bluuebunny/crossref_metadata_2025 huggingface dataset:
In vitro and in vivo actions of zinc ion affecting cellular substances which influence host metabolic responses to inflammation
Authors: Mapes Carol A. , Bailey Paul T. , Matson Charles F. , Hauer Edward C. , Sobocinski Philip Z.
Published: February 2005

Glycogen‐stimulated rabbit peritoneal exudate cells (polymorphonuclear leukocytes, PMN) produce prostaglandins (PG) and substances which induce alterations (mediators) in experimental animals characteristics of host metabolic responses to infection and other acute inflammatory stresses. The effect of Zn2+ on mediator production and PG synthesis was examined because: Zn homeostasis is perturbed during infection, Zn is known to regulate some cellular functions, and there appears to be an interrelationship between PG synthesis and mediator production

Using exudate cells, 2 mM Zn2+ causec complete inhibition of in vitro PG synthesis as assessed by conversion of [1−14C] arachidonic acid into PG. This concentration of Zn2+ also inhibited production of substances mediating plasma Zn depression, hepatic amino acid "uptake," fever, and neutrophil release from bone marrow. Conversely, Zn2+ did not inhibit in vivo metabolic responses to these mediators. Zn‐pretreatment of rabbits or simultaneous injection of Zn2+ and crude PMN‐derived pyrogenic activity resulted in prolongation of fever. It is suggested that this action of Zn2+ may be attributed to either stabilization of cyclic AMP through inhibition of phosphodiesterase or a Zn‐mediator interaction which stabilizes crude endogenous pyrogen

The potential physiological significance of these results includes: possible potentiation of the host's defense mechanisms by Zn2+ and its utilization for prolongation of fever to determine its effect on potentially temperature‐dependent host defense mechanisms.
Effect of extreme amino acid starvation on the protein synthetic machinery of CHO cells
Authors: Stanners C. P. , Wightman T. M. , Harkins J. L.
Published: February 2005

When CHO cells are incubated under conditions of extreme amino acid starvation, effected by withdrawal of an amino acid from the medium together with genetic or chemical interference with the activity of the corresponding aminoacyl‐tRNA synthetase, there is a rapid and profound decline in the functional capacity of the protein synthetic machinery. The effect was observed for all amino acids tested including leucine, asparagine, histidine, methionine and glutamine. This decline in protein synthetic potential appears to be due to a progressive permanent inactivation of the specific aminoacyl‐tRNA synthetase concerned, as shown by a decline in the amount of cellular, specific aminoacyl‐tRNA and a decline in the cell‐free enzyme activity, measured after reversal of the starvation conditions. When cells are left for more than several hours under these starvation conditions, they shrink in size, lose viability and eventually disintegrate, with anomalous rapidity. We suggest that the progressive loss of protein synthetic capacity of the cells is the prime cause of these subsequent events.

If the starvation conditions are reversed before cell death, regeneration of the protein synthetic potential occurs rapidly but requires protein synthesis itself, implying the existence of strong control mechanisms for cellular aminoacyl‐tRNA synthetase activities.
Co‐cultivation of tumorigenic mouse melanoma cells with cells of a non‐tumorigenic subclone inhibits plasminogen activator expression by the melanoma cells
Authors: Kyner David , Christman Judith , Acs George , Silagi Selma , Newcomb Elizabeth W. , Silverstein Samuel C.
Published: February 2005

Clone B5 59 mouse melanoma cells are highly tumorigenic and produce plasminogen activator. Cells of clone C3471, a line obtained by continued growth of B559 cells in medium containing 5‐bromodeoxyuridine (1 μg/ml), have no plasminogen activator and are non‐tumorigenic. When B559 cells are co‐cultivated with C3471 cells, the ability of B559 cells to activate plasminogen is suppressed. Under these conditions cell fusion occurs. Lack of expression of plasminogen activators is not a consequence of cell fusion, inhibition of cell division or release of soluble inhibitors of either plasminogen activators or plasmin. No inhibitors of plasminogen activators could be demonstrated in association with subcellular fractions of C3471 cells or with the C‐type viral particles released from C3471 cells. Close contact between cells of the two lines is shown to be essential for suppression of plasminogen activation.
Significance of the cell cycle in commitment of murine erythroleukemia cells to erythroid differentiation
Authors: Geller Ruth , Levenson Robert , Housman David
Published: February 2005

The relationship between differentiation of murine erythroleukemia cells (MEL) induced by DMSO and the cell division cycle has been analyzed. We demonstrate that incubation in the presence of DMSO increases the length of the G1 phase of the cell cycle. A method of synchronization of MEL cells by unit gravity sedimentation has been developed and characterized. Using this method, a series of synchronized cell populations covering the entire cell division cycle can be generated simultaneously. Cells synchronized by this technique were challenged with DMSO and analyzed for kinetics of commitment to the differentiation program. Our results indicate that populations of cells in G1 or G2 at the time of addition of inducer give rise to a greater proportion of committed cells than an unfractionated population, while cells in S phase result in a lower percentage of committed cells than the unfractionated population when cultured in DMSO.
The development of lysosomal apparatus I. Lysosomal enzyme activities in the liver of mice at perinatal stages and those of their mothers
Authors: Mazzei Héctor R. , Bertini Francisco
Published: February 2005

The enzymatic activity of five acid hydrolases: acid phosphatase, arylsulfatase A, deoxyribonuclease, β‐glucuronidase, and cathepsin D, was assayed in fetal (fifteenth and eighteenth days of pregnancy) and neonatal (Days 0, 5, 10, and 15 post‐partum) mouse liver. With the exception of cathepsin D, the activity increased around birth to levels varying according to the enzyme. Histochemical observations of other authors appear to justify, at least in part, the present results, which indicate that late days of fetal development and early neonatal life may constitute a transitional stage to full lysosomal enzyme functionality of the adult organ

