bluuebunny/crossref_metadata_2025 huggingface dataset:
Epidermal growth factor initiates DNA synthesis after a time‐dependent sequence of regulatory events in Swiss 3T3 cells—interactions with hormones and growth factors
Authors: Otto Angela M. , Ulrich Marie‐Odile , de Asua Luis Jimenez
Published: February 2005

Epidermal growth factor (EGF) stimulates the initiation of DNA synthesis in Swiss 3T3 cells after a constant prereplicative period of 14–15 hours. The final rate of initiation follows apparent first‐order kinetics and can thus be quantified by a rate constant k. The value of k can be changed by later additions during the prereplicative period: When cells stimulated by a very low concentration of EGF, alone or with insulin, which results in a relatively low value of k, receive a saturating amount of EGF at 15 hours, then k is markedly increased after 4–6 hours. Insulin alone (up to 200 ng/ml) is unable to set the lag phase, but does have a synergistic effect on the value of k given by EGF. When added at 15 hours, insulin also increases k, but after a delay of 4–6 hours. In contrast, both hydrocortisone and prostaglandin E1 (PGE1) inhibit the stimulation of DNA synthesis by EGF only during the first 8 hours of the prereplicative period of decreasing the value of k.

Prostaglandin F2α (PGF2α), which stimulates DNA synthesis in a similar mode as EGF, when added with EGF has a synergistic effect on DNA synthesis. This suggests that EGF and PGF2α, nevertheless, act through different regulatory events.
Fibroblast colonies in monolayer cultures of human bone marrow
Authors: Nagao Tadami , Komatsuda Mitsumoto , Yamauchi Kunihiko , Arimori Shigeru
Published: February 2005

In a liquid culture of human bone marrow, the development of fibroblast colonies takes place on days 6 to 9. Twenty percent fetal calf serum is used as the stimulus for fibroblast colony growth. Human bone marrow cells are plated as 2 × 105 cells in the culture. Normal human bone marrow yields 47 ± 4 fibroblasts colonies per 2 × 105 cells plated. Bone marrow fibroblast cultures using agar or methylcellulose restrict colony formation. Marked colony suppression was observed in acute leukemia, and a discrete colony number was observed in hypoplastic anemia. This fibroblast culture method should be applied to a larger number of patients to determine whether it has a pathognomonic value and clinical significance.
Cyclic AMP‐dependent protein kinases from Balb 3T3 cells and other 3T3 derived lines
Authors: Wehner Jeanne M. , Malkinson Alvin M. , Wiser Mark F. , Sheppard J. R.
Published: February 2005

Cyclic AMP‐dependent protein kinase and 3H‐cAMP‐binding activities were determined in normal Balb 3T3 cells and compared with the same preparations from SV40, chemical, and spontaneous transformants of 3T3 cells. The cytosolic protein kinase activities and protein kinase activity ratios were similar in all cell lines, although when the normal 3T3 cytosol was prepared by homogenization it contained less 3H‐cAMP binding activity than the transformed 3T3 cytosols. The Triton X‐100 treated particulate fractions from the normal and transformed 3T3 cells contained similar protein kinase and binding activities.

The isozymic profile of cAMP‐dependent protein kinases was examined by DEAE‐chromatography. The 3T3 cells contained only type II isozyme in either cytosolic or membrane fractions. All transformants of the 3T3 cells contained both type I and type II isozymes. Other cell cultures, including chicken embryo fibroblasts, rat kidney cells, and human or calf endothelial cells contained type I and type II isozymes.

Binding of the photoaffinity analogue of cAMP, 8‐N3 cAMP, to the regulatory subunits of protein kinases in sonicates obtained from Balb 3T3 and SV 3T3 cells followed by separation on SDS polyacrylamide electrophoresis showed that the amount of RII subunit was approximately equal in the two cell lines. RI in Balb 3T3 cells was detectable but in a much lower quantity than in SV 3T3 cells.

The cyclic AMP dependent‐protein kinases from Balb 3T3 cells appear to be different from SV 3T3 cells by three criteria: 3H‐cAMP binding in homogenates, DEAE chromatographic separation of isozymes, and 8‐N3 cAMP binding.
Effect of aminonucleoside on transcription, methylation, and maturation of ribosomal RNA in SV40‐transformed human lung fibroblasts
Authors: Tunkel Allan R. , Studzinski George P.
Published: February 2005

The effects of puromycin aminonucleoside (AMS) have been studied in the nucleoli of SV40‐transformed human lung fibroblasts with attention to the relationships between transcription, methylation, and maturation of ribosomal RNA (rRNA). Inhibition of the transcription of pre‐rRNA became evident between 1 and 2 hr of exposure to AMS, and the degree of inhibition remained approximately constant throughout the remainder of the 18‐hr period of study. Methylation of the ribose units of pre‐rRNA was inhibited by AMS approximately as much as RNA transcription, but later, suggesting that the inhibition of methylation is a consequence of the lowered rate of RNA transcription. It was also noted that ribose methylation of nucleolar RNA occurs in the absence of concurrent RNA transcription, indicating that rRNA may be methylated post‐transcriptionally. Exposure of the fibroblasts to fresh serum had the capacity to increase the rate of nucleolar RNA transcription if the fibroblasts were treated with AMS for periods of less than 2 hr but was ineffective thereafter, confirming that the inhibitory effect of AMS on pre‐rRNA transcription is established in approximately 2 hr. On the other hand, processing of the primary transcript (45 S RNA) to the mature rRNA species (28 and 18 S) was inhibited more gradually, with a complete inhibition of rRNA maturation being noted after 18 hr of AMS treatment. These data are consistent with the view that the primary effect of AMS is on the rate of rRNA transcription, with a later effect on its maturation, while RNA methylation is reduced only because of the diminished availability of the RNA substrate.
Evaluation of Mg2+ as an intracellular regulator of uridine uptake
Authors: Vidair C. , Rubin H.
Published: February 2005

