bluuebunny/crossref_metadata_2025_split huggingface dataset:
Detection of colony‐stimulating factor messenger RNA in single T cells by in situ hybridization
Authors: Williamson D. James , Owens Trevor , Pearse Martin 
Published: February 2005

AbstractIn situ hybridization has been used to study the accumulation of colonystimulating factor (CSF) mRNA in single cells of a T lymphocyte clone (E9.D4) following antibody‐mediated (F23.1) activation via the Ti‐T3 complex in fillerindependent bulk cultures. The specificity of hybridization for cellular RNA was demonstrated by pretreating the cells with the Ca2+‐dependent enzyme micrococcal nuclease by using a novel protocol developed for use with riboprobes. Maximal levels of granulocyte‐macrophage (GM) and multipotential‐CSF (interleukin 3) mRNA were detected after 8–10 h, with GM‐CSF mRNA being detected earlier and at a lower concentration of stimulus. The rise in intracellular mRNA was accompanied by an increase in the corresponding CSF bioactivity in the supernatant. In situ hybridization was of comparable sensitivity to Northern blot analysis and revealed significant heterogeneity in the accumulation of CSF mRNA within individual cells of the clone following stimulation with F23.1. This could account for the corresponding heterogeneity in CSF production by single cells. Under optimal conditions at least 25% of cells contained both transcripts. The method has been used to examine CSF production by normal spleen cells and should be useful in the further analysis of lymphokine gene regulation in single T cells.
Effects of recombinant human granulocyte‐macrophage colony‐stimulating factor (GM‐CSFrh) on transmembrane electrical potentials in granulocytes: Relationship between enhancement of ligand‐mediated depolarization and augmentation of superoxide anion (O) production
Authors: Sullivan Richard , Fredette Joseph P. , Leavitt Julie L. , Gadenne Anne‐Sophie , Griffin James D. , Simons Elizabeth R. 
Published: February 2005

AbstractWhen human granulocytes that have been primed with recombinant human granulocyte–macrophage colony‐stimulating factor (GM‐CSFrh) are activated by ligands that stimulate the respiratory burst, the amount of superoxide anion (O−2) they generate is significantly increased. We have found that the accelerated rate of O−2 release occurring under these conditions is accompanied by an antecedent increase in membrane depolarization. We examined the nature of the enhancement of membrane depolarization in GM‐CSFrh‐primed granulocytes and investigated its relationship to the increase in O generation by N‐formyl methionylleucylphenylalanine (fMLP)‐activated granulocytes. We found that augmented depolarization could not be accounted for by a change in the resting membrane potential induced by the growth factor and was still present after either blocking passive transmembrane Na+ movement with dimethylamiloride or by increasing the membrane's permeability to K+ with valinornycin. When their ability to depolarize was virtually eliminated by dissipating the transmembrane K+ gradient, GM‐CSFrh‐pretreated cells continued to generate more O after fMLP than did control cells. These results indicate that augmentation of the granulocyte's ability to generate O anions, which is induced by priming with GM‐CSFrh, is independent both of the resting transmembrane potential and of alterations in the extent of membrane potential change induced by stimuli such as fMLP.
Centriole ciliation and cell cycle variability during G1 phase of BALB/c 3T3 Cells
Authors: Ho Peter T. C. , Tucker Robert W. 
Published: February 2005

AbstractAlthough variability in the duration of the cell cycle is thought to reflect growth‐regulatory processes that control cell cycle progression, the precise timing of the variable period within the GI phase of the cell cycle has not been defined. In particular, the timing of cell cycle variability in relation to the cell's commitment (R point) to the initiation of DNA synthesis remains controversial. In order to investigate cell cycle variability, indirect immunofluorescence was used to measure the formation of the primary cilium as a possible marker of G1 events in both stimulated quiescent and exponentially growing cells. The primary cilium, an internal “9 + 0” nonmotile structure formed by one of the interphase centrioles, was first detected in postmitotic BABL/c 3T3 cells 5 hr before the initiation of DNA synthesis, an interval similar to that for the reassembly of the primary cilium in serum‐stimulated quiescent fibroblasts. This similarity in the timing of ciliation suggests that serum‐stimulated quiescent cells reenter the cell cycle in early G1 and recapitulate much of G1. Moreover, the rate of cilia formation in both postmitotic and serum‐stimulated quiescent cells was identical to the rate of DNA synthesis initiation. Thus, cell cycle variability occurs before ciliation in both stimulated quiescent and exponentially growing cells. Furthermore, since ciliation also precedes the R point, variability in the centriole cycle occurs before the R point and thus may reflect processes controlling the cell's commitment to the initiation of DNA synthesis.
Cell cycle regulation of human diploid fibroblasts: Possible mechanisms of platelet‐derived growth factor
Authors: Larsson Olle , Latham Carolyn , Zickert Peter , Zetterberg Anders 
Published: February 2005