The livers of the mothers were also assayed for the same enzymes. Each activity showed a peculiar pattern which was, in turn, different from that found in the liver of the litter for the same enzyme, probably as a cause of the metabolic requirement of the gland

The hypothesis that the lysosomes are heterogeneous in their enzyme composition is suggested by the variety of enzymatic patterns found in the liver of the litters and their mothers.
Ontogeny of human neutrophil granulocyte alkaline phosphatase
Authors: Kelemen Endre , Gulya Ernö , Vass Katalin
Published: February 2005

Human embryonic neutrophils (N) in the liver (from 8.5 mm crown‐rump length) are alkaline phosphatase (AP) negative during the first trimester of pregnancy. Early bone marrow granulocytes (from eleventh to sixteenth weeks of gestation) behave similarly. Only a small percentage of slightly AP positive cells could be found. Occasional cells with strong NAP reaction appear in the second trimester. NAP positivity greatly increases in the third trimester and term‐babies have a somewhat higher than normal NAP activity in circulating blood. Unlike NAP reaction, naphthol‐AS‐D‐chloroacetate esterase and peroxidase reactions are positive even in the earliest (AP negative) neutrophils.
Regulation of megakaryocytes in W/Wv mice
Authors: Ebbe Shirley , Phalen Eizabeth
Published: February 2005

W/Wv mice were injected with antiplatelet serum to produce thrombocytopenia or with platelet transfusions to induce thrombocytosis. The responses of their platelets and megakaryocytes were followed to determine if proliferative abnormalities of the megakaryocytic system would be detected.

W/Wv mice responded normally to the stimulation from thrombocytopenia with rebound thrombocytosis, macromegakaryocytosis, and macrothrombocytosis. The megakaryocytes of these mice became smaller than normal in response to post‐thrombocytopenic rebound thrombocytosis but not to transfusion‐induced thrombocytosis. Thus, endogenous thrombocytosis appeared to be a more potent suppressor of megakaryocyte growth than exogenous.

These results failed to reveal an effective abnormality of the thrombocytopoietic regulatory system of W/Wv mice in spite of their intrinsically reduced numbers of megakaryocytes and the well known defect of stem cell proliferation. Thrombocytopoietic regulation appeared, therefore, to occur mainly at the committed, rather then pluripotential, stem cell level, and normal responses of the platelet system were observed in spite of severe abnormalities at the pluri‐potential stem cell level.
Control of glycolysis and the pentose phosphate shunt in transformed 3T3 cultures rendered permeable by ATP
Authors: Makan Nizar R. , Heppel Leon A.
Published: February 2005

Exogenous ATP has been shown earlier to activate a permeability change in transformed 3T3 cultures leading to massive efflux of the acidsoluble pools. This leads to reduction of the basal rate of glycolysis to a very low level so that glycolysis becomes almost totally dependent on the addition to the medium of glucose, inorganic phosphate and ADP in order to restore the rate to that of untreated cells. No such depression of glycolysis is observed in untreated transformed cells or in ATP‐treated normal 3T3 cells. In such permeabilized cultures, phosphorylated intermediates such as glucose‐6‐phosphate and fructose‐1,6‐diphosphate can serve as effective substrates for lactic acid formation. ATP treatment of cultured cells also allows molecules as big as NADP to enter the cells and participate in the pentose phosphate shunt pathway. This ability to temporarily and differentially render transformed cells permeable allows a review of several aspects of cellular metabolism and biosynthesis in the intact cell where the cellular organization is maintained. Furthermore, it deserves serious consideration as a means to achieve differential cytotoxicity of transformed cells by chemotherapeutic agents which, on their own, are indiscriminate in their action.
Characterization of a guanine‐sensitive mutant defective in adenylo‐succinate synthetase activity
Authors: Tu Alice S. , Patterson David
Published: February 2005

A contingent auxotrophic mutant of CHO‐Kl cell is described. This mutant grow in minimal medium. Its growth is inhibited by the exogenous addition of guanine at levels which do not affect the wild type parent. Adenine reverses the guanine effect. This mutant does not complement ade−H (defective in adenylosuccinate synthetase) and has been denoted as ade−HG because of its guanine sensitivity. Some partial revertants of ade−H are found to be also sensitive to guanine, suggesting a close relationship between the ade−H locus and the guanine sensitivity. Studies of 14C‐hypoxanthine incorporation into nucleotides indicated that ade−HG has some adenylosuccinate synthetase activity whether it is pre‐exposed to guanine or not. Early de novo purine synthesis in ade−HG, however, is greatly inhibited when pre‐exposed to guanine. This inhibition of purine synthesis by guanine is reversible and its recovery is facilitated by adenine.
The isolation of a low molecular weight serum factor which enhances the viability of sv3t3 cells
Authors: Young Delano V. , Nakano Eileen T. , Wiggins Douglas S. , McElaney Mary A.
Published: February 2005

A serum factor which enhances the growth/viability of SV40 transformed 3T3 mouse fibroblasts, but not untransformed 3T3 cells in tissue culture has been partially purified from calf serum. The purification, which involves acidification to pH 2, chromatography on Sephadex G‐100 at pH 2 followed by either Sephadex G‐25 or BioGel P‐2, results in material exhibiting two ninhydrin positive spots on thin layer chromatography and five to six dansylatable bands on SDS gels. This substance, termed Peak III or Serum Factor III, has been found in every serum examined thus far (including calf, rat, mouse, goat, lamb, rabbit, pig, horse, and chicken) and appears to be a low molecular weight, heat‐insensitive molecule. Attempts to characterize it by chemical analyses and specific enzymatic inactivation have been either negative or inconclusive.

