bluuebunny/crossref_metadata_2025 huggingface dataset:
Megakaryocytopoiesis in culture: Modulation by cholinergic mechanisms
Authors: Burstein Samuel A. , Adamson John W. , Harker Laurence A.
Published: February 2005

Treatment of murine bone marrow cultures with the cholinergic agonist carbamylcholine enhanced megakaryocytic colony growth by as much as 65%. In contrast, adrenergic agonists had no such effect. Addition to cultures of dibutyryl cyclic GMP (db‐cGMP) also enhanced megakaryocytic colonies up to 50%, whereas dibutyryl cyclic AMP (db‐cAMP) had no effect. Sodium nitroprus‐side and sodium nitrite, putative guanyl cyclase activators, also enhanced colony numbers, as did imidazole, a postulated cGMP phosphodiesterase inhibitor. Preincubation of marrow for two hours with carbamylcholine resulted in both an increase in colony numbers (58%) and percent of progenitors in DNA synthesis (48%, compared to 14% for controls) as determined by tritiated thymidine suicide studies. Treatment of mice with the acetylcholinesterase inhibitor neostigmine resulted in an increase in CFU‐M/humerus (62%) and percent in DNA synthesis (45%). These data indicate that (1) cholinergic, but not adrenergic, agonists modulate megakaryocytopoiesis in culture; (2) this effect may be mediated by cyclic GMP; and (3) only a brief period of exposure of marrow cells to agonist results in enhancement of megakaryocytic colonies.
Senescence of cultured human diploid fibroblasts. Are mutations responsible?
Authors: Gupta Radhey S.
Published: February 2005

Two predictions of the error/mutation hypothesis of cellular sensecence (Orgel, '73) namely, (a) exponential accumulation of somatic mutations during the replicative lifespan and (b) shortening of culture lifespan upon treatment with mutagens have been examined experimentally in a strain of cultured human diploid fibroblasts. Our studies show that as cells traverse the replicative lifespan (from 10 to 75 mean population doublings (MPD); total lifespan ⋍ 95 MPD), no rapid and exponential increase occurs in the accumulation of mutations measured by the frequencies of Thgr (thioguanine resistance) and Dipr (diphtheria toxin resistance) mutants. Furthermore, repeated cycles of treatment (from 1‐ to 14‐times) of human fibroblasts with two mutagens, ethyl methane sulfonate (EMS) and N‐methyl‐N′nitro‐nitrosoguanidine, which led to a marked increase in the mutation frequency for the Dipr marker (⋍ 100‐fold), failed to shorten the lifespan of cultured fibroblasts. On the contrary, repeated mutagen treatment (12 times with EMS) prolonged the lifespan of one replicative culture (110 MPD versus 94–98 MPD). These results strongly indicate that mutations are unlikely to be the primary event in cellular senescence and suggest instead that senescence is probably controlled by one or more (specific) gene(s) whose expression can be modified by mutations.
Decay of hormone responsiveness in mouse melanoma cells in culture as a function of cell density
Authors: Fuller Bryan B. , Lebowitz Joyce
Published: February 2005

Cloudman S91 mouse melanoma cells lose their ability to demonstrate an MSH‐induced increase in tyrosinase activity as cell density increases. This loss in hormone responsiveness occurs before confluency is reached and cannot be reversed by exposure of cells to increasing concentrations of MSH. The failure of high‐density cultures to respond to MSH is apparently not the result of an inability of MSH to stimulate cAMP production, since either low‐ or high‐density cultures exposed to MSH demonstrate equivalent increases in intracellular levels of cAMP. Further, neither theophylline (1mM), dibutyryl cyclic AMP (10–4M), or prostaglandin E1 (10–6M) is effective in stimulating tyrosinase activity in melanoma cells cultured at densities exceeding 6 ± 104 cells/cm2. This finding suggests that the decay of hormone responsiveness occurs at a cellular site distal to cAMP production. The decrease in tyrosinase stimulation by MSH as cell density increases is also apparently not the result of an increase in activity of any soluble inhibitor of the enzyme, for cytosol preparations from high‐density cultures (105 cells/cm2) fail to inhibit tyrosinase activity in cell homogenates from low‐density cultures treated with MSH.
Fidelity of protein synthesis does not decline during aging of cultured human fibroblasts
Authors: Wojtyk Roman I. , Goldstein Samuel
Published: February 2005

To ascertain whether the fidelity of protein synthesis declines during cellular aging in vitro, we have developed a cell‐free protein synthesizing system from cultured human fibroblasts which actively incorporates phenylalanine into acid‐insoluble material upon addition of poly (U). The accuracy of poly(U)‐directed protein synthesis was determined by comparing the ratio of leucine to phenylalanine incorporation in extracts of early‐ and late‐passage fibroblasts derived from normal persons and from subjects with two genetic disorders of premature aging, progeria, and Werner syndrome. The results show no decline in translational fidelity at late passage or in prematurely aging cells, and thus fail to support the error catastrophe theory of cellular aging.
Self‐maintenance capacity of CFU‐S
Authors: Schofield R. , Lord B. I. , Kyffin S. , Gilbert C. W.
Published: February 2005