The rate of uptake of the nucleoside uridine increases within minutes after adding a growth stimulus to quiescent 3T3 cells. We have previously shown this uptake rate to be highly sensitive to changes in the intracellular concentration of Mg2+. In the present paper, the alteration of uptake by Mg2+ is shown to occur at the phosphorylation step — the same point at which serum acts to modulate uridine uptake. The serum stimulation of uridine uptake can be mimicked by Mg2+ alone or blocked by partially depleting cells of their Mg2+. Work with cell‐free extracts shows that the uridine kinase enzyme responds to Mg2+ in a manner similar to that exhibited by whole cells whose concentrations of Mg2+ have been raised. In addition, the enzyme's inhibition by ATP is relieved by raising the Mg2+ concentration. Thymidine uptake, a reaction which does not respond quickly to mitogenic stimulation, is unaffected by alterations in Mg2+ concentration. These results are discussed in terms of a possible role for Mg2+ as an intracellular regulator of uridine uptake and other reactions of the coordinate response of cells to external effectors.
Procaine inhibits the erythroid differentiation of MEL cells by blocking commitment: Possible involvement of calcium metabolism
Authors: Tsiftsoglou Asterios S. , Mitrani Alberto A. , Housman David E.
Published: February 2005

The action of procaine on the terminal erythroid differentiation of murine erythroleukemia (MEL) cells has been investigated at the level of individual cells. At concentrations (7 × 10−4 M) which had no inhibitory effect on cell growth, pretreatment of these cells with procaine for 12–24 hr caused a pronounced inhibition (> 90%) of commitment to terminal erythroid differentiation of dimethyl sulfoxide (DMSO)‐treated cells. Simultaneous treatment of MEL cells with DMSO and procaine, however, resulted to only slight inhibition (< 20%) of commitment. Blockade of commitment by procaine pretreatment appears to be general since it was observed in cells treated with other inducers (6‐thioguanine, dimethylformamide). Procaine pretreatment did not abolish the ability of MEL cells to complete the “latent period” and commit upon the removal of the block. Reversal of procaine inhibition of commitment was obtained by the addition of either CaCl2 (1.0 mM), calcium ionophore A23817 (1 μg/ml), but not of MgCl2 (1.0 mM). From these data we conclude that procaine inhibits the terminal erythroid differentiation of MEL cells by blocking an event or process required for commitment which occurs prior to commitment itself. Our results suggest that this process involves calcium metabolism.
Mechanism of synergistic induction of DNA synthesis by epidermal growth factor and tumor promoters
Authors: Matrisian Lynn M. , Bowden George T. , Magun Bruce E.
Published: February 2005

Evidence is presented to support our previously proposed hypothesis that the hyperplastic effect of tumor promoters is related to their ability to alter existing physiological levels of growth factors in target tissues. Epidermal growth factor and phorbol ester tumor promoters acted synergistically at low (0.001–0.05 ng/ml) but not high (> 0.1 ng/ml) EGF concentrations to induce DNA synthesis in cultured Rat‐1 fibroblast cells. The degree of synergism correlated with the tumor‐promoting ability of the compound. The tumor promoters decreased 125I‐EGF binding to cellular receptors in a dose‐dependent manner that also correlated with the tumor‐promoting ability of the compound. The inhibition of EGF binding by phorbolester compounds resulted in a decrease in the amount of EGF degraded as compared to control cultures. At limiting EGF concentrations, the sparing of EGF degradation resulted in an increase in the amount of EGF remaining in the culture medium after 12 h of incubation and a concomitant increase in the amount of EGF bound to phorbol ester‐treated cells at this time as compared to control cultures. The ability of a phorbol ester compound to alter EGF degradation and to stimulate DNA synthesis synergistically with EGF correlated with the tumor‐promoting ability of the compound and occurred only at low EGF concentrations.
Protection of heat induced cytoxicity by glycerol
Authors: Lin P.‐S. , Kwock L. , Hefter K.
Published: February 2005

Glycerol, at concentrations of 2–10% is a potent hyperthermic (43°–45°C) protector of cultured Chinese hamster cells, V79. Furthermore, the sensitization effect of low pH on heat death is also drastically reduced by the addition of glycerol into the culture medium. Together with the known cellular effects of heat and the role of glycerol in various cellular structures and functions, the data suggest that microtubules and membranes may be involved in the expression of heat‐induced cell death.
Growth, biochemistry, and morphology of isolated rabbit aortic smooth muscle cells maintained in the presence or absence of serum
Authors: Yau‐Young Annie O. , Shio Helen , Fowler Stanley
Published: February 2005