AbstractCell‐cycle regulation of human diploid fibroblasts (HDF) is located in the proximal half of G1, designated G1‐pm (G1‐postmitosis). In order to traverse this subphase, cells require serum factors or PDGF. However, when cells have traversed into the distal half of G1, designated G1‐ps (G1‐pre‐DNA synthesis), they become independent of serum or PDGF and progress through the remainder of the cell cycle at an invariable rate. From this, it follows that a specific G1‐pm block can be induced by serum depletion. A similar G1‐pm block could also be induced by a moderate inhibition of overall protein synthesis following treatment with CHM. Even this block could be prevented by the addition of PDGF, suggesting that a high level of protein synthesis in itself is not necessary for sustaining cell‐cycle traverse. Nevertheless, a critical accumulation of some specific proteins might be required for the G1‐pm/G1‐ps‐transition. However, the underlying mechanisms of modulation of the accumulation of such proteins by PDGF must involve alternative regulatory events (e.g., gene expression, protein stabilization) rather than protein synthesis. Among the possible cell cycleregulatory proteins, the present study focused on 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG CoA) reductase. This enzyme is regulated by various kinds of control mechanisms and regulates the biosynthesis of sterols and nonsterol isoprenes, some of which are proposed to be necessary for mammalian cell growth (Brown and Goldstein, 1980). The present results suggest that regulation of HMG CoA reductase may be involved in the control of the G1‐pm/G1‐ps‐progression in HDF.
Serum‐free mouse embryo cells: Growth responses in vitro
Authors: Loo Deryk , Rawson Cathleen , Helmrich Angela , Barnes David 
Published: February 2005

AbstractWe have derived serum‐free mouse embryo (SFME) cultures in a basal nutrient medium supplemented with insulin, transferrin, epidermal growth factor (EGF), high‐density lipoprotein (HDL), and fibronectin. These cells are nontumorigenic, lack gross chromosomal aberrations, and exhibit several other unique properties, including dependence on EGF for survival and growth inhibition by serum. We have examined the concentration dependence of the growth stimulatory effects of protein supplements used in the SFME medium formulation and surveyed other supplements that might act as alternative or complementary additions to the culture medium. Insulin could be replaced by insulin‐like growth factor I and EGF could be replaced by transforming growth factor alpha in the same concentration range. Transferrin could be replaced by higher concentrations of lactoferrin. Deterioration of cultures in the absence of EGF began within 8 hours of the removal of the growth factor, and could be prevented by the addition of fibroblast growth factor/heparin‐binding growth factor. Attachment proteins other than fibronectin were effective on SFME cells, but limited success was obtained when substituting other lipid preparations for HDL. These data introduce a precise system for exploring the unusual characteristics of SFME cells and contribute additional information that may be useful in the extension of these approaches to other cell types and species.
Frequency dependence of increased cell proliferation, in vitro, in exposures to a low‐amplitude, low‐frequency electric field: Evidence for dependence on increased mitogen activity released into culture medium
Authors: Fitzsimmons Robert J. , Farley John R. , Adey W. R. , Baylink David J. 
Published: February 2005