The numbers of CFU‐S which developed in spleen colonies were measured 11 days after injection of irradiated mice with marrow from normal mice or mice which had been treated in one of a variety of ways. The broad spread of CFU‐S numbers, seen by other authors, in colonies derived from normal marrow was confirmed. However, the range and distribution of CFU‐S per colony was generally different in colonies derived from the marrow of mice which were recovering or had recovered from some form of depopulation. From the data obtained, the mean CFU‐S/colony, M1, and the probability of self‐renewal, p, of the CFU‐S were calculated. These values are used to calculate the number of cell cycles undergone during development of the colony and, by making certain assumptions, the cell cycle time of the CFU‐S. The plot of p against log M for the various samples measured should be linear if all CFU‐S proliferate at the same rate in a growing colony. It is not linear, however, so that CFU‐S obtained under different experimental conditions do not all undergo the same number of cycles. In general, treatments given to the mice result in a lowering of the capacity for self‐renewal of their CFU‐S and also to a shortening of their cell cycle time. Some of the possible implications of these findings are discussed.
Effects of linoleic acid on capping, lectin mediated mitogenesis, surface antigen expression, and fluorescent polarization in lymphocytes and BHK cells
Authors: Hoover Richard L. , Bhalla Deepak K. , Yanovich Saul , Inbar Michael , Karnovsky Morris J.
Published: February 2005

The incubation of linoleic acid with cells causes profound effects on membrane associated phenomenon. Using the fluorescent probe diphenyl hexatriene (DPH) to monitor lipid changes in the microenvironment of the cell surface, we find that linoleic acid reduces the polarization values (P) in mouse lymphocytes and BHK cells. Measurements on lipids extracted from the cells grown in linoleic acid produce similar results. We also find in the mouse lymphocyte that capping of Ig is inhibited and con A stimulated mitogenesis is unaffected. In contrast to the latter effect, LPS and PHA stimulated mitogenesis is inhibited and in the rat lymph node, con A stimulated mitogenesis, greatly enhanced. We also show that linoleic acid alters the binding of antibodies to the cell surface of EL‐4 lymphoma cells. These observations suggest that linoleic acid alters cellular function by interfering with protein/lipid interactions within the surface membrane.
Water permeability of mammalian cells as a function of temperature in the presence of dimethylsulfoxide: Correlation with the state of the membrane lipids
Authors: Rule G. S. , Law P. , Kruuv J. , Lepock J. R.
Published: February 2005

The water permeability of V‐79 Chinese hamster lung fibroblasts was determined by measuring the rate of cell shrinkage in hypertonic medium using a cell sizer. The water permeability appears to follow Arrhenius kinetics as a function of temperature with a sharp discontinuity at 21°C. An activation energy of 7.0±1.6 kcal/mole was found below 21°C and 22.8±3.1 kcal/mole above 21°C. The correlation time of rotation of the spin label 2,2‐dimethyl‐5‐dodecyl‐5‐methyloxazolidine‐N‐oxide was measured as a function of temperature in the cellular membranes, and shows a break at 20°C. A discontinuity was also found in the membrane to water partitioning of the spin label 2,2‐dimethyl‐5‐pentyl‐5‐butyloxazolidine‐N‐oxide near 20°C. These breaks may correspond to a membrane lipid phase transition. Dimethylsulfoxide, in the concentration range of 0.2–0.5 M, decreases the water permeability by a factor of two.
Differential effect of interferon on DNA synthesis, 2‐deoxyglucose uptake and ornithine decarboxylase activity in 3T3 cells stimulated by polypeptide growth factors and tumor promotors
Authors: Sreevalsan T. , Rozengurt E. , Taylor‐Papadimitriou J. , Burchell J.
Published: February 2005

Quiescent 3T3 cells can be stimulated to enter S by defined factors. When used in combination, three polypeptide hormones (EGF, vasopressin, and insulin), or a tumor promotor and insulin, are very effective in stimulating DNA synthesis. Like serum, the defined factors also stimulate deoxyglucose uptake and induce the synthesis of ornithine decarboxylase during G1. The second stage of deoxyglucose uptake and the induction of ornithine decarboxylase are protein synthesis‐dependent events. When added with the growth factors, mouse interferon inhibits the synthesis of DNA and the induction of ornithine decarboxylase but has no effect on the uptake of deoxyglucose. Kinetic experiments comparing the effect of inhibitors of translation or transcription on induction of ornithine decarboxylase with the effect of interferon suggest that interferon may affect the synthesis of enzyme by inhibiting both transcription and translation of message. The findings provide further support for the proposition that interferon exerts a differential effect on mitogen‐stimulated events which are dependent on continuous protein synthesis.
X‐ray‐induced production of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) by mouse spleen cells in culture
Authors: Onoda M. , Shinoda M. , Tsuneoka K. , Shikita M.
Published: February 2005

Spleen cells were collected from normal mice and cultured in a medium containing 20% calf serum. Addition of lipopolysaccharide (LPS) in the culture significantly increased the production of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), and a maximum induction was attained in 5 days. Irradiation of the spleen cells with 300 to 3,000R X‐rays also enhacned the production of GM‐CSF, but there was a latent period of about 5 days before the factor appeared in the culture medium. The observed difference between LPS and X‐rays in the timing of inducing GM‐CSF production in the spleen cell culture was consistent with the difference in timing of the increase of spleen cell proliferation observed in animals after the administration of LPS or during recovery from damages by X‐irradiation. It was observed furthermore that the X‐ray‐induced GM‐CSF differed from the LPS‐induced GM‐CSF in its molecular properties; the X‐ray‐induced factor was represented by an acidic (pI=3.0) 70,000‐dalton species, while the LPS‐induced factor was much smaller in size (M.W. 20,000) and less acidic (pI=5.4). These results suggest that different mechanisms of GM‐CSF production operate in the spleen in response to either LPS or X‐rays.
Cyclic AMP levels in migrating and non‐migrating newt epidermal cells
Authors: Dunlap Mary K.
Published: February 2005