Arterial smooth muscle cells undergo marked biochemical and morphological changes upon culturing. We have studied the time course of these changes in smooth muscle cells isolated from normal rabbit aortas by enzymic digestion and then maintained in Dulbecco's modified Eagle's medium with or without 10% rabbit serum. Subcultured smooth muscle cells were also examined. Isolated cells cultured in the presence of serum multiply rapidly and by 9 days exhibit features typical of subcultured cells including multilayered growth, elevated marker enzyme activities of subcellular organelles, and proliferation of organelles. In contrast, isolated cells cultured in the absence of serum remain quiescent, as indicated by the low level (<10%) of 3H‐thymidine incorporation into nuclei and constant DNA content of the cultures. These cells spread slowly to form a monolayer of randomly oriented cells and they retain differentiated morphological features. Their enzyme activities remain at the levels of those of freshly isolated cells initially, but by 5 days some enzyme activities increase, in particular those of the acid hydrolases and catalase. Rates of pinocytosis and protein synthesis in these cells are comparable to those of cells maintained in serum‐supplemented medium for the same period, but are significantly less than those measured in subcultured cells. Within 5 days, morphological alterations in the serum‐deprived cells occur including the presence of increased numbers of lysosomes. Quiescent cultures of enzymically isolated cells may be a useful tool for short‐term biochemical and physiological studies of differentiated arterial smooth muscle cells.
Effects of prior sterol depletion on neurite outgrowth in neuroblastoma cells
Authors: Maltese William A. , Reitz Beverly A. , Volpe Joseph J.
Published: February 2005

The effect of decreasing cellular sterol content on neurite outgrowth in C1300 (Neuro 2A) neuroblastoma cells in serum‐free medium has been studied. Sterol‐depleted, undifferentiated neuroblastoma cells were obtained by growing cells for 24 h in medium containing lipoprotein‐poor serum and 25‐hydroxy‐cholesterol (25‐OHC). Under these conditions the activity of 3‐hydroxy‐3‐methyl‐glutaryl‐CoA reductase and the incorporation of [14C] acetate into sterols were almost completely suppressed, and the sterol/phospholipid ratio of the cells declined to 60% of that in cultures grown without 25‐OHC. The sterol‐depleted cells were viable and exhibited rates of DNA, RNA, protein and fatty acid synthesis comparable to those measured in control cultures.

Sterol depletion had no detectable effect on the number of cells that were able to undergo morphological differentiation within 3 h after removal of serum from the medium. However, by 24 h most of the sterol‐depleted cells had retracted their neurites. The observation that addition of low‐density lipoprotein was able to restore neurite outgrowth in cultures treated with 25‐OHC indicates that the inability of sterol‐depleted cells to maintain their neurites is related specifically to the decline in the sterol content rather than to a general cytotoxic effect of 25‐OHC. Our findings suggest that incorporation of cholesterol into the cell membrane is important for long‐term maintenance and elongation of neuroblastoma neurites, but that the initial morphological change (i.e., within 3 h after removal of serum) is apparently a separate and distinct event, not dependent on the availability of cholesterol.
Regulation of granulopoiesis and distribution of granulocytes in early phase of bacterial infection
Authors: Hartmann D. W. , Entringer M. A. , Robinson W. A. , Vasil M. L. , Drebing C. J. , Morton N. J. , Ture L.
Published: February 2005

Studies have been carried out to determine the effect of bacterial infection on CSF production, CFU‐C activation, and bacterial clearance by mature granulocytes in mice infected with Escherichia coli. These studies have shown that immediately after bacterial infection (5 minutes), serum colony‐stimulating factor (CSF) levels and bone marrow colony‐forming units in culture (CFU‐C) levels are elevated. This is followed by oscillator rises in both of these parameters and the appearance of granulocytes in the infected site. With clearance of bacteria, CSF and CFU‐C levels return to normal. These studies have indicated further that bacterial infection is a major stimulus for granulocyte production through the CSF‐CFU‐C system and that clearance of bacteria by mature granulocytes may serve as a negative feedback regulatory arm.
Regulation of murine B lymphocyte plasma membrane protein turnover and shedding
Authors: Emerson Stephen G. , Cone Robert E.
Published: February 2005

Lactoperoxidase‐catalyzed cell surface radioiodination was employed to radiolabel murine splenic B‐cell membrane immunoglobulins (IgM and IgD) and alloantigens encoded by the Major Histocompatibility Complex (I‐Ak, I‐Ek, H‐2Kk, H‐2Dk). The fate of the radiolabeled proteins was monitored by in vitro culture of labeled cells and isolation of labeled antigens from detergent lysates of the cells or culture fluids obtained at different times during culture. The effects of temperature, antimetabolites, colchicine, and cytochalasins on membrane protein catabolism demonstrated heterogeneity in rate, energy dependence, and cytoskeletal control of turnover suggesting that functional domains of turnover control exist in the B lymphocyte membrane.
Stimulation of DNA synthesis by transient exposure of cell cultures to TPA or polypeptide mitogens: Induction of competence or incomplete removal?
Authors: Dicker Phillip , Rozengurt Enrique
Published: February 2005

A transient exposure of cell cultures to 12‐0‐tetradecanoyl‐phorbol‐13‐acetate (TPA) is sufficient to stimulate DNA synthesis during a subsequent incubation TPA‐free medium. We show that (1) a substantial fraction of TPA remains bound to cultures following a transient exposure to TPA and thorough washing, (2) the ability of TPA to induce DNA synthesis is a function of the amount of TPA bound to cell cultures irrespective of whether it is incubated continuously with cultures or transiently exposed to cultures under various conditions, and that (3) a transient exposure of cultures to phorbol 12‐13‐dibuytrate (PDB), a mitogenic phorbol ester which binds reversibly to cell cultures, does not stimulate DNA synthesis during a subsequent incubation in PDB‐free medium. Therefore the persisting effects of TPA are due to it binding to cultures in a manner resistant to washing and not due to the induction of a stable cellular change prerequisite for mitogenesis. Further, we show that certain combinations of polypeptide growth factors induce DNA synthesis in the absence of any such stable cellular change. Evidence is also presented that the persisting effects on DNA synthesis following transient exposure of cultures to other polypeptide growth factors (e.g., platelet‐derived growth factor) reflect tenacious binding rather than induction of a lasting biological event.
Induction of human malignant T‐lymphoblastic cell lines MOLT‐3 and jurkat by 12‐O‐tetradecanoylphorbol‐13‐acetate: Biochemical, physical, and morphological characterization
Authors: Nagasawa Kohei , Howatson Allan , Mak Tak W.
Published: February 2005

