzed by Northern blot. While the greatest majority of these cell populations are phenotypically normal and express low levels of human myc transcripts, the rare neoR clones which display anchorage‐independence express the human gene at high levels. This correlation was confirmed by the analysis of two neoR lines which contain low levels of c‐myc RNA but gave rise to agar colonies at a frequency of about 10−5. These agar derivatives were found to have greatly increased levels of c‐myc mRNA when compared to the respective parental lines. The clones expressing high levels of myc RNA were found to be tumorigenic upon inoculation into young syngeneic rats. These results suggest that constitutive high expression of the normal c‐myc gene is sufficient to confer anchorage‐independence and tumorigenic potential to established rat fibroblasts.
The effects of epidermal growth factor and the state of confluence on enzymatic activities of cultured rat liver epithelial cells
Authors: Tsao Ming‐Sound , Earp H. Shelton , Grisham Joe W. 
Published: February 2005

AbstractIn cultured normal rat liver epithelial cells, the specific activity and/or isozyme expression of NADH‐diaphorase (NADH‐D), pyruvate kinase (PK), glucose‐6 phosphate dehydrogenase (G6PD), gamma‐glutamyl transpeptidase (GGT), and alkaline phosphatase (AP) were markedly dependent on the growth state of the cultures. Proliferating, preconfluent cells had higher specific activities of PK, NADH‐D, and G6PD but lower activities of GGT and AP than did the more stationary confluent cells. Addition of epidermal growth factor [EGF] to the media of proliferating cells enhanced the specific activities of PK, NADH‐D, G6PD, GGT, and lactate dehydrogenase (LDH) of these cells, but the specific activity of AP was markedly depressed. The increase in activity of PK and GGT by EGF appeared to involve new protein synthesis, whereas the effect of EGF on AP appeared to involve the EGF‐directed suppression of the synthesis of a form of AP that is produced exclusively by cells in confluent cultures. Furthermore, the preconfluent cells were more responsive to the action of EGF on AP than were confluent cells, i.e., the EGF‐mediated decrease in AP activity was seen at lower concentration in preconfluent than in confluent cells. Paradoxically, confluent cells exhibited a two‐to threefold higher capacity to bind [125I]EGF because of an increase in surface receptor number. The results of this study indicate that enzymatic or other biochemical studies performed on cultured cells must take into account the growth‐state of the cultures. EGF can modulate enzyme activity in growing and nongrowing cells; one effect of EGF is to maintain higher activity of glycolytic enzymes, suggesting that EGF or EGF‐like factors may contribute to the high rate of glycolysis in certain neoplasms.
Reduced pinosome formation in a cell‐fusion‐impaired mutant of mouse lymphocytic cell line L5178Y
Authors: Pauw Peter G. , Biagi Karen G. , Stadler Joan K. 
Published: February 2005

AbstractAccumulation and release of a fluid‐phase marker ([14C]‐sucrose) were studied in a subline of the mouse lymphocytic cell line L5178Y and in a polyethylene glycol‐resistant, intercellular‐fusion‐impaired mutant of this line. The mutant was found to accumulate [14C]‐sucrose at a significantly slower rate than the parent. Analysis of release of preloaded label shows that the reduced rate of accumulation is due to a correspondingly low level of internalization, i.e., pinosome formation, rather than to a reduction in delivery of label to lysosomes. Cell‐to‐cell fusion and pinosome formation both involve a fusion event initiated at the extracellular surface of the plasma membrane, and we propose that the coordinate reduction in both processes suggests that they are mechanistically related.
Heat shock proteins within the mammalian cell cycle: Relationship to thermal sensitivity, thermal tolerance, and cell cycle progression
Authors: Rice Glenn , Laszlo Andrei , Li Gloria , Gray Joe , Dewey William 
Published: February 2005