Cyclic AMP (cAMP) levels were measured in 8‐hour migrating wound epithelial and non‐migrating epithelial cells of the newt. Tissues were collected in vivo and in vitro with and without epidermal‐dermal separation by collagenase. Regardless of manner of collection and treatment, cAMP levels were always significantly higher in the migrating cells. Levels were also measured in 28‐hour and 36‐hour wound epithelia. There was a progressive decline in levels in wound epithelia between 8, 28, and 36 hours, suggesting that levels were in the process of returning to normal. When cells were treated with a dose of cAMP and theo‐phylline previously shown to inhibit migration, levels of cAMP were much higher than any migrating epithelium. The fact that cAMP inhibits migration, yet migrating cells have higher cAMP levels, seems contradictory at first, but possible explanations are advanced to account for the apparent discrepancy.
Sugar transport in the LLC‐PK1 renal epithelial cell line: Similarity to mammalian kidney and the influence of cell density
Authors: Mullin James M. , Weibel Josef , Diamond Leila , Kleinzeller Arnost
Published: February 2005

Cells of confluent cultures of the established pig renal epithelial line, LLC‐PK1, accumulate α‐methyl‐D‐glucoside against a concentration gradient. This transport system is strongly inhibited by phlorizin and 6‐deoxy‐D‐glucose, moderately inhibited by phloretin, and only weakly inhibited by 3‐0‐methyl‐D‐glucose, paralleling the situation in mammalian kidney. The time course for the uptake of α‐methyl‐D‐glucoside and for the carrier‐mediated but passive uptake of 3‐0‐methyl‐D‐glucose are identical to those seen in mammalian kidney. Subconfluent cultures of LLC‐PK1 cells are unable to accumulate α‐methyl‐D‐glucoside, and their transport of this glucose analog is less sensitive to phlorizin inhibition than is the transport system in confluent cultures. Transmission electron micrographs show that cells from subconfluent cultures lack the microvillous surface seen in cells from confluent cultures. Cell density is thus a factor in the occurrence of structural and functional differentiated properties related to transport in these cells.
Lactate: A major product of glutamine metabolism by human diploid fibroblasts
Authors: Zielke H. Ronald , Sumbilla Carlota M. , Sevdalian David A. , Hawkins Robert L. , Ozand Pinar T.
Published: February 2005

Human diploid fibroblasts metabolize up to 13% of the glutamine in tissue culture medium to lactate. Four μCi of glutamine‐U‐14C were added to media containing 5 mM or 65 μM glucose or medium containing no added glucose, but supplemented with purine and pyrimidine nucleosides (HGTU). Aliquots of the media were taken at daily intervals and were assayed for glucose, lactate, pyruvate, malate, citrate, aspartate, glutamine, and glutamate. The label incorporation into these compounds was determined, except for glutamine and glucose. The distribution of label from glutamine‐U14C in 5 mM glucose medium by day 4 was lactate (10.2%), glutamate (15.2%), citrate (1.9%), pyruvate (2.0%), malate (1.1%), and aspartate (< 0.1%). The accumulation of label in lactate and glutamate occurred continuously during the growth cycle. Malate, citrate, and aspartate accumulation occurred primarily in confluent cultures. The label in aspartate was seen only in stationary phase cells or when the glucose concentration was decreased to 65 μM or less; net aspartate accumulation was increased twofold in low glucose media. These data demonstrate an actively functioning pathway for the conversion of 4‐carbon TCA‐cycle intermediates to 3‐carbon glycolytic intermediates in human diploid fibroblasts.
Temperature dependent expression of polyoma virus in murine embryonal carcinoma cells
Authors: Dandolo L. , Vasseur M. , Kress C. , Aghion J. , Blangy D.
Published: February 2005

At 37°C, undifferentiated murine teratocarcinoma cells (PCC4) are resistant to infection with SV40 and Polyoma virus. When infection is carried out at 31°C, these cells become fully susceptible to a variety of polyoma virus strains, including wt, ts‐a, and hr‐t; they also display an increased susceptibility to polyoma virus mutants (PyE.C.) which have been selected for their ability to develop in PCC4 cells at 37°C (Vasseur et al., '80). However, expression of SV40 is still restricted at 31°C and no T antigen can be detected. PCC4 cells grown at 31°C express the characteristic embryonal surface antigen(s), but no H2 antigen, and do not produce plasminogen activator.

PyE.C. mutants and other polyoma virus strains cannot develop at 37°C in nullipotent F9 embryonal carcinoma cells and restriction is not abolished at 31°C.

The results indicate that: (i) Resistance of PCC4 cells to polyoma virus and to SV40 are not mediated by the same process; (ii) loss of restriction of polyoma in PCC4 cells does not require cell differentiation; (iii) F9 and PCC4 cells control polyoma virus expression through different mechanisms.
Differentiation and production of colony‐stimulating factor induced by immunostimulants in a leukemia cell line
Authors: Maeda Michiyuki , Ichikawa Yasuo , Azuma Ichiro
Published: February 2005

Immunostimulants from various microorganisms were tested on a myeloid leukemia cell line (Ml) for the ability to induce production of CSF and to cause differentiation of these cells. Based on their activities, the compounds were divided into two general classes: those inducing extensive cellular differentiation and those devoid of this effect. The stimulants which were active in this regard always produced large quantities of CSF, whereas those devoid of a differentiating effect did not cause CSF production. Even the potent stimulants had no effect on the D− subline, in which the differentiation was not inducible.
Expression of the teratocarcinoma phenotype in hybrids between totipotent mouse teratocarcinoma and myeloma cells
Authors: Correani Alessandro , Croce Carlo M.
Published: February 2005