AbstractIn order to investigate the mechanism(s) through which an electric field can increase bone cell proliferation, we have developed an in vitro model incorporating a low‐amplitude (estimated 10−7 V/cm in the serum‐free culture medium), low‐frequency, capacitively coupled electric field. In previous studies with this model, we have shown that electric field exposure can increase bone cell proliferation both in chick tibiae organ cultures and in calvaria‐derived monolayer cell cultures. The current in vitro studies demonstrate that skeletal tissue responses to a 30 min electric field exposure are characterized by (a) a frequency window for both increased cell proliferation and increased release of mitogen activity into the cell‐conditioned medium, with a peak near 16 Hz; (b) a dependence on conditioned medium from exposed cells for increased cell proliferation; and (c) a correlation between the alkaline phosphatase content of the bone cell cultures and effects of electric field exposure on both cell proliferation and release of mitogen activity into the conditioned medium.
Prolactin is transported across the epithelium of the jejunum and ileum of the suckling rat
Authors: Gonnella Patricia A. , Harmatz Paul , Walker W. Allan 
Published: February 2005

AbstractMilk prolactin is transferred from the gastrointestinal tract to the circulation of the suckling rat. To identify the site of prolactin penetration and to determine the mechanism by which the hormone traverses the mucosal barrier, we followed the uptake of prolactin from ligated loops of jejunum or ileum in vivo by three methods: autoradiography, transport of prolactin‐gold conjugates, and immuno‐cytochemistry. Autoradiographic studies demonstrated specific binding sites for 125l‐prolactin on apical membranes of the jejunum and ileum. Excess cold pro‐lactin reduced radiolabel in apical and basal compartments. Gel autoradiography of portal sera showed the presence of intact prolactin and a prolactin fragment following jejunal transport but only a prolactin fragment following ileal transport. Uptake of prolactin‐gold conjugates demonstrated that, in the jejunum, label was present at the luminal surface, within endosomal compartments and lysosomes, in basal coated and smooth vesicles, within basal coated pits, and beyond the basolateral surface. In the ileum, label was found at the luminal surface; within the tubulocisternae, endosomal vesicles, lysosomes, and basal smooth vesicles; and beyond the basolateral surface. Immunoreactive prolactin was present throughout the transepithelial pathways. This study demonstrates that prolactin is selectively and nonselectively absorbed in the jejunum and ileum and that the hormone is directed either to the lysosome for degradation or across the epithe‐lium by means of a transcellular pathway.
Characterization of transforming growth factors produced by the insulin‐independent teratoma‐derived cell line 1246‐3A
Authors: Yamada Yukio , Serrero Ginette 
Published: February 2005

AbstractThe 1246‐3A cell line is an insulin‐independent variant derived from the adipogenic cell line 1246. Data presented in this paper indicate that the 1246‐3A cell line releases in its culture medium two types of transforming growth factors, TGF‐α and TGF‐β‐like polypeptides, and a growth inhibitor. TGF‐α like polypeptide eluted from Biogel P60 column into two fractions with an apparent molecular weight of 50 kDa and 13 kDa. These high‐molecular‐weight TGF‐α‐like factors competed with 125l‐EGF for binding to epidermal growth factor (EGF) receptors and were specifically immunoprecipitated by incubation with antirat TGF‐α antibody, not by incubation with anti‐EGF antibody. Both fractions promoted anchorage‐independent growth of normal rat kidney NRK cells in the absence of EGF and stimulated DNA synthesis in quiescent Balb/c‐3T3 cells in a fashion similar to EGF and synthetic TGF‐α. In addition to secreting TGF‐α‐like polypeptides, 1246‐3A cells produce TGF‐β. This polypeptide, eluted from Biogel P60 chromatography with an apparent molecular weight of 25 kDa, promoted anchorage‐independent growth of NRK cells in the presence of EGF and was growth inhibitory for Chinese hamster lung fibroblasts CCL 39 cells. Interestingly, another growth inhibitory activity was detected in Biogel P60 fractions and eluted with an apparent molecular weight of between 9.5‐11 kDa. This fraction was different from TGF‐β and TGF‐α as determined by specific radioreceptor competition assays. TGF‐α and TGF‐β‐like polypeptides could represent autocrine growth stimulators for the insulin‐independent 1246‐3A cells and act in synergy with insulin‐related factor (IRF) for an optimal stimulation of 1246‐3A cell proliferation in serum‐free medium.
Effect of volume and pH on surface receptor number in macrophages
Authors: Buys Saundra S. , Novak Jeanne M. , Gren Lisa H. , Kaplan Jerry 
Published: February 2005

