bluuebunny/crossref_metadata_2025 huggingface dataset:
Penetration of mouse fibroblasts by 2′‐deoxyadenosine 5′‐phosphate and incorporation of the nucleotide into DNA
Authors: Plunkett William , Cohen Seymour S.
Published: February 2005

The metabolism of 2′‐deoxyadenosine 5′‐phosphate (dAMP) by exponentially growing mouse fibroblasts (L‐cells) in suspension culture has been studied. Cells incubated for four hours with 0.10 μmole [3H, 32P]dAMP/ml in medium containing 9.6 μmole 31Pi/ml incorporate both activities at nearly linear rates into acid‐soluble and acid‐insoluble fractions. The 3H/32P value increased about 30‐fold in each fraction during the incubation, indicating extensive dephosphorylation of dAMP. The DNA from treated cells was degraded enzymatically to 5′‐mono‐nucleotides, which were fractionated by ion‐exchange chromatography. 3H was associated exclusively with dAMP (53%) and dGMP (47%). 32P, associated with all deoxynucleotides, was at a 20‐fold higher specific activity in dAMP than in either dGMP or dCMP. The specific activity of [32P]dAMP incorporated into DNA in four hours was 24‐fold greater than that of 32Pi in the cellular pool. In experiments in which cells were incubated with 32Pi plus or minus 0.10 μmole dAMP/ml, the specific activity of dAMP was slightly less than that of dGMP or dCMP, which were equal. These results suggest that the higher specific activity of [32P]dAMP in the DNA of cells after incubation with doubly‐labeled dAMP was due to the intact penetration of some dAMP into the cells with its subsequent incorporation into DNA. Calculations, based on the amount of exogenously added [3H]thymidine incorporated into the cellular DNA in parallel cultures and the 32P of dAMP isolated from this DNA, suggest that 1% of the total dAMP residues incorporated during the 4‐hour incubation were derived directly from intact, exogenously added dAMP.
Cation specificity of propranolol‐induced changes in RBC membrane permeability: Comparative effects in human, dog and cat erythrocytes
Authors: Müller‐Soyano Aixa , Glader Bertil E.
Published: February 2005

Propranolol, in the presence of calcium, causes marked K efflux from human red blood cells (high K, low Na). The studies reported here indicate this effect of propranolol is specific for K and does not represent a nonspecific permeability increase for intracellular cations to leave the cell. Amphotericin‐treated human RBC's (high Na, low K) and dog RBC's (high Na, low K) both gain K and increase in size when incubated in a K‐medium containing propranolol and calcium. No effect was noted when cat RBC's (high Na, low K) were similarly treated. Propranolol, independent of added calcium, also inhibited the normally increased Na efflux observed when dog RBC's are suspended in K‐medium. These species differences in response to propranolol thus may serve as a focus for elucidating the mechanism by which this drug alters normal membrane physiology. The unique drug effect on Na permeability of canine erythrocytes also may be a useful probe for the study of dog RBC volume regulation.
Colloidal iron hydroxide‐binding to the surfaces of chick embryo fibroblasts transformed by wild‐type and a temperature‐sensitive mutant of Rous sarcoma virus
Authors: Subjeck J. R. , Weiss L. , Warren L.
Published: February 2005

The densities of colloidal iron hydroxide (CIH) particles binding to the surfaces of chick embryo fibroblasts were determined before and after transformation with wild type Rous sarcoma virus and a temperature sensitive (ts) mutant of this virus. On the basis of in vitro behavior, cells transformed by the ts virus manifest a malignant phenotype at 36°C (permissive temperature) and appear normal at 41°C (non‐permissive temperature). At the permissive temperatures there is a significant increase in CIH particle‐binding to spaces of cell surface between microvilli on the wild type and ts transformed cells. At the non‐permissive temperature this significant increase in binding is only observed on the wild type transformant, while the density found on the ts transformant is not significantly different from the untransformed state. Therefore, in vitro characteristics of normalcy and malignancy are reflected in changes in the CIH binding properties of the cell surface spaces between microvilli.

The CIH densities observed on the microvilli are significantly different from the density on the spaces between them for each of the classes of cells studied at either temperature. The microvilli are found to bind a lower density of particles in five of the six cases. No correlations between microvilli particle density and transformation to in vitro malignant characteristics were observed.
Electron microbeam analysis of calcium distribution in the ciliated protozoan, Spirostomum ambiguum
Authors: Osborn Dustan , Hamilton T. C.
Published: February 2005

Electron microprobe analyses of calcium distribution in the ciliated protozoan, Spirostomum ambiguum, indicated several calcium rich sites. One site was an endoplasmic distribution of calcium coincident with phosphorus which corroborates previous findings of hydroxyapatite deposits within Spirostomum. These apatite deposits were distributed throughout the endoplasm, but not within the nuclei or the contractile vacuole. Calcium was also detected within the cortical region. Cortical calcium was in greater concentration in the anterior portion of the organism and decreased towards the posterior end (region containing the contractile vacuole). Phosphorus and potassium were also detected as gradients from the anterior end, whereas magnesium was detected in the same density throughout the cortical region. Line scans of cortical regions suggested (1) distributions of calcium within mitochondria and/or vesicles, and (2) calcium associated with bundles of microfilaments.
Hormonal regulation of initiation of DNA synthesis and of differentiated function in Y‐1 adrenal cortical cells
Authors: Gill Gordon N. , Weidman E. Russell
Published: February 2005

