bluuebunny/crossref_metadata_2025 huggingface dataset:
Ionophore‐induced disassembly of blood platelet microtubules: Effect of cyclic AMP and indomethacin
Authors: Kenney Dianne M. , Chao Francis C.
Published: February 2005

Cytoplasmic calcium levels are believed to be important in blood platelet activation. Upon activation, the discrete marginal microtubule band, which maintains the discoid shape of non‐activated platelets, becomes disrupted. Present studies demonstrate that the extent of assembly of the marginal microtubule band is related to cytoplasmic calcium levels. The divalent cationophore, A23187, causes platelet aggregation, secretion, and contraction by promoting calcium transport from intraplatelet storage sites into the cytoplasm. A23187 caused disassembly of platelet microtubules. Quantitation of electron micrographs revealed that numbers of microtubules were reduced by approximately 80% after A23187 treatment. Secondly, assembled microtubules in homogenates of platelets, in which microtubules were stabilized prior to homogenization, were decreased in favor of free tubulin in A23187‐treated platelets. Thirdly, A23187 increased 14C‐colchicine binding by intact platelets; this also indicated a shift in the microtubule subunit equilibrium to favor free, colchicine‐binding tubulin subunits. In control experiments, A23187 did not affect the stability of platelet tubulin, the colchicine binding reaction, or the total tubulin content of platelets. Stimulation of colchicine binding depended on A23187 concentration (0.05–0.5 μM) and did not require extracellular calcium. A23187‐stimulation of colchicine binding was blocked by dibutyryl cyclic AMP (0.80 mM) and/or 3‐isobutyl‐1‐methylxanthine (50 μM) and by indomethacin (10 μM). Cyclic AMP or indomethacin also interferes with A23187‐induced platelet activation, but indomethacin is not likely to completely inhibit the perturbation of intraplatelet calcium gradients by A23187. It is suggested that A23187‐induced microtubule disassembly may be an indirect effect of calcium on microtubules.
Colony formation in agar by adult bone marrow multipotential hemopoietic cells
Authors: Johnson G. R.
Published: February 2005

Erythroid colony formation in agar cultures of CBA bone marrow cells was stimulated by the addition of pokeweed mitogen‐stimulated spleen conditioned medium (SCM). Optimal colony numbers were obtained when cultures contained 20% fetal calf serum and concentrated spleen conditioned medium. By 7 days of incubation, large burst or unicentric erythroid colonies occurred at a maximum frequency of 40–50 per 105 bone marrow cells. In CBA mice the cells forming erythroid colonies were also present in the spleen, peripheral blood, and within individual spleen colonies. A marked strain variation was noted with CBA mice having the highest levels of erythroid colony‐forming cells.

In CBA mice erythroid colony‐forming cells were mainly non‐cycling (12.5% reduction in colony numbers after incubation with hydroxyurea or 3H‐thymidine). Erythroid colony‐forming cells sedimented with a peak of 4.5 mm/hr, compared with CFU‐S, which sedimented at 4.25 mm/hr. The addition of erythropoietin (up to 4 units) to cultures containing SCM did not alter the number or degree of hemoglobinisation of erythroid colonies.

Analysis of the total number of erythroid colony‐forming cells and CFU‐S in 90 individual spleen colonies gave a correlation coefficient of r = 0.93 for these two cell types.

In addition to benzidine‐positive erythroid cells, up to 40% of the colonies contained, in addition, varying proportions of neutrophils, macrophages, eosinophils, and megakaryocytes. Taken together with the close correlation between the numbers of CFU‐S in different adult hemopoietic tissues, including individual spleen colonies, the data indicate that the erythroid colony‐forming cells expressing multiple hemopoietic differentiation are members of the hemopoietic multipotential stem cell compartment.
Polyadenylate‐deficient analogues of poly(A)‐containing mRNA sequences in cultured AKR mouse embryo cells
Authors: Siegal G. P. , Hodgson C. P. , Elder P. K. , Stoddard L. S. , Getz M. J.
Published: February 2005

Five to six percent (by mass) of AKR‐2B mouse embryo cell polysomal RNA consists of messenger RNA sequences which may exist in polyadenylated form. In the steady state, however, only 30–40% of these molecules are retained by extensive passage over oligo(dT)‐cellulose, the remainder being present in the form of poly(A)‐deficient analogues. Within experimental limits, these poly(A)‐deficient analogues contain representatives of all poly(A)‐containing mRNA sequences in these cells. An analysis of the kinetics of hybridization of cDNA probes enriched for either abundant or rare poly(A)‐containing mRNA sequences suggests that the frequency distributions of poly(A)‐containing and poly(A)‐deficient analogues are dissimilar, and that a relationship exists between the intracellular frequency of a given mRNA sequence and the number of poly(A)‐deficient analogues of that sequence. High frequency sequences appear to be enriched in the poly(A)‐containing fraction, while low frequency sequences are predominately associated with the poly(A)‐deficient fraction, thus, poly(A) may play a role in the regulation of mRNA frequency in the cytoplasm.
Cell‐surface glycosaminoglycans: Turnover in cultured human embryo fibroblasts (IMR‐90)
Authors: Vogel Kathryn G. , Kendall Vaughan F.
Published: February 2005