The process of induction of human malignant T‐lymphoblastic cell line MOLT‐3 by the phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) was examined. It was found that the induction process by TPA, which included increase in cells with receptors to sheep red blood cells (E‐rosette positive‐E+) and decrease in the levels of the marker enzyme terminal deoxynucleotidyl transferase (TdT) was not affected by the presence of DNA synthesis inhibitor arabinofuranosylcytosine (Ara‐C). The exposure time to TPA required to elicit these changes was found to be short, in the order of 1 hour or less. The kinetics of the increase in E+ cells, decrease in the levels of TdT in these cells, or decrease in the ability to proliferate as measured by colony formation were similar with exposure to TPA for 1, 6, 24, or 96 hours. We have examined the effect of antitumor promoter compounds on their ability to block induction of MOLT‐3 cells by TPA. Results indicated that none of these compounds, dexamethasone, antipain, retinoic acid, and L‐1‐tosylamide‐2‐phenylethylchloromethyl ketone (TPCK), was effective in reducing the number of E+ cells induced by TPA.

Examination of three other leukemic T‐cell lines indicated that, in addition to MOLT‐3, the leukemic T‐cell line Jurkat also responded to TPA, whereas two other leukemic T‐cells lines CCRF‐CEM and CCRF‐HSB‐2 did not. Certain physical and morphological changes were also observed after stimulation of MOLT‐3 cells and Jurkat cells by TPA. We found that, following the addition of TPA, the cell volumes of MOLT‐3 cells decreased from an average of 1150 μm3 to about 500 μm3, whereas those of Jurkat were reduced to about 700 μm3 from 1100 μm3. Electron microscopic studies of these lymphoblasts also revealed that after treatment with TPA the induced cells were generally smaller in size with increase in the density of the nuclear materials and condensation of the chromatin structures.
Effect of cycloheximide and dexamethasone phosphate on hyaluronic acid synthesis and secretion in cultured human skin fibroblasts
Authors: Mapleson J. L. , Buchwald M.
Published: February 2005

We have studied some aspects of the regulation of hyaluronic acid synthesis in cultured human skin fibroblasts derived from normal individuals. In these cells 85% of radiolabeled glucosamine is incorporated into two types of macromolecules, hyaluronic acid and glycoproteins, characterized by district partitions between the cell and medium compartments. Treatment of cells with cycloheximide under conditions in which protein and glycoprotein synthesis is inhibited by at least 90% has no effect on hyaluronic acid synthesis. This result is consistent with the hypothesis that protein synthesis is not necessary for the synthesis of hyaluronic acid. Conversely, 5 × 10−6M dexamethasone phosphate inhibits hyaluronic acid by 60% but has no effect on protein or glycoprotein or glycoprotein synthesis. Concomitant treatment with dexamethasone phosphate and cycloheximide restores the synthesis of hyaluronic acid to normal values. The dexamethasone phosphate effect on hyaluronic acid synthesis is therefore not a primary induction response since it requires protein synthesis. The secretion of hyaluronic acid and glycoproteins from the cells into the medium is not affected by either of the two drugs. This experimental approach will be useful in the study of the regulation of glycosaminoglycan metabolism in human cells especially with respect to disorders of connective tissue.
Small nuclear rnas in cellular growth and differentiation. I: Metabolic alterations seen in friend erythroleukemic cells
Authors: Kuncio Gerald S. , Goldstein Lester
Published: February 2005

Electrophoretic analysis of near steady‐state labeled nuclear RNA obtained from Friend virus‐transformed murine erythroleukemic cells reveals the presence of at least 15 small nuclear RNAs (snRNAs) distinct from ribosomal 5.8S or 5S. Identical qualitative distributions were obtained from logarithmically growing, stationary‐phase, and dimethyl sulfoxide‐induced, terminally differentiated cultures, indicating the constitutive synthesis of all snRNAs regardless of the proliferative or differentiated state of the cells. However, several quantitative differences in nuclear snRNA levels were observed. Progression from rapidly growing to stationary‐phase cultures was accompanied by the marked reduction in accumulation of all snRNAs except the 4.5S snRNAs. Particularly striking were the decreases in levels of U3 and the U1 group, snRNAs that are relatively abundant. Similar reductions were noted when cells were induced to differentiate, except that decreases in the levels of U2 and 4.5S were more dramatic than those seen for cells entering stationary‐phase. The data thus demonstrate that snRNA levels may be regulated both in association with changes in proliferative capacity of cells and with changes in gene expression that occur during terminal differentiation.
Adherence of murine t cells to solid substrata in the absence of serum
Authors: Barker Robert H. , O'Shea Patricia F. , Strauss Phyllis R.
Published: February 2005

