bluuebunny/crossref_metadata_2025 huggingface dataset:
Intracellular calcium pools and their metabolic dependence in normal versus simian virus 40‐transformed human fibroblasts
Authors: Smith Janet W. , Tupper Joseph T.
Published: February 2005

Previous studies indicate that although normal and Simian virus (SV40)‐transformed WI38 human fibroblasts have similar levels of intracellular Ca++ on a per mg protein basis, their ability to maintain this intracellular Ca++ against a low concentration of extracellular Ca++ differs markedly. The transformed but not the normal cells rapidly lose Ca++ when exposed to low extracellular Ca++, suggesting Ca++ transport and/or sequestration differ in the two cell types. In this study we have extended our investigations of Ca++ metabolism in the two cell types. We observe that normal WI38 cells, when exposed to metabolic inhibitors to deplete intracellular ATP, undergo a twofold increase in intracellular Ca++ levels. Under similar conditions and over the same time course, no comparable change in Ca++ level is observed in the SV40‐transformed cell, despite the extensive depletion of ATP. 45Ca++ desaturation curves indicate that the bulk of the net increase in cell Ca++ following ATP depletion of the normal WI38 cell comes to reside in a slowly exchanging Ca++ pool. The data also indicate that glycolysis, and not oxidative phosphorylation, drives the active extrusion of Ca++ from these cells, an observation consistent with previous studies on the Na+‐K+ pump in other cell types. Finally, the data indicate that in these cells mitochondria do not appear to be the major subcellular organelle responsible for regulation of at least the two cellular Ca++ pools measurable using isotope desaturation analysis. This is based on the inability of the respiratory inhibitor rotenone to alter significantly the size of either of these Ca++ pools. These pools compose 80–90% of total cell Ca++ in both cell types.
Adenosine‐resistant chinese hamster fibroblast variants with hyperactive adenosine‐deaminase: An analysis of the protection against exogenous adenosine afforded by increased activity of the deamination pathway
Authors: Fernandez‐Mejia C. , Debatisse M. , Buttin G.
Published: February 2005

The activity of purine salvage and interconversion enzymes was examined in two sublines of Chinese hamster cells–RA11 and RA41–isolated on the basis of their resistance to adenosine concentrations toxic to wild‐type CCL39 cells. Adenosine deaminase (ADA) activity was found to be two times higher in RA11 and three times higher in RA41 than in CCL39. Inhibition of ADA activity by coformycin reduced the level of adenosine resistance but did not restore wild‐type sensitivity, indicating that a second defect contributes to the adenosine‐resistant phenotype of these variants; evidence was indeed obtained for the presence in both lines of additional alterations protecting them against the lethal depletion of phosphoribosylpyrophosphate (Ishii and Green, 1973) imposed by adenosine to wild‐type cells. To gain better insight into the influence of ADA hyperactivity on adenosine resistance, a procedure was developed for the specific isolation of variants with increased levels of ADA activity. Cell lines with 3–5 times and then 100–500 times the wild‐type ADA activity were stepwise recovered. These investigations confirmed that amplification of ADA can efficiently contribute in protecting cells against high concentrations of exogenous adenosine. The variants isolated by this procedure again manifested, in addition to amplification of ADA activity, another alteration decreasing their sensitivity to adenosine. A possible mechanism accounting for the frequent isolation of variants that coexpress ADA hyper‐activity and a second defect contributing protection against adenosine toxicity are considered.
Factors modifying 3‐aminobenzamide cytotoxicity in normal and repair‐deficient human fibroblasts
Authors: Boorstein Robert J. , Pardee Arthur B.
Published: February 2005

3‐Aminobenzamide (3‐AB), an inhibitor of poly(ADP‐ribosylation), is lethal to human fibroblasts with damaged DNA. Its cytotoxicity was determined relative to a number of factors including the types of lesions, the kinetics of repair, and the availability of alternative repair systems. A variety of alkylating agents, UV or gamma irradiation, or antimetabolites were used to create DNA lesions. 3‐AB enhanced lethality with monofunctional alkylating agents only. Within this class of compounds, methylmethanesulfonate (MMS) treatments made cells more sensitive to 3‐AB than did treatment with methylnitrosourea (MNU) or methylnitronitrosoguanidine (MNNG). 3‐AB interfered with a dynamic repair process lasting several days, since human fibroblasts remained sensitive to 3‐AB for 36–48 hours following MMS treatment. During this same interval, 3‐AB caused these cells to arrest in G2 phase. Alkaline elution analysis also revealed that this slow repair was delayed further by 3‐AB. Human mutant cells defective in DNA repair differed in their responses to 3‐AB. Among mutants sensitive to monofunctional alkylating agents, ataxia telangiectasia cells were slightly more sensitive to 3‐AB than control cells, while Huntington's disease cells had a near‐normal response. Among UV‐sensitive strains, xeroderma pigmentosum variant (XPV) cells were more sensitive to 3‐AB after MMS than were XP complementation group A (A) cells, which responded normally. Greater lethality with 3‐AB could be dependent on inability of the mutant cells to repair damage by other processes.
Flat revertants derived from kirsten murine sarcoma virus‐transformed cells produce transforming growth factors
Authors: Salomon David S. , Zwiebel James A. , Noda Makoto , Bassin Robert H.
Published: February 2005