As constituents of both extracellular matrix and the cell surface, glycosaminoglycans are in a strategic position to influence several basic cell features. The localization and turnover of glycosaminoglycans was investigated in cultured normal human embryo fibroblasts of lung origin (IMR‐90). Attention was directed particularly toward that compartment of the culture which could be released by gentle proteloysis (trypsin, 0.1 mg/ml, 15 min) and is considered to represent the cell surface. In the presence of Na2SO4, sulfated glycosaminoglycans (S‐GAGs) of the cell surface were labeled rapidly, but within 30 min some 35S‐GAG appeared in the extracellular medium. The intracellular pool of S‐GAGs labeled during a 10‐min period was lost during the first hr of chase with a half‐life of 18 min, compared with 16 hr for S‐GAGs labeled over a 48‐hr period. Pulse‐labeled S‐GAGs of the surface turned over with an initial half‐life of 60 min, compared with 7 hr for surface material labeled over a 48‐hr period. These rapid movements of the early chase period were followed by similar movement at a much slower rate. The results are consistent with a model in which most of the S‐GAGs synthesized in the cell move rapidly to the surface. The surface GAGs are then released immediately to the medium or accumulate at the membrane to be shed more slowly at a later time or to be degraded. The S‐GAG which left the cell layer most rapidly during chase was dermatan sulfate, while heparan sulfate made up an increasing percentage of the cell layer as chase progressed. These cultures produce a fibrillar matrix of fibronectin, but the kinetics of this study suggest that the S‐GAGs of the surface are membrane‐bound, and an extracellular glycosaminoglycan matrix does not form.
Transmembrane potential and amino acid transport in rat hepatocytes in primary monolaver culture
Authors: Wondergem Robert , Harder David R.
Published: February 2005

Transmembrane potential (Em) and alpha‐aminoisobutyric acid (AIB) transport were measured in primary monolayer cultures of rat hepatocytes obtained from unoperated control rats and from rats 12 hr following partial hepatectomy. Measurements were performed 20–24 hr after plating the cells. The capacity of both kinds of cells to concentrate AIB depended upon extracellular sodium; however, the steady‐state accumulation in regenerating cells was twice that of control cells. Transmembrane potentials, recorded with glass microelectrodes, were –13 ± 0.6 mV and –27 ± 1.6 mV in control and regenerating cells, respectively. Ouabain (1 mM) depolarlized regenerating cells to –18 ± 1.0 mV, but it had no effect on control cells. The initial rates of 1 mM AIB transport into control and regenerating cells were 1.2 ± 0.1 and 3.1 ± 0.1 nanomoles/mg protein × 4 min, respectively. Ouabain (1 mM) reduced the initial rate of AIB transport into regenerating cells to 2.7 ± 0.1 nanomoles/mg protein × 4 min, but it had no effect on AIB transport into control cells. Glucagon (10−7 M) added to control cells 12 hr before measurements hyperpolarized Em to –31 ± 1.3 mV and increased AIB transport rate to 3.1 nanomoles/mg protein × 4 min. The results suggest a relationship between increases in Em and increases in AIB transport in rat hepatocytes. An electrogenic Na‐K pump may be involved in both of these events.
Age‐dependent decrease of growth responsiveness in density inhibited 3T3 cell populations and their interactions with SV 40‐3T3 cells
Authors: van Der Bosch Jürgen
Published: February 2005

An aging process has been detected in stationary 3T3 cell cultures, especially in the presence of plasma‐derived serum (PDS) from adult bulls. It leads to irreversible conversion of an increasing percentage of initially responsive cells of a stationary population into cells unresponsive to growth stimulation by newborn calf serum (NBCS) or reseeding at low cell density in the presence of NBCS. These unresponsive cells are viable in the sense that, following trypsinization, they reattach and spread on a new culture plate and can be maintained for many days. The conversion process is accelerated by increasing PDS concentration. It is antagonized by NBCS. It is accompanied by enhancement of growth‐inhibiting interactions exerted by stationary 3T3 cell populations on SV 40–3T3 cells.
Control and virus‐transformed baby hamster kidney cells resistant to ethidium bromide. I. Characterization and the respiratory enzymes
Authors: Soslau Gerald , Zavodny Paul J.
Published: February 2005

Cell lines resistant to ethidium bromide have been developed from cultured mammalian BHK21/C13 cells and these same cells transformed by Rous sarcoma virus (C13/B4). Cells resistant to 2 μg ethidium bromide per milliliter have been cloned. One clone of the control and one of the virus‐transformed cell lines has been employed for characterization. The resistant cells, in the presence of 2 μg ethidium bromide/ml, grow at approximately the same rate as the untreated parental cells. The control cells possess a “normal” karyotype (44 chromosomes), while the corresponding ethidium bromide mutant has a reduced chromsome number of 41 and a number of translocations. The mitochondria displayed morphological alterations compared to the parental lines during the transition phase prior to the isolation of the ethidium bromide‐resistant cells. The mitochondria of the ethidium bromide‐resistant mutants appear somewhat enlarged with a normal morphology.

The effect of ethidium bromide on selected respiratory enzymes in normal and virus‐transformed ethidium bromide‐resistant baby hamster kidney cells was determined. Ethidium bromide‐resistant cells exhibited a depressed level of cytochrome aa3. This depression could not be reversed by growth in ethidium bromide‐free media. Ethidium bromide‐resistant cells possessed the same cytochrome b, c, and c1 levels per cell as their corresponding parental lines.

