bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies on a fractionated murine fibrosarcoma: Proliferative potential of the separated cells
Authors: Sheridan J. W. , Finlay‐Jones J. J.
Published: February 2005

A transplantable methylcholanthrene‐induced fibrosarcoma of female BALB/c mice (the MC‐2 fibrosarcoma) was dissociated by combined mechanical and enzymatic means, then fractionated by isopycnic centrifugation in linear albumin gradients.

In some experiments recovered cells were both cultured in soft nutrient agar and inoculated subcutaneously into syngeneic recipients. In these experiments a highly significant correlation was observed between subsequent colony number and rapid growth phase tumor size suggesting identity of clonigenic and tumorigenic cells.

It was consistently found that clonigenic cells were markedly depleted from the low density extremes of the cell density distribution profiles suggesting that the low density neoplastic cells had irreversibly left the growth fraction.

With increasing tumor age, sequential studies showed that both total and clonigenic cell density distribution profiles were variable, showing no obvious trend, suggesting that in the age (13–35 days) and size (2–8 g) range studied growth fraction changes had little selective effect on cells of any specific density. These results imply that a marked selective depletion of low density clonigenic cells (or selective accumulation of low density non‐proliferative cells) must mainly occur during an earlier phase of tumor growth. Studies on several other murine solid tumors also showed maximal depletion of clonigenic cells from the least dense fractions, suggesting that this situation may be common.
Perturbation of growth and differentiation of friend murine erythroleukemia cells by 5‐bromodeoxyuridine incorporation in early S phase
Authors: Brown Eric H. , Schildkraut Carl L.
Published: February 2005

Cultured Friend murine erythroleukemia cells (Friend cells) are induced to undergo erythroid differentiation when grown in the presence of dimethylsulfoxide (DMSO) and other compounds. The effects of unifilar substitution of bromouracil (BU) for thymidine in the DNA (BU‐DNA) of Friend cells were examined. Cells were grown in the presence of 5‐bromodeoxyuridine (BrdU) for one generation, then centrifuged and resuspended in medium containing DMSO without BrdU. These cells exhibited a delay in the appearance of heme‐producing, benzidine‐reactive (B+) cells and a decreased rate of cell proliferation in comparison to the control not containing BU‐DNA. A transient inhibition of entry into S phase was observed when control cells or cells containing BU‐DNA were grown in the presence of DMSO for 10 to 20 hours. This transient inhibition was increased in the BrdU culture. Thus, BU‐substitution in Friend cells alters other cellular functions in addition to erythroid differentiation. The rate of increase in the percent of cells committed to differentiate (those forming B+ colonies in plasma clots) was similar in the BrdU and control cultures until 40 to 50 hours. After this time, a delay in the appearance of committed cells was observed in the BrdU culture. The effect of BrdU on the appearance of B+ cells was more pronounced and occurred earlier than its effect on the rate of commitment. Therefore, the delay in the appearance of B+ cells in the BrdU culture was due primarily to perturbation of post‐commitment events such as the accumulation of hemoglobin.

We also examined the effect on growth and differentiation after BrdU was incorporated during different intervals of S phase in cells synchronized by centrifugal elutriation or by double thymidine block and hydroxyurea treatment. The delay in the appearance of B+ cells and inhibition of cell proliferation were only observed when BrdU was incorporated in the first half of S phase. BrdU (10 μM) had no effect on growth or differentiation when present during late S or G1 and G2. These results, using two very different methods to achieve cell synchrony, indicate that the effects of BrdU on growth and differentiation described above are due to its incorporation into DNA sequences replicating during early S.
Production of factors required for cell attachment and spreading is a constitutive property in mouse A9 cells
Authors: Dairkee Shahnaz Hashmi , Gilbert Michael W.
Published: February 2005

It is demonstrated here that cells in a suspension culture of an established mammalian cell line release non‐dialyzable factors into their growth medium. These factors are capable of promoting the adhesion and spreading of these cells on a generally non‐attachable substratum and also promote spreading on an adhering substrate. Evidence in presented which demonstrates that the spreading promotion activity of the conditioned medium is dependent on the cell density of the culture from which it was derived. Dilution of the conditioned medium results in a proportionate dilution of the spreading promotion activity. The results clearly demonstrate that the production of this spreading promotion factor is continued even in the absence of cell to substrate attachment.
Neurotrophic effect of nerve extract on development of tetrodotoxin‐sensitive spike potential in skeletal muscle cells in culture
Authors: Kano Masaakira , Suzuki Nobuyuki , Ojima Hisayuki
Published: February 2005

The effect of the presence of nerve extracts on the development of tetrodotoxin (TTX)‐sensitive sodium channels in cultures of dissociated embryonic chick skeletal muscle cells was examined by measuring the maximum rate of rise of TTX‐sensitive spike potential. The addition of the nerve extract prepared from brain or spinal cord of chick embryos to the culture medium caused an increase in the channel density. Extracts of non‐neural tissues, i.e., lung, kidney, and muscle, were ineffective Liver extract, however, produced an effect similar to the nerve extracts. These resutls suggest that the TTX‐sensitive sodium channels in the muscle cell membrane are regulated by a diffusible chemical substance independently of innervation, and that this substance resides in neural tissues, and perhaps also in liver.
Endogenous membrane phosphorylation increases in serum‐stimulated 3T3 cells
Authors: Mastro Andrea M.
Published: February 2005