Two flat cellular revertant cell lines, F‐2 and C‐11, which were originally selected from the DT line of Kirsten murine sarcoma virus (Ki‐MuSV)‐transformed NIH/3T3 cells, were examined for the production of transforming growth factors (TGFs). The revertant cells fail to grow in semisolid medium as colonies and exhibit a markedly reduced level of tumorigenicity in nude mice, although they are known to express high levels of p21ras, the product of the Kirsten sarcoma virus oncogene, ras, and they contain a rescuable transforming virus. TGF activity associated with the transformed, revertant, and non‐transformed cell lines was measured by the ability of concentrated conditioned medium (CM) from these cells to induce normal rat kidney (NRK) and NIH/3T3 cells to form colonies in semisolid agar suspension cultures and to inhibit the binding of 125I epidermal growth factor (EGF) to specific cell surface receptors. CM from the transformed DT cells and from both the F‐2 and C‐11 revertants contains TGF activity, in contrast to CM obtained from normal NIH/3T3 cells. Furthermore, unlike NIH/3T3 cells, neither the DT nor the revertant cells were able to bind 125I EGF. All four cell lines were able to proliferate in serum‐free medium supplemented with transferrin, insulin, EGF, and Pedersen fetuin. However, in basal medium lacking these growth factors, only DT cells and, to a lesser extent, the revertant cells were able to grow. These results suggest that the F‐2 and C‐11 revertants fail to exhibit all of the properties associated with transformation because the series of events leading to the transformed phenotype is blocked at a point(s) distal both to the expression of the p21 ras gene product and also to the production of TGFs and that the production of TGFs may be necessary but not sufficient for maintaining the transformed state.
Glucose metabolism in murine fetal cortical brain cells: Lack of insulin effects
Authors: Gorus Frans K. , Hooghe‐Peters Elisabeth L. , Pipeleers Daniël G.
Published: February 2005

Glucose uptake and oxidation were markedly higher in cultured than in freshly isolated neural cells, prepared from murine fetal brain cortices. The hexose transport process–measured as 3‐O‐methyl‐D‐glucose uptake–appeared comparable in both conditions, and proceeded proportionally to the extracellular sugar concentration up to 6 mM. In contrast, glucose oxidation occurred independently of the prevailing glucose concentration from 1.4 mM on. Acute or chronic exposure to insulin exerted no effect upon cellular glucose uptake or oxidation. These results suggest that glucose handling by maturing fetal cortical cells is mainly determined by the rate of cellular glucose breakdown rather than by the rate of glucose transport into the cell; the marked rise in cellular glucose metabolism during culture might result from the synthesis and/or activation of a key enzyme in glucose catabolism. Our observations also indicate that the previously described neurotrophic effects of insulin are not mediated via enhanced glucose handling.
Regulation of the rate of cell cycle progression in quiescent cytolytic T cells by T cell growth factor: Analysis by flow microfluorometry
Authors: Sekaly Rafick P. , MacDonald H. Robson , Nabholz Markus , Smith Kendall A. , Cerottini Jean‐Charles
Published: February 2005

We have previously shown that >90% of B6.1 cells, a murine cytolytic T lymphocyte (CTL) cloned line which is solely dependent on T cell growth factor (TCGF) for continuous growth in vitro, accumulates in the G1 phase of the cell cycle after transfer into culture medium containing no TCGF. Moreover, when such quiescent cells are exposed again to TCGF, >85% reenter the S phase and subsequently divide in a relatively synchronous fashion. In this study, the regulation of the rate of cell cycle progression of quiescent B6.1 cells after exposure to TCGF was analyzed using two complementary DNA staining techniques, namely, the propodium iodide method (to enumerate cells entering the S phase) and the Hoechst 33342‐bromodeoxyuridine substitution technique (to enumerate cells which have gone through mitosis). After TCGF addition, quiescent B6.1 cells resumed DNA synthesis and divided after a lag phase of 10 and 20 h, respectively. The duration of the lag phase was found to be dependent on the length of time during which quiescent B6.1 cells had been deprived of TCGF, but was independent of the concentration of TCGF used for restimulation. In contrast, the proportion of cells responding to TCGF as well as the rate of their first passage through mitosis was dependent on TCGF concentration. The presence of TCGF for at least 6 h was required for a maximal response. Moreover, direct evidence was obtained that TCGF by itself was able to stimulate proliferation of quiescent B6.1 cells in the absence of other growth factors and serum constituents other than bovine serum albumin, transferrin, and lipids.
Effects of temperature on aggregation and the mitogen‐induced exit of lymphocytes from the resting state
Authors: Hall David J. , O'Leary James J. , Rosenberg Andreas
Published: February 2005

We have determined the temperature dependence of the kinetics of entry into the first S phase of phytohemagglutinin‐stimulated lymphocytes under conditions varying the stability of substrata over which the cells have settled. An exponential model was used to characterize entry into S phase. This model yields as parameters duration of lag period, t0, apparent first order rate constant for entry, k, and the number of cells committed to enter the first S phase, NA(t0). Values of t0 and NA(t0) show a 1.5‐fold and 2.0‐fold decrease and increase, respectively, over a 4°C temperature range and are independent of variation in substrate stability. The temperature dependence of the apparent first‐order rate constant, k, however, is strongly influenced by stability. The observed activation energy increases from 3.0 kcal to 37 kcal when the substratum is agitated. This correlates well with reduced adherence of multicellular aggregates in agitated samples. The temperature dependencies for these three parameters are all numerically different, indicating that these parameters are determined by different rate‐limiting processes. We propose that the mechanism mirrored by k is linked to the adherence of multicellular aggregates to the substratum.
Desensitization of the serum effect on Na+ influx in cultured human fibroblasts
Authors: Villereal Mitchel L. , Owen Nancy E.
Published: February 2005