Purified mitochondria isolated from virus‐transformed ethidium bromide‐resistant cells exhibited a depression in cytochrome oxidase‐specific activity, while the ethidium bromide‐resistant control cells did not. All cell lines studied showed a depression in NADH‐ferricyanide and NADH‐cytochrome c reductase‐specific activities relative to parental BHK21/C13 cells. An increase in succinate dehydrogenase and succinate cytochrome c reductase‐specific activities was observed in ethidium bromide‐resistant control cells relative to their parental BHK21/C13 cells. No increase was observed in virus‐transformed ethidium bromide‐resistant cells.

Ethidium bromide‐resistant control cells exhibited a two‐fold increase in oligomycin‐insensitive adenosine triphosphatase activity relative to their parental cells. All of the cell lines studied possessed equivalent oligomycin‐sensitive adenosine triphosphatase‐specific activity except for the virus‐transformed, dyeresistant mutant, whose activity was increased.
Sodium butyrate affects expression of fibronectin on CHO cells: Specific increase in antibody‐complement‐mediated cytotoxicity
Authors: Milhaud Pierre , Yamada Kenneth M. , Gottesman Michael M.
Published: February 2005

Cellular fibronectin is expressed only at very low levels on the surface of the spontaneously transformed Chinese Hamster Ovary (CHO) cell line, as detected by immunofluorescence studies and confirmed by resistance of CHO cells to complement‐mediated lysis in the presence of anti‐fibronectin antibody. Treatment of CHO cells with sodium butyrate results in a marked susceptibility to anti‐fibronectin complement‐mediated killing. Using selected complement‐containing sera from rabbits, it is possible to demonstrate that killing of butyrate‐treated CHO cells is absolutely and specifically dependent on the presence of antibody to fibronectin. The increased susceptibility of butyrate‐treated cells to complement‐mediated killing correlates with an increase in cell surface fibronectin detected by immunofluorescence studies. This increased antigen expression should allow the isolation of mutants with altered regulation of fibronectin expression in a cell line of proven value for somatic cell genetic studies.
Variant endothelial cells. Fibronectin as a transducer of signals for migration and neovascularisation
Authors: McAuslan B. R. , Hannan G. N. , Reilly W. , Stewart F. H. C.
Published: February 2005

A morphologic and growth control variant of bovine aortal endothelial cells has been isolated and shown to synthesise factor VIII antigen (McAuslan and Reilly '79). The variant also possesses the endothelial surface markers angiotensin converting enzyme and α2‐macroglobulin. The normal cell synthesises fibronectin and deposits it underneath the cells; the variant also synthesises fibronectin. At least three times more fibronectin is distributed over the upper cell surface of variants. This correlates with the three‐fold increased binding of the replication inhibitor Con A and suggests a role of fibronectin in endothelial cell growth control. When stimulated to migrate by CuII ions, the variant leaves deposits of fibronectin in its trail; in contrast, migrating normal cells do not, but they do redistribute their surface fibronectin. As revealed by scanning electron microscopy, variant cells are unusual in that they grow over or under cultured normal endothelial cells. It is proposed that during the process of neovascularisation, variant cells have a special function as lead cells that lay down fibronectin on which an endothelium can become established.
Differences in pattern and level of plasminogen activator production between a cloned cell line from an ethylnitrosourea‐induced glioma and one from normal adult rat brain
Authors: Hince Trevor A. , Roscoe Joan P.
Published: February 2005

The production of plasminogen activator (PA) by two cloned cell lines, one from an ethylnitrosourea‐induced glioma (Al5A5) and the other from normal adult rat brain (ARBO C9), has been investigated. Three assays were used to detect and measure PA in harvest fluids, cells and cell lysates. Similar levels were detected in harvest fluids from both cell lines. However, the cell and lysate assays indicated much higher levels in the tumor line. When actively growing cells were compared Al5A5 cells had approximately 16 × more fibrinolytic activity than the control cells with a limit of detection in the order of 103 cells or 1 μg protein (cell lysate). In contrast for the control cells PA could only be detected when upwards of 104 cells or 5 ‐ 10 μg protein were assayed. Plaminogen activator in as few as 103 tumor cells could be detected in the presence of 104 non‐tumor cells. Plasmingoen activator in 26 μg protein of Al5A5 cell lysate could also be detected in the presence of 44 μg protein from ARBO C9 lysates indicating no inhibitory activity in the control cell lysates. Levels of PA in both harvest fluids and cell lysates wre determined as cultures progressed through the growth cycle. For cell lysates this showed a build‐up of PA in the normal cell line as the cells approached and attained confluence. A much higher level was measured in the tumor cells soon after seeding and maintained to confluence. No differences in growth cycle‐associated changes in secreted PA could be determined in harvest fluids: both cell lines showing similar levels at confluence.
Evidence for control of protein synthesis in HeLa cells via the elongation rate
Authors: Nielsen Peter J. , McConkey Edwin H.
Published: February 2005

