bluuebunny/crossref_metadata_2025 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

In 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

When 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

Although 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

Cell‐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

We 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

In 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

Milk 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

The 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

Incubation 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

Human 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

The 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

Platelet‐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

Conjugates 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

The 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.
Vasopressin rapidly stimulates protein kinase C in digitonin‐permeabilized swiss 3T3 cells: Involvement of a pertussis toxin‐insensitive guanine nucleotide binding protein
Authors: Erusalimsky Jorge D. , Rozengurt Enrique
Published: February 2005

Guanine nucleotides and pertussis toxin were used to test for the involvement of a guanine nucleotide binding protein in the vasopressin V1 receptor‐mediated stimulation of protein kinase C activity in Swiss 3T3 cells. Addition of vasopressin in the presence of [γ‐32P]ATP and digitonin caused a marked and rapid increase (8 ± 1‐fold after 1 min) in the phosphorylation of an Mr = 80,000 cellular protein (80K), a specific marker for protein kinase C activation. This phosphorylation was selectively blocked by the V1 receptor antagonist Pmp1‐0‐Me‐Tyr2 [Arg8]vasopressin, indicating that the effect was mediated through the vasopressin V1 receptor. Down regulation of protein kinase C by prior prolonged pretreatment of intact cells with phorbol 12, 13‐dibutyrate (PBt2) blocked the ability of vasopressin to stimulate the phosphorylation of 80K in digitonin‐permeabilized cells. Addition of a submaximal concentration of vasopressin together with the GTP analogue GTP‐γ‐S caused a synergistic stimulation of 80K phosphorylation. The GDP analogue GDP‐β‐S caused a 50% inhibition of the phosphorylation of 80K induced by a saturating concentration of vasopressin and shifted the vasopressin dose‐response curve to the right. GDP‐β‐S had no effect on the dose‐response for the stimulation of 80K phosphorylation induced by PBt2. Prior incubation of intact quiescent cultures of Swiss 3T3 cells with pertussis toxin did not impair either vasopressin‐induced increase in cytosolic [Ca2+] or activation of protein kinase C. These findings provide functional evidence for the involvement of a pertussis toxin‐insesitive G protein in the vasopressin V1 receptor‐mediated stimulation of protein kinase C in Swiss 3T3 cells.
Evidence for increased αMSH receptor binding in the F1 variant of B16 melanoma cells grown in dialyzed fetal calf serum
Authors: McQuillan A. , Hutchinson M. , Panasci L. C.
Published: February 2005

The specific binding of an αMSH analogue (Ac‐[Nle4, D‐Phe7] αMSH4–11 NH2) was enhanced in the presence of 10% dialyzed fetal calf serum (FCS) as compared with 10% FCS (nondialyzed) in the F1 variant of B16 melanoma cells. The replenishment of dialyzed serum with adrenocorticotrophic hormone (ACTH) or insulin had no effect on the increased level of αMSH receptor binding in these cells. However, 10 nM αMSH or 1 μM ACTH under identical conditions significantly decreased the level of αMSH binding. Competitive binding studies involving the αMSH analogue revealed that the specificity of the receptor was restricted to the complete molecule of αMSH, our analogue, βMSH and ACTH1–24. ACTH4–10, which contains the amino acid sequence responsible for biological activity, showed a very low affinity for the receptor. Furthermore, we observed an interesting phenomenon unique to dialyzed FCS in that once the cells were grown to confluence and melanin was produced, the cells were no longer viable. However, in McCoy's medium, which is deficient in tyrosine, the cells did not produce melanin and remained viable.
Kinetics of insulin internalization and processing in adipocytes: Effects of insulin concentration
Authors: Jochen Albert , Hays Judith , Lee Martha
Published: February 2005