Stimulation of an amiloride‐sensitive Na+ influx pathway, which mediates Na+/H+ exchange, has been postulated to be an important step in the initiation of DNA synthesis in quiescent human fibroblasts. If the elevation of intracellular Na+ or the alkalinization of intracellular pH resulting from the activation of this system is a trigger for subsequent mitogenic events, then its inactivation may also be important to cellular functions. We investigated the duration of the activation of Na+ influx by serum in human foreskin fibroblasts (HSWP). It was found that activation of Na+ influx by 10% serum was transient, declining with a t1/2 = 15 min. Similarly, the Na+ content of the cells rose rapidly following serum addition and decreased with a t1/2 = 15 min. In addition, both the lys‐bradykinin‐ and the vasopressin‐stimulated Na+ influx and Na+ content declined with a t1/2 of approximately 15 min. Similar results were obtained using both Tris‐buffered and Hepes‐buffered, amino‐acid‐free EMEM. Finally, the above experiments were repeated under conditions normally used to assess the mitogenic response of cells. It was found that in cells arrested in G0 by serum deprivation in CO2‐buffered EMEM, the serum activated Na+ flux was also transient with a t1/2 of approximately 20 min. The desensitization of cells to serum could be readily (t1/2 = 20′) reversed by a subsequent incubation of cells in serum‐free medium. Stimulation of Na+ influx by both the divalent cation ionophore A23187 and the phospholipase activator melittin in also desensitized rapidly, suggesting the process is independent of receptor downregulation. The desensitization during serum preincubation occurred in both low Na+ and low pH medium suggesting that the process is not due to negative feedback on the transport system via a rise in cellular Na+ concentration or a rise in intracellular pH. Although the mechanism of desensitization is at present not known, it is likely to be a physiologically important event.
Sodium‐potassium exchange in sea urchin egg. II. Ionic events stimulating the Na+‐K+ pump activity at fertilization
Authors: Ciapa B. , Allemand D. , Payan P. , Girard J. P.
Published: February 2005

The preceding paper (Ciapa et al., 1984) provided biochemical and kinetic characterization of the Na+‐K+ exchange in Paracentrotus lividus eggs. The present work is a study of the ionic events involved in the stimulation of the Na+‐K+ transporter after fertilization. Fertilization in low Na+‐external medium containing amiloride (0.1 mM) suppresses the stimulation of the net efflux of H+ and 86Rb uptake. Activation of eggs with the ionophore A23187 leads to stimulation of both Na+‐H+ exchange and ouabain‐sensitive 86Rb influx. When eggs were activated with A23187 in artificial seawater, 86Rb uptake and 24Na influx showed similar saturable kinetics with respect to the external Na+. A23187 treatment of eggs in Na+ ‐free artifical seawater did not stimulate the Na+‐K+ exchange until 10 mEq Na+ was added. Activation of eggs by NH4Cl (5 mM) stimulated 86Rb influx and Na+ exit; both fluxes were ouabain sensitive. Monensin increased cell Na+ of unfertilized eggs without any significant increase in intracellular pH: a condition in which 86Rb influx was not markedly stimulated. Addition of 10 mEq Na+ to unfertilized eggs in Na+‐free artificial seawater stimulated 86Rb uptake but to a lower extent that did 10 mEq Na+ plus sperm. It is concluded that (1) the stimulation of the Na+‐K+ pump at fertilization has an absolute requirement for the Na+‐H+ exchange; (2) the alkalinization of eggs resulting from the acid efflux is a prerequisite for the enhancement of the Na+‐K+ pump; (3) the amount of Na+ entering eggs at fertilization determines the intensity of the Na+‐K+ exchange; (4) early events of fertilization such as exocytosis and calcium release which may be involved in the stimulation of the Na+‐K+ pump must necessarily be coupled to cell alkalinization.
Effects of inhibitors of protein degradation on the rate of protein synthesis in Chinese Hamster Ovary cells
Authors: Scornik Oscar A.
Published: February 2005

In the absence of serum and amino acids, cultured Chinese Hamster Ovary cells released to the medium two thirds of the leucine produced by protein degradation. Because protein synthesis requires all the amino acids, the loss of leucine implies incomplete reincorporation of the other amino acids as well. Leupeptin (0.45 mg/ml) and chloroquine (up to 40 μM) inhibited protein breakdown by 21 and up to 41%, respectively, and resulted in proportional decreases in protein synthesis. Chloroquine abolished the stimulation of protein breakdown by amino acid deprivation. From the values of protein synthesis and leucine output with and without chloroquine, it is estimated that the stimulation of protein degradation not only permitted continuing protein synthesis but also increased amino acid output. In the presence of serum or amino acids protein breakdown was slower than in their absence and less sensitive to inhibition by chloroquine, but proportional effects on synthesis and degradation were still observed. It is suggested that protein degradation may be necessary for the maintenance of optimum intracellular concentrations of amino acids even in the presence of extracellular amino acids.
An in vitro clonal assay of adherent stem cells (ASC) in mouse marrow
Authors: Reincke Ursula , Rosenblatt Margery , Hellman Samuel
Published: February 2005