The effects of fresh medium and serum on protein synthesis in suspension‐cultured HeLa cells after growth to high cell density (>5 × 105 cells/ml) were studied. Cells which were resuspended in fresh medium plus serum and grown for 24 hours (control) were compared with cells grown for 2 hours after resuspension (stimulated). The spectrum of proteins being synthesized by control and stimulated cells does not appear to be grossly different; that is, the weight and number average molecular weights of newly synthesized whole‐cell protein are about the same in both cultures. Also, no significant differences were observed in the number of ribosomes per polysome or in the fraction of total ribosomes in polysomes. However, the transit times (combined elongation and termination times) were found to differ significantly; the average transit time for control cells was 2.24 minutes, while the average transit time for stimulated cells was 1.26 minutes. (An appendex evaluating the methodology involved in measuring the transit time is included.) In agreement with the difference in transit time, the absolute rate of protein synthesis in stimulated cells was approximately 1.8 times the rate measured in control cells. These data are taken as evidence that under certain conditions, the rate of elongtion and/or termination of polypeptide chains limits the overall rate of translation, and that cells can respond to growth conditions by changing the elongation and/or termination rate of protein synthesis.
Coordinate secretion of mouse alphafetoprotein, mouse albumin and rat albumin by mouse hepatoma‐rat hepatoma hybrid cells
Authors: Cassio Doris , Hassoux René , Dupiers Michèle , Uriel José , Weiss Mary C.
Published: February 2005

Mouse hepatoma cells that secrete large amounts of α‐fetoprotein (AFP) and albumin have been crossed with rat hepatoma cells that secrete only albumin, and in relatively small amounts, to investigate the influence of each parental genome upon the expression of serum proteins. All of the ten independent hybrid clones examined produce mouse AFP and both mouse and rat albumin; none produces rat AFP. The absence of production of rat AFP by the hybrids suggests that different mechanisms are involved in the initiation and in the maintenance of expression of this function. The secretion of the three proteins by the hybrid cells is coordinate: Whatever the growth phase (exponential or stationary) and irrespective of the amounts produced over a wide range, the ratio secreted of mouse AFP to mouse albumin is near to one, and that of mouse albumin to rat albumin is near to five. In addition, even though the pattern of protein secretion during the growth cycle of hybrid cells is different from those of both parents, the products of both parental genomes conform to the new hybrid pattern. Finally, some hybrids secrete less of the proteins with increasing numbers of cell generations, yet all three continue to be secreted in coordinate fashion. Since the rates of secretion of serum proteins probably reflect their rates of synthesis, we conclude that coordinate secretion indicates coordinate synthesis, and may reflect coordinate transcription of the relevant genes.
Spreading of human fibroblasts in serum‐free medium: Inhibition by dithiothreitol and the effect of cold insoluble globulin (plasma fibronectin)
Authors: Grinneli Frederick , Feld Marian K.
Published: February 2005

We have tested the effect of dithiothreitol (DTT) treatment on the initial spreading of human fibroblasts in serum‐free medium in tissue culture dishes. Cell spreading was inhibited following treatment of these cells with 10 mM DTT. Inhibition occurred when the cells were treated at 37°C but not at 4° and was reversible metabolically but not by the addition of sulfhydryl oxidizing reagents. The inhibition was overcome when DTT‐treated human fibroblasts were plated on cold insoluble globulin (plasma fibronectin)—coated dishes. Under these conditions spreading appeared to be completely normal, including the formation of focal adhesions. Analysis of the fibronectin concentrations in the human fibroblasts following DTT treatment indicated that there was little decrease in the absolute level of activity as determined in a biological assay for BHK cell spreading on culture dishes. Analysis of the fibronectin distribution on the DTT‐treated human fibroblasts by indirect immunofluorescence using a specific anti‐CIG antiserum revealed that fibronectin was no longer deposited onto the culture dish surfaces. Even when the DTT‐treated human fibroblasts spread in the presence of fetal calf serum, the cell fibronectin remained for the most part in a perinuclear location. These results indicate that DTT treatment of human fibroblasts prevents the normal translocation of fibronectin from a perinuclear location to the surface of the culture dish. This study further supports our hypothesis that the initial spreading in serum‐free medium of fibroblasts from cell strains depends upon secretion of fibronectin onto the culture dish surface.
Regulation of prostaglandin synthesis during differentiation of cultured mouse myeloid leukemia cells
Authors: Honma Yoshio , Kasukabe Takashi , Hozumi Motoo , Koshihara Yasuko
Published: February 2005

Mouse myeloid leukemia cells (Ml) were induced to differentiate into mature macrophages and granulocytes by various inducers. The differentiated Ml cells synthesized and released prostaglandins, whereas untreated Ml cells did not. When the cells were prelabelled with [14C]arachidonate, the major prostaglandins released into the culture media were found to be prostaglandin E2, D2, and F2α in an early stage of differentiation, but the mature cells produced predominantly prostaglandin E2. The synthesis and release of prostaglandins were completely inhibited by indomethacin. Dexamethasone, a potent inducer of differentiation of Ml cells, did not induce production of prostaglandins in resistant Ml cells that could not differentiate even with a high concentration of dexamethasone. These results suggest that production of prostaglandins in Ml cells is closely associated with differentiation of the cells. Homogenates of dexamethasone‐treated Ml cells converted arachidonate to prostaglandins, but this conversion was scarcely observed with homogenates of untreated Ml cells. Dexamethasone and the other inducers stimulated the release of arachidonate from phospholipids. Therefore, induction of prostaglandin synthesis during differentiation of Ml cells may result from induction of prostaglandin synthesis activity and stimulation of the release of arachidonate from cellular lipids. Lysozyme activity, which is a typical biochemical marker of macrophages, was induced in Ml cells by prostaglandin E2 or D2 alone, as well as by inducers of differentiation of the cells, but it was not induced by arachidonate or prostaglandin F2α. These results suggest that prostaglandin synthesis is important in differentiation of myeloid leukemia cells.
Synergistic effects of epidermal growth factor and thrombin on the growth stimulation of diploid chinese hamster fibroblasts
Authors: Cherington Paul V. , Pardee Arthur B.
Published: February 2005