Its biological mode of action is presently obscure. While it does not affect DNA synthesis, it does greatly increase the viability of SV3T3 cultures growing in low serum (0.15–0.30% calf serum) medium and, when added to “stationary phase” cells, restores healthy proliferation. This and evidence reported by others suggest that Peak III may affect SV3T3 cells in either overcoming a potentially lethal block in G1 or in passage through G2/M.
Transport and accumulation of purine bases by Crithidia fasciculata
Authors: Kidder G. W. , Dewey Virginia C. , Nolan Linda L.
Published: February 2005

The capacity for the rapid transport of purine bases by Crithidia fasciculata is found only in cells starved for purines. Cells grown in complete medium transport poorly. Rapid transport capability appears and then disappears during growth of purine‐depleted cultures. This rapid transport appears to occur by a process of mediated diffusion. Two mechanisms are involved, one of low velocity and high affinity, the other of high velocity and low affinity. Accumulation of the bases within the cell occurs by their rapid conversion to ribonucleotides by phosphoribosyltransferases.
The modulation of the induction of ornithine decarboxylase by spermine, spermidine and diamines
Authors: Heller John S. , Chen Kuang Yu , Kyriakidis Dimitri A. , Fong Wang F. , Canellakis E. S.
Published: February 2005

Extremely low concentrations of putrescine, spermidine and spermine added to the extracellular medium of cultures of mammalian cells inhibit the induction of ornithine decarboxylase activity despite 100‐ to 1,000‐fold greater intracellular polyamine concentrations. The diamines, 1,2‐diaminoethane, 1,3‐diaminopropane, 1,5‐diaminopentane, 1,7‐diaminoheptane, 1,10‐diaminodecane, 1,12‐diaminododecane also inhibit ornithine decarboxylase at all concentrations tested (greater than 10−6 M). In contrast, 10−6 M to 10 −3 M 1,8‐diaminooctane, the alkyl analog of spermidine, enhances ornithine decarboxylase activity.

The concentraton of putrescine required to inhibit the activity of ornithine decarboxylase by 50% is a characteristic of each cell line; however, it varies by as much as 1,000‐fold among the five cell lines we have tested (L1210 leukemic, H35 hepatoma, N18 neuroblastoma, W256 carcinosarcoma and 3T3 fibroblasts).

The antizyme to ornithine decarboxylase can be induced in all these cells by high (di)(poly)amine concentrations.

Based on these and other experiments we suggest a working hypothesis: that the polyamines regulate ornithine decarboxylase activity through two different sites that may be interrelated; a sensitive membrane‐mediated site that responds to minute fluctuations of extracellular polyamine levels and a coarse site which may be intracellular or membrane associated that responds to larger fluctuations of intracellular polyamine levels. The consequences of such a control mechanism operating within the whole organism are discussed.
Platelet derived growth factor(s) for a hormone‐responsive rat mammary tumor cell line
Authors: Eastment Caroline T. , Sirbasku David A.
Published: February 2005

Mammary tumor cell growth factor(s) has been identified in extracts of platelets from both male and female rats, as well as in extracts prepared from pooled outdated human platelets. When assayed by the growth promotion of MTW9/PL rat mammary tumor cells in culture, platelet extracts alone were able to support growth 50–75% as well as whole serum. The mitogenic activity from crude human platelet lysates was shown to be trypsin sensitive, relatively stable to extremes of pH, labile to heat treatment at 70°, non‐dialysable, ammonium sulfate precipitable, not removed by 56° charcoal treatment, and of apparent molecular weight of 30,000 to 50,000 daltons as estimated by G‐100 Sephadex chromatography. The platelet derived mammary growth factor activity was not replaced or potentiated by thrombin or known hormones and growth factors such as prolactin, insulin, 17‐β‐estradiol, progesterone, hydrocortisone, L‐thyroxine, and mouse epidermal growth factor. The experimental report demonstrates that platelets are a rich source growth factor activity for rat epithelial mammary tumor cells, and that the activity appears to be a polypeptide(s) different from other mitogenic activities known to influence growth of mammary tissue.
Ribonucleotide reductase from wild type and hydroxyurea‐resistant chinese hamster ovary cells
Authors: Lewis William H. , Wright Jim A.
Published: February 2005

The kinetic properties of partially purified ribonucleotide reductase from Chinese hamster ovary cells have been investigated. Double reciprocal plots of velocity against substrate concentration were found to be linear for three the substrates tested, and yielded apparent Km values of 0.12 mM for CDP, 0.14 mM for ADP and 0.026 mM for GDP. Hydroxyurea, a potent inhibitor of ribonucleotide reduction, was tested against varying concentrations of ribonucleotide substrates and inhibited the enzyme activity in an uncompetitive fasion. Intercept replots were linear and exhibited Ki values for hydroxyurea of 0.08 mM for CDP reduction, 0.13 mM for ADP reduction and 0.07 mM for GDP reduction. Guanazole, another inhibitor of ribonucleotide reductase, interacted with the enzyme in a similar manner to hydroxyurea showing an uncompetitive pattern of inhibition with CDP reduction and yielding a Ki value of 0.57 mM.