Hematopoietic stem cells with high proliferative capacity can be assayed when stromal bone marrow cultures are overlaid with limiting dilutions of marrow samples. This leads to hematopoietic growth after 4 weeks in a fraction of cultures, consistent with expectations based on Poisson statistics. It will be shown that monoclonal cultures are obtained that last from 2 to 15 weeks and that can generate up to several million mature granulocytes. The originating clone‐forming cell is named adherent stem cell (ASC) because of its adherence to plastic or stromal surfaces. The ASC is comparable to the CFU‐S in frequency, proliferative capacity and in its ability to give rise to CFU‐S. As an unexpected additional finding we report that a mode of “clonal succession” was apparent in cultures which expressed more than one clone.
Flexible association of hemopoietic differentiation programs in multilineage colonies
Authors: Lim B. , Jamal N. , Messner H. A.
Published: February 2005

Pluripotent hemopoietic progenitors lose potentialities during the process of differentiation. We have examined events that lead to lineage restriction by determining the cellular composition of 785 multilineage colonies grown from peripheral blood samples of glucose‐6‐phosphate‐dehydrogenase (G‐6‐PD) heterozygous volunteers. Of these colonies, 762 contained only one isoenzyme type and were considered to be of clonal origin. A considerable heterogenity was observed. Some colonies were composed of cells belonging to two different lineages, while other colonies contained three or more different cell types. A small number of colonies consisted—in addition to myeloid cells—of T‐lymphocytes. The variable association within individual colonies of members belonging to different hemopoietic lineages suggests a flexible determination and expression of differentiation programs by early progenitors.
Interaction of circulating cell‐derived and plasma growth factors in stimulating cultured smooth muscle cell replication
Authors: Clemmons David R.
Published: February 2005

Multiple growth factors that circulate in plasma have been shown to stimulate cellular growth in vitro. The plasma growth factors appear to stimulate DNA synthesis in cultured fibroblasts only after prior exposure of cell growth factors derived from circulating cell types, such as platelets and macrophages. The purpose of these studies was to investigate the role of the plasma growth factors in stimulating smooth muscle cell replication following exposure to platelet‐derived growth factor (PDGF). Following transient exposure to PDGF, insulin stimulated smooth muscle cell replication but only when supraphysiologic concentrations were used (i.e., greater than 1.0 μg/ml). Somatomedin‐C (Sm‐C), in contrast, was found to stimulate a 320% increase in [3H]thymidine incorporation when concentrations that are present in extracellular fluids were used (i.e., 0.5–10 ng/ml). Epidermal growth factor (EGF), an important mitogen for multiple cell types, caused a 70% increase in [3H]thymidine incorporation when added to quiescent cells following PDGF exposure, and EGF caused a substantial increase in the absolute level of [3H]thymidine incorporation when coincubated with Sm‐C. When EGF (1 ng/ml) was incubated simultaneously with concentrations of Sm‐C between 1 and 10 ng/ml plus Sm‐C‐deficient plasma, maximal [3H]thymidine incorporation was 2.1‐fold greater in the presence of EGF. In contrast, insulin (20 ng/ml), when coincubated with Sm‐C under similar conditions, had no enhancing effect on the cellular response to Sm‐C. None of the plasma factors tested was an effective stimultant of replication when incubated either in serum‐free medium or in the presence of Sm‐C‐deficient plasma without prior PDGF exposure. Hydrocortisone was shown to inhibit smooth muscle cell replication in concentrations between 10−7and 10−5M. In summary, multiple plasma growth factors can stimulate the smooth muscle cell replication, and Sm‐C appears to be most effective of those tested. Insulin and EGF appear to work by different mechanisms; that is, EGF can facilitate the cellular response to Sm‐C, whereas insulin is effective only at supraphysiologic concentrations at which it will directly bind to Sm‐C receptors.
Interferon inhibition of thymidine incorporation into DNA through effects on thymidine transport and uptake
Authors: Pfeffer Lawrence M. , Tamm Igor
Published: February 2005

Replenishment of medium after 72 hr of growth of HeLa‐S3 cells in dense suspension cultures increased [3H]‐thymidine uptake into cells and incorporation into DNA, with the levels reaching a peak ∼ 12 hr following medium change; β interferon inhibits the enhanced uptake of [3H]‐thymidine and labeling of DNA in a dose‐dependent manner. Some reduction in these processes is observed at a concentration as low as 1 u/ml, and ∼ 75% inhibition at 640 u/ml. Kinetic analysis has revealed that the rate of labeling of the acid‐soluble pool with [3H]‐thymidine, measured either at 22°C, or 37°C, is reduced in interferon‐treated (640 u/ml, 24 hr) HeLa‐S3 cells. At 22°C, the initial rate of thymidine transport at a high (500 μM) thymidine concentration, determined within the first 30 sec of [3H]‐thymidine addition was depressed by 44% in interferon‐treated HeLa cells. At 37°C, labeled precursors accumulate in acid‐soluble material for ∼ 8 min after the addition of [3H]‐thymidine, after which an apparent equilibrium level is attained. At this temperature, the rate of thymidine uptake and the apparent equilibrium level attained were depressed by 70% in interferon‐treated HeLa cells. The reduced incorporation of [3H]‐thymidine into DNA in interferon‐treated HeLa‐S3 cells can be largely explained by interferon inhibition of thymidine transport and phosphorylation.
Treatment of lymphoblastoid cells with interferon decreases insulin binding
Authors: Faltynek Connie R. , McCandless Sarah , Baglioni Corrado
Published: February 2005