The serum supplement used in the culture of a variety of mammalian cells can be replaced by known growth factors. Diploid Chinese hamster fibroblasts (CHEF/18) will grow for several days in a medium (4F) supplemented with four growth factors: epidermal growth factor (EGF), insulin, fibroblast growth factor (FGF), and transferrin. The growth rate is only about 50% as fast as when fetal calf serum is added. This difference is eliminated by thrombin (10–100 ng/ml; 0.3–3 nM). The CHEF/18 cell line is unique in that no other cell line responds to thrombin in this concentration range.

Thrombin acts synergistically with other growth factors to stimulate CHEF/18 cell growth. By itself, thrombin is only mitogenic at elevated concentrations. Thrombin can largely compensate for the absence of EGF and partly for the absence of insulin in serum‐free media. Chemically and “spontaneously” transformed cell lines related to CHEF/18 have lost requirements for both EGF and thrombin, and have retained requirements for insulin and transferrin expressed by CHEF/18. No CHEF cells in this work required FGF. These results suggest that the mechanisms by which EGF and thrombin stimulate cells to grow are related.
Ribonucleotide reduction in intact human diploid fibroblasts
Authors: Dick John E. , Wright Jim A.
Published: February 2005

There have been very few studies on ribonucleotide reductase activity in human tissue. In this report we describe a rapid and convenient procedure for determining purine and pyrimidine ribonucleotide reduction in normal human diploid fibroblasts and use the method to examine some general properties of the activity in these cells. ADP and CDP reductase was characterized for its response to the positive effectors, ATP and dGTP, the negative effector dATP, and the reducing agent dithiothreitol. Apparent Km values for ADP and CDP were determined to be 0.1 mM and 0.04 mM respectively. The antitumor agent hydroxyurea inhibited both purine and pyrimidine reductase in a noncompetitive fashion, giving Ki values of 0.40 mM and 0.41 mM for ADP and CDP respectively. These Ki estimates are about four to five times higher than those reported for some permanent cell lines. An examination of the cytotoxic effects of hydroxyurea indicated a close correlation between the concentration of drug which inhibited enzyme activity and decreased colony‐forming ability.

Clearly the ability to investigate ribonucleotide reduction in low numbers of normal human diploid cells will be useful for genetic and biochemical studies.
Osmotic properties of differentiating bone marrow precursor cells: Membrane permeability to non‐electrolytes
Authors: Cicoria A. D. , Hempling H. G.
Published: February 2005

Membrane permeability to the non‐electrolyte probes urea and thiourea was measured in a population of differentiating bone marrow precursor cells (Cicoria and Hempling, '80). The phenomenologic equations of Kedem and Katchalsky ('58) were applied. The population of cells, which can be separated by velocity sedimentation, was examined over the maturation range of 400 to 1,000 cubic micra. Several membrane properties were observed: (1) Solute permeability (R̃) increased linearly as the cell volume increased, while the reflection coefficients (σ) remained constant. (2) Membrane permeability to urea was significantly greater than to thiourea; however, their respective sigmas did not differ significantly. Solute permeability was independent of sigma at 22 degrees C. (3) In the two maturation phases observed, wr̃ varied directly with the volume of osmotically active water. (4) The permeability to urea and to thiourea has provided information on the characteristics of the pathways used for water and non‐electrolytes, as well as a description of the phenomenologic membrane events accompanying cellular maturation.
Stimulus secretion coupling: Role of cyclic GMP and calcium in the regulation of secretion from rat exocrine pancreas
Authors: Rochette‐Egly C. , Launay J. F. , Grenier J. F.
Published: February 2005

In rat pancreatic fragments, stimulation of amylase and labeled protein release by carbachol, caerulein, and ionophore A 23187 results within minutes in a short rise in cyclic GMP levels. Cyclic AMP levels do not change significantly. The secretory response elicited by each secretagogue is not modified when combined in pairs. Under intracellular calcium depleting conditions, both the cyclic GMP and the secretory responses to secretagogues are inhibited in parallel, suggesting a good correlation between both processes. Furthermore, 8‐Bromocyclic GMP induces pancreatic secretion, but to a lesser extent, and fails to alter the increase in secretion caused by the various secretagogues. However, other agents such as imidazole, ascorbic acid, phenylhydrazine, and sodium azide also increase cyclic GMP levels but fail to stimulate pancreatic secretion. On the other hand, dibutyryl cyclic AMP also stimulates amylase and labeled protein discharge and potentiates the increase caused by cabachol, caerulein, and ionophore A 23187. These results do not permit conclusions regarding a cause and effect relationship between cyclic GMP and secretion. A role for calcium seems to be the most likely.
Effects of beta adrenergic agents and prostaglandin E1 on erythroid colony (CFU‐E) growth and cyclic AMP formation in friend erythroleukemic cells
Authors: Beckman Barbara , Mirand Edwin , Fisher James W.
Published: February 2005