It is generally assumed that lymphocytes do not adhere firmly to solid substrata. However, in attempting to culture murine spleen and thymus cells in RPMI 1640 without serum, we observed that some cells adhered to glass or plastic surfaces. As a minimum estimate, 10–12% of the applied spleen cells and 22% of those from thymus attached between 1 and 24 hours after plating. The cells remained attached despite extensive and vigorous washing. Viability of 70% was maintained between 4 hours and 3 days in culture. Readdition of 10% mouse or horse serum for 2 hours resulted in removal of 80% of the attached cells. The percentage of adherent cells was not affected by cell density, but was greatly reduced when cells were cultured at 4 °C. Glutaraldehyde‐fixed cells did not adhere. Adherent cells were primarily T lymphocytes. The cell‐plate distance would indicate a focal contact mode of adherence; however, the absence of filamentous material at the adherent surface and the broad, continuous surface apposition would imply a close contact mode. We conclude that attachment modes described for fibroblasts in culture are not applicable for lymphocytes.
The development of lysosomal apparatus. II. Incorporation, subcellular distribution, and intraparticulate hydrolysis of 131 I‐albumin by liver of mice at perinatal stages
Authors: Bertini Francisco , Mayorga Luis , Gonzalez Mercedes
Published: February 2005

Mouse fetuses at 15th and 18th day of gestation, and newborns aging 0, 5, 10, and 15 days were injected i.p. with 131 I‐albumin (RISA). The degree of incorporation by liver and the intraparticulate hydrolysis of RISA in 27,000g × 10 min. particles increased after birth. By this time, changes in subcellular distribution of RISA and marker enzymes were also observed. Following an i.p. injection of India ink, numerous hepatic cells stained with carbon particles were observed at the light microscope from day 5 of life. The results suggest that the lysosomal apparatus acquires full capacity to incorporate colloidal particles and to degrade foreign macromolecules in the first week of life.
Growth of C6 glioma cells in serum‐containing medium decreases β‐adrenergic receptor number
Authors: Dibner Mark D. , Insel Paul A.
Published: February 2005

Rat C6 glioma cells were grown in 5% fetal bovine serum‐containing medium and under serum‐free, defined conditions. In order to ask whether cells grown in serum‐free medium are phenotypically identical to cells grown in serum, we examined effects of cell growth under both conditions on the β‐adrenergic receptor, a cell surface marker that activates adenylate cyclase and thereby regulates these cells. β‐Adrenergic receptors were qualitatively similar in cells grown in serum‐containing and serum‐free media, but the number of receptors was 30‐50% less in cells grown with serum. This effect required several days to occur or to be reversed. The decreased number of receptors appeared to be caused by an inhibitory effect of serum on receptor number and not by a stimulatory action of the constituents of the serum‐free medium. Growth medium with 5% fetal bovine serum maximally inhibited β‐adrenergic receptor numbers with 1% serum causing a half‐maximal inhibition. The ability of serum to inhibit the expression of β‐adrenergic receptors could be blocked by dialyzing but not by boiling fetal bovine serum. β‐Adrenergic receptor‐stimulated cyclic AMP accumulation was also decreased by growth in medium containing serum. These studies demonstrate that compared to growth of cells in serum‐free medium, growth in serum‐containing medium can inhibit expression of cell surface β‐adrenergic receptors. These results imply that the presence of serum in medium in which cells are grown alters properties in the plasma membrane and may alter hormonal responses in such cells.
Megakaryocytopoiesis in the mouse: Response to varying platelet demand
Authors: Burstein Samuel A. , Adamson John W. , Erb Susan K. , Harker Laurence A.
Published: February 2005

The response of murine megakaryocytopoiesis was studied under conditions of varying platelet demand. Twenty‐four hours after mice were given a single injection of rabbit anti‐platelet serum, megakaryocyte number and volume were increased, becoming maximal at 65 and 40 hr, respectively. Total body megakaryocytic colony‐forming unit (CFU‐M) numbers did not change until 90 hr, when a 35% increase in the experimental group was noted. The percentage of CFU‐M in DNA synthesis in the experimental group was 38 ± 2% at 24 hr, 49 ± 1% at 40 hr, and returned to normal (11 ± 3%) at 90 hr. When mice were made thrombocytotic by platelet transfusions, both megakaryocyte number and volume were decreased compared to controls, while no difference was noted in the number and percentage of CFU‐M in DNA synthesis. Finally, experiments were performed to examine the effect of platelet transfusions on regenerating marrow. Experimental mice were given platelet transfusions while control animals received platelet buffer solution. At sacrifice the number and volume of megakaryocytes in the experimental group (platelet count 2.568 × 106/μl) were less than controls (platelet count 0.363 × 106/μl), while the number and percentage of CFU‐M in DNA synthesis were similar in both groups. These results demonstrate that CFU‐M are not immediately responsive to acute changes in platelet demand. The data suggest that megakaryo‐cytopoiesis is structured on at least two levels which are independently regulated.
The relationship between tumor promoter binding and epstein‐barr virus induction in human lymphoblastoid cell lines
Authors: Yamamoto Naoki , Bauer Georg
Published: February 2005

The tumor promoter TPA (12‐0‐tetradecanoyl‐phorbol‐13‐acetate) efficiently induces the synthesis of Epstein‐Barr virus (EBV) antigens in EBV‐genome‐harboring human lymphoblastoid cells. We attempted to obtain information on the binding of TPA to cells and on the relationship between TPA‐binding and EBV induction by the use of tritiated TPA (3H‐TPA). Our data show: (1) In the absence of cells TPA can bind to serum completely within 60 minutes. (2) Cells can compete for a proportion of serum‐bound TPA. (3) Binding of TPA to cells reaches equilibrium within 60 minutes and is higher at 37° than 0°C. In the absence of serum, the rate of binding is about twice as high as in the presence of serum. (4) Dissociation of TPA from cells also seems to be rapid. When the cells are incubated with cold TPA after prior treatment with 3H‐TPA, followed by washing, a much higher rate of release of labeled TPA is observed than in cultures to which fresh medium is added exclusively. Dissociation is higher in the presence of serum than in the absence of it. If radiolabeled cells are analyzed after serial washing, the rate of cell‐associated/noncell‐associated radioactivity indicates that the proportion of molecules required for EBV induction is dissociated rapidly.
The role of protein accumulation in the cell cycle control of human NHIK 3025 cells
Authors: Rønning Ø. W. , Lindmo T. , Pettersen E. O. , Seglen P. O.
Published: February 2005

The cell cycle kinetics of NHIK 3025 cells, synchronized by mitotic selection, was studied in the presence of cycloheximide at concentrations (0.125‐1.25 μM) which inhibited protein synthesis partially and slowed down the rate of cell cycle traverse.