AbstractIncubation of rabbit alveolar macrophages in hypo‐osmotic solutions transiently increases cell volume and inhibits membrane internalization, resulting in an increase in surface receptor number. Since recent reports suggest that hypo‐osmotic treatment decreases intracellular pH, and that reduced pH inhibits receptor internalization, pH was measured in hypo‐osmotically treated macro‐phages. We found that cells incubated in iso‐osmotic solutions of pH less than 7.2 exhibited a decrease in intracellular pH upon exposure to hypo‐osmotic solutions, while cells in iso‐osmotic solutions of pH greater than 7.2 had an increase in pH upon exposure to hypo‐osmotic solutions. The relative increase in surface receptor number was unaffected by the initial pH or by the direction of change in pH. Incubation of cells in high K+/low Na+ hypotonic buffers induced a persistent increase in cell volume and surface receptor number. Cell volume and surface receptor number fell to baseline values after restoration of isotonicity by the addition of hypertonic sucrose. These manipulations had little effect on intracellular pH. We conclude that the inhibition of membrane internalization observed in cells exposed to hypo‐osmotic solutions is independent of changes in intracellular pH. The inhibition of internalization observed in this system may be due directly to forces produced as a consequence of cell swelling.
Production of transforming growth factor beta by human peripheral blood monocytes and neutrophils
Authors: Grotendorst Gary R. , Smale Georgeann , Pencev Dobromir 
Published: February 2005

AbstractHuman peripheral blood monocytes and neutrophils secrete TGF‐beta. Activation of monocytes with LPS stimulates the secretion of TGF‐beta; however, the production of TGF‐beta by neutrophils was not altered by treatment with LPS. The secreted TGF‐beta appears to be in a fully active form since acid treatment of the conditioned medium does not increase the amount of TGF‐beta activity. TGF‐beta 1 transcripts were detected at similar levels in both activated and nonacti‐vated monocytes and neutrophils, suggesting that the increase in TGF‐beta secretion after activation of monocytes is regulated by a posttranscriptional mechanism. Western blot analysis with anti‐N‐terminal TGF‐beta 1 peptide antibodies indicate the leukocyte‐derived TGF‐beta is beta 1. In addition, TGF‐beta 1 transcripts were detected in rat peritoneal macrophages and in a differentiating human hematopoietic tumor cell line (HEL). The ability of inflammatory cells such as monocytes and neutrophils to produce TGF‐beta may play an important role in the function of these cells in wound repair, in the immune response, and in the pathogenesis of fibrotic diseases.
Oxygen gradients in CHO cells: Measurement and characterization by electron spin resonance
Authors: Glockner James F. , Swartz Harold M. , Pals Mark A. 
Published: February 2005

AbstractThe concentration of oxygen within cells is important in many physiological and pathological processes, but such oxygen‐dependent processes are generally studied as a function of the concentration of extracellular oxygen, due to a lack of suitable methods. Using a newly developed technique based on ESR spectroscopy, we show that respiration stimulation of a cell suspension can result in a significant difference between average intracellular and extracellular concentrations of oxygen. These results indicate that studies of oxygen‐dependent phenomena in cells may require measurement of intracellular oxygen concentrations and imply that there are mechanisms in cells that restrict the free diffusion of oxygen.
Both homodimeric isoforms of PDGF (AA and BB) have mitogenic and chemotactic activity and stimulate phosphoinositol turnover
Authors: Hosang Markus , Rouge Marianne , Wipf Beat , Eggimann Bernhard , Kaufmann Franz , Hunziker Willi 
Published: February 2005