We have studied the effect of insulin concentration on the kinetics of insulin internalization and efflux in isolated rat adipocytes. To determine internalization rates adipocytes were incubated with 125l‐insulin at 37°C; and at frequent, early time points surface‐bound and intracellular insulin were quantitated. Surface‐bound and intracellular insulin were discriminated by the sensitivity of the former to rapid dissociation by a pH 3.0 buffer at 4°C. From this data the endocytotic (internalization) rate constant (ke) was calculated for six insulin concentrations ranging from 0.3 to 100 ng/ml. Ke was found to decrease in an insulin concentration‐dependent manner (P < .001). Thus, values for ke were 0.121 ± 0.006 min−1 versus 0.074 ± 0.011 min−1 at 0.3 ng/ml and 100 ng/ml, respectively. The decrease in ke did not parallel insulin concentration‐dependent changes in insulin receptor affinity indicating it was not the result of an inability of low affinity receptors to be internalized. The kinetics of insulin efflux were determined by loading various concentrations of 125l‐insulin into the adipocyte interior, washing away surface‐bound and extracellular insulin, and then monitoring the subsequent efflux of pre‐loaded insulin into medium that contained the same concentration of insulin used in the loading step. The overall rate of efflux was independent of insulin concentration. In summary, these results show that at high insulin concentrations the efficiency of insulin internalization is impaired. In contrast, the rate of insulin efflux is unaffected.
Cell cycle dependent growth factor regulation of gene expression
Authors: Morgan Claudia J. , Pledger W. J.
Published: February 2005

The expression of the proto‐oncogenes c‐fos and c‐myc is a rapid response of G0‐arrested fibroblasts to serum and peptide growth factors; however, the role of the c‐fos and c‐myc gene products in subsequent cell cycle transit is not understood. We examined the expression of c‐fos and c‐myc mRNA in Balb/c 3T3 murine fibroblasts in response to platelet‐derived growth factor (PDGF) and platelet‐poor plasma, using arrest points associated with density dependent growth inhibition or metabolic inhibition to synchronize cells in S phase of the cell cycle. The expression of c‐fos and c‐myc mRNA in Balb/c 3T3 cells was differentially regulated with respect to growth factor dependence and cell cycle dependence, c‐fos expression was induced in the presence of PDGF and was unaffected by plasma. The induction of c‐fos expression in response to PDGF was cell cycle independent, occurring in cells transiting S phase and G2 as well as in G0 arrest. In contrast, c‐myc expression was both growth factor and cell cycle dependent. In G0 arrested cells, c‐myc expression was PDGF‐dependent and plasma‐independent, and PDGF was required for maintenance of elevated c‐myc levels during G1 transit. In cells transiting S phase, c‐myc mRNA was induced in response to PDGF, but was also plasma‐dependent in S phase cells that had been “primed” by exposure to PDGF during S phase.
Activation and proliferation signals in murine macrophages: Relationships among c‐fos and c‐myc expression, phosphoinositide hydrolysis, superoxide formation, and DNA synthesis
Authors: Hamilton John A. , Veis Nurin , Bordun Anne‐Marie , Vairo Gino , Gonda Thomas J. , Phillips Wayne A.
Published: February 2005

Murine bone marrow‐derived macrophages (BMM) undergo DNA synthesis in response to growth factors such as colony stimulating factor‐1 (CSF‐1) and granulocyte‐macrophage CSF (GM‐CSF). These macrophages can also be “activated,” but without subsequent DNA synthesis, by a number of other agents, including lipopolysaccharide (LPS), concanavalin A, zymosan, formyl‐methionyl‐leucylphenylalanine (FMLP), and the Ca2+ ionophore, A23187. When BMM are treated with a range of stimuli, there is some, although not perfect, correlation between transient elevations in both c‐myc mRNA and c‐fos mRNA levels and increases in DNA synthesis. However, enhanced DNA synthesis and oncogene expression are readily dissociated from rises in inositol phosphates and, by implication, phospholipase C‐mediated hydrolysis of phosphatidyl inositol 4,5‐bisphosphate. Superoxide formation in BMM can also be dissociated from the other responses and does not necessarily depend on protein kinase C activation.
Appearance of basic fibroblast growth factor receptors upon differentiation of rat mammary epithelial to myoepithelial‐like cells in culture
Authors: Fernig David G. , Smith John A. , Rudland Philip S.
Published: February 2005