Lymphoblastoid Daudi cells, which are highly sensitive to growth inhibition by interferon (IFN), can be grown in a defined serum‐free medium containing insulin, transferrin, and albumin as the only proteins. We examined whether the growth inhibition by IFN could be in part due to a change in receptors for insulin or transferrin. Cells treated for at least 2 days with 100 units/ml of IFN‐α2 bound less 125I‐insulin and after 3 days of treatment this binding was reduced by more than 50%. No change in the binding of 125I‐transferrin was observed. Treatment with IFN of Raji cells, which are resistant to growth inhibition by IFN, resulted in a similar decrease in 125I‐insulin binding. Growth inhibition of Daudi cells by serum deprivation had no effect on 125I‐insulin binding. Therefore, the IFN‐induced loss of insulin binding sites is not a consequence of growth inhibition.
Self‐inhibition of chloride transport in ehrlich ascites tumor cells
Authors: Levinson Charles
Published: February 2005

Previous studies have shown that mediated Cl− transport which occurs by at least two processes (Cl−‐dependent cation cotransport and Cl− self‐exchange) becomes progressively inhibited when extracellular Cl− exceeds about 60 mM (Hoffmann et al., 1979). To account for this type of kinetic behavior, that is, self‐inhibition, an anion transport system possessing two sites, a high affinity transport site and a lower affinity modifier site is suggested (Dalmark, 1976). In the present experiments we have attempted to determine which of the mediated transport pathways is susceptible to self‐inhibition by studying the dependence of the steady state Cl− flux on the extracellular Cl− concentration and how DIDS, an inhibitor of Cl− self‐exchange, and H+ affect this relationship. Addition of DIDS to Ehrlich cells results in inhibition of Cl− transport at every Cl− concentration tested (40–150 mM). Moreover, the Cl− flux/Cl− concentration relationship no longer exhibits self‐inhibition, suggesting that this phenomenon is a characteristic of the Cl− self‐exchanger rather than of the Cl−‐dependent cation cotransport system. Lowering the extracellular pH (pHo) from 7.35 to 5.30 stimulates Cl− transport by a process that saturates with respect to [H+]. Half‐maximal stimulation occurs at pHo 6.34. A comparison of the kinetic parameters, Ks and Jmax, calculated from the ascending limb of the Cl− flux/Cl− concentration curve at pHo 7.30 to those at pHo 5.50 show that the values for Ks are almost identical (23.6 mM and 21.3 mM, respectively), while the values for Jmax [(22.2 mEq/Kg dry wt). min] differ by only 15%. This finding along with the observation that DIDS completely blocks H+ stimulation of Cl− transport is compatible with the suggestion that H+ interact at the modifer site of the Cl− self‐exchanger and thereby prevents self‐inhibition.
Apparent desensitization of swiss 3T3 cells to the mitogens FGF and vasopressin
Authors: Richmond K. M. V. , Riddle P. N. , Brooks R. F.
Published: February 2005

The growth factors FGF and vasopressin were found to have only a transient effect on confluent quiescent monolayers of Swiss 3T3 cells. Whether measured as cumulative entry into S‐phase by autoradiography, or as cell division by time‐lapse filming, the elevated rate of cell proliferation was maintained only over 10‐15 hr. Several trivial or artifactual explanations for this transience were ruled out, including toxicity of 3H‐thymidine; exhaustion or degradation of medium components, nutrients or growth factors (although some medium depletion was observed); and the generation during quiescence of cells incapable of division. We have also eliminated heritable variation in the capacity to respond to individual growth factors. However, unstable phenotypic heterogeneity in growth factor requirements between cells may play some part, as found elsewhere for the response to low concentrations of serum (Brooks et al, 1984). Cell populations that had ceased to respond to vasopressin recovered their sensitivity after 2‐3 days' incubation in conditioned medium lacking vasopressin. The phenomenon thus resembles the mitogen‐induced desensitization described by Collins and Rosengurt (1982, 1983). However, in our case, the loss of sensitivity was not selective for vasopressin but applied also to epidermal growth factor (EGF) and to prostaglandin F2α. Furthermore, changes in responsiveness to vasopressin with time were associated with changes in cell density. Although some element of selective desensitization has not been ruled out, the transient response in our experiments can be accounted for in terms of unstable heterogeneity in growth factor requirements and/or in terms of density‐dependent regulation of growth.
Variant (MDCK) kidney epithelial cells altered in response to inducers of dome formation and differentiation
Authors: Lever Julia E.
Published: February 2005

Confluent cultures of the MDCK kidney epithelial cell line exhibit dome formation, a result of transepithelial fluid transport influenced by cell‐cell and cell‐substratum interaction. Dome formation was inducible by hexamethylene bisacetamide (HMBA) or dimmethylformamide (DMF), compounds known as inducers of cell differentiation (Lever, 1979b). Analysis of the incidence of the dome‐forming phenotype in colonies derived nonselectively from the MDCK cell line suggested that inducers recruit an increased fraction of the cell population to express dome formation. Variant MDCK cell lines were isolated which differed from the parental line in response to inducers while retaining cuboidal epithelial morphology. In five independently isolated and cloned MDCK variants, dome formation was not inducible by DMF and only marginally increased by HMBA. This phenotype was also associated with increased cell adhesiveness to a plastic substratum. Results from cocultivation experiments suggested that the DMF‐unresponsive phenotype of variant cells may be partially overcome by cell‐cell contact with wild‐type cells. Sodium pump transport activity assessed by ouabain‐sensitive Rb+ uptake was partially inhibited by HMBA and by DMF in a “wild‐type” inducer‐responsive clone. By contrast, DMF did not inhibit ouabain‐sensitive Rb+ uptake in DMF‐unresponsive variant clones, and sodium pump inhibition by HMBA was greatly diminished. This close correspondence between altered sodium pump modulation by inducers in variant clones and their altered dome‐forming response reinforces our previous conclusions (Kennedy and Lever, 1984) that sodium pump modulation is closely associated with mechanisms of inducer action. Taken together, these findings implicate cell‐cell interaction, cell‐substratum interaction and sodium pump modulation in regulation of the differentiated phenotype of this cell line.
The requirements for ionized calcium and magnesium in lymphocyte proliferation
Authors: Abboud C. N. , Scully S. P. , Lichtman A. H. , Brennan J. K. , Segel G. B.
Published: February 2005