We have produced somatic cell hybrids between totipotent mouse teratocarcinoma and myeloma cells. These hybrids behave like the teratocar‐cinoma cell parent and express teratocarcinoma embryonal antigens. However, they also express the myeloma H‐2 antigens. The availability of these hybrids should make it possible to study the expression of already rearranged immunoglobulin genes during mouse development.
Growth response to insulin in mouse melanoma cells and fibroblast × melanoma hybrids
Authors: Coppock Donald L. , Covey Lori R. , Straus Daniel S.
Published: February 2005

PG19 mouse melanoma cells arrest growth when they become confluent in medium containing low concentrations of serum. Under these conditions, insulin does not stimulate DNA synthesis in the mouse melanoma cells, whereas it does in mouse embryo fibroblats and fibroblast × melanoma hybrids. A detailed examination of the binding of insulin to the melanoma cells and fibroblast × melanoma hybrids in the presence of bacitracin has shown that they have approximately equal numbers of insulin receptors, and that these receptors have similar affinities for insulin. These results indicate that the unresponsiveness of the melanoma cells to the mitogenic action of insulin is not attributable to a lack of insulin receptors. Although insulin does not stimulate incorporation of 3Hthymidine into DNA in confluent cultures of the melanoma cells, it does stimulate uptake of α‐aminoisobutyrate, indicating that the insulin receptors are functional and that insulin elicits an acute response in these cells. In hormone‐supplemented serum‐free medium, insulin does not stimulate growth of the melanoma cells, while it does stimulate growth of the fibroblast × melanoma hybrid. This suggests that an acute response to insulin is not sufficient for stimulation of growth either in confluent growth‐arrested cells or in exponentially growing cells in serum‐free medium.
Vasopressin rapidly stimulates NA entry and NA‐K pump activity in quiescent cultures of mouse 3T3 cells
Authors: Mendoza Stanley A. , Wigglesworth Noel M. , Rozengurt Enrique
Published: February 2005

Addition of (Arg) vasopressin to quiescent cultures of Swiss 3T3 cells rapidly stimulates an ouabain‐sensitive 86Rb uptake. In contrast the hormone has no significant effect on the rate of efflux of this cation from preloaded cells. The stimulation of 86Rb uptake is cycloheximide‐insensitive, occurs within minutes of hormone addition and results from an increase in the Vmax of the uptake system. Vasopressin stimulates ion uptake in a concentration‐dependent fashion (1‐100 ng/ml); oxytocin also stimulated the Na‐K pump but at significantly higher concentrations. The stimulation of the Na‐K pump by vasopressin is apparently mediated by an increase in Na entry into the cells, since the hormone (1) strikingly shifts the concentration dependence on Na+ of the Na‐K pump, (2) increases 22Na uptake, and (3) increases intracellular Na contents when the efflux of this ion is blocked by ouabain. Since vasopressin is a potent mitogen for Swiss 3T3 cells, the results provide further evidence in support of a possible role of monovalent ion fluxes in signalling the initiation of growth stimulation.
Intracellular analysis of sodium, potassium, and chloride in mouse erythrocytes
Authors: Benos D. J.
Published: February 2005

Recently, Cameron et al. ('79) published measurements of intracellular solute amounts (expressed as mmoles per kilogram dry cell solids) obtained by energy dispersive electron probe microanalysis in different rodent tissues. In this communication, I wish to compare Cameron et al.'s ('79) erythrocyte values of Na, K, and Cl with those I have made using more conventional techniques of elemental analysis. This comparison is necessitated by Cameron et al.'s ('79) observation of extremely high intracellular sodium levels. If their findings are accurate, the possibility of a polymorphism with respect to intracellular Na levels therefore presents itself. If a polymorphism between different inbred strains of mice exists, and if, as in ruminants, this trait has a genetic basis, an examination of the genetic aspects of the control, differentiation, and the ultimate expression of the ion transport mechanisms responsible would undoubtedly provide insight into the molecular basis as well as the adpative dynamics of transport systems in general.
Oxidation of glucose by mouse peritoneal macrophages: A comparison of suspensions and monolayers
Authors: Lazdins Janis K. , Koech Davy K. , Karnovsky Manfred L.
Published: February 2005

Macrophages, when maintained in vitro, take up glucose from the medium and oxidize it to CO2. The rate of oxidation of glucose varies considerably, depending on the physical state of the cell preparation. Cells in suspension oxidize glucose at a level six‐fold that of cells in monolayers. The differences cannot be attributed to change in the rates of transport of glucose. On the other hand, an increse in intracellular glycogen (about three‐fold) and free glucose plus glucose‐6‐P (many‐fold) was found in the cells prepared as monolayers. During subsequent incubation with glucose‐14C, this could be the cause of an isotope dilution effect and could explain the lower production of 14CO2 by the adherent cells. Since oxidation of glucose‐1‐14C to 14CO2 is used by many investigators to indicate the functional state of macrophages, we suggest close attention be paid to the system used, i.e., monolayers vs. suspensions.
Enzyme induction in a temperature‐sensitive cell cycle mutant of chinese hamster fibroblasts
Authors: Landy‐Otsuka Fyllis , Scheffler Immo E.
Published: February 2005

A temperature‐sensitive (ts) cell cycle mutant of Chinese hamster fibroblasts with a block in G1 was investigated. Attention was on the expression of the activity of three enzymes: ornithine decarboxylase (ODC), S‐adenosylmethionine decarboxylase (SAMDC), and thymidine kinase (TK). ODC and SAMDC activities are normally induced in the middle of, or late in, the G1 phase, while TK activity starts to appear at the G1/S boundary. In the ts mutant released from serum starvation at the nonpermissive temperature (40.8°C), we find no effect on the expression of SAMDC activity, a significantly reduced level of ODC activity compared to the control at the permissive temperature (34°C), and no induction of TK activity. Results presented here and in a previous publication (Landy‐Otsuka and Scheffler, '78) suggest that the decrease in ODC activity is due to an effect of the nonpermissive temperature on a post‐transcriptional step, possibly a very rapid inactivation of the enzyme. The absence of TK activity, on the other hand, appears to be due to a block in transcription at the nonpermissive temperature.
Protein synthesis and degradation during expression of the temperature‐sensitive defect in ts A1S9 mouse L‐cells
Authors: Sparkuhl Joachim , Sheinin Rose
Published: February 2005