Partially purified ribonucleotide reductase from hydroxyurea‐resistant cells was compared to enzyme activity from wild type cells. Significant differences were observed in the hydroxyurea Ki values with the three ribonucleotide substrates that were tested. Also, CDP reductase activity from the drug‐resistant cells yielded a significantly higher Ki value for guanazole inhibition than the wild type activity. The properties of partially purified ribonucleotide reductase from a somatic cell hybrid constructed from wild type and hydroxyurea‐resistant cells was also examined. The Ki value for hydroxyurea inhibition of CDP reductase was intermediate between the Ki values of the parental lines and indicated a codominant expression of hydroxyurea‐resistance at the enzyme level. The most logical explanation for these results is that the mutant cells contain a structurally altered ribonucleotide reductase whose activity is less sensitive to inhibition by hydroxyurea or guanazole.
Studies on the nature of protease‐induced growth stimulation in normal and transformed BHK cells
Authors: Kiehn Don , Brown McKay
Published: February 2005

In the presence of growth‐limiting serum concentrations trypsin displays mitogenic activity on actively‐growing but not quiescent BHK cells. These results suggest that BHK cells arrested in G1 (G0) are not sensitive to protease‐induced growth stimulation. Previous work strongly suggested that the trypsin active‐site is not directly involved in its mitogenic activity on BHK cells. Additional studies on denatured trypsin fragments further indicate that the molecular conformation and size of native trypsin may not be absolutely required for mitogenic activity.

Cellular multiplication induced by the addition of fresh serum to quiescent BHK cultures is not inhibited by high concentrations of soybean trypsin inhibitor. Similar to our previous findings with trypsin, it has been further observed that plasmin is not sufficient to initiate the growth of BHK cells in soft agar. Trypsin also fails to enhance the growth of a thermosensitive polyoma‐transformed BHK line in soft agar at the restrictive temperature. Finally, the growth of transformed BHK cells in soft agar does not display a requirement for plasminogen and is not inhibited by soybean trypsin inhibitor. These studies argue against the involvement of plasmin or other exogenous trypsin‐like enzymes in the growth and transformation of BHK cells.
Regulation of cell‐cycle initiation in yeast by nutrients and protein synthesis
Authors: Shilo B. , Simchen G. , Pardee A. B.
Published: February 2005

Arrested Saccharomyces cerevisiae cells initiate the cell cycle in an asynchronous mode. The asynchronous manner of cycle initiation generates variability in cell‐cycle times of individual cells. Limiting concentrations of adenine, methionine or histidine regulate the rate of cycle initiation in auxotrophs. A sigmoidal curve of rate vs. concentration is obtained for each of the three substances. Moreover, the three curves have similar Hill coefficients of 2.4, suggesting that a common intermediate requiring adenine, methionine and histidine regulates cell‐cycle initiation in yeast.

Low concentrations of cycloheximide reduce the rate of cycle initiation of arrested cells that are released from the block in a similar way as limiting nutrients. It thus appears that the common intermediate that requires the limiting nutrients depends upon protein synthesis. The rate of cycle initiation is more sensitive to cycloheximide or nutrient limitation than is protein synthesis. It is also affected by limiting nutrients to a much greater extent than is the overall rate of protein accumulation (i.e., net protein synthesis). Hence the mechanism that controls cycle initiation does not depend on the overall synthesis or accumulation of proteins in the cell. It may depend on synthesis of particular proteins whose production or function requires the limiting nutrients.

The high sensitivity of cycle initiation to a decrease in the rate of protein synthesis could explain the ability of yeast cells to complete the cycle and arrest at stationary phase upon depletion of medium components. The cells cannot initiate the cycle although their protein synthesis capacity remains sufficiently high to allow traversal of the rest of the cell cycle.
Responsiveness to insulin is a dominant characteristic in somatic cell hybrids
Authors: Straus Daniel S. , Williamson Robert A.
Published: February 2005

The mouse melanoma cell line PG19 has been found to be unresponsive to the growth‐stimulatory action of insulin, although it responds well to other growth factors present in serum. Insulin stimulates DNA synthesis in mouse embryo fibroblasts, and responsiveness to insulin has been found to be a dominant characteristic in mouse fibroblast x PG19 hybrids. To examine the possibility that the unresponsiveness to insulin of the melanoma cells is attributable to a lack of insulin receptors, we have measured the binding of 125I‐labeled insulin to the fibroblasts, melanoma cells, and fibroblast x melanoma hybrids. Insulin binds to the surface of the melanoma cells; however, the binding affinity appears to be lower than that observed for binding to diploid fibroblasts. In addition, the dissociation of insulin from the melanoma cells is not accelerated by excess unbound insulin, a kinetic effect observed in the dissociation of insulin from the fibroblasts and fibroblast x melanoma hybrids. This suggests that the class of insulin receptors characterized by this effect is absent on the PG19 cells, and present on the fibroblasts and fibroblast x PG19 hybrids.
Presence of spectrin in untreated friend erythroleukemic cells. Its accumulation upon treatment of the cells with dimethyl sulfoxide
Authors: Rossi Giovanni B. , Aducci Patrizia , Gambari Roberto , Minetti Maurizio , Vernole Patrizia
Published: February 2005

Friend leukemia cells (FLC) are nucleated erythroid precursors, and are markedly stimulated towards more advanced stages of differentiation by treatment with dimethyl sulfoxide (DMSO). The presence of spectrin, an erythrocyte membrane protein, has been investigated in untreated and in DMSO‐treated FLC by indirect immunofluorescence and by analysis in SDS‐polyacrylamide gel electrophoresis of low‐ionic‐strength cell extracts immuno‐precipitated with a monospecific anti‐spectrin serum.