The formation of erythroid colonies from bone marrow and spleen cells infected with the polycythemic strain of the Friend virus (FV‐P) was characterized in an in vitro methyl cellulose colony‐forming system in response to prostaglandin E1 and the beta‐2 adrenergic agonist, albuterol. Both drugs markedly inhibited the formation of CFU‐E colonies of FV‐P‐infected bone marrow and spleen in the absence or presence of erythropoietin. The albuterolmediated inhibition of CFU‐E colonies (FV‐P‐infected) was selectively blocked by butoxamine, a beta‐2 antagonist. Adenylate cyclase (AC) activity was also determined in FV‐P spleen membrane preparations in response to albuterol and PGE1. Both agents stimulated enzyme activity, and butoxamine blocked the stimulation seen with albuterol. The ability of albuterol and PGE1 to stimulate AC activity in the FV‐P‐infected cells suggests that the effects of these agents on CFU‐E formation may be mediated by specific beta‐2 adrenergic and PG receptors through the adenylate cyclase‐cyclic AMP system.
The use of lanthanum and cytochalasin B to study calcium effects on skeletal muscle differentiation in vitro
Authors: Morris Glenn E.
Published: February 2005

A dual effect of external Ca2+ on creatine kinase (CPK) accumulation during myogenesis has recently been demonstrated (Morris and Cole, '79). Ca2+ inhibits muscle‐specific CPK accumulation at intermediate (50–100 μ) concentrations compared with both lower (no added Ca2+) and higher (2–3 μ) concentrations. Myoblast fusion, however, requires high Ca2+ and is inhibited at both low and intermediate Ca2+ levels.

These effects are now investigated further by studying the effects of lanthanum ion (La3+), which interferes with Ca2+‐binding to membranes and Ca2+‐transport, and cytochalasin B, which affects the cell membrane and prevents cell fusion without inhibiting CPK accumulation.

The results show that low concentrations (10–100 μ) of La3+ inhibit the appearance of the muscle‐specific (MM) CPK isoenzyme during myogenesis without significantly affecting cell fusion or intracellular cyclic AMP levels. Three further observations are consistent with the existence of myotube‐specific membrane‐binding sites for Ca2+, which are involved in the stimulation of CPK accumulation on increasing external Ca2+ from intermediate to high concentrations.

(1) CPK levels are not affected by La3+ at 0–50 μ external Ca2+.

(2) CPK levels in cytochalasin B treated myoblasts are hardly affected by La3+ at any Ca2+ concentration.

(3) In cytochalasin B treated cultures, CPK levels are not increased by raising external Ca2+ from intermediate to high levels.

In contrast, the stimulation of CPK accumulation on decreasing external Ca2+ from intermediate to very low concentrations is not affected by either La3+ or cytochalasin B.

Some alternative interpretations of the data are also considered, including direct disruption of a membrane Ca2+‐binding site by cytochalasin B.
Hemoglobin biosynthesis in individual bursts from human adult peripheral and umbilical cord blood: Analysis of the relative rates of synthesis of Gγ and Aγ globin chains
Authors: Terasawa Takashi , Ogawa Makio
Published: February 2005

We cultured human adult peripheral and umbilical cord blood mononuclear cells in methylcellulose and measured the synthetic rates of globin chains in individual erythropoietic bursts. Globin chains were labeled with 14C‐amino acids in culture, separated by isoelectric focusing in slab gels containing 8 M urea and 3% Nonidet P‐40, and quantitated by fluorographic methods. All bursts exhibited Gγ, Aγ, and β chains in varying ratios. Cumulative frequency distributions of individual bursts differing in the ratios of Gγ/(Gγ + Aγ) and γ/(γ + γ) approached normal distributions. These results indicated that perinatal hemoglobin switching and the hemoglobin switching of cultured adult erythropoietic precursors do not involve population changes in the precursors. Further, a positive correlation between γ/(γ + γ) and Gγ/(Gγ + Aγ) ratios of individual bursts existed in all of the samples. Switching between γ and β chains and switching of Gγ:Aγ ratios may share common regulatory mechanisms.
Effect of changes in the ionic environment of reticulocytes on the uptake of transferrin‐bound iron
Authors: Paterson Suzanne , Morgan E. H.
Published: February 2005

Rabbit reticulocytes were incubated with rabbit transferrin labelled with 59Fe and 125I in media in which the NaCl was replaced by other electrolytes or sucrose. Iron and transferrin uptake by the cells was affected by changes in the pH, ionic strength, ionic composition, and the osmolarity of the medium. Uptake was maximal at pH 7.4. A reduction in ionic strength produced by replacing NaCl with sucrose inhibited the uptake in a concentration‐dependant manner, greatest inhibition occurring at lowest salt concentration. Similar results were obtained when KCl, LiCl, RbCl, Na2SO4, or K2SO4 were used instead of NaCl. Low ionic strength was found to inhibit the endocytotic uptake of transferrin labelled with colloidal gold, but had only a small effect on transferrin binding to cell membrane receptors. It was concluded that low ionic strength inhibits iron uptake primarily by blocking the endocytosis of transferrin. Three salts, NH4Cl, CaCl2, and MgCl2, produced different results from the above. NH4Cl inhibited iron uptake at all concentrations used. This action was due to an effect on the release of iron from transferrin, which appeared to be taken up by the cells in a normal manner. When the ionic strength of the sucrose medium was increased by adding low concentrations of CaCl2, iron uptake was greater than with equivalent concentrations of NaCl. However, with CaCl2 concentrations above 10 mM, iron uptake was inhibited, due to inhibition of transferrin uptake, possibly by blocking endocytosis. By contrast, MgCl2 stimulated iron uptake at all concentrations used. The results are discussed in terms of the possible effects of ionic strength, pH, and ionic composition of the extracellular fluid on the three main steps involved in iron uptake by immature erythroid cells: transferrin‐receptor interaction, endocytosis, and iron release from transferrin.
Na+, K+‐ATPase in HeLa cells after prolonged growth in low K+ or ouabain
Authors: Pollack Lewis R. , Tate Emily H. , Cook John S.
Published: February 2005