AbstractWe have measured endogenous and induced rates of 70‐kD, 89‐kD, and 110‐kD heat shock proteins in highly pure G1‐, S‐, or G2‐M phase fractions of Chinese hamster fibroblasts (CHO) separated by fluorescence‐activated cell sorting (FACS). Relative rates of synthesis of all three polypeptides as measured by two‐dimensional gel electrophoresis were similar throughout the cell cycle, and therefore, endogenous levels were unlikely to explain the thermal sensitivity of S‐phase cells. Distinct heterogeneity in induced rates of these polypeptides was noted in all phase fractions. Enhanced rates of 70‐kD polypeptide were measured in S and G2‐M as compared to G1 following heat shock. Little increase in either the 89‐kD or 110k‐kD heat shock proteins was observed in heated G1 cells. This heterogeneity in induced rates of synthesis was in contrast to the similarity in thermal tolerance expression kinetics between each phase. Finally, enhanced synthesis of these polypeptides appeared unrelated to regulation of either heat‐induced cell cycle delay or to the resumption of phase‐specific progression after heat shock as measured by simultaneous flow cytometric measurement of incorporated BrdUrd and DNA content.
Binding and metabolism of leukotriene B4 by neutrophils and their subcellular organelles
Authors: O'Flaherty Joseph , Kosfeld Scott , Nishihira Jun 
Published: February 2005

AbstractThe subcellular distribution of leukotriene (LT)B4 binding and metabolizing sites was investigated in human neutrophils. Cells were disrupted by nitrogen cavitation and fractionated by Percoll density gradient centrifugation to yield cytoplasm, membranes, azurophilic granules, and specific granules. Only membrane fractions contained high affinity [3H]LTB4 binding sites. Binding of radiolabeled ligand to membranes was rapid, reversible, and saturable; it was blocked by a series of LTB4 analogues at concentrations corresponding to their respective potencies in (1) blocking [3H]LTB4 binding to whole cells and (2) stimulating neutrophil degranulation responses. In contrast, [3H]LTB4 was metabolized by fractions enriched with markers for cytoplasm plus endoplasmic reticulum. The metabolic activity was sedimented by ultracentrifugation, enhanced by NADPH, and inhibited at 4°C. The cell‐free system, like intact cells, metabolized [3H]LTB4 to ω‐oxidized product rapidly and quantitatively at 37°C but was inactive at 4°C. Whole cells converted radiolabel to 20‐hydroxy (∼ 30% of product) and 20‐carboxy (∼ 70% of product) derivatives; the cell‐free system formed principally 20‐hydroxy‐[3H]LTB4. These products were less bioactive than LTB4. Nevertheless, metabolism of LTB4 played little role in limiting the cells' response to the ligand: neutrophils completed degranulation and became desensitized to LTB4 within 3–5 min of exposure. Within this time frame, they oxidized less than 30% of the stimulus, and the extracellular fluid of these neutrophil suspensions was fully capable of activating fresh cells. We conclude that neutrophils transmit bioactions of LTB4 via a specific receptor integrally associated with their plasmalemma and/or endoplasmic reticulum. They inactivate the stimulus via a particulate ω‐oxidase. At the level of the individual cell, receptor down‐regulation, rather than ligand metabolism, appears to limit functional responses such as degranulation.
Heparin inhibits proliferation of fetal vascular smooth muscle cells in the absence of platelet‐derived growth factor
Authors: Benitz William E. , Lessler Daniel S. , Coulson John D. , Bernfield Merton 
Published: February 2005