ACTH, 8‐Br‐cAMP, and serum deprivation arrested Y‐1 functional mouse adrenal tumor cells in the G1 phase of the cell cycle. Though ACTH and 8‐Br‐cAMP treated cells were larger with increased macromolecular synthetic rates compared to cells arrested in G1 by serum removal, a similar 8‐ to 10‐hour lag to initiation of DNA synthesis was observed after either ACTH or 8‐Br‐cAMP removal or after serum addition. After the 8‐ to 10‐hour lag period, cells entered S phase exponentially. ACTH or 8‐Br‐cAMP opposed serum induced DNA synthesis initiation only when added prior to S. Once commitment to DNA synthesis occurred, ACTH or 8‐Br‐cAMP addition did not inhibit DNA synthesis although 8‐Br‐cAMP induced a secondary block in G2. Though ACTH and 8‐Br‐cAMP inhibited serum induced initiation of DNA synthesis and did not affect serum induced cellular hypertrophy, both substances increased the steroidogenic capacity of the cell. ACTH and 8‐Br‐cAMP thus appear to specifically oppose the stimulatory effects of serum on initiation of DNA synthesis while inducing the differentiated function of the cell.
Control of normal differentiation of myeloid leukemic cells. XII. Isolation of normal myeloid colony‐forming cells from bone marrow and the sequence of differentiation to mature granulocytes in normal and D+ myeloid leukemic cells
Authors: Lotem Joseph , Sachs Leo
Published: February 2005

An enriched population of early myeloid cells has been obtained from normal mouse bone marrow by injection of mice with sodium caseinate and the removal of cells with C3 (EAC) rosettes by Ficoll‐Hypaque density centrifugation. This enriched population had no EAC or Fc (EA) rosettes and contained 87% early myeloid cells stained for myeloperoxidase and/or AS‐D‐chloroacetate esterase, 7% cells in later stages (ring forms) of myeloid differentiation and 6% unstained cells, 2% of which were small lymphocytes. After seeding in agar with the macrophage and granulocyte inducer MGI, the enriched population showed a cloning efficiency of 14% when removed from the animal and of 24% after one day in mass culture. Both the enriched and the unfractionated bone marrow cells gave the same proportion of macrophage and granulocyte colonies.

The normal early myeloid cells were induced to differentiate by MGI in mass culture in liquid medium to mature granulocytes and macrophages. The sequence of granulocyte differentiation was the formation of EA and EAC rosettes followed by the synthesis and secretion of lysozyme and morphological differentiation to mature cells. D+ myeloid leukemic cells with no EA or EAC rosettes had a similar morphology to normal early myeloid cells and showed the same sequence of differentiation. The induction of EA and EAC rosettes occurred at the same time in both the normal and D+ leukemic cells, but lysozyme synthesis and the formation of mature granulocytes was induced later in the leukemic than in the normal cells. The results indicate that selection for non‐rosette‐forming normal early myeloid cells also selected for myeloid colony forming cells, that these normal early myeloid cells can form colonies with differentiation to macrophages and granulocytes, that normal and D+ myeloid leukemic cells have a similar sequence of differentiation and that the normal cells had a greater sensitivity for the formation of mature cells by MGI.
The effect of TLCK on transcription and its role in modifying cell growth
Authors: Noonan Norine E. , Noonan Kenneth D.
Published: February 2005

The synthetic protease inhibitor N‐tosyl‐L‐lysine‐chloromethyl ketone (TLCK) acts to inhibit transcription when added to cell lines growing in vitro. This inhibition of transcription is most pronounced in transformed cells where TLCK is very toxic at concentrations as low as 25 μg/ml of culure medium. Non‐transformed cells are more resistant to the effect of TLCK, requiring ten times more TLCK to produce a comparable inhibition of transcription. The effect of this protease inhibitor on transcription can be prevented by preincubation of the cells in reduced glutathione or cysteine; however, the cells can not be rescued from the effect of TLCK even if glutathione or cysteine are added to the culture medium within five minutes of the addition of TLCK.
Phagocytosis by rabbit polymorphonuclear leukocytes: The effect of albumin and polyamino acids on latex uptake
Authors: Deierkauf Frans A. , Beukers Harm , Deierkauf Martha , Riemersma Jelle C.
Published: February 2005

Albumin in low concentrations (0.001–0.01 weight percent) was found to be an effective inhibitor of phagocytosis of polystyrene latex beads by rabbit polymorphonuclear leukocytes. Polyglutamic acid proved to be an inhibitor of latex uptake at even lower concentrations. Polylysine stimulates phagocytosis, maximal stimulation occurring at 0.002% polylysine. These findings are discussed with reference to the surface properties of latex particles and leukocytes, and particularly with reference to electrostatic interactions in phagocytosis.
Membrane transport by macrophages in suspension and adherent to glass
Authors: Pofit Joseph F. , Strauss Phyllis R.
Published: February 2005

We have compared membrane transport of lysine and adenosine by rabbit lung macrophages in suspension and adherent to glass cover slips. The rapid sampling techniques employed permitted measurements to be made over intervals as short as 10 seconds. Suspended cells transported both nutrients at a faster rate, although the Km values for the two transport systems remained unchanged. Colchicine, cytochalasin B and adenosine did not interfere with the enhancement.
Pyruvate carboxylase and phosphoenolpyruvate carboxykinase in cultured human fibroblasts
Authors: Raghunathan Rengachari , Russell James D. , Arinze Ifeanyi J.
Published: February 2005