The involvement of altered protein metabolism in the expression of the temperature‐sensitive (ts) pleiotropic phenotype of ts A1S9 cells was investigated. Cells are ts in growth and DNA replication. They undergo decondensation of their heterochromatin, interruptions of chromatin synthesis, and changes in cell size and morphology at the non‐permissive temperature (npt) of 38.5°C. Whereas the rates of incorporation of 3H‐leucine, 35S‐methionine, and 3H‐fucose into proteins were unaffected at 38.5°C, net protein accumulation was greatly reduced. This imbalance resulted from a rapid increase in the rate of protein degradation at the npt. Enhancement of protein degradation was detected within 2‐4 hours after temperature upshift and constitutes the earliest metabolic alteration thus far observed during expression of the temperaturesensitive phenotype. The average half‐life of proteins preformed in ts A1S9 cells at 34°C was decreased four‐fold at the npt, and all major cytoplasmic proteins were affected equally. Enhanced protein degradation at the npt was shown to be sensitive to cycloheximide, ammonia, chloroquine, and vinblastine at concentrations that did not affect the basal protein degradation of normally cycling cells. Increased protein degradation at 38.5°C did not involve an equivalent increase in total cellular protease activity. The data obtained are compatible with a model that suggests that temperature inactivation of the ts A1S9 gene product results in activation of a lysosome‐mediated mechanism for the rapid degradation of cytoplasmic proteins.
Intracellular NAD+ content and ADP‐ribose polymerase activity of serum‐stimulated baby hamster kidney fibroblasts
Authors: Furneaux Henry M. , Pearson Colin K.
Published: February 2005

We have examined a number of events relating to ADP‐ribose metabolism during serum‐stimulated growth of BHK‐21/C13 fibroblasts. Both the intracellular NAD+ content and the ADP‐ribose polymerase activity were found to increase after serum stimulation of cells that were previously arrested by growth in low‐serum medium. NAD+ content increased about two‐fold, reaching a maximum of 4.2 nmol/μg of DNA 8 hr after serum step‐up. The polymerase exhibited a sharp rise in activity, reaching a peak at about 5 hr after step‐up; the activity declined below initial values by 10 hr, and then increased again to reach a plateau at 20 hr. We also report evidence which suggests a possible effect of ADP‐ribosylation on the activity of DNA‐dependent RNA polymerase I. The activity of this enzyme is diminished in isolated nuclei, and in a subsequent (NH4)2SO4 extract, when the nuclei are incubated with NAD+, the substrate for poly(ADP‐ribose) polymerase. This inhibitory effect on the RNA polymerase is abolished when nuclei are incubated also with nicotinamide, a powerful inhibitor of the poly(ADP‐ribose) polymerase.
A study of binding‐solubilization of some glycolytic enzymes in striated muscle in situ
Authors: Ross Robert E. , Hultin Herbert O.
Published: February 2005

The solubilization of lactate dehydrogenase (LDH), glyceralde‐hyde‐3‐phosphate dehydrogenase (GAPDH), and alpha‐glycerophosphate dehydrogenase (aGPDH) was studied in pressed muscle as a function of ionic strength and NADH concentration.

The results indicate that these factors affect the binding‐solubilization of LDH and GAPDH in a similar way to their effect in dilute homogenized tissue. Alpha‐glycerophosphate dehydrogenase was included as a typical soluble enzyme, since we have been unable to demonstrate its binding to subcellular fractions under any conditions. As with homogenized tissue, LDH was less susceptible to solubilization by ionic strength than GAPDH. It was demonstrated that LDH isozymes richer in heart‐type subunits were more easily removed from muscle by centrifugation‐imbibition than isozymes richer in the muscle‐type subunits. This was interpreted as indicating that binding of the enzyme to subcellular structures was a major factor in the restricted removal of these enzymes from muscle, since only the muscle‐type subunit is capable of binding to subcellular particles.

It was further demonstrated that LDH could be taken up into muscle tissue, depleted in the enzyme, against an apparent concentration gradient. This was also interpreted as binding of the enzyme to the particulate structure of the muscle. Furthermore, this uptake of LDH occurred under conditions of ionic strength (0.25) and pH (7.5) that would prevent binding of the enzyme to the particulate fraction of a dilute suspension of homogenized muscle tissue. Thus, physiological conditions of pH and ionic strength do not necessarily induce solubilization of chicken breast muscle LDH in situ. Data obtained with dilute tissue homogenates, therefore, may not necessarily be easily and safely extrapolated to conditions in situ.
Measurement of intracellular pH in sea urchin eggs by 31P NMR
Authors: Inoue Hiroko , Yoshioka Tohru
Published: February 2005