Spectrin is detectable in significant amounts in the “inducible” clones prior to DMSO stimulation, and accumulates 4‐ to 5‐fold upon addition of this compound to the cultures. Spectrin accumulation occurs rather early (24 hours after cell seeding) and reaches its peak on the third day, to decline thereafter. Semiquantitative determinations of spectrin amounts present in DMSO‐stimulated 745A and A°1 cells on the third day after treatment were 2.4 × 105 and 3.0 × 105 molecules/cell, respectively.

Spectrin is also detectable in very low amounts in an “uninducible” line of FLC, and is not accumulated upon DMSO treatment thereof, whereas treatment with hemin does cause a significant increase of spectrin‐positive cells.

These data indicate that spectrin is a convenient “early” marker for in vitro studies of erythropoiesis.
Detection of in vitro macrophage colony‐forming cells (M‐CFC) in mouse bone marrow, spleen, and peripheral blood
Authors: Macvittie T. J. , Porvaznik M.
Published: February 2005

In vitro macrophage colony‐forming cells (M‐CFC) have been detected in bone marrow (BM) (317/105 cells), spleen (SPL) (81/105), and peripheral blood leukocytes (PBL) (242/105) of the mouse. These M‐CFCs were similar to those previously detected in thymus (T) (30/106) and lymph node (LN) (22/106) tissue in several respects. BM‐ and SPL‐derived M‐CFC required PMUE to consistently initiate colony formation, whereas PBL‐derived M‐CFC formed colonies with stimulation by either PMUE or L‐cell‐conditioned medium. All colonies formed showed a singular macrophage line of differentiation, a lag of 13 to 18 days prior to initiating colony formation, a marked ability to survive in culture in the absence of PMUE, and markedly slow rates of appearance in culture once colony formation was initiated. The macrophage progeny were identified on the basis of morphology, glass adherence, the phagocytosis of agar, bacteria and SRBC, and the presence of receptors for IgG. These characteristics are also shared by those macrophage CFCs observed within stimulated peritoneal exudate, pleural effusion, and alveolar space. These M‐CFCs are most likely members of a large, heterogeneous population of macrophage progenitor cells distributed throughout the hemato‐lymphopoietic organs, serosal cavities and surfaces, and inflammatory and alveolar tissue sites. The degree of heterogeneity may be determined in part by the influence of tissue‐specific microenvironment.
Effect of dibutyryl cyclic AMP on leucine incorporation in L5178Y cells II. Effect of actinomycin and heat shock
Authors: Fuhr Joseph E.
Published: February 2005

3H‐leucine incorporation was assayed in L5178Y cells as a measure for protein synthesis. Protein synthesis was inhibited by short duration heat shock, actinomycin, cordycepin or a combination of these agents. Dibutyryl cyclic AMP had little or no effect upon protein synthesis in control cells. Dibutyryl cyclic AMP, however, stimulated protein synthesis in cells previously heat shocked and/or inhibited by actinomycin or cordycepin. The results have been interpreted to indicate that the cyclic nucleotide stimulated protein synthesis by influencing the metabolism of some species of RNA which may regulate translation.
Measurements of metabolites during cAMP oscillations of Dictyostelium discoideum
Authors: Geller Joshua S. , Brenner Michael
Published: February 2005

During aggregation the cellular slime mold Dictyostelium discoideum synthesizes and releases pulses of cAMP about every five minutes. Current models proposed to explain this phenomenon postulate that oscillating levels of some key intracellular metabolite control the oscillatory synthesis of cAMP. We have assayed the levels of likely candidates for this metabolite during a cAMP oscillation, but have found them to remain constant. Compounds measured include ATP, GTP, glucose‐1‐phosphate, glucose‐6‐phosphate, isocitrate, α‐ketoglutarate, amino acids, and other aminated metabolites. On the basis of this negative data, as well as results described elsewhere (Geller and Brenner, 1978), we question whether the proposed models are correct, and discuss several alternatives.
Altered cyclic AMP‐dependent protein kinase activity in a mutant adrenocortical tumor cell line
Authors: Gutmann Norman S. , Rae Patricia A. , Schimmer Bernard P.
Published: February 2005

A somatic cell genetic approach has been used to evaluate the role of cyclic AMP‐dependent protein kinase in ACTH action on adrenal steroidogenesis. A mutant clone, 8BrcAMPr‐1, previously was isolated from an ACTH‐sensitive adrenocortical tumor cell line (clone Y1) following mutagenesis and selective growth in 8‐bromoadenosine 3′, 5′‐monophosphate. This study demonstrates that the 8BrcAMP4‐1 cells have an altered cyclic AMP‐dependent protein kinase. The protein kinase in the cytosol of the mutant characteristically requires, for half‐maximal activity, concentrations of cyclic AMP 7‐fold higher than those required by the enzyme in preparations from the parent. The cytosolic cyclic AMP‐dependent protein kinases of Y1 and 8BrcAMPr‐1 cells chromatograph similarly on columns of DEAE‐cellulose. From each cell line, a major peak of activity (≥ 70% of recovered activity), designated as Peak I, elutes with 0.04–0.06 M NaCl; a second peak of activity, designated as Peak II, elutes with 0.12–0.14 M NaCl. Protein kinase activity in the Peak I fraction of mutant cells has a decreased apparent affinity (4‐fold) for cyclic AMP relative to the corresponding fraction of parental Y1 cells. The protein kinase activities present in Peak II fractions from Y1 and mutant cells are indistinguishable. The protein kinase mutant exhibits poor steroidogenic responses to added ACTH and cyclic AMP; and as shown previously does not display the growth arrest and morphological changes produced in Y1 by these agents. These results suggest that cyclic AMP‐dependent protein kinase is important in the regulation of adrenal steroidogenesis, morphology and growth by ACTH.
The effect of oxygen tension on haemopoietic and fibroblast cell proliferation in vitro
Authors: Bradley T. R. , Hodgson G. S. , Rosendaal M.
Published: February 2005