The activities of pyruvate carboxylase and phosphoenolpyruvate carboxykinase were measured in cultured human fibroblasts. Considerable variation was observed in strains derived from different individuals. The activities of both enzymes throughout the culture cycle were measured in two strains. In these strains the specific activities of the enzymes increased during log phase and remained constant during the stationary phase. However, one cell strain exhibited a high activity of pyruvate carboxylase which remained unchanged throughout the culture cycle, suggesting that this enzyme may be regulated differently in different strains of cultured human cells. Familial studies suggest that the observed variations in pyruvate carboxylase activity may be due to a genetic polymorphism.
Protein metabolism during growth of vero cells
Authors: Lee Gloria T‐Y. , Engelhardt Dean L.
Published: February 2005

Protein synthesis and degradation were studied throughout a growth cycle of Vero cells. The rate of protein synthesis, measured as the rate of amino acid incorporation, reached a maximum at the mid‐exponential phase and declined to 10–30% of the maximum in the stationary phase. The rate of protein degradation, measured as the release of radioactive amino acids from uniformly labelled cellular proteins, did not vary in the growth cycle. The amount of protein per cell, measured by an isotopic method, remained constant when normalized to account for the variation in the proportion of actively dividing cells in the cell population during the growth cycle. Cellular protein was determined using this method since it was found that the chemical determination of the amount of protein in the monolayer was not accurate during the early stage of the growth cycle. This was due to a significant amount of serum protein adsorbed to the cells. In this study we were able to show that, in Vero cells, protein synthetic activity is correlated with the rate of cell division, and variations in the rate of synthesis alone are sufficient to meet the changing requirements for cellular protein in a growth cycle.
Decreased in vivo and in vitro colony stimulating activity responses to bacterial lipopolysaccharide in C3H/HeJ mice
Authors: Russo Momtchilo , Lutton John D.
Published: February 2005

Mice of the C3H/HeJ strain exhibit low inflammatory and immunological responses to certain bacterial lipopolysaccharide (LPS) preparations. Lymphocytes from C3H/HeJ mice also show defective LPS‐induced mitogenesis. We tested the colony stimulating activity (CSA) response of C3H/HeJ mice. As controls we used mice of the congenic C3HeB/FeJ strain, which are good responders to LPS. Serum was obtained three hours after intravenous administration of LPS. Serum CSA was determined in agar cultures of bone marrow cells from AKR mice. The serum CSA response of C3H/HeJ to 10 μg LPS was approximately 8‐fold lower than that of control C3HeB/FeJ mice. In contrast, both strains showed similar serum CSA‐induced by Poly I:poly C. Peritoneal macrophage cultures were also incubated with 0.1–10.0 μg LPS and culture media assayed for CSA. The response of C3H/HeJ macrophages was about 6‐fold lower than that of macrophages obtained from the control mice. The results show that the lower responsiveness of C3H/HeJ mice to LPS also extends to the production of CSA. The in vitro findings indicate that the postulated defect in the LPS receptor of B lymphocytes may also be present on macrophages.
Time of replication of genes responsible for a temperature‐sensitive function in a cell cycle‐specific ts mutant from a hamster cell line
Authors: Chang Helena L. , Baserga Renato
Published: February 2005

A method involving short pulses of 5‐bromodeoxyuridine (brUdRib) followed by irraidation with 313 nm light was used to locate the time of replication of certain genes during the cell cycle of two cell lines, AF8 and AL106. AF8, a temperature‐sensitive mutant of BHK21/13 cells, grows at 33°C but not at 39.5°C. AL106, a hybrid clone of tsAF8 and SV‐40 transformed Lesch‐Nyhan fibroblasts (LNSV), which retains all hamster chromosomes and one human chromosome (No. 3), has the ability to grow at 39.5°C. AF8 and AL106 cells synchronized at the G1‐S boundary were released from their block and pulsed with brUdRib for 2‐hour periods during the S phase. The cells were subsequently irradiated with 313 nm light. Colony‐forming efficiency and revertants frequency were studied. Incorporation of brUdRib during the early S phase (0–4 hours from the begining of S), decreased the colony‐forming efficiency of AL106 cells both at 33°C and 39.5°C, and also of AF8 cells at 33°C. No AF8 colonies grew at the nonpermissive temperature regardless of the treatment. Thus the time of replication of genes responsible for colony‐forming ability was the same in tsAF8 at the permissive temperature and in AL106 at both temperatures. The time of replication of the genes responsible for the ts function in AF8 cells was located by determining the revertants frequency in synchronized AF8 cells pulsed with brUdRib and irradiated during 1‐ to 2‐hour periods of the S phase. Back‐mutants were scored by counting the number of clones capable of growing at 39.5°C (nonpermissive for AF8 cells). The highest frequency of induced back‐mutations occurred in synchronized AF8 cells pulsed with brUdRib (and irradiated) between two to four hours from the begining of the S phase. Exposure to brUdRib during other periods of the S phase or during G1 had no effect on the reversion rate.