The binding of [125I]‐epidermal growth factor (EGF) and [125I]‐basic fibroblast growth factor (bFGF) to a number of single‐cell cloned rat mammary cell lines was measured using a saturation assay. Similar numbers of high‐affinity [125I]‐EGF binding sites (KD 1.3 nM) were found in epithelial and myoepithelial‐like cell lines. In contrast, high‐affinity (KD 35–276 pM) [125I]‐bFGF binding sites were present on fibroblastic and myoepithelial‐like cell lines but were not detectable on epithelial cell lines. A series of cell lines representing stages in the differentiation pathway of epithelial cells to an elongated myoepithelial‐like morphology showed a graded increase in the number of bFGF receptors. The sensitivity of a cell line to stimulation of DNA synthesis by bFGF correlated with the level of expression of bFGF receptors on the cellular surface. Complexes of cell surface receptors affinity‐cross‐linked to [125I]‐bFGF were analysed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). In each case two distinct complexes having apparent molecular weights of 180 kDa and 160 kDa were observed.
Male germ cell specific sulfogalactoglycerolipid is recognized and degraded by mycoplasmas associated with male infertility
Authors: Lingwood Clifford , Schramayr Susan , Quinn Patricia
Published: February 2005

Sulfogalactosylglycerolipid (SGG) is the major mammalian male germ cell glycolipid and has been implicated in sperm/egg binding. Mycoplasma pulmonis, a species of Mollicutes, is associated with male infertility in rodents.

Purified SGG incubated in the presence of M. pulmonis was enzymatically degraded by both desulfation and deacylation. Desulfation occurred primarily at alkaline pH, and deacylation also increased with increased pH, indicating that these represent novel enzymatic activities. Digestion was facilitated, but not dependent on, the presence of detergent. Rat spermatozoa exposed to M. pulmonis showed a reduction in SGG content which was particularly marked for cauda (mature) spermatozoa. With the aid of tic overlay binding procedure, intact M. pulmonis were found to bind specifically to sulfated glycolipids and thus SGG may provide the cell membrane receptor for this organism. The topology of mycoplasma binding to rat sperm was consistent with the known topology of sperm SGG. The reduced binding (and subsequent digestion) of caput spermatozoan SGG correlates with the membrane colocalization of SGG and its endogenous binding protein at this stage. Separation of SGG and its binding protein during epididymal sperm maturation appears to facilitate M. pulmonis binding to and digestion of cauda sperm SGG.

The binding and degradation of the sperm SGG by M. pulmonis may play a role in the induction of infertility which follows infection with these organisms by interfering in sperm/egg receptor recognition.
Morphology of the differentiation and maturation of LLC‐PK1 epithelia
Authors: Pfaller Walter , Cstraunthaler Gerhard , Loidl Peter
Published: February 2005