AbstractProliferation of smooth muscle cells from the pulmonary arteries and aortas of fetal calves is inhibited by heparin in vitro. This effect is reversible and dose dependent. Comparisons with effects of other polysaccharides indicate that only extensively sulfated polysaccharides inhibit proliferation of smooth muscle cells but that specific structural features of heparin are required to achieve maximum effect. Heparin‐Sepharose chromatography of medium containing fetal calf serum reduces the ability of that medium to promote growth of smooth muscle cells from fetal pulmonary arteries, suggesting that heparin may remove soluble growth factors in serum. However, inhibition of fetal pulmonary artery smooth muscle cell proliferation by heparin is identical in media supplemented either with serum prepared from fetal calf plasma, in which platelet‐derived growth factor (PDGF) is not detectable, or with fetal calf serum, which contains relatively abundant PDGF (114 pg/ml). Thus, inhibition of fetal pulmonary artery smooth muscle cell proliferation by heparin is not mediated solely by decreased availability or activity of exogenous PDGF. These studies suggest that morphogenesis of the smooth muscle investment of the pulmonary arteries could be regulated by local production of heparinlike inhibitors of smooth muscle cell growth.
Effects of retinoids on human bronchial epithelial cells: Differential regulation of hyaluronate synthesis and keratin protein synthesis
Authors: Wu Reen , Wu Mary M. J. 
Published: February 2005

AbstractRespiratory tract epithelia are one type of tissue targeted by vitamin A. In this study the effects of vitamin A and its analogs (retinoids) on human bronchial epithelial (HBE) cells have been investigated in a serum‐free hormone‐supplemented medium. This serum‐free medium, which was developed for the longterm cultivation of protease‐dissociated HBE cells, consists of Ham's F12 nutrient medium supplemented with insulin, transferrin, epidermal growth factor, hydrocortisone, cholera toxin, and bovine hypothalamus extract. Under these in vitro conditions, retinoids specifically stimulate the synthesis and secretion of hyaluronate (HA) and alter the pattern of synthesis of keratin proteins. In regard to HA, the degree of stimulation ranges from two‐fold to ten‐fold and is concentration dependent. In regard to keratin proteins, the most prominent effects of retinoids are inhibition of synthesis of the 48 kd and 50 kd keratin proteins (corresponding to cytokeratins 16 and 14, respectively, in the catalog of human cytokeratins; Moll et al., 1982) and stimulation of synthesis of the 40 kd and 52–54 kd proteins. The data indicate that retinoid effects on HA and keratin protein synthesis occur at different levels. The stimulation of HA synthesis occurs immediately after the addition of retinoid and cannot be prevented by pretreatment with actinomycin D, whereas the alterations in the pattern of keratin protein synthesis appear later and are inhibited by treatment with actinomycin D at or before the administration of retinoid. This study demonstrates that HBE cultures maintained in the serum‐free condition can serve as an in vitro model to elucidate the mechanisms of retinoid actions.
Heparin potentiation of 3T3‐adipocyte stimulated angiogenesis: Mechanisms of action on endothelial cells
Authors: Castellot John J. , Kambe Arbetta M. , Dobson Deborah E. , Spiegelman Bruce M. 
Published: February 2005

AbstractWe have examined the cellular mechanisms by which heparin potentiates the ability of 3T3‐adipocytes to stimulate the formation of new blood vessels. Both anticoagulant and non‐anticoagulant heparin species enhanced the angiogenic activity of adipocyte‐secreted products in the chick chorioallantoic membrane assay, indicating that the angiotropic effect of this glycosaminoglycan is independent of its effect on the coagulation cascade. Heparin alone was unable to produce a neovascular response. The ability of heparin to modulate three endothelial functions in vitro thought to be related to angiogenesis were examined: protease activity, motility, and mitogenesis. Heparin caused a 100% increase in the adipocyte‐induced stimulation of endothelial cell plasminogen activator activity and motility, but had no effect on proliferation. The enhancement of plasminogen activator and chemoattractant activities had a similar ED50 (1‐2 μg/ml) and optimum dose (10‐30 μg/ml). When we examined the direct effect of heparin on the activity of two distinct plasminogen activator enzymes–urokinase and tissue‐type–a dual action of heparin was observed: tissue‐type enzyme activity was stimulated 100% by heparin at 10 μg/ml, whereas urokinase activity was inhibited by 77% at this dose. These data suggest that heparin potentiates angiogenesis in vivo by stimulating endothelial cell plasminogen activator, motility, or both. Our results further suggest that for adipocyte‐induced blood vessel formation, in contrast to other angiogenesis systems, heparin does not appear to affect the mitogenic activity.
Fetuin: A serum component associated with rat sertoli and spermatogenic cells in coculture
Authors: Abdullah Munir , Crowell James A. , Tres Laura L. , Kierszenbaum Abraham L. 
Published: February 2005