This method can be used to locate the time of replication (in S) of ts genes in other temperature‐sensitive mutants or of other specific genes in other conditional mutants.
Steroid hormone receptors and the differentiation of myeloid leukemic cells
Authors: Krystosek Alphonse , Sachs Leo
Published: February 2005

Receptors for the steroid hormone dexamethasone have been analyzed in clones of myeloid leukemic cells which differ in their ability to be induced by dexamethasone to form C3 and Fc rosettes, to synthesize and secrete lysozyme and form macrophages. The receptor dependent nuclear binding of (3H)‐dexamethasone has been measured in intact cells. As found with other glucocorticoid receptors, saturation of nuclear binding occurred at 4 × 10−8 M (3H)‐dexamethasone. The specificity of the receptors for only certain steroids was shown by the competition of nuclear binding by prednisolone, progesterone and testosterone, but not by the inactive steroid androstenedione. Nuclear bound hormone‐receptor complexes were released from the nucleus by treatment with deoxyribonuclease, indicating that the complexes were associated with DNA‐containing structures.

The amount of nuclear binding of receptors was similar in all clones examined, including those which could not be induced for rosette, lysozyme or macrophages. The non‐responding clones were also similar to the responding clones in the kinetics of binding, hormone concentration dependence of binding and association with DNA‐containing structures. The results indicate that the non‐responding clones of myeloid leukemic cells did not have detectable defects in the number, nuclear transport, or association with DNA of their steroid receptors.
Intracellular protein degradation in growing, in density‐inhibited, and in serum‐restricted fibroblast cultures
Authors: Hendil Klavs B.
Published: February 2005

Exponentially growing Balb/3T3 mouse fibroblasts contain protein populations with slow and fast turnover. These two stability classes were labelled selectively with 3H‐leucine. The intracellular degradation of the proteins was then followed as the release into the medium of radioactive leucine.

The degradation rate of both stability classes of protein is increased by about 55% in cultures whose growth is inhibited by high cell density. Serum‐deprivation, which also halts cell growth, accelerates protein breakdown to a smaller extent, the increase for relatively stable and unstable proteins being 30% and 13%, respectively.

The density‐dependent increase in protein breakdown is also found in BHK21 cells but not in chick fibroblasts. Protein degradation in Balb/3T3 cells transformed by simian virus 40 is affected by serum‐deprivation but not by cell density.

The proteins which are relatively stable during growth were shown to become less stable in density‐inhibited or serum‐deprived cultures, and vice versa.

Cycloheximide inhibits protein degradation to a variable extent. Dibutyryl adenosine‐3′,5′‐cyclic monophosphate has no effect on the protein degradation under the conditions investigated here.
Coexpression of mutant and wild type protein kinase in lymphoma cells resistant to dibutyryl cyclic AMP
Authors: Lemaire Irma , Coffino Philip
Published: February 2005

A mutant clone resistnat to dibutyryl cyclic AMP was isolated from S49 mouse lymphoma cells. The mutant expressed a form of cyclic AMP‐dependent protein kinase distinguishable from wild type kinase by its decreased sensitivity to activation by cyclic AMP and its increased thermal lability. Hybrids formed between mutant and wild type cells were resistant to dibutyryl cyclic AMP and expresed both mutant and wild type activities in about equal amount. The parent mutant cells also appeared to express wild type kinase activity, but at a lower level. We conclude that wild type S49 cells have and expression two identical alleles for the regulatory subunit of protein kinase, one of which has undergone mutation in the mutant cells.
On the regulation of DNA synthesis in a line of adrenocortical tumor cells: Effect of serum, adrenocorticotropin and pituitary factors
Authors: Armelin Mari C. S. , Gambarini Angelo G. , Armelin Hugo A.
Published: February 2005

Y‐1 adrenal cells responded to serum step down by a several fold decrease in DNA synthesis. Serum starved cells resumed DNA synthesis upon serum step up. ACTH and cAMP inhibited DNA synthesis both at low and high serum concentrations, a fact previously known. Pituitary, brain and liver crude extracts stimulated DNA synthesis in serum starved cells. Purified pituitary factors preparations contained two activities: one specific for Y‐1 cells and another active with both fibroblasts and Y‐1 cells. The kinetics of restimulation of DNA synthesis by serum and pituitary factors was studied. DNA synthesis restimulation occurred after a lag of 11 hours. This lag did not vary irrespective of the type of stimulator or its concentration. Cells entered S phase continuously at a rate which increased with increasing concentrations of the stimulator. Cells became refractory to the inhibitory action of ACTH five hours before entering S phase. The implications of these data to the understanding of cell growth control are considered.
Fatty acid and glucose oxidation by cultured rat heart cells
Authors: Rosenthal Miriam D. , Warshaw Joseph B.
Published: February 2005

Monolayer cultures of fetal rat myocardial cells can be utilized to examine substrate preferences and interactions. The specific activity of glucose oxidation by myocardial cell cultures was high in sparse cultures but decreased with increased cell density. In contrast, palmitate oxidation was independent of initial cell density. Palmitate inhibited glucose oxidation by 50% in rat heart cultures. Glucose had only a slight sparing effect on palmitate oxidation. This suggests that fetal and newborn rat myocardial cells in culture preferentially oxidize palmitate similar to adult heart. The sparing effect of palmitate on glucose oxidation is accounted for by inhibition of the glycolytic‐aerobic pathway and not by inhibition of the pentose phosphate pathway. Data on oxidation of 14C‐pyruvate specifically labelled suggest that palmitate or a product of its oxidation such as acetyl‐CoA may be acting directly to inhibit the pyruvate dehydrogenase complex. Palmitate oxidation per mg of cell protein was constant from 15 days gestational age to 2 days postnatal age. The observed differences between cultured cells and the intact heart may relate to decreased aerobic metabolism in monolayer cell culture and suggest that the increase in fatty acid oxidation observed in vivo is controlled by the oxygen environment of the cell. These studies show that heart cells in monolayer culture can be utilized to obtain metabolic information similar to an adult organ perfusion model.
The stability of tyrosine aminotransferase and other proteins in enucleated rat hepatoma tissue culture cells
Authors: Gopalakrishanan T. V. , Brad Thompson E.
Published: February 2005