The median cell cycle duration was equal to the protein doubling time in both the control cells and in the cycloheximide‐treated cultures at all drug concentrations. This conclusion was valid whether protein synthesis was continuously depressed by cycloheximide throughout the entire cell cycle, or temporarily inhibited during shorter periods at various stages of the cell cycle. These results may indicate that cell division does not take place before the cell has reached a critical size, or has completed a protein accumulation‐dependent sequence of events.

When present throughout the cell cycle, cycloheximide increased the median G1 duration proportionally to the total cell cycle prolongation. However, the entry of cells into S, once initiated, proceeded at an almost unaffected rate even at cycloheximide concentrations which reduced the rate of protein synthesis 50%.

The onset of DNA synthesis seemed to take place in the cycloheximide‐treated cells at a time when the protein content was lower than in the control cells. This might suggest that DNA synthesis in NHIK 3025 cells is not initiated at a critical cell mass.
Mechanism of glucocorticoid‐mediated inhibition of murine erythroid colony formation in vitro
Authors: Gidari Anthony S.
Published: February 2005

The mechanism by which glucocorticoids influence erythroid colony formation by murine bone marrow cells was studied in a plasma clot culture system. All glucocorticoids tested, including 9α‐fluorocortisol (> 10−8M), cortisol (≥ 10−8M), dexamethasone (> 10−7M), and corticosterone (≥ 10−7M), inhibited erythroid colony formation; none enhanced colony formation. Inhibition by a prototypic glucocorticoid, dexamethasone, was evident at all concentrations of erythropoietin (25–200 mU) in the clots. The percent dexamethasone‐sensitive cells killed by high specific activity [3H]‐TdR (∼ 78%) was the same as the percent erythroid colony‐forming cells killed by this treatment, and the inhibition appears to represent a direct effect on the erythroid colony‐forming cell since: (1) Cells exposed (1 hour) to dexamethasone, washed, and cultured in steroid‐free medium formed a reduced number of colonies (67% of control); (2) dexamethasone decreased colony formation by marrow depleted of adherent cells or obtained from either nude or neonatally thymectomized mice; and (3) co‐cultivation studies of normal marrow with that obtained from glucocorticoid‐treated mice, which forms a reduced number of colonies in vitro, indicated that this inhibition was not the result of enhanced suppressor cell or reduced stimulatory cell activity.

The proliferative rate of colony‐forming cells obtained from dexamethasone‐treated mice, as determined with high specific activity [3H]‐TdR, was reduced ( ∼ 38% of control), suggesting that a reduction in the proliferative rate of the colony‐forming cell is the major mechanism of the glucocorticoid‐mediated inhibition of erythroid colony formation. In addition, dexamethasone, added 24 hours after the initiation of the cultures, inhibited the growth of developing erythroid colonies, indicating a potential inhibitory action on the proliferation of the progeny of the colony‐forming cell.
Differential conjugation of polyamines to calf nuclear and nucleolar proteins
Authors: Haddox Mari K. , Russell Diane Haddock
Published: February 2005

Nuclei and nucleoli isolated from calf liver contain acid‐precipitable putrescine, spermidine and spermine conjugates. The polyamines are released upon peptide bond hydrolysis. All of the nuclear putrescine conjugate and a major portion of the polyamine conjugates are localized within the nucleolus. Nuclei and nucleoli also contain, in proportions consistent with the nucleolar/nuclear protein ratio, the putative conjugating enzyme, transglutaminase, as well as amine acceptor substrates to which radiolabeled putrescine can be conjugated by endogenous enzyme. Extraction of the isolated organelles with saline solutions of increasing ionic strength showed a differential distribution of the polyamine derivatives: all the covalently linked putrescine was associated with the less soluble components of the chromatin residue, while the spermidine and spermine conjugates were associated with several salt‐extractable protein fractions as well as tightly bound to the chromatin pellet. Mono‐γ‐glutamyl putrescine was detected after proteolytic digestion of the 600 mM NaCl fraction, further suggesting the enzymatic action of transglutaminase(s) in the conjugation process.
SV40‐transformed human cells fail to grow in zinc concentrations which permit normal human fibroblast proliferation
Authors: Epstein J.
Published: February 2005

Zinc is a metal known to be required for normal growth of both cells and organisms. When normal and SV40‐transformed human tumor cells are plated and grown in medium containg zinc sulfate, a significant fraction of the transformed cells fail to grow at zinc concentrations which are relatively nontoxic to the normal fibroblasts. Although cultured cells respond to certain metallic ions by incresing their metallothionein content, no difference in the ability of normal and transformed cells to produce metallothionein in response to zinc exposure could be detected, thus ruling out this mechanism as a basic for the differing abilities of the cells to grwo in zinc in vitro. These results suggest that zinc may be capable of differntially regulating the growth of normal and SV40− transformed human fibroblasts.
A biochemical investigation of the adenovirus‐induced G1 to S phase progression: Thymidine kinase, ornithine decarboxylase, and inhibitors of polyamine biosynthesis
Authors: Cheetham Brian F. , Bellett Alan J. D.
Published: February 2005