Autophosphorylation of 3T3 cells, utilizing endogenous membrane protein kinase, can be detected by incubating the cells with μgM32P‐ATP. The phosphorylation activity of growing cells is two to four‐fold greater than quiescent ones. In this study, the increased phosphorylation activity of serum‐stimulated cells was examined. Phosphorylation, measured at times after serum stimulation of quiescent cultures, was found to increase in early G1 and to reach a maximum prior to DNA synthesis. This increase in stimulated cells was dependent on RNA and protein synthesis but not on DNA synthesis. The increased activity decayed quickly (half‐life approximately 2–3 hours) in the presence of cycloheximide, while the basal activity in quiescent cells was relatively unchanged. Insulin, prostaglandin E1 or prostaglandin F2α were also found to bring about the same increase in phosphorylation as serum, although in contrast with serum they caused only a small percentage of the culture to synthesize DNA. The results suggest that enhanced phosphorylation activity is a G1 event. It does not depend on subsequent DNA synthesis. Phosphorylation may be one of the biochemical steps in G1, necessary but not sufficient for cells to move into S phase.
Tyrosinase maturation and pigment expression in B16 melanoma: Relation to theophylline treatment and intracellular cyclic AMP
Authors: White Roger , Hanson Gary C. , Hu Funan
Published: February 2005

We have studied the effects of theophylline treatment on pigmentation characteristics and growth of two B16 melanoma cell lines, HFH‐18 and P/140. Cell counts of control and theophylline‐treated cultures confirmed that the drug inhibits cell growth. Light and electron microscope cytochemistry with the L‐dopa reaction indicated that the two cell lines differ in their ability to transfer Golgi‐associated tyrosinase to developing premelanosomes. The results of these experiments, considered with results of electrophoretic analyses and activity measurements by the Pomerantz method, also provide evidence that increased tyrosinase synthesis occurs in response to theophylline treatment. In addition, results indicate that theophylline induces changes in the rate of synthetic or degradative posttranslational modification of tyrosinase. Measurements of intracellular cyclic AMP levels by radioimmunoassay in control cultures and in theophylline‐ and α‐MSH‐treated cultures were made. Although the hormone induced spectacular increases in cyclic AMP levels, theophylline produced no detectable change. These results indicate that theophylline differs from α‐MSH because theophylline‐induced changes in pigmentation may not require the participation of intracellular cyclic AMP.
Specific esterase activity induced in mouse Friend erythroleukemia cells by dimethylsulfoxide
Authors: Revoltella Roberto P. , Bertolini Luisa , Fioritoni Giuseppe , Torlontano Glauco
Published: February 2005

Three cell lines of mouse erythroleukemia transformed by Friend virus (FLC), namely 745, F4‐1, and 3BM‐78, were grown for six days in the absence or in the presence of 1.5% (v/v) dimethylsulfoxide (DMSO) and compared cytochemically for naphtol‐AS D‐chloroacetate esterase (E), alkalinephosphatase (AP), myeloperoxidase (MP) and periodic acid Schiff (PAS) reaction activity. In the absence of inducer only 1–2% of slightly E positive cells could be found. E positivity greatly increased in 3BM‐78 and F4‐1 but poorly in 745 cells, after treatment with DMSO. Unlike E reaction, AP and MP reactions were positive in about 5% 3BM‐78 and F4‐1 cells without DMSO, but there were no positive cells after DMSO treatment. All three lines were always PAS negative. Hemoglobin synthesis (benzidine staining) was intensively induced by DMSO in all three lines. Morphologically after DMSO treatment, FLC matured displaying characteristics of basophilic megaloblastoid cells. The emergence of specific esterase activity, a marker of granulocytes, in FLC differentiating along the erythroid pathway, suggests that in these leukemia cells the genetic determinants for leukopoietic differentiation are retained and capable of being expressed phenotypically.
Degradation of proteins microinjected into cultured mammalian cells
Authors: Zavortink Michael , Thacher Thomas , Rechsteiner Martin
Published: February 2005

Iodinated proteins were degraded after injection into HeLa cells at first‐order rates with half‐lives varying from three hours for the trout nonhistone chromosomal protein, HMG‐T, to 60 hours for whale myoglobin. Fluoresceinated‐bovine serum albumin (fl‐BSA) was degraded almost twice as fast as unmodified BSA. The rate of degradation of 125I‐BSA was very similar in eight cell lines of mouse, human, monkey and rat origin. Microinjected proteins were analyzed on SDS‐acrylamide gels after injection, and for BSA and immunoglobin G, all remaining intracellular 125I migrated at the molecular weight of the injected proteins. By contrast, more than 80% of the extracellular 125I chromatographed as iodotyrosine. With the exception of fl‐BSA, which exhibited perinuclear accumulation in approximately one‐half of the injected cells, autoradiography showed that throughout the period of study the injected proteins remained dispersed in the cytoplasm.
α‐Amanitin and 5‐fluorouridine inhibition of serumstimulated DNA synthesis in quiescent AKR‐2B mouse embryo cells
Authors: Wells David J. , Stoddard Linda S. , Getz Michael J. , Moses Harold L.
Published: February 2005