The role of the nucleus in bringing about the induction of tyrosine aminotransferase (TAT) by glucocorticosteroid hormone and its deinduction upon steroid removal has been studied in enucleated rat hepatoma tissue culture cells (FU5–5). Both processes require the presence of the nucleus. However, cytoplasts from preinduced cells show an intial rapid decline in enzyme activity immediately after enucleation followed by maintenance of a constant level of activity. This initial decline in enzyme activity can be partially prevented by trypan blue, an inhibitor of lysosomal activity. This suggests that the early fall in enzyme activity could be due to an increase in the level of lysosomal activity immediately after enucleation. The subsequent constant level of activity seems due to maintenance rather than synthesis and degradation since it is not affected by cycloheximide. The absence of degradation applies to other kinds of proteins in enucleated FU5–5 cells and enucleated mouse fibroblast L cells. Thsese experiments suggest that some kind of labile RNA or protein dependent on the presence of the nucleus is required for the degradation of all classes of proteins in diferent kinds of cells.
Alteration of membrane electrical activity in rat myocardial cells following selective laser microbeam irradiation
Authors: Kitzes Margarita , Twiggs Gary , Berns Michael W.
Published: February 2005

Laser microirradiation of neonatal rat (1 to 2‐day‐old) ventricular cells in tissue culture results in overt changes in contractility. The intracellular study of their ongoing electrical activity prior to, during, and after laser microirradiation demonstrates that definite membrane alteration occurs concomitantly with induced contractile responses. Although all ventricular cells are depolarized by laser microirradiation, the ultimate response elicited seems to differ according to the type of myocardial cell impaled. Typical fibrillation potentials were induced mainly in pacemaker cells.
Separation of proximal tubule cells from suspensions of rat kidney cells by free‐flow electrophoresis
Authors: Kreisberg Jeffrey I. , Sachs George , Pretlow Thomas G. , McGuire Robert A.
Published: February 2005

Suspensions of rat kidney cells obtained by disaggregation of the kidney with 0.25% trypsin were separated by electrophoresis. Previously, we found a correlation between cells with histochemically demonstrable alkaline phosphatase (HDAP) and cells with brush borders which established that HDAP is a useful marker for rat proximal tubule cells (Kreisberg et al., '77). The starting suspension of cells for electrophoresis consisted of 38.4 ± 5.7% nucleated cells with HDAP, 39.8 ± 5.7% nucleated cells without HDAP, and 20.8 ± 9.2% red blood cells. After electrophoresis, the purest fraction contained 85.8 ± 3.5% nucleated cells with HDAP, 8.4 ± 2.2% nucleated cells lacking HDAP, and 5.8 ± 2.8% red blood cells; 91.9 ± 2.4% of the nucleated cells in the purest fractions had HDAP.
Expression of nervous system antigen‐3 by lines of mouse fibroblasts and kidney cells and continued expression in hybrid cells
Authors: Doyle Jane M. , Schachner Melitta , Davidson Richard L.
Published: February 2005

In a survey of the expression on cultured mouse cells of the cell surface antigen known as nervous system antigen‐3 (NS‐3), it was found that RAG, a renal adenocarcinoma line, expressed that antigen. It was also observed that 3T3, a fibroblast line of unknown tissue origin, expressed NS‐3. Cells of these two lines were hybridized with cells of two mouse L cell lines that did not express NS‐3. Four hybrid clones were tested for both the 3T3 × L cell cross and the RAG × L cell cross, and all the hybrids were found to be NS‐3 positive. All the hybrids had at least 40% as much activity as the NS‐3 positive parent. Of the four parental mouse cell lines used, only 3T3 expressed Thy‐1.2 antigen on the cell surface. In contrast to the continued expression of NS‐3 on hybrid cells, Thy‐1.2 antigen was not detectable on two clones of 3T3 × L cell hybrids that were tested.
Glucocorticoid binding and mechanism of resistance in some clones of mouse myeloid leukemic cells resistant to induction of differentiation by dexamethasone
Authors: Honma Yoshio , Kasukabe Takashi , Okabe Junko , Hozumi Motoo
Published: February 2005

Several clones of dexamethasone‐resistant cells, which could not differentiate even in a high concentration of dexamethasone, were isolated from glucocorticoid‐sensitive myeloid leukemic cells. Some of them were shown to be deficient in steroid binding to specific cytoplasmic receptors, while the others contained glucocorticoid‐specific cytoplasmic receptors that might be the same as those in sensitive cells. One of the resistant clones was found to be almost completely deficient in nuclear acceptor sites for cytoplasmic steroid‐receptor complexes. The remaining clones were also characterized by significantly reduced amounts of nuclear‐bound glucocorticoid. These results suggest that resistibility to glucocorticoids in the resistant clones of myeloid leukemic cells is due mainly to a defect in some steps of intracellular transfer of the steroid. Dexamethasone‐sensitive cells, which could differentiate in the presence of dexamethasone, could be also induced to differentiate by protein factor(s) in ascitic fluid. Although all the resistant cells showed a low response to ascitic fluid, some of them showed 10‐fold enhancement of phagocytic activity which is a typical character of differentiated cells. These results suggest that response to steroids is not directly correlated with that to protein inducer(s).
Reversible inhibition of the norepinephrine induction of lactate dehydrogenase by cytochalasin B in rat glial C6 cells
Authors: Bennett Kimberly , De Vellis Jean
Published: February 2005