Effects of long‐term, subtotal inhibition of Na+‐K+ transport, either by growth of cells in sublethal concentrations of ouabain or in low‐K+ medium, are described for HeLa cells. After prolonged growth in 2 × 10−8 M ouabain, the total number of ouabain molecules bound per cell increases by as much as a factor of three, mostly due to internalization of the drug. There is only about a 20% increase in ouabain‐binding sites on the plasma membrane, representing amodest induction of Na+, K+‐ATPase.

In contrast, after long‐term growth in low K+ there can be a twofold or greater increase in ouabain binding per cell, and in this case the additional sites are located in the plasma membrane. The increase is reversible. To assess the corresponding transport changes, we have separately estimated the contributions of increased intracellular [Na+] and of transport capacity (number of transport sites) to transport regulation. During both induction and reversal, short‐term regulation is achieved primarily by changes in [Na+]i. More slowly, long‐term regulation is achieved by changes in the number of functional transporters in the plasma membrane as assessed by ouabain binding, Vmax for transport, and specific phosphorylation.

Parallel exposure of cryptic Na+, K+‐ATPase activity with sodium dodecyl sulfate in the plasma membranes of both induced and control cells showed that the induction cannot be accounted for by an exposure of preexisting Na+, K+‐ATPase in the plasma membrane. Analysis of the kinetics of reversal indicates that it may be due to a post‐translational event.
The use of fluorescent antimyosin and DNA labeling for the estimation of the myoblast and myocyte population of primary rat heart cell cultures
Authors: Masse Marie J. O. , Harary Isaac
Published: February 2005

Cell division in heart muscle cells progressively ceases during the development of the rat heart, leading to an adult stage with muscle cells incapable of cell division. We have quantitatively determined the number of dividing and nondividing heart muscle cells in cultures derived from different stages of the developing rat heart with the use of 3HTdR continuous labeling and fluorescent antimyosin staining. The cultures were derived from 14 and 17 day postcoital (dPC) rat embryos and from 1 and 4 day postnatal (dPN) rats. The percent nondividing cells increased with development and the age of the postnatal rat. The percent nondividing cells in 14 dPC equalled 21%, 17 dPC equalled 25%, 1 dPN equalled 44%, and 4 dPN equalled 60%. This method for the quantitative determination of dividing and nondividing cells in the developing rat heart provides a model that is useful for the study of the mechanism of the loss of cell division capacity.
Oxygen enhances fusion of cultured chick embryo myoblasts
Authors: Hollenberg Milton , Honbo Norman , Ghani Q. Perveen , Samorodin Albert J.
Published: February 2005

Fusion of mononucleate myoblasts to form multinucleated myotubes increases when skeletal muscle cells are grown in progressively higher oxygen concentrations (5%, 20%, and 40% oxygen). At four days of growth fusion of myoblasts (as expressed by the percent of all muscle nuclei that are located in myotubes) is 57 ± 2% in 5% oxygen, 68 ± 1% in 20% oxygen, and 78 ± 2% in 40% oxygen (P<0.001). However, at a concentration of 40%, oxygen depresses the rate of cell division and thereby affects the number of myoblasts available for fusion. Thus, oxygen concentration significantly modifies growth of skeletal muscle in vitro. Its net effect on myotube formation results from the interaction of its separate effects to enhance cell fusion and to depress cell proliferation.
The effect of sarcomere non‐uniformity on the sarcomere length‐tension relationship of skinned fibers
Authors: Iwazumi Tatsuo , Pollack Gerald H.
Published: February 2005

It has proved difficult to activate skinned muscle fibers to produce high tension (3 kg/cm2 level) without loss of clear striations. A new method was developed which permits high tension production in skinned muscle fibers while retaining clear striations. Clear striations allow reliable measurement of the sarcomere lengths during contraction by microscopy and diffractometry. The method is to increase the Ca++ concentration of the bathing solution very gradually over a time period of 5 to 10 minutes. Once the skinned fiber is conditioned by this slow activation, subsequent contractions can be elicited by ordinary quick activations without loss of striations. When the experiments are carried out with careful controls for the uniformity of the sarcomere length distribution along the entire length of the fiber, contractions are highly repeatable. Using the new method and stringent quality control of fibers, the sarcomere length‐isometric tension relationship of skinned rabbit soleus fibers was obtained. The results differ from those previously obtained by conventional activation methods in that tension increases with sarcomere length not only at low (pCa = 5.8), but also at high (pCa = 5.2), calcium concentration.
Variable coupling of active NA+ + K+ transport in Ehrlich ascites tumor cells: Regulation by external NA+ and K+
Authors: Smith Thomas C. , Robinson Susan C.
Published: February 2005