The extracellular ionized calcium and magnesium requirements for lectin‐induced lymphocyte DNA synthesis were measured in a serum‐free system. The use of this system permitted measurements of the ionized calcium and magnesium concentrations with ion‐selective electrodes. Maximal DNA synthesis was observed at 270 μ ionized calcium and at 100 μ ionized magnesium in phytohemagglutinin‐treated lymphocytes. Lymphocyte DNA synthesis was much more sensitive to reduction of external ionized calcium than to reduction of ionized magnesium. In calcium‐free medium (ionized calcium 25 μM), DNA synthesis was reduced by 90%, but in magnesium‐free medium (ionized magnesium concentration 7 μM) DNA synthesis was reduced by only 30%. Fifty percent of DNA synthesis stimulated by phytohemagglutinin (PHA) and concanavalin A (Con A) was observed at external ionized calcium concentrations of 97 and 43 μM, respectively. When lymphocytes were stimulated with PHA and the external calcium was chelated with EGTA, 50% inhibition of DNA synthesis was observed at 98 μM ionized calcium. This value agreed well with the free calcium required for PHA activation of DNA synthesis (97 μM). Cytoplasmic calcium, measured with the fluorescent probe Quin 2, increased following lectin exposure if the extracellular ionized calcium concentration was greater than 80 μM. No increase in cytoplasmic calcium could be detected in lectin‐treated lymphocytes below 80 μM extracellular ionized calcium, although substantial DNA synthesis was sustained.
Amino acid analogs while inducing heat shock proteins sensitize CHO cells to thermal damage
Authors: Li Gloria C. , Laszlo Andrei
Published: February 2005

Amino acid analogs have been shown to induce heat shock proteins (HSPs). We have examined the effect of these analogs on the thermal sensitivity of Chinese hamster fibroblasts (HA‐1) and their stable heat‐resistant variants. We found that exposure of HA‐1 cells and their heat‐resistant variants to canavanine or L‐azetidine‐2‐carboxylic acid cause enhanced synthesis of the three major mammalian HSPs (molecular weight 70,000, 87,000, and 110,000 kd). Although the synthesis of HSPs was increased, the analogs did not induce thermotolerance, a transient ability to protect cells from thermal damage. On the contrary, the analog treatment increased the thermal sensitivity of HA‐1 cells, but not of the heat‐resistant strains, when these cells were exposed subsequently to elevated temperatures. Our tentative explanation for these findings is that the incorporation of amino acid analogs into HSPs or other cellular proteins sensitizes HA‐1 cells to heat. The heat‐resistant strains contain higher levels of constitutive HSPs. The additional functional HSPs in the heat‐resistant variants may protect these cells from thermal stress. The presence of some newly synthesized analog‐substituted, perhaps nonfunctional, HSPs need not affect this thermal protection.
Differential responsiveness of cultured suckling and adult rat hepatocytes to growth‐promoting factors: Entry into s phase and mitosis
Authors: Baribault Hélène , Leroux‐Nicollet Isabelle , Marceau Normand
Published: February 2005

The capacity of suckling and adult rat hepatocytes in culture to enter into S phase and mitosis in response to EGF, insulin, and glucagon was measured. Both cell types were isolated in high yield and purity and cultured in the absence of serum under identical conditions. At the time of isolation, suckling rat hepatocytes were all diploid and in the G1 phase of the cell cycle. Adult rat hepatocytes constituted a population of mixed ploidy level, as shown by flow cytometry. Upon stimulation, both suckling and adult rate hepatocytes entered S phase after a minimum lag period of 24 h. For suckling rat hepatocytes EGF was required, but its stimulating action was dependent on insulin and/or glucagon. In contrast, adult rat hepatocytes entered into S phase in response to EGF alone; insulin and glucagon did not significantly potentiate its effect. Under optimal hormonal stimulation for entry into S phase a large proportion of suckling rat hepatocytes underwent mitosis, whereas only a few mitoses were observed in the case of adult rat hepatocytes. Therefore, there is a differential response of suckling and adult rat hepatocytes to growth factors which correlates with ploidy level, and this difference may be associated with the degree of maturation.
Cyclic AMP‐independent processes mediate Kirsten sarcoma virus‐induced changes in collagen production and other properties of cultured cells
Authors: Majmudar Gopa , Peterkofsky Beverly
Published: February 2005