The intracellular pH (pHi) of sea urchin eggs was measured using 31P nuclear magnetic resonance (NMR) spectroscopy. The mean value of pHi of unfertilized eggs was about 6.2 (H. pulcherrimus and A. crassispina) to 6.3 (P. depressus). In contrast to results of other studies, pHi of sea urchin eggs was not changed after fertilization. During exposure of the eggs to NH4Cl or procaine‐containing natural sea water (NSW), however, pHi rose about 0.4‐0.8 pH units; the pHi fell to its initial value upon washing the eggs with weak basefree NSW. These changes of pHi by weak base treatment agreed well with the data obtained by other workers. In order to understand the discrepancy of pHi changes in fertilized eggs between NMR data and other measuring procedures, we measured acid production and O2 uptake, so that CO2 accumulation and proton release did not result in alkalinization. The invariance of the fertilized eggs under anaerobic conditions; otherwise NMR showed a different answer from other measuring procedures, because of its particular characteristics such as non‐destructivity and compartmentation of pH.
Regulation of metallothionein synthesis in HeLa cells by heavy metals and glucocorticoids
Authors: Karin Michael , Slater Emily P. , Herschman Harvey R.
Published: February 2005

Metallothioneins (MTs) are low molecular weight, cysteine‐rich proteins that bind heavy metals. MT induction occurs in liver in response to either heavy metal (Zn++ or Cd++) administration or stress. The synthesis of MT can also be induced by either heavy metals or glucocorticoid hormones in HeLa cells cultured in serum‐free medium. Induction of MT by zinc is subject to “desensitization.” In contrast, dexamethasone (dex) induction results in a continued elevation in the rate of MT synthesis. The stability of MT is dependent on the availability of metal; consequently, MT induced by dex is degraded much more rapidly (half‐life of 11 to 12 hours) than MT induced by elevated zinc levels (half‐life of 36 to 38 hours). Removal of either inducer results in biphasic degradation curves, as apothionein and zinc come into balance. In contrast, deinduction kinetics for MT synthesis following removal of the two inducers (zinc and dex) are the same, with a half‐life of two and one‐half hours. Inhibition of RNA synthesis blocks deinduction after removal of inducer. Induction of MT occurs in a wide variety of species, from blue‐green algae to man. This system should provide an excellent model for the comparative biochemistry of regulation of gene expression.
External cell surface protein phosphorylation in normal and Rous sarcoma virus transformed chick embryo fibroblasts
Authors: Yee Siu‐Pok , Branton Philip E.
Published: February 2005

Normal and Rous sarcoma virus (RSV)‐transformed chick embryo fibroblasts growing on plastic dishes were incubated with ATP (γ32P) in situ to detect external cell surface protein kinase activity. Under the conditions employed, 32P was incorporated exclusively into proteins, specifically those at the external cell surface, as radioactivity was removed by tryspin treatment of labeled whole cells. In addition, exogenous histones were phosphorylated when added to the reaction mixture. Cyclic nucleotides had virtually no effect on 32P incorporation, suggesting that little or no cyclic nucleotide‐dependent protein kinase activity was present at the external cell surface. Cell surface protein kinase activity was higher in transformed than in normal cells, and, using a temperature‐sensitive RSV src mutant, this difference was shown to be transformation‐specific. Several differences were observed in the cell surface proteins phosphoryllated in normal and transformed cells and at least two of these were transformation‐specific. These data suggest that changes in external cell surface protein physphorylation are associated with RSV transformation and thus could play a role in the formation of the transformed cell phenotype.
Enhancement of melanotic expression in cultured mouse smelanoma cells by retinoids
Authors: Lotan Reuben , Lotan Dafna
Published: February 2005

Retinoic acid (RA), which reduces the rate of cell proliferation in S91 mouse melanoma clone C2 cells, was found to stimulate the expression of their melanotic phenotype. RA treatment also induced the extension of long cellular processes. The RA effects on melanogenesis included stimulation of tyrosinase activity and augmentation of cellular melanin content to levels 3‐ to 4‐fold higher than in untreated cultures at similar cell densities. These effects became apparent after 48 hours of exposure to 10−5 M RA and increased thereafter. Halfmaximal stimulation in cells treated for 6 days occurred at 5 × 10−7 M RA. Although the degrees of melanogenesis enhancement by RA (10−5 M) and by α‐melanocyte stimulatory hormone (2 × 10−7 M) were similar, the former did not alter the intracellular cAMP level, whereas the latter induced a transient 4‐fold increase. In high‐passage (p28) cells, as well as in low‐passage cells (< p10) treated with tyrosinase inhibitor phenylthiocarbamate, melanin synthesis was suppressed in the absence and presence of RA, yet the ability of RA to inhibit cell proliferation was not compromised. In the presence of the tumor promotor phorbol myristate acetate (> 5 × 10−9 M) melanin synthesis in control as well as in cells exposed to RA was dramatically inhibited. Phorbol which is not active in tumor promotion had no effect on melanogenesis. In addition to RA, other retinoids, such as 13‐cis‐retinoic acid, retinyl acetate, the TMMP analog of RA and the phenyl analog of RA, but not the pyridyl analog of RA or retinyl palmitate, also inhibited cell growth and enhanced melanin synthesis.
Effects of parathyroid hormone and cyclic AMP analogues on the activity of ornithine decarboxylase and expression of the differentiated phenotype of chondrocytes in culture
Authors: Takigawa M. , Takano T. , Suzuki F.
Published: February 2005

Parathyroid hormone (PTH) greatly increased the level of adenosine 3′,5′ cyclic monophosphate (cAMP) in rabbit costal chondrocytes in culture 2 minutes after its addition. PTH, as well as N6 O2' dibutyryl adenosine 3′,5′ cyclic monophosphate (DBcAMP) and 8 Bromo adenosine 3′,5′ cyclic monophosphate (8 Br‐cAMP) induced ornithine decarboxylase (ODC; L‐ornithine carboxylyase; EC 4.1.1.17), which reached a maximum 4 hours after their addition. Neither cAMP, N6 O2′ dibutyryl guanosine 3′,5′ cyclic monophosphate (DBcGMP), nor sodium butyrate increased the activity of the enzyme. PTH had no effect on DNA synthesis, while DBcAMP and 8 Br‐cAMP decreased DNA synthesis. Expression of the differentiated phenotype of chondrocytes in culture was also induced by PTH, DBcAMP, and 8 Br‐cAMP, but not by cAMP, DBcGMP, or sodium butyrate, as judged by morphological change. Glycosaminoglycan synthesis, a characteristic of the cartilage phenotype, began to increase 8 hours after addition of PTH or DBcAMP, reaching a plateau 32 hours after their addition.