The effects of altered external sodium and potassium concentrations on steady state, active Na+ + K+ transport in Ehrlich ascites tumor cells have been investigated. Membrane permeability to Na+ and K+, intracellular [Na+] and [K+], and membrane potential were measured. Active cation fluxes were calculated as equal and membrane potential were measured. Active cation fluxes were calculated as equal and opposite to the net, diffusional leak fluxes. Elevation of external K+ (6–60 Mm)by equivalent replacement of Na+ (154–91 mM) inhibits both active Na+ and K+ fluxes, but not proportionally. This results in a decrease of the coupling ratio (rp = ‐Jkp/J) as external K+ is increased. Elevation of external K+ (3–68 mM) at constant Na+ (92mM) inbibits J, but is without effect on J. The coupling ratio declines from 1.01 ± 0.14 to 0.07 ± 0.05, a 14‐fold alteration. Reduction of external Na+ (154–25 mM) at constant K+ (6mM) depresses J, but is without effect on J. The coupling ratio increases from 0.63 ± 0.04 at 154 mM Na+ to 4.5 ± 2.04 at 25 mM Na+. The results of this investigation are consistent with the independent regulation of active cation fluxes by the transported species. Kinetic analysis of the data indicates that elevation of external sodium stimulates active sodium efflux by interacting at “modifier sites” at the outer cell surface. Similarly, external potassium inhibits active potassium influx by interaction at separate modifier sites.
Cation transport and growth regulation in neuroblastoma cells. Modulations of K+ transport and electrical membrane properties during the cell cycle
Authors: Boonstra Johannes , Mummery Christine L. , Tertoolen Leon G.J. , Van Der Saag Paul T. , De Laat Siegfried W.
Published: February 2005

Cation transport and membrane potential were studied during the cell cycle of neuroblastoma cells (clone Neuro‐2A) to investigate the role of these parameters in growth regulation. The cells were synchronized by selective detachment of mitotic cells. The membrane potential and intracellular K+ activity were measured with conventional and K+‐selective microelectrodes respectively. Both the membrane potential and K+ activity were high in mitosis, decreased to half maximal in G1 phase, and rose again during S phase.

K+ efflux across the plasma membrane was studied with 42K+ as a radioactive tracer using a washing method for cells grown in monolayer and a continuous efflux method for mitotic cells in suspension. The intracellular K+ content and unidirectional K+ efflux rate obtained from these measurements showed modulations during the cell cycle similar to those of the membrane potential. Using equations of electrodiffusion theory the membrane permeabilities to K+ and Na+ were calculated. These permeabilities were high in mitosis, decreased rapidly in G1 phase and increased during S phase, followed by a transient decrease in G2 phase. A rapid increase was observed between G2 phase and the next mitosis. A similar pattern was obtained for the K+ conductance. K+ resistance changes during the cell cycle were similar to changes in the specific membrane resistance, measured by microelectrodes, except for the early cell cycle phases (mitosis and G1).

These studies clearly demonstrate large modulations of the passive membrane permeability properties during the cell cycle. These modulations can be correlated with physicochemical membrane variations during the cell cycle, such as membrane fluidity and lateral mobility of lipids.
Replication pattern of a large homogenously staining chromosome region in antifolate‐resistant Chinese hamster cell lines
Authors: Hamlin Joyce L. , Biedler June L.
Published: February 2005

We have investigated the replication pattern of a large, homogenously staining chromosome region (HSR) in two antifolate‐resistant Chinese hamster cell lines. This region is believed to be the location of an amplified genetic sequence which includes at least the gene coding for dihydrofolate reductase and which may be present in as many as 200 copies. It is shown that the HSR in both cell lines is among the first chromosome regions to begin DNA synthesis after reversal of an early G1 block. In cells synchronized in the S period with hydroxyurea, it is also clear that the HSR in both cell lines begins replication at many sites within its length in early S. The replicons comprising the HSR therefore may respond to a common initiation signal in early S. In one cell line (A3), replication of the HSR requires, at most, 3 hours of a 7‐hour S period; in a second line (MQ19), replication proceeds for approximately 5 hours. In neither line does replication of the HSR occur concomitantly with synthesis of characteristic late replicating regions. These results were confirmed in exponential cultures using a retroactive labeling technique.

The significance of these findings is discussed with reference to the possible origin and arrangement of the amplified sequence in these two cell lines.
Nuclear magnetic resonance study of muscle water protons in muscular dystrophy of chickens
Authors: Chang D. C. , Misra L. K. , Beall P. T. , Fanguy R. C. , Hazlewood C. F.
Published: February 2005

Using the pulsed nuclear magnetic resonance (NMR) spectroscopy, the spin‐lattice (T1) and the spin‐spin (T2) relaxations times of water protons from samples of pectoralis major muscles of normal (line 412) and homozygous dystrophic (line 413) chickens were measured. Both the T1 and T2 were significantly increased (P < 0.05) in the dystrophic muscles. The mean values of the relaxation times are given ± S.D. The T1 values were 654 ± 22 msec in normal and 692 ± 41 msec in dystrophic muscles. The T2 values for normal and dystrophic muscles were 39 ± 4 msec and 52 ± 7 msec, respectively. Although the water content of dystrophic muscles (78.9 ± 0.6%) determined by gravimetric methods was significantly higher than normal muscles (74.9 ± 1.1%), this difference in tissue hydration could not explain quantitatively the increase of T1 and T2 values in the dystrophic muscles. The results of the measurements of the relaxation times seem to suggest that there are changes in the composition and/or conformational state of the proteins.