The effects of providing low oxygen tension in the gas phase of two different types of cell culture systems were investigated. The clonal growth of granulocyte‐macrophage progenitor cells in an agar culture system was improved markedly by incubation within a low oxygen tension gas phase (48 mmHg – 6.8%) instead of the conventional air (135 mmHg – 19%), the effects being measured by increases in numbers of colony forming cells detected and in the colony sizes. The increased efficiency of colony formation was observed both with mouse and human marrow cells. A similar effect was observed in a liquid adherence culture system with primary cultures of foetal mouse fibroblasts both at clonal and higher cell densities.
The effects of dopamine on prostaglandin E1‐elicited electrophysiological changes in a neuronal somatic cell hybrid
Authors: Myers Paul R.
Published: February 2005

PGE1 elicited a slow, dose‐dependent membrane depolarization with an increase in membrane conductance in the somatic cell hybrid TCX11. The ED 50 was 1‐2 × 10−8 M with maximal responses at 1‐5 × 10−7 M. Dopamine (DA) reversed the effect of PGE1 and caused the membrane potential and resistance to return to control levels. Chronic exposure of cells (measured in minutes) to DA alone would not cause this hyperpolarization. 5‐HT was also tested and failed to consistently reverse the PGE1 effects. Chlorpromazine antagonized the effects of DA on the PGE1 response. The electrophysiological results reported here using TCX11 cells are discussed in light of previously reported biochemical results describing interactions of PGE1 and DA, and the electrophysiological effects of DA alone.
Isolation and cell cycle analysis of temperature‐sensitive mutants from chinese hamster cells
Authors: Melero Jose A.
Published: February 2005

Mutants temperature‐sensitive for growth have been isolated from the established line of Chinese hamster fibroblasts Wg1A. These mutants, together with the ones previously isolated by Roscoe et al. ('73), have been characterized with regard to their cell cycle properties. Most of them become arrested in the G1 phase of the cell cycle when incubated under restrictive conditions. By performing temperature shift experiments with synchronous cultures, the execution steps of most of the mutated functions have been located within the second half of the G1 phase.
Cell cycle control by Ca++‐ions in mouse 3T3 cells and in transformed 3T3 cells
Authors: Paul Dieter , Ristow Hans‐J.
Published: February 2005

Total cellular calcium levels do not change when 3T3‐4a cells stop proliferating due to serum depletion, or when serum‐arrested quiescent cells are incubated for up to 44 hours in calcium‐deficient medium (∼10 μM Ca++). Upon stimulation with dialyzed serum cells enter S and progress through at least one cycle even at extremely low calcium levels in the culture medium (≥10 μM). Cells divide until a final cell density is attained which is proportional to the calcium concentration in the medium and cells reversibly arrest in G1. Cells which arrested in G1 in medium containing ≤26 μM Ca++ in the presence of excess serum can be stimulated to enter S in response to added calcium after a prereplicative phase of 14 to 16 hours. Serum does not affect 45Ca‐uptake in these cells. Benzo[a]pyrene transformed 3T3 (BP3T3) cells have a 100–200 times lower Ca++‐requirement than 3T3 cells but arrest in G1 at low Ca++ levels. In contrast, SV40‐virus transformed 3T3 (SV3T3) cells that grow without restriction in monolayer cultures have even lower Ca++‐requirements for growth than BP3T3 cells and have no Ca++‐sensitive restriction point. Therefore, 3T3 and BP3T3 cells have retained the capacity to sense intracellular Ca++‐pool sizes and to arrest in G1 at subthreshold cellular Ca++‐levels.
Rate of potassium‐sodium exchange by human lymphocytes: Prediction of the cooperative adsorption model
Authors: Negendank William , Karreman George
Published: February 2005

The equilibrium parameters of potassium‐sodium distribution in human lymphocytes, determined experimentally in the preceding study (Negendank and Shaller, '79), were incorporated into a stochastic treatment of the cooperative adsorption model in order to predict the kinetics of “active” potassium‐sodium exchange. The rate of uptake of potassium, in potassium‐depleted, sodium‐loaded cells, is complex and deviates markedly from simple exponential functions. The sigmoid form of the exchange data closely followed the predicted curve. This result enhances one's confidence in the usefulness and applicability of the cooperative adsorption model, and adds further support to the association‐induction hypothesis as a coherent theory of cell physiology.
Properties of protein kinase and adenylate cyclase‐deficient variants of a macrophage‐like cell line
Authors: Rosen Neal , Piscitello Jeanne , Schneck Jonathan , Muschel Ruth J. , Bloom Barry R. , Rosen Ora M.
Published: February 2005