AKR‐2B mouse embryo cells undergoing the serum‐stimulated transition from a quiescent to a proliferating state exhibit an increase in the rate of hnRNA synthesis which appears to be mediated through an increase in the actual number of RNA polymerase II molecules. α‐Amanitin, administered early in the prereplication interval following stimulation, effectively inhibits hnRNA synthesis, polysomal mRNA accumulation, polyribosome formation, and subsequent DNA synthesis, and cell division. Unexpectedly, α‐amanitin treatment also produces almost complete inhibition of the synthesis of 45SrRNA precursor and the increase in accumulation of cytoplasmic rRNA following serum stimulation. In order to determine whether the inhibition of new ribosomal synthesis might in itself be sufficient to prevent serum‐stimulated DNA synthesis, the effects of 5‐fluorouridine (5‐FU), a specific inhibitor of 45SrRNA processing, were investigated. If added within eight hours following serum stimulation, 5‐FU was found to completely inhibit subsequent DNA synthesis. These results suggest that quescent AKR‐2B cells do not contain a sufficient excess of ribosomes to support the synthesis of proteins which are required for DNA synthesis in response to serum growth factors. Furthermore, an early polymerase II mediated synthesis of mRNA (s) coding for some factor(S) necessary for ribosomal gene transcription may be an essential step in the serum‐stimulated synthesis of new ribosomes.
Cell cycle dependence of transformation expression in mouse cells transformed by a thermosensitive mutant (Ts‐121) of polyoma virus
Authors: Okada Yasuhiro S. , Hakura Akira
Published: February 2005

Change in division capability as a phenotypic expression of cellular transformation was investigated by using one of the temperature‐sensitive (ts) mutants of the polyoma virus‐transformed cell line, the 121‐6‐5 cells of BALB/3T3. When contact ‐inhibited cells were treated with hyaluronidase at 39°C, a single round of cell division was induced after which cell growth was inhibited by cell density. However, if the cells were incubated at 35°C, after the enzyme treatment, density‐inhibition block disappeared and the cells entered a second division. This indicates that the release of cells from density‐inhibition depends on the low temperature incubation. The ability of cells to complete a second division was examined by shifting the cells from 39°C to 35°C during different phases of the first division cycle after the enzyme‐treatment. A 6‐hour incubation of S phase cells at 35°C resulted in a second cycle of division, while the 24‐hour incubation of G1 cells at 35°C did not induce a second round of division. These results suggest that expression of the transformed phenotype in 121‐6‐5 cells is clearly dependent upon both the temperature and the phase of the division cycle.
A growth factor from spinal cord
Authors: Jennings Thomas , Jones R. David , Lipton Allan
Published: February 2005

Mitogenic activity is present at a variety of sites in the central nervous system. A growth factor was purified from neonatal bovine spinal cord. It has a pI of 9.5‐9.8 and a molecular weight of about 11,000 daltons. Spinal cord growth factor is a basic polypeptide that is inactivated by extremely acid or basic conditions. Its mobility on SDS polyacrylamide gels suggests that this factor is different from pituitary FGF and brain FGF‐1.
Insulin effects on protein synthesis and secretion in primary cultures of amphibian hepatocytes
Authors: Stanchfield James E. , Yager James D.
Published: February 2005

The objective of the present study was to determine the effects of insulin on amphibian hepatocytes in primary culture. Hepatocytes were isolated from adult bullfrogs by collagenase perfusion and maintained as monolayers in serum‐free medium. Cells cultured in the continuous presence of insulin exhibited a relatively constant rate of protein secretion over the first four to five days, whereas controls showed an almost three‐fold decrease over the same time period. The decline in secreted proteins was equally represented in most exported proteins, except that serum albumin secretion showed twice as much of a decrease relative to the other proteins.

The maintenance of protein secretion by insulin was the result of its effect on protein synthesis. The rate of protein synthesis was measured by the incorporation of (3H)‐leucine into protein using culture medium containing 0.5 mM leucine, a condition where the specific radioactivity of leucyl‐tRNA was shown to be equal to that of (3H)‐leucine in the medium. Cultures maintained with insulin for 60 hours synthesized protein at two to three times the rate found in non‐insulin treated controls whose rate of protein synthesis was first detectably decreased after nine hours of culture in the insulin‐free medium.

Sedimentation profiles of polyribosomes from hepatocytes maintained for 60 hours without insulin showed proportionately fewer ribosomes in large polysomes and more in monosomes and free ribosomal subunits than ribosomes from cells cultured with insulin. This result suggests that the decrease in protein synthesis found in the absence of insulin is due to a defect in initiation. Insulin does not exert its effect by regulating cellular levels of ATP; no change in ATP content was found in cells maintained with or without insulin.