AbstractPlatelet‐derived growth factor (PDGF) occurs as three dimeric isoforms, AA, BB, and AB, which were previously shown to bind to two receptors with different isoform‐specificity, the A/B‐type (binds all three isoforms) and the B‐type (binds only PDGF‐BB). Results from competition binding experiments with Swiss 3T3 cells suggest the existence of a third receptor type, which recognizes PDGF‐AB and PDGF‐BB. Furthermore, Swiss 3T3 cells and human dermal fibroblasts express different relative and absolute levels of these receptor types. In particular, Swiss 3T3 cells express 90,000 PDGF‐AA binding sites (A/B‐receptors) per cell, whereas human fibroblasts express only 20,000 A/B‐receptors per cell. All three PDGF isoforms were tested in either cell type for their effect on DNA synthesis. PDGF‐BB and PDGF‐AA were also tested in Swiss 3T3 cells for their effect on inositol phospholipid metabolism and chemotaxis. Each isoform promoted all three processes dose‐dependently, but there were differences in the maximum cellular responses elicited. These responses reflect the capacity of the cells to bind the individual isoforms. These results demonstrate that the previous distinctions in responsiveness to the different PDGF isoforms are primarily a consequence of the differences in the levels of surface expression of the various isoform‐specific receptor types, rather than of the differences in the intrinsic activity of these isoforms. Furthermore, these results suggest that all types of PDGF receptors are capable of responding to their respective ligands by mediating phosphoinositide breakdown, chemotactic responses, and DNA synthesis. Whether they exhibit other functional differences remains to be seen.
Cytotoxicity of gelonin and its conjugates with antibodies is determined by the extent of their endocytosis
Authors: Goldmacher Victor S. , Scott Charles F. , Lambert John M. , McIntyre Gordon D. , Blättler Walter A. , Collinson Albert R. , Stewart Jean K. , Chong Lisa D. , Cook Sherrilyn , Slayter Henry S. , Beaumont Elisabeth , Watkins Simon 
Published: February 2005

AbstractConjugates of the single‐chain ribosome‐inactivating protein gelonin with ligands that bind to cell surface molecules vary greatly in their cytotoxicity. Conjugates that are not endocytosed after binding to cells exhibit low cytotoxicity similar to that of free gelonin, while conjugates that are endocytosed demonstrate enhanced cytotoxicity relative to free gelonin. However, the number of internalized gelonin molecules needed to intoxicate cells to the same degree has been found to be similar for all conjugates and for free gelonin. The intracellular concentration of gelonin has to be between 2,000–10,000 molecules/cell to achieve a surviving fraction of 0.37. Our studies revealed the presence of three distinct categories of cell surface molecules, those that are efficient in mediating endocytosis of im‐munotoxins, those that are only moderately efficient, and those that seem not to cause internalization of bound immunotoxins.
Subcellular distribution of protein kinase C/phorbol ester receptors in differentiating mouse keratinocytes
Authors: Isseroff R. Rivkah , Stephens Lynn Enders , Gross Janet L. 
Published: February 2005

AbstractThe activation of protein kinase C (PKC) by diacylglycerol or tumor promoters plays a pivotal role in signal transduction and subsequent activation of cellular processes. Since the activity of this enzyme is dependent on its immediate lipid domain, its relative distribution within the cell may be an important regulatory mechanism. We report here a relative decrease in PKC/phorbol ester receptor associated with the particulate fraction of mouse keratinocytes induced to differentiate by two separate systems. First, proliferating keratinocytes maintained in low Ca2 + (0.09 mM) serum‐free medium were induced to differentiate rapidly by the addition of Ca2+ (1.8 mM). A 1.4‐fold decrease in the percent of total phorbol receptor binding activity present in the particulate fraction and concomitant increase in binding in the cytosol fraction was evident 20 min after the Ca2 + addition. Second, in keratinocytes that differentiate over a 6 day cultivation period in serum‐containing medium with Ca2+ concentration of 1.8 mM, a significant decrease in the percent of the phorbol receptor binding activity present in the particulate fraction was observed as the culture begins to differentiate on days 3 and 4. Maximal phorbol ester binding in the particulate fraction corresponded to the proliferative phase of the culture (day 2), while lower levels of PKC/phorbol ester binding to particulate fractions were noted during the early differentiative phase (days 3 and 4). Addition of the synthetic diacylglycerols 1‐oleoyl‐2‐acetyl‐glycerol or L‐α‐1,2 dioctanyl glycerol at 30 μg/ml to proliferating keratinocyte cultures induced a modest increase in two markers of terminal differentiation: cornified envelope formation and transglutaminase levels. These findings, taken together, support the hypothesis that PKC activation plays a role in the initial signalling events for keratinocyte differentiation.