Previous studies suggested that the decreased collagen production observed in Kirsten sarcoma virus (Ki‐MSV)‐transformed BALB 3T3 cells could be reversed by treating cells with Bt2cAMP. We examined the relationship between intracellular cAMP, collagen production, and other properties in NRK and BALB 3T3 cells transformed by Ki‐MSV. Two 3T3 transformants (Ki‐3T3‐234 and Ki‐3T3Cl1) had lower cAMP levels than nontransformed cells. The level in a temperature‐sensitive transformant, tsKi‐3T3‐714, was similar to the level in its parent, 3T3‐714, and when it was shifted to a temperature nonpermissive for transformation (40°C), intracellular cAMP did not increase although the growth and morphological properties were normal. The relative rate of collagen production also increased to the normal level. These results indicate that transformation‐induced changes were regulated independently of cAMP. Further observations supported this conclusion. Intracellular cAMP in a flat revertant of Ki‐3T3Cl1 was lower, rather than higher, than in the transformant, although the relative rate of collagen production was higher. Treatment of Ki‐3T3‐234 and tsKi‐3T3‐714 with cholera toxin plus isobutylmethylxanthine increased intracellular cAMP concentrations to 2–20 times the level in untreated cells, levels much higher than in nontransformed 3T3. In spite of this, collagen production was not increased by these agents in tsKi‐3T3‐714 and it was only partially restored in Ki‐3T3‐234 relative to the level in the nontransformed cells. In contrast, these agents inhibited growth on a substratum or in soft agar and produced a flattened morphology in both lines. Similarly, collagen production in transformed NRK cells (K‐NRK) was only 3% of normal but treatment with Bt2cAMP or cholera toxin plus isobutylmethylxanthine increased production to only one‐third the normal level while increasing cAMP to four times the normal level. We conclude that in Ki‐MSV‐transformed BALB 3T3 cells, changes in cAMP may be secondary effects and not related to maintenance of the transformed phenotype. The high levels of cAMP induced by exogenous agents may act on similar targets as those affected by transformation, but reversal of the transformed phenotype by these agents probably occurs by a different mechanism than that originally used to impose the changes.
Hormonal regulation of the system a amino acid transport adaptive response mechanism in a kidney epithelial cell line (MDCK)
Authors: Boerner Paula , Saier Milton H.
Published: February 2005

When mamalian cells are starved for amino acids, the activity of the A amino acid transport system increases, a phenomenon called adaptive regulation. We have examined the effects of those factors which support Madin‐Darby canine kidney (MDCK) cell growth in a defined medium on the derepression of System A activity. Of the five factors which supported MDCK cell growth, insulin was found to be an absolute requirement for derepression. In contrast, PGE1 was a negative controlling factor for the transport system. Growth of MDCK cells in the absence of PGE1 resulted in elevated System A activity which derepressed poorly upon amino acid starvation. Kinetic analysis of α‐(methylamino) isobutyric acid (mAIB) uptake as a function of substrate concentration showed that the elevated A activity observed when cells were grown in the absence of PGE1 was kinetically similar to the activity induced by starvation for amino acids. Transport of mAIB by amino‐acid‐fed cells grown in the presence of PGE1 was characterized by a linear Eadie‐Hofstee graph and by a relatively low Vmax. Transport by cells starved for amino acids or by cells grown in the absence of PGE1 was characterized by biphasic kinetics for mAIB transport and by elevated Vmax values. An influence of growth factors on the inactivation of derepressed A activity was also observed. In the presence of cycloheximide the rate of loss of A activity in amino‐acid‐starved cells was 1/4–1/2 that of amino‐acid‐fed cells. Insulin slowed inactivation in the absence of most amino acids in a protein‐synthesis‐independent manner, but insulin did not influence the more rapid inactivation observed in amino‐acid‐fed cells. These results indicate that the level of System A activity observed in response to regulation by amino acids represents a balance between carrier synthesis and inactivation, which can be positively or negatively influenced by growth factors.
Synthesis of extracellular matrix glycoproteins by cultured microvascular endothelial cells isolated from the dermis of neonatal and adult skin
Authors: Kramer R. H. , Fuh G. M. , Bensch K. G. , Karasek M. A.
Published: February 2005

We examined the synthesis of extracellular matrix macromolecules by human microvascular endothelial cells isolated from the dermis of neonatal (foreskin) and adult (abdominal) skin. Electron microscopy showed that both cell types produced an extracellular matrix that was strictly localized to the subendothelial space. The subendothelial matrices were initially deposited as a single discontinuous layer of filamentous, electron‐dense material that progressively became multilayered. Biosynthetic studies indicated that 2–4% of the newly synthesized protein was deposited in the subendothelial matrices by both cell types. Approximately 15–20% of the radiolabeled protein was secreted into the culture medium, and the remainder was confined to the cellular compartment. Biochemical and immunochemical analyses demonstrated the extracellular secretion of type IV collagen, laminin, fibronectin, and thrombospondin by the newborn and adult cells. Whereas type IV collagen was the predominant constituent of the matrix, fibronectin was secreted into the medium, with only small amounts being deposited in the matrix. Thrombospondin was a major constituent of the matrix produced by the newborn foreskin cells but was virtually absent in the matrix elaborated by the adult cells. However, both cell types did release comparable amounts of thrombospondin into their medium. Immunoperoxidase staining for type IV collagen revealed a fibrillar network in the subendothelial matrices produced by both adult and neonatal cells. In contrast, thrombospondin, which was detected only in the matrix of newborn cells, exhibited a spotty and granular staining pattern. The results indicate that the extracellular matrices synthesized by cultured human microvascular endothelial cells isolated from anatomically distinct sites and different stages of development and age are similar in ultrastructure but differ in their macromolecular composition.
The effects of taxol, a microtubule‐stabilizing drug, on steroidogenic cells
Authors: Rainey W. E. , Kramer R. E. , Mason J. I. , Shay J. W.
Published: February 2005