Biochemical events were investigated in the G1 to S phase progression induced in quiescent rodent cells by human adenovirus type 5 (Ad5) and by serum. Thymidine kinase activity increased after infection of cells with Ad5 or addition of 10% serum. These stimulations were additive. An early viral gene was respnsible for induction by Ad5, but the early mutants ts36, ts37, and ts125 induced thymidine kinase at the permissive and nonpermissive temperatures. Several differences were found between cells stimulated by serum compared with Ad5. Induction of thymidine kinase was delayed in Ad5‐ infected cells, insensitive to 0.01 μ/ml actinomycin D and relatively resistant to reduced Ca2+ compared with induction by serum. Ornithine decarboxylase was induced by serum, but not by Ad5. α‐Methylornithine had little effect on the induction of thymidine kinase by Ad5, but reduced the induction of thymidine kinase by serum, suggesting that Ad5‐induced entry into S phase is uncoupled from polyamine biosynthesis. Methylglyoxal bis(guanylhydrazone), however, prevented the induction of thymidine kinase by both serum and Ad5. Adenovirus infection appears to induce cellular DNA synthesis and thymidine kinase in G1‐arrested cells by a mechanism different from serum, and by passes events in the normal G1 to S phase progression.
Intracellular localization of certain membrane glycoproteins in mouse T‐lymphoma cells using immunoferrtin staining of ultrathin frozen sections
Authors: Bourguignon Lilly Y. W. , Butman Bryan T.
Published: February 2005

Comparative studies on the cellular morphology of cultured mouse T‐lymphoma cells (with particular emphasis on organelles and membrane‐associated materials) were conducted using both frozen thin sections and epon thin sections. Due to the fact that the frozen thin sectioning technique allows antigenicity to be retained and also permits good accessbility of the external macromolecular reagents to the interior of the cell, we have been able to explore the intracellular localization of some membrane glycoproteins such as Con A‐binding sites and viral membrane glycoprotein, gp 69/71. Our data indicate that most of the membranous cellular structures (e.g., rough endoplamic reticulum, vesicles, Golgi and nuclear envelope) contain the Con A‐specific sugars, mannose, and glucose. In addition, we have found that intracellular gp 69/71 molecules exist in an aggregated form at the terminal region of cisternae of rough endoplasmic reticulum and in vesicles of two size ranges (0.1 to 0.15 μ and 0.3 to 0.4μm) as wells as in the cytoplasm close to the plasma membrane. These findings have not only confirmed close some of the previous biochemical data but have also provided new information concening the biochemical nature of intracellular membrane components and the possible biosynthetic fate of membrane precursor molecules.
Effect of growth stage on histone H1 metabolism in human diploid fibroblasts
Authors: Sakagami Hiroshi , Mitsui Youji , Murota Sei‐Itsu , Yamada Masa‐Atsu
Published: February 2005

Metabolism of histone H1 at different stages of cell growth was investigated in order to get a better understanding of the role of histone H1 in the cell growth of human diploid fibroblasts. Histone H1 content exhibited some fluctuation during the culture stage of cell growth. When cells entered confluent phase, the ratio of histone H1 to total histones decreased significantly. Histone H1 had a tunover half‐life of 80 hours whereas nucleosomal histones did not significantly turn‐over regardless of the growth stage. DNA synthesis was drastically diminished with increase cell density whereas histone synthesis was less sensitive to contact inhibition. The gradual decline of histone H1 content with increased cell density suggest that its degradation is slightly superior to its residual synthesis. When the confluent cells were seeded at low density, cell proliferation resumed and histone H1 was synthesized and deposited to chromatin in a greater amoun than nucleosomal histones, thus resulting in an abrupt increase of histone H1 content. The possible role of histone H1 metabolism in normal cell is discussed.
The effect of metabolic inhibition on lon contents and sodium exchange in human lymphocytes
Authors: Negendank William , Shaller Calvin
Published: February 2005

Lymphocytes depleted of ATP by incubation in iodoacetate (IAA) and nitrogen (N2) lost K and gained Na. Isotopic Na exchange showed a fast fraction and a slower exponential fraction, the latter conventionally assumed to reflect surface membrane properties. The gain of cell Na was not accounted for by a decrease in 22Na efflux in either the slow or the fast fraction. After 3–5 hours, Na efflux increased. These results led us to question the concept that normal cell ion levels are maintained by an ATPase pump and could not be explained by exchange diffusion, cotransport, countertransport, or other inherently dissipative mechanisms. The data are, on the other hand, consistent with the concept that cell ion contents are determined by their relative exclusion from cell water coupled with selective adsorption onto fixed macromolecular anionic sites within the cell. In this view, the IAA, N2‐induced rise in cell Na is due to the occupancy of adsorption sites losing K, while the increased isotopic exchange is due to a decreased activation energy for ion‐site interaction.
Hormonal and nutritional factors influencing glycogen deposition in primary cultures of rat liver parenchymal cells
Authors: Weber Charles A. , Kletzien Rolf F.
Published: February 2005