These findings suggest that PTH induces increase of ODC activity and expression of the differentiated phenotype of chondrocytes through increase of cAMP and that induction of ODC is closely related to expression of the differentiated phenotype of chondrocytes.
Somatomedin‐C and platelet‐derived growth factor stimulate human fibroblast replication
Authors: Clemmons David R. , Van Wyk Judson J.
Published: February 2005

Previous studies have demonstrated that serum contains mitogens, such as platelet‐derived growth factor (PDGF), which may alter fibroblast responsiveness to growth factors contained in plasma. Somatomedin‐C (SM‐C) has been identified as one of the plasma growth factors required for mouse Balb/c 3T3 fibroblasts to initiate DNA synthesis. The present experiments were undertaken to explore the interaction between PDGF, human growth hormone (hGH), SM‐C, and other growth‐promoting agents in stimulating the growth of human fibroblasts. Proliferation of human dermal fibroblasts plated at low density (3,000 cells/cm2) was found to be equally stimulated by continuous exposure to either normal or somatomedin‐C‐deficient serum. In contrast, when confluent monolayers were sequentially exposed to PDGF, followed either by normal platelet poor plasma (PPP) or hypopituitary PPP, the cells exposed to normal PPP entered the “S” phase of the cell cycle 50% faster. This difference could be abolished by a 6‐hour incubation with growth hormone (10 ng/ml) or somatomedin‐C (5 ng/ml) preceding the addition of plasma. When medium containing either hGH or Sm‐C was changed frequently so as to remove factors secreted by fibroblasts, only those cells exposed to exogenous somatomedin‐C entered DNA synthesis. This finding is in agreement with previous findings that human fibroblasts are capable of making Sm‐C in response to hGH. These findings support the hypothesis that somatomedin is required for fibroblast replication in vitro, and that growth hormone appears to stimulate replication indirectly through somatomedin production.
Gluconeogenesis in rat liver parenchymal cells in primary culture: Permissive effect of the glucocorticoids on glucagon stimulation of gluconeogenesis
Authors: Stumpo Deborah J. , Kletzien Rolf F.
Published: February 2005

Primary cultures of parenchymal cells isolated from adult rat liver by a collagenase perfusion procedure and maintained as a monolayer in a serum‐free culture medium were used to study glucoeogenesis and the role that the glucocorticoids play in the control of this pathway. These cells carried out gluconeogenesis from three‐carbon precursors (alanine and lactate) in response to glucagon and dexamethasone added alone or in combination. Maximum glucose production was observed with cells pretreated for several hours with dexamethasone and glucagon prior to addition of substrate and glucagon (8‐ to 12‐fold increase over basal glucose production). Half‐maximum stimulation of gluconeogenesis was seen with 3.6 × 10−10 M glucagon and 3.6 × 10−8 M dexamethasone. Maximum stimulation was oberved with 10−7 M glucagon and 10−6 M dexamethasone. The length of time of dexamethasone pretreatment was found to be important in demonstrating the effect of glucocorticoids on glucagon‐stimulated gluconeogenesis. Treeatment of cells with dexamethasone for 2 hours did not result in an increase in glucose production over identical experimental conditions in the absence of dexamethasone, wherease pretreatment for 5 hours (1.2‐fold increase) or 15 hours (1.7‐fold increase) did result in an increase in glucose production. The results establish that the adult rat liver parenchymal cells in primary culture are a valid model system to study hepatic gluconeogenesis. In addition, we have established directly that the glucocorticoids amplify the glucagon stimulation of gluconeogenesis.
Proliferative characteristics of clonal endothelial cell strains
Authors: Rosen Eliot M. , Mueller Stephen N. , Noveral James P. , Levine Elliot M.
Published: February 2005

We have utilized clonal strains of bovine fetal aortic endothelial cells to study cellular senescence in a differentiated cell type of physiological significance. Serial subcultivation of nine endothelial clones derived from three fetal calf aortas revealed proliferative life‐spans in vitro of 53–125 population doublings (PDs), compared with 60 and 143 PDs for two lines of bovine fetal lung cells and 85 and 147 PDs for two lines of bovine vascular smooth muscle cells. Serial growth curves showed marked reductions associated with endothelial cellular senescence both in cellular growth rate and culture plateau density. Studies of the 24‐hour [3H]‐thymidine labeling index versus percentage of proliferative life‐span completed indicated that clonal endothelial cultures contained a large proportion (greater than 90%) of rapidly cycling cells until about 75% of the life‐spans were completed. Senescent endothelial cells showed evidence of large increases in cell area, cell volume, and protein content. In those clones examined, one specialized endothelial function, Factor VIII antigen expression, was retained qualitatively throughout the life‐spans.
Cellular control of ornithine decarboxylase activity by its antizyme
Authors: Heller John S. , Canellakis Evangelos S.
Published: February 2005

Conditions have been established under which the antizyme of ornithine decarboxylase (E.C. 4.1.1.17, L‐ornithine carboxy‐lyase, ODC) a noncompetitive protein inhibitor of ODC, can be detected in cells in response to as little as 10−7 M putrescine. The maintenance of intracellular antizyme activity depends upon the continued presence of putrescine in the medium. Removal of putrescine results in a rapid decline of antizyme activity. These phenomena are unaffected by the presence of cycloheximide and are comparable to the requirement of L‐asparagine for the maintenance of ODC activity.