The results show that insulin maintains high levels of protein synthesis and secretion in amphibian hepatocytes. The hepatocytes in monolayer culture provide a system to study the molecular mechanisms involved in the translational control of protein synthesis by insulin.
Polyene macrolide antibiotic cytotoxicity and membrane permeability alterations. II. Phenotypic expression in intraspecific and interspecific somatic cell hybrids
Authors: Fisher Paul B. , Bryson Vernon , Schaffner Carl P.
Published: February 2005

Cytotoxicity and membrane permeability alterations induced by the polyene macrolide antibiotics filipin (FIL) and pimaricin (PIM) have been compared in parental intraspecific and interspecific somatic cell hybrids. B82 (mouse) and B1 (hamster) cells were found to be more resistant than RAG (mouse) parental cells to both polyene macrolides as indicated by 24‐hour survival, 72‐hour viability, and growth rate. Analysis of both intraspecific and interspecific somatic cell hybrids indicated that polyene macrolide resistance was being expressed even in the presence of the polyene macrolide‐sensitive (RAG) genome. Where one of the two parental cell types is relatively polyene macrolide resistant, the use of specific polyene macrolides may prove efficacious as half‐selective agents in cell hybridization.
Characterization of factor(s) in culture supernatants affecting cell social behavior
Authors: Weston James A. , Yamada Kenneth M. , Hendricks Karen L.
Published: February 2005

Treatment of embryonic chick heart fibroblast cultures with 0.2 M urea reversibly increases cellular overlap. The increase in cellular overlapping over that in control cultures may be quantitated by the overlap ratio (R), the ratio of the number of superimposed nuclei observed, to the number expected to occur when cells are assumed to be distributed randomly over the culture substratum (R = observed/expected overlaps). Reversal of the urea‐induced increase in R is blocked by 0.2 μg/ml cycloheximide. In the presence of cycloheximide, normal (low) overlap ratios are restored to urea‐treated cultures by adding non‐dialyzable material recovered by washing fibroblast monolayers with serum‐free medium. The overlap ratio assay revealed no effect of supernatant material added either to urea‐treated cultures in the continued presence of urea, or to untreated cultures. Although unfiltered supernatants were shown by SDS‐polyacrylamide gel electrophoresis to contain fibronectin (CSP; LETS; MWappar. = 220,000 d) and smaller proteins, the ability to reverse the urea‐induced increase in overlap ratio was present in Diaflo and Millipore filtrates of culture supernatants in which fibronectin was greatly depleted or absent. In contrast, purified fibronectin preparations failed to lower urea‐induced increases in overlap‐ratio. Partially purified, biologically active supernatants, prepared from 14C‐leucine or 125I‐labeled cultures, contained several macromolecules smaller than fibronectin that were labeled by both radioisotopes. In particular, one band (MWappar. = 58‐60,000 d) was present in polyacrylamide gels of active supernatant and also depleted in gels of homogenates from urea‐treated cultures. These results indicate that external macromolecules other than fibronectin are synthesized by culture fibroblasts and can affect cell social behavior or culture morphology.
A calcium requirement for movement of cultured cells
Authors: Moore Leon , Pastan Ira
Published: February 2005

When calcium is removed from culture medium, motility of cultured cells is decreased. The effect is rapid, reversible and pronounced. Decreased motility is observed with normal mouse Balb/c 3T3 cells, mouse L929 cells, rat kidney fibroblasts and chick embryo fibroblasts. The calcium dependence of movement can be observed both with individual cells and with the movement of the margin of a monolayer into a wound. Magnesium will not substitute for calcium to maintain motility. Strontium will substitute, but is not as effective as calcium for maintaining cell movement. Low concentrations of the divalent cation ionophore A23187 (0.5–1 μm) partially reverse the reduced migration observed at low calcium concentrations. These results are consistent with the hypothesis that movement of non‐muscle cells occurs through mechanisms similar to those important in the contraction of muscle.
Selective high metabolic lability of uridine, guanosine and cytosine triphosphates in response to glucose deprivation and refeeding of untransformed and polyoma virus‐transformed hamster fibroblasts
Authors: Rapaport Eliezer , Christopher C. William , Svihovec Sandra K. , Ullrey Donna , Kalckar Herman M.
Published: February 2005

Sugar deprivation of hamster fibroblasts (NIL

Abbreviations: ATP, ADP and AMP, adenosine 5′‐tri‐, di‐ and monophosphate respectively; CTP, CDP and CMP, cytosine 5′‐tri‐, di and monophosphate respectively; GTP, GDP and GMP, guanosine 5′‐tri‐, di‐ and monophosphate respectively; IMP, inosine 5′‐monophosphate; Gal‐1‐P, α‐D‐galactose‐1‐phosphate; UDP‐Gal, uridine 5′‐diphosphogalactose; D‐MEM, Dulbecco's modified Eagle's minimal essential medium; D‐PBS, Dulbecco's phosphate‐buffered saline (pH 7.2); NIL, a line of Syrian hamster fibroblasts; PyNIL, a polyoma virus‐transformed line of NIL cells.

) affected the steady state levels (pool sizes) of cellular acid soluble nucleotides in the following fashion; the pools of UTP, GTP and CTP decreased to a much greater extent than the cellular ATP pools, with the UTP pools undergoing the most dramatic reduction. Sugar deprivation of polyoma‐transformed NIL cells (PyNIL) yielded even sharper decreases in the nucleoside triphosphate pools with relative changes similar to those of the untransformed cells. Inhibition of protein synthesis by cycloheximide, initiated at the onset of (and continued during) sugar deprivation, prevented the reduction in pool sizes and yielded values slightly higher than those observed for pool sizes in cells cultured in sugar‐supplemented medium. Refeeding glucose to sugar‐depleted hamster fibroblasts led to rapid increases (within 1 hour) in the UTP and CTP pools to levels well above the pool sizes observed in cells which were continuously cultured (16 hours) in sugar supplemented medium. Feeding NIL or PyNIL cells with fructose instead of glucose as the only hexose source did not appreciably affect any of the ribonucleoside triphosphate pool sizes. Measurements of hexose uptake by NIL and PyNIL cells under a variety of conditions suggest that hexose transport is not regulated by the total cellular pools of ATP or any of the other ribonucleoside triphosphates.
Adhesion of murine cells to glass microbeads: Substrate‐adsorbed serum proteins
Authors: Haas Robert , Culp Lloyd A.
Published: February 2005