The effects of taxol on steroid production and microtubule polymerization were examined using Y‐1 adrenocortical tumor cells, MLTC‐1 Leydig tumor cells, and primary cultures of bovine adrenocortical cells. Taxol inhibited the following steroidogenic processes within the Y‐1 and MLTC‐1 cells: (1) hormonal increase of steroid production, (2) dibutyryl cyclic AMP‐increased steroid production, and (3) hormone‐stimulated pregnenolone production. The inhibitory action of taxol was concentration dependent and also resulted in an increase in cytoplasmic microtubules. In addition, the inhibitory action of taxol on hormone‐stimulated steroid production was reversible. Taxol appeared to inhibit cholesterol movement to the mitochondrial site of cholesterol side‐chain cleavage enzyme but did not affect overall protein synthesis. Interestingly, taxol did not affect hormone‐stimulated steroid production in bovine adrenocortical cells. This lack of inhibition may correspond to the ultrastructural observation that microtubule bundling after taxol treatment was observed in the tumor cells but not in similarly treated bovine adrenal cells. With this conflicting information between cell types, a direct relationship between taxol treatment and inhibition of steroid production has not been established. However, these results suggest that taxol alters the rate of transport of cholesterol to the cholesterol side‐chain cleavage enzyme within the steroidogenic tumor cells.
Stochastic branching model for hemopoietic progenitor cell differentiation
Authors: Kurnit David M. , Matthysse Steven , Papayannopoulou Thalia , Stamatoyannopoulos George
Published: February 2005

We present algebraic expressions describing the predictions of a stochastic branching model for differentiation of hemopoietic progenitor cells. The model assumes that there is a fixed probability, p (0 ⩽ p ⩽ 1), that commitment to a differentiative event occurs per progenitor cell division for each daughter cell. The model describes properties of in vitro hemopoietic cell differentiation including the population structure at the time the first progenitor cell becomes committed, the number of committed progenitor cells engendered by a single progenitor cell, and the probability of eventual commitment of all daughter cells derived from a single progenitor or stem cell. Application of the model to experimental data obtained from erythroid cultures suggests that the observed data can be explained by the stochastic branching model alone without making the deterministic assumption that there is a differentiative hierarchy in the lineage of the progenitors of erythropoiesis (BFU‐E). The qualitative and quantitative aspects of the proposed stochastic model are discussed in conjunction with other analogous stochastic branching models.
Pseudomonas aeruginosa cytotoxin stimulates prostacyclin production in cultured pulmonary artery endothelial cells: Membrane attack and calcium influx
Authors: Suttorp Norbert , Seeger Werner , Uhl Jürgen , Lutz Frieder , Roka Ladislaus
Published: February 2005

The effects of highly purified Pseudomonas aeruginosa cytotoxin were investigated on cultured pulmonary artery endothelial cells. This toxin dose‐dependently (7.5–60 μg/ml) and time‐dependently (20–75 minutes) stimulated the release of radiolabeled arachidonic acid and metabolites and the synthesis of prostacyclin in the absence of overt cell damage (no enhanced lactate dehydrogenase [LDH] release). Preincubation of the toxin with neutralizing antibodies abolished the effect. The toxin response on endothelial cells required extracellular calcium but not magnesium and was accompanied by a calcium influx. Interference with intracellular calcium function by TMB 8 or with (calcium)‐calmodulin function by trifluoperazine and W7 dose‐dependently reduced the cytotoxin mediated synthesis of prostacyclin. Calcium channel blockers (nimodipine, diltiazem, verapamil, D 888), however, were ineffective in this system. Following addition of cytotoxin to endothelial cells, an increased passive permeability for small marker molecules (potassium, 45calcium, 3H‐sucrose), but not for large ones (3H‐inulin, 3H‐dextran, LDH) was noted, suggesting that cytotoxin creates discrete hydrophilic transmembrane lesions of about 0.5‐1.5 nm in diameter. These data are compatible with the notion that Pseudomonas aeruginosa cytotoxin triggers the arachidonic acid pathway in cultured pulmonary artery endothelial cells by calcium influx and suggest that this calcium influx may proceed through toxin created transmembrane lesions.
A comparison of protein synthetic patterns of MDCK cells grown in serum and hormonally defined serum‐free medium
Authors: Jefferson Douglas M. , Yang Chia‐Ping H. , Cobb Melanie H. , Scott Walter N.
Published: February 2005

The development of hormonally defined serum‐free media (HDM) for the culture of mammalian cells has allowed the study of specific cellular functions in a totally defined environment. Recently, several reports have indicated that there are differences in the basic cellular physiology of cells cultured in HDM when compared to cells cultured by the more traditional method of using serum‐supplemented culture media (SM). We report here that there are significant changes in the protein synthetic pattern in MDCK cells grown in HDM. There were no changes exhibited during the first passage in HDM, but following 10 passages in HDM there was an increased isotope ratio of (1) plasma membrane proteins with molecular weights of 12,000, 36,000 and 68,000 and (2) endoplasmic reticulum proteins with molecular weights of 12,000 and 37,000. Additionally, the incorporation of methionine and uridine were significantly increased in cells cultured in HDM for 10 passages. At present, we believe that these changes in the protein synthetic patterns are due at least partially to increased protein synthesis as indicated by monosome/polysome ratios. Therefore, though the use of HDM offers a completely defined system for studying cellular function, results obtained using HDM must be interpreted with caution when comparing them to previous studies that have used SM.