In the present study, a stereologic approach was utilized to quantitatively assess morphological changes during the differentiation of LLC‐PK1 cells into an epithelial membrane. This renal epithelial cell line has been described to undergo morphological changes during differentiation and maturation from subconfluent culture to a confluent epithelial layer. An increase in the number of apical microvilli, interpreted as an areal increase in this membrane domain, was reported. This morphological differentiation was found to be accompanied by an increase in the expression of apical Na+‐dependent hexose transport and the activities of certain brush border enzymes. Since no data are available that quantify the morphologic changes during LLC‐PK1 differentiation, a quantitative morphologic—stereologic—investigation was performed for an early (6 days) and a late (12 days) state of confluence of LLC‐PK1 monolayer cultures. The following morphological parameters were determined by light and electron microscopic morphometry: volume fractions (Vv) of nuclei, mitochondria, and lysosomes, and surface densities(Sv) of the apical and basolateral cell membrane domains. For the apical membrane surface, the microvillous fraction has been measured separately. Since the stereologic approach used in the present study allows the determination of absolute cell volumes, the absolute measures of organelle volumes (V)and membrane surfaces (S) per average cell can be calculated from volume and surface densities. Although no changes in cell density were found for 6 and 12 day old LLC‐PK1 monolayers, indicating ceased cell proliferation due to contact inhibition, remarkable changes were found concerning the absolute cell volume and apical membrane surface. The observed increase in the apical cell surface was exclusively due to the enlarged microvillous surface fraction. This finding is in good agreement with the increased number of Na+‐dependent hexose transporters as well as with the increased expression of apical membrane marker enzymes observed during the differentiation of LLC‐PK1 monolayers.
Tumor promoters retard the loss of a transient subpopulation of cells in low passage Syrian hamster cell cultures
Authors: Ueo Hiroaki , Nakano Shuji , Paul O. P. Ts'O , Bruce Sarah A.
Published: February 2005

Early passage normal diploid Syrian hamster (SH) fetal cell cultures contain a transient subpopulation of contact‐insensitive (CS−) cells which lack density‐dependent inhibition of cell division. The size of this CS− subpopulation decreases during in vitro passage by conversion of the CS− cells to contact‐sensitive (CS+) cells. Approximately 10—15 population doublings after the frequency of the CS− cells has declined to below 0.001%, mass cultures cease proliferating and exhibit cellular senescence. Cultures with higher initial numbers of CS− cells exhibit longer in vitro proliferative life spans than cultures with smaller initial numbers of CS− cells. Active tumor promoting phorbol esters (12‐O‐tetra‐decanoyl‐phorbol‐13‐acetate [TPA] and phorbol‐12, 13‐didecanoate [PDD]) retard the decline in the proportion of CS− cells during in vitro passage, while the inactive tumor promoting phorbol ester, 4α‐phorbol‐12, 13‐didecanoate (4αPDD) has no effect on the rate of loss of the CS− cells. In addition, continuous treatment from secondary culture with TPA or PDD extends by approximately twofold the in vitro proliferative life span of SH fetal cell cultures. Treatment must, however, begin at passage 1 or 2 when the CS− cells are still present. After the proportion of the CS− cells has decreased to <0.001%, as in passage 6 cultures, promoters have no effect on the life span of the culture. This finding that exposure to promoters results in both a prolonged maintenance of the CS− cellular subpopulation, as well as an extention of in vitro proliferative life span, suggests that the conversion of CS− cells to CS+ cells is involved in the mechanism of in vitro senescence.
Chloride action potentials and currents in embryonic skeletal muscle of the chick
Authors: Steele Joy A.
Published: February 2005

Chloride‐dependent action potentials were elicited from embryonic skeletal muscle fibers of the chick during the last week of in ovo development. The duration of the action potentials was extremely long (>8 sec). The action potentials were reversibly blocked by the stilbene derivative, SITS, a specific blocker of chloride permeability. Using patch clamp pipettes, in which the intracellular chloride concentration was controlled and with other types of ion channels blocked, the membrane potential at the peak of the action potential closely coincided with the chloride equilibrium potential calculated from the Nernst equation. These data indicate that activation of a chloride‐selective conductance underlies the long duration action potential. The occurrence of the chloride‐dependent action potential was found to increase during embryonic development. The percentage of fibers that displayed the action potential increased from ∼︁20% at embryonic day 13 to ∼︁ 70% at hatching. Chloride‐dependent action potentials were not found in adult fibers. The voltage and time‐dependent currents underlying the action potential were recorded under voltage clamp using the whole‐cell version of the patch pipette technique. The reversal potential of the currents was found to shift with the chloride concentration gradient in a manner predicted by the Nernst equation, and the currents were blocked by SITS. These data indicate that chloride ions were the charge carriers. The conductance was activated by depolarization and exhibited very slow activation and deactivation kinetics.
Differentiation of simian virus 40 transformed human mammary epithelial stem cell lines to myoepithelial‐like cells is associated with increased expression of viral large T antigen
Authors: Rudland Philip S. , Barraclough Roger
Published: February 2005