Small (1 mm diameter) glass beads treated with acid and alkali provided a satisfactory substratum for the attachment and growth of normal and SV‐40 transformed Balb/c 3T3 murine cells. Cells attached to the beads with similar, though slower kinetics as to flat glass surfaces, and spread and grew with their usual morphology; when detached by EGTA treatment, they left behind cellular substrate‐attached material (SAM) which was similar electrophoretically to that obtained from tissue culture plastic. Because of their small size and rough etched surfaces, the beads have a large surface area and high adsorptive capacity, and so are a useful tool to isolate specific serum proteins adsorbed from the culture medium that may be important for cell attachment and spreading. The adsorbed serum proteins were solubilized with SDS and analyzed by SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) under reducing and non‐reducing conditions. They included all reduced species adsorbed to tissue culture plastic, and only small amounts of one other major protein extracted from a “bacteriological” polystyrene surface on which cells could not grow. Profiles of unreduced samples differed considerably. The profile of adsorbed proteins varied little with tiem (5 minutes–4 days), temperature (4°–37°C), pH (5–9), presence of the protease inhibitor PMSF, or serum concentration (0.1–10%). Much of the adsorbed protein, qualitatively similar to the SDS‐extracted material, could be eluted with H2O or phosphate‐buffered saline. Purified albumin and fibrinogen bound avidly to the beads; the material adsorbed from serum contained a large amount of albumin, however, little fibrinogen and no cold‐insoluble globulin (as a 220 K protein) could be detected by Coomassie blue stained SDS‐PAGE gels.
Role of cytoplasmic ATP in the restoration and maintenance of a membrane permeability barrier in transformed mammalian cells
Authors: Makan Nizar R.
Published: February 2005

Addition of ATP to medium surrounding intact, transformed 3T3 cells activates the formation of aqueous channels in the plasma membrane. This results in efflux of nucleotide pools and ions and entry into the cytosol of charged, phosphorylated species. In such permeabilized cells, glycolysis is totally dependent on the external addition of glucose, inorganic phosphate, ADP, K+, Mg2+ and NAD+ which restore lactic acid formation to levels found in untreated cells. As expected, such reconstitution of glycolytic activity is found to restore intracellular ATP levels. This is accompanied by sealing of the membrane channels so that efflux of nucleotide pools ceases. Pyruvate, a substrate for mitochondrial ATP synthesis, when provided along with ADP and inorganic phosphate also produces sealing of the membrane channels. On the other hand, reactivation of pentose phosphate shunt activity, which does not lead to ATP synthesis, does not induce restoration of the membrane permeability barrier. Furthermore, compounds which lower the internal ATP pool prevent sealing, and also render the plasma membrane more sensitive to external ATP (Roxengurt and Heppel, 1979). Sealing of aqueous channels following restoration of the internal ATP pool is associated with phosphorylation of the inner membrane surface, and is unaffected by inhibitors of protein synthesis, microfilament or microtubular assembly. These results indicate the probable role of intracellular ATP in the restoration and/or maintenance of an active membrane barrier against efflux of small molecules and ions in transformed 3T3 cells.
Ribosome biosynthesis in Tetrahymena thermophila. IV. Regulation of ribosomal RNA synthesis in growing and growth arrested cells
Authors: Sutton Claudia A. , Sylvan Paul , Hallberg Richard L.
Published: February 2005

Three parameters involved in the production of new ribosomal RNA (rRNA) were measured in Tetrahymena thermophilia: (i) the rate of synthesis of the rRNA precursor, (ii) the rate of processing of the RNA precursor and rRNA intermediates and (iii) the efficiency of utilization of the rRNA precursor in producing mature ribosomal RNA. These parameters were measured in cells in exponential growth and in cells starved in a dilute salt solution. Growing cells synthesize rRNA 20 times faster and process rRNA precursors and intermediates 10 to 15 times more rapidly than do starved cells. Both utilize their rRNA precursors with an efficiency of one in converting them to mature rRNA.
A possible role for cyclic AMP in the initiation of DNA synthesis by isoproterenol‐activated parotid gland cells
Authors: Tsang B. K. , Rixon R. H. , Whitfield J. F.
Published: February 2005