Stable variants of the macrophage‐like cell line J774.2, defective in adenylate cyclase and protein kinase activities, were selected by cloning cells resistant to the growth‐inhibitory effect of cholera toxin and 8‐bromoadenosine 3′:5′ cyclic monophosphoric acid (8 Br‐cAMP), respectively. These variants were analyzed for their ability to respond to cyclic AMP‐mediated enhancement of phagocytosis and cyclic AMP‐mediated inhibition of plasminogen activator secretion and growth. The adenylate cyclase variants were unaffected by cholera toxin but were sensitive to 8 Br‐cAMP‐mediated inhibition of plasminogen activator secretion and growth. One of these variants exhibited a defect in phagocytosis that could be corrected by 8 Br‐cAMP. The protein kinase variants exhibited normal basal phagocytosis that could not be stimulated by either 8 Br‐cAMP or cholera toxin; they were also insensitive to cyclic AMP‐mediated inhibition of plasminogen activator secretion and growth. The studies demonstrate that the three effects of cyclic AMP in J774.2–inhibition of growth and plasminogen activator secretion, and enhancement of basal Fc‐mediated phagocytosis–are mediated by a cyclic AMP‐dependent protein kinase. The results support the usefulness of variants in cyclic nucleotide metabolism in understanding the regulation of differentiated cell function by cyclic AMP.
Requirements for RNA and protein synthesis in the induction of several differentiation‐markers in a myeloid leukemia cell line
Authors: Nagata Kazuhiro , Ichikawa Yasuo
Published: February 2005

In a myeloid leukemia cell line, the inducibilities of the Fc receptor, phagocytosis and cell motility were compared. Thymidine analogues such as BUdR, BCdR and IUdR blocked the induction of phagocytosis and motility but not induction of the Fc receptor. This BUdR susceptibility in the induction of phagocytosis and motility was lost in a BUdR resistant line which was isolated for its growth capability in a high concentration of BUdR. Actinomycin D and puromycin brought about a marked decrease in the inducibility of phagocytosis but not in that of the Fc receptor.

This led us to the following conclusion: There is a genetic control in the inducibility of phagocytosis and motility in this cell line, and the incorporation of BUdR into cellular DNA results in the DNA becoming unresponsive to a differentiation‐stimulating factor. In contrast, gene activation does not seem to be necessary for induction of the Fc receptor.

The order of induction of several differentiation markers was also discussed.
Establishment of a rat hepatoma cell line which has ornithine carbamoyltransferase activity and grows continuously in arginine‐deprived medium
Authors: Niwa Akira , Yamamoto Katsuhiko , Yasumura Yosihiro
Published: February 2005

The epithelial cell line, H4‐II‐E derived from Reuber hepatoma H35 has no significant activity of ornithine carbamoyltransferase (OCT, EC 2.1.3.3) and is not able to grow in arginine‐deprived medium.

A multi‐step selection procedure is described which selects from H4‐II‐E populations, cells with OCT activity which can grow in arginine‐deficient, ornithine‐supplemented media.
Patterns of peptide synthesis in senescent and presenescent human fibroblasts
Authors: Engelhardt Dean L. , Lee Gloria T‐Y. , Moley Jeffrey F.
Published: February 2005

Peptide production in senescent and presenescent human foreskin fibroblasts was measured using 2‐dimensional polyacrylamide gel electrophoresis. This procedure permits the visualization of a cohort of the major peptides being produced. Among this cohort of over 500 peptides only two were found to differ in relative amount in that more was being produced in senescent cells. This difference was confirmed by measurements of the relative intensity of the peptide spot. This difference was senescent cell‐specific and not due to the differences in rate of growth of senescent and non‐senescent cells.
Elevation of HeLa cell 3‐hydroxy‐3‐methylglutaryl coenzyme a reductase activity by glucocorticoids: Possible relationship to the cell cycle
Authors: Cavenee Webster K. , Melnykovych George
Published: February 2005

Elevation of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase (HMG‐CoA reductase, EC 1.1.1.34) activity by glucocorticoids was shown to be dependent on the concentration of hormone in the medium over a range of 5 × 10−10 to 1 × 10−8 M, although the presence of steroid in the assay at 10−5 M elicited no increase in activity. There was a demonstrated time dependence for the addition of dexamethasone i.e., from zero to six hours after serum removal, addition of hormone resulted in the same peak activity; addition at 12 hours gave slight elevation but resulted in an extended maintenance of the peak level of activity; addition at 24 hours showed no effect. When cycloheximide was added at the above times, subsequent kinetics showed identical decay of the enzyme activities from control and treated cultures at 6 and 24 hours, but at 12 hours the activity from dexamethasone treated cells exhibited an extended lag before the onset of decay, which then proceeded at the same rate as the control. The continuous presence of the hormone was not necessary for the induction to continue and the addition of Actinomycin D to cultures incubated in the presence of hormone resulted in an immediate decay of catalytic activity without evidence of “superinduction”. The addition of progesterone at the same time as dexamethasone resulted in a concentration‐dependent inhibition of the augmentation, suggesting the involvement of the glucocorticoid receptor in the aug‐mentation, suggesting the involvement of the glucocorticoid receptor in the elevation of HMG‐CoA reductase activity. Flow microfluorometric (FMF) analysis of hormone treated cells indicated a delayed entrance into the DNA synthesis (S) phase of the cell cycle. The temporal relationships between this cell cycle perturbation and HMG‐CoA reductase elevation are discussed.
Frequency differences between nuclear and polysomal sequences of poly(A)‐containing RNA from cultured akr mouse embryo cells
Authors: Siegal Gene P. , Quinlan Thomas J. , Moses Harold L. , Getz Michael J.
Published: February 2005