Cloned simian virus 40 (SV40)‐transformed human breast epithelial cell lines can differentiate to myoepithelial‐like cells, and these can be isolated as clonal cell lines. Immunofluorescent and immunocytochemical analysis of such cell lines growing on plastic surfaces, collagen gels, and as tumor‐nodules in nude mice indicate that all the cell lines produce SV40 large T antigen, but that the production of this antigen is qualitatively increased in the myoepithelial‐like cells and cell lines. The myoepithelial‐like cell lines produce 4—6 times more immunoprecipitable large T antigen than the parental epithelial cells. The amount of mRNA for large T antigen is also increased by 3.5—5‐fold in the myoepithelial‐like cell lines when analysed by dot‐blot or by Northern hybridisations. Thus, differentiation along the myoepithelial‐like cell pathway is associated in these SV40‐transformed cells with increased expression of the viral large T antigen. It is suggested that immortalization of primary breast epithelial cell cultures may be, in part, due to the expression of large T antigen preventing processes of terminal keratinization.
Extracellular calcium affects the membrane currents of cultured human keratinocytes
Authors: Mauro Theodora M. , Pappone Pamela A. , Isseroff R. Rivkah
Published: February 2005

Electrophysiologic properties of cultured human keratinocytes were studied using the patch voltage‐clamp technique. Undifferentiated, proliferative keratinocytes grown in low Ca2+ medium had an average resting membrane potential of −24 mV. Voltage‐clamp experiments showed that these cells had two membrane ionic currents: a large voltage‐independent leak conductance, and a smaller voltage‐dependent Cl− current that activated with depolarization. Increasing the extra‐cellular Ca2+ concentration from 0.15 to 2 mM resulted in a doubling of the magnitude of the voltage‐gated current and a shift in current activation to more negative potentials. Since levels of extracellular Ca2+ can alter the morphology and differentiation state of keratinocytes, the finding of a Ca2+ ‐activated Cl− current in these cells suggests a role for this conductance in the initiation of differentiation.
Bradykinin induces superoxide anion release from human endothelial cells
Authors: Holland James A. , Pritchard Kirkwood A. , Pappolla Miguel A. , Wolin Michael S. , Rogers Nancy J. , Stemerman Michael B.
Published: February 2005

The time‐dependent release of superoxide anion (O) from bradykinin (Bk)‐stimulated human umbilical vein endothelial cells (EC) was measured as the superoxide dismutase‐inhibitable reduction of ferricytochrome C employing a novel application of microspectrophotometry. In the absence of Bk, O release by EC was not detectable. EC exposure to Bk (10−6 to 10−5 M) resulted in a rapid release of O. The release of O occurred within 5 minutes of exposure. O release was partially inhibited by indomethacin (63 ± 6%), thus suggesting that arachidonic acid metabolism, through cyclooxygenase, contributes to EC O production. EC O release may be an important component in the pathophysiologic actions of Bk on vascular function.
Contraction of vascular smooth muscle in cell culture
Authors: Murray Thomas R. , Marshall Bryan E. , Macarak Edward J.
Published: February 2005