An intraperitoneal injection of the β‐adrenergic agonist dl‐isoproterenol hydrochloride (100 mg/Kg body weight) into a rat caused an early, very large (400‐fold) cyclic AMP surge (peaking at 10 minutes) in the parotid gland which was followed by a second, much smaller (two‐fold) surge 12 to 16 hours later. DNA synthesis began about 16 to 20 hours after the isoproterenol injection and peaked between 24 and 28 hours. The maximum level of DNA‐synthetic activity at 24 hours was correlated positively to the magnitude of the small cyclic AMP surge at 12 hours, but not to the size of the much larger cyclic AMP surge at 10 minutes. An intraperitoneal injection of dl‐propranolol hydrochloride (59 mg/Kg body weight) at 8 hours after isoproterenol injection abolished the second cyclic AMP surge at 12 hours and markedly (65‐75%) reduced the incorporation of [3H]‐thymidine into DNA. Injection of dibutyryl cyclic AMP (6.3 mg/Kg body weight) and theophylline (25 mg/Kg body weight) at 8 hours prevented propranolol from inhibiting DNA synthesis. Propranolol appeared specifically to affect the cyclic AMP‐dependent pre‐DNA‐synthetic step because it did not reduce [3H]‐thymidine incorporation when injected after the second cyclic AMP surge had passed and DNA synthesis had just begun. Thus, the initial, large cyclic AMP surge following β‐adrenergic stimulation may not be necessary for the initiation of prereplicative development, while the much smaller second surge may be needed for the initiation of DNA synthesis.
Regulation of amino acid transport in chicken embryo fibroblasts by purified multiplication‐stimulating activity (MSA)
Authors: Derr James T. , Smith Gary L.
Published: February 2005

Enhanced amino acid transport is observed when quiescent cultures of chicken embryo fibroblasts are stimulated to proliferate by the addition of purified multiplication‐stimulating activity (MSA). This increase in amino acid transport is an early event occuring prior to the onset of DNA synthesis in stimulated cells. Results indicate that the changes in transport activity, as measured by α‐aminoisobutyric acid (AIB) uptake, are due to stimulation of only the Na+‐dependent A transport system. There is little or no change in the activities of transport systems ASC, L, or Ly+ upon exposure to MSA. A kinetic analysis shows this increased activity is due to a change in Vmax while Km remains unaltered. Continuous exposure to the stimulus is required to maintain the increased level of transport activity and the presence of inhibitors of RNA and protein synthesis significantly inhibits the response. Results also indicate that a similar specific increase in the A transport system is initiated when RSV tsNY68 infected cells are shifted to the permissive temperature. It appears that the A system of mediation is emerging as a strategic regulatory site for cell function.
Chemosensitivity of single smooth muscle cells to acetylcholine, noradrenaline, and histamine in vitro
Authors: Sinback Christopher Nelson , Shain William
Published: February 2005

Electrical responses to acetylcholine, noradrenaline, and histamine were recorded from solitary smooth muscle cells. Iontophoresis of each transmitter elicited three fast responses: a hyperpolarization, a depolarization, or a biphasic hyperpolarization‐depolarization. Each transmitter activated a specific receptor since responses were specifically blocked by antagonists, two transmitters elicited different responses in solitary cells, and desensitization of response to one transmitter did not cause desensitization of responses to other transmitters. Responses were due to increased ion conductances since input resistance decreased during responses and reversal potentials were measured for deplarizing responses (−5 mV) and hyperpolarizing responses (−60 mV). Regional differences in transmitter sensitivity were mapped on solitary cells. Biphasic responses were due to simultaneous activation of receptors mediating hyperpolarizing responses and receptors mediating depolarizing responses which were segregated in the cell membrane. Noradrenaline enhanced action potential amplitude by regulation of voltage‐dependent ion conductances. Finally, noradrenaline and histamine elicited periodic hyperpolarizing potentials, which may be due to increased intracellular Ca++.
The maturation state of three types of granulocyte/macrophage progenitor cells from mouse bone marrow
Authors: Bol Simon , Williams Neil
Published: February 2005

The progenitor cells of neutrophil granulocytes and macrophages which are able to proliferate and differentiate in vitro (CFU‐c) form a heterogeneous population. By the use of specific colony stimulating activities and cell separation by equilibrium density centrifugation, three subpopulations of CFU‐c can be detected. These three CFU‐c are characterized by buoyant densities of 1.070, 1.075 and 1.080 g.cm−3 and by their proliferative response to 18 h postendotoxin serum, colony stimulating factor from extracts of mouse embryos and uteri (CSF‐pmue) and erythrocyte lysate, respectively. The three CFU‐c are compared with respect to their differentiation potential, the maturation rate of their progeny cells and their proliferation capacity. It is shown that with increasing density of the CFU‐c the maturation rate increases (sequential maturation of colonies derived from CFU‐c with densities of 1.080, 1.075, 1.070 g.cm−3) and the proliferation capacity decreases (colony size decreases in the sequence of CFU‐c with densities 1.070, 1.075, 1.080 g.cm−3). Concerning the differentiation potential it is shown that all three CFU‐c detected have the capacity to form granulocytes as well as macrophages. On the basis of these results it is concluded that the CFU‐c with densities of 1.070, 1.075 and 1.080 g.cm−3 represent a maturation sequence.
Hexose uptake enhancing factor released from rous sarcoma cells
Authors: Lawrence David A. , Jullien Pierre
Published: February 2005

Conditioned media from Rous sarcoma virus transformed chicken embryo fibroblasts stimulate the uptake of 2‐deoxyglucose in normal chicken fibroblasts. The factor responsible for this effect, which is also shed in very low amount by non‐transformed fibroblasts, is destroyed by trypsin and not linked to the protease and plasminogen activator activities present in the media. Its apparent molecular weight, determined by gel filtration, is about 20.000 daltons. The factor released by transformed cells might be related to the monomeric form of a family of glucose binding and transport proteins recently reported by Lee and Lipmann ('78) to be detached by detergents from normal and transformed cells.
Production of a colony‐stimulating factor following differentiation of leukemic myeloblasts to macrophages
Authors: Maeda Michiyuki , Ichikawa Yasuo
Published: February 2005

Leukemic cells in the myeloblastic stage from a murine myeloid leukemia cell line (M1) were induced to differentiate to macrophages by lipopolysaccharide (LPS) from Gram‐negative bacteria. A granulocyte‐macrophage colony‐stimulating factor (CSF) was produced only during differentiation. After induction of differentiation, the continued presence of LPS was necessary to stimulate the macrophages to release CSF.