The cyclic AMP mediated induction of lactate dehydrogenase (LDH: E.C. 1.1.1.27) activity by norepinephrine in the rat glial cell line C6 is inhibited by cytochalasin B. Doses of 5, 15, and 25 μg/ml of cytochalasin B inhibited the induction equally. Twenty‐five μg/ml of cytochalasin B inhibited the induction reversibly, and had no effect on basal enzyme level. No effect of cytochalasin B on general protein synthesis was found, nor did it increase the rate of decline of enzyme activity in deinduced cells. It therefore appears to block LDH induction by selectively inhibiting its synthesis. Cytochalasin B had no effect on the transient (intracellular and extracellular) rise in cyclic AMP generated in response to norepinephrine treatment. Cytochalasin B was effective when added during the transcription dependent phase (first 3 hours) but not during the translation dependent phase (after 3 hours) of LDH induction. The suggestion is discussed that cytochalasin B inhibits one of the early events of the inductive process.
Gel filtration analysis of vanadium in Ascidia nigra blood cell lysate
Authors: Gilbert Katharine , Kustin Kenneth , McLeod Guy C.
Published: February 2005

Fractions from a Sephadex gel filtration of homogenized Ascidia nigra blood cell lysate were analyzed for vanadium by atomic absorption spectroscopy. The results were unaffected by temperature from 4–21°C, and by ionic strength in the range 0.09‐1.0 M (NaCl). Appreciable loss of vanadium in the supernatant was experienced above pH 2.2. Experiments at pH 2.1 under anaerobic conditions show that the green chromogen and the vanadium‐containing bands elute separately. Under these experimental conditions, the vanadium‐containing species is of relatively low (⋍ 1,000) molecular weight.
On the mechanism of the crabtree effect in mouse ascites tumor cells
Authors: Sauer Leonard A.
Published: February 2005

The addition of glucose to ELD and ELT/B1 mouse ascites tumor cell suspensions caused a 2.3‐fold increase in the phosphorylation state ratio, (ATP)/(ADP)(Pi), because of a decrease in the intracellular Pi concentration. The addition of glucose to these cell suspensions has been reported by Chance and Hess ('59) to cause an increase in the steady state reduction of cytochrome b and an increase in the steady state oxidation of cytochrome c. On a quantitative basis these two independent measurements suggest that a near equilibrium exists between the oxidation‐reduction state of the mitochondrial electron carriers and the reactions of ATP synthesis (as expressed by the phosphorylation state ratio) both before and after glucose addition. We conclude that the mechanism of the inhibition of respiration by glycolysis (the Crabtree effect) is a decrease in the rate of electron transport caused by the mass action effect of the elevated phosphorylation state ratio.
Modulation of protein synthesis during the growth cycle of a culture of scarlet rose
Authors: Ramagopal S. , Huang B. , Marcus A.
Published: February 2005

Dilution of a stationary phase culture of Scarlet Rose results in an increased rate of protein synthesis. This study compares the time course of this increase with the changes in polyribosome content and the levels of adenine and guanine nucleotides.

During the first two hours after dilution, protein synthesis increases 2‐ to 3‐fold; much of the large monoribosome pool that characterizes the stationary state disappears and a steady state situation is reached in which 70% of the ribosomes are in polyribosomes. Between two and eight hours, there is no further change in polyribosome content although the rate of protein synthesis increases an additional 2‐ to 3‐fold. During this initial 8‐hour period there is little change in the levels of ATP and GTP. An explanation consistent with these observations is that the initial activation (within the first 2 hours), characterized by the monoribosome to polysome transition, is at the level of a component(s) of the initiation system, and that between two and eight hours, since neither mRNA availability nor energy level are primary determinants, protein synthesis is augmented by the activation of a translational component, perhaps an elongation factor.

After 24 hours, there is a proliferative phase characterized by the onset of ribosome accumulation. By day 5, maximum ribosome levels, 5‐fold that of 24‐hour cells, are reached, but the rate of protein synthesis increases only 2.5‐fold during this period. The lack of quantitative coincidence between the changes in polyribosome content and the rates of protein synthesis again suggests that factors other than mRNA availability are involved in determining the overall rate of protein synthesis. Finally at days 6–8, while the growth of the culture is still in the exponential phase, the rate of protein synthesis per unit fresh weight drops markedly concomitant with a decline in ribosome content. At days 11–12, the monoribosome to polysome ratio begins to change with the monoribosome pool increasing.