The use of cultured vascular smooth muscle cells for the study of events related to excitation and contraction of smooth muscle has been limited by the inability to reliably induce contractile responses after subculturing of the cells. This limitation has been overcome by the cell culture preparation described herein. We demonstrate that appropriate responses to both smooth muscle agonists and vasodilators were preserved in cells that were serially subcultured. Fetal bovine pulmonary artery and aortic cell cultures were established following enzymatic dispersion of the medial portion of freshly harvested vessels. At various times after isolation, cells were transferred to microscope coverslips coated with a polymerized silicone preparation (polydimethyl siloxane). Tension forces generated by the cells were manifested as wrinkles and distortions of this flexible growth surface. Visual evidence of cell contraction in the form of increased wrinkling was documented for cells exposed to angiotensin II, carbachol, and KCl. Decreases in cell tension occurred following treatment with isoproterenol, and those relaxing effects were overcome by subsequent treatment with the agonist carbachol. The contractile responses did not diminish with prolonged maintenance in culture or repeated subculturing. Phosphorylation of the light chains on the contractile protein myosin was also measured as a biochemical index of agonist‐induced contraction. Cells depolarized with KCl or exposed to carbachol showed increased myosin phosphorylation when analyzed by 2‐dimensional gel electrophoresis. The responses remained intact through 7 passages and 9 weeks in culture. These results show that cultured vascular smooth muscle cells do not necessarily undergo a phenotypic modulation with loss of contractility under prolonged maintenance in culture.
Reprogramming of gene expression in postischemic rat liver: Induction of proto‐oncogenes and hsp 70 gene family
Authors: Schiaffonati Luisa , Rappocciolo Emilia , Tacchini Lorenza , Cairo Gaetano , Bernelli‐Zazzera Aldo
Published: February 2005

Steady‐state levels of messenger RNA (mRNA) for different members of the heatshock protein 70 gene family were studied in rat livers reperfused after nonnecrogenic ischemia. The expression of constitutive hsc 73 gene decreases during ischemia, returns to normal upon reperfusion, and increases 4 hr after restoration of blood flow. Reperfusion induces the expression of another hsp 70 gene family member (the so‐called inducible hsp 70 gene), which remains at high levels for at least 7 hr. The induction of hsp 70 family genes is preceded by activation of the cellular oncogene c‐fos, the most prompt change in gene expression detected in reperfused liver. Run‐on experiments demonstrate that the increased expression of these genes is largely dependent on activation of transcription. Changes in the amount of c‐myc and ornithine decarboxylase mRNA are not evident, while the level of the mRNA for glucose‐regulated protein GRP 78 increases later, concurrent with the onset of the acute phase response to surgical trauma. Analysis of polysomal and nonpolysomal fractions from sucrose gradients indicates that in postischemic liver, hsp 70 and hsc 73 mRNA are rapidly engaged on light polysomal or nonpolysomal complexes and are later shifted to polysomes. Albumin mRNA displays the same behavior, indicating that hsp 70 mRNA are not preferentially translated and that increased transcription is the major mechanism for enhanced hsp synthesis in postischemic liver. Damage by active oxygen species, pressure overload, and derangements of protein synthesis is likely to include the causative factors of increased expression of c‐fos and the hsp 70 gene family in postischemic reperfused liver.
Acidic fibroblast growth factor (HBGF‐1) stimulates DNA synthesis in primary rat hepatocyte cultures
Authors: Houck Keith A. , Zarnegar Reza , Muga Stephanie J. , Michalopoulos George K.
Published: February 2005

Acidic fibroblast growth factor (aFGF) stimulated DNA synthesis in primary rat hepatocyte cultures in a dose‐dependent manner with maximal effect at 10–50 ng ml−1. This activity was dependent on the presence of heparin at a concentration of 10–50 μ·ml−1. Insulin interacted synergistically with aFGF, as it did with epidermal growth factor (EGF). The response to aFGF was only 50% that found with EGF. The disparity was not due to different kinetics of DNA synthesis, since the peak response for both growth factors occurred at 36–72 hr after plating of the hepatocytes. The potential relevance of this novel hepatocyte mitogen to normal and pathological liver growth is discussed.