In contrast, a macrophage cell line (Mm‐1) derived from the M1 line produced CSF without LPS‐stimulation, but CSF release was stimulated by the presence of LPS.
Hemopoietic colonies on the chorioallantoic membrane of the chick embryo: Induction by embryonic, adherent, non‐hemopoietic spleen cells
Authors: Keller Gordon , Longenecker Michael , Diener Erwin
Published: February 2005

Granulocytic and erythrocytic colonies developed on the chick embryo chorioallantoic membrane (CAM) following the inoculation of chick embryo spleen cells. Dose response and kinetic experiments showed that the colonies were derived from cell aggregates present in the inoculum. Dissociation and reaggregation studies of the CAM colony‐inducing cells (CAM‐CIC) indicated that these cells must be present as aggregates in order to form colonies. Results from the morphology and cell marker experiments suggested that the colony‐inducing aggregates (CAM‐CIA) attract and support the differentiation of primitive host hemopoietic cells. The physical characteristics of the CAM‐CIC, which are different from those of the hemopoietic progenitor cells, indicated that they represent a stromal cell population of the chick embryo spleen. Further evidence supporting this notion was provided by the radiation studies which showed that the colony‐inducing ability of the CAM‐CIC is radioresistant. The above characteristics of the CAM‐CIC strongly suggest that they represent the stromal cells of the chick embryo spleen which influence hemopoiesis.
NAD turnover in microplasmodia of physarum polycephalum
Authors: Manser Tim , Olivera Baldomero M. , Haugli Finn B.
Published: February 2005

The rate of NAD turnover in microplasmodia of Physarum polycephalum was investigated using a double labeling technique with (14C)‐adenine or adenosine and (3H)‐nicotinamide.

The half‐life of an NAD molecule in Physarum was estimated to be 25 min, which is shorter than in either E. coli or human cell lines. The half‐life of NAD in the presence of an inhibitor of NADase and poly ADPR synthase, 5‐methyl‐nicotinamide, was also investigated, but found to be indistinguishable from controls. The possible reasons for this and for the rapid turnover is discussed in the light of the known functions for NAD in prokaryotes and eukaryotes.
Inhibition of growth of cells in culture by L‐phenylalanine as a model system for the analysis of phenylketonuria. I. Amino acid antagonism and the inhibition of protein synthesis
Authors: Dillehay L. , Bass R. , Englesberg E.
Published: February 2005

Phenylalanine in high concentrations inhibits the growth of mouse A9 cells. Protein synthesis is inhibited earlier and more severely than RNA or DNA synthesis. Phenylalanine inhibits the uptake and decreases the intracellular pool of several amino acids. Certain amino acids added in excess reverse the phenylalanine inhibition. The strongest reversing amino acids appear to function by excluding phenylalanine. The phenylalanine inhibition does not appear to be due to a deficiency of any amino acid, but to the high intracellular phenylalanine concentration and/or an amino acid imbalance resulting from the large ratio of phenylalanine to other amino acids.
Initiation of DNA synthesis in mammary epithelium and mammary tumors by lithium ions
Authors: Ptashne Kay , Stockdale Frank E. , Conlon Sandra
Published: February 2005

The growth promoting effects of lithium and insulin on cultures of mammary gland epithelium and CZF mouse mammary tumor cells were investigated. Lithium chloride exerts a 450‐fold increase in the rate of DNA synthesis in mammary epithelium from mid‐pregnant mice in organ culture or monolayer culture. There is an increase in both the percentage of cells initiating DNA synthesis and the net accumulation of DNA. The most effective lithium concentration is 10 mM, and the maximally effective rate of stimulation is reached 48 hours after addition. The magnitude of response to lithium varies with the physiological state of the mammary epithelial cell donor: epithelium from non‐pregnant or lactating mice is less responsive than that from mid‐pregnant mice. In combination, insulin and lithium produce either a synergistic or an additive effect on the growth of epithelium dependent upon the physiological state of the donor animal. Lithium also promotes the growth of mammary tumor cells in the absence of serum or other mitogens. The action of lithium on DNA synthesis appears to be a direct effect on the epithelial cells.
Inhibition of proliferation of cloudman S91 melanoma cells by insulin and characterization of some insulin‐resistant variants
Authors: Kahn Richard , Murray Marilyn , Pawelek John
Published: February 2005

Insulin is a potent, reversible inhibitor of proliferation in Cloudman S91 mouse melanoma cells. The inhibition seems to be unique to the Cloudman line since five other cell lines, including the mouse B16 and hamster Greene melanomas, were unaffected by insulin under the same culture conditions. Variants of Cloudman S91 cells were isolated which differed from wildtype (WT) cells in their response to insulin. Most of these variants were resistant to insulin (INSres) and had the same generation time independent of the presence of the hormone. One line (INSdep) was found to require insulin for growth. This line was about 15 times more sensitive to the proliferative effects of insulin than the WT. Revertants of the INSdep line were selected for their ability to proliferate in the absence of insulin. Five out of five such revertants were insulin resistant, suggesting that the INSdep line arose as a result of at least two separate mutations.

Both WT and INSdep cells showed enhanced uptake of 14C‐α‐aminoisobutyric acid (AIB) when exposed to insulin. Dose‐response curves of the stimulation of AIB uptake in WT and INSdep cells were superimposable. Stimulation of AIB uptake and stimulation of proliferation by insulin were not under coordinate control since AIB uptake was increased equally in the wild‐type cells when proliferation was inhibited and in INSdep cells when proliferation was enhanced.

Binding of 125I‐insulin was used to demonstrate the presence of specific, high affinity insulin receptors on the cells. INSres variants generally had fewer receptors than WT, but in no case did the magnitude of this effect appear to be sufficient to explain the insensitivity to insulin. The INSdep variant showed a greater than two‐fold increase in the number of insulin receptors per cell, compared to WT. Revertants of the INSdep line had the same number of receptors as WT. The specificity for both binding and for the effects on proliferation were the same in WT and INSdep cells. Since the effects of insulin on proliferation were opposite in the two lines, we propose at least two distinct sites of insulin action on the cells. Further isolation and analyses of Cloudman lines with unusual responses to insulin should be useful for understanding the molecular basis of action of this hormone.
Intracellular concentration of elements in normal and dystrophic skeletal muscles of the chicken
Authors: Misra L. K. , Smith N. K. R. , Chang D. C. , Sparks R. L. , Cameron I. L. , Beall P. T. , Harrist R. , Nichols B. L. , Fanguy R. C. , Hazlewood C. F.
Published: February 2005

In this study, the intracellular concentrations of six elements (mmole/kg dry weight) were directly measured in the muscle fibers of pectoralis major muscles of eight week old, genetically dystrophic and normal chickens by the X‐ray microanalysis technique. The extent of muscle degeneration was evaluated by morphometric measurements of muscle fiber diameter and other histological changes. A significant increase in the concentration of intracellular sodium and chlorine was evident in dystrophic muscles. The concentration of intracellular sodium was 127.0 ± 35.0 in the muscle fibers of dystrophic chicks compared to 65.7 ± 16.5 in normal controls. The concentration of chlorine was 90.5 ± 27.5 and 54.1 ± 5.5 in the muscle fibers of dystrophic and normal chicks respectively. The intracellular concentrations of potassium, magnesium, phosphorous, and sulfur remained unchanged in the dystrophic condition. Morphometric studies revealed that the dystrophic pectoralis muscles contain fewer but thicker fibers per unit area compared to normal pectoralis muscles. The importance of these findings are discussed in relation to the results of earlier investigations.
Limitation of macrophage production in long‐term marrow cultures containing prostaglandin E
Authors: Williams N. , Jackson H.
Published: February 2005

Addition of prostaglandin E2 (PGE2) significantly altered the cellular composition of murine long‐term bone marrow cultures. After 4–5 weeks of culture, increased cellularity in the suspension phase was observed in all cultures containing prostaglandin. These suspension cells contained markedly higher proportions of differentiated neutrophils than did cells cultured in the absence of PGE2. Granulocyte‐macrophage progenitor cell levels in the suspension layer were increased 3–20 fold after five weeks in prostaglandin‐containing cultures compared with control cultures. Fewer cells comprised the adherent layer in cultures containing prostaglandin. The number of macrophages in this layer was reduced 3–8 fold in these cultures compared with control cultures, while the number of granulocytes was increased 2–3 fold. The progenitor cells biased toward macrophage development were selectively inhibited in the cultures with PGE2. There was no significant effect of PGE2 on pluripotent stem cell levels or on the longevity of the cultures. It is concluded that excessive monopoiesis in bone marrow may be limited by PGE2 without influencing either stem cell maintenance or the development of other marrow‐derived cell types.
Stimulation of DNA synthesis and myo‐inositol incorporation in mammalian cells
Authors: Ristow Hans‐Jürgen , Messmer Trudy O. , Walter Sephanie , Paul Dieter
Published: February 2005

Phosphatidylinositol (PI) synthesis and its role in controlling the cell cycle has been investigated using fibroblasts and liver cells in culture. PI synthesis as measured by incorporation of [3H]‐myo‐inositol into trichloroacetic acid precipitable material during 0–60 min after serum or growth factor stimulation of serum‐starved cells is increased in primary fetal rat liver cells, rat embryo fibroblasts, and 3T3 mouse cells. In contrast, growth stimulation of 3T3 cells and hepatocytes rendered quiescent in G1 by amino acid starvation is not accompanied by increased incorporation of [3H]‐myo‐inositol into trichloroacetic acid precipitable material. This suggests that those cells might be arrested at a different point in G1 than cells arrested by serum depletion. Inhibition of PI synthesis by δ‐hexachlorocyclohexane (HCH), a steric analog of myo‐inositol, during early times (e.g., 0–4 hr) after growth stimulation, reversibly blocks initiation of DNA synthesis in 3T3 cells. The results support the idea that increased PI synthesis in response to growth stimulation in the cell types studied here is a prerequisite for progression through G1 and subsequent entry into S phase.