The involvement of post‐transcriptional mechanisms in the determination of the frequency distribution of messenger RNA sequences has been studied in cloned mouse embryo cells in culture. Hybridization kinetic experiments between poly(A)‐containing RNA and complementary DNA have been used to study the alterations in frequencies which occur in those nuclear poly(A)‐containing RNA sequences which are conserved in cytoplasmic polyribosomes. Sequences adjacent to nuclear poly(A) tracts are present in a much narrower frequency distribution in the nucleus than in polysomes, with a large proportion of the nuclear sequences present in an average frequency of about one molecule per cell. Few nuclear sequences appear to be present in more than ten copies per cell. A minimum of 70% of these sequences are also found in polyribosomal RNA but in greatly altered frequencies. Abundant sequences which comprise a major fraction of the poly(A)‐containing polyribosomal RNA are derived from a small fraction of the nuclear poly(A)‐adjacent RNA sequences. Very few nuclear poly(A)‐adjacent sequences are present in a frequency characteristic of high abundance polysomal sequences. Conversely, poly(A)‐containing polyribosomal RNA appears to contain few sequences which are present in as low a frequency as the majority of nuclear poly(A)‐adjacent sequences. These observations suggest that post‐transcriptional mechanisms play a major role in determining the steady‐state frequency of polyribosome‐associated messenger RNAs.
The effect of amino acids on the temperature sensitive phenotype of the mammalian leucyl‐tRNA synthetase mutant ts Hl and its revertants
Authors: Molnar S. J. , Rauth A. M.
Published: February 2005

The temperature sensitive leucyl‐tRNA synthetase mutant tsHl and two revertants have been compared to the parental Chinese hamster ovary cells with respect to the effects of amino acid concentrations in the medium on growth. Elevating the leucine concentration 30‐ or 100‐fold allowed tsHl to grow exponentially at 38.5°C, normally the nonpermissive temperature. Partial revertants that had recovered some enzyme activity required smaller supplements for growth. Measurements of the leucine pools indicated that they respond directly to the extracellular leucine concentration and may mediate the effect. Use of combinations of amino acids confirmed that isoleucine has a similar though weaker effect on tsHl and identified an even weaker protection by valine. The triple combination of leucine, isoleucine and valine was a much more efficient medium supplement and three times normal concentrations of these amino acids supported growth of tsHl at 38.5°C. It is postulated that they are acting at their respective aminoacyl‐tRNA synthetases to help stabilize a complex which also contains the mutant leucyl‐tRNA synthetase. The pool size measurements also showed that the leucine pools of tsHl and a revertant increased 2‐fold more in a response to increased temperature than those of WT. It is suggested that this is a regulatory response to low leucyl‐tRNA synthetase activity and is important in determining growth phenotypes.
Isolation and characterization of revertants of the mammalian temperature sensitive leucyl‐tRNA synthetase mutant tsHI
Authors: Molnar S. J. , Thompson L. H. , Lofgren D. J. , Rauth A. M.
Published: February 2005

Nine spontaneous and seven ethyl methanesulfonate induced revertants of the Chinese hamster ovary cell line mutant (tsHl), which possesses a temperature sensitive leucyl‐tRNA synthetase, were isolated and characterized with respect to growth rate, leucyl‐tRNA synthetase activity and thermolability, intracellular leucine pool size, and rRNA content. Although most revertants had increased leucyl‐tRNA synthetase activity, and of those tested, all but one had increased thermostability, each appears to be unique. One revertant may be an intergenic suppressor since it appears to contain an elevated level of tsHl‐like synthetase. There was no evidence for any of the revertants having increased rRNA and tRNA contents, however, many showed leucine pools two to three times larger than wild type cells. Since similar increases have been observed in tsHl cells they are believed to result from regulation of leucine pool size by the leucyl‐tRNA synthetase and are of a magnitude sufficient to affect significantly the growth of revertants at 38.5°C.
Comparison of chloride transport in mouse erythrocytes and friend virus‐transformed erythroleukemic cells
Authors: Harper Patricia A. , Knauf Philip A.
Published: February 2005

Friend erythroleukemic cells, which grow continuously in tissue culture, resemble in many respects early precursors of mouse erythrocytes. To determine whether or not the membranes of these cells exhibit the rapid and selective exchange of chloride, a specialized feature of the mature erythrocyte membrane, anion fluxes were compared in Friend cells and mouse erythrocytes. The chloride flux in Friend cells at 37°C was about 800‐fold lower than in mouse erythrocytes (extrapolated from data at lower temperatures). This difference could not be accounted for by the somewhat lower chloride concentration in Friend cells relative to erythrocytes. Comparison of chloride and sulfate fluxes revealed that the Friend cells had over a 1,000‐fold lower selectivity for chloride versus sulphate than did the mouse red cells. The temperature dependence of chloride fluxes in Friend cells corresponded to an Arrhenius activation energy of 17.9 kcal/mol, in contrast to over 30 kcal/mol for mature red cells. The chloride flux in Friend cells was also 10‐fold less sensitive to the inhibitor, furosemide, than was the flux in mature red cells. The selective chloride exchange system of the mature erythrocyte therefore does not seem to be functional at the stage represented by the Friend cell, and must appear at some later stage of erythroid maturation.
The nature of cells generating human myeloma colonies in vitro
Authors: Hamburger Anne W. , Kim Mary B. , Salmon Sydney E.
Published: February 2005

Freshly explanted human myeloma cells formed colonies of monoclonal plasma cells in soft agar in the presence of medium conditioned by the adherent spleen cells of mineral oil‐primed BALB/c mice. The medium showed peak activity at a dilution of 1:4. 2‐mercaptoethanol or monothioglycerol was necessary for colony formation. Other thiols tested were ineffective in promoting colony growth. Colony‐forming cells adhered to nylon wool, but not glass beads or plastic dishes. The presence of E‐rosetting cells was not required for myeloma colony formation. Antibody prepared against a human myeloma cell line, RPMI 8226, reduced colony formation. These studies demonstrate the usefulness of this bioassay for determining functional properties of the myeloma colony‐forming cell.