The extent to which the antizyme level is increased is inversely related to the preexisting level of intracellular ODC at the time of addition of putrescine. The time of appearance of free antizyme is delayed in cells that have high levels of ODC; the amount of free antizyme that can be assayed for in these cells, at any particular time is correspondingly less. The converse is also true. In cells that have high levels of antizyme, the delay in appearance of ODC is greater and the amount of ODC that can be assayed for is correspondingly less than in cells with low levels of antizyme.

These experiments, as well as others, indicate that the ODC antizyme and ODC interact in vitro with each other to modify their respective activities.
Patients with myotonic dystrophy, a possible segmental progeroid syndrome, and duchenne muscular dystrophy have fibroblasts with normal limits for in vitro lifespan and growth characteristics
Authors: Wertz Richard L. , Hartwig Geoffrey B. , Frost Arlo P. , Brophy Jere J. , Atwater Susan K. , Roses Allen D.
Published: February 2005

Myotonic dystrophy (MyD) has been suggested to be a segmental progeroid syndrome in man, as this syndrome has some clinical manifestations of premature aging. Fibroblasts from patients with other progeroid syndromes have been shown to have diminished in vitro lifespans or growth characteristics; therefore, it was of interest to study cellular senescence in fibroblasts from patients with MyD. Fibroblast cultures from patients with Duchenne muscular dystrophy (DMD) were used as additional controls, as premature aging is not associated with this genetic disorder. Primary skin fibroblast cultures obtained from patients with MyD or DMD and from age‐sex matched controls were grown in DMEM plus 10% FBS. The in vitro lifespan was determined by either a 1:4 split ratio or with a constant initial inoculum of 1 × 104 cells/cm2, followed by determination of the final density at weekly intervals. Our results demonstrate that there is no difference in the limits of the in vitro lifespan for either the MyD or DMD fibroblast strains compared to the controls. Likewise, no difference could be detected in the growth characteristics of these cells. The only observable difference was that the pooled age‐matched controls and MyD cultures had a shorter in vitro lifespan than the DMD group and their pooled controls, a finding expected because of the age of the patients in each group. Unlike the other progeroid syndromes, MyD fibroblasts have normal limits for in vitro lifespan. MyD is probably not closely related to the other premature aging syndromes, although there is an increasing phenotypic expression as a function of age.
The tumor promoter 12‐0‐tetradecanoylphorbol‐13‐acetate induces ornithine decarboxylase in proliferating basal cells but not in differentiating cells from mouse epidermis
Authors: Lichti Ulrike , Patterson Elroy , Hennings Henry , Yuspa Stuart H.
Published: February 2005

The cultivation of mouse epidermal cells in medium of reduced calcium concentration (0.02–0.1 mM) selects for basal cell growth. Elevation of medium calcium levels above 0.1 mM results in rapid and well defined differentiative changes. This model was utilized to determine which cell type in mouse epidermis responds to the phorbol ester tumor promoter, 12‐0‐tetradecanoyl‐phorbol‐13‐acetate (TPA), by an induction of the enzyme ornithine decarboxylase (ODC). Previous data had shown that TPA induces ODC in primary mouse epidermal cells only during the first 36 hr after plating in medium containing 1.44 mM Ca2+. In contrast, the induction in cells grown in low calcium medium was 2–10‐fold greater, and inducibility persisted for at least 4 weeks. The greater inducibility of ODC in low calcium cells is not paralleled by increased thymidine incorporation after TPA treatment, probably because these cells are already proliferating at a maximum rate. When low calcium cells grown in 0.07 mM Ca2+ medium were switched to 1.2 mM Ca2+, there was a rapid loss of ODC inducibility. These results strongly suggest that the basal cells of the epidermis constitute the major target cells for the induction of ODC by TPA. The induction of ODC by ultraviolet light was not enhanced by growth of cells in low calcium medium, indicating that extracellular calcium concentration per se does not determine ODC inducibility. When epidermal cells grown in 1.2 mM or 0.07 mM Ca2+ medium were exposed to both UV light and TPA, there was a significant synergistic effect of combined treatment over the sum of each individual response, suggesting that factors in addition to differentiation determine the extent of ODC induction.
Demonstration of a hematopoietic stem cell antigen (SC‐1) on a murine lymphoma and isolation of variants lacking the antigen
Authors: Basch Ross S. , Panagiotatos Thomas , Berman Joan W. , Buxbaum Joel N.
Published: February 2005

Murine multipotential hematopoietic stem cells (CFU‐s) bear an antigen (SC‐1) which is recognized by heterologous antisera to mouse brain. We have found that cloned Thy‐1 negative variants of the T‐cell lymphoma RL ♂1 are sensitive to complement‐mediated cytolysis by anti‐brain serum and can absorb the anti‐stem cell activity from the antiserum. We have isolated several subclones derived from a primary Thy‐1 negative variant which are not susceptible to anti‐brain serum. The surface of the resistant lines has little or no antigen capable of binding anti‐mouse brain antibodies as measured by either immunofluorescence or a radioimmunoassay. These lines are also unable to absorb the anti‐bodies responsible for the cytotoxic effect of rabbit anti‐mouse brain serum against CFU‐s. We conclude that the predominant antigen, serologically detectable on Thy‐1 negative variants of RL ♂1, is SC‐1.