The effect of the glucocorticoids, insulin, and glucose concentration on glycogen deposition in adult rat liver parenchymal cells maintained in a chemically defined, serum‐free medium has been studied. Increasing the medium concentration of glucose from 5.6 mM to 30.6mM in the absence of hormones increased cellular glycogen content from 6.5 to 51 μg of glycogen per mg of cell protein. Treatment of the cells with insulin increased the glycogen content by 15 to 30% at medium glucose concentrations above 10.6 mM. The addition of the synthetic glucocorticoid, dexamethasone, to the culture medium resulted in 40 to 105% increases in glycogen content at glucose concentrations greater than 5.6 mM. The addition of dexamethasone and insulin together in the culture medium resulted in an increase in glycogen content that was greater than the additive effect of each hormone alone. This established that glucose concentrations above 10.6 mM stimulate glycogen deposition in the absence of any hormonal stimulus. In addition, glucocorticoids directly stimulate glycogen deposition at glucose concentrations which are greater than physiological (5.6 mM).
Effects of pyruvate on the growth of normal and transformed hamster embryo fibroblasts
Authors: Sens Donald A. , Hochstadt Bruce , Amos Harold
Published: February 2005

The growth of NIL and NILpy hamster embryo fibroblasts was determined in the presence and absence of pyruvate as a component of the growth medium. It was demonstrated that NIL cells respond to the presence of pyruvate by decreasing cell doubling time, glucose utilization, glutamine utilization, and increasing lactate production with the effects being more pronounced at low inoculum densities. Polyoma‐virus transformed NIL cells (NILpy) demonstrate none of the above effects upon pyruvate addition regardless of initial cell inoculum density.
Modulation of epithelial cell proliferation in culture by dissolved oxygen
Authors: Taylor William C. , Camalier Richard F.
Published: February 2005

Modulation of epithelial cell proliferation by the dissolved oxygen concentration (PO2) of the growth medium was assessed with primary human foreskin epithelium and a continuous monkey kidney epithelial cell line (LLC‐MK2). Direct measurement of the growth medium PO2 provides the first quantitative evaluation of epithelial cell proliferation as a function of PO2 provides the first quantitative evaluation of epithelial cell proliferation as a function of PO2. Sustained proliferation of LLC‐MK2 cells occurs in serum‐free medium equilibrated with a gas phase containing 18% or 30% O2 v/v. Mid‐logarithmic phase cultures rapidly consume dissolved oxygen; this results in a 60–70 mm Hg decline in PO2 and leads to a stable growth medium PO2 between 70 and 100 mm Hg, well above anoxic values. In contrast, if culture medium is equilibrated with a gas phase containing 0% or 1% O2 v/v to yield a growth medium PO2 ∼ 20–40 mm Hg, proliferation of LLC‐MK2 and primary foreskin epithelial cells is retarded, and LLC‐MK2 cells use little dissolved oxygen. Gentle, continuous rocking to prevent diffusion gradient formation enhances proliferation slightly at the higher PO2, but neither periodic fluid renewals nor continued rocking stimulates cells retarded by a lowered oxygen concentration to resume proliferation. The data collectively demonstrate that epithelial cell proliferation requires a PO2 > 40 mm Hg, and threshold requirements are probably closer to 70 mm Hg. Glycolysis continues at a PO2 insufficient for proliferation, but more lactic acid accumulates in actively proliferating cultures than in cultures equilibrated with 0% oxygen. We conclude that epithelial cells in vitro both consume more oxygen and require a higher PO2 for continued proliferation, and that the oxygen requirement for epithelial cell proliferation exceeds that of a comparable population of fibroblasts for which low oxygen may enhance survival and proliferation.
Rous sarcoma virus transformed cells are resistant to cyclic AMP
Authors: Roth Charles W. , Singh Toolsee , Pastan Ira , Gottesman Michael M.
Published: February 2005

A nontransformed line of Chinese hamster ovary (CHO) cells (Pollard and Stanners, 1979) has been transformed by the Schmidt‐Ruppin subgroup D strain of Rous sarcoma virus (SR‐RSV). SR‐RSV transformed CHO cells are shown to differ from spontaneously transformed cells in that the virally transformed cells are more resistant to growth inhibition or changes in cell shape by 8‐Br‐cyclic AMP or cholera toxin. SR‐RSV transformed rat (NRK) cells also have a reduced sensitivity to growth inhibition by 8‐Br‐cyclic AMP. Cyclic AMP‐dependent protein kinase was examined in SR‐RSV transformed CHO cells, but no differences in enzyme level, activation by cyclic AMP, chromatographic behavior, or its ability to phosphorylate endogenous proteins in whole cells could be detected. It is concluded that transformation of CHO and NRK cells by SR‐RSV alters the cells in a manner different from spontaneous transformation, and that this alteration does not affect cAMP‐dependent protein kinase activity.
Separation of isolated hamster intestinal epithelial cells by velocity sedimentation on Ficoll gradients
Authors: Carter Julia H. , Poretz R. D. , Eichholz Alexander
Published: February 2005

Isolated hamster intestinal epithelial cells can be separated by velocity sedimentationion on 2–10% Ficoll gradients into three subpopulations of cells which differ in morphology, biochemistry, physiology, and membrane components. These subpopulations are not pure but are enriched in a single cell type to the extent that differences in cell function can be observed. The proliferative crypt cells are separated from the digestive‐absorptive villus cells. A third subpopulation with a distinctive morphology is also obtained. Quantitation of DNA recoveries from the gradients indicates that this population constitutes approximately one‐third of the epithelial cell population. These carrot‐shaped cells are found adjacent to the digestive‐absorptive columnar epithelial cells on the villus. The two types of villus cells differ in glycolipid or glycoprotein components of the brush border as shown by lectin binding experiments with the isolated cells. The gradient data also suggest that only one‐third of the intestinal epithelial cell population is responsible for most monosaccharide absorption in hamster small intestine.