AbstractCocultures of rat Sertoli‐spermatogenic cells plated in a culture medium supplemented with 10% fetal bovine serum for 6–12 h and then maintained in serum free, hormone/growth factor‐supplemented medium accumulated an acidic glycoprotein of molecular weight of 68,000 dalton (68 kD) and isoelectric point range of about 4.2–3.5. Anion exchange chromatography has allowed the partial purification of this protein, which consists of a major protein band of 68 kD and two minor, low molecular weight components. A rabbit antiserum raised against the 68 kD component also crossreacts with the two low molecular weight components, thus suggesting that these two minor components are antigenically related to the 68 kD protein. The 68 kD protein has been identified as fetuin, the major component of fetal bovine serum, based on similar molecular weight, isoelectric point, immunoreactivity and trypsin inhibitory activity. Labeling experiments with [14C]amino acid mixture show that 68 kD protein is not synthesized by cocultured rat Sertoli and spermatogenic cells. Immunocytochemistry and Western blot approaches carried out under various experimental conditions support the view that the fetuin‐68 kD protein is taken up from serum by both Sertoli cells and pachytene spermatocytes. Because fetuin (1) behaves as a carrier protein for growth factors, (2) has protease inhibitory activity, (3) is preferentially internalized by Sertoli cells and pachytene spermatocytes and (4) fetal bovine serum‐supplemented medium impairs spermatogenic cell viability, there is a need to further define appropriate conditions for optimizing long‐term viability and differentiation of spermatogenic cells in vitro.
Effects of bacterial lipopolysaccharide on protein synthesis in murine peritoneal macrophages: Relationship to activation for macrophage tumoricidal function
Authors: Hamilton Thomas A. , Jansen Marilyn M. , Somers Scott D. , Adams Dolph O. 
Published: February 2005

AbstractEarly biochemical events in the response of murine peritoneal macrophages to bacterial lipopolysaccharide (LPS) have been examined (i.e., 0–4 hr after initiation of treatment). At concentrations of 10 ng/ml or less, LPS stimulated the new or enhanced synthesis of a series of at least six polypeptides of 85, 80, 75, 65, 57, and 38 kD. This effect was dependent upon the lipid A moiety of LPS as lipid A itself could induce the changes and the effect of LPS could be blocked by inclusion of polymixin B sulfate in the culture medium. The effect was specific for LPS in that other endotoxin‐free agents known to alter macrophage physiology could not produce the same changes. The time course of LPS stimulation of macrophage protein synthesis was remarkable in that the synthesis of all six proteins was transient even in the continued presence of LPS, being first detected approximately 1 hr after exposure and no longer apparent by 8–10 hr after treatment was initiated. Furthermore, both pulse‐chase and cumulative radiolabeling studies indicated that at least two of the proteins (85 and 38 kD) were short‐lived and did not accumulate in LPS‐treated cells, suggesting the possibility that they participate in a regulatory rather than a functional role. Macrophage tumoricidal activation involves cooperation in response to two independent signals; interferon gamma and LPS. Pretreatment of macrophages with interferon gamma increased the sensitivity of macrophages to LPS‐stimulated protein synthesis by one to two orders of magnitude documenting such cooperativity in molecular terms. The LPS‐induced stimulation of specific protein synthesis could be reproduced by treatment of macrophages with heat killed Listeria monocytogenes, a gram‐positive, endotoxin‐negative bacterial stain which has been shown to substitute effectively for LPS in macrophage tumoricidal activation. Furthermore, reversible inhibition (i.e., treatment with cycloheximide) of protein synthesis during LPS treatment abrogated the acquisition of tumoricidal function. These results identify an early biochemical response to LPS which may be a necessary component of the intracellular transduction of signals which regulate macrophage functional development.