Presence of either actinomycin D or cordycepin inhibits increased protein synthesis in direct relation to the ability of these compounds to inhibit RNA synthesis. This suggests that the protein synthetic processes occurring after dilution require either the synthesis of the mRNA that is being translated or of an RNA functioning in a closely linked reaction.
Inhibition of mammalian ribonucleotide reductase by a dinucleotide produced in eucaryotic cells
Authors: Lewis William H. , McNaughton David R. , Goh Swee Han , Lejohn Herb B. , Wright Jim A.
Published: February 2005

HS3, a highly phosphorylated dinucleoside originally purified from the fungus Achlya, has been isolated from Chinese hamster ovary cells undergoing glutamine starvation. The HS3 compounds obtained from the fungal and mammalian sources exhibited similar physical and chemical properties. This unusual dinucleotide may be an important regulator of eucaryotic ribonucleoside diphosphate reductase activity; for 50 μm HS3, isolated from either mammalian or fungal cells, significantly inhibited CDP reduction in Achlya or hamster cell preparations, but only marginally affected the activity of the enzyme from E. coli. Studies with HS3 isolated from Achlya and partially purified mammalian ribonucleotide reductase indicated that the compound noncompetitively inhibited the reduction of varying concentrations of the substrates CDP, ADP and GDP with Ki values of 23 μm, 14 μM and 16 μM respectively. These inhibitor concentrations are well below the estimated intracellular levels of HS3 in glutamine starved cells and suggest that HS3 inhibition of ribonucleotide reduction may be responsible for the rapid inhibition of DNA synthesis seen under these culture conditions.
Possible regulation of macromolecular biosynthesis in mammalian cells by a novel dinucleoside polyphosphate (HS3) produced during step‐down growth conditions
Authors: Goh Swee Han , Wright Jim A. , LéJohn Herb B.
Published: February 2005

Shortly after the withdrawal of L‐glutamine from the growth medium of Chinese hamster ovary (CHO) cells, the rate of synthesis of a bizarre dinucleoside polyphosphate, HS3, increased by 5‐ to 6‐fold. This elevated rate of synthesis was maintained for six hours before it gradually declined to basal level 22 hours later. The pool size of HS3 increased and decreased coincidentally with rate changes. Withdrawal of L‐isoleucine did not affect HS3 biosynthesis. A glycine, adenosine, thymidine (GAT−) auxotroph of CHO cells accumulated HS3 when adenosine, not glutamine, was withdrawn. Replenishment of either glutamine (“wild type” cells) or adenosine (GAT− cells) caused an immediate depletion of HS3 intracellularly.

When HS3 accumulated in CHO cells, DNA and RNA synthesis decreased and, vice versa. A similar correlation was not seen for protein synthesis. But, inhibition of protein synthesis by either puromycin or cycloheximide, and of RNA biosynthesis by actinomycin D facilitated HS3 depletion in L‐glutamine starved cells.

Mutant CHO cells that are deficient in purine salvage metabolism, HGPRT− (hypoxanthine‐guanine phosphoribosyltransferase) failed to deplete their accumulated HS3 when fed with hypoxanthine, whereas the “wild type” CHO cells responded accordingly. The available data suggest that HS3 metabolism is connected with de novo and salvage pathways of nucleotide biosynthesis, and may play a crucial role in regulating nucleic acid metabolism in CHO cells under conditions of nutritional stress.
Serum‐mediated regulation of amino acid transport in cultured chick embryo fibroblasts
Authors: Tramacere Mariarosaria , Borghetti Angelo F. , Guidotti Guido G.
Published: February 2005

When chicken serum was added to serum‐deprived quiescent cultures of chick embryo fibroblasts the activity of amino acid transport by means of the A system, as measured by α‐aminoisobutyric acid and L‐proline uptake after discrimination of the contribution of interacting systems, increased with time of exposure to serum between 30 and 120 minutes (remaining constantly high thereafter). Under the same conditions, DNA synthesis, as measured by thymidine incorporation, increased abruptly six to eight hours after the addition of serum. Serum‐mediated increases of transport activity by the A system have also been detected with glycine, L‐alanine and L‐serine. Transport activities of systems ASC, L and Ly+ did not change appreciably (or decreased slightly) after the addition of serum. The stimulation of amino acid transport was apparently proportional to the length of exposure to serum; its rate declined progressively with time after withdrawal of serum from the culture medium. Kinetic analysis indicated that stimulation of the activity of transport system A by serum occurred through a mechanism affecting Vmax rather than Km; stimulation was prevented by inhibitors of protein synthesis. Our results indicate that the A transport system is the only system which is regulated by serum in cultured avian fibroblasts. Remarkably, the A transport system appears to be the target on which widely different factors and conditions converge to regulate amino acid transport in eukaryotic cells.
Development of hormonal regulation of ion transport in embryonic chick red cells
Authors: Wacholtz M. , Chan L. N. , Sha'Afi R. I.
Published: February 2005

We have found that cation transport in red cells from chick embryos is stimulated by the hormone epinephrine and that this response develops as the embryonic definitive cells mature. Sodium efflux and potassium influx are significantly stimulated (50%) by epinephrine in red cells from embryos incubated ten days or longer, whereas cation fluxes in erythroid cells from 8‐ or 9‐day embryos are stimulated little or not at all.

The effect of epinephrine may be mediated by cyclic AMP as adenylate cyclase activity in membranes isolated from embryonic red cells is only slightly stimulated at nine days, but the response increases as the cells mature to a maximum of about 180%. Also the stimulation of cation transport by epinephrine is blocked by propranolol, but not by phentolamine. Although the younger cells respond poorly to epinephrine, cyclic AMP significantly stimulates transport.

The enhancement of cation fluxes by epinephrine or cyclic AMP occurs even in the presence of ouabain. Since both K influx and Na efflux are enhanced by these agents, their action is most likely on some form of the “Na‐K” pump which is not ouabain sensitive resulting in a significant increase in the maximum velocity of the pump. We suggest the hypothesis that there are two classes of “Na‐K” pump in these embryonic cells. One pump is similar to that found in many erythrocytes including mammalian cells in that it selectively pumps potassium in and sodium out, is ouabain‐sensitive, and is primarily involved in maintaining intracellular cation concentrations. The second pump is enhanced by epinephrine via cyclic AMP, is not inhibited by ouabain, and may have lower ion selectivity. This hormone sensitive pump activity is lost as the cells mature, a process which is completed when the animal is fully grown and no longer has significant numbers of embryonic cells in its circulation.
Effects of cyclic AMP on the growth of differentiating and undifferentiated friend erythroleukemic cells
Authors: Rubin Charles S.
Published: February 2005

Elevated concentrations of cyclic AMP elicit only minor reductions in growth rate and saturation density in undifferentiated Friend erythroleukemic cells. During the course of dimethylsulfoxide (DMSO)‐induced differentiation, Friend cells convert from a cyclic AMP‐tolerant state to a phenotype characterized by a high degree of sensitivity to cyclic AMP‐mediated growth arrest.

Conversion to cyclic AMP sensitivity is detectable after 30 hours growth in medium containing 2% DMSO, and either 0.5 mM 8‐Br‐cyclic AMP or 5 nM cholera toxin. Cultures of differentiating Friend cells achieved a stationary phase density that was approximately 8‐fold higher than the cell density observed in parallel, differentiating cultures treated with 0.5 mM 8‐Br‐cyclic AMP. Temporally, the appearance of cyclic AMP‐sensitivity corresponds to the early expression of in vitro erythroid differentiation (Ross et al., ′74), but growth arrest does not alter the subsequent accumulation of hemoglobin in non‐dividing DMSO‐induced cells. Since growth arrest is preceded by a round of cell division, these observations are consistent with the concept that DMSO must be present during DNA replication for the subsequent expression of hemoglobin synthesis (McClintock and Papaconstantinou, ′74; Levy et al., ′75; Harrison, ′76).
Polyamine metabolism in regenerating livers from normal and hypocalcemic rats
Authors: Walker P. Roy , Whitfield James F. , Sikorska Marianna
Published: February 2005

There is a marked increase in the concentration of putrescine during the first ten hours following partial hepatectomy in rats. The concentration of spermidine also increases but to a smaller degree. Putrescine levels return to normal between 10 and 24 hours after the operation, whereas the increased spermidine level is maintained. The production of putrescine and spermidine appears to be initiated by the induction of ornithine decarboxylase which shows a single peak of activity at four hours after hepatectomy. The activity of S‐adenosylmethionine decarboxylase shows little change following hepatectomy. The changes in polyamine levels and the activities of the enzymes of polyamine metabolism are not affected by thyroparathyroidectomy 72 hours prior to hepatectomy. Thus although these hypocalcemic conditions considerably reduce and delay DNA synthesis and mitosis, the prereplicative changes in polyamine metabolism still occur. These data suggest that the hepatocytes in hypocalcemic animals have become activated and moved to an advanced stage of prereplicative development before being blocked.
Different red cell populations in newborn, genetically low potassium sheep: Relation to hematopoietic, immunologic and physiologic differentiation
Authors: Valet G. , Franz G. , Lauf P. K.
Published: February 2005

Three red cell populations have been distinguished in genotypically low potassium (LK) newborn sheep by an improved electrical sizing method and were best approximated by a logarithmic normal distribution. Labeling studies with 51Cr and 59Fe exclude transformation of the three red cell populations into each other. Population I, consisting of large red cells (mean volume 36 μm3), with a comparatively slow electrophoretic mobility is present at birth and disappears within three to four weeks from circulation. These cells possess a high potassium (HK) steady state concentration, a K+ pump influx activity at least 5‐fold greater than observed in adult LK red cells, very low amounts of the L antigens generally associated with the LK property, and do not respond to the stimulatory action of the L antibody. The first population is gradually replaced by population II comprising small red cells (mean volume 28 μm3) of intermediate electrophoretic mobility and with a peak production around day 20 after birth. The potassium concentration, [K+]c, in these cells appears to be lower than in the cells of population I but the L antigen content is increased. Formation of population III (mean volume 30 μm3 and comparatively fast electrophoretic mobility) follows closely that of population II and is preceded by a sharp increase in reticulocytosis. The red cells of population III exhibit parameters characteristic for adult LK cells: low [K+]c and K+ pump activity, fully developed L antigen content, and an almost maximal response to the K+ pump stimulating effect of anti‐L. In L and M antigen positive LK red cells of newborn sheep, the development of the M antigen parallels that of the L antigen. The data are consistent with the hypothesis that cellular replacement and not maturation is the major factor in controlling the HK‐LK transition in newborn sheep.
Growth requirements of calf lens epithelium in culture
Authors: Taylor‐Papadimitriou J. , Shearer M. , Watling D.
Published: February 2005

The in vitro life span and rate of growth of calf lens cells cultured in serum‐supplemented 199 medium can be markedly increased by growing the cells with a layer of mitomycin‐killed 3T6 feeders. In the absence of feeders, the epithelial cells are partially blocked in the S period of the cell cycle but show a normal distribution of cells in G1 and G2 when grown with fibroblasts. Increased growth rates and division potential can also be achieved by growing the lens in 199 medium containing 10−5 M thymidine, and cells grown under these conditions show a normal growth cycle. Our results suggest that lens epithelial cells cultured in medium 199 show a deficient endogenous synthesis of thymidylic acid, and fibroblast feeders or exogenously added thymidine enable them to overcome this deficiency. When grown in the presence of 10−5 M thymidine, the lens epithelial cells show a very low serum requirement for